Intrinsic Mitochondrial Apoptotic Pathway Research Articles

Effect of Valproic Acid on the Class I Histone Deacetylase 1, 2 and 3, Tumor Suppressor Genes p21WAF1/CIP1 and p53, and Intrinsic Mitochondrial Apoptotic Pathway, Pro- (Bax, Bak, and Bim) and anti- (Bcl-2, Bcl-xL, and Mcl-1) Apoptotic Genes Expression, Cell Viability, and Apoptosis Induction in Hepatocellular Carcinoma HepG2 Cell Line.

Hepatocellular carcinoma (HCC), Primary liver cancer, is the fifth most common cancer in men. Histone deacetylation causes chromatin condensation resulting in gene silencing and tumorigenesis. These enzymes have become a novel target for the treatment of cancer. Histone deacetylase inhibitors (HDACIs) can reactivate tumor suppressor genes (TSGs) by inhibition of histone deacetylases (HDACs) activity leads to apoptosis induction in cancer cells. Further, these compounds can induce apoptosis through the intrinsic/mitochondrial pathway. Previously, we reported the effect of valproic acid (VPA) and trichostatin A (TSA) on TSGs p21WAF1/CIP1 (p21), p27Kip1 (p27), and p57Kip2 (P57) and also HDAC1 in colon cancer. The present study was designed to investigate the effect of VPA on the class I histone deacetylase (HDAC) 1, 2 and 3, TSGs p21and p53, and intrinsic mitochondrial pathway, pro- (Bax, Bak, and Bim) and anti- (Bcl-2, Bcl-xL, and Mcl-1) apoptotic genes, viability, and apoptosis in HCC HepG2 cell line. The HepG2 cells were cultured and treated with VPA. To determine viability, apoptosis, and the relative expression level of the mentioned genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. VPA downregulated class I histone deacetylase (HDAC) 1, 2, and 3, Bcl-2, Bcl-xL, and Mcl-1 and upregulated p21, p53, Bax, Bak, and Bim resulting in apoptosis induction. VPA can induce apoptosis via activation of the intrinsic mitochondrial apoptotic pathway and also epigenetic reactivation of p21 and p53 through inhibition of class I HDAC 1, 2 and 3, activity.<br />.

Protective Effect of γ-mangostin Isolated from the Peel of Garcinia mangostana against Glutamate-Induced Cytotoxicity in HT22 Hippocampal Neuronal Cells.

The aim of the present study was to examine the protective effect of γ-mangostin, a component of the mangosteen shell, against oxidative damage to nerve cells induced by excessive glutamate, a known excitatory neurotransmitter. To investigate the effect of γ-mangostin on apoptosis, 5 mM of glutamate was used to induce apoptotic cell death in mouse hippocampal HT22 cells. In this study, γ-mangostin was found to exert a stronger protection than N-acetyl cysteine against glutamate-induced cell damage. γ-Mangostin showed prevented glutamate-induced apoptosis in HT22 cells by reducing the production of reactive oxygen species and stimulating the expression of heme oxygenase-1 protein. In addition, glutamate significantly induced the accumulation of intracellular calcium ions, whereas treatment with γ-mangostin markedly reduced it. Hoechst 33342 staining showed an improvement in glutamate-induced nuclear condensation following γ-mangostin treatment. Furthermore, the number of annexin V-positive cells was significantly reduced following treatment with γ-mangostin. Western blot analysis showed the inhibition of glutamate-induced mitogen-activated protein kinase phosphorylation by γ-mangostin. γ-mangostin also inhibited the regulation of the intrinsic mitochondrial apoptotic pathway. Thus, the results of this study suggest that γ-mangostin is an active ingredient of mangosteen and exerts neuroprotective activities in HT22 cells.

Deoxyelephantopin, a novel naturally occurring phytochemical impairs growth, induces G2/M arrest, ROS-mediated apoptosis and modulates lncRNA expression against uterine leiomyoma

Deoxyelephantopin (DOE), a phytochemical, extracted and purified from Elephantopus scaber, has been shown to exhibit antitumor activities. Objective of the present study was to investigate anti-tumor and apoptosis-inducing properties of DOE against uterine leiomyoma (UL) and to explore their molecular mechanisms. Primary cell cultures from fresh UL tissue were established and maintained up to 12 passages. The cells exhibited continuous proliferation with 24 -h doubling time until 12 passages and was then subjected to molecular characterization. The growth inhibitory effect of DOE on UL cells was confirmed by colony formation, cellular senescence, AO/PI and DAPI staining. Fluorescent-activated cell sorting (FACS) assay for apoptosis and cell cycle arrest analysis revealed that DOE significantly (p < 0.05) inhibited the UL cell proliferation via cell cycle arrest at G2/M phase and induced apoptosis via ROS production by lowering mitochondrial membrane potential. Subsequently, the DOE induced ROS was markedly attenuated by co-treatment of N-Acetyl-Cysteine (NAC). Our quantitative RT-PCR and western blot results showed up-regulation of Bax, Caspase-3 and down-regulation of Bcl2, P53, αSMA, COL4A2, VEGF, PCNA, Cyclin B1 and oncogenic lncRNAs (H19, HOTAIR, BANCR and ROR) in DOE treated UL cells which further strengthen our findings. In conclusion, DOE inhibits growth of UL cells via cell cycle arrest at G2/M phase, induces ROS-dependent caspase-3-mediated mitochondrial intrinsic apoptotic pathway and down-regulation of oncogenic lncRNA in UL cells. Our findings suggest that DOE deserves for further systematic investigation in the uterine leiomyoma animal model as a novel apoptosis inducer for potential applications in the prevention or treatment of uterine leiomyoma.

Novel 3,4-diarylpyrazole as prospective anti-cancerous agents

Cancer is a leading cause of death globally. Despite therapeutic advancements the mortality rate of cancer is continuously increasing. Thus, it is important to identify and design potential therapeutic agents which can specifically bind with most common targets of cancer and inhibit tumor progression. The present work discloses the potential therapeutic application of the novel 3,4-diaryl 1H-pyrazoles as prospective anti-cancerous agent. The in silico molecular docking studies performed with 3,4-disubstituted pyrazoles as ligand with targets including DNA, BCL-2 and F1-ATP Synthase revealed strong binding affinity with DNA (-7.5 kcal/mol), BCL-2 (-8.1 kcal/mol) and F1-ATP Synthase (-7.2 kcal/mol). Furthermore, the in silico finding was validated with the in vitro cytotoxicity assay with human breast cancer cell line (MDA-MB-231). MDA-MB-231 cells treated with 3,4-diarylpyrazole resulted in an increase in annexin-V positive cells, production of reactive oxygen species (ROS), dissipation of the mitochondrial membrane potential and activation of caspase-3. Taken together, this study demonstrate that a novel synthesized 3,4-diarylpyrazoles, showed strong binding affinity against DNA, anti-proliferative activity and executed apoptosis through ROS-dependent caspase-3-mediated mitochondrial intrinsic apoptotic pathway against MDA-MB-231 cells. These findings increase our understanding of the molecular mechanism (s) by which 3,4-diarylpyrazoles can exert their anticancer activity and may contribute towards development of novel therapeutic agent against breast cancer.

Anticancer potentiality and mode of action of low-carbohydrate proteins and peptides from mushrooms.

Severe side effects of chemotherapy as well as drug resistance highlight the ongoing need to discover novel natural bioactive compounds with anticancer potentiality. Mushroom-derived proteins are among the naturally occurring compounds that have been the subject of a body of research on their potentiality in cancer therapy. The greatest attention in relevant review articles has been paid to well-known mushroom-derived glycoproteins such as lectins and protein-bound polysaccharide complexes such as polysaccharide-K (PSK) or krestin and polysaccharopeptide (PSP), which contain substantial amounts of carbohydrates (50-90%). These complex compounds exert their anticancer activity mainly by binding to cell membranes leading to extrinsic (death receptor) apoptosis or intrinsic (mitochondrial) apoptotic pathways. However, several other research studies have reported pure, well-characterized, proteins or peptides from mushrooms, which are carbohydrate-free or have very low amounts of carbohydrate. These proteins may fall into four categories including fungal immunomodulatory proteins, ubiquitin-like proteins, enzymes, and unclassified proteins. Well-defined chemical structure, elucidated full amino acid or N-terminal sequences, purity, and having some distinct and specific pathways compared to glycoproteins have made these low-carbohydrate proteins attractive for cancer research. The aim of this review was therefore to improve the current understanding of mushroom-derived low-carbohydrate proteins and to consolidate the existing knowledge of the most promising mushroom species from which low-carbohydrate proteins have been derived, characterized, and examined for their anticancer activity. In addition, molecular targets and mechanisms of action of these proteins have been discussed. Key points • Mushroom-derived low-carbohydrate proteins lack or have low carbohydrate. • Low-carbohydrate proteins show potent anticancer activities in vitro and in vivo. • There are specific pathways for low-carbohydrate proteins to inhibit cancer cells.

Neuroprotective Effect of Chlorogenic Acid on Mitochondrial Dysfunction-Mediated Apoptotic Death of DA Neurons in a Parkinsonian Mouse Model.

Mitochondrial dysfunction and oxidative stress characterize major factors involved in the activation of complex processes corresponding to apoptosis-mediated neuronal senescence of dopaminergic neurons (DA) in Parkinson's disease (PD). Here, we evaluated the molecular mechanisms participating in the treatment of a 1-methyl-4-phenyl-1,2,3,6-tetrahydopyridine- (MPTP-) intoxicated PD mouse model in response to chlorogenic acid (CGA). The results indicate that CGA treatment significantly improved the motor coordination of the MPTP-intoxicated mice. CGA also alleviated the fall in activity of mitochondrial complexes I, IV, and V in accordance with ameliorating the level of superoxide dismutase and mitochondrial glutathione in the midbrain of MPTP-induced mice. CGA inhibited the activation of proapoptotic proteins including Bax and caspase-3, while elevating the expression of antiapoptotic protein like Bcl-2 consequently preventing the MPTP-mediated apoptotic cascade. The study also revealed the improved phosphorylation state of Akt, ERK1/2, and GSK3β which was downregulated as an effect of MPTP toxicity. Our findings signify that CGA may possess pharmacological properties and contribute to neuroprotection against MPTP induced toxicity in a PD mouse model associated with phosphorylation of GSK3β via activating Akt/ERK signalling in the mitochondrial intrinsic apoptotic pathway. Thus, CGA treatment may arise as a potential therapeutic candidate for mitochondrial-mediated apoptotic senescence of DA neurons in PD.

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

Plumbagin engenders apoptosis in lung cancer cells via caspase-9 activation and targeting mitochondrial-mediated ROS induction.

Plumbagin is a naturally-derived phytochemical which exhibits promising medicinal properties, including anticancer activities. In the present study, the anticancer potential of plumbagin has been demonstrated in lung cancer cells by targeting reactive oxygen species (ROS) and the intrinsic mitochondrial apoptotic pathway. Plumbagin showed impressive cytotoxic, anti-proliferative, and anti-migratory activities with IC50 3.10 ± 0.5μM and 4.10 ± 0.5μM in A549 and NCI-H522 cells, respectively. Plumbagin treatment significantly reduced the size of A549 tumor spheroids in a concentration-dependent manner. Plumbagin enhanced ROS production and arrested lung cancer cells in S and G2/M phase. Expression of antioxidant genes such as glutathione S-transferase P1 and superoxide dismutase-2 were found to be upregulated with plumbagin treatment in A549 cells. Plumbagin induced dissipation in mitochondrial membrane potential and affected the expression of intrinsic apoptotic pathway proteins. Increased expression of cytochrome c promotes the activation of pro-apoptotic protein Bax with decreased expression of anti-apoptotic protein Bcl-2. Further, plumbagin activated the mitochondrial downstream pathway protein caspase-9 and caspase-3 leading to apoptosis of A549 cells. Collectively, plumbagin could be a promising future phytotherapeutic candidate for lung cancer treatment via targeting intrinsic mitochondrial apoptotic pathway and ROS.

Synthesis of Substituted Cinnamido Linked Quinazolinone Congeners as Potential Anticancer Agents via Mitochondrial Dependent Intrinsic Apoptotic Pathway.

The synthesis of novel heterocyclic scaffolds with amide functionality is a key research area due to their plethora of medicinal applications. The present study aims to explore the synthesis of new cinnamido linked quinazolinone congeners and their potential as anticancer agents. Cytotoxicity evaluation, Cell cycle analysis, JC-1 staining, ROS, Annexin V assays, AO/EB, DAPI nuclear staining, Colony-forming assay and Western blot analysis. Among the synthesized compounds, 5eb and 5fc have shown promising cytotoxic activity with an IC50 value of 3.89±1.01µM and 4.05±0.62µM against HeLa cell lines. The flow-cytometry analysis demonstrated that the compound 5eb arrested the sub-G1 phase of the cell cycle and induced apoptosis. Furthermore, the compound 5eb triggered the collapse of mitochondrial membrane potential (ΔΨm), which was assessed by JC-1 staining and also induced the generation of Reactive Oxygen Species. An increase in the expression of proapoptotic proteins such as Bax, p53, cleaved PARP and cleaved caspase-3 by 5eb confirmed the activation of the mitochondrial-dependent intrinsic apoptosis pathway. Our results suggest that compound 5eb and 5fc of cinnamido linked quinazolinone derivatives could serve as potential leads in the development of novel chemotherapeutic agents.

SpBOK inhibits WSSV infection by regulating the apoptotic pathway in mud crab (Scylla paramamosain)

B-cell lymphoma 2 (Bcl-2) related ovarian killer (BOK) is a member of the Bcl-2 family, which has a similar function to BAX and BAK in the process of apoptosis. However, how BOK activates the intrinsic (mitochondrial) apoptotic pathway remains poorly understood in invertebrates. In this study, SpBOK identified in mud crab is an important effector responsible for the anti-WSSV (White Spot Syndrome Virus) infection by activating the apoptotic pathway. The SpBOK gene encoded a 282 amino acid peptides (molecular mass of 29 kD), which contained four distinct Bcl-2 family homology (BH) domains. SpBOK was widely expressed in all tested tissues and up-regulated after WSSV infection in vivo. The role of SpBOK on the anti-WSSV response in mud crab was investigated by using the RNAi approach in vivo. SpBOK exerted a regulatory role in changing the mitochondrial membrane potential (⊿ψm) and activating the caspase signaling and thus induced apoptosis. Moreover, the results showed that WSSV replication in mud crab could be effectively inhibited by SpBOK. Therefore, the results of this study demonstrated that SpBOK can inhibit WSSV infection by regulating the intrinsic apoptosis pathway in mud crab.

Synthesis of 5-Alkynyltetrandrine Derivatives and Evaluation of their Anticancer Activity on A549 Cell Lines.

Lung cancer is one of the most prevalent malignancies and thus the development of novel therapeutic agents for managing lung cancer is imperative. Tetrandrine, a bis-benzyltetrahydroisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, has been found to exert cytotoxic effects on cancerous cells. A series of 5-alkynyltetrandrine derivatives was synthesized via the Sonogashira cross-coupling reactions and evaluated as potential anti-tumor agents. The anti-tumor activities of 12 compounds on lung cancer cells (A549) were evaluated using the MTT method. The population of apoptotic cells was measured using a TUNEL assay. Real-time PCR quantified the gene expression levels of Bcl-2, Bax, survivin and caspase-3. The content of Cyt-C was detected using a Human Cyt-C ELISA kit. Most of these compounds exhibited better activities than tetrandrine itself on A549 cells. Among them, compound 7 showed the highest cytotoxicity among the tested compounds against human lung adenocarcinoma A549 cells with an IC50 of 2.94 µM. Preliminary mechanistic studies indicated that compound 7 induced apoptosis of human lung cancer A549 cells and increased the level of the proapoptotic gene Bax, release of Cyt-C from mitochondria and activation of caspase-3 genes. The results suggest that compound 7 exerts its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway. These findings will contribute to the future design of more effective anti-tumor agents in lung cancer therapy.

Fabrication and Antitumor Mechanism of a Nanoparticle Drug Delivery System: Graphene Oxide/Chitosan Oligosaccharide/γ‐Polyglutamic Acid Composites for Anticancer Drug Delivery

AbstractThis study focused on the design of a novel pH responsive nanoparticle drug delivery system (NDDS) with high biocompatibility and water solubility. The designed system was used for doxorubicin (DOX) delivery at tumor sites in a controlled manner. These innovative nanoparticles are soluble in physiological solutions besides water. In this study, pretreated γ‐polyglutamic acid (γ‐PGA) aqueous solutions were loaded into composites of graphene oxide (GO) covalently linked with chitosan oligosaccharide (CO) to fabricate GO‐CO‐γ‐PGA via amidation. The characterizations of the synthesized samples were carried out by using fourier transform infrared spectrometer (FTIR), X‐ray photoelectron spectrometer (XPS), ultraviolet– visible spectrophotometer (UV‐Vis), transmission electron microscopy (TEM), atomic force microscope (AFM) and zeta potential. Furthermore, drug loading and release behaviors from GO‐CO‐γ‐PGA were studied. The DOX loading in the constructed nanocarriers (GO‐CO‐γ‐PGA‐DOX) resulted in controlled and sustained release. The results of cell experiments revealed that the synthesized composites, which were easily transferred into cells, showed low toxicity and exhibited excellent antitumor effect due to its good connection with DOX. In addition, the composite nanoparticles caused the cell cycle arrest of Hela cells at the G2/M phase. Western blot results indicated that GO‐CO‐γ‐PGA‐DOX downregulated the expression of Bcl‐2 protein but upregulated Bax protein expression, which triggered the release of cytochrome C (Cytc). This condition further activated caspase‐3, cleaved the substrate of poly ADP‐ribose polymerase (PARP), and caused cell apoptosis. These results demonstrated that GO‐CO‐γ‐PGA‐DOX induces apoptosis via the intrinsic mitochondrial apoptotic pathway. Hence GO‐CO‐γ‐PGA composites have promising application in the field of biomedicine.

Combination of BCL-2 Inhibition and Triptolide Causes Synthetic Lethality in Acute Myeloid Leukemia through Preventing Reciprocal Resistance

BCL-2 inhibition exerts effective pro-apoptotic activities in acute myeloid leukemia (AML) but clinical efficacy as a monotherapy was limited in part due to the treatment-induced MCL-1 increase. Triptolide (TPL) exhibits anti-tumor activities in part by upregulating pro-apoptotic BCL-2 proteins and decreasing MCL-1 expression in various malignant cells. We hypothesized that combined BCL-2 inhibition and TPL exert synergistic anti-leukemia activities and prevent the resistance to BCL-2 inhibition in AML. We here report that TPL combined with BCL-2 inhibitor ABT-199 synergistically induced apoptosis in leukemic cells regardless of p53 status through activating the intrinsic mitochondrial apoptotic pathway in vitro. Although ABT-199 or TPL alone inhibited AML growth in vivo, the combination therapy demonstrated a significantly stronger anti-leukemic effect. Mechanistically, TPL significantly upregulated BH3 only proteins including PUMA, NOXA, BID and BIM and decreased MCL-1 but upregulated BCL-2 expression in both p53 wild type and p53 mutant AML cell lines, while the combination decreased both BCL-2 and MCL-1 and further increased BH3 only BCL-2 proteins. MCL-1 and BCL-2 increases associated with respective ABT-199 and TPL treatment and resistance were also observed in vivo. Significantly downregulating MCL-1 and elevating BH3 only proteins by TPL could not only potentially block MCL-1-mediated resistance but also enhance anti-leukemic efficacy of ABT-199. Conversely, BCL-2 inhibition counteracted the potential resistance of TPL mediated by upregulation of BCL-2. The combination further amplified the effect, which likely contributed to the synthetic lethality. This mutual blockade of potential resistance provides a rational basis for the promising clinical application of TPL and BCL-2 inhibition in AML independent of p53 status. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding.

Chrysophanol exhibits inhibitory activities against colorectal cancer by targeting decorin.

Colorectal cancer (CRC) is a common human malignancy that accounts for 600,000 deaths annually worldwide. Chrysophanol, a naturally occurring anthraquinone compound, exhibits anti-neoplastic effects in various cancer cells. The aim of this study was to explore the biological effects of chrysophanol on CRC cells, and determine the underlying mechanism. Chrysophanol inhibited proliferation of and promoted apoptosis in CRC cells by activating the intrinsic mitochondrial apoptotic pathway. In addition, chrysophanol also suppressed tumor growth in vivo and increased the percentage of apoptotic cells in tumor xenografts, without general toxicity. Proteomic iTRAQ analysis revealed decorin (DCN) as the major target of chrysophanol. DCN was upregulated in the tumor tissues following chrysophanol treatment, and ectopic DCN expression markedly augmented the pro-apoptotic effects of chrysophanol in CRC cells. In contrast, DCN knockdown significantly abrogated chrysophanol-induced apoptosis in CRC cells. Taken together, chrysophanol exerts anti-neoplastic effects in vitro and in vivo in CRC cells by modulating DCN, there by highlighting its therapeutic potential in CRC.

Chloroquine promotes gefitinib‑induced apoptosis by inhibiting protective autophagy in cutaneous squamous cell carcinoma.

Aberrant expression of the epidermal growth factor receptor (EGFR) plays vital roles in tumor development and progression. In the present study, ultraviolet irradiation induced the upregulation of EGFR in skin-derived keratinocytes, which may contribute to the development of cutaneous squamous cell carcinoma (CSCC). This was supported by the high expression of EGFR in CSCC clinical samples. Treating A431 CSCC cells with gefitinib, a tyrosine kinase inhibitor, activated the intrinsic mitochondrial apoptotic pathway while inducing protective autophagy. Combined application of chloroquine with gefitinib enhanced the treatment efficacy of gefitinib against CSCC by inhibiting autophagic flux. These findings demonstrated that autophagy inhibition may be an effective strategy for enhancing the sensitivity of EGFR-expressing cells to tyrosine kinase inhibitor treatment. Manipulating pro-survival autophagy by the combined application of chloroquine with gefitinib is a promising approach for improving the efficacy of EGFR inhibitors in cancer treatment. This may contribute to novel EGFR-targeted therapeutic strategies in the near future.

Abstract 325: A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition

Abstract Therapeutics targeted to specific proteins known to drive or modulate progression of acute myeloid leukemia (AML) have shown limited effectiveness as resistance frequently develops during their clinical use. Similar to many cancers, a stalled process of programmed cell death in apoptosis-primed cells is one of the prevalent mechanisms of drug resistance in AML. The intrinsic mitochondrial apoptotic pathway is controlled by a rheostat of anti-apoptotic and pro-apoptotic proteins and perturbing the balance toward pro-apoptotic responses remains a focus of many small molecular therapeutics. Venetoclax, an inhibitor of anti-apoptotic gene BCL2, approved for treatment of a certain subtypes of chronic lymphocytic leukemia (CLL), shows limited success as a monoagent in AML clinical trials due to an inherent resistance in approximately 37% of all patients (IC50 &amp;gt; 5μM). To identify mechanisms underlying intrinsic and acquired resistance to the BCL2 inhibitor, venetoclax, we used a genome-wide CRISPR/Cas9 screen to identify genes whose inactivation results in loss of venetoclax sensitivity in MOLM13 cells, derived from an AML patient that harbored mutation in FLT3-ITD, one of the major genetic landscapes of AML. Interrogation with two independently derived genome-wide libraries yielded identical top candidates TP53, BAX and PMAIP1. We also observed other gene targets that implicate the mitochondrial intrinsic, TP53-controlled pro-apoptotic pathway in the acquisition of venetoclax resistance. Correlatively, in a large AML cohort (Beat AML dataset), patients with loss of function mutations in TP53, or either low expression of TP53 or BAX exhibited statistically significant lower levels of responses to venetoclax ex vivo. Resultant venetoclax resistant cells with inactivation of TP53 or BAX had significantly reduced capacity for apoptosis when treated with venetoclax and exhibited elevated pro-survival signaling exemplified by increased levels of MAPK and AKT. In addition, loss of TP53 led to changes in the ratio of anti-apoptotic proteins BCL2, BCL-xL and MCL1 implicating TP53 as a major transcriptional regulator of the apoptotic rheostat in MOLM13 cells. Evaluation of the TP53 and BAX knockout cells for sensitivities to a panel of small molecule inhibitors revealed loss of sensitivities to a wide range of additional inhibitors including FLT3, AKT, multi-kinase inhibitors as well as a surprising gain of sensitivity to NTRK inhibitors relying on alternative pathways of cell death. Our results provide an independent confirmation of TP53 and its apoptotic network involvement to the venetoclax response in AML cells and suggest strategies to overcome resistance. Citation Format: Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney. A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition [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 325.

Molecular Targeted Therapies Elicit Concurrent Apoptotic and GSDME-Dependent Pyroptotic Tumor Cell Death.

The induced death signals following oncogene inhibition underlie clinical efficacy of molecular targeted therapies against human cancer, and defects of intact cell apoptosis machinery often lead to therapeutic failure. Despite potential importance, other forms of regulated cell death triggered by pharmacologic intervention have not been systematically characterized. Pyroptotic cell death was assessed by immunoblot analysis, phase-contrast imaging, scanning electron microscopy, and flow cytometry. Tumor tissues of patients with lung cancer were analyzed using IHC. Functional impact of pyroptosis on drug response was investigated in cell lines and xenograft models. We showed that diverse small-molecule inhibitors specifically targeting KRAS-, EGFR-, or ALK-driven lung cancer uniformly elicited robust pyroptotic cell death, in addition to simultaneously invoking cellular apoptosis. Upon drug treatment, the mitochondrial intrinsic apoptotic pathway was engaged and the mobilized caspase-3 protease cleaved and activated gasdermin E (GSDME, encoded by DFNA5), which permeabilized cytoplasmic membrane and executed cell-lytic pyroptosis. GSDME displayed ubiquitous expression in various lung cancer cell lines and clinical specimens, including KRAS-mutant, EGFR-altered, and ALK-rearranged adenocarcinomas. As a result, cooccurrence and interplay of apoptosis and pyroptosis were widespread in lung cancer cells, succumbing to genotype-matched regimens. We further demonstrated that pyroptotic cell death partially contributed to the drug response in a subset of cancer models. These results pinpoint GSDME-dependent pyroptosis as a previously unrecognized mechanism of action for molecular targeted agents to eradicate oncogene-addicted neoplastic cells, which may have important implications for the clinical development and optimal application of anticancer therapeutics.

Peroxisomicine A1 (toxin T-514) induces cell death of hepatocytes in vivo by triggering the intrinsic apoptotic pathway

Karwinskia parvifolia possesses the highest concentration levels of the anthracenone T-514 (PA1). Studies have demonstrated the induction of apoptosis by PA1 in cancer cell lines. The aim was to investigate the effects of PA1 on the apoptosis of the mouse liver in vivo and its underlying pathway. Sixty CD-1 mice were divided into three groups: untreated, vehicle, and treated with PA1. The animals were euthanized at 4, 8, 12, and 24 h post-treatment. To confirm the toxic effect of PA1 we determined the activity of catalase. Liver sections were prepared for morphological examination and for immunohistochemical evaluation of anti and pro-apoptotic markers. DNA fragmentation was detected by TUNEL assay and electrophoresis. Pre-apoptotic mitochondrial alterations and cytochrome c oxidase activity were analyzed by transmission electron microscopy. PA1 induced pre-apoptotic mitochondrial alterations, a high activity of the cytochrome oxidase, and apoptosis in hepatocytes. PA1 caused p53 over-expression and down regulation of PCNA. PA1 also increased the expression levels of the pro-apoptotic markers Bax and Bak, whereas the anti-apoptotic molecule Bcl-2 was decreased. PA1 induces apoptosis by activating the intrinsic mitochondrial apoptotic pathway. These results will be useful for studies regarding the use of PA1 as an antineoplastic agent.

Ganoderma atrum polysaccharide improves doxorubicin-induced cardiotoxicity in mice by regulation of apoptotic pathway in mitochondria

The present study aimed to determine the cardioprotective effect of Ganoderma atrum polysaccharide (PSG-1) in doxorubicin (DOX)-treated mice and its underlying mechanism. Results indicated that PSG-1 treatment significantly alleviated DOX-induced myocardial damage via attenuating apoptosis and maintaining the structure of myocardial mitochondria. Meanwhile, PSG-1-evoked cardioprotection was associated with an increase of manganese superoxide dismutase activity and decrease of caspases activities. Moreover, administration of PSG-1 suppressed DOX-induced mitochondrial disorders, which was evidenced by reducing reactive oxygen species, elevating mitochondrial membrane potential and inhibiting the opening of mitochondrial permeability transition pore. PSG-1 was also found to reduce the release of cytochrome c from mitochondria to cytoplasm in mice subjected to DOX. Finally, our findings have provided comprehensive evidence for the cardioprotective effects of PSG-1 via reduction of apoptosis mediated by modification of the mitochondrial intrinsic apoptotic pathway, indicating that PSG-1 could be developed as an effective therapeutic strategy to prevent DOX-induced cardiotoxicity in clinical settings.

Downregulation of GSDMD attenuates tumor proliferation via the intrinsic mitochondrial apoptotic pathway and inhibition of EGFR/Akt signaling and predicts a good prognosis in non‑small cell lung cancer.

Gasdermin D (GSDMD) is a newly discovered pyroptosis executive protein, which can be cleaved by inflammatory caspases and is essential for secretion of IL-1β, making it a critical mediator of inflammation. However, the precise role of GSDMD in carcinogenesis remains nearly unknown. Considering the vital role of inflammation in tumorigenesis, we investigated the biological function of GSDMD in non-small cell lung cancer (NSCLC). Our study demonstrated that the GSDMD protein levels were significantly upregulated in NSCLC compared to these levels in matched adjacent tumor specimens. Higher GSDMD expression was associated with aggressive traits including larger tumor size and more advanced tumor-node-metastasis (TNM) stages. In addition, high GSDMD expression indicated a poor prognosis in lung adenocarcinoma (LUAD), but not in squamous cell carcinoma (LUSC). Knockdown of GSDMD restricted tumor growth in vitro and in vivo. Notably, intrinsic and extrinsic activation of pyroptotic (NLRP3/caspase-1) signaling in GSDMD-deficient tumor cells induced another type of programmed cell death (apoptosis), instead of pyroptosis. GSDMD depletion activated the cleavage of caspase-3 and PARP, and promoted cancer cell death via intrinsic mitochondrial apoptotic pathways. In addition, co-expression analyses indicated a correlation between GSDMD and EGFR/Akt signaling. Collectively, our results revealed a crosstalk between pyroptotic signaling and apoptosis in tumor cells. Knockdown of GSDMD attenuated tumor proliferation by promoting apoptosis and inhibiting EGFR/Akt signaling in NSCLC. In conclution, GSDMD is an independent prognostic biomarker for LUAD.