Transactivation and expression patterns of Jun and Fos/AP-1 super-family proteins in human oral cancer

Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers and considered as an important target for modern therapeutics. But the role of AP-1 during development of human oral cancers is still poorly understood. Because oral cancer is one of the most common cancers in India and south-east Asia, we studied the activation and expression pattern of AP-1 family of proteins and mRNA in different stages of oral carcinogenesis. Gel-shift assay, western blotting, immunohistochemistry and northern blotting have been used to assess the binding activity and expression pattern of AP-1 family (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) proteins and mRNA transcripts in a total of 100 fresh oral tissue specimens comprising precancer (n = 40), cancer (n = 50) and healthy control (n = 10). Constitutive activation of AP-1 with concomitant upregulated expression of majority of AP-1 family of proteins and mRNA was observed in cancer cases. Interestingly, almost all precancerous cases showed JunD homodimers, whereas c-Fos/JunD was the most prevalent complex found in cancer tissues. The overexpression of EGFR mRNA, p50:p50/NF-?B homodimer formation, together with overexpression of pERK and c-Fos proteins in this study suggests an interesting cross talk between AP-1 and NF-?B pathways in oral cancers. Thus, this study demonstrates differential expression and activation of AP-1 super-family proteins in relation to severity of lesion and their crucial role in human oral carcinogenesis

Differential expression and activation of NF-κB family proteins during oral carcinogenesis: role of high risk human papillomavirus infection

Oral cancer is one of the most common cancers in India and south-east Asian region consisting of more than 50% of all malignant tumors. Along with many known risk factors, infection of Human Papillomavirus (HPV) has been associated with the development of oral cancer and is suggested to modulate host cell transcription. Reciprocally, cellular transcription factors, such as NF-κB and AP-1 are known to modulate the expression of viral and other genes involved in the development of cancer. In the absence of data on NF-κB in relation to HPV in oral cancer, we studied the DNA binding activity and expression pattern of NF-κB family of proteins in different stages of oral cancer and correlated with HPV infection that has been associated with better prognosis of the disease. A total of 110 fresh oral tissue biopsies were collected comprising 10 normal controls, 34 precancer and 66 oral cancer lesions prior to chemotherapy/radiotherapy. Diagnosis of HPV was done by both consensus and type-specific PCR. Electrophoretic mobility shift assays, western blots and immunohistochemical analysis were performed to assess the binding activity and expression pattern of NF-κB family of proteins (p50, p65, p52, c-Rel, RelB and Bcl-3) in oral tissue biopsies. Twenty seven percent (18/66) of the oral cancer biopsies showed the presence of HPV infection exclusively of high risk HPV type 16, which was primarily associated with the well differentiated squamous cell carcinomas (WDSCC). We observed a high constitutive activation of NF-κB with concomitant upregulated expression of all the NF-κB members in oral cancer tissues. Expression of NF-κB components gradually increased as the severity of lesion increased from precancer to invasive cancer. NF-κB p50 was found to be the major DNA binding component, which is indicative of homodimerization of p50 subunits. Interestingly, in HPV16 infected oral cancers although p50 showed high binding activity, p65 also showed a partial involvement as evidenced in supershift assay. Both by western blotting and immunohistochemistry, a differential overexpression and nuclear localization of p50, p65 and partially of Bcl-3 were observed in HPV16 positive oral cancer patients that also showed an over-expression of p21. We therefore, demonstrate a constitutive activation and differential expression of NF-κB proteins, which change as a function of severity of oral lesions during development of oral cancer. The NF-κB DNA binding is primarily due to homodimerization of p50 but infection of high risk HPV promotes participation of p65 in NF-κB complex formation, leading to heterodimerization of p50/p65. We propose that the involvement of p65 in HPV infected oral cancer may be linked to improved differentiation and better prognosis of the disease when treated

Efficacy of plasma vascular endothelial growth factor in monitoring first-line chemotherapy in patients with advanced non-small cell lung cancer

Background: Along with the development of new cancer therapeutics, more effective tools for the estimation of response to therapy and prediction of disease progression are required for the better management of inoperable cancer patients. Methods: We studied 134 newly diagnosed and primarily untreated advanced non-small cell lung cancer patients and 100 controls. Forty two patients received platinum-based chemotherapy. Plasma VEGF levels were quantified in all samples at baseline and also before second and third chemotherapy cycle in 42 patients and correlated with response to therapy as assessed by computed tomography after the third chemotherapy cycle. Results: We observed that, patients who went into remission had significantly lower baseline VEGF levels before second and third cycles of chemotherapy when compared with patients with no change and progression. Plasma VEGF levels showed a greater decrease from cycle 1 to 2 and from cycle 1 to 3 in patients who showed remission in comparison to those with no change or progression. Plasma VEGF levels before the second cycle detected poor response to therapy with a sensitivity and specificity of 76.9% and 75.0%, respectively (area under the ROC curve = 0.724). Early prediction of disease progression was achieved with a sensitivity and specificity of 71.4% for plasma VEGF before cycle 2 (area under the ROC curve = 0.805). The kinetics of VEGF form cycle 1 to 2 and cycle 1 to 3 also gave significant information for predicting disease progression as well as insufficient therapy response. Conclusion: Monitoring of plasma VEGF levels during the course of first-line chemotherapy could identify patients who are likely to have insufficient response to therapy and disease progression at an early stage. This may help in individualizing treatment and could lead to better management of the advanced stage lung cancer

Berberine modulates AP-1 activity to suppress HPV transcription and downstream signaling to induce growth arrest and apoptosis in cervical cancer cells

<p>Abstract</p> <p>Background-</p> <p>Specific types of high risk Human papillomaviruses (HR-HPVs) particularly, HPV types 16 and 18 cause cervical cancer and while the two recently developed vaccines against these HPV types are prophylactic in nature, therapeutic options for treatment and management of already existing HPV infection are not available as yet. Because transcription factor, Activator Protein-1 (AP-1) plays a central role in HPV-mediated cervical carcinogenesis, we explored the possibility of its therapeutic targeting by berberine, a natural alkaloid derived from a medicinal plant species, <it>Berberis </it>which has been shown to possess anti-inflammatory and anti-cancer properties with no known toxicity; however, the effect of berberine against HPV has not been elucidated.</p> <p>Results-</p> <p>We studied the effect of berberine on HPV16-positive cervical cancer cell line, SiHa and HPV18-positive cervical cancer cell line, HeLa using electrophoretic mobility gel shift assays, western and northern blotting which showed that berberine could selectively inhibit constitutively activated AP-1 in a dose- and time-dependent manner and downregulates HPV oncogenes expression. Inhibition of AP-1 was also accompanied by changes in the composition of their DNA-binding complex. Berberine specifically downregulated expression of oncogenic c-Fos which was also absent in the AP-1 binding complex. Treatment with berberine resulted in repression of E6 and E7 levels and concomitant increase in p53 and Rb expression in both cell types. Berberine also suppressed expression of telomerase protein, hTERT, which translated into growth inhibition of cervical cancer cells. Interestingly, a higher concentration of berberine was found to reduce the cell viability through mitochondria-mediated pathway and induce apoptosis by activating caspase-3.</p> <p>Conclusion-</p> <p>These results indicate that berberine can effectively target both the host and viral factors responsible for development of cervical cancer through inhibition of AP-1 and blocking viral oncoproteins E6 and E7 expression. Inhibition of AP-1 activity by berberine may be one of the mechanisms responsible for the anti-HPV effect of berberine. We propose that berberine is a potentially promising compound for the treatment of cervical cancer infected with HPV.</p

Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM) substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) and TIMP-2 (-418 G/C or C/C). Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs) are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities

Infection of human papillomaviruses in cancers of different human organ sites

Clinico-epidemiological and molecular studies have established the casual link between Human Papillomavirus (HPV) infection and cervical cancer as also association of HPV infection with several other cancers. In India, cervical cancer is a leading cancer among women and almost all cases of cervical cancer show prevalence of High Risk (HR)-HPV infection. HPV has been also detected in a significant proportion of oral, esophageal, anal, vaginal, vulvar, and penile cancer and in a small percentage of lung, laryngeal, and stomach cancer in India. Due to lack of organized HPV screening program, insufficient infrastructure and trained manpower and inadequacy in cancer registries, there are not much data available on the countrywide HPV prevalence and its type distribution in different cancers in India. Forthcoming introduction of recently developed HPV vaccines in India given a new urgency to know the prevalence and distribution of various HPV types in different organ sites for the management and monitoring of vaccination program and its impact on prevalence of other cancers. This review, summarizes studies on the prevalence of HPV infection in cancers of different organ sites in India

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

<p>Abstract</p> <p>Background</p> <p><it>Bryophyllum pinnata </it>(<it>B. pinnata</it>) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of <it>B. pinnata </it>we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle.</p> <p>Methods</p> <p>A chloroform extract derived from a bulk of botanically well-characterized pulverized <it>B</it>. <it>pinnata </it>leaves was separated using column chromatography with step- gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells.</p> <p>Results</p> <p>Results showed presence of growth inhibitory activity in the crude leaf extracts with IC₅₀at 552 μg/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50:50) and showed IC₅₀at 91 μg/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A.</p> <p>Conclusions</p> <p>Our study therefore demonstrates presence of anticancer and anti-HPV an activity in <it>B</it>. <it>pinnata </it>leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.</p

Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection

<p>Abstract</p> <p>Background</p> <p>Recent observations indicate potential role of transcription factor STAT3 in cervical cancer development but its role specifically with respect to HPV infection is not known. Present study has been designed to investigate expression and activation of STAT3 in cervical precancer and cancer in relation to HPV infection during cervical carcinogenesis. Established cervical cancer cell lines and prospectively-collected cervical precancer and cancer tissues were analyzed for the HPV positivity and evaluated for STAT3 expression and its phosphorylation by immunoblotting and immunohistochemistry whereas STAT3-specific DNA binding activity was examined by gel-shift assays.</p> <p>Results</p> <p>Analysis of 120 tissues from cervical precancer and cancer lesions or from normal cervix revealed differentially high levels of constitutively active STAT3 in cervical precancer and cancer lesions, whereas it was absent in normal controls. Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells. Expression and activity of STAT3 were found to change as a function of severity of cervical lesions from precancer to cancer. Expression of active pSTAT3 was specifically high in cervical precancer and cancer lesions found positive for HPV16. Interestingly, site-specific accumulation of STAT3 was observed in basal and suprabasal layers of HPV16-positive early precancer lesions which is indicative of possible involvement of STAT3 in establishment of HPV infection. In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades.</p> <p>Conclusion</p> <p>We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.</p

Differentially localized survivin and STAT3 as markers of gastric cancer progression: Association with Helicobacter pylori

BackgroundLocalization and differential expression of STAT3 and survivin in cancer cells are often related to distinct cellular functions. The involvement of survivin and STAT3 in gastric cancer has been reported in separate studies but without clear understanding of their kinetics in cancer progression.MethodsWe examined intracellular distribution of STAT3 and survivin in gastric adenocarcinoma and compared it with normal and precancer tissues using immunoblotting and immunohistochemistry.ResultsAnalysis of a total of 156 gastric samples comprising 61 histologically normal, 30 precancerous tissues (comprising intestinal metaplasia and dysplasia), and 65 adenocarcinomas, collected as endoscopic biopsies from treatment naïve study participants, revealed a significant (P < .001) increase in overall protein levels. Survivin expression was detectable in both cytoplasmic (90.8%) and nuclear (87.7%) compartments in gastric adenocarcinomas lesions. Precancerous dysplastic gastric lesions exhibited a moderate survivin expression (56.7%) localized in cytoplasmic compartment. Similarly, STAT3 and pSTAT3 expression was detected at high level in gastric cancer lesions. The levels of compartmentalized expression of survivin and STAT3/pSTAT3 correlated in precancerous and adenocarcinoma lesions. Although overexpression of these proteins was found associated with the tobacco use and alcohol consumption, their expression invariably and strongly correlated with concurrent Helicobacter pylori infection. Receiver operating characteristic analysis of nuclear survivin, STAT3, and pSTAT3 in different study groups showed acceptable positive and negative predictive values with area under the curve above 0.8 (P < .001).ConclusionOverall, our results suggest that overall increase in survivin and STAT3 and their subcellular localization are key determinants of gastric cancer progression, which can be collectively used as potential disease biomarkers and therapeutic targets for gastric cancer.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144680/1/cnr21004-Supplementary_Methods_20180313.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144680/2/cnr21004-sup-0001-F1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144680/3/cnr21004_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144680/4/cnr21004.pd

Comparative study between the Hybrid Capture II test and PCR based assay for the detection of human papillomavirus DNA in oral submucous fibrosis and oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Oral malignancy is a major global health problem. Besides the main risk factors of tobacco, smoking and alcohol, infection by human papillomavirus (HPV) and genetic alterations are likely to play an important role in these lesions. The purpose of this study was to compare the efficacy of HC-II assay and PCR for the detection of specific HPV type (HPV 16 E6) in OSMF and OSCC cases as well as find out the prevalence of the high risk HPV (HR-HPV) in these lesions.</p> <p>Methods and materials</p> <p>Four hundred and thirty patients of the potentially malignant and malignant oral lesions were taken from the Department of Otorhinolaryngology, Moti Lal Nehru Medical College, Allahabad, India from Sept 2007-March 2010. Of which 208 cases were oral submucous fibrosis (OSMF) and 222 cases were oral squamous cell carcinoma (OSCC). The HC-II assay and PCR were used for the detection of HR-HPV DNA.</p> <p>Result</p> <p>The overall prevalence of HR-HPV 16 E6 DNA positivity was nearly 26% by PCR and 27.4% by the HC-II assay in case of potentially malignant disorder of the oral lesions such as OSMF. However, in case of malignant oral lesions such as OSCC, 32.4% HPV 16 E6 positive by PCR and 31.4% by the HC-II assay. In case of OSMF, the two test gave concordant result for 42 positive samples and 154 negative samples, with an overall level of agreement of 85.4% (Cohen's kappa = 66.83%, 95% CI 0.553-0.783). The sensitivity and specificity of the test were 73.7% and 92.05% (p < 0.00). In case of OSCC, the two test gave concordant result for 61 positive samples and 152 negative samples, with an overall level of agreement of 88.3% (Cohen's kappa = 79.29, 95% CI 0.769-0.939) and the sensitivity and specificity of the test were 87.14% and 92.76% (p < 0.00).</p> <p>Conclusion</p> <p>This study concluded that slight difference was found between the positivity rate of HR-HPV infection detected by the HC-II and PCR assay in OSMF and OSCC cases and the HC II assay seemed to have better sensitivity in case of OSCC.</p