Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel

https://doi.org/10.1016/j.ejca.2005.12.018Get rights and content

Abstract

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

Introduction

Carcinoma of the cervix, the second most common neoplasm in women worldwide, constitutes 12% of all female cancer and causes 250,000 deaths per annum.1 The incidence of cervical cancer is the highest in developing countries and is the leading cause of female cancer mortality.2 Patients with advanced, persistent, or recurrent squamous cell carcinoma of the cervix, which cannot be treated with surgical resection or radiation therapy, have a low survival rate.3 The need for effective chemotherapy has yet to be defined by increasing our understanding of the molecular effect of chemotherapeutic agents in cancer cells.

Paclitaxel (Taxol), a naturally anti-neoplastic agent, is widely used in the treatment of a variety of solid tumours.4, 5, 6 Trials have been performed to expand the list of tumour diseases that can be treated with paclitaxel, including cervical cancer.5, 7, 8, 9 At the cellular level, paclitaxel binds to β-tubulin subunits in microtubules, thus promoting the polymerization of tubulin and disrupting microtubule dynamics, leading to mitotic arrest and, subsequently, to apoptotic cell death.10 Two signalling pathways for the initiation of apoptosis are well known. One is mediated by a dead receptor on the cell surface, which is called an “extrinsic pathway”, and the other, the “intrinsic pathway”, is mediated by mitochondria.11, 12 In both pathways, the induction of apoptosis leads to the activation of the initiator caspase: caspase-8 for the extrinsic pathway and caspase-9, which is activated at the apoptosome, for the intrinsic pathway.13 These initiator caspases then activate caspase-3 to execute apoptotic cell death, DNA fragmentation, and chromatin condensation.14, 15 Caspase-3 and caspase-9 have been shown to cleave the 116 kDa nuclear protein poly (ADP-ribose) polymerase “PARP” into an 85 kDa apoptotic fragment.16, 17

It is accepted that the mechanism of drug-induced apoptosis is governed not only by the upregulation of pro-apoptotic factors or tumour suppressors but also by the modulation of the survival-signalling pathways.18 One of the important survival-signalling pathways is mediated by phosphoinositide 3-OH kinase “PI3K” and its downstream target, Akt.19 Recent studies suggest that paclitaxel affects the activities of Akt in lung, oesophageal, ovarian and pancreatic cancer cells and that the inhibition of Akt activities enhances the cytotoxic effect of chemotherapy agents.20, 21, 22 The mammalian target of rapamycin (mTOR) is one critical target of Akt in survival-signalling. Akt phosphorylates tuberous sclerosis complex 2, leading to the dissociation of tuberous sclerosis complexes 1 and 2. Together, tuberous sclerosis complexes 1 and 2 compose a GTPase complex for Rheb, which, in turn, activates mTOR.23 In addition, Akt may directly activate mTOR by phosphorylating an auto-inhibitory region of mTOR.24 The best characterised function of mTOR is the regulation of translation by phosphorylating ribosomal S6 kinase (S6K1) and the eukaryote initiation factor 4E-binding protein (4E-BP1).25

Although numerous reports have determined the molecular mechanism of paclitaxel, its effects on the survival-signalling pathway, especially, in cervical cancer cells, are not well understood. In this study we investigated the different sensitivity of human cervical cancer cell lines to paclitaxel and if it could induce apoptosis in those cells. Further, we used HeLa and CaSki cells to evaluate the possible involvement of several apoptosis regulator proteins. We also analysed the effect of paclitaxel on the expression of survival-signalling pathway proteins, such as Akt and mTOR. Additionally, LY294002 (PI3K inhibitor) or rapamycin (mTOR inhibitor) were used to determine whether pre-treatment with these inhibitors would be more effective in exerting the cytotoxic effects of paclitaxel in human cervical cancer cell lines.

Section snippets

Cell lines and culture conditions

Four human cervical cancer cell lines, HeLa, SiHa, ME180, and CaSki, were purchased from the American Type Culture Collection (Manassas, VA). HeLa and SiHa cells were maintained in Eagle’s Minimum Essential Medium (EMEM) obtained from Sigma Chemical Co. (St. Louis, MO), supplemented with 2 mM l-glutamine, 1.0 mM sodium pyruvate, and 10% heat-inactivated fetal bovine serum (FBS) obtained from Gibco (BRL, Grand Island, NY). ME180 cells were maintained in McCoy5a Modified Medium (Gibco) supplemented

Sensitivity of human cervical cancer cells to paclitaxel

To investigate the sensitivity of human cervical cancer cells to paclitaxel, the cells were treated for 24 h in a medium containing varying concentrations of paclitaxel. Exposure to paclitaxel (0.5–25 μM) produced a dose- and time-dependent reduction in cell growth in HeLa, ME180, SiHa, and CaSki cells (Fig. 1a–c). HeLa cells demonstrated the highest sensitivity among all cell types; at 24 h, they showed decreases of 44.6% and 80.1% in cell viability at 0.5 and 25 μM, respectively, whereas CaSki

Discussion

Paclitaxel, one of the broadest-spectrum anticancer agents, is currently used in the treatment of many types of advanced cancer, including carcinoma of the cervix.7, 8, 9 Until now, the prognosis of patients with advanced, persistent, or recurrent squamous cell carcinoma of the cervix has been poor.3 Resistance to chemotherapy is the most frequent obstacle to effective treatment. Although the molecular mechanisms of paclitaxel in the mediation of cell death are well characterised, its effects

Conflict of interest statement

None declared.

Acknowledgements

This work has supported by Grants-in-Aid for Scientific Research (C) 15591730 from the Ministry of Education, Culture, Sports, Science and Technology, Japan for L.S.F.; Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan for A.F. We thank Dr. Helen M. Coley (University of Surrey, Guildford, UK) for valuable discussion.

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