Sanjiv Kumar Yadav

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.

Automated laser scanning cytometry: A powerful tool for multi‐parameter analysis of drug‐induced apoptosis

Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi‐parameter analysis of drug‐induced tumor cell apoptosis.

A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3’ ,4’ -dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

ABSTRACT Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca2+. This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3′,4′-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production

AIMS We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. RESULTS HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. INNOVATION AND CONCLUSION These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Abstract 2605: Selective targeting of KRAS mutant cancer cells by a novel small molecule compound

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We report here the molecular mechanism(s) underlying the anti-tumor activity of the novel small molecule, C1, which was previously shown by our group to trigger autophagy-associated apoptosis via a ROS-dependent manner in a variety of human cancer cell lines and primary cells derived from patients. Our results indicate the paradoxical involvement of mutant K-RAS as a specific target of this drug and its highly selective preference for cells with a mutant KRAS phenotype; HCT116 colorectal carcinoma cells that harbor an inherent mutated KRAS allele (V13D) are significantly more sensitive to this drug than HT29 cells that express wild type RAS. Exposure of HCT116 cells to C1 resulted in activation of KRAS which in turn activated downstream crucial effector, AKT. Interestingly, activated AKT was responsible for downstream ROS production, which sensitized HCT116 cells to cell death. Corroborating the upstream involvement of KRAS in the anti-tumor activity of C1, transient gene silencing of KRAS abrogated ROS generation and death inducing activity of C1 in HCT116 cells. In contrast, activation of KRAS and AKT as well as downstream ROS production were absent in HT29 cells. To confirm the specificity of C1 for KRAS mutant cells, we evaluated the effect of the small molecule compound on RWPE-1 prostate epithelial cells transformed with mutant KRAS. Interestingly, exposure to C1 resulted in a significant reduction in colony forming ability in mutant KRAS prostate cells (RWPE-1-KRAS), compared to the control RWPE-1-Vector cells. Importantly, ROS generation was also significantly higher in RWPE-1-KRAS cells than the RWPE-1-Vector cells. To provide further evidence for the specific involvment of mutant RAS signaling in the anti-cancer activity of C1, we made use of HCT116 mutant KRAS knock-out cells (KO), which have its mutant KRAS allele deleted and carries a single wild-type KRAS allele and phenotype. Notably, C1 could not trigger mitochondrial ROS production in KO cells. Furthermore, our results show that C1 could trigger KRAS-dependent Bax translocation effectively in HCT116 cells as well as RWPE-1 KRAS cell lines. We postulate that this novel small molecule compound exploits the vulnerabilities of KRAS mutant cells by targeting directly on the active KRAS, further activating it and impacting its downstream effectors, AKT in particular, with the resultant increase in intracellular ROS and execution of Bax translocation that signal for death. Taken together, these data could have tremendous therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting cancers with mutated KRAS. Citation Format: Kartini Iskandar, Majidah Rezlan, Sanjiv K. Yadav, Shazib Pervaiz. Selective targeting of KRAS mutant cancer cells by a novel small molecule compound. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2605. doi:10.1158/1538-7445.AM2014-2605

Abstract 4611: Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In addition to being investigated as probes for diagnostic imaging, lanthanide family of rare earth compounds were shown to be tumor-selective, however the mechanism of anti-cancer activity remains unclear. We synthesized a conjugate of the lanthanide, praseodymium (141Pr), and observed remarkable anti-tumor activity in 2 murine xenograft models (colorectal and prostate cancer), with minimal off target effects. Importantly, Pr-mercaptopyridine oxide (Pr-MPO) elicited strong anti-cancer activity (IC50∼2.5uM) against a host of human cancer cell lines including cisplatin resistant, p53-/- and/or Bax-/- variants. Intrigued by this, we investigated the mechanisms underlying the anti-cancer activity of Pr-MPO. Our earlier findings indicated weak PPARγ agonist-like activity despite any structural similarity to known PPARγ agonists, therefore, we first investigated the effect of Pr-MPO on cell metabolism. An increase in glucose uptake and a significant decrease in pyruvate levels were observed. Indeed, pyruvate delayed cell death while restoring a regulated activity similar to non-treated conditions. Despite the PPARγ agonist-like activity, the anti-cancer effect of Pr-MPO was not dependent on PPARγ signaling. Furthermore, while exposure to Pr-MPO resulted in a drastic reduction in tumor colony forming ability, cell death was caspase independent and associated with a strong autophagic phenotype. Despite the induction of mToR-dependent autophagy, confirmed by western blot analysis of LC3II, p62, and puncta formation upon GFP-LC3 transfection, neither pharmacological inhibition nor the knockdown of Atg7/5 blocked Pr-MPO-induced cell death. Interestingly, Pr-MPO induced a significant increase in intracellular calcium fluorescence, measured by time resolved live imaging, however, unlike thapsigargin, the increase in calcium was not mediated by ER calcium stores. Instead, a significant increase in calcium fluorescence (puncta) was observed within minutes, a pattern distinctly different from the diffuse cytosolic increase upon depleting ER calcium. Notably, chelation of extracellular calcium abrogated the increase in intracellular calcium as well as rescued cells from Pr-MPO-induced cell death, thus suggesting the involvement of store operated calcium entry. Further analysis indicated these puncta being associated with the lysosomes. Therefore, we turned our focus on lysosomes as the “organelle prefere” in Pr-MPO induced cell death. Indeed, Pr-MPO resulted in the induction of lysosomal membrane permeability, which was associated with an overall increase in cathepsin L activity. As the increase in lysosomal calcium fluorescence is rapidly induced by Pr-MPO, a possible target could be a membrane ATPase or the rapid increase in fluorescence could be an indicator for the subsequent release of calcium. These possibilities are the current focus of investigation. Citation Format: Dan Liu, Gregory L. Bellot, Sanjiv K. Yadav, Mark Fivaz, Charnjit Kaur, Gautam Sethi, Shazib Pervaiz. Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4611. doi:10.1158/1538-7445.AM2014-4611