Background
Secondary metabolites have been developed by plants to combat the harsh environmental conditions [ 1 ]. For several centuries, plant extracts rich in secondary metabolites have been used to treat human disease and, in China, plant extracts are used as traditional medicines. Improved methods in chemical extraction have resulted in the isolation of bioactive molecules from plants and their subsequent use as purified forms of herbal medicines [ 2 ]. Sesquiterpene lactones are a large and diverse group of plant metabolites with potent bioactivity [ 3 ]. The number of new sesquiterpene lactones from plants is increasing, and currently, there are approximately 500 sesquiterpenes that have been identified [ 4 ]. The potential anticancer properties of sesquiterpene lactones have begun to be investigated in clinical trials [ 5 ].
Parthenolide is a bioactive sesquiterpene lactone that has the potential to be used in cancer chemotherapy regimens [ 6 ]. Parthenolide has been reported to trigger apoptosis in colorectal cancer cells in vitro [ 7 ], and to enhance the chemosensitivity of breast and lung cancer cells to paclitaxel [ 8 , 9 ]. In combination with docetaxel, parthenolide has been shown to chemosensitize HBL-100 and MDA-MB-231 breast cancer cells in vitro and reduced tumor spread in vivo in a mouse xenograft model [ 10 ] . Radiosensitivity of prostate cancer cells is increased by pretreatment with parthenolide [ 11 ]. Also, parthenolide has been reported to halt the growth of cells in a multiple myeloma cell line in vitro [ 12 ].
However, the effects of parthenolide have not been previously examined in human thyroid cancer cells in vitro . Thyroid cancers are rare, but their incidence is increasing worldwide, resulting in increased mortality rates for thyroid cancer [ 12 ]. The most common type of thyroid cancer is papillary thyroid carcinoma [ 12 ]. Late diagnosis and the presence of metastases make advanced thyroid cancer difficult to manage clinically [ 13 ]. Surgical excision and chemotherapy are the first-line treatment for early-stage and advanced thyroid cancer, respectively [ 14 ]. However, currently available chemotherapy drugs impair the quality of life of the patients due to their adverse toxic effects [ 15 ].
Therefore, this study aimed to examine the effects of the sesquiterpene lactone, parthenolide, on migration, autophagy, and apoptosis of MDA-T32 human papillary thyroid carcinoma cells in vitro and in mouse tumor xenografts.
Material and Methods
Cell viability assay
The MTT assay was used to assess the viability and proliferation of MDA-T32 human papillary thyroid carcinoma cells, compared with a normal human thyrocyte cell line. At 70% confluence, the MDA-T32 cells were seeded in 96-well plates and treated with 0–100 μM of parthenolide. After an incubation of 24 hours, the cells were incubated with MTT for 4 h. Then, the media was removed and the colored formazan product was solubilized by 200 μl of dimethyl sulfoxide (DMSO). The viability of the MDA-T32 human papillary thyroid carcinoma cells and the normal human thyrocytes was then determined by measuring absorbance at 570 nm.
Acridine orange (AO) and ethidium bromide (EB) staining
The MDA-T32 cells were cultured in 6-well plates (0.6×10 6 cells/well) and incubated for 12 h. MDA-T32 cells were treated with parthenolide for 24 h at 37°C, and slides were prepared by placing 10 μl of cell culture onto glass slides. A drop of the AO and EB solution was added and a coverslip was placed on the slides, which were examined using a fluorescence microscope.
Transmission electron microscopy (TEM)
The MDA-T32 human papillary thyroid carcinoma cells were fixed in the solution of 4% glutaraldehyde and 0.05 M sodium cacodylate, postfixed in 1.5% osmium tetroxide (OsO4), and dehydrated in alcohol. The samples were then embedded in Epon 812 and observed using a Zeiss CEM 902 electron microscope (Zeiss, Oberkochen, Germany).
Cell migration and Invasion assay
Briefly, 1×10 4 MDA-T32 human papillary thyroid carcinoma cells were maintained in the upper chamber of transwells with 8 μm pores. In the lower chamber, RPIM-1640 medium was added, followed by incubation for 24 h at 37°C. Extracellular matrix gel was used for the cell invasion assay. The non-migrated and non-invasive cells were removed by swabbing. The cells that invaded and migrated to the lower side of the transwell chamber were fixed and stained the crystal violet and observed under the light microscope.
Western blot
The MDA-T32 human papillary thyroid carcinoma cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). RIPA buffer was used for lysis of the cell pellet. A BCA assay was used to determine the protein content of each sample. From each sample, around 30 μg of protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being treated with TBS, the membranes were incubated with primary antibody at 4°C and then with secondary antibody. The bands of interest were visualized using enhanced chemiluminescence (ECL) reagent.
Mouse tumor xenografts from MDA-T32 human papillary thyroid carcinoma cells
The National Institutes of Health (NIH) standards for the care and use of laboratory animals were approved by the Sichuan Provincial Cancer Hospital, Chengdu, Sichuan Province, China. Mice were injected with 5×10 6 MDA-T32 human papillary thyroid carcinoma cells subcutaneously in the left flank. Mice in the control group (n=5) were injected intraperitoneally with DMSO (0.1%) in normal saline, and mice in the treated group (n=5) were injected with parthenolide diluted with 100 μL normal saline at a dose of 20 mg/kg body weight. Parthenolide treatment and control injections were given three times per week. At the end of six weeks, the mice were euthanized and the xenograft tumors were harvested for further assessment.
Statistical analysis
The experiments were performed in triplicate and data were presented as the mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. A P-value <0.05 was considered to be statistically significant.
Results
Parthenolide reduced cell viability of MDA-T32 human papillary thyroid carcinoma cells
The MTT assay examined the effects of parthenolide on cell viability ( Figure 1A ) of the MDA-T32 human papillary thyroid carcinoma cells. The results showed that parthenolide significantly decreased the proliferation rate of thyroid cancer cells ( Figure 1B ). The IC 50 of parthenolide was found to be 12 μM. These effects of parthenolide were found to exhibit a dose-dependent trend, as the proliferation of the MDA-T32 cells decreased with increase in the dose of parthenolide.
Parthenolide induced apoptosis of the MDA-T32 cells
Acridine orange (AO) and ethidium bromide (EB) staining showed that parthenolide caused apoptosis of the MDA-T32 human papillary thyroid carcinoma cells, as shown by the increase in AO-stained cells ( Figure 2 ). The induction of apoptosis by parthenolide was accompanied by the increased expression levels of Bax protein and decreased levels of Bcl-2 ( Figure 3 ), which was dose-dependent.
Parthenolide induced autophagy in the MDA-T32 cells
Electron microscopy showed that parthenolide treatment of the MDA-T32 cells resulted in the development of autophagosomes ( Figure 4 ). The number of autophagosomes increased as the dose of parthenolide was increased. Parthenolide treatment increased the expression of apoptosis-associated proteins, LC3-II and beclin-1, with no significant effects on LC3-I expression ( Figure 5 ).
Parthenolide inhibited cell migration of the MDA-T32 cells
The transwell assay showed that parthenolide inhibited the migration of the MDA-T32 cells at an IC 50 of 12 μM ( Figure 6 ). The migration rate of the MDA-T32 cells was decreased to 32% compared with the control.
Parthenolide inhibited cell invasion of the MDA-T32 cells
The transwell assay showed that parthenolide decreased the invasion of the MDA-T32 cells at an IC 50 of 12 μM ( Figure 7 ). The invasion rate of the MDA-T32 cells was decreased to 25% compared with the control.
Parthenolide inhibited the mTOR/PI3K/AKT pathway
Parthenolide decreased the phosphorylation of components of the mTOR/PI3K/AKT pathway and included the expression of p-mTOR, p-PI3K, and p-AKT ( Figure 8 ). The total protein levels of mTOR, PI3K, and AKT remained constant, as determined by Western blot analysis.
Parthenolide inhibited MDA-T32 cell xenograft tumor growth
The effects of parthenolide were also assessed on the growth of the MDA-T32 cell mouse tumor xenografts at a dose of 20 mg/kg. Parthenolide suppressed the tumor volume ( Figure 9A ) and the tumor weight ( Figure 9B ) of the MDA-T32 cell mouse tumor xenografts.
Discussion
Sesquiterpene lactones have shown potential as anticancer agents [ 16 ]. The findings of the present study demonstrated the tumor suppressor effects of the sesquiterpene lactone, parthenolide, on MDA-T32 human papillary thyroid carcinoma cells. Parthenolide suppressed the growth of human MDA-T32 thyroid cancer cells with a half-maximal inhibitory concentration (IC 50 ) of 12 μM. Previous studies have also shown the potent anticancer effects of parthenolide. Parthenolide has been shown to reduce the proliferation rate of gastric cancer cells in vitro [ 17 ]. Parthenolide has also been reported to target NF-kB to suppress the growth of pancreatic cancer cells in vitro [ 18 ]. In bladder cancer cells, parthenolide treatment resulted in cell cycle arrest and also increased cell apoptosis [ 19 ]. Parthenolide has also been reported to inhibit cell proliferation in human nasopharyngeal carcinoma cells in vitro [ 20 ]. The findings of the present study have shown that the inhibitory effects of parthenolide on MDA-T32 human papillary thyroid carcinoma cells included increased cell apoptosis, which was also associated with upregulation of Bax and down-regulation of Bcl-2.
The findings from previously reported studies support the findings of the present study. Parthenolide has previously been shown to cause apoptosis of HepG2 human liver cancer cells [ 21 ]. Parthenolide has also been shown to target the PI3K/AKT cascade to suppress the growth of cervical cancer cells in vitro [ 22 ]. In the present study, electron microscopy showed that parthenolide treatment caused the development of autophagosomes in the MDA-T32 human papillary thyroid carcinoma cells. The increased expression of LC3-II triggered by parthenolide in the MDA-T32 cells also confirmed that parthenolide induced autophagy in these cells. Previous studies also supported this finding, as parthenolide has been shown to induce autophagy in osteosarcoma cells via the induction of autophagy [ 23 ]. Also, parthenolide has been shown to cause autophagy in breast cancer cells in vitro [ 24 ], and to suppress the migration and invasion of breast cancer cells [ 25 ].
The findings of the present study showed that parthenolide reduced the migration and invasion of MDA-T32 human papillary thyroid carcinoma cells in vitro by downregulating the PI3K/AKT/mTOR signaling pathway. Sesquiterpene lactones, including parthenolide, have become recognized as having anticancer effects via multiple signaling pathways [ 26 ]. This study investigated the cell line, MDA-T32, which was isolated in 2014 from a 74-year-old man with papillary thyroid carcinoma and was previously shown to be suitable for the generation of mouse xenograft tumors [ 27 ]. In this study, parthenolide was shown to downregulate the PI3K/AKT/mTOR signaling pathway and inhibited the growth of MDA-T32 mouse tumor xenografts in vivo .
Conclusions
Parthenolide suppressed the growth of MDA-T32 human papillary thyroid carcinoma cells in vitro by the induction of autophagy and apoptosis. Parthenolide also reduced the migration of MDA-T32 cells. The effects of parthenolide were due to downregulation of the PI3K/AKT/mTOR signaling pathway. Further in vivo studies are required to evaluate the effects of parthenolide at different doses and on the different subtypes of human thyroid cancer.
Footnotes
Source of support: Departmental sources
Conflict of interest
None.
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