An antitumor effect of oncolytic adenovirus capable of selectively replicating in CEA-expressing cancer cells and its enhancement by 5-FU

In recent years, many have focused on the use of oncolytic virus capable of lysing cancer cells by a cancer-selective replication for a new treatment modality of cancer. In the present study, we used oncolytic adenovirus, AdCEAp/Rep, genetically modified to selectively replicate in CEA-expressing cancer cells, and investigated whether AdCEAp/Rep could induce selective cytotoxicity to CEA expressing cancer cells, and where the AdCEAp/Rep induced cytotoxic effect could be enhanced by 5 FU in using human gastric cancer cell lines, MKN45 (CEA-positive) and MKN74 (CEA-negative). The results showed that AdCEAp/Rep showed cytotoxicity against MKN45 in a dose-dependent manner, while it did not against MKN74. Furthermore, 5-FU remarkably enhanced AdCEAp/Rep-induced cytotoxicity against MKN45 by being added 3 days after adenovirus infection. These findings strongly suggest that AdCEAp/Rep may be applicable as a new therapeutic agent for clinical cancers expressing CEA in combination with chemotherapeutic agents such as 5-FU.     

The effects of gamma-interferon combined with 5-fluorouracil or 5-fluoro-2'-deoxyuridine on proliferation and antigen expression in a panel of human colorectal cancer cell lines.

Gamma-Interferon (IFN-gamma) and the antimetabolites 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FUdR) were investigated as individual agents and in combination for their in vitro antiproliferative capacity and for their effect on the expression of HLA class-I antigen, carcinoembryonic antigen (CEA) and the intracellular tumor-associated antigen CTA-I in 7 human colorectal cancer cell lines: WiDr, HT29, Colo 205, SW116, LS174T, SW1398, and LoVo. Growth inhibition by IFN-gamma at clinically relevant concentrations (50-100 U/ml) was found in 4/7 cell lines. The cell lines were equally sensitive to 5-FU (IC50 in a range of 2-10 microM), while sensitivity to FUdR varied considerably (IC50 in a range of 0.01-90 microM). When 50 U/ml IFN-gamma were combined with 5-FU or FUdR, the antiproliferative effects were synergistic in those cell lines with sensitivity to IFN-gamma as a single agent, but not in the IFN-gamma-insensitive cell lines. IFN-gamma was able to enhance the expression of HLA Class I and CEA in 4/7 and 3/7 cell lines, respectively, as measured by flow cytometry. CTA-I expression could not be enhanced with IFN-gamma. The expression of the 3 antigens tested was also increased by 5-FU and FUdR. This effect was concentration-dependent in most instances and varied between the individual cell lines. The combination of 50 U/ml IFN-gamma with 25% growth-inhibitory concentration of 5-FU or FUdR for each cell line resulted in an additional increase in antigen expression in 4/7 cell lines. No relation was found between the enhancement of antigen expression and the sensitivity to IFN-gamma or the anti-metabolites. The enhancement in antigen expression also did not show a relationship with changes in cell-cycle distribution upon exposure to IFN-gamma or the anti-metabolites. These results suggest independent mechanisms for the antiproliferative and antigen-enhancing effects of IFN-gamma, 5-FU and FUdR.     

Detection of circulating tumor cells is improved by drug-induced antigen up-regulation: preclinical and clinical studies

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.     

Growth inhibitory effects of pegylated IFN-alpha2b and 5-fluorouracil in combination on renal cell carcinoma cell lines in vitro and in vivo

We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) alone and PEG-IFN-alpha2b plus 5-fluorouracil (5-FU) in vitro on the proliferation of renal cell carcinoma (RCC) cell lines. After the transplantation of RCC cells into nude mice, we administered IFN (PEG-IFN-alpha2b or IFN-alpha2b) alone, 5-FU alone, or IFN (PEG-IFN-alpha2b or IFN-alpha2b) plus 5-FU; and investigated tumor volume, tumor weight, the numbers of apoptotic cells and artery-like blood vessels, relative mRNA expression levels of enzymes which relate to 5-FU metabolism, angiogenesis factor, and type I interferon receptor. RCC cells in vitro were generally and relatively resistant to the anti-proliferative effects of PEG-IFN-alpha2b, but the addition of 5-FU augmented IFN-induced anti-proliferative effects with the induction of apoptosis. PEG-IFN-alpha2b in vivo presented stronger anti-tumor effects than IFN-alpha2b, and its combination with 5-FU augmented the effects. The significant anti-tumor effect of the combination treatment was the increase in apoptotic cell number, but there were no significant differences in the suppression of angiogenesis, expression of IFN receptor, and the actions of metabolic enzymes of 5-FU. In conclusion, PEG-IFN-alpha2b presents stronger anti-tumor effects than non-pegylated IFN, and the effects are augmented in the combination with 5-FU. Our findings suggest the clinical usefulness of PEG-IFN-alpha2b in the treatment of RCC.     

曲妥珠单抗联合氟尿嘧啶或顺铂治疗胃癌的实验研究

目的：探讨曲妥珠单抗（Herceptin,HCP）联合氟尿嘧啶（5-FU）或顺铂（PDD）治疗胃癌的效果及机制。方法：采用流式细胞检查确定胃癌细胞株的HER-2/neu（HER2）表达情况,并测定药物对细胞周期的影响;以MTT法评价药物的细胞毒性作用;以Westernblot法分析药物对HER2-PI3K-AKT信号转导的影响;以RT-PCR分析药物对靶蛋白酶mRNA的影响。结果：HCP单抗可增强化疗药对HER2阳性细胞的毒性作用。先予5-FU后予HCP单抗、先HCP单抗和PDD联合再序贯使用HCP单抗的方案效果最佳,但先予HCP单抗再和5-FU联合可拮抗5-FU的化疗效果。HCP单抗单用或与化疗药联用均可抑制AKT的磷酸化,HCP单抗与PDD同时使用可抑制NF-κB的核移位;HCP单抗不能增强5-FU对胸苷酸合成酶（TS）mRNA表达的抑制,但可使PDD上调的核苷酸切除修复交叉互补基因（ERCC1）mRNA恢复至正常水平;联合方案对细胞周期的改变影响明显。结论：HCP单抗更适于与PDD联合用于HER2阳性胃癌的治疗,HCP单抗与PDD同时使用再序贯使用HCP单抗的给药方法可能是最佳联合模式;而HCP单抗与5-FU联合则要注意正确的用药次序,以免减弱5-FU的化疗效果。     

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.     

Treatment of colon and breast carcinoma cells with 5-fluorouracil enhances expression of carcinoembryonic antigen and susceptibility to HLA-A(*)02.01 restricted,CEA-peptide-specific cytotoxic T cells in vitro

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines.     

The use of carcinoembryonic antigen for identification of human tumor cells in malignant effusions

A specific anti-carcinoembryonic antigen (CEA) antiserum was used to identify CEA-positive tumor cells in peritoneal and pleural effusions obtained from patients with various malignant neoplasia. In 7 out of 10 fluids in which tumor cells were detected by cytological examinations, cytoplasmic CEA-positive cells were also detected by an indirect immunofluorescence test. In addition, out of 11 fluid samples cytologically negative for tumor cells, CEA-positive cells were found in 8 cases. When both staining for cytoplasmic CEA and soluble fluid CEA levels were considered in combination, 81% of the samples were found to be positive by either one or both of these markers, whereas only 54% were positive by using cytological criteria. The data suggest that CEA marker may be used to identify tumor cells and to assess malignancy in pleural and peritoneal effusions in patients with certain types of cancer. The CEA marker was also used for identifying tumor cells grown in tissue cultures and for separating viable CEA-positive and CEA-negative cells from a mixed cell population using the fluorescence-activated cell sorter.     

Anti-tumor effects of various interferons-alpha +5-fluorouracil via downregulation of dihydropyrimidine dehydrogenase in hepatoma cells

BACKGROUND AND AIMS: Systemic chemotherapy has not seen widespread use in the treatment for hepatocellular carcinoma (HCC). Recently, it was reported that combination treatment of 5-fluorouracil (5-FU) and interferon (IFN)-alpha was effective for non-resectable HCC. The effect of 5-FU treatment is influenced by the activities of pyrimidine catabolic enzymes. The aim of this study was to investigate which IFN-alphais most effective in combination therapy via downregulation of dihydropyrimidine dehydrogenase (DPD). METHODS: Cell proliferation assay in human hepatoma cells (HepG 2) was performed using 5-FU and/or various IFNs-alpha (C-IFN, Intron A, OIF). At the same time,DPD mRNA levels were quantified by real-time polymerase chain reaction. RESULTS: 5-FU alone but not all the IFNs showed anti-cancer effects, and the combination of each IFN +5-FU had a greater anti-cancer effect than 5-FU alone. C-IFN +5-FU most effectively prevented cancer cell proliferation (p < 0.05). Also, the combination of C-IFN +5-FU most downregulated DPD mRNA expression. CONCLUSION: The combination of each IFN-alpha +5-FU showed anti-cancer effects for HCC via downregulation of DPD. C-IFN may be most effective in the combination therapy of IFN-alpha +5-FU for HCC.     

Epigenetic regulation of chemosensitivity to 5-fluorouracil and cisplatin by zebularine in oral squamous cell carcinoma

Epigenetic alterations such as histone acetylation and DNA methylation play an important role in the regulation of gene expression for cell cycles and apoptosis that may affect the chemosensitivity of cancers. Previously, we have reported that the combination of suberoylanilide hydroxamic acid (SAHA), a newly developed histone deacetylase inhibitor, with cisplatin (CDDP) possessed synergistic cytotoxicity against human oral squamous cell carcinoma (OSCC) cell line HSC-3. In this study, we used a novel DNA methyltransferase inhibitor, zebularine (Zeb), to investigate the epigenetic influence on the sensitivity of carcinoma cell lines to 5-fluorouracil (5-FU) or CDDP by evaluating apoptotic inducibility. Treatment with CDDP or 5-FU either alone or in combination with Zeb or SAHA continued for 48 or 72 h. In HSC-3 cells, Zeb had chemosensitive efficacy with CDDP, but not 5-FU, whereas SAHA showed efficacy with both CDDP and 5-FU. We showed that Zeb has strong anti-proliferative activity against HSC-3 cells, shown by decreased cellular growth and G2/M cell cycle phase accumulation. Furthermore, DNA methylation could be a regulatory mechanism for dihydropyrimidine dehydrogenase (DPD), known to be a principal factor in 5-FU resistance. CDHP (5-chloro-2,4-dihydroxypyridine), an inhibitor of DPD, had an enhancing effect on the apoptotic ability of 5-FU alone or 5-FU/Zeb combination. In conclusion, the present study suggests that low-dose (IC20) Zeb may sensitize cancer cells to CDDP, which may be an important characteristic for solid cancer treatment, and that DPD and other agents activated by Zeb in cancer cells could be an inhibitory factor in the response to apoptosis induced by 5-FU.     

Immuno-chemotherapy of advanced colorectal cancer with alpha-2a interferon and 5-fluorouracil. Immunopharmacological studies.

Twelve patients with metastatic colorectal cancer received alternating cycles of low immunomodulating doses of alpha-IFN + 5-Fluorouracil (5-FU) or 5-FU alone. Hematological, biochemical and physical evaluation showed that both treatment cycles were well tolerated. However, transient fever and moderate flu-like symptoms were observed following alpha-IFN administration. Treatment with 5-FU alone produced long-lasting inhibition of CD8+ T lymphocytes, but did not depress NK activity (NKA). Combined treatment with alpha-IFN produced a short-term increase of NKA and antagonized the effect of 5-FU on CD8+ cells on day 5 of the cycle. Parallel studies on in vitro models showed antiproliferative effects of 5-FU on PHA-stimulated MNC and confirmed the preferential inhibition of CD8+ cells. Pretreatment with alpha-IFN did not reverse the effect of 5-FU on CD8+ lymphocytes, but partially protected MNC from the toxic effects of the drug. This was presumably due to the cytostatic effects induced by alpha-IFN on MNC before exposure to the cycle-specific antineoplastic agent. This investigation suggests that alpha-IFN could play a positive role in immuno-chemotherapy of colorectal cancer through multiple mechanisms not entirely related to direct antitumor effects of the agent.     

Increase in carcinoembryonic antigen release from cancer cells by combined treatment with retinoic acid and low-temperature hyperthermia

The growth of a human gastric adenocarcinoma cell line, MKN-45, was inhibited and the amount of carcinoembryonic antigen (CEA) in both the culture medium and the cell extract was increased in the presence of retinoic acid at a concentration of 75 (1?5×)-125 μM (1?9×), which did not substantially affect cell survival. Treatment using a combination of retinoic acid (125 μM) and low-temperature hyperthermia (40°C, 30 min) was more effective in increasing CEA compared with retinoic acid alone (extracellular 1?9-2?4×, intracellular 1?5-1?9×). The inhibition of cell growth was reversed after the retinoic acid was removed from the medium. Cells treated with both retinoic acid and (low-temperature) hyperthermia, however, could be induced to release a significant amount of CEA at about 48 h after retinoic acid removal. The induced CEA increase in the cells, but not in the medium, was suppressed by actinomycin D (1 ng/ml) or cyclohexamide (0-2μg/ml). These results suggest that retinoic acid, used alone or in combination with hyperthermia, enhances the production and release of CEA in human gastric cancer cells.     

Increase in carcinoembryonic antigen release from cancer cells by combined treatment with retinoic acid and low-temperature hyperthermia

The growth of a human gastric adenocarcinoma cell line, MKN-45, was inhibited and the amount of carcinoembryonic antigen (CEA) in both the culture medium and the cell extract was increased in the presence of retinoic acid at a concentration of 75 (1.5x)-125 microM (1.9x), which did not substantially affect cell survival. Treatment using a combination of retinoic acid (125 microM) and low-temperature hyperthermia (40 degrees C, 30 min) was more effective in increasing CEA compared with retinoic acid alone (extracellular 1.9-2.4x, intracellular 1.5-1.9x). The inhibition of cell growth was reversed after the retinoic acid was removed from the medium. Cells treated with both retinoic acid and (low-temperature) hyperthermia, however, could be induced to release a significant amount of CEA at about 48 h after retinoic acid removal. The induced CEA increase in the cells, but not in the medium, was suppressed by actinomycin D (1 ng/ml) or cyclohexamide (0.2 microgram/ml). These results suggest that retinoic acid, used alone or in combination with hyperthermia, enhances the production and release of CEA in human gastric cancer cells.     

Chemoimmunosensitization of the T24 human bladder cancer line to Fas-mediated cytotoxicity and apoptosis by cisplatin and 5-fluorouracil.

Previous studies have demonstrated that cisplatin (CDDP) sensitizes bladder cancer cells to Fas-mediated cytotoxicity and that CDDP also enhances the cytotoxic effect of 5-fluorouracil (5-FU). These agents mediate apoptosis and may share common intracellular pathways leading to cell killing. We reasoned that combination treatment with CDDP, 5-FU and anti-Fas monoclonal antibody (mAb) might overcome the drug-resistance. We investigated whether CDDP, 5-FU and anti-Fas mAb synergize in cytotoxicity and apoptosis against the T24 bladder cancer line. Cytotoxicity was monitored by a one day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with CDDP or 5-FU resulted in a synergistic cytotoxic effect. In addition, combination treatment of T24 cells with CDDP, 5-FU and anti-Fas mAb further enhanced the synergistic cytotoxicity achieved by two agents. The synergy achieved in cytotoxicity with CDDP, 5-FU and anti-Fas mAb was also achieved in apoptosis. The current study demonstrates that combination treatment of bladder cancer cells with CDDP, 5-FU and anti-Fas mAb overcomes their resistance. In addition, the sensitization required low concentrations of CDDP and 5-FU, and thus supporting the potential in vivo application of combination of CDDP, 5-FU and immunotherapy in the treatment of drug-resistant bladder cancer.     

Interferon-alpha does not improve the antineoplastic efficacy of high-dose infusional 5-fluorouracil plus folinic acid in advanced colorectal cancer: First results of a randomized multicenter study by the Association of Medical Oncology of the German Cancer Society(AIO)

Background: High-dose 5-FU given weekly as a 24-h infusion incombination with folinic acid (FA) has been associated withlow toxicity and a high response rate. Interferonalpha (IFN)either alone or in combination with FA has also improved treatmentresults by modulating 5-FU activity. We therefore initiateda randomized multicenter trial comparing the ability of FA orEFN to modulate infusional 5-FU. The statistical design usinga sequential analysis allows us to report on the comparisonof 5-FU/FA vs. 5-FU/FA/IFN while randomization of patients into5-FU/FA vs. 5 FU/TFN continues. MethodsChemotherapy-naive patientswith advanced progressive colorectal cancer and measurable metastaticlesions were randomized to receive 5-FU 2600 mg/m² i.v. as a24-h infusion, combined with either FA 500 mg/m² as a 2-h infusion(A), or IFN 3 x10⁶ U s.c. 3 x/week (B), or the combination ofFA plus IFN as in arms A and B (C). Treatment arms were repeatedweekly for 6 weeks followed by a 2-week rest period. These 8-weekcycles were administered until tumor progression. Because ofthe occurrence of 2 toxic deaths among the first 17 patientstreated in arm C, 5-FU was reduced to 2000 mg/m² for all patientsin arm C. Sequential analysis according to Whitehead for objectiveresponse was planned with     

术前中性粒细胞与淋巴细胞比值联合癌胚抗原检测对直肠癌患者预后的评估价值

目的探讨术前中性粒细胞与淋巴细胞比值(NLR)联合癌胚抗原(CEA)对直肠癌预后的评估价值。方法选取268例直肠癌患者,依据术后5年生存情况,分为生存组和死亡组,比较两组患者的临床特征,影响直肠癌患者预后的独立影响因素采用Cox回归分析,采用受试者工作特征(ROC)曲线和曲线下面积(AUC)评估NLR及CEA单独及联合检测对直肠癌预后的评估价值。结果随访结束,268例直肠癌患者中,生存156例,病死112例,直肠癌患者术后5年生存率为58.21%。将生存患者和病死患者分别作为生存组和死亡组,生存组和死亡组直肠癌患者性别、年龄比较,差异均无统计学意义(P﹥0.05);生存组和死亡组直肠癌患者TNM分期、分化程度、肿瘤直径、神经侵犯情况、浸润深度、远处转移情况、淋巴结转移情况、CEA水平及NLR比较,差异均有统计学意义(P﹤0.05)。Cox回归分析结果显示,TNM分期、肿瘤直径、远处转移、CEA水平和NLR是直肠癌患者预后不良的独立危险因素(P﹤0.05)。CEA、NLR及二者联合预测直肠癌预后的AUC分别为0.991、0.923、0.997,CEA和NLR联合检测对直肠癌患者的预后评估具有较高的准确性,二者联合检测的AUC高于单独预测的AUC(P﹤0.05)。结论术前NLR联合CEA检测对直肠癌预后具有较高的预估价值,二者联合能够为评估直肠患者预后提供重要的参考信息。