RESUMO
mTORC2 is aberrantly activated in cancer and therefore is considered to be an important therapeutic target. The hedgehog pathway, which is also often hyperactivated, regulates transcription of several genes associated with angiogenesis, metastasis, cellular proliferation and cancer stem cell (CSC) regeneration. However, the contribution of mTORC2 toward hedgehog pathway activity has not been explored yet. Here we have addressed the molecular cross talk between mTORC2 and hedgehog pathway activities in the context of glioblastoma multiforme, a malignant brain tumor using as a model system. We observed that higher mTORC2 activity enhanced the expression of a few hedgehog pathway molecules (Gli1, Gli2 and Ptch1) and amplified its target genes (Cyclin D1, Cyclin D2, Cyclin E, Snail, Slug and VEGF) both in mRNA and protein levels as corroborated by increased metastasis, angiogenesis, cellular proliferation and stem cell regeneration. Inhibition of mTORC2 formation decreased hedgehog pathway activity and attenuated all these above-mentioned events, suggesting their cross talk with each other. Further investigations revealed that mTORC2 inhibited ubiquitination of Gli2 by inactivating GSK3ß, and thus it promotes stability to Gli2 and its nuclear translocation. Moreover, enhanced mTORC2 activity led to the increased clonogenic properties and CD133+ cells, indicating its role in CSC regeneration. mTORC2 inhibitor directed the reduction of hedgehog pathway proteins and also reduced CSCs. Thus, our observations support a role for elevated mTORC2 activity in regulating angiogenesis, metastasis, cellular proliferation and CSC regeneration via hedgehog pathway activity. Taken together, it provides a rationale for including the mTOR2 inhibitor as part of the therapeutic regimen for CSCs.
Assuntos
Núcleo Celular/metabolismo , Proteínas Hedgehog/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteína Gli2 com Dedos de Zinco/metabolismo , Carbazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina E/genética , Ciclina E/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteína Companheira de mTOR Insensível à Rapamicina/antagonistas & inibidores , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Earlier we have established a carbazole alkaloid (mahanine) isolated from an Indian edible medicinal plant as an anticancer agent with minimal effect on normal cells. Here we report for the first time that mahanine-treated drug resistant and sensitive virulent Leishmania donovani promastigotes underwent apoptosis through phosphatidylserine externalization, DNA fragmentation and cell cycle arrest. An early induction of reactive oxygen species (ROS) suggests that the mahanine-induced apoptosis was mediated by oxidative stress. Additionally, mahanine-treated Leishmania-infected macrophages exhibited anti-amastigote activity by nitric oxide (NO)/ROS generation along with suppression of uncoupling protein 2 and Th1-biased cytokines response through modulating STAT pathway. Moreover, we have demonstrated the interaction of a few antioxidant enzymes present in parasite with mahanine through molecular modeling. Reduced genetic and protein level expression of one such enzyme namely ascorbate peroxidase was also observed in mahanine-treated promastigotes. Furthermore, oral administration of mahanine in acute murine model exhibited almost complete reduction of parasite burden, upregulation of NO/iNOS/ROS/IL-12 and T cell proliferation. Taken together, we have established a new function of mahanine as a potent antileishmanial molecule, capable of inducing ROS and exploit antioxidant enzymes in parasite along with modulation of host's immune response which could be developed as an inexpensive and nontoxic therapeutics either alone or in combination.