DRAM1 blocking peptide

Induction of nitrosative stress has been observed in various cancer types and in tumor environment. However, it is still unclear how cancer cells combat the effect of nitrosative stress. The main targets of nitrosative stress in cells are cellular lipids, proteins and DNA. Autophagy or self-cleaning generates energy for cell survival under stress conditions. In the present study we investigated the role of autophagy under nitrosative stress in MCF7, a breast cancer cell line. Interestingly, we observed induction of autophagy associated with cell death when MCF7 cells were treated with NO donor compound DETA-NONOate for eight hours. While investigating the mode of cell death under nitrosative stress in MCF7 cells, it was found that it was neither apoptotic nor necrotic. Moreover, nitrosative stress did not alter mitochondrial membrane potential and cellular redox status in MCF7 cells. But we observed an increase in NAD(+)/NADH and a drop in NADH level in MCF7 cells following NO donor treatment. Sirtuins having NAD(+) dependent deacetylase activity, play an important role in cell survival mechanisms. So we further checked the status of SIRT1 under nitrosative stress in MCF7 cells. Surprisingly, we observed an induction of SIRT1, phospho-AMPK and p53 in MCF7 cells under nitrosative stress. Interestingly, autophagy markers were down regulated in MCF7 cells upon treatment with nicotinamide, an inhibitor of SIRT1 activity and dorsomorphin, a phospho-AMPK inhibitor when treated separately under nitrosative stress. To further confirm the role of SIRT1 in the induction of autophagy associated cell death, it was knocked down using si-RNA and nitrosative stress was applied. SIRT1 knock down led to increase in MCF7 cell viability along with down regulation of autophagic markers and phospho-AMPK as well as accumulation of acetylated p53. The increase in p53 controlled DRAM1 mRNA expression in MCF7 cells under nitrosative stress further confirmed a complex interplay between p53, SIRT1 and AMPK under nitrosative stress in MCF7 cells. Altogether our work for the first time suggests a complex inter-twined partnership between AMPK, SIRT1 and p53 in regulating autophagy in response to nitrosative stress in MCF7 cells.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. The mechanisms underlying NSCLC tumorigenesis are incompletely understood. Transfer RNA (tRNA) modification is emerging as a novel regulatory mechanism for carcinogenesis. However, the role of tRNA modification in NSCLC remains obscure. In this study, HPLC/MS assay was used to quantify tRNA modification levels in NSCLC tissues and cells. tRNA-modifying enzyme genes were identified by comparative genomics and validated by qRT-PCR analysis. The biological functions of tRNA-modifying gene in NSCLC were investigated in vitro and in vivo. The mechanisms of tRNA-modifying gene in NSCLC were explored by RNA-seq, qRT-PCR, and rescue assays. The results showed that a total of 18 types of tRNA modifications and up to seven tRNA-modifying genes were significantly downregulated in NSCLC tumor tissues compared with that in normal tissues, with the 2'-O-methyladenosine (Am) modification displaying the lowest level in tumor tissues. Loss- and gain-of-function assays revealed that the amount of Am in tRNAs was significantly associated with expression levels of FTSJ1, which was also downregulated in NSCLC tissues and cells. Upregulation of FTSJ1 inhibited proliferation, migration, and promoted apoptosis of NSCLC cells in vitro. Silencing of FTSJ1 resulted in the opposite effects. In vivo assay confirmed that overexpression of FTSJ1 significantly suppressed the growth of NSCLC cells. Mechanistically, overexpression of FTSJ1 led to a decreased expression of DRAM1. Whereas knockdown of FTSJ1 resulted in an increased expression of DRAM1. Furthermore, silencing of DRAM1 substantially augmented the antitumor effect of FTSJ1 on NSCLC cells. Our findings suggested an important mechanism of tRNA modifications in NSCLC and demonstrated novel roles of FTSJ1 as both tRNA Am modifier and tumor suppressor in NSCLC.