Elsevier

Biochemical Pharmacology

Volume 90, Issue 3, 1 August 2014, Pages 265-275
Biochemical Pharmacology

Autophagy inhibition switches low-dose camptothecin-induced premature senescence to apoptosis in human colorectal cancer cells

https://doi.org/10.1016/j.bcp.2014.05.009Get rights and content

Abstract

Recently, several studies indicated that senescent tumor cells are resistant to apoptosis in chemotherapy. They may return to cell cycle, thus act as stumbling blocks in anticancer treatments. In the present study, we found that, in human colorectal cancer cells, low-dose camptothecin (CPT) simultaneously induced autophagy and premature senescence through AMPK-TSC2-mTOR pathway and ATM-Chk2-p53-p21 pathway respectively. What's important is the suppression of autophagy substantially increased apoptosis and greatly attenuated senescence possibly by blocking p53/p21 pathway, which suggests that autophagy plays an indispensable role in sustaining cell senescence caused by low-dose CPT. The combination of low-dose CPT and autophagy inhibitor, a way to lead senescent cells to die, would be potentially valuable in cancer therapy.

Introduction

In recent years, a novel chemotherapy named “low-dose chemotherapy” has come into focus. It refers to administration of comparatively low doses of chemotherapeutic agents. Using this method, toxic side effects are drastically reduced and the risk of multidrug resistance by tumor cells is greatly avoided, and all of those advantages distinguish it from conventional chemotherapy [1], [2], [3]. Recently, several studies have reported that low-dose chemotherapy can induce premature senescence (also known as accelerated or stress-induced senescence) which has been shown to contribute to successful cancer therapy [4], [5], [6], [7], [8]. However, senescence does not mean death, and some problems arise from senescence-inducing therapies should be considered: (1) senescence-associated secretory phenotypes (SASP) components secreted by senescent tumor cells might stimulate the malignant phenotypes of nearby tumor cell [9]; (2) a noticeable possibility exists that one day the senescent cells could become active again as long as they are not cleared by the system [10], [11]; (3) senescent tumor cells are resistant to cell death [12]. Therefore, how to find a chemotherapeutic approach that specifically switches senescent tumor cells into apoptotic seems to have great potential value in cancer therapy.

Autophagy, a lysosome-dependent degradation pathway that is activated by a variety of stress such as starvation and oxidative stress, regulates cell fate by selectively degrading long-lived proteins and damaged organelles and maintaining cellular homeostasis and genomic integrity [13], [14]. More recently, accumulated evidence suggested that autophagy was implicated in cellular senescence. Increased autophagic vacuoles and senescence-associated β-galactosidase (SA-β-gal) activity were observed in aging fibroblasts [15]. Young et al. found that autophagy-related genes were up-regulated in the process of oncogene-induced senescence and blockade to autophagy delayed the senescence phenotype [16]. Autophagy is usually considered to be a cytoprotective response to stress. In our previous study and others, the inhibition of high dose chemotherapeutic drugs-induced autophagy sensitized tumor cells to apoptotic cell death [17], [18], [19]. In this study, we hypothesized that autophagy could be induced by low dose chemotherapeutic agents and the inhibition of autophagy might switch senescence to apoptosis. We found that premature senescence and autophagy were contemporarily induced by low-dose CPT. What's more, the combination treatment of low-dose CPT and autophagy inhibitor substantially increased apoptosis, which may have great implication for chemotherapy.

Section snippets

Materials

CPT was purchased from Sigma (USA), dissolved in sterile double distilled water, stored at −20 °C until dilution in culture media for treatments. KU55933 (Merck, Germany) was dissolved at 10 mM in DMSO (Sigma, USA) and stored at −20 °C. 3-MA and CQ were obtained from Sigma (USA) and dissolved in sterile double distilled water. Anti-γH2AX, p21, AMPK, p-AMPK, TSC2, p-TSC2 and Cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (USA). Antibodies for ATM, p-ATM, Chk2, p-Chk2,

Low-dose CPT induces premature senescence and autophagy in CRC cells

CPT is a nuclear topoisomerase I inhibitor. CPT is widely used in colorectal cancer therapy as it can induce replication-dependent DNA double-strand breaks (DSBs) to kill the cells [20]. In this study, the low-doses CPT for HCT116 and RKO colorectal cancer (CRC) cells were 20 nM and 50 nM respectively, both of which induced the least detectable cell death (Fig. 1A and B).

Premature senescence, different from replicative senescence and oncogene-induced senescence, is associated with accumulation of

Discussion

The conventional drug regimens at “maximum tolerated doses” (MTDs) of the cytotoxic agents have been designed to kill as many tumor cells as possible. Due to the severe side effects causing damage to normal cells, the patients receiving such chemotherapy regiments often require extended treatment-free periods to allow normal cells to repair some of the damage conflicted by the chemotherapy [34]. However, during the recovery periods, endothelial cells within the tumor can also recover from

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This project was supported by Key Basic Research Project of China (Grant NO.2012CBA01303, 2011CB966200, 2010CB945600, 2011CB965100); Key project of National Natural Science Foundation of China (Grant NO. 81030041); National Natural Science Foundation of China (Grant NO. 31171321, 81101622, 81372330); Special Funds for National key Sci-Tech Sepcial Project of China (Grant NO. 2012ZX10002-016, 2012ZX10002011-011); Shanghai Science and Technology Committee (Grant NO. 10ZR1439600, 11ZR1449500);

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