HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

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.

In silico analysis unveiling potential biomarkers in gallbladder carcinogenesis.

Gallbladder cancer (GBC) is a rare but very aggressive most common digestive tract cancer with a high mortality rate due to delayed diagnosis at the advanced stage. Moreover, GBC progression shows asymptomatic characteristics making it impossible to detect at an early stage. In these circumstances, conventional therapy like surgery, chemotherapy, and radiotherapy becomes refractive. However, few studies reported some molecular markers like KRAS (Kirsten Rat Sarcoma) mutation, upregulation of HER2/neu, EGFR (Epidermal Growth Factor Receptor), and microRNAs in GBC. However, the absence of some specific early diagnostic and prognostic markers is the biggest hurdle for the therapy of GBC to date. The present study has been designed to identify some specific molecular markers for precise diagnosis, and prognosis, for successful treatment of the GBC. By In Silico a network-centric analysis of two microarray datasets; (GSE202479) and (GSE13222) from the Gene Expression Omnibus (GEO) database, shows 50 differentially expressed genes (DEGs) associated with GBC. Further network analysis revealed that 12 genes are highly interconnected based on the highest MCODE (Molecular Complex Detection) value, among all three genes; TRIP13 (Thyroid Receptor Interacting Protein), NEK2 (Never in Mitosis gene-A related Kinase 2), and TPX2 (Targeting Protein for Xklp2) having highest network interaction with transcription factors and miRNA suggesting critically associated with GBC. Further survival analysis data corroborate the association of these genes; TRIP13, NEK2, and TPX2 with GBC. Thus, TRIP13, NEK2, and TPX2 genes are significantly correlated with a greater risk of mortality, transforming them from mere biomarkers of the GBC for early detections and may emerge as prognostic markers for treatment.

The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic.

Glycine betaine (GB) is an important osmolyte synthesized in response to different abiotic stresses, including salinity. The two known pathways of GB synthesis involve: 1) two step oxidation of choline (choline → betaine aldehyde → GB), generally found in plants, microbes and animals; and 2) three step methylation of glycine (glycine → sarcosine → dimethylglycine → GB), mainly found in halophilic archaea, sulphur bacteria and the cyanobacterium Aphanothece (Ap.) halophytica. Here, we transformed a salt-sensitive freshwater diazotrophic filamentous cyanobacterium Anabaena (An.) doliolum with N-methyltransferase genes (ApGSMT-DMT) from Ap. halophytica using the triparental conjugation method. The transformed An. doliolum synthesized and accumulated GB in cells, and showed increased salt tolerance and protection to nitrogenase activity. The salt responsiveness of the transformant was also apparent as GB synthesis increased with increasing concentrations of NaCl in the nutrient solution, and maximal [12.92 µmol (g dry weight)(-1)] in cells growing at 0.5 M NaCl. Therefore, the transformed cyanobacterium has changed its behaviour from preferring freshwater to halophily. This study may have important biotechnological implications for the development of stress tolerant nitrogen-fixing cyanobacteria as biofertilizers for sustainable agriculture.

Haematological and biochemical analysis of blood samples from early and late stage breast cancer patients in India.

Breast cancer is the most prevalent cancer with the maximum number of cases worldwide. Early diagnosis of the cancer is necessary for an effective treatment plan. Due to a lack of awareness, diagnosis of breast cancer at an early stage is difficult. The present study aims to evaluate and compare the haematological and biochemical profiles of the early and late-stage breast cancer patient's data records. A retrospective cohort study was conducted on 56 breast cancer patients at the Institute of Medical Sciences, Banaras Hindu University India. Patient data records were obtained and haematological and biochemical parameters were arranged on an Excel sheet and analyzed. Random blood sugar (RBS), alkaline phosphates (ALP) levels, and urea levels were significantly high in patients with late-stage breast cancer (Tumor stage III and IV). At the advanced stage of breast cancer hemoglobin level falls and patients became anemic. Further large-scale studies with a greater number of patient data can help establish these parameters individually or in combination as prognostic and diagnostic markers in breast cancer staging.

Olaparib modulates DNA repair efficiency, sensitizes cervical cancer cells to cisplatin and exhibits anti-metastatic property.

PARP1 trapping at DNA lesion by pharmacological inhibitors has been exploited in several cancers exhibiting defects in DNA repair mechanisms. PARP1 hyperactivation is involved in therapeutic resistance in multiple cancers. The role of PARP1 in cervical cancer (CC) resistance and implication of PARP inhibitor is yet to be elucidated. Our data demonstrates significantly higher expression of PARP1 in primary cervical tumors and CC cell lines SiHa and ME180. Upon cisplatin treatment CC cells display significant overexpression of PARP1 and its hyperactivation. PARP inhibitor olaparib shows significant anti-proliferative effect on CC cells and drive loss of clonogenic survival and enhanced cell death in combination with cisplatin. PARP inhibited cells show delay in resolution of γH2A.X foci and prolonged late S and G2-M phase arrest resulting in apoptosis. Further, PARP inhibition disrupts the localization of base excision repair (BER) effector XRCC1 and non-homologous end joining (NHEJ) proteins Ku80 and XRCC4. Due to disrupted relocation of repair factors, cisplatin induced stalled replication forks collapse and convert into double strand breaks (DSBs). Interestingly, PARP inhibition also shows anti-migratory and anti-invasive properties in CC cells, increases anchorage independent cell death and induces anoikis. Collectively, our data demonstrates therapeutic potential of PARP inhibitor in cervical cancer.

CXCL12 is a key regulator in tumor microenvironment of cervical cancer: an in vitro study.

CXCL12 is a small pro-inflammatory chemo-attractant cytokine which signals through chemokine receptor CXCR4. The importance of CXCL12/CXCR4 axis is coming to the fore in several divergent signaling pathway-initiating signals related to cell survival and/or proliferation and cancer metastasis. In the present study we have investigated whether deregulation in CXCR4 signaling (as a consequence of deregulated expression of CXCL12) modulate the metastatic potential of cervical carcinoma cells. We demonstrate that CXCL12 is frequently down regulated and its promoter is hypermethylated in cervical cancer cell lines and primary tumor biopsies. Exogenous treatment of cervical cancer cell lines (HeLa, SiHa and C-33A) with recombinant CXCL12 inhibited the metastasis promoting cell migration, cell invasion and anchorage independent cell growth events. Although this study will need further in vivo validation, our observations suggest that (a) silencing of CXCL12 in cervical cancer cells may be critical in migration and invasion, the key events in cancer cell metastases; (b) cervical cancer cells having down regulated CXCL12 are more prone to being attracted to CXCL12 expressed at secondary sites of metastases; and (c) CXCL12 inhibits anchorage independent cell growth via anoikis. These findings suggest the tumor suppressor functions of CXCL12 in cervical cancer.

Cyclin D1 and cyclin E2 are differentially expressed in gastric cancer.

Cell cycle regulators cyclin D1 and cyclin E2 function in G1/S transition by activating downstream cyclin-dependent kinases. Deregulated expression of these cyclins has been reported in various cancers. However, little is known about their clinical significance in gastric carcinoma. We aimed to explore that whether there is differential expression of these cyclins in clinically distinct gastric cancer patients. In this study we recruited a total of 92 subjects including 20 controls and 72 cases of histopathologically proven gastric carcinoma. Expression profiling at transcript level was done by semiquantitative RT-PCR and of protein by immunohistochemistry. Receiver operator characteristics analysis was done for determining diagnostic utility of cyclin D1 and cyclin E2. We demonstrate that cyclins D1 and E2 are frequently overexpressed in early stages of gastric carcinoma. Interestingly, expression of cyclins D1 and E2 significantly correlates with different clinical parameters such as gender, histological type (intestinal and diffuse), tumor location (proximal, middle, and distal), tumor differentiation (differentiated and undifferentiated), tumor invasion (serosal, lymphatic, and venous) and tumor metastasis (lymph node, peritoneal, ascites, and liver). Cyclin D1 has significantly higher sensitivity and specificity as diagnostic biomarker than cyclin E2. Our results suggest that overexpression of cyclin D1 and cyclin E2 is an early event in gastric carcinogenesis. The differential expression of these cyclins may be useful as diagnostic biomarkers for early detection of gastric carcinoma.

Anti-tumor activity of staurosporine in the tumor microenvironment of cervical cancer: An in vitro study.

AIM: The fundamental events for cancer progression and metastases include loss of cell adhesion, cell proliferation, anchorage-independent cell growth (evading anoikis), cell migration and cell invasion. All these events leading to cancer progression happen in a favorable nurturing tumor microenvironment. This study was designed to explore the anti-tumor activity of staurosporine (a nonspecific protein kinase inhibitor) in the tumor microenvironment of cervical cancer. MAIN METHODS: The anti-tumor activity of staurosporine was investigated by cell adhesion assay, colony formation assay, apoptosis assay and quantitative real-time polymerase chain reaction (PCR) in cervical cancer cell lines. KEY FINDINGS: The cell adhesion assay showed that staurosporine induces adhesion of cervical cancer cells to the extracellular matrix (ECM) protein fibronectin. The soft agar colony formation assay showed that staurosporine inhibits both the number and size of colony formation in a dose dependent manner and also induces adherent tendency in the cancer cells. Staurosporine also induces prominent apoptosis in single cell suspensions compared to adherent cells. Stroma cell induced transcription of matrix metalloprotease 1 (MMP1) and matrix metalloprotease 2 (MMP2) in cervical cancer cells was inhibited by staurosporine. SIGNIFICANCE: Our results indicate that staurosporine induces anti-tumor response in the cervical tumor microenvironment by inhibiting the fundamental events for cancer progression and metastases. The present study represents an attractive area for further research and opens up new avenues towards the understanding of cervical cancer therapeutics.

PI3K/AKT pathway-mediated regulation of p27(Kip1) is associated with cell cycle arrest and apoptosis in cervical cancer.

BACKGROUND: The cyclin-dependent kinase inhibitor p27(Kip1) is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27(Kip1) may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27(Kip1) down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27(Kip1) 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 p27(Kip1) 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 p27(Kip1) localization analyses were carried out using immunofluorescence assays. RESULTS: We observed p27(Kip1) 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 p27(Kip1), and a cytoplasmic p27(Kip1) 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 p27(Kip1) 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.

Epigenetic silencing of CXCR4 promotes loss of cell adhesion in cervical cancer.

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.

Down Regulation of FOXO1 Promotes Cell Proliferation in Cervical Cancer.

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.

Over expression of minichromosome maintenance genes is clinically correlated to cervical carcinogenesis.

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.