BackgroundRecent 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.ResultsAnalysis 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.ConclusionWe 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.

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.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor constitutively active and aberrantly expressed in cervical cancer. However, the functional role of STAT3 in regulation of HPV's viral oncogene expression and downstream events associated with cervical carcinogenesis is not known. Our present study performed on HPV16-positive cervical cancer cell lines (SiHa and CaSki) and primary tumor tissues revealed a strong positive correlation of constitutively active STAT3 with expression of HPV16 E6 and E7 oncoproteins and a negative association with levels of p53 and pRB. Pharmacologic targeting of STAT3 expression in cervical cancer cell lines either by STAT3-specific siRNA or blocking its tyrosine phosphorylation by AG490 or curcumin led to dose-dependent accumulation of p53 and pRb in cervical cancer cells. Interestingly, the suppression of STAT3 expression or activation was associated with the gradual loss of HPV16 E6 and E7 expression and was accompanied by loss of cell viability. The viability loss was specifically high in HPV16-positive cells as compared to HPV negative C33a cells. These findings substantiate the regulatory role of STAT3 in HPV16-mediated cervical carcinogenesis. Leads obtained from the present study provide a strong rationale for developing novel STAT3-based approaches for therapeutic interventions against HPV infection to control cervical cancer.

Purpose: Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer "stem-cell like" characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer.Experimental Design: Isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis.Results: CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation.Conclusions: Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1.

Aberrantly expressed survivin and STAT3 signaling have emerged as major determinants of chemoresistance in gastric cancer. We evaluated effects of potent herbal derivatives curcumin, berberine, and quercetin on STAT3 signaling, survivin expression, and response to 5-fluorouracil (5-FU) treatment in gastric cancer cells (AGS). Cytotoxic and inhibitory effects of berberine, curcumin, and quercetin alone or in combination with 5-FU were examined by MTT assay, and their effect on survivin, STAT3, and the phosphorylated active STAT3 (pSTAT3) expression was examined by western blotting. Effect of these herbal derivatives on STAT3 DNA binding activity was measured by electrophoretic mobility shift assay. Curcumin, berberine, and quercetin effectively downregulated pSTAT3 levels, survivin expression, and gastric cancer cells viability in a dose-dependent manner (with corresponding IC50 values of 40.3μM, 29.2μM and 37.5μM, respectively). Berberine was more effective in inhibiting survivin expression as compared to other herbal agents. 5-FU in combination with berberine or curcumin showed a synergistic inhibition of survivin and STAT3 level resulting in enhanced cell death in gastric cancer cells. Overall, our data suggest use of berberine and curcumin as adjunct therapeutics to overcome chemoresistance during treatment of gastric malignancies.

Background Bryophyllum pinnata ( B. pinnata ) 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 B. pinnata 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. Methods A chloroform extract derived from a bulk of botanically well-characterized pulverized B . pinnata 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. Results Results showed presence of growth inhibitory activity in the crude leaf extracts with IC 50 at 552 μg/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50:50) and showed IC 50 at 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. Conclusions Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B . pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.

Plant products of Phyllanthus emblica Linn. are traditionally consumed for its immense nutritive and medicinal values. However, the molecular mechanism(s) by which it exerts it effects is less understood. In this study, we investigated mechanism of action of P. emblica fruit extract (PE) by studying its effect on activator protein-1 (AP-1) activity and human papillomavirus (HPV) transcription that are essential for tumorigenicity of cervical cancer cells. PE resulted in a dose-and time-dependent inhibition of DNA binding activity of constitutively active AP-1 in both HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cells. PE-induced AP-1 inhibition was found mediated through downregulation of constituent AP-1 proteins, c-Jun, JunB, JunD, and c-Fos; however, the kinetics of their inhibition varied in both the cell types. Inhibition of AP-1 by PE was accompanied by suppression of viral transcription that resulted in growth inhibition of cervical cancer cells. Growth inhibitory activity of PE was primarily manifested through induction of apoptotic cell death. These results suggest that P. emblica exhibits its anticancer activities through inhibition of AP-1 and targets transcription of viral oncogenes responsible for development and progression of cervical cancer thus indicating its possible utility for treatment of HPV-induced cervical cancers.