Introduction
Multiple myeloma (MM) is a cancer in which malignant plasma cells accumulate in the bone marrow [1] , which can result in typical disease manifestations, including anaemia, hypercalcaemia, immunosuppression, and end-organ damage, such as renal impairment and bone destruction [2] .
Although therapeutic advances have been achieved with the introduction of proteasome inhibitors, monoclonal antibodies, and immunomodulators, MM remains an incurable disease owing to intrinsic or acquired drug resistance [3] . Bortezomib (BTZ), a first-in-class selective proteasome inhibitor, has been validated for use in the treatment of MM [4] . Nevertheless, almost all patients with MM inevitably relapse or progress and develop resistance and/or intolerance to BTZ. Consequently, the discovery of novel therapeutic targets that can counter BTZ resistance in MM is urgently needed.
The JAK2/STAT3 pathway plays a central role in regulating cellular proliferation, apoptosis, drug resistance, and other critical hallmarks involved in the pathophysiology of MM [5] . Signal transducer and activator of transcription 3 (STAT3) is one of the seven members of the STAT protein family and is present in the cytoplasm as a monomer in its inactive state. STAT3 can be activated by phosphorylation of tyrosine 705, which leads to homodimerization followed by nuclear translocation, DNA binding, and gene transcription [6] . In the nucleus, STAT3 binds to consensus response elements in the promoter regions of target genes, thereby modulating the transcription of various genes that regulate cellular proliferation (such as cyclin D1 and c-Myc), survival (myeloid cell leukemia 1, MCL-1), and immunosuppressive growth factors (IL-10) [7] .
Several studies have shown that the PI3K/Akt/mTOR pathway controls biological processes critical to tumorigenesis, including apoptosis, translation, and the cell cycle [8] . Inhibition of this pathway could prolong overall survival and improve the quality of life of MM patients. Thus, the hyperactivated PI3K/Akt/mTOR pathway serves as a vital prognostic biomarker and therapeutic target in MM. Moreover, the downstream target oncogene Myc, which is also targeted by the JAK2/STAT3 pathway, regulates the cell cycle by promoting the expression of several G1 phase-correlated cyclins and cyclin-dependent catalytic kinases (CDKs), including CDK4, cyclin D1, and CDK6 [9] .
To identify a potential therapeutic agent, we performed virtual biological screening of our internal compound library and subsequent cell viability tests. We found that a novel compound, DCZ0358, exhibited potent activity against BTZ-resistant MM. The aim of the present study was to further investigate the effect and mechanism of action of DCZ0358 against BTZ-resistant MM.
Materials and Methods
Cell lines and culture conditions
The BTZ-resistant MM cell lines NCI-H929R and RPMI-8226R5 were obtained by gradually increasing concentrations of BTZ over a period of 10 months as previously decribed [10] . BTZ-sensitive cell lines were routinely cultured in RPMI-1640 medium (Gibco, Grand Island, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. To maintain the BTZ-resistant characteristics of the applicable cell lines, they were cultured in the presence of 20 nM BTZ. BTZ treatment ended at least two weeks prior to each experiment. All cells were incubated in a humidified atmosphere at 37°C and 5% CO 2 . Primary CD138 + plasma cells were isolated from diagnostic MM patient bone marrow (BM) samples using CD138 microbeads (Miltenyi Biotech, Auburn, USA). All human samples were collected after obtaining written informed consent from patients with MM, in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of the Shanghai Tenth People’s Hospital, Tongji University.
Reagents and antibodies
DCZ0358 was synthesized by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China) according to a previously described method [11] . Bortezomib was purchased from Sigma (St Louis, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Shanghai Yeasen Biotechnology Co., Ltd. (Shanghai, China). The Annexin V/PI apoptosis detection kit was purchased from BD Pharmingen (San Diego, USA). Antibodies against JAK2, phosphor (p)-JAK2 (Tyr1007/1008), p-PI3K (Tyr607), Akt, p-Akt (Ser473), STAT3, p-STAT3 (Tyr705), mTOR, p-mTOR (Ser2481), cyclin D1, CDK4, CDK6, and c-Myc were purchased from Abcam (Cambridge, UK). Antibodies against caspase-3, caspase-9, and cleaved caspase-8 were purchased from Cell Signaling Technology (Beverly, USA).
Cell viability assay
Cells were seeded in 96-well plates (100 μL/well) at a density of 2×10 5 cells/mL and treated with the indicated concentrations (5, 10, 20, 40, and 80 μM) of DCZ0358 for 24, 48, and 72 h. At these time points, 10 μL of CCK-8 kit reagent was added to each well and incubated at 37°C for 2 h. The final absorbance was measured at 450 nm using a microplate reader. The CalcuSyn software program (CalcuSyn; Biosoft, Cambridge, UK) was used to analyze the half-maximal inhibitory concentration (IC 50 ) of DCZ0358.
Apoptosis measurement
RPMI-8226R5 and NCI-H929R cells were seeded at a density of 2×10 5 cells/mL in 12-well plates and treated with DCZ0358 (0, 15, 30, and 60 μM) for 48 h. Then, cell apoptosis was detected by flow cytometry with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (BD Pharmingen, San Diego, USA). Annexin V-FITC was employed to stain cells at 4°C for 40 min in the dark and subsequently stained with PI for another 10 min. A BD FASC Canto II flow cytometer (BD Biosciences, San Jose, USA) was used for analysis. Annexin V + /PI – (early apoptosis) and Annexin V + /PI + (late apoptosis) were identified as apoptotic cells.
Soft agar assay
A total of 3000 cells were mixed with RPMI-1640 medium containing 20% FBS and 0.33% agar, placed on top of the base layer of 0.5% agar and seeded in 12-well plates. After 14 days of incubation with or without DCZ0358, the formed colonies were stained with 0.5% crystal violet for 60 min at room temperature. Next, a digital camera was used to record images of colonies, and ImageJ 1.53 was used to count the colonies (National Institutes of Health, Bethesda, USA).
Cell cycle analysis
BTZ-resistant MM cells were treated with DCZ0358 for the indicated time, harvested, and fixed with precooled 75% ethanol overnight. The cells were then washed with cold PBS, stained with 500 μL of PI/RNase staining buffer (BD Pharmingen, Franklin Lakes, USA) at 4°C for 30 min, and finally subject to cell cycle analysis with a BD FACS Canto II flow cytometer (BD Biosciences). The results were analyzed using ModFit LT 5.0 software (Verity Software House, Inc., Topsham, USA).
EdU incorporation assay
The 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was performed using an EdU kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. In brief, cells were exposed to DCZ0358 for 48 h, and before collection, cells were treated with 50 μM EdU at 37°C for 2 h. The cells were then harvested, resuspended in PBS, and treated with Triton X-100. Next, the cells were washed three times with PBS. Thereafter, the cells were exposed to 100 μL of 1× Apollo reaction cocktail (deionized water, Apollo® reaction buffer [reagent B], Apollo® catalytic buffer [reagent C], Apollo® fluorescent dye solution buffer [reagent D], Apollo® buffer additives [reagent E]) at 37°C for 30 min and incubated with DAPI at room temperature for 5 min to stain the cell nuclei. A fluorescence microscope was used to detect positive cells which were red-colored.
Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay
BTZ-resistant cells (2×10 5 cells/mL) were treated with medium or DCZ0358 (20 μM) for 48 h and then fixed with 4% paraformaldehyde (Sigma) for 15 min. A one-step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to detect DNA fragmentation following the manufacturer′s instructions. The cell nuclei were stained with DAPI, and images were acquired using a confocal laser scanning microscope (LSM710; Carl Zeiss, Jena, Germany).
Western blot analysis
BTZ-resistant cells (2×10 5 cells/mL) were treated with various concentrations of DCZ0358 and lysed with lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol) to extract proteins. A BCA kit (Beyotime Institute of Biotechnology) was used to detect the protein concentrations. Protein samples (30 μg) were subject to 8%–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with the corresponding primary antibodies at 4°C overnight. The membranes were then sequentially incubated with specific secondary antibodies at room temperature for 1 h, and immunoreactive bands were detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, USA). Primary antibodies against caspase-3, cleaved caspase-8, caspase-9, cyclin D1, CDK4, CDK6, JAK2, p-JAK2, STAT3, p-STAT3, p-PI3K, Akt, p-Akt, c-Myc, mTOR, p-mTOR and β-actin were used. Secondary antibodies used were Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories Inc, West Grove, USA).
Animal study
Six-week-old male BALB/c nude mice were purchased from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China), raised in a standard animal laboratory and fed with a standard diet. Human BTZ-resistant cells (3×10 7 ) were suspended in 100 μL serum-free culture medium and subcutaneously injected into the upper flank region of nude mice. When the tumors were measurable, mice were randomly divided into a control group ( n =4) that was treated with vehicle (200 μL of saline containing 10 μL dimethyl sulfoxide) and a DCZ0358 treatment group ( n =4) that was treated with 10 mg/kg DCZ0358 in vehicle. Mice were administered with DCZ0358 or vehicle via intraperitoneal injection for 15 days. Tumor size and body weight were measured daily, and the tumor volume was calculated as (length×width 2 )×0.5. All mice were euthanized at the end of the experiment, and the tumors and vital organs, including the heart, liver, spleen, and kidney, were harvested for hematoxylin-eosin (H&E) staining, TUNEL assay, and immunohistochemical staining. All animal-related experimental procedures were approved by the Animal Care and Use Committee of the Tenth People’s Hospital of Tongji University (Shanghai, China). This study was approved by the Science and Technology Commission of Shanghai Municipality (ID: SYXK 2018–0034).
Immunohistochemistry
The paraffin-embedded tumor tissue sections were dewaxed and rehydrated for immunohistochemistry (IHC) analysis, and the antigens were retrieved by heating in an autoclave in 0.01 M sodium citrate buffer (pH 6.0). After pre-incubation with normal goat serum at 37°C for 40 min to block nonspecific binding sites, the slides were incubated with primary antibodies at 4°C overnight. On the second day, slides were incubated with secondary antibodies at room temperature for 1 h, then observed using diaminobenzidine peroxide solution and counterstained with hematoxylin. Images of representative fields were taken by the Aperio Image Scope (Leica Biosystems, Wetzlar, Germany).
Statistical analysis
All data are presented as the mean±standard deviation (SD) of three independent experiments. Statistical significance was determined by Student’s t test or one-way ANOVA using SPSS v22.0 (IBM, Armonk, USA). Statistical significance was set at P <0.05.
Results
DCZ0358 treatment exhibited potent cytotoxicity against BTZ-resistant MM cells
The chemical structure of DCZ0358, which has a molecular weight of 417.8, is shown in Figure 1 A. The CCK-8 assay revealed that DCZ0358 potently inhibited the proliferation of BTZ-resistant MM cells in a concentration-dependent manner, with IC 50 values of 14.68 μM and 23.37 μM in RPMI-8226R5 and NCI-H929R cells, respectively ( Figure 1 B). In addition, the anti-proliferative effect of DCZ0358 on BTZ-resistant cells increased with prolonged treatment ( Figure 1 C). In addition, after DCZ0358 treatment, colony formation and EdU incorporation were lower than those in the control. These results illustrated that DCZ0358 had a potent inhibitory effect on BTZ-resistant MM cells ( Figure 1 D,E).
DCZ0358 induced apoptosis via caspase activation
Drug resistance is conferred by dysfunctional apoptosis in myeloma cells. As shown in Figure 2 A, DCZ0358 treatment for 48 h caused a drastic increase in the proportion of early-stage (Annexin V + /PI – ) and late-stage (Annexin V + /PI – ) apoptotic cells, and this increase varied consistently with DCZ0358 concentrations. In addition, TUNEL assay revealed an increase in TUNEL-positive MM cells upon DCZ0358 treatment ( Figure 2 B). As shown in Figure 2 C, western blot analysis revealed that DCZ0358 treatment significantly enhanced the cleavage of caspase-3, caspase-8, and caspase-9 in a dose-dependent manner. We also validated this inhibitory effect in patient-derived cells from three refractory/relapsed MM patients and the results showed that DCZ0358 induced significant apoptosis in primary MM cells ( Figure 2 D).
DCZ0358 induced G0/G1 phase arrest in BTZ-resistant MM cells
Many antitumor drugs exert a therapeutic effect by interfering with cell cycle progression via inhibition of cell cycle regulation. Therefore, to explore the effects of DCZ0358 on cell cycle progression, flow cytometry was performed to investigate changes in the cell cycle distribution after DCZ0358 treatment. The results showed that the ratio of BTZ-resistant MM cells in the G0/G1 phase clearly increased in a dose-dependent manner ( Figure 3 A). Next, we evaluated proteins that promote the G0/G1 phase of the cell cycle, and found that, consistent with the flow cytometry results, the expression levels of the key regulatory proteins cyclin D1, CDK4, and CDK6 were dramatically down-regulated ( Figure 3 B,C).
DCZ0358 blocked the JAK2/STAT3 pathway in BTZ-resistant cells
To decipher the mechanism underlying the DCZ0358-induced anti-myeloma effect, the expression levels of proteins essential to a few major signaling pathways were measured. The results showed that DCZ0358 decreased the phosphorylation levels of JAK2 and its downstream effector STAT3 in BTZ-resistant cells ( Figure 4 A). In addition, upregulation of the c-Myc oncogene is regarded as the trigger for the aberrant genomic event leading to MM progression [12] , and DCZ0358 drastically downregulated its expression level. Additionally, PI3K/Akt/mTOR, another vital pathway that is downstream of JAK, was also inactivated after DCZ0358 treatment ( Figure 4 A,B).
DCZ0358 suppressed tumor growth in vivo
Our in vitro experiments have verified the anti-proliferative effect of DCZ0358 in BTZ-resistant cells. To determine whether DCZ0358 can also kill tumors in vivo , a myeloma xenograft model was established by subcutaneously inoculating NCI-H929R cells into mice. We then analysed the anti-tumor effect of DCZ0358 in vivo by observing changes in specific indicators, such as tumor size, between the drug treatment group and control group. We found that continuous application of DCZ0358 for 15 consecutive days remarkably inhibited tumor growth, as demonstrated by tumor size ( Figure 5 A) and tumor volume ( Figure 5 B). Meanwhile, no significant difference was observed in mouse weights between the two groups ( Figure 5 C). Ki67 is strongly associated with cell proliferation. Immunofluorescence staining demonstrated that the number of Ki67-positive cells was markedly reduced in the treatment group, compared with that in the control group ( Figure 5 D). In addition, the proportion of apoptotic cells was markedly increased in the treatment group compared with that in the control group, as assessed by TUNEL staining ( Figure 5 E). The safety and tolerability of DCZ0358 in vivo were further investigated by H&E staining ( Figure 5 F). The results showed that the enlargement of necrotic proportions depended on DCZ0358 treatment, while no obvious differences were observed in organs such as the heart, liver, kidney and spleen. Consistent with our in vitro findings, decreased p-JAK2 expression was detected in the DCZ358-treated group compared with that in the vehicle control group ( Figure 5 G). These results suggest that DCZ0358 may exert powerful antitumor effects in vivo .
Discussion
Drug resistance may be resulted from mutations or alternative splicing during treatment and the activation of alternative compensatory signaling pathways or other abundant adaptive responses [13] . Although BTZ-based first-line chemotherapy has improved the survival rates of MM patients, resistance to BTZ remains a major challenge. After acquiring drug resistance, most patients eventually relapse, followed by the development of the disease [14] . In this study, we illustrated the potential mechanism by which DCZ0358 counteracts MM resistance by culturing isogenic sensitive cell lines in BTZ for an extended time to establish two BTZ-resistant MM cell lines.
Through virtual screening of our group’s compound library, we identified and synthesized a new aryl guanidine compound, DCZ0358. We previously reported that this compound showed potent antiproliferative activity in MM [15] . In the present study, DCZ0358 displayed a significant dose- and time-dependent antiproliferative effect on BTZ-resistant MM cell lines. DCZ0358 has very similar remarkable cytotoxicity towards primary MM cells and MM cell lines, suggesting that it could also kill MM cells in clinical samples.
In most malignancies, impaired apoptosis is the main factor contributing to chemoresistance and radioresistance, and resistance to chemotherapy is partly mediated by an inherent defect in apoptosis [ 16 , 17 ] . Therefore, proper modulation of the apoptotic pathway could attenuate resistance and enhance the efficacy of chemotherapy. Apoptosis can be initiated through two alternative pathways: the intrinsic and extrinsic pathways, which ultimately converge on the proteolytic activation of procaspase-3 [18] . Then, the executioner caspase-3 further catalyzes the hydrolysis of numerous proteins, leading to cell death [ 19 , 20 ] . In the current study, flow cytometry data indicated that DCZ0358 promoted cell apoptosis, and the ratio of apoptosis increased as the concentration increased. In addition, this also demonstrated the activation of caspase-3, caspase-8, and caspase-9 proteins in DCZ0358-treated BTZ-resistant MM cells. We also observed caspase-dependent apoptosis and necrosis in xenotransplanted tumors following DCZ0358 treatment in vivo . The sensitivity of BTZ-resistant cell lines was partially restored when the two apoptotic signaling pathways were induced by DCZ0358.
As dysregulation of the cell cycle is involved in MM progression, drug resistance, and altered immune surveillance, it has become a novel and important therapeutic target for MM [ 21 , 22 ] . A cyclin regulatory component and CDK are essential in the main checkpoints involved in cell cycle regulation. Activated cyclin D1 interacts with CDK4 and CDK6 to form a holoenzyme complex, which then acts as an executor to initiate a downstream cascade response and drive cells to cross the G1/S checkpoint [ 23 , 24 ] . In this study, DCZ0358 rendered the cell cycle stagnant in the G0/G1 phase by inhibiting the expressions of the regulatory proteins cyclin D1, CDK4, and CDK6.
Activation of the JAK/STAT signaling pathway is strongly associated with MM progression and resistance. STAT3 is generally located in the cytoplasm and can be phosphorylated by JAK2, which results in its dimerization and eventual translocation into the nucleus [25] . Upon translocation, STAT3 regulates the expressions of its target genes involved in proliferation, apoptosis, and cancer immunoediting [26] . It has been reported that inhibiting the JAK2/STAT3 signaling pathway can sensitize head and neck cancers quamous carcinoma and non-small cell lung cancer to tyrosine kinase inhibitors [ 27 , 28 ] . Moreover, the inhibition of STAT3 can reverse the drug resistance of hematological malignancies, such as leukemia [29] . In this study, we found that DCZ0358 inhibited not only the phosphorylation of JAK2 and STAT3 but also the expressions of STAT3 regulatory genes, including cyclin D1 and anti-apoptotic genes. Persistent activation of STAT3 drives tumorigenesis via protecting MM cells against apoptosis, implying that the suppression of STAT3 activity by agents such as DCZ0358 could increase the susceptibility of cancer cells to apoptosis.
Interestingly, the PI3K/Akt/mTOR pathway acts upstream of STAT3 phosphorylation and promotes tumorigenesis. At the apex of this pathway, activated PI3K phosphorylates and activates Akt, which then localizes to the plasma membrane [30] . Subsequently, activated Akt initiates a cascade of downstream effectors, particularly p-mTOR (Ser-2448). mTOR directly activates STAT3 via phosphorylation at the S727 residue. Meanwhile, as a pivotal kinase downstream of PI3K/Akt, mTOR plays a key role in the regulating cell proliferation, survival, and apoptosis.
This is particularly significant because published reports have demonstrated the crosstalk between the STAT3 and PI3K/Akt/mTOR pathways when driving resistance to targeted therapies, and that JAK2 is an upstream activator of both pathways [31] . In addition, it has been reported that inhibition of JAK and mTOR functions as a therapeutic method to inhibit dysregulated myeloid hyperplasia caused by excessive activation of STAT3 [32] . In this study, we explored the possible changes involved in the above-mentioned pathways and found that DCZ0358 suppressed the phosphorylation of PI3K, Akt, mTOR, JAK2 and STAT3, which most likely resulted in the observed anti-myeloma effects. c-Myc, a key growth factor correlated with tumor, is regulated by STAT3, and we found that DCZ0358 could downregulate the expression of this growth factor. These results suggest that suppression of STAT3 activity by DCZ0358 may lead to increased apoptosis and chemosensitivity and decreased proliferation in BTZ-resistant MM cells.
Finally, we established a xenograft mouse model and found that DCZ0358 prevented tumor growth in vivo and did not exert toxic effects in mice. This provides evidence for the potential application of DCZ0358 in the treatment of BTZ-resistant MM.
In summary, we demonstrated the pharmacological safety and therapeutic efficacy of DCZ0358 in BTZ-resistant MM cells by downregulating the expressions of pivotal genes involved in the cell cycle, cell survival, and chemoresistance. Our results provide a rationale and ideas for the development and application of DCZ0358 in the treatment of BTZ-resistant MM, and suggest that further testing in relapsed and drug-resistant patients should be performed in the future.
COMPETING INTERESTS
The authors declare that they have no conflicts of interest.
Funding Statement
This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81900211, 81971529, 81870158 and 82070224), the Natural Science Foundation of Shanghai, China (No. 19ZR1467800), and Shanghai Sailing Program, China (No. 21YF1435000).
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