Cancer Letters

Cancer Letters

Volume 320, Issue 2, 28 July 2012, Pages 171-179
Cancer Letters

Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells

https://doi.org/10.1016/j.canlet.2012.03.002Get rights and content

Abstract

Induction of cell death and inhibition of cell growth are the main targets of cancer therapy. Here we evaluated the role of autophagy on chemoresistance of human hepatocarcinoma (HCC) cell lines, focusing on its crosstalk with cell apoptosis and proliferation. In this study, a chemotherapeutic agent (cisplatin or 5FU) induced the formation of autophagosomes in three human HCC cell lines and upregulated the expression of autophagy protein LC3-II. Inhibition of autophagy by 3-methyladenine or si-beclin 1 increased chemotherapy-induced apoptosis in HCC cells. Meanwhile, increased damage of the mitochondrial membrane potential was also observed in HCC cells when autophagy was inhibited. Furthermore, inhibition of autophagy reduced clone formation and impaired cell growth of HCC cells when treated with chemotherapy. Co-administration of an autophagy inhibitor (chloroquine) and chemotherapy significantly inhibited tumor growth in a mouse xenograft tumor model, with greater extent of apoptosis and impaired proliferation of tumor cells. This study suggests that autophagy is a potential novel target to improve therapy efficiency of conventional chemotherapeutics towards HCC.

Introduction

Hepatocarcinoma (HCC) is one of the most common malignant tumors in China [1]. Surgical resection and liver transplantation are potentially curative therapies [2]. Unfortunately, most of hepatocarcinomas are too advanced at the time of diagnosis to benefit from these surgical approaches. Currently, chemotherapy is ineffective for HCC because of the inherent chemoresistance. However, the exact mechanism of chemotherapy resistance in hepatocarcinoma is largely unknown. Recently, accumulated evidence suggested that autophagy can promote cancer resistance to chemotherapy [3], [4], [5], [6].

Autophagy is an evolutionarily conserved catabolic process. The autophagic pathway consists of isolation of membrane, formation of autophagosomes and autolysosomes. Isolation membrane engulfs cellular macromolecules and organelles to form autophagosomes. Subsequently, the autophagosome fuses with the lysosome to form autolysosomes, leading to breakdown of macromolecules and organelles by the lysosomal enzymes [7], [8], [9]. Dysregulation of autophagy contributes to a number of diseases including tumorigenesis [7]. However, the role of autophagy in cancer is still controversial. Recent studies suggested that autophagy was required for cancer survival [10] and tumorigenesis [11], [12], [13]. On the other hand, prolonged autophagy has been suggested to cause non-apoptotic type II programmed cell death [14], [15]. Some pharmacologic inhibitors have been used to evaluate the physiologic relevance of autophagy. For example, 3-methyladenine (3-MA), an inhibitor of phosphatidylinositol 3-kinase, blocks autophagosome formation to inhibit autophagy.

Induction of cell death and inhibition of growth are the main targets of cancer therapy. It was found that some types of cancers including hepatocarcinoma were still resistant to chemotherapy. Thus, drugs which reinforce the function of chemotherapy in cell death induction and growth inhibition are beneficial to anti-cancer therapy. Here we assessed the role of autophagy on chemotherapy-induced apoptosis and growth inhibition. We investigated the effect of autophagy on HCC cells exposed to chemotherapeutic agents. Then we detected whether inhibition of autophagy could affect chemotherapy-induced apoptosis and growth inhibition.

Section snippets

Cell culture

Human hepatocarcinoma cell lines SMMC-7721, Hep3B and HepG2 were maintained in Dulbecco’s modified Eagle’s medium (high glucose) (GIBCO, Invitrogen) and supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator under 5% CO2 at 37 °C.

Regents

Cisplatin and 5-fluorouracil (5FU) were purchased from Qilu Pharmaceutical Co., Ltd. (JiNan, Shandong, China). 3-Methyladenine (3MA, M9281) was obtained from Sigma–Aldrich and dissolved in sterile double

Chemotherapeutic agents induces autophagy in hepatocarcinoma cells

Autophagy occurs at low basal levels in all cells and will be rapidly upregulated when cell faces stress. To determine the role of autophagy on chemosensitivity, we first examined the effect of chemotherapy on autophagy. Hepatocarcinoma cells were cultured for 24 h with or without cisplatin (8 μg/ml) or 5FU (120 μg/ml). LC3-I to LC3-II protein processing is considered as a hallmark of autophagy. As shown in Fig. 1A, expression levels of endogenous LC3-II were markedly increased in hepatocarcinoma

Discussion

The results described here demonstrated that autophagy functions as a chemoresistant mechanism in HCC cells. We report here that chemotherapeutic agents (cisplatin or 5FU) could induce autophagy and inhibition of autophagy rendered HCC cells susceptibility to chemotherapy -induced apoptosis and cell growth inhibition. Meanwhile, inhibition of autophagy increased damage of the mitochondrial membrane potential in HCC cells. Furthermore, combined treatment of autophagy inhibitor and cisplatin or

Acknowledgements

This project was supported by the National Natural Science Foundation of China (Grant Nos.: 30870974, 30801347, 30901722, 30901722, 81000970, 81030041). Shanghai Science and Technology Committee (Grant Nos.: 11ZR1449500, 10411963100, 10ZR1439900, 10ZR1439600). Key Basic Research Project of China (Grant Nos.: 2010CB945600, 2011CB966200). Science Fund for Creative Research Groups, NSFC, China (Grant No.: 30921006) and Stem Cell and Medicine Research Center’s Innovation Research Program, The

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    These authors contributed equally to this work.

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