Dysregulation of Reg gene expression occurs early in gastrointestinal tumorigenesis and regulates anti-apoptotic genes

Expression of anti-apoptotic genes is frequently elevated in tumors, where they increase resistance to chemotherapeutic agents and predict poor patient outcomes. However, key cellular factors regulating anti-apoptotic genes in tumors remain unknown. Increased expression of the regenerating (Reg) genes is commonly observed in gastrointestinal (GI) malignancies including colorectal cancer (CRC). We therefore examined Reg gene expression and associated changes in anti-apoptotic genes in an animal model of GI tumorigenesis. Using real time RT-PCR, we measured expression of Reg genes in human colorectal adenocarcinoma specimens, colon adenocarcinoma cell lines and adenomas from multiple intestinal neoplasia (min) mice heterozygous for a germ-line mutation of the adenomatous polyposis coli (APC) gene. Expression of Reg genes is increased in human colorectal adenocarcinomas and in the intestine of APCmin/+ mice at four weeks of age, a time preceding the spontaneous second mutation in the APC gene. Individual Reg genes exhibited regional expression profiles across the GI tract in mice. Adenomas from 14-week old mice had significant increases in at least one member of the Reg gene family, most commonly Reg IV and an associated increase in expression of the anti-apoptotic gene, Bcl-2. Addition of exogenous recombinant human Reg IV to human colon adenocarcinoma cells significantly increased Bcl-2 and Bcl-xL expression and induced resistance to ionizing radiation. These results show that dysregulation of Reg genes occur early in tumorigenesis. Furthermore, increased expression of Reg genes, specifically Reg IV contribute to adenoma formation and lead to increased resistance to apoptotic cell death in CRC.     

Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression

INTRODUCTION: Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2alpha, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer. MATERIAL AND METHODS: Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV). RESULTS: DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes. CONCLUSION: The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.     

Tumor invasiveness and liver metastasis of colon cancer cells correlated with cyclooxygenase-2 (COX-2) expression and inhibited by a COX-2-selective inhibitor, etodolac

Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to reduce the risk and mortality of colorectal cancer (CRC). Although the exact mechanisms remain unclear, the inhibition of cyclooxygenase (COX) by NSAIDs appears to abort, if not prevent, CRC carcinogenesis or metastatic tumor progression. The aim of our study was to investigate the association between COX-2 expression and CRC tumor cell invasiveness. The differences in immunoblot-detectable COX-2 protein contents in primary CRCs, metastatic hepatic lesions and corresponding normal mucosa from the same individual were evaluated in 17 patients. Three different colon cancer cell lines, SW620, Lovo, HT-29 and a metastatic variant of HT-29, HT-29/Inv3, were employed to evaluate COX-2 expression and prostaglandin E(2) (PGE2) production in relation to their invasive abilities in vitro. The effects of a COX-2-selective inhibitor, etodolac, on cell proliferation and invasive activity were also determined. The results showed that 15 of 17 (88%) metastatic CRC cells from the liver and 14 of 17 (82%) primary CRC tissue exhibited much higher levels of COX-2 than corresponding adjacent normal mucosa from the same patient. Among those patients with relatively high COX-2 expression in the primary tumors, almost all exhibited even higher levels of COX-2 in their hepatic metastases. Among the 4 colon cancer cell lines, HT-29/Inv3 manifested the highest COX-2 expression, PGE2 production and in vitro invasive activity. The selective COX-2 inhibitor, etodolac, could especially exert cytotoxicity and markedly suppress the invasive property and PGE(2) production, although not the COX-2 protein level, in HT-29/Inv3 cells. Our results imply that COX-2 expression may be associated with the invasive and metastatic properties of CRC tumor cells.     

那可丁对人结肠癌5-Fu耐药细胞株HT-29/5-Fu、LoVo/5-Fu及SW480/5-Fu耐药性的影响

目的 探讨那可丁（Nos）对人结肠癌5-氟尿嘧啶（5-Fu）耐药株耐药性的影响及其机制。方法 构建人结肠癌耐药株HT-29/5-Fu、LoVo/5-Fu及SW480/5-Fu，MTT法筛选出Nos对各组细胞的半数抑制浓度（IC50），观察细胞形态变化，利用流式细胞仪检测细胞周期及凋亡水平；RT-qPCR及Western blot检测细胞P38表达和激活水平，以及耐药相关蛋白的表达。结果 成功构建耐药细胞株HT-29/5-Fu、LoVo/5-Fu和SW480/5-Fu。与对照组比较，Nos干预后的结肠癌耐药细胞株HT-29/5-Fu、LoVo/5-Fu及SW480/5-Fu细胞周期明显阻滞在G₀/G₁期（P<0.01），凋亡率升高，而P38表达及磷酸化受到显著抑制（P<0.01），与耐药相关的多药耐药相关蛋白、肺耐药相关蛋白及P-glycoprotein表达显著降低（P<0.01）。结论 Nos对耐5-Fu的人结肠癌细胞具有一定的毒性作用，降低了耐药细胞耐药性，作用机制可能与抑制P38的表达和磷酸化有关。     

MicroRNA-99a促进结肠癌细胞的增殖和迁移及其可能机制

目的:探讨结肠癌患者组织中microRNA-99a表达水平以及对结肠癌细胞增殖和迁移的影响.方法:取南京医科大学附属常州二院胃肠病中心49例结肠癌患者肿瘤组织、癌旁组织(癌旁5cm)标本以及结肠癌细胞HCT-116、HT-29、SW-480、Caco-2和正常结肠上皮细胞HCoePic,采用实时荧光定量PCR检测结肠癌患者肿瘤组织和结肠癌细胞中microRNA-99a表达水平;结肠癌细胞株HT-29转染microRNA-99a抑制剂后,CCK-8法检测microRNA-99a对结肠癌细胞增殖的变化;transwell法观察microRNA-99a对结肠癌细胞迁移的影响;Western blotting检测了HT-29中FGFR-3的表达水平.结果:microRNA-99a表达在结肠癌组织中明显高于癌旁组织(6.27±0.48 vs 1.34±0.54,P＜0.05)、在肿瘤细胞中明显高于正常结肠上皮细胞(5.48±0.34,7.67±0.24,5.78±0.22,6.28±0.44 vs 1.45±0.37,P＜0.05).转染microRNA-99a抑制剂后,HT-29细胞的增殖和迁移能力均明显下降(P＜0.05);同时,HT-29中FGFR-3显著降低(P＜0.05).结论:microRNA-99a在结肠癌组织中高表达,低表达microRNA-99a可减弱结肠癌细胞的增殖和迁移能力,且可能通过FGFR-3信号通路发挥作用.     

Pharmacokinetic profile of the microtubule stabilizer patupilone in tumor-bearing rodents and comparison of anti-cancer activity with other MTS in vitro and in vivo

Introduction  Patupilone is a microtubule stabilizer (MTS) currently in clinical development. Here, we evaluate the anti-cancer activity in vitro and in vivo in comparison to paclitaxel and describe the pharmacokinetics (PK) of patupilone in tumor-bearing nude mice and rats. Methods  The potency in vitro of patupilone and two other MTS, paclitaxel and ixabepilone, was determined using human colon carcinoma cell lines with low (HCT-116, HT-29, RKO) and high (HCT-15) P-glycoprotein expression (P-gp), as well as two multi-drug resistance (MDR) model cell pairs, MCF7/ADR and KB-8511 cells and their respective drug-sensitive parental counterparts. The PK of patupilone was investigated in nude mice bearing HCT-15 or HT-29 xenografts and in rats bearing s.c. pancreatic CA20498 tumors or A15 glioma tumors. Anti-cancer activity in vivo was compared to that of paclitaxel using three different human tumor colon models. The retention and efficacy of patupilone was compared in small and large HT-29 xenografts whose vascularity was determined by non-invasive magnetic resonance imaging. Results  Patupilone was highly potent in vitro against four different colon carcinoma cell lines including those showing multi-drug-resistance. In contrast, paclitaxel and ixabepilone displayed significantly reduced activity with markedly increased resistance factors. In both rats and mice, a single i.v. bolus injection of patupilone (1.5–4 mg/kg) rapidly distributed from plasma to all tissues and was slowly eliminated from muscle, liver and small intestine, but showed longer retention in tumor and brain with no apparent elimination over 24 h. Patupilone showed significant activity against three human colon tumor models in vivo, unlike paclitaxel, which only had activity against low P-gp expressing tumors. In HT-29 tumors, patupilone activity and retention were independent of tumor size, blood volume and flow. Conclusions  The high potency of patupilone, which is not affected by P-gp expression either in vitro or in vivo, and favorable PK, independent of tumor vascularity, suggest that it should show significant activity in colorectal cancer and in other indications where high P-gp expression may compromise taxane activity.     

Regenerating islet-derived family member, 4 modulates multiple receptor tyrosine kinases and mediators of drug resistance in cancer

Regenerating islet-derived family member, 4 (Reg IV) is a secreted protein and member of the C-type lectin superfamily. Expression analyses have characterized Reg IV as a prognostic marker for certain cancers; however, the functional role of Reg IV in cancer, including downstream signaling, has only begun to be elucidated. To investigate the biological role of Reg IV in cancer, phosphorylation events were studied in cancer cell lines in the context of either Reg IV stimulation (HCT116 cells) or knockdown of endogenous Reg IV (PC3 and KM12 cells). In addition to the previously observed impact on epidermal growth factor receptor and Akt phosphorylation, we observed modulation in the phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor as well as their downstream effectors, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways. Furthermore, knockdown of Reg IV impacted the ability of insulin and EGF to stimulate downstream tyrosine phosphorylation. Knockdown of Reg IV in cancer cell lines inhibited anchorage-dependent and anchorage-independent (both soft-agar and spheroid assays) cell growth and induced cell cycle arrest. This was accompanied by upregulation of p21 and p27. Transiently silencing Reg IV in cancer cells induced apoptosis and downregulated Bcl-2. Conversely, stimulation of HCT116 cells with recombinant Reg IV induced Bcl-2. Hsp27, a molecule implicated in drug resistance, was similarly modulated by Reg IV. Consistent with our observations with Reg IV siRNA-mediated knockdown, monoclonal antibodies directed against Reg IV inhibited PC3 and KM12 cell growth. Collectively, Reg IV plays an important role in cancer by modulation of key signaling molecules including Hsp27, Bcl-2 and multiple RTKs.     

Expression of stress protein gp96, a tumor rejection antigen, in human colorectal cancer

Preparations of stress protein gp96 from tumor cells are active as tumor vaccines by eliciting immune responses against mixtures of individual tumor peptide antigens which are complexed to gp96. Due to the individual antigenicity of tumors, a vaccine consisting of tumor-derived gp96 has to be prepared individually for each patient from autologous tumor tissue. So far, gp96 expression by human tumors has not been analyzed. Here, we report stable and mostly homogenous expression of gp96 by colorectal cancer, which was enhanced compared to surrounding tumor stroma in 70% to 80% of colorectal cancer specimens. Fewer non-metastatic than metastatic primary cancer specimens showed enhanced gp96 expression. Glucose deprivation increased gp96 protein and RNA expression in the human colon cancer cell line HT-29 in accordance with the role of gp96 as a glucose-regulated stress protein. Additionally, TNF-alpha, interferons and other cytokines induced an increase of gp96 RNA expression in HT-29 cells, suggesting that gp96 expression by colorectal cancer cells can be influenced by different methods of immunomodulation. The stable and homogenous expression of gp96 in 19 primary and metastatic colorectal cancer specimens and the up-regulation of gp96 in colon cancer cells by glucose deprivation point to an essential role of this stress protein in colorectal cancer, presumably by protecting against hostile conditions of the tumor micro-environment like glucose deprivation. In view of these results, loss of gp96 expression by colorectal cancer cells as an immune escape mechanism is unlikely.     

改变细胞膜脂肪酸组成防治大肠癌的基因治疗研究*

目的:利用脂肪酸去饱和酶基因fat- 1 改变细胞膜脂肪酸组成,进行大肠癌的基因治疗研究。方法:将fat- 1 基因插入腺病毒载体中,与骨架载体同源重组,构建腺病毒重组载体（Ad-GFP-fat1）,通过包装细胞系（293）产生的腺病毒,感染人大肠癌株HT- 29细胞。提取细胞的总RNA,以fat- 1 基因的反义mRNA 作探针,用Northern Blot检测fat- 1 基因在HT- 29细胞内的表达。以流式细胞仪对HT- 29细胞G0/G1 期、S 期、G2/M期所占比例进行检测,分析fat- 1 基因对HT- 29细胞增殖和凋亡的影响。以气相色谱分析仪分析fat- 1 基因对HT- 29细胞细胞膜n-6 PUFAs 和n-3 PUFAs 含量及n-6/n- 3PUFAs 比例的影响。将HT- 29细胞皮下接种于裸鼠右前肢腋下,建立裸鼠HT- 29大肠癌细胞皮下移植瘤模型。成瘤后进行治疗实验,经连续5 次治疗,于最后一次治疗后第3 天处死小鼠,取肿瘤称重。分析fat- 1 基因裸鼠体内抗肿瘤效果。结果:通过基因重组技术,得到高滴度的含fat- 1 基因的重组病毒;腺病毒介导的fat- 1 基因能够在HT- 29细胞中有效表达;fat- 1 基因的表达可降低HT- 29细胞膜n-6/n- 3PUFAs 的比例,有效抑制HT- 29细胞增殖,促进细胞凋亡并能抑制裸鼠移植瘤的发展。结论:fat- 1 基因的表达,可抑制HT- 29细胞的体内外增殖并诱导细胞凋亡,在大肠癌基因治疗中可能具有良好利用价值。      

Inhibition of cell proliferation, induction of apoptosis, reactivation of DLC1, and modulation of other gene expression by dietary flavone in breast cancer cell lines

BACKGROUND: Dietary flavone was previously shown to increase the expression of deleted in liver cancer-1 gene (DLC-1) in HT-29 colon carcinoma cell line [Herzog A, Kindermann B, Doring F, Daniel H, Wenzel U. Pleiotropic molecular effects of the pro-apoptotic dietary constituent flavone in human colon cancer cells identified by protein and mRNA expression profiling. Proteomics 2004;4:2455-64]. DLC-1 that encodes a Rho GTPase-activating protein, functions as a tumor suppressor gene and is frequently inactivated or down-regulated in several common cancers. Restoration of DLC-1 expression suppresses in vitro tumor cells proliferation and tumorigenicity in vivo. METHODS: Here, the effect of flavone was examined in several DLC-1-deficient cell lines derived from different types human cancer using assays for cell proliferation, gene expression and transfer. RESULTS: We show that exposure to 150 microM flavone increased DLC1 expression in breast but not in liver or prostate carcinoma cells or a nonmalignant breast epithelial cell line. Flavone restored the expression of DLC1 in the breast carcinoma cell lines MDA-MB-468, MDA-MB-361, and BT20 as well as in the colon carcinoma cell line HT-29 all of which are DLC-1-negative due to promoter hypermethylation. We further show that flavone inhibited cell proliferation, induced cell cycle arrest at G(2)-M, increased p21(Waf1) gene expression, and caused apoptosis. Microarray analysis of these aggressive and metastatic breast carcinoma cells revealed 29 flavone-responsive genes, among which the DNA damage-inducible GADD genes were up-regulated and the proto-oncogene STMN1 and IGFBP3 were down-regulated. CONCLUSIONS: Flavone-mediated alterations of genes that regulate tumor cell proliferation, cell cycle, and apoptosis contribute to chemopreventive and antitumoral effects of flavone. Alone or in combination with demethylating agents, flavone may be an effective adjunct to chemotherapy in preventing breast cancer metastasis.     

Radiotherapy Enhancement with Electroporation in Human Intestinal Colon Cancer HT-29 Cells

Background: The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. Previousstudies have shown that electroporation (EP) can sensitize some cancer cell lines to ionizing radiation (IR). HT-29is a radiation resistant colorectal cancer cell line, representative of a cancer type which is the second cause of cancermortalities in developed countries. The present study aimed to evaluate radiosensitizing effects of EP on HT-29 cellsin vitro exposed to 6 MV X-ray photon beams. Methods: HT-29 cells were exposed to a 6 MV X-ray photon beamas the control or to a combination of electroporation and irradiation. The response of cells was evaluated by colonyformation assay and survival curves. Results: The survival fraction of the HT-29 cells was significantly decreased byelectroporation prior to radiotherapy. A single electric pulse increased colorectal HT-29 cancer cell sensitivity to megavoltageradiation by a factor of 1.36. Conclusion: Our findings showed that EP before radiotherapy can significantlyenhance tumor cell sensitivity. This combined treatment modality should be assessed for its applicability in clinic settingsfor employment against radioresistant cancers. However, to facilitate achieving this goal, many different tumors witha broad range of radiosensitivities should be evaluated.     

Determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines

PURPOSE: Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target. RESULTS: The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells. CONCLUSIONS: Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.     

Effects of altered expression and activity levels of CK1δ and ɛ on tumor growth and survival of colorectal cancer patients

Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1‐specific inhibitors. Furthermore, expression of CK1δ and ? is changed in colorectal tumors compared to normal bowel epithelium, and high CK1? expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and ? expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients' survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer.     

Modulation of colon tumor oncogene expression by cancer patient-derived lipids

In an effort to understand the role of specific fats on carcinogenesis, we have studied the effects of lipids derived from cancer patients on components associated with the regulation of proliferation. The treatment of tumor cells with patient-derived fats produced increased cell proliferation, as indicated by shorter doubling times. The effects of patient-derived lipids on the expression of ras, c-jun, c-erbB-2, and p53 gene products were examined. The cellular expression of the ras proto-oncogene product was increased in both colon tumor cell lines, following lipid treatment. However, c-jun proto-oncogene expression was elevated in HT-29 cells and appeared unchanged in SK-Co-1 cells after lipid treatment. Treatment of HT-29 tumor cells with patient-derived fats produced an enhancement of the p53 gene product, whereas fat treatment reduced p53 expression in SK-Co-1 tumor cells. Further separation of the patient-derived fats indicated that the amplification of p53 gene expression in HT-29 cells could be achieved primarily by addition of the diacylglycerides fraction. Addition of the purified fatty acids, comprising the diglyceride fraction, indicated that the fatty acids, 16:1, 18:0, and 18:1, induced the most significant increases in p53 expression by HT-29 cells. These alterations caused by cancer patient-derived fats are consistent with the loss of normal growth regulation and may explain the epidemiologic association between certain fats and carcinogenesis. © 1996 Wiley-Liss, Inc.