Effect of targeting STAT3 expression by RNA interference on the expression of p53 & pRb and E6 & E7 and on viability of cervical cancer cells.

<p>(A) Specific STAT3 siRNA inhibits STAT3 and pSTAT3 expression in cervical cancer cells. Cellular proteins (50 µg) isolated from SiHa cells (2×10⁵ cells) treated with the indicated concentrations of STAT3 and control siRNA (Scr) for 48 h were examined for pSTAT3 (Y705), STAT3, p53, or pRb by immunoblotting as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067849#s2" target="_blank">Methods</a>’. Blots were stripped and reprobed with β-actin as loading control. (B) Nuclear proteins (10 µg/lane) isolated from SiHa cells treated with STAT3 or control siRNA were incubated with radiolabeled STAT3 oligos to check STAT3-specific DNA binding activity by EMSA. NS- Non specific band. (C) Blocking STAT3 by specific siRNA abrogates expression of HPV16 oncogenes E6 and E7 in cervical cancer cells. Cellular proteins isolated from SiHa cells (2×10⁵ cells) treated with the STAT3 and control siRNA (160 nM) for 48 h were examined for expression of HPV16 oncogenes E6 and E7 by immunoblotting. Blots were stripped and reprobed with β-actin as loading control. (D) STAT3 inhibition was accompanied with reduced proliferation/ loss of cell viability in HPV16 positive cervical cancer cells. HPV16 positive SiHa cells and HPV negative C33a cells (2×10⁵ cells) treated with the indicated concentration of STAT3-specific and control siRNA for 48 h were harvested by trypsinization and counted for live cells using Trypan blue vital dye. Experiment was performed in triplicate, error bars indicates mean±SD. Lower panel indicates photomicrographs of respective control and treated SiHa and C33a cell cultures prior to their harvesting for cell counting.</p

Increased STAT3 activity in cervical cancer cells is associated with elevated expression of HPV16 E6 & E7 and corresponding loss of expression of their respective cellular targets, p53 and pRb.

<p>(<b>A</b>) Representative immunoblots indicating differential expression of active pSTAT3 (Y705), STAT3, HPV16 E6 & E7, p53 and pRb in HPV negative (C33a), and HPV16 positive (SiHa & Caski) cervical cancer cell lines. (<b>B</b>) Expression of HPV16 E6, E7, p53 and pRb proteins in representative cervical cancer cases (#1–4) with differential pSTAT3/STAT3 expression [low (Case #1 and #2) vs. high (Case #3 and #4)]. Total cellular proteins (50 µg) isolated from C33a, SiHa and Caski cells (1×10⁶ cells) and cervical cancer tissues were resolved on 10 or 15% SDS-PAGE and immunoblotted for phospho-STAT3, STAT3, HPV16 E6, HPV16 E7, p53 and pRb proteins by specific antibodies as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067849#s2" target="_blank">Methods</a>’. Blots were stripped and reprobed for β-actin as loading control. (<b>C</b>) Representative photomicrographs of immunohistochemical analysis of STAT3, pSTAT3 (Y705), HPV16 E6, E7, cellular p53 and pRb in cancer lesions with differential pSTAT3/STAT3 expression. Freshly fixed, paraffin-embedded sections (5 μm) of cervical tissues from cancerous lesion of the cervix were processed for IHC and probed for indicated antibodies and detected by HRP-DAB as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067849#s2" target="_blank">Methods</a>’. Brown precipitate indicates immune reactive-positive cells, blue stain represents nuclei. Black and grey arrows indicate strong and weak immunopositive cells respectively. (Original magnification: 200×). (<b>D</b>) Cumulative immunohistochemical data indicating association of level of active STAT3 with expression of HPV16 oncogenes E6 and E7 and their cellular targets p53 and pRb. Arbitrary staining intensity grades of respective proteins in immunohistochemistry were categorized into Strong, Moderate, Weak or Nil/not detectable as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067849#s2" target="_blank">Methods</a>. Values indicate the distribution of specimens in each category. ^a<i>p</i> value between pSTAT3 nil/weak or Moderate/Strong versus HPV16 E6 nil/weak or moderate/strong; ^b<i>p</i> value between pSTAT3 nil/weak or Moderate/Strong versus HPV16 E7 nil/weak or moderate/strong; ^c<i>p</i> value between pSTAT3 nil/weak or Moderate/Strong versus p53 nil/weak or moderate/strong; ^d<i>p</i> value between pSTAT3 nil/weak or Moderate/Strong versus pRb nil/weak or moderate/strong, as determined by two tailed Fischer’s Exact Test.</p

Both, curcumin and AG490, rescue expression of p53 and pRb through suppression of HPV16 E6 and E7 expression.

<p>(<b>A–B</b>) Effect of curcumin and AG490 on p53, pRb expression in cervical cancer cells. SiHa cells (1×10⁶ cells) treated with the curcumin (50 µM) or AG490 (100 μM) for indicated durations were tested for p53 and pRb expression by western blotting as described. (<b>C</b>) Curcumin and AG490-mediated abrogation of HPV16 E6 and E7 expression. SiHa cells (1×10⁶ cells) treated with curcumin (50 µM) or AG490 (100 μM) for 24 h were tested for HPV16 E6 and E7 expression by immunoblotting as described. Blots were stripped and re-probed with β-actin as loading control.</p

Effect of curcumin and AG490 on STAT3 expression and phosphorylation in cervical cancer cells.

<p>Chemical structure of curcumin (<b>A</b>) and AG490 (<b>B</b>). Curcumin and AG490 inhibit constitutive STAT3 phosphorylation in cervical cancer cells. (<b>C, E</b>) Dose-dependent inhibition of STAT3 phosphorylation by curcumin (<b>C</b>) and by AG490 (<b>E</b>). SiHa cells (1×10⁶ cells) treated with indicated concentration of curcumin and AG490 for 24 h were tested for expression of phospho-STAT3 by immunoblotting. Subsequently the blots were stripped and reprobed with STAT3 and β-actin. (<b>C, E</b>) Time kinetics of inhibition of STAT3 phosphorylation in curcumin- (<b>D</b>) and AG490- (<b>F</b>) treated cervical cancer cells. SiHa cells (1×10⁶ cells) treated with curcumin (50 µM) or AG490 (100 µM) for indicated durations were tested for expression of phospho-STAT3 by immunoblotting. Subsequently the blots were stripped and reprobed with STAT3 and β-actin. (<b>G</b>) Downregulation of STAT3 phosphorylation by curcumin and AG490 is associated with abrogation of STAT3 DNA binding activity. Nuclear proteins (10 μg) isolated from SiHa cells (1×10⁶ cells) treated with the indicated concentrations of curcumin (<i>left panel</i>), or with AG490 (<i>right panel</i>) for 24 h were incubated with specific hSIE probes, assayed for STAT3 binding by EMSA as described and intensities of STAT3-specific band quantified are indicated. NS- Non-specific bands.</p

Schematic presentation of possible mechanism of STAT3 mediated transcriptional control of HPV16 E6 and E7 oncogene expression and effect of STAT3 targeting.

<p>Activated STAT3 binds to the HPV16 LCR and control the aberrant expression of E6 and E7 which further binds to cellular p53 and pRb and leads to their degradation during natural history of HPV16 infection (<i>left panel</i>). Control of STAT3 expression by specific siRNAs or STAT3 activation by specific pharmacological agents leads to the inhibition of STAT3 binding to the HPV16 LCR which results into suppression of E6 and E7 expression. Loss of E6 and E7 causes p53 and pRb accumulation and inhibition of cell proliferation with induction of apoptosis in cervical cancer cells (<i>right panel</i>). <i>Key; Solid thick arrows indicates positive upregulation; Solid thin arrows indicates loss of expression; dashed arrows indicates inhibition. small circles and boxes inidcates decreased level; big circles and boxes indicates upregulation.</i></p

Values and sources for input parameters in the Goals Model used for modeling in Tamil Nadu, Maharashtra and Andhra Pradesh.

<p>Values and sources for input parameters in the Goals Model used for modeling in Tamil Nadu, Maharashtra and Andhra Pradesh.</p

Adult HIV prevalence curves for Andhra Pradesh, Maharashtra and Tamil Nadu generated by the Goals Model and EPP/AIM.

<p>Adult HIV prevalence curves for Andhra Pradesh, Maharashtra and Tamil Nadu generated by the Goals Model and EPP/AIM.</p

Map of Bihar showing study districts (Source: www.bihar.gov.in).

<p>Map of Bihar showing study districts (Source: <a href="http://www.bihar.gov.in/" target="_blank">www.bihar.gov.in</a>).</p

Number of Kala-azar cases captured by snowballing and house-to-house survey approach.

<p>Number of Kala-azar cases captured by snowballing and house-to-house survey approach.</p

Estimates of annual incidence of Kala-azar in the study area based on total number of cases captured during survey.

<p>Estimates of annual incidence of Kala-azar in the study area based on total number of cases captured during survey.</p