The Mechanism Of Arsenic Trioxide Induced Human Gastric Cancer Cell Death

Apoptosis is a cell-autonomous programmed cell death mechanism that is utilized extensively during development, tissue homeostasis and maintenance of the immune system in adult organisms. These are two pathways for apoptosis are commonly referred to as the intrinsic and extrinsic pathways. Multiple signals converge on mitochondria, including DNA damage, microtubule disruption, and growth-factor deprivation. The intrinsic pathway centers on mitochondria as initiators of cell death seem to be very important for anticancer drugs.Bc1-2 family proteins are the most prominent of the intrinsic pathway in regulating cell apoptosis, Mitochondria is central to the apoptosis activities pathway in many physiological and pathological conditions. The mitochondrial inner transmembrane potential (â–³(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). Apoptosis stimuli cause these organelles to release cytochrome c (cyt c) and other apoptotic proteins into cytosol. In the cytosol, cyt-e binds the caspase-activating protein, apoptotic protease-activating factorl (Apaf 1), triggering its oligomerization into a hepatmeric complex that binds procaspase-9, forming a multiprotein structure known as the "apoptosome." Physical binding of Apafl to procaspase-9 is mediated by their caspase recruitment domains (CARDs), through homotypic CARD-CARD binding. Activation of apoptosome associated cell death protease caspase-9 then initiates a proteolytic cascade, where activated caspase-9 cleaves and activates downstream effectors proteases, such as procaspase-3. The Bc1-2 family of pro- and antiapoptotic proteins constitutes a critical control point for apoptosis, and the family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, involving dimerization, phosphorylation, proteolytic cleavage and mitochondrial translocation. The mitochondrial inner transmembrane potential (â–³(?)m) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore (PTP). But the mechanisms in anti-apoptosis are conversal.The intrinsic and extrinsic pathways for apoptosis converge on downstream effectors caspases. Certain effectors caspases are targets of suppression by an endogenous family of anti apoptotic proteins called inhibitor of apoptosis proteins (IAPs). The human genome encodes 8 lAP family members Survivin, X-linked inhibitor of apoptosis (X-IAP) and so on. Pathologic over expression of lAPs has been documented in cancer and leukemia. The functional importance of IAPs for apoptosis suppression in cancers has been documented by antisense experiments, in which knocking-down expression of Survivin, X-IAP or others induced apoptosis of tumor cell lines in culture or sensitized to apoptosis induced by anticancer drugs.Gastric cancer is one of the most common cancers in China. Conventional chemotherapy to most patients with advanced stage is usually not satisfactory. Therefore, discovery of new drugs for the treatment of gastric cancer is urgent.As₂O₃ is an active ingredient of a traditional Chinese medicine (TCM) has shown substantial efficacy in treating both newly diagnosed and relapsed patients with acute promyelocytic leukemia (APL) with few adverse effects and only minimal myelosuppression As₂O₃ has been shown to be an effective inducer of apoptosis in some solid cancer cells, such as gastric cancer, esophageal, prostate and ovarian carcinoma, neuroblastoma, hepatocarcinoma as well as head and neck cancers. Reports showed that As₂O₃ was also effective in inhibiting the growth of these cancers and which has been shown to be an effective inducer of apoptosis in some solid cancer cells. But the mechanism by which arsenic kills cancer cells remains unclear. Some reports have shown that arsenic-induced apoptosis is caused by a direct effect on the mitochondrial permeability transition pore, resulting in loss of the mitochondrial transmembrane potential. Other studies have demonstrated that arsenic compounds can disrupt mitosis, ROS, Bc1-2 family proteins, inhibitor of apoptosis proteins (lAPs) and therefore induce apoptosis in a variety of cell systems Because arsenic affects so many cellular and physiological pathways, a wide variety of malignancies, including both hematologic cancer and solid tumors derived from several tissue types, may be susceptible to therapy with arsenic trioxide. These multiple actions of arsenic trioxide also highlight the need for additional mechanistic studies to determine which actions mediate the diverse biological effects of this agent.In the present study, we will investigate the mechanisms of As₂O₃, mainly the role of Bc1-2, ROS and Survivin in As₂O₃ induced in human gastric cancer cell death.Materials and Methods1. Growth inhibition assay in vitro growth inhibition effect of As₂O₃ on gastric cancer cells was determined by MTT assay.2. Cell morphology and mitotic analysis ware performed by cytospin preparation with Wright-Giemsa stain. The slides were viewed and photographed under a light microscope.3. Flow cytometric analysis of mitochondrial transmembrane potential (â–³(?)m), membrane integrity, ROS, and Cell cycle.4. Expression of Bcl-2,Bax,Survivin,Smac/DIABLO and Caspase-3 were analyzed by Western blot.5. Statistical Analysis. All assays were set up in triplicate experiments, the results were showed as the mean±SD. Statistical analysis was determined by Student's t test.Results1. As₂O₃ inhibited human gastric cancer cells growth through different mechanisms. The MGC803, BCG823, SGC7901 gastric cancer cell lines exhibited susceptibility to As₂O₃ and showed in time and dose dependent manner, The IC₅₀ value for As₂O₃ in MGC803, BCG823, SGC7901 gastric cancer cells was about 2.8,3.1,10.2uM. MGC803, BCG823 was more susceptibility to As203 than SGC7901, the susceptibility was associated with the production of ROS but not with the internal ROS level. After treatment with SuM for 24h, the peak level of ROS was 100.8,103.3,56.5 respectively. The ROS in SGC7901 did not increase. Low concentrations of As₂O₃ can induce ROS production and reach peak level at 12h, and which also induce lose MPT, after treatment with 2, 5, 25uMAs₂O₃ for12-24h, the cells with lower MPT were 8.2%, 16.1%, 23% and in time and dose dependent manner.2. NAC can relieve the inhibition of growth induced by As203 and in combined with NAC the IC50 (48h) is more than 25uM, six times than As₂O₃ alone. NAC can decrease the ROS level induced by As₂O₃, the apoptosis and cell cycle was recovered. The ROS level was decreased to 7.2. The cell cycle was similar to the control.3.5μM As₂O₃ can induced typical apoptosis appearance, mitosis arrest and apoptosis body. But 25μM As₂O₃ did not induced cell cycle arrest and no apoptosis body, cells show swelling in cytosol. All different concentration As203 released Cyto-C from mitochondrial to Cytosol. Treatment with 5μM As₂O₃ 24h induced apoptotic cell froml.2% to 32.8%, necrotic cells reached to 12%. But treatment with 25μM As₂O₃ for 24h induced necrotic cells from 5.9% to72.2%.4. After treatment with 5uM As₂O₃ for 24h, the apoptotic cells increase to 20.37% and necrosis cells increase from 5.7%to 14.36%. 5uM As203 combined with 10uM BSO can switch cell from apoptosis to necrosis and the necrotic cell was about 46.11%. As203 combined with BSO killed MGC803 cells through caspase-3 independent manner, 12KD active subunit ofprcaspase-3 disappear and 17KD active subunit was in a half.5.5μM As₂O₃ can induce Bc1-2 protein phosphorylation from 12 and reached peak level at 36h and decreased after 48h, treatment with 5μM As₂O₃ results in Caspase-3 active and be cleaved into small parts, but treatment with 25μM As₂O₃ doesn't induce Bc1-2 protein phosphorylation and Pro-Caspase-3 was actived. The level of Pro-Caspase-3 decrease.6. After treatment with 5μM As₂O₃ for 6-36h, we separate the attaching and floating cell by lightly shaking the culture bottles, the floating cells show the decrease ofâ–³(?)m and which was not seen in the attaching cells, the floating cells display Bc1-2 phosphorylation, up regulation of survivin and caspase-3 was actived. The attaching cells show nothing, after washed with culture media 3 times, the floating and attaching cells were continue cultured for future 12 and 24h,the floating cells show that most of them appear apoptosis, Bcl-2 protein de-phosphorylation and the expression of survivin protein decrease, the attaching cell display small part apoptosis followed the decrease of the cells in G₂ phase, but Bc1-2 phosphorylation, up regulation of survivin and active caspase-3 was not induced. The level of Bcl-2, survivin and caspase-3 protein show no change.7.500nM Okadaic acid can keep Bcl-2 protein phosphorylation than the control in the floating cells for 4h. 100nM Staurosporine can promote Bcl-2 protein de-phosphorylation than the control in the floating cells for 4h.the cells treatment with 500nM Okadaic acid showed more apoptosis than the cells treatment with Staurosporine, the apoptotic cells in treatment with Okadaic acid 2 and 4h was 19.51%, 40.8% and in that with Staurosporine was 10.61%, 11.2% respectively for 2 and 4h.8.5uM As₂O₃ can increase the expression of Survivin protein from12h and reach the peak after treatment with As203 36h ,the expression of Survivin protein is 2.5 times higher than that of the control and 1.4 times in 48h, 25 uM As203 decrease the expression of surviving, Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As₂O₃ which enhanced the apoptosis and G₂/M phase arrest, he sub G0/GI phase cells was increased from 8.2% to 14.8% in 24h and from 15.9%to 26.7% in 48h, the cells in G₂/M phase were 32.38% to 56.4% in 24h and from 26.72% to 42.5% in 48h.DiscussionCell death can occur by either of two distinct mechanisms, apoptosis or necrosis. Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis is the pathological process which occurs when cells are exposed to a serious physical or chemical insult. Necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response. In the last few years, interest in apoptosis has increased greatly. Great progress has been made in the understanding of the basic mechanisms of apoptosis and the gene products involved. This genetic program is vital for normal development, for maintenance of tissue homeostasis, and for an effective immune system. Not surprisingly therefore, its disturbance is implicated in numerous pathological conditions, ranging from degenerative disorders to autoimmunity and cancer. Mitochondria are pivotal in controlling cell life and death. Reduced mitochondrial membrane potential (â–³(?)m) is considered as an initial and irreversible step towards apoptosis. The PTP is a multiprotein complex spanning the inner and outer mitochondrial membranes whose activation results in dissipation of the mitochondrial inner membrane potential, and eventual disruption of the outer mitochondrial membrane. In most cases, Bcl-2 family proteins are postulated to play a central role by controlling the permeabilization of mitochondrial membranes, either as pore forming sub-units or as regulators of intrinsic mitochondrial channels. Anti-apoptotic members of Bcl-2 family (e.g. Bcl-2 and Bcl-xL) act primarily to preserve mitochondrial integrity by suppressing the release of cytochrome c and Smac/DIABLO. Mitochondrial efflux of Cyt-C, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bc1-2 over expressing cells. Thus, in addition to modulating apoptosis- associated mitochondrial cytochrome c release.Arsentie has potential as an effective chemotherapeutic agent in treatment of a variety of cancers, but arsenties's chemotherapeutic mechanism is poorly understood. In many countries, As203 is approved for the treatment of APL, which is most often characterized by the presence of the oncogenic fusion protein PML-RARa. it is thought that arsentie exerts its proapoptotic and differentiate effects on these cells by inducing PML-RARo degradation and downregulation Bcl-2 protein. However, degradation of the oncoprotein is not the sole mechanism of arsenite action, because arsenite does induce apoptosis in cell lines that lack the fusion protein. Therefore, arsenite may be useful in the treatment of cancers other than APL. Recently, studies were carried out in the treatment of solid tumors. Now, the molecular mechanism of action by which As203 induces cell death remains poorly understood. In terms of a mechanism for the effect of on APL cells, it has been suggested based on experiments, with NB4 cells of APL that arsenic caused apoptosis directly through down-regulation of Bcl-2, some investigator reported that Bcl-2 was not down-regulated in arsenic trioxide-treated HL-60 cells. Our results showed that low concentration 5μM As₂O₃ induced Bcl-2 phosphorylation from 12 to 48h and reached peak level at 36h and the level of Bc1-2 protein did not change, procaspase-3 was cleavage into small molecular parts from 24h, high concentration 25μM As₂O₃ did not induced Bcl-2 protein phosphorylation and activated Caspase 3, mainly increased Rh123⁺ PI⁺ necrotic cells. As₂O₃ can induce ROS production and associated with the sensitivity to As₂O₃. BSO can switch cell from apoptosis to necrosis. AS₂O₃ combined with BSO killed MGC803 cells through caspase-3 independent manner, Okadaic acid can keep Bcl-2 protein phosphorylation, cells are more susceptible to a death signal during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2.Staurosporine can promote Bc1-2 protein de-phosphorylation and inhibit apoptosis. Ly294002, an inhibiter of PI3 Kinase inhibiter can inhibit the up-regulation of survivin induced by As203 enhanced the apoptosis and G2/M phase arrest, this result is consistent with us. Bc1-2 phosphorylation may lose it anti-apoptosis function and activated Caspase-3 results in MGC803 cells apoptosis.ConclusionIn summary, the present results indicate that ROS production play an important role in As₂O₃ killed human gastric cancer cell death, As203 killed human gastric cancer cell death via apoptosis and necrosis, Both apoptosis and necrosis are share the same mitochondrial pathway. Cells are more susceptible to As203 during G2/M and that characteristic can be attributed to phosphorylation of Bcl-2, As₂O₃ can increase the expression of Survivin protein may confer drug resistance through promoting cells through the mitosis. Inhibition the up-regulation of the Survivin expression sensitized gastric cancer cells to apoptosis induced by As203, so survivin may be a target for cancer therapy.