KINK-1, a Novel Small-Molecule Inhibitor of IKK[beta], and the Susceptibility of Melanoma Cells to Antitumoral Treatment

Background Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-[kappa]B) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-[kappa]B is regulated by inhibitory [kappa]B (I[kappa]B) proteins, which are in turn phosphorylated by the I[kappa]B kinase (IKK) complex. Methods The effect on NF-[kappa]B activity of a novel small-molecule inhibitor of the [beta] subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-[kappa]B-1) was assessed by measuring phosphorylation of the [alpha] subunit of I[kappa]B by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-[kappa]B using immunofluorescence. Regulation of NF-[kappa]B-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-[kappa]B activity in melanoma cells. It reduced the expression of NF-[kappa]B-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-[kappa]B activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 1