Mitochondrial Outer Membrane Permeabilization Research Articles

Achillea fragrantissima (Forssk.) Sch.Bip Flower Dichloromethane Extract Exerts Anti-Proliferative and Pro-Apoptotic Properties in Human Triple-Negative Breast Cancer (MDA-MB-231) Cells: In Vitro and In Silico Studies.

The aggressive triple-negative breast cancer (TNBC) is a challenging disease due to the absence of tailored therapy. The search for new therapies involves intensive research focusing on natural sources. Achillea fragrantissima (A. fragrantissima) is a traditional medicine from the Middle East region. Various solvent extracts from different A. fragrantissima plant parts, including flowers, leaves, and roots, were tested on TNBC MDA-MB-231 cells. Using liquid chromatography, the fingerprinting revealed rich and diverse compositions for A. fragrantissima plant parts using polar to non-polar solvent extracts indicating possible differences in bioactivities. Using the CellTiter-Glo™ viability assay, the half-maximal inhibitory concentration (IC50) values were determined for each extract and ranged from 32.4 to 161.7 µg/mL. The A. fragrantissima flower dichloromethane extract had the lowest mean IC50 value and was chosen for further investigation. Upon treatment with increasing A. fragrantissima flower dichloromethane extract concentrations, the MDA-MB-231 cells displayed, in a dose-dependent manner, enhanced morphological and biochemical hallmarks of apoptosis, including cell shrinkage, phosphatidylserine exposure, caspase activity, and mitochondrial outer membrane permeabilization, assessed using phase-contrast microscopy, fluorescence-activated single-cell sorting analysis, Image-iT™ live caspase, and mitochondrial transition pore opening activity, respectively. Anticancer target prediction and molecular docking studies revealed the inhibitory activity of a few A. fragrantissima flower dichloromethane extract-derived metabolites against carbonic anhydrase IX, an enzyme reported for its anti-apoptotic properties. In conclusion, these findings suggest promising therapeutic values of the A. fragrantissima flower dichloromethane extract against TNBC development.

Role of released mitochondrial DNA in acute lung injury.

Acute lung injury(ALI)/acute respiratory distress syndrome(ARDS) is a form of acute-onset hypoxemic respiratory failure characterised by an acute, diffuse, inflammatory lung injury, and increased alveolar-capillary permeability, which is caused by a variety of pulmonary or nonpulmonary insults. Recently, aberrant mitochondria and mitochondrial DNA(mtDNA) level are associated with the development of ALI/ARDS, and plasma mtDNA level shows the potential to be a promising biomarker for clinical diagnosis and evaluation of lung injury severity. In mechanism, the mtDNA and its oxidised form, which are released from impaired mitochondria, play a crucial role in the inflammatory response and histopathological changes in the lung. In this review, we discuss mitochondrial outer membrane permeabilisation (MOMP), mitochondrial permeability transition pore(mPTP), extracellular vesicles (EVs), extracellular traps (ETs), and passive release as the principal mechanisms for the release of mitochondrial DNA into the cytoplasm and extracellular compartments respectively. Further, we explain how the released mtDNA and its oxidised form can induce inflammatory cytokine production and aggravate lung injury through the Toll-like receptor 9(TLR9) signalling, cytosolic cGAS-stimulator of interferon genes (STING) signalling (cGAS-STING) pathway, and inflammasomes activation. Additionally, we propose targeting mtDNA-mediated inflammatory pathways as a novel therapeutic approach for treating ALI/ARDS.

The Anticancer Effects of the Pro-Apoptotic Benzofuran-Isatin Conjugate (5a) Are Associated With p53 Upregulation and Enhancement of Conventional Chemotherapeutic Drug Efficiency in Colorectal Cancer Cell Lines.

The present study aimed to investigate in-depth a cytotoxic novel benzofuran-isatin conjugate (5a, 3-methyl-N'-(2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide) with promising potential anticancer activities in colorectal adenocarcinoma HT29 and metastatic colorectal cancer (CRC) SW620 cell lines. Thus, the primary cell events involved in tumorigenicity, tumor development, metastasis, and chemotherapy response were explored. Both CRC cell lines were exposed to different concentrations of Compound 5a and then subjected to real-time cell viability, migration, and invasion assays, colony formation and cytotoxicity assays, and flow cytometry for cell cycle analysis and apoptosis determination. Western blot and RT-qPCR were performed to assess the protein and transcript expression levels of epithelial-mesenchymal transition (EMT), cell cycle, and apoptosis markers. We showed that the Compound 5a treatment exhibited anticancer effects through inhibition of HT29 and SW620 cell viability, migration, and invasion, in a dose-dependent manner, which were associated with the upregulation of the tumor suppressor p53. Compound 5a also inhibited the colony formation ability of HT29 and SW620 cells and reversed EMT markers E-cadherin and N-cadherin expression. CRC cell exposure to Compound 5a resulted in a cell cycle arrest at the G1/G0 phase in HT29 cells and at the G2/M phase in SW620 cells, along with the downregulation of cyclin A1 expression, described to be involved in the S phase entry. Furthermore, Compound 5a-induced apoptosis was associated with the downregulation of the anti-apoptotic Bcl-xl marker, upregulation of pro-apoptotic Bax and cytochrome c markers, and increased mitochondrial outer membrane permeability, suggesting the involvement of mitochondria-dependent apoptosis pathway. In addition, the combination studies of Compound 5a with the main conventional chemotherapeutic drugs 5-fluorouracil, irinotecan, and oxaliplatin showed a more potent cytotoxic effect in both CRC cells than a single treatment. In conclusion, our findings described the interesting in vitro anticancer properties of Compound 5a, shown to have possible antitumor, antimetastatic, and pro-apoptotic activities, with the enhancement of the cytotoxic efficiency of conventional chemotherapeutic drugs. In vivo studies are requested to confirm the promising anticancer potential of Compound 5a for CRC therapy.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Mitochondrial sirtuin 3 and various cell death modalities.

Sirtuin 3, a member of the mammalian sirtuin family of proteins, is involved in the regulation of multiple processes in cells. It is a major mitochondrial NAD+-dependent deacetylase with a broad range of functions, such as regulation of oxidative stress, reprogramming of tumor cell energy pathways, and metabolic homeostasis. One of the intriguing functions of sirtuin 3 is the regulation of mitochondrial outer membrane permeabilization, a key step in apoptosis initiation/progression. Moreover, sirtuin 3 is involved in the execution of various cell death modalities, which makes sirtuin 3 a possible regulator of crosstalk between them. This review is focused on the role of sirtuin 3 as a target for tumor cell elimination and how mitochondria and reactive oxygen species (ROS) are implicated in this process.

Modulating mitofusins to control mitochondrial function and signaling

Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.

MiR-183-5p-PNPT1 Axis Enhances Cisplatin-induced Apoptosis in Bladder Cancer Cells

ObjectiveIt has been reported that intrinsic apoptosis is associated with the progression of bladder cancer (BC). Recent evidence suggests that polyribonucleotide nucleotidyltransferase 1 (PNPT1) is a pivotal mediator involved in RNA decay and cell apoptosis. However, the regulation and roles of PNPT1 in bladder cancer remain largely unclear.MethodsThe upstream miRNA regulators were predicted by in silico analysis. The expression levels of PNPT1 were evaluated by real-time PCR, Western blotting, and immunohistochemistry (IHC), while miR-183-5p levels were evaluated by qPCR in BC cell lines and tissues. In vitro and in vivo assays were performed to investigate the function of miR-183-5p and PNPT1 in apoptotic RNA decay and the tumorigenic capability of bladder cancer cells.ResultsPNPT1 expression was decreased in BC tissues and cell lines. Overexpression of PNPT1 significantly promoted cisplatin-induced intrinsic apoptosis of BC cells, whereas depletion of PNPT1 potently alleviated these effects. Moreover, oncogenic miR-183-5p directly targeted the 3′ UTR of PNPT1 and reversed the tumor suppressive role of PNPT1. Intriguingly, miR-183-5p modulated not only PNPT1 but also Bcl2 modifying factor (BMF) to inhibit the mitochondrial outer membrane permeabilization (MOMP) in BC cells.ConclusionOur results provide new insight into the mechanisms underlying intrinsic apoptosis in BC, suggesting that the miR-183-5p-PNPT1 regulatory axis regulates the apoptosis of BC cells and might represent a potential therapeutic avenue for the treatment of BC.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

Singlet Oxygen, Photodynamic Therapy, and Mechanisms of Cancer Cell Death.

Photodynamic therapy (PDT) can be developed into an important arsenal against cancer; it is a minimally invasive therapy, which is used in the treatment or/and palliation of a variety of cancers and benign diseases. The removal of cancerous tissue is achieved with the use of photosensitizer and a light source, which excites the photosensitizer. This excitation causes the photosensitizer to generate singlet oxygen and other reactive oxygen species. PDT has been used in several types of cancers including nonmelanoma skin cancer, bladder cancer, esophageal cancer, head and neck cancer, and non-small cell lung cancer (NSCLC). Although it is routinely used in nonmelanoma skin cancer, it has not been widely adopted in other solid cancers due to a lack of clinical data showing the superiority of PDT over other forms of treatment. Singlet oxygen used in PDT can alter the activity of the catalase, which induces immunomodulation through HOCl signaling. The singlet oxygen can induce apoptosis through both the extrinsic and intrinsic pathways. The extrinsic pathway of apoptosis starts with the activation of the Fas receptor by singlet oxygen that leads to activation of the caspase-7 and caspase-3. In the case of the intrinsic pathway, disruption caused by singlet oxygen in the mitochondria membrane leads to the release of cytochrome c, which binds with APAF-1 and procaspase-9, forming a complex, which activates caspase-3. Mechanisms of PDT action can vary according to organelles affected. In the plasma membrane, membrane disruption is caused by the oxidative stress leading to the intake of calcium ions, which causes swelling and rupture of cells due to excess intake of water, whereas disruption of lysosome causes the release of the cathepsins B and D, which cleave Bid into tBid, which changes the mitochondrial outer membrane permeability (MOMP). Oxidative stress causes misfolding of protein in the endoplasmic reticulum. When misfolding exceeds the threshold, it triggers unfolding protein response (UPR), which leads to activation of caspase-9 and caspase-3. Finally, the activation of p38 MAPK works as an alternative pathway for the induction of MOMP.

P534: AZACITIDINE PLUS VENETOCLAX FOR THE TREATMENT OF RELAPSED AND FIRST-LINE AML PATIENTS

Background: Venetoclax (VEN) belongs to a novel BH3-mimetic class of small molecules that selectively targets BCL-2, activating the apoptosis effectors BAX and BAK to drive mitochondrial outer membrane permeabilization, cytochrome c release and cell death. Combination of VEN and the hypomethylating agent azacitidine (AZA) has deeply changed the paradigm of treatment of newly diagnosed (ND) AML patients ineligible for high dose chemotherapy because of older age or comorbidities. There is scarce evidence for the utilization of VEN-AZA for relapsed or refractory (R/R) AML, a category of patients classically associated with an extremely poor outcome. Aims: The objective of our study was to describe a R/R AML cohorts of AML patients treated with VEN-AZA in our institution and to compare the clinical and molecular characteristics predicting response in R/R AML versus ND AML Methods: This retrospective study included consecutive patients treated with VEN-AZA for R/R AML and ND AML. Patients received AZA at standard dose of 75 mg/m2 QD for seven days and VEN was administrated either at 400 mg or at 100 mg when associated with strong CYP3P450 A3 inhibitors after three days ramp up. Response was determined using the ELN 2017 criteria. The ORR was defined as the combination of complete response (CR), CR with incomplete hematologic recovery (CRi), and morphologic leukemia-free state (MLFS). Results: We compared the outcome of 39 R/R AML and 38 concomitant ND AML patients treated in our institution between Jan. 2020 and Dec. 2021. The median age was 69 (22-86) and 73 (61-81) in the R/R and ND groups, respectively. Thirty-five percent of patients had MRC-AML. Adverse cytogenetics was found in 36% of patients in the R/R group and 59% of patients in the ND group. Most frequent mutations were ASXL1, RUNX1, TET2, IDH1/2 and TP53 found in 33%, 33%, 28%, 24% and 22% of patients, respectively. Overall response rate was lower in R/R AML (37% versus 56%) including 13% CR, 8% CRi, 3% PR and 13% MLFS in the R/R AML group and 32% CR, 13% CRi and 13% MLFS in the ND AML group. Adverse cytogenetics was associated with treatment failure only in the R/R group (Relative Risk=0.10, p=0.005). ASXL1, IDH1/2 and SFSR2 mutations were associated with a trend in a higher response rate in the R/R group. Median overall survival (OS) were 5.9 months in the R/R group and 9.4 months in the ND group. In the R/R group, median OS were 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group (p=0.02). Median leukemia-free survival of responding patient was not different between the two groups (9 months), indicating that VEN-AZA can be efficient as a salvage treatment for selected R/R AML patients. Summary/Conclusion: We described one of the largest series of R/R AML patients treated wiht VEN-AZA. By a doing a direct comparison between R/R AML and ND AML treated concomitantly, we found that adverse cytogenetics was associated with treatment failure only in the R/R group suggesting that this subgroup of patients should not be treated with VEN-AZA. Further analyses including more patients are needed to determine which subgroup may benefit from the VEN-AZA as a salvage treatment.

Ca2+-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak.

The ability of mitochondria to buffer a rapid rise in cytosolic Ca2+ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca2+ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca2+ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca2+. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca2+ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca2+ release from ER stores. Therefore, loss of Ca2+ intracellular balance and mitochondrial Ca2+ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.

CircHIPK3 prevents chondrocyte apoptosis and cartilage degradation by sponging miR‐30a‐3p and promoting PON2

Osteoarthritis (OA) is a common joint disease featured by the deterioration of articular cartilage and chondrocyte death. Emerging evidence has indicated that circular RNAs (circRNAs) play an essential role in OA progress. Here, we found that the expression of circHIPK3 was significantly decreased in human and mouse OA cartilage. Knocking down circHIPK3 increased apoptosis and intracellular ROS level in HC‐a chondrocytes. We performed proteomic studies and identified that circHIPK3 regulated chondrocyte apoptosis through the mitochondrial pathway. Results of JC‐1 staining and western blot further confirmed that mitochondrial outer membrane permeabilization was promoted in HC‐a chondrocytes transfected by circHIPK3 siRNA. In terms of mechanism, we showed that PON2 functioned as a potential target of circHIPK3 to regulate chondrocyte apoptosis. Moreover, we revealed that circHIPK3 interacted with miR‐30a‐3p to regulate PON2 expression in chondrocytes. Taken together, our findings suggested that circHIPK3 regulated chondrocyte apoptosis by mitochondrial pathway, and targeting the circHIPK3/miR‐30a‐3p/PON2 axis might be a potential strategy for OA treatment.

Abstract 2133: In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355

Abstract Appropriate control of cell death is a fundamental biological process which is frequently dysregulated during tumor development and therapeutic resistance. Apoptosis is a form of regulated cell death initiated by either the extracellular environment (extrinsic) or following internal cellular damage (intrinsic). It is controlled by the BCL-2 family which includes anti-apoptotic regulators like BCL-2, BCL-XL and MCL-1 that bind and sequester various pro-apoptotic BH3-only proteins (BIM, BAD, BID, NOXA, PUMA, etc.), and the pro-apoptotic effectors (BAK, BAX, etc.) responsible for mitochondrial pore formation and MOMP (mitochondrial outer membrane permeabilization). MOMP results in intermembrane space protein release, leading to caspase activation in an irreversible path to programmed cell death. Of the anti-apoptotic regulators, MCL-1 is one of the most frequently and highly amplified genes in human cancers such as myeloid leukemia making it a compelling therapeutic target. Since BCL-2 proteins interact through protein-protein interactions, they have long been elusive targets. The success of selective BCL-2 protein inhibitor Venetoclax in the treatment of various hematological cancers, however spurred interest in MCL-1 as an oncology target. Using structure-based drug design, major breakthroughs were made in the development of MCL-1 inhibitors, with several candidates entering clinical studies in the past five years. JNJ-4355, a highly potent 1,4-indolyl macrocycle (MCL-1 Ki = 18 pM, Cell (MOLP8) AC50 = 8.7 nM) was optimized to address shortcomings from first generation MCL-1 inhibitors: it has improved physicochemical properties (CHI LogD7.4 = 2.35, EPSA = 151 Å2), resulting in greatly improved equilibrium solubility (3.14 mM in buffer pH 7) and reduced protein binding (99.93%). JNJ-4355 showed promising in vitro potency data in cancer cell lines and AML patient-derived samples (cell killing AC50 0.29-75 nM in 25/27 evaluable samples). In vivo MCL-1:BAK complex disruption was confirmed in a mouse MOLM13 (AML) xenograft. Efficacy was demonstrated in a mouse MOLP8 (multiple myeloma) xenograft resulting in complete tumor regression after a single IV dose of JNJ-4355. Citation Format: Frederik J. Rombouts, Lento William, Ingrid Velter, Ann Vos, Aldo Peschiulli, Reuillon Tristan, Maria Dominguez Blanco, Matthieu Jouffroy, Lisa McQueen, Helena Steyvers, Mariette Bekkers, Cristina Altrocchi, Beth Pietrak, Seong Joo Koo, Lawrence Szewczuk, David Walker, Kathryn Packman, Ruud Bueters, Petra Vinken, Amy Johnson, Ricardo Attar, Ulrike Philippar. In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355 [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 2133.

Abstract 1846: In vitro studies of the effects of a novel ceramide analog on chemo- resistant breast cancer cells

Abstract Globally, breast cancer is the most frequently diagnosed cancer in women, with an estimated 281,550 new cases in the U.S.A in 2021. About 43,600 women in the U.S. are expected to die from the disease by the end of the year. Chemo- and radio-therapies, primarily, exert anti-tumor effects through the activation of apoptosis, or programmed cell death pathways. As many current therapies have severe side effects, and/or lose their effectiveness over time due to developed resistance, there is a need for novel agents that can improve patient outcomes including the quality and length of life. Ceramide-mimicking drugs have such potential. Ceramide, a bioactive sphingolipid, is a powerful tumor suppressor molecule that is thought to induce apoptosis and inhibit proliferation. As part of our ongoing efforts toward the development of a potent anti-cancer drug, ceramide analog 315, (S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecylpropanamide, was synthesized. Ceramide analog 315 has been shown to induce apoptosis in vitro. In the present study, an attempt is made to determine the cell death pathway induced during the treatment of chemo-resistant breast cancer cells with this analog. The MCF-7TN-R cell line is a derivative of the MCF-7 cell line obtained after prolonged exposure to increasing concentrations of TNF-α. These chemo-resistant cells, 24 hours after plating, were treated with a 40 µM solution of ceramide analog 315. Caspase 9 and cytochrome c Elisa assays were performed according to the vendor protocols. Cytochrome c and caspase 9 are believed to activate several pro-apoptotic proteins like APAF-1, BAX, and BCL-2, supporting the intrinsic or mitochondrial pathway. Caspase 9 and cytochrome c are both released from the mitochondrial intermembrane space, increasing the mitochondrial outer membrane permeability. Cytochrome c is a heme protein that, when released, binds to apoptotic protease activating factor 1 (APAF-1). APAF-1 then creates an apoptosome that acts as a platform for caspase 9 activation. Caspase 9, in turn, activates the cascade pathway and induces apoptosis. In this study, cytochrome c levels were shown to be decreased. Controls were found to have a concentration of 8.8 +/- 0.268 ng/mL, while treated cells had a concentration of 7.46 +/- 0.181 ng/mL, with a P value of less than .05. Caspase 9 levels were shown to be even more significantly decreased with controls having a concentration of 678,800 +/- 24,462 ng/mL, while treated cells had a concentration of 496,444 +/- 49,542 ng/mL, with a P value of less than 0.10.We further determined the protein level expression in these cells using microarrays, which revealed the upregulation of death receptors TNFR1, and TRAIL. Our findings help us in better understanding the mechanisms of action of ceramide-mimicking agents, and in working toward the development of strategies to improve the efficacy of anti-cancer drugs. Citation Format: Teresa C. Beamon, Royce Hooks, Degrick Cheatham, Navneet Goyal, Tulasi Ponnapakkam, Shahensha Shaik, Maryam Foroozesh. In vitro studies of the effects of a novel ceramide analog on chemo- resistant breast cancer cells [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 1846.

Mitochondrial dynamics regulate genome stability via control of caspase-dependent DNA damage.

Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming. Underlying this process, we reveal a key role for mitochondrial dynamics in the regulation of DNA damage and genome instability. The ability of mitochondrial dynamics to regulate oncogenic DNA damage centers upon the control of minority mitochondrial outer membrane permeabilization (MOMP), a process that enables non-lethal caspase activation leading to DNA damage. Mitochondrial fusion suppresses minority MOMP and its associated DNA damage by enabling homogeneous mitochondrial expression of anti-apoptotic BCL-2 proteins. Finally, we find that mitochondrial dysfunction inhibits pro-apoptotic BAX retrotranslocation, causing BAX mitochondrial localization and thereby promoting minority MOMP. Unexpectedly, these data reveal oncogenic effects of mitochondrial dysfunction that are mediated via mitochondrial dynamics and caspase-dependent DNA damage.

Synthesis, characterization, molecular docking and antimicrobial activity studies of novel oxadiazole derivatives

In order to generate new chemotherapeutics with significant anticancer and bioactivity, a series of new 6-(4-((5- (substituted phenyl)-1,3,4-oxadiazol-2-yl)methyl)piperazin- 1-yl)-3-nitroimidazo[1,2-b]pyridazine (4a-f) were designed and synthesized. The structures of compounds were confirmed using elemental analysis, NMR and Mass spectral data. On the basis of molecular docking with the BAX protein, the chemicals were evaluated for their anticancer potential. The anti-apoptotic protein Bcl-2 is over expressed in a variety of cancer cells inhibiting apoptosis. Bcl-2 family members are mostly known as mitochondrial outer membrane integrity regulators. Anti and pro-apoptotic members of the Bcl-2 protein family regulate mitochondrial outer-membrane permeabilization (MOMP) and mitochondrial dysfunction in an antagonistic manner. BAX docked well into ligand 3d and docking score was -7.3 kcal/mol. Also, all the derivatives were screened for antibacterial and antifungal activity (disc diffusion method).

Tissue homeostasis in sponges: Quantitative analysis of cell proliferation and apoptosis.

Tissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Sponges are early branching metazoans essential to understanding the key mechanisms of tissue homeostasis. This article is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea). Labeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. Quantitative DNA staining reveals the classic cell cycle distribution curve. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant throughout various areas of sponge bodies that contain choanocytes. The EdU tracking experiments conducted in H. dujardinii indicate that choanocytes may give rise to mesohyl cells through migration. The number of apoptotic cells in tissues of both species is insignificant, although being comparable to the renewing tissues of other animals. In vivo studies with tetramethylrhodamine ethyl ester and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization. Altogether, a combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell proliferation and apoptosis in sponges displaying either rapid growth or cell turnover.

Involvement of Akt and the antiapoptotic protein Bcl-xL in the inhibition of apoptosis of dendritic cells by Leishmania mexicana.

The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

Mitochondrial dynamics in the neonatal brain - a potential target following injury?

The impact of birth asphyxia and its sequelae, hypoxic–ischaemic (HI) brain injury, is long-lasting and significant, both for the infant and for their family. Treatment options are limited to therapeutic hypothermia, which is not universally successful and is unavailable in low resource settings. The energy deficits that accompany neuronal death following interruption of blood flow to the brain implicate mitochondrial dysfunction. Such HI insults trigger mitochondrial outer membrane permeabilisation leading to release of pro-apoptotic proteins into the cytosol and cell death. More recently, key players in mitochondrial fission and fusion have been identified as targets following HI brain injury. This review aims to provide an introduction to the molecular players and pathways driving mitochondrial dynamics, the regulation of these pathways and how they are altered following HI insult. Finally, we review progress on repurposing or repositioning drugs already approved for other indications, which may target mitochondrial dynamics and provide promising avenues for intervention following brain injury. Such repurposing may provide a mechanism to fast-track, low-cost treatment options to the clinic.

Disruption of mitochondrial quality control genes promotes caspase-resistant cell survival following apoptotic stimuli

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed “minority MOMP.” In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.