磷烯菌素诱导HL-60细胞凋亡及其与增殖周期的关系

目的　探讨一种新型的DNA拓扑酶 Ⅱ抑制剂磷烯菌素 (FST)对人急性粒细胞白血病细胞株HL 60的杀伤机理。方法　采用流式细胞术 (FCM )、TdT联合FCM及LadderDNA三种方法研究FST对HL 60细胞凋亡的诱导作用及其与增殖周期的关系。结果 :FCM (乙醇抽提法 )显示 :1 0 μmolFST作用 6h未引起HL 60凋亡 ,1 0 0 μmol/L时出现明显“亚G1 峰” ,但1 0 0 0 μmol/L诱导时 ,却不出现凋亡而呈“冰冻”现象。TdT联合FCM表明 :1 0 μmol/LFST作用 6h ,即有明显细胞凋亡 ,且凋亡发生在整个细胞周期。LadderDNA测定提示 :1 0 0 μmol/LFST作用HL 60细胞 6h ,其DNA凝胶电泳出现典型梯状图谱。结论　 1 FST诱导HL 60细胞死亡具有异质性 :低剂量触发凋亡 ,高剂量诱导坏死。 2 TdT联合FCM是一种灵敏的凋亡检测手段 ,它能同时测定细胞DNA含量及断裂 ,故能用以研究细胞凋亡及其与增殖周期的关系 ;3 FST诱导的凋亡似乎没有细胞周期特异性     

In vivo inhibition of etoposide-mediated apoptosis,toxicity, and antitumor effect by the topoisomerase II-uncoupling anthracycline aclarubicin

A number of clinically important drugs such as the epipodophyllotoxins etoposide (VP-16) and teniposide (VM-26), the anthracyclines daunorubicin and doxorubicin (Adriamycin), and the aminoacridine amsacrine exert their cytotoxic action by stabilizing the cleavable complex formed between DNA and the nuclear enzyme topoisomerase II. We have previously demonstrated in several in vitro assays that the anthracycline aclarubicin (aclacinomycin A) inhibits cleavable-complex formation and thus antagonizes the action of drugs such as VP-16 and daunorubicin. The present study was performed to validate these in vitro data in an in vivo model. At nontoxic doses of 6 and 9 mg/kg, aclarubicin yielded a marked increase in the survival of non-tumor-bearing mice given high doses of VP-16 (80–90 mg/kg) in six separate experiments. In therapy experiments on mice inoculated with Ehrlich ascites tumor cells, aclarubicin given at 6 mg/kg roughly halved the increase in median life span induced by VP-16 at doses ranging from 22 to 33 mg/kg. An attempt to determine a more favorable combination of VP-16 and aclarubicin by increasing VP-16 doses failed, as the two drugs were always less effective than VP-16 alone. The way in which VP-16-induced DNA strand breaks lead to cell death remains unknown. However, VP-16 has been reported to cause apoptosis (programmed cell death) in several cell lines. To ascertain whether the protection given by aclarubicin could have a disruptive effect on the apoptotic process, we used the small intestine as an in vivo model. Whereas VP-16-induced apoptosis in crypt stem cells was detectable at a dose as low as 1.25 mg/kg, aclarubicin given at up to 20 mg/kg did not cause apoptosis. Indeed, aclarubicin caused a statistically significant reduction in the number of cells rendered apoptotic by VP-16. The present study thus confirms the previous in vitro experiments and indicates the value of including an in vivo model in a preclinical evaluation of drug combinations.This work was supported by the Danish Cancer Society     

Defining functional drug-interaction domains on topoisomerase II by exploiting mechanistic differences between drug classes

Topoisomerase II is the primary cellular target for a variety of antineoplastic drugs that are active against human cancers. These drugs exert their cytotoxic effects by stabilizing covalent topoisomerase II-cleaved DNA complexes that are fleeting intermediates in the catalytic cycle of the enzyme. Despite this common feature of drug action, a number of mechanistic differences between drug classes have been described. These mechanistic differences (including effects on DNA cleavage/religation, DNA strand passage, and adenosine triphosphate hydrolysis) were used as the basis for a series of competition experiments to determine whether different compounds share a common site of action on topoisomerase II or interact at distinct sites. Results of the present study strongly suggest that at least four structurally disparate antineoplastic drugs, etoposide, amsacrine, genistein, and the quinolone CP-115,953, share an overlapping interaction domain on the enzyme.Paper presented at the Topoisomerase Inhibitors Conference, University of Maryland Cancer Center, 27–30 October 1993, Monterey, California, USA. Supported in part by Bristol Myers Oncology Division     

Hypophosphorylation of Topoisomerase IIα in Etoposide (VP-16)-resistant Human Carcinoma Cell Lines Associated with Carboxy-terminal Truncation

Topoisomerase Ilα is a target for many chemotherapeutic agents in clinical use. To define mechanisms of resistance and regions crucial for the function of topoisomerase IIα, drug-resistant cell lines have been isolated following exposure to topoisomerase II poisons. Two resistant sublines, T47D-VP and MCF-7-VP, were isolated from human carcinoma cell lines following exposure to 300 or 500 ng/ml etoposide (VP-16). Cytotoxicity studies confirmed resistance to etoposide and other topoisomerase II poisons. KCl-sodium dodecyl sulfate (K-SDS) precipitation assays using intact cells showed reduced DNA-topoisomerase II complex formation following VP-16 or amsacrine (m-AMSA). RNAse protection analysis identified a deletion of 200 base pairs in the topoisomerase Ilα cDNA of T47D-VP and "AA insertion" in the topoisomerase Ilα cDNA of MCF-7-VP. Reduced topoisomerase Ila mRNA and protein levels were observed in both cell lines. It was somewhat surprising to find that nuclear extracts from T47D-VP and MCF-7-VP cells had comparable topoisomerase II activity to that of parental cells. Analysis of the extent of phosphorylation demonstrated that topoisomerase Ila from the resistant cells was relatively hypophosphorylated compared to that of parental cells. In these cell lines, hypophosphorylation secondary to loss of a portion of the C-terminal domain of topoisomerase Ilα mediated the restored activity, despite a fall in topoisomerase Ila mRNA and protein, and this resulted in cross resistance to topoisomerase II poisons.     

白屈菜红碱对大鼠的长期毒性试验研究

目的： 观察连续给予抗肿瘤药物白屈菜红碱对大鼠的长期毒性。方法：采用Wistar大鼠,共分成4个白屈菜红碱组(12.6、8.4、5.6和3.7 mg/kg)及溶剂对照组,每组20只。腹腔注射给药6 d,停药8 d为1个周期,连续给药3个周期,间歇给药结束后12.6 mg/kg剂量组解剖大鼠6只,8.4 mg/kg剂量组解剖大鼠8只,其余每组解剖大鼠12只,停药观察4周后解剖各组剩余大鼠,进行一般状况、体质量、饲料摄入量、血液学、血清生化学、尸体解剖和脏器系数以及病理组织学检查。结果：大鼠在给药后,12.6 和8.4 mg/kg剂量组立即出现腹膜刺激征,一周之内出现消瘦和被动体态等,体质量增长缓慢,摄食量减少,甚至死亡。与对照组比较,白屈菜红碱5.6 mg/kg以上剂量组大鼠红细胞系各项指标降低,白细胞数及白细胞分类异常,凝血时间延长,葡萄糖降低,碱性磷酸酶增加(P<0.05或P<0.01);并可观察到肺脏瘀血,腹腔大量血性腹水,脏器粘连并严重变形,肝脏和脾脏明显增大,前列腺和睾丸系数明显减小(P<0.05或P<0.01),睾丸生精上皮细胞和精子明显损伤等。结论：白屈菜红碱在 ≥5.6 mg/kg剂量下,可引起大鼠局部刺激和药物毒性所引起的全身性异常反应,以致部分大鼠死亡。     

Etoposide: current status and future perspectives in the management of malignant neoplasms

Etoposide has demonstrated highly significant clinical activity against a wide variety of neoplasms, including germ-cell malignancies, small-cell lung cancer, non-Hodgkin's lymphomas, leukemias, Kaposi's sarcoma, neuroblastoma, and soft-tissue sarcomas. It is also one of the important agents in the preparatory regimens given prior to bone marrow and peripheral stem-cell rescue. Despite its high degree of efficacy in a number of malignancies, the optimal dose, schedule, and dosing form remain to be defined. It is possible that continuous or prolonged inhibition of the substrate, i.e., topoisomerase II, may be the key factor for the cytotoxic effects of etoposide. Clinical studies have shown the activity of etoposide to be schedule-dependent, with prolonged dosing, best accomplished by the oral dosing form, offering a therapeutic advantage. This benefit awaits validation by prospective randomized studies, some of which are in progress. Recent clinical investigations have focused on the use of etoposide in combination with (a) cytokines to ameliorate myelosuppression, the dose-limiting toxicity of etoposide; (b) agents such as cyclosporin A and verapamil to alter the p-glycoprotein (mdr 1) function; and (c) topoisomerase I inhibitors to modulate the substrate upon which it acts. There is continued interest in the development of etoposide to its maximal clinical dimensions and in the examination of alternative biochemical and mechanistic approaches to further our understanding of this highly active agent.Paper presented at the Topoisomerase Inhibitors Conference, University of Maryland Cancer Center, 27–30 October 1993, Monterey, California USA. Supported in part by Bristol Myers Oncology Division     

Effect of phenazine compounds XR11576 and XR5944 on DNA topoisomerases

Purpose  Previous in vitro cleavage data showed that XR11576 and XR5944 stabilised topoisomerase I and topoisomerase II complexes on DNA in a dose-dependent fashion. However, some studies indicated a possible topoisomerase-independent mechanism of action for these drugs. Methods  Three methods, the TARDIS assay, immunoband depletion and the K⁺/SDS assay have been used to assess topoisomerase complex formation induced by XR11576 or XR5944 in human leukaemic K562 cells. Results  TARDIS and immunoband depletion assays demonstrated that XR11576 and XR5944 induced complex formation for both topoisomerase I and topoisomerase II (alpha and beta) in a dose- and time-dependent manner, following exposure times of 24 and 48 h at concentrations of 1 or 10 μM. The K⁺/SDS assay showed the formation of protein/DNA complexes after a 1 h exposure to 1 or 10 μM XR11576. Conclusion  Our data confirm that XR11576 or XR5944 can form topoisomerase complexes, after long periods of exposure.     

Cisplatin-induced renal toxicity and toxicity-modulating strategies: a review

Summary We have attempted to outline the fundamental statistical aspects of pharmacodynamic modeling. Unexpected yet substantial variability in effect in a group of similarly treated patients is the key motivation for pharmacodynamic investigations. Pharmacokinetic and/or pharmacodynamic factors may influence this variability. Residual variability in effect that persists after accounting for drug exposure indicates that further statistical modeling with pharmacodynamic factors is warranted. Factors that significantly predict interpatient variability in effect may then be employed to individualize the drug dose.In this paper we have emphasized the need to understand the properties of the effect measure and explanatory variables in terms of scale, distribution, and statistical relationship. The assumptions that underlie many types of statistical models have been discussed. The role of residual analysis has been stressed as a useful method to verify assumptions. We have described transformations and alternative regression methods that are employed when these assumptions are found to be in violation. Sequential selection procedures for the construction of multivariate models have been presented. The importance of assessing model performance has been underscored, most notably in terms of bias and precision.In summary, pharmacodynamic analyses are now commonly performed and reported in the oncologic literature. The content and format of these analyses has been variable. The goals of such analyses are to identify and describe pharmacodynamic relationships and, in many cases, to propose a statistical model. However, the appropriateness and performance of the proposed model are often difficult to judge. Table 1 displays suggestions (in a checklist format) for structuring the presentation of pharmacodynamic analyses, which reflect the topics reviewed in this paper.This study was supported in part by grant N01-CM-07301 and Cancer Center Core grant CA-14599     

Part 3: Pharmacogenetic variability in phase II anticancer drug metabolism

Equivalent drug doses may lead to wide interpatient variability in drug response to anticancer therapy. Known determinants that may affect the pharmacological response to a drug are, among others, nongenetic factors, including age, gender, use of comedication, and liver and renal function. Nonetheless, these covariates do not explain all the observed interpatient variability. Differences in genetic constitution among patients have been identified to be important factors that contribute to differences in drug response. Because genetic polymorphism may affect the expression and activity of proteins encoded, it is a key covariate that is responsible for variability in drug metabolism, drug transport, and pharmacodynamic drug effects. We present a series of four reviews about pharmacogenetic variability. This third part in the series of reviews is focused on genetic variability in phase II drug-metabolizing enzymes (glutathione S-transferases, uridine diphosphoglucuronosyl transferases, methyltransferases, sulfotransferases, and N-acetyltransferases) and discusses the effects of genetic polymorphism within the genes encoding these enzymes on anticancer drug therapy outcome. Based on the literature reviewed, opportunities for patient-tailored anticancer therapy are proposed.     

Topoisomerase IIα content and topoisomerase II catalytic activity cannot explain drug sensitivities to topoisomerase II inhibitors in lung cancer cell lines

 Purpose: Topoisomerase IIα content, topoisomerase II catalytic activity and drug sensitivities to the topoisomerase II inhibitors, doxorubicin and etoposide, were examined in a panel of 14 unselected human lung cancer cell lines in order to determine the relationship between topoisomerase II and drug sensitivities to the topoisomerase II inhibitors. Methods: Drug sensitivities were determined using a microculture tetrazolium assay. The topoisomerase IIα levels were determined by Western blot analysis and the topoisomerase II catalytic activity was determined using a decatenation assay of kinetoplast DNA, using nuclear protein from cells of each cell line. Results: Drug sensitivity tests revealed that small-cell lung cancer (SCLC) cell lines were more sensitive to drugs than non-small-cell lung cancer (NSCLC) cell lines. The relative topoisomerase IIα levels and relative topoisomerase II catalytic activity from SCLC cell lines (mean± SD 0.89±0.54 and 5.3±3.4, respectively) were slightly higher than those from NSCLC cell lines (0.78±0.56 and 4.0±2.8, respectively), but the differences were not statistically significant, and not sufficient to account for the variation in drug sensitivities. Moreover, no clear association was observed between the topoisomerase IIα levels or the topoisomerase II catalytic activity and drug sensitivities in the cell lines studied. Conclusions: These findings suggest that the difference in drug sensitivities to doxorubicin and etoposide in human lung cancer cell lines might not be explainable by the topoisomerase IIα levels and topoisomerase II catalytic activity. Moreover, our results suggest that the topoisomerase IIα levels and topoisomerase II catalytic activity may play a minor role in the determination of clinical drug resistance of human lung cancers. Received: 11 July 1995/Accepted: 18 May 1996     

Enhanced Topoisomerase I Activity and Increased Topoisomerase IIα Content in Cisplatin-resistant Cancer Cell Lines

Although the combined effects of cisplatin (CDDP) and DNA topoisomerase (Topo) inhibitors have been described in recent literature, little is known about the combined effects and their biological basis in CDDP-resistant cells. The aim of the present study was to elucidate the combined effect of CDDP and Topo inhibitors on CDDP-resistant cells as well as to investigate the biological factors involved in the sensitivity to these anti-cancer agents. We found synergistic actions between CDDP and SN-38 (a Topo I inhibitor) or VP-16 (a Topo II inhibitor) in KFr cells, a CDDP-resistant subline of the KF epithelial ovarian carcinoma cell line, but not in the parent KF cells. We subsequently assayed Topo protein levels and enzymatic activities in two sets of CDDP-sensitive and -resistant cell lines: KF and KFr, and HeLa and HeLa/CDDP. The levels of Topo I protein in the CDDP-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to CDDP. Topo I enzymatic activity, however, was 2- to 4-fold higher in the CDDP-resistant cell lines than in their respective parent cell lines. In contrast, higher levels of Topo lice protein were observed both before and after CDDP exposure in the CDDP-resistant cells than in their controls. However, no difference in Topo II catalytic activity was observed between the CDDP-resistant and -sensitive cells.     

Sequence effect of irinotecan (CPT-11) and topoisomerase II inhibitors in vivo

The DNA topoisomerases I and II are the target of several clinically important antineoplastic agents which produce DNA cleavage by stabilization of the covalent DNA-protein bond with resultant cell death after DNA synthesis is attempted. Depletion of the target topoisomerase and reciprocal changes in the other occur with drug treatment. Purpose and methods: To develop empiric treatment regimens of combinations and sequences of agents directed against topoisomerase I (irinotecan/CPT-11) and II (etoposide and doxorubicin), in vivo studies were performed in mice bearing the EMT-6 mammary tumor to assess efficacy, host tolerance and the resultant biochemical changes in topoisomerase mRNA and protein. Results: At 24 h after therapy, depletion of the target topoisomerase mRNA and protein with reciprocal increases in the alternate topoisomerase mRNA and, to a lesser extent, protein were noted. No therapeutic antagonism was found with any combination or sequence of agents, and therapeutic antagonism was noted with concurrent irinotecan/etoposide and sequential doxorubicin/irinotecan. Depletion of target topoisomerases by combined therapy beyond a threshold necessary for therapeutic efficacy produced no additional benefit. Conclusions: Antineoplastic therapy with combinations of topoisomerase I and II agents is feasible and may produce therapeutic synergy. The appropriate sequence may depend on the particular agents used. The rationale for such therapy, that topoisomerases I and II may have reciprocal and compensatory interactions, is supported by the biochemical data. Received: 8 September 1997 / Accepted: 15 January 1998     

Topoisomerase II and the Response to Antileukemic Therapy

A number of recent studies have investigated the expression of topoisomerase II in clinical leukemia specimens. Here we outline the rationale for these studies, identify potential pitfalls, summarize recent results, and discuss unanswered questions in this area.     

Renal function in children and adolescents following 72 g/m2 of ifosfamide

A detailed analysis of the renal function of 18 children and adolescents aged 7–20 years (median, 16 years) was performed at least 3 months following the completion of a non-platinum-containing chemotherapy regimen with a total dose of 72 g/m² of ifosfamide. Ifosfamide had been given as a 1-h infusion of 1.8 g/m² daily for 5 days at 5- to 6-week intervals along with mesna uroprotection. The mean glomerular filtration rate (GFR) as determined by inulin clearance was 100 ml/min/1.73 m². Although 6 of 18 patients had GFRs below normal, the lowest was only 18% less than the lower limit of normal and would not account for any clinical compromise. The renal plasma flow and filtration fraction were normal. Proximal tubular function evaluation revealed normal fractional excretion (FE) of glucose; normal mean tubular maximum phosphate reabsorption per GFR (TMP)/GFR values; high FE of urate (17%); and mild, generalized aminoaciduria in 6 of the 18 patients. Distal tubular function evaluation showed normal 24-h urinary calcium levels and FE of magnesium as well as normal urinary osmolality after water deprivation. Two patients had mild proteinuria. The findings in this study are encouraging in terms of the lack of clinically significant renal abnormalities observed in patients who had received a cumulative dose of 72 g/m² of ifosfamide.This work was supported by a grant from the Eagles Foundation and a Career Development Award from the American Cancer Society     

灯盏花素对顺铂肾损害的防治及抗氧化的实验

目的观察灯盏花素对小鼠顺铂（cisplatin,DDP）肾损害的防治及抗氧化作用,探究其可能的作用机制。方法以 DDP 8 mg/kg单次腹腔注射制备小鼠肾脏损害模型,再以不同剂量的灯盏花素灌胃,1次/天,连续给药7 d后采样,观察肾脏结构变化,检测血清中肌酐（Scr）、尿素氮（BUN）、丙二醛（MDA）含量及超氧化物歧化酶（SOD）活性,肾皮质 SOD 及 MDA 的变化。 结果灯盏花素可明显改善顺铂肾损害的小鼠肾脏结构,降低血清Scr、BUN及肾皮质 MDA含量（P<0.05,P<0.01）,而 SOD 活性明显升高（P<0.05）。 结论灯盏花素可明显减轻顺铂引起的肾毒性,其机制可能与抑制顺铂肾损害所致血液和肾皮质脂质过氧化反应增强有关。     

Antitumor Activity of a Novel Quinoline Derivative, TAS-103, with Inhibitory Effects on Topoisomerases I and II

A novel quinoline derivative, TAS-103 (6-[[2-(dimethyIamino)ethyl]amino]-3-hydroxy-7 H -indeno[2,l-c]quinolin-7-one dihydrochloride), was developed as an anticancer agent targeting topoisomerases (topo) I and II, with marked efficacy in solid tumors. TAS-103 inhibited topo I and II (IC₅₀: 2 μM, 6.5 μM ) at a concentration similar to or lower than those of previous agents, and had a strong cytotoxic effect on P388 and KB cells (IC₅₀,: 0.0011 μM, 0.0096 μM ). TAS-103 stabilized topo I and II-DNA cleavable complexes in KB cells, generating a similar amount of topo II-DNA complex to that induced by etoposide (VP-16) but a smaller amount of topo I-DNA complex than that produced by camptothecin (CPT). In the in vivo study, intermittent i.v. administration was markedly effective against s.c.-implanted murine tumors. Furthermore, TAS-103 had marked efficacy against various lung metastatic tumors, and a broad antitumor spectrum in human tumor xenografts (derived from lung, colon, stomach, breast, and pancreatic cancer). The efficacy of TAS-103 was generally greater than that of irinotecan (CPT-11), VP-16, or cis -diamminedichloroplatinum (CDDP).     

Biomodulation by Hyperthermia of Topoisomerase II-Targeting Drugs in Human Colorectal Cancer Cells

We examined whether heat stress could enhance the sensitivity of human colon cancer WiDr cells to topoisomerase II-targeting anticancer agents, etoposide (VP-16) and teniposide (VM-26), and also determined the most effective timing for the drug administration after exposure to hyperthermia. Both topoisomerase II contents and topoisomerase II activity were significantly increased in WiDr cells 3 to 12 h after heat stress at 43°C for 1 h, in comparison with those immediately after the heat stress. Cytotoxicity by VP-16 was most significantly enhanced 3 to 12 h after exposure to 43°C for 1 h, but no synergistic effect was observed when the drug was administered immediately after the heat stress. A combination of VM-26 with heat stress, but not that of a topoisomerase I-targeting camptothecin derivative (CPT-11), or vincristine, showed a synergistic cytotoxic effect on WiDr cells. VP-16 alone induced cellular accumulation at the G2+M phase, whereas the combination of VP-16 and heat stress further increased the cell population at the G2+M phase, and decreased S-phase cells. A possible application of the combination of VP-16 and hyperthermia in clinical use is discussed.     

Expression of Drug Resistance Genes in VP-16 and mAMSA-selected Human Carcinoma Cells

The cell lines described in the present study were isolated as part of an effort to understand resistance to topoisomerase (topo) II inhibitors. To that end, 50 sublines were isolated from four human breast cancer cell lines, i.e., MCF‐7, T47D, MDA‐MB‐231, and ZR‐75B. As an initial step, a concentration that would be lethal to the majority of cells (IC99) was selected for both VP‐16 and mAMSA, for each cell line. The identification of an increasing number of putative drug resistance‐related proteins provided the opportunity to examine expression of the corresponding genes in the selected cell lines. Northern blot analysis revealed different responses to the selecting agents in the different cell lines. Previous studies examining expression of multidrug resistance (MDR)‐l in resistant cell lines had found undetectable levels in all cells. In the ZR‐75B sublines, increased expression of MDR‐associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) was observed, and when the relative levels of overexpression were compared, a high correlation was found. In contrast, increased expression of MRP was observed in some of the MDA‐MB‐231 sublines, without a concomitant increase in cMOAT expression. Finally, in both T47D and MCF‐7 sublines, increased expression of cMOAT or MRP was observed infrequently, and where it occurred, was of a much smaller magnitude. In the analysis of expression of MRP, the highest levels were found in the ZR‐75B and MDA‐MB‐231 sublines, with lower levels in the MCF‐7 and T47D clones. Similarly, differences in the expression of topo IIα were observed among the sublines. Although the differences in expression appear to depend on the parental cell line from which the resistant sublines were derived, a strong correlation was observed between the expression of MRP and the levels of topo IIα. Cell lines with low levels of MRP had lower levels of topo IIα, while those with high levels of MRP maintained higher levels of topo IIα. While a reduced topo IIα level was common, there did not appear to be a compensating increase in the expression of topo IIβ or topo I or casein kinase (CK) IIα in any of the cell lines. While the possibility that such compensation could occur has been discussed and even reported in some cell lines, such an adaptation was not observed in the present study, suggesting that it is not common.