The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.     

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.     

紫草素对肝癌细胞SMMC-7721凋亡的影响及其机制

目的:探讨紫草素对肝癌SMMC-772细胞的作用及分子机制.方法:SMMC-7721细胞分别经0、5、20、80、320 ng/ml的紫草素作用0h、24 h、48 h和72 h后,适时采用CCK8法观察该细胞增殖的活性,hoechst染色分析细胞的核型变化,流式细胞仪检测细胞凋亡水平,Western blot证实细胞...     

Protein A-activated macrophages induce apoptosis in Ehrlich's ascites carcinoma through a nitric oxide-dependent pathway

Protein A (PA) of Staphylococcus aureus has been demonstrated to possess anti-tumor activity against a wide variety of tumors. In the current study we endeavored to obtain a mechanistic insight into PA-mediated Ehrlich's ascites carcinoma (EAC) killing. Our results indicate that PA stimulates generation of nitric oxide (NO) from murine peritoneal macrophages. Nitric oxide in turn induces cytotoxic damage to the tumor cells. Analysis of the morphological features and cell cycle phase distribution pattern of nuclear DNA revealed an induction of apoptosis (appearance of sub-G0/G1 population) in EAC after PA treatment. We have further elaborated the alterations in the expressions of the proto-oncoproteins p53 and Bax, together with a change in the ratio of Bcl-2/Bax in the treated tumor cells, which favor apoptosis. PA-induced apoptosis and changes in the expression of oncoproteins in the tumor cells was prevented by the suppression of NO release by the addition of L-NAME, the competitive NOS inhibitor, suggesting a possible mechanism by which PA exerts its anti-tumor activities involving nitric oxide through the alteration in the expressions of pro-apoptotic proteins.     

Influence of sCD40L on gastric cancer cell lines

The CD40 signaling pathway plays a key role in tumor cell proliferation, differentiation, and apoptosis. Gastric cancer usually possesses a higher level of CD40 expression than normal tissue. We evaluated inhibition of soluble CD40 ligand (sCD40L) in apoptosis induction of gastric cancer cells. Gastric cancer cells (AGS and BGC-823) were incubated with sCD40L. Cell viability and cell cycle were determined by methyl-tetrazolium (MTT) assay and flow cytometry, respectively. The results showed that sCD40L hindered cell growth, arrested cells at G0/G1 phase and induced apoptosis. In conclusion, sCD40L suppress growth of gastric cancer cells through apoptosis induction and cell cycle quiescence. This work will provided a new approach to gene therapy of gastric cancer.     

Salinomycin suppresses T24 cells by regulating KDM1A and the unfolded protein response pathway

In recent years, salinomycin has been shown to exert an anticancer effect in a variety of tumors; however, its function and mechanism in bladder cancer (BC) remain unclear. This study examined the effect of salinomycin on bladder cancer and analyzed its regulatory mechanism. T24 cells were treated with different concentrations of salinomycin to detect subsequent changes in cell proliferation, apoptosis, oxidative stress, H3K4 methylation, and related gene expression by the CCK8 assay, Edu staining, Tunel staining, ELISA, RT-qPCR, and western blotting, respectively. A KDM1A overexpression plasmid, catalytically inactive KDM1A overexpression plasmid, or short hairpin RNA (shRNA) plasmid was transfected into T24 cells to evaluate their effects. A xenograft tumor model was used to further confirm the anti-tumor effect of salinomycin. Our results showed that salinomycin significantly inhibited cell proliferation, promoted apoptosis, increased MDA levels, decreased SOD levels, induced H3K4 histone methylation, and suppressed KDM1A expression. Furthermore, the sh-KDM1A plasmid had effects similar to those of salinomycin and also activated the unfolded protein response pathway. The KDM1A overexpression plasmid had effects opposite to those of the sh-KDM1A plasmid, and the catalytically inactive KDM1A overexpression plasmid had no effect. Meanwhile, KDM1A overexpression reversed the effects of salinomycin on T24 cells. Finally, in vivo experiments confirmed the above results. In the salinomycin treatment group, tumor growth and KDM1A expression were suppressed and cell apoptosis and UPR were induced, while treatment with the KDM1A overexpression plasmid produced the opposite effects. Collectively, our study revealed that salinomycin suppressed T24 cell proliferation and promoted oxidative stress and apoptosis by regulating KDM1A and the UPR pathway.

     

ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma,interacts directly with metallothionein 2A and links to apoptosis

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.     

Nitric oxide promotes resistance to tumor suppression by CTLs

Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CTLs. We propose that NO production could initially act as an autocrine suicide or paracrine killing mechanism in cells undergoing malignant transformation. However, once failed, the outcome is fatal. NO promotes tumor formation by enhancing the selection of cells that can evade immune attack by acquiring apoptosis resistance.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Celastrol suppresses nitric oxide synthases and the angiogenesis pathway in colorectal cancer

The thunder god vine (Tripterygium wilfordii Hook. F) is traditionally used for inflammation-related diseases in traditional Chinese medicine. In recent years, celastrol (a natural compound from the root of the thunder god vine) has attracted great interest for its potential anticancer activities. The free radical nitric oxide (NO) is known to play a critical role in colorectal cancer growth by promoting tumour angiogenesis. However, how celastrol influences the NO pathway and its mechanism against colorectal cancer is largely unknown. In this study, we investigated the effects and mechanism of celastrol on nitric oxide synthase (NOS) and the angiogenesis pathway in colorectal cancer. Our data show that celastrol inhibited HT-29 and HCT116 cell proliferation, migration, and NOS activity in the cytoplasm. The antiproliferation activity of celastrol was associated with the inhibition of iNOS and eNOS in colorectal cancer cells. Treatment with celastrol inhibited colorectal cancer cell growth and migration, and was associated with suppression of the expression of key genes (TYMP, CDH5, THBS2, LEP, MMP9, and TNF) and proteins (IL-1b, MMP-9, PDGF, Serpin E1, and TIMP-4) involved in the angiogenesis pathway. In addition, combinational use of celastrol with 5-fluorouracil, salinomycin, 1400 W, and L-NIO showed enhanced inhibition of colorectal cancer cell proliferation and migration. In sum, our study suggests that celastrol could suppress colorectal cancer cell growth and migration, likely through suppressing NOS activity and inhibiting the angiogenesis pathway.     

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines

Summary Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [³H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.     

Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.     

RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo

This study aimed to investigate the anti-tumor activity of RY10-4, a small molecular that was designed and synthesized based on the structure of protoapigenone. A previous screening study showed that RY10-4 possessed anti-proliferative effects against HepG2 human hepatocellular carcinoma cells. However, the full range of RY10-4 anti-cancer effects on liver tumors and the underlying mechanisms have not been identified. Herein, employing flow cytometry, and Western blot analysis, we demonstrate that RY10-4 can induce cell cycle arrest, intracellular reactive oxygen species (ROS) production and apoptosis in HepG2 cells. In HepG2 cell xenograft tumor model, RY10-4 significantly inhibited the growth of tumors and induced apoptosis in tumor cells, with little side effects. Moreover, RY10-4 caused the suppression of STAT3 activation, which may be involved the apoptosis induction. In addition, RY10-4 inhibited the proliferation of Hep3B and HuH-7 human hepatocellular carcinoma cells in a concentration-dependent manner. Taken together, our results suggest that RY10-4 has a great potential to develop as chemotherapeutic agent for liver cancer.     

IL-21 enhances tumor-specific CTL induction by anti-DR5 antibody therapy

Tumor cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful anti-tumor immunotherapies. In this study, we show that induction of tumor cell apoptosis by agonistic mAb against DR5, combined with delayed IL-21 treatment, suppressed tumor growth and pre-established tumor metastases. Synergistic effects of the combination were observed in several tumor models where the target tumor was sensitive to DR5-mediated apoptosis. IL-21 promoted tumor-specific CTL activity and enhanced memory responses to tumor rechallenge. These results indicate that a rational combination of Ab-based therapy that causes tumor cell apoptosis and a cytokine that promotes T cell memory is a useful new strategy for cancer immunotherapy.     

Contribution of nitric oxide-mediated apoptosis to cancer metastasis inefficiency

Metastasis is largely an unsuccessful process, because the majority of disseminating tumor cells demise shortly after reaching distant organs. Therefore, survival is essential for disseminating tumor cells to establish metastases. During metastasis, interaction between tumor and host cells leads to the production of nitric oxide (NO). An increasing amount of evidence suggests that NO regulates tumor-cell survival and influences cancer metastasis. The ultimate effect of NO on tumor-cell survival is dictated by multiple factors, including the levels of NO production and genetic and epigenetic makeup of the tumor cells. Also, expression of inducible nitric oxide synthase (NOS) II has the potential to produce NO at a toxic level and tumor-cell death via apoptosis. Yet, impaired NOS II expression during tumor progression may lead to decreased NO production, which may be insufficient to produce significant cytotoxic effects, and the subsequent low level of NO production may cause induction of NO resistance, and the NO-resistant tumor cells may usurp NO to undergo progression. Thus, restoration of NOS II expression and reversal of NO resistance may prevent tumor growth and metastasis.     

Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation

The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G1 and G2/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages.     

Inhibition of cell growth and induction of apoptosis in non-small cell lung cancer cells by delta-tocotrienol is associated with notch-1 down-regulation

Lung cancer is the leading cause of death among all cancers. Non-small cell lung cancer accounts for 80% of lung cancer with a 5-year survival rate of 16%. Notch pathway, especially Notch-1 is up-regulated in a subgroup of non-small cell lung cancer patients. Since Notch-1 signaling plays an important role in cell proliferation, differentiation, and apoptosis, down-regulation of Notch-1 may exert anti-tumor effects. The objective of this study was to investigate whether delta-tocotrienol, a naturally occurring isoform of Vitamin E, inhibits non-small cell lung cancer cell growth via Notch signaling. Treatment with delta-tocotrienol resulted in a dose and time dependent inhibition of cell growth, cell migration, tumor cell invasiveness, and induction of apoptosis. Real-time RT-PCR and western blot analysis showed that antitumor activity by delta-tocotrienol was associated with a decrease in Notch-1, Hes-1, Survivin, MMP-9, VEGF, and Bcl-XL expression. In addition, there was a decrease in NF-κB-DNA binding activity. These results suggest that down-regulation of Notch-1, via inhibition of NF-κB signaling pathways by delta-tocotrienol, could provide a potential novel approach for prevention of tumor progression in non-small cell lung cancer.     

Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells

Matrine, one of the main components extracted from Sophora flavescens Ait, has a wide range of pharmacological effects including anti-tumor activities on a number of cancer cell lines. This study has investigated whether matrine could also display anti-tumor action on rat C6 glioma cells. Exposure of C6 cells to matrine resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner, as measured by the MTT assay and Flow cytometry. The Annexin V/PI staining further detected the apoptotic cells at both early and late phases of apoptosis. We used AO/EB staining to examine the programmed cell death of matrine-treated C6 cells, and showed that the death rate detected by AO/EB staining was higher than the apoptosis rate measured by Annexin V/PI staining, suggesting that autophagy, the Type II programmed cell death, may be involved in matrine-induced cell death, which was further confirmed by electronic microscopy. To explore the molecular mechanism, an apoptosis real-time PCR array was performed, which has demonstrated that 57 genes were at least 2-fold upregulated, and 11 genes were at least 2-fold downregulated in matrine-treated C6 cells, compared with untreated cells. However, the gene expression profiles could only partly and roughly explain molecular mechanisms of apoptosis and autophagy in matrine-treated C6 cells, thus further investigations are required to confirm the specific molecular pathways and related molecules responsible for the programmed cell death.