Release Of Pro-apoptotic Proteins Research Articles

Transcutaneous Auricular Vagus Nerve Stimulation Ameliorates Preeclampsia-Induced Apoptosis of Placental Trophoblastic Cells Via Inhibiting the Mitochondrial Unfolded Protein Response.

Preeclampsia is a serious obstetric complication. Currently, there is a lack of effective preventive approaches for this disease. Recent studies have identified transcutaneous auricular vagus nerve stimulation (taVNS) as a potential novel non-pharmaceutical therapeutic modality for preeclampsia. In this study, we investigated whether taVNS inhibits apoptosis of placental trophoblastic cells through ROS-induced UPRmt. Our results showed that taVNS promoted the release of acetylcholine (ACh). ACh decreased the expression of UPRmt by inhibiting the formation of mitochondrial ROS (mtROS), presumably through M3AChR. This reduced the release of pro-apoptotic proteins (cleaved caspase-3, NF-κB-p65, and cytochrome C) and helped preserve the morphological and functional integrity of mitochondria, thus reducing the apoptosis of placental trophoblasts, improving placental function, and relieving preeclampsia. Our study unravels the potential pathophysiological mechanism of preeclampsia. In-depth characterization of the UPRmt is essential for developing more effective therapeutic strategies for preeclampsia targeting mitochondrial function.

Apoptotic proteins with non-apoptotic activity: expression and function in cancer.

Apoptosis is a process of programmed cell death in which a cell commits suicide while maintaining the integrity and architecture of the tissue as a whole. Apoptosis involves activation of one of two major pathways: the extrinsic pathway, where extracellular pro-apoptotic signals, transduced through plasma membrane death receptors, activate a caspase cascade leading to apoptosis. The second, the intrinsic apoptotic pathway, where damaged DNA, oxidative stress, or chemicals, induce the release of pro-apoptotic proteins from the mitochondria, leading to the activation of caspase-dependent and independent apoptosis. However, it has recently become apparent that proteins involved in apoptosis also exhibit non-cell death-related physiological functions that are related to the cell cycle, differentiation, metabolism, inflammation or immunity. Such non-conventional activities were predominantly reported in non-cancer cells although, recently, such a dual function for pro-apoptotic proteins has also been reported in cancers where they are overexpressed. Interestingly, some apoptotic proteins translocate to the nucleus in order to perform a non-apoptotic function. In this review, we summarize the unconventional roles of the apoptotic proteins from a functional perspective, while focusing on two mitochondrial proteins: VDAC1 and SMAC/Diablo. Despite having pro-apoptotic functions, these proteins are overexpressed in cancers and this apparent paradox and the associated pathophysiological implications will be discussed. We will also present possible mechanisms underlying the switch from apoptotic to non-apoptotic activities although a deeper investigation into the process awaits further study.

Potential of Moringa oleifera extract-incorporated with folic acid-conjugated gold nanoparticles as an oral squamous cell carcinoma therapy by modulating intrinsic apoptotic pathway: A narrative review

Background: Oral squamous cell carcinoma (OSCC) is the most common oral cancer worldwide. Surgery, radiotherapy and chemotherapy are the most common treatments, despite their side effects including toxicity, metastasis and multidrug resistance, thus evoking the need to develop safer treatment. Moringa oleifera (Mo) acts as anticancer agent but has poor bioavailability then incorporated with folic acid-conjugated gold nanoparticles (AuNPs) as drug carriers may enhance the action of Mo in OSCC treatment. Purpose: To describe the potential of Mo extract incorporated with folic acid-conjugated AuNPs as an OSCC therapy by modulating intrinsic apoptosis pathway. Review: Mo-AuNPs was injected to the body and reached the target cell. Folic acid in AuNPs bound to folic acid receptors in the cell membrane thus promoting endocytosis and encapsulation of Mo. AuNPs along with irradiation using near infrared light converted light into heat thus promoting pro-apoptotic protein release. This condition was also supported by Mo's ability to downregulate Akt thus upregulating Bad. Bad induces translocation of Bax into the outer mitochondrial membrane then induces the opening of mitochondrial pores. This condition manifests in formation of apoptosomes thus activating caspase-3 and inducing formation of apoptotic bodies. Conclusion: Mo extract-incorporated with folic acid-conjugated AuNPs may potential as an OSCC therapy by modulating intrinsic apoptosis pathway.

Mitochondrial Dysfunction Induced by Zinc Oxide Nanoparticles

The constant evolution and applications of metallic nanoparticles (NPs) make living organisms more susceptible to being exposed to them. Among the most used are zinc oxide nanoparticles (ZnO-NPs). Therefore, understanding the molecular effects of ZnO-NPs in biological systems is extremely important. This review compiles the main mechanisms that induce cell toxicity by exposure to ZnO-NPs and reported in vitro research models, with special attention to mitochondrial damage. Scientific evidence indicates that in vitro ZnO-NPs have a cytotoxic effect that depends on the size, shape and method of synthesis of ZnO-NPs, as well as the function of the cells to which they are exposed. ZnO-NPs come into contact with the extracellular region, leading to an increase in intracellular [Zn2+] levels. The mechanism by which intracellular ZnO-NPs come into contact with organelles such as mitochondria is still unclear. The mitochondrion is a unique organelle considered the “power station” in the cells, participates in numerous cellular processes, such as cell survival/death, multiple biochemical and metabolic processes, and holds genetic material. ZnO-NPs increase intracellular levels of reactive oxygen species (ROS) and, in particular, superoxide levels; they also decrease mitochondrial membrane potential (MMP), which affects membrane permeability and leads to cell death. ZnO-NPs also induced cell death through caspases, which involve the intrinsic apoptotic pathway. The expression of pro-apoptotic genes after exposure to ZnO-NPs can be affected by multiple factors, including the size and morphology of the NPs, the type of cell exposed (healthy or tumor), stage of development (embryonic or differentiated), energy demand, exposure time and, no less relevant, the dose. To prevent the release of pro-apoptotic proteins, the damaged mitochondrion is eliminated by mitophagy. To replace those mitochondria that underwent mitophagy, the processes of mitochondrial biogenesis ensure the maintenance of adequate levels of ATP and cellular homeostasis.

Abstract 3707: Moonlighting function of mitochondrial protein VDAC2 provides a potent pharmacological strategy against selective killing of hepatocellular carcinoma

Abstract Voltage dependent anion channels (VDACs) family of proteins are the most abundant proteins in the outer mitochondrial membrane (OMM). There are three isoforms of VDAC, that are structurally and functionally conserved as far as their ion transport function is concerned. However, there are isoform-specific functions that support distinct pathways. For VDAC2 isoform, an established isoform-specific function is the targeting of the pro-apoptotic Bak protein to the OMM, which stabilizes Bak, leading to its elevated cellular levels. On the surface of OMM, activation and oligomerization of Bak and Bax, another pro-apoptotic protein leads to OMM perforation and release of pro-apoptotic proteins from intermembrane space to the cytoplasm. This is an irreversible step that stimulates the downstream events of apoptotic pathway. Hepatocellular carcinoma (HCC) ranks third in cancer mortality and in the advanced stages is associated with poor prognosis. We observed that unexpectedly, HCC cells are vastly more sensitive to tBid induced apoptosis compared to normal hepatocytes. tBid is a BH3-only pro-apoptotic Bcl-2 family protein, the effect of which is primarily mediated by Bak. We have shown that, in the absence of VDAC2, the Bid-Bak apoptosis pathway is suppressed. VDAC2 protein expression is the lowest in liver and lung among different tissues. We also determined the protein abundances in the HCC lines and normal hepatocytes and found elevated VDAC2 and Bak levels in HCC. Our findings are in line with multiple datasets that show an elevation of VDAC2/Bak expression in human HCC suggesting a potential clinical relevance. We found that combination of an inhibitor of the anti-apoptotic proteins (S63845, a selective Mcl-1 inhibitor), which normally suppresses Bak activation, with an activator of the tBid pathway (TRAIL) enhanced the death of hepatocarcinoma cells with little effect on normal hepatocytes. This treatment strategy was also effective at reducing tumor growth in vivo, but only in tumors expressing VDAC2. Thus, we postulate that increased VDAC2 expression in HCC leads to elevated Bak recruitment to the OMM, which in turn sensitizes HCC to tBid. Therefore, this differential expression provides an excellent opportunity for selective elimination of HCC cells. Citation Format: Piyush Mishra. Moonlighting function of mitochondrial protein VDAC2 provides a potent pharmacological strategy against selective killing of hepatocellular carcinoma [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 3707.

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.

Indole propionic acid induced Ca2+ -dependent apoptosis in Candida albicans.

Indole propionic acid (IPA) which majorly influences the modulation of cellular respiration is a metabolite generated by gut microbiota. The antimicrobial effects of IPA have not been previously demonstrated. Therefore, this study focused on investigating the antimicrobial activity of IPA. Initially, antifungal activity of IPA against Candida albicans was observed, accompanied by variations in mitochondrial respiration indicating modulation of NAD+ /NADH ratios. Consumption of O2 contributes to the respiratory regulation and triggered by Ca2+ overloading. After treatment with IPA, the cells were monitored, and Ca2+ increases leading to membrane depolarization and reactive oxygen species (ROS) accumulation in mitochondria were noted. Depolarization of mitochondria membrane induced release of proapoptotic proteins in mitochondria. Oxidative stress exerted by ROS contributed to glutathione depletion and oxidation of glutathione (GSH). Fragmentation of DNA is a characteristic event leading to apoptosis and accompanies major hallmarks of apoptosis including phosphatidylserine exposure and metacaspase activation. In addition, phosphatidylserine exposure and metacaspase activation were detected in the cell treated with IPA. In conclusion, IPA triggered apoptosis in C. albicans under the influence of Ca2+ .

ASPP2 k, a Dominant-Negative Splicing Variant of the Apoptosis-Stimulating Protein of p53-2 (ASPP2), Modulates Treatment Response Towards BCL-Signaling Inhibitors in Acute Myeloid Leukemia

In recent years, it has become increasingly apparent that BCL-2 inhibition in AML is a clinically highly effective approach - and the first BCL-2 inhibitor, venetoclax, has gained FDA and EMA approval. However, drug resistance frequently occurs and mechanisms or biological markers to predict response to BCL-2 inhibition are urgently needed. ASPP2 plays a central role to orchestrate induction of apoptosis via binding of p53 - but also via binding of antiapoptotic BCL-2, allowing release of proapoptotic proteins. We recently described an alternatively spliced oncogenic ASPP2 isoform (ASPP2k) (Schittenhelm et al., 2019), which is characterized by truncation of the C-terminus allocating the BCL2- as well as the p53 binding sites (in analogy to major TP53 mutations lacking the ASPP2 binding motifs). We therefore hypothesized that expression of ASPP2k attenuates efficacy of pro-apoptotic compounds:AML cell line models, including MOLM13/14 (FLT3 ITD+), OCI-AML3 (NPM1 A+) and HL60, as well as freshly isolated native leukemia blasts (n=40) are used in dose-dilution assays to assess for pro-apoptotic efficacy in annexin V-based assays. Bone marrow donors served as a control population. A lentiviral approach was used for isoform-specific ASPP2k-shRNA transduction. A HisMax vector was used to forcely express ASPP2k. Several compounds targeting BCL-2 signaling, which are under clinical investigation, were tested (BCL-2: venetoclax, BCL-2/Xl: AZD4320, MCL-1: AZD5991 and CDK9: AZD4573).To summarize, leukemia cells demonstrated variable and preferential sensitivity profiles towards the tested compounds: MOLM cells were sensitive towards all tested compounds. In contrast, OCI-AML3 proofed reduced sensitivity towards BCL-2/Xl inhibition - whereas MCL-1 and CDK9 inhibition (indirectly targeting MCL-1 signaling) showed IC50s in the nanomolar range (AZD5991: 307nM, AZD4573: 16nM). Furthermore, HL60 were relatively resistant towards both BCL-2/Xl and MCL-1 inhibition - however remained high sensitivity towards CDK9 inhibition (IC50 25nM). Priming with a hypomethylating agent (HMA, decitabine) resulted in additive (CI close to 1) to synergistic (CI 0.25 - 0.7) proapoptotic effects in isobologram analysis and led to a release of drug resistance in primary resistant cell lines.Exposure of native leukemia cells towards all inhibitors confirmed (individually differing) sensitivity in the nanomolar ranges - whereas bone marrow donor controls were relatively resistant towards the tested compounds, which argues for a therapeutic clinical window.In a last step, we addressed, whether ASPP2k functionally impedes the proapoptotic efficacy observed for the tested compounds. Indeed, we demonstrate that isoform-specific ASPP2k interference similarly results in a significant increase of pro-apoptotic capacities of all tested BCL-2, BCL-2/XL, MCL-1 and CDK9 inhibitors, (resp. attenuation thereof after forcely expressing the dominant-negative splicing variant).To summarize, we show that inhibition of BCL-2 signaling is a promising approach to target acute leukemia - as a monoagent as well as in combination with HMA: Further, we provide a path for exploration of ASPP2k as a predictive tool as well as a therapeutic sensitizer of pro-apoptotic drugs, which will be addressed in future studies. DisclosuresSchittenhelm: BMS: Other: advisory board; Astellas: Other: advisory board; Takeda: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.

Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis.

Angiotensin II- (Ang II-) induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Choline exerts cardioprotective effects; however, its effects on Ang II-induced cardiomyocyte apoptosis are unclear. In this study, the role and underlying mechanism of choline in regulating Ang II-induced cardiomyocyte apoptosis were investigated using a model of cardiomyocyte apoptosis, which was induced by exposing neonatal rat cardiomyocytes to Ang II (10−6 M, 48 h). Choline promoted heat shock transcription factor 1 (HSF1) nuclear translocation and the intracellular domain of Notch1 (NICD) expression. Consequently, choline attenuated Ang II-induced increases in mitochondrial reactive oxygen species (mtROS) and promotion of proapoptotic protein release from mitochondria, including cytochrome c, Omi/high-temperature requirement protein A2, and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P. The reversion of these events attenuated Ang II-induced increases in cardiomyocyte size and numbers of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive cells, presumably via type 3 muscarinic acetylcholine receptor (M3AChR). Indeed, downregulation of M3AChR or Notch1 blocked choline-mediated upregulation of NICD and nuclear HSF1 expression, as well as inhibited mitochondrial apoptosis pathway and cardiomyocyte apoptosis, indicating that M3AChR and Notch1/HSF1 activation confer the protective effects of choline. In vivo studies were performed in parallel, in which rats were infused with Ang II for 4 weeks to induce cardiac apoptosis. The results showed that choline alleviated cardiac remodeling and apoptosis of Ang II-infused rats in a manner related to activation of the Notch1/HSF1 pathway, consistent with the in vitro findings. Taken together, our results reveal that choline impedes oxidative damage and cardiomyocyte apoptosis by activating M3AChR and Notch1/HSF1 antioxidant signaling, and suggest a novel role for the Notch1/HSF1 signaling pathway in the modulation of cardiomyocyte apoptosis.

Osthole suppresses the proliferation and induces apoptosis via inhibiting the PI3K/AKT signaling pathway of endometrial cancer JEC cells.

Osthole, a natural product extracted mainly from fruits of Fructus Cnidii, possesses multiple pharmacological functions, including anti-inflammatory, anti-convulsant and anticancer effects. However, the effects of osthole in endometrial cancer (EC) is not fully understood. In the present study, EC cell lines, including JEC, KLE and Ishikawa cells and normal human cervical epithelial cells (HcerEpic) were applied to detect the anticancer effect of osthole. The present study demonstrated that osthole inhibited the proliferation of JEC, KLE and Ishikawa cells, but had no cytotoxic effect on HcerEpic. Furthermore, treatment of osthole induced JEC cell apoptosis, while osthole promoted the release of pro-apoptotic proteins, Bax and activated the cleaved caspase-3, caspase-9 and PARP. Additionally, osthole significantly increased the expression of PETN and decreased the phosphorylated form of PI3K and AKT in a concentration-dependent manner. Furthermore, osthole treatment suppressed the JEC tumor cell growth in a nude mouse xenograft model in vivo, and neither renal toxicity nor hepatotoxicity was induced by the indicated concentration. Taken together, the results of the present study suggested that osthole may be a novel and potential therapeutic agent of EC.

VDAC1 at the Intersection of Cell Metabolism, Apoptosis, and Diseases.

The voltage-dependent anion channel 1 (VDAC1) protein, is an important regulator of mitochondrial function, and serves as a mitochondrial gatekeeper, with responsibility for cellular fate. In addition to control over energy sources and metabolism, the protein also regulates epigenomic elements and apoptosis via mediating the release of apoptotic proteins from the mitochondria. Apoptotic and pathological conditions, as well as certain viruses, induce cell death by inducing VDAC1 overexpression leading to oligomerization, and the formation of a large channel within the VDAC1 homo-oligomer. This then permits the release of pro-apoptotic proteins from the mitochondria and subsequent apoptosis. Mitochondrial DNA can also be released through this channel, which triggers type-Ι interferon responses. VDAC1 also participates in endoplasmic reticulum (ER)-mitochondria cross-talk, and in the regulation of autophagy, and inflammation. Its location in the outer mitochondrial membrane, makes VDAC1 ideally placed to interact with over 100 proteins, and to orchestrate the interaction of mitochondrial and cellular activities through a number of signaling pathways. Here, we provide insights into the multiple functions of VDAC1 and describe its involvement in several diseases, which demonstrate the potential of this protein as a druggable target in a wide variety of pathologies, including cancer.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Synergistic Activity of the MCL-1-Specific Inhibitor S63845 with Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

Deregulated cell death and survival pathways contribute to leukemogenesis and treatment failure of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The intrinsic apoptosis pathway is regulated at the mitochondrial level by different pro- and anti-apoptotic molecules. Members of the BCL-2 family are key regulators of mitochondrial apoptosis signaling. Pro-apoptotic BH3-only proteins like BIM and BID activate pro-death proteins such as BAX and BAK leading to cell death. Anti-apoptotic BCL-2 family members including BCL-2, BCL-XL and MCL-1 bind to and sequester pro-apoptotic molecules, prevent activation of pro-death proteins and counter-regulate apoptosis induction. Small molecule inhibitors have been developed that block binding to anti-apoptotic molecules like BCL-2, leading to release of pro-apoptotic proteins and cell death induction. In particular, the BCL-2-specific inhibitor venetoclax (VEN) has demonstrated substantial anti-cancer activity and became an approved drug for the treatment of CLL patients. Investigating different, individual BCP-ALL samples, we and others recently identified heterogeneous sensitivities for VEN, suggesting that BCP-ALL cells might also depend on other pro-survival BCL-2 family proteins including MCL-1, leading to VEN insensitivity and resistance. A novel BH3-mimetic, S63845, that selectively targets MCL-1 has been reported.Here, we assessed the activity of S63845 and addressed a potential synergism of simultaneous blockage of BCL-2 and MCL-1 by VEN and S63845 (S) in BCP-ALL.The activity of the MCL-1 inhibitor was analyzed in a panel of BCP-ALL cell lines (N=6) and a series of primary, patient-derived BCP-ALL primograft samples (N=27) determining half-maximal effective concentrations (EC50) upon exposure to increasing concentrations of S and analysis of cell death induction. We observed heterogeneous sensitivities to S with EC50 values ranging from 16 nM to almost 10 µM. Protein expression of MCL-1 and other BCL-2 family members BCL-2, BCL-XL and BCL-W was assessed by western blot analysis and quantified, however neither association of MCL-1 levels nor expression of the other regulators and S sensitivity was found in cell lines and primograft leukemias. Moreover, we also compared sensitivities for both inhibitors but found independent activities of S and VEN in individual ALL samples.Next, we addressed the role of MCL-1 for VEN sensitivity and generated two MCL-1 knock out BCP-ALL cell lines by CRISPR/Cas9 gene editing. In both lines, clearly increased VEN sensitivities were observed upon depletion of MCL-1, indicating that MCL-1 is contributing to activity of the BCL-2 inhibitor VEN.Based on these findings, we investigated the effects of pharmacological MCL-1 inhibition for VEN sensitivity and incubated all 6 cell lines with VEN and S at increasing concentrations and observed clear synergistic effects upon combined BCL-2 and MCL-1 inhibition indicated by combination indices (CI) below 0.1. Moreover, we investigated 7 primograft BCP-ALL samples and found that MCL-1 inhibition by S clearly synergized with VEN activity (CI < 0.3).To investigate the anti-leukemia activity of co-targeting BCL-2 and MCL-1 in vivo in a pre-clinical setting, a high-risk leukemia derived from an infant, MLL/ENL rearranged pro-B ALL case was transplanted onto NOD/SCID mice. Upon ALL manifestation (presence of >5% human blasts in blood), recipients were treated with either VEN, S, the combination of both, or vehicle for 10 days. After treatment, leukemia loads were analyzed showing significantly reduced loads in the co-treated group as compared to vehicle, VEN or S alone in spleen, bone marrow, and central nervous system (p-values < 0.05), indicating synergistic activity of co-inhibition of BCL-2 and MCL-1 in vivo.Taken together, our data show heterogeneous sensitivity of individual BCP-ALL samples to MCL-1 inhibition by S, which is not associated with MCL-1 protein expression levels or VEN sensitivity. Both, genetic depletion and inhibition of MCL-1 by S synergizes with VEN leading to increased anti-leukemia activity in vitro and ex vivo. Importantly, co-targeting BCL-2 and MCL-1 significantly reduced leukemia infiltration in spleen, BM and CNS in a pre-clinical model of high-risk BCP-ALL, warranting further evaluation and possible clinical application of targeting MCL-1 alone and in combination with BCL-2 inhibition. DisclosuresNo relevant conflicts of interest to declare.

Astragaloside IV reduces neuronal apoptosis and parthanatos in ischemic injury by preserving mitochondrial hexokinase-II

Cerebral ischemia induces neuronal cell death in different ways and mitochondrial dysfunction is an important cause. Astragaloside IV (AIV) is a natural saponin abandent in Astragalus membranaceus and this study aims to find if AIV protects neuronal survival via preserving mitochondrial hexokinase-II (HK-II). Glutamate stimulation induced HK-II dissociation from mitochondria and impaired mitochondrial function, indicated by the opening of the mitochondrial permeability transition pore, the collapse of mitochondrial membrane potential and reduced mitochondrial oxygen consumption ratio in neurons. Accompanied with apoptosis, oxidative DNA damage, PAR formation and nuclear translocation of apoptosis inducing factor (AIF) indicated the presence of parthanatos. AIV activated Akt and protected mitochondrial HK-II via promoting the binding of Akt to HK-II and protected hexokinase activity with improved glycolysis. As a consequence of preserved mitochondrial HK-II, AIV reduced the release of pro-apoptotic proteins and AIF, resultantly protected neurons from apoptosis and parthanatos. Moreover, the neuroprotective effects of AIV were also reproduced in mice subjected to middle cerebral artery occlusion to support the findings in vitro. Together, these results showed that glutamate excitotoxicity impaired mitochondrial HK-II and simultaneously induced apoptosis and parthanatos owing to mitochondrial dysfunction. AIV activated Akt to promote HK-II binding to mitochondria, and the structural and functional integrity of mitochondria contributed to protecting neurons from apoptosis and DNA damage. These findings address the important role of mitochondrial HK-II in neuronal protection.

Astragaloside IV Reduces Neuronal Apoptosis and Parthanatos in Ischemic Injury by Preserving Mitochondrial Hexokinase-II

Background: Cerebral ischemia induces neuronal cell death in different ways and mitochondrial dysfunction is an important cause. Astragaloside IV (AIV) is a natural saponin abandent in Astragalus membranaceus and this study aims to find if AIV protects neuronal survival via preserving mitochondrial hexokinase-II (HK-II). Methods: The primary cortical neuron were prepared to observe the effects of AIV on neuroprotection with focus on the regulation of mitochondrial HK-II via Akt activation. Meanwhile, we also observed the protection in mice MCAO model. Results: Glutamate stimulation induced HK-II dissociation from mitochondria and impaired mitochondrial function, indicated by the opening of the mitochondrial permeability transition pore and the collapse of mitochondrial membrane potential. Accompanied with apoptosis, oxidative DNA damage, PAR formation and nuclear translocation of apoptosis inducing factor (AIF) indicated the presence of parthanatos. AIV activated Akt and protected mitochondrial HK-II via promoting the binding of Akt to HK-II. As a consequence of preserved mitochondrial HK-II, AIV reduced the release of pro-apoptotic proteins and AIF, resultantly protected neurons from apoptosis and parthanatos. Moreover, the neuroprotective effects of AIV were also reproduced in mice subjected to middle cerebral artery occlusion to support the findings in vitro. Interpretation: Glutamate excitotoxicity impaired mitochondrial HK-II and simultaneously induced apoptosis and parthanatos owing to mitochondrial dysfunction. AIV activated Akt to promote HK-II binding to mitochondria, and the structural and functional integrity of mitochondria contributed to protecting neurons from apoptosis and DNA damage. These findings address the important role of mitochondrial HK-II in neuronal protection. Funding:This study is funded by National Natural Science Foundation of China (Grant No. 81703738) and Foundation of The Science and Technology Department of Henan Province (Grant No. 182102310169) Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The animal care and all experimental procedures were approved by Animal Ethics Committee of China Pharmaceutical University.

Uncoupling Protein Overexpression in Metabolic Disease and the Risk of Uncontrolled Cell Proliferation and Tumorigenesis.

In metabolic diseases such as obesity, metabolic syndrome and type II diabetes, the over-expression of uncoupling proteins (UCPs) in a response to increased reactive oxygen species (ROS) generation by mitochondrial respiratory complexes, and to the excess of free fatty acid (FFA) supply from adipose tissue, may protect cells from oxidative stress, lipotoxicity and in turn from death. UCPs by reducing superoxide anion and H2O2 generation trigger several signals to cell for their adaptation to the lipotoxic microenvironment. In mitochondria, a decrease of cytochrome c (cyt c) and proapoptotic protein release promotes cell survival and proliferation. The altered lipid metabolism also affects cardiolipin susceptibility to the peroxidation, a process involved in the dissociation of cyt c from mitochondrial inner membrane and its release, a key step of apoptosis. Therefore, UCPs by attenuating ROS generation and lipotoxicity may downregulate programmed cell death, a well-known physiological process controlling cell proliferation contributing to uncontrolled cell proliferation and tumorigenesis. In addition, tumor cells over-expressed UCPs, by inhibiting ROS generation acquire resistance to death during pharmacological treatment with oxidative stress drug inducers. Therefore, the aim of this review is to discuss recent findings regarding the role that UCPs play in cell survival by protecting against ROS generation and maintaining bioenergetic metabolism homeostasis to promote cell proliferation.

XIAP impairs mitochondrial function during apoptosis by regulating the Bcl-2 family in renal cell carcinoma.

Efficient apoptosis requires Bcl-2 family-mediated mitochondrial outer membrane permeabilization (MOMP), which releases pro-apoptotic proteins to the cytosol, activating apoptosis and inhibiting X-linked inhibitor of apoptosis protein (XIAP). XIAP is a member of the inhibitors of apoptosis protein family whose expression is elevated in many cancer types and participates in the release of pro-apoptotic proteins. To explore the association between XIAP and the Bcl-2 family, and the influence of XIAP on mitochondria, RNA interference of XIAP was performed in Caki-1 cells and the dynamic change in the levels of related proteins was compared with the original Caki-1 cells upon induction of apoptosis. Upon knockdown of XIAP, the release of cytochrome c (Cyt-c), second mitochondria-derived activator of caspase (Smac) and apoptotic protease activating factor 1 (Apaf-1) from mitochondria proceeded normally, whereas in Caki-1 cells, the release of these pro-apoptotic proteins was significantly prolonged, and incomplete. Downregulation of XIAP through small interfering RNA resulted in an increase of apoptosis and a marked decrease in Bcl-2 and Bcl-xl levels at 3 h. Additionally, the regulation of the level of XIAP protein affected the specific ratios of Bcl-2/Bax and Bcl-xl/Bax, which play decisive roles in cell death. In the present study, it was revealed that XIAP can feed back to mitochondria, delaying Cyt-c and Apaf-1 release. Furthermore, XIAP can limit the release of its inhibitor Smac with the involvement of Bcl-2 family proteins.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

Summary The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.