Human Colorectal HCT116 Cells Research Articles

ASK1-JNK signaling cascade mediates Ad-ST13-induced apoptosis in colorectal HCT116 cells

ST13, a co-factor of heat shock protein, has shown potential antitumor efficacy for colorectal cancer in our previous study. However, the molecular mechanisms governing ST13-induced apoptosis are poorly understood. Here, we demonstrate that Ad-ST13 (ST13 mediated by adenovirus) activates apoptosis signal-regulated kinase (ASK1) and c-Jun N-terminal kinase (JNK) but not p38 (mitogen-activated protein kinase) in human colorectal HCT116 cells. Ad-ST13 also increases extracellular-regulated kinase (ERK) phosphorylation levels, but the change is due to adenovirus replication. Overexpression of ST13 also increases the transcription activity of AP-1. Blocking ASK1-JNK pathway affects Ad-ST13-mediated colorectal cell apoptosis, decreases the release of cytochrome c in cytoplasm and caspase activation. Because ASK1 is known to contain a tetratricopeptide repeat (TPR)-acceptor site and ST13 has TPR domain, we found the interaction between ST13 and ASK1. These results strongly indicate Ad-ST13 triggers colorectal cell apoptosis via ASK1-JNK signaling cascade.

Abstract 2605: CE-TOFMS metabolome analysis for pharmacological responses to CPT-11 in colorectal cancer cells with different chemosensitivities

Abstract Purpose: The topoisomerase I inhibitor irinotecan (CPT-11) is widely used for the treatment of metastatic colorectal cancer. However, mechanism to determine pharmacological response to CPT-11 is still unclear. We reported the application of a novel metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) on the study of metabolic responses to anticancer agents (AACR #2563 and #4785, 2008 and #3764, 2009). The purpose of this study is to compare the metabolomic responses to SN-38, the active metabolite of CPT-11, between high-sensitive and low-sensitive colorectal cancer cells, for finding new biomarkers that can predict CPT-11 response. Experimental Procedures: To elucidate metabolomic responses to SN-38, human colorectal carcinoma, HCT116 and HT29 cells were exposed to 50 nM SN-38. At time 0, 3, 8, and 24 h after SN-38 exposure, all intracellular metabolites were extracted by liquid-liquid extraction and simultaneously analyzed by CE-TOFMS. To find out low molecular weight compounds related to the sensitivity to SN-38, the metabolomic profiles were compared across the 8 colorectal cancer cell lines and sensitivity to SN-38 was investigated. Results: The HCT116 cells were more sensitive to SN-38 than HT29. The intracellular metabolites of key pathways for cellular activity were quantitatively analyzed by CE-TOFMS, which included glycolysis, tricarboxylic acid (TCA) cycles, urea cycles, amino acid metabolism and pyrimidine metabolism. The intracellular levels of endogenous deoxyribonucleotides (dTTP and dCTP) increased after SN-38 exposure in both cells. In contrast, ribonucleotides such as CTP and UTP of RNA synthesis pathway were not affected by SN-38 exposure in either cell. These results imply the change in deoxyribonucleotide pool due to the disruption of DNA synthesis by topoisomerase I inhibition, the main anti-cancer mechanism of SN-38. It was also found that lactate and glycerol-3-phosphate were accumulated to a greater extent in HCT116 than in HT29. Furthermore, correlation analysis using 8 colorectal cancer cell lines revealed that the intracellular level of γ-aminobutyric acid (GABA) strongly correlated with cellular sensitivity to SN-38. Conclusions: The present CE-TOFMS metabolome analysis created a new finding on metabolomic mechanisms for response to SN-38 by colorectal cancer cells with different chemosensitivities. GABA was found to be a potential low molecular weight biomarker that may be used for predicting CPT-11 response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2605.

인간 대장암 세포주에서 capsaicin 처리에 의한 차별적인 유전자 발현의 p53 의존성 분석

본 연구에서는 대장암 세포주 모델에서 파이토케미칼 capsaicin에 의한 항 생장 활성과 유전체 수준에서의 유전자 발현 변화를 연구하였다. 그 결과, 처리한 capsaicin 농도 의존적으로 세포 생존율이 감소함을 확인하였고, capsaicin은 다양한 유전자의 발현 변화를 유도하였다. DNA microarray 실험결과 <TEX>$100\;{\mu}M$</TEX> capsaicin의 처리에 의해 2배 이상 증가되는 유전자 103개가 확인된 반면, 2배 이상 발현이 감소되는 유전자 153개가 확인되었다. 발현이 증가되는 유전자 중 4개(NAG-1, DDIT3, GADD45A 그리고 PCK2)를 선택하여 RT-PCR을 수행한 결과, DNA micorarray 실험과 일치함을 확인하였다. 또한 <TEX>$100\;{\mu}M$</TEX> capsaicin의 처리에 의해 암 억제유전자인 p53의 발현이 증가됨을 RT-PCR과 real-time PCR 방법으로 확인하였다. 게다가, NAG-1, DDIT3 그리고 GADD45A 유전자는 p53의 존재에 관계없이 발현이 증가되는 반면, PCK2 유전자는 반드시 p53에 의해 발현이 유도됨을 확인할 수 있었다. 이러한 연구는 대장암 세포주에서 capsaicin에 의한 항암 기전을 이해하는데 도움을 줄 것으로 기대된다. In the present study, we investigated anti-proliferative activities of capsaicin and gene expression changes in response to capsaicin treatment in human colorectal HCT116 cells. The results showed that capsaicin decreased cell viabilities in a dose dependent manner and induced global gene expression changes. We found that 103 genes were up-regulated more than twofold, whereas 153 genes were down-regulated more than twofold by <TEX>$100\;{\mu}M$</TEX> capsaicin treatment. Among the up-regulated genes, we selected 4 genes (NAG-1, DDIT3, GADD45A and PCK2) and performed RT-PCR to confirm the microarray data. We found that <TEX>$100\;{\mu}M$</TEX> of capsaicin increased tumor suppressor p53 gene expression. In addition, the results showed that NAG-1, DDIT3 and GADD45A expressions were not dependent on p53 presence, whereas PCK2 expression. The results of this study may help to increase our understandings of the molecular mechanism of anti-proliferative activity mediated by capsaicin in human colorectal cancer cells.

Caffeic acid phenethyl ester의 처리에 의한 NSAID activated gene-1의 과대발현

파이토케미칼의 일종인 CAPE가 암세포 생장에 미치는 영향과 유전자 발현을 연구하기 위하여, 인간 대장암 세포주 HCT116에 CAPE를 처리하였다. CAPE의 처리는 농도 의존적으로 암 세포 생존율을 감소시키고, 세포사멸을 유도함을 확인하였다. CAPE에 의해 차별적으로 발현되는 유전자를 분석하기 위하여, oligo DNA microarray 실험을 수행하였다. 그 결과, <TEX>$20{\mu}M$</TEX> CAPE를 24시간 동안 처리한 경우, 2배 이상 발현이 증가되는 유전자 266개, 2배 이상 발현이 감소되는 유전자 143개를 확인하였다. 발현이 증가되는 유전자중 3개(NAG-1, p21, GADD45A)를 선택하여, RT-PCR을 수행하였다. 그 결과, 모든 유전자의 발현이 마이크로어레이 실험결과와 일치하였다. 또한, CAPE를 농도 의존적으로 처리한 후, NAG-1 유전자와 단백질의 발현을 확인한 결과, mRNA 수준과 단백질 수준에서의 발현양상이 동일함을 확인하였다. 게다가, CAPE를 포함한 5개의 다른 종류의 파이토케미칼(resveratrol, genistein, daidzein, capsaicin)을 처리한 경우, 처리한 모든 파이토케미칼에 의해 NAG-1 유전자의 발현이 증가됨을 확인하였다. 이중 CAPE가 가장 낮은 농도의 처리임에도 불구하고 NAG-1의 발현을 가장 강하게 유도하였다. 결론적으로 이러한 연구결과는 CAPE에 의한 세포사멸은 항암유전자인 NAG-1의 과대발현과 밀접한 관련이 있음을 의미한다. To investigate whether caffeic acid phenethyl ester (CAPE) could affect cancer cell viabilities and gene expression, human colorectal HCT116 cells were incubated with CAPE. CAPE decreased cancer cell viabilities and induced apoptosis in a dose-dependent manner. To analyse differently expressed genes by CAPE, we performed oligo DNA microarray analysis. We found that 266 genes were up-regulated more than twofold, whereas 143 genes were down-regulated more than twofold by 24 hr of treatment with <TEX>$20{\mu}M$</TEX> CAPE. Among the up-regulated genes, we selected 3 genes (NSAID activated gene-1 [NAG-1], cyclin-dependent kinase inhibitor 1A [CDKN1A, p21] and growth arrest and DNA-damage-inducible alpha [GADD45A]) and performed reverse-transcription PCR to confirm microarray data. In addition, we found that CAPE increased NAG-1 gene and NAG-1 protein expression in a dose-dependent manner. And, several other phytochemicals (resveratrol, genistein, daidzein and capsaicin) also could induce NAG-1 expression in human colorectal HCT116 cells. However, CAPE was the highest inducer of NAG-1, even in low concentrations. Overall, these results imply that cancer cell death by CAPE is closely related with over-expression of NAG-1.

대장암 세포주에서 genistein과 daidzein의 병합처리에 의한 상승적인 세포독성 효과

콩의 대표적인 이소플라본인 genistein과 daidzein에 의해 암세포 생존율에 미치는 영향을 확인하기 위하여, HCT116 세포주에 genistein과 daidzein을 농도 의존적으로 처리하였다. Genistein은 처리한 농도 의존적으로 암세포 생존율을 감소시켰으며, 이에 반해 daidzein은 세포생존율에 큰 변화를 보여주지는 못하였다. 이전의 마이크로어레이 실험 결과에 의하면, <TEX>$50{\mu}M$</TEX>의 genistein에 의해 2배 이상 증가되는 유전자 71개, 2배 이상 감소되는 유전자 64개가 검색되었다. 이중 3개의 유전자(DKK-1, ATF3 그리 고 NAG-1)를 선택하여, 마이크로어레이 실험 결과를 검증하기 위하여 RT-PCR을 수행하였다. RT-PCR 결과 마이크로어레이 결과와 모두 일치함을 증명하였다. 한편, genistein과 daidzein에 의한 병합처리에 의해 암세포생존에 미치는 영향을 확인하였다. 그 결과 병합처리에 의한 상승적인 세포독성 효과를 확인하였다. RT-PCR과 real-time PCR의 결과 genistein과 daidzein의 병합처리에 의해 항암유전자인 NAG-1 유전자가 상승적으로 발현이 증가됨을 확인하였다. 이러한 결과는 이소플라본뿐만 아니라 대두제품에 의한 암 화학예방법의 기전을 이해하는 도움을 줄 것으로 생각된다. To investigate whether isoflavone genistein and daidzein could affect cancer cell viability, human colorectal HCT116 cells were incubated with genistein or daidzein in a dose-dependent manner. Genistein decreased cancer cell viability in a dose-dependent manner, whereas daidzein did not show dramatic cytotoxic effects. We also found that 71 genes were up-regulated more than 2-fold, whereas 64 genes were down-regulated more than 2-fold with 24 hr of <TEX>$50{\mu}M$</TEX> genistein treatment by our previous microarray data. Among the up-regulated genes, we selected 3 genes (DKK1, ATF3 and NAG-1) and performed RT-PCR to confirm microarray data. The results of RT-PCR were highly correlated with those of the microarray experiment. In addition, we investigated whether a combination treatment of genistein and daidzein could affect cancer cell viability. Surprisingly, the combination treatment did show synergistic cytotoxic effects detected by MTS assay. The results of RT-PCR and real-time PCR indicate that a combination of genistein and daidzein can synergistically induce NAG-1 expression in HCT116 cells. This result implies that NAG-1 induction is highly associated with synergistic cytotoxic effects induced by a combination treatment of genistein and daidzein. Overall, these results may provide a clue in explaining the anti-cancer activity of soy bean in human colorectal cancer.

Membrane Microarray를 이용한 Resveratrol에 의해 차별적으로 발현되는 유전자 군의 분석

본 연구에서는 다섯 종류의 phytochemical (resveratrol, genistein, epicatechin gallate, diaIly disulfide, caffeic acid phenetyl ester)과 sulindac sulfide가 암 억제 단백질 p53을 유도할 수 있는지에 대해 연구하였다. 처리한 모든 phytochemical에 의해 p53 단백질의 발현이 강하게 유도된 반면, sulindac sulfide에 의해서는 p53 단백질이 유도되지 않았다. 처리한 phytochemical 중 포도껍질이나 와인에 많이 들어있는 resveratrol에 의해 p53 단백질이 농도의존적 혹은 처리시간 의존적으로 증가 발현되는 것을 확인하였다. 암 억제 단백질인 p53 하위 단계의 유전자들만 집적되어 있는 membrane microarray를 이용하여 실험을 수행한 결과, 25개의 유전자가 up-regulation 된 반면, 2개의 유전자가 down-regulation 되는 것을 확인하였다. Up-regulation 되는 유전자중 4개를 선택하여, RT-PCR을 수행한 결과 모두 membrane microarray 실험의 결과와 일치하였다. 게다가 p53 null인 HCT116 세포주를 이용한 RT-PCR을 통하여 TSP-1 유전자의 발현은 p53 의존적이지 않은 반면, MASPIN 유전자는 p53 의존적임을 확인하였다. 이러한 연구 결과는 resveratrol에 의한 화학적 암 예방법의 분자생물학적 기전을 이해하는데 도움을 줄 것으로 기대된다. In the present study, we investigated whether several phytochemicals (resveratrol, genistein, epicatechin gallate, dially disulfide, caffeic acid phenetyl ester) and sulindac sulfide could induce expression of tumor suppressor p53 protein in human colorectal HCT116 cells. We found that p53 was dramatically induced by all phytochemical treatments except sulindac sulfide. Among treated phytochemicals, we selected resveratrol for further experiments because it is one of the highest p53 inducer. Using a Western blot analysis, we found that resveratrol induced p53 in a dose- and time-dependent manner. Additionally, using membrane-based microarray analysis, we found that twenty-five genes were up-regulated and two genes were down-regulated by resveratrol treatment. Among the up-regulated genes, we selected 4 genes and performed reverse-transcription-PCR to confirm microarray data. The results of RT-PCR were highly accorded with those of membrane microarray. In addition, we found that thrombospondin-1 (TSP-1) expression was not dependent on p53 presence, whereas mammary serine protease inhibitor (MASPIN) expression was dependent on p53 expressed by resveratrol treatment. The results of this study may help to promote our understandings of the molecular mechanisms of chemoprevention that are mediated by resveratrol in human colorectal cancer.

WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

Indomethacin suppresses growth of colon cancer via inhibition of angiogenesisin vivo

It has been reported that regular consumption of nonsteroidal anti-inflammatory drugs like indomethacin decreases the incidence and mortality rate of a number of gastrointestinal cancers. We aimed to explore the efficacy and possible mechanisms of indomethacin on tumor growth and tumor angiogenesis of human colon cancer xenografts in nude mice. MTT (thiazolyl blue) assay was used to assess the effect of indomethacin on cultured human colorectal cancer cell line HCT116. HCT116 cells were inoculated subcutaneously into BALB/c-nu/nu mice. After oral administration of indomethacin, 3 mg/kg.d for 4 wk, animals were sacrificed by cervical dislocation. Immunohistochemical staining was employed to determine the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in tumor tissues. Indomethacin, a non-selective COX inhibitor, significantly decreased the viability of HCT116 cells in a dose-dependent manner (P<0.05) with 50% inhibition at approximately 318.2+/-12.7 mumol/L. Growth of HCT116 cell tumor was significantly suppressed by indomethacin. The tumor volume was significantly decreased in the treated group (458.89+/-32.07 mm(3)) compared to the control group (828.21+/-31.59 mm(3)) (P<0.05). The MVD of the treated group (19.50+/-5.32) was markedly decreased compared to the control group (37.40+/-4.93) (P<0.001). The VEGF expression of the treated group (1.19+/-0.17) was obviously reduced as compared to the control group (1.90+/-0.48) (P<0.01). The decrease in MVD was positively correlated with the decrease of VEGF expression (r(s) = 0.714, P<0.05). We did not see gastrointestinal complications in the treated group and no differences were noted in the body weight of the mice between the two groups throughout the study (P>0.05). Indomethacin can significantly decrease the viability of cultured HCT116 cells and retard human colorectal HCT116 cell tumor growth via inhibiting tumor angiogenesis, which might be through reduction of VEGF expression.

Diallyl Disulfide (DADS) Induces the Antitumorigenic NSAID-Activated Gene (NAG-1) by a p53-Dependent Mechanism in Human Colorectal HCT 116 Cells

Garlic is appealing as an anti-carcinogenic agent due to its ability to induce apoptosis in vitro and inhibit the formation and growth of tumors in animals in vivo. Diallyl disulfide (DADS) is a constituent of garlic that suppresses neoplastic cell growth and induces apoptosis. We examined the effects of DADS on various cancer cell lines to better understand its effect on apoptosis and apoptosis-related genes. The nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) has proapoptotic and antitumorigenic activities and is upregulated by anticancer agents such as NSAIDs. In this study, human colorectal HCT-116 (wild-type p53), HCT-15 (p53 mutant) and human prostate PC-3 (p53 mutant) cells were exposed to DADS. DADS inhibited cell proliferation in all cell lines albeit to a lesser extent in HCT-15 and PC-3 cells at 11.5 and 23 micromol/L. In HCT-116 cells, DADS induced p53 and NAG-1 in a dose-dependent manner and the induction of p53 preceded that of NAG-1. In HCT-116 cells, NAG-1 protein expression was increased 2.4-fold +/- 0.6 at 4.6 micromol/L and 6.1-fold +/- 1.7 at 23 micromol/L DADS, whereas p53 was induced 1.5-fold +/- 0.1 and 2.3-fold +/- 0.4. DADS did not induce NAG-1 or p53 in p53 mutant cell lines; however, NAG-1 expression was induced by sulindac sulfide. HCT-116 cells treated with 4.6 and 23 micromol/L DADS resulted in a 1.9- and 2.9-fold increase in apoptosis, respectively. In contrast, 23 micromol/L DADS induced apoptosis only 1.8-fold in HCT-15 cells and not at all in PC-3 cells. Thus, DADS-induced apoptosis and NAG-1 protein expression appear to occur via p53.

Growth suppression mediated by transfection of wild-type hMLH1 in human cancer cells expressing endogenous truncated hMLH1 protein.

Many genes that are frequently mutated in human cancer are known to be involved in the control of normal cellular proliferation. One of the genes involved in DNA mismatch repair is hMLH1, defective mutations of which are found in some familial and various sporadic cancers. Although the DNA mismatch repair activity of hMLH1 has been identified, other biological functions of hMLH1 have not been well investigated. To investigate the effect of wild-type hMLH1 in cellular proliferation, wild-type hMLH1 cDNA was introduced into human colorectal carcinoma cell line HCT116 and human gastric carcinoma cell line SNU-1, each containing a homozygous non-sense mutation at codon 252 and 226 in hMLH1, repectively. The hMLH1-transfected stable clones showed mRNA and protein expression of transfected hMLH1. Three in vitro cell growth experiments demonstrated that compared with parental and vector-transfected control counterparts, both hMLH1-transfected HCT116 and SNU-1 clones displayed: i) decreased cellular proliferation; ii) a significant decrease in the rate of DNA synthesis and iii) a dramatic reduction of anchorage-independence and the size of colonies in semisolid medium. In addition to DNA repair activity, these results suggest that hMLH1 may play a role in the negative regulation of HCT116 and SNU-1 cell growth.