Tonic suppression of PCAT29 by the IL-6 signaling pathway in prostate cancer: Reversal by resveratrol.

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. A better understanding of the molecular basis of prostate cancer proliferation and metastasis should enable development of more effective treatments. In this study we focused on the lncRNA, prostate cancer associated transcript 29 (PCAT29), a putative tumor suppressive gene. Our data show that the expression of PCAT29 was reduced in prostate cancer tumors compared to paired perinormal prostate tissues. We also observed substantially lower levels of PCAT29 in DU145 and LNCaP cells compared to normal prostate (RWPE-1) cells. IL-6, a cytokine which is elevated in prostate tumors, reduced the expression of PCAT29 in both DU145 and LNCaP cells by activating signal transducer and activator of transcription 3 (STAT3). One downstream target of STAT3 is microRNA (miR)-21, inhibition of which enhanced basal PCAT29 expression. In addition, we show that resveratrol is a potent stimulator of PCAT29 expression under basal condition and reversed the down regulation of this lncRNA by IL-6. Furthermore, we show that knock down of PCAT29 expression by siRNA in DU145 and LNCaP cells increased cell viability while increasing PCAT29 expression with resveratrol decreased cell viability. Immunohistochemistry studies showed increased levels of STAT3 and IL-6, but low levels of programmed cell death protein 4 (PDCD4), in prostate tumor epithelial cells compared to adjacent perinormal prostate epithelial cells. These data show that the IL-6/STAT3/miR-21 pathway mediates tonic suppression of PCAT29 expression and function. Inhibition of this signaling pathway by resveratrol induces PCAT29 expression and tumor suppressor function

Image_1_Targeting CXCL1 chemokine signaling for treating cisplatin ototoxicity.jpg

Cisplatin is chemotherapy used for solid tumor treatment like lung, bladder, head and neck, ovarian and testicular cancers. However, cisplatin-induced ototoxicity limits the utility of this agent in cancer patients, especially when dose escalations are needed. Ototoxicity is associated with cochlear cell death through DNA damage, the generation of reactive oxygen species (ROS) and the consequent activation of caspase, glutamate excitotoxicity, inflammation, apoptosis and/or necrosis. Previous studies have demonstrated a role of CXC chemokines in cisplatin ototoxicity. In this study, we investigated the role of CXCL1, a cytokine which increased in the serum and cochlea by 24 h following cisplatin administration. Adult male Wistar rats treated with cisplatin demonstrated significant hearing loss, assessed by auditory brainstem responses (ABRs), hair cell loss and loss of ribbon synapse. Immunohistochemical studies evaluated the levels of CXCL1 along with increased presence of CD68 and CD45-positive immune cells in cochlea. Increases in CXCL1 was time-dependent in the spiral ganglion neurons and organ of Corti and was associated with progressive increases in CD45, CD68 and IBA1-positive immune cells. Trans-tympanic administration of SB225002, a chemical inhibitor of CXCR2 (receptor target for CXCL1) reduced immune cell migration, protected against cisplatin-induced hearing loss and preserved hair cell integrity. We show that SB225002 reduced the expression of CXCL1, NOX3, iNOS, TNF-α, IL-6 and COX-2. Similarly, knockdown of CXCR2 by trans-tympanic administration of CXCR2 siRNA protected against hearing loss and loss of outer hair cells and reduced ribbon synapses. In addition, SB225002 reduced the expression of inflammatory mediators induced by cisplatin. These results implicate the CXCL1 chemokine as an early player in cisplatin ototoxicity, possibly by initiating the immune cascade, and indicate that CXCR2 is a relevant target for treating cisplatin ototoxicity.</p

IL-6 induces STAT3 phosphorylation in prostate cancer cells.

<p><b>(A)</b> RWPE-1 cells were transfected with either scramble or siSTAT3 (10 mM) for 24 h then subjected to IL-6 treatment (10 ng/ml) for 30 min, then cells lysate were used for Western blotting analysis. IL-6 showed no significant effect on STAT3 phosphorylation on this cell. <b>(B, C)</b> LNCaP and DU145 cells were transfected with a scrambled siRNA sequence or siSTAT3 (10 nM) for 24 h then treated with vehicle or IL-6 (10 ng/ml) for 5 min (DU145) or 30 min (LNCaP). Cells were then lysed and used for Western blotting analysis. Bar graph represents mean ± SEM of 3 independent experiments. Asterisks (*) (**) indicate statistically significantly difference (p < 0.05) from scramble or IL-6, respectively.</p

Levels of pSTAT3, PDCD4 and IL-6 in human prostate specimens.

<p><b>(A, B)</b> Immunohistochemistry studies were performed to determine the levels of pSTAT3 in formalin-fixed paraffin embedded prostate tumor specimens, as compared to adjacent normal prostate specimen. Diaminobenzidine tetrahydrochloride (DAB) staining revealed high expression of pSTAT3 <b>(B)</b> as dark-brown labeling in epithelial cells of tumor samples (marked by red arrows) while low expression was observed in paired perinormal prostate tissues <b>(A)</b>. In contrast, high expression of PDCD4 was observed in perinormal prostate tissues (as evidenced by dark-brown labeling, indicated by yellow arrows) <b>(C)</b>, as compared to prostate tumors <b>(D)</b>. Immunolabling of prostate tumor revealed high expression of IL-6 <b>(F)</b>, compared to perinormal prostate specimens. Representative images show immunohistochemical studies performed in four different specimens obtained from three different patients. Pictures magnification is 400x. Scale bar is 20 μm.</p

Reduced expression of <i>PCAT29</i> expression in prostate cancer cells and tumor tissues.

<p><b>(A)</b> Relative mRNA levels of <i>PCAT29</i> in normal human prostate epithelial cells (RWPE-1) and prostate cancer cells (LNCaP and DU145). The levels of <i>PCAT29</i> were significantly reduced in prostate cancers cells compared to RWPE-1 cells. (n≥3) <b>(B)</b> Significant reductions in <i>PCAT29</i> levels in normal prostate tissues compared to prostate cancer. Data are presented as the mean ± SEM of 4 prostate samples. Asterisks (*) indicate statistically significantly difference (p < 0.05) from RWPE-1 and from normal prostate tissue, respectively. (**) indicate statistically significantly difference (p < 0.05) from LNCaP cells.</p

<i>PCAT29</i> tonically suppressed prostate cancer cell proliferation.

<p><b>(A, B)</b> DU145 and LNCaP cells were transfected with scrambled siRNA or siRNA against <i>PCAT29</i> (si<i>PCAT29</i>, 30 nM) for 24 h, followed by treatment with IL6 (10 ng/ml) for 24 h. Cells were then used to determine <i>PCAT29</i> levels. <b>(C, D)</b> DU145 and LNCaP cells were transfected with scrambled siRNA or si<i>PCAT29</i> (30 ng/ml) for 24 h, followed by treatment with IL-6 for 24 h and cells proliferation was determined by MTS assay. IL-6 treatment significantly increased cells proliferation, which was mimicked by si<i>PCAT29</i>. Knockdown of <i>PCAT29</i> did not increase cell proliferation above that observed with IL-6 alone in DU145 but not in LNCaP cells. Resveratrol treatment significantly reduced cell proliferation, even in presence of IL-6. Data are presented as the mean ± SEM of at least 3 independent experiments. Asterisks (*) and (**) indicate statistically significantly difference (p < 0.05) from scramble and from scramble + IL-6, respectively.</p

Tonic suppression of <i>PCAT29</i> by the IL-6 signaling pathway in prostate cancer: Reversal by resveratrol

<div><p>Prostate cancer (PCa) is the second leading cause of cancer deaths in men. A better understanding of the molecular basis of prostate cancer proliferation and metastasis should enable development of more effective treatments. In this study we focused on the lncRNA, prostate cancer associated transcript 29 (<i>PCAT29</i>), a putative tumor suppressive gene. Our data show that the expression of <i>PCAT29</i> was reduced in prostate cancer tumors compared to paired perinormal prostate tissues. We also observed substantially lower levels of <i>PCAT29</i> in DU145 and LNCaP cells compared to normal prostate (RWPE-1) cells. IL-6, a cytokine which is elevated in prostate tumors, reduced the expression of <i>PCAT29</i> in both DU145 and LNCaP cells by activating signal transducer and activator of transcription 3 (STAT3). One downstream target of STAT3 is microRNA <i>(miR)-21</i>, inhibition of which enhanced basal <i>PCAT29</i> expression. In addition, we show that resveratrol is a potent stimulator of <i>PCAT29</i> expression under basal condition and reversed the down regulation of this lncRNA by IL-6. Furthermore, we show that knock down of <i>PCAT29</i> expression by siRNA in DU145 and LNCaP cells increased cell viability while increasing <i>PCAT29</i> expression with resveratrol decreased cell viability. Immunohistochemistry studies showed increased levels of STAT3 and IL-6, but low levels of programmed cell death protein 4 (PDCD4), in prostate tumor epithelial cells compared to adjacent perinormal prostate epithelial cells. These data show that the IL-6/STAT3/<i>miR-21</i> pathway mediates tonic suppression of <i>PCAT29</i> expression and function. Inhibition of this signaling pathway by resveratrol induces <i>PCAT29</i> expression and tumor suppressor function.</p></div

Resveratrol blocks IL-6 suppression of PDCD4 and <i>PCAT29</i> and its induction of <i>miR-21</i>.

<p><b>(A, B)</b> Western blotting analysis of DU145 and LNCaP cells showed that IL-6 reduced the levels of PDCD4. Resveratrol (25 μM) treatment for 24 h blocked this reduction and increased the levels of PDCD4 above that of control. This effect of resveratrol persisted even in the presence of IL-6. <b>(C, D)</b> Resveratrol also increased <i>PCAT29</i> by greater than 2-fold and this effect was partly reduced by concurrent IL-6 treatment. <b>(E, F)</b> Resveratrol restored IL-6 stimulated expression of <i>miR-21</i> in LNCaP and DU145 cells. Data are presented as the mean ± SEM of at least 3 independent experiments. Asterisks (*), (**), and (***) indicate statistically significantly difference (p < 0.05) from vehicle, from vehicle+IL-6 and from resveratrol, respectively.</p

Image_4_Targeting CXCL1 chemokine signaling for treating cisplatin ototoxicity.jpg

Cisplatin is chemotherapy used for solid tumor treatment like lung, bladder, head and neck, ovarian and testicular cancers. However, cisplatin-induced ototoxicity limits the utility of this agent in cancer patients, especially when dose escalations are needed. Ototoxicity is associated with cochlear cell death through DNA damage, the generation of reactive oxygen species (ROS) and the consequent activation of caspase, glutamate excitotoxicity, inflammation, apoptosis and/or necrosis. Previous studies have demonstrated a role of CXC chemokines in cisplatin ototoxicity. In this study, we investigated the role of CXCL1, a cytokine which increased in the serum and cochlea by 24 h following cisplatin administration. Adult male Wistar rats treated with cisplatin demonstrated significant hearing loss, assessed by auditory brainstem responses (ABRs), hair cell loss and loss of ribbon synapse. Immunohistochemical studies evaluated the levels of CXCL1 along with increased presence of CD68 and CD45-positive immune cells in cochlea. Increases in CXCL1 was time-dependent in the spiral ganglion neurons and organ of Corti and was associated with progressive increases in CD45, CD68 and IBA1-positive immune cells. Trans-tympanic administration of SB225002, a chemical inhibitor of CXCR2 (receptor target for CXCL1) reduced immune cell migration, protected against cisplatin-induced hearing loss and preserved hair cell integrity. We show that SB225002 reduced the expression of CXCL1, NOX3, iNOS, TNF-α, IL-6 and COX-2. Similarly, knockdown of CXCR2 by trans-tympanic administration of CXCR2 siRNA protected against hearing loss and loss of outer hair cells and reduced ribbon synapses. In addition, SB225002 reduced the expression of inflammatory mediators induced by cisplatin. These results implicate the CXCL1 chemokine as an early player in cisplatin ototoxicity, possibly by initiating the immune cascade, and indicate that CXCR2 is a relevant target for treating cisplatin ototoxicity.</p

IL-6 reduced the expression of <i>PCAT29</i> in prostate cancer but not normal cells.

<p>DU145 and LNCaP cells were treated with IL-6 (10 ng/ml) for 24 h, following which the expression of <i>PCAT29</i> was determined by real-time RT-PCR. Both DU145 and LNCaP cells showed decreased <i>PCAT29</i> expression after IL-6 treatment. There was no difference in <i>PCAT29</i> expression following IL-6 treatment in RWPE-1 cells. Data are presented as the mean ± SEM of at least 3 independent samples. Asterisks (*) indicates statistically significantly difference (p < 0.05) from vehicle groups.</p