Mitochondrial Apoptotic Signaling Research Articles

Involvement of Nrf2 and mitochondrial apoptotic signaling in trehalose protection against cadmium-induced kidney injury.

Cadmium (Cd) poisoning is characterized by multiple organ dysfunction in organisms, and the kidney is the main target organ of Cd toxicity. Trehalose (Tr), a multifunctional bioactive disaccharide, possesses potential kidney protective properties. Nevertheless, the specific biological function of Tr in antagonizing kidney injury induced by Cd remains to be elucidated. Herein, an in vivo model of Tr antagonizing Cd nephrotoxicity was established and the indictors related to kidney function, oxidative stress, and apoptosis were detected to investigate the molecular mechanism underlying the Tr-protection against Cd-induced kidney injury of rats. Firstly, Tr significantly declined the levels of blood urea nitrogen (BUN) and serum creatinine, and partially restored renal pathological changes caused by Cd. Secondly, Cd exposure significantly increased the malondialdehyde (MDA) content, and decreased the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione (GSH) in serum. However, Tr significantly ameliorated these abnormal alterations. Moreover, Tr regulated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway to suppress the Cd-induced nuclear translocation of Nrf2 and the up-regulation of heme oxygenase-1 (HO-1) and NAD (P) H quinone reductase-1 (NQO1). Meanwhile, Tr significantly reversed the increased Sequestosome-1(SQSTM1/p62) and decreased Kelch-like ECH associated protein-1 (Keap1) protein levels induced by Cd. Thirdly, further mechanistic exploration suggested that Tr inhibited the mitochondrial apoptotic signaling pathway induced by Cd. Collectively, the results indicated that Tr exerts antioxidant and anti-apoptosis functions involving the Nrf2 and mitochondrial apoptotic signaling pathways to protect against Cd-induced kidney injury in rats.

Pre-Clinical Validation of a Novel Erk1/2 and CDK4/6 Inhibitor Combination in Multiple Myeloma (MM)

Whole-genome sequencing analysis of newly diagnosed and relapsed multiple myeloma (MM) samples identified recurrent mutations in genes involved in the MAPK pathway, highlighting the potential of RAS/RAF/MEK/ERK signaling as a therapeutic target. Genomic studies identified translocations that involve IGH and set of partner genes MMSET, FGFR3, and CCND1 as primary events in MM. CDK4/CDK6 is overexpressed in MM, and CDK6 overexpression correlates with poor OS, suggesting that CDK4/6 are promising targets for MM therapy. Recent studies demonstrated synergistic activity of combined novel ERK1/2i inhibitor LY3214996 and CDK4/6i LY2835219 in solid tumors, but analogous studies have not been done in MM. Here we used preclinical models of MM to investigate inhibiting Erk1/2, CDK4/6, or both using ERK1/2i, CDK4/6i, or combination therapy. MM cell lines, RAS mutated or wild type (WT), were sensitive to ERK1/2i at IC50<0.5uM, and CDK4/6i at IC50<3uM. Synergistic effects of the Erk1/2i and CDK4/6i were noted in both RAS mutated and WT MM cell lines when ERK1/2i combined with CDK4/6i. Combination of ERK1/2i+CDK4/6i resulted in dose-dependent G0/G1 arrest in RAS mutated and WT MM cells. Similar effects were seen in RAS mutated cells treated with ERK1/2i or CDK4/6i as a single agent. ERK1/2i + CDK4/6i treatment triggered modest early apoptosis in RAS mutated MM cells, while in RAS WT MM cells this effect was more evident. Using dynamic BH3 profiling assay, we found that short-term treatment of MM cell with ERK1/2i and CDK4/6i led to increased overall mitochondrial priming in response to promiscuous BIM peptide in all MM cell lines. Even single agent treatment with ERK1/2i and CDK4/6i was able to enhance priming of RAS mutated or WT cells. Thus, ERK1/2i and CDK4/6i may activate mitochondrial apoptotic signaling in MM cells alone or in combination, consistent with observed synergistic cytotoxicity. HD PBMC and ARH77 cells were tested as controls. These cells were resistant to ERK1/2i and CDK4/6i at a broad range of concentrations, suggesting a favorable therapeutic index. The clinical potential of CDK4/6i+ERK1/2i was supported by an in vivo study demonstrating a significant (P=0.0004) decrease of the MM burden in CDK4/6i+ERK1/2i treated mice, without adverse effects. Proliferation and apoptosis studies of PCs from MM patient BM samples in the presence and absence of autologous BMSC/BMSCI-CM suggest potent and strong synergistic effects of ERK1/2i+CDK4/6i in MM and may allow successful use in clinic. To address the underlying mechanism of the synergism between Erk1/2i and CDK4/6i, we evaluated their cellular and transcriptional activity in MM cells. Gene expression profiling showed significant downregulation of RAS and CDK4/6 signaling pathway genes in MM cells as a result of ERK1/2i and CDK4/6i treatment at specific concentration ratios (3:1/1:3). Further evaluation of functional effects of ERK1/2i and CDK4/6i, alone or in combination, demonstrated that the synergistic effect of these inhibitors in MM cells is achieved through inhibition of p-S6, downregulation of c-myc, and correlate with ERK1/2i+CDK4/6i induced cell arrest in the G1 cell cycle phase. We noted increased ERK1/2 phosphorylation, which generally results in compensatory activation of parallel signaling pathways or in the loss of negative feedback. Regardless, ERK1/2i+CDK4/6i retained the inhibitory activity of the downstream signaling network, as demonstrated by the inhibition of cytoplasmic (p-RSK1) and nuclear (c-myc) targets of ERK at protein and mRNA levels. Treatment with ERK1/2i+CDK4/6i significantly decreased the levels of p-Rb and E2F1, downstream targets of CDK4/6. Recent studies shown that, in addition to cell cycle regulation, CDK4 and CDK6 induce tumorigenesis through regulation of inflammatory cytokines that are induced via NFκB pathway activation. CDK4/6i functional effects on MM cells cannot be limited to cell cycle arrest, CDK4/6i might also inhibit cytokines, which are produced in MM cells by NFκB activation. Overall, we shown that ERK1/2i+CDK4/6i induced cell proliferation and led to the key target molecule (p-c-myc, p-RSK, p-S6, p-RB, and E2F1) downregulations suggesting on-target activity of these inhibitors in MM cells. Importantly, our studies demonstrate strong synergistic anti-MM activity with ERK1/2+CDK4/6 therapy, providing a preclinical framework for clinical trials to improve patient outcome in MM. Disclosures Letai: Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees; Chugai: Other: Lecture Fees. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics..

Dynamic BH3 Profiling As Pharmacodynamic Biomarker for the Activity of BH3 Mimetics

Pharmacodynamic (PD) biomarkers provide information about the pharmacologic effects of a drug on its target, i.e. to assess whether a given agent is engaging its target in the expected manner. There is currently no convenient PD marker for BH3 mimetics. BH3 profiling (BP) is a functional assay developed to measure cells susceptibility to apoptosis and cell's dependence on certain Bcl-2 anti-apoptotic proteins for survival. Dynamic BH3 profiling (DBP) measures the changes of BP signals after drug perturbations. In this study, we studied whether DBP with lymphoid cells can serve as a PD biomarker for the activity of BH3 mimetics. Using DBP and cell lines with defined dependency, we first showed that BH3 mimetics BCL201 (S55746) and S63845 are highly selective for Bcl-2 or Mcl-1, respectively. Next, we treated primary human lymphoid cells with BCL201 and then conducted DBP. We asked if pretreatment with BCL201 altered mitochondrial sensitivity to different BH3 peptides. After treatment with 1 μM BCL201, an amount consistent with achievable levels in vivo, T cell sensitivity to the Mcl-1 selective MS1 peptide, but not Bad peptide or Bfl-1 selective FS1 peptide, increased significantly, from 20% to 51%. These results suggest that BCL201 treatment increases T cell dependency on Mcl-1 and that DBP of T cells with MS1 peptide may serve as a PD marker for BCL201 activity. Similar results were also observed in B cells. Next, we conducted DBP on healthy T cells treated with S63845. T cell mitochondrial sensitivity to MS1 peptide or Bcl-xL selective HRK peptide did not change. However, the treatment increased mitochondrial sensitivity to the Bad and FS1 peptides significantly, boosting the signal from 10.2% to 57.2% and 46.8% respectively, suggesting DBP of T cells with Bad or FS1 peptides can be robust PD markers for S63845 activity. This results also indicate that S63845 treatment augmented T cell dependency on Bcl-2 and Bfl-1 for survival. Currently, we are investigating whether DBP can be used as a PD biomarker for in vivo studies. Our preliminary data suggest that DBP works well across different species (rat, mouse, and human). More importantly, DBP can detect changes in mitochondrial apoptotic signaling of ex vivo rat cells after in vivo treatment with BH3 mimetics. Conclusions: Exposure to BH3 mimetics causes changes in mitochondrial apoptotic signaling of T/B cells which are readily detectable by DBP. DBP with lymphoid cells may provide a robust PD biomarker for clinical trials testing BH3 mimetics, either as monotherapy or in combination with other agents. Disclosures Wang: Novartis Institutes for Biomedical Research: Current Employment. Qiu:Novartis Institutes for Biomedical Research: Current Employment. Halilovic:Novartis Institutes for Biomedical Research: Current Employment, Current equity holder in publicly-traded company.

Marine algal polysaccharides alleviate aflatoxin B1-induced bursa of Fabricius injury by regulating redox and apoptotic signaling pathway in broilers

Aflatoxin B1 (AFB1) causes toxic effect and leads to organ damage in broilers. Marine algal polysaccharides (MAP) of Enteromorpha prolifera exert multiple biological activities, maybe have a potential detoxification effect on AFB1, but the related research in broilers is extremely rare. Therefore, the purpose of this study was to investigate whether MAPs can alleviate AFB1-induced oxidative damage and apoptosis of bursa of Fabricius in broilers. A total of 216 five-week-old male indigenous yellow-feathered broilers (with average initial body weight 397.35 ± 6.32 g) were randomly allocated to one of three treatments (6 replicates with 12 broilers per replicate), and the trial lasted 4 wk. Experimental groups were followed as basal diet (control group); basal diet mixed with 100 μg/kg AFB1 (AFB1 group, the AFB1 is purified form); basal diet with 100 μg/kg AFB1 + 2,500 mg/kg MAPs (AFB1 + MAPs group). The results showed that the diet with AFB1 significantly decreased the relative weight of bursa of Fabricius (P < 0.05), antioxidant enzymes activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and total antioxidation capacity (T-AOC), while increased malondialdehyde (MDA) content (P < 0.05). Besides, compared with AFB1 group, dietary MAPs improved the relative weight of bursa of Fabricius and activities of antioxidant enzymes (T-SOD, GSH-Px, CAT, GST) with decreased MDA contents (P < 0.05). Moreover, the consumption of AFB1 downregulated the mRNA expression of SOD1, SOD2, GSTA3, CAT1, GPX1, GPx3, GSTT1, Nrf2, HO-1, and p38MAPK (P < 0.05). Dietary MAPs upregulated the mRNA expression of SOD2, GSTA3, CAT1, GPX1, GSTT1, p38MAPK, Nrf2, and HO-1 in comparison with AFB1 group (P < 0.05). The histological analysis confirmed restoration of apoptotic cells of bursa of Fabricius (P < 0.01), which seen with MAPs supplemented broilers. Besides, dietary MAPs down-regulated the mRNA expression of caspase-3 and Bax (P < 0.05), while up-regulated the mRNA expression of Bcl-2 (P < 0.05) compared with AFB1 group. In addition, according to protein expression results, dietary MAPs up-regulated the protein expression level of antioxidant and apoptosis-associated proteins (Nrf2, HO-1, p38MAPK, Bcl-2) (P < 0.01), but down-regulated the protein expression level of caspase-3 and Bax (P < 0.01). In conclusion, dietary MAPs alleviated AFB1-induced bursa of Fabricius injury through regulating Nrf2-mediated redox and mitochondrial apoptotic signaling pathway in broilers.

Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence. Refractory/relapsed RMS patients present a bad prognosis that combined with the lack of specific biomarkers impairs the development of new therapies. Here, we utilize dynamic BH3 profiling (DBP), a functional predictive biomarker that measures net changes in mitochondrial apoptotic signaling, to identify anti-apoptotic adaptations upon treatment. We employ this information to guide the use of BH3 mimetics to specifically inhibit BCL-2 pro-survival proteins, defeat resistance and avoid relapse. Indeed, we found that BH3 mimetics that selectively target anti-apoptotic BCL-xL and MCL-1, synergistically enhance the effect of clinically used chemotherapeutic agents vincristine and doxorubicin in RMS cells. We validated this strategy in vivo using a RMS patient-derived xenograft model and observed a reduction in tumor growth with a tendency to stabilization with the sequential combination of vincristine and the MCL-1 inhibitor S63845. We identified the molecular mechanism by which RMS cells acquire resistance to vincristine: an enhanced binding of BID and BAK to MCL-1 after drug exposure, which is suppressed by subsequently adding S63845. Our findings validate the use of DBP as a functional assay to predict treatment effectiveness in RMS and provide a rationale for combining BH3 mimetics with chemotherapeutic agents to avoid tumor resistance, improve treatment efficiency, and decrease undesired secondary effects.

Triggering of Apoptosis in Osteosarcoma 143B Cell Line by Carbon Quantum Dots via the Mitochondrial Apoptotic Signal Pathway.

Objectives Carbon-based nanomaterials have gained attention in the field of biomedicine in recent years, especially for the treatment of complicated diseases such as cancer. Here, we report a novel carbon-based nanomaterial, named carbon quantum dots (CQDs), which has potential for cancer therapy. We performed a systematic study on the effects of CQDs on the osteosarcoma 143B cell line in vitro and in vivo. Methods Cell counting assay, the neutral red assay, lactic dehydrogenase assay, and fluorescein isothiocyanate (FITC) Annexin V/Propidium iodide (PI) were used to detect the cytotoxicity and apoptosis of CQDs on the 143B cell line. Intracellular reactive oxygen species (ROS) were detected by the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate. The JC-10 assay was used to detect the mitochondrial membrane potential (MMP) of 143B cells incubated with CQDs. The effects of CQDs on the 143B cell line were evaluated by Western blot and immunofluorescence analysis of apoptosis-related proteins Bax, Bcl-2, cytochrome-C, caspase-3, cleaved-caspase-3, PARP1, and cleaved-PARP1. Male tumor-bearing BALB/c nude mice were used to investigate the antitumor effects of CQDs, and the biosafety of CQDs in vivo was tested in male BALB/c mice by measuring weight changes, hematology tests, and histological analyses of major organs. Results CQDs exhibited a high cytotoxicity and induced apoptosis toward the 143B cell line. CQDs can also significantly increase the intracellular level of ROS and lower the mitochondrial membrane potential levels of 143B cells. CQDs increase apoptotic protein expression to induce apoptosis of 143B cells by triggering the mitochondrial apoptotic signaling pathway. The tumor volume in the CQD-treated mice was smaller than that in the control group, the tumor volume inhibition rate was 38.9%, and the inhibitory rate by tumor weight was 30.1%. All biosafety test indexes were within reference ranges, and neither necrosis nor inflammation was observed in major organs. Conclusions CQDs induced cytotoxicity in the 143B cell line through the mitochondrial apoptotic signaling pathway. CQDs not only showed an antitumor effect but also high biocompatibility in vivo. As a new carbon-based nanomaterial, CQDs usage is a promising method for novel cancer treatments.

Histone Deacetylase Inhibitor Trichostatin A Suppresses Cell Proliferation and Induces Apoptosis by Regulating the PI3K/AKT Signalling Pathway in Gastric Cancer Cells.

Gastric cancer, a common malignant tumour worldwide, has a relatively poor prognosis and is a serious threat to human health. Histone Deacetylase Inhibitors (HDACi) are anticancer agents that are known to affect the cell growth of different cancer types. Trichostatin A (TSA) selectively inhibits the class I and II mammalian Histone Deacetylase (HDAC) family enzymes and regulates many cell processes. Still, the underlying mechanisms of HDACs are not fully understood in gastric cancer. This study aims to investigate the antitumor effect and the mechanism of growth modulation of gastric cancer cells by TSA. The cell proliferation of gastric cancer cells was measured by MTT and BrdU immunofluorescence assays. Soft agar assay was used to detect the colony formation ability of gastric cancer cells. Flow cytometry was used to examine cell cycle and apoptosis. Western blot was employed to detect protein expression of target factors. TSA inhibits the proliferation of MKN-45 and SGC-7901 cells and leads to significant repression of colony number and size. Flow cytometry assays show TSA induces cell cycle arrest at G1 phase and apoptosis, and TSA effects the expression of related factors in the mitochondrial apoptotic signalling and cell cycle-related regulatory pathways. Furthermore, TSA increased histone H3K27 acetylation and downregulated the expression of PI3K and p-AKT. Downregulating PI3K/AKT pathway activation is involved in TSA-mediated proliferation inhibition of gastric cancer.

High-throughput dynamic BH3 profiling may quickly and accurately predict effective therapies in solid tumors.

Despite decades of effort, the sensitivity of patient tumors to individual drugs is often not predictable on the basis of molecular markers alone. Therefore, unbiased, high-throughput approaches to match patient tumors to effective drugs, without requiring a priori molecular hypotheses, are critically needed. Here, we improved upon a method that we previously reported and developed called high-throughput dynamic BH3 profiling (HT-DBP). HT-DBP is a microscopy-based, single-cell resolution assay that enables chemical screens of hundreds to thousands of candidate drugs on freshly isolated tumor cells. The method identifies chemical inducers of mitochondrial apoptotic signaling, a mechanism of cell death. HT-DBP requires only 24 hours of ex vivo culture, which enables a more immediate study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Effective compounds identified by HT-DBP induced tumor regression in genetically engineered and patient-derived xenograft (PDX) models of breast cancer. We additionally found that chemical vulnerabilities changed as cancer cells expanded ex vivo. Furthermore, using PDX models of colon cancer and resected tumors from colon cancer patients, our data demonstrated that HT-DBP could be used to generate personalized pharmacotypes. Thus, HT-DBP appears to be an ex vivo functional method with sufficient scale to simultaneously function as a companion diagnostic, therapeutic personalization, and discovery tool.

PAWI-2: A novel inhibitor for eradication of cancer

Cancer is a major worldwide public health problem and is still the leading cause of death in the United States. There are many types of cancer treatment but completely successful results are oftentimes not attained. It remains a challenge to develop efficacious clinically useful cancer therapies. Therapies targeting dysregulated signal transduction pathways in cancer can be efficacious anti-cancer therapies with minimal adverse effects. In this study, we focus on novel small molecule p53 Activator Wnt Inhibitor-2 (PAWI-2) that was developed by optimizing potency and pharmaceutical properties. PAWI-2 is a nontoxic DNA-damage pathway inhibitor that shows a broad spectrum of potency and significant efficacy in vitro and in vivo. This study focuses on the application of PAWI-2 to four major types of cancers including colorectal cancer (CRC), breast cancer (BC), prostate cancer (PCa), and pancreatic cancer (PC). PAWI-2 shows a novel mechanism of action (MOA) by modulating two mechanisms of cancer invasion. In cancer with unimpaired p53, PAWI-2 activates DNA-damage checkpoint and mitochondrial p53-dependent apoptotic signaling. Consistently observed in most cancer types, PAWI-2 induces phosphorylation of optineurin (OPTN) to cause G2/M cell cycle arrest. These two mechanisms operate regardless of p53 variants and/or KRAS mutation status and also manipulate the effect of PAWI-2 to overcome tumor stemness and drug resistance in PC stem cells (PCSCs). This study summarizes the development of PAWI-2 as an attractive targeted therapeutic for mechanism-driven anti-cancer drug discovery.

Mild microwave ablation combined with HSP90 and TGF‑β1 inhibitors enhances the therapeutic effect on osteosarcoma.

Osteosarcoma is the most common malignant bone tumour and the second leading cause of cancer-related death in children and adolescents. Microwave ablation has an excellent therapeutic effect on bone tumours by instantaneously increasing the temperature in the tumour; however, there is a risk of damaging the surrounding healthy tissues by exposure to a high temperature when the treatment power is too large. In the present study, two anti-tumour reagents, a heat shock protein 90 (HSP90) inhibitor (PF-04929113) and a transforming growth factor-β1 (TGF-β1) inhibitor (SB-525334) were employed to enhance the therapeutic effect of mild-power microwave ablation. It was revealed that microwaving to 48°C combined with HSP90 and TGF-β1 inhibitors significantly increased the apoptotic rate of VX2 cells. The same results were observed during in vivo experiments using New Zealand rabbits to model osteosarcoma. In addition, the results indicated that the expression of cytochrome c, caspase-3 and caspase-9 were upregulated in response to the treatment, which indicated that the mitochondrial apoptotic signalling pathway had been activated. These findings may provide a novel strategy for the development of microwave ablation in osteosarcoma treatment, which could effectively kill tumour cells without damaging the surrounding normal tissues.

Promotion of ubiquitination-dependent survivin destruction contributes to xanthohumol-mediated tumor suppression and overcomes radioresistance in human oral squamous cell carcinoma

BackgroundOverexpression of survivin plays a crucial role in tumorigenesis and correlates with poor prognosis in human malignancies. Thus, survivin has been proposed as an attractive target for new anti-tumor interventions.MethodsA natural product library was used for natural compound screening through MTS assay. The expression of survivin in oral squamous cell carcinoma (OSCC) and the inhibitory effect of xanthohumol (XN) on OSCC were examined by anchorage-dependent and -independent growth assays, immunoblot, immunofluorescence, immunohistochemical staining, ubiquitination analysis, co-immunoprecipitation assay, CRISPR-Cas9-based gene knockout, and xenograft experiment.ResultsSurvivin is highly expressed in OSCC patient-derived tissues and cell lines. Knockout of survivin reduced the tumorigenic properties of OSCC cells in vitro and in vivo. With a natural compound screening, we identified that xanthohumol inhibited OSCC cells by reducing survivin protein level and activating mitochondrial apoptotic signaling. Xanthohumol inhibited the Akt-Wee1-CDK1 signaling, which in turn decreased survivin phosphorylation on Thr34, and facilitated E3 ligase Fbxl7-mediated survivin ubiquitination and degradation. Xanthohumol alone or in combination with radiation overcame radioresistance in OSCC xenograft tumors.ConclusionOur findings indicate that targeting survivin for degradation might a promising strategy for OSCC treatment.

Protective Role of Spirulina platensis Against Bifenthrin-Induced Reprotoxicity in Adult Male Mice by Reversing Expression of Altered Histological, Biochemical, and Molecular Markers Including MicroRNAs.

The potential reprotoxicity of bifenthrin remains unclear if only the common clinical indicators of reproductive disease are examined. The present study aimed to investigate the efficacy of Spirulina platensis, a microalga rich in antioxidant compounds, against bifenthrin-induced testicular oxidative damage in male mice. At the first, we demonstrate that administration of bifenthrin resulted in a decline of testosterone level and in deterioration of sperm quality that was correlated with significant transcription changes of some specific mRNA and microRNA involved in cholesterol transport, testosterone synthesis, and spermatogenesis. At the biochemical level, we found that oxidative stress was obvious in the bifenthrin group, as evidenced by increase in malondialdehyde (MDA), protein carbonyls (PCO), reactive oxygen species (ROS), and nitrite oxide (NO) that was correlated with activation of genes related to mitochondrial apoptotic signal pathways. We then brought, for the first time to our knowledge, solid and complete experimental evidences that administration of mice with Spirulina extract was sufficient to protect against deleterious effects BF in testicular tissues by abrogating the change in antioxidant enzyme activities; the increase in MDA, PCO, and NO concentrations; and the altered expression level of miRNA and mRNA involved in spermatogenesis. We finally demonstrate that Spirulina restores the production of testosterone in mice as well as epididymal sperm viability and motility. These results suggest a potential antitoxic activity of Tunisian Spirulina deserving further attention.

Corilagin induces laryngeal cancer antiproliferation and inhibits growth factor and cytokine signaling pathways in vitro and in vivo

Laryngeal cancer is a prevalent malignancy of the head and neck with a high rate of mortality. Corilagin, a natural gallic acid derivative, was isolated from PhmLlanthi Fructus, a “drug homologous food”, and its in vitro and in vivo activity were investigated. Results of our study demonstrate that corilagin has significant anti-laryngeal cancer activity in vitro and in vivo. It was observed that corilagin inhibits proliferation and migration of cancerous laryngeal cells Hep-2 and TU212 in a concentration-dependent manner and inhibits growth of transplanted tumors in nude mice without apparent signs of toxicity. The anti-cancer effects of corilagin may be attributed to the inhibition of human laryngeal cancer proliferation by activation of mitochondrial apoptotic and endoplasmic reticulum stress signaling pathways and inhibition of Akt and STAT3 pathways.

Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation

IntroductionMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.Hypothesisp66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.Aimto test whether p66Shc can associate with Pyk2.MethodsMammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.ResultsWe confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.Conclusionp66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.Support or Funding InformationApart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

Protective effects of luteolin against oxidative stress and mitochondrial dysfunction in endothelial cells

Background and aimsLuteolin is a common flavonoid that is abundantly present in various edible plants, it is known to exhibit beneficial effects on cardiovascular system. However, the mechanisms which underlie the protective effects of luteolin on endothelial cell damage caused by oxidative stress remains unclear. The purpose of this study is to test the hypothesis which states that luteolin protects against H2O2-induced oxidative stress via modulating ROS-mediated P38 MAPK/NF-κB and calcium-evoked mitochondrial apoptotic signalling pathways. Methods and resultsHuman umbilical vein endothelial cells (HUVECs) were pretreated with luteolin prior to being stimulated by 600 μM H2O2 for another 24 h. The expression of native and phosphorylated-P38, IκB, NF-κB, native eNOS, phosphorylated-eNOS, iNOS and several apoptosis-related proteins were analyzed by Western blot. In addition, intracellular calcium was determined by fura-2 AM and mitochondrial membrane potential was examined by using JC1. Using the data gathered, we found indications that H2O2 induced P38 MAPK/NF-κB activation. H2O2 downregulated the expression of eNOS and upregulated iNOS, which in turn contribute to an elevated NO generation and protein nitrosylation. However, pretreatment with luteolin markedly reversed all of these alterations dose-dependently. Additionally, an intracellular calcium rise and subsequent mitochondrial membrane potential collapse, P53 phosphorylation, reduced BcL-2/Bax ratio in the mitochondrial membrane, release cytochrome c from mitochondria, leading to the subsequent activation of caspase 3 activation by H2O2 were all markedly suppressed in the presence of luteolin. ConclusionResults from this study may provide the possible molecular mechanisms underlying cardiovascular protective effects of luteolin.

C1q/TNF-related protein 5 contributes to diabetic vascular endothelium dysfunction through promoting Nox-1 signaling

ObjectiveDysregulated adipokine profiles contribute to the pathogenesis of diabetic cardiovascular complications. Endothelial cell (EC) dysfunction, a common pathological alteration in cardiovascular disorders, is exaggerated in diabetes. However, it is unclear whether and how dysregulated adipokines may contribute to diabetic EC dysfunction. Methods and resultsSerum C1q/TNF-Related Protein 5 (CTRP5) were determined in control/diabetes patients, and control/diabetic mice (high-fat diet, HFD). We observed for the first time that serum total CTRP5 was increased, high molecular weight (HMW) form was decreased, but the globular form (gCTRP5) was significantly increased in diabetic patients. These pathological alterations were reproduced in diabetic mice. To determine the pathological significance of increased gCTRP5 in diabetes, in vivo, ex vivo and in vitro experiments were performed. Diabetic atherosclerosis and EC dysfunction were significantly attenuated by the in vivo administration of CTRP5 neutralization antibody (CTRP5Ab). EC apoptosis was significantly increased in diabetic EC (isolated from HFD animal aorta) or high glucose high lipid (HGHL) cultured HUVECs. These pathological alterations were further potentiated by gCTRP5 and attenuated by CTRP5Ab. Pathway specific discovery-driven approach revealed that Nox1 expression was one of the signaling molecules commonly activated by HFD, HGHL, and gCTRP5. Treatment with CTRP5Ab reversed HFD-induced Nox1 upregulation. Finally, Nox1siRNA was used to determine the causative role of Nox1 in gCTRP5 induced EC apoptosis in diabetes. Results showed that gCTRP5 activated the mitochondrial apoptotic signal of EC in diabetes, which was blocked by the silencing Nox1 gene. ConclusionWe demonstrated for the first time that gCTRP5 is a novel molecule contributing to diabetic vascular EC dysfunction through Nox1-mediated mitochondrial apoptosis, suggesting that interventions blocking gCTRP5 may protect diabetic EC function, ultimately attenuate diabetic cardiovascular complications.

Effect of Hsp90 Inhibitor KW-2478 on HepG2 Cells.

The objectives of this study were to investigate the effects of proliferation, apoptosis, cell cycle, invasion, and senescence of KW-2478 on HepG2 cells, and to explore the related mechanism of apoptosis and the cell cycle. HepG2 cells (hepatocellular carcinoma cells) were cultured with KW-2478, at different doses and for different times, in vitro. The MTT assay was used to detect the effect of KW-2478 on proliferation of HepG2 cells. Flow cytometry was used to determine the effects of KW-2478 on the cell cycle and apoptosis of HepG2 cells. The Transwell assay was used to determine the effect of KW-2478 on cell invasion. The β-galactosidase assay tested the effect of low-dose KW-2478 on the senescence of HepG2 cells. Western blotting and the quantitative polymerase chain reaction were used respectively to assess changes in protein and mRNA levels of related factors in HepG2 cells after the KW-2478 treatment. KW-2478 significantly inhibited proliferation of HepG2 cells. KW-2478 induced apoptosis and cell cycle arrest of HepG2 cells, and inhibited the invasion of HepG2 cells; low dose KW-2478 promoted HepG2 senescence. KW-2478 inhibited the proliferation of HepG2 cells, induced apoptosis and cell cycle arrest, inhibited invasion, and promoted senescence. KW-2478 affected the expression of related factors in the mitochondrial apoptotic signaling and cell cycle-related regulatory pathways. KW-2478 downregulated the expression of STAT3, which is a key factor in the JAK-STAT pathway, indicating that KW-2478 may affect the function of HepG2 cells by downregulating STAT3.

Oxidative DNA damage and multi-organ pathologies in male mice subchronically treated with aflatoxin B1

Although the acute and/or chronic exposure to AFB1 has been widely investigated, the study on the toxic effects resulted from the subchronic exposure of AFB1 which is more close to the real scenario in view of the regional and seasonal characters of aflatoxin-producing strains is still limited. To understand the subchronically toxic effects of AFB1, we studied the AFB1-induced oxidative damage, reproductive impairment as well as their potential correlations and mechanisms at the molecular level. Generally, our results showed that subchronic exposure of AFB1 gave rise to pathological and oxidative damages in mice, disrupted oxidation-reduction homeostasis, activated mitochondrial apoptotic and p53-regulated signaling pathways, induced DNA and chromosomal damages and increased the rate of sperm malformation. Importantly, reproductive toxic effects were detected in AFB1–treated mice under a subchronic exposure, which was evidenced by the ascended sperm malformation. Based on our pilot study, it's speculated that the partial mechanism of reproductive toxicity may be the oxidative damages, especially DNA damages directly induced by AFB1. In short, our study demonstrated that severe damages can be caused even by a subchronic exposure as well as hinted that reproductive toxicity also should be taken into consideration when conducting risk assessments of the subchronic exposure of AFB1.

Atorvastatin Exerts Antileukemia Activity via Inhibiting Mevalonate-YAP Axis in K562 and HL60 Cells.

Novel therapeutic strategies are still urgently expected for leukemia despite undisputed success of various targeted therapeutics. The antileukemia activity of Atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on human leukemia cells was investigated. Atorvastatin inhibited K562 and HL60 cell proliferation, induced G2/M cell cycle arrest in K562 cells by down-regulating cyclinB1 and cdc2, but G0/G1 arrest in HL60 cells by up-regulating p27 and down-regulating cyclinD1 and p-pRb. Atorvastatin also induced apoptosis in both cell lines, in which the reactive oxygen species (ROS)-related mitochondrial apoptotic signaling might be involved, with increase of ROS and Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (MMP), release of cytochrome C into cytosol, and activation of Bax/Caspase-9/Caspase-3/PARP pathway. Inhibition of YAP nuclear localization and activation by Atorvastatin was reversed by the addition of mevalonate, GGPP, or FPP. Further, the effects on cell cycle arrest- and apoptosis- related proteins by Atorvastatin were alleviated by addition of mevalonate, suggesting the antileukemia effect of Atorvastatin might be through mevalonate-YAP axis in K562 and HL60 cells. Our results suggest that Atorvastatin might be used for leukemia therapy while evidence of clinical efficacy is required.

Antileukemic activity and mechanism of ardisianone-a crucial role of mitochondrial stress in apoptosis and necroptosis

Abstract Background Ardisianone was reported to induce apoptosis in prostate cancer cells, but necroptosis in leukemia cell lines. It is needed to clarify whether ardisianone could cause apoptosis and necroptosis in leukemia cells and investigate its underlying mechanisms. Methods Cytotoxicity was examined using the MTT assay. Cytoflowmetric analysis of JC-1 and PI staining was used to examine mitochondrial membrane potential and cell cycle progression, respectively. Apoptosis was examined by annexin V-PI staining assay. Protein expression was detected by Western blot and apoptosis antibody array chip. Immunofluorescence staining detected necroptosis effect. Results Ardisianone inhibited cell viability in HL-60, a promyelocytic leukemia cell line, with IC50 value of 1.87 µ M in a 24h exposure. Further detection showed that ardisianone induced time- and concentration-dependent apoptosis. JC-1 staining demonstrated that ardisianone caused a profound loss of mitochondrial membrane potential. Western blot also confirmed the decrease of pro-survival Bcl-2 family protein and the downregulation of necroptosis relatives (e.g., RIPK1 and RIPK3). The caspase cascade was profoundly activated by ardisianone. Notably, the specific pan-caspase inhibitor Q-VD-OPh significantly blunted ardisianone induced loss of membrane potential and apoptosis, suggesting that caspase cascade activation may further amplify mitochondrial damage and apoptotic signaling cascade. The expression of RIPK1/RIPK3/MIKL as significantly reduced by pre-treatment of necroptosis inhibitor (Necrostatin-1, Nec-1), suggesting that Nec-1 attenuates ardisianone-induced necroptosis. Conclusion The data suggest that ardisianone induces mitochondrial dysfunction and apoptosis in leukemia cells through modification of Bcl-2 family members and causes necrotic cell death.