Melatonin enhances SIRT1 to ameliorate mitochondrial membrane damage by activating PDK1/Akt in granulosa cells of PCOS.

Mitochondrial injury in granulosa cells (GCs) is associated with the pathophysiological mechanism of polycystic ovary syndrome (PCOS). Melatonin reduces the mitochondrial injury by enhancing SIRT1 (NAD-dependent deacetylase sirtuin-1), while the mechanism remains unclear. Mitochondrial membrane potential is a universal selective indicator of mitochondrial function. In this study, mitochondrial swelling and membrane defect mitochondria in granulosa cells were observed from PCOS patients and DHT-induced PCOS-like mice, and the cytochrome C level in the cytoplasm and the expression of BAX (BCL2-associated X protein) in mitochondria were significantly increased in GCs, with p-Akt decreased, showing mitochondrial membrane was damaged in GCs of PCOS. Melatonin treatment decreased mitochondrial permeability transition pore (mPTP) opening and increased the JC-1 (5,5',6,6'-tetrachloro1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide) aggregate/monomer ratio in the live KGN cells treated with DHT, indicating melatonin mediates mPTP to increase mitochondrial membrane potential. Furthermore, we found melatonin decreased the levels of cytochrome C and BAX in DHT-induced PCOS mice. PDK1/Akt played an essential role in improving the mitochondrial membrane function, and melatonin treatment increased p-PDK 1 and p-Akt in vivo and in vitro. The SIRT1 was also increased with melatonin treatment, while knocking down SIRT1 mRNA inhibiting the protective effect of melatonin to activate PDK1/Akt. In conclusion, melatonin enhances SIRT1 to ameliorate mitochondrial membrane damage by activating PDK1/Akt in granulosa cells of PCOS.

The protective role of Coenzyme Q10 in metallothionein-3 expression in liver and kidney upon rats' exposure to lead acetate.

Metallothionein-3 (MT3) is an antioxidant protein that alters after exposure to heavy metals. In this study, we investigated the hepatic and renal expression of MT3 gene following exposure to lead acetate (PbAc) alone and PbAc plus CoQ10 as an adjuvant antioxidant. Twenty-four rats were allocated into three groups, including control, PbAc (free access to drinking water contaminated with PbAc at 1 g/100 ml), and PbAc plus CoQ10 (10 mg/kg/day Oral). After 28 consecutive days of treatment, the mRNA expression of MT3 and Cyt-c genes and MT3 protein levels were assessed using real-time PCR and immunosorbent assay. The serum lipid profile was also monitored in the three groups. PbAc exposure significantly reduced the hepatic and renal MT3 mRNA and protein expression compared to the control group. This reduction was significantly increased with addition of CoQ10 to levels near those of the control group. The hepatic and renal expression of Cyt-c mRNA increased after treatment with PbAc, while such effect was reversed after addition of CoQ10. Alteration in lipid profile including increased cholesterol and low-density lipoprotein levels were observed after PbAc exposure which were counteracted by CoQ10. Our results confirm the cytotoxic effects of acute lead exposure manifested as changes in the serum lipid profile and cellular levels of Cyt-c mRNA. These cytotoxic effects may have been caused by decreased MT3 gene expression and be reduced by the protective role of CoQ10.

Roles of ERK/Akt signals in mitochondria-dependent and endoplasmic reticulum stress-triggered neuronal cell apoptosis induced by 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a major active metabolite of bisphenol A.

Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toxicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoxicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5-15 µM) exhibited greater neuronal cytotoxicity than BPA (50-100 µM). The mitochondria-dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the increase in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 µM)-induced Neuro-2a cell death. Exposure of Neuro-2a cells to MBP (10 µM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-box binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 µM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pretreatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoxicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.

Progesterone induces apoptosis by activation of caspase-8 and calcitriol via activation of caspase-9 pathways in ovarian and endometrial cancer cells in vitro.

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12 h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24 h. Caspase 9 activity was increased in all cell lines after 24 h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Benzyl Isothiocyanate Induces Apoptosis via Reactive Oxygen Species-Initiated Mitochondrial Dysfunction and DR4 and DR5 Death Receptor Activation in Gastric Adenocarcinoma Cells.

Benzyl isothiocyanate (BITC) is known to inhibit the metastasis of gastric cancer cells but further studies are needed to confirm its chemotherapeutic potential against gastric cancer. In this study, we observed cell shrinkage and morphological changes in one of the gastric adenocarcinoma cell lines, the AGS cells, after BITC treatment. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, a cell viability assay, and found that BITC decreased AGS cell viability. Reactive oxygen species (ROS) analyses using 2',7'-dichlorofluorescin diacetate (DCFDA) revealed that BITC-induced cell death involved intracellular ROS production, which resulted in mitochondrial dysfunction. Additionally, cell viability was partially restored when BITC-treated AGS cells were preincubated with glutathione (GSH). Western blotting indicated that BITC regulated the expressions of the mitochondria-mediated apoptosis signaling molecules, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cytochrome c (Cyt c). In addition, BITC increased death receptor DR5 expression, and activated the cysteine-aspartic proteases (caspases) cascade. Overall, our results showed that BITC triggers apoptosis in AGS cells via the apoptotic pathways involved in ROS-promoted mitochondrial dysfunction and death receptor activation.

α-Hederin Induces Apoptosis of Esophageal Squamous Cell Carcinoma via an Oxidative and Mitochondrial-Dependent Pathway.

BACKGROUND: α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable. AIM: To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism. METHODS: Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin. RESULTS: Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety. CONCLUSION: α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

Fipronil induces apoptosis and cell cycle arrest in porcine oocytes during in vitro maturation.

Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 µM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 µM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.

Indirubin and NAD+ prevent mitochondrial ischaemia/reperfusion damage in fatty livers.

BACKGROUND: Fatty livers are considerably more susceptible to acute stressors, such as ischaemia/reperfusion (I/R). As the incidence of I/R is high due to surgical events and some pathologies, there is an urgent need to find strategies against I/R injury (I/RI) in fatty livers. We postulate that an acute pretreatment with indirubin-3'-oxime (Ind) or NAD+ prevents mitochondrial dysfunction associated with warm I/RI in fatty livers. MATERIALS AND METHODS: Zucker fatty rats were subjected to warm ischaemia and 12 hours of reperfusion. Ind or NAD+ was administered in the hepatic artery 30 minutes before ischaemia. Hepatic mitochondrial isolation was performed, and functional assays as well as molecular analysis were performed. RESULTS: Pretreatment decreased markers of liver injury while preserving mitochondrial cytochrome c content, which is related to the prevention of calcium-induced mitochondrial permeability transition (mPT), the decline in mitochondrial respiratory state 3 and ATP content. The generation of reactive oxygen species (ROS) was also diminished. Inhibition of GSK-3ß by Ind resulted in the prevention of cyclophilin-D (CypD) phosphorylation, unabling it to bind to the adenine nucleotide translocator (ANT), thus, preventing mPT induction. Furthermore, deacetylation of CypD at Lys residue by sirtuin 3 (SIRT3) caused its dissociation from ANT, contributing to an increase in mPT threshold in NAD+ -pretreated animals. CONCLUSIONS: Pretreatment with Ind or NAD+ protects fatty livers by maintaining mitochondrial calcium homoeostasis, thus, preserving mitochondrial function and energetic balance. As such, CypD might be a new protective target against I/RI in fatty livers.

Susceptibility of Mycobacterium tuberculosis Cytochrome bd Oxidase Mutants to Compounds Targeting the Terminal Respiratory Oxidase, Cytochrome c.

We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

14,15-EET Suppresses Neuronal Apoptosis in Ischemia-Reperfusion Through the Mitochondrial Pathway.

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia-reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia-reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen-glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia-reperfusion by 14,15-EET.

Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes.

The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome c (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression.

Lifelong quercetin enrichment and cardioprotection in Mdx/Utrn+/- mice.

Duchenne Muscular Dystrophy (DMD) is associated with progressive cardiac pathology; however, the SIRT1/PGC1-α activator quercetin may cardioprotect dystrophic hearts. We tested the extent to which long-term 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in Mdx/Utrn+/- mice. At 2 mo, Mdx/Utrn+/- mice were fed quercetin-enriched (Mdx/Utrn+/--Q) or control diet (Mdx/Utrn+/-) for 8 mo. Control C57BL/10 (C57) animals were fed a control diet for 10 mo. Cardiac function was quantified by MRI at 2 and 10 mo. Spontaneous physical activity was quantified during the last week of treatment. At 10 mo hearts were excised for histological and biochemical analysis. Quercetin feeding improved various physiological indexes of cardiac function in diseased animals. Mdx/Utrn+/--Q also engaged in more high-intensity physical activity than controls. Histological analyses of heart tissues revealed higher expression and colocalization of utrophin and α-sarcoglycan. Lower abundance of fibronectin, cardiac damage (Hematoxylin Eosin-Y), and MMP9 were observed in quercetin-fed vs. control Mdx/Utrn+/- mice. Quercetin evoked higher protein abundance of PGC-1α, cytochrome c, ETC complexes I-V, citrate synthase, SOD2, and GPX compared with control-fed Mdx/Utrn+/- Quercetin decreased abundance of inflammatory markers including NFκB, TGF-ß1, and F4/80 compared with Mdx/Utrn+/-; however, P-NFκB, P-IKBα, IKBα, CD64, and COX2 were similar between groups. Dietary quercetin enrichment improves cardiac function in aged Mdx/Utrn+/- mice and increases mitochondrial protein content and dystrophin glycoprotein complex formation. Histological analyses indicate a marked attenuation in pathological cardiac remodeling and indicate that long-term quercetin consumption benefits the dystrophic heart. NEW & NOTEWORTHY: The current investigation provides first-time evidence that quercetin provides physiological cardioprotection against dystrophic pathology and is associated with improved spontaneous physical activity. Secondary findings suggest that quercetin-dependent outcomes are in part due to PGC-1α pathway activation.

Nanoparticle-Mediated Delivery of Mitochondrial Division Inhibitor 1 to the Myocardium Protects the Heart From Ischemia-Reperfusion Injury Through Inhibition of Mitochondria Outer Membrane Permeabilization: A New Therapeutic Modality for Acute Myocardial Infarction.

BACKGROUND: Mitochondria-mediated cell death plays a critical role in myocardial ischemia-reperfusion (IR) injury. We hypothesized that nanoparticle-mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization (MOMP), which causes mitochondrial-mediated cell death. METHODS AND RESULTS: We formulated poly (lactic-co-glycolic acid) nanoparticles containing Mdivi1 (Mdivi1-NP). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H2O2-induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1-NP ameliorated H2O2-induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half-maximal effective concentration and greater maximal effect on cell survival. Mdivi1-NP treatment of Langendorff-perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1-NP treatment also inhibited Drp1-mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP, in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD-KO) mice, which lack the mitochondrial permeability transition pore (MPTP) opening. Intravenous Mdivi1-NP treatment in vivo at the time of reperfusion reduced IR injury in wild-type and CypD-KO mice, but not Bax-KO mice. CONCLUSIONS: Mdivi1-NP treatment reduced IR injury through inhibition of MOMP, even in the absence of a CypD/MPTP opening. Thus, nanoparticle-mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury.

Cytotoxic Effects of One-step Self-etching Dental Adhesives on Human Periodontal Ligament Fibroblasts In Vitro.

PURPOSE: To evaluate the potential cytotoxic effects of four one-step self-etching dental adhesives [Adper Easy One (AEO), iBond (IB), Clearfil S³ Bond (CSB), and G-Bond (GB)] on cultured human periodontal ligament fibroblasts. MATERIALS AND METHODS: Cured adhesives were immersed in complete DMEM or deionized water and maintained at 37°C for 24 h, followed by sterilization. The deionized water-based extract was used for Fourier transform infrared spectroscopy analysis. The DMEM-based extract was diluted into various concentrations for cytotoxicity tests. The viability, integrity, and apoptosis of cultured human periodontal ligament fibroblasts upon treatment with the extracts were determined using the CCK-8 assay, microscopy, and flow cytometry. RESULTS: All of the four adhesives induced cell viability loss, cell morphology alteration, and cell death. GB showed the greatest cytotoxicity by inducing cell apoptosis and necrosis, while IB had the weakest cytotoxic effect on the cultured cells. CONCLUSION: All tested dental adhesives have significant adverse effects on cell viability. Therefore, precautions should be taken to protect the periodontal tissues when dental adhesives are applied in the clinic.

Protective Effects of Isorhamnetin on Cardiomyocytes Against Anoxia/Reoxygenation-induced Injury Is Mediated by SIRT1.

It has been reported that apoptosis plays a very important role on anoxia/reoxygenation (A/R)-induced injury, and human silent information regulator type 1 (SIRT1) can inhibit the apoptosis of cardiomyocytes. It has been proved that isorhamnetin (IsoRN), 3'-O-methyl-quecetin, can protect the cardiomyocytes, but the mechanism is still not clear. The aim of the study was to explore whether the protective effects of IsoRN on the cardiomyocytes against the A/R-induced injury are mediated by SIRT1. The effects of IsoRN on cardioprotection against A/R injury in neonatal rat cardiomyocytes were monitored by cell viability, the levels of mitochondrial membrane potential (Δψm), apoptosis, and intracellular reactive oxygen species (ROS), the levels of lactate dehydrogenase (LDH), creatine phosphokinase (CPK) and mitochondrial permeability transition pores (mPTP). The effects on protein expression were measured by western blot assay. The results showed that IsoRN can reduce A/R-induced injury by decreasing the level of lactate dehydrogenase and creatine phosphokinase release from the cardiomyocytes, increasing cell viability and expression of SIRT1, reducing the generation of reactive oxygen species, inhibiting opening of mitochondrial permeability transition pores and loss of Δψm and activation of caspase-3, and decreasing the release of cytochrome c, and reducing apoptosis. In addition, sirtinol, a SIRT1 inhibitor, drastically reduced the protective effects of IsoRN on cardioprotective effects in cardiomocytes. In conclusion, we firstly demonstrated that SIRT1 may be involved in the protective effects of IsoRN on cardiomocytes against the A/R-induced injury.

[Dose-effect relationship between vitamin C andparaquat poisoning rats].

OBJECTIVE: To explore the dose-effect relationship between vitamin C and paraquat (PQ) poisoning rats.
 Methods: A total of 40 Sprague-Dawley (SD) rats were randomly divided into 4 groups: a control group, a PQ poisoning group, a vitamin C group 1 and a vitamin C group 2 (n=10 in each group). 150 mg/kg PQ was perfused into rat stomach to establish PQ poisoning rat model. In PQ poisoning group, 30 mg/kg methylprednisolone and 2.5 mg/kg cyclophosphamide were injected peritoneally on the basis of PQ poisoning rat model. In vitamin C1 and C2 group, vitamin C was injected at a dosage of 5 or 500 mg/kg, respectively. The control group only received normal saline (NS). The malondialdehyde (MDA), liver and kidney function as well as arterial blood gas in the blood were examined 36 h later. At the end, the rats were killed and took the liver tissues for pathological examination and weight ratio calculation. The glutathione peroxidase (GSH-PX), ctychrome C (Cyt C) in the liver tissues were detected by chromatometry, and the Bcl-2 was detected by Western blot.
 Results: Compared with the PQ poisoning group, the MDA and Cyt C were decreased, the GSH-PX was increased, and liver and kidney functions were improved in the vitamin C group 1 (all P<0.01); but in the vitamin C group 2, the MDA increased and liver/kidney functions were impaired (all P<0.01). The expression of Bcl-2 in the PQ poisoning group was lower than that in the control group; compared with the PQ poisoning group, it was increased in the vitamin C1 group, while it was decreased in the vitamin C group 2 (both P<0.01). There was no obvious difference in the lung function, wet/dry weight ratio and pathological changes between the poisoning group and experimental groups (all P>0.05).
 Conclusion: Vitamin C at the low dose shows a certain degree of protection for the liver and kidney in the PQ poisoning rats model through it antioxidative activity and anit-apoptosis activity, while vitamin C at the high does may promote oxidation. Meanwhile, vitamin C doesn't show protective effect on lung in the PQ poisoning rats.

10-Hydroxycamptothecin induces apoptosis in human neuroblastoma SMS-KCNR cells through p53, cytochrome c and caspase 3 pathways.

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, remains one of the most challenging types of cancer to treat. Therefore, the search for novel effective drugs for its treatment is essential. The present study used 10-hydroxycamptothecin (HCPT), which is a naturally occurring alkaloid anticancer agent extracted from the Chinese tree, Camptotheca acuminata, and has a strong anticancer activity in vitro and in vivo. HCPT is able to induce apoptosis in cells of various tumor types. However, few studies have been conducted on its efficacy in NB, and its apoptosis-inducing mechanism has not been elucidated. In the present study, the in vitro effects of HCPT on apoptosis in the human NB cell line, SMS-KCNR, and its underlying molecular mechanisms were investigated. Cell proliferation was measured by an MTT assay and apoptosis was measured using DAPI staining and flow cytometric analysis. In addition, western blot analysis was used to evaluate the apoptosis-associated signaling pathways. HCPT was observed to markedly inhibit cell proliferation and induce apoptosis in SMS-KCNR cells at a relatively low concentration (2.5-20 nM). DAPI staining revealed typical apoptotic feature, namely apoptotic body formation. The flow cytometric analysis revealed that the number of apoptotic cells increased from 20.89% (for 2.5 nM) to 97.66% (for 20 nM) following HCPT treatment for 48 h. Western blot analysis revealed that p53, cytoplasmic cytochrome c, cleaved caspase-3 and poly ADP-ribose polymerase (PARP) proteins were significantly upregulated, while the mitochondrial cytochrome c and pro-caspase-3 proteins were downregulated. However, the B-cell lymphoma 2 and Bcl-2-associated X proteins were unaffected. The results indicated that HCPT may inhibit proliferation and induce apoptosis in the SMS-KCNR cells. The possible mechanism of apoptosis induction is the p53-mediated mitochondrial apoptotic signaling pathway, which promotes cytochrome c release and induces apoptosis by activating caspase-3 and PARP. Our study provides experimental evidence for HCPT as a potent therapeutic drug in NB treatment.

Pemetrexed induces apoptosis in malignant mesothelioma and lung cancer cells through activation of reactive oxygen species and inhibition of sirtuin 1.

Pemetrexed is a multitargeted antifolate used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). However, the mechanism by which pemetrexed induces apoptosis remains unclear. In the present study, we investigated the involvement of reactive oxygen species (ROS) and sirtuin 1 (SIRT1) in pemetrexed-induced apoptosis in MSTO-211 malignant mesothelioma cells and A549 NSCLC cells. Pemetrexed enhanced caspase-dependent apoptosis, induced intracellular ROS generation, and downregulated SIRT1 in the MSTO-211 and A549 cells. Pemetrexed-induced apoptosis, which was prevented by pretreatment with N-acetyl-cysteine (NAC), was mediated by effects on the mitochondria, including mitochondrial membrane potential transition (MPT) and cytosolic release of cytochrome c, and also involved regulation of SIRT1 expression. Interference with SIRT1 expression using siRNA enhanced pemetrexed-induced apoptosis through mitochondrial dysfunction and ROS generation, whereas resveratrol, an activator of SIRT1, protected against pemetrexed-induced apoptosis. These results show that pemetrexed induces apoptosis in MSTO-211 mesothelioma cells and A549 NSCLC cells through mitochondrial dysfunction mediated by ROS accumulation and SIRT1 downregulation.

Methanol extract of Flacourtia indica aerial parts induces apoptosis via generation of ROS and activation of caspases in human colon cancer HCT116 cells.

Different plant parts of Flacourtia indica have long been used in Ayurvedic medicine. Previous studies have demonstrated that the methanolic extract of F. indica possess anti-inflammatory properties. The present study was aimed at investigating the anticancer effects of methanol extract of Flacourtia indica (FIM) aerial parts in human colon cancer (HCT116) cells. Treatment of cells with FIM at a concentration of 500 µg/ml for 24 hours significantly reduced cell viability and induced apoptosis, which was associated with the increased cytoplasmic expression of cytochrome c, activation of caspase-3, and the cleavage of poly-(ADP-ribose) polymerase. Incubation with FIM also inhibited the levels of Bcl-2, Bcl-xl and survivin, which are the markers of cell proliferation, whereas the expression of Bax remained unchanged. Treatment with FIM led to the generation of reactive oxygen species (ROS) in a concentration-dependent manner. Pharmacological inhibition of ROS generation by pretreatment of cells with N-acetyl cysteine abrogated FIM-induced apoptosis in HCT116 cells. Thus, these results demonstrate that FIM has anti-proliferative and pro-apoptotic effects in HCT116 cells and the effects are, at least in part, due to the ROS dependent activation of caspases.

ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.