Heat shock protein 27, a novel regulator of 5-fluorouracil resistance in colon cancer

The resistance of colon cancer to 5-fluorouracil (5-FU) is a critical issue, and the cause of this resistance cannot always be explained based on the known molecules. Heat shock protein 27 (HSP27) mRNA expression has recently been shown to be correlated with 5-FU resistance in 5-FU-resistant cell lines. In this study, we attempted to elucidate the functional mechanism of HSP27 in 5-FU resistance in colon cancer. HSP27 protein levels in several human colon cancer cell lines (LoVo, HCT15, WiDr, HCT116, HT-29 and SW480) were determined by immunoblot and densitometry analysis. The in vitro growth inhibition rates (IR) of the cell lines at various concentrations of 5-FU were assessed by MTT assay. The degree of 5-FU resistance was estimated as the drug concentration inducing 50% IR (IC50). The HSP27 protein level and IC50 were significantly correlated in these cell lines (p=0.010). The effect of HSP27 overexpression on IC50 was evaluated in LoVo cells. HSP27 transfectants significantly increased IC50 and reduced HSP27 resistance. The effect of HSP27 down-regulation by HSP27 siRNA on IC50 was confirmed in HCT15 cells. HSP27 siRNA suppressed HSP27 protein levels and reduced IC50 in a dose-dependent manner. These data indicated that HSP27 is closely connected with 5-FU resistance in colon cancer and suggested that HSP27 levels predicted 5-FU resistance. HSP27 down-regulation overcame 5-FU resistance and HSP27 may be a clinical target in patients with 5-FU-resistant colon cancer.     

Molecular characterizations of derivatives of HCT116 colorectal cancer cells that are resistant to the chemotherapeutic agent 5-fluorouracil

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.     

5'-Nitro-indirubinoxime, an indirubin derivative, suppresses metastatic ability of human head and neck cancer cells through the inhibition of Integrin β1/FAK/Akt signaling

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 μM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.     

Down-regulation of cIAP2 enhances 5-FU sensitivity through the apoptotic pathway in human colon cancer cells

Currently 5-fluorouracil (5-FU) plays a central role in the chemotherapeutic regimens for colorectal cancers and thus it is important to understand the mechanisms that determine 5-FU sensitivity. The expression profiles of human colon cancer cell line DLD-1, its 5-FU-resistant subclone DLD-1/FU and a futher 21 types of colon cancer cell lines were compared to identify the novel genes defining the sensitivity to 5-FU and to estimate which population of genes is responsible for 5-FU sensitivity. In the hierarchical clustering, DLD-1 and DLD-1/FU were most closely clustered despite over 100 times difference in their 50% inhibitory concentration of 5-FU. In DLD-1/FU, the population of genes differentially expressed compared to DLD-1 was limited to 3.3%, although it ranged from 4.8% to 24.0% in the other 21 cell lines, thus indicating that the difference of 5-FU sensitivity was defined by a limited number of genes. Next, the role of the cellular inhibitor of apoptosis 2 ( cIAP2) gene, which was up-regulated in DLD-1/FU, was investigated for 5-FU resistance using RNA interference. The down-regulation of cIAP2 efficiently enhanced 5-FU sensitivity, the activation of caspase 3/7 and apoptosis under exposure to 5-FU. The immunohistochemistry of cIAP2 in cancer and corresponding normal tissues from colorectal cancer patients in stage III revealed that cIAP2 was more frequently expressed in cancer tissues than in normal tissues, and cIAP2-positive patients had a trend toward early recurrence after fluorouracil-based chemotherapy. Although the association between drug sensitivity and the IAP family in colorectal cancer has not yet been discussed, cIAP2 may therefore play an important role as a target therapy in colorectal cancer. ( Cancer Sci 2009; 100: 903–913)     

结肠癌 COLO 耐药株的构建及其与肿瘤干细胞的关系

目的：构建结肠癌 COLO 耐药株,探索其功能特性及其与肿瘤干细胞的关系。方法通过逐渐递增化疗药5-氟尿嘧啶（5-FU）浓度的方法,构建了耐0．10μmol／ml 和0．20μmol／ml 5-FU 的结肠癌 COLO 耐药株各1株,分别命名为 COLO／5-FU-1、COLO／5-FU-2。从增殖能力、克隆形成能力、迁移和侵袭能力、微球体形成能力、干性基因的表达、交叉耐药等方面与亲本结肠癌细胞株 COLO 进行对比。结果在存活检测中,常规培养后第4天,结肠癌耐药株 COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 细胞的 A 值分别为0．61±0．13、0．54±0．07、0．41±0．09,差异有统计学意义（F ＝63．43,P ＝0．033）。随着耐5-FU 药物浓度的增加,COLO 耐药株的克隆形成率逐渐增强,COLO／5-FU-2、COLO／5-FU-1、亲本 COLO的克隆形成率依次为（87．6±12．7）％、（65．3±9．7）％、（38．5±7．6）％,差异有统计学意义（F ＝33．64, P ＝0．017）。COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 每高倍视野中迁移穿过基底膜的细胞数分别为（482±39）个、（434±45）个、（373±38）个,侵袭穿过基底膜的细胞数分别为（174±42）个、（112±31）个、（87±29）个,差异均有统计学意义（F ＝109．61,P ＝0．009;F ＝67．31,P ＝0．032）。与亲本结肠癌细胞株COLO 比较,耐药株表达更高的干性基因,差异有统计学意义（F ＝47．31,P ＝0．042）;且耐药株对其他化疗药如米托蒽醌存在交叉耐药[如给药96 h,米拖蒽醌对亲本 COLO 的抑制率高于 COLO／5-FU-1、COLO／5-FU-2（0．749±0．042、0．423±0．024、0．342±0．018）,差异有统计学意义（F ＝12．61,P ＝0．028）]。COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 细胞的微球体形成率依次为（8．90±0．97）％、（6．20±0．75）％、（3．90±0．32）％,差异有统计学意义（F ＝164．32,P ＝0．006）。结论结肠癌 COLO耐药株有肿瘤干细胞样细胞的特点,是富集了的肿瘤干细胞。     

Pyrotinib Sensitizes 5-Fluorouracil-Resistant HER2⁺ Breast Cancer Cells to 5-Fluorouracil

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent for breast cancer. However, acquired chemoresistance leads to a loss of its efficacy; methods to reverse are urgently needed. Some studies have shown that pyrotinib, an ErbB receptor tyrosine kinase inhibitor, is effective against HER2+ breast cancer. However, whether pyrotinib sensitizes 5-FU-resistant breast cancer cells to 5-FU is unknown. We hypothesized that the combination of pyrotinib and 5-FU would show synergistic antitumor activity, and pyrotinib could reverse 5-FU resistance in HER2+ breast cancer cells in vitro and in vivo. Our data showed that pyrotinib inhibited the growth of 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. 5-FU remarkably suppressed the growth of SKBR-3 and MAD-MB-453 cells. However, SKBR-3/FU and MADMB-453/FU cells showed resistance to 5-FU. A combination of pyrotinib and 5-FU resulted in the synergistic inhibition of the growth of the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. Pyrotinib decreased significantly the IC50 values of 5-FU and the thymidylate synthase (TS) mRNA expression levels in the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines and increased significantly the intracellular concentration of 5-FU in SKBR-3/FU and MDA-MB-453/FU cells. In addition, pyrotinib reduced the ABCG2 mRNA and protein expression levels in SKBR-3/FU and MDA-MB- 453/FU cells and downregulated the protein expression levels of pAKT, pHER2, and pHER4 in all four cell lines. After TS or ABCG2 in 5-FU-resistant breast cancer cells was knocked down, the sensitivity of SKBR-3/ FU and MDA-MB-453/FU cells to 5-FU was restored. Moreover, in vivo experiments demonstrated that pyrotinib in combination with 5-FU more effectively inhibited SKBR-3/FU tumor growth than either pyrotinib or 5-FU alone. In conclusion, our findings suggest that pyrotinib could restore sensitivity of 5-FU-resistant HER2+ breast cancer cells to 5-FU through downregulating the expression levels of TS and ABCG2.     

Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells

Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor kappaB (NFkappaB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFkappaB DNA-binding activity. Cotransfection of NFkappaB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFkappaB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G(1)-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G(1), and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.     

Prediction of chemosensitivity of colorectal cancer to 5-fluorouracil by gene expression profiling with cDNA microarrays

5-Fluorouracil (5-FU) is the most widely used anticancer agent for gastrointestinal cancers. Because many tumors show primary resistance, it is clinically meaningful to predict tumor sensitivity to the drug before treatment. cDNA microarrays containing 21,168 clones were used to identify genes associated with sensitivity to 5-FU. Gene expression profiling of 3 colorectal cancer cell lines (DLD-1, HT-29 and NUGC-3) and the corresponding 5-FU-resistant sublines (DLD-1/FU, HT-29/FU and NUGC-3/5FU/L) showed 81 genes that were differentially expressed. The gene set thus identified successfully predicted the sensitivities of 5 other colorectal cancer cell lines and could also separate 5-FU resistant clinical samples from sensitive ones.     

Predicting the chemosensitivity of pancreatic cancer cells by quantifying the expression levels of genes associated with the metabolism of gemcitabine and 5-fluorouracil

Gemcitabine (GEM) is the standard treatment for advanced/metastatic pancreatic cancer. However, there is a substantial subset of patients in whom the efficacy of GEM, when used as a single agent, is inadequate. Recently, the 5-fluorouracil (5-FU) prodrugs capecitabine and S-1 have been used as an alternative, either alone or in combination with GEM. The aim of the present study was to investigate the expression pattern of genes that render pancreatic cancer cells sensitive to GEM and 5-FU, and to identify markers for individualized chemotherapy, even in patients who have developed resistance. We investigated the correlation between the expression of genes associated with the metabolism of GEM and 5-FU, and sensitivity to these drugs in 15 human pancreatic cancer cell lines. We also established GEM- and 5-FU-resistant pancreatic cancer cell lines to investigate changes in the expression levels of these genes and the effects of one drug on cells resistant to the other. We found no correlation between pancreatic cancer cell sensitivity to either GEM- or 5-FU. GEM-resistant cells did not become resistant to 5-FU and vice versa. High expression of RRM1 (P=0.048) and TS x DPD (P=0.035) correlated significantly with sensitivity to GEM and 5-FU, respectively. 5-FU-resistant cells expressed significantly higher levels of TP than parental cells (P<0.05). In conclusion, pancreatic cancer cells showed no cross-resistance to GEM and 5-FU. Quantitative analyses of RRM1, TP, DPD and TS mRNA levels in pancreatic cancer cells may be useful for predicting their sensitivity to GEM and 5-FU.     

Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery

Background  

Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure.     

Expression of rTSbeta as a 5-fluorouracil resistance marker in patients with primary breast cancer

Expression of thymidylate synthase (TS) in tumor cells is frequently suggested as an important prognostic factor for patients scheduled for chemotherapy with 5-fluorouracil (5-FU). However, clinical evidence does not fully support such an anticipation. We studied the expression of rTSbeta, a reverse orientation gene of TS, as a 5-FU resistance marker in patients with primary breast cancer. Expression of rTSbeta was examined in 129 patients with newly diagnosed breast cancer and five breast cancer cell lines by immunohistochemistry, immunocytochemistry and immunoblotting. Clinically, expression of rTSbeta was found to correlate with survival of the patients (p=0.023) when patients received chemotherapeutic regimen containing 5-FU. In vitro, rTSbeta expression was found to correlate with 5-FU resistance in breast cancer cell lines. Notably, in the 5-FU-resistant cells, rTSbeta was identified in the nucleus, whereas in the 5-FU-sensitive cells, rTSbeta was found in the cytoplasm. Nuclear localization of rTSbeta was further found to be associated with protein farnesylation. Therefore, nuclear expression of rTSbeta could be a novel 5-FU resistance marker in patients with primary breast cancer.     

Anti-tumor effects of carmofur on human 5-FU resistant cells]

As a part of studies to clarify the efficacy mechanism of carmofur (HCFU), a 5-FU analog, the degree of cross-resistance to HCFU of cell lines of cultured human colon cancer DLD-1 and a stomach cancer NUGC-3, both of which had acquired resistance to 5-FU, was studied in vitro and in vivo. In vitro, both resistant cell lines showed no or little cross-resistance to HCFU under a short-term drug exposure, and very low cross-resistance to HCFU, as compared to resistance to 5-FU, under a long-term drug exposure. In therapeutic experiments using nude mice bearing parent or subcutaneously transplanted 5-FU-resistant DLD-1 cells, HCFU exerted almost the same growth-inhibitory effects on both tumors. These results suggest that HCFU itself may have its own anti-tumor mechanism, different from that of 5-FU.     

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.     

In Vitro Generation of Cytotoxic T Lymphocytes Against HLA-A2.1 -Restricted Peptides Derived from Human Thymidylate Synthase

Abstract

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses In Vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1 + healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity In Vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.     

Mechanisms of acquired chemoresistance to 5-fluorouracil and tomudex: thymidylate synthase dependent and independent networks

Purpose Thymidylate synthase (TS) over-expression is widely accepted as a major molecular mechanism responsible for 5-fluorouracil (5-FU) and tomudex (TDX) resistance. In this study, the importance of TS in 5-FU and TDX resistance was evaluated. Methods The sensitivity of TS-over-expressing 5-FU (3) and TDX (3) resistant cell lines to 5-FU and TDX was analysed. The cross-resistance between 5-FU and TDX resistant cell lines was determined. The relationship between p53 and NF-κB status and the sensitivity to 5-FU and TDX was evaluated. Results Compared to relevant parental sensitive cell lines, the 5-FU resistant cell lines were highly cross-resistant to TDX (over 20,000-fold). In contrast, over-expression of TS did not significantly confer 5-FU resistance on the TDX resistant cell lines (0.8- to 1.3-fold). Thymidine (20 μM) rescue induced TDX resistance in TDX sensitive cell lines (over 10,000-fold) but only moderately influenced 5-FU sensitivity in 5-FU sensitive cell lines (1.1- to 2.4-fold). Uridine moderately protected one cancer cell line (RKO) from 5-FU-induced, but not TDX-induced, cytotoxicity. NF-κB transfected MCF-7 and p53 knockout HCT116 cells were resistant to 5-FU (4.4- and 2.4-fold, respectively) but not to TDX. TS protein expression in NF-κB transfected and p53 knockout cell lines was comparable to the relevant parental cell lines. Conclusion In some cancer cell lines, TS-independent molecular events may play a key role in 5-FU resistance. Loss of p53 function and NF-κB over-expression may be involved in TS-independent 5-FU chemoresistance in some cancer cell lines.