Apoptotic pathway induced by diallyl trisulfide in pancreatic cancer cells

AIM:To investigate the effects of diallyl trisulfide(DATS),a garlic-derived organosulfur compound,in pancreatic cancer cells.METHODS:Human pancreatic cancer cells with wildtype p53 gene(Capan-2)and normal pancreatic epithelial cells(H6C7)were cultured in RPMI1640.DATS was prepared at a concentration of 100μmol/L.Cell viability was determined via the methyl thiazolyl tetrazolium assay.Apoptotic cells were detected by TUNEL assay.Cell cycle analysis was performed using flow cytometry.Protein expression was determined by Western blot.Bax and Bcl-2 expression was detected by immunofluorescence.Apoptosis genes and cell cycle were assessed by quantitative real-time polymerase chain reaction.RESULTS:DATS suppressed the viability of cultured human pancreatic cancer cells(Capan-2)by increasing the proportion of cells in the G2/M phase and induced apoptotic cell death.Western blot analysis indicated that DATS enhanced the expression of Fas,p21,p53and cyclin B1,but downregulated the expression of Akt,cyclin D1,MDM2 and Bcl-2.DATS induced cell cycle inhibition which was correlated with elevated levels of cyclin B1 and p21,and reduced levels of cyclin D1 in Capan-2 cells and H6C7 cells.DATS-induced apoptosis was markedly elevated in Capan-2 cells compared with H6C7 cells,and this was correlated with elevated levels of cyclin B1 and p53,and reduced levels of Bcl-2.DATS-induced apoptosis was correlated with downregulation of Bcl-2,Akt and cyclin D1 protein levels,and up-regulation of Bax,Fas,p53 and cyclin B protein levels in Capan-2 cells.CONCLUSION:DATS induces apoptosis of pancreatic cancer cells(Capan-2)and non-tumorigenic pancreatic ductal epithelial cells(H6C7).     

Inhibition of DNA primase and induction of apoptosis by 3,3′-diethyl-9-methylthia-carbocyanine iodide in hepatocellular carcinoma BEL-7402 cells

AIM:To evaluate the effects of 3,3′-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.RESULTS:DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09μmol/L. Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53,Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.     

Significance of Bcl-xL in human colon carcinoma

AIM：To investigate the clinical significance of Bcl-xL gene in the pathogenesis of human colon carcinoma. METHODS：Fifty-six pair tissue samples from patients with colon cancer were collected, and protein level of the Bcl-xL gene was measured by immunohistochemistry method. The correlation of Bcl-xL expression with clinical index was evaluated. After human colon cancer cell line HT29 was transfected with Bcl-xL small interfering RNA （siRNA）, the anchorage-independent growth of cancer cells was detected by colony formation in soft agar and invasion ability of cancer cells was determined by a transwell model.
RESULTS：The Bcl-xL expression was higher in cancerous tissue samples than in normal tissue samples （38.78 ± 11.36 vs 0.89 ± 0.35, P 〈 0.001）, and was associated with the pathological grade, lymphnode metastasis and Duke＇s stage of colorectal carcinoma. Transfection with Bcl-xL siRNA inhibited the colony formation and invasion ability of human colon cancer cell line HT29 in vitro.
CONCLUSION：Bcl-xL gene plays an important role in carcinogenesis of human colorectal carcinoma and is associated with malignant biological behaviors of human colorectal carcinoma.     

Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating peroxisome proliferator-activated receptor γ

AIM:To examine whether and how rosiglitazone enhances apoptosis induced by fluorouracil in human colon cancer(HT-29)cells.METHODS:Human colon cancer HT-29 cells were cultured in vitro and treated with fluorouracil and/or rosiglitazone.Proliferation and growth of HT-29 cells were evaluated by MTT assay and trypan blue exclusion methods,respectively.The apoptosis of HT-29 cells was determined by acridine orange/ethidium bromide staining and flow cytometry using PI fluorescence staining.The expressions of peroxisome proliferator-activated receptor γ(PPARγ),Bcl-2 and Bax in HT-29 cells were analyzed by Western blot.RESULTS:Although rosiglitazone at the concentration below 30 μmol/L for 72 h exerted almost no inhibitory effect on proliferation and growth of HT-29 cells,it could significantly enhance fluorouracil-induced HT-29 cell proliferation and growth inhibition.Furthermore,10 μmol/L rosilitazone did not induce apoptosis of HT-29 cells but dramatically enhanced fluorouracil-induced apoptosis of HT-29 cells.However,rosiglitazone did not improve apoptosis induced by fluorouracil in HT-29 cells pretreated with GW9662,a PPARγ antagonist.Meanwhile,the expression of Bax and PPARγ was up-regulated,while the expression of Bcl-2 was down regulated in HT-29 cells treated with rosiglitazone in a time-dependent manner.However,the effect of rosiglitazone on Bcl-2 and Bax was blocked or diminished in the presence of GW9662.CONCLUSION:Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating PPARγ.     

Apoptosis induction with polo-like kinase-1 antisense phosphorothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM:To investigate the effects of polo-like kinase-1 (PLKl) antisense phosphorothioate oligodeoxynudeotide (ASODN) on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS:After SW480 colon cancer cells were transfected with PLKl ASODN, Northern and Western blot analyses were used to examine PLKl gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynudeotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS:The levels of PLKl mRNA and protein were greatly inhibited by PLKl ASODN in SW480 cancer cells transfected with PLKl ASODN. Apoptosis index (AI) induced PLKl ASODN in a time-and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G_2/M compared with control groups. CONCLUSION:PLKl ASODN can induce apoptosis of human colon cancer cell line SW480.     

Mechanisms of trichostatin A inhibiting AGS proliferation and identification of lysine-acetylated proteins

AIM: To explore the effect of lysine acetylation in related proteins on regulation of the proliferation of gastric cancer cells, and determine the lysine-acetylated proteins and the acetylated modified sites in AGS gastric cancer cells. METHODS: The CCK-8 experiment and flow cytometry were used to observe the changes in proliferation and cycle of AGS cells treated with trichostatin A (TSA). Real time polymerase chain reaction and Western blotting were used to observe expression changes in p21, p53, Bax, Bcl-2, CDK2, and CyclinD1 in gastric cancer cells exposed to TSA. Cytoplasmic proteins in gastric cancer cells before and after TSA treatment were immunoprecipitated with anti-acetylated lysine antibodies, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and silver-stained to detect the proteins by mass spectrometry after removal of the gel. The acetylated proteins in AGS cells were enriched with lysine-acetylated antibodies, and a high-resolution mass spectrometer was used to detect the acetylated proteins and modified sites. RESULTS: TSA significantly inhibited AGS cell proliferation, and promoted cell apoptosis, leading to AGS cell cycle arrest in G0/G1 and G2/M phases, especially G0/G1 phase. p21, p53 and Bax gene expression levels in AGS cells were increased with TSA treatment duration; Bcl-2, CDK2, and CyclinD1 gene expression levels were decreased with TSA treatment duration. Two unknown protein bands, 72 kDa (before exposure to TSA) and 28 kDa (after exposure to TSA), were identified by silver-staining after immunoprecipitation of AGS cells with the lysine-acetylated monoclonal antibodies. Mass spectrometry showed that the 72 kDa protein band may be PKM2 and the 28 kDa protein band may be ATP5O. The acetylated proteins and modified sites in AGS cells were determined. CONCLUSION: TSA can inhibit gastric cancer cell proliferation, which possibly activated signaling pathways in a variety of tumor-associated factors. ATP5O was obviously acetylated in AGS cells following TSA treatm     

Mechanisms of acupuncture and moxibustion in regulation of epithelial cell apoptosis in rat ulcerative colitis

AIM:To investigate the effect of acupuncture and moxibustionon epithelial cell apoptosis and expression of Bcl-2,Bax,fasand FasL proteins in rat ulcerative colitis.METHODS:A rat model of ulcerative colitis was estabelishedby immunological methods and local stimulation.All ratswere randomly divided into model control group (MC),electro-acupuncture group (EA),herbs-partition moxibustiongroup (HPM).Normal rats were used as normal controlgroup (NC).Epithelial cell apoptosis and expression ofBcl-2,Bax,fas and FasL proteins were detected by TUNELand immunohistochemiscal method respectively.RESULTS:The number of epithelial cell apoptosis in MCwas significantly higher than that in NC,and was markedlydecreased after the treatment with herbs-partitionmoxibustion or electro-acupuncture.The expression of Bcl-2,Bax,fas and FasL in colonic epithelial cells in MC was higherthan that in NC,and was markedly down-regulated by herbs-partition moxibustion or electro-acupuncture treatment.CONCLUSION:The pathogenesis of ulcerative colitis in ratsinvolves abnormality of apoptosis.Acupuncture andmoxibustion can regulate the expression of Bcl-2,Bax,fasand FasL proteins and inhibit the apoptosis of epithelial cellsof ulcerative colitis in rats by Bcl-2/Bax,fas/FasL pathways.     

Early apoptosis and cell death induced by ATX-S10Na ( Ⅱ)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (Ⅱ).METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting.RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-XL,were decreased in Bax- and p53-independent manner.CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizingphotosensitizer ATX-S10Na (Ⅱ) are mediated by p53-Bax network and Iow levels of Bcl-2 and Bcl-XL proteins.Our results might help in formulating new therapeutic approaches in photedynamic therapy.     

Expression of wild-type p53 and Bcl-2 family genes oscillates with recurrent remission and relapse in an unusual case of low-grade lymphoma

Downregulation of apoptosis has been proposed as a mechanism of clonal expansion in low-grade B cell neoplasms. We have previously described an unusual case of CD5+ B cell lymphoma characterized by cycles of leukemic phase alternating with spontaneous remission. In the present study, we examined the involvement of apoptosis-related proteins in the progression of this cyclic lymphoma ex vivo. During the leukemic phases, the clonal cells were activated blasts expressing elevated levels of wild-type (wt) p53, Bcl-2, Bcl-x(L), and Bax, while Bak expression increased during the decline of lymphocytosis. Bax heterodimerized with Bcl-2 but not with Bcl-x(L). The anti-apoptotic Bcl-2/Bax heterodimers peaked during early leukemic phases and declined during regression. The elevation in Bcl-2, Bcl-x(L) and Bax expression during early leukemic phases seems to result from cell activation since a similar increase was induced by activating the remission phase leukemic cells in culture. The data suggest that wt p53, Bcl-x(L), and Bcl-2/Bax heterodimers support the accumulation of activated leukemic cells during the leukemic phases, while Bax and Bak may be involved in their decline during regression.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x_L and Bcl-x_S, which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma.   In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Decreased Bax expression by mucosal T cells favours resistance to apoptosis in Crohn's disease

BACKGROUND: Activated T cells are more susceptible to apoptosis than resting T cells. As intestinal T cells normally exhibit a higher state of activation, increased apoptosis may be necessary to maintain immune homeostasis in the specialised microenvironment of the mucosa. On the other hand, in Crohn's disease (CD) mucosal T cells are resistant to apoptosis, suggesting abnormal regulation of cell death mechanisms. AIMS: To investigate differences in expression of anti- and proapoptotic Bcl-2 family proteins, key regulators of apoptosis, between circulating and mucosal T cells, and possible alterations in CD. PATIENTS AND METHODS: Lamina propria T cells (LPT) were isolated from 10 control, seven CD, and eight ulcerative colitis (UC) patients, and peripheral blood T cells (PBT) from healthy volunteers. Purified T cells were stained intracellularly for Bcl-2, Bcl-x(L), and Bax, and mean fluorescence intensity measured by flow cytometry. RESULTS: Compared with PBT, the expression level of Bcl-2 and Bax, but not Bcl-x(L), was significantly greater in LPT, resulting in lower Bcl-x(L)/Bax ratios. In PBT, Bax expression was highly and significantly correlated with both Bcl-2 and Bcl-x(L), but correlation with Bcl-2 was absent in LPT. Bax expression in CD, but not UC, LPT was significantly lower than in control LPT, resulting in a significantly higher Bcl-x(L)/Bax ratio. The significant correlation of Bcl-x(L) to Bax was preserved in CD, but not UC, LPT. CONCLUSIONS: Regulation of Bcl-2 family protein expression differs between circulating and mucosal T cells, probably underlying diverse survival potentials. In CD LPT, a low Bax expression and a high Bcl-x(L)/Bax ratio favour resistance to apoptosis and may contribute to the chronicity of inflammation.     

Expression of apoptosis-regulating proteins p53, Bcl-2, and Bax in primary resected esophageal squamous cell carcinoma

Apoptosis plays a key role in the pathogenesis, aggressiveness, and therapy responsiveness of cancer. Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. The present study retrospectively examines the expression of apoptosis-regulating proteins in primary resected esophageal squamous cell carcinoma (ESCC) and the correlation between the outcome of patients' treatment and the expression of the proteins. We used antibodies specific for the human p53, Bcl-2 and Bax proteins to examine the expression of these apoptosis-regulating proteins in 40 archival specimens of patients with primary resected esophageal squamous cell carcinoma. The overall expression of p53, Bcl-2, and Bax was 73%, 18%, and 100%, respectively. No significant correlations were found between the expression of p53, Bcl-2, and Bax. The expression of Bcl-2 had a negative influence on survival in this population of primary resected ESCC patients (p=0.03). But no differences in survival were observed in relation to the expression of p53 or Bax. In conclusion, Bcl-2 expression may provide additional and prognostic information for the clinical course of the disease and therefore to be developed as a prognostic indicator for primary resected esophageal squamous cell carcinoma.     

The Bcl-2/Bax system and apoptosis in ulcerative colitis

BACKGROUND/AIMS: Ulcerative colitis (UC) constitutes a chronic inflammatory process of the colon of unknown etiology. Current data support a pivotal role of apoptosis in the evolution of pathogenesis of UC. We performed a prospective study in order to determine the role of Bcl-2, Bax and Bcl-x in the apoptotic pathway in UC. METHODOLOGY: We included 23 patients with UC and 11 controls. Histological severity of the disease was assessed according to the Sidney classification system. Patients in the UC group were divided in 2 groups according to histological severity of the disease. The TUNEL method was used for the in situ evaluation of apoptosis. Immunohistochemical staining was used for the detection of Bax, Bcl-2, Bcl-x. For the assessment of cellular proliferation we used the monoclonal antibody Ki67. Appropriate statistical methods were applied. RESULTS: Overall 77 specimens were assessed; 57 from UC patients and 20 from controls. Bcl-2, Bax and Bcl-x were upregulated in the group of patients with UC compared to controls. Nevertheless, Bax in epithelial cells and Bcl-x in lymphocytes were downregulated in patients with moderate/severe disease (p = 0.029 and 0.04 respectively). A weak correlation between epithelial apoptosis and Bcl-x expression in lymphocytes (r = 0.31, p = 0.02) was found. An even weaker correlation was also noticed between the epithelial component apoptosis and Bax in lymphocytes (r = 0.02, p = 0.07). CONCLUSIONS: Bcl-2/Bax system does not appear to be involved in the induction of apoptosis in UC. Activation of intraepithelial lymphocytes may be associated with epithelial apoptosis or simply represent epiphenomena related to the inflammatory process.     

Trichomonas vaginalis-induced apoptosis in RAW264.7 cells is regulated through Bcl-xL, but not Bcl-2

The purpose of this study was to determine whether anti-apoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L), proteins that confer resistance to apoptotic death from some stimuli, block apoptotic cell death in RAW264.7 cells upon treatment with Trichomonas vaginalis. In this study, the expression level of Bcl-2 was unchanged throughout the course of apoptotic cell death, and overexpressed Bcl-2 did not prevent release of cytochrome c, the significant change of the membrane potential, activation of caspases, and PARP cleavage in T. vaginalis-treated RAW264.7 cells. On the other hand, Bcl-x(L)expression was decreased after T. vaginalis treatment accompanied with Bax activation. Furthermore, we showed that release of mitochondrial cytochrome c, cleavage of caspase-9 and PARP during apoptosis in T. vaginalis-treated RAW264.7 cells were considerably diminished by transfection with overexpressed Bcl-x(L), and overexpressed Bcl-x(L)could inhibit T. vaginalis-induced apoptosis in RAW264.7 cells. In addition, interestingly, pre-treatment with caspase inhibitors, Boc-D-FMK and Z-DEVD-FMK, significantly abolished T. vaginalis-induced down-regulation of Bcl-x(L), suggesting that caspase-3 may play a pivotal role in the process of apoptosis as well as the down-regulation of Bcl-x(L)by T. vaginalis. Therefore, these results suggest that T. vaginalis-induced apoptosis in RAW264.7 cells can occur via a Bcl-x(L)-dependent apoptotic mechanism.     

斑蝥素诱导人胰腺癌细胞凋亡的实验研究

目的 探讨斑蝥素（Cantharidin）对人胰腺癌细胞凋亡的影响及其作用机制。方法采用MTT法观察斑蝥素对人胰腺癌细胞株SW1990细胞增殖的抑制作用。采用Hoechst33258染色、TUNNEL染色、流式细胞术检测细胞凋亡改变，并以RT—PCR和Westernblot检测凋亡调节基因p53和Bcl-2、Bax的表达。结果 5mol／L斑蝥素能明显抑制人胰腺癌SW1990细胞的生长，呈现凋亡特征。RT—PCR和Westernblot检测可见Bax、p53基因表达显著增加，而Bcl—2基因表达减少。结论 斑蝥素能诱导人胰腺癌细胞凋亡，其作用可能与上调p53、Bax基因和下调Bcl-2基因有关。