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  • br Materials and methods br Results br Discussion


    Materials and methods
    Discussion Although autophagy is known to be implicated in the therapeutic effect of DNA alkylating agents such as MNNG [28], [29], [30], the molecular mechanisms underlying autophagy induced by MNNG remain largely elusive. In this study, we present evidence showing that PARP-1 activation following MNNG-induced DNA damage leads to pro-survival autophagy via the AMPK-mTOR signaling pathway. Therefore, PARP-1 appears to have dual roles in determining the cell fate in response to MNNG (summarized in Fig. 5G): (i) PARP-1 activation is the cause of non-apoptotic cell death via ATP depletion depletion [4], [31]; and (ii) PARP-1 activation is able to elicit a self-protective mechanism by induction of autophagy. Our findings are indeed generally consistent with a very recent report on MNNG-induced necrotic cell death in human embryonic kidney HEK293 via PARP1 activation, ATP depletion, AMPK activation and mTOR suppression [32], although the role of autophagy was less studied. One of the key findings of this study is the critical role of AMPK in MNNG-induced autophagy and cell death, downstream of PARP-1 activation and ATP depletion. AMPK is a well-conserved heterotrimeric kinase complex composed of a catalytic (α) subunit and two regulatory (β and γ) subunits. Serving as a cellular fuel gauge, AMPK is activated under stress conditions, particularly those that decrease ATP levels while increasing the AMP:ATP ratio, such as hypoxia, ischeamia and pitavastatin starvation [33]. At present, AMPK has been increasingly appreciated as one of the key tumor suppressors via suppression of mTOR and activation of autophagy [24], [25]. AMPK serves as an important kinase that negatively regulates mTOR activity, through the following two pathways: (i) activated AMPK phosphorylates and stimulates the GAP activity of TSC1/TSC2 complex, causing conversion of GTPase Rheb to its inactive GDP from, and subsequently leads to the downregulation of mTOR activity [33]; and (ii) activated AMPK suppresses mTOR function by direct phosphorylation of raptor, an essential component of the mTOR complex I (mTORC1), on two well-conserved serine residues [34]. On the other hand, there is strong evidence setting AMPK as a key upstream regulator of autophagy. First, AMPK activation may lead to autophagy via suppression of mTORC1 as pitavastatin discussed above. Second, recent studies have set AMPK as the direct upstream activator of autophagy via its direct effect on ULK1, the ATG1 homolog in mammalians that plays a key role in autophagy initiation stage [35], [36]. Consistently, data from our study demonstrate that activation of AMPK leads to suppression of mTORC1 and induction of autophagy in cells exposing to MNNG. In fact, the effect of MNNG is found be similar to that of hydrogen peroxide which is known to elicit a pro-survival autophagy via activation of PARP-1-AMPK and suppression of mTOR in both DKO MEFs and human cancer cells [37]. Therefore, our findings may represent a general mode of reaction in cancer cells in response to DNA damage. After establishing the signaling pathway in MNNG-mediated autophagy, we then studied the function role of autophagy in MNNG-mediated necrotic cell death. Data from this study clearly suggest that autophagy is a cell survival mechanism in MNNG-induced cell death, based on the observations that suppression of autophagy by knockdown of ATG7 or blockage of lysosomal function by CQ sensitizes MNNG-induced cell death. Our data are found to be consistent with the earlier report that alkylating agent induces the pro-survival autophagy [13]. The pro-survival function of autophagy was further supported by the findings that induction of autophagy by rapamycin was able to offer significant protection against MNNG-mediated cell death. At present, there is growing understanding that autophagy is a protective mechanism under stress conditions, especially against necrotic cell death, with evidence from a series of in vivo studies in which autophagy is found to inhibit necrotic cell death of cancer cells under metabolic stress [38], [39], [40]. Therefore, targeting autophagy or its upstream signaling pathways (PARP-1-AMPK-mTOR) should be considered in developing more effective cancer therapeutic approaches.