Pro-apoptotic Molecules Research Articles

Cryo-Electron Microscopy to Study Bax Pores and MOMP.

During apoptosis, mitochondria permeabilize the outer membranes to release apoptogenic proteins from the intermembrane space. This process termed mitochondrial outer membrane permeabilization (MOMP) is regulated by Bcl-2 family proteins. Bax is an effector proapoptotic molecule that permeabilizes the lipid membranes when it is activated by activator BH3-only proteins. We investigated this critical event by developing simple but faithful vesicle systems-outer membrane vesicles (OMVs) and liposomes-to visualize the pores in the membrane by using cryo-electron microscopy (cryo-EM). We have revealed the morphology of the pore, determined the localization of Bax labeled with nanogold and have performed image analysis to help understand the mechanisms of pore formation induced by Bax.

IL-39 acts as a friend to pancreatic cancer.

Pancreatic cancer is the most lethal digestive cancer and the fourth leading cause of cancer death in the US. IL-39, a heterodimer of p19 and EBI3, is a newly found cytokine and its role in the pathogenesis of neoplasia has not been studied yet. This study was designed to investigate the direct role of IL-39 in the growth of pancreatic cancer. Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the direct effects of IL-39 on cell survival, proliferation and apoptosis of the widely studied pancreatic cancer cell line MiaPaCa-2. We further investigated the possible molecular mechanisms by using RT-PCR and IHC. The percentage of colonies of pancreatic cancer cells increased significantly in the presence of IL-39. This was paralleled with the increase in the OD value of cancer cells in the presence of IL-39. Interestingly, the relative caspase-3 activity in cancer cells decreased significantly in the presence of IL-39. Furthermore, the pro-tumor effect of IL-39 on pancreatic cancer cells correlated with decreased anti-proliferative molecule p21.The anti-apoptotic effect of IL-39 correlated with decreased pro-apoptotic molecule TRAILR1. These results suggest that IL-39 favors growth of pancreatic cancer by promoting growth and inhibiting apoptosis of cancer cells. This suggests that IL-39 acts as a friend to pancreatic cancer. Thus, inhibition of effect of IL-39 on cells might be a promising strategy to treat pancreatic cancer.

Characterization of Mechanisms Underlying Acquired Venetoclax-Resistance in Mantle Cell Lymphoma: BDA-366 As a Potential Treatment Option

In many cancers the equilibrium of pro- versus anti-apoptotic BCL-2 proteins is deregulated. BCL-2 inhibitors like Venetoclax (VEN) have been shown to be highly active drugs in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Despite being highly efficient in cell killing, resistance to VEN can be acquired over time. In addition to understanding the underlying mechanisms of resistance to VEN it is important to identify additional treatment options. BDA-366 is a BCL-2 inhibitor with a different mode of action than the BH3 mimetic VEN. BDA-366 acts by inhibiting the BH4 domain and thereby inducing a conversion of anti-apoptotic BCL-2 into a pro-apoptotic protein.BDA-366 showed high effectivity in inducing apoptosis in CLL cells, in primary as well as in cell lines, while all of the CLL cell lines (n=7) tested were resistant to VEN. Furthermore all of the MCL cell lines (n=5) tested were sensitive to the treatment with BDA-366 while only a subset (3 out of 5) responded to treatment with VEN.In order to investigate whether BDA-366 would be a treatment option for VEN-resistant patients, we generated VEN-resistant MCL cell lines (MINO and MAVER-1) by chronic exposure to the drug. In the resistant cell lines, BCL-2 protein levels were not deregulated. In variance to previous reports in diffuse large B cell lymphoma (DLBCL) (Choudhary et al, Cell Death Dis 2015), resistance in MCL cell lines was not mediated by MCL-1 upregulation. In VEN-resistant MINO cells, MCL-1 expression was similar to the parental cells, while MCL-1 was significantly downregulated in VEN-resistant MAVER-1 cells. In contrast, VEN-resistant MCL cell lines showed BCL-XL upregulation as compared to parental cells, which is in line with results obtained in DLBCL (Choudhary et al, Cell Death Dis 2015). Furthermore, dynamic BH3 profiling validated a dependency on BCL-XL in resistant cells and confirmed that resistance was not mediated by MCL-1. The significance of BCL-XL in mediating resistance to VEN was underlined by additional experiments using navitoclax. In contrast to VEN, navitoclax inhibits BCL-2, BCL-XL and BCL-W and was sufficient to induce apoptosis in both parental and resistant cells. In contrast to the BH3 domain inhibitor VEN, the BCL-2 inhibitor BDA-366 acts by converting BCL-2 into a pro-apoptotic molecule. BDA-366 efficiently induced dose dependent apoptosis in VEN-resistant cells. MINO as well as MINO VEN-resistant cells showed the same sensitivity to BDA-366 while VEN-resistant MAVER-1 cells showed reduced sensitivity to BDA-366 as compared to the parental cells. However, with increased BDA-366 concentrations efficient cell killing was achieved in the VEN-resistant cell linesOverall, these results suggest that VEN-resistance is mostly mediated by permanent upregulation of BCL-XL. BCL-2 levels are not deregulated upon development of resistance to VEN. The inhibition of the BH4 domain and thereby converting BCL-2 into a pro-apoptotic protein proved to be a promising therapeutic option even in cancers with acquired resistance to VEN. DisclosuresDöhner:Pfizer: Research Funding; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Pfizer: Research Funding; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia

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.

Neuroprotective Effects of Purple Sweet Potato Balinese Cultivar in Wistar Rats With Ischemic Stroke.

BACKGROUND:Purple sweet potato (Ipomoea Batatas L.) is one of the sources for anthocyanin, which promotes the health through antioxidant, anti-inflammatory, anti-cancer, neuroprotection, and anti-apoptosis activities. Oxidative stress has been shown to be the cause of apoptosis in ischemic stroke.AIM:The objective of this research was to delineate the pleiotropic effects of anthocyanin for neuroprotection during an acute stroke event.METHODS:Anthocyanin was extracted from Balinese cultivar of purple sweet potato and subsequently administered to rat models of induced ischemic stroke (labelled as treatment group), as well as a placebo (labelled as a control group). Several parameters were in turn evaluated, i.e. the activities of anti-apoptotic (Bcl-2) as well as pro-apoptotic (cytochrome c, caspase-3) molecules, and apoptosis rate. Bcl-2 levels were determined using the histochemical method, cytochrome c and caspase-3 via ELISA method, while apoptosis rate was measured by TdT-medicated Dutp-Nick End Labeling (TUNEL) assay.RESULTS:Bcl-2 expression demonstrated significantly higher Bcl-2 expression in the treatment compared with control group (median 31.2 vs. 1.1; p = 0.001). Accordingly, pro-apoptotic cytochrome c and caspase-3 levels were also found significantly lower in the treatment as opposed to control group (mean 4.17 vs. 8.06; p = 0.001; mean 3.81 vs. 8.02; p = 0.001). Ultimately, apoptosis rate was found markedly lower among treatment than control groups (mean 3.81 vs. control 21.97; p = 0.003).CONCLUSION:The results of this study indicated a significant neuroprotective effect of anthocyanin derived from Balinese cultivar of PSP. Anthocyanin was able to increase and reduce anti-apoptotic and pro-apoptotic protein levels, respectively, resulting in lesser cellular apoptotic rate when compared with placebo. The potential mechanism was thought mainly due to its anti-oxidant properties.

Silencing of C3G increases cardiomyocyte survival inhibition and apoptosis via regulation of p-ERK1/2 and Bax.

Experimental studies have shown that overexpression of Rap guanine nucleotide exchange factor 1 (C3G) plays pro-survival and anti-apoptotic roles through molecule phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2) in cardiomyocytes. However, it is still unclear if silencing of C3G may increase cell survival inhibition and apoptosis in cardiomyocytes, and whether C3G silence induced injuries are reduced by the overexpression of C3G through regulation of p-ERK1/2 and pro-apoptotic molecule Bax. In this study, the rat-derived H9C2 cardiomyocytes were infected with C3G small hairpin RNA interference recombinant lentiviruses, which silenced the endogenous C3G expression in the cardiomyocytes. Then, contrary experiments were conducted using C3G overexpression. The cell proliferation and apoptosis were analyzed in the cardiomyocytes which were treated with or without hypoxia/reoxygenation (H/R). Silencing of C3G leaded to significant increase in cell survival inhibition and apoptosis, combined with aggravated the injuries induced by H/R. Overexpression of C3G reduced the injuries induced by the silencing of C3G in the cardiomyocytes via regulation of p-ERK1/2 and Bax. In conclusion, our results provide new experimental evidence that silencing of C3G can increase cell survival inhibition and apoptosis in cardiomyocytes via regulation of p-ERK1/2 and Bax.

Nur77 is a promoting factor in traumatic brain injury-induced nerve cell apoptosis.

Traumatic brain injury (TBI) poses a serious threat to human health. TBI has a high mortality rate, resulting in a great burden on the affected individual's family as well as society as a whole. The incidence of craniocerebral fractures continues to rise as both the economy and transportation options grow, making it imperative that the mortality and disability rate of craniocerebral trauma be reduced. Nur77 is a transcription factor of the nuclear receptor superfamily. Following stimulation of extracellular apoptosis, Nur77 is involved in a variety of diseases as a powerful pro-apoptotic molecule. Here, we determined the effect and mechanism of Nur77 in TBI-induced nerve cell apoptosis in vitro and in vivo. We found that Nur77 and Bcl-2 protein expression increased as nerve cell apoptosis increased in TBI tissues. Furthermore, inhibition of Nur77 improved nerve cell injury by regulation of Bcl-2 and downstream pathways in vitro and in vivo.

Molecular hydrogen protects against ischemia-reperfusion injury in a mouse fatty liver model via regulating HO-1 and Sirt1 expression

Fatty liver has lower tolerance against ischemia-reperfusion (I/R) injury in liver operations, including liver transplantation. Seeking to ameliorate liver injury following I/R in fatty liver, we examined the protective effect of hydrogen (H2) saline on I/R liver injury in a methionine and choline-deficient plus high fat (MCDHF) diet-induced fatty liver mouse model. Saline containing 7 ppm H2 was administrated during the process of I/R. Livers were obtained and analyzed. Primary hepatocytes and Kupffer cells (KCs) were obtained from fatty liver and subjected to hypoxia/reoxygenation. Apoptosis-related proteins and components of the signaling pathway were analyzed after treatment with hydrogen gas. The MCDHF I/R group showed higher levels of AST and ALT in serum, TUNEL-positive apoptotic cells, F4/80 immunopositive cells, mRNA levels of inflammatory cytokines, constituents of the signaling pathway, pro-apoptotic molecules in liver, and KCs and/or primary hepatocytes, compared to the control group. In contrast, H2 treatment significantly suppressed the signs of I/R injury in fatty liver. Moreover, the expression of Bcl-2, HO-1, and Sirt1 in liver, KCs, and hepatocytes by hydrogen gas were increased, whereas caspase activation, Bax, and acetylation of p53 were suppressed by hydrogen gas. These results demonstrated that H2 treatment ameliorated I/R liver injury in a fatty liver model by reducing hepatocyte apoptosis, inhibiting macrophage activation and inflammatory cytokines, and inducing HO-1 and Sirt1 expression. Taken togather, treatment with H2 saline may have a protective effect and safe therapeutic activity during I/R events, such as in liver transplantation with fatty liver.

Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway

Although anlotinib, a multi-targeted receptor tyrosine kinase inhibitor has been reported have antitumor effects in many preclinical and clinical trials, little is known about its effect on hepatocellular carcinoma (HCC). Here, we have shown the antitumor effects of anlotinib on HCC. Data indicated that anlotinib application significantly inhibited HCC cell viability, proliferation, colony formation, and prompted apoptosis in vitro. Furthermore, animal experiments also illustrated that anlotinib alleviated HCC progression. Mechanically, we demonstrated that anlotinib treatment downregulated the anti-apoptotic protein Bcl-2 and Survivin, but upregulated pro-apoptotic molecule Bax, which accounts for its therapeutic effect on HCC. Pathway analysis has shown decreased phosphorylation levels of Erk and Akt. Together, this study suggests that anlotinib may have a direct antitumor progression effect on HCC by inhibiting Bcl-2 and Survivin expression, promoting Bax expression via inactivating Erk and Akt pathways and could be a promising agent treating HCC.

Bixin Triggers Apoptosis of Human Hep3B Hepatocellular Carcinoma Cells: An Insight to Molecular and IN SILICO Approach

Hepatocellular carcinoma (HCC) is the most common liver cancer and is known to be resistant to conventional chemotherapy. The use of herbal medicine and supplements has increased over recent decades following side effects and resistant to conventional chemotherapy. The seeds of Bixa orellana L. commonly known as annatto have recently gained scientific attention due to presence of a carotenoid bixin for its substantial anticancer properties. However, molecular mechanisms underlying bixin-induced apoptosis are still unclear. Treatment of bixin significantly decreased the number of Hep3B cells and morphological study revealed the change in cellular and nuclear morphology that trigger the events of apoptosis confirmed by annexin V/PI staining. Further DCFDA and rhodamine 123 spectrofluorimetry study showed elevation in reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (MMP), respectively. ROS production caused DNA damage and apoptosis was marked by cell cycle arrest, up-regulation of Bax and FasL protein as well as cleavage of caspase-9, caspase-8 and caspase-3 protein. Docking study with pro-apoptotic molecule Bax and surface Fas ligand exhibited energetically favourable binding interaction. Collectively, these results suggest that bixin capable of modulating the extrinsic and intrinsic molecules of apoptosis indicating its potential for development of promising candidate for hepatocellular carcinoma.

TAZ inhibition promotes IL-2-induced apoptosis of hepatocellular carcinoma cells by activating the JNK/F-actin/mitochondrial fission pathway

BackgroundCytokine-based cancer therapies have attracted a great deal of attention in recent years. Unfortunately, resistance to treatment limits the efficacy of these therapeutics. Therefore, the aim of our study was to explore the mechanism of IL-2-based therapy for hepatocellular carcinoma in an attempt to increase the efficiency of this treatment option.MethodsHepG2 cells were treated with IL-2. Then, siRNA against TZA was used to transfected into HepG2 cells. Cellular apoptosis was measured via MTT assay, TUNEL assay and caspase-3 activity. Cellular proliferation was evaluated via EdU assay and western blotting. Cellular migration was detected via Transwell assay. Mitochondrial function was monitored by mitochondrial potential analysis, ROS staining, immunofluorescence and western blotting. Pathway blocker and activator were used to establish the role of JNK/F-actin/mitochondrial fission signaling pathway in HepG2 cells stress response.ResultsOur study found that IL-2 treatment significantly reduced the viability, mobility and proliferation of HepG2 cells in vitro. We also demonstrated that IL-2 treatment was accompanied by an increase in the expression of transcriptional co-activator with PDZ-binding motif (TAZ). Interestingly, genetic ablation of TAZ in the presence of IL-2 further promoted apoptosis, inhibited mobility, and arrested proliferation in HepG2 cells. At the molecular level, IL-2 administration activated excessive mitochondrial fission via the JNK/F-actin pathway; these effects were further enhanced by TAZ deletion. Mechanistically, TAZ knockdown further increased the expression of mitochondrial fission-related proteins such as Drp1, Mff and Fis. The augmented mitochondrial fission stimulated ROS overproduction, mediated redox imbalance, interrupted mitochondrial energy generation, reduced mitochondrial membrane potential, promoted leakage of the pro-apoptotic molecule cyt-c into the nucleus, and initiated caspase-9-related mitochondrial death. Further, we demonstrated that the anti-proliferative and anti-metastatic effects of IL-2 in HepG2 cells were enhanced by TAZ deletion, suggesting that IL-2 sensitizes HepG2 cells to IL-2-based cytokine therapy. However, JNK/F-actin pathway blockade could abrogate the inhibitory effects of TAZ deletion on HepG2 migration, proliferation and survival.ConclusionsTaken together, our data indicate that the anti-tumor effects of IL-2-based therapies may be enhanced by TAZ deletion in a JNK/F-actin pathway-dependent manner. This finding provides a novel combinatorial therapeutic approach for treating hepatocellular carcinoma that might significantly increase the efficacy of cytokine-based therapies in a clinical setting.

The venom of spider Haplopelma hainanum suppresses proliferation and induces apoptosis in hepatic cancer cells by caspase activation in vitro

Ethnopharmacological relevanceSpiders and spider venoms have been used in traditional Chinese medicine to treat various ailments for more than 1000 years. For instance, several large spiders have been utilized by the Li People, who mainly live in Hainan Island of China, in their own unique traditional Chinese medicine therapy. Recent studies have indicated that spider venoms may be an important source of bioactive compounds for anti-tumor treatments. However, the specific mechanisms underlying these activities are not yet completely understood. Aim of the studyThe present study investigated how the venom of the spider Haplopelma hainanum regulate proliferation and apoptosis in HepG2 cells via the underlying molecular mechanisms. Materials and methodsWe treated HepG2 cells with various concentrations of the spider venom (0, 10, 50, 100 and 200 μg/mL) for 48 h, and then analyzed anti-proliferation activity, apoptosis-inducing effects, mitochondrial membrane potential (Δψm) and changes in the pro-apoptotic pathway. The anti-proliferation activity was detected by the MTT assay and Western blotting. Flow cytometry was used to analyze both apoptosis and mitochondrial membrane potential. The key pro-apoptotic molecules in the caspase-3 and −9 dependent mitochondrial pathway, including Bcl2 family, were assessed through realtime PCR, Western blotting and enzymatic test. ResultsObvious morphological changes induced by the spider venom included decreased cell numbers, shorter cell length and reduced cell adhesion. MTT and Western blotting demonstrated that the spider venom potently suppressed cell proliferation in a dose- and time-dependent manner with IC50 of 126.00 μg/mL for 48 h. In addition, the spider venom caused a reduction in the mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm under the participation of Bax. Finally, cytochrome c activated caspase-3 and caspase-9, and induced the apoptosis in the HepG2 cells. ConclusionThe results indicated that the venom of H. hainanum exhibited potent inhibition effects in HepG2 cells through suppressing proliferation, reducing the mitochondrial membrane potential, activating caspase-3 and caspase-9, and inducing the apoptosis through a mitochondrial-dependent pathway.

Mangiferin Ameliorates Cisplatin Induced Acute Kidney Injury by Upregulating Nrf-2 via the Activation of PI3K and Exhibits Synergistic Anticancer Activity With Cisplatin

Occurrence of oxidative stress is the principal cause of acute kidney injury induced by cisplatin. Mangiferin, a naturally occurring antioxidant molecule, is found to ameliorate several oxidative stress mediated pathophysiological conditions including cancer. Cisplatin induced cytotoxicity was measured in NKE cells by MTT assay and microscopic analysis. Induction of oxidative stress and regulation of proapoptotic molecules were subsequently investigated by using different spectrophotometric analyses, FACS and immunocytochemistry. Induction of nephrotoxicity was determined by analyzing different serum biomarkers and histological parameters in vivo using swiss albino mice. Activation of NF-κB mediated pro-inflammatory and caspase dependent signaling cascades were investigated by semi-quantitative RT-PCR and immunoblotting. Mangiferin was found to ameliorate cisplatin induced nephrotoxicity in vitro and in vivo by attenuating the induction of oxidative stress and upregulating Nrf-2 mediated pro-survival signaling cascades via the activation of PI3K. Additionally, mangiferin showed synergistic anticancer activity with cisplatin in cancer cell lines (MCF-7 and SKRC-45) and EAC cell induced solid tumor bearing experimental mice. The ameliorative effect of mangiferin is primarily attributed to its anti-oxidant and anti-inflammatory properties. It acts differentially in normal tissue cells and tumor cells by modulating different cell survival regulatory signaling molecules. For the first time, the study reveals a mechanistic basis of mangiferin action against cisplatin induced nephrotoxicity. Since Mangiferin shows synergistic anticancer activity with cisplatin, it can be considered as a promising drug candidate, to be used in combination with cisplatin.

BST106 Protects against Cartilage Damage by Inhibition of Apoptosis and Enhancement of Autophagy in Osteoarthritic Rats.

Chrysanthemum zawadskii var. latilobum (CZ) has been used as a traditional medicine in Asian countries for the treatment of inflammatory diseases. Recently, CZ extract was shown to inhibit differentiation of osteoclasts and provide protection against rheumatoid arthritis. The aim of this study was to investigate the molecular mechanisms of BST106, the ethanol extract of CZ, for cartilage protection in monosodium iodoacetate (MIA)-induced osteoarthritis (OA), particularly focusing on apoptosis and autophagy. BST106 (50, 100, and 200 mg/kg) was orally administered once daily to MIA-induced OA rats. Swelling, limping, roentgenography, and histomorphological changes were assessed 28 d after MIA injection. Biochemical parameters for matrix metalloproteinase (MMP), apoptosis, and autophagy were also assessed. BST106 ameliorated the severity of swelling and limping after MIA injection. Roentgenographic and histomorphological examinations revealed that BST106 reduced MIA-induced cartilage damage. BST106 decreased MIA-induced increases in MMP-2 and MMP-13 mRNA levels. Increased levels of serum cartilage oligomeric matrix protein and glycosaminoglycan release were attenuated by BST106. Furthermore, BST106 suppressed the protein expression of proapoptotic molecules and increased the protein expression of autophagosome- and autolysosome-related molecules. These findings indicate that BST106 protects against OA-induced cartilage damage by inhibition of the apoptotic pathway and restoration of impaired autophagic flux.

Bioactive Compounds of Kimchi Inhibit Apoptosis by Attenuating Endoplasmic Reticulum Stress in the Brain of Amyloid β-Injected Mice

This study investigated the inhibitory effects of kimchi bioactive compounds against endoplasmic reticulum (ER) stress-induced apoptosis in amyloid beta (Aβ)-injected mice. Mice received a single intracerebroventricular injection of Aβ25-35, except for the normal group. Mice were subjected to oral administration of 10 mg of capsaicin, 50 mg of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA), 50 mg of quercetin, 50 mg of ascorbic acid, or 200 mg of kimchi methanol extract (KME) per kilogram of body weight for 2 weeks ( n = 7 per group). In the in vitro blood-brain barrier (BBB) permeability test, all bioactive compounds penetrated the BBB except ascorbic acid. The protein expression level of APP, BACE, and p-Tau elevated by Aβ injection was decreased by kimchi bioactive compounds ( P < 0.05). Quercetin, HDMPPA, and KME decreased oxidative stress, as indicated by ROS and TBARS levels ( P < 0.05). The protein expression level of ER stress markers GRP78, p-PERK, p-eIF2α, XBP1, and CHOP and the proapoptotic molecules Bax, p-JNK, and cleaved caspases-3 and -9 decreased ( P < 0.05). In contrast, the protein expression level of antiapoptotic molecules Bcl2 and cIAP increased ( P < 0.05). These results were supported by histological analysis.

Indoxyl Sulfate Induces Apoptosis and Hypertrophy in Human Kidney Proximal Tubular Cells.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with declining kidney function. Although generally thought of as a consequence of declining kidney function, emerging evidence demonstrates direct cytotoxic role of IS on endothelial cells and cardiomyocytes, largely through the expression of pro-inflammatory and pro-fibrotic factors. The direct toxicity of IS on human kidney proximal tubular epithelial cells (PTECs) remains a matter of debate. The current study explored the effect of IS on primary cultures of human PTECs and HK-2, an immortalized human PTEC line. Pathologically relevant concentrations of IS induced apoptosis and increased the expression of the proapoptotic molecule Bax in both cell types. IS impaired mitochondrial metabolic activity and induced cellular hypertrophy. Furthermore, statistically significant upregulation of pro-fibrotic (transforming growth factor-β, fibronectin) and pro-inflammatory molecules (interleukin-6, interleukin-8, and tumor necrosis factor-α) in response to IS was observed. Albumin had no influence on the toxicity of IS. The results of this study suggest that IS directly induced a pro-inflammatory and pro-fibrotic phenotype in proximal tubular cells. In light of the associated apoptosis, hypertrophy, and metabolic dysfunction, this study demonstrates that IS may play a role in the progression of chronic kidney disease.

The role of apoptosis of enterocytes in inflammatory bowel diseases

Purpose. Study the topography of cells, expressing villin, p53, Bcl-2, and PUMA in the intestinal mucosa in the non-infectious colitis. Materials and methods. We studied the morphology and localization of the immunoreactive markers of apoptosis in small intestine and bowel with ulcerative colitis and Crohn’s disease, using the material of 20 autopsies and 40 biopsies. Results. In the focus of the specific inflammation in the mucosa, we discovered numerous cells, in various degrees expressing anti- and proapoptotic molecules. In the control material, the antiapoptotic factors (villin and Bcl-2) localized mainly in the enterocytes, while the proapoptotic factors (p53 and PUMA) were expressed in the cells of the lamina propria of the mucosa. The reverse correlation between the number of cells expressing p53/PUMA and the villin/Bcl-2 positive cells had been discovered. At the same time proapoptotic molecules were discovered in the enterocytes and in the cells of the lamina propria of intestinal mucosa. Conclusion. The obtained data points to the important role of apoptosis in pathomorphogenesis of chronic non-infectious intestinal inflammation. The chronization of the process and the nature of the intestinal damage in the UC and CD are connected with balance of the regulative anti- and proapoptotic factors. The cells, expressing those factors, demonstrate the reverse number dependence.

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

BackgroundEffects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts.MethodsUnder treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression.ResultsMTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high.ConclusionsWe present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.

Type I interferons promote the survival and proinflammatory properties of transitional B cells in systemic lupus erythematosus patients.

A hallmark of systemic lupus erythematosus (SLE) is the breaking of B-cell tolerance with the generation of high-affinity autoantibodies; however, the antibody-independent features of the B-cell compartment in SLE are less understood. In this study, we performed an extensive examination of B-cell subsets and their proinflammatory properties in a Chinese cohort of new-onset SLE patients. We observed that SLE patients exhibited an increased frequency of transitional B cells compared with healthy donors and rheumatoid arthritis patients. Plasma from SLE patients potently promoted the survival of transitional B cells in a type I IFN-dependent manner, which can be recapitulated by direct IFN-α treatment. Furthermore, the effect of IFN-α on enhanced survival of transitional B cells was associated with NF-κB pathway activation and reduced expression of the pro-apoptotic molecule Bax. Transitional B cells from SLE patients harbored a higher capacity to produce proinflammatory cytokine IL-6, which was also linked to the overactivated type I IFN pathway. In addition, the frequency of IL-6-producing transitional B cells was positively correlated with disease activity in SLE patients, and these cells were significantly reduced after short-term standard therapies. Thus, the current study provides a direct link between type I IFN pathway overactivation and the abnormally high frequency and proinflammatory properties of transitional B cells in active SLE patients, which contributes to the understanding of the roles of type I IFNs and B cells in the pathogenesis of SLE.