Autophagy inhibition promotes paclitaxel-induced apoptosis in cancer cells
Introduction
Chemotherapy is an important option in curing or controlling various cancers including lung cancer. Paclitaxel, which stabilizes microtubule and causes apoptosis, offers both symptomatic and survival benefits for lung adenocarcinoma. The paclitaxel-based combination therapies are standard treatments for nearly all patients diagnosed with non-small cell lung carcinoma (NSCLC) [1]. However, clinical treatment with paclitaxel often encounters a number of undesirable side effects as occurred using other anticancer agents. The dose increment of systemic administration of paclitaxel would generate unacceptable levels of toxicity to normal cells, especially of bone marrow, gastrointestinal tract, and the hair follicles [2]. Therefore, many attempts have been made to enhance its therapeutic effectiveness, simultaneously reducing its toxicity. In an effort to search for strategies that could enhance cancer cell killing mediated by paclitaxel, we have investigated possible pro-survival pathways that are activated in response to paclitaxel. Herein, we report the induction of autophagy by paclitaxel.
Autophagy is an evolutionary conserved process in which cell engulfs cytoplasmic constituents within a double-membrane vacuole (named autophagosome) and delivers them to the lysosome for degradation [3]. Autophagy contributes to maintaining cellular homeostasis as a result of quality control of both proteins and organelles. In addition to its basic role in the turnover of proteins and organelles, autophagy has multiple physiological and pathophysiological functions [4], [5]. When cells encounter environmental stressors such as nutrient starvation and pathogen infection, autophagy is induced to provide nutrients and energy required for cell survival. So autophagy is recognized as a cytoprotective process against environmental stress [6], [7]. Meanwhile, autophagy is also an alternative route of programmed cell death, called type-2 programmed cell death or autophagic cell death [8]. In tumor cells, the role of autophagy may depend on the type of tumor, the stage of tumorigenesis, and the nature and extent of the insult. Appropriate modification of autophagy, that is, inhibition of cytoprotective autophagy or promotion of cyto-killing autophagy could augment cytotoxicity caused by anticancer therapy in tumor cells [9], [10], [11]. Thus, in addition to apoptotic response, it would be very useful to determine if an antitumor agent can induce autophagy and what type of autophagies it is. In this study, we found that paclitaxel-induced autophagy in cancer cells, and inhibition of autophagy could lead to enhancement of paclitaxel-mediated cytotoxicity through increasing apoptosis.
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Cell lines and cell culture
A549, PC-3, and HT-29 cell lines were obtained from The Cell Bank of Chinese Academy of Sciences (Shanghai). Human glioma cancer cell line U87 which stably expresses GFP-LC3 protein was kindly gifted by Yan bing (Shan Dong university). A549 cells were cultured with F12 medium supplemented with 10% foetal bovine serum and antibiotics (100Ā U/mL penicillin and 100Ā Ī¼g/mL streptomycin). PC-3, HT-29 and GFP-LC3 transfected U87 cells were maintained in DMEM medium supplemented with 10% foetal bovine
Paclitaxel induces apoptosis in A549 cells
In order to determine whether the observed cell death caused by paclitaxel was due to apoptosis, apoptosis parameters were analyzed by DAPI staining and Western-blot assay. As shown in Fig. 1A, nuclear morphological changes of apoptosis were observed in paclitaxel-treated cells. Paclitaxel caused significant increases in apoptotic cell number with condensed and fragmented DNA (as indicated by a strong blue fluorescence). In addition, alternations in the expression levels of Bax and Bcl-2
Discussion
Despite having diverse mechanisms of action, many frontline anticancer agents would be predicted to stimulate autophagy including arsenic trioxide, 5-FU, histone deacetylase (HDAC) inhibitors, tamoxifen, imatinib, and ionizing radiation [18], [19], [20], [21], [22], [23]. Paclitaxel is a widely used agent that is effective in the treatment of a variety of human cancers. Despite its capability to stabilize microtubules and impair mitosis, paclitaxel also exhibits induction of apoptosis, and
Conflicts of interest
None of the authors has any financial or other interest with regard to the submitted manuscript that might be constructed as a conflict of interest.
Acknowledgments
The authors thank Xuejun Jiang (Institute Microbiology Chinese Academy of Sciences) for providing the GFP-LC3 expressing vector.
This work was supported by the National Natural Science Foundation of China (30973551), and Shandong Scientific Technology Program (2008GG10002042).
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