Browsing by Author "Mitali Das"

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Down regulation of FOXO1 promotes cell proliferation in cervical cancer
(Ivyspring International Publisher, 2014) Shyam Babu Prasad; Suresh Singh Yadav; Mitali Das; H.B. Govardhan; Lakshmi Kant Pandey; Sunita Singh; Satyajit Pradhan; Gopeshwar Narayan
The Forkhead transcription factor FOXO1, an important downstream target of phosphatidylinositol- 3-kinase (PI3K)/AKT signaling pathway, regulates cellular homeostasis by maintaining cell proliferation, apoptosis and viability in normal cells. Though, the function and regulation of FOXO1 is well documented in many cancers, the molecular mechanism of its regulation in cervical cancer is largely unknown. In the present study we have investigated the role of PI3K inhibition on FOXO1 regulation. Expression profiling of primary tumors and cell lines show over expression of PIK3CA and AKT1; and down regulation of FOXO1. Lack of FOXO1 promoter methylation and inability of hypomethylating drug 5-Aza-2'-deoxycytidine and HDAC inhibitor trichostatin A to reactivate FOXO1 expression suggest that loss of FOXO1 expression is due to mechanisms other than promoter methylation/ acetylation. Inhibition of PI3K by LY294002 decreased the level of p-AKT1 and activated FOXO1 transcription factor. We demonstrate that activation of FOXO1 induces apoptosis, cell proliferation arrest, and decreased cell viability in cervical cancer cell lines. Our data suggest that frequent down regulation of FOXO1 and its functional inactivation may be due to post-translational modifications in cervical cancer. Together, these observations suggest that activation of FOXO1 and its nuclear sequestration is critical in the regulation of cell proliferation, cell viability and apoptosis in cervical cancer. Hence, PI3K/AKT pathway may be a potential molecular target for cervical cancer therapy. © Ivyspring International Publisher.
Epigenetic silencing of CXCR4 promotes loss of cell adhesion in cervical cancer
(Hindawi Publishing Corporation, 2014) Suresh Singh Yadav; Shyam Babu Prasad; Mitali Das; Soni Kumari; Lakshmi Kant Pandey; Sunita Singh; Satyajit Pradhan; Gopeshwar Narayan
In the network of chemokine signaling pathways, recent reports have described the SDF-1α/CXCR4 axis and its role in cancer progression and metastasis. Interestingly, we found downregulation of CXCR4 at both transcript and protein level in cervical cancer cell lines and primary tumors. We also found CXCR4 promoter hypermethylation in cervical cancer cell lines and primary biopsy samples. DNA hypomethylating drug 5-AZA-2′-deoxycytidine and histone deacetylase inhibitor Trichostatin A treatments in cell lines reactivate both CXCR4 transcription and protein expression. Cell adhesion assay demonstrated that autocrine SDF-1α promotes the loss of cell adhesion while paracrine SDF-1α predominantly protects the normal cervical cells from loss of cell adhesion. Cervical cancer cell line C-33A having increased expression of CXCR4 after TSA treatment showed increased cell adhesion by paracrine source of SDF-1α in comparison to untreated C-33A. These findings demonstrate the first evidence that epigenetic silencing of CXCR4 makes the cells inefficient to respond to the paracrine source of SDF-1α leading to loss of cell adhesion, one of the key events in metastases and progression of the disease. Our results provide novel insight of SDF-1α/CXCR4 signaling in tumor microenvironment which may be promising to further delineate molecular mechanism of cervical carcinogenesis. © 2014 Suresh Singh Yadav et al.
HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4
(Springer Science and Business Media B.V., 2015) Mitali Das; Shyam Babu Prasad; Suresh Singh Yadav; Arusha Modi; Sunita Singh; Satyajit Pradhan; Gopeshwar Narayan
Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2–7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4−/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells. © 2015, International Society of Oncology and BioMarkers (ISOBM).
Over Expression of Minichromosome Maintenance Genes is Clinically Correlated to Cervical Carcinogenesis
(2013) Mitali Das; Shyam Babu Prasad; Suresh Singh Yadav; H.B. Govardhan; Lakshmi Kant Pandey; Sunita Singh; Satyajit Pradhan; Gopeshwar Narayan
Minichromosome Maintenance (MCM) proteins play important roles in cell cycle progression by mediating DNA replication initiation and elongation. Among 10 MCM homologues MCM 2-7 form a hexamer and assemble to the pre-replication complex acting as replication licensing factors. Binding and function of MCM2-7 to pre-replication complex is regulated by MCM10 mediated binding of RECQL4 with MCM2-7. The purpose of this study is to explore the role of MCMs in cervical cancer and their correlation with the clinical parameters of cervical cancer. We have investigated sixty primary cervical cancer tissue samples, eight cervical cancer cell lines and thirty hysterectomised normal cervical tissue. The expression profiling of MCMs was done using semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. MCM2, 4, 5, 6, 7, 10 and RECQL4 are significantly over-expressed in cervical cancer. Among these, MCM4, 6 and 10 show increased frequency of over expression along with advancement of tumor stages. MCM4, 5 and 6 also show differential expression in different types of lesion, while MCM2 and MCM10 are over expressed in cervical cancer irrespective of clinico-pathological parameters. Our data indicates the role of MCM4, MCM5, MCM6, MCM10 and RECQL4 in the progression of cervical cancer. © 2013 Das et al.
PI3K/AKT pathway-mediated regulation of p27Kip1 is associated with cell cycle arrest and apoptosis in cervical cancer
(Kluwer Academic Publishers, 2015) Shyam Babu Prasad; Suresh Singh Yadav; Mitali Das; Arusha Modi; Soni Kumari; Lakshmi Kant Pandey; Sunita Singh; Satyajit Pradhan; Gopeshwar Narayan
Background: The cyclin-dependent kinase inhibitor p27Kip1 is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27Kip1 may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27Kip1 down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27Kip1 and its effects on proliferation, cell cycle progression and (inhibition of) apoptosis in cervical cancer cells. Methods: Primary cervical cancer samples (n = 70), normal cervical tissue samples (n = 30) and cervical cancer-derived cell lines (n = 8) were used to assess the expression of p27Kip1 and AKT1 by RT-PCR, Western blotting and immunohistochemistry, respectively. The effects of the PI3K inhibitor LY294004 and the proteasome inhibitor MG132 on cervical cancer cell proliferation were investigated using a MTT assay. Apoptosis and cell cycle analyses were carried out using flow cytometry, and sub-cellular p27Kip1 localization analyses were carried out using immunofluorescence assays. Results: We observed p27Kip1 down-regulation (p = 0.045) and AKT1 up-regulation (p = 0.046) in both the primary cervical cancer samples and the cervical cancer-derived cell lines, compared to the normal cervical tissue samples tested. Treatment of cervical cancer-derived cell lines with the PI3K inhibitor LY294002 resulted in a reduced AKT1 activity. We also observed a dose-dependent inhibition of cell viability after treatment of these cell lines with the proteasome inhibitor MG132. Treatment of the cells with LY294002 resulted in a G1 cell cycle arrest, a nuclear expression of p27Kip1, and a cytoplasmic p27Kip1 accumulation after subsequent treatment with MG132. Additionally, we found that the synergistic effect of MG132 and LY294002 resulted in a sub-G1 cell cycle arrest and apoptosis induction through poly (ADP-ribose) polymerase (PARP) cleavage. Conclusion: Our data suggest that p27Kip1 down-regulation in cervical cancer cells is primarily regulated through PI3K/AKT-mediated proteasomal degradation. The observed synergistic effect of the MG132 and LY294002 inhibitors may form a basis for the design of novel cervical cancer therapies. © 2015, International Society for Cellular Oncology.