Cervical cancer is one of the three major malignant tumors in the female reproductive system. It has the fourth highest incidence and mortality rate of all cancers in women worldwide, and seriously harms their physical and mental health. A large number of studies have shown that human papillomavirus (HPV) infection is the primary trigger for cervical cancer, and more than 90% of cervical cancer cases are associated with HPV infection. The carcinogenic mechanism mainly occurs via the E6 and E7 oncogenes,[3,4] and the activation of oncogenes, as well as the inactivation of tumor suppressor genes, is associated with HPV infection. The tumor protein of the E6 virus can cause p53 ubiquitination and proteasomal degradation, and the E7 virus can block the function of the retinoblastoma (Rb) protein. Although high-risk HPV infection is an important factor for cervical cancer, simple high-risk HPV infection is not sufficient to induce tumor progression.[7,8] The pathogenesis of cervical cancer has not yet been elucidated. Despite the recent developments in surgery and chemoradiotherapy, there is no satisfactory treatment for advanced or metastatic cervical cancer, and there is an urgent need to find more effective treatments or therapies. Molecular targeted therapies, which have emerged in recent years, will provide new ideas for the treatment of such patients.
Kim et al found that Taurine (Tau) can act synergistically with the anticancer drug Cisplatin (DDP), increasing the drug's effect while decreasing the toxicity, thus inhibiting the growth of cervical cancer cells; however, the detailed mechanisms underlying these effects are still unclear. The chemical name of Tau is 2-aminoethanesulfonic acid; it is the most abundant β-amino acid in the human body. It has many biological functions, such as regulating the intracellular osmotic pressure and calcium balance, mediating anti-oxidation and anti-inflammation effects, and immune regulation. Tau is an important endogenous anti-injury substance in the human body and is a good endogenous cytoprotective agent.[12–14] In clinics, it is mainly used for the prevention and treatment of cardiovascular diseases, diabetes, cataract, and hepatobiliary and ophthalmic diseases, such as myocarditis, liver cell damage, and ischemia-reperfusion injury. Our earlier studies[15–17] have found that Tau can induce the apoptosis of lung cancer, breast cancer, and liver cancer cells by upregulating the expressions of the proapoptotic protein Bax, p53 upregulated modulator of apoptosis (PUMA), and P53, while downregulating the expression of the anti-apoptotic protein Bcl-2; furthermore, Tau shows more pronounced inhibitory effects on the proliferation of p53 −/− tumor cells than p53+/+ tumor cells, suggesting that the Tau-induced apoptosis of cancer cells is not only related to the mitochondrial apoptotic pathway, but also associated with other apoptotic signaling pathways.
Our recent studies[18,19] have found that regulating the MST1-Hippo signaling pathway can induce the apoptosis of lung cancer and colon cancer cells. Human mammalian sterile 20-like kinase 1 (MST1) is one of the core members of the Hippo pathway; the gene for MST1 is located on chromosome 20q11 and encodes a 59-K protein. Yes-associated protein (YAP) is a major downstream effector molecule of the Hippo signaling pathway, as well as a transcriptional coactivator, which can regulate cell proliferation and apoptosis. Clinical studies have shown that high YAP expression in ovarian cancer tissues is associated with malignant clinicopathological features, suggesting that YAP may be a potential target for the targeted therapy of ovarian cancer. Chiang and Liang[22,23] found that the MST-Hippo pathway and the PI3K/AKT/mTOR pathway have common regulatory roles towards cell proliferation and organ size, and MST1 is the molecular target that crosses between these two pathways, and acts as a tumor target molecule. However, the effects of Tau on the apoptosis of cervical cancer cells and MST1 expression have rarely been reported. In this study, the effects of exogenous MST1 on the Tau-induced apoptosis of cervical cancer SiHa cells were observed, aiming to investigate the roles and mechanism of MST1 in the Tau-induced apoptosis of cervical cancer cells.
Cell lines and plasmid vector
Cells from the human cervical cancer cell line SiHa were purchased from the Cell Bank of Type Culture Collection Committee, Chinese Academic Science (Shanghai, China). The overexpression plasmid p-EGFP-MST1 carrying Enhanced Green Fluorescent Protein (EGFP) of human wild-type MST1 and the empty vector p-EGFP-N1 were constructed by our lab.
The SiHa cells were cultured in complete DMEM containing 100 U/mL penicillin, 100 U/mL streptomycin, and 10% FBS at 37°C, 5% CO2, and saturated humidity. The cells in the logarithmic growth phase were then harvested for the MTT assay. The experiment was performed by dividing the cells into seven groups: Control, Tau 10 mmol/L, Tau 20 mmol/L, Tau 40 mmol/L, Tau 80 mmol/L, Tau 160 mmol/L, and Tau 320 mmol/L.
The SiHa cells were collected, and single-cell suspensions were prepared using complete H-DMEM, and inoculated (200 μL) into 96-well plates, yielding a cell density of 1 × 103–1 × 104 cells/well (the marginal wells were filled with sterile PBS) making the cell density as about 1 × 103–1 × 104 cells/well. Three replicate wells were set up. The cells were cultured for 24 h, 48 h, or 72 h, after which 20 μL of MTT solution (Beyotime, Haimen, China) was added to each well. The reaction with MTT was terminated after another 4 h of incubation. The plates were shaken 10 min for full dissolution. The absorbance of each well was continuously measured 3 to 5 times at 490 nm, and the results were recorded to calculate the inhibition rate of cell proliferation using the following formula: Cell proliferation inhibition rate (%) = 1 − cell viability rate = (1 − A experiment/A Control) × 100%.
The cell apoptosis was detected by flow cytometry. First, 1−5 × 105 SiHa cells were collected after 48 h of drug treatment, followed by the addition of 500 μL of Binding Buffer to suspend the cells. Then, 5 μL of annexin V-FITC (MultiSciences, Hangzhou, China) was added, and the cell suspension was mixed. Next, 5 μL of propidium iodide was added to the cell suspension, followed by mixing; the reaction was allowed to occur for 5 to 15 min at room temperature in the dark. Apoptosis was detected using a Fascallbar flow cytometer (Becton Dickinson, USA). The clinically used first-line antitumor drug cisplatin (DDP) was used as the positive control.
Plasmid and transfection
The PolyJetTM in vitro DNA transfection reagent was used to transfect the pEGFP-N1-MST1 recombinant plasmid (Cat.SL100688; SignaGen, Rockville, MD, USA) and empty vector plasmid pEGFP-N1 (Clontech, Mountain View, CA, USA) into the SiHa cells. First, the SiHa cells to be transfected were cultured in six-well plates until the cell density reached 60% to 70%; the old medium was discarded before the transfection. Next, 900 μL of fresh complete medium was then added to each well and the cells were cultured for 30 to 60 min. Then, 50 μL of transfection solution was added into each well, followed by gentle mixing; the cells were then cultured for 5 h at 37°C and 5% CO2 conditions. The culture medium was then replaced by DMEM containing 10% FBS (TransGen Biotech, Beijing, China), followed by incubation for another 24 to 48 h. The transfection efficiency was then observed; confirmation of the transfection was performed by visualizing the cells using fluorescence microscopy.
Preparation of transfection solution: Solution A: 1 μg of the plasmid was dissolved in 50 μL of serum-free DMEM, followed by gentle pipetting 3 to 4 times; solution B: 3 μL of PolyJetTM reagent was dissolved in 50 μL of serum-free DMEM, followed by gentle pipetting 3 to 4 times; solution C (transfection solution): solution B was quickly added to solution A, followed by pipetting 3 to 4 times. This solution was allowed to rest at room temperature for 15 min.
The total cell RNA was extracted in accordance with the instructions of the TRIzol kit (Beijing Tiangen Biotech Co., Ltd, China). The RT-qPCR primers were selected from http://pga.mgh.harvard.edu/primerbank/, and the primer sequences used are shown in Table 1. The primers were synthesized by Kingsray Biotech, and β-actin was used as the internal reference. The operation procedure was performed after referring to the instructions of the SYBR®Premix Ex TaqTM kit. The II (Tli RNase H Plus).
The cells were harvested and the total proteins were extracted. The proteins were then denatured and subjected to SDS-PAGE (30 μg of total protein content per well). The proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane by the wet transfer method. The membrane was then incubated with MST1 monoclonal antibodies (mAbs) (ab124787, Abcam), Bcl-2 mAbs (12789-1-AP, Proteintech), p73 (ab40658, Abcam), p53 antibodies (#21086-1, SAB), PUMA antibodies (ab33906, Abcam), phospho-YAP1 (Ser127) mAbs (ab76252, Abcam), caspase-3 mAbs (Cat. #9665, Cell Signaling Technology), Bax polyclonal antibodies (50599-2-lg, Proteintech) Company), YAP polyclonal antibodies (Cat. #4912, Cell Signaling Technology), and subsequently, other antibodies (1:1200 or 1:1500) overnight at 4°C; then, the membrane was treated with horseradish peroxidase-conjugated secondary antibodies (ZDR-5307, Beijing ZSGB-Bio Origene Co., Ltd, China 1:2000) for 1.5 h. Finally, ECL immunodetection was performed in the dark. The gray values of the protein bands were scanned and analyzed using Image-Pro Plus 6.0 gel image analysis software for semi-quantitative analysis using β-actin (internal reference, AF7018, Santa Cruz, CA) as the control.
The experimental data were analyzed by SPSS 19.0 statistical software (SPSS Inc., USA). One-way analysis of variance (ANOVA) or unpaired t test was used for the comparison of various parameters among the groups. The Q test was used for the pairwise comparison among multiple groups; A value of P < 0.05 was considered statistically significant.
Inhibition of cell proliferation by Tau
After the SiHa cells were treated with Tau (concentrations: 0 (control), 10, 20, 40, 80, 160, and 320 mmol/L for 24 h, 48 h, and 72 h), the results of the MTT assay for examining the cell proliferation showed that Tau significantly inhibited the proliferation of SiHa cells (P < 0.05) in a dose- and time-dependent manner [Figure 1]. The IC50 values of Tau for SiHa cells at 24 h, 48 h, and 72 h were 239.87 mmol/L, 113.05 mmol/L, and 92.62 mmol/L, respectively. It was seen that Tau has a strong inhibitory effect on the proliferation of cervical cancer cells.
Effect of Tau on apoptosis
The apoptotic rates in SiHa cells treated with different concentrations of Tau for various time periods were detected using the Annexin-V-FITC/PI double staining method, with Cisplatin (DDP) as the positive control. The results [Figure 2] showed that no significant difference between the apoptotic rates in cells from the Tau 20 mmol/L, Tau 40 mmol/L, and Tau 80 mmol/L and the control group was observed. The apoptotic rate in the Tau 160 mmol/L group was 21.95%, and that in the Tau 320 mmol/L group was 30%, showing statistical significance when compared with the results for the control group (P < 0.01). The inductive effects of Tau on apoptosis were second only to those of DDP, indicating that high concentrations of Tau can induce the apoptosis of cervical cancer cells.
Effect of Tau on the expressions of MST1, Bax, and Bcl-2 proteins
Figure 3 shows that compared with the control group, the expression of MST1 protein and Bax protein in the groups treated with 40 to 320 mmol/L of Tau increased gradually with the increase of Tau concentration, and the differences were statistically significant (P < 0.01). On the contrary, the expression of Bcl-2 gradually decreased with the increase of Tau concentration, and the difference was statistically significant (P < 0.01).
Effects of MST1 overexpression on Tau-induced apoptosis
The previous results show that Tau can induce the apoptosis of SiHa cells and upregulate the expression of MST1 protein. But what is the role of MST1 in the Tau-induced apoptosis of SiHa cells? We used gene transfection and Annexin-V-FITC/PI double staining to observe the effects of MST1 overexpression on the apoptosis of SiHa cells. The results shown in Figure 4 reveal that the apoptotic rates of the Tau group, the p-EGFP-MST1 group, and the p-EGFP-MST1+Tau group were 20.52%, 33.19%, and 64.1%, respectively, showing statistical significance when compared with the results of the control group or the p-EGFP-NC group (P < 0.01). The results of the p-EGFP-MST1+ Tau group also showed statistical significance when compared with those of the p-EGFP-MST1 group (P < 0.01). These results indicate that the overexpression of MST1 can increase the Tau-induced apoptosis of SiHa cells.
Effects of MST1 overexpression on the expressions of p73, p53, and PUMA mRNA
Based on the effects of MST1 overexpression on Tau-induced apoptosis, it can be seen than MST1 overexpression can increase the Tau-induced apoptosis of SiHa cells, but the mechanism underlying this phenomenon was not clear. RT-qPCR was used to observe the effects of high MST1 expression on the mRNA expression of certain apoptosis-related genes and cell proliferation-related genes (p73, p53, PUMA, caspase-3, yap, CTGF (connective tissue growth factor), and amphiregulin (Amphiregulin, AREG)) in SiHa cells. The results for this RT-qPCR analysis are shown in Figure 5. Compared with the control group and the p-EGFP-N1 group, the expression levels of MST1 mRNA in the p-EGFP-MST1 group and the p-EGFP-MST1+DDP group increased significantly by 40 folds and 70 folds, respectively, (P < 0.01). In addition, the mRNA expression levels of p73 (P < 0.01), p53 (P < 0.05), PUMA (P < 0.01), and caspase-3 (P < 0.01) also increased to varying degrees (p73 by about 3.5 fold, p53 by about 2 fold, and caspase-3 by about 5 fold); these increases were also statistically significant when compared with the results of the control group and the p-EGFP-N1 group (P < 0.05 or P < 0.01). The mRNA expression levels of CTGF, AREG, and yap in the p-EGFP-MST1 group decreased to 0.2, 0.6, and 0.4 times those in the control group, respectively, and showed statistical significance when compared with the results of the control group and the p-EGFP-N1 group (P < 0.05). The mRNA expression levels of CTGF, AREG, and yap in the p-EGFP-MST1+DDP group decreased to about 0.2 times those in the control group, and showed statistical significance when compared with the p-EGFP-MST1 group (P < 0.05) and the DDP group (P < 0.05). These results indicate that the overexpression of MST1 can upregulate the mRNA expression of p73, p53, PUMA, and caspase-3 and downregulate the mRNA expression of CTGF, AREG, and yap.
Effects of Mst1 overexpression on the expressions of p73, p53, and PUMA
The expression levels of the MST1, YAP, Phospho-YAP1 (Ser127), p73, p53, PUMA, and caspase-3 proteins in SiHa cells were detected by Western blotting. The results [Figure 6] showed that compared with the control group or the p-EGFP-N1 group, the total MST1 protein expression in the cells increased by 2.2-fold after the transfection of the p-EGFP-MST1 plasmid [Figure 6A and 6B]. The expression of the YAP protein decreased significantly (P < 0.01), while that of the Phospho-YAP protein increased significantly; the ratio of the Phospho-YAP1 (Ser127) expression to YAP expression increased by about 4.5 fold [Figure 6A–6C]. The expression levels of the p73, p53, PUMA and caspase-3 proteins increased significantly (P < 0.05 or P < 0.01; Figure 6A, 6D and 6E).
Compared with the p-EGFP-MST1 group, the expression levels of the proapoptotic proteins p73, p53, PUMA, and caspase-3 in the p-EGFP-MST1+DDP group increased significantly (P < 0.01; Figure 6A, 6D and 6E). There may be some synergy between MST1 and DDP, suggesting that MST1 may increase the chemosensitivity of SiHa cells to DDP.
The occurrence and development of cervical cancer involves multiple biological mechanisms and many signaling factors. Apoptosis imbalance is one of the most important aspects related to the development of cervical cancer, and apoptosis-related gene expression changes are one of the most important causes of apoptosis imbalance. There are mainly two types of gene expression alterations: one is the activation of proto-oncogenes such as Bcl-2, which can promote cell proliferation and inhibit apoptosis, and the other is the inactivation of tumor suppressor genes such as Bax, which can promote apoptosis. Although surgery and concurrent chemoradiotherapy can cure 80% to 95% of patients with early-stage cervical cancer, there is no satisfactory treatment for patients with locally advanced cancer or those with recurrent metastasis. The efficacy of the cisplatin plus paclitaxel or topotecan chemotherapy regimens that are widely used against advanced cervical cancer fluctuates between 28.0% and 46.3%, with the estimated overall survival being less than 12 months. Therefore, there is an urgent need to find effective and low-toxic treatment methods for recurrent or metastatic cervical cancer; molecularly targeted therapies represent an important direction for future developments in this field.
Studies have shown that[10,11,25–27] Tau can not only act synergistically with cisplatin, increasing the drug's effect while decreasing its toxicity, but also downregulate matrix metalloproteinase 2 (MMP-2) and upregulate N-acetylgalactosamine transferase, thus inhibiting the invasion and metastasis of glioma cells. Vanitha has confirmed that Tau also has good therapeutic effects against dimethylbenzopyrene (DMBA)-induced breast cancer, as well as good inhibitory effects against the in vitro invasion and migration of breast cancer cells. A recent study has reported that Tau can also downregulate the expressions of the N-cadherin, Snail, and Vimentin proteins in prostate cancer cells and inhibit the epithelial-mesenchymal transition (EMT) of human prostate cancer cells; EMT is the key event whereby cells acquire the ability of invasion and metastasis. Our earlier studies[15–17] have shown that Tau can induce the apoptosis of tumor cells such as those of liver cancer, lung cancer, and breast cancer. The results of this study showed that Tau can induce the apoptosis of SiHa cells, and increase the expressions of the MST1 and Bax proteins and downregulate the expression of the Bcl-2 protein in SiHa cells [Figure 3]. The Bcl2/Bax ratio is a classic indicator of apoptosis, and Bcl2/Bax plays an important role in the apoptosis of a variety of cells.
Abnormalities of MST1 can be found in various tumors, such as liver cancer,[28,29] gastric cancer, and soft-tissue sarcoma, and have become a research hotspot with regards to tumor-targeted therapies. One of our early studies had found that exogenous MST1 can increase the protein levels of MST1, caspase-3, and Phospho-YAP (Ser127) in HepG2 cells, downregulate the mRNA expression levels of CTGF, AREG, and Survivin, and inhibit HepG2 cell proliferation and promote the apoptosis of these cells. The results of this study confirmed that the overexpression of MST1 can not only enhance Tau-induced apoptosis, but also upregulate the expression of p73, p53, PUMA, and caspase-3 in SiHa cells [Figures 5 and 6], as well as downregulate the expression of CTGF, AREG, and YAP [Figure 5D], thereby inhibiting the cell proliferation. Therefore, MST1 plays an important role in the process whereby Tau inhibits the proliferation and induces the apoptosis of cervical cancer cells. The mechanism of action may be related to, in addition to the Hippo pathway, the p53 signaling pathway and PUMA-mediated mitochondrial apoptosis pathway. Tumor occurrence and development is the result of the combination of multiple signaling pathways, and there is a link between the Hippo signaling pathway and the p53 apoptotic signaling pathway. It has been found that in adrenal pheochromocytoma PC12 cells, YAP can be phosphorylated at serine 127 and then enter the nucleus to interact with the p53 family protein p73, thus leading to apoptosis. In addition, p73 can activate the apoptosis-associated protein PUMA in the cytoplasm in a transcription-dependent manner, which in turn promotes the release of cytochrome c from the mitochondria and leads to apoptosis. Therefore, we have reasons to believe that there may be certain relationships between the Hippo signaling pathway, p53 signaling pathway, and PUMA-mediated mitochondrial apoptosis signaling pathway in cervical cancer cells, and that Tau affects these signaling pathways through key molecules, such as MST1, and exhibits anti-tumor effects. The specific mechanism underlying this phenomenon, however, needs to be elucidated in future studies.
Currently, targeted therapies against cervical cancer have limited efficacy, and their long-term effects and side effects are yet to be studied. With deepened research on targeted therapies against cervical cancer, we expect that more molecular targeted drugs, especially those targeting MST1, can be developed successfully; these drugs can be used alone or in combination with other chemotherapeutic drugs so as to improve the survival and quality of life of patients with advanced cervical cancer.
This study was supported by the grants from the National Natural Science Foundation (No. 81360032) and Jiangxi Provincial Natural Science Foundation (No. 20171BAB215059).
Conflicts of interest
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