Activated mTOR kinase regulates mitochondrial biogenesis both at the transcriptional level, through activation of PGC-1/Yin Yang 1 (YY1) signaling leading to mitochondrial gene expression, and at the translational level, through repression of 4E-BPs that downregulate nuclear-encoded mitochondrial protein translation60. and iNOS, have been identified as potential targets of the anti-inflammatory action of cryptolepine22C24. The anticancer effects of cryptolepine have been reported as due to its direct interactions with DNA, inhibition of DNA synthesis and inhibition of topoisomerase functions25C27. However, the molecular mechanisms underlying the potential cytotoxicity against cancer cells and in particular melanoma are not known and have not been explored. Therefore, we investigated the anti-cancer potential of cryptolepine using human melanoma cells. We report that treatment of human melanoma cells with cryptolepine inhibits the growth and viability of melanoma cells in culture and in an mouse xenograft model and does so by targeting the mechanisms that regulate mitochondrial dynamics and mitochondrial biogenesis. Results Cryptolepine reduces the viability of melanoma cells but has less effect on normal human 5-hydroxymethyl tolterodine (PNU 200577) melanocytes We first determined the short-term effects of cryptolepine on the viability of various human melanoma cell lines (and the numbers of Rhodamine 123-stained cells quantified using flow cytometry. We found a significant decrease (studies are translatable to an system, we determined the effects of administration of cryptolepine in a melanoma xenograft model. The A375 cell line was chosen as a representative melanoma cell line as we had found similar effects of cryptolepine on the viability of the different melanoma cell lines (Fig.?1). The A375 melanoma cells were implanted in the flanks of athymic nude mice and cryptolepine was administered intraperitoneally (conditions and suggest that it does so by 5-hydroxymethyl tolterodine (PNU 200577) modulating 5-hydroxymethyl tolterodine (PNU 200577) cross-talk between AMPK1/2 and mTOR cross-talk. Western blot analysis revealed that administration of cryptolepine to A375 xenograft-bearing mice resulted in a decrease in the levels of phosphorylated form of Drp1 protein that is involved in maintenance of mitochondrial dynamics (Fig.?7d). Further, the levels of c-Myc, SIRT1 and PGC-1 protein were reduced in the tumor samples from mice treated with cryptolepine as compared with the tumor samples from vehicle-treated control mice (Fig.?7d). These results verified our findings and demonstrated that cryptolepine-induced effects in melanoma cells are translatable to conditions. Discussion The balance between mitochondrial energy production and physiological functions required for cell survival is regulated by mitochondrial dynamics41. Maintenance of mitochondrial mass and the numbers of mitochondria in cells is regulated by the processes of mitochondrial biogenesis, fission, fusion and mitophagy. Uncontrolled mitochondrial function and dysregulated Rabbit Polyclonal to RAB38 mitochondrial dynamics contribute to the pathogenesis of various diseases42. Thus, the targeting of mitochondrial biogenesis and mitochondrial functions has emerged as a novel preventive and therapeutic strategy for various metabolic diseases including cancer6, 43. Cryptolepine has been shown to possess anti-inflammatory activity and cytotoxic potential that is mediated by direct and indirect interactions with DNA22C27, 44, 45. In the current study, we found that cryptolepine treatment induced a highly significant decrease in melanoma cell viability and growth demonstrating that this compound possesses strong anti-melanoma activity. Furthermore, we found that cryptolepine targets mitochondrial dynamics and biogenesis in melanoma cells and that these effects were accompanied by activation of AMPK1/2-LKB1, inhibition of mTOR signaling, and a reduction in the levels of c-Myc, SIRT1 and PGC-1 protein. AMPK1/2 is recognized as a central energy-sensing protein that regulates glucose and lipid metabolism and can be activated by various stress-related factors such as ATP depletion, low glucose levels, exercise and fasting13, 46. A growing body of evidence demonstrates that loss of AMPK1/2 expression is associated with enhanced tumorigenesis whereas induction of AMPK1/2 expression is related to reduced cancer cell 5-hydroxymethyl tolterodine (PNU 200577) growth13, 14. Activation of AMPK1/2 has emerged as a novel strategy for prevention and treatment of cancer and several metabolic diseases13, 14, 47. Our data demonstrate that cryptolepine reduces ATP production in melanoma cells and enhances both the levels of AMPK1/2 protein and its phosphorylation. We also found that manifestation of LKB1, an upstream regulator of AMPK1/213, 48, was enhanced in melanoma cells after cryptolepine treatment. It has been shown that in response to energy-deprived conditions, activation of AMPK1/2 inhibits protein synthesis through inhibition of mTOR signaling35, 36. Our results are in line with these observations in that we found that cryptolepine treatment induced activation of AMPK1/2 in melanoma cells and caused inhibition of the 5-hydroxymethyl tolterodine (PNU 200577) protein synthesis machinery by reducing the phosphorylation of mTOR, p70S6K and 4E-BP1. Collectively, these results suggest that the ability of.