Search

T-2 toxin is one of the most toxic trichothecenes and harmful to human health and animal husbandry. The mechanism underlying its growth suppression remains unclear, especially for mitochondrial damage in human gastric epithelial cells. In the present study, we investigated cell death caused by T-2 toxin in a human gastric epithelial cell line (GES-1) and the possible mechanism of T-2-induced cytotoxicity. T-2 strongly reduced the viability of GES-1 cells in a time- and dose-dependent manner within a small range of concentrations. However, when the concentrations of T-2 were >40 nM, there was no concentration dependence, only time dependence. Moreover, T-2 induced apoptosis, with the activation of caspase-3 in GES-1 and mitochondrial membrane potential (MMP) decrease and cytochrome c release. T-2 also resulted in the accumulation of reactive oxygen species (ROS) and DNA damage with a positive signal of p-H2A.X in GES-1 cells. While T-2 caused a MMP decrease, DNA damage and cell death were not blocked by pretreatment with 3 mM glutathione (GSH), a typical scavenger of ROS. The induction of mitochondrial permeability transition pore (mPTP) regulators voltage-dependent anion channel (VDAC1) and cyclophilin D (CypD) were also observed in T-2-treated cells. Interestingly, cyclosporine A (CsA), a CypD inhibitor, significantly reversed the drop in MMP and the DNA damage, as well as ROS accumulation caused by T-2. Additionally, GES-1 cell death could also be protected to some extent by 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), an inhibitor of VDAC1, especially the combination of CsA and DIDS, and 3 mM GSH could further enhance the effect of CsA + DIDS on cell viability. In conclusion, our present findings indicate that the T-2 induced MMP decrease, DNA damage and cell death, as well as ROS accumulation in GES-1 cells, starts with T-2 directly perturbing the mitochondria triggering ROS generation by acting on CypD and VDAC1. This study presents a new viewpoint for evaluating the toxicity of T-2 toxin., (© 2020 John Wiley & Sons, Ltd.)

Hepatic injury is one of the most common digestive system diseases worldwide in clinic. Guanylic acid or guanosine monophosphate (GMP) was an important component of nucleotides, which is mainly in the form of sodium salt (disodium guanylate, GMP-Na 2 ). However, its effect on hepatic injury has not yet been investigated. This study is to investigate the protective effects of GMP-Na 2 on acute hepatic injury induced by carbon tetrachloride (CCl 4 ), and to explore its mechanism. The hepatic injury models of mice and HL-7702 cells were induced by CCl 4 . The alanine transaminase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidant capacity (T-AOC) were determined by biochemical method. Hematoxylin-eosin staining were used to determine the morphological changes on liver tissue in mice. The mRNA and protein expressions of caspase-3, Bax, and Bcl-2 were detected by RT-PCR and Western blot analysis. Our results show that GMP-Na 2 treatment significantly decreased the activities of ALT and AST, and the levels of MDA as well as increased the levels of SOD, GSH-Px, and T-AOC. Importantly, GMP-Na 2 effectively enhanced the antiapoptosis function by upregulating Bcl-2 expression and downregulating caspase-3 and Bax expressions in vivo and in vitro. Moreover, the histopathological changes of liver tissue were obviously improved after GMP-Na 2 treatment. These findings suggest that GMP-Na 2 has protective effects on hepatic injury, and its mechanisms may be associated with antioxidative stress and antiapoptosis., (© 2019 Institute of Food Technologists®.)

In current study, we investigated the anti-tumor effect of luteolin in human ESCC cell lines in vitro and in vivo and tried to explore the potential mechanisms. Results from flow cytometry showed that luteolin could induce apoptosis and caspase-3 activation and induce cell cycle arrest at G2/M phase in a dose- and time-dependent manner in EC1 and KYSE450 cells. JC-1 test results showed that membrane potential of mitochondria after luteolin treatment was down-regulated and this was an indicator for intrinsic apoptosis. Western Blot results showed the expression of cell cycle regulatory protein p21 and p53 increased and three apoptosis related proteins that participate in mitochondrial apoptotic pathway, namely, Bim, CYT-c and cPARP, also increased in luteolin treated cells compared with control groups. We further confirmed that luteolin could significantly inhibit the growth of ESCC tumors in xenograft mouse models and no evidence of systemic toxicity was observed. Our results suggest that luteolin can induce cell apoptosis and cell cycle arrest in G2/M phase through mitochondrial pathway in EC1 and KYSE450 cell lines and proper utilization of luteolin might be a practical approach in ESCC chemotherapy.

Ent-kaurane diterpene compounds have attracted considerable attention in recent years due to its antitumor, antibacterial, and antiviral activities. However, the clinical development of natural kaurane diterpenes, for example, oridonin for cancer therapy has been hampered by its relatively moderate potency, limited bioavailability. Herein, we report a newly synthetic analog of natural ent-kaurane diterpene, DS2, which exhibits significantly improved activity of antiproliferation against various cancer cell lines relative to oridonin. DS2 treatment triggers the mitochondria-mediated apoptosis and cell cycle arrest in human esophageal cancer cell lines (EC9706, EC109). Interestingly, normal human esophageal epithelial cells (HEECs) and normal human liver cells (HL-7702) are both significantly more resistant to the growth inhibition by DS2 compared with esophageal cancer cells. The DS2-induced apoptosis in EC9706 cells correlated with the drop of mitochondrial membrane potential (MMP), release of cytochrome c into the cytosol and activation of caspase-9 and -3. The induction of proapoptotic proteins p21 and Bax were also observed in DS2-treated cells. The DS2-induced apoptosis was significantly attenuated by knockdown of Bax proteins. Meanwhile, the DS2 treatment caused generation of reactive oxygen species (ROS) in human esophageal cancer cells, but not in HEECs, which was attenuated by pretreatment with ROS scavenger N-acetylcysteine (NAC). More interestingly, the antioxidants pretreatment completely attenuated DS2 mediated loss of the MMP and apoptosis, as well as Bax expression and growth inhibition. In conclusion, the present study reveals that the mitochondria-mediated cell death by DS2 is associated with Bax regulation and ROS generation, and understanding the function and mechanism of DS2 will help us to design better anti-cancer drugs.

Aim: To investigate anti-apoptotic effects of glycyrrhizic acid (GA) against fibrosis in carbon tetrachloride (CCl4)-induced liver injury and its contributing factors., Methods: Liver fibrosis was induced by administration of CCl4 for 8 wk. Pathological changes in the liver of rats were examined by hematoxylin-eosin staining. Collagen fibers were detected by Sirius red staining. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3, Bax, α-SMA, connective tissue growth factor (CTGF), matrix metalloproteinase (MMP) 2 and MMP9 proteins were evaluated by western blot analysis, and α-SMA mRNA, collagen type I and III mRNA were estimated by real-time PCR., Results: Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group. TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group. The expression levels of cleaved caspase-3, Bax, α-SMA, CTGF, MMP2 and MMP9 proteins, and α-SMA mRNA, collagen type I and III mRNA were also significantly reduced by GA compared with the CCl4-treated group (P < 0.05)., Conclusion: GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.

Objective: To investigate the role of p38 MAPK on ischemic postconditioning (IPO) attenuating pneumocyte apoptosis after lung ischemia/reperfusion injury (LIRI)., Methods: Forty adult male SD rats were randomly divided into 5 groups based upon the intervention (n = 8): control group (C), LIR group (I/R), LIR + IPO group (IPO), IPO + solution control group (D), IPO + SB203580 group (SB). Left lung tissue was isolated after the 2 hours of reperfusion, the ratio of wet lung weight to dry lung weight (W/D), and total lung water content (TLW) were measured. The histological structure of the left lung was observed under light and electron transmission microscopes, and scored by alveolar damage index of quantitative assessment (IQA). Apoptosis index (AI) of lung tissue was determined by terminal deoxynuleotidyl transferase mediated dUTP nick end and labeling (TUNEL) method. The mRNA expression and protein levels of and Bax were measured by RT-PCR and quantitative immunohistochemistry (IHC)., Results: Compared with C group, W/D, TLW, IQA, AI and the expression of Bax of I/R were significantly increased, the expression of Bcl-2 and Bcl-2/Bax were significantly decreased (P < 0.05, P < 0.01), and was obviously morphological abnormality in lung tissue. Compared with I/R group, all the indexes of IPO except for the expression of Bcl-2 and Bcl-2/ Bax were obviously reduced, the expression of Bcl-2 and Bcl-2/Bax were increased (P < 0.05, P < 0.01). All the indexes between D and IPO were little or not significant( P > 0.05). The expression of Bcl-2 and Bcl-2/Bax of SB were significantly increased and other indexes were reduced than those of IPO (P < 0.05, P < 0.01)., Conclusion: IPO may attenuate pneumocyte apoptosis in LIRI by inactivation of p38 MAPK, up-regulating expression of Bcl-2/Bax ratio.

Objective: To investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats., Methods: Adult male SD rats were randomly divided into 3 groups based upon the intervention (n = 8): control group (C), lung ischemic reperfusion group (LIR), LIR+ IPostC group (IPostC). At the end of the experiment, blood specimens drawn from the arteria carotis were tested for the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO); the pneumocyte apoptosis index (AI) was achieved by tennrminal deoxynucleotidyl transferase mediated dUTP nick end abeling (TUNEL); the expression of Bcl-2, Bax protein in lung tissue was accessed by quantitative immunohistochemistry (MHC) and Bcl-2, Bax mRNA by RT-PCR., Results: IPostC could significantly attenuate the MDA level, MPO activity and improve SOD activity in blood serum which was comparable to I/R and significantly reduced the number of TUNEL-positive cells compared with I/R group, expressed as Al (% total nuclei) from (39.0 +/- 3.46) to (8.0 +/- 0.88) (P < 0.01). The protein and mRNA expression of Bcl-2 and Bax showed that IPO significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein but improved the expression of Bcl-2 that improved the Bcl-2/Bax ratio (P < 0.01) ., Conclusion: IPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.

Isodon rubescens, a Chinese herb, has been used as a folk, botanical medicine in China for inflammatory diseases and cancer treatment for many years. Recently, we isolated a new ent-kaurene diterpenoid, named Jaridonin, from Isodon rubescens. The chemical structure of Jaridonin was verified by infrared (IR), nuclear magnetic resonance (NMR), and mass spectrum (MS) data as well as X-ray spectra. Jaridonin potently reduced viabilities of several esophageal cancer cell lines, including EC109, EC9706 and EC1. Jaridonin treatment resulted in typical apoptotic morphological characteristics, increased the number of annexin V-positive staining cells, as well as caused a G2/M arrest in cell cycle progression. Furthermore, Jaridonin resulted in a significant loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and then activation of Caspase-9 and -3, leading to activation of the mitochondria mediated apoptosis. Furthermore, these effects of Jaridonin were accompanied by marked reactive oxygen species (ROS) production and increased expression of p53, p21(waf1/Cip1) and Bax, whereas two ROS scavengers, N-acetyl-L-cysteine (LNAC) and Vitamin C, significantly attenuated the effects of Jaridonin on the mitochondrial membrane potential, DNA damage, expression of p53 and p21(waf1/Cip1) and reduction of cell viabilities. Taken together, our results suggest that a natural ent-kaurenoid diterpenoid, Jaridonin, is a novel apoptosis inducer and deserves further investigation as a new chemotherapeutic strategy for patients with esophageal cancer.

Objective: To study the effect of Triptolide (TL) on the growth of prostate carcinoma cell line, and analyze its function and mechanism in anti-prostate cancer., Methods: MTT experiments were performed to examine the inhibiting effect of TL on the proliferation of RM-1 cells, cell morphological changes observed by acridine staining, cellular cycles and apoptosis peak analyzed by flow cytometry, the apoptosis fracture zone investigated with DNA electrophoresis, and the expressions of caspase-3 and bcl-2 mRNA in RM-1 cells examined by RT-PCR., Results: The results of MTT experiments showed that after the treatment of TL (5, 10, 20, 40 and 80 ng/ml), the RM-1 cell proliferation inhibition rates were 9.8%, 25.1%, 39.2%, 48.8% and 53.2% respectively; 12, 24, 36 and 48 hours after the treatment of TL (10 and 20 ng/ml), the cell proliferation inhibition rates were 8.4%, 25.1%, 36.1%, 42.4% and 10.2%, 39.2%, 50.2% and 58.5% respectively. Acridine staining after the TL treatment revealed nucleus condensation, cell membrane invagination, irregular orange particles in the cells and apoptosis morphological changes; flow cytometry tests showed that 48 hours after the TL treatment (10, 20 ng/ml) of RM-1 cells, an obvious apoptosis peak appeared before the G1 stage; 24, 36 and 48 hours after it, DNA "trapezoid" strips could be seen; the caspase-3 mRNA expression in the TL treated cells was higher, and the bcl-2 mRNA expression was lower than in the controls., Conclusion: TL can decrease bcl-2 expression, increase caspase-3 expression, induce apoptosis of prostate carcinoma cells, and consequently inhibit the proliferation of RM-1 cells in mice.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.