Targeting the two-pore channel 2 in cancer progression and metastasis.

Introduction

Ca 2+ is a major second messenger in cells and alterations in intracellular Ca 2+ signaling regulate a variety of biological processes, including exocytosis/endocytosis, cell proliferation, invasion, migration, and apoptosis. Dysregulation of Ca 2+ signaling is increasingly being demonstrated to contribute to the development of cancer [ 1 ], and has been implicated in each of the hallmarks of cancer originally identified by Hanahan and Weinberg [ 2 ].

For the most part, alterations in intracellular Ca 2+ occur due to the opening of Ca 2+ -channels. Many different types of Ca 2+ -channels are found in cells; some are expressed on the plasma membrane [e.g., voltage-gated Ca 2+ channels, transient receptor potential (TRP) channels, and store-operated channels], whereas others are expressed on the membrane of intracellular organelles, such as ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3 R) located on the sarcoplasmic and endoplasmic reticulum. Two-pore channels (TPCs) are unique in that they are located on the acidic organelles of the endolysosomal system [ 3 ].

The endolysosomal system is a complex pathway of dynamic organelles responsible for the delivery of cargo from the cell surface to internal lysosomes via trafficking through early endosomes and late endosomes. Final transfer to lysosomes is achieved by the fusion of late endosomes with lysosomes to form transient hybrid organelles known as endolysosomes, from which lysosomes are eventually reformed. These acidic, protease-laden organelles were referred to as “suicide bags” by the scientist who discovered them [ 4 ], making them attractive targets for cancer therapies that could permeabilize their membranes to allow toxic hydrolases to escape into the cytosol. But it is now known that the endosomal network is used for a multitude of cellular functions, including recycling and cell signaling, cell death and survival, and ultimately maintaining cell homoeostasis [ 5 ]. Thus, the seemingly simple concept of lysosome membrane destabilization as a therapeutic option has taken on more complexity, but also offers more potential.

The recently identified TPC2 (also known as TPCN2) Ca 2+ -channel provides the seemingly perfect anti-cancer target. Although the full range of TPC2 functions are only beginning to be understood, it has already emerged as an intracellular ion channel of significant pathophysiological relevance, including in a variety of cancer-related functions. This review will summarize developments in our understanding of TPC2 in pathophysiological conditions, with a focus on cancer, and highlight the pre-clinical evidence for targeting TPCs as a novel anti-cancer therapeutic strategy.

TPC structure and activation

TPCs comprise a family of voltage- and ligand-gated Na + /Ca 2+ ion channels exclusively located in endolysosomes. TPC1 and 3 are voltage-gated channels, whereas TPC2 opens in response to binding phosphatidylinositol-3,5-diphosphate [PI(3,5)P 2 ] and nicotinic acid adenine dinucleotide phosphate (NAADP) [ 6 – 8 ]. However, the role of TPC2 in NAADP-mediated Ca 2+ release has been controversial [ 9 , 10 ]. In an effort to resolve this controversy, Jha et al. [ 11 ] identified regulators of TPC2, and demonstrated that Mg 2+ can inhibit these NAADP-mediated Ca 2+ currents, but that when Mg 2+ is absent, NAADP can activate TPC2, which may account for the conflicting findings.

While three distantly related genes ( TPC1 – 3 ) have been identified, humans and rodents only express two isoforms, TPC1 and TPC2 [ 12 ], encoded by genes in humans located on chromosomes 12 and 11, respectively. TPC2 expression is localized to the late endosome and lysosomal membranes, whereas TPC1 shows a broader distribution throughout the endolysosomal system, particularly in earlier, less acidic endosomal compartments [ 7 , 13 ]. While the name “TPC” suggests an ion channel with two separate pores, TPCs actually consist of two subunits forming a dimer. Each TPC subunit is comprised of two homologous shaker-like six transmembrane domain repeats (labeled IS1–S6 and IIS1–S6) ( Figure 1 ), with the two-pore loop domains located in IS5–S6 and IIS5–S6 ( Figure 1 ). PI(3,5)P 2 binds to the first 6 transmembrane domains to activate the channel independently of voltage changes, inducing a structural change at IS6 [ 14 ]. However, the binding region of NAADP remains unknown. TPC1 and TPC2 subunits form homo- and hetero-dimers, with four pore domains forming the central channel in a two-fold symmetric arrangement [ 15 , 16 ].

TPC interactome

While NAADP can endogenously regulate Ca 2+ release via TPC2, how it does so remains unknown, and it has been suggested that a separate unidentified NAADP-binding protein acts to accessorize TPC2 activation. As a result, the identification of NAADP-TPC2 interactomes is currently an area of great interest ( Figure 2 ), and proteomic characterisation has unsurprisingly revealed that TPC2 complexes with proteins involved in Ca 2+ homeostasis, trafficking and membrane organisation, including scaffold Rab GTPases, where Rab binding was essential for NAADP-evoked TPC2 Ca 2+ release [ 18 ]. TPC2 can also associate with the transient receptor potential mucolipin 1 (TRPML1) ion channel, but despite this interaction, TRPML1 and TPC2 function as independent ion channels [ 19 ]. Although the physiological relevance of this interaction remains unknown, it is most likely related to the control of lysosomal pH. Additionally, TPC2 can associate with the leucine-rich repeat kinase 2 (LRRK2), mutations in which are linked to late-onset Parkinson’s disease [ 20 ], and the mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that when hyperactivated leads to increased growth and proliferation [ 10 ]. These findings suggest that TPC2 may be involved in the pathological processes of both Parkinson’s disease and cancer. Silencing of endogenous expression of TPC2 , but not TPC1 , reduces store-operated Ca 2+ entry (SOCE), which is modulated through the endoplasmic reticulum Ca 2+ sensor stromal interaction molecule-1 (STIM1) and plasma membrane Orai1 Ca 2+ channels, both of which were found to associate with TPC2 [ 21 ]. A yeast 2 hybrid assay identified hematopoietic cell-specific protein 1 (HS1)-associated protein X-1 (Hax-1) as a novel binding partner for both TPC1 and TPC2 [ 22 ]. Hax-1 has recently been implicated in apoptotic signaling [ 23 ], and the TPC/Hax-1 interaction may potentially play a role in modulating apoptosis. A recent study has identified a largely uncharacterized Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2 [ 24 ], thus potentially identifying the missing accessory protein that has been postulated to exist for NAADP-mediated activation of TPC2. A better understanding of the TPC interactome may potentially lead to the identification of effective pharmacological modulators of TPC-mediated cellular functions, which will have broad implications for a variety of disease processes.

TPC2 functions

TPC2 plays important roles in various diseases and physiological conditions, including cell proliferation, differentiation, development, autophagy, membrane trafficking, endolysosomal degradation pathway, phagocytosis, angiogenesis, low-density lipoprotein (LDL)-cholesterol trafficking, T cell activation, hormone secretion from the pancreas, and cardiac function [ 25 – 34 ]. Consequently, NAADP/TPC2/Ca 2+ signaling has been shown to play a critical role in a variety of associated pathophysiological processes, including the life cycle of Ebola virus, human immunodeficiency virus (HIV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Parkinson’s disease, Alzheimer’s disease, non-alcoholic fatty liver disease, and cardiac dysfunction [ 28 , 31 , 35 – 42 ], thus highlighting the importance of the endolysosomal system in a variety of pathophysiological processes. Importantly, pharmacological inhibitors of TPC2 are potentially beneficial for the treatment of Ebola virus, SARS-CoV-2, Parkinson’s disease, and Alzheimer’s disease [ 35 , 40 , 41 , 43 ], highlighting that TPC2 may be a suitable drug target for a plethora of pathophysiological processes.

Of particular note, TPC2 is overexpressed or mutated in several cancer types. Additionally, TPC2 has recently been implicated in a variety of cancer-related processes (such as proliferation, adhesion, migration, invasion, angiogenesis, and autophagy) that underpin the key hallmarks of cancer [ 2 ] ( Figure 3 ).

TPC2 function in cancer-related processes

Cell growth and differentiation

A role for TPC2 in proliferation was first postulated based on the finding that TPC2 function could be regulated by the protein kinases, p38 mitogen activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), and the mTOR complex 1 (mTORC1) [ 11 ]. Since then, TPC2 has been directly implicated in controlling cancer cell growth and differentiation in a variety of different cell types. Knockdown of TPC2 expression in pulmonary artery smooth muscle cells, MNT-1 human melanoma cells, RIL175 murine hepatocellular carcinoma cells, and 4T1 murine breast cancer cells significantly decreased proliferation in vitro [ 29 , 44 – 46 ], demonstrating that TPC2 can control both normal and cancerous cell proliferation. Additionally, decreasing TPC2 expression in hepatocellular carcinoma cells reduced glycolysis and respiratory activity in vitro [ 45 ], potentially accounting for these proliferative effects, and completely abrogated hepatocellular carcinoma tumor growth in vivo [ 45 ]. Further, knockout of the TPCN2 gene in murine primary melanoma cells decreased initial proliferation rates by slowing G 2 phase progression [ 47 ]. By contrast, TPC2 overexpression in 4T1 breast cancer cells did not influence proliferation [ 29 ], suggesting that this control of proliferation may not be related to the level of TPC2 expression, but rather to the presence of TPC2. Taken together, these results indicate that TPC2 controls cancer cell proliferation highlighting that it may be a useful target for controlling cancer cell growth.

Changes in TPC2 expression have been observed at various stages in differentiation in multiple cell types, suggesting that TPC2 may control differentiation. For example, TPC2 mRNA expression was highest in the first 3 days of differentiation of C2C12 myoblast cells but was reduced thereafter, and TPC2 knockdown inhibited differentiation of these myoblasts [ 48 ]. Similarly, TPC2 mRNA was increased in osteoclast precursor cells upon receptor activator of nuclear factor kappa B (NFκB) ligand (RANKL)-induced osteoclast differentiation, and downregulation of TPC2 suppressed osteoclastogenesis [ 49 ]. By contrast, in mouse day 3 embryonic stem cells, TPC2 was markedly decreased during the early stages of neural differentiation but increased at later times during neuronal differentiation [ 50 ]. As a result, TPC2 knockdown accelerated the initial commitment of neural progenitors, but inhibited later neuronal differentiation. Additionally, overexpression of TPC2 induced cell death and prevented embryonic stem cells from differentiating into early neural lineages [ 50 ]. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes or oligodendrocytes [ 50 ]. Taken together, these studies demonstrate that the role of TPC2 in promoting differentiation is cell type specific.

Cancer cell migration, invasion, and metastasis

Emerging evidence has also implicated TPC2 in all the major steps of tumor metastasis, namely adhesion, migration, and invasion. Knockdown of TPC2 expression in T24 bladder cancer and CHL1 and B16-F10 melanoma cells, and pharmacological inhibition in T24, HUH7 liver cancer and 4T1 breast cancer cells, significantly reduced adhesion [ 47 , 51 ]. Mechanistically, the knockout of the TPC2 gene in CHL1 and B16-F10 melanoma cell lines decreased adhesion to collagen type I matrix and was associated with a decrease in α2β1 integrin expression on the plasma membrane [ 47 ]. While pharmacological inhibition resulting in reduced adhesion to collagen type I matrix may initially appear to be counterintuitive and to promote metastasis, several studies have shown that metastasis is instead promoted by increased adhesion of mda-9/syntenin protein to collage type I and activation of β1 integrin signaling complexes [ 52 ]. Whether decreasing adhesion to collagen via pharmacological inhibition of TPC2 promotes or inhibits metastasis remains to be seen, highlighting that the future application of pharmacological inhibitors of TPC2 needs to be cautiously evaluated to ensure that they do not promote metastasis in patients.

In support of TPC2 having a metastasis promoting role, knockdown of TPC2 expression in MNT-2 melanoma cells, T24 bladder cancer cells, RIL175 hepatocellular carcinoma, 4T1 breast cancer, Hela cervical cancer and pulmonary artery smooth muscle cells, and pharmacological inhibition in T24, HUH7 and 4T1 breast cancer cells led to decreased migration in vitro [ 29 , 44 – 46 , 51 ]. Mechanistically, the downregulation of TPC2 resulted in accumulation of enlarged acidic vesicles, as general recycling was impaired, which halted β1-integrin trafficking, and prevented leading-edge formation [ 51 ], providing further support that pharmacological inhibition of TPC2-mediated inhibition of β1-integrin signaling will prevent, rather than promote, metastasis. By contrast, TPC2 overexpression in 4T1 breast cancer cells had no effect on migration in vitro [ 29 ]. Taken together, these studies suggest that TPC2 can control cancer cell migration in vitro , however, similarly as to what was observed for proliferation, the presence of TPC2, rather than an increased level of expression, may be the only requirement for the regulation of migration.

TPC2 has also been implicated in invasion in melanoma cells. Knockout of TPC2 in MNT-1 human melanoma cells significantly reduced invasion in vitro [ 44 ], providing further support for TPC2 possessing a metastasis promoting role . By contrast, knockout of the TPC2 gene in CHL1 and B16-F10 primary murine melanoma cells increased invasion, with an associated increase in matrix metalloproteinase 9 (MMP-9) expression [ 47 ]. These conflicting studies highlight that additional examination of the precise role of TPC2 in cancer, particularly melanoma, is required to identify whether TPC2 promotes or prevents invasion and metastasis.

Taken together, these in vitro studies demonstrate the importance of TPC2 in adhesion, migration, and invasion, and suggest that TPC2 may promote metastasis in vivo . Indeed, knockdown of TPC2 expression and pharmacological inhibition in 4T1 orthotopic breast cancer and RIL175 hepatocellular carcinoma mouse xenograft models reduced the formation of lung metastases in vivo [ 45 , 51 ], providing additional evidence that TPC2 is an important enhancer of metastasis in vivo , particularly for breast and hepatocellular cancer, and suggest that TPC2 may be a potential therapeutic target for preventing cancer metastasis, at least in breast and hepatocellular cancers. Because the expression levels of TPC2 seem to be less important than its presence for most cancer-associated functions, further investigation into TPC2 binding partners in specific cancer cell types will ultimately be required to unravel the pathways responsible for the varying effects observed.

Angiogenesis

Tumor cells are highly dependent on the formation of new blood vessels to sustain their insatiable growth requirements. This pathological process of neoangiogenesis involves stimulation of normally quiescent endothelial cells through their VEGF receptors to induce proliferation, migration, and capillary branch formation. Production of VEGF by tumor cells is the key mediator of this process, but VEGF also affects immune cells in the tumor micro-environment as well as providing an autocrine pro-malignancy signal that promotes epithelial-mesenchymal transition of the cancer cells [ 53 ]. VEGF inhibitors initially appeared promising as anti-cancer therapies but were ultimately overcome by tumor cells that developed compensatory ways to survive [ 54 ]. The discovery that signaling through the VEGFR2 receptor subtype involves NAADP and TPC2-dependent lysosomal Ca 2+ release [ 33 ] offers alternative strategies for targeting this pathway. In these studies, TPC2, but not TPC1, knockdown in endothelial cells inhibited VEGF-induced Ca 2+ release and angiogenesis in vitro [ 33 ]. Further, pharmacological inhibition of TPC2 decreased angiogenesis in glioma and breast cancer models in vivo [ 55 – 57 ]. Additionally, Tpcn2 −/− mice or pharmacological inhibition of TPC2 inhibited vascularisation of VEGF-containing matrigel plugs in vivo [ 33 , 58 ]. TPC2 has also been implicated in controlling VEGF-dependent neovascularization in a mouse model of age-related macular degeneration [ 59 ], demonstrating that these angiogenic pathways are likely to be a normal response to VEGF-VEGFR2 signaling in endothelial cells. In contrast, autocrine VEGF signaling in cancer cells is expected to be dysregulated through alterations in TPC2 function. VEGFR2 is expressed by a multitude of cancers, ranging from solid tumors (such as brain, gynaecological, gastrointestinal, lung) to those of the haematopoietic system (multiple myeloma, myeloid leukaemias) [ 3 ]. Thus, pharmacological targeting of TPC2 will have many effects on many cell types, with the potential to alter both the tumor and its microenvironment.

Autophagy

Autophagy is a lysosomal dependent mechanism used to naturally rid the cell of dysfunctional or redundant elements and is a critical regulator of cellular homeostasis. In extreme cases of starvation, a cell will use autophagy as a source of energy to maintain survival, and defects in this pathway have been associated with numerous human diseases, including cancer [ 60 ]. However, autophagy plays a dual role in cancer, where it can play a tumor suppressive role in normal tissue but promote tumorigenesis in cancer tissue, by increasing proliferation, angiogenesis, metastasis, and decreasing apoptosis [ 61 ]. The lysosomal location of TPC2 implies involvement in autophagic pathways, but this is enigmatic and there is debate as to whether TPC2 promotes or hampers autophagy.

Several studies have indicated that TPC2 is a positive regulator of autophagic flux. Overexpression of TPC2 in astrocytes increased the expression of the autophagic markers LC3-II and beclin-1, as well as acidic vesicular organelle formation [ 62 ]. Conversely, expression of a dominant-negative TPC2 construct led to decreased LC3-II levels in these astrocytes, and knockdown of TPC2 expression decreased autophagy in astrocytes in an mTOR-independent manner [ 63 ]. Furthermore, in cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 expression that paralleled increased autophagy [ 64 ]. Additionally, silencing of TPC2 alone or in combination with TPC1 in nutrient-rich conditions induced autophagy, whereas under starvation, silencing of either TPC1 or TPC2 induced an autophagic block [ 64 ]. This study suggests that TPC2 is mainly required for basal autophagy, while both TPC1 and TPC2 are required for starvation-induced autophagy. Additionally, one of the identified TPC2 binding partners, LRRK2, is a regulator of autophagy, involving activation of the calcium/calmodulin-stimulated protein kinase kinase β (CaMKKβ)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway [ 20 ]. These LRRK2-mediated effects on autophagy could be completely abrogated by the expression of a dominant-negative, but not wild-type, TPC2 construct [ 20 ], identifying a potential functional role for the LRRK2/TPC2 interaction.

By contrast, there is also evidence that TPC2 can act as a negative regulator of autophagic flux. Overexpression of TPC2, but not an inactive mutant form of TPC2, in Hela cervical cancer and 4T1 breast cancer cells decreased autophagosomal-lysosomal fusion, resulting in the accumulation of autophagosomes [ 29 , 65 ]. Conversely, TPC2 knockdown in mouse embryonic stem cells promoted autophagosomal-lysosomal fusion [ 65 ], and autophagy-related gene 5 ( ATG5 ) knockdown, but not mTOR activity inhibition, abolished the TPC2-induced accumulation of autophagosomes [ 65 ]. Further, skeletal muscles from Tpcn2 −/− mice exhibited enhanced autophagy flux characterized by increased accumulation of autophagosomes upon starvation and treatment with the microtubule inhibitor, colchicine [ 66 ]. Taken together, the studies demonstrating a positive regulation of autophagy effect for TPC2 have largely involved examination in normal cells, whereas the studies showing a negative regulation of autophagy mostly investigated cancer or embryonic stem cells, suggesting that the cell type will influence the TPC2-mediated effect on autophagy.

Further controversy surrounds the mechanisms by which TPC2 may mediate autophagy. While the TPC-mediated effects in astrocytes occurred in an mTOR-independent manner [ 63 ], pharmacological inhibition of TPC2 in HGC-27 gastric cancer, T24 and 5637 bladder cancer cells and MDA-MB-231 breast cancer cells induced autophagy by inhibiting mTOR signaling pathway [ 67 – 69 ], suggesting that TPC2- mediated positive regulation of autophagy in normal cells may occur in an mTOR-independent manner, but that TPC-mediated negative regulation of autophagy may occur in an mTOR-dependent manner.

TPC2 alterations and expression in cancer

As TPC2 has been implicated in several cancer related processes, it raises the question as to whether abnormalities in TPC2 gene coding or protein expression may be observed in cancer. Inherited polymorphisms in TPC2 have been identified as predisposition factors for a range of cancer types [ 70 – 72 ], most notably melanoma due to the role of TPC2 in controlling pigmentation through regulation of melanosomes [ 73 ]. However, acquired abnormalities within the TPC2 coding region appear restricted to rare instances of gene fusions in breast cancer and uterine leiomyomas [ 74 , 75 ]. The chromosomal region harbouring TPC2 (11q13.2) is commonly amplified in cancer [ 76 ], and overexpression of the TPC2 gene has been identified as the potential driver of this amplification by providing growth advantage in oral squamous cell carcinoma [ 77 , 78 ]. Hepatocellular carcinoma patient samples show highly positive staining for TPC2, and the majority of breast cancer and non-tumorigenic breast samples express TPC2 [ 51 , 79 ]. Additionally, increased expression of TPC2 has been observed in cell lines derived from blood, liver and bladder cancer relative to MDA-MB-231 breast cancer cells [ 51 ].

TPC2 expression is also a potential prognostic biomarker for several types of solid tumors. TPC2 was part of a gene signature for biomarkers of lymph node metastasis in oral squamous cell carcinoma, recurrence in prostate cancer, and decreased overall survival in bladder cancer [ 77 , 80 , 81 ], and has been associated with increased risk of melanoma [ 82 ]. Taken together, these studies indicate that TPC2 is highly expressed in hepatocellular carcinoma, oral squamous cell carcinoma and blood cancers, however, whether this expression is observed in additional cancer types, remains to be seen. Additionally, increased TPC2 expression may be a biomarker for poor prognosis for several types of cancer, including oral, prostate and bladder cancers. These negative effects on patient prognosis are most likely due to the central role of TPC2 in regulating cancer cell proliferation and metastasis and highlight that inhibiting TPC2 activity is likely to be an anti-cancer therapeutic strategy for a range of cancer types.

Pharmacological inhibition of TPC as an anti-cancer strategy

Due to the importance of TPC2 in controlling cancer-related functions ( Figure 3 ), its suitability as an anti-cancer target is beginning to be appreciated. Several pharmacological inhibitors of TPC2 have been identified ( Table 1 ) and their pre-clinical efficacy in a range of cancer types has recently begun to be explored ( Table 2 ).

Naringenin

Naringenin, a natural flavonoid found in citrus and tomatoes, is one of the main flavonoids present in the human diet [ 84 ]. Naringenin has a wide range of potential applications, including anti-viral, anti-ageing, anti-inflammatory, lipid lowering, anti-microbial, blood thinning and cardioprotective properties [ 84 ]. Anecdotally, higher serum concentrations of flavonoids, including naringenin, are significantly associated with lower breast, lung, and gastric cancer risk [ 85 – 89 ], suggesting that naringenin may possess anti-cancer properties. Mechanistically, naringenin can inhibit TPC2 and dampen the NAADP-stimulated Ca 2+ response [ 58 ], and as such has been explored as a TPC2 inhibitor. However, naringenin also has other non-TPC2 related actions, and is predicted to act as an estrogen receptor α (ERα) agonist [ 90 ], so its effects cannot be attributed solely to TPC2 inhibition.

Naringenin exhibits anti-proliferative and pro-apoptotic effects in glioma, melanoma, breast, colorectal, hepatic, prostate, lung, and pancreatic cancer in vitro [ 91 – 114 ], and in glioma, melanoma, breast, colorectal, hepatic, and lung cancer xenografts in vivo [ 91 , 96 – 98 , 115 – 121 ] ( Table 2 ). By contrast, HCT116, HT29, and T84 colorectal cancer cells were only slightly sensitive to naringenin treatment in vitro but combined with 5-fluorouracil synergistically enhanced cell death [ 122 ]. Additionally, combining naringenin with paclitaxel synergistically increased cytotoxicity in prostate cancer cells in vitro [ 123 ], and when combined with histone deacetylase inhibitors, synergistically suppressed neuroblastoma tumor progression in vivo [ 124 ]. Taken together, these studies suggest that naringenin can act as a chemosensitiser. Indeed, naringenin has been demonstrated to alleviate multidrug resistance against gemcitabine and anthracyclines in a range of cancer types [ 106 , 125 ]. It is likely that naringenin will be most beneficial clinically in a combinatorial setting to alleviate multi-drug resistance in advanced cancers.

Further, naringenin inhibits migration of glioma, melanoma, breast, prostate pancreatic, and lung cancer cells in vitro [ 97 , 104 – 106 , 109 , 112 – 114 , 128 ] ( Table 2 ), suggesting that naringenin may act as an anti-metastatic agent, and that pharmacologically inhibiting TPC2 will inhibit, rather than promote metastasis. Indeed, naringenin can decrease lung metastases in 4T1 breast cancer and B16F10 melanoma xenografts in vivo [ 110 , 126 , 127 ]. Anti-cancer agents that are effective against metastases are rare, and these pre-clinical findings highlight that naringenin, and inhibiting TPC2, may be a promising avenue for the treatment of metastatic cancers.

Several studies have investigated the mechanisms underlying naringenin-mediated anti-cancer effects and have identified a variety of mechanisms ( Table 2 ), including immunomodulation, NFκB-signaling, ROS-mediated induction of apoptosis, PI3K/Akt pathway, and TGFβ-mediated signaling pathways. Many of these pathways are known to be activated in cancer cells, thus providing additional support for naringenin being a suitable anti-cancer agent.

In an attempt to improve the effectiveness of naringenin, derivatives modified at position 7 have been developed. Initial examination has shown that HCT116 colon cancer cells were more sensitive to these derivatives than to naringenin [ 181 ], however, examination in additional clinically relevant laboratory models is required to ascertain whether cancer cells in general are more sensitive to these derivatives. Further, additional flavonoids from a Southeast Asian plant extract ( Dalbergia parviflora ) block TPC2 activity in melanoma cells and inhibit melanoma cell proliferation, migration, and invasion in vitro [ 44 ]. Further evaluation of these flavonoids is required; however, they may be an additional avenue of promising research.

Despite the abundance of pre-clinical evidence indicating that naringenin is a potent anti-cancer agent with low toxicity ( Table 2 ), this has not been investigated clinically in cancer. Importantly, bioavailability of ingested natural compounds may be lower than required to exert an anti-cancer effect at a cellular level. However, clinical trials evaluating the safety and pharmacokinetics of naringenin in healthy adults have shown that naringenin ingestion had no related adverse events or alterations in blood safety markers [ 182 ]. Most promising, maximal blood concentrations of 48.45 ± 7.88 μmol/L were achieved within 4 h, with a half-life of 2–3 h. These concentrations are compatible with those required for inhibitory effects against cancer cells in vitro , with reported IC 50 values ranging from 2.2–178 μmol/L for most cell lines [ 183 ]. Future efforts should involve focusing on clinical trials of naringenin, both alone and as a chemosensitizing agent, in advanced cancers, particularly breast cancers.

Tetrandrine

Tetrandrine is isolated from the plant Stephania tetrandra and belongs to the class of bisbenzylisoquinoline alkaloids [ 184 ]. Tetrandrine has been used as a traditional medicine in China for decades to treat patients with autoimmune and inflammatory pulmonary disease, silicosis, cardiovascular diseases, and hypertension [ 185 ]. Tetrandrine acts as a Ca 2+ channel blocker, and recently, tetrandrine was shown to block TPC2 currents elicited by both NAADP and PI(3,5)P 2 [ 35 ]. Additionally, tetrandrine inhibits the downstream regulator CaMKII [ 165 ], a kinase that has been implicated in cancer progression and metastasis [ 1 , 186 , 187 ]. This raises the question as to whether tetrandrine is blocking TPC2 itself or inhibiting downstream Ca 2+ -signaling pathways. Tetrandrine can also suppress another Ca 2+ -mediated process, specifically the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome activation via Sirt-1 [ 188 , 189 ]. Taken together, these studies highlight that tetrandrine is not a specific TPC2 inhibitor, but rather inhibits Ca 2+ -signaling related processes. Nevertheless, emerging evidence indicates that tetrandrine possesses anti-cancer efficacy against a range of cancer types ( Table 2 ).

Tetrandrine displayed anti-proliferative and cytotoxic effects as a monotherapy against AML, glioma, neuroblastoma, osteosarcoma, nasopharyngeal, lung, oral, gastric, prostate, bladder, breast, renal cell, hepatocellular, colorectal, gallbladder, pancreatic, and ovarian cancer cells in vitro [ 55 , 56 , 67 , 68 , 129 – 135 , 137 – 146 , 148 – 150 , 152 , 154 – 157 , 160 – 162 , 164 , 165 , 167 – 175 ], and decreased tumor growth in glioma, osteosarcoma, AML, breast, hepatocellular, cervical, pancreatic, colorectal, and ovarian cancer xenograft models in vivo [ 55 , 56 , 130 , 132 , 140 , 147 , 156 , 159 , 162 , 163 , 168 , 169 , 172 , 174 , 175 ] ( Table 2 ). By contrast, despite nasopharyngeal cancer cells exhibiting sensitivity to tetrandrine in vitro , nasopharyngeal carcinoma xenografts were not sensitive to tetrandrine as a monotherapy in vivo , however tetrandrine was able to sensitise these tumors to irradiation [ 190 ], suggesting that it may act as a radiosensitising agent. Further, tetrandrine can also synergistically enhance the effects of numerous chemotherapeutics, including 5-flourouracil, imatinib, paclitaxel, vincristine, daunorubicin, cisplatin, sorafenib, arsenic trioxide, chloroquine, and the protein kinase inhibitor H89 both in vitro and in vivo [ 169 , 175 , 191 – 198 ], indicating that like naringenin, tetrandrine is also a chemosensitizing agent.

There is also substantial pre-clinical evidence that tetrandrine may be effective against metastatic disease. Tetrandrine treatment decreases invasion and migration of a range of cancer types in vitro [ 51 , 133 , 134 , 136 , 150 , 151 , 153 , 158 , 169 , 171 , 172 ], and importantly, the formation of lung metastases in an orthotopic 4T1 breast cancer and an intravenous CT-26 colorectal cancer model in vivo [ 51 , 57 , 166 ] ( Table 2 ), indicating that tetrandrine may be an effective agent for the treatment of metastatic disease.

Several studies have investigated the mechanisms underlying tetrandrine-mediated anti-cancer effects ( Table 2 ). Although a variety of different mechanisms were identified, several were repeatedly observed, including ROS-mediated induction of apoptosis, PI3K/Akt/mTOR signaling, and caspase activation, suggesting that these may underpin the anti-proliferative and pro-apoptotic tetrandrine-mediated effects observed in a variety of cancer types.

Taken together, these studies highlight that tetrandrine is an interesting drug candidate for the treatment of cancer as a monotherapy and in combination with a variety of chemotherapeutics. While a large amount of pre-clinical evidence demonstrated that tetrandrine is effective in both in vitro and in vivo cancer models ( Table 2 ), the majority of these studies were performed in two-dimensional cell culture models in only a single cancer cell line. Not all promising in vitro effects will translate into in vivo or clinical effects, therefore further examination in clinically relevant in vivo models is required before future clinical examination is performed. Additionally, it is likely that tetrandrine will be more beneficial clinically when combined with existing chemotherapeutics.

Nevertheless, a clinical trial examining the combination of intravenous tetrandrine with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed AML showed that this combination was relatively well tolerated. Forty-two percent of patients achieved complete remission or restored chronic phase, 23% achieved a partial response, and 34% failed therapy [ 199 ], this provides the first evidence that tetrandrine may be a suitable therapy for the treatment of cancer. An additional clinical trial examined tetrandrine in combination with gemcitabine and cisplatin for patients with advanced non-small cell lung cancer. While the addition of tetrandrine to this regimen did not significantly improve the objective response rate, it did improve quality of life scores and mitigated adverse reactions to the chemotherapy [ 200 ], providing support for tetrandrine as a chemosensitiser.

Despite these promising results, several animal models have identified that tetrandrine can induce substantial liver and lung damage [ 201 – 203 ], which has raised concerns as to the use of tetrandrine in cancer patients. As such, analogues of tetrandrine, with lower toxicity profiles are under development [ 184 ]. Several of these analogues (RMS1–2, RMS4, RMS7–8) can inhibit proliferation and in some cases (RMS1, RMS4, RMS8) induce apoptosis of leukaemia cells that are resistant to a variety of chemotherapeutic drugs [ 184 ]. An additional analogue (H1) decreases proliferation and clonogenicity of colorectal, non-small cell lung cancer cells, and doxorubicin-resistant breast cancer cells in vitro [ 204 – 206 ]. Tetrandrine analogues that are potent TPC2 specific inhibitors have been identified by screening a library of synthesised benzyltetrahydroisoquinoline derivatives. One of these analogues, SG-094, suppresses tumor growth of RIL175 hepatocellular carcinoma xenografts in vivo [ 45 ]. While these initial results are promising, examination in additional more clinically relevant laboratory models is required.

Ned-19

Ned-19 is a selective membrane-permeable non-competitive NAADP antagonist [ 207 ], which likely inhibits TPCs in an indirect manner [ 208 ]. Ned-19 has primarily been used as an experimental tool to study the functions of TPCs, however, several studies have demonstrated pre-clinical efficacy for Ned-19 ( Table 2 ). Ned-19 treatment decreased melanoma and colorectal cancer cell proliferation and induced apoptosis in B16 melanoma cells in vitro [ 176 , 177 ]. These studies further highlight the role of TPC2 in controlling important cancer-related functions and provide further evidence for the suitability of TPC2 as a potential anti-cancer target.

Further evidence indicates that Ned-19 may be a potential inhibitor of metastasis. Importantly, Ned-19 decreased migration of melanoma, hepatic, bladder, and breast cancer cells in vitro [ 51 , 177 ], and decreased the formation of lung metastases in 4T1 breast cancer and B16 melanoma xenograft models in vivo [ 51 , 176 ]. While these results are promising, additional examination of Ned-19 is required before its clinical utility can be examined.

Verapamil

Verapamil is a Ca 2+ -channel blocker [ 209 ], used clinically to manage angina, arrhythmia and hypertension. Verapamil has also been shown to block NAADP-induced Ca 2+ release [ 210 ], and hence antagonise TPCs. Based on the pre-clinical evidence for the other TPC inhibitors, it would be expected that verapamil also possesses anti-cancer properties. However, controversy exists concerning the potential association between Ca 2+ -channel blockers, particularly verapamil, and increased risk for the development of breast cancer [ 211 ]. Indeed, verapamil has a growth stimulatory effect on breast cancer cells in vitro [ 178 ], suggesting that it may indeed act as a tumor promoter for breast cancers. By contrast, verapamil inhibited proliferation ex vivo of tumor cells collected from AML, but not CML patients in blast phase [ 179 , 180 ]. Taken together, these studies highlight that verapamil exhibits dramatically different effects in different cell types, emphasising that a better understanding of the signaling pathways altered by verapamil in different cancer types is required.

Despite these conflicting results on cancer cell proliferation, verapamil has been widely established to act as a chemosensitiser, by inhibiting the function of p-glycoprotein [ 212 – 214 ]. While in vitro evidence spanning several decades indicates that verapamil can sensitise a range of cancer cells to treatment with chemotherapeutics, including doxorubicin, daunorubicin, vincristine, paclitaxel, mitomycin and amlodipine [ 215 – 221 ], this efficacy has not always translated into improved survival benefit or tumor reduction in in vivo models. For example, verapamil + doxorubicin combination treatment did not increase survival or reduce metastatic burden in a 4T1-resistant breast cancer xenograft mouse model in vivo [ 215 ]. Additionally, orally administered verapamil significantly increased doxorubicin toxicity in mice [ 222 ]. Further, intravenous administration of verapamil in combination with doxorubicin and vincristine in patients with advanced and refractory breast cancer potentiated neurotoxicity and haemotoxicity [ 223 ]. By contrast, arterial infusion of verapamil alone and in combination with chemotherapeutics has been demonstrated to improve medical imaging parameters without increasing toxicity in primary liver cancer patients [ 224 ]. Taken together, these pre-clinical and clinical studies indicate that the route of administration of verapamil can influence the toxicity and chemosensitizing activity, and that arterial infusion of verapamil is the optimal clinical administration method. In support of this, targeted arterial perfusion of verapamil and chemotherapeutics (platinum therapy in combination with paclitaxel, docetaxel, 5-fluorouracil, and paclitaxel + gemcitabine + 5-fluorouracil) in advanced lung cancer patients [ 225 ] was clinically effective, where 85% of patients achieved a complete or partial remission, and all side-effects experienced with the combinatorial treatments resolved quickly [ 225 ]. In addition to being well tolerated, combining arterial verapamil with platinum and 5-fluorouracil and an anthracycline, or with docetaxel and anthracycline in advanced gastric cancer patients, significantly improved the efficacy over chemotherapy alone, and also improved progression-free and overall survival rates compared to chemotherapy alone [ 226 ]. These clinical studies identify that verapamil is particularly amenable to combination with platinum agents and also indicates that the route of administration of verapamil may impact its toxicity profile.

Conclusion

TPC2 is an attractive anti-cancer target as it is expressed in a range of cancer types and is vital in the control of cancer cell proliferation, survival, metastasis, and angiogenesis. Several drugs that inhibit TPC2, including the naturally occurring naringenin and tetrandrine, have been identified which demonstrate pre-clinical anti-cancer efficacy, including reducing metastatic burden in vivo . These agents have all been shown to act as chemosensitizing agents, suggesting that they will be most beneficial clinically when used in combination with existing chemotherapeutics. Indeed, several clinical studies have shown that tetrandrine and verapamil can potentiate the effects of chemotherapeutics in advanced cancer patients. However, both of these drugs can induce toxicity, most likely due to their Ca 2+ -channel blocking effects, so different routes of administration and analogues with improved safety profiles are being examined. Overall, a large body of evidence indicates that TPC2 is an attractive anti-cancer target that warrants further investigation.

Abbreviations

acute myeloid leukaemia

hematopoietic cell-specific protein 1-associated protein X-1

c-jun N-terminal kinase

leucine-rich repeat kinase 2

mitogen activated protein kinase

matrix metalloproteinase 9

mammalian target of rapamycin

nicotinic acid adenine dinucleotide phosphate

nuclear factor kappa B

phosphatidylinositol-3,5-diphosphate

phosphoinositide-3-kinase

reactive oxygen species

transforming growth factor β

two-pore channels

transient receptor potential mucolipin 1

vascular endothelial growth factor

vascular endothelial growth factor receptor 2

Declarations

Author contributions

All authors contributed to the drafting and editing of the manuscript. DLB and DZT created the figures. KAS and LFL contributed conception and design of the review. KAS, LFL, DLB and DZT contributed to manuscript revision, read, and approved the submitted version.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Availability of data and materials

Not applicable.

Funding

Not applicable.

Copyright

© The Author(s) 2022.

References

1. Brzozowski JS Skelding KA. The multi-functional calcium/calmodulin stimulated protein kinase (CaMK) family: emerging targets for anti-cancer therapeutic intervention . Pharmaceuticals (Basel). 2019 ; 12 : 8 . 10.3390/ph12010008 30621060 PMC6469190

2. Hanahan D Weinberg RA. The hallmarks of cancer . Cell. 2000 ; 100 : 57 – 70 . 10.1016/S0092-8674(00)81683-9 10647931

3. Zhu MX Ma J Parrington J Calcraft PJ Galione A Evans AM. Calcium signaling via two-pore channels: local or global, that is the question . Am J Physiol Cell Physiol . 2010 ; 298 : C430 – 41 . 10.1152/ajpcell.00475.2009 20018950 PMC2838574

4. de Duve C. Lysosomes, a new group of cytoplasmic particles . In: Hayashi T , editor. Subcellular particles . New York : Ronald Press Co ; 1959 . pp. 129 – 59 .

5. Repnik U Cesen MH Turk B. The endolysosomal system in cell death and survival . Cold Spring Harb Perspect Biol . 2013 ; 5 : a008755 . 10.1101/cshperspect.a008755 23284043 PMC3579391

6. Kintzer AF Stroud RM. On the structure and mechanism of two-pore channels . FEBS J . 2018 ; 285 : 233 – 43 . 10.1111/febs.14154 28656706 PMC5745306

7. Calcraft PJ Ruas M Pan Z Cheng X Arredouani A Hao X NAADP mobilizes calcium from acidic organelles through two-pore channels . Nature. 2009 ; 459 : 596 – 600 . 10.1038/nature08030 19387438 PMC2761823

8. Zong X Schieder M Cuny H Fenske S Gruner C Rotzer K The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores . Pflugers Arch. 2009 ; 458 : 891 – 9 . 10.1007/s00424-009-0690-y 19557428 PMC2719734

9. Wang X Zhang X Dong XP Samie M Li X Cheng X TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes . Cell . 2012 ; 151 : 372 – 83 . 10.1016/j.cell.2012.08.036 23063126 PMC3475186

10. Cang C Zhou Y Navarro B Seo YJ Aranda K Shi L mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state . Cell . 2013 ; 152 : 778 – 90 . 10.1016/j.cell.2013.01.023 23394946 PMC3908667

11. Jha A Ahuja M Patel S Brailoiu E Muallem S. Convergent regulation of the lysosomal two-pore channel-2 by Mg 2+ , NAADP, PI(3,5)P 2 and multiple protein kinases . EMBO J . 2014 ; 33 : 501 – 11 . 10.1002/embj.201387035 24502975 PMC3989630

12. Brailoiu E Rahman T Churamani D Prole DL Brailoiu GC Hooper R An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca 2+ signals . J Biol Chem . 2010 ; 285 : 38511 – 6 . 10.1074/jbc.M110.162073 20880839 PMC2992283

13. Brailoiu E Churamani D Cai X Schrlau MG Brailoiu GC Gao X Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling . J Cell Biol . 2009 ; 186 : 201 – 9 . 10.1083/jcb.200904073 19620632 PMC2717647

14. She J Zeng W Guo J Chen Q Bai XC Jiang Y. Structural mechanisms of phospholipid activation of the human TPC2 channel . Elife . 2019 ; 8 : e45222 . 10.7554/eLife.45222 30860481 PMC6424560

15. Kintzer AF Stroud RM. Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana . Nature. 2016 ; 531 : 258 – 62 . 10.1038/nature17194 26961658 PMC4863712

16. Rietdorf K Funnell TM Ruas M Heinemann J Parrington J Galione A. Two-pore channels form homo- and heterodimers . J Biol Chem . 2011 ; 286 : 37058 – 62 . 10.1074/jbc.C111.289835 21903581 PMC3199452

17. Pettersen EF Goddard TD Huang CC Couch GS Greenblatt DM Meng EC UCSF Chimera—a visualization system for exploratory research and analysis . J Comput Chem . 2004 ; 25 : 1605 – 12 . 10.1002/jcc.20084 15264254

18. Lin-Moshier Y Keebler MV Hooper R Boulware MJ Liu X Churamani D The two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation . Proc Natl Acad Sci U S A . 2014 ; 111 : 13087 – 92 . 10.1073/pnas.1407004111 25157141 PMC4246952

19. Yamaguchi S Jha A Li Q Soyombo AA Dickinson GD Churamani D Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels . J Biol Chem . 2011 ; 286 : 22934 – 42 . 10.1074/jbc.M110.210930 21540176 PMC3123061

20. Gomez-Suaga P Luzon-Toro B Churamani D Zhang L Bloor-Young D Patel S Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP . Hum Mol Genet. 2012 ; 21 : 511 – 25 . 10.1093/hmg/ddr481 22012985 PMC3259011

21. Lopez J Dionisio N Berna-Erro A Galan C Salido GM Rosado JA. Two-pore channel 2 (TPC2) modulates store-operated Ca(2+) entry . Biochim Biophys Acta . 2012 ; 1823 : 1976 – 83 . 10.1016/j.bbamcr.2012.08.002 23077736

22. Lam AKM Galione A Lai FA Zissimopoulos S. Hax-1 identified as a two-pore channel (TPC)-binding protein . FEBS Lett . 2013 ; 587 : 3782 – 6 . 10.1016/j.febslet.2013.10.031 24188827

23. Sharp TV Wang HW Koumi A Hollyman D Endo Y Ye H K15 protein of Kaposi’s sarcoma-associated herpesvirus is latently expressed and binds to HAX-1, a protein with antiapoptotic function . J Virol . 2002 ; 76 : 802 – 16 . 10.1128/JVI.76.2.802-816.2002 11752170 PMC136811

24. Zhang J Guan X Shah K Yan J. Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles . Nat Commun. 2021 ; 12 : 4739 . 10.1038/s41467-021-24735-z 34362892 PMC8346516

25. Webb SE Kelu JJ Miller AL. Role of two-pore channels in embryonic development and cellular differentiation . Cold Spring Harb Perspect Biol . 2020 ; 12 : a035170 . 10.1101/cshperspect.a035170 31358517 PMC6942120

26. Marchant JS Patel S. Two-pore channels at the intersection of endolysosomal membrane traffic . Biochem Soc Trans. 2015 ; 43 : 434 – 41 . 10.1042/BST20140303 26009187 PMC4730950

27. Ramos I Reich A Wessel GM. Two-pore channels function in calcium regulation in sea star oocytes and embryos . Development . 2014 ; 141 : 4598 – 609 . 10.1242/dev.113563 25377554 PMC4302940

28. Grimm C Holdt LM Chen CC Hassan S Muller C Jors S High susceptibility to fatty liver disease in two-pore channel 2-deficient mice . Nat Commun. 2014 ; 5 : 4699 . 10.1038/ncomms5699 25144390

29. Sun W Yue J. TPC2 mediates autophagy progression and extracellular vesicle secretion in cancer cells . Exp Cell Res . 2018 ; 370 : 478 – 89 . 10.1016/j.yexcr.2018.07.013 29990474

30. Hamilton A Zhang Q Salehi A Willems M Knudsen JG Ringgaard AK Adrenaline stimulates glucagon secretion by Tpc2-dependent Ca 2+ mobilization from acidic stores in pancreatic α-Cells . Diabetes. 2018 ; 67 : 1128 – 39 . 10.2337/db17-1102 29563152 PMC6258900

31. Capel RA Bolton EL Lin WK Aston D Wang Y Liu W Two-pore channels (TPC2s) and nicotinic acid adenine dinucleotide phosphate (NAADP) at lysosomal-sarcoplasmic reticular junctions contribute to acute and chronic β-adrenoceptor signaling in the heart . J Biol Chem . 2015 ; 290 : 30087 – 98 . 10.1074/jbc.M115.684076 26438825 PMC4705968

32. Davis LC Morgan AJ Galione A. NAADP-regulated two-pore channels drive phagocytosis through endo-lysosomal Ca 2+ nanodomains, calcineurin and dynamin . EMBO J . 2020 ; 39 : e104058 . 10.15252/embj.2019104058 32510172 PMC7360967

33. Favia A Desideri M Gambara G D'Alessio A Ruas M Esposito B VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca 2+ signaling . Proc Natl Acad Sci U S A . 2014 ; 111 : E4706 – 15 . 10.1073/pnas.1406029111 25331892 PMC4226099

34. Davis LC Morgan AJ Chen JL Snead CM Bloor-Young D Shenderov E NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing . Curr Biol . 2012 ; 22 : 2331 – 7 . 10.1016/j.cub.2012.10.035 23177477 PMC3525857

35. Sakurai Y Kolokoltsov AA Chen CC Tidwell MW Bauta WE Klugbauer N Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment . Science . 2015 ; 347 : 995 – 8 . 10.1126/science.1258758 25722412 PMC4550587

36. Millet JK Whittaker GR. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein . Proc Natl Acad Sci U S A . 2014 ; 111 : 15214 – 9 . 10.1073/pnas.1407087111 25288733 PMC4210292

37. Gunaratne GS Yang Y Li F Walseth TF Marchant JS. NAADP-dependent Ca 2+ signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system . Cell Calcium . 2018 ; 75 : 30 – 41 . 10.1016/j.ceca.2018.08.003 30121440 PMC6251489

38. Ou X Liu Y Lei X Li P Mi D Ren L Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV . Nat Commun. 2020 ; 11 : 1620 . 10.1038/s41467-020-15562-9 32221306 PMC7100515

39. Khan N Halcrow PW Lakpa KL Afghah Z Miller NM Dowdy SF Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation . FASEB J . 2020 ; 34 : 4147 – 62 . 10.1096/fj.201902534R 31950548 PMC7079041

40. Hockey LN Kilpatrick BS Eden ER Lin-Moshier Y Brailoiu GC Brailoiu E Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition . J Cell Sci . 2015 ; 128 : 232 – 8 . 10.1242/jcs.164152 25416817 PMC4294771

41. Tong BCK Wu AJ Huang AS Dong R Malampati S Iyaswamy A Lysosomal TPCN (two pore segment channel) inhibition ameliorates beta-amyloid pathology and mitigates memory impairment in Alzheimer disease . Autophagy . 2021 ;[Epub ahead of print]. 10.1080/15548627.2021.1945220 34313551 PMC9037479

42. Garcia-Rua V Otero MF Lear PV Rodriguez-Penas D Feijoo-Bandin S Noguera-Moreno T Increased expression of fatty-acid and calcium metabolism genes in failing human heart . PLoS One . 2012 ; 7 : e37505 . 10.1371/journal.pone.0037505 22701570 PMC3368932

43. Riva L Yuan S Yin X Martin-Sancho L Matsunaga N Pache L Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing . Nature. 2020 ; 586 : 113 – 9 . 10.1038/s41586-020-2577-1 32707573 PMC7603405

44. Netcharoensirisuk P Abrahamian C Tang R Chen CC Rosato AS Beyers W Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2 . Sci Rep. 2021 ; 11 : 8515 . 10.1038/s41598-021-88196-6 33875769 PMC8055690

45. Muller M Gerndt S Chao YK Zisis T Nguyen ONP Gerwien A Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth . Cell Chem Biol . 2021 ; 28 : 1119 – 31.e27 . 10.1016/j.chembiol.2021.01.023 33626324

46. Jiang Y Zhou Y Peng G Tian H Pan D Liu L Two-pore channels mediated receptor-operated Ca 2+ entry in pulmonary artery smooth muscle cells in response to hypoxia . Int J Biochem Cell Biol . 2018 ; 97 : 28 – 35 . 10.1016/j.biocel.2018.01.012 29355755

47. D’Amore A Hanbashi AA Di Agostino S Palombi F Sacconi A Voruganti A Loss of two-pore channel 2 ( TPC2 ) expression increases the metastatic traits of melanoma cells by a mechanism involving the hippo signalling pathway and store-operated calcium entry . Cancers (Basel). 2020 ; 12 : 2391 . 10.3390/cancers12092391 32846966 PMC7564716

48. Aley PK Mikolajczyk AM Munz B Churchill GC Galione A Berger F. Nicotinic acid adenine dinucleotide phosphate regulates skeletal muscle differentiation via action at two-pore channels . Proc Natl Acad Sci U S A . 2010 ; 107 : 19927 – 32 . 10.1073/pnas.1007381107 21041635 PMC2993425

49. Notomi T Ezura Y Noda M. Identification of two-pore channel 2 as a novel regulator of osteoclastogenesis . J Biol Chem . 2012 ; 287 : 35057 – 64 . 10.1074/jbc.M111.328930 22833668 PMC3471725

50. Zhang ZH Lu YY Yue J. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells . PLoS One . 2013 ; 8 : e66077 . 10.1371/journal.pone.0066077 23776607 PMC3680454

51. Nguyen ONP Grimm C Schneider LS Chao YK Atzberger C Bartel K Two-pore channel function is crucial for the migration of invasive cancer cells . Cancer Res. 2017 ; 77 : 1427 – 38 . 10.1158/0008-5472.CAN-16-0852 28108508

52. Hwangbo C Park J Lee JH. mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen . J Biol Chem . 2011 ; 286 : 33601 – 12 . 10.1074/jbc.M110.206789 21828040 PMC3190898

53. Goel HL Mercurio AM. VEGF targets the tumour cell . Nat Rev Cancer. 2013 ; 13 : 871 – 82 . 10.1038/nrc3627 24263190 PMC4011842

54. Itatani Y Kawada K Yamamoto T Sakai Y. Resistance to anti-angiogenic therapy in cancer-alterations to anti-VEGF pathway . Int J Mol Sci. 2018 ; 19 : 1232 . 10.3390/ijms19041232 29670046 PMC5979390

55. Chen Y Chen JC Tseng SH. Tetrandrine suppresses tumor growth and angiogenesis of gliomas in rats . Int J Cancer . 2009 ; 124 : 2260 – 9 . 10.1002/ijc.24208 19165864

56. Ma JW Zhang Y Li R Ye JC Li HY Zhang YK Tetrandrine suppresses human glioma growth by inhibiting cell survival, proliferation and tumour angiogenesis through attenuating STAT3 phosphorylation . Eur J Pharmacol . 2015 ; 764 : 228 – 39 . 10.1016/j.ejphar.2015.06.017 26086859

57. Gao JL Ji X He TC Zhang Q He K Zhao Y Tetrandrine suppresses cancer angiogenesis and metastasis in 4T1 tumor bearing mice . Evid Based Complement Alternat Med. 2013 ; 2013 : 265061 . 10.1155/2013/265061 23762115 PMC3677646

58. Pafumi I Festa M Papacci F Lagostena L Giunta C Gutla V Naringenin impairs two-pore channel 2 activity and inhibits VEGF-induced angiogenesis . Sci Rep. 2017 ; 7 : 5121 . 10.1038/s41598-017-04974-1 28698624 PMC5505983

59. Li Y Schon C Chen CC Yang Z Liegl R Murenu E TPC2 promotes choroidal angiogenesis and inflammation in a mouse model of neovascular age-related macular degeneration . Life Sci Alliance . 2021 ; 4 : e202101047 . 10.26508/lsa.202101047 34183443 PMC8321671

60. Huang T Song X Yang Y Wan X Alvarez AA Sastry N Autophagy and hallmarks of cancer . Crit Rev Oncog . 2018 ; 23 : 247 – 67 . 10.1615/CritRevOncog.2018027913 30311559 PMC6447410

61. Singh SS Vats S Chia AY Tan TZQ Deng S Ong MS Dual role of autophagy in hallmarks of cancer . Oncogene. 2018 ; 37 : 1142 – 58 . 10.1038/s41388-017-0046-6 29255248

62. Pereira GJS Hirata H Fimia GM do Carmo LG Bincoletto C Han SW Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes . J Biol Chem . 2011 ; 286 : 27875 – 81 . 10.1074/jbc.C110.216580 21610076 PMC3151033

63. Pereira GJS Antonioli M Hirata H Ureshino RP Nascimento AR Bincoletto C Glutamate induces autophagy via the two-pore channels in neural cells . Oncotarget . 2017 ; 8 : 12730 – 40 . 10.18632/oncotarget.14404 28055974 PMC5355049

64. Garcia-Rua V Feijoo-Bandin S Rodriguez-Penas D Mosquera-Leal A Abu-Assi E Beiras A Endolysosomal two-pore channels regulate autophagy in cardiomyocytes . J Physiol. 2016 ; 594 : 3061 – 77 . 10.1113/JP271332 26757341 PMC4887684

65. Lu Y Hao B Graeff R Yue J. NAADP/TPC2/Ca(2+) Signaling inhibits autophagy . Commun Integr Biol . 2013 ; 6 : e27595 . 10.4161/cib.27595 24753792 PMC3984295

66. Lin PH Duann P Komazaki S Park KH Li H Sun M Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling . J Biol Chem . 2015 ; 290 : 3377 – 89 . 10.1074/jbc.M114.608471 25480788 PMC4319008

67. Bai XY Liu YG Song W Li YY Hou DS Luo HM Anticancer activity of tetrandrine by inducing pro-death apoptosis and autophagy in human gastric cancer cells . J Pharm Pharmacol . 2018 ; 70 : 1048 – 58 . 10.1111/jphp.12935 29770446

68. Guo Y Pei X. Tetrandrine-Induced Autophagy in MDA-MB-231 Triple-negative breast cancer cell through the inhibition of PI3K/AKT/mTOR signaling . Evid Based Complement Alternat Med. 2019 ; 2019 : 7517431 . 10.1155/2019/7517431 30713576 PMC6332987

69. Kou B Liu W Xu X Yang Y Yi Q Guo F Autophagy induction enhances tetrandrine-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells . Oncol Rep . 2017 ; 38 : 3137 – 43 . 10.3892/or.2017.5988 29048631

70. Kosiniak-Kamysz A Marczakiewicz-Lustig A Marcinska M Skowron M Wojas-Pelc A Pospiech E Increased risk of developing cutaneous malignant melanoma is associated with variation in pigmentation genes and VDR, and may involve epistatic effects . Melanoma Res . 2014 ; 24 : 388 – 96 . 10.1097/CMR.0000000000000095 24926819

71. Pho LN Leachman SA. Genetics of pigmentation and melanoma predisposition . G Ital Dermatol Venereol . 2010 ; 145 : 37 – 45 . 20197744

72. Alharbi AF Parrington J. The role of genetic polymorphisms in endolysosomal ion channels TPC2 and P2RX4 in cancer pathogenesis, prognosis, and diagnosis: a genetic association in the UK Biobank . NPJ Genom Med. 2021 ; 6 : 58 . 10.1038/s41525-021-00221-9 34253731 PMC8275681

73. Ambrosio AL Boyle JA Aradi AE Christian KA Di Pietro SM. TPC2 controls pigmentation by regulating melanosome pH and size . Proc Natl Acad Sci U S A . 2016 ; 113 : 5622 – 7 . 10.1073/pnas.1600108113 27140606 PMC4878521

74. Howarth KD Mirza T Cooke SL Chin SF Pole JC Turro E NRG1 fusions in breast cancer . Breast Cancer Res. 2021 ; 23 : 3 . 10.1186/s13058-020-01377-5 33413557 PMC7788813

75. Hodgson A Swanson D Tang S Dickson BC Turashvili G. Gene fusions characterize a subset of uterine cellular leiomyomas . Genes Chromosomes Cancer . 2020 ;[Epub ahead of print]. 10.1002/gcc.22888 32677742

76. Wilkerson PM Reis-Filho JS. The 11q13-q14 amplicon: clinicopathological correlations and potential drivers . Genes Chromosomes Cancer . 2013 ; 52 : 333 – 55 . 10.1002/gcc.22037 23225572

77. Sugahara K Michikawa Y Ishikawa K Shoji Y Iwakawa M Shibahara T Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma . Int J Oncol . 2011 ; 39 : 761 – 9 . 10.3892/ijo.2011.1094 21701773

78. Huang X Godfrey TE Gooding WE McCarty KS Jr Gollin SM. Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma . Genes Chromosomes Cancer . 2006 ; 45 : 1058 – 69 . 10.1002/gcc.20371 16906560

79. Jahidin AH Stewart TA Thompson EW Roberts-Thomson SJ Monteith GR. Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells . Biochem Biophys Res Commun . 2016 ; 477 : 731 – 6 . 10.1016/j.bbrc.2016.06.127 27353380

80. Li F Ji JP Xu Y Liu RL. Identification a novel set of 6 differential expressed genes in prostate cancer that can potentially predict biochemical recurrence after curative surgery . Clin Transl Oncol. 2019 ; 21 : 1067 – 75 . 10.1007/s12094-018-02029-z 30637711

81. Shivakumar M Lee Y Bang L Garg T Sohn KA Kim D. Identification of epigenetic interactions between miRNA and DNA methylation associated with gene expression as potential prognostic markers in bladder cancer . BMC Med Genomics. 2017 ; 10 : 30 . 10.1186/s12920-017-0269-y 28589857 PMC5461531

82. Kocarnik JM Park SL Han J Dumitrescu L Cheng I Wilkens LR Pleiotropic and sex-specific effects of cancer GWAS SNPs on melanoma risk in the population architecture using genomics and epidemiology (PAGE) study . PLoS One . 2015 ; 10 : e0120491 . 10.1371/journal.pone.0120491 25789475 PMC4366224

83. Kim S Chen J Cheng T Gindulyte A He J He S PubChem in 2021: new data content and improved web interfaces . Nucleic Acids Res. 2021 ; 49 : D1388 – 95 . 10.1093/nar/gkaa971 33151290 PMC7778930

84. Salehi B Fokou PVT Sharifi-Rad M Zucca P Pezzani R Martins N The therapeutic potential of naringenin: a review of clinical trials . Pharmaceuticals (Basel). 2019 ; 12 : 11 . 10.3390/ph12010011 30634637 PMC6469163

85. Feng XL Zhan XX Zuo LSY Mo XF Zhang X Liu KY Associations between serum concentration of flavonoids and breast cancer risk among Chinese women . Eur J Nutr. 2021 ; 60 : 1347 – 62 . 10.1007/s00394-020-02331-z 32683480

86. Khan N Afaq F Mukhtar H. Cancer chemoprevention through dietary antioxidants: progress and promise . Antioxid Redox Signal . 2008 ; 10 : 475 – 510 . 10.1089/ars.2007.1740 18154485

87. Valko M Leibfritz D Moncol J Cronin MTD Mazur M Telser J. Free radicals and antioxidants in normal physiological functions and human disease . Int J Biochem Cell Biol . 2007 ; 39 : 44 – 84 . 10.1016/j.biocel.2006.07.001 16978905

88. Le Marchand L Murphy SP Hankin JH Wilkens LR Kolonel LN. Intake of flavonoids and lung cancer . J Natl Cancer Inst . 2000 ; 92 : 154 – 60 . 10.1093/jnci/92.2.154 10639518

89. Vitelli Storelli F Molina AJ Zamora-Ros R Fernandez-Villa T Roussou V Romaguera D Flavonoids and the risk of gastric cancer: an exploratory case-control study in the MCC-Spain study . Nutrients. 2019 ; 11 : 967 . 10.3390/nu11050967 31035601 PMC6566880

90. Pang X Fu W Wang J Kang, Xu L Zhao Y Identification of estrogen receptor α antagonists from natural products via in vitro and in silico approaches . Oxid Med Cell Longev. 2018 ; 2018 : 6040149 . 10.1155/2018/6040149 29861831 PMC5971309

91. So FV Guthrie N Chambers AF Moussa M Carroll KK. Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices . Nutr Cancer. 1996 ; 26 : 167 – 81 . 10.1080/01635589609514473 8875554

92. Bulzomi P Bolli A Galluzzo P Acconcia F Ascenzi P Marino M. The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background . IUBMB Life . 2012 ; 64 : 690 – 6 . 10.1002/iub.1049 22692793

93. Ayob Z Mohd Bohari SP Abd Samad A Jamil S. Cytotoxic activities against breast cancer cells of local justicia gendarussa crude extracts . Evid Based Complement Alternat Med. 2014 ; 2014 : 732980 . 10.1155/2014/732980 25574182 PMC4276683

94. Abaza MSI Orabi KY Al-Quattan E Al-Attiyah RJ. Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer . Cancer Cell Int. 2015 ; 15 : 46 . 10.1186/s12935-015-0194-0 26074733 PMC4464250

95. Chandrika BB Steephan M Kumar TRS Sabu A Haridas M. Hesperetin and naringenin sensitize HER2 positive cancer cells to death by serving as HER2 tyrosine kinase inhibitors . Life Sci . 2016 ; 160 : 47 – 56 . 10.1016/j.lfs.2016.07.007 27449398

96. Ke JY Banh T Hsiao YH Cole RM Straka SR Yee LD Citrus flavonoid naringenin reduces mammary tumor cell viability, adipose mass, and adipose inflammation in obese ovariectomized mice . Mol Nutr Food Res . 2017 ; 61 : 1600934 . 10.1002/mnfr.201600934 28370954

97. Zhao Z Jin G Ge Y Guo Z. Naringenin inhibits migration of breast cancer cells via inflammatory and apoptosis cell signaling pathways . Inflammopharmacology. 2019 ; 27 : 1021 – 36 . 10.1007/s10787-018-00556-3 30941613

98. El-Kersh DM Ezzat SM Salama MM Mahrous EA Attia YM Ahmed MS Anti-estrogenic and anti-aromatase activities of citrus peels major compounds in breast cancer . Sci Rep. 2021 ; 11 : 7121 . 10.1038/s41598-021-86599-z 33782546 PMC8007834

99. Woo Y Shin SY Hyun J Lee SD Lee YH Lim Y. Flavanones inhibit the clonogenicity of HCT116 cololectal cancer cells . Int J Mol Med . 2012 ; 29 : 403 – 8 . 22160193 10.3892/ijmm.2011.857

100. Song HM Park GH Eo HJ Lee JW Kim MK Lee JR Anti-proliferative effect of naringenin through p38-dependent downregulation of cyclin D1 in human colorectal cancer cells . Biomol Ther (Seoul) . 2015 ; 23 : 339 – 44 . 10.4062/biomolther.2015.024 26157550 PMC4489828

101. Moreno-Jimenez MR Lopez-Barraza R Cervantes-Cardoza V Perez-Ramirez IF Reyna-Rojas JA Gallegos-Infante JA Mechanisms associated to apoptosis of cancer cells by phenolic extracts from two canned common beans varieties (Phaseolus vulgaris L.) . J Food Biochem . 2019 ; 43 : e12680 . 10.1111/jfbc.12680 31353616

102. Storniolo CE Sacanella I Lamuela-Raventos RM Moreno JJ. Bioactive compounds of Mediterranean cooked tomato sauce (sofrito) modulate intestinal epithelial cancer cell growth through oxidative stress/arachidonic acid cascade regulation . ACS Omega . 2020 ; 5 : 17071 – 7 . 10.1021/acsomega.9b04329 32715192 PMC7376686

103. Arul D Subramanian P. Naringenin (citrus flavonone) induces growth inhibition, cell cycle arrest and apoptosis in human hepatocellular carcinoma cells . Pathol Oncol Res. 2013 ; 19 : 763 – 70 . 10.1007/s12253-013-9641-1 23661153

104. Lim W Park S Bazer FW Song G. Naringenin-induced apoptotic cell death in prostate cancer cells is mediated via the PI3K/AKT and MAPK signaling pathways . J Cell Biochem . 2017 ; 118 : 1118 – 31 . 10.1002/jcb.25729 27606834

105. Gumushan Aktas H Akgun T. Naringenin inhibits prostate cancer metastasis by blocking voltage-gated sodium channels . Biomed Pharmacother . 2018 ; 106 : 770 – 5 . 10.1016/j.biopha.2018.07.008 29990870

106. Lou C Zhang F Yang M Zhao J Zeng W Fang X Naringenin decreases invasiveness and metastasis by inhibiting TGF-β-induced epithelial to mesenchymal transition in pancreatic cancer cells . PLoS One . 2012 ; 7 : e50956 . 10.1371/journal.pone.0050956 23300530 PMC3530567

107. Park HJ Choi YJ Lee JH Nam MJ. Naringenin causes ASK1-induced apoptosis via reactive oxygen species in human pancreatic cancer cells . Food Chem Toxicol . 2017 ; 99 : 1 – 8 . 10.1016/j.fct.2016.11.008 27838343

108. Stompor M Uram L Podgorski R. In vitro effect of 8-prenylnaringenin and naringenin on fibroblasts and glioblastoma cells-cellular accumulation and cytotoxicity . Molecules. 2017 ; 22 : 1092 . 10.3390/molecules22071092 28665345 PMC6152326

109. Chen YY Chang YM Wang KY Chen PN Hseu YC Chen KM Naringenin inhibited migration and invasion of glioblastoma cells through multiple mechanisms . Environ Toxicol . 2019 ; 34 : 233 – 9 . 10.1002/tox.22677 30431227

110. Lentini A Forni C Provenzano B Beninati S. Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the in vivo metastatic potential . Amino Acids. 2007 ; 32 : 95 – 100 . 10.1007/s00726-006-0304-3 16699821

111. Nasr Bouzaiene N Chaabane F Sassi A Chekir-Ghedira L Ghedira K. Effect of apigenin-7-glucoside, genkwanin and naringenin on tyrosinase activity and melanin synthesis in B16F10 melanoma cells . Life Sci . 2016 ; 144 : 80 – 5 . 10.1016/j.lfs.2015.11.030 26656314

112. Choi J Lee DH Jang H Park SY Seol JW. Naringenin exerts anticancer effects by inducing tumor cell death and inhibiting angiogenesis in malignant melanoma . Int J Med Sci . 2020 ; 17 : 3049 – 57 . 10.7150/ijms.44804 33173425 PMC7646117

113. Chang HL Chang YM Lai SC Chen KM Wang KC Chiu TT Naringenin inhibits migration of lung cancer cells via the inhibition of matrix metalloproteinases-2 and -9 . Exp Ther Med . 2017 ; 13 : 739 – 44 . 10.3892/etm.2016.3994 28352360 PMC5348669

114. Shi X Luo X Chen T Guo W Liang C Tang S Naringenin inhibits migration, invasion, induces apoptosis in human lung cancer cells and arrests tumour progression in vitro . J Cell Mol Med . 2021 ; 25 : 2563 – 71 . 10.1111/jcmm.16226 33523599 PMC7933922

115. Leonardi T Vanamala J Taddeo SS Davidson LA Murphy ME Patil BS Apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats . Exp Biol Med (Maywood) . 2010 ; 235 : 710 – 7 . 10.1258/ebm.2010.009359 20511675 PMC2885760

116. Subramanian P Arul D. Attenuation of NDEA-induced hepatocarcinogenesis by naringenin in rats . Cell Biochem Funct . 2013 ; 31 : 511 – 7 . 10.1002/cbf.2929 23172681

117. Arul D Subramanian P. Inhibitory effect of naringenin (citrus flavonone) on N -nitrosodiethylamine induced hepatocarcinogenesis in rats . Biochem Biophys Res Commun . 2013 ; 434 : 203 – 9 . 10.1016/j.bbrc.2013.03.039 23523793

118. Sabarinathan D Mahalakshmi P Vanisree AJ. Naringenin, a flavanone inhibits the proliferation of cerebrally implanted C6 glioma cells in rats . Chem Biol Interact . 2011 ; 189 : 26 – 36 . 10.1016/j.cbi.2010.09.028 20932960

119. Sabarinathan D Vanisree AJ. Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats . Mol Cell Biochem. 2013 ; 375 : 171 – 8 . 10.1007/s11010-012-1539-9 23263903

120. Lian GY Wang QM Mak TS Huang XR Yu XQ Lan HY. Inhibition of tumor invasion and metastasis by targeting TGF-β-Smad-MMP2 pathway with asiatic acid and naringenin . Mol Ther Oncolytics . 2021 ; 20 : 277 – 89 . 10.1016/j.omto.2021.01.006 33614911 PMC7873580

121. Bodduluru LN Kasala ER Madhana RM Barua CC Hussain MI Haloi P Naringenin ameliorates inflammation and cell proliferation in benzo(a)pyrene induced pulmonary carcinogenesis by modulating CYP1A1, NFκB and PCNA expression . Int Immunopharmacol . 2016 ; 30 : 102 – 10 . 10.1016/j.intimp.2015.11.036 26655880

122. Fernandez J Silvan B Entrialgo-Cadierno R Villar CJ Capasso R Uranga JA Antiproliferative and palliative activity of flavonoids in colorectal cancer . Biomed Pharmacother . 2021 ; 143 : 112241 . 10.1016/j.biopha.2021.112241 34649363

123. Erdogan S Doganlar O Doganlar ZB Turkekul K. Naringin sensitizes human prostate cancer cells to paclitaxel therapy . Prostate Int . 2018 ; 6 : 126 – 35 . 10.1016/j.prnil.2017.11.001 30505814 PMC6251953

124. Ling D Marshall GM Liu PY Xu N Nelson CA Iismaa SE Enhancing the anticancer effect of the histone deacetylase inhibitor by activating transglutaminase . Eur J Cancer . 2012 ; 48 : 3278 – 87 . 10.1016/j.ejca.2012.02.067 22459762

125. Zhang FY Du GJ Zhang L Zhang CL Lu WL Liang W. Naringenin enhances the anti-tumor effect of doxorubicin through selectively inhibiting the activity of multidrug resistance-associated proteins but not P-glycoprotein . Pharm Res. 2009 ; 26 : 914 – 25 . 10.1007/s11095-008-9793-y 19067124

126. Qin L Jin L Lu L Lu X Zhang C Zhang F Naringenin reduces lung metastasis in a breast cancer resection model . Protein Cell. 2011 ; 2 : 507 – 16 . 10.1007/s13238-011-1056-8 21748601 PMC4875175

127. Zhang F Dong W Zeng W Zhang L Zhang C Qiu Y Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation . Breast Cancer Res. 2016 ; 18 : 38 . 10.1186/s13058-016-0698-0 27036297 PMC4818388

128. Han KY Chen PN Hong MC Hseu YC Chen KM Hsu LS Naringenin attenuated prostate cancer invasion via reversal of epithelial-to-mesenchymal transition and inhibited uPA activity . Anticancer Res . 2018 ; 38 : 6753 – 8 . 10.21873/anticanres.13045 30504386

129. Jang BC Lim KJ Paik JH Cho JW Baek WK Suh MH Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells . Biochem Pharmacol . 2004 ; 67 : 1819 – 29 . 10.1016/j.bcp.2004.01.018 15130759

130. Liu T Men Q Wu G Yu C Huang Z Liu X Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells . Oncotarget . 2015 ; 6 : 7992 – 8006 . 10.18632/oncotarget.3505 25797266 PMC4480730

131. Liu T Zhang Z Yu C Zeng C Xu X Wu G Tetrandrine antagonizes acute megakaryoblastic leukaemia growth by forcing autophagy-mediated differentiation . Br J Pharmacol . 2017 ; 174 : 4308 – 28 . 10.1111/bph.14031 28901537 PMC5715583

132. Wu G Liu T Li H Li Y Li D Li W. c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation . Cell Death Dis. 2018 ; 9 : 473 . 10.1038/s41419-018-0498-9 29700286 PMC5920096

133. Wu Z Wang G Xu S Li Y Tian Y Niu H Effects of tetrandrine on glioma cell malignant phenotype via inhibition of ADAM17 . Tumour Biol. 2014 ; 35 : 2205 – 10 . 10.1007/s13277-013-1293-y 24185966

134. Zhang Y Wen YL Ma JW Ye JC Wang X Huang JX Tetrandrine inhibits glioma stem-like cells by repressing β-catenin expression . Int J Oncol . 2017 ; 50 : 101 – 10 . 10.3892/ijo.2016.3780 27909720

135. Ma JW Zhang Y Ye JC Li R Wen YL Huang JX Tetrandrine exerts a radiosensitization effect on human glioma through inhibiting proliferation by attenuating ERK phosphorylation . Biomol Ther (Seoul) . 2017 ; 25 : 186 – 93 . 10.4062/biomolther.2016.044 27829269 PMC5340544

136. Jiang YW Cheng HY Kuo CL Way TD Lien JC Chueh FS Tetrandrine inhibits human brain glioblastoma multiforme GBM 8401 cancer cell migration and invasion in vitro . Environ Toxicol . 2019 ; 34 : 364 – 74 . 10.1002/tox.22691 30549224

137. Jin Q Kang C Soh Y Sohn NW Lee J Cho YH Tetrandrine cytotoxicity and its dual effect on oxidative stress-induced apoptosis through modulating cellular redox states in Neuro 2a mouse neuroblastoma cells . Life Sci. 2002 ; 71 : 2053 – 66 . 10.1016/S0024-3205(02)01989-6 12175898

138. Chen Y Chen JC Tseng SH. Effects of tetrandrine plus radiation on neuroblastoma cells . Anticancer Res . 2009 ; 29 : 3163 – 71 . 19661330

139. Tao LJ Zhou XD Shen CC Liang CZ Liu B Tao Y Tetrandrine induces apoptosis and triggers a caspase cascade in U2-OS and MG-63 cells through the intrinsic and extrinsic pathways . Mol Med Rep . 2014 ; 9 : 345 – 9 . 10.3892/mmr.2013.1761 24173687

140. Tian DD Zhang RX Wu N Yuan W Luo SH Chen HQ Tetrandrine inhibits the proliferation of human osteosarcoma cells by upregulating the PTEN pathway . Oncol Rep . 2017 ; 37 : 2795 – 802 . 10.3892/or.2017.5560 28393256

141. Sun X Xu R Deng Y Cheng H Ma J Ji J Effects of tetrandrine on apoptosis and radiosensitivity of nasopharyngeal carcinoma cell line CNE . Acta Biochim Biophys Sin (Shanghai) . 2007 ; 39 : 869 – 78 . 10.1111/j.1745-7270.2007.00349.x 17989878

142. Lin YJ Peng SF Lin ML Kuo CL Lu KW Liao CL Tetrandrine induces apoptosis of human nasopharyngeal carcinoma NPC-TW 076 cells through reactive oxygen species accompanied by an endoplasmic reticulum stress signaling pathway . Molecules. 2016 ; 21 : 1353 . 10.3390/molecules21101353 27754332 PMC6273859

143. Cho HS Chang SH Chung YS Shin JY Park SJ Lee ES Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells . J Vet Sci . 2009 ; 10 : 23 – 8 . 10.4142/jvs.2009.10.1.23 19255520 PMC2801106

144. Chen Z Zhao L Zhao F Yang G Wang JJ. Tetrandrine suppresses lung cancer growth and induces apoptosis, potentially via the VEGF/HIF-1alpha/ICAM-1 signaling pathway . Oncol Lett . 2018 ; 15 : 7433 – 7 . 10.3892/ol.2018.8190 29849794 PMC5962877

145. Huang AC Lien JC Lin MW Yang JS Wu PP Chang SJ Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-I and LC3-II activation- dependent autophagy . Int J Oncol . 2013 ; 43 : 485 – 94 . 10.3892/ijo.2013.1952 23695424

146. Yu FS Yu CS Chen JC Yang JL Lu HF Chang SJ Tetrandrine induces apoptosis via caspase-8, -9, and -3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin-1/LC3-I, II signaling pathways in human oral cancer HSC-3 cells . Environ Toxicol . 2016 ; 31 : 395 – 406 . 10.1002/tox.22053 25266202

147. Lien JC Lin MW Chang SJ Lai KC Huang AC Yu FS Tetrandrine induces programmed cell death in human oral cancer CAL 27 cells through the reactive oxygen species production and caspase-dependent pathways and associated with beclin-1-induced cell autophagy . Environ Toxicol . 2017 ; 32 : 329 – 43 . 10.1002/tox.22238 26822499

148. Qin R Shen H Cao Y Fang Y Li H Chen Q Tetrandrine induces mitochondria-mediated apoptosis in human gastric cancer BGC-823 cells . PLoS One . 2013 ; 8 : e76486 . 10.1371/journal.pone.0076486 24098511 PMC3788110

149. Chaudhary P Vishwanatha JK. c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine . Biochem Pharmacol . 2014 ; 91 : 457 – 73 . 10.1016/j.bcp.2014.08.014 25181458 PMC4171211

150. Liu W Kou B Ma ZK Tang XS Lv C Ye M Tetrandrine suppresses proliferation, induces apoptosis, and inhibits migration and invasion in human prostate cancer cells . Asian J Androl . 2015 ; 17 : 850 – 3 . 10.4103/1008-682X.142134 25677131 PMC4577603

151. Kou B Liu W He W Zhang Y Zheng J Yan Y Tetrandrine suppresses metastatic phenotype of prostate cancer cells by regulating Akt/mTOR/MMP-9 signaling pathway . Oncol Rep . 2016 ; 35 : 2880 – 6 . 10.3892/or.2016.4649 26935264

152. Li X Su B Liu R Wu D He D. Tetrandrine induces apoptosis and triggers caspase cascade in human bladder cancer cells . J Surg Res . 2011 ; 166 : e45 – 51 . 10.1016/j.jss.2010.10.034 21176918

153. Zhang Y Liu W He W Zhang Y Deng X Ma Y Tetrandrine reverses epithelial-mesenchymal transition in bladder cancer by downregulating Gli-1 . Int J Oncol . 2016 ; 48 : 2035 – 42 . 10.3892/ijo.2016.3415 26983576

154. Xu W Debeb BG Lacerda L Li J Woodward WA. Tetrandrine, a compound common in Chinese traditional medicine, preferentially kills breast cancer tumor initiating cells (TICs) in vitro . Cancers (Basel). 2011 ; 3 : 2274 – 85 . 10.3390/cancers3022274 24212809 PMC3757417

155. Wong VKW Zeng W Chen J Yao XJ Leung ELH Wang QQ Tetrandrine, an activator of autophagy, induces autophagic cell death via PKC-α inhibition and mTOR-dependent mechanisms . Front Pharmacol . 2017 ; 8 : 351 . 10.3389/fphar.2017.00351 28642707 PMC5462963

156. Wang CH Yang JM Guo YB Shen J Pei XH. Anticancer activity of tetrandrine by inducing apoptosis in human breast cancer cell line MDA-MB-231 in vivo . Evid Based Complement Alternat Med. 2020 ; 2020 : 6823520 . 10.1155/2020/6823520 32714412 PMC7345956

157. Chen T Ji B Chen Y. Tetrandrine triggers apoptosis and cell cycle arrest in human renal cell carcinoma cells . J Nat Med. 2014 ; 68 : 46 – 52 . 10.1007/s11418-013-0765-0 23564030

158. Chen S Liu W Wang K Fan Y Chen J Ma J Tetrandrine inhibits migration and invasion of human renal cell carcinoma by regulating Akt/NF-κB/MMP-9 signaling . PLoS One . 2017 ; 12 : e0173725 . 10.1371/journal.pone.0173725 28288190 PMC5348026

159. Yoo SM Oh SH Lee SJ Lee BW Ko WG Moon CK Inhibition of proliferation and induction of apoptosis by tetrandrine in HepG2 cells . J Ethnopharmacol. 2002 ; 81 : 225 – 9 . 10.1016/S0378-8741(02)00082-X 12065155

160. Oh SH Lee BH. Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase- dependent Bid cleavage and cytochrome c release . Biochem Pharmacol. 2003 ; 66 : 725 – 31 . 10.1016/S0006-2952(03)00397-6 12948852

161. Ng LT Chiang LC Lin YT Lin CC. Antiproliferative and apoptotic effects of tetrandrine on different human hepatoma cell lines . Am J Chin Med. 2006 ; 34 : 125 – 35 . 10.1142/S0192415X06003692 16437745

162. Liu C Gong K Mao X Li W. Tetrandrine induces apoptosis by activating reactive oxygen species and repressing Akt activity in human hepatocellular carcinoma . Int J Cancer . 2011 ; 129 : 1519 – 31 . 10.1002/ijc.25817 21128229

163. Gong K Chen C Zhan Y Chen Y Huang Z Li W. Autophagy-related gene 7 ( ATG7 ) and reactive oxygen species/extracellular signal-regulated kinase regulate tetrandrine-induced autophagy in human hepatocellular carcinoma . J Biol Chem . 2012 ; 287 : 35576 – 88 . 10.1074/jbc.M112.370585 22927446 PMC3471698

164. Yu VWL Ho WS. Tetrandrine inhibits hepatocellular carcinoma cell growth through the caspase pathway and G2/M phase . Oncol Rep . 2013 ; 29 : 2205 – 10 . 10.3892/or.2013.2352 23525490

165. Huang T Xu S Deo R Ma A Li H Ma K Targeting the Ca 2+ /Calmodulin-dependent protein kinase II by Tetrandrine in human liver cancer cells . Biochem Biophys Res Commun . 2019 ; 508 : 1227 – 32 . 10.1016/j.bbrc.2018.12.012 30554655

166. Chang KH Liao HF Chang HH Chen YY Yu MC Chou CJ Inhibitory effect of tetrandrine on pulmonary metastases in CT26 colorectal adenocarcinoma-bearing BALB/c mice . Am J Chin Med. 2004 ; 32 : 863 – 72 . 10.1142/S0192415X04002478 15673192

167. Chen XL Ren KH He HW Shao RG. Involvement of PI3K/AKT/GSK3beta pathway in tetrandrine-induced G1 arrest and apoptosis . Cancer Biol Ther . 2008 ; 7 : 1073 – 8 . 10.4161/cbt.7.7.6142 18437054

168. Wu JM Chen Y Chen JC Lin TY Tseng SH. Tetrandrine induces apoptosis and growth suppression of colon cancer cells in mice . Cancer Lett . 2010 ; 287 : 187 – 95 . 10.1016/j.canlet.2009.06.009 19586712

169. He BC Gao JL Zhang BQ Luo Q Shi Q Kim SH Tetrandrine inhibits Wnt/β-catenin signaling and suppresses tumor growth of human colorectal cancer . Mol Pharmacol . 2011 ; 79 : 211 – 9 . 10.1124/mol.110.068668 20978119 PMC3033706

170. Wu K Zhou M Wu QX Yuan SX Wang DX Jin JL The role of IGFBP-5 in mediating the anti-proliferation effect of tetrandrine in human colon cancer cells . Int J Oncol . 2015 ; 46 : 1205 – 13 . 10.3892/ijo.2014.2800 25524807

171. Tsai SC Wu WC Yang JS. Tetrandrine inhibits epithelial-mesenchymal transition in IL-6-induced HCT116 human colorectal cancer cells . Onco Targets Ther . 2021 ; 14 : 4523 – 36 . 10.2147/OTT.S324552 34456573 PMC8387317

172. Zhang H Xie B Zhang Z Sheng X Zhang S. Tetrandrine suppresses cervical cancer growth by inducing apoptosis in vitro and in vivo . Drug Des Devel Ther . 2018 ; 13 : 119 – 27 . 10.2147/DDDT.S187776 30587932 PMC6304242

173. Zhu R Liu T Tan Z Wu X Li M Jiang L Tetrandrine induces apoptosis in gallbladder carcinoma in vitro . Int J Clin Pharmacol Ther. 2014 ; 52 : 900 – 5 . 10.5414/CP202123 25074868

174. Singh K Dong Q TimiriShanmugam PS Koul S Koul HK. Tetrandrine inhibits deregulated cell cycle in pancreatic cancer cells: differential regulation of p21 Cip1/Waf1 , p27 Kip1 and cyclin D1 . Cancer Lett . 2018 ; 425 : 164 – 73 . 10.1016/j.canlet.2018.03.042 29605511

175. Zhang Y Wang C Wang H Wang K Du Y Zhang J. Combination of tetrandrine with cisplatin enhances cytotoxicity through growth suppression and apoptosis in ovarian cancer in vitro and in vivo . Cancer Lett . 2011 ; 304 : 21 – 32 . 10.1016/j.canlet.2011.01.022 21333438

176. Favia A Pafumi I Desideri M Padula F Montesano C Passeri D NAADP-dependent Ca(2+) signaling controls melanoma progression, metastatic dissemination and neoangiogenesis . Sci Rep. 2016 ; 6 : 18925 . 10.1038/srep18925 26733361 PMC4702115

177. Faris P Pellavio G Ferulli F Di Nezza F Shekha M Lim D Nicotinic acid adenine dinucleotide phosphate (NAADP) induces intracellular Ca 2+ release through the two-pore channel TPC1 in metastatic colorectal cancer cells . Cancers (Basel). 2019 ; 11 : 542 . 10.3390/cancers11040542 30991693 PMC6521149

178. Correale P Tagliaferri P Celio L Genua G Montagnani S Bianco AR. Verapamil upregulates sensitivity of human colon and breast cancer cells to LAK-cytotoxicity in vitro . Eur J Cancer. 1991 ; 27 : 1393 – 5 . 10.1016/0277-5379(91)90018-9 1835854

179. Bruserud O Pawelec G. Effects of dipyridamole and R-verapamil on in vitro proliferation of blast cells from patients with acute myelogenous leukaemia . Leuk Res. 1993 ; 17 : 507 – 13 . 10.1016/0145-2126(93)90126-6 7685052

180. Bruserud O. Effect of dipyridamole, theophyllamine and verapamil on spontaneous in vitro proliferation of myelogenous leukaemia cells . Acta Oncol. 1992 ; 31 : 53 – 8 . 10.3109/02841869209088266 1586506

181. Yoon H Kim TW Shin SY Park MJ Yong Y Kim DW Design, synthesis and inhibitory activities of naringenin derivatives on human colon cancer cells . Bioorg Med Chem Lett . 2013 ; 23 : 232 – 8 . 10.1016/j.bmcl.2012.10.130 23177257

182. Rebello CJ Beyl RA Lertora JJL Greenway FL Ravussin E Ribnicky DM Safety and pharmacokinetics of naringenin: a randomized, controlled, single-ascending-dose clinical trial . Diabetes Obes Metab . 2020 ; 22 : 91 – 8 . 10.1111/dom.13868 31468636 PMC6956701

183. Tundis R Loizzo MR Menichini F Bonesi M Colica C Menichini F. In vitro cytotoxic activity of extracts and isolated constituents of Salvia leriifolia Benth. Against a panel of human cancer cell lines . Chem Biodivers . 2011 ; 8 : 1152 – 62 . 10.1002/cbdv.201000311 21674787

184. Schutz R Muller M Geisslinger F Vollmar A Bartel K Bracher F. Synthesis, biological evaluation and toxicity of novel tetrandrine analogues . Eur J Med Chem . 2020 ; 207 : 112810 . 10.1016/j.ejmech.2020.112810 32942071 PMC7473156

185. Liu T Liu X Li W. Tetrandrine, a Chinese plant-derived alkaloid, is a potential candidate for cancer chemotherapy . Oncotarget . 2016 ; 7 : 40800 – 15 . 10.18632/oncotarget.8315 27027348 PMC5130046

186. Chi M Evans H Gilchrist J Mayhew J Hoffman A Pearsall EA Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells . Sci Rep. 2016 ; 6 : 33132 . 10.1038/srep33132 27605043 PMC5015093

187. Hoffman A Carpenter H Kahl R Watt LF Dickson PW Rostas JA Dephosphorylation of CaMKII at T253 controls the metaphase-anaphase transition . Cell Signal . 2014 ; 26 : 748 – 56 . 10.1016/j.cellsig.2013.12.015 24407174

188. Wang J Guo M Ma R Wu M Zhang Y. Tetrandrine alleviates cerebral ischemia/reperfusion injury by suppressing NLRP3 inflammasome activation via Sirt-1 . PeerJ . 2020 ; 8 : e9042 . 10.7717/peerj.9042 32419986 PMC7211409

189. Song MY Wang JX Sun YL Han ZF Zhou YT Liu Y Tetrandrine alleviates silicosis by inhibiting canonical and non-canonical NLRP3 inflammasome activation in lung macrophages . Acta Pharmacol Sin. 2021 ;[Epub ahead of print]. 10.1038/s41401-021-00693-6 34417574 PMC9061833

190. Wang J Yao Z Lai X Bao H Li Y Li S Tetrandrine sensitizes nasopharyngeal carcinoma cells to irradiation by inducing autophagy and inhibiting MEK/ERK pathway . Cancer Med . 2020 ; 9 : 7268 – 78 . 10.1002/cam4.3356 32780562 PMC7541130

191. Xu XH Gan YC Xu GB Chen T Zhou H Tang JF Tetrandrine citrate eliminates imatinib-resistant chronic myeloid leukemia cells in vitro and in vivo by inhibiting Bcr-Abl/β-catenin axis . J Zhejiang Univ Sci B . 2012 ; 13 : 867 – 74 . 10.1631/jzus.B1200021 23125079 PMC3494025

192. Zhu X Sui M Fan W. In vitro and in vivo characterizations of tetrandrine on the reversal of P-glycoprotein- mediated drug resistance to paclitaxel . Anticancer Res . 2005 ; 25 : 1953 – 62 . 16158930

193. Jin J Wang FP Wei H Liu G. Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine . Cancer Chemother Pharmacol. 2005 ; 55 : 179 – 88 . 10.1007/s00280-004-0868-0 15378274

194. Liu ZL Hirano T Tanaka S Onda K Oka K. Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells . J Pharm Pharmacol. 2003 ; 55 : 1531 – 7 . 10.1211/0022357022115 14713364

195. Wan J Liu T Mei L Li J Gong K Yu C Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species (ROS)/Akt signaling . Br J Cancer . 2013 ; 109 : 342 – 50 . 10.1038/bjc.2013.334 23807172 PMC3721403

196. Chen Y Li P Yang S Tong N Zhang J Zhao X. Tetrandrine enhances the anticancer effects of arsenic trioxide in vitro . Int J Clin Pharmacol Ther. 2014 ; 52 : 416 – 24 . 10.5414/CP201939 24548979

197. Mei L Chen Y Wang Z Wang J Wan J Yu C Synergistic anti-tumour effects of tetrandrine and chloroquine combination therapy in human cancer: a potential antagonistic role for p21 . Br J Pharmacol . 2015 ; 172 : 2232 – 45 . 10.1111/bph.13045 25521075 PMC4403090

198. Yu M Liu T Chen Y Li Y Li W. Combination therapy with protein kinase inhibitor H89 and tetrandrine elicits enhanced synergistic antitumor efficacy . J Exp Clin Cancer Res. 2018 ; 37 : 114 . 10.1186/s13046-018-0779-2 29866132 PMC5987653

199. Xu WL Shen HL Ao ZF Chen BA Xia W Gao F Combination of tetrandrine as a potential-reversing agent with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia . Leuk Res . 2006 ; 30 : 407 – 13 . 10.1016/j.leukres.2005.08.005 16219352

200. Liu W Zhang J Ying C Wang Q Yan C Jingyue Y Tetrandrine combined with gemcitabine and cisplatin for patients with advanced non-small cell lung cancer improve efficacy . Int J Biomed Sci . 2012 ; 8 : 28 – 35 . 23675254 PMC3614858

201. Jin H Li L Zhong D Liu J Chen X Zheng J. Pulmonary toxicity and metabolic activation of tetrandrine in CD-1 mice . Chem Res Toxicol. 2011 ; 24 : 2142 – 52 . 10.1021/tx200290s 21992520

202. Tian Y Shen S Jiang Y Shen Q Zeng S Zheng J. CYP3A5 mediates bioactivation and cytotoxicity of tetrandrine . Arch Toxicol. 2016 ; 90 : 1737 – 48 . 10.1007/s00204-015-1584-8 26302866

203. Shi JP Li SX Ma ZL Gao AL Song YJ Zhang H. Acute and sub-chronic toxicity of tetrandrine in intravenously exposed female BALB/c mice . Chin J Integr Med. 2016 ; 22 : 925 – 31 . 10.1007/s11655-015-2303-2 26514966

204. Zhang YX Liu XM Wang J Li J Liu Y Zhang H Inhibition of AKT/FoxO3a signaling induced PUMA expression in response to p53-independent cytotoxic effects of H1: a derivative of tetrandrine . Cancer Biol Ther . 2015 ; 16 : 965 – 75 . 10.1080/15384047.2015.1040950 25893985 PMC4622009

205. Wei N Sun H Wang F Liu G. H1, a novel derivative of tetrandrine reverse P-glycoprotein-mediated multidrug resistance by inhibiting transport function and expression of P-glycoprotein . Cancer Chemother Pharmacol. 2011 ; 67 : 1017 – 25 . 10.1007/s00280-010-1397-7 20640574

206. Lin Y Wang Y Liu X Yan J Su L Liu X. A novel derivative of tetrandrine (H1) induces endoplasmic reticulum stress-mediated apoptosis and prosurvival autophagy in human non-small cell lung cancer cells . Tumour Biol. 2016 ; 37 : 10403 – 13 . 10.1007/s13277-016-4950-0 26846103

207. Naylor E Arredouani A Vasudevan SR Lewis AM Parkesh R Mizote A Identification of a chemical probe for NAADP by virtual screening . Nat Chem Biol . 2009 ; 5 : 220 – 6 . 10.1038/nchembio.150 19234453 PMC2659327

208. Jin X Zhang Y Alharbi A Hanbashi A Alhoshani A Parrington J. Targeting two-pore channels: current progress and future challenges . Trends Pharmacol Sci . 2020 ; 41 : 582 – 94 . 10.1016/j.tips.2020.06.002 32679067 PMC7365084

209. Simpson WG. The calcium channel blocker verapamil and cancer chemotherapy . Cell Calcium. 1985 ; 6 : 449 – 67 . 10.1016/0143-4160(85)90021-1 3912055

210. Genazzani AA Mezna M Dickey DM Michelangeli F Walseth TF Galione A. Pharmacological properties of the Ca 2+ -release mechanism sensitive to NAADP in the sea urchin egg . Br J Pharmacol . 1997 ; 121 : 1489 – 95 . 10.1038/sj.bjp.0701295 9257932 PMC1564845

211. Pahor M Furberg CD. Is the use of some calcium antagonists linked to cancer? Evidence from recent observational studies . Drugs Aging. 1998 ; 13 : 99 – 108 . 10.2165/00002512-199813020-00002 9739499

212. Solary E Bidan JM Calvo F Chauffert B Caillot D Mugneret F P-glycoprotein expression and in vitro reversion of doxorubicin resistance by verapamil in clinical specimens from acute leukaemia and myeloma . Leukemia . 1991 ; 5 : 592 – 7 . 1677057

213. Ledwitch KV Gibbs ME Barnes RW Roberts AG. Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein . Biochem Pharmacol . 2016 ; 118 : 96 – 108 . 10.1016/j.bcp.2016.08.013 27531061 PMC5341695

214. Muller C Bailly JD Goubin F Laredo J Jaffrezou JP Bordier C Verapamil decreases P-glycoprotein expression in multidrug-resistant human leukemic cell lines . Int J Cancer . 1994 ; 56 : 749 – 54 . 10.1002/ijc.2910560523 7906257

215. McCarthy M Auda G Agrawal S Taylor A Backstrom Z Mondal D In vivo anticancer synergy mechanism of doxorubicin and verapamil combination treatment is impaired in BALB/c mice with metastatic breast cancer . Exp Mol Pathol . 2014 ; 97 : 6 – 15 . 10.1016/j.yexmp.2014.04.013 24780744

216. Shiozaki A Katsurahara K Kudou M Shimizu H Kosuga T Ito H Amlodipine and verapamil, voltage-gated Ca 2+ channel inhibitors, suppressed the growth of gastric cancer stem cells . Ann Surg Oncol. 2021 ; 28 : 5400 – 11 . 10.1245/s10434-021-09645-0 33566246

217. Maruyama Y Murohashi I Nara N Aoki N. Effects of verapamil on the cellular accumulation of daunorubicin in blast cells and on the chemosensitivity of leukaemic blast progenitors in acute myelogenous leukaemia . Br J Haematol . 1989 ; 72 : 357 – 62 . 10.1111/j.1365-2141.1989.tb07716.x 2765404

218. Tsuruo T Iida H Yamashiro M Tsukagoshi S Sakurai Y. Enhancement of vincristine- and adriamycin-induced cytotoxicity by verapamil in P388 leukemia and its sublines resistant to vincristine and adriamycin . Biochem Pharmacol. 1982 ; 31 : 3138 – 40 . 10.1016/0006-2952(82)90097-1 7150340

219. Li P Zhong D Gong PY. Synergistic effect of paclitaxel and verapamil to overcome multi-drug resistance in breast cancer cells . Biochem Biophys Res Commun . 2019 ; 516 : 183 – 8 . 10.1016/j.bbrc.2019.05.189 31204050

220. Wang X Wang Z Wang K Gao M Zhang H Xu X. Metabolomics analysis of multidrug resistance in colorectal cancer cell and multidrug resistance reversal effect of verapamil . Biomed Chromatogr . 2021 ; 35 : e4976 . 10.1002/bmc.4976 32852057

221. Schuldes H Dolderer JH Zimmer G Knobloch J Bickeboller R Jonas D Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts . Eur J Cancer. 2001 ; 37 : 660 – 7 . 10.1016/S0959-8049(00)00450-0 11290442

222. Candussio L Decorti G Crivellato E Granzotto M Rosati A Giraldi T Toxicologic and pharmacokinetic study of low doses of verapamil combined with doxorubicin . Life Sci. 2002 ; 71 : 3109 – 19 . 10.1016/S0024-3205(02)02175-6 12408877

223. Ries F Dicato M. Treatment of advanced and refractory breast cancer with doxorubicin, vincristine and continuous infusion of verapamil. A phase I–II clinical trial . Med Oncol Tumor Pharmacother . 1991 ; 8 : 39 – 43 . 10.1007/BF02988570 2041381

224. Pingsheng F Tengyue Z Qiang H Qiang W Xin S Liting Q. Basic and clinical research on the therapeutic effect of intervention in primary liver cancer by targeted intra-arterial verapamil infusion . Cell Biochem Biophys. 2012 ; 62 : 59 – 67 . 10.1007/s12013-011-9259-4 21830124

225. Huang J Zhang T Ma K Fan P Liu Y Weng C Clinical evaluation of targeted arterial perfusion of verapamil and chemotherapeutic drugs in interventional therapy of advanced lung cancer . Cancer Chemother Pharmacol. 2013 ; 72 : 889 – 96 . 10.1007/s00280-013-2271-1 23975244 PMC3784059

226. Fan GF Pan JJ Fan PS Zhang TY Liu YB Huang J The clinical observation of verapamil in combination with interventional chemotherapy in advanced gastric cancer . Eur Rev Med Pharmacol Sci. 2018 ; 22 : 5508 – 18 . 10.26355/eurrev_201809_15811 30229822