Bhudev C. Das

Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin

The transcription factor AP‐1 plays a central role in the transcriptional regulation of specific types of high‐risk human papillomaviruses (HPVs) such as HPV16 and HPV18, which are etiologically associated with the development of cancer of the uterine cervix in women. In our study, we investigated the AP‐1 binding activity and the expression pattern of different members of the AP‐1 transcription factor family (c‐Jun, JunB, JunD, c‐Fos, FosB, Fra‐1 and Fra‐2) in different grades of cervical lesions starting from mild dysplasia to invasive cervical tumors, including normal control tissues, using specific antibodies raised against each of the AP‐1 members. Results indicate that though AP‐1 showed high binding activity and the majority of its members were highly expressed in tumor tissues, there is a distinct pattern of gradual increase of c‐fos and a concomitant decrease of fra‐1 expression that perfectly match the progression of cervical lesions. While c‐fos is highly expressed in invasive cervical tumor, the expression of fra‐1 becomes almost nil or absent, but the reverse is true in both controls and early precancerous lesions. These findings corroborate the results obtained in the cervical cancer cell line, HeLa. Interestingly, despite very low or absent AP‐1 binding in normal as well as in premalignant lesions, AP‐1 transcription and its binding activity was found to be very high in malignant tissues showing a preferential heterodimerization of c‐fos with JunB instead of its canonical dimerization partner c‐jun. Both in vivo and in vitro studies demonstrate that the overexpression of c‐fos and downregulation of fra‐1 expression as well as a change in the dimerization pattern of the AP‐1 complex seem to play a crucial role during progression to malignancy. In a previous study, we demonstrated that a synthetic antioxidant, pyrrolidine dithiocarbamate (PDTC) can selectively downregulate HPV expression in human keratinocytes and cervical cancer cell lines. Since a redox regulatory pathway is involved in the expression of HPV that can be modulated by an antioxidant‐induced reconstitution of the AP‐1 transcription complex, we have used curcumin (diferuloylmethane), an active component of the perennial herb turmeric, which is a potent antioxidant and is well‐known for its antiinflammatory and anticarcinogenic activity, to modulate the transcription of AP‐1 and HPV. We demonstrate for the first time that curcumin can selectively downregulate HPV18 transcription as well as the AP‐1 binding activity in HeLa cells. Most interestingly, curcumin can reverse the expression dynamics of c‐fos and fra‐1 in this tumorigenic cell line, mimicking the expression pattern observed in normal controls or precancerous lesions. Observation of curcumin‐mediated complete downregulation of AP‐1 binding activity and reversal of c‐fos/fra‐1 transcription to a normal state in tumorigenic HeLa cells represents a novel mechanism that can control transcription of pathogenic HPVs during keratinocyte differentiation and progression of cervical cancer. Our study thus provides a basis for developing a novel therapeutic approach to control pathogenic HPV infection by using potent antioxidative agents, such as curcumin.

Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions

Integration of human papillomavirus (HPV) DNA into the host cell genome is believed to be essential for malignant progression. However unambiguous detection of the physical state of HPV is a difficult and time-consuming procedure. To resolve this issue a simple, rapid and highly sensitive technique of polymerase chain reaction (PCR) has been utilized for detecting the physical state of HPV-16 DNA. Investigations were carried out in 122 cervical specimens comprising the whole spectrum of cervical lesions starting from cervical dysplasia to invasive carcinoma including HPV-16-positive normal controls. A pair of oligonucleotide primers specific to the E2 open reading frame, which is often deleted or disrupted following HPV integration, was used for the study. Distinction between episomal and integrated forms of viral DNA was accomplished by detecting amplification of the E2-specific fragment (1139 bp) in the PCR product. The PCR results were compared with those obtained by the conventional methods of Southern blotting, two-dimensional gel electrophoresis and chromosomal in situ hybridization; a high degree of agreement was observed between the methods. The findings indicate that although integrated forms of HPV-16 DNA were detected in more than 70% of cervical cancer specimens, integration was less frequent (23%) in severe dysplasia and carcinoma in situ. Only 2.5% of cases showed both episomal and integrated forms of HPV-16 DNA. The difference between episomal and integrated forms was statistically significant (P less than 0.01). The absence of integration in about 30% of cancer cases suggests that integration of HPV may not be necessary for malignant progression and alternative mechanism(s) of malignant transformation may occur without HPV integration. The PCR test thus provides an effective complement to Southern blotting and two-dimensional gel electrophoresis for accurate detection of the integration of HPV DNA.

Hepatitis E virus infection and fulminant hepatic failure during pregnancy.

Background and Aim:  Hepatitis E virus (HEV) infection leading to fulminant hepatic failure (FHF) and high mortality is a common feature in Indian women during the second and third trimesters of pregnancy. An altered status of hormones and immunity are observed during pregnancy but the actual cause of high mortality is still unknown. The present study was carried out to analyze CD3, CD4 and CD8 T cell counts and to assay the level of pregnancy‐related hormones such as estrogen, progesterone and β‐HCG in order to discover the role played by these factors.

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.

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

Background-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, Berberis 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.Results-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.Conclusion-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.

Does Hepatitis E Viral Load and Genotypes Influence the Final Outcome of Acute Liver Failure During Pregnancy

BACKGROUND:Hepatitis E is a major health problem in developing countries including India. The incidence and mortality rate in pregnant women with fulminant hepatic failure (FHF) due to hepatitis E virus (HEV) has been reported to be significantly higher, specifically in Asian women. Pregnancy is usually associated with an altered status of sex steroid hormones and immunity. Steroid hormones directly influence the replication through their effects on viral regulatory elements. Moreover, pregnant women in Asia generally suffer from folate deficiency, which is known to cause reduced immunocompetence leading to greater risk of multiple viral infections and higher viral load.OBJECTIVES:To correlate and analyze the viral load and genotypes of HEV in acute liver failure with that of acute viral hepatitis among pregnant and nonpregnant women.MATERIALS AND METHODS: A total of 100 FHF and 150 acute viral hepatitis (AVH) patients (50, 75 pregnant and 50, 75 nonpregnant, respectively), were included in the study. These cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test of hepatitis A, B, C, and E using commercially available ELISA kits. Quantification of HEV RNA-positive samples was carried out.RESULTS:Out of 100 FHF and 150 acute viral hepatitis (AVH) patients, 28 (56%) and 22 (29.3%) pregnant and 7 (14%) and 8 (16%) nonpregnant, respectively, were HEV RNA-positive. HEV viral load in FHF pregnant women was 5.87 × 104± 1.5 × 105μ L/mL as compared to AVH pregnant women 343.29 ± 216.44 μL/mL and FHF and AVH nonpregnant 199.2 ± 225.5 μL/mL and 13.83 ± 7.8 μL/mL, respectively. Sequencing data of all the positive samples of FHF and AVH pregnant and nonpregnant women showed genotype 1.CONCLUSION:HEV viral load was found to be significantly higher (P < 0.05) in pregnant patients compared to the nonpregnant. Pregnancy appears to be a risk factor for viral replication. The viral copies of HEV in FHF pregnant women were comparatively higher when compared to AVH pregnant women, which may be related to the severity of the disease in these patients. We could detect only one genotype (genotype 1) in our study population. Thus in the absence of other genotypes in this population, the impact of genotype could not be adequately assessed in this study.

Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction

SummaryOncogenic human papillomavirus (HPV) types 16 and 18 commonly associated with cervical cancer are found in many epithelial malignancies at extra-genital sites including breast. The transforming gene products of HPV have also been shown to immortalize breast epithelial cellsin vitro. But the findings of HPV DNA in breast carcinoma are found to be contradictory. In the present study fine needle aspirate cell (FNAC) samples from 26 breast cancer patients and four breast tumour biopsies were analysed for the presence of HPV 16 and 18 DNA sequences by both polymerase chain reaction (PCR) and Southern blot hybridization. Of 26 fine needle aspi rate cell samples and four breast cancer biopsies, not a single sample was found to be positive by either PCR or Southern blot hybridization. The observation of complete absence of HPV DNA sequences in breast cancer refute the possibility of any role for oncogenic genital HPV types 16 and 18 in the pathogenesis of breast cancer.

Human papillomavirus type 16 variant analysis of E6, E7, and L1 genes and long control region in biopsy samples from cervical cancer patients in north India.

ABSTRACT High-risk human papillomaviruses (HPVs), particularly HPV types 16 and 18 (HPV-16 and HPV-18, respectively), play a cardinal role in the etiology of cervical cancer. The most prevalent type, HPV-16, shows intratypic sequence variants that are known to differ in oncogenic potential and geographic distribution. This study was designed to analyze sequence variations in E6, E7, and L1 genes and the LCR (for long control region) of HPV-16 in cervical cancer patients to identify the most prevalent and novel HPV-16 variants and to correlate them with the severity of the disease. Cervical biopsies from 60 HPV-16-positive cancer cases were analyzed by PCR and DNA sequencing. The most frequently observed variations were T350G (100%) in E6, T789C (87.5%) in E7, A6695C (54.5%) in L1, and G7521A (91.1%) in the LCR. In addition, only one novel variant (T527A) in E6 and four new variants each in L1 (A6667C, A6691G, C6906T, and A6924C) and in the LCR (C13T, A7636C, C7678T, and G7799A) were identified. While E7 was found to be highly conserved, the variant 350G of E6 was the most prevalent in all of the histopathological grades. The majority of LCR variants were found at the YY1 transcription factor binding sites. Interestingly, a complete absence of the Asian lineage and a high prevalence of European lineages in E6, E7, L1, and the LCR (85, 86.7, 67.7, and 63.3%, respectively) indicate a possible epidemiological linkage between Europe and India with regard to the dissemination of HPV-16 infections in India.

High viral load and deregulation of the progesterone receptor signaling pathway: Association with Hepatitis E-related poor pregnancy outcome

BACKGROUND & AIMS Hepatitis E virus (HEV) infection is associated with high maternal and fetal mortalities. A prospective study was undertaken to evaluate the role of viral and host factors in HEV related pregnancy outcomes. METHODS The study included HEV infected pregnancy cases; acute viral hepatitis (AVH), n=100 and fulminant hepatic failure (FHF), n=43, and healthy pregnancy cases, n=50. HEV genotypes and viremia were studied by nucleotide sequencing and real time PCR, respectively. Progesterone receptor (PR) gene mutations (PROGINS) were studied by PCR, PR expression at the mRNA and protein levels in the placenta were studied by semi-quantitative RT-PCR and immunohistochemistry, respectively. Progesterone induced blocking factor (PIBF) expression was studied by RT-PCR in blood. Serum interleukin-10 (IL-10) and interleukin-12 (IL-12) levels were assayed by ELISA. RESULTS HEV viral load was significantly higher in FHF than AVH (p<0.001) and in cases with fetal mortality in AVH (p=0.001) and FHF (p=0.018). PROGINS were predominant in FHF compared to AVH (p=0.26) and showed reduced mRNA and protein expression. The risk of fetal mortality in AVH was two times higher (OR, 2.190; CI, 0.303-15.85) and maternal and fetal mortalities in FHF were 4-fold (OR, 4.0; CI, 0.363-44.113) increased in PROGINS carriers. PR and PIBF expression was lower in AVH and even lower in FHF compared to healthy controls. The higher IL-12/IL-10 ratio observed in FHF compared to other groups correlated with fetal mortality in AVH and FHF (p<0.001). CONCLUSIONS In conclusion, reduced expression of PR and PIBF, a higher IL-12/IL-10 ratio, and a high viral load results in poor pregnancy outcome in Hepatitis E.

Profile of hepatocellular carcinoma in India: An insight into the possible etiologic associations

Several etiologic factors including hepatitis viruses, alcohol and aflatoxin have been implicated in the pathogenesis of hepatocellular carcinoma (HCC). There is, however, limited information from the Indian subcontinent.