Molecular signaling cascade of indubin-3-monoxime in apotosis, paraptosis and inflammation

Abstract

Chapter2. The indirubin derivative, indirubin 3′-monoxime (I3M) has already exhibited its anticancer effects by leading to cell death in different cancer cell lines and may be a novel therapeutic approach for treatment-resistant cancers. Since many cancers resistant to apoptosis, the present study reports for the first time that I3M-induced cell death by targeting paraptosis as a nonapoptotic programmed cell death mechanism. I3M significantly inhibited the cell viability in different types of cancer cell lines. However, H_(2)O_(2) -induced apoptosis was attenuated by pretreatment of z-VAD-fmk, a pan-caspase inhibitor; however, this inhibition was not observed for I3M-treated MDA-MB-231 cells. The expression of LC3-I, LC3-II, p62, and ATG-7 was not upregulated upon I3M treatment and also I3M did not induced Beclin 1 protein expression level by indicating autophagy process may not involve to cell death in MDA-MB-231 cells. As a key feature of paraptosis, I3M enhanced formation of vacuoles which are derived from mitochondria and/or the ER. Nevertheless, exposure to I3M remarkably increased ER stress protein markers such as ATF4 and p-eIF2α by activating mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, I3M treatment significantly enhanced CHOP protein and m-RNA expression level, indicating that one of the major roles of CHOP is to increase I3M-mediated paraptosis. In concentration dependent treatment of I3M subsequently enhanced accumulation of poly-ubiquitinated proteins confirming I3M-induced paraptosis occurred via proteasome dysfunction. More importantly, we have shown that involvement of intra-mitochondrial Ca^(2+) in I3M-induced paraptosis in MDA-MB-231 cells. Our results demonstrate that mitochondrial Ca^(2+) accumulation possesses induction of ROS generation thus induced paraptosis followed by I3M treatment. Flow cytometry analysis showed that pretreatment with 4 μM ruthenium red remarkably decreased I3M-induced mitochondrial Rhod-2 staining intensity suggesting that Ca^(2+) accumulation into mitochondria mediate via mitochondrial channel uniporters. Collectively, our results show that accumulation of Ca^(2+) into mitochondria via mitochondrial channel uniporters through ER stress which regulate by proteasome dysfunction mainly involved in I3M-induced paraptosis in MDA-MB-231 breast cancer cells. Chapter3. Indirubin-3′-monoxime (I3M) is a derivative of indirubin which is the active component of Danggui-Long-Hui-Wan, a traditional Chinese recipe used exhibits promising anticancer effects in wide range of cancers. I3M exhibits anticancer properties by modulating cell cycle arrest, apoptosis, cell invasion and metastasis, lack of understanding of their mode of action. Therefore, in this study, we present the I3M as a promising anticancer agent based on the induction of the cell death via apoptosis and paraptosis in Hep3B hepatocarcinoma cells. Also, we investigated the role of p21 and ROS regulate NFκB signaling pathway in I3M-midiated cell death in Hep3B cells. I3M treatment significantly reduced viability of Hep3B cells and gradually upregulated Bax, and Bid protein expression level by indicating I3M-induced apoptosis in Hep3B cells. Nevertheless, treatment of I3M-increased numerous small ER and mitochondria derived fluorescent vacuoles and enhanced the Rhod-2 staining intensity suggested that I3M-induced cell death in Hep3B cells partially enhanced via paraptosis. We observed that I3M-enhanced the expression of p53 and p21 protein and mRNA level in Hep3B cells. The cells transfection with sip53 markedly decreased I3M-induced p21 fluorescence intensity, demonstrate that I3M mediate p21 expression regulate by p53 in Hep3B cells. Further, we investigated that I3M-induced expression of p21 mediate the ROS accumulation in Hep3B cells. Treatment with 15μM I3M significantly induced NFκB luciferase activity, whereas treatment of NAC downregulated I3M-induced NFκB luciferase reporter activity in Hep3B cells. Therefore, ROS play an important role in activation of NFκB thereby reduced apoptosis in Hep3B cells. Nevertheless, our results demonstrate that I3M mediate p53 regulate both apoptosis and paraptosis processes in Hep3B cells. Taken together, our findings illustrate the anticancer activity of I3M basis of its ability to simultaneously induce apoptosis and paraptosis in Hep3B hepatoma cancer cells, proving the potential of I3M in developing as an anticancer drug in near future. Chapter4. Indirubin is the active ingredient of the traditional Chinese medicine Dang Gui Long Hui Wan, a mixture of plants, acts a potent inducer of apoptosis in a variety of cell types. In the present study, we have shown the anticancer effect of indirubin-3'-monoxime (I3M), a derivative of indirubin, by targeting nitric oxide (NO). Our data showed that I3M treatment triggers the apoptotic signaling pathways in prostate cancer cells. Considering the NO-targeted apoptosis, transcriptional and translational regulation of NO regulate by various factors such as nuclear factor-κB (NF-κB), reactive oxygen species (ROS), Nuclear factor erythroid 2-related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress etc: through alterations in iNOS activity. I3M induces NF-κB activity by enhancing the nuclear translocation of p65/p50 and degradation of IκBα, and consequently, induces the expression of iNOS in LNCaP cells. Except NF-κB, Nrf2 also involve in regulation of NO-mediated apoptosis by I3M. Treatment of I3M induced ROS generation and ROS act as key regulators of Nrf2 activity in the I3M-mediated apoptosis. Nevertheless, Nrf2 is an important factor that regulate ROS level thereby regulate the NO expression. On the other hand, NO importantly involve in nuclear translocation of Nrf2. Our data showed that I3M induces NF-κB activity in LNCaP cells via the ROS and consequently, regulates the expression of NO production by regulating iNOS expression. Moreover, through NF-κB and NO production potently regulate ROS level by I3M treatment in the LNCaP prostate cancer cells. The concentration dependent manner treatment of I3M remarkably induced ER stress which potently regulate by ROS in LNCaP cells. The current study indicated that the ER stress, NFκB and ROS are important factors that regulate NO expression by I3M. Further, the induction of NO regulates apoptosis via intrinsic and extrinsic pathways in LNCaP prostate cancer cells. Hence, NO-mediated cell death could be explored as a crucial mode for apoptosis process; I3M may offer a 'NO targeting' approach as a potential chemotherapeutic agent. Chapter5. Indirubin-3′-monoxime (I3M), is a synthetic derivative of indirubin, was originally accepted as potent anticancer agent. Recent reports have suggested I3M exhibits anticancer properties by modulating cell cycle arrest, apoptosis, cell invasion and metastasis, very lack of data about the mode of action. Despite, in the current study, we have shown that I3M suppresses cellular invasiveness of LNCaP prostate carcinoma cells and to further investigate the underlying mechanisms of the I3M-inhibited invasiveness of cancer cells via down regulating Matrix Metalloproteinase-9 (MMP-9) activity. I3M does not affect the viability of LNCaP prostate cancer cells upto 5μM of concentration. According to the western blot and RT-PCR analysis, concentration dependent manner treatment of I3M reduced PMA-stimulated protein and mRNA expression level of MMP-9 in LNCaP and DU145 cells. Further, Matrigel invasion assay showed that I3M substantially reduced the PMA-induced cell invasion. Nevertheless, I3M significantly enhanced the protein and m-RNA expression level of HO-1 in LNCaP cells. Accordance to the induction of HO-1 expression, I3M resulted in remarkably induction of Nrf2 expression in LNCaP cells at 24 h. Moreover, I3M treatment notably enhanced PI3K and Akt phosphorylation, suggesting that the I3M-induced Nrf2 expression associated with the PI3K/Akt signaling pathway. Treatment with I3M reduced PMA-induced AP-1 activity by providing evidence that AP-1 may critically require to induce the MMP-9 transcription in prostate cancer cells. We also investigated that I3M-decreased c-Jun and c-Fos activation by blocking their respective phosphorylation. Considering above facts our results indicated that I3M attenuates PMA-induced expression of MMP-9 at mRNA and protein level by suppressing AP-1 activity via Nrf2 signaling pathway in LNCaP prostate carcinoma cells. Chapter6. We investigated the molecular mechanism that Indirubin 3-monoxime (I3M) induce apoptosis via CHOP, DR5 upregulation in p53^(+/+)HCT-116 cells sensitize to tumor necrosis factor-related apoptosis inducing ligand (TRAIL). We have shown that treatment of I3M induce apoptosis in HCT-116 cells depends on the p53 status. I3M significantly induced Ras levels in p53^(-/-) HCT-116 cells whereas reduced the expression of Ras in p53^(+/+) HCT-116 cells. These results suggested that the effect of I3M on regulation of Ras expression in colon cancer cells independent on the status of p53. Further, I3M remarkably enhanced m-RNA and protein level of DR5. We found that p53 controls the sensitivity to TRAIL-induced apoptosis through enhancing the expression of DR5 in p53^(+/+) HCT-116 cells. Our results showed that I3M-induced the expression of CHOP, with optimum induction occurring at around 24 h. The p53^(+/+) HCT-116 cells transfection with CHOP siRNA significantly abrogated the up-regulation of DR5 protein and m-RNA level by confirming that I3M-induced up-regulation of DR5 is mediated via the induction of CHOP. The H_(2)DCFDA and HE-based fluorescence analysis showed that concentration dependent manner treatment of I3M increased ROS generation by increasing intracellular superoxide anion and hydrogen peroxide levels respectively. Moreover, fluorometric data indicated that silencing of p53 had more dramatic effect on I3M-induced ROS production. Pretreatment of NAC remarkably blocked I3M-induced ROS production. Nevertheless, I3M treatment induced the TRAIL expression in time dependent manner in p53^(+/+)HCT-116 cells. Treatment of TRAIL synergistically enhanced I3M-induced DR5 expression thereby induced the TRAIL-induced apoptosis. Concentration dependent manner addition of a DR5 specific blocking antibody significantly blocked I3M/TRAIL-induced apoptosis. Taken together, our data have shown that I3M potently enhances TRAIL-induced apoptosis by upregulating DR5 expression via activating p53. We suggest that relationship between p53 and oxidative stress indirectly regulate I3M/TRAIL induced apoptosis in p53^(+/+) HCT-116 cells. Chapter7. Indirubin is the active ingredient of the traditional Chinese medicine Dang Gui Long Hui Wan, a mixture of plants, was reported used as an anti-leukemic, anti-inflammatory, and detoxification treatment. Nevertheless, the anti-inflammatory properties of Indirubin-3-monoxime (I3M) occurred via inflammasomes components against LPS- and ATP-stimulated responses have not yet been studied. In this study, we elucidated whether I3M has the ability to attenuate the expression of pro-inflammatory cytokine IL-1β by suppression of inflammasomes activity in BV2 microglial cells. Moreover, we have shown the relation between reactive oxygen species (ROS) and autophagy process in regulation of inflammasomes activation. I3M has the ability to attenuate the expression of IL-1β and caspase-1 in LPS- and ATP-stimulated BV2 microglial cells. Pretreatment of caspase-1 inhibitor significantly decreased the expression levels of LPS- and ATP-induced caspase-1 and IL-1β genes suggests IL-1β expression is associated with activation of caspase-1. Furthermore, we found that I3M suppresses LPS- and ATP-induced NLRP1 and NLRP3 inflammasomes expression in BV2 microglial cells. The protein expression levels of Beclin-1 and Atg7 was attenuated by I3M treatment where as an autophagy inhibitor, 3-Methyladenine (3-MA) suppressed the LPS- and ATP-induced Beclin-1, and Atg7 protein level. Fluorometric analysis showed that approximately 30% of the relative fluorescence intensity of DCFDA was increased in response to LPS and ATP. The LPS- and ATP-enhanced Beclin-1 and Atg7 protein notably decreased with the treatment of NAC and GSH, therefore these results indicate that LPS- and ATP-enhanced autophagy process regulate via ROS production in BV2 microglial cells. Taken together our results demonstrated that the I3M is a potent inhibitor of LPS-and ATP-induced caspase-1 activation and IL-1β secretion by suppression of NLRP1, NLRP3 inflammasomes, ROS production and autophagy process. Considering above facts, I3M may be useful as an anticancer agent to treat various inflammatory diseases.