Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.     

Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model

Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU–induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.     

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)     

Inhibition of p38 MAPK increases the sensitivity of 5-fluorouracil-resistant SW480 human colon cancer cells to noscapine

A major cause of treatment failure in advanced colon cancer is resistance to chemotherapy. p38 mitogen-activated protein kinase (MAPK) has been associated with cellular apoptosis and plays an important role in multidrug resistance (MDR) in cancer cells. In the present study the effect of p38 MAPK on the sensitivity of 5-fluorouracil (5-FU)-resistant SW480 (SW480/5-FU) human colon cancer cells to noscapine was investigated. Following p38 MAPK interference, the inhibitory effect of noscapine on cell viability and proliferation was increased in the SW480/5-FU cells and there was also a decrease in the expression level of minichromosome maintenance proteins, recombinant Ki-67 and proliferating cell nuclear antigen. Inhibition of p38 MAPK also enhanced noscapine-induced G₁-phase cell cycle arrest in the SW480/5-FU cells and there was also a decrease in the protein and mRNA expression level of cyclin D, cyclin E and cyclin-dependent kinase 2, and an increase in the expression level of P57. Furthermore, p38 MAPK interference increased noscapine-induced apoptosis of the SW480/5-FU cells and there was an increase in the protein and mRNA expression level of caspases-3 and 8 and Bax, and decreased Bcl-2 expression level. The sensitivity of the SW480/5-FU cells to noscapine was also increased following p38 MAPK interference, as demonstrated by MDR inhibition via decreased Akt activity and reduced protein expression level of the MDR proteins P-glycoprotein, multidrug resistance protein 1 and ATP-binding cassette G2. These observations indicated that inhibition of p38 MAPK increased the sensitivity of the SW480/5-FU cells to noscapine by suppressing proliferation, induction of cell cycle arrest and apoptosis, and reversal of MDR in the SW480/5-FU cells.     

Cyclin A correlates with the sensitivity of human cancer cells to cytotoxic effects of 5-FU

We investigated the role of cyclin A in the cytotoxic effect of 5-fluorouracil (5-FU) on cancer cell lines. Experiments were performed using gastric cancer chemosensitive NUGC3, and chemoresistant NUGC3/5FU/L established by repeated exposure to 5-FU. 5-FU inhibited cell growth of NUGC3 in a dose-dependent manner. Low concentrations of 5-FU did not inhibit cell growth of NUGC3/5FU/L, while high concentrations slightly inhibited cell growth. Examination of the cell cycle pattern of NUGC3 cells showed accumulation at S-phase at 10 micro M and at G1-S-phase at 100 micro M of 5-FU. Cell cycle pattern of NUGC3/5FU/L cells did not change 5-FU concentrations. 5-FU increased cyclin A mRNA level in NUGC3 cells but not NUGC3/5FU/L cells. In the presence of 100 micro M 5-FU, cyclin A protein level increased 2.6-fold in NUGC3 and 1.47-fold in NUGC3/5FU/L. 5-FU dose-dependently increased the percentage of cyclin A-positive NUGC3 cells, but not NUGC3/5FU/L cells. The percentage of cyclin A-positive cells in other 5-FU sensitive esophageal, colon and gastric cancer cell lines (T.Tn, LOVO, DLD-1, MKN-7), increased in the presence of 1 and 10 micro M 5-FU, while cyclin A-positive cells in 5-FU resitant hepatocellular and colon carcinoma cell lines (HCC50 and C-1), did not increase with the same treatment. Our results indicate that the cytotoxic effects of 5-FU in human cancer cell lines correlate with cyclin A and it may be used as a predictive factor for chemotherapy response.     

Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells

5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.     

The transfection of thymidylate synthase antisense suppresses oncogenic properties of a human colon cancer cell line and augments the antitumor effect of fluorouracil

5-Fluorouracil (5-FU), a fluoropyrimidine analogue, is one of the most commonly used anticancer drugs for the treatment of gastrointestinal malignancies. Some studies reported that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the thymidylate synthase (TS), an essential DNA synthetic enzyme. The aim of this study was to determine if antisense TS technology could augment the chemosensitivity of human cancer cells to 5-FU. The full length coding region of TS cDNA was inversely cloned into the eukaryotic expression vector pCDL81 and transfected into DLD-1 cells. The expression and activity of TS were significantly suppressed in the antisense TS transfected cells. Interestingly, the transfection of antisense TS alone inhibited the cellular growth in vitro. The chemosensitivity to 5-FU was significantly increased in the transfected cells. The 50% inhibition values of 5-FU on DLD-1/anti-TS were approximately one forth that on parental cells. The augmentation of chemosensitivity to 5-FU was also confirmed in a nude mice model. The tumor growth of DLD-1/anti-TS cells was suppressed significantly more than that of DLD-1 cells by the 5-FU. The expression and activity of TS in human colon cancer cells were effectively inhibited by TS antisense treatment and the effect of 5-FU to cancer cells can be augmented. The antisense TS technology could be promising for treatments of gastrointestinal cancers.     

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.     

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.     

Chromosomal imbalances associated with acquired resistance to fluoropyrimidines in human colorectal cancer cells

The chromosomal aberrations underlying the development of resistance to fluoropyrimidines have not yet been identified. To characterise the genomic changes that induce the development of resistance to fluoropyrimidines, we used comparative genomic hybridisation (CGH) to analyse and compare the parent DLD-1 human colorectal cancer cell line and two cell lines, DLD-1/5-FU and DLD-1/FdUrd, which were resistant to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd), respectively. Both resistant cell lines showed a genetic aberration derived from the parental cell line DLD-1. Losses of 3p and 3q were also detected as additional genetic changes in the two resistant cell lines. Both resistant cell lines showed decreased orotate phosphoribosyltransferase (OPRT) activity, which is associated with the activity of the uridine monophosphate (UMP) synthase gene (3q13). These results suggested that the loss of 3q might be a genetic change responsible for the decreased OPRT activity and fluoropyrimidine cytotoxic response in cancer cells. Amplification of 18p11.2-p11.3 containing the thymidine synthase (TS) gene (18p11.32) was observed only in the DLD-1/FdUrd-resistant cell line, which overexpresses TS. These findings suggested that 18p amplification represents a genetic change associated with the overexpression of the TS protein. Our results indicate that chromosomal aberrations identified by CGH could explain, at least in part, acquired fluoropyrimidine resistance.     

Proteomic differential display analysis for TS-1-resistant and -sensitive pancreatic cancer cells using two-dimensional gel electrophoresis and mass spectrometry

TS-1 is an oral anticancer agent containing two biochemical modulators for 5-fluorouracil (5-FU) and tegafur (FT), a metabolically activated prodrug of 5-FU. TS-1 has been recognized as an effective anticancer drug using standard therapies for patients with advanced pancreatic cancer along with gemcitabine. However, a high level of inherent and acquired tumor resistance to TS-1 induces difficulty in the treatment. To identify proteins linked to the TS-1-resistance of pancreatic cancer, we profiled protein expression levels in samples of TS-1-resistant and -sensitive pancreatic cancer cell lines by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytotoxicity of a 5-FU/5-chloro-2,4-dihydroxypyridine (CDHP) combination towards pancreatic cancer cell lines was evaluated by MTS assay. Panc-1, BxPC-3, MiaPaCa-2 and PK59 showed high sensitivity to the 5-FU/CDHP combination (TS-1-sensitive), whereas PK45p and KLM-1 were much less sensitive (TS-1-resistant). Proteomic analysis showed that eleven spots, including T-complex protein 1 subunit beta, ribonuclease inhibitor, elongation factor 1-delta, peroxiredoxin-2 and superoxide dismutase (Cu-Zn), appeared to be down-regulated, and 29 spots, including hypoxia up-regulated protein 1, lamin-A/C, endoplasmin, fascin and annexin A1, appeared to be up-regulated in TS-1-resistant cells compared with -sensitive cells. These results suggest that the identified proteins showing different expression between TS-1-sensitive and -resistant pancreatic cancer cells possibly relate to TS-1-sensitivity. These findings could be useful to overcome the TS-1-resistance of pancreatic cancer cells.     

CDK inhibitor enhances the sensitivity to 5-fluorouracil in colorectal cancer cells

Thymidylate synthase (TS) is a dNTP synthetic enzyme and is also a target enzyme of 5-fluorouracil (5-FU). 5-FU is one of the anticancer agents most frequently used for the treatment of colorectal cancers. However, the clinical rate of response to its use as a single agent is not exceptionally high. Therefore, various combination chemotherapies have been devised. The elevated expression of TS in cancer cells is a serious obstacle in the clinical use of 5-FU. In the present study, TS expression was up-regulated by the knockout of the p21WAF1/CIP1 gene in human colorectal cancer HCT116 cells, suggesting that TS expression is mediated through the inhibition of cyclin-dependent kinase (CDK). Based on these findings, we tested whether the CDK inhibitor (CDKI) SU9516, acted as a suppressor of TS. SU9516 effectively reduced the expression of TS in a dose-dependent manner. Furthermore, the reduction of TS expression resulted in enhancement of the sensitivity to 5-FU in human colon cancer DLD-1 cells. Thus, SU9516 might be a promising compound for combination chemotherapy with 5-FU.     

Effect of 5-Fluorouracil on Cell Cycle Regulatory Proteins in Human Colon Cancer Cell Line

We investigated the effects of 5-fluorouracil (5-FU) on cell cycle-regulating proteins in RPMI 4788 cells. 5-FU inhibited cell growth dose-dependently and this growth inhibition was accompanied with cell cycle accumulation in early S phase and increased expression of cyclin A. When cells were released from short-term treatment (3 or 24 h) with 5-FU, the cell cycle started to progress again and cyclin A protein levels decreased. Cyclin A-associated kinase activity assay showed that cyclin A-cyclin-dependent kinase (Cdk) 2 kinase activity was altered by 5-FU treatment concomitantly with the changes in cell cycle state seen in flow cytometric analysis. Furthermore, the elevation of cyclin A protein level by 5-FU treatment was observed in three other human cancer cell lines, DLD-1, H226Br and T.Tn. These results suggest that cyclin A protein levels in cancer cells are increased by 5-FU, and the cyclin A function and degradation mechanism remain normal.     

Effect of 5-fluorouracil on cell cycle regulatory proteins in human colon cancer cell line.

We investigated the effects of 5-fluorouracil (5-FU) on cell cycle-regulating proteins in RPMI 4788 cells. 5-FU inhibited cell growth dose-dependently and this growth inhibition was accompanied with cell cycle accumulation in early S phase and increased expression of cyclin A. When cells were released from short-term treatment (3 or 24 h) with 5-FU, the cell cycle started to progress again and cyclin A protein levels decreased. Cyclin A-associated kinase activity assay showed that cyclin A-cyclin-dependent kinase (Cdk) 2 kinase activity was altered by 5-FU treatment concomitantly with the changes in cell cycle state seen in flow cytometric analysis. Furthermore, the elevation of cyclin A protein level by 5-FU treatment was observed in three other human cancer cell lines, DLD-1, H226Br and T.Tn. These results suggest that cyclin A protein levels in cancer cells are increased by 5-FU, and the cyclin A function and degradation mechanism remain normal.     

Role for alpha1,2-fucosyltransferase and histo-blood group antigen H type 2 in resistance of rat colon carcinoma cells to 5-fluorouracil

5-Fluorouracil (5-FU) is a drug of standard use in chemotherapy of colon carcinoma. However, its efficacy is limited by inherent and acquired cell resistance. Major changes in histo-blood group antigenic expression, at times associated with poor prognosis, occur on colon cancer cells. To assess whether these antigens might play a role in the resistance to 5-FU, a rat model of colon carcinoma was used. We observed that in vivo treatment of tumors with the drug increased expression of antigen H type 2. The increase was also observed after in vitro short-term exposure to 5-FU, as well as on a cell-resistant variant selected by continuous exposure to the drug, and was accompanied by an increase in alpha1,2-fucosyltransferase activity, the key enzyme involved in synthesis of H antigens. Transfection of cells devoid of this enzymatic activity by an alpha1, 2-fucosyltransferase cDNA allowed expression of H type 2 antigen and increased resistance to 5-FU. Inversely, transfection of cells which possess enzymatic activity by a cDNA in anti-sense orientation reduced both H type 2 cell-surface antigen and resistance to the drug. These results demonstrate that, in this experimental model, alpha1,2-fucosyltransferase and H type 2 antigen are involved in cellular resistance to 5-FU.     

Expression of CYP2A6 in Tumor Cells Augments Cellular Sensitivity to Tegafur

To examine the role of cytochrome P450 2A6 (CYP2A6) in the cellular sensitivity to an anti-tumor prodrug, tegafur (FT), a CYP2A6 cDNA construct was transfected into cells of a colon cancer cell line, DLD-1. CYP2A6-expressing cells (DLD-1/CYP2A6 cells) more efficiently catalyzed the conversion of FT to 5-fluorouracil (5-FU) (2.6-fold) and the 7-hydroxylation of coumarin (7.9-fold) than cells transfected with a null construct (DLD-1/null cells). These results indicated that the expressed CYP2A6 was functionally active. The extent of growth inhibition of the DLD-1/CYP2A6 cells by FT was greater than that of DLD-1/null cells; the difference between the DLD-1/CYP2A6 and DLD-1/null cells was statistically significant at the concentrations of 250, 500 and 1000 μ M. 5-FU, an active metabolite of FT, inhibited the growth of both types of cells to the same extent. Thus, intracellular expression of CYP2A6 can sensitize cells to FT.     

Expression of CYP2A6 in tumor cells augments cellular sensitivity to tegafur.

To examine the role of cytochrome P450 2A6 (CYP2A6) in the cellular sensitivity to an anti-tumor prodrug, tegafur (FT), a CYP2A6 cDNA construct was transfected into cells of a colon cancer cell line, DLD-1. CYP2A6-expressing cells (DLD-1 / CYP2A6 cells) more efficiently catalyzed the conversion of FT to 5-fluorouracil (5-FU) (2.6-fold) and the 7-hydroxylation of coumarin (7.9-fold) than cells transfected with a null construct (DLD-1 / null cells). These results indicated that the expressed CYP2A6 was functionally active. The extent of growth inhibition of the DLD-1 / CYP2A6 cells by FT was greater than that of DLD-1 / null cells; the difference between the DLD-1 / CYP2A6 and DLD-1 / null cells was statistically significant at the concentrations of 250, 500 and 1000 mM. 5-FU, an active metabolite of FT, inhibited the growth of both types of cells to the same extent. Thus, intracellular expression of CYP2A6 can sensitize cells to FT.     

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.     

干预自噬影响DPD的表达促进5-FU抗结直肠癌疗效的实验研究

背景与目的：结直肠癌作为常见的消化系统恶性肿瘤之一,其化疗与耐药一直以来备受关注,5-氟尿嘧啶（5-fluorouracil,5-FU）是结直肠癌的一线化疗药物,其疗效常因耐药或不良反应而受到影响。二氢嘧啶脱氢酶（dihydropyrimidine dehydrogenase,DPD）是5-FU代谢的关键限速酶,其表达或降解可能成为影响5-FU疗效的因素。自噬是细胞内蛋白质代谢的重要途径,其在化疗诱导的细胞死亡或增殖抑制中的作用仍存在争议。本研究旨在探讨自噬在结直肠癌化疗过程中的作用以及干预自噬影响5-FU耐药的机制。方法：体外细胞培养人结肠癌HCT-8、COLO205、LOVO和SW480细胞系,观察不同细胞系中DPD的表达对5-FU敏感性的影响,通过药物敏感性实验筛选出5-FU敏感细胞,检测5-FU敏感细胞株及其相对应的耐药细胞株中DPD的表达、自噬水平及自噬关键因子微管相关蛋白轻链3（light chain 3,LC3）、P62的表达,并通过诱导/抑制细胞自噬,观察调控自噬状态对DPD的表达变化以及肿瘤细胞生物学行为和化疗抵抗能力的影响,通过UbiBrowser数据库筛选及免疫共沉淀实验（co-inmunoprecipitation,Co-IP）实验验证DPD降解过程中的E3连接酶,探讨自噬降解DPD逆转5-FU耐药的分子机制。结果：DPD低表达的细胞系对5-FU的敏感性更强,其中COLO205细胞系在4种细胞中DPD表达量最高,并对5-FU的耐药性最强,而HCT-8细胞DPD表达最低并对5-FU最为敏感。与HCT-8细胞相比,HCT-8/FU耐药细胞表达较高水平的DPD,以及较低的基础自噬水平;雷帕霉素（rapamycin,RAPA）介导的自噬激活增强了细胞的自噬水平,降低了DPD表达,同时降低了细胞增殖、侵袭和5-FU耐药性;3甲基腺嘌呤（3-methyladenine,3-MA）及羟氯喹（hydroxychloroquine,HCQ）介导的自噬抑制减弱了细胞的自噬水平,增加了DPD表达,增强了5-FU耐药性。5-FU与自噬激活剂联合应用对细胞的抑效果要远远强于单用5-FU及与自噬抑制剂联合应用的效果;DPD的降解需要完整的自噬流的参与,其中自噬溶酶体的形成是DPD降解的关键步骤之一,而单一的自噬体无法对DPD进行降解。E3连接酶NEDD4在HCT-8/5-FU细胞中与DPD和P62蛋白免疫共沉淀,在DPD的自噬降解中发挥作用。结论：自噬参与DPD降解影响结直肠癌细胞对化疗药物的敏感性,激活自噬可促进DPD降解和抑制5-FU的分解代谢,可能成为逆转结直肠癌5-FU耐药的新途径。