Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel
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.
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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.
References (45)
- et al.
Microtubules and actin filaments: dynamics target for cancer chemotherapy
Curr Opin Cell Biol
(1998) - et al.
Regulation of death receptor-mediated apoptosis pathways
Int J Biochem Cell Biol
(2000) - et al.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade
Cell
(1997) - et al.
ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B
J Biol Chem
(1996) - et al.
Potentiation of paclitaxel cytotoxicity in lung and esophageal cancer cells by pharmacologic inhibition of the phosphoinositide 3-kinase/protein kinase B (Akt)
J Thorac Cardiovasc Surg
(2004) - et al.
Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel
J Biol Chem
(2002) - et al.
Inhibition of AKT abrogates chemotherapy-induced NFκB survival mechanisms: implications for therapy in pancreatic cancer
J Am Coll Surg
(2004) - et al.
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry
J Immunol Meth
(1991) - et al.
In rat hepatocytes glucagons increases mammalian target of rapamycin phosphorylation on serine 2448 but antagonizes the phosphorylation of its downstream targets induced by insulin and amino acids
J Biol Chem
(2004) - et al.
The mammalian target of rapamycin-p70 ribosomal S kinase but not phosphatidylinositol 3-kinase-Akt signalling is responsible for fibroblast growth factor-9-induced cell proliferation
J Biol Chem
(2005)
Globocan 2000: Cancer incidence, mortality, and prevalence worldwide
Estimates of the worldwide mortality from 25 cancers in 1990
Int J Cancer
Current status of chemotherapy for cancer of the cervix
Oncology
The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents
Annu Rev Med
Paclitaxel has moderate activity in squamous cervix cancer: a Gynecologic Oncology Group study
J Clin Oncol
Phase I clinical and pharmacokinetics study of taxol
Cancer Res
Phase III study of cisplatin with or without paclitaxel in stage IVB, recurrent, or persistent squamous cell carcinoma of the cervix: a Gynecologic Oncology Group Study
J Clin Oncol
Phase II trial of paclitaxel and cisplatin in metastatic and recurrent carcinoma of the uterine cervix
J Clin Oncol
Paclitaxel and cisplatin as first-line therapy in recurrent or advanced squamous cell carcinoma of the cervix: a Gynecologic Oncology Group study
J Clin Oncol
CD95 (APO-1/Fas)-mediated apoptosis: live and let die
Adv Immunol
Death and anti-death: tumour resistance to apoptosis
Nat Rev Cancer
Mitochondria and apoptosis
Science
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