Klotho has dual protective effects on cisplatin-induced acute kidney injury.

Klotho protects the kidney from ischemia-reperfusion injury, but its effect on nephrotoxins is unknown. Here we determined whether Klotho protects the kidney from cisplatin toxicity. Cisplatin increased plasma creatinine and induced tubular injury, which were exaggerated in Klotho haplosufficient (Kl/+) and ameliorated in transgenic Klotho overexpressing (Tg-Kl) mice. Neutrophil gelatinase-associated lipocalin and active caspase-3 protein and the number of apoptotic cells in the kidney were higher in Kl/+ and lower in Tg-Kl compared with wild-type mice. Klotho suppressed basolateral uptake of cisplatin by the normal rat kidney cell line (NRK), an effect similar to cimetidine, a known inhibitor of organic cation transport (OCT). A decrease in cell surface and total OCT2 protein and OCT activity by Klotho was mimicked by ß-glucuronidase. The Klotho effect was attenuated by ß-glucuronidase inhibition. On the other hand, OCT2 mRNA was reduced by Klotho but not by ß-glucuronidase. Moreover, cimetidine inhibited OCT activity but not OCT2 expression. Unlike cimetidine, Klotho reduced cisplatin-induced apoptosis from either the basolateral or apical side and even when added after NRK cells were already loaded with cisplatin. Thus, Klotho protects the kidney against cisplatin nephrotoxicity by reduction of basolateral uptake of cisplatin by OCT2 and a direct anti-apoptotic effect independent of cisplatin uptake. Klotho may be a useful agent to prevent and treat cisplatin-induced nephrotoxicity.

The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection.

Although the role of the erythropoietin (EPO) receptor (EpoR) in erythropoiesis has been known for decades, its role in nonhematopoietic tissues is still not well defined. Klotho has been shown and EPo has been suggested to protect against acute ischemia-reperfusion injury in the kidney. Here we found in rat kidney and in a rat renal tubular epithelial cell line (NRK cells) EpoR transcript and antigen, and EpoR activity signified as EPo-induced phosphorylation of Jak2, ErK, Akt, and Stat5 indicating the presence of functional EpoR. Transgenic overexpression of Klotho or addition of exogenous recombinant Klotho increased kidney EpoR protein and transcript. In NRK cells, Klotho increased EpoR protein, enhanced EPo-triggered phosphorylation of Jak2 and Stat5, the nuclear translocation of phospho-Stat5, and protected NRK cells from hydrogen peroxide cytotoxicity. Knockdown of endogenous EpoR rendered NRK cells more vulnerable, and overexpression of EpoR more resistant to peroxide-induced cytotoxicity, indicating that EpoR mitigates oxidative damage. Knockdown of EpoR by siRNA abolished Epo-induced Jak2, and Stat5 phosphorylation, and blunted the protective effect of Klotho against peroxide-induced cytotoxicity. Thus in the kidney, EpoR and its activity are downstream effectors of Klotho enabling it to function as a cytoprotective protein against oxidative injury.

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells.

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.

Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line.

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-alpha led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-alpha-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.

Conjugated linoleic acid inhibits DNA synthesis and induces apoptosis in TSU-Pr1 human bladder cancer cells.

BACKGROUND: Conjugated linoleic acid (CLA) has strong chemoprotective properties in experimental animal models. The insulin-like growth factor (IGF) system has been implicated as a risk factor for the development of bladder cancer. The present study examined CLA regulation of TSU-Pr1 bladder cancer cell proliferation and apoptosis and the influence of CLA on IGF-I receptor (IGF-IR) signaling. MATERIALS AND METHODS: TSU-Pr1 cells were cultured in serum-free medium with 0, 2, 5, or 10 microM CLA and/or 10 nM IGF-I. [3H]Thymidne incorporation, DNA laddering, FACS analysis, immunoprecipitation and Western blotting were performed. RESULTS: CLA decreased DNA synthesis and induced apoptosis in TSU-Pr1 cells dose-dependently. Exogenous IGF-I alone increased viable cell numbers but did not counteract growth inhibition induced by CLA. CLA decreased IGF-IR and insulin receptor substrate (IRS)-1 protein levels. In addition, CLA decreased IGF-I-induced phosphorylation of IGF-IR and IRS-1, recruitment of the p85 subunit of phosphoinositide 3-kinase to IRS-1 and phosphorylation of Akt and extracellular signal-regulated kinase-1/2. CONCLUSION: These results suggest that CLA inhibits cell proliferation and stimulates apoptosis of TSU-Pr1 cells via its inhibition of the IGF-IR signaling pathway.