Induction of Apoptotic Signaling Pathways by 3' methyl ATP in Different Malignant Cells: in vitro Study.

Extracellular ATP is a dynamic signaling molecule that modulates myriad of cellular functions through P2 purinergic receptors activation and is cytotoxic to a variety of cells at high concentration. But the mechanism of this extracellular ATP/ATP analogs- elicited cytotoxicity is not fully understood. In this study we aim to investigate whether there is differential sensitivity towards induction of apoptosis by ATP analogs (2'-Me ATP and 3'-Me ATP) and its effect on receptor mediated or extrinsic and mitochondria mediated or intrinsic apoptotic signaling pathways. Our findings demonstrated that the IC50 values for 2'-Me ATP and 3'-Me ATP were 3mM and 2mM, respectively, in Hep2, and SiHa cells. The downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, along with a significant increase in the expression of the pro-apoptotic protein Bax (p<0.05), indicated the involvement of both pro- and anti-apoptotic factors in HeP2 cells, whereas in SiHa cells, a downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. Furthermore, an upregulation of p53 and apoptosis-inducing factor (AIF) was observed in HeP2 cells (p<0.05) whereas, an upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF) was observed in SiHa cells. Additionally, there was a notable rise in caspase-3 and -9 activities, PARP cleavage, and the release of cytochrome c (p<0.05) from the mitochondria to the cytosol in both cells. Collectively, our study suggests that 3'-Me ATP induces apoptosis in Hep2 and SiHa cells through the intrinsic mitochondrial pathway.

Molecular approaches to lung cancer prevention.

Lung cancer is generally diagnosed at advanced stages when surgical resection is not possible. Late diagnosis, along with development of chemoresistance, results in high mortality. Preventive approaches, including smoking cessation, chemoprevention and early detection are needed to improve survival. Smoking cessation combined with low-dose computed tomography screening has modestly improved survival. Chemoprevention has also shown some promise. Despite these successes, most lung cancer cases remain undetected until advanced stages. Additional early detection strategies may further improve survival and treatment outcome. Molecular alterations taking place during lung carcinogenesis have the potential to be used in early detection via noninvasive methods and may also serve as biomarkers for success of chemopreventive approaches. This review focuses on the utilization of molecular biomarkers to increase the efficacy of various preventive approaches.

Single nucleotide polymorphisms in the DNA repair genes in HPV-positive cervical cancer.

Genetic variation in DNA repair genes can modulate DNA repair capacity and may be related to the risk of cancer. The human papillomavirus is considered to be a necessary but not sufficient cause for cervical cancer and, therefore, other factors contribute to the carcinogenesis. A hereditary component for this neoplasia has been reported. Evaluation of the association of six polymorphisms was carried out in the following DNA repair genes: XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), ERCC1 (Asp118Asp), ERCC2 (Lys751Gln), and ERCC4 (Arg415Gln). The cases (n=110) included 65 squamous cell carcinomas (SCCs) and 45 squamous intraepithelial lesions (SIL). Controls (n=68) were recruited from among women without cervical abnormalities. Genotypes were determined by PCR-restriction fragment length polymorphism and DNA sequencing. A positive association was observed between the polymorphisms of XRCC1 genes, that is, in codons 194 [P=0.001, odds ratio (OR)=20.1, 95% confidence interval (CI)=5.9-68.8], 280 (P=0.001, OR=5.4, 95% CI=2.3-12.6), and 399 (P=0.008, OR=4.2, 95% CI=1.5-12.1) and cervical cancer. SIL patients also showed a significant association with codon 194 (P=0.012, OR=3.8, 95% CI=1.3-10.6), but not with 280 (P=0.35) and 399 (P=0.81). A positive correlation was also found in ERCC4 Gln415Gln in both SCCs and SILs (P=0.001, OR=21.3, 95% CI=7.1-64.0 and P=0.001, OR=7.8, 95% CI=2.9-20.9, respectively). For ERCC2 Gln751Gln, the association was significant for both SCCs (P=0.001, OR=10.1, 95% CI=2.6-37.9) and SILs (P=0.001, OR=8.9, 95% CI=2.8-28.3). However, the risk of SCC did not appear to differ significantly among individuals with the ERCC1 Asp118Asp genotype (P=0.404). For SILs, it appeared to be a protective genotype (95% CI=0.1-0.7). This study indicates that variant types of DNA repair genes play an important role in modifying individual susceptibility to SCC.

Serum one-carbon metabolites and risk of cervical cancer.

Most cases of cervical cancer are associated with human papilloma virus (HPV) infection of high risk types. In folate deficiency, heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) interferes with HPV16 viral capsid protein synthesis. We aimed to study the importance of 1-carbon metabolism in cervical carcinogenesis by examining serum vitamin B12 (cobalamin), homocysteine, folate levels, and the RNA and protein expression of HPV16 L1, L2, E6, E7, and to correlate them with hnRNP-E1 expression and HPV infection in normals, squamous intraepithelial lesions (SILs), and cervical cancer subjects. Serum cobalamin, folate, and homocysteine were estimated using kits, RNA by real time PCR and proteins by Western blotting. We observed that lower folate and vitamin B12 levels were associated with HPV infection. hnRNP-E1 progressively decreased from normals (100%) to SILs (75%) to cervical cancer (52.6%). The findings show that HPV16 E6 and E7 are overexpresed whereas HPV16 L1 and L2 are downregulated at mRNA and protein levels in cervical cancer as compared to normals and SILs. The results indicate that perhaps the reduced expression of hnRNP-E1 might be involved with the cervical cancer pathogenesis, with folate playing a role in the natural history of HPV infection.

Decreased expression of DNA repair genes (XRCC1, ERCC1, ERCC2, and ERCC4) in squamous intraepithelial lesion and invasive squamous cell carcinoma of the cervix.

Reduced DNA repair might affect the risk of progression from infection with carcinogenic human papillomavirus (HPV), the etiologic agent for cervical cancer (CC), to persistent HPV infection, and hence to cervical pre-cancer and cancer. We assessed the variation in baseline expression of base excision repair gene XRCC1 and three nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and the risk of developing cervical cancer. A hospital-based case-control study was designed with 50 invasive cervical cancer patients, 40 squamous intraepithelial lesions (SIL) patients and 85 controls subjects. RT-qPCR and Western blotting was used to quantitate in vitro the mRNA and protein levels in fresh CC, SIL and normal cervix tissue. The levels of XRCC1, ERCC2, ERCC4, and ERCC1 transcripts and their respective proteins were lower in cervical cancer and SILs as compared to controls (p ≤ 0.001, 0.001, 0.001, and 0.025, respectively). In multivariate logistic regression analysis (adjusting for parity, age at first child birth, use of oral contraceptives, smoking status), low expression of XRCC1, ERCC2, ERCC4, and ERCC1 was associated with a significant increased risk for CC and SIL. Our results suggest that individuals whose expression of XRCC1, ERCC4, ERCC2, and ERCC1 are reduced may be at a higher risk of developing SIL which eventually leads to invasive cervical carcinoma. Moreover, independently also the reduced expression of these genes can directly lead to cervical cancer progression.

Cervical cancer pathogenesis is associated with one-carbon metabolism.

Cervical cancer is the most common cancer among women in India and a leading cause of death in these women. Most cases of cervical cancer are associated with human papillomavirus (HPV) infection of the high-risk type. It has been reported that aberrant DNA methylation can be associated with HPV infection and cervical cancer, and folate is directly involved in DNA methylation via one-carbon metabolism. We aimed to study the importance of one-carbon metabolism in the progression of cervical carcinogenesis by examining serum levels of vitamin B(12) (cobalamin), homocysteine, and folate and DNA methylation of tumor suppressor genes CDH1, HIC1, and Retinoic acid receptor beta (RARß) amid these women ranging from normal to squamous intraepithelial neoplastic lesions (SIL) to cervical cancer. Blood and tissue samples were collected from normal (n = 35), SILs (n = 27), and cervical cancer patients (n = 38) in the age group of 26-70 years. Measurement of serum vitamin B(12), folate, and homocysteine were done using kits (Immulite). Promoter methylation was examined using methylation-specific PCR. The frequency of promoter hypermethylation for all the three tumor suppressor genes CDH1, HIC1, and RARß showed an increasing trend from normal to dysplastic to invasive cervical cancer (p < 0.05). We observed that lower folate and vitamin B(12) status were associated with HPV infection. Taken together, our findings suggest a role of folate and vitamin B(12) in modulating the risk of cervical cancer and HPV infection. CDH1, HIC1, and RARß genes can be used as potential biomarkers of cervical cancer risk assessment.

Rare developmental abnormalities of thyroid gland, especially multiple ectopia: A review and our experience.

BACKGROUND: Developmental structural abnormalities of the thyroid gland are relatively rare. There are scanty reports of hemiagensis, dual and triple ectopia of the thyroid in the literature MATERIALS AND METHODS: We did a retrospective analysis of 236 patients referred to us for Tc-99m Pertechnetate thyroid scan over period of four months (May 2010 to Sept 2010). Twenty of these 236 patients aged less than 20 years found to have developmental abnormality of the thyroid gland on thyroid scan. Diagnosis was correlated with anatomical imaging (USG/CT scan), fine needle aspiration cytology (FNAC) and histopathology. RESULTS: Out of the 20 patients, 8 were diagnosed with thyroglossal cyst, 4 with ectopic thyroid gland, 4 with dual ectopia, two had agenesis of thyroid gland, one case each with hemiagenesis and triple ectopia. CONCLUSION: The study has emphasized the indispensable role of Tc-99m Pertechnetate thyroid scan in the evaluation of midline neck swellings of childhood and diagnosing developmental anomalies of thyroid gland.

Apoptotic signaling induced by benzamide riboside: an in vitro study.

Benzamide riboside (BR) is a novel anticancer agent exhibiting potent cytotoxic activity in malignant cell lines. However, the mechanism of induction of apoptosis is not clear. The purpose of this study was to elucidate the apoptotic signaling induced by BR on different human cancer cell lines. Our results revealed that BR at a dose of 50 microM induces apoptosis in SiHa, Hep2, and Ca Ski cells as studied by morphology and flow cytometry. A downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. An upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF). A significant increase in caspase-3 and -9 activities was seen, which was accompanied by PARP cleavage. Release of cytochrome c from the mitochondria to the cytosol was also observed. Taken together, the findings suggest that BR induces apoptosis in SiHa, Hep2, and Ca Ski cells via the intrinsic mitochondrial pathway.

Percutaneous endoscopic gastrostomy site metastases in head and neck cancer: use of FDG PET-CT.

PURPOSE: To retrospectively evaluate the utility of positron emission tomography-computed tomography (PETCT) in the diagnosis of percutaneous endoscopic gastroscopy (PEG) site metastases in head and neck cancer. MATERIALS AND METHODS: From the database of 250 patients of head and neck cancer who were referred for PET-CT over 2 years (from January 2005 to January 2007), 6 patients who had PEG tube placement were considered for the study. Imaging was performed on a GE Discovery ST PET-CT system after intravenous injection of 370 MBq (10 mCi) of < sup > 18 < /sup > F-fluorodeoxyglucose (FDG). RESULTS: Intense FDG uptake with an associated soft tissue mass was seen at the PEG site in 3 patients and mild uptake was seen in 2 patients. Biopsy revealed PEG site metastases in 2 patients, abscess in 1 patient, and granulation tissue in 1 patient. Intense uptake with an associated soft tissue mass suggested the diagnosis of metastasis. Stranding of the peristomal fat seen on the CT component of the PET-CT indicated an infective/inflammatory pathology. PET-CT findings showed local recurrence in 3 patients and disseminated metastases (excluding the PEG site) in 1 patient. CONCLUSION: The functional information provided by PET combined with the morphologic detail of CT can improve characterizing of the stoma site abnormality and help in distinguishing recurrence from infective/inflammatory changes. Whole body combined PET-CT is a useful modality for evaluating gastrostomy site metastases and for detecting coexisting local recurrences and distant metastases in head and neck cancer patients. In addition it can detect early asymptomatic recurrences at the gastrostomy site.