Lead induces Siberian tiger fibroblast apoptosis by interfering with intracellular homeostasis

Lead (Pb²⁺) is a poisonous heavy metal that causes many pathophysiological effects in living systems. Its toxicological effects are well known as it causes apoptosis of several cell types and tissues. This study aimed to determine the criteria required for early diagnosis of Pb²⁺ poisoning in the Siberian tiger using a tiger population in China, to identify a safety Pb²⁺ concentration threshold, and to provide suggestions for preventing Pb²⁺ poisoning in Siberian tigers. We investigated the apoptotic effects of Pb²⁺ (0, 32, 64, and 125?μM) for 12–48?h on Siberian tiger fibroblasts in vitro. Typical apoptotic effects were observed after Pb²⁺ exposure. Pb²⁺ strongly blocked DNA synthesis in the G0/G1 phase and induced cell apoptosis in a dose- and time-dependent manner. Intracellular free calcium (Ca²⁺) levels, reactive oxygen species levels, and efflux of extracellular Ca²⁺ were increased. The mitochondrial membrane potential was lowered. Caspase-3, -8, and -9 activities were increased when fibroblasts were treated with 32, 64, and 125?μM Pb²⁺. The gene expression levels of Bax, caspase-3, -8, Fas, and p53 were increased, while that of Bcl-2 was decreased. Calcium homeostasis and mitochondrial function were disturbed. Ca²⁺ efflux, oxidative damage, activation of caspases, and regulation of Bax, Bcl-2, caspase-3, -8, Fas, and p53 gene expression played an important role in the apoptotic effects. The disorder of intracellular homeostasis was the trigger for apoptosis in Siberian tiger fibroblasts.     

Effect of protriptyline on [Ca2+]i and viability in MG63 human osteosarcoma cells

Tricyclic antidepressants (TCA) have been clinically prescribed in the auxiliary treatment of cancer patients. Although protriptyline, a type of TCA, was used primarily in the clinical treatment of mood disorders in cancer patients, the effect of protriptyline on physiology in human osteosarcoma is unknown. This study examined the effect of protriptyline on cytosolic free Ca^2+?concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Protriptyline between 50 and 250?μM evoked [Ca²⁺]_i rises concentration-dependently. Protriptyline induced influx of Mn²⁺, indirectly implicating Ca^2+?influx. Protriptyline-evoked Ca^2+?entry was inhibited by nifedipine by 20% but was not altered by econazole, SKF96365, GF109203X, and phorbol-12-myristate-13-acetate (PMA). In Ca²⁺-free medium, treatment with protriptyline inhibited the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin-evoked [Ca²⁺]_i rises. Conversely, treatment with thapsigargin inhibited 45% of protriptyline-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter protriptyline-evoked [Ca²⁺]_i rises. Protriptyline at 50–250?μM decreased cell viability, which was not reversed by pretreatment with the Ca^2+?chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in MG63 cells, protriptyline induced [Ca²⁺]_i rises by evoking Ca^2+?release from the endoplasmic reticulum and other stores in a PLC-independent manner, and Ca^2+?entry via a nifedipine-sensitive Ca^2+?pathway. Protriptyline also caused Ca²⁺-independent cell death.     

Effect of 2,5-dimethylphenol on Ca2+ movement and viability in PC3 human prostate cancer cells

The phenolic compound 2,5-dimethylphenol is a natural product. 2,5-Dimethylphenol has been shown to affect rat hepatic and pulmonary microsomal metabolism. However, the effect of 2,5-dimethylphenol on Ca^2+?signaling and cyotoxicity has never been explored in any culture cells. This study explored the effect of 2,5-dimethylphenol on cytosolic free Ca^2+?levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500?μM and 1000?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca^2+?signal was inhibited by approximately half by the removal of extracellular Ca²⁺. 2,5-Dimethylphenol-induced Ca^2+?influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pretreatment with the protein kinase C (PKC) inhibitor GF109203X, nifedipine or the store-operated Ca^2+?entry inhibitors (econazole or SKF96365) inhibited 2,5-dimethylphenol-induced Ca^2+?signal in Ca²⁺-containing medium by ～30%. Treatment with the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin in Ca²⁺-free medium abolished 2,5-dimethylphenol-induced [Ca²⁺]_i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca²⁺]_i rises by ～80%. 2,5-Dimethylphenol killed cells at concentrations of 350–1000?μM in a concentration-dependent fashion. Chelation of cytosolic Ca^2+?with 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid/AM (BAPTA/AM) did not prevent 2,5-dimethylphenol’s cytotoxicity. Together, in PC3 cells, 2,5-dimethylphenol induced [Ca²⁺]_i rises that involved Ca^2+?entry through PKC-regulated store-operated Ca^2+?channels and PLC-dependent Ca^2+?release from the endoplasmic reticulum. 2,5-Dimethylphenol induced cytotoxicity in a Ca²⁺-independent manner.     

藏药蕨麻对原代培养小鼠肝细胞酒精损伤保护作用研究

目的 研究藏药蕨麻对原代培养酒精损伤肝细胞的保护作用。方法 原代肝细胞经分离纯化培养后,MTT法评价藏药蕨麻对酒精损伤肝细胞存活率的影响;荧光染色法测定藏药蕨麻对酒精损伤肝细胞活性氧物质(ROS)含量、细胞内钙离子浓度的影响;流式细胞仪检测藏药蕨麻对酒精损伤肝细胞凋亡的影响;免疫印迹法检测藏药蕨麻对Bcl-2和Bax表达的影响。结果 经酒精损伤后,肝细胞存活率降低;细胞内ROS含量和钙离子浓度增高;凋亡抑制基因Bcl-2减弱、促凋亡基因Bax表达增强。藏药蕨麻可明显提高细胞存活率;降低细胞内ROS含量和钙离子浓度;改善凋亡情况,增强Bcl-2表达,抑制Bax表达,且作用呈剂量依赖性。结论 藏药蕨麻对原代培养小鼠肝细胞酒精损伤具有一定的保护作用。     

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways

Context and objective: Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells.

Materials and methods: EL4 cells were exposed to POX for 0-16?h. EGTA was used to chelate Ca^2+? in extracellular medium, and heparin and procaine were used to inhibit Ca^2+?efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis.

Results and discussion: POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78.

Conclusions: Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.     

迷迭香酸抗过氧化氢诱导血管平滑肌细胞凋亡作用的研究

目的探讨迷迭香酸对过氧化氢(H2O2)诱导血管平滑肌细胞凋亡的保护作用及其机制。方法流式细胞术检测细胞凋亡率;吖啶橙(acridine orange,AO)染色观察细胞形态学变化;MTT法检测细胞活性;Western blot检测细胞中Bcl-2、Bax、Fas和FasL蛋白的表达。结果经500μmol·L-1H2O2处理24h后,细胞凋亡率为34.9%±2.55%,出现核固缩、核碎裂等典型的凋亡形态学改变,所以随后实验选择500μmol·L-1H2O2为诱导血管平滑肌细胞凋亡的最佳浓度。①500μmol·L-1的H2O2处理血管平滑肌细胞24h能引起细胞活性明显降低;细胞凋亡率明显增加,达35.7%±1.33%;细胞中Bcl-2蛋白表达降低,Bax蛋白表达升高,Bcl-2/Bax蛋白比值减少,Fas和FasL蛋白表达升高。②用迷迭香酸(10、20、40μmol·L-1)预处理细胞30min,可以增加细胞活性;降低细胞凋亡率,呈浓度依赖性;细胞中Bcl-2/Bax蛋白比值升高,Fas和FasL蛋白表达降低。结论迷迭香酸能拮抗H2O2诱导血管平滑肌细胞凋亡;其作用机制可能与升高细胞中Bcl-2/Bax蛋白比值,减少Fas、FasL蛋白表达有关。     

The role of apoptosis in the pathogenesis of dermatitis herpetiformis

Apoptosis is a form of cell death that is claimed to be involved in a number of chronic inflammatory and malignant skin diseases. The aim of this study was to investigate whether apoptosis may contribute to the pathogenesis of epidermal changes in dermatitis herpetiformis (DH) and, in particular, whether certain apoptosis-related markers such as Bax, Bcl-2, Fas and Fas ligand (FasL) take part in this process. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed on cryostat sections. Skin lesions from six and perilesional skin from four DH patients were stained with monoclonal antibodies to Bax, Bcl-2, Fas and FasL. The same evaluation was also performed on three patients affected by bullous pemphigoid (BP) and in two healthy donors. Using TUNEL technique, a remarkable increase in the apoptotic rate within the epidermal compartment was observed in DH and BP patients in comparison with normal controls. In our immunohistochemical analysis, Bax/Bcl-2 ratio was almost the same in the epidermis of perilesional/lesional DH, BP and healthy skin specimens. In DH and BP specimens both Bax and Bcl-2 proteins were increased in the dermal perivascular compartment. Fas showed a prevalently epidermal staining, both in DH and BP lesions, while FasL was distributed in perivascular and subjunctional dermis; some FasL+ cells infiltrated the DEJ and the basal layer of epidermis. This study allowed us to highlight conspicuous apoptotic phenomena in basal and suprabasal keratinocytes within lesional and perilesional skin of DH. We conclude that in DH, as well as in BP, apoptosis plays a role in the pathogenesis of cutaneous lesions in concert with other pathogenetic mechanisms.     

EGCG inhibits Cd2+-induced apoptosis through scavenging ROS rather than chelating Cd2+ in HL-7702 cells

Context and objective: Epigallocatechin-3-gallat (EGCG), the major catechin in green tea, shows a potential protective effect against heavy metal toxicity to humans. Apoptosis is one of the key events in cadmium (Cd²⁺)-induced cytotoxicity. Nevertheless, the study of EGCG on Cd²⁺-induced apoptosis is rarely reported. The objective of this study was to clarify the effect and detailed mechanism of EGCG on Cd²⁺-induced apoptosis.

Methods: Normal human liver cells (HL-7702) were treated with Cd²⁺ for 21?h, and then co-treated with EGCG for 3?h. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP) and caspase-3 activity were detected. On the other hand, the chelation of Cd²⁺ with EGCG was tested by UV-Vis spectroscopy analysis and Nuclear Magnetic Resonance (¹H NMR) spectroscopy under neutral condition (pH 7.2).

Results and conclusion: Cd²⁺ significantly decreased the cell viability and induced apoptosis in HL-7702 cells. Conversely, EGCG co-treatment resulted in significant inhibition of Cd²⁺-induced reduction of cell viability and apoptosis, implying a rescue effect of EGCG against Cd²⁺ poisoning. The protective effect most likely arises from scavenging ROS and maintaining redox homeostasis, as the generation of intracellular ROS and MDA is significantly reduced by EGCG, which further prevents MMP collapse and suppresses caspase-3 activity. However, no evidence is observed for the chelation of EGCG with Cd²⁺ under neutral condition. Therefore, a clear conclusion from this work can be made that EGCG could inhibit Cd²⁺-induced apoptosis by acting as a ROS scavenger rather than a metal chelating agent.     

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

Ouabain, a cardiotonic steroid and specific Na⁺/K⁺‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca²⁺, and mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca²⁺ production, but decreased the levels of ΔΨ_m in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATM^Ser1981, p‐H2A.X^Ser139, and p‐p53^Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERK^Thr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.     

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U‐2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell‐cycle distribution, apoptotic cells in sub‐G1 phase, reactive oxygen species (ROS), Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). Comet assay and 4′‐6‐diamidino‐2‐phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis‐associated protein levels in U‐2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U‐2 OS cells. CECF‐stimulated activities of caspase‐8, caspase‐9, and caspase‐3, ROS, and Ca²⁺ production, decreased ΔΨ_m levels of in U‐2 OS cells. CECF increased protein levels of caspase‐3, caspase‐9, Bax, cytochrome c, GRP78, AIF, ATF‐6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell‐cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U‐2 OS cells via ROS‐modulated caspase‐dependent and ‐independent pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1020–1031, 2014.     

Effects of NaF on the expression of intracellular Ca2+ fluxes and apoptosis and the antagonism of taurine in murine neuron

Sodium fluoride (NaF) has been shown to be cytotoxic and produces inflammatory responses in humans. However, the cellular mechanisms underlying the neurotoxicity of fluoride are unclear. The present study aims to define a possible mechanism of NaF-induced neurotoxicity with respect to apoptosis and intracellular Ca²⁺ fluxes. Meanwhile, the cytoprotective role of taurine in intervention, the toxic effects of NaF on neurons, is also investigated. The primary mouse hippocampal neurons were incubated with 5.0, 10.0, 15.0, 20.0, and 40.0?mg NaF/L in vitro and Kunming mice were exposed to 0.7, 2.8, and 11.2?mg NaF/kg and 7.5 and 15.0?mg taurine/kg in vivo. Intracellular Ca²⁺ fluxes and apoptosis were assayed. Compared with the control, the significant differences of intracellular Ca²⁺ concentration and apoptotic peaks were found in 5.0–40.0?mg NaF/L groups in vitro (p?<?0.01) and in the groups of 0.7–11.2?mg NaF/kg in vivo (p?<?0.01). Instantaneously, taurine can minimize F-induced neurotoxicity significantly at doses of 7.5 and 15.0?mg/kg (p?<?0.01). The present study herein suggested that NaF could increase intercellular Ca²⁺ concentration leading to apoptosis. Meanwhile, taurine could minimize neurotoxicity caused by fluoride through decreasing intercellular Ca²⁺ concentration and cell apoptosis.     

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase‐8/Bid and the reactive oxygen species‐dependent mitochondrial pathway

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC‐induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose‐dependent manners. Results from flow cytometric assay indicated that BITC induced sub‐G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca²⁺ release, but decreased the mitochondrial membrane potential (ΔΨ_m) and promoted caspase‐8, ‐9, and ‐3 activates. After cells were pretreated with Z‐IETD‐FMK, Z‐LEHD‐FMK, and Z‐DEVD‐FMK (caspase‐8, ‐9, and ‐3 inhibitors, respectively) led to decrease in the activities of caspase‐8, ‐9, and ‐3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase‐dependent pathways. Western blotting indicated that BITC induced Fas, Fas‐L, FADD, caspase‐8, caspase ‐3, and pro‐apoptotic protein (Bax, Bid, and Bak), but inhibited the ant‐apoptotic proteins (Bcl‐2 and Bcl‐x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP‐1, and ATF‐6β, IRE‐1α, IRE‐1β, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC‐induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase‐3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC‐caused growth inhibition and induced apoptotic cell death of GBM 8401 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1751–1760, 2016.     

Novel roles for ceramides, calpains and caspases in kidney proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies

Apoptosis is a tightly regulated physiological process, which can be initiated by toxic stimuli, such as cadmium (Cd²⁺). Cd²⁺ (10-50 μM) induces a rapid increase in reactive oxygen species (ROS) (≥30 min) in a cell line derived from the S1 segment of rat kidney proximal tubule, without any apparent mitochondrial dysfunction. The sphingolipid ceramide is an important second messenger in apoptosis. Short exposure to Cd²⁺ (3 h) causes an increase in ceramides, which occurs downstream of ROS formation, and may interact with cellular components, such as endoplasmic reticulum and mitochondria. Following apoptosis initiation, execution must take place. The classical executioners of apoptosis are caspases, a family of cysteine proteases. However, increasing studies report caspase-independent apoptosis, which questions the essentiality of caspases for apoptosis implementation. With low micromolar Cd²⁺ concentrations (<10 μM), caspases are only activated after 24 h and not at earlier time points, which supports the notion of caspase-independent apoptosis. Due to increased cytosolic Ca²⁺ under pathological conditions, a role for the Ca²⁺-dependent proteases, calpains, has emerged. Calpain activation by Cd²⁺ (3-6 h) seems to be regulated by ceramide levels, in order to induce apoptosis. Calpain and caspase substrates overlap but yield different fragments, which may explain their diverse downstream targets. Furthermore, calpains and caspases may interact with one another to enhance, as seen by Cd²⁺, or diminish apoptosis.In this review, we discuss novel roles for ceramides, calpains and caspases as part of Cd²⁺-induced apoptotic signalling pathways in the kidney proximal tubule and their in vivo relevance to Cd²⁺-induced nephrotoxicity.     

Schizandra chinensis extracts induce apoptosis in human gastric cancer cells via JNK/p38 MAPK activation and the ROS-mediated/mitochondria-dependent pathway

Abstract

Context: Schizandra chinensis Baill (Magnoliaceae) fruit extract (SCE) is considered a traditional herbal medicine for the treatment and alleviation of various diseases. Gastric cancer is the second most common cause of cancer-related death worldwide, and the first most common in Korea.

Objectives: This study investigates the mechanism of SCE-induced apoptosis in AGS human gastric cancer cells.

Materials and methods: SCE concentrations from 100 to 400?µg/ml were used. Cell viabilities were determined using MTT assay. Members of the Bcl-2 family and Bax were detected by Western blotting. RT-PCR was performed to measure the expression level of the Fas/FasL pro-apoptotic genes.

Results: SCE inhibited the proliferation AGS cells for 24 or 72?h (inhibition by 3.1%?±?5.2% at 100?µg/ml and 87.3%?±?7.6% at 400?µg/ml at 24?h and by 40.2%?±?5.3% 100?µg/ml and 95.3%?±?1.3% 400?µg/ml at 72?h) and increased the sub-G1 phase (25.3%?±?5.2% at 100?µg/ml and 370.2%?±?7.2% at 400?µg/ml) and the mitochondrial membrane depolarization (11.2%?±?2.1% at 100?µg/ml and 311.5%?±?6.1% at 400?µg/ml). The SCE-induced apoptotic cell death showed the down-regulation of Bcl-2, but up-regulation of Bax. Subsequently, SCE increased the expression level of Fas/FasL, activated caspase-9 and -3, and increased reactive oxygen species generation. Also, JNK II inhibitor or a p38 MAPK inhibitor inhibited SCE-induced cell death.

Discussion and conclusion: These results indicate that SCE might be an effective chemotherapeutic for the treatment of human gastric cancer.     

Cadmium perturbs calcium homeostasis in rat osteosarcoma (ros 17/2.8) cells; a possible role for protein kinase c

The mechanism of the toxic effects of Cd²⁺ on bone cell function is not completely understood at this time. This study was designed to characterize the effect of Cd²⁺ on Ca²⁺ metabolism in ROS 17/2.8 cells. Cells were labeled with ⁴⁵Ca (1.87 mM Ca) for 20 h in the presence of 0.01, 0.1, or 1.0 μM Cd²⁺ and kinetic parameters were determined from ⁴⁵a efflux curves. Three kinetic compartments described the intracellular metabolism of ⁴⁵Ca. Cd²⁺ (0.01 μM) caused an approximate 9 × increase in Ca²⁺ flux across the plasma membrane and a decrease in the most rapidly exchanging intracellular Ca²¹ compartment (S₁). However, there was no change in total cell Ca²⁺, indicating an increased cycling of Ca²⁺ across the plasma membrane. Flux between S₁, and the intermediate Ca²⁺ compartment (83) was also increased and S₂ increased significantly. All Cd²⁺ induced changes in Ca²⁺ homeostasis were obliterated by concurrent treatment with 0.1 μM calphostin C (CC), a potent protein kinase C (PKC) inhibitor. This data suggests that Cd²⁺ perturbs Ca²⁺ metabolism via a PKC dependent process.     

双酚A体外诱导雄性小鼠生殖细胞凋亡及其分子机制

目的采用双酚A(BPA,一种典型的环境雌激素)体外诱导雄性小鼠生殖细胞凋亡,并探讨其分子机制。方法对体外培养的雄性小鼠生殖细胞以不同剂量的BPA(10-7、10-6和10-5mol/L)进行诱导,检测细胞的凋亡状况;并应用RT-PCR和免疫蛋白印记技术检测凋亡相关基因(Bax/Bcl-2、Fas/FasL)的表达变化。结果低剂量的BPA(10-7mol/L)即能够引起支持细胞和精子细胞凋亡,但精原细胞对于BPA的作用却不敏感;随着BPA处理浓度的升高,支持细胞和精子细胞中Fas/FasL、Bax表达上调,Bcl-2表达下调,而精原细胞中基因表达变化不明显。结论BPA诱导的睾丸生殖细胞凋亡,不仅是依靠调节Fas/FasL系统起作用,Bax/Bcl-2基因也能够参与这一过程的调节。     

Senna leaf extracts induced Ca+2 homeostasis in a zoonotic tapeworm Hymenolepis diminuta

Context Plants and plant products have been used in traditional medicine as anthelmintic agents in human and veterinary medicine. Three species of Senna plant, S. alata (L), S. alexandrina (M) and S. occidentalis (L.) Link (Fabaceae) have been shown to have a vermicidal/vermifugal effect on a zoonotic tapeworm Hymenolepis diminuta (Rudolphi) (Cyclophyllidean).

Objective The present study validates the mode of action of these Senna plants on the parasite. The alcoholic leaf extract was determined to obtain information on the intracellular free calcium concentration level.

Materials and methods Hymenolepis diminuta was maintained in Sprague–Dawley rat model for 2 months. Live parasites collected from infected rat intestine were exposed to 40?mg/mL concentration of each plant extracts prepared in phosphate buffer saline at 37?°C, till parasite gets paralyzed. The rate of efflux of calcium from the parasite tissue to the medium and the level of intracellular Ca^2+?concentration were determined by an atomic absorption spectroscopy.

Results This study revealed that exposure of the worms to the plant extract leads to disruption in intracellular calcium homeostasis. A significant increase (44.6% and 25%) of efflux in Ca^2+?from the tissue to the incubated medium was observed. Senna alata showed high rate of efflux (5.32?mg/g) followed by S. alexandria and S. occidentalis (both 4.6?mg/g) compared with control (3.68?mg/g).

Discussion and conclusion These results suggest that leaf extracts caused membrane permeability to Ca^2+?after vacuolization of the tegument under stress and the extracts may contain compound that can be used as a chemotherapeutic agent.     

Sulforaphane‐induced apoptosis in human leukemia HL‐60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN‐induced cytotoxic effects and whether or not they went through cell‐cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL‐60 cells. Cell viability, cell‐cycle distribution, sub‐G1 (apoptosis), reactive oxygen species (ROS) and Ca²⁺ production, levels of mitochondrial membrane potential (ΔΨ_m), and caspase‐3, ?8, and ?9 activities were assayed by flow cytometry. Apoptosis‐associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub‐G1 phase development. Furthermore, SFN increased ROS and Ca²⁺ production and decreased the levels of ΔΨ_m and activated caspase‐3, ?8, and ?9 activities in HL‐60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas‐L, caspase‐8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl‐x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase ?8, ?3, ?4, ?6, and ?7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL‐60 cells via Fas‐ and mitochondria‐dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311–328, 2017.     

Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro

Purpose: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells.

Materials and methods: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10?g/L to 0.078125?g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm.

Results: Tetracaine at concentrations above 0.15625?g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF).

Conclusions: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.