Silymarin prevents endothelial dysfunction by upregulating Erk-5 in oxidized LDL exposed endothelial cells.

Extracellular signal-regulated kinase (Erk)-5 is a key mediator of endothelial cell homeostasis, and its inhibition causes loss of critical endothelial markers leading to endothelial dysfunction (ED). Circulating oxidized low-density lipoprotein (oxLDL) has been identified as an underlying cause of ED and atherosclerosis in metabolic disorders. Silymarin (Sym), a flavonolignan, possesses various pharmacological activities however its preventive mechanism in ED warrants further investigation. Here, we have examined the effects of Sym in regulating the expression of Erk-5 and ameliorating ED using in vitro and in vivo models. Primary human umbilical vein endothelial cells (pHUVECs) viability was measured by MTT assay; mRNA and protein expression by RT-qPCR and Western blotting; tube-formation assay was performed to examine endothelialness. In in-vivo experiments, normal chow-fed mice (control) or high-fat diet (HFD)-fed mice were administered Sym or Erk-5 inhibitor (BIX02189) and body weight, blood glucose, plasma-LDL, oxLDL levels, and expression of EC markers in the aorta were examined. Sym (5 µg/ml) maintained the viability and tube-formation ability of oxLDL exposed pHUVECs. Sym increased the expression of Erk-5, vWF, and eNOS and decreased ICAM-1 at transcription and translation levels in oxLDL-exposed pHUVECs. In HFD-fed mice, Sym reduced the body weight, blood glucose, LDL-cholesterol, and oxLDL levels, and increased the levels of vWF and eNOS along with Erk-5 and decreased the level of ICAM-1 in the aorta. These data suggest that Sym could be a potent anti-atherosclerotic agent that could elevate Erk-5 level in the ECs and prevent ED caused by oxidized LDL during HFD-induced obesity in mice.

EGFR-mediated Rad51 expression potentiates intrinsic resistance in prostate cancer via EMT and DNA repair pathways.

AIM: To study the role of EGFR signaling in regulation of intrinsic resistance in prostate cancer. MATERIALS AND METHODS: Radioresistant prostate carcinoma DU145 and PC-3 cells were used to study the effect of shRNA-mediated knockdown of EGFR on intrinsic radioresistance mechanisms. Semi-quantitative PCR, western blotting, growth kinetics, colony formation, transwell migration, invasion and trypan blue assays along with inhibitors erlotinib, NU7441, B02, PD98059 and LY294002 were used. KEY FINDINGS: EGFR knock-down induced morphological alterations along with reduction in clonogenic potential and cell proliferation in DU145 cells. Migratory potential of prostate cancer cells were reduced concomitant with upregulation of epithelial marker, E-cadherin and decreased expression of mesenchymal markers, vimentin and snail. Further, EGFR knock-down decreased the expression of Rad51 and DNA-PK at mRNA as well as protein levels. Likewise, erlotinib, an EGFR inhibitor, and NU7441, a DNA-PK inhibitor increased the expression of E-cadherin and decreased the level of vimentin. Both these inhibitors also decreased the levels of DNA damage regulatory protein Rad51. Further, Rad51 inhibitor, B02, inhibited the clonogenic potential, cell migration and reduced the expression of vimentin, Ku70 and Ku80, and also, B02 radiosensitized DU145 cells. EGFR-regulated expression of Rad51 was found to be mediated via PI3K/Akt and Erk1/2 pathways. SIGNIFICANCE: EGFR was found to regulate DNA damage repair, survival and EMT responses in prostate cancer cells through transcriptional regulation of Rad51. A novel role of EGFR-Erk1/2/Akt-Rad51 axis through modulation of EMT and DNA repair pathways in prostate cancer resistance mechanisms is suggested.