Tetrandrine Induces Apoptosis in HepG2 by Modulating Hippo Signalling Pathway

Fuling Wang1,2, Changlin Yue2, Hong Li2, Wenjing Yu1, Wenlan Li1* and Guosong Xin1*

1College of Pharmacy, Harbin University of Commerce, Harbin 150076, China

2Zhejiang Shangyao Jiuxu Pharmaceutical Co., Ltd., Jinhua 321016, China

ABSTRACT

Tetrandrine (TET) has been known to possess anti-cancer properties in wide variety of cancers, however, the underlying mechanism for hepatocellular carcinoma (HCC) have not been fully elucidated. We focused our investigation the effect of TET on programmed cell death and growth of HepG2 cancer cell live. TET was found to significantly inhibit the proliferation of HepG2 cells, with an half maximal inhibitory concentration (IC50) of 7.76 μmol/L. Fluorescence microscopy showed that after 48 h of TET exposure, the cells presented morphological alterations typical of apoptosis while normal cellular morphology was preserved in the control. Staining with propidium iodide (PI), followed by flow cytometry showed that following 48 h TET treatment at 3.75, 7.5, and 15 μmol/L, the rate of apoptosis in HepG2 was 5.1%, 19.7%, and 36.9%, respectively. Western blotting was done to study the effect of TET on the expression profile of proteins involved in Hippo signalling pathway. We observed that in response to 48 h incubation with TET, there was a significant upregulation of the expression levels of MST1 (mammalian sterile 20-like kinase 1), LATS1 (large tumor suppressor kinase 1), and P-YAP1 while the expression levels of YAP1 (Yes-associated protein 1) and TAZ (transcriptional coactivator with PDZ-binding motif) were downregulated. Taken together, these results indicated that the anti-cancer activity of TET on HepG2 cells may be due to the modulation of Hippo signalling pathway.

Article Information

Received 10 April 2023

Revised 18 October 2023

Accepted 01 November 2023

Available online 28 February 2024

(early access)

Published 11 November 2024

Authors’ Contribution

Methodology, GX and FW. Validation, WY. Help and advice, HL and CY. Data analysis, GX and FW. Writing original draft, FW. Writing review and editing, FW. Supervision, WL. All authors have read the manuscript.

Key words

Tetrandrine, HepG2 cells, Proliferation, Apoptosis, Hippo pathway, Molecular mechanisms

DOI: https://dx.doi.org/10.17582/journal.pjz/20230410080440

* Corresponding author: 13766801150@163.com

0030-9923/2024/0000-3383 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The hepatocellular carcinoma (HCC) is one of the predominant cancers accounting for about a third of cancer related mortality (Torre et al., 2015). Surgical resection remains the first choice of treatment for early-stages of HCC (Zhang and Sun, 2015), however, its efficiency is limited due to delayed clinical diagnosis, which usually occurs when the cancer has progressed to intermediate or advanced stage. Recurrence rate of HCC upon curative hepatectomy is high and it is then tackled using targeted drug therapy with sorafenib, which although suffers from issues of incomplete suppression of HCC and drug resistance (Llovet et al., 2008). Therefore, the focus is towards the development of new and effective drugs.

One of the drugs being explored is tetrandrine (TET), extracted from the roots of Stephania tetrandra (Wang et al., 2004; Bhagya and Chandrashekar, 2018). TET is an alkaloid belonging to the bis-benzylisoquinoline family and induces apoptosis and autophagy in mammalian oral and gastric cancer cells (Liu et al., 2008; Lien et al., 2017; Bai et al., 2018). It also induces apoptosis as well as hinders the proliferation of human bladder and prostate cancer cells (Li et al., 2011; Liu et al., 2015). Despite the studies, underlying mechanism of anti-cancer activity against the HCC and other cancer types is not fully understood. One of the hypotheses suggests the dysregulation of Hippo signalling pathway as a possible cause of HCC (Zender et al., 2006; Kowalik et al., 2011). In normal cells, this pathway is responsible for regulation of cell growth, apoptosis, and differentiation (Irvine, 2012; Camargo et al., 2007). In mammalian cells, it comprises protein kinases MST1/2 and LATS1/2 along with its downstream effectors YAP, and TEADs, and the paralog TAZ (Ma et al., 2015; Hong et al., 2016; Edgar, 2006). Activation of MST1/2 triggers the LATS1/2 to induce phosphorylation of YAP/TAZ followed by degradation (Zhao et al., 2007; Yi et al., 2016; Song et al., 2018). Elevated levels of YAP have been found in samples of hepatitis B virus-induced HCC (Pan, 2010; Lu et al., 2013), thus supporting the previous hypothesis. The present work was carried out to study the antiproliferative and apoptotic effects of TET in human liver cancer cell line, HepG2. We inferred that TET may have an impact on the biological functions such as the proliferation and apoptosis of HepG2 cells through the Hippo signalling pathway, and provide new ideas for the treatment of HepG2.

MATERIALS AND METHODS

Cell culture

HepG2 cells were procured from the Engineering Research Centre for Medicine at Harbin University of Commerce (Harbin, China). RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% (v/v) foetal bovine serum (Tianhang Biological Technology Co. Ltd., China) was used to culture the cells at 37 ℃ in a 5% CO2 environment. A cell density of about 80% indicated the logarithmic growth phase.

Assay for cell viability (CCK-8 assay)

One hundred microliters of the HepG2 cells (cell density 3×104 cells/mL) digested with 0.25% pancreatin, were inoculated in a 96-well plate. Six parallel wells were set up for each group. After 24 h of incubation, 100 μL each of TET (purity, ≥98%; Aladdin Reagent Co., Ltd., China) or hydroxy camptothecin (HCPT; purity, ≥ 98%; Medican Pharmaceutical Co., Ltd., China) were added to each well to get a final concentration of 2, 4, 8, 16, and 32 μmol/L or 1, 2, 4, 8, and 16 μmol/L, respectively, while sterile RPMI-1640 (100 μL) served as control, and incubated further for 72 h at 37 ℃. To each well, 10 μL of CCK-8 solution (Beyotime Institute of Biotechnology, China) was added and incubation was carried out at 37 ℃ for 2 h (Shang et al., 2021). A microplate reader was used to measure the absorbance at 450 nm, which was used to determine the inhibition rate and IC50.

Apoptotic morphology assay

A 6-well plate was inoculated with HepG2 cells (3×104 cells/mL) grown in RPMI-1640, with a coverslip inside each well. After 24 h, TET (3.75, 7.5, or 15 μmol/L) was added to each well, HCPT (7 μmol/L) served as positive control, while in negative control sterile RPMI-1640 was added. Incubation was carried out for 48 h after which sterile phosphate-buffered saline (PBS) was used to wash the HepG2 cells, followed by fixing in 1 mL of 4% buffered paraformaldehyde at 4 ℃ for 1 h. PBS was used to wash the cells and the cells were stained with 5 mg/L Hoechst 33258 fluorescent probe (Sigma-Aldrich, St. Louis, MO, USA) by incubation in dark for 30 min at 37 ℃ (Ji and Yu, 2015). The excess stain was removed by washing the cells with PBS. The coverslip was removed from each well and examined under a fluorescence microscope (CKX-41-32, Olympus Corporation, Japan).

Apoptosis rate assay

A 6-well plate was inoculated with HepG2 cells (3×104 cells/mL grown in RPMI-1640) and TET (3.75, 7.5, or 15 μmol/L) was added at 24 h. The incubation was continued for 48 h after followed by overnight incubation in 70% ethanol at 4 ℃ to fix the cells. After incubation, PBS was used to wash the cells. Staining with PI (Sigma-Aldrich) 50 μg/mL was carried out for 30 min at 37 ℃. The apoptosis was analysed by flow cytometry (EPICS-XL; Beckman Coulter, Inc., USA).

Western blotting

HepG2 cells were plated in culture flasks (1×106 cells/mL) followed by addition of TET (3.75, 7.5, or 15 μmol/L) at 24 h. After 48 h, the collected cells were lysed and cellular protein was extracted. Protein quantification was done by the bicinchoninic acid method (Solarbio Science and Technology Co., Ltd., Beijing, China). For western blotting (Wang et al., 2021), 12% SDS-PAGE gel electrophoresis was carried out with 50 μg of protein (20 μL loading volume). Protein transfer was done onto nitrocellulose membrane and blocking was carried out with 5% skim milk for 1 h after which incubation was done with primary antibodies (rabbit anti-human binding immunoglobulin protein; MST1, bs-3504R; LATS1, bs-2904R; P-LATS1, bs-7913R; YAP1, bs-52418R; P-YAP1, bs-3475R; TAZ, bs-12367R; BIOSS) at 4 ℃ overnight. Upon washing, secondary antibodies were added to the membrane and incubation was carried out at room temperature (Goat anti-rabbit IgG horseradish peroxidase; A0192; Beyotime Institute of Biotechnology) for 2 h. ECL Chemiluminescence Kit (P0018AS-1,2; Beyotime Institute of Biotechnology) was used to visualise the bands which were photographed and analysed with a gel imaging system (GIS-2019; Tanon Science and Technology Co., Ltd., China).

Statistical analysis

The data from each experiment were recorded as mean ± standard deviation. The results were evaluated by One-way ANOVA. To analyse the data, SPSS software for Windows version 18.0 (SPSS, Inc., USA) was used. Statistically significant differences between groups were defined as P values less than 0.05.

RESULTS

Antiproliferative effects of TET

Treatment with TET was found to inhibit the proliferation of HepG2 cells. The inhibition followed a dose-dependent pattern. The IC50 was calculated to be 7.76 μmol/L (Table I, Fig. 1), in comparison to the positive control HCPT, which was 7.18 μmol/L (Table I).

 

Table I. Inhibitory effect of TET and HCPT treatment on cell proliferation of 24 h old HepG2 cells.

Groups	Concentration (μmol/L)	Optical density	Inhibition rate (%)
Control	–	0.801 ±0.088	–
TET	2	0.654 ±0.095**	18.21
	4	0.487 ±0.091**	39.19
	8	0.337 ±0.051**	57.89
	16	0.253 ±0.031**	68.36
	32	0.256 ±0.046**	68.41
HCPT	1	0.675 ±0.083**	15.76
	2	0.565 ±0.052**	29.49
	4	0.549 ±0.039**	31.41
	8	0.397 ±0.016**	50.41
	16	0.248 ±0.034**	69.29

 

Compared with control group, **P<0.01. Date was expressed as means ± SD (n=6).

 

 

TET induces morphological changes in HepG2 cells

Fluorescence microscopic examination of control group shows uniform fluorescence in the cell nuclei. Incubation with various concentrations of TET or 7 μmol/L HCPT for 48 h, showed the presence of apoptosis related changes in the HepG2 cells, such as chromatin condensation and formation of apoptotic bodies (Fig. 2).

 

TET induces apoptosis in HepG2 cells

TET administration showed a dose dependent increase in the total number of apoptotic cells as compared to control, and the effect was found to be statistically significant. The results are summarised in (Table II, Fig. 3).

 

Table II. TET causes an increased apoptosis in HepG2 cells.

Groups	Concentration (μmol/L)	Apoptosis rate (%)
Control	–	1.579 ±0.197
HCPT	7	21.197 ±1.459**
TET	3.75	5.125 ±0.797**
	7.5	19.698 ±1.860**
	15	36.882 ±1.511**

 

Compared with control group, **P<0.01. Date was expressed as means ± SD (n=3).

 

TET treatment affects the expression of MST1, LATS1, P-LATS1, and P-YAP1

Treatment with 3.75, 7.5, and 15 μmol/L TET or 7 μmol/L HCPT for 48 h caused a significant upregulation (P<0.01) of the MST1, LATS1, P-LATS1, and P-YAP1 expression. The effects were found to be concentration-dependent (Fig. 4).

 

 

TET downregulates the expression of YAP1 and TAZ

Treatment with 3.75, 7.5, and 15 μmol/L TET or 7 μmol/L HCPT for 48 h caused a significant downregulation (Fig. 5) in the expression levels of YAP1 and TAZ in comparison to the control group (P<0.05). The decrease was gradual and coincided with the dose of TET used.

 

DISCUSSION

The disturbance of Hippo signalling pathway disrupts the balance between the cellular proliferation and apoptosis and may lead the cell towards cancerous state (Zhao et al., 2008). Therefore, Hippo signalling pathway is being considered a potential target for anticancer drug research. The Hippo signalling pathway was first discovered in Drosophila, and it is highly conserved during evolution. It is homologous to MST1/2, human Salvador 1 (hSAV1), LATS1/2, Mps One Binder inaseactivator-like1 (MOB1), YAP / TAZ and TEA domain family member (TEAD) in mammals (Dong et al., 2007). The core components of the Hippo signal pathway include LATS and MST, both of which have important regulatory roles in the Hippo signal pathway. The LATS gene was identified from Drosophila in 1995 and was found to express LATS1 and LATS2. MST is a serine/threonine kinase-like enzyme found in yeast cells. Subsequent studies have found that LATS and MST can significantly inhibit tumor cell proliferation and promote tumor cell apoptosis, suggesting that these may act as tumor suppressor genes (Furth et al., 2015). YAP gene is highly expressed in many human cancer tissues and cells, which accelerates the proliferation of tumor cells and inhibits apoptosis. A high level of YAP expression corresponds to a lower the degree of tumor differentiation as well as with a shorter survival period of the patient. Therefore, reducing the expression level of YAP in tumor cells may help in tumor treatment. YAP, as a transcription factor of the TEAD/TEF family, is a downstream target gene of the Hippo pathway and a nuclear auxiliary transcription factor. In mammals, the Hippo signalling pathway inhibits YAP nuclear translocation to achieve cell apoptosis in mammals. The mechanism is that dephosphorylated YAP combines with TAZ to transfer into and accumulate in the nucleus to form transcription factor complexes and induce the expression of target genes.

The results of this study show that TET can activate MST1 and increase the level of protein expression. LATS1 was phosphorylated under the influence of MST1, and the activated LATS1 phosphorylates the downstream targets YAP and TAZ, resulting in the nucleus knocking out the inactive YAP and TAZ. Phosphorylated YAP and TAZ cannot combine with the transcription factor TEAD to convey growth and development signals, thereby inhibiting the proliferation of HepG2 cells through the Hippo signaling pathway.

CONCLUSION

In the present study a possible mechanism for the action of TET on HepG2 cells and consequently on the Hepatocellular carcinoma was investigated. We found an inhibition in cell proliferation as well as induction of apoptosis in HepG2 cells following TET treatment. Incubation with TET could also inhibit cell growth and cause a dose-dependent increase in the rate of apoptosis. One of the possible mechanisms proposed for the development of cancer is the disruption of the Hippo signalling pathway. TET treatment could efficiently obstruct the expression of YAP HepG2 cells which was evident in the expression profile. Cumulatively, these results suggest that anti-cancer effects of TET against HepG2 cells can be attributed to modulation of the Hippo signalling pathway. We will continue to explore the anti-tumor mechanism of TET, use quantitative RT-PCR to verify the expression protein-related genes, and explore the underlying targets of TET on HepG2 cells through proteomics.

Funding

This study was supported by the Key Research and Development Plan Project of Heilongjiang Province (No. 2022ZX02C08), the Talents Training Project Supported by the Central Government for the Reform and Development of Local Colleges and Universities, the Excellent Youth Project of Heilongjiang Natural Science Foundation (No. YQ2022H002) and the Doctoral Research Support Program of Harbin University of Commerce (No. 22BQ42).

Statement of conflicts of interest

The authors have declared no conflicts of interest.

REFERENCES

Bai, X.Y., Liu, Y.G., Song, W., Li, Y.Y., Hou, D.S., Luo, H.M. and Liu, P., 2018. Anticancer activity of tetrandrine by inducing pro-death apoptosis and autophagy in human gastric cancer cells. J. Pharm. Pharmacol., 70: 1048-1058. https://doi.org/10.1111/jphp.12935

Bhagya, N. and Chadrashekar, K.R., 2018. Tetrandrine and cancer. An overview on the molecular approach. Biomed. Pharmacother., 97: 624-632. https://doi.org/10.1016/j.biopha.2017.10.116

Camargo, F.D., Gokhale, S., Johnnidis, J.B., Fu, D., Bell, G.W., Jaenisch, R., and Brummelkamp, T.R., 2007. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr. Biol., 17: 2054-2060. https://doi.org/10.1016/j.cub.2007.10.039

Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S.A., Gayyed M.F., Anders, R.A., Maitra, A. and Pan, D., 2007. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell, 130: 1120-1133. https://doi.org/10.1016/j.cell.2007.07.019

Edgar, B.A., 2006. From cell structure to transcription: Hippo forges a new path. Cell, 124: 267-273. https://doi.org/10.1016/j.cell.2006.01.005

Furth, N., Bossel Ben-Moshe, N., Pozniak, Y., Porat, Z., Geiger, T., Domany, E., Aylon, Y. and Oren, M., 2015. Down-regulation of LATS kinases alters p53 to promote cell migration. Genes Dev., 29: 2325-2330. https://doi.org/10.1101/gad.268185.115

Hong, A.W., Meng, Z.P. and Guan, K.L., 2016. The Hippo pathway in intestinal regeneration and disease. Nat. Rev. Gastroenterol. Hepatol., 13: 324-337. https://doi.org/10.1038/nrgastro.2016.59

Irvine, K.D., 2012. Integration of intercellular signalling through the Hippo pathway. Semin. Cell Dev. Biol., 23: 812-817. https://doi.org/10.1016/j.semcdb.2012.04.006

Ji, Y.B. and Yu, L., 2015. In vitro analysis of the role of the mitochondrial apoptosis pathway in CSBE therapy against human gastric cancer. Exp. Ther. Med., 10: 2403-2409. https://doi.org/10.3892/etm.2015.2779

Kowalik, M.A., Saliba, C., Pibiri, M., Perra, A., Ledda-Columbano, G.M., Sarotto, I., Ghiso, E., Giordano, S. and Columbano, A., 2011. Yes-associated protein regulation of adaptive liver enlargement and hepatocellular carcinoma development in mice. Hepatology, 53: 2086-2096. https://doi.org/10.1002/hep.24289

Li, X.D., Su, B., Liu, R.M., Wu, D.P. and He, D.L., 2011. Tetrandrine induces apoptosis and triggers caspase cascade in human bladder cancer cells. J. Surg. Res., 166: e45-51. https://doi.org/10.1016/j.jss.2010.10.034

Lien, J.C., Lin, M.W., Chang, S.J., Lai, K.C., Huang, A.C., Yu, F.S. and Chung, J.G., 2017. 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., 32: 329-343. https://doi.org/10.1002/tox.22238

Liu, B.R., Wang, T.T., Qian, X.P., Liu, G.X., Yu, L.X. and Ding, Y.T., 2008. Anticancer effect of tetrandrine on primary cancer cells isolated from ascites and pleural fluids. Cancer Lett., 268: 166-175. https://doi.org/10.1016/j.canlet.2008.03.059

Liu, W., Kou, B., Ma, Z.K., Tang, X.S., Lv, C., Ye, M., Chen, J.Q., Li, L., Wang, X.Y. and He, D.L., 2015. Tetrandrine suppresses proliferation, induces apoptosis, and inhibits migration and invasion in human prostate cancer cells. Asian J. Androl., 17: 850-853. https://doi.org/10.4103/1008-682X.142134

Llovet, J.M., Di Bisceglie, A.M., Bruix, J., Kramer, B.S., Lencioni, R., Zhu, A.X., Sherman, M., Schwartz, M., Lotze, M., Talwalkar, J. and Gores, G.J., 2008. Design and endpoints of clinical trials in hepatocellular carcinoma. J. natl. Cancer Inst., 100: 698-711. https://doi.org/10.1093/jnci/djn134

Lu, J., Wang, F., Dai, W.Q., Zhou, Y.Q., Xu, L., Shen, M., Cheng, P. and Guo, C.Y., 2013. The hippo-yes association protein pathway in liver cancer. Gastroenterol. Res. Pract., 2013: 187070. https://doi.org/10.1155/2013/187070

Ma, Y.L., Yang, Y.Z., Wang, F., Wei, Q. and Qin, H.L., 2015. Hippo-YAP signaling pathway: A new paradigm for cancer therapy. Int. J. Cancer, 137: 2275-2286. https://doi.org/10.1002/ijc.29073

Pan, D.J., 2010. The hippo signaling pathway in development and cancer. Dev. Cell, 19: 491-505. https://doi.org/10.1016/j.devcel.2010.09.011

Shang, Y., Jiang, Y.L., Ye, L.J., Chen, L.N. and Ke, Y., 2021. Resveratrol acts via melanoma-associated antigen A12 (MAGEA12)/protein kinase B (Akt) signalling to inhibit the proliferation of oral squamous cell carcinoma cells. Bioengineered, 12: 2253-2262. https://doi.org/10.1080/21655979.2021.1934242

Song, M.J., Bao, H.Y. and Bau, T., 2018. FPOA induces the apoptosis of HepG2 cells. Exp. Ther. Med., 15: 2649-2654. https://doi.org/10.3892/etm.2018.5718

Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J. and Jemal, A., 2015. Global cancer statistics, 2012. CA Cancer J. Clin., 65: 87-108. https://doi.org/10.3322/caac.21262

Wang, G., Lemos, J.R. and Iadecola, C., 2004. Herbal alkaloid tetrandrine: from an ion channel blocker to inhibitor of tumor proliferation. Trends Pharmacol. Sci., 25: 120-123. https://doi.org/10.1016/j.tips.2004.01.009

Wang, G.Q., Yu, X.W., Xia, J.J., Sun, J., Huang, H.Y. and Liu, Y.Q., 2021. MicroRNA-9 restrains the sharp increase and boost apoptosis of human acute myeloid leukemia cells by adjusting the Hippo/YAP signaling pathway. Bioengineered, 12: 2906-2914. https://doi.org/10.1080/21655979.2021.1915727

Yi, J., Lu, L., Yanger, K., Wang, W., Sohn, B.H., Stanger, B.Z., Zhang, M., Martin, J.F., Ajani, J.A., Chen, J., Lee, J.S., Song, S. and Johnson, R.L., 2016. Large tumor suppressor homologs 1 and 2 regulate mouse liver progenitor cell proliferation and maturation through antagonism of the coactivators YAP and TAZ. Hepatology, 64: 1757-1772. https://doi.org/10.1002/hep.28768

Zender, L., Spector, M.S., Xue, W., Flemming, P., Cordon-Cardo, C., Silke, J., Fan, S.T., Luk, J.M., Wigler, M., Hannon, G.J., Mu, D., Lucito, R., Powers, S. and Lowe, S.W., 2006. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell, 125: 1253-1267. https://doi.org/10.1016/j.cell.2006.05.030

Zhang, W.J. and Sun, B.C., 2015. Impact of age on the survival of patients with liver cancer: an analysis of 27,255 patients in the SEER database. Oncotarget, 6: 633-641. https://doi.org/10.18632/oncotarget.2719

Zhao, B., Lei, Q.Y. and Guan, K.L., 2008. The Hippo-YAP pathway: new connections between regulation of organ size and cancer. Curr. Opin. Cell Biol., 20: 638-646. https://doi.org/10.1016/j.ceb.2008.10.001

Zhao, B., Wei, X., Li, W., Udan, R.S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z.C. and Guan, K.L., 2007. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev., 21: 2747-2761. https://doi.org/10.1101/gad.1602907