5-Fluorouracil modulation of radiosensitivity in cultured human carcinoma cells

We evaluated conventional pulse exposure versus continuous exposure models of 5-fluorouracil (5-FU) radiosensitization in HT-29 (human colon adenocarcinoma) and DU-145 (human prostate cancer adenocarcinoma) cell lines. Cell survival following treatment with drug and/or radiation was determined by colony formation assays. Radiation was delivered either by itself, approximately midway through a 1-hr exposure to 5-FU (10 micrograms/ml), or at various times following initiation of exposure to 5-FU (0.5 microgram/ml) present throughout the entire period of incubation. Drug concentrations were selected to approximate those achieved in vivo in humans. HT-29 cells showed a plating efficiency of 87% and similar cytotoxicity (survival reduced to 0.57-0.71) for all 5-FU conditions. The Do's of the radiation survival curves were not different for 1 hr of 5-FU exposure versus radiation alone. However, continuous exposure conditions demonstrated statistically significantly different Do's from radiation alone and pulse 5-FU exposure. DU-145 cells displayed a plating efficiency of 17% and cytotoxicities of 0.10-0.91 for the 5-FU conditions. DU-145 cells showed different radiation 5-FU interactions: 5-FU produced statistically significant changes in Do well as the differences between cell lines insofar as their radiosensitization by 5-FU underscore the caution required in extrapolating these radiobiologic models to the clinical setting.     

5-Fluorouracil incorporation in DNA of human breast carcinoma cells

We have demonstrated previously the presence of 5-fluorouracil (FUra) residues in L1210 DNA. These findings have been extended to the MCF-7 human breast carcinoma cell line. Cesium sulfate gradient centrifugation has been used to separate the MCF-7 RNA and DNA fractions. Alkali and RNase digests have also been used to remove any possible RNA contaminating the DNA fraction. The purified DNA has been analyzed by high-pressure liquid chromatography following digestion to nucleotides and nucleosides. The results demonstrate that FUra residues are detectable in the DNA of these human breast carcinoma cells following exposure to either FUra of 5-fluorodeoxyuridine. Further, the extend of FUra incorporation in both MCF-7 RNA and DNA is similar with either fluorinated pyrimidine. We also demonstrate that the FUra incorporation in DNA from this human cell line can be enhanced by concurrent incubation with thymidine.     

RNA-Seq profiling of circular RNAs in human colorectal cancer 5-fluorouracil resistance and potential biomarkers

BACKGROUNDColorectal cancer (CRC) is a commonly diagnosed cancer of the digestive system worldwide. Although chemotherapeutic agents and targeted therapeutic drugs are currently available for CRC treatment, drug resistance is a problem that cannot be ignored and needs to be solved. AIMTo explore the relationship between circular RNA (circRNA) and CRC drug resistance. circRNA plays a key role in the occurrence and development of cancers, but its function in the process of drug resistance has not been widely revealed. METHODSTo explore the role of circRNA in 5-fluorouracil (5-Fu) resistance, we performed the circRNA expression profile in two CRC cell lines and their homologous 5-Fu resistant cells by high-throughput sequencing.RESULTSWe validated the differentially expressed circRNAs in other two paired CRC cells, confirmed that circ_0002813 and circ_0000236 could have a potential competitive endogenous RNA mechanism and be involved in the formation of 5-Fu resistance. And we combined the sequencing results of mRNA to construct the regulatory network of circRNA-miRNA-mRNA. CONCLUSIONOur study revealed that circ_0002813 and circ_0000236 may as the biomarkers to predict the occurrence of 5-Fu resistance in CRC.     

5-Fluorouracil up-regulates interferon pathway gene expression in esophageal cancer cells

5-Fluorouracil (FU) is a chemotherapeutic agent commonly used against esophageal cancer. Recently, interferons (IFNs) have been administered together with cytotoxic chemotherapy to patients with this cancer, although the mechanisms of synergy are unknown. We reconsidered the mechanisms for the effects of 5-FU in this context, aiming to refine combination therapy. After three cell lines (T.T, TE-2, and TE-6), derived from human esophageal squamous cell carcinoma (SCC), were exposed to 5-FU, the expression profiles were analyzed using high-density oligonucleotide microarrays representing about 12,000 genes. Among them, three IFN-related genes, an IFN receptor gene (IFNAR2) and two IFN-stimulated genes (ISG15K, ISG-54K), that were up-regulated following addition of 5-FU, were investigated. Up-regulation was confirmed by RT-PCR. Based on these results, the antitumor effects of exposure to 5-FU simultaneously with IFN-alpha, -beta and -gamma were investigated. The growth of esophageal SCC cells with 5-FU was suppressed synergistically or semi-additively by IFN-alpha and -beta, but not by IFN-gamma. These findings indicated that 5-FU stimulated IFN pathways in esophageal SCC cells following up-regulation of the IFN type I receptor. A combination of 5-FU and an IFN, therefore, may be particularly efficacious in esophageal cancer.     

Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis-regulating genes

OBJECTIVES: Resistance to chemotherapy is a major cause of treatment failure and poor prognosis in pancreatic cancer. Inasmuch as most effects of chemotherapeutic agents are mediated via the activation of apoptosis, the cytotoxic effects of gemcitabine and 5-fluorouracil (FU) in correlation with apoptosis-regulating genes in pancreatic cancer cell lines were analyzed. METHODS: The cytotoxic effects of 5-FU and gemcitabine in AsPC-1, Capan-1, Mia-PaCa-2 and T3M4 pancreatic cancer cell lines were assessed by growth assays, and mRNA expression levels of pro-apoptotic and anti-apoptotic genes of the Bcl-2 family were analyzed by RNAse protection assays. RESULTS: Pancreatic cancer cells displayed a wide range of responses towards 5-FU (IC(50) 0.22-4.63 microM) and gemcitabine (11.51-42.2 nM). After repeated treatment with 5-FU, the IC(50) values in Capan-1 and T3M4 cells increased 2.1- and 1.8-fold, respectively, compared to their parental cells. Following recurrent treatment with gemcitabine, the IC(50) values in Capan-1 cells increased significantly (1.5-fold, p < 0.01). RNase protection assay showed a negative correlation between bcl-x(L) and mcl-1 mRNA expression levels and the sensitivity to 5-FU and gemcitabine after 5-FU and gemcitabine treatment. The bax/bcl-2 ratio maintained relatively stable following 5-FU/gemcitabine treatment and reflected the chemotherapeutic sensitivity of these cell lines. CONCLUSIONS: These findings reveal that pancreatic cancer cell lines are generally resistant to 5-FU and are more sensitive towards gemcitabine. The bax/bcl-2 ratio is predictive of chemotherapy sensitivity, whereas bcl-x(L) and mcl-1 mRNA levels following repeated exposure to 5-FU or gemcitabine are associated with resistance to these drugs. These findings suggest that the activation of anti-apoptotic genes after repeated drug exposure contributes to chemoresistance of pancreatic cancer cells and that blockage of anti-apoptotic genes might enhance chemosensitivity in pancreatic cancer.     

5-Fluorouracil plus 5-methyltetrahydrofolate in advanced pancreatic cancer

A total of 20 patients with advanced pancreatic adenocarcinoma were enrolled in a phase II trial testing the activity of 5-fluorouracil given at 370 mg/m² as a rapid i. v. bolus for 5 consecutive days, preceded by a rapid i. v. bolus of 200 mg/m² 5-methyltetrahydrofolic acid. The treatment was repeated every 4 weeks. The median age of the patients was 68 years and their median Eastern Cooperative Oncology Group (ECOG) performance status was 1. There were 7 patients with locally advanced disease and 13 with distant metastases (median, 2 sites). A median of 3 monthly cycles of treatment (range, 1–7) were given, with a corresponding dose intensity of 396 mg/m² per week (86% of that planned). No complete response, 1 partial response, and 8 cases of disease stabilization were obtained. In general the regimen was well tolerated, with only 2 patients suffering from grade 3 stomatitis or diarrhea; the most common toxicity was nausea, which was experienced by almost 50% of the patients. The combination of 5-methyltetrahydrofolate plus 5-fluorouracil appears as little effective in this disease as 5-fluorouracil plus 5-formyltetrahydrofolate (leucovorin). It is suggested that bolus 5-fluorouracil is so inactive as an effector agent against pancreatic cancer that its biochemical modulation with exogenous high-dose reduced folates cannot improve the therapeutic outcome produced by the fluoropyrimidine in these patients.Abbreviations FUra 5-fluorouracil - LV 5-formyltetrahydrofolic acid - ME-THF 5-methyltetrahydrofolic acid Supported by CNR grant 92.01289.PF70, 92.02271.PF39 and AIRC 1994. A Guglielmi is a fellow of the Associazione Italiana Ricerca Cancro     

5-Fluorouracil and mitomycin C in advanced breast cancer

Fifty-seven patients with advanced breast cancer were treated with a combination of 5-fluorouracil and mitomycin C (FuMi). Fifty-three were previously treated with hormones and/or cytotoxic drugs. Complete and partial remissions (CR, PR) were seen in 16%, stationary disease in 46%, and progressive disease in 38%. The mean time to remission was 4.5 (CR) to 4.0 (PR) months. The toxicity was mild and consisted mainly of thrombocytopenia. The FuMi-regimen may be a useful alternative in advanced breast cancer, at least in progression after previous systemic therapy, and if a moderate toxicity is considered important.     

Thapsigargin resistance in human prostate cancer cells

BACKGROUND: Thapsigargin (TG) is a potent inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). TG-based prodrugs are being developed for the treatment of prostate cancer (PC). To develop optimal TG-based therapeutics it is important to understand the mechanisms of resistance to TG that may potentially occur in cancer cells. METHODS: DU145/TG and PC3/TG cells were derived from human PC DU145 and PC3 cells, respectively, by incremental exposure to TG. Growth assays, Western blot analyses, cDNA microarrays, semiquantitative and real-time polymerase chain reaction (PCR), Northern blot analyses, and immunohistochemistry were used to study these cells. RESULTS: DU145/TG cells are 1100-fold and PC3/TG cells are 1350-fold resistant to TG. Although expression of both SERCA and p-glycoprotein can mediate TG resistance in hamster cells, neither is modulated in DU145/TG cells. In contrast, in PC3/TG cells, SERCA, and not p-glycoprotein, is significantly overexpressed but cannot by itself account for the 1350-fold resistance to TG in these cells. Several genes not previously identified to be altered by TG selection are modulated in DU145/TG and PC3/TG cells. Furthermore, the spectrum of genes modulated in DU145/TG cells are distinct from that in PC3/TG cells, even though both cells are of prostate origin and share the same TG-resistant phenotype. CONCLUSIONS: PC cells can adapt to SERCA inhibition by TG. However, they demonstrate cell type-specific plasticity with respect to gene expression upon TG selection. Further, previously not described mechanisms of resistance appear to be recruited in the TG-resistant PC cells, which provide a novel model to study mechanisms of resistance and adaptation in PC on TG-mediated dysregulation of Ca2+ homeostasis.     

Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells

For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.     

5-Fluorouracil and cisplatin in the treatment of advanced oral cancer

The benefit of the effect of chemotherapy in patients with advanced head and neck squamous cell tumors have been demonstrated by recent meta-analyses of randomized studies. However, the role of chemotherapy-especially in advanced oral cancer-is not fully clear, because of the very small amount of phase II literature available. From January 1994 to December 2000, a total of 44 pts aged 33-75 years (mean age 60 years) with advanced and histologically proved squamous cell carcinoma's of the oral cavity received at least one chemotherapy course. Seven patients had stage III and 37 stage IV disease. The chemotherapy was the initial therapy in a group of 21 patients. In a second group of 23 patients the chemotherapy was delivered after relapse of their disease. The pre-chemotherapy treatment of the second group was radiotherapy in 11, surgery in 4, combination of radiotherapy and surgery in 8 patients. The chemotherapy regimen consisted of cisplatin 100 mg/m(2) in 3-h infusion, day 1 and 5-FU 1000 mg/m(2) in 24-h infusion, days 1-5. Treatment was repeated every 21 days. A total of 154 treatment courses (3.5 per patient, ranged 1-10) were administered. Myelotoxicity, nausea and vomiting were the major treatment complications. The overall response rate to the induction chemotherapy was 52.3%, with 19% complete (CR), and 33.3% partial response's (PR) and to the chemotherapy for recurrent/metastatic disease 30.4% with 8.7% CR, and 21.7% PR. No difference was found in the median survival of the two subgroups (12 months). The median survival of the responders was 15 months (95% CI 11.3-18.7 months), and of the non-responders 9 months (95% CI 5.6-12.4 months) (P = 0.0067). Chemotherapy with cisplatin and 5-FU combination is effective in pts with advanced squamous cell oral cancer and appears to improve the survival of patients who have a good response.     

乳腺癌干细胞抵抗放射治疗及化疗的调控机制

据中国城市癌症报告统计,乳腺癌是中国城市女性发病率最高的癌症。尽管乳腺癌早期原位切除及放射治疗和化疗(简称放化疗)后,患者的5年和10年生存率较好,但依旧有小部分癌细胞逃逸、潜伏和扩散,导致乳腺癌复发和转移。未治愈乳腺癌中,约70%发生肺转移,60%发生骨转移和肝转移,15%发生脑转移。肿瘤干细胞是肿瘤组织中存在的少量具有干细胞特征的细胞亚群,有极强的再生肿瘤能力以及天然放化疗抵抗能力,被视作乳腺癌复发、转移的主要原因。靶向肿瘤干细胞的研究,为人类彻底攻克恶性肿瘤带来了希望。乳腺癌组织中具备乙醛脱氢酶(＋)、CD44^＋ CD24^－Lin^－等特征的细胞亚群已经被证实是乳腺肿瘤干细胞。笔者总结了乳腺肿瘤干细胞的鉴定、起源、特征、调控机制、放化疗不敏感的原因及临床靶向治疗的研究进展,供同行参考。     

5-氟尿嘧啶术前化疗对胃癌细胞凋亡和增殖的影响

目的:观察应用5-氟尿嘧啶术前化疗对胃癌细胞凋亡和增殖的影响。方法:30例进展期胃癌患者术前应用5-氟尿嘧啶,周围静脉给药,750mg/d,连用5d后手术。对比分析术前胃镜活检及术后切取肿瘤组织细胞凋亡和增殖情况,分别应用TUNEL标记和免疫组化方法。结果:化疗后胃癌细胞凋亡总指数为(10·1±5·3)%,较化疗前凋亡总指数(6·9±3·0)%显著增高,P=0·026。高中分化腺癌组、肿瘤直径<4cm组、未浸及浆膜层组化疗前后凋亡指数差异明显。化疗后增殖总指数为(48·7±13·4)%,较化疗前增殖总指数(58·4±14·8)%显著降低,P=0·0035。化疗前后各组均差异有统计学意义。结论:5-氟尿嘧啶术前静脉化疗可诱导胃癌细胞凋亡,并有效抑制胃癌细胞增殖。5-氟尿嘧啶对分化程度高的胃癌诱导凋亡作用明显。     

5-Fluorouracil rechallenge by protracted infusion in refractory breast cancer

The authors assessed the value of protracted low-dose 5-fluorouracil (5-FU) infusion (250 mg/m2/day) in refractory breast cancer. Thirty-six patients with prior 5-FU exposure who were not eligible for other investigational therapeutic measures mainly due to extensive prior chemotherapy (median, four regimens) and poor performance status (median of 3, Zubrod scale) were enrolled. Thirty-four patients were evaluable for toxicity and 32 for response. Concurrent radiotherapy was given to 17 patients. The median duration of 5-FU treatment was 65 days (range, 19-508+) delivering a median cumulative 5-FU dose of 13.75 g/m2 (4.75-144.5+). The median number of interruptions of infusion was 1 (0-16) for a median of 7 days (1-25). One patient achieved complete remission, and four a partial response, for a median of 129 days (101-412) and a response rate of 16%. Five additional patients had a minor objective response (16%), whereas 15 (48%) had no change, and seven progressed (22%). The main toxicities seen were oral mucositis in 13 patients, hand-foot syndrome in six, Coombs'-positive hemolytic anemia in two, and progressive liver dysfunction in two patients who also had cirrhotic changes on computed tomography. Myelosuppression was uncommon and transient. Elective temporary interruptions of the 5-FU infusion diminished the incidence of mucocutaneous toxicity. Hemoglobin indices consistently displayed macrocytic features which did not readily revert to baseline after discontinuation of 5-FU infusion. Despite the adverse characteristics of the treated population, 5-FU infusion demonstrated moderate activity in refractory breast cancer with clinical resistance to prior 5-FU-containing regimens. Intermittent scheduled interruptions of infusion rendered this method of 5-FU delivery nontoxic.     

Mechanistic analysis of the antitumor efficacy of human natural killer cells against breast cancer cells

We investigated the role of human natural killer (NK) cells in the peripheral blood (PB) and liver in controlling breast cancer. The proportion of NK cells among liver mononuclear cells was significantly higher than among PB mononuclear cells. Liver NK cells inductively expressed higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) than PB NK cells in response to interleukin-2 (IL-2). Liver NK cells displayed higher cytotoxicity against various breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, and MCF-7) after IL-2 stimulation than did PB NK cells. Anti-HER2 monoclonal antibody (mAb) promoted the cytotoxicity of both the types of NK cells toward HER2-expressing cell lines. All breast cancer cell lines highly expressed death-inducing TRAIL receptors, death receptor 4, but did not express death-inhibitory receptors (DcR1 and DcR2). Both PB and liver NK cell-induced cytotoxicity was inhibited partially by anti-TRAIL mAb and more profoundly by the combination of anti-TRAIL mAb and concanamycin A, indicating that TRAIL and perforin are involved. IL-2-stimulated liver and PB NK cells exhibited upregulated expression of CXCR3, which bind to the chemokines CXCL9, CXCL10, and CXCL11 secreted by breast cancer cells. We also found that IFN-γ promoted the production of CXCL10 from breast cancer cells. The results of this study show that IFN-γ secreted from NK cells likely promotes the production of CXCL10 from breast cancer cells, which in turn accelerates the migration of CXCR3-expressing NK cells into the tumor site. These findings suggest the possibility of a therapeutic approach by either activation of endogenous PB and liver NK cells or adoptive transfer of in vitro-activated autologous NK cells.     

5-Fluorouracil resistant colon cancer cells are addicted to OXPHOS to survive and enhance stem-like traits

Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.     

Prevention of peritoneal metastasis of human gastric cancer cells in nude mice by S-1, a novel oral derivative of 5-Fluorouracil

OBJECTIVE: Recent clinical trials have suggested that oral administration of a new anti-cancer agent, S-1, seems a promising therapy for advanced gastric cancer. In this study, we assessed the efficacy of S-1 against peritoneal dissemination of gastric cancer in a newly developed animal model and investigated the efficacy of S-1 from a pharmacokinetic angle. METHODS: Human gastric cancer cells (MKN-45) were injected into the peritoneal cavity of nude mice. The cancer cells were transduced using an enhanced green fluorescent protein (EGFP)-expressing plasmid vector, enabling micrometastatic foci to be accurately assessed with a high level of detection sensitivity. To investigate pharmacokinetics, the concentration of 5-FU was determined in tumor, peritoneum and plasma. RESULTS: Fourteen and 21 days after intraperitoneal injection, a significant difference in the number of fluorescent foci was observed between the control group and the S-1 group (p = 0.02 and p = 0.0024, respectively. The therapeutic effect of S-1 was significantly greater than that of 5-FU. Furthermore, S-1 treatment greatly improved the survival time and cachexia. The area under the curve of 5-FU in tumor was higher than in the peritoneum and plasma. CONCLUSION: Oral S-1 is a promising chemotherapy for peritoneal dissemination of gastric cancer.