Circumvention of 5-Fluorouracil Resistance in Human Stomach Cancer Cells by Uracil Phosphoribosyltransferase Gene Transduction

A human stomach cancer cell line with acquired resistance to 5-fluorouracil (5-FU), NUGC-3/5FU/L, has been found to possess reduced ability to convert 5-FU into active metabolites. We attempted in vitro gene therapy for this 5-FU-resistant cell line. NUGC-3 and NUGC-3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase ( UPRT ) gene driven by CAG promoter (CA), AdCA-UPRT, and changes in their 5-FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC-3 and NUGC-3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5-FU nucleotide level in the acid-insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC-3 cells on infection with AdCA-UPRT, and in NUGC-3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth-inhibition concentration (IC50) was 12.7 μmol/liter for NUGC-3 and much higher than 100 μmol/liter for NUGC-3/5FU/L, indicating over 8-fold resistance. NUGC-3/5FU/L transfected with the UPRT gene showed very high sensitivity to 5-FU with an IC₅₀ of 3.2 μmol/liter. The high resistance in this metabolic activation-deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5-FU-anabolizing enzyme.     

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.     

蝙蝠葛碱在人乳腺癌MCF-7细胞对5氟尿嘧啶敏感性的研究

目的 探讨人乳腺癌MCF-7细胞受蝙蝠葛碱(Dauricine,Dau)作用后,对5氟尿嘧啶(5-FU)敏感性的影响。方法 分别以终浓度为2.5 μg/mL的蝙蝠葛碱、50 μg/mL的5-FU、2.5 μg/mL的蝙蝠葛碱和50 μg/mL的5-FU作用于人乳腺癌细胞MCF-7,运用MTT法检测细胞增殖活性,Transwell实验分析细胞的迁移,DAPI染色检测细胞的凋亡,Western blot法检测cyclinD1和Bcl-2蛋白的表达。结果 MTT实验结果显示联合使用阈下浓度的蝙蝠葛碱增强了5-FU对细胞增殖的抑制;Transwell实验表明联合应用阈下浓度的蝙蝠葛碱进一步加剧了5-FU对细胞迁移的抑制;DAPI染色说明联合应用阈下剂量的蝙蝠葛碱加强了5-FU对细胞凋亡的诱导,Western blot实验表明联合应用阈下浓度的蝙蝠葛碱进一步抑制了cyclinD1和Bcl-2蛋白的表达。结论 蝙蝠葛碱可有效增强人乳腺癌MCF-7细胞对5-FU的敏感性。     

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.     

Circumvention of 5-fluorouracil resistance in human stomach cancer cells by uracil phosphoribosyltransferase gene transduction.

A human stomach cancer cell line with acquired resistance to 5-fluorouracil (5-FU), NUGC-3/5FU/ L, has been found to possess reduced ability to convert 5-FU into active metabolites. We attempted in vitro gene therapy for this 5-FU-resistant cell line. NUGC-3 and NUGC-3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA-UPRT, and changes in their 5-FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC-3 and NUGC-3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5-FU nucleotide level in the acid-insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC-3 cells on infection with AdCA-UPRT, and in NUGC-3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth-inhibition concentration (IC50) was 12.7 micromol/liter for NUGC-3 and much higher than 100 micromol/liter for NUGC-3/5FU/L, indicating over 8-fold resistance. NUGC-3/ SFU/L transfected with the UPRT gene showed very high sensitivity to 5-FU with an IC50 of 3.2 micromol/liter. The high resistance in this metabolic activation-deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5-FU-anabolizing enzyme.     

Expression of the bifunctional suicide gene CDUPRT increases radiosensitization and bystander effect of 5-FC in prostate cancer cells

Purpose

To test the hypothesis that, with 5-fluorocytosine (5-FC) treatment, the co-expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) can lead to greater radiosensitization and bystander effect than CD-expression alone.

Methods and materials

R3327-AT cell lines stably expressing CD or CDUPRT were generated. The 5-FC and 5-FU cytotoxicity, and the radiosensitivity with/without 5-FC treatment, of these cells were evaluated under both aerobic and hypoxic conditions. The bystander effect was assessed by apoptosis staining and clonogenic survival. The pharmacokinetics of 5-FU and 5-FC metabolism was monitored in mice bearing CD- or CDUPRT-expressing tumors using ¹⁹F MR spectroscopy (MRS).

Results

CDUPRT-expressing cells were more sensitive to 5-FC and 5-FU than CD-expressing cells. CDUPRT-expression further enhanced the radiosensitizing effect of 5-FC, relative to that achieved by CD-expression alone. A 25-fold lower dose of 5-FC resulted in the same magnitude of radiosensitization in CDUPRT-expressing cells, relative to that in CD-expressing cells. The 5-FC cytotoxicity in co-cultures of parental cells mixed with 10-20% CDUPRT cells was similar to that in 100% CDUPRT cells. ¹⁹F MRS measurements showed that expression of CDUPRT leads to enhanced accumulation of fluorine nucleotide (FNuc), relative to that associated with CD-expression alone.

Conclusion

Our study suggests that CDUPRT/5-FC strategy may be more effective than CD/5-FC, especially when used in combination with radiation.     

Synergistic antitumor effect of 5-fluorouracil in combination with parthenolide in human colorectal cancer

Parthenolide (PT), a NF-κB inhibitor, has recently been demonstrated as a promising anticancer agent that promotes apoptosis of cancer cells. 5-fluorouracil (5-FU) has been a drug of choice for treatment of colorectal cancer (CRC). Unfortunately, many of the therapies that use 5-FU alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigated the antitumor effect of PT combined with 5-FU on a human CRC cell line, SW620. The results demonstrated that combination of PT and 5-FU induced apoptosis which was determined using MTT, cell cycle analysis, annexin-V assay, and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome C release, and activation of caspase 3 and 9. Moreover, intra-peritoneal injection of PT and 5-FU showed significant inhibition of tumor growth in the xenograft model. These results demonstrate that PT exhibits anticancer activity in human colorectal cancer in vitro and in vivo. These findings provide an efficacious strategy to overcome 5-FU resistance in certain CRC.     

Relationships between the expression of thymidylate synthase,dihydropyrimidine dehydrogenase,and orotate phosphoribosyltransferase and cell proliferative activity and 5-fluorouracil sensitivity in colorectal carcinoma

Background: The site of action of the 5-fluorouracil (5-FU) antitumor effect has been explicated in recent years. Many studies have investigated enzymes involved in 5-FU metabolism in attempts to predict this effect, and a correlation of enzyme activity with the 5-FU drug sensitivity test has been reported. The aim of this study was to identify the biochemical response determinants of 5-FU. Additionally, we aimed to clarify the association between cell proliferative activity and the response to 5-FU of colorectal cancer. Methods: Our research subjects were 54 patients with colorectal carcinoma who had undergone operations between August 1999 and July 2001 in our department. Assays of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) activities in colorectal carcinoma tissue and assays of 5-FU sensitivity by the collagen gel droplet embedded culture drug sensitivity test (CD-DST) were conducted to investigate the relationships between each enzyme activity and 5-FU sensitivity. In addition, the proliferative activity of cancer cells was evaluated with Ki-67 antibody, and the relationship of this activity to each enzyme activity and 5-FU sensitivity were investigated. Results: 5-FU sensitivity was high in the low-TS-activity group and in the high-OPRT-activity group. Cancers with high cell proliferative activity showed good sensitivity to 5-FU, and TS and OPRT activities were high in such cancers. Conclusion: The results suggest that OPRT activity can predict sensitivity to 5-FU, and high OPRT activity may cause good 5-FU sensitivity in cancers with high cell proliferative activity. Received: April 23, 2002 / Accepted: January 20, 2003 Correspondence to:R. Fujii     

mdr1启动子调控CD::UPP基因对紫杉醇耐药卵巢癌细胞的杀伤作用

目的探讨腺病毒介导的mdr1启动子调控胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶(CDUPP)融合基因联合5-氟胞嘧啶(5-FC)对紫杉醇耐药卵巢癌细胞的特异性杀伤作用.方法扩增、纯化含有mdr1-CDUPP基因的重组腺病毒,转染人卵巢癌紫杉醇耐药细胞株A2780/Taxol和亲本细胞株A2780,RT-PCR检测mdr1和CDUPP基因的表达水平;之后加入5-FC,MTT法检测细胞抑制情况及旁观者效应,并观察腺病毒转染后裸鼠移植瘤的生长情况.结果mdr1和CDUPP基因在A2780/Taxol细胞中可稳定表达,转染后A2780/Taxol组的细胞生长明显低于A2780组;转基因的A2780/Taxol细胞联合5-FC后可通过旁观者效应杀伤周围未转基因的耐药细胞;耐药组移植瘤生长明显受到抑制,肿瘤体积为(569.10±187.93)mm3,对照组肿瘤体积为(2 111.98±230.82)mm3,差异有统计学意义(P＜0.01).结论mdr1启动子可调控CDUPP基因特异性表达并特异性杀伤紫杉醇耐药卵巢癌细胞.     

In-vitro effect of a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) on human gastric cancer cell lines: timing of cisplatin treatment

Abstract. Background: To date, we have few effective chemotherapeutic agents against advanced and recurrent gastric cancer. 5-Fluorouracil (5-FU) and cisplatin (CDDP) are the most widely used drugs, and their combination has demonstrated favorable outcomes. However, the method (especially the timing) of CDDP administration is not well established. Methods: We examined the in-vitro effect of a combination of 5-FU and CDDP on four human gastric cancer cell lines: MKN-1, MKN-28, MKN-45, and MKN-74. The cell lines were exposed to 50% of the inhibitory concentrations of 5-FU and CDDP for 72 h and 8 h, respectively. CDDP was applied before, simultaneously with, and after the start of treatment with 5-FU. Results: When CDDP was applied after 5-FU, the cytotoxic activity against MKN-28, MKN-45, and MKN-74 was significantly potentiated. Against MKN-1, the earlier the initiation of CDDP treatment, the stronger was the cytotoxic effect. Conclusion: Our results suggest that the cytotoxicity of a combination of 5-FU and CDDP against human gastric cancer cells is both cell-line- and schedule-dependent and is especially affected by the timing of the CDDP treatment. Received: August 22, 2001 / Accepted: December 7, 2001     

Gene therapy for intraperitoneally disseminated pancreatic cancers by Escherichia coli uracil phosphoribosiltransferase (UPRT) gene mediated by restricted replication-competent adenoviral vectors

Although patients with unresectable pancreatic tumors have been treated with 5-fluorouracil (5FU)-based combination chemotherapy, the drug resistance of cancer cells presents a crucial therapeutic problem. It was reported that UPRT overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits thymidylate synthetase (TS). We first demonstrated that injecting an E1-deficient adenoviral vector (Adv) expressing UPRT (AxCAUPRT) followed by 5-FU treatment resulted in a volume reduction of xenotransplanted human tumors. In examining the therapeutic effect of AxCAUPRT/5-FU against peritoneal dissemination, we found that non-selective gene transduction of AxCAUPRT caused severe adverse effects arising from the increase of F-dUMP in normal intestine. Because the therapeutic gene delivered by a restricted replication-competent Adv lacking 55 kDa E1B protein (AxE1AdB) is speculated to be expressed selectively in tumors, mice with established tumors were injected with AxE1AdB and E1-deleted Adv expressing the lacZ reporter gene (AxCAlacZ). The expression of the reporter gene (lacZ) was selectively enhanced in disseminated tumors. The therapeutic advantage of restricted replication competent Adv that expresses UPRT (AxE1AdB-UPRT) was evaluated in an intraperitoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the Adv, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. Our results showed that the AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.     

The role of levamisole in the adjuvant treatment of stage III colon cancer patients: a randomized trial of 5-fluorouracil and levamisole versus 5-fluorouracil alone

Adjuvant 5-fluorouracil (5FU) and levamisole (Lev) have been considered standard treatment for stage III colon cancer patients. However, the uncertain contribution of Lev to the efficacy of treatment has led many oncologists to prefer the 5FU/leucovorin combination. To establish the role of Lev, we conducted a randomized trial comparing the 5FU/Lev combination with 5FU alone in patients with Dukes' C colon cancer. Patients with stage III colon cancer were randomized to receive 5FU alone (450 mg/m² IV bolus daily for 5 days and then, beginning at day 28, weekly for 48 weeks) or the same plus Lev (50 mg orally three times/day for 3 days, repeated every 2 weeks for 1 year). From December 1994 to March 1998, 92 patients were assigned to receive 5FU/Lev, and 93 were assigned to receive 5FU alone. Leukopenia and hepatic toxicity were more frequent in patients receiving 5FU/Lev as compared with those receiving 5FU (respectively, p=0.003 and p=0.039), whereas other toxicities were equivalent and mild in both arms. After a median follow-up time of 48 months, 80 patients have had recurrences (40 in each arm) and no advantages in terms of disease-free survival and overall survival could be demonstrated for the combination arm. The addition of Lev to 5FU does not seem to be relevant for the clinical activity of this adjuvant regimen, whereas toxicity related to Lev should be considered when an adjuvant treatment for stage III colon cancer patients is proposed.     

Enhancement of 5-fluorouracil cytotoxicity on human colon cancer cells by retrovirus-mediated interferon-alpha gene transfer.

Gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Several reports documented the clinical effects of interferon-alpha (IFN-alpha) in management of patients with advanced colorectal carcinoma. We report for the first time, the successful transduction of human IFN-alpha gene into colon cancer cells, COLO 201 using a replication-defective retroviral vector. Retrovirus-containing supernatant from PA 317 packaging cells was used to infect colon cancer cells, COLO 201 and NIH 3T3 cells. Transient infection showed that cell proliferation and cell viability were significantly suppressed in colon cancer cells transduced with IFN-alpha gene. Moreover, IFN-alpha-transduced cells acquired less resistance to 5-FU induced apoptosis. These data demonstrate that IFN-alpha gene transfer may have a clinical application and can be combined with chemotherapy for treatment of advanced colorectal cancer.     

Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(Phenylthio)acyclouridine, a uridine phosphorylase inhibitor

Purpose: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host-toxicity and enhancing its chemotherapeutic efficacy against human colon tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. Methods: SCID mice bearing human colon DLD-1 or HCT-15 tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24 and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg) or their combination was administered orally 2 or 4 h after FUra injection. Another four administrations of PTAU + uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. Results: Administration of the maximum tolerated dose (50 mg/kg) of FUra reduced DLD-1 and HCT-15 tumor weights by 48 and 59%, respectively, at day 38 post implantation. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not alleviate FUra host-toxicity as all the mice died. Administration of 120 mg/kg PTAUresulted in partial rescue from this lethal dose of FUra as 63% of mice survived and tumor weights were reduced by approximately 60%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra as 100% of the mice survived and tumor weights were reduced by 81–82%. Delaying the administration of the combination of PTAU plus uridine to 4 h post FUra treatment was less effective in rescuing from FUra toxicity as only 88% of the mice survived and tumor weights were reduced by only 62%. Administration of PTAU alone, under the same conditions, resulted in a 38% survival rate while the tumor weights were reduced by 47%. Treatment with uridine alone did not protect from FUra toxicity at the dose of 200 mg/kg as all mice died. At the higher dose of 300 mg/kg FUra, neither uridine nor PTAU alone, administered 2 h following the treatment with FUra, had any rescuing effect. On the other hand, the use of the PTAU plus uridine combination reduced the tumor weight by 79%, although this reduction in the tumor weight was accompanied by 37% mortality. There was no significant difference between DLD-1 and HCT-15 in their response to the different regimens employed in this study despite the fact that the tumors have different levels of UrdPase. Conclusions: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host-toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combinationin raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al., Cancer Chemo Pharmacol 55:541–551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the large doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side-effects associated with such doses of uridine. This combination may also allow for the escalation of FUra doses for better chemotherapeutic efficacy against human colon carcinoma while avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.This paper is dedicated to the memory of Daniel S. Martin, a colleague and a dear friend. Daniel S. Martin pioneered the use of combination chemotherapy against solid tumors.     

胞嘧啶脱氨酶基因联合5-Fc治疗结肠癌的实验研究

目的研究胞嘧啶脱氨酶基因(CD)联合前体药物5-氟胞嘧啶(5-Fc)对结肠癌细胞的生长抑制作用。方法应用逆转录病毒介导的方法,将逆转录病毒载体pLXSN中含有大肠杆菌CD基因的重组质粒pCD2转染到病毒包装细胞PA317后,感染人结肠癌细胞株SW1116。对转基因的细胞进行RT-PCR检测和体内外药物敏感实验。结果获得了稳定表达EC—CD基因的结肠癌细胞株SWCD2,与野生型SW1116细胞相比,SWCD2细胞对基本无毒性的原药5-Fc的敏感性增加,50％细胞生长抑制率(IC50)浓度由野生型SW1116细胞的16000μmol／L降低到SWCD2的66μmol／L。经5-Fc治疗后,SWCD2细胞的裸鼠移植瘤生长明显缩小。结论CD基因联合5-Fc对人结肠癌细胞具有实验性基因治疗作用。     

Notoginseng enhances anti-cancer effect of 5-fluorouracil on human colorectal cancer cells

Purpose Panax notoginseng is a commonly used Chinese herb. Although a few studies have found that notoginseng shows anti-tumor effects, the effect of this herb on colorectal cancer cells has not been investigated. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of colorectal cancer that interferes with the growth of cancer cells. However, this compound has serious side effects at high doses. In this study, using HCT-116 human colorectal cancer cell line, we investigated the possible synergistic anti-cancer effects between notoginseng flower extract (NGF) and 5-FU on colon cancer cells. Methods The anti-proliferation activity of these modes of treatment was evaluated by MTS cell proliferation assay. Apoptotic effects were analyzed by using Hoechst 33258 staining and Annexin-V/PI staining assays. The anti-proliferation effects of four major single compounds from NGF, ginsenosides Rb1, Rb3, Rc and Rg3 were also analyzed. Results Both 5-FU and NGF inhibited proliferation of HCT-116 cells. With increasing doses of 5-FU, the anti-proliferation effect was slowly increased. The combined usage of 5-FU 5 μM and NGF 0.25 mg/ml, significantly increased the anti-proliferation effect (59.4 ± 3.3%) compared with using the two medicines separately (5-FU 5 μM, 31.1 ± 0.4%; NGF 0.25 mg/ml, 25.3 ± 3.6%). Apoptotic analysis showed that at this concentration, 5-FU did not exert an apoptotic effect, while apoptotic cells induced by NGF were observed, suggesting that the anti-proliferation target(s) of NGF may be different from that of 5-FU, which is known to inhibit thymidilate synthase. Conclusions This study demonstrates that NGF can enhance the anti-proliferation effect of 5-FU on HCT-116 human colorectal cancer cells and may decrease the dosage of 5-FU needed for colorectal cancer treatment.     

Surviving cells after treatment with gemcitabine or 5-fluorouracil for the study of de novo resistance of pancreatic cancer

One of the hallmarks of pancreatic cancer is its inherent insensitivity to chemotherapy. This study was undertaken to develop a cell model for the study of de novo resistance of pancreatic cancer. The surviving pancreatic cancer cells after a 3-day exposure to gemcitabine or 5-fluorouracil followed by another 7-day recovery were potentially drug-resistant. They had similar morphology and comparable growth and tumorigenic potentials to their untreated parental cells. Repeated subculture affected the cell-cycle profile and growth characteristics of the surviving cells. Our data suggest that surviving pancreatic cancer cells after drug treatment are a useful model for exploring intrinsic resistance.     

DUOX2对结直肠癌细胞氟尿嘧啶敏感性的影响

目的：探讨双氧化酶 2（dual oxidase 2,DUOX2）对结直肠癌（colorectal cancer,CRC）细胞5-氟尿嘧啶（5-fluorouracil,5-FU）药物敏感性的影响。方法：选用CRC细胞系DLD-1、SW480、HCT116、SW620与正常肠上皮细胞株NCM460,用qPCR法检测细胞中DUOX2的表达水平。利用慢病毒感染技术,稳定敲降HT-29与HCT116细胞中DUOX2表达,qPCR法和WB法检测敲降效率。用不同浓度（0、5、10、20、40、80、120 μg/ml）5-FU处理sh-Control组与sh-DUOX2组细胞 ,用 CCK-8 法、流式细胞术分别检测5-FU对细胞增殖、细胞凋亡和细胞周期的影响。构建裸鼠 HT29 细胞移植瘤模型,观察DUOX2基因对5-FU疗效的影响。结果：DUOX2 mRNA 在 CRC 细胞中的表达水平显著高于 NCM460 细胞（P<0.05 或 P<0.01）。 敲 降 DUOX2 基 因后,与sh-Control 组 相 比 ,sh-DUOX2 组 HT29和HCT116细胞中DUOX2 mRNA和蛋白表达水平明显下降（均P<0.01）;细胞对5-FU的敏感性增强,细胞凋亡率明显升高（均 P<0.01）,G0/G1 期比例显著升高、G2 期与 S 期比例显著下降（均P<0.01）。未经5-FU治疗的sh-Control组与sh-DUOX2组裸鼠移植瘤体积及质量比较差异均无统计学意义（均P>0.05）,经5-FU治疗的sh-DUOX2+5-FU组移植瘤体积及质量明显低于sh-Control+5-FU组（均P<0.01）。结论：敲降DUOX2基因可显著增强CRC细胞对5-FU的敏感性。     

Pilot study of continuous low-dose 5-fluorouracil and cisplatin (FP regimen) for the treatment of metastatic breast cancer

Background. The effect of low-dose 5-fluorouracil (FU) and cisplatin therapy (FP regimen) against metastatic breast cancer was investigated. Methods. A pilot study of the FP regimen was performed in 11 patients with metastatic breast carcinoma who had previously received chemotherapy, including adriamycin, and/or hormonal therapy. Their median age was 56 years (range, 48–72 years). Visceral metastases were present in all patients. FU, at a dose of 170 mg/m² per day, was administered for 28 days by continuous intravenous infusion. Cisplatin (7 mg/m² per day) was given intravenously on days 1–5, 8–12, 15–19, and 22–26. After a 2-week interval, this treatment was repeated. Results. Of the 11 patients assessable for tumor response to the FP regimen, 4 patients (36%; 95% confidence intervals [CI], 8%–64%) achieved an objective response, with 1 showing a complete response and 3 showing a partial response. Median time to progression was 6.5 months (range, 4–25 months). The median survival time from the initiation of the FP regimen was 11 months (range, 3–25 months). Gastrointestinal and hematologic toxicity was mild. Conclusion. The FP regimen is promising for and has acceptable tolerance in patients with metastatic breast carcinoma refractory to previous anthracycline-containing chemotherapy. Received: July 27, 1998 / Accepted: September 22, 1999     

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells

Purpose  Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Methods  Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Results  Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20–100 μM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 μM) for 24 and 48 h. Panaxadiol (25 μM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 μM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Conclusions  Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.