Jayshree L. Hirpara

Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation

Background Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions. Methodology/Principal Findings We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H2O2) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H2O2-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H2O2. Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells. Conclusions/Significance Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.

Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells

Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H2O2)-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H2O2-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H2O2 or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H2O2-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H2O2-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.

Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria

AbstractAt least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the β-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.

Phase I and biomarker study of OPB-51602, a novel signal transducer and activator of transcription (STAT) 3 inhibitor, in patients with refractory solid malignancies

BACKGROUND The aim of this study was to determine the maximum-tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of OPB-51602, an oral, direct signal transduction activator of transcription 3 (STAT3) inhibitor, in patients with refractory solid tumors. PATIENTS AND METHODS Three cohorts were studied: cohort A, a sequential dose escalation of OPB-51602 administered intermittently (days 1-14 every 21 days); cohort B, an expansion cohort evaluating the dose lower than the MTD; cohort C, evaluating continuous daily dosing. RESULTS Fifty-one patients were studied at 2, 4, and 5 mg per day dosing. The MTD was 5 mg; first-cycle dose-limiting toxicities (DLTs) were grade 3 hyponatremia in one patient, and grade 3 dehydration in another. Intermittent dosing of both 2 and 4 mg doses were tolerable, and the recommended phase II dose was 4 mg. Cohort B investigated 4 mg intermittently, whereas cohort C investigated 4 mg continuously. Common toxicities included fatigue, nausea/vomiting, diarrhea, anorexia, and early-onset peripheral neuropathy. Drug-induced pneumonitis occurred in two patients in cohort C. Continuous dosing was associated with a higher incidence of peripheral neuropathy and a lower mean relative dose intensity, compared with intermittent dosing. Steady-state pharmacokinetics was characterized by high oral clearance, mean elimination half-life ranging from 44 to 61 h, and a large terminal-phase volume of distribution. An active metabolite, OPB-51822, accumulated to a greater extent than OPB-51602. Flow cytometry of peripheral blood mononuclear cells demonstrated pSTAT3 (Tyr(705)) inhibition following exposure. Two patients achieved partial responses at 5 mg intermittently and 4 mg continuously; both had epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) with prior EGFR tyrosine kinase inhibitor exposure. CONCLUSION OPB-51602 demonstrates promising antitumor activity, particularly in NSCLC. Its long half-life and poorer tolerability of continuous dosing, compared with intermittent dosing, suggest that less frequent dosing should be explored. CLINICALTRIALSGOV IDENTIFIER NCT01184807.

The small GTPase Rac1 is a novel binding partner of Bcl-2 and stabilizes its antiapoptotic activity

The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase-fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.

LY303511 amplifies TRAIL-induced apoptosis in tumor cells by enhancing DR5 oligomerization, DISC assembly, and mitochondrial permeabilization

Certain classes of tumor cells respond favorably to TRAIL due to the presence of cell surface death receptors DR4 and DR5. Despite this preferential sensitivity, resistance to TRAIL remains a clinical problem and therefore the heightened interest in identifying compounds to revert tumor sensitivity to TRAIL. We recently demonstrated that the phosphatidylinositide-3-kinase (PI3K) inhibitor, LY294002, and its inactive analog LY303511, sensitized tumor cells to vincristine-induced apoptosis, independent of PI3K/Akt pathway. Intrigued by these findings, we investigated the effect of LY303511 on TRAIL-induced apoptosis in HeLa cells. Preincubation of cells with LY30 significantly amplified TRAIL signaling as evidenced by enhanced DNA fragmentation, caspases 2, 3, 8, and 9 activation, and reduction in the tumor colony formation. This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the egress of cytochrome c and second mitochondrial activator of caspase/direct IAP-binding protein with low PI, cleavage of X-linked inhibitor of apoptosis protein, and activation of caspase 9. We link this execution signal to the ability of LY30 to downregulate cFLIPS and oligomerize DR5, thus facilitating the signaling of the death initiating signaling complex. The subsequent exposure to TRAIL resulted in processing/activation of caspase 8 and cleavage of its substrate, the BH3 protein Bid. These data provide a novel mechanism of action of this small molecule with the potential for use in TRAIL-resistant tumors.

Do STAT3 inhibitors have potential in the future for cancer therapy

Signal transduction activator of transcription (STAT) 3 is a member of the STAT protein family which transduces intracellular and extracellular signals to the nucleus, controlling the expression of...

Cleavage of misfolded nuclear receptor corepressor confers resistance to unfolded protein response-induced apoptosis.

We have recently reported that accumulation of misfolded nuclear hormone receptor corepressor (N-CoR) as insoluble protein aggregates in acute promyelocytic leukemia (APL) cells induces endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). Although accumulation of misfolded proteins is known to trigger UPR-induced cytotoxic cell death in several neurodegenerative disorders, APL cells are notably resistant to UPR-induced apoptosis. The molecular basis for the paradoxical response of APL cells to UPR is not known. Here, we report that a glycoprotease, selectively expressed in APL cells, regulates the response of APL cells to UPR-induced apoptosis through processing of misfolded N-CoR protein. Results show that misfolded N-CoR is cleaved selectively in APL cells, and cellular extracts of APL cells and human primary APL cells contain activity that cleaves N-CoR protein. Purification and spectrometric analysis of N-CoR cleaving activity from an APL cell line reveals that it is a glycoprotein endopeptidase known as OSGEP. Furthermore, the cleavage of N-CoR in APL cells could be blocked by the broad-spectrum protease inhibitor AEBSF and by RNA interference-mediated down-regulation of OSGEP expression. AEBSF selectively inhibits growth and promotes apoptosis of APL cells possibly through a mechanism involving AEBSF-induced accumulation of insoluble N-CoR protein and by triggering ER stress. Taken together, these findings suggest that selective induction of protease activity in APL cells may represent a novel cytoprotective component of UPR, which could be exploited by tumor cells to survive the toxic insult of misfolded protein(s).

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•−). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•− blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•− production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.