Pan-caspase Inhibitor zVAD-fmk Research Articles

SAT069 Targeting Human β-Cell G Protein-coupled Receptors To Alter Cytokine-mediated Caspase Activation

Abstract Disclosure: K. Rodrigues dos Santos: None. N. Mukherjee: None. L.F. Barella: None. F. Armoo: None. D.L. Eizirik: None. A.T. Templin: None. M.A. Kalwat: None. In type 1 diabetes (T1D) autoimmune destruction of pancreatic islet β-cells causes a lack of insulin, leading to hyperglycemia. The development of therapies that can prevent β-cell death is critical. β-cell proliferation and survival pathways are affected by signaling through G protein-coupled receptors (GPCRs). Using high-depth RNA-seq, we found altered expression of 123 different GPCRs in human islets exposed to cytokines (IL1β 50U/ml + IFNγ 1000U/ml, 48 h) to model the T1D milieu. We hypothesized these candidates may be exploited for β-cell protection. Among the candidates, adenosine receptors (ADORA1 and ADORA2A), calcium-sensing receptor (CASR), and metabotropic glutamate receptor 4 (GRM4) stood out. Cytokines decreased the expression of ADORA1 and CASR, but increased ADORA2A and GRM4. To gauge the impact of agonizing/antagonizing these receptors, we pre-treated human EndoC-β H1 β-cells or islets with receptor ligands and co-treated with cytokines (IL1β 50U/ml + IFNγ or IL1β + 1000U/ml IFNγ + 1000U/ml TNFɑ, 48 h) and measured glucose-stimulated insulin secretion, cell viability, and gene expression. Each cytokine combination induced target gene expression (e.g. CXCL10), but only the combination of IL1β + IFNγ + TNFɑ reduced glucose-stimulated insulin secretion. Preliminary experiments indicated that adenosine receptor ligands had little impact in our assays. However, ligands for CASR and GRM4 exhibited significant suppression of cytokine-induced caspase 3/7 activation in EndoC-βH1 cells. Interestingly, suppressed caspase activation did not result in observable impacts on live/dead cell ratios or live-cell analysis using Incucyte in EndoC-βH1 cells. Consistently, we also observed cytokine-mediated cell death in the presence of the pan-caspase inhibitor ZVAD-fmk. We conclude that a variety of β-cell GPCR-mediated signaling pathways may converge to prevent cytokine-induced caspase activation, but additional measures are needed to enable protection from eventual cell death. Our results and this line of investigation is significant because understanding mechanisms of GPCR-cytokine crosstalk in β-cells will inform strategies to protect islets in other models of T1D. Funding: JDRF 1-INO-2022-1113-A-N and Diabetes Research Connection #33. Presentation: Saturday, June 17, 2023

The Histone Deacetylase Inhibitor ITF2357 (Givinostat) Targets Oncogenic BRAF in Melanoma Cells and Promotes a Switch from Pro-Survival Autophagy to Apoptosis.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death.

Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.

Abstract PO-110: Plant isolate dibenzyl trisulfide induces caspase-independent death in triple negative breast cancer cells derived from patients of West African descent

Abstract Patients with triple negative breast cancer (TNBC) possess tumors that lack estrogen receptor, progesterone receptor, and human epidermal growth receptor expression. While these patients traditionally receive chemotherapy to combat this aggressive breast cancer subtype, others use natural remedies. Dibenzyl trisulfide (DTS) is derived from Petiveria alliacea, a perennial shrub that grows in tropical regions of the world. Many TNBC patients residing in the tropics are of West African descent. Therefore, we evaluated the anticancer actions of DTS in TNBC cells, including those derived from patients of West African descent. We found that DTS inhibited TNBC cell viability, migration and proliferation in a dose-dependent manner. Interestingly, DTS blocked the propensity of pro-carcinogen benzo-A-pyrene to induce proliferation of immortalized breast epithelial cells. Moreover, we found that DTS induced early apoptosis in TNBC cells, which was only partially attenuated following pretreatment with pan-caspase inhibitor zVAD-fmk. Though DTS induced pro-apoptotic gene and protein expression along with PARP cleavage, it failed to produce appreciable caspase 3 cleavage and promote significant apoptotic body formation. This suggests that this plant isolate induces caspase-independent and non-apoptotic death of TNBC cells. Furthermore, DTS promoted lysosomal membrane destabilization and cathepsin B release in TNBC cells. Taken together, DTS exhibits promising chemotherapeutic and chemopreventive ability by inducing non-apoptotic TNBC cell death and thwarting TNBC progression, supporting its evaluation in clinical trials as an agent to combat TNBC among patients of West African descent. Citation Format: Jonathan Wooten, Shaniece Wooten, Cristina Araújo, Joyce Aja, Nicole Mavingire, Rupika Delgoda, Eileen Brantley. Plant isolate dibenzyl trisulfide induces caspase-independent death in triple negative breast cancer cells derived from patients of West African descent [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-110.

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

The Performance of HepG2 and HepaRG Systems through the Glass of Acetaminophen-Induced Toxicity.

Investigation of drug-induced liver injuries requires appropriate in vivo and in vitro toxicological model systems. In our study, an attempt was made to compare the hepatocarcinoma HepG2 and the stem cell-derived HepaRG cell lines both in two- and three-dimensional culture conditions to find the most suitable model. Comparison of the liver-specific characteristics of these models was performed via the extent and mechanism of acetaminophen (APAP)-induced hepatotoxicity. Investigating the detailed mechanism of APAP-induced hepatotoxicity, different specific cell death inhibitors were used: the pan-caspase inhibitor zVAD-fmk and dabrafenib significantly protected both cell lines from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and primary hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression.

Discovery of a Necroptosis Inhibitor Improving Dopaminergic Neuronal Loss after MPTP Exposure in Mice.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit—Oxa12—that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.

Abstract 6553: Plant power: Plant isolate dibenzyl trisulfide induces death of triple-negative breast cancer cells derived from an African American patient

Abstract Triple negative breast cancer (TNBC), characterized by tumors that lack estrogen receptor, progesterone receptor, and human epidermal growth receptor expression, represents one of the most aggressive subtypes of breast cancer. TNBC carries a poor prognosis due, in part, to a lack of clinically available targeted therapy. Novel therapeutic strategies to combat TNBC primarily involve the use of synthetic small molecules. However, the World Health Organization reports that there is increased use of natural remedies and traditional medicine to treat diseases. Our lab has investigated the chemotherapeutic and chemopreventive effects of a variety of plant isolates. Dibenzyl trisulfide (DTS) is derived from Petiveria alliacea, a perennial shrub that grows in tropical regions of the world. Previous investigations surrounding DTS are limited to anticancer actions in European American breast cancer cells. The purpose of this study was to evaluate the anticancer actions of DTS in CRL-2335 cells which are derived from an African American patient with TNBC. We found that DTS inhibited TNBC cell migration and proliferation using wound healing and colony forming assays. Furthermore, we found DTS induced apoptotic body formation and nuclear fragmentation using relief contrast and fluorescence microscopy respectively. Moreover, using the Annexin V/PI assay we found that DTS induced early apoptosis, which was partially attenuated in cells pretreated with pan-caspase inhibitor zVAD-fmk. Finally, quantitative polymerase chain reaction analyses revealed that DTS induced the expression of pro-apoptotic genes BAK1, LTA and GADD45A. Our data suggest that DTS effectively induces caspase-independent death of TNBC cells derived from an African American patient and represents a promising agent to treat more refractory forms of breast cancer. Citation Format: Jonathan Wooten, Cristina Araújo, Shaniece Wauchope, Joyce Aja, Nicole Mavingire, Rupika Delgoda, Eileen Brantley. Plant power: Plant isolate dibenzyl trisulfide induces death of triple-negative breast cancer cells derived from an African American patient [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6553.

YB-1 Mediates TNF-Induced Pro-Survival Signaling by Regulating NF-κB Activation.

Cell fate decisions regulating survival and death are essential for maintaining tissue homeostasis; dysregulation thereof can lead to tumor development. In some cases, survival and death are triggered by the same receptor, e.g., tumor necrosis factor (TNF)-receptor 1 (TNFR1). We identified a prominent role for the cold shock Y-box binding protein-1 (YB-1) in the TNF-induced activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65. In the absence of YB-1, the expression of TNF receptor-associated factor 2 (TRAF2), a central component of the TNF receptor signaling complex required for NF-κB activation, is significantly reduced. Therefore, we hypothesized that the loss of YB-1 results in a destabilization of TRAF2. Consistent with this hypothesis, we observed that YB-1-deficient cells were more prone to TNF-induced apoptotic cell death. We observed enhanced effector caspase-3 activation and could successfully rescue the cells using the pan-caspase inhibitor zVAD-fmk, but not necrostatin-1. Taken together, our results indicate that YB-1 plays a central role in promoting cell survival through NF-κB activation and identifies a novel mechanism by which enhanced YB-1 expression may contribute to tumor development.

Homeostatic Rho GTPase activation governs thymopoiesis

Abstract The thymus is extremely sensitive to damage, resulting in attenuated T cell reconstitution and failure to elicit appropriate responses to infection, cancer and immunization. Although the thymus has a remarkable regenerative capacity, the mechanisms underlying this endogenous regeneration remain poorly understood. Previously we have identified two critical secreted factors that drive thymic regeneration, including BMP4 from endothelial cells (ECs) and IL-22 from dendritic cells (ILCs), which can be used as therapeutic regenerative strategies. Additionally, we have shown that steady-state CD4+CD8+ thymocytes (DPs) suppress levels of IL-23 (Science 336:91), a key regulator of IL-22; and as developing thymocytes undergo high levels of apoptosis, we hypothesize that apoptotic DPs mediate suppression of regenerative factors under homeostatic conditions, and damage-induced DP depletion removes this suppression, triggering thymic recovery. Here, we demonstrate a DP-specific reduction in cl-caspase 3 levels after acute injury in mice (total body irradiation, 550 cGy), and reduced Bmp4 expression in ECs co-cultured with apoptotic thymocytes, an effect rescued by apoptosis inhibition, using the pan-caspase inhibitor zVAD-FMK. We identified that apoptotic thymocytes mediate their suppression via TAM receptors in ECs and DCs, which subsequently activate Rho GTPases. Importantly, we show that inhibition of Rac1 activation increases Bmp4 and Il23 expression in ECs and DCs, respectively, and in vivo inhibition of Rac1 with EHT1864 enhances thymus cellularity in models of acute injury and age. These data describe a novel regulatory pathway that can be therapeutically targeted to boost T cell reconstitution after damage.

DMAMCL exerts antitumor effects on hepatocellular carcinoma both in vitro and in vivo.

Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis. Dimethylaminomicheliolide (DMAMCL) is a novel antitumor agent that has been tested in phase I clinical trials; however, little is known regarding its effects in HCC. In this study, we found that DMAMCL reduces the viability of HCC cells in a dose- and time-dependent manner. In addition, DMAMCL causes cell cycle arrest at the G2/M phase and inhibits cell invasion and epithelial-mesenchymal transition (EMT). DMAMCL treatment also induces apoptosis via the intrinsic apoptotic pathway in HCC cells, which could be blocked by the pan-caspase inhibitor zVAD-fmk and silencing of Bax/Bak or overexpression of Bcl-2. Furthermore, DMAMCL treatment inactivates the PI3K/Akt pathway and leads to the generation of reactive oxygen species (ROS), which regulate apoptosis and inhibition of PI3K/Akt induced by DMAMCL. In vivo, DMAMCL inhibits tumor growth in mice bearing xenograft HCC tumors without noticeable toxicity. In summary, DMAMCL exerts antitumor effects both in vitro and in vivo and therefore may be applied as a potential therapeutic agent for HCC.

Heterocyclic organobismuth(III) compound induces nonapoptotic cell death via lipid peroxidation.

Heterocyclic organobismuth compounds, such as N-tert-butyl-bi-chlorodibenzo[c,f][1,5]azabismocine (compound 1) and bi-chlorodibenzo[c,f ][1,5]thiabismocine (compound 3), exert potent antiproliferative activities in vitro in human cancer cell lines. We showed that compound 3 induced both apoptotic and nonapoptotic cell death via reactive oxygen species production and mitotic arrest in a dose-dependent manner. The mechanisms underlying the dose-dependent effect of these organobismuth compounds were not clear. In the present study, we examined the dose-dependent mechanism underlying cell death induced by compound 1 in a human pancreatic cancer cell line, SUIT-2, and a human colorectal cancer cell line, DLD-1. Compound 1 inhibited cell growth in a dose-dependent manner and induced cell death. Treatment with the pan-caspase inhibitor zVAD-fmk reduced cell death induced by compound 1, whereas the inhibitory effect of zVAD-fmk was limited. Moreover, compound 1 significantly induced lipid peroxidation with concomitant induction of caspase-independent cell death. Our results suggested that eight-membered ring organobismuth compounds induce nonapoptotic cell death via lipid peroxidation.

Homeostatic Regulation of Apoptosis Governs Thymus Regeneration

Although the thymus has a remarkable capacity for repair following acute injury, such as that caused by the conditioning required for successful hematopoietic cell transplant (HCT), the mechanisms underlying this endogenous regeneration remain poorly understood. Delayed T cell reconstitution occurs following thymus insult and can exceed more than a year post-transplant due to a delay in full recovery of thymic output, function and T cell repertoire. Therefore, strategies to enhance T cell reconstitution post-transplant represents a rational approach to significantly improve the overall outcome of allo-HCT. We propose that enhancing thymic function will boost T cell reconstitution and substantially increase immune responses following allo-HCT. Our recent studies have identified two critical pathways that govern thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors (RAFs) after damage remain unclear. Given that our prior work revealed that the presence of DP thymocytes suppresses the production of RAFs like IL-23, a key downstream mediator of IL-22; and the high basal rate of thymocyte apoptosis, as apoptotic thymocytes form the bulk of developing T cells, we hypothesized that apoptotic DP thymocytes were mediating this suppression of RAFs under homeostatic conditions. Upon injury, loss of DP thymocytes leads to reduced apoptotic signaling and reduced suppression of RAFs, triggering thymic recovery (Fig 1A). Consistent with this hypothesis, our preliminary data shows a significantly reduced number of apoptotic thymocytes after total body irradiation (TBI, 550 cGy), as measured by cleaved caspase 3 levels (Fig 1B). Additionally, co-culture of apoptotic thymocytes results in reduced Bmp4 expression in ECs, which is rescued by inhibition of thymocyte apoptosis using the pan-caspase inhibitor zVAD-FMK (Fig 1C). One way in which apoptotic thymocytes could induce this suppression of RAFs is via TAM receptor activation, which is supported by our data demonstrating increased Bmp4 expression in ECs treated with a pan-TAM receptor antagonist and subsequently co-cultured with apoptotic thymocytes (Fig 1D). Interestingly, TAM receptors can activate Rac1, a Rho GTPases involved in actin cytoskeletal rearrangement; converging neatly on our previous data showing that inhibition of Rac1 with small molecule inhibitors led to robust induction of Bmp4 and Il23 expression. Therefore, we propose that in steady-state, apoptotic thymocytes activate TAM receptors on ECs and DCs and induce intracellular activation of Rac1, which ultimately suppresses the production of BMP4 and IL-23; but after damage, when the number of apoptotic thymocytes drops precipitously, this suppression is abrogated, allowing for thymic regeneration (Fig 1E). Importantly, we demonstrate here that this pathway can be therapeutically targeted, as inhibition of Rac1 in vivo with EHT1864 enhances thymus cellularity in models of acute injury (Fig. 1F), and age (Fig. 1G). As post-transplant T cell deficiency is associated with an increased risk of infections, relapse of malignancy, and the development of secondary malignancies, identifying molecular targets to enhance thymic recovery will aid in the development of therapeutics with imminent clinical need. These findings not only reveal a novel molecular mechanism governing tissue regeneration, but also offer a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by allo-HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

Abstract 701: Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer

Abstract Platinum compounds are an inherent part of chemotherapy for patients with high grade serous ovarian cancer (HGSC). Platinum based therapeutics, such as cisPt, induce DNA-damage by directly binding to nuclear DNA. Interestingly, cisPt also critically affects mitochondrial DNA. Elucidating the role of mitochondria in cisplatin induced cell death of HGSC cells likely identifies new efficient therapy approaches to overcome resistance. Flow cytometric analysis of cisPt sensitive and resistant HGSC cell lines from the NCI60 panel revealed higher mitochondrial mass and higher levels of mitochondrial ROS (mtROS) in cells that are sensitive to cisPt-induced cell death. In clonal sub-lines derived from OVCAR3 the mitochondrial mass correlates with basal oxygen consumption rate and survival after cisPt exposure. The correlation of mitochondrial mass and sensitivity to cisPt-induced apoptosis is corroborated by data from the human protein atlas (www.proteinatlas.org) that indicates high expression of the mitochondrial transcription factor TFAM and the mitochondrial inner membrane protein TIMM23 as favorable prognostic factors for the survival of ovarian cancer patients. Together, this shows the importance of mitochondrial mass in cellular response to cisPt treatment. Furthermore, exposure to cisPt enhances TFAM expression and increases mitochondrial mass as well as mitochondrial ROS. Incubation with cisPt in the presence of the ROS scavenger glutathione (GSH) or pan-caspase inhibitor zVAD-fmk reduces cisPt induced apoptosis while mitochondrial mass is not affected. However, the oxygen consumption rate in cells exposed to cisPt and GSH shows that increased ROS levels induce mitochondrial dysfunction. In line with a sensitivity-determining role of mitochondrial mass, knock-down of key-regulators of mitochondrial biogenesis, i.e. PGC-1α or TFAM, blocks mitochondrial ROS and protects cells from cisPt-induced apoptosis. Mitochondrial ROS is also reduced in an autoregulatory feedback loop by so-called uncoupling proteins (UCPs). Inhibition of UCP2 by Genipin results in increased induction of mitochondrial ROS by cisPt and consequently results in enhanced apoptosis. A comparable sensitization to cisPt-induced apoptosis and ROS production is evident in cells incubated with the iron chelator VLX600.Mitochondria are a critical element in cell response to cisPt since mitochondrial mass correlates with induction of ROS and apoptosis. We show the relevance of cellular mitochondrial content by manipulation of mitochondrial mass and function. We propose the relative mitochondrial content as a biomarker that indicates the response of ovarian cancer cells to cisPt-induced apoptosis. Furthermore, our data provides evidence that increasing mitochondrial mass or induction of mtROS enhances sensitivity to cisPt-induced apoptosis and therefore is a reliable strategy to overcome resistance of ovarian cancer. Citation Format: Markus Kleih, Kathrin Böpple, Andrea Gaißler, Meng Dong, Walter E. Aulitzky, Frank Essmann. Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 701.

Repurposing the Electron Transfer Reactant Phenazine Methosulfate (PMS) for the Apoptotic Elimination of Malignant Melanoma Cells through Induction of Lethal Oxidative and Mitochondriotoxic Stress.

Redox-directed pharmacophores have shown potential for the apoptotic elimination of cancer cells through chemotherapeutic induction of oxidative stress. Phenazine methosulfate (PMS), a N-alkylphenazinium cation-based redox cycler, is used widely as an electron transfer reactant coupling NAD(P)H generation to the reduction of tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a superoxide generating chemotherapeutic for the pro-oxidant induction of cancer cell apoptosis. In a panel of malignant human melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1–10 μM, 24 h) displayed pronounced apoptogenicity as detected by annexin V-ITC/propidium iodide flow cytometry, and PMS-induced cell death was suppressed by antioxidant (NAC) or pan-caspase inhibitor (zVAD-fmk) cotreatment. Gene expression array analysis in A375 melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced superoxide production was substantiated by MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating NADPH-driven (SOD-suppressible) superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375 melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of malignant melanoma cells.

SAT-354 Characterization of Cell Death Induced by SOAT1 Inhibition in Adrenocortical Carcinoma Cells

Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). We have found that mitotane inhibits Sterol-O-Acyl transferase 1 (SOAT1) which leads to depletion of cholesterol esters and increase of free cholesterol in the ACC cell line NCI-H295. We demonstrated activation of endoplasmic reticulum response pathway that resulted in decreased cell viability. Aim: To characterize the mechanisms underlying cell death resulting from SOAT1 inhibition with inhibitors mitotane, ATR101, AZD3988 and Sandoz58-035. Methods: SOAT1 inhibition was quantified in vitro, ER-stress marker expression by qPCR, cell viability by cell titer glo assay, lipid composition by GC-MS, and lipid peroxidation with BODIPY 581/591 C11. Cell death in NCIH295R cells induced by mitotane was determined by mitotane treatment alone and in combination with either the pan caspase inhibitor zvad-fmk, necroptose inhibitor necrostatin-1, ferroptosis inhibitor liproxstatin or ferroptosis activators erastin and RSL-3. Results: Mitotane and ATR101 but not AZD3988 and Sandoz58-035 induced lipid peroxidation in NCI-H295R cells in a concentration dependent manner. ATR101, AZD3988 and Sandoz58-035 were inhibitors of SOAT1 with IC50 of 3 nM, 13 nM and 0.9 nM. Correspondingly, expression of ER-stress marker CHOP and splicing of X-box protein 1 mRNA was activated by mitotane, ATR101 and poorly by AZD3988. Mitotane and ATR101 but not AZD3988 and Sandoz58-035 treatment induced the accumulation of free cholesterol in NCI-H295 cells after 6h. Cell viability was impaired with mitotane, ATR101 and AZD3988 but not Sandoz58-035. None of the inhibitors could reverse the effects of mitotane while synergistic effects were observed for RSL3. Erastin was shown to reverse the effects of mitotane treatment. Conclusion: Although SOAT1 inhibition was confirmed for all compounds, downstream effects on ER-stress markers and cell viability exhibit marked differences. Hence it is likely that targets different from SOAT1 are relevant for in vitro cytotoxic activity of mitotane and ATR101. Caspases do not seem to be predominantly involved in mitotane-induced cell death. Although we observed a synergistic effect of mitotane with RSL3, ferroptosis inhibitors were not able to block mitotane-induced cell death. The precise mechanism of cell death and immunogenic cell death pathways require further investigation.

Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

BackgroundGrowth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs.MethodsPrimary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry.ResultsExpression of the GADD45γ gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part.ConclusionsTo our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

Bufalin induces apoptosis via mitochondrial ROS-mediated caspase-3 activation in HCT-116 and SW620 human colon cancer cells

Objective: Bufalin has been reported to kill various types of cancer including human colorectal cancer. Our previous study demonstrated that bufalin induced cell death via autophagy in HT-29 and Caco-2 colon cancer cells, but the action of bufalin remains unclear. This study was conducted to investigate the role of bufalin in other colon cancer HCT-116 and SW620 cells as well as its potential mechanism.Methods: The effect of bufalin in HCT-116 and SW620 colon cancer cells was detected by assessing cell viability and cell death. Apoptotic cells were analyzed by Western blot and trypan blue dye exclusion assay. Mitochondrial ROS production was analyzed by flow cytometry after DCFDA and DHR-123 staining. The potential mechanism was investigated via pharmacological inhibitors.Results: Bufalin had high potency against HCT-116 and SW620 cells with IC50 values of 12.823 ± 1.792 nM and 26.303 ± 2.498 nM in HCT-116 and SW620 cells, respectively. Bufalin decreased cell viability, increased cell death as well as caspase-3 downstream target (cleaved PARP) accumulation, and these actions were significantly blocked by pan-caspase inhibitor zVAD-FMK. Mechanistically, ROS production, but neither the NAD(P)H oxidase, AMPK, ERK nor p38, is responsible for bufalin-induced apoptotic cell death. Moreover, bufalin-induced ROS generation is derived from mitochondria.Conclusion: Bufalin significantly induces apoptosis in HCT-116 and SW620 colon cancer cells via mitochondrial ROS-mediated caspase-3 activation. We believe that our novel findings will greatly alter our current understanding on the anti-cancer mechanism of bufalin in colon cancer cells and will pave the way for further exploiting the clinical application.

Caspase-2 is a negative regulator of necroptosis.

The role of caspase-2 in cell death regulation remains largely unknown. In this study we have analyzed the involvement of caspase-2 in RIPK1-regulated necrosis (necroptosis) in human ovarian carcinoma cells. We show that these cells undergo necroptosis upon treatment with the DNA damaging drug cisplatin in combination with the pan-caspase inhibitor zVAD-fmk. Downregulation of caspase-2 leads to an increase of necroptosis in CAOV-4 cells. Interestingly, an association of caspase-2 to the necrosome complex was not detected. Importantly, downregulation of caspase-2 with shRNA or CRISPR/Cas9 system led to an enhanced phosphorylation of RIPK1 and MLKL. Taken together, our data strongly indicate that caspase-2 negatively regulates necroptotic cell death, which might play an important role in further therapeutic applications.