Inhibition of STAT3

(as an important factor in the formation of skin lesions) has the potential to be one of the pathogenic mechanisms underlying the dermatological side effects induced by treatment with molecular target drugs. In the present study, we investigated the effects of STAT3 and related mechanisms on everolimus-mediated cell growth inhibition in human epidermal keratinocyte cell lines. Our findings suggest that STAT3 activity in keratinocytes may be a biomarker of everolimus-induced dermatological events. Materials and methods Chemicals Everolimus (Figure 1), a derivative of sirolimus and an mTOR inhibitor, was purchased from Sigma-Aldrich Chemical, Co. (St Louis, MO, USA). Stattic, a small-molecule inhibitor of STAT3 activation [16], was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). STA-21, a STAT3 inhibitor [17], was purchased from Santa Cruz Biotechnology (Santa EPZ015666 supplier SB525334 Cruz, CA, USA). Z3, an inhibitor of the autophosphorylation of Janus kinase 2 (JAK2) [18], was obtained from Calbiochem (Darmstadt, Germany). SB203580,

a specific blocker of p38 mitogen-activated protein kinase (MAPK) activity, and SP600125, a selective and NVP-HSP990 in vitro reversible inhibitor of the c-Jun N-terminal kinase 1 (JNK1), JNK2, and JNK3, were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). U0126, a selective inhibitor of mitogen-induced extracellular kinase 1 (MEK1) and MEK2, was purchase from Cell Signaling Technology, Inc. (Boston, MA, USA). Figure 1 Chemical structure of everolimus. Antibodies Rabbit anti-phosphorylated (anti-phospho)-STAT3 at tyrosine 705 (Tyr705) and serine 727 (Ser727), mouse anti-STAT3 antibodies, rabbit anti-phospho-extracellular signal-regulated kinase (Erk) 1/2, rabbit anti-Erk 1/2 antibodies, rabbit anti-phospho-p38 MAPK, rabbit anti-p38 antibodies, anti-phospho-S6 kinase

(Thr389) and anti-p70 S6 kinase antibodies were purchased from Cell Signaling Technology. Mouse anti-phospho-JNK and rabbit anti-JNK antibodies, as well as anti-mouse HRP-conjugated IgG, Idoxuridine anti-rabbit HRP-conjugated IgG, and anti-rabbit FITC-conjugate IgG, were purchased from Santa Cruz Biotechnology. A rabbit anti-β-actin antibody was obtained from Sigma-Aldrich. Cells and cell culture HaCaT cells, the human immortalized keratinocyte cell lines, were kindly provided by Professor Norbert Fusenig (German Cancer Research Centre, Heidelberg, Germany) [19]. HepG2 cells, the human hepatocarcinoma cell lines, were purchased from JCRB (Osaka, Japan). HaCaT and HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (lot. No. 9866 J; MP Biomedicals, Solon, OH, USA), 100 units/mL of penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Caki-1 cells, the human renal cell carcinoma cell lines, were purchased from JCRB.