淫羊藿素通过线粒体通路增强索拉非尼的诱导凋亡作用

doi:10.5246/jcps.2022.12.078

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (12): 928-937.DOI: 10.5246/jcps.2022.12.078

淫羊藿素通过线粒体通路增强索拉非尼的诱导凋亡作用

石阿茜¹, 沈阗阗², 夏文彬³, 席莉莉⁴, 王利军¹, 魏玉辉¹^,^*()

1. 兰州大学第一医院药剂科, 甘肃兰州 730000
2. 兰州大学第一临床医学院, 甘肃兰州 730000
3. 兰州大学药学院, 甘肃兰州 730000
4. 兰州大学第一医院国家药物临床试验机构办公室, 甘肃兰州 730000

收稿日期:2022-08-23 修回日期:2022-09-15 接受日期:2022-09-26 出版日期:2022-12-27 发布日期:2022-12-27
通讯作者: 魏玉辉
作者简介:
+ Tel./Fax: +86-931-8616392, E-mail: yhwei@lzu.edu.cn

Icaritin enhances sorafenib-induced apoptosis through a mitochondria-dependent pathway

Axi Shi¹, Tiantian Shen², Wenbin Xia³, Lili Xi⁴, Lijun Wang¹, Yuhui Wei¹^,^*()

1 Department of Pharmacy, the First Hospital of Lanzhou University, Lanzhou 730000, China
2 The First Clinical Medical College, Lanzhou University, Lanzhou 730000, China
3 School of Pharmacy, Lanzhou University, Lanzhou 730000, China
4 Office of the National Institute for Clinical Trials of Drugs, First Hospital of Lanzhou University, Lanzhou 730000, China

Received:2022-08-23 Revised:2022-09-15 Accepted:2022-09-26 Online:2022-12-27 Published:2022-12-27
Contact: Yuhui Wei

摘要/Abstract

摘要：

索拉非尼是晚期人肝细胞癌(human hepatocellular carcinoma, HCC)的标准全身治疗药物, 但在治疗中出现的低应答率、高复发率和高进展限制了其疗效。因此, 以加强索拉非尼的抗癌作用为目的的联合其他药物的治疗策略被广泛研究。本研究的目的是考察淫羊藿素增强索拉非尼对人肝癌细胞的抑制作用, 并阐明其潜在机制。采用MTT法检测细胞存活率, 并通过计算联合用药指数(combination index, CI)确定索拉非尼和淫羊藿素是否对细胞生长具有协同抑制作用。细胞克隆形成试验和流式细胞术分别考察药物对细胞增殖和凋亡的影响, 采用流式细胞术检测线粒体膜电位(mitochondrial membrane potential, MMP), 通过western blot技术分析凋亡通路相关蛋白的表达。结果显示, 索拉非尼联合淫羊藿素对肝癌细胞的生长具有协同抑制作用(CI < 1)。淫羊藿素一方面可增强索拉非尼对肝细胞集落形成的抑制作用, 另一方面可增加索拉非尼诱导的肝细胞凋亡。联合治疗后, 抗凋亡蛋白Bcl-2表达降低, 而促凋亡蛋白Bax表达升高。 PARP、caspase-9和caspase-3的剪切型表达显著增加。此外, 与单用索拉非尼相比, 联合用药使线粒体膜电位进一步丢失, 进而导致线粒体通透性发生变化, 细胞色素c从线粒体释放到胞质中。结果表明, 淫羊藿素可增加索拉非尼诱导的细胞毒作用, 并通过线粒体依赖性途径增加索拉非尼诱导的细胞凋亡。

关键词: 索拉非尼, 淫羊藿素, 人肝癌细胞

Abstract:

Sorafenib remains the standard systemic treatment for advanced human hepatocellular carcinoma (HCC). However, the low response rate, high recurrence, and high progression limit the therapeutic efficacy. Therefore, a combination therapy strategy was advanced to strengthen the antitumor effects of sorafenib. In the present study, we aimed to evaluate whether icaritin could enhance the inhibitory effects of sorafenib on HCC cells and clarify the underlying mechanism. The cell viability was evaluated via MTT assay, and the synergistic inhibitory effects of sorafenib and icaritin were verified by calculating the combination index (CI). Their combined effects on cell proliferation or apoptosis were investigated using colony formation assay and flow cytometry. Mitochondrial membrane potential (MMP) was detected by flow cytometric assay. The protein expressions associated with the apoptotic pathway were determined by Western blotting analysis. The data demonstrated that sorafenib and icaritin exerted synergistic inhibitory effects on cell viability (CI < 1). Icaritin enhanced the inhibitory effect of sorafenib on colony formation and sorafenib-induced apoptosis of HCC cells. We discovered a reduced level of antiapoptotic Bcl-2 and an elevated level of proapoptotic protein Bax when the cells were exposed to the combination. The effect of cleaved and activated PARP was also enhanced. Cleaved caspase-9 and cleaved caspase-3 were increased markedly in the combination group. Furthermore, the combination of icaritin and sorafenib significantly increased the loss of MMP compared with the single treatment group and induced the release of cytochrome c from the mitochondria to the cytosol. These findings indicated that icaritin could enhance sorafenib-induced cytotoxicity and trigger sorafenib-induced apoptosis through a mitochondria-dependent pathway.

Key words: Sorafenib, Icaritin, Human hepatocellular carcinoma

Supporting:

石阿茜, 沈阗阗, 夏文彬, 席莉莉, 王利军, 魏玉辉. 淫羊藿素通过线粒体通路增强索拉非尼的诱导凋亡作用[J]. 中国药学(英文版), 2022, 31(12): 928-937.

Axi Shi, Tiantian Shen, Wenbin Xia, Lili Xi, Lijun Wang, Yuhui Wei. Icaritin enhances sorafenib-induced apoptosis through a mitochondria-dependent pathway[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(12): 928-937.

图/表 7

Figure 1. Sorafenib or/and icaritin inhibit HCC cell viability in vitro. Hep G2 (A–C) and Huh-7 (D–F) cell lines were treated with a series of concentrations of sorafenib or/and icaritin for 72 h. Cell viability was measured by MTT assay, and IC50 value was calculated by GraphPad Prism 7.0. Data were represented by mean ± SD (n = 3).

Table 1. . Synergistic interaction of sorafenib and icaritin in Hep G2 and Huh-7 cell lines.

Figure 2. Colony formation of Huh-7 (A) and Hep G2 (B) cell lines treated with sorafenib and/or icaritin. Cells were treated with sorafenib (10 μM) or icaritin (80 μM) alone or in combination for 10 d.

Figure 3. Apoptotic cells are significantly increased following sorafenib and icaritin co-treatment in Hep G2 and Huh-7 cell lines. Cells were treated with sorafenib (10 μM) or icaritin (80 μM) alone or in combination for 48 h. The cells were stained with Annexin V?FITC and PI, followed by flow cytometry analysis. The percentages of cell population in Q1, Q2, Q3, and Q4 are presented, respectively. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4. Protein expression levels of apoptosis-related proteins in Hep G2 and Huh-7 cell lines treated with sorafenib and/or icaritin.

Figure 5. Flow cytometric assay of MMP changes detected with Rhodamine 123 dye in Hep G2 and Huh-7 cells following exposure to sorafenib or icaritin for 24 h. The change in the ?uorescence intensity of Rhodamine 123 indicated the loss of MMP.

Figure 6. Protein expression levels of cytochrome c in Hep G2 and Huh-7 cell lines treated with sorafenib and/or icaritin.

参考文献 24

[1]	Villanueva, A. Hepatocellular carcinoma. N Engl J. Med. 2019, 380, 1450–1462.
[2]	Liu, L.; Cao, Y.; Chen, C.; Zhang, X.; McNabola, A.; Wilkie, D.; Wilhelm, S.; Lynch, M.; Carter, C. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006, 66, 11851–11858.
[3]	Wörns, M.A.; Galle, P.R. HCC therapies—lessons learned. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 447–452.
[4]	Wo, Y.B.; Zhu, D.Y.; Hu, Y.; Wang, Z.Q.; Liu, J.; Lou, Y.J. Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells. J. Cell Biochem. 2008, 103, 1536–1550.
[5]	Liao, J.Y.; Liu, Y.; Wu, H.J.; Zhao, M.; Tan, Y.X.; Li, D.; Long, H.; Dai, Y.; Yung, S.; Chan, T.M.; Lu, Q.J. The role of icaritin in regulating Foxp3/IL17a balance in systemic lupus erythematosus and its effects on the treatment of MRL/lpr mice. Clin. Immunol. 2016, 162, 74–83.
[6]	Wang, Z.; Zhang, X.; Wang, H.; Qi, L.; Lou, Y. Neuroprotective effects of icaritin against beta amyloid-induced neurotoxicity in primary cultured rat neuronal cells via estrogen-dependent pathway. Neuroscience. 2007, 145, 911–922.
[7]	Qin, L.; Yao, D.; Zheng, L.Z.; Liu, W.C.; Liu, Z.; Lei, M.; Huang, L.; Xie, X.H.; Wang, X.L.; Chen, Y.; Yao, X.S.; Peng, J.; Gong, H.; Griffith, J.F.; Huang, Y.P.; Zheng, Y.P.; Feng, J.Q.; Liu, Y.; Cheng, C.Y. Phytomolecule icaritin incorporated PLGA/TCP scaffold for steroid-associated osteonecrosis: proof-of-concept for prevention of hip joint collapse in bipedal emus and mechanistic study in quadrupedal rabbits. Biomaterials. 2015, 59, 125–143.
[8]	Yang, X.J.; Xi, Y.M.; Li, Z.J. Icaritin: a novel natural candidate for hematological malignancies therapy. Biomed. Res. Int. 2019, 2019, 4860268.
[9]	Liu, P.; Jin, X.; Lv, H.; Li, J.; Xu, W.; Qian, H.H.; Yin, Z. Icaritin ameliorates carbon tetrachloride-induced acute liver injury mainly because of the antioxidative function through estrogen-like effects. Vitro Cell Dev. Biol. Animal. 2014, 50, 899–908.
[10]	Zhang, W.; Xing, B.; Yang, L.; Shi, J.; Zhou, X. Icaritin attenuates myocardial ischemia and reperfusion injury via anti-inflammatory and anti-oxidative stress effects in rats. Am. J. Chin. Med. 2015, 43, 1083–1097.
[11]	Hao, H.B.; Zhang, Q.; Zhu, H.; Wen, Y.X.; Qiu, D.; Xiong, J.; Fu, X.L.; Wu, Y.Z.; Meng, K.; Li, J. Icaritin promotes tumor T-cell infiltration and induces antitumor immunity in mice. Eur. J. Immunol. 2019, 49, 2235–2244.
[12]	He, J.; Wang, Y.; Duan, F.; Jiang, H.; Chen, M.F.; Tang, S.Y. Icaritin induces apoptosis of HepG2 cells via the JNK₁ signaling pathway independent of the estrogen receptor. Planta Med. 2010, 76, 1834–1839.
[13]	Sun, L.; Chen, W.; Qu, L.; Wu, J.; Si, J. Icaritin reverses multidrug resistance of HepG2/ADR human hepatoma cells via downregulation of MDR1 and P‑glycoprotein expression. Mol. Med. Rep. 2013, 8, 1883–1887.
[14]	Chen, S.H.; Wang, G.; Niu, X.J.; Zhao, J.Y.; Tan, W.X.; Wang, H.B.; Zhao, L.J.; Ge, Y.B. Combination of AZD2281 (Olaparib) and GX15-070 (Obatoclax) results in synergistic antitumor activities in preclinical models of pancreatic cancer. Cancer Lett. 2014, 348, 20–28.
[15]	Keating, G.M. Sorafenib: a review in hepatocellular carcinoma. Target. Oncol. 2017, 12, 243–253.
[16]	Liu, J.X.; Cui, X.P.; Qu, L.S.; Hua, L.; Wu, M.M.; Shen, Z.Y.; Lu, C.H.; Ni, R.Z. Overexpression of DLX2 is associated with poor prognosis and sorafenib resistance in hepatocellular carcinoma. Exp. Mol. Pathol. 2016, 101, 58–65.
[17]	Chen, W.L.; Hsieh, C.L.; Chen, J.H.; Huang, C.S.; Chen, W.T.; Kuo, Y.C.; Chen, C.Y.; Hsu, F.T. Amentoflavone enhances sorafenib-induced apoptosis through extrinsic and intrinsic pathways in sorafenib-resistant hepatocellular carcinoma SK-Hep1 cells in vitro. Oncol. Lett. 2017, 14, 3229–3234.
[18]	Wang, H.; Zhang, C.; Chi, H.; Meng, Z. Synergistic anticancer effects of bufalin and sorafenib by regulating apoptosis associated proteins. Mol. Med. Rep. 2018, 17, 8101–8110.
[19]	Feng, X.Q.; Rong, L.W.; Wang, R.X.; Zheng, X.L.; Zhang, L.; Zhang, L.; Lin, Y.; Wang, X.; Li, Z.P. Luteolin and sorafenib combination kills human hepatocellular carcinoma cells through apoptosis potentiation and JNK activation. Oncol. Lett. 2018, 16, 648–653.
[20]	Yu, Z.; Guo, J.F.; Hu, M.Y.; Gao, Y.Q.; Huang, L. Icaritin exacerbates mitophagy and synergizes with doxorubicin to induce immunogenic cell death in hepatocellular carcinoma. ACS Nano. 2020, 14, 4816–4828.
[21]	Pan, X.W.; Li, L.; Huang, Y.; Huang, H.; Xu, D.F.; Gao, Y.; Chen, L.; Ren, J.Z.; Cao, J.W.; Hong, Y.; Cui, X.G. Icaritin acts synergistically with epirubicin to suppress bladder cancer growth through inhibition of autophagy. Oncol. Rep. 2016, 35, 334–342.
[22]	Tait, S.W.; Green, D.R. Mitochondrial regulation of cell death. Cold Spring Harb. Perspect. Biol. 2013, 5, a008706.
[23]	International, B.R. Retracted: apoptosis and molecular targeting therapy in cancer. Biomed Res. Int. 2020, 2020, 2451249.
[24]	Antonsson, B.; Montessuit, S.; Sanchez, B.; Martinou, J.C. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 2001, 276, 11615–11623.

淫羊藿素通过线粒体通路增强索拉非尼的诱导凋亡作用

Icaritin enhances sorafenib-induced apoptosis through a mitochondria-dependent pathway

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 24

相关文章 0

编辑推荐

Metrics

本文评价