利用中效原理观察抗癌药物的相互作用

目的 :利用中效原理体外定量分析抗癌药物苦参素、5 氟尿嘧啶联合应用对人肝癌细胞株(HepG2 )的相互作用。方法 :采用MTT法及中效原理判断联合用药的效果。结果 :两种药物单独及联合应用时随着药物剂量增加 ,其效应也增加。当fa =0 4 1时 ,CI =1,两药作用相加 ;fa <0 4 1时 ,CI <1,两药为协同作用 ;fa >0 4 1时 ,CI>1,两药拮抗。两药合用时给药时间先后次序不同 ,合用效应差异无显著意义 ,而合用时药物浓度比例变化能够影响合用效应。结论 :两种药物合用小剂量为协同 ,大剂量是拮抗 ,其效应大小与药物浓度比例有关 ,而与给药时间先后次序无关。     

利用中效原理观察紫杉醇和木黄酮对子宫内膜癌JEC细胞的作用

[目的]利用中效原理体外定量分析紫杉醇及木黄酮对子宫内膜癌JEC细胞的作用。[方法]采用MTT法观察两药单用及合用时对JEC细胞的抑制率,用中效方程计算两药的中效浓度,并计算两药合用时的合用指数（CI）。[结果]两药单独使用时随着药物剂量的增加,其效应也增加,紫杉醇的中效浓度为5.33μmol/L,木黄酮的中效浓度为190.98μmol/L。两药合用时的中效浓度为49.31μmol/L。两药在高效应剂量时合用效应为协同（CI〉1）,在低效应剂量时合用效应为拮抗（CI〈1）。[结论]紫杉醇联合木黄酮对子宫内膜癌JEC细胞有抑制作用,两种药物大剂量合用时效应为协同,小剂量合用时效应为拮抗。     

紫杉醇联合染料木黄酮对人肝癌细胞株SMMC-7721的作用

目的 利用中效原理体外定量分析紫杉醇及染料木黄酮联合应用对人肝癌细胞株SMMC-7721的作用.方法 采用MTT法观察两种药物单用及合用对肝癌细胞株SMMC-7721的抑制率,然后用中效方程计算各自的中效浓度,并计算两种药物合用时的合用指数(CI).结果 两种药物单独使用时随着药物剂量的增加,其效应也增加,紫杉醇的中效浓度为8.912μmol/L,染料木黄酮的中效浓度为163.680 μmol/L.两种药物合用时的中效浓度为58.340μmol/L.大剂量合用(大于中效浓度)时CI＜1,表示协同;小剂量合用(小于中效浓度)时CI＞1,表示拮抗.结论 紫杉醇联合染料木黄酮对人肝癌细胞株SMMC-7721有抑制作用,两种药物大剂量合用时具有协同作用,小剂量合用时则相互拮抗.     

姜黄素联合木黄酮对人胰腺癌细胞株BXPC-3的作用

目的　体外观察姜黄素及木黄酮对人胰腺癌细胞株 ( BXPC- 3)增殖的抑制作用及两者联合应用时的抑制率及相互作用。方法　采用 MTT法测定两者对人胰腺癌细胞株 ( BXPC- 3)的增殖抑制率 ,利用中效原理判定两药合用的效果。结果　两种药单独应用时随着药物剂量增加 ,其效应也增加 ,它们的相关系数分别为 0 .95 3和0 .998,姜黄素的中效浓度为 2 89.16 μm ol/L,木黄酮的中效浓度为 10 7.5 5 μmol/L;两药合用时它们的相关系数为0 .987,中效浓度为 2 2 5 .2 0 ( 171.6 0 + 5 3.6 0 ) μm ol/L。大剂量 (大于中效剂量 )合用时是协同作用 ( CI<1) ,小剂量(小于中效剂量 )合用时是拮抗作用 ( CI>1)。结论　姜黄素联合木黄酮对人胰腺癌细胞株 BXPC- 3的生长有抑制作用 ,它们合用时大剂量是协同 ,而小剂量则拮抗     

姜黄素和阿霉素联合应用对人肝癌细胞SMMC—7721抑制作用的研究

目的：观察姜黄素和阿霉素联合应用对体外培养的人肝癌细胞SDMMC－7721生长的影响。方法：采用MTT法应用Webb系数判定2药合用效果,应用流式细胞仪和透射电镜观察药物对细胞周期分布及超微结构的影响。结果：2种药物单独应用时随着药物判量增加,其效应也增加,2药大剂量合用时可产生拮抗作用,小剂量合用时可产生协同作用。结论：在体外,2．5mg/L-5mg/L的姜黄素和0．125mg/L-0.5mg/L的阿霉素联合应用,对SMMC－7721具有协同抑制作用。姜黄素对阿霉素有化疗增效减毒作用。     

氟尿嘧啶、顺铂和表柔比星对胰腺癌细胞(Aspc-1)的相互作用

目的观察氟尿嘧啶(5-FU)、顺铂(DDP)和表柔比星(表阿霉素,E-ADM)体外对胰腺癌细胞(Aspc-1)的作用和药物联合应用时的相互作用.方法采用MTT法测定5-FU、DDP和E-ADM单用及合用时对细胞的抑制作用,并应用中效原理判定药物合用的效果.结果单用或合用时随着药物浓度的增加其对细胞的抑制作用也增加.5-FU和DDP合用时小剂量时产生协同作用(CI<1),大剂量时产生拮抗作用(CI>1).5-FU和E-ADM、DDP和E-ADM合用时产生轻度拮抗作用(CI>1).结论 5-FU和DDP合用时的相互作用与药物浓度有关,5-FU和E-ADM、DDP和E-ADM合用时在不同浓度表现为轻度拮抗作用.     

Celecoxib与5-FU联用对人肝癌细胞系HepG2细胞增殖的影响

目的体外观察氟尿嘧啶（5-FU）与Celecoxib联用对人肝癌细胞系HepG2细胞增殖的相互作用。方法采用MTT法观察不同浓度Celecoxib、5-FU单独或联合应用对HepG2细胞生长的抑制作用,并利用中效原理判定两药合用的效果。结果两种药物单独应用时,对HepG2细胞的抑制作用呈明显的剂量效应依赖关系;Celecoxib的中效浓度（IC50）为93.55μmol/L,5-FU的中效浓度为734.32μmol/L。这两种药物联用时存在“加速抑制作用”。应用中效原理分析显示,两种药物联用在很宽的效应范围内存在协同效应,增大Celecoxib的用量可在更宽的效应范围内获得两种药物的协同效应,而与药物应用的先后顺序无关。结论Celecoxib与5-FU在联合应用时的相互作用为协同效应,研究结果对肝癌的治疗具有一定的临床意义。     

苦参碱和顺铂联合应用对人骨肉瘤细胞株MG-63增殖抑制作用的研究

[目的]观察苦参碱和顺铂单药及联合应用对人骨肉瘤细胞株MG-63细胞增殖的抑制作用。[方法]采用MTT法（四甲基偶氮唑蓝比色法）,利用中效原理判断药物联合应用的效应。[结果]苦参碱与顺铂联合应用对骨肉瘤细胞株MG-63细胞增殖抑制作用加强,低浓度（≤1．0mg／ml）苦参碱与顺铂联合应用时两者为拮抗作用,〉1．5mg／ml苦参碱与顺铂合用时为相加作用。在苦参碱浓度〉2．0mg／ml联合用药的抑制率接近95％。顺铂以20μg／ml的浓度作用于MG-63细胞48h．增殖抑制率是86．7％,而顺铂以5μg／ml的浓度与2mg／ml的苦参碱联合作用48h,增殖抑制率是92．6％,说明苦参碱和顺铂联用可使顺铂在小剂量时即有较好的杀伤作用。[结论]苦参碱和顺铂联合应用可使顺铂在小剂量时有较好的作用,从而避免了顺铂的毒副作用。     

利用中效原理评价蛇床子素与5-FU相互作用

目的研究中药单体蛇床子素（Osthole,Ost）与5-FU（Fluorouracil,5-FU）在人胃癌细胞株BGC-823以及人大肠癌细胞株LOVO中的体外相互作用。方法采用MTT法检测单药及联合用药对两株肿瘤细胞株的作用;利用中效原理评价两药之间的相互作用;利用流式细胞仪检测单药及联合用药对肿瘤细胞凋亡和周期的影响。结果Ost和5-FU对两株肿瘤细胞株均具有增殖抑制作用;作用于BGC-823细胞时,当Fa〈0．65时,CI〈1,两药合用效应为协同作用;作用于LOVO细胞时,当0．05〈Fa〈0．90时,CI〈1,两药合用效应为协同作用。结论Ost联合5-Fu作用于BGC-823细胞在低剂量合用时产生协同效应,协同机制可能与诱导细胞凋亡及G2／M期阻滞相关。     

羟基喜树碱联合奥沙利铂对人肺癌细胞作用的体外研究

目的: 体外观察羟基喜树碱(hydroxycamptothecine,HCPT)联合奥沙利铂(oxaliplatin,L-OHP)对人肺癌A549细胞增殖的协同抑制作用,并初步探讨其机制.方法: 采用SRB法测定HCPT和L-OHP对A549细胞增殖的抑制作用,根据中效原理判断两药合用时的效应.采用显微镜、流式细胞仪观察两药对A549细胞的诱导凋亡作用.结果: HCPT和L-OHP均能明显抑制A549细胞增殖和诱导细胞凋亡,两药合用具有协同作用.结论: HCPT联合L-OHP能协同抑制A549细胞增殖,其机制可能是通过协同诱导凋亡.     

APPLICATION OF MEDIAN-EFFECT IN QUANTITATIVE ANALYSIS OF ANTITUMOR DRUGS IN VITRO

Chemotherapy is one of the main clinical treatments for malignant tumors. It can bring about long-term remittent effect or even recovery to many sensitive tumors. Nowadays, administration of combined antitumor agents is often adopted to enhance the curative effect without increasing their toxicity while reducing the possibility of tolerance. In chemotherapy, integrated administration of medicines with different functioning mechanisms is preferred so as to bring about its synergistic effect. Wh…     

Application of median-effect in quantitative analysis of antitumor drugs in vitro

Objective: To analyze quantitatively the synergistic and antagonistic effects of combined oxymatrine (OMT) and 5-fluorouracil (5-GU) on a cell line of human liver cancer (HepG2) with median-effect principle in vitro. Methods: The median-effect principle and MTT method were used in the quantitative analysis of effects of the two drugs. Results: Cytotoxic activity of the individual drugs enhanced as drug concentration increased. As fa=0.41, a CI equal to 1 indicated additivity; fa<0.41, a CI less than 1 indicated synergy; and fa>0.41, a CI greater than 1 indicated antagonism. The sequence of administration did not influence the cytotoxic activity of the combined antitumor drugs. The ratio of drug concentration was a factor that can influence the killing effect. Conclusion: The combined drugs interaction (CI<1) was synergistic at lower concentration and antagonistic at higher concentration. The ratio of drug concentration is a factor that can influence the killing effect. Biography: HE Song(1965–), male, doctor of medicine, associate professor, Chongqing Medical University, majors in gastroenterology.     

Sequence-dependent growth-inhibitory effects of the in vitro combination of fluorouracil,cisplatin, and dipyridamole

The present study was designed to analyze the growth-inhibitory effects of the combination of fluorouracil (FUra), cisplatin (CDDP), and dipyridamole (DP). These toxic effects were assessed on the human breast-carcinoma cell line MCF-7 using the MTT (tetrazolium bromide) assay in 96-well culture dishes. Data were analyzed using the median-effect principle. The drug combinations tested included FUra concentrations ranging from 0.8 to 800 nmol/l, CDDP concentrations of 0.3–30 mol/l, and DP concentrations of 2–200 mol/l. A total of 189 different experimental conditions were tested, including different sequences of administration, with being DP applied before, simultaneously with, or after the two antitumor drugs. Synergistic cytotoxic interactions were found between FUra and CDDP, FUra and DP, and CDDP and DP as well as when the three drugs were combined. The sequence of exposure did not influence the growth-inhibitory activity of the combination FUra-CDDP but altered the effect of combinations of either FUra or CDDP with DP, since at lower concentrations the effect shifted from synergism to antagonism when DP was added simultaneously with CDDP and after the two antitumor drugs. However, the interaction was shown to be truly synergistic by median-effect analysis when the two antitumor drugs were simultaneously associated, with no change in the synergistic effect being observed for the three DP administration sequences.Abbreviations FUra fluorouracil - CDDP cisplatin,cis-diamminedichloroplatinum(II) - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (azolyl blue); IC₅₀, concentration inducing 50% inhibition of cell growth; fa, fraction affected; fu, fraction unaffected - Cl combination index     

Synergistic and additive combinations of several antitumor drugs and other agents with the potent alkylating agent adozelesin

Adozelesin is a highly potent alkylating agent that undergoes binding in the minor groove of double-stranded DNA (ds-DNA) at A-T-rich sequences followed by covalent bonding with N-3 of adenine in preferred sequences. On the basis of its highpotency, broad-spectrum in vivo antitumor activity and its unique mechanism of action, adozelesin has entered clinical trial. We report herein the cytotoxicity for Chinese hamster ovary (CHO) cells of several agents, including antitumor drugs, combined with adozelesin. The additive, synergistic, or antagonistic nature of the combined drug effect was determined for most combinations using the median-effect principle. The results show that in experiments using DNA- and RNA-synthesis inhibitors, prior treatment with the DNA inhibitor aphidicolin did not affect the lethality of adozelesin. Therefore, ongoing DNA synthesis is not needed for adozelesin cytotoxicity. Combination with the RNA inhibitor cordycepin also did not affect adozelesin cytotoxicity. In experiments with alkylating agents, combinations of adozelesin with melphalan or cisplatin were usually additive or slightly synergistic. Adozelesin-tetraplatin combinations were synergistic at several different ratios of the two drugs, and depending on the schedule of exposure to drug. In experiments using methylxanthines, adozelesin combined synergistically with noncytotoxic doses of caffeine or pentoxifylline and resulted in several logs of increase in adozelesin cytotoxicity. In experiments with hypomethylating agents, adozelesin combined synergistically with 5-azacytidine (5-aza-CR) and 5-aza-2-deoxycytidine (5-aza-2-CdR). Combinations of adozelesin with tetraplatin or 5-ara-2-CdR were also tested against B16 melanoma cells in vitro and were found to be additive and synergistic, respectively. The synergistic cytotoxicity to CHO cells of adozelesin combinations with tetraplatin, 5-aza-CR, or pentoxifylline was not due to increased adozelesin uptake or increased alkylation of DNA by adozelesin.     

Synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells

Summary We have previously shown that the toxicity of valinomycin (VM), a membrane-active agent with antineo-plastic activity, can be dramatically reduced with no loss of the antitumor efficacy of the drug by incorporating it into liposomes. In the present study, we investigated the interaction betweencis-diamminedichloroplatinum(II) (CDDP) and VM in terms of in vitro cytotoxicity to human ovarian tumor cells. Using the MTT assay and analyzing the data using the median-effect principle, we showed that synergistic cytotoxic interactions exist between CDDP and VM in their liposomal form. The degree of cytotoxic synergism was influenced by the duration of drug exposure and the dose ratio. The cellular accumulation of platinum by ovarian cells at 37°C was slightly higher after exposure to VM as compared with controls; however, it is not clear that this accounts for the cytotoxic synergism. These results suggest that the combination of liposomal VM and CDDP may have merit as a form of localized drug delivery for the treatment of ovarian cancer disseminated within the peritoneal space.Abbreviations CDDP cisplatin,cis-diamminedichloroplatinum(II) - VM valinomycin - MLV-VM liposomal valinomycin - MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (thiazolyl blue) - alpha-MEM alpha minimal essential medium - CHO Chinese hamster ovary - IC₅₀ concentration causing 50% inhibition of cell growth - IC₁₀ concentration causing 10% inhibition of cell growth - SF surviving fraction - fa fraction affected - CI combination index     

Schedule-dependent cytotoxic interaction between epidoxorubicin and gemcitabine in human bladder cancer cells in vitro.

PURPOSE: The aim of the study was to evaluate the activity of epidoxorubicin (EPI) and gemcitabine (GEM) and to define the most effective schedule in human bladder cancer cells. EXPERIMENTAL DESIGN: The study was performed on HT1376 and MCR cell lines. Cells were exposed for 1 and 24 h to drugs used in different schemes. Cytotoxic activity was evaluated by the sulforhodamine B assay, potential clinical activity was estimated by relative antitumor activity, and the type of drug interaction was assessed using the method of Chou and Talalay. Cell cycle perturbations and apoptosis were assessed by flow cytometry; BAX, BCL-2, and P53 expression was evaluated by Western blot; and DNA damage was assessed using the alkaline Comet assay. RESULTS: EPI and GEM produced a cytotoxic effect in both cell lines, with 50% inhibitory concentration and relative antitumor activity values suggestive of a high clinical activity. Simultaneous treatment with EPI and GEM and the sequence GEM-->EPI caused an antagonistic interaction (combination index > 1) after both 1- and 24-h treatments. Conversely, the inverse sequence, EPI-->GEM, produced a synergistic interaction that was more pronounced in MCR cells than in HT1376 cells. The increase in DNA-damaged cells from 10% to 20% after single-drug exposure to 40-60% at the end of EPI-->GEM treatment may explain the synergistic interaction produced by the anthracycline-antimetabolite sequence. CONCLUSIONS: Our findings show that the efficacy of the EPI and GEM combination is highly schedule dependent and indicate that the most active scheme is EPI followed by GEM, which is currently being validated in an ongoing intravesical Phase I-II clinical protocol.     

Determination of drug synergism between the tyrosine kinase inhibitors NSC 680410 (adaphostin) and/or STI571 (imatinib mesylate,Gleevec) with cytotoxic drugs against human leukemia cell lines

The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells. In order to prevent this problem and to augment their antitumor efficacy, these agents could be administered in combination with cytotoxic antineoplastic drugs. We hypothesized that the combination of the antiangiogenesis tyrosine kinase inhibitors with cytotoxic drugs would produce synergistic drug regimens. Two human T-lymphoblastic leukemia cell lines that express VEGF-R1, CEM/0 (wild-type, WT) and the drug-resistant clone CEM/ara-C/I/ASNase-0.5-2, were utilized in the drug combination studies. NSC 680410, a tyrosine kinase inhibitor given at 0.1 to 1 microM for 72 h, inhibited VEGF secretion and leukemic cell growth at 90% of vehicle-treated control cultures with an IC50 value of less than 1 microM. The cytotoxic drugs idarubicin (IDA), fludarabine (Fludara), and cytosine arabinoside (ara-C) were used for the various drug combinations. One-, two-, three-, and four-drug treatments were tested. Cell viability was documented by the MTT assay and photomicrographic estimation of apoptotic cells. Both the combination index (CI) and isobologram evaluations demonstrated strong synergism between these drugs and the tyrosine kinase inhibitor. NSC 680410 was highly synergistic with IDA, IDA + ara-C, and IDA + Fludara + ara-C, over the respective cytotoxic drug regimens at concentrations easily achieved in patient plasma. NSC 680410 potentiated the activity of IDA in both leukemia cell lines by 17.8- and 221.4-fold in the WT and drug-resistant line, respectively. The activity of NSC 680410 + IDA + ara-C was also potentiated by 58.8-fold in the WT line, and the activity of NSC 680410 + IDA + Fludara + ara-C by 2.4- and 6.47x10(6)-fold in the WT and drug-resistant lines, respectively. The results suggest that IDA was not needed for optimal synergistic activity in the CEM/0 cells, but IDA was a necessary component to obtain drug synergism in the drug-resistant clone. Similarly, STI571 (imatinib mesylate, Gleevec), the p210(bcr/abl) tyrosine kinase inhibitor, demonstrated synergism with Fludara + ara-C or IDA + ara-C. Most importantly STI571 showed synergism with NSC 680410, suggesting that these drugs inhibit different tyrosine kinase domains in human leukemia cells. Lastly, pretreatment of leukemic cells with NSC 680410 showed additivity with gamma radiation in comparison to either treatment modality alone. The data, taken together, suggest that by inhibiting the pro-survival signal transduction pathway (VEGF-R1) and DNA replication by cytotoxic drugs, leukemic cells undergo apoptosis in a synergistic manner. In conclusion, the combinations of antiangiogenesis and DNA-damaging cytotoxic drugs are highly synergistic regimens in both WT and drug-resistant leukemic cell lines and they should be examined further.     

In vitro evaluation of the anticancer drug modulatory effect of hyaluronidase in human gastrointestinal cell lines

In an attempt to establish whether the combination of anticancer drugs with hyaluronidase would result in enhanced cytotoxicity, we have tested a range of 6 continuous cell lines against 4 different chemotherapeutic drugs with or without the addition of various concentrations of the enzyme. Measurement of cytotoxic drug effects has been performed using the Bactec system, a new semiautomated radiometric technique. In only 15 of a total of 144 experiments (11%) was a significant hyaluronidase-mediated potentiation of the single agents' activity seen. In the large majority of experiments, the antiproliferative effect of the combined treatment was classified as additive or subadditive, while in 23% it was antagonistic. Evaluation of the drug modulatory mechanism of hyaluronidase suggested that the combined drug-hyaluronidase effects were independent of the nature of the drug, the exposure mode and the concentration of the enzyme employed. Among the various tumor cell lines tested there was a marked heterogeneity in the sensitivity to the combined effect (P less than 0.0001). In summary, we have not been able to confirm the promising results of early reports of in vitro and in vivo enhancement of the cytotoxicity of antitumor agents by hyaluronidase. Our data emphasize the need for further controlled clinical studies in order to prove or disprove this new therapeutic approach.     

Increased preclinical efficacy of irinotecan and floxuridine coencapsulated inside liposomes is associated with tumor delivery of synergistic drug ratios

Whether anticancer drug combinations act synergistically or antagonistically often depends on the ratio of the agents being combined. We show here that combinations of irinotecan and floxuridine exhibit drug ratio-dependent cytotoxicity in a broad panel of tumor cell lines in vitro where a 1:1 molar ratio consistently provided synergy and avoided antagonism. In vivo delivery of irinotecan and floxuridine coencapsulated inside liposomes at the synergistic 1:1 molar ratio (referred to as CPX-1) lead to greatly enhanced efficacy compared to the two drugs administered as a saline-based cocktail in a number of human xenograft and murine tumor models. When compared to liposomal irinotecan or liposomal floxuridine, the therapeutic activity of CPX-1 in vivo was not only superior to the individual liposomal agents, but the extent of tumor growth inhibition was greater than that predicted for combining the activities of the individual agents. In contrast, liposome delivery of irinotecan:floxuridine ratios shown to be antagonistic in vitro provided antitumor activity that was actually less than that achieved with liposomal irinotecan alone, indicative of in vivo antagonism. Synergistic antitumor activity observed for CPX-1 was associated with maintenance of the 1:1 irinotecan:floxuridine molar ratio in plasma and tumor tissue over 16-24 h. In contrast, injection of the drugs combined in saline resulted in irinotecan:floxuridine ratios that changed 10-fold within 1 h in plasma and sevenfold within 4 h in tumor tissue. These results indicate that substantial improvements in the efficacy of drug combinations may be achieved by maintaining in vitro-identified synergistic drug ratios after systemic administration using drug delivery vehicles.