Differential effects of retinoic acid on growth and apoptosis in human colon cancer cell lines associated with the induction of retinoic acid receptor beta

Retinoids are well known as potential chemopreventive and chemotherapeutic agents against a variety of human cancers. Here, we report that retinoic acid (RA) induced differential growth inhibition in human colon cancer cell lines: while DLD-1, HT-29, and WiDr were relatively resistant, HCT-15 and Colo201 were relatively sensitive. All-trans-retinoic acid caused morphological and biochemical changes such as membrane shrinkage, chromatin condensation, and DNA cleavage, which are typical features of cells undergoing apoptosis in sensitive cell lines. Although retinoic acid receptor (RAR)alpha, beta, gamma and retinoid X receptor alpha were expressed in all cell lines examined, a significant induction of RARbeta by all-trans-RA was observed only in sensitive cell lines, suggesting important roles of RARbeta in RA sensitivity. When a vector containing the RARbeta gene was introduced into a relatively resistant cell line, DLD-1, the cells acquired RA sensitivity. Further, we found that the RARbeta transfectants of DLD-1 expressed an enhanced level of c-Myc and Bax proteins, which may result in the increased susceptibility of the cells to all-trans-RA-induced apoptosis. In summary, our data demonstrated that RA induced growth inhibition and apoptosis in human colon cancer cells and that the induction of RAR3 may mediate the retinoid action.     

Celecoxib inhibits the expression of survivin via the suppression of promoter activity in human colon cancer cells

We investigated the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on human colon cancer cell lines to clarify the mechanisms underlying the chemopreventive effect of NSAIDs. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, induced apoptosis and strongly reduced the expression of an anti-apoptotic protein, survivin, in both protein and mRNA levels in HCT-116 cells. Subsequently, we conducted luciferase reporter assay using a reporter gene driven by the human survivin promoter. A series of analyses using luciferase reporter constructs containing fragments of the survivin promoter and electrophoretic mobility shift assay indicated that the -75/-66 bp region relative to the initiating codon was involved in celecoxib action to suppress survivin promoter activity. Celecoxib also suppressed the activity of TOPflash, T-cell factor reporter plasmid, and the reporter gene driven by the human cyclin D1 promoter, suggesting that this compound inhibited the expression of Wnt/beta-catenin signaling target genes. Further, we found that other NSAIDs including indomethacin, resveratrol, and SC-560 induced apoptosis and suppressed the expression of survivin and the Wnt/beta-catenin signaling pathway in HCT-116 cells, indicating that these effects were likely to be common among NSAIDs. Moreover, NSAIDs (celecoxib, SC-560 and indomethacin) also suppressed the expression of cyclin D1 and survivin on other colon cancer cell lines (DLD-1 and SW-620). Our results suggested that NSAIDs could inhibit proliferation and induce apoptosis in colon cancer cells by inhibition of survivin expression and the Wnt/beta-catenin signaling pathway.     

DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine.

The retinoic acid receptor beta (RARbeta), a putative tumor suppressor gene, has been reported to be poorly expressed in breast cancer. In this report using the methylation-specific PCR reaction we observed DNA methylation in the promoter region of RARbeta in several primary breast tumors. DNA sequence analysis showed that the positions of 5-methylcytosine in the RARbeta promoter region was almost identical to that reported previously by our laboratory for human DLD-1 colon carcinoma cells (Anti-Cancer Drugs 1998; 9: 743). Several other cancer-related genes have been also reported to be silenced by DNA methylation, including the p16 tumor suppressor gene, E-cadherin, an invasion suppressor gene and the estrogen receptor gene in breast cancer cell lines. Since breast cancer cells have several potential target genes for the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), we investigated the in vitro antineoplastic activity of this analog on the human breast cancer cell line MDA-MB-231. We report that 5-Aza-CdR is a potent growth inhibitor and a potent cytotoxic agent against the breast carcinoma cells. These results suggest that 5-Aza-CdR may be an interesting agent to investigate in patients with breast cancer resistant to conventional chemotherapy.     

4- [3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) induced fas expression and activated caspase-3 and -8 in a DLD-1 colon cancer cell line

BACKGROUND: 4-[3,5-Bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a novel retinobenzoic acid derivative which has a specific binding affinity to the retinoic acid receptors (RAR)alpha and RARbeta. Using time-dependent FACScan analysis, it was observed that TAC-101 induced apoptosis in a DLD-1 human colon cancer cell line. In this study, the induction of apoptosis-related proteins and the activities of caspases in a DLD-1 cell line under medication with TAC-101 were investigated. MATERIALS AND METHODS: DLD-1 cells were cultured with different concentrations of TAC-101 for 12, 24 and 48 h. The expressions of Fas, TNF-R1, DR3, bcl-2, Bax and Bid were measured using a Western blot analysis. The activities of caspase-3, -8 and -9 were measured using a colorimetric protease assay kit. RESULTS: The Western blot analysis showed that TAC-IO1 had almost no effect on the level of Bcl-2, Bax or Bid protein. Although TAC-101 did not change the expression of TNF-R1 and DR3, TAC-101 increased the expression of Fas in both a time- and a dose-dependent manner. A 3-fold increase in caspase-3 activity and a 1.5-fold increase in caspase-8 activity were observed in cells treated with TAC-101 in comparison to the control cells (p<0.01). CONCLUSION: Our data indicate that the death receptor root of the apoptotic signal transduction in DLD-1 cells mainly participates in the apoptotic induction of TAC-101. Because the compounds inducing apoptotic activity are frequent targets of cancer therapy, TAC-101 may be a good candidate for use in the treatment of colon cancer.     

Activities of purine-metabolizing enzymes in human colon carcinoma cell lines and xenograft tumors

The activities of eleven enzymes of purine metabolism have been determined in extracts of four human colon carcinoma cell lines (clones A and D of the heterogeneous DLD-1 line, HCT-15 and DLD-2) in culture and as xenografts in nude mice. Activities of the enzyme adenine phosphoribosyltransferase (APRT), hypoxanthine phosphoribosyltransferase (HPRT), adenosine kinase, 5'-deoxy-5'-methylthioadenosine (MTA) phosphorylase, adenylate (AMP) kinase, guanylate (GMP) kinase, and nucleoside diphosphokinase (NDP kinase) changed little from line to line in vitro. Adenosine deaminase (ADA) activity varied more than 5-fold between HCT-15 and clone A cells. Purine nucleoside phosphorylase (PNP) activity was about 3-fold higher in DLD-2 cells than in the other three lines. Guanine deaminase (guanase) activity was 4- to 6-fold higher in clone A and D cells than in HCT-15 and DLD-2 cells. Xanthine oxidase was not detected in any of the lines grown in vitro or in vivo. The activity of APRT was highest in clone D xenografts. HPRT was lowest in DLD-2 tumors. ADA activity in DLD-2 tumors was more than 2-fold elevated over that for clone A and HCT-15 xenografts. PNP activity in clone A and HCT-15 xenografts was lower than that for clone D or DLD-2 tumors. Guanase was lower in HCT-15 and DLD-2 tumors than in those of clones A and D. Clone D tumor GMP kinase activity was elevated more than 4-fold above that for the other three tumors. The activity of NDP kinase was highest in clone D tumors. MTA phosphorylase activity was similar in all four xenografts. In general, the activity of a given enzyme was similar in cells growing in monolayer culture and as xenografts in nude mice (37/44 comparisons). Enzyme activities were also measured in normal human colon and compared to those in the xenografts. Where significant differences were seen, the values for normal tissue were almost always lower than those of the tumors. The striking similarities between the in vitro and in vivo enzymic profiles for each of these four human colon carcinoma lines indicate that, at least on this basis, responses of these cells to purines and purine analogs in the two-dimensional monolayer culture system should be predictive of in vivo responses.     

Role of nonsteroidal anti-inflammatory drug-activated gene-1 in docetaxel-induced cell death of human colorectal cancer cells with different p53 status

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression is upregulated not only by NSAIDs such as sulindac sulfide, but also by several antitumorigenic dietary compounds, suggesting that NAG-1 is a specific target for the development of effective anticancer agents. Despite being a downstream target of p53, NAG-1 induction is both p53-dependent and p53-independent. It is not clear whether NAG-1 induction is the responsible factor in cancer cell apoptosis with mutated p53. In this study, we report that NAG-1 induction alone cannot determine apoptotic cell fate in colon cancer cells. Although docetaxel induced an increase in NAG-1 and apoptosis in both HCT-116 (wild-type p53) and HT-29 (mutant p53) colon cancer cells, NAG-1 knockdown with siRNA prevented docetaxel-induced cell death in only HCT-116 cells. Docetaxel decreased Bcl-2 in HCT-116 cells, which have functionally active p53, according to luciferase reporter gene analyses, and docetaxel-induced cell death and changes in Bcl-2 and NAG-1 expression were blocked by PFT-α, a p53 inhibitor. In HT-29 cells with functionally inactive p53, the docetaxel-induced Bcl-xL decrease, NAG-1 increase, and cell death were not blocked by PFT-α. On the other hand, sulindac sulfide at concentrations that significantly induced NAG-1 did not decrease cell viability comparable to docetaxel, and it did not affect the level of p53, Bax, Bcl-2, and Bcl-xL in either cell line. The present study demonstrates that p53-dependent NAG-1 induction is linked to cell death and that NAG-1 induction without accompanying alteration of antiapoptosis protein Bcl-2 family members may not lead to cancer cell death.     

Different involvement of DNA methylation and histone deacetylation in the expression of solute-carrier transporters in 4 colon cancer cell lines

The purpose of this study on the involvement of epigenetic control of the expression of solute carrier (SLC) transporters by DNA methylation and histone deacetylation in 4 colon cancer cells is to find the epigenetic control mechanisms of drug transporters in colon cancers. Human colon cancer cell lines (HCT116, HT29, SW48, SW480) were treated with 5-aza-2'-deoxycytidine (DAC), as a DNA methyltransferase inhibitor, followed by trichostatin A (TSA), as a histone deacetylase inhibitor. The mRNA expression and DNA methylation of several SLC transporters were analyzed by real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. Among 12 SLC transporters possessing cytosine-phosphate-guanine (CpG) islands, thiamine transporter 2 (THTR2) (SLC19A3) gene showed a correlation between its mRNA expression level and DNA methylation status. TSA treatment increased histone H3 acetylation of THTR2 promoter region in all 4 colon cancer cell lines examined. HCT116 and SW48 cells showed a lack of THTR2 mRNA expression and methylation of its promoter, and DAC treatment induced its re-expression. In addition, the co-treatment with DAC and TSA increased THTR2 mRNA expression more markedly than DAC treatment in HCT116 and SW48 cells. In HT29 and SW480 cells that showed little methylation of THTR2 promoter, TSA treatment induced THTR2 mRNA expression markedly, but DAC treatment did not. In the 4 colon cancer cells examined, THTR2 mRNA expression is down-regulated by DNA methylation and/or histone deacetylation.     

Characterization of the N-methoxyindole-3-carbinol (NI3C)--induced cell cycle arrest in human colon cancer cell lines.

Recent results have shown that indole-3-carbinol (I3C) inhibits the cellular growth of human cancer cell lines. In some cruciferous vegetables, another indole, N-methoxyindole-3-carbinol (NI3C), is found beside I3C. Knowledge about the biological effects of NI3C is limited. The aim of the present study was to show the effect of NI3C on cell growth of two human colon cancer cell lines, DLD-1 and HCT-116. For the first time it is shown that NI3C inhibits cellular growth of DLD-1 and HCT-116 and that NI3C is a more potent inhibitor of cell proliferation than I3C. In addition to the inhibition of cellular proliferation, NI3C caused an accumulation of HCT-116 cells in the G2/M phase, in contrast to I3C, which led to an accumulation of the colon cells in G0/G1 phase. Furthermore, NI3C delays the G1-S phase transition of synchronized HCT-116 cells. The indole-mediated cell-cycle arrest may be related to the increased levels of the CDK-inhibitors p21 and p27 (only induced by NI3C). Only an initial increase of cdc2 protein was observed, whereas prolonged treatment with NI3C or I3C downregulates the mRNA and proteins of cyclin-dependent kinases and cyclins. These results indicate that both NI3C and I3C inhibit the proliferation of human colon cells but via different mechanisms.     

Retinoids activate RXR/CAR-mediated pathway and induce CYP3A

Retinoids and carotenoids are frequently used as antioxidants to prevent cancer. In this study, a panel of retinoids and carotenoids was examined to determine their effects on activation of RXR/CAR-mediated pathway and regulation of CYP3A gene expression. Transient transfection assays of HepG2 cells revealed that five out of thirteen studied retinoids significantly induced RXRα/CAR-mediated activation of luciferase activity that is driven by the thymidine kinase promoter linked with a PXR binding site in the CYP3A4 gene [tk-(3A4)₃-Luc reporter]. All-trans retinoic acid (RA) and 9-cis RA were more effective than CAR agonist TCBOPOP in induction of the tk-(3A4)₃-Luc reporter. Addition of retinoid and TCBOPOP further enhanced the inducibility and the induction was preferentially mediated by RXRα/CAR and RXRγ/CAR heterodimer. Chromatin immunoprecipitation assay showed that retinoids recruit RXRα and CAR to the proximal ER6 and distal XREM nuclear receptor response elements of the CYP3A4 gene promoter. The experimental data demonstrate that retinoids can effectively regulate CYP3A gene expression through the RXR/CAR-mediated pathway.     

Gene Expression Profiles of ABC Transporters and Cytochrome P450 3A in Caco-2 and Human Colorectal Cancer Cell Lines

Purpose. The mRNA levels of MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), cytochrome P450 3A (CYP3A) and villin in human colorectal cell lines (HCT-15, LoVo, DLD-1, HCT-116 and SW620) were quantitatively compared with those in Caco-2 cells. Methods. The mRNA levels were determined by real time quantitative polymerase chain reaction and expressed as the relative concentrations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Results. MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2. The expression of MRP1 mRNA in the cell lines tested was comparable with Caco-2. MRP2 mRNA was detected only in HCT-116 and SW620 at significantly lower level than Caco-2. CYP3A mRNA was detected in HCT-15, LoVo, DLD-1 and SW620 at similar level to Caco-2. Conclusions. HCT-15 LoVo and DLD-1 cells express proteins important for regulating the intestinal absorption of drugs, i.e., MDR1, MRP1 and CYP3A, whereas HCT-116 and SW620 cells were not acceptable for evaluation of absorption properties of drug candidates.     

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes are peroxisome proliferator-activated receptor gamma agonists but decrease HCT-116 colon cancer cell survival through receptor-independent activation of early growth response-1 and nonsteroidal anti-inflammatory drug-activated gene-1

1,1-Bis-(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents decrease survival of HCT-116 colon cancer cells and activate peroxisome proliferator-activated receptor (PPAR) gamma in this and other cancer cell lines. These PPARgamma-active compounds had minimal effects on expression of cell cycle proteins and did not induce caveolin-1 in HCT-116 cells. However, these compounds induced nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) and apoptosis in HCT-116 cells, and in time-course studies, the PPARgamma agonists maximally induced early growth response-1 (Egr-1) protein within 2 h, whereas a longer time course was observed for induction of NAG-1 protein. These data, coupled with deletion and mutation analysis of both the Egr-1 and NAG-1 gene promoters, indicate that activation of NAG-1 by these compounds was dependent on prior induction of Egr-1, and induction of these responses was PPARgamma-independent. Results of kinase inhibitor studies also demonstrated that activation of Egr-1/NAG-1 by methylene-substituted diindolylmethanes (C-DIMs) was phosphatidylinositol 3-kinase-dependent, and this represents a novel receptor-independent pathway for C-DIM-induced growth inhibition and apoptosis in colon cancer cells.     

微小 RNA-4478下调鼠双微体 -2对结肠癌细胞增殖、迁移、侵袭的影响

目的 探讨微小RNA-4478(miR-4478)对结肠癌细胞增殖、迁移、侵袭的作用并阐明相关机制.方法 实时定量聚合酶链式反应(PCR)检测miR-4478在结肠癌组织中的表达.结肠癌SW1116细胞转染miR-4478 mimics及阴性对照后,分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)和小室(Transwell)法检测细胞的增殖、迁移及侵袭.预测miR-4478的靶基因,双荧光素酶报告基因实验验证靶基因.结肠癌SW1116细胞转染miR-4478 mimics或抑制物后,蛋白质免疫印迹(Western blot)法检测细胞靶基因表达.靶基因过表达验证细胞增殖、迁移及侵袭.Western blot法检测细胞周期蛋白D1(Cy-clin D1)、基质金属蛋白酶2(MMP-2)、P21、钙粘附蛋白E(E-cadherin)蛋白表达.结果 相对于癌旁组织,miR-4478在结肠癌组织中的表达显著降低[(0.84±0.07)比(0.26±0.02),t=48.461,P<0.05];转染miR-4478 mimics可显著抑制细胞增殖、迁移及侵袭(P<0.001).miR-4478可负向调控MDM2表达(P<0.001).miR-4478过表达可抑制由MDM2过表达导致的结肠癌细胞增殖、迁移及侵袭(P<0.001).结论 miR-4478通过抑制MDM2表达而抑制结肠癌细胞增殖、迁移及侵袭.