Regulation Of Bcl-2 Family Proteins Research Articles

Evaluation of Efficiency of Liposome-Entrapped Iridium(III) Complexes Inhibiting Tumor Growth In Vitro and In Vivo.

In this paper, three new iridium(III) complexes: [Ir(piq)2(DFIPP)]PF6 (piq = deprotonated 1-phenylisoquinoline, DFIPP = 3,4-difluoro-2-(1H-imidazo[4,5-f][1,10]phenenthrolin-2-yl)phenol, 3a), [Ir(bzq)2(DFIPP)]PF6 (bzq = deprotonated benzo[h]quinoline, 3b), and [Ir(ppy)2(DFIPP)]PF6 (ppy = deprotonated 1-phenylpyridine, 3c), were synthesized and characterized. The complexes were found to be nontoxic to tumor cells via 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide (MTT) assay. Surprisingly, its liposome-entrapped complexes 3alip, 3blip, and 3clip on B16 cells showed strong cytotoxicity (IC50 = 13.6 ± 2.8, 9.6 ± 1.1, and 18.9 ± 2.1 μM). Entry of 3alip, 3blip, and 3clip into B16 cells decreases mitochondrial membrane potential, regulates Bcl-2 family proteins, releases cytochrome c, triggers caspase family cascade reaction, and induces apoptosis. In addition, we also found that 3alip, 3blip, and 3clip triggered ferroptosis and autophagy. In vivo studies demonstrated that 3blip inhibited melanoma growth in C57 mice with a high inhibitory rate of 83.95%, and no organic damage was found in C57 mice.

Nuclear export of PML promotes p53-mediated apoptosis and ferroptosis

Promyelocytic leukemia protein (PML), a tumor suppressor protein, plays a key role in cell cycle regulation, apoptosis, senescence and cellular metabolism. Here, we report that PML promotes apoptosis and ferroptosis. Our data showed that PML over-expression inhibited cell proliferation and migration. PML over-expression increased apoptotic cells, nuclear condensation and the loss of mitochondrial membrane potential, accompanied by regulation of Bcl-2 family proteins and reactive oxygen species (ROS) level, suggesting that PML enhanced apoptosis. Meanwhile, PML over-expression not only increased lipid ROS accumulation and Malondialdehyde (MDA) content but also downregulated solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression, indicating that PML enhanced ferroptosis. Additionally, knockdown of p53 attenuated the effect of PML on SLC7A11 and GPX4, and inhibited the increase of lipid ROS and ROS by PML over-expression. Moreover, translocation of PML from nucleus to cytoplasm not only promoted apoptosis and ferroptosis, but also inhibited cell proliferation. Taken together, PML promotes apoptosis and ferroptosis, in which the mediation of p53 and the nuclear export of PML play important roles.

Dysregulation of BCL-2 family proteins in blood neoplasm: therapeutic relevance of antineoplastic agent venetoclax

During cellular stress, the master regulators of intrinsic self-death (apoptosis) are BCL-2 family proteins. The BCL-2 family proteins play a key role in apoptosis and are tightly regulated via other BCL-2 family proteins, non-BCL-2 protein suppressors, and epigenetic modifications. As the name implies, these proteins possess one or two of the four BCL-2 homology domains (BH1–BH4). According to their roles, they are classified as pro-apoptotic or pro-survival proteins. BH-3-only proteins possess a single BH3 domain and are specific/key effector proteins for intracellular death commitment, particularly in the context of cell survival and programmed cell death. This delicate interplay among the BCL-2 family members is essential for maintaining the primary hemostasis, or balance, of cell fate. The anti-apoptotic proteins, such as BCL-2 and BCL-XL, promote cell survival by inhibiting apoptosis. On the other hand, the pro-apoptotic proteins, such as BAX and BAK, drive apoptosis. It ensures that cells are able to respond appropriately to various internal and external signals, ultimately determining whether a cell survives or undergoes programmed cell death. Understanding and targeting this delicate balance is a promising avenue for developing therapeutic strategies to modulate cell fate and treat various diseases. The molecular pathogenesis of BCL-2 family proteins in blood disorders involves differential expression of these components resulting in the dysregulation of the pathway contributing to cell survival and resistance to apoptosis as observed in follicular lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and acute myeloid leukemia. Such dysregulation is a major impediment to standard therapies and aids in chemo resistance. Studies show some promising clinical outcomes with antineoplastic agent venetoclax either as a monotherapy or in combination with other agents. This review discusses recent studies on the regulation of BCL-2 family proteins which might provide a molecular landscape for their clinical implications in blood disorders.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Neuroprotective effects of chlorogenic acid by controlling the Bcl-2 family protein in a stroke animal model.

Ischemic stroke causes severe brain damage and high mortality. Chlorogenic acid is a phenolic compound that has neuroprotective properties. B-cell lymphoma-2 (Bcl-2) family proteins are important for apoptosis regulation. Bcl-2 and Bcl-xL are proteins that inhibit apoptosis, and Bax and Bad induce apoptosis. In this study, we investigated whether chlorogenic acid exerts a neuroprotective effect against ischemic stroke damage by regulating Bcl-2 family proteins. We performed middle cerebral artery occlusion (MCAO) to induce ischemic stroke in adult male rats. The animals were intraperitoneally injected with normal saline as a vehicle or chlorogenic acid (30 mg/kg) 2 hr after MCAO. Cerebral cortex tissue was collected 24 hr after MCAO damage. MCAO damage caused histopathological changes and increased the number of terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling-positive cells, while chlorogenic acid attenuated these changes. RT-qPCR and Western blot results showed decreases in Bcl-2 and Bcl-xL expression and an increase in Bax and Bad expression in MCAO animals. However, chlorogenic acid treatment attenuated these changes due to MCAO damage. The interaction of Bax with Bcl-2 or Bcl-xL decreased in MCAO animals, and the binding of Bad with Bcl-2 or Bcl-xL increased. However, chlorogenic acid treatment reduced these changes. Chlorogenic acid also prevented MCAO-induced increases in caspase-3 and caspase-9 expression. This study provides evidence that chlorogenic acid has neuroprotective effects against MCAO damage by modulating Bcl-2 family proteins including Bcl-2, Bcl-xL, Bax, and Bad. Furthermore, chlorogenic acid regulates the interaction between Bcl-2 family proteins. In conclusion, chlorogenic acid contributes to neuroprotection against ischemic stroke damage by controlling Bcl-2 family proteins.

Apoptosis Regulators Bcl-2 and Caspase-3

Apoptosis, programmed cell death, has a central role in developmental biology and in maintaining the equilibrium of renewing tissues. A founding member of the Bcl-2 family of regulatory proteins for apoptosis is Bcl-2, which is encoded by the BCL2 gene. Caspase-3 shares typical features with all caspases, including the role of acting as a crucial mediator of apoptosis.

Abstract 2150: LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade

Abstract The recent approval of the KRAS G12C inhibitor sotorasib (AMG 510) for non-small cell lung cancer (NSCLC) marked a milestone in the development of targeted therapies for KRAS mutant cancers. While sotorasib and other KRAS G12C inhibitors have demonstrated rapid and durable responses in the clinic, some patients do not achieve responses. The identification of specific vulnerabilities conferred by recurrent co-occurring mutations may enable the development of biomarker-driven combination therapies with enhanced activity in distinct subsets of patients. We screened a panel of KRAS-mutant NSCLC cell lines as well as patient-derived xenograft (PDX) mouse models and observed that loss of the tumor suppressor STK11/LKB1 is associated with increased sensitivity to combined MAPK (either the KRAS G12C inhibitor sotorasib or MEK inhibitor trametinib) and MCL-1 inhibition (AMG 176). Restoration of LKB1 expression in LKB1-deficient cell lines and PDX tumors blunted the apoptotic response to MAPK + MCL-1 inhibition; conversely, deletion of LKB1 in LKB1 wild-type models increased sensitivity. Mitochondrial apoptotic cell death is regulated by interactions between pro- (e.g., BIM) and anti-apoptotic (e.g., MCL-1, BCL-XL) BCL-2 family members. MAPK inhibition increases BIM, while MCL-1 inhibition prevents BIM sequestration by MCL-1, resulting in apoptosis. LKB1 deficient cells exhibit increased association of BIM and MCL-1 upon MAPK inhibition, effectively priming cells for death upon inhibition of MCL-1. Mechanistically, LKB1 deficiency and associated loss of NUAK phosphorylation leads to hyperactivation of the JNK phospho-kinase network. JNK phosphorylates MCL-1 at S64 and T163, which enhances BIM: MCL-1 protein-protein interaction. Conversely, JNK phosphorylates BCL-XL at S62 and prevents sequestration of BIM. This series of phosphorylation events increases MCL-1 dependence and creates a specific vulnerability of KRAS-LKB1 tumors to MAPK + MCL-1 inhibition. Consistent with this mechanism, ex vivo treatment of tumor tissue from a KRAS-LKB1 mutant NSCLC patient with sotorasib or trametinib increased MCL-1 dependent priming. These results reveal a novel link between LKB1 and the regulation of BCL-2 family proteins and provide preclinical rationale for evaluation of combined KRAS G12C + MCL-1 inhibitors for KRAS-LKB1 mutant NSCLC. Citation Format: Chendi Li, Mohammed Usman Syed, Yi Shen, Audris Oh, Cameron Fraser, Johannes Kreuzer, Christopher Nabel, Kaitlyn Webster, Robert Morris, Sean Caenepeel, Anne Y. Saiki, Karen Rex, J. Russell Lipford, Wilhelm Hass, Kristopher Sarosiek, Paul E. Hughes, Aaron Hata. LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade [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 2150.

Regulation of Bcl-2 Family Proteins in Estrogen Receptor-Positive Breast Cancer and Their Implications in Endocrine Therapy.

Simple SummaryBreast cancer is the leading cause of cancer-related deaths in the female population, with estrogen receptor (ER)-positive breast cancer accounting for two-third of incidents. Endocrine therapy has proven beneficial in treating ER-positive breast cancer. However, resistance acquired toward therapy remains a drawback in treatment. The activation of signaling pathways regulated by estrogen receptors has been linked with the evasion of cell death and drug resistance. An important role in regulating signals determining cell survival or death is assigned to the Bcl-2 family of proteins. Since the upregulation of anti-survival Bcl-2 proteins has been associated with decreased endocrine therapy efficacy and resistance, this review focuses on the molecular regulation of this group of proteins in ER-positive breast cancer and their implications in endocrine therapy treatment. Furthermore, advancements in the development of agents targeting the Bcl-2 family proteins have been overviewed, and their application in ER-positive breast cancer is presented.Estrogen receptor (ER)-positive breast cancer accounts for around two-thirds of breast cancer occurrences, with endocrine therapy serving as first-line therapy in most cases. Targeting estrogen signaling pathways, which play a central role in regulating ER+ breast cell proliferation and survival, has proven to improve patient outcomes. However, despite the undeniable advantages of endocrine therapy, a subset of breast cancer patients develop acquired or intrinsic resistance to ER-targeting agents, limiting their efficacy. The activation of downstream ER signaling pathways upregulates pro-survival mechanisms that have been shown to influence the response of cells to endocrine therapy. The Bcl-2 family proteins play a central role in cell death regulation and have been shown to contribute to endocrine therapy resistance, supporting the survival of breast cancer cells and enhancing cell death evasion. Due to the overexpression of anti-apoptotic Bcl-2 proteins in ER-positive breast cancer, the role of these proteins as potential targets in hormone-responsive breast cancer is growing in interest. In particular, recent advances in the development of BH3 mimetics have enabled their evaluation in preclinical studies with ER+ breast cancer models, and BH3 mimetics have entered early ER+ breast cancer clinical trials. This review summarizes the molecular mechanisms underlying the regulation of Bcl-2 family proteins in ER+ breast cancer. Furthermore, an overview of recent advances in research regarding the efficacy of BH3 mimetics in ER+ breast cancer has been provided.

Functionalized Selenium Nanotherapeutics Synergizes With Zoledronic Acid to Suppress Prostate Cancer Cell Growth Through Induction of Mitochondria-Mediated Apoptosis and Cell Cycle S Phase Arrest.

The use of established drugs in new therapeutic applications has great potential for the treatment of cancers. Nanomedicine has the advantages of efficient cellular uptake and specific cell targeting. In this study, we investigate using lentinan-functionalized selenium nanoparticles (LET-SeNPs) for the treatment of prostate cancer (PCa). We used assays to demonstrate that a combination of LET-SeNPs and zoledronic acid (ZOL) can reduce PCa cell viability in vitro. Stability and hemocompatibility assays were used to determine the safety of the combination of LET-SeNPs and ZOL. The localization of LET-SeNPs was confirmed using fluorescence microscopy. JC-1 was used to measure the mitochondrial membrane potential, while the cellular uptake, cell cycle and apoptosis were evaluated by flow cytometry. Finally, cell migration and invasion assays were used to evaluate the effects of the combination treatment on cell migration and invasion. Under optimized conditions, we found that LET-SeNPs has good stability. The combination of LET-SeNPs and ZOL can effectively inhibit metastatic PCa cells in a concentration-dependent manner, as evidenced by cytotoxicity testing, flow cytometric analysis, and mitochondria functional test. The enhanced anti-cancer effect of LET-SeNPs and ZOL may be related to the regulation of BCL2 family proteins that could result in the release of cytochrome C from the inner membranes of mitochondria into the cytosol, accompanied by induction of cell cycle arrest at the S phase, leading to irreversible DNA damage and killing of PCa cells. Collectively, the results of this study suggest that the combination of SeNPs and ZOL can successfully inhibit the growth of PCa cells.

Retinoic acid exerts neuroprotective effects against focal cerebral ischemia by preventing apoptotic cell death

Cerebral ischemia is a neurological disorder that leads to cognitive decline and high mortality. Retinoic acid is a metabolite of vitamin A that has anti-inflammatory and anti-apoptotic effects. This study investigated whether retinoic acid prevents neuronal cell damage on focal cerebral ischemia through modulating apoptosis signaling pathway. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in adult male rats. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally for 4 days prior to MCAO. Neurological behavior deficit tests were performed 24 h after MCAO. Brain edema and infarct volume were measured, and TUNEL histochemistry was carried out. We also investigated the changes in apoptosis-related proteins including bcl-2 family proteins and caspases. MCAO injury induced severe neurological behavior deficits and brain edema. It also increased infarct volume, histopathological damages, and the number of TUNEL-positive cells in cerebral cortex. However, retinoic acid pretreatment attenuated MCAO-induced neurological behavior deficits, brain edema, and infarction. It also alleviated histopathological lesion and decreased the number of TUNEL-positive cells. Bcl-2 and bax proteins are representative bcl-2 family proteins. MCAO injury induced a decrease in bcl-2 expression and an increase in bax expression, and retinoic acid pretreatment alleviated these changes. MCAO injury caused a decrease in bcl-2/bax expression ratio in cerebral cortex, while retinoic acid restored this decrease by MCAO. Moreover, our result showed increases in caspase-9, caspase-3, PARP protein levels in MCAO-operated animals. Retinoic acid pretreatment prevented these increases. We identified the changes in cleaved forms of these proteins, similar to the changes in full-length protein. Activation of caspases and PARP proteins are considered to be representative apoptosis indicators. This study showed that retinoic acid regulates bcl-2 family proteins and caspase proteins in focal cerebral ischemia. Thus, our findings demonstrate that retinoic acid exhibits a neuroprotective effect against ischemic damage by modulating apoptosis signaling pathway.

Danggui Buxue Tang Rescues Folliculogenesis and Ovarian Cell Apoptosis in Rats with Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is a common female endocrine disease that is closely linked to ovarian function. Danggui Buxue Tang (DBT) is a classic prescription of traditional Chinese medicine that is helpful for rescuing ovarian function. However, the mechanism by which DBT rescues ovarian function remains unclear. This study explored the molecular mechanism of DBT with respect to apoptosis and related signals in ovarian cells. The quality control of DBT was performed by HPLC. After DBT intervention in the POI rat model, serum AMH/FSH/LH/E2 levels were detected by ELISA, follicles at various developmental stages were observed by HE staining, apoptosis was detected by TUNEL, and the expression profiles of Bcl-2 family proteins and key proteins in the Jak2/Foxo3a signaling pathway were evaluated by western blot. The results demonstrated that DBT could encourage the development of primary/secondary/antral follicles and increase the secretion of AMH. Moreover, DBT might inhibit Foxo3a by upregulating Jak2, thereby mediating Bcl-2 family activities and inhibiting apoptosis in ovarian cells. In conclusion, DBT promotes follicular development and rescues ovarian function by regulating Bcl-2 family proteins to inhibit cell apoptosis, which could be related to the Jak2/Foxo3a signaling pathway.

Achyranthes aspera L. leaf extract induced anticancer effects on Dalton's Lymphoma via regulation of PKCα signaling pathway and mitochondrial apoptosis

Ethnopharmacological relevanceEpidemiological studies promote the inclusion of natural-products in diet due to their inhibitory effects on various types of cancer. Among them, Achyranthes aspera L. (Family Amaranthaceae) is a medicinal plant in Ayurvedic pharmacopeia, found in India, Southeast Asia, America, and Sub-Saharan Africa. It is endowed with anti-inflammatory, anti-oxidant, and anti-cancer activities. However, its potential effect on Non-Hodgkin lymphomas (NHLs), has not yet been clarified. Aim of the studyIn the present study, we aimed to investigate the effect of Achyranthes aspera L. leaf extracts on highly aggressive murine NHL called Dalton's Lymphoma (DL) in vitro and in vivo. Material and methodsGC-HRMS analysis was carried out for the identification of compounds present in A. aspera leaf extract. The cytotoxicity of various A. aspera leaf extracts was evaluated on DL cells by MTT assay. Chromatin condensation, nuclear fragmentation, and morphological changes were observed by microscopy technique. Flow cytometry was used to measure the changes in mitochondrial membrane potential (ΔΨm) and apoptosis. In addition, the expressions of apoptosis-related proteins were detected by western blotting. Meanwhile, the in vivo anti-tumor effect of leaf extract was tested in DL induced Balb/c mice. ResultGC-HRMS analysis of A. aspera methanolic leaf extract (AAML) revealed the presence of ten pharmacologically active compounds. The results showed that AAML suppressed cell proliferation, decreased mitochondrial membrane potential, changed the morphological structure, and induced apoptosis. Moreover, AAML could promote the release of cytochrome c by regulating Bcl-2 family proteins and then activated caspase-9/ -3 to triggered cell apoptosis. At the same time in DL cells treated with AAML, the protein kinase Cα (PKCα) pathway was inhibited in a concentration-dependent manner. Remarkably, in vivo, AAML mediated suppression of DL growth in Balb/c mice was accompanied by attenuation of the PKCα pathway and induction of apoptosis. Our result suggested that AAML promotes mitochondrial apoptotic cascade in DL cells by suppressing the PKCα signaling pathway. ConclusionThe study suggests that AAML could potently suppress DL progression by promoting apoptosis via mitochondrial-cascade and attenuation of the PKCα signaling pathway.

Apigenin 7-O-glucoside promotes cell apoptosis through the PTEN/PI3K/AKT pathway and inhibits cell migration in cervical cancer HeLa cells

Epidemiologic evidence promote the inclusion of flavones in diet due to their inhibitory effects on certain types of cancers, particularly in women. Among the naturally occurring plant flavonoids, Apigenin 7-O-glucoside (AGL) is endowed with anti-inflammatory, anti-oxidant, and anti-cancer activities. However, its mechanism of action on cervical cancer, the fourth largest cancer in women, has not yet been clarified. In the current study, we have determined the effect of AGL on human cervical cancer cells and studied its molecular mechanism against cervical cancer. The results showed that AGL inhibited the proliferation of HeLa cells (IC50 was 47.26 μM at 48 h) by inducing apoptosis. Furthermore, AGL treatment caused G0/G1 phase arrest, reduced mitochondrial membrane potential (MMP), and upgraded intracellular ROS production. AGL could promote the release of cytochrome c by regulating Bcl-2 family proteins, and then activated caspase 9/3 to promote cell apoptosis. Moreover, AGL treatment promoted the expression of p16 INK4A, while inhibited the expression of Cyclin A/D/E and CDK2/6. At the same time in HeLa cells treated with AGL, the PTEN/PI3K/AKT pathway was inhibited in a concentration-dependent manner, and cell migration was also impeded correspondingly through the matrix metalloproteinase 2 and 9. Our study may provide a new research direction for harnessing the novel natural compounds in cervical cancer treatment.

SP6616 as a Kv2.1 inhibitor efficiently ameliorates peripheral neuropathy in diabetic mice.

BackgroundDiabetic peripheral neuropathy (DPN) is a common complication of diabetes severely afflicting the patients, while there is yet no effective medication against this disease. As Kv2.1 channel functions potently in regulating neurological disorders, the present work was to investigate the regulation of Kv2.1 channel against DPN-like pathology of DPN model mice by using selective Kv2.1 inhibitor SP6616 (ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate) as a probe.MethodsSTZ-induced type 1 diabetic mice with DPN (STZ mice) were defined at 12 weeks of age (4 weeks after STZ injection) through behavioral tests, and db/db (BKS Cg-m+/+Leprdb/J) type 2 diabetic mice with DPN (db/db mice) were at 18 weeks of age. SP6616 was administered daily via intraperitoneal injection for 4 weeks. The mechanisms underlying the amelioration of SP6616 on DPN-like pathology were investigated by RT-PCR, western blot and immunohistochemistry technical approaches against diabetic mice, and verified against the STZ mice with Kv2.1 knockdown in dorsal root ganglion (DRG) tissue by injection of adeno associated virus AAV9-Kv2.1-RNAi. Amelioration of SP6616 on the pathological behaviors of diabetic mice was assessed against tactile allodynia, thermal sensitivity and motor nerve conduction velocity (MNCV).FindingsSP6616 treatment effectively ameliorated the threshold of mechanical stimuli, thermal sensitivity and MNCV of diabetic mice. Mechanism research results indicated that SP6616 suppressed Kv2.1 expression, increased the number of intraepidermal nerve fibers (IENFs), improved peripheral nerve structure and vascular function in DRG tissue. In addition, SP6616 improved mitochondrial dysfunction through Kv2.1/CaMKKβ/AMPK/PGC-1α pathway, repressed inflammatory response by inhibiting Kv2.1/NF-κB signaling and alleviated apoptosis of DRG neuron through Kv2.1-mediated regulation of Bcl-2 family proteins and Caspase-3 in diabetic mice.InterpretationOur work has highly supported the beneficial of Kv2.1 inhibition in ameliorating DPN-like pathology and highlighted the potential of SP6616 in the treatment of DPN.FundingPlease see funding sources.

Combining triptolide with ABT-199 is effective against acute myeloid leukemia through reciprocal regulation of Bcl-2 family proteins and activation of the intrinsic apoptotic pathway

Bcl-2 inhibitors display an effective activity in acute myeloid leukemia (AML), but its clinical efficacy as a monotherapy was limited in part owing to failure to target other antiapoptotic Bcl-2 family proteins, such as Mcl-1. In this context, the combination strategy may be a promising approach to overcome this barrier. Here, we report the preclinical efficacy of a novel strategy combining ABT-199 with triptolide (TPL), a natural product extracted from a traditional Chinese medicine, in AML. Combination treatment exhibited markedly increased cytotoxicity in leukemic cells irrespective of p53 status while largely sparing normal cells of the hematopoietic lineage. Moreover, co-administration of ABT-199 with TPL dramatically suppressed leukemia progression as well as prolonged animal survival in a xenograft AML model. The potentiated effect of ABT-199 and TPL against AML was associated with activation of the mitochondrum-related intrinsic apoptotic pathway through a mechanism reciprocally modulating Bcl-2 family proteins. In this case, TPL not only downregulated Mcl-1 but also upregulated proapoptotic BH3-only proteins, thereby overcoming the resistance toward ABT-199. Conversely, ABT-199 abrogated Bcl-2-mediated cytoprotection against TPL. Together, these findings suggest that the regimen combining TPL and ABT-199 might be active against AML by inducing robust apoptosis through reciprocal regulation of anti- and proapoptotic Bcl-2 family proteins, therefore providing a strong rationale for the clinical investigation of this combination regimen for the treatment of AML.

Liquiritin inhibits proliferation and induces apoptosis in HepG2 hepatocellular carcinoma cells via the ROS-mediated MAPK/AKT/NF-κB signaling pathway.

Liquiritin (LIQ), a major constituent of Glycyrrhiza Radix, exhibits various pharmacological activities. In this study, to explore the potential anti-cancer effects and its underlying molecular mechanisms of LIQ in hepatocellular carcinoma (HCC) cells. LIQ significantly decreased viability and induced apoptosis in HepG2 cells by decreasing mitochondrial membrane potential and regulating Bcl-2 family proteins, cytochrome c, cle-caspase-3, and cle-PARP. The cell cycle analysis and western blot analysis revealed that LIQ induced G2/M phase arrest through increased expression of p21 and decreased levels of p27, cyclin B, and CDK1/2. The flow cytometry and western blot analysis also suggested that LIQ promoted the accumulation of ROS in HepG2 cells and up-regulated the phosphorylation expression levels of p38 kinase, c-Jun N-terminal kinase (JNK), and inhibitor of NF-κB (IκB-α); the phosphorylation levels of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), signal transducer activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) were down-regulated. However, these effects were reversed by N-acetyl-L-cysteine (NAC), MAPK, and AKT inhibitors. The findings demonstrated that LIQ induced cell cycle arrest and apoptosis via the ROS-mediated MAPK/AKT/NF-κB signaling pathway in HepG2 cells, and the LIQ may serve as a potential therapeutic agent for the treatment of human HCC.

Evaluation of toxicity of 4-octylphenol in TM4 Sertoli cells: an in vitro study

The reproductive toxicity of 4-octylphenol (4-OP) has been studied in animals such as mouse and fish. In humans, the exposure of sperm to 4-OP has been shown to decrease motility and viability. In this study, we performed an in vitro assessment of the toxic effects of 4-OP on mouse TM4 Sertoli cells and investigated the underlying molecular mechanisms. TM4 cells were treated with four concentrations (0, 10, 30, and 50 µM) of 4-OP at the following time points: 24, 48, and 72 h. Cell viability and apoptosis assays were conducted following 4-OP exposure. We found that 4-OP significantly decreased cell viability in a concentration- and time-dependent manner, and increased apoptosis. Quantitative PCR analysis showed that the mRNA expression levels of BCL2 Associated X, Apoptosis Regulator (Bax) and BCL2 Antagonist/Killer (Bak) increased while that of BCL2 Apoptosis Regulator (Bcl-2) decreased in 4-OP-exposed cells compared with that in the controls. Western blotting revealed that 4-OP induced caspase-3 activity and Bad phosphorylation in a concentration- and time-dependent manner. Additionally, cytochrome C protein did not colocalize with mitochondrial marker dye by 24 h. Cytochrome c protein expression increased in a time-dependent manner upon exposure to 50 µM 4-OP. These results suggest that 4-OP induces mitochondria-mediated apoptosis through regulation of Bcl-2 family proteins and caspase-3 activation in male Sertoli cells.

CFTR interacts with Hsp90 and regulates the phosphorylation of AKT and ERK1/2 in colorectal cancer cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF cells and tissues exhibit various mitochondrial abnormalities. However, the underlying molecular mechanisms remain elusive. Here, we examined the mechanisms through which CFTR regulates Bcl‐2 family proteins, which in turn regulate permeabilization of the mitochondrial outer membrane. Notably, inhibition of CFTR activated Bax and Bad, but inhibited Bcl‐2. Moreover, degradation of phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) and AKT increased significantly in CFTR‐knockdown cells. Dysfunction of CFTR decreased heat‐shock protein 90 (Hsp90) mRNA levels, and CFTR was found to interact with Hsp90. Inhibition of Hsp90 by SNX‐2112 induced the degradation of phosphorylated AKT and ERK1/2 in Caco2 and HRT18 cells. These findings may help provide insights into the physiological role of CFTR in CF‐related diseases.

Protective Effect of Anthocyanin on Neurovascular Unit in Cerebral Ischemia/Reperfusion Injury in Rats.

Treating cerebral ischemia continues to be a clinical challenge. Studies have shown that the neurovascular unit (NVU), as the central structural basis, plays a key role in cerebral ischemia. Here, we report that anthocyanin, a safe and natural antioxidant, could inhibit apoptosis and inflammation to protect NVU in rats impaired by middle cerebral artery occlusion/reperfusion (MCAO/R). Administration of anthocyanin significantly reduced infarct volume and neurological scores in MCAO/R rats. Anthocyanin could also markedly ameliorate cerebral edema and reduce the concentration of Evans blue (EB) by inhibiting MMP-9. Moreover, anthocyanin alleviated apoptotic injury resulting from MCAO/R through the regulation of Bcl-2 family proteins. The levels of inflammation-related molecules including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), which were over-expressed with MCAO/R, were decreased by anthocyanin. In addition, Nuclear factor-kappa B (NF-κB) and the NLRP3 inflammasome pathway might be involved in the anti-inflammatory effect of anthocyanin. In conclusion, anthocyanin could protect the NVU through multiple pathways, and play a protective role in cerebral ischemia/reperfusion injury.

Structure-guided design of pyridoclax derivatives based on Noxa / Mcl-1 interaction mode

Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.