P53 status plays no role in radiosensitizing effects of SN-38, a camptothecin derivative

Purpose: Topoisomerase inhibitors including camptothecin are being studied as potential radiosensitizers. CPT-11 is a derivative of camptothecin and is clinically available. In this study, we investigated the effects of SN-38 (an active metabolite of CPT-11) on four nonirradiated and irradiated murine fibroblast cell lines with different p53 statuses to clarify the role of p53 in the radiosensitizing activity of SN-38. Materials and methods: Four fibroblast cell lines, MT158, MT158/neo, MT158/wtp53 and MT158/mp53 with the same genetic background but with different p53 statuses, were used. Exponentially growing cells were treated with SN-38 (200 nM) and incubated with the drug for 30 min. Cells were then irradiated (0 to 12 Gy) and further incubated with the drug for 2 h. The cell survival rate was determined using a conventional clonogenic assay. The effects of the treatments on the cell cycle were analyzed with a flow cytometric assay. Apoptosis after these treatments was also detected by an annexin V assay. Results: There were no significant differences in sensitivity to radiation or SN-38 treatment among these cell lines. The combined treatment of irradiation and SN-38 showed supraadditive effects in all four cell lines independent of their p53 status. Transient arrest in G₂ with a decreased percentage of cells in both the S and G₁ phases was observed 8 h after treatment with either SN-38 alone, radiation or their combination, regardless of the p53 status. No significant differences in frequency of apoptosis were observed between treatment and control groups in two cell lines with or without wild-type p53. Conclusion: The combination of irradiation and SN-38 treatment showed supraadditive effects in all four cell lines tested, and the p53 status did not play a role in the combination effect. Received: 19 May 1999 / Accepted: 19 November 1999     

Modulation of glucuronidation of SN-38, the active metabolite of irinotecan, by valproic acid and phenobarbital

 Purpose: Irinotecan (CPT-11) is hydrolyzed to its active metabolite SN-38 which is subsequently conjugated by uridine diphosphate glucuronosyl transferase (UDP-GT) to the glucuronide (SN-38G). Both preclinical and clinical data indicate that conjugation is a primary clearance mechanism for SN-38 with the plasma glucuronide levels being substantially higher than those of SN-38. This investigation was designed to determine the possibility of modulation of glucuronidation of SN-38 and its effect on the disposition of the parent drug and metabolites. Methods: Female Wistar rats were pretreated with 200 mg/kg valproic acid (VPA), an inhibitor of glucuronidation, 5 min prior to the administration of 20 mg/kg irinotecan. The control rats were given 20 mg/kg irinotecan only. To study the effect of inducers of UDP-GT activity, rats were pre- treated with phenobarbital (PB) before irinotecan administration. Results: Pretreatment with VPA caused a 99% inhibition in the formation of SN-38G leading to a 270% increase in the area under plasma concentration-time curve (AUC) of SN-38 compared with the control rats. The irinotecan estimations were unchanged in the two groups. PB pretreatment caused a 1.7-fold increase in the AUC of SN-38G and a concomitant 31% and 59% reduction in the AUCs of SN-38 and irinotecan, respectively. Conclusions: The most plausible explanation for the alterations in SN-38G disposition is inhibition of SN-38 conjugation by VPA and induction of the conjugation by PB. Received: 5 February 1996 / Accepted: 30 July 1996     

Molecular changes to HeLa cells on continuous exposure to SN-38, an active metabolite of irinotecan hydrochloride

It is important to clarify the molecular characteristics of tumor cells showing multidrug resistance (MDR) and to identify the novel targets or biomarkers for chemotherapy. The aim of this study is to establish resistant HeLa sublines through exposure to SN-38, an active metabolite of irinotecan hydrochloride, and to investigate their molecular changes. HeLa cells were exposed to SN-38 at 1, 10, or 100 nM, and resistant clones were isolated and named HeLa/SN1, HeLa/SN10, and HeLa/SN100, respectively. Their cellular changes were examined based on growth inhibition assays, the function of ABCG2/BCRP, and a RT-PCR analysis of MDR-related protein. The sublines showed a decrease in sensitivity to not only SN-38 but also other chemotherapeutic agents as compared with HeLa cells. mRNA and protein levels of ABCG2/BCRP were increased, and the transport activity of ABCG2/BCRP was enhanced, in the resistant cells. In addition, the expression levels of ABCC1/MRP1, ABCC3/MRP3, and ABCC5/MRP5 were higher than in HeLa cells. The mRNA levels of GGT1 encoding a γ-glutamyl transferase, but not GCS encoding a γ-glutamyl cysteine synthetase, were also higher. Other factors examined, i.e., topoisomerase, SLCO1B1, and apoptosis-regulating factors, were comparable among the cells. The overexpression of ABCG2/BCRP was involved in the mechanism of resistance in SN-38-tolerant cells, and ABCC1/MRP1, ABCC3/MRP3, ABCC5/MRP5, and GGT1 may also have participated.     

Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11.

It is known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a semisynthesized derivative of camptothecin (CPT), has a potent antitumor activity in vivo, but 7-ethyl-10-hydroxycamptothecin (SN-38), a metabolite of CPT-11, shows much stronger cytotoxicity in vitro than CPT-11. In this study, we demonstrated that the relaxation of SV40 DNA plasmids by type I DNA topoisomerase prepared from P388 murine leukemia cells was inhibited by 50% by SN-38 at approximately 1 microM, although CPT-11 at 1 mM slightly inhibited the relaxation. SN-38 and CPT showed strong, time-dependent inhibitory activity against DNA synthesis of P388 cells. However, CPT-11 weakly inhibited DNA synthesis independently of time with coincident inhibition of the total thymidine uptake by the cells. By alkaline and neutral elution assays, it was demonstrated that SN-38 caused much more frequent DNA single-strand breaks in P388 cells than did CPT-11. The same content of SN-38 and a similar frequency of single-strand breaks were detected in the cells treated with SN-38 at 0.1 microM or with CPT-11 at 100 microM. Therefore, single-strand breaks by CPT-11 seem to be due to SN-38 produced from CPT-11 in cells. These results indicate that CPT-11 itself possesses a marginal antiproliferative effect but that SN-38 plays an essential role in the mechanism of action of CPT-11.     

Inhibition of intestinal microflora β-glucuronidase modifies the distribution of the active metabolite of the antitumor agent, irinotecan hydrochloride (CPT-11) in rats

Purpose: SN-38, a metabolite of irinotecan hydrochloride (CPT-11), is considered to play a key role in the development of diarrhea as well as in the antitumor activity of CPT-11. We have previously found that the inhibition of β-glucuronidase, which hydrolyzes detoxified SN-38 (SN-38 glucuronide) to reform SN-38, in the lumen by eliminating the intestinal microflora with antibiotics, markedly ameliorates the intestinal toxicity of CPT-11 in rats. In this study we compared the disposition of CPT-11 and its metabolites in rats treated with and without antibiotics. Methods: Rats were given drinking water containing 1 mg/ml penicillin and 2 mg/ml streptomycin from 5 days before the administration of CPT-11 (60 mg/kg i.v.) and throughout the experiment. CPT-11, SN-38 glucuronide and SN-38 concentrations in the blood, intestinal tissues and intestinal luminal contents were determined by HPLC. Results: Antibiotics had little or no effect on the pharmacokinetics of CPT-11, SN-38 glucuronide or SN-38 in the blood, or in the tissues or contents of the small intestine, which has less β-glucuronidase activity in its luminal contents. In contrast, antibiotics markedly reduced the AUC_1–24 h of SN-38 (by about 85%) in the large intestine tissue without changing that of CPT-11, and this was accompanied by a complete inhibition of the deconjugation of SN-38 glucuronide in the luminal contents. Conclusions: These results suggest that SN-38, which results from the hydrolysis of SN-38 glucuronide by β-glucuronidase in the intestinal microflora, contributes considerably to the distribution of SN-38 in the large intestine tissue, and that inhibition of the β-glucuronidase activity by antibiotics results in decreased accumulation of SN-38 in the large intestine. Received: 8 August 1997 / Accepted: 16 January 1998     

Effective Irinotecan (CPT-11)-containing Liposomes: Intraliposomal Conversion to the Active Metabolite SN-38

Irinotecan hydrochloride (CPT-11) is a prodrug of SN-38, which is an active metabolite with anti-tumor activity and side toxicity. The activities of CPT-11 and SN-38 depend on the closed lactone ring form of SN-38. We have examined the tissue distributions of the closed and open forms of CPT-11 and SN-38 in Lewis lung carcinoma-bearing mice after the administration of liposomal CPT-11 (S-Lip) and polyethyleneglycol (PEG)-modified S-Lip (S-PEG). The plasma concentrations of closed CPT-11 and SN-38 were increased by liposomalization, and their blood circulation was prolonged by the PEG modification. The concentrations of closed CPT-11 and SN-38 in tumors were elevated by both the liposomalization and PEG modification. The closed/total ratio of SN-38 in the tumors of the S-PEG group was greater than that of the CPT-11 solution (Sol) group. Thus, SN-38 was thought to be generated in intact liposomes containing CPT-11. The bile concentration of closed SN-38, which is responsible for CPT-11-induced intestinal disorder, was decreased by liposomalization. In an in vitro experiment, the SN-38/CPT-11 ratio in the tumor cells of the S-Lip group was found to be higher than that of the Sol group, and the ratio of the closed form of SN-38 was increased by the liposomalization. Laser scanning confocal microscopy showed the generation of SN-38 in the liposomal membrane after the incubation of S-Lip with carboxylesterase. It is therefore considered that a part of CPT-11 is converted to SN-38 in the intact liposomes.     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

Paclitaxel and irinotecan are important new anticancer agents. The combination of these two agents has been considered for use against a variety of advanced solid tumors. Since the schedule-dependent effects of this combination may be crucial to its use, we studied the interaction of paclitaxel and SN-38 (the active metabolite of irinotecan) in various schedules in four human cancer cell lines in culture. Cell growth inhibition after 5 days was determined using an MTT assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. Simultaneous exposure to paclitaxel and SN-38 for 24 h produced antagonistic (subadditive and protective) effects in the human lung cancer cell line A549, the breast cancer cell line MCF7, and the colon cancer cell line WiDr, and produced additive effects in the ovarian cancer cell line PA1. Sequential exposure to paclitaxel for 24 h followed by SN-38 for 24 h, and the reverse sequence, produced additive effects in all four cell lines. These findings suggest that sequential administration, not simultaneous administration, may be the appropriate schedule for the therapeutic combination of paclitaxel and irinotecan. Continued preclinical and clinical studies should provide further insights and assist in determining the optimal schedule for this combination in clinical use. Received: 25 February 1997 / Accepted: 6 November 1997     

Relationship between Development of Diarrhea and the Concentration of SN-38, an Active Metabolite of CPT-11, in the Intestine and the Blood Plasma of Athymic Mice Following Intraperitoneal Administration of CPT-11

Severe diarrhea occurred during daily intraperitoneal administration of 7-ethyl-10-[4-(l-piperidino)-l-piperidino]carbonyloxycamptothecin (CPT-11) at a dose of 50 mg/kg in athymic mouse. Serial determination of CPT-11 and 7-ethyl-10-hydroxycamptothecin (SN-38), with the use of an on-line solid extraction HPLC system, demonstrated that much higher levels of the compounds are retained in the intestine and the blood plasma after five consecutive daily injections than after a single injection. Histologic examination of the gastrointestinal tract showed hemorrhagic colitis on day 7 and later after five consecutive daily injections of CPT-11. The direct cause of diarrhea associated with CPT-11 administration is considered to be enterocolitis caused by high levels of SN-38 and/or CPT-11 retained for a long period in the intestine.     

RNA干扰技术逆转神经胶质瘤细胞多药耐药性

目的 探讨利用RNA干扰（RNAi）技术逆转神经胶质瘤细胞多药耐药性。方法 根据多药耐药基因1（MDR1）的碱基序列设计并合成短发夹RNA（shRNA）,构建逆转录病毒质粒载体,用阳离子脂质体法体外转染BT325细胞株,以增强型绿色荧光蛋白（EGFP）表达作为对照。采用定量PCR、Northern blot检测转染前后MDR1 mRNA的表达,Western blot检测蛋白表达;使用CCK-8试剂盒对转染后的细胞进行化疗药物敏感性试验,评价RNAi对多药耐药性的逆转作用。结果 成功构建RNAi质粒载体。共转染实验组RT-PCR定量MDR1 mRNA相对表达水平均有所下降（P〈0．05）;Northern blot表明,转染48h细胞干扰最强;Western blot显示,siRNA各转染组P-糖蛋白（P-gp）的表达分别降低12．9％、30．3％和4．8％,在48h抑制最强;而药物敏感试验显示,转染siRNA后细胞对药物的敏感性明显增强。结论 RNAi能够明显抑制神经胶质瘤细胞系MDR1 mRNA和P-gp蛋白的表达,进而对多药耐药性发挥明显的逆转作用,为基因治疗提供了一种新的手段。     

免疫效应细胞促进阿霉素诱导乳腺癌耐药细胞凋亡

目的 探讨免疫效应细胞促进阿霉素（ADR）诱导乳腺癌耐药细胞MCF7／ADR凋亡的作用机制。方法 采用干扰素-γ（IFN-γ）、CD3单抗、白介素（IL）-1和IL-2诱导并扩增免疫效应细胞。利用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）技术和P-糖蛋白（P-gP）免疫组织化学染色,观察P-gP表达与凋亡的关系。用流式细胞术检测乳腺癌相关抗原P185蛋白的表达,以及Annexin V-FITC／PI阳性率。荧光显微镜下观察ADR在靶细胞内的分布和Annexin V的表达。结果 免疫效应细胞使MCF7／ADR细胞P185和P-gP表达明显下降,CIK细胞作用后,MCF7／ADR细胞P185标记荧光几何均数下降了91．9％。免疫效应细胞增加了ADR在MCF7／ADR细胞内的积累及其在细胞核的分布。联合应用免疫效应细胞和ADR后,MCF7／ADR细胞凋亡率达78．9％,比单纯加入ADR组增高了10倍,较单纯加入免疫效应细胞组增高了13倍。结论 免疫效应细胞可同时下调P185蛋白和P-gp蛋白的表达,协同ADR可提高MCF7／ADR细胞的凋亡率。     

Effects of SN-38 (an active metabolite of CPT-11) on responses of human and rodent cells to irradiation

Purpose: Modulators of the DNA-unwinding enzyme, topoisomerase I (Topo I), inhibit DNA repair and have been reported to increase the lethal effects of X rays, which create breaks in DNA. CPT-11 is a derivative of camptothecin, a Topo I inhibitor, and is clinically available. In this study, we tested the in vitro combination effects of SN-38, an active form of CPT-11, and irradiation on several cell lines.Materials and Methods: Exponentially growing or confluent cultures of CHO cells were treated with SN-38 for 30 min. Cells were then irradiated. Thereafter, the cells were further incubated with the drug for 0 to 3 h. Exponentially growing other cell lines were exposed to 200 nM SN-38 for 30 min before, during, and 3 h after irradiation. The cell survival rate was determined using a conventional clonogenic assay.Results: SN-38 (200 nM to 4 μM) alone showed slight toxicity to CHO cells in the confluent culture after a 3.5-h incubation. When the cells were treated with the lower doses of SN-38 (50 to 800 nM) during the exponentially growing phase, the cell survival rates were much lower. In combination with irradiation, SN-38 showed only additive effects to irradiation when cells were treated in confluent cultures. However, higher combination effects of SN-38 and irradiation were observed in the cells treated in the exponentially growing phase. When cells were irradiated during the exponentially growing phase, a significant combination effect of 200 nM SN-38 and irradiation was also observed in some cell lines, but not in others.Conclusion: SN-38 and irradiation showed supraadditive effects in some cell lines, when treated in the exponentially growing phase, but not in other cell lines or when cells were treated in the confluent phase.     

Combined effects of SN-38 and pentoxifylline on the induction of apoptosis through the activation of CPP-32 in pancreatic adenocarcinoma cell lines

Background. Pentoxifylline (PENT) is a theophylline derivative that enhances cytotoxic effects against tumor cells pretreated with antitumor agents. It has also been reported that chemotherapy can induce apoptosis in some carcinoma cells. We investigated the effects of PENT on human pancreatic adenocarcinoma cells pretreated with SN-38, an active form of CPT-11 (a camptothecin analogue) and we also examined the participation of CPP-32, a member of the interleukin 1β-converting enzyme (ICE) family proteases, in chemotherapeutic agent-induced apoptosis. Methods. Human pancreatic adenocarcinoma cells (PK-1, PK-8) were cultured in RPMI 1640. Lethal effects were examined by MTT assay; DNA fragmentation was analyzed by agarose gel electrophoresis; and Western blot analysis was performed with anti-CPP-32 monoclonal antibody. Results. Pretreatment with SN-38 followed by PENT increased the cytotoxic effect compared with that seen for treatment with SN-38 alone. Isobologram analysis of the IC₅₀ value revealed that PENT had supra-additive effects when administed after SN-38, but not when administered prior to or simultaneously with SN-38. Agarose gel electrophoresis showed typical DNA ladders in the DNA of cells treated with SN-38 and PENT. The acridine orange (AO) staining method was used to observe the morphological changes characteristic of apoptosis. Western blot analysis verified that activation of CPP-32 accompanied the development of apoptosis. In addition, SN-38-induced apoptosis was prevented by pretreatment with Ac-DEVD-CHO (DEVD), an inhibitor of CPP-32. Conclusions. These results indicate that the antitumor activity of SN-38 is attributable to apoptosis through the activation of CPP-32, and that combined treatment with PENT enhances the induction of apoptosis by SN-38. Accordingly, the use of PENT may provide a combined modality treatment for pancreatic cancer. Received: August 15, 1997 / Accepted: November 27, 1998     

cis-Diamminedichloroplatinum(II)-induced cell death through apoptosis in sensitive and resistant human ovarian carcinoma cell lines

 We have studied the effects of the chemother-apeutic drug cis-diamminedichloroplatinum(II) (cisplatin) on three human ovarian carcinoma cell lines – one sensitive to the drug (CH1), one with acquired resistance (CH1cisR) and one with intrinsic resistance (SKOV-3). Previous work has shown that the 50% inhibitory concentrations (IC₅₀ values) after a 2-h exposure to the drug are: CH1, 2.5 μM; CH1cisR, 7.5 μM; and SKOV-3, 33 μM. Despite the variation in sensitivity, the amount of Pt bound to DNA and the rate of removal of Pt was similar for the three lines. There were significant differences in the rates of formation of DNA cross-links but these were not large enough to account for the high resistance of the SKOV-3 line. We have reported that in the L1210 murine leukaemia cell line there are two mechanisms of cisplatin-induced cell death – one of which involves apoptosis. In this paper, we report on an investigation into whether sensitivity to apoptosis played a role in the resistance of these ovarian lines towards cisplatin. After a 2-h incubation with the drug, cells from the three lines showed evidence of death through apoptosis. The cells detached from the culture dish in a time- and dose-dependent fashion. These cells morphologically were quite distinctive from the attached cells and showed changes in their chromatin structure indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kilobase pairs, kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the quantity of cells undergoing apoptosis was similar for the three cell lines. The most prominent effect on cell-cycle kinetics was a slowdown in S-phase transit during which the cells underwent apoptosis. Cells that successfully completed the S phase subsequently suffered a temporary G2 block. We propose that the sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA and that the major mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis. Received: 13 December 1994/Accepted: 6 June 1995     

Alleviation of side effects induced by irinotecan hydrochloride (CPT-11) in rats by intravenous infusion

Purpose Irinotecan hydrochloride (CPT-11) is a potent topoisomerase I inhibitor and is established and used widely as an antitumor agent. However, it sometimes causes severe side effects such as myelosuppression and diarrhea. These dose-limiting toxicities prevent the adoption of CPT-11 in aggressive chemotherapy. Thus we sought to determine in a rat model whether extending the period of infusion of CPT-11 would ameliorate the adverse reactions.Methods CPT-11 was administered intravenously (i.v.) to rats at a dose of 60 mg/kg per day for four consecutive days as a bolus injection or as 3-, 8- or 24-h infusions, and then blood cell counts and the incidence of acute and delayed-onset diarrhea were monitored.Results Serious acute and delayed-onset diarrhea and marked decreases in the number of neutrophils and lymphocytes were observed in the bolus injection group. These symptoms were alleviated in the infusion groups with the degree of alleviation dependent on infusion time. In the bolus injection group, mucosal impairment of the cecal epithelium including wall thickening, edema, a decrease in the number and size of crypts, and the formation of a pseudomembrane-like substance was observed, whereas these changes were less severe in the infusion groups. Areas under the plasma concentration-time curves (AUC_pla) of CPT-11 and its metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), differed little among the bolus injection group, and the 3-h and 8-h infusion groups. However, the AUC_pla values of CPT-11 and SN-38 were significantly decreased and increased, respectively, in the 24-h infusion group. The maximum plasma concentrations (C_max) of CPT-11 decreased with increasing infusion time, but those of SN-38 did not.Conclusions It was confirmed that the side effects of CPT-11 were alleviated by extending the infusion time. The pharmacokinetic parameters suggested that the C_max of CPT-11 is closely related to the incidence and severity of adverse reactions such as myelosuppression and acute and delayed-onset diarrhea.Abbreviations AUC_cec Area under the cecal tissue concentration-time curve - AUC_mar Area under the bone marrow tissue concentration-time curve - AUC_pla Area under the plasma concentration-time curve - C_max Maximum concentration - CL_tot Total clearance - CPT Camptothecin - CPT-11 Irinotecan hydrochloride [7-ethyl-10-(4-(piperidino)-1-piperidino)carbonyloxycamptothecin] - G-CSF Granulocyte colony-stimulating factor - HPLC High-performance liquid chromatography - i.v. Intravenous(ly) - MRT Mean residence time - SN-38 7-Ethyl-10-hydroxycamptothecin - SN-38G SN-38 glucuronide - T_1/2 Half-life - UGT UDP-glucuronosyltransferase     

Establishment and characterization of human gastric and colonic xenograft lines resistant to CPT-11 (A new derivative of camptothecin)

CPT-11-resistant human gastric and colonic xenograft lines were established by direct intratumoral injection of CPT-11 into subcutaneous SC-1-NU and CC-2-NU tumors in nude mice once a week for 10 months. The resistance of these xenograft lines to CPT-11 was confirmed by growth inhibition rate, to be 36.3% and 45.4%, respectively, compared to each parent cell line. DNA topoisomerase I activity of the nuclear extracts of SC-1-NU/CPT-11 and CC-2-NU/CPT-11, as assayed by relaxation of supercoiled DNA Col-E1, was significantly less than those of the parent lines. The cellular levels of topoisomerase I in those resistant lines measured by Western blot analysis were 0.57- and 0.79-fold lower than those of the parental lines, respectively. However, the activity of DNA topoisomerase II of those resistant cell lines assayed by decatenation of kinetoplast DNA was higher than that of the parental lines and the cellular levels of topoisomerase II in the resistant lines measured by Western blot analysis were 10.8- and 8.1-fold higher than those of the parent lines. Intracellular accumulation of CPT-11 in CPT-11-resistant tumors was not changed as compared to that of the parental lines, but hydrolysis of CPT-11 to more active SN-38 was reduced in the resistant tumors.     

Bcl-2 gene prevents induction of apoptosis in l1210 murine leukemia-cells by sn-38, a metabolite of the camptothecin derivative cpt-11

New camptothecin (CPT) derivatives have recently been synthesized following the finding that CPT has strong antitumor activity due to its inhibition of topoisomerase I through the formation of stable topoisomerase I-DNA cleavable complexes, but has not been clinically used due to its pronounced toxicity. 7-ethyl-10-hydroxy-CPT (SN-38), a metabolite of the CPT derivative 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-CPT(CPT-11), plays an essential role in mediating the antitumor effect of CPT-11. However, the reasons for the cytotoxicity of SN-38 remain unclear. In this study, we demonstrated using results of DNA fragmentation assay and cell cycle analysis that SN-38 and CPT both induce apoptosis in L1210 murine leukemia cells. We demonstrated in addition that enforced expression of the bcl-2 gene in L1210 cells by MPZenNeo (bcl-2) retroviral gene transfer increased resistance to the apoptosis induced by SN-38 and CPT. These findings suggest the possibility that the bcl-2 gene impedes the activity of a common pathway for apoptosis induced by SN-38 and CPT.     

Blocking of PI3K/Akt pathway enhances apoptosis induced by SN-38, an active form of CPT-11, in human hepatoma cells

Hepatocellular carcinoma (HCC) is a common malignancy and often resistant to chemotherapy. Many chemotherapy regimens have been tried to control advanced HCC, but have produced a low response rate and no clear impact. CPT-11, a derivative of camptothecin, works as type-I DNA topoisomerase inhibitor and showed a major objective response rate in patients with metastatic colorectal cancer. In this study, the mechanism underlying chemo-resistance to SN-38, an active form of CPT-11, in HCC was investigated in relation to anti-apoptotic pathways NF-kappaB and PI3K/Akt. Hep3B was the most resistant to SN-38 among three hepatoma cell lines. NF-kappaB was constitutively activated in Hep3B, and SN-38 further enhanced the nuclear translocation of NF-kappaB. However, inactivation of NF-kappaB by adenovirus expressing IkappaB super-repressor or MG-132, proteasome inhibitor, did not sensitize Hep3B to SN-38-induced apoptosis. On the other hand, SN-38 phosphorylated Akt and pretreatment with PI3K inhibitors increased SN-38-induced apoptosis, indicating that resistance to SN-38 in Hep3B occurs partly through the PI3K/Akt not the NF-kappaB pathway. Blocking of PI3K/Akt may thus be helpful for overcoming chemo-resistance of HCC.     

In vivo antitumor activity of S 16020-2, a new olivacine derivative

 The antitumor activity of S 16020-2, a new olivacine derivative, was investigated in vivo and compared with that of Adriamycin and elliptinium acetate in a panel of murine (P388 leukemia, M5076 sarcoma, Lewis lung carcinoma, and B16 melanoma) and human (NCI-H460 non-small-cell lung and MCF7 breast carcinomas) tumor models. S 16020-2 given i.v. was active against P388 leukemia implanted i.p., s.c., or intracerebrally. The therapeutic effect of an intermittent schedule (administration on days 1, 5, 9) was superior to that of single-dose treatment, allowing the i.v. administration of high total doses of S 16020-2 and resulting in the cure of 60% of mice in the i.p. P388 model. In this model, S 16020-2 was more active than elliptinium acetate and showed a better therapeutic index than Adriamycin:≥8 versus 2. A good therapeutic effect of S 16020-2 was also observed in three P388 leukemia sublines displaying the classic multidrug-resistance phenotype, namely, P388/VCR, P388/VCR-20, and P388/MDRC.04, the latter being totally insensitive to vincristine and Adriamycin. However, S 16020-2 was not active against the P388/ADR leukemia, a model highly resistant to adriamycin in vivo. S 16020-2 was both more active than Adriamycin and curative in the M5076 sarcoma and Lewis lung carcinoma implanted s.c. In the B16 melanoma implanted i.p. or s.c., S 160202 was less active than Adriamycin. Against the NCI-H460 human tumor xenograft, S 16020-2 demonstrated activity superior to that of Adriamycin (T/C=20% versus 43% on day 21). Against the MCF7 breast cancer xenograft, S 16020-2 was active, but less so than Adriamycin (T/C=23% versus 9% on day 21), whereas elliptinium acetate was marginally active (T/C=49% on day 24). The hematological toxicity of S 16020-2 given to B6D2F1 mice at pharmacological dose appeared to be less severe than that of Adriamycin, particularly in bone-marrow stem cells. These results demonstrate that S 16020-2 is a highly active antitumor drug in various experimental tumor models and is markedly more efficient than elliptinium acetate. Because of its pharmacological profile, which is globally different from that of Adriamycin, S 16020-2 is considered an interesting candidate for clinical trials. Received: 21 October 1995/Accepted: 4 March 1996     

The human cell multiprotein DNA replication complex (MRC): the effect of camptothecin on its ability to support in vitro DNA synthesis

 Purpose : We have previously reported on the isolation and characterization of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability to replicate DNA in a cell-free environment (devoid of other cellular processes) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor. Methods : We examined the effects of increasing concentrations of camptothecin on HeLa cell survival, intact HeLa cell DNA synthesis and MRC-mediated in vitro DNA replication. We also performed topoisomerase I assays in the presence of increasing concentrations of camptothecin to study the direct effects of the agent on MRC-associated topoisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleavable complexes in the presence of increasing concentrations of camptothecin. Results : We found a close correlation between the IC₅₀ values for intact HeLa cell DNA synthesis (0.15 μM) and MRC-mediated in vitro DNA synthesis (0.05 μM). Similarly, we found that 0.05 μM camptothecin inhibited MRC-associated topoisomerase I activity by approximately 50%. In addition, we found that the formation of MRC-associated topoisomerase I-cleavable complexes increased linearly with increasing concentrations of camptothecin. Conclusions: The data presented in this report support the use of the MRC as a model system to study the mechanism of action of camptothecin. We anticipate that future studies with the MRC will help elucidate the cellular consequences of camptothecin-cleavable complex formation. Received: 24 March 1995/Accepted: 22 March 1996