1-Benzyl-indole-3-carbinol is a highly potent new small molecule inhibitor of Wnt/ß-catenin signaling in melanoma cells that coordinately inhibits cell proliferation and disrupts expression of microphthalmia-associated transcription factor isoform-M.

1-Benzyl-indole-3-carbinol (1-benzyl-I3C), a synthetic analogue of the crucifer-derived natural phytochemical I3C, displayed significantly wider sensitivity and anti-proliferative potency in melanoma cells than the natural compound. Unlike I3C, which targets mainly oncogenic BRAF-expressing cells, 1-benzyl-I3C effectively inhibited proliferation of melanoma cells with a more extensive range of mutational profiles, including those expressing wild-type BRAF. In both cultured melanoma cell lines and in vivo in melanoma cell-derived tumor xenografts, 1-benzyl-I3C disrupted canonical Wnt/ß-catenin signaling that resulted in the downregulation of ß-catenin protein levels with a concomitant increase in levels of the ß-catenin destruction complex components such as glycogen synthase kinase-3ß (GSK-3ß) and Axin. Concurrent with the inhibition of Wnt/ß-catenin signaling, 1-benzyl-I3C strongly downregulated expression of the melanoma master regulator, microphthalmia-associated transcription factor isoform-M (MITF-M) by inhibiting promoter activity through the consensus lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factor (TCF) DNA-binding site. Chromatin immunoprecipitation revealed that 1-benzyl-I3C downregulated interactions of endogenous LEF-1 with the MITF-M promoter. 1-Benzyl-I3C ablated Wnt-activated LEF-1-dependent reporter gene activity in a TOP FLASH assay that was rescued by expression of a constitutively active form of the Wnt co-receptor low-density lipoprotein receptor-related protein (LRP6), indicating that 1-benzyl-I3C disrupts Wnt/ß-catenin signaling at or upstream of LRP6. In oncogenic BRAF-expressing melanoma cells, combinations of 1-benzyl-I3C and Vemurafenib, a clinically employed BRAF inhibitor, showed strong anti-proliferative effects. Taken together, our observations demonstrate that 1-benzyl-I3C represents a new and highly potent indolecarbinol-based small molecule inhibitor of Wnt/ß-catenin signaling that has intriguing translational potential, alone or in combination with other anti-cancer agents, to treat human melanoma.

Inhibition of oncogenic BRAF activity by indole-3-carbinol disrupts microphthalmia-associated transcription factor expression and arrests melanoma cell proliferation.

Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. © 2016 Wiley Periodicals, Inc.

Antioxidants and lipid peroxidation in gestational diabetes--a preliminary study.

Increased free radical activity in gestational diabetes (GDM) can lead to a host of damaging and degenerative maternal and fetal complications. Hence antioxidant levels in blood of GDM mothers and cord blood were estimated. Erythrocyte glutathione (GSH), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS), plasma vitamins C and E and serum total glutathione-S-transferase (GST), protein thiols and ceruloplasmin (Cp) were estimated spectrophotometrically in maternal blood of age matched controls and mothers with GDM and also in cord blood samples of the above. There was a significant increase in the erythrocytic GSH, serum total GST and protein thiols in GDM maternal blood when compared to controls whereas erythrocytic SOD exhibited a marked decrease in GDM cases. The changes in plasma vitamins C and E, Cp and erythrocytic TBARS in GDM were not significantly different from controls. Cord blood levels of protein thiols were also significantly increased in GDM. No significant changes were observed in the serum Cp and GST levels of the same. Hence, elevated glucose levels can induce oxidative stress in GDM mothers.

The antiproliferative response of indole-3-carbinol in human melanoma cells is triggered by an interaction with NEDD4-1 and disruption of wild-type PTEN degradation.

UNLABELLED: Human melanoma cells displaying distinct PTEN genotypes were used to assess the cellular role of this important tumor-suppressor protein in the antiproliferative response induced by the chemopreventative agent indole-3-carbinol (I3C), a natural indolecarbinol compound derived from the breakdown of glucobrassicin produced in cruciferous vegetables such as broccoli and Brussels sprouts. I3C induced a G1-phase cell-cycle arrest and apoptosis by stabilization of PTEN in human melanoma cells that express wild-type PTEN, but not in cells with mutant or null PTEN genotypes. Importantly, normal human epidermal melanocytes were unaffected by I3C treatment. In wild-type PTEN-expressing melanoma xenografts, formed in athymic mice, I3C inhibited the in vivo tumor growth rate and increased PTEN protein levels in the residual tumors. Mechanistically, I3C disrupted the ubiquitination of PTEN by NEDD4-1 (NEDD4), which prevented the proteasome-mediated degradation of PTEN without altering its transcript levels. RNAi-mediated knockdown of PTEN prevented the I3C-induced apoptotic response, whereas knockdown of NEDD4-1 mimicked the I3C apoptotic response, stabilized PTEN protein levels, and downregulated phosphorylated AKT-1 levels. Co-knockdown of PTEN and NEDD4-1 revealed that I3C-regulated apoptotic signaling through NEDD4-1 requires the presence of the wild-type PTEN protein. Finally, in silico structural modeling, in combination with isothermal titration calorimetry analysis, demonstrated that I3C directly interacts with purified NEDD4-1 protein. IMPLICATIONS: This study identifies NEDD4-1 as a new I3C target protein, and that the I3C disruption of NEDD4-1 ubiquitination activity triggers the stabilization of the wild-type PTEN tumor suppressor to induce an antiproliferative response in melanoma. Mol Cancer Res; 12(11); 1621-34. ©2014 AACR.