Synergistic antitumor effect of TRATL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosisinducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms. METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT)assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detectapoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50＞1 mg@L1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25+3.48μmol@L-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg.L-1),combined with subtoxic doxorubicin (0.86 μmol@L1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+2.67%, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P＞0.05). CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein i snot involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.     

藤梨根提取物通过调控miR-192-5p/ARPP19轴影响结直肠癌细胞的增殖和凋亡

背景 藤梨根提取物(rattan root extract,RRE)可抑制胃癌、肺癌等肿瘤细胞增殖,具有一定抗肿瘤作用,但其是否影响结直肠癌细胞的恶性表型还未知.miR-192-5p在结直肠癌组织中表达降低,且其低表达与肿瘤大小等临床病理特征相关,可作为结直肠癌诊治的潜在生物学标志物.StarBase生物信息学软件预测...     

姜黄素对结肠癌细胞SW480 FasL基因表达和侵袭能力的影响

目的研究姜黄素对人结肠癌SW480细胞FasL mRNA表达及对其侵袭能力的影响,为中药抗肿瘤提供实验依据。方法根据MTT法得到姜黄素对SW480细胞的半数有效抑制浓度（IC50）,确定药物作用浓度。SW480细胞分别经姜黄素不同浓度（0．5 IC50、IC50）作用后,应用逆转录-聚合酶链反应（RT-PCR）法检测姜黄素作用前后人结肠癌SW480细胞FasL mRNA的变化;应用Transwell细胞侵袭试验检测姜黄素对SW480细胞侵袭能力的影响。结果姜黄素处理后SW480细胞FasL mRNA表达水平均明显高于对照组（P〈0．01）;而且FasL mRNA表达水平随姜黄素作用浓度增加显著上调,姜黄素不同浓度组比较均有显著性差异（P〈0．01）;随姜黄素作用浓度升高,SW480细胞侵袭能力明显增强,不同浓度组比较均有显著性差异（P〈0．01）。结论姜黄素在一定时间内均可上调人结肠癌SW480细胞FasL mRNA的表达,而且这种上调作用在一定范围内呈剂量依赖性,可使结肠癌细胞的侵袭能力增强。     

Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer

AIM:To explore the effect of antisense oligocleoxynucletide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.RESULTS:The telomerase activity in SW480 cells transfected with 1.0μmol/L of As-ODN for 2-5days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis.The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes.CONCLUSION:As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.     

Mechanism of counterattack of colorectal cancer cell by Fas/Fas ligand system

AIM: To determine the role of Fas/Fas ligand (FasL) in the immune escape of colon cancer cells. METHODS: Immunohistochemistry was used to observe the expression of Fas and FasL in the tissues of colon cancer patients. In situ hybridization was used to detect the localization of FasL mRNA expression in cancer tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and CD45 staining were performed to detect the apoptosis of tumor-infiltrating lymphocytes (TILs). Co-culture assays of colon cancer cells (SW480) and Jurkat cells (Fas-sensitive cells) were performed to observe the counterattack of colon cancer cells to lymphocytes. RESULTS: Of 53 cases of colon carcinomas, 23 cases (43.4%) expressed Fas which was significantly lower as compared to the normal colonic mucosa (73.3%, P<0.01), and 45 cases (84.9%) of colon carcinomas expressed FasL, whereas only two cases (3.75%) in normal mucosa expressed FasL. FasL expression in the colon cancer cells was found to be associated with increased cell death of TILs. The apoptotic rate of TIL in the FasL-positive staining regions of tumor cells was significantly higher than that in the FasL-negative staining region (54.84±2.79% vs 25.73±1.98%, P<0.01). The co-culture of SW480 cells and Jurkat cells confirmed the function of FasL on the SW480 cells. The apoptotic rates of Jurkat cells were found to be related with the amount of SW480 cells. CONCLUSION: Colon cancer cells can escape the immune surveillance and killing via decreasing Fas expression, and can counterattack the immune system via increasing FasL expression. Fas/FasL can serve as potential targets for effective antitumor therapy.     

Effect of indomethacin on cell cycle proteins in colon cancer cell lines

AIM: To study the effect of indomethacin (IN) on human colon cancer cell line SW480 with p53 mutant and SW480 transfected wild-type p53 (wtp53/SW480) in vitro and investigate molecular mechanism of anti-tumor effect of IN on colon cancer. METHODS: SW480 cells and wtp53/SW480 cells were treated with different concentrations of IN respectively, the expressions of CDK2, CDK4 and p21WAF1/CIP1 protein were detected by Western blotting. RESULTS: IN gradually down-regulated the expression of CDK2, CDK4 protein of wtp53/SW480 cells in a dose-dependent manner, and inhibitory effect reached the maximum level at 600 μmol/L; IN up-regulated the expression of p21WAF1/CIP1 protein in a dose-dependent manner at a certain concentration range, and the expression reached the maximum level at 400 μmol/L, and returned to the base level at 600 μmol/L. The expression of CDK2, CDK4 and P21WAF1/CIP1 protein of SW480 cells did not change. CONCLUSION: IN exerts antitumor effect partly through down regulation of the expression of CDK2, CDK4, protein and up regulation of the expression of p21WAF1/PIC1.     

Adenovirus-mediated tumor suppressor p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.     

New anti-proliferative agent, MK615, from Japanese apricot ＂Prunus mume＂ induces striking autophagy in colon cancer cells in vitro

AIM: To investigate the anti-neoplastic effects of MK615, an extract from the Japanese apricot (Prunus mume), against colon cancer cells. METHODS: Three colon cancer cell lines, SW480, COLO, and WiDr, were cultured with MK615. Growth inhibition was evaluated by cell proliferation assay and killing activity was determined by lactate dehydrogenase assay. Induction of apoptosis was evaluated by annexin Ⅴ flow cytometry. Morphological changes were studied by light and electron microscopy, and immunofluorescence staining with Atg8. RESULTS: MK615 inhibited growth and lysed SW480, COLO and WiDr cells in a dose-dependent manner. Annexin Ⅴ flow cytometry showed that MK615 induced apoptosis after 6 h incubation, at which point the occurrence of apoptotic cells was 68.0%, 65.7% and 64.7% for SW480, COLO, and WiDr cells, respectively. Light and electron microscopy, and immunofluorescence staining with Atg8 revealed that MK615 induced massive cytoplasmic vacuoles (autophagosomes) in all three cell lines. CONCLUSION: MK615 has an anti-neoplastic effect against colon cancer cells. The effect may be exerted by induction of apoptosis and autophagy.     

Mechanisms underlying aspirin-mediated growth inhibition and apoptosis induction of cyclooxygenase-2 negative colon cancer cell line SW480

AIM： To investigate the effects of aspirin （acetylsalicylic acid） on proliferation and apoptosis of colorectal cancer cell line SW480 and its mechanism. METHODS： Cyclooxygenase （COX）-2 negative colorectal cancer cell line SW480 was treated with aspirin at concentrations of 2.5 mmol/L, 5.0 mmol/L, 10.0 mmol/L for different periods in v/tro. Anti-proliferation effect of aspirin on SW480 was detected by 3-（4,5-dimeth- ylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Cell cycle and apoptosis were observed by flow cytometry （FCM）. Transmission electron microscope （TEM） was used for morphological study. Apoptosis-associated genes were detected by immunohistochemical staining and Western blotting. RESULTS： Aspirin inhibited SW480 proliferation and induced apoptosis in a dose- and time-dependent manner. Treatment with different concentrations of aspirin significantly increased the proportions of cells at the G0/G1 phase and decreased the proportions of cells at the S- and G2/M phases in a concentration-dependent manner. Aspirin not only induced apoptosis but also caused cell necrosis at a high concentration as well. After treatment with aspirin, SW480 cells displayed typically morphological features of apoptosis and necrosis under TEM, and increased the Bcl-2 expression in cells, but the expression of Bax was down regulated. CONCLUSION： Aspirin inhibits proliferation and induces apoptosis of SW480 cells. Its anti-tumor mechanism may arrest cell cycle and shift Bax/Bcl-2 balance in cells.     

Inhibitory effect of caffeic acid phenethyl ester on the growth of SW480 colorectal tumor cells involves β-catenin associated signaling pathway down-regulation

AIM: To study the anti-tumor effect of caffeic acid phenethyl ester (CAPE) and the influence of CAPE on β-catenin associated signaling pathway in SW480colorectal cancer (CRC) cells.METHODS: SW480 cells were treated with CAPE at serial concentrations. The proliferative status of cells was measured by methabenzthiazuron (MTT) assay. Cell cycle and cell apoptosis were analyzed using flow cytometry (FCM). Western blotting assay was used to evaluate the protein level of β-catenin, c-myc and cyclinD1.β-catenin localization was determined by indirect immunofluorescence.RESULTS: CAPE displayed a strong inhibitory effect in a significant dose- and time-dependent manner on SW480cell growth. FCM analysis showed that the ratio of G0/G1 phase cells increased, S phase ratio decreased and apoptosis rate increased after SW480 cells were exposed to CAPE for 24 h. Pretreatment of SW480 cells with CAPE significantly suppressed β-catenin, c-myc and cyclinD1protein expression. CAPE treatment was associated with decreased accumulation of β-catenin protein in nucleus and cytoplasm, and concurrently increased its accumulation on the surface of cell membrane.CONCLUSION: CAPE can inhibit SW480 cell proliferation by inducing cell cycle arrest and apoptosis. Decreased β-catenin and the associated signaling pathway target gene expression may mediate the anti-tumor effects of CAPE.     

Inhibitory effect of caffeic acid phenethyl ester on the growth of SW480 colorectal tumor cells involves β-catenin associated signaling pathway down-regulation

AIM: To study the anti-tumor effect of caffeic acid phenethyl ester (CAPE) and the influence of CAPE on beta-catenin associated signaling pathway in SW480 colorectal cancer (CRC) cells. METHODS: SW480 cells were treated with CAPE at serial concentrations. The proliferative status of cells was measured by methabenzthiazuron (MTT) assay. Cell cycle and cell apoptosis were analyzed using flow cytometry (FCM). Western blotting assay was used to evaluate the protein level of beta-catenin, c-myc and cyclinD1. Beta-catenin localization was determined by indirect immunofluorescence. RESULTS: CAPE displayed a strong inhibitory effect in a significant dose- and time-dependent manner on SW480 cell growth. FCM analysis showed that the ratio of G0/G1 phase cells increased, S phase ratio decreased and apoptosis rate increased after SW480 cells were exposed to CAPE for 24 h. Pretreatment of SW480 cells with CAPE significantly suppressed beta-catenin, c-myc and cyclinD1 protein expression. CAPE treatment was associated with decreased accumulation of beta-catenin protein in nucleus and cytoplasm, and concurrently increased its accumulation on the surface of cell membrane. CONCLUSION: CAPE can inhibit SW480 cell proliferation by inducing cell cycle arrest and apoptosis. Decreased beta-catenin and the associated signaling pathway target gene expression may mediate the anti-tumor effects of CAPE.     

氧化苦参碱对人结肠癌细胞株SW1116杀伤作用的实验研究

目的探讨氧化苦参碱(OM)对于人结肠癌细胞株SW1116的杀伤作用及其抗肿瘤作用的机制。方法采用四甲基偶氮唑蓝(MTT)比色法、流式细胞仪、PCR—ELISA和RT-PCR法检测OM对SW1116细胞的杀伤作用、细胞周期分布、端粒酶活性和端粒酶逆转录酶(hTERT)及其上游调控基因(c-myc、p53、mad1)表达的影响。结果0M对SW1116细胞具有剂量依赖性杀伤作用；可以引起G1／G0期细胞增加、S期细胞减少；OM可抑制SW1116细胞的端粒酶活性，其作用呈剂量和时间依赖性；OM可引起SW1116细胞hTERT基因表达下调，pS3和mad1基因的表达升高，对c-myc基因的表达无影响。结论OM对人结肠癌细胞株SW1116具有杀伤作用，可能通过影响hTERT及其上游调控基因的表达来抑制肿瘤细胞端粒酶活性，发挥其抗肿瘤作用。     

阿霉素与Fas抗体对提高肺癌细胞凋亡的实验研究

目的 研究阿霉素(ADM)与Fas抗体在肺癌细胞的细胞毒作用中是否具有协同作用。方法 MTT法检测细胞毒作用，荧光染色法测定癌细胞凋亡效应。结果 NCL446人类肺癌细胞株及12例手术新鲜肺癌标本与ADM及Fas抗体共同培养显示有显的细胞毒协同效应。Fas抗体与ADM亦有协同的诱导凋亡效应。Fas抗体可以提高NCL446细胞内ADM的聚集量。ADM可以增强NCL446细胞表面的Fas表达。结论 实验结果表明，Fas抗体与ADM共培养可以克服肺癌细胞的耐药性。协同效应不仅在肺癌细胞株中发现，而且存在于手术切除的肺癌标本中。说明ADM与Fas相关免疫制剂联合有潜在的临床应用前景。     

Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor –related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAILinduced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.     

Additive effect of Apo2L/TRAIL and Adeno-p53 in the induction of apoptosis in myeloma cell lines

OBJECTIVE: We have previously shown that Adenovirus-p53 (Ad-p53) is a potent inducer of apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2 and that Apo2L/TRAIL is a potent inducer of apoptosis, independent of bcl-2. A study was designed to test the synergy between Ad-p53 and Apo2L/TRAIL in the induction of apoptosis in relation to the expression of DR4/DR5 and DcR1, in cells undergoing Ad-p53-induced apoptosis. METHODS: Replication deficient Ad-p53 and human recombinant Apo2L/TRAIL were used. Myeloma cells with mutated/w.t. p53 and varying expression of bcl-2 were used to test the effect of Ad-p53, Apo2L/TRAIL, or both, on apoptosis, measured by annexin V. RESULTS: Treatment with Ad-p53 resulted in a dose-dependent apoptosis concomitant with a dose-dependent increase in the expression of DR4/DR5 and a decrease in the expression of DcR1, in Ad-p53-sensitive cell lines. In these cells, addition of Apo2L/TRAIL to cells treated with Ad-p53 resulted in a dose-dependent increase in apoptosis. Myeloma cells resistant to Ad-p53 had high levels of DR4/DR5 and high levels of DcR1 and treatment with Ad-p53 did not reduce the expression of DcR1. Also, addition of Apo2L/TRAIL to Ad-p53 did not affect the level of apoptosis beyond the level of apoptosis observed with Apo2L/TRAIL alone. CONCLUSIONS: 1) Cotreatment with Ad-p53 and Apo2L/TRAIL resulted in additive apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2. 2) Resistance to Ad-p53 or to the combination of Ad-p53 and Apo2L/TRAIL was not due to the lack of adenovirus receptor (CAR) or low expression of DR4/DR5 but rather due to the relatively high expression of DcR1 receptor.     

p300 regulates p53-dependent apoptosis after DNA damage in colorectal cancer cells by modulation of PUMA/p21 levels

Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptosis, but what determines the choice between cytostasis and death is not clear. In this report, we show that the E1A-binding p300 nucleoprotein is a key determinant of p53-dependent cell fate in colorectal cancer cells: absence of p300 increases apoptosis in response to DNA damage. In addition, p300-deficient (p300(-)) cells fail to undergo G(1)/S arrest after UV irradiation. These abnormalities are associated with prolongation of p53 stability, reduced p53-acetylation, blunting of MDM2 activation, failure to transactivate p21, and a disproportionate increase in PUMA levels. When xenografted, p300(-) cells are more sensitive to chemotherapy with doxorubicin. These results show that p300 is a key regulator of the p53 response and suggest that p300 inhibition could be used to modulate chemotherapy.     

Less cytotoxicity to combination therapy of 5—fluorouracil and cisplatin than5—fluorouracil alone in human colon cancer cell lines

AIM：Our previous studies showed increased sensitivity to 5-FU in colon cancer cell lines with microsatellite instability,and considered that mutations of TGFβ-RⅡ,IGFⅡR,RIZgene might enhance the potentials of cell growth and proliferation,which increased the sensitivity to 5-FU.Here we compared the distribution of cell cycle and P53 status between two human colon cancer cell lines with P53status between two human colon cancer cell lines with different sensitivity to5-FU.Because mechanistic differences exist between 5-FUand CDDP,we also analyzed the efficacy of CDDPand combination therapy on two human colon cancer cell lines.METHODS:We compared the sensitivity to CDDP of these two cell lines by MTT assay,Distributon of cell cycle under treatment of5-FU,cddp alone or both was analyzed by Flow Cytometry,and expression of P53was detected by immunocytochemical staining.RESULTS:SW480 cells were more sensitive to CDDP than LoVo cells at the concentrations above 16μmol/l(Ratio of absorption is 0.64and0.79at16μmol/l.respectively;P&lt;0.010,Efficacy of combination therapy was conversely lower than that of single-therapy of 5-FU(Ratio of absorption in LoVo+5-FU,SW480+5-FU,LoVo+5-FU+CDDPand SW480+5-FU+CDDPis0.53,0.54,0.72,0.78,respectively;P&lt;0.01).LoVo cells were negative whereas wsw480 cells positive inP53expression,5-FU induced G1-phase arrest in both cell lines,butLoVo cells peaked 24hours earlier than SW480cells,and 48hours earlier for an apparent hypodiploidDNA.However,CDDP showedthe contrary,inducingS-phase arrest,andSW480cells peaking 36hours earlier,Both cell lines showed hypodipliod nuclei 48hours after CDDP treatment.Percentage of cells in G1-phase and 5-phase dominated alternatively under conbination therapy in both cell lines.CONCLUSION:There results suggest that colon cancer cells with microsatellite instability are mor sensitive to 5-FU,whereas more resistant to CDDP.Combination therapy of5-FU and CDDPshows fewer efficacies than5-FU single-therapy,although it can render a cell cycle arrest.P53may be involoved in the shift of G1-phase to S-phase,but inessentially.