Apoptosis-deficient Cells Research Articles

Phytochemical Profile of the Ethanol Extract of Malvaviscus arboreus Red Flower and Investigation of the Antioxidant, Antimicrobial, and Cytotoxic Activities

Flowers are rich sources of bioactive antimicrobial, antioxidant, and anticancer components. This study aimed to determine the constituents of the ethanol extract of Malvaviscus arboreus red flower (ERF) by GC-MS analysis and HPLC identification of phenolic compounds and flavonoids, in addition to the 1HNMR fingerprint. The antimicrobial, antioxidant, and cytotoxic activities of the ERF were investigated. The GC-MS analysis revealed twenty-one components, while HPLC analysis revealed the presence of phenolic and flavonoid compounds. The ERF showed antifungal and antibacterial activity. The highest antibacterial activity was found against Vibrio damsela where a time-kill assay revealed a decline in the amount of viable V. damsela. For fungi, the highest activity was observed against Aspergillus terreus. Using the SRB test on HepG2, the anti-proliferative efficacy of the ERF was evaluated. Cell cycle analysis was utilized to determine autophagic cell death. The ERF prevented the proliferation of the HepG2 cell line with an IC50 of 67.182 µg/µL. The extract primarily promoted apoptosis in HepG2 cells by accumulating hypodiploid cells in the sub-G0/G1 phase, increased caspase 3/7 activity, and caused considerable autophagic cell death in apoptosis-deficient cells. Finally, the observed elevation of cancer cell death indicated that ERF had substantial anticancer potential against HepG2 cells.

Abstract 2825: Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines

Abstract Multiple myeloma (MM) is a disease of malignant plasma cells that resides in the bone marrow microenvironment (BMM). While advances in therapy have improved patient outcomes, the majority of patients relapse. Clinical trials have shown that BCMA targeted CAR-T therapy induces MM remission in most patients with heavily pretreated relapsed refractory MM; however, the duration of the response has been disappointing. CAR-T cells induce target cell death through two primary mechanisms, granzyme B and FASL. To define the role of FAS-induced cell death in CAR-T therapy in MM, we first evaluated if FAS-induced cell death was important for CAR-T activity in MM. We utilized CRISPR-Cas9 to generate FAS KOs in three MM cell lines: RPMI8226, KMS18, and OCIMY5. These lines were utilized in vitro in cytotoxic CAR-T assays where MM cells were cultured with BMCA CAR-T cells at five effector to target (E:T) ratios (range: 0:1 to 1:1). The loss of FAS protected KMS18 and OCIMY5 from CAR-T induced cell death, changing the E:T ratio needed to induce 50% MM cell death 3.2 and 3.4 fold respectively, but did not protect RPMI8226. We determined the expression of FAS negatively correlated with the EC50 of rFASL, suggesting that FAS expression levels are important for this response. To determine the role of the intrinsic apoptotic pathway in FASL-induced death, we knocked out BAK and BAX in KMS12 PE and OCIMY5. This did not protect KMS12 PE or OCIMY5 from rFASL-induced cell death, suggesting that FAS uses the type I mitochondria-independent pathway in these cell lines. The BMM supports MM progression and promotes drug resistance in part by altering the apoptotic threshold. Therefore, we examined the impact of the BMM on FASL-induced cell death. Coculture of MM cells with the HS5 stromal cell line protected KSM12 PE and KMS18 from rFASL, with the EC50 of rFASL changing 2.72 and 3.1 fold respectively, but did not protect OCIMY5. To determine if the protection observed was cell contact dependent, or only required soluble factors, we utilized HS5 conditioned media (CM). HS5 CM protected KMS12 PE, KMS18, and surprisingly OCIMY5 from rFASL, causing a change in EC50 from control of 1.65 - 2.23 fold. CM lacking IL6 did not protect MM cells from rFASL. Therefore, we tested the effects of IL6 addition IL6(10 ng/ml) and observed protection from rFASL in KMS12 PE and KMS18 cells with a change in EC50 of 2.03 and 3.84 fold respectively; however, we did not see protection in OCIMY5. To identify the mechanism of IL6-induced resistance to rFASL, we cultured BAK/BAX KO cells in HS5 CM and observed protection of these apoptosis-deficient KMS12 PE and OCIMY5 cells from rFASL. This suggests that soluble factors in the BMM can convert cells from type I to type II (mitochondrial-dependent) death receptor signaling and that inhibiting BMM signals such as IL6 may enhance FASL-induced cell death in MM. However, the data also demonstrate that the role of FAS in CAR-T killing is cell context dependent. Citation Format: James C. Ackley, Samuel McCachren, Sagar Lonial, Damien J. Green, Stanley R. Riddell, Geoffrey R. Hill, Madhav V. Dhodapkar, Lawrence H. Boise. Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2825.

The Smac mimetic BV6 cooperates with STING to induce necroptosis in apoptosis-resistant pancreatic carcinoma cells

Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2′3′-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2′3′-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2′3′-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.

Crosstalk between autophagy and apoptosis induced by camphor in Schizosaccharomyces pombe.

Camphor is widely used in pharmacy, the food industry, and cosmetics. In this study, we evaluate inhibitory and cytotoxic effects of camphor in the fission yeast (Schizosaccharomyces pombe), which presents a unicellular model in mechanistic toxicology and cell biology. Low-dose camphor exposure (0.4 mg/mL) activated autophagy, which was shown by GFP-Atg8 dots and transcriptional upregulation of Atg6 (Beclin-1 ortholog). Autophagy was also confirmed by using autophagy-deficient cells, which showed reduction in GFP-Atg8 dot formation. However, high-dose camphor exposure (0.8 mg/mL) caused dramatic cell death ratios, demonstrated by spot and colony-forming assays, even in autophagy-deficient cells. To unravel the underlying mechanism, this time, apoptosis-deficient cells were exposed to low- and high-dose camphor. Apoptosis was also confirmed by acridine orange/ethidium bromide staining. Among yeast apoptosis mediators, Aif1 was found to mediate camphor-induced cell death. In conclusion, differential regulation of autophagy and apoptosis, and switches between them, were found to be dose-dependent. The potential effects of camphor on autophagy and apoptotic cell death and underlying mechanisms were clarified in basic unicellular eukaryotic model, S. pombe.

Short-term activation of the Jun N-terminal kinase pathway in apoptosis-deficient cells of Drosophila induces tumorigenesis

In Drosophila, the JNK pathway eliminates by apoptosis aberrant cells that appear in development. It also performs other functions associated with cell proliferation, but analysis of the latter is hindered by the pro-apoptotic activity. We report the response of apoptosis-deficient cells to transient activation of JNK and show that it causes persistent JNK function during the rest of the development. As a consequence, there is continuous activity of the downstream pathways JAK/STAT, Wg and Dpp, which results in tumour overgrowths. We also show that the oncogenic potential of the Ras-MAPK pathway resides largely on its ability to suppress apoptosis. It has been proposed that a hallmark of tumour cells is that they can evade apoptosis. In reverse, we propose that, in Drosophila, apoptosis-deficient cells become tumorigenic due to their property of acquiring persistent JNK activity after stress events that are inconsequential in tissues in which cells are open to apoptosis.

The pro-death role of autophagy and apoptosis in cell death induced by the BH3 mimetic gossypol

Gossypol has been identified as a natural BH3 mimetic and a small-molecule inhibitor of Bcl-2 that can induce apoptosis in various cancer cell lines. In this study, we evaluated the effect of gossypol on apoptosis-proficient (wild-type [WT] mouse embryonic fibroblasts, MEFs) and apoptosis-deficient cells (Bax–/–/Bak–/– MEFs) in order to determine whether apoptosis indeed plays an essential death-promoting role in gossypol-mediated cell death. Unexpectedly, we found that Bax–/–/Bak–/– double-knockout (DKO) MEFs were more sensitive than the WT cells to gossypol-induced cell death. Gossypol caused progressive emergence of both apoptotic and necrotic cells in DKO MEFs; this result suggests that gossypol induces Bax/Bak-independent apoptosis. Conversely, fluorescence-activated cell sorting profiles showed no apoptotic cell death in WT MEFs. Rather, incubation of WT MEFs with gossypol markedly induced autophagy, as evidenced by punctate patterns of LC3 immunoreactivity, indicative of apoptosis-independent autophagic cell death. Nonetheless, there was only limited induction of autophagy in DKO MEFs. No significant differences between WT and DKO MEFs were observed in cell cycle profiles. Gossypol caused a weak increase of the percentage of cells at the G2/M checkpoint in the two cell lines, with a concomitant small decrease in the number of cells in the S phase. Taken together, our results suggest that apoptotic cell death is induced by gossypol in DKO MEFs, whereas autophagy plays a death-promoting role in gossypol-mediated death of WT MEFs cells.

Persephone/Spätzle Pathogen Sensors Mediate the Activation of Toll Receptor Signaling in Response to Endogenous Danger Signals in Apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as damage-associated molecular patterns (DAMPs) and a noninfectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. In this study, we found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spätzle (Spz). We speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. We demonstrated that Toll signaling activation required the action of Persephone, a CLIP domain serine protease that usually reacts to microbial proteolytic activities. Our findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants.

Requirement of evading apoptosis for HIF-1α-induced malignant progression in mouse cells

Tumor hypoxia is correlated with genetic alteration and malignant progression. Our previous studies indicated that the hypoxia-inducible transcription factor, HIF-1α, is responsible for hypoxic suppression of DNA repair in tumor cells by a non-canonical mode of action that requires the HIF-1α PAS-B subdomain. The involvement of HIF-1α in genetic alteration has raised an intriguing question as to whether normal cells would respond to hypoxic stress differently to avert genetic alteration. In this study, we chose several mouse cell types ranging from benign to malignant, apoptosis-proficient to apoptosis-deficient, and determined their responses to HIF-1α expression. In agreement with our previous findings, transient hypoxia and HIF-1α expression inhibited DNA repair and induced DNA damage in all cell types examined; however, cumulative DNA damage only occurred in apoptosis-deficient, malignant cells transduced for sustained expression of HIF-1α or HIF-1α PAS-B itself. In keeping with the theory of apoptosis as a cancer barrier, only these apoptosis-deficient cells acquired anchorage-independent growth and epithelial-mesenchymal transition. Furthermore, these cells exhibited increased Akt activity and resistance to etoposide by inhibiting autophagy. Altogether, our results define an essential role for apoptosis to prevent HIF-1α-induced genetic alteration and thereby malignant progression.

Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Photodynamic therapy (PDT) uses a photosensitizer, light, and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7+ cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.

An Essential Role of the HIF‐1α–c‐Myc Axis in Malignant Progression

Cancer is a disease of genomic aberration. The hypoxic microenvironment is believed to promote tumor progression via the induction of genetic instability. To understand how hypoxia drives tumor progression, we have shown recently that the hypoxia-inducible transcription factor, HIF-1alpha, is critical for transcriptional repression of DNA repair genes by a noncanonical mode of action referred to as the "HIF-1alpha-c-Myc axis." HIF-1alpha action via the HIF-1alpha-c-Myc axis is independent of its DNA-binding and transactivation domains; instead it requires the PAS-B domain to displace the transcription activator c-Myc from the target gene promoter for gene repression. Owing to the functional compromise on DNA repair, tumor cells with activated HIF-1alpha-c-Myc axis display persistent DNA damage, genetic alterations, and malignant progression. However, apoptosis-proficient cells are resistant to such changes. These findings argue that the hypoxic microenvironment plays a critical role in driving genetic alterations especially in apoptosis-deficient cells for malignant progression.

A Novel Inhibitory Mechanism of Mitochondrion-Dependent Apoptosis by a Herpesviral Protein

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine γ-herpesvirus 68 (γHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient γHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that γHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.

Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy

Non-apoptotic forms of programmed cell death are targets for novel approaches in anticancer therapy. Indeed, cancer cells often present with mutations in the apoptotic machinery that result in resistance to most anticancer therapies and contribute to a relatively low response rate to therapies based on the use of pro-apoptotic strategies. (Macro-)autophagy can be a highly efficient mode of cell death induction by excessive self-digestion as demonstrated by our experiments that studied the effect of radiation to induce autophagy cell death in apoptosis-deficient cells. Despite current controversies on the possible role of autophagy in the process of carcinogenesis and cancer progression by promoting cell survival, autophagy can be seen as a backup cell death mechanism, when other cell death mechanisms fail. This review will focus on the pathways linking autophagy and cancer that are relevant for target identification and on pharmaceuticals that can be utilized to improve cancer therapy by targeting the autophagic pathway.

The Death of Human Cancer Cells Following Photodynamic Therapy: Apoptosis Competence is Necessary for Bcl‐2 Protection but not for Induction of Autophagy†

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.

T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2)-producing, T cells results in impaired ZAP70/CD3-zeta association.

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.