In vitro pharmacology of cryptophycin 52 (LY355703) in human tumor cell lines

Purpose: Cryptophycin 52 (LY355703) is a new member of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The mechanism of action of the cryptophycin class of compounds is associated with an action on microtubules. This report details the pharmacological profile of this new clinical compound in a panel of human tumor cell lines. Methods: Antiproliferative effects of cryptophycin 52 were measured indirectly by detection of the metabolic reduction of alamarBlue^®. Cytoxicity was assessed by enzymatic dye activation (calcein AM) combined with dye exclusion (ethidium homodimer) and by clonogenicity assay. Cell cycle effects were evaluated using flow cytometry and fluorescence microscopy. Results: Both antiproliferative and cytotoxic effects of cryptophycin 52 were concentration- and time-dependent. IC₅₀ values for antiproliferative activity in both solid and hematologic tumor cell lines were in the low picomolar range, and without exception, were significantly below values for the antimitotic agents paclitaxel and vinblastine. Flow cytometry and microscopic examination of tumor cells treated with cryptophycin 52 indicated that they accumulated in the mitotic phase of the cell cycle. Cryptophycin 52 was tested for its sensitivity to multidrug-resistance in several paired cell lines in which a sensitive parental line was matched with a multidrug-resistant derivative line. The resistant lines have been shown to over express Pgp and/or MRP multidrug-resistance transport factors. Compared to other antimitotic agents (paclitaxel, vinblastine, vincristine), the potency of cryptophycin 52 was shown to be minimally affected in multidrug-resistant cells compared to their sensitive parental lines. Conclusion: Cryptophycin 52 has potent antimitotic, antiproliferative and cytotoxic activity in in vitro human tumor cell models. It is significantly more potent and less sensitive to multidrug resistance mechanisms than other antimitotic antitumor agents currently used in cancer therapy. These characteristics may translate into therapeutic advantages for the clinical use of cryptophycin 52 in cancer chemotherapy.     

Resistance to paclitaxel mediated by P-glycoprotein can be modulated by changes in the schedule of administration

Purpose: Increasing use of paclitaxel in clinical oncology has stimulated interest in its mechanisms of resistance and ways to overcome these. Studies were performed with paclitaxel to determine the role of P-glycoprotein in drug sensitivity, and the effect of schedule on relative resistance. We have previously reported that prolonged exposure to P-glycoprotein substrates decreases relative resistance in multidrug resistant cells. Methods: Using both unselected and drug-selected cell lines, cross-resistance and cytotoxicity reversal studies using cyclosporin A were performed. In multidrug-resistant cells, cross-resistance was evaluated after 3-, 24-, and 96-h exposures to paclitaxel. Results: Cross-resistance to paclitaxel in P-glycoprotein-expressing sublines was shown to be comparable to that of other drugs transported by P-glycoprotein. Sensitivity to paclitaxel could be modulated by cyclosporin A in unselected cell lines expressing P-glycoprotein and not in P-glycoprotein-negative cell lines. Resistance to paclitaxel was reduced tenfold by increasing the duration of exposure in P-glycoprotein-expressing cells. This effect was not observed in a paclitaxel-resistant cell line which does not express P-glycoprotein. Conclusions: These studies extend observations on the schedule dependence of paclitaxel cytotoxicity and the role of P-glycoprotein in mediating paclitaxel sensitivity. The schedule dependence of relative resistance suggests that infusional paclitaxel may help in overcoming P-glycoprotein-mediated resistance. Received: 12 February 1996 / Accepted: 16 November 1996     

Activity of dolastatin 10 against small-cell lung cancer in vitro and in vivo: induction of apoptosis and bcl-2 modification

Purpose: Dolastatin 10 is a natural cytotoxic peptide which acts through the inhibition of microtubule assembly. Studies have suggested that such agents can induce apoptosis in association with bcl-2 phosphorylation. Since bcl-2 overexpression is common in small-cell lung cancer (SCLC), we evaluated the activity of dolastatin 10 in SCLC cell lines and xenografts. Methods: In vitro growth inhibition was evaluated with a standardized MTT assay and apoptosis with fluorescent microscopy and a TUNEL assay. Immunoblot analysis and phosphatase digestion were used to determine bcl-2 modification. In vivo activity was evaluated in subcutaneous and metastatic SCLC xenograft models in SCID mice. Results: Dolastatin 10 had growth inhibitory activity against four SCLC cell lines (NCI-H69, -H82, -H446, -H510) with IC₅₀ values ranging from 0.032 to 0.184 nM. All four cell lines exhibited evidence of apoptosis after 48 h of exposure to 1.3 nM dolastatin 10. Immunoblot analysis revealed that 1.3 nM dolastatin 10 altered the electrophoretic mobility of bcl-2 in NCI-H69 and -H510 cells within 16 h of treatment. Incubation of protein extract from dolastatin 10-treated NCI-H69 and -H510 cells with calcineurin resulted in the disappearance of the altered mobility species, suggesting dolastatin 10-induced bcl-2 phosphorylation. In in vivo studies, 450 μg/kg of dolastatin 10 IV × 2 given after intravenous injection of NCI-H446 cells completely inhibited tumor formation. In established subcutaneous NCI-H446 xenografts, 450 μg/kg of dolastatin 10 IV induced apoptosis in the majority of tumor cells within 96 h, resulting in a log₁₀ cell kill of 5.2 and an increase in median survival from 42 to 91 days. Conclusions: These findings suggest that dolastatin 10 has potent activity against SCLC and that the modulation of apoptotic pathways deserves further evaluation as an anticancer strategy. Received: 14 July 1998 / Accepted: 3 September 1998     

Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents

Purpose: Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resistance to paclitaxel. Methods: The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy. Results: Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3–5-fold for the PTX analog versus 25–30-fold for PTX. Conclusion: Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitaxel resistance. Received: 30 October 1996 / Accepted: 9 December 1996     

Antitumor activity of magainin analogues against human lung cancer cell lines.

Magainin 1 and magainin 2, originally isolated from African clawed frog Xenopus laevis skin, inhibit the growth of bacteria and fungi. Synthetic magainin A (MAG A) and magainin G (MAG G) are more potent against bacteria and protozoa. In order to determine the antitumor activity of these analogues, we have tested these two analogues against six small cell lung cancer (SCLC) cell lines NCI-H82, NCI-H526, NCI-H678, NCI-H735, NCI-H841, and NCI-H889, which were known to differ by more than 10-fold in their sensitivity to different chemotherapeutic agents, and four normal human fibroblast cell lines. Semiautomated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays of the six SCLC cell lines revealed average concentrations producing 50% inhibition (IC50) values of 2.6 microM (range, 0.49-9.30 microM) for cisplatin, 2.5 microM (range, 0.39-6.00 microM) for etoposide, and 138.8 nM (range, 55.0-450.0 nM) for doxorubicin. The average IC50 of MAG A was 8.64 microM (range, 6.23-11.7 microM) and that of MAG G was 8.82 microM (range, 4.44-12.5 microM) against the SCLC cell lines. Despite a 10-fold difference in sensitivity to standard chemotherapeutic agents, the IC50 of MAG A and MAG G differs by less than 3-fold. The average IC50 against four normal human fibroblast cell lines was 21.1 microM (range, 12.7-25.6 microM) for MAG A and 29.2 microM (range, 21.3-34.8 microM) for MAG G. Combined exposure to the IC50 concentration of MAG A or MAG G plus IC50 of etoposide or cisplatin decreased the percentage of surviving SCLC cells to 29.0% (range, 26.1-31.7%). MAG A or MAG G had an additive effect when used with standard chemotherapeutic agents. These data suggest that MAG A and MAG G have in vitro antitumor activity against SCLC cell lines.     

Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel

Purpose: To determine, as we did for paclitaxel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drug's antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. Methods: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxel's antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. Results: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4 +/− 0.4% to 25.1 +/− 0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3 +/− 1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. Conclusions: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxel's antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors. Received: 3 March 1998 / Accepted: 14 May 1998     

Human tumor models in the severe combined immune deficient (scid) mouse

Purpose: To test a number of established human tumor cell lines and early passage breast cancer (UACC2150) and melanoma cells (UACC1273) for growth in the scid mouse and the tumors' response to conventional chemotherapeutic drugs. Methods: Established melanoma (A375, C81-61), colon (SW480), lung (A549), lymphomoblastoid leukemia (LCL-B), promyelocytic leukemia (HL60), prostate (PC-3, DU145), and breast (MCF7) cell lines were injected at subcutaneous (s.c.), intraperitoneal (i.p.), or mammary fat pad (MFP) sites. Tumor volume growth curves and survival curves were established for the various tumor cell lines. Carmustine (BCNU), cisplatin (CDDP), cyclophosphamide (CPA), doxorubicin, dacarbazine (DTIC), tamoxifen and vincristine were injected s.c. or i.p.. The chemotherapeutic drug effects on tumor volumes and survival were determined. Results: Tumor growth occurred with each cell type. After i.p. injection, 90% mortality occurred within 26 to 60 days except for the early passage melanoma cell line UACC1273 with which mortality occurred within approximately 90 days. In the MCF7 breast model, treatment with tamoxifen (P < 0.001) and CPA (P < 0.0001) resulted in significant tumor growth delay compared with control groups. BCNU and CDDP resulted in significant tumor growth delays relative to control in SW480 colon cancer (P < 0.0014) and A375 melanoma (P < 0.0001) models, respectively. CPA and doxorubicin improved survival in the HL60 leukemia model (P = 0.0018). Conclusions: These scid mouse human tumor models appear to reflect the clinical situation in that clinically active chemotherapeutic drugs are similarly active in the scid mouse models. Therefore, the scid mouse models may be useful for testing new chemotherapeutic agents against various human cancer types. Received: 6 March 1996 / Accepted: 3 October 1996     

重组人血管内皮抑制素联合EP方案对小细胞肺癌细胞株NCI-H446的增殖抑制和促细胞凋亡作用

目的:本研究旨在评估依托泊苷(etoposide,VP-16)、顺铂(cisplatin,DDP)(EP方案)联合重组人血管内皮抑制素对小细胞肺癌(small-cell lung cancer,SCLC) NCI-H446细胞的促凋亡和抑制细胞增殖的协同作用.方法:EP方案、重组人血管内皮抑制素单药以及EP方案联合重组人血管内皮抑制素作用于NCI-H446细胞72 h后,采用CCK-8(cell counting kit-8)法测定药物对NCI-H446细胞的增殖抑制作用,FCM检测药物对NCI-H446细胞的细胞周期分布的影响,ELISA法检测药物对NCI-H446细胞分泌血管内皮细胞生长因子(vascularendothelial cell growth factor,VEGF)水平的影响.结果:EP方案联合重组人血管内皮抑制素的细胞增殖抑制率明显高于EP方案组(P＜0.01).EP方案组和EP方案联合重组人血管内皮抑制素组的NCI-H446细胞大多被阻滞于G1期,EP方案联合重组人血管内皮抑制素组的NCI-H446细胞凋亡率显著高于EP方案组(P＜0.01).与EP方案和重组人血管内皮抑制素单药相比,EP方案联合重组人血管内皮抑制素可显著抑制NCI-H446细胞分泌VEGF(P＜0.05).结论:EP方案联合重组人血管内皮抑制素在抑制SCLC NCI-H446细胞增殖和促细胞凋亡方面,具有协同作用.     

Combination effects of cisplatin,vinorelbine and irinotecan in non-small-cell lung cancer cell lines in vitro

 Purpose: Isobologram analysis has been widely used for evaluating the combined effect of two antitumor drugs in vitro as a pre-clinical screening test. In this study, we tried to extend two-dimensional isobologram analysis to three dimensions for evaluating the effects of a three-drug combination. Methods: We selected three anticancer agents, cisplatin, vinorelbine and irinotecan. Each of them has been classified as having good single-agent activity against non-small-cell lung cancer (NSCLC). Human NSCLC cell lines (EBC-1, PC-3, RERF-LC-MS) were incubated for 4 days in the presence of the three drugs and cytotoxic activities were determined by a tetrazolium-based colorimetric assay (MTT assay). The data were analyzed by three dimensional isobologram analysis. Results: The effects of the three drugs were additive against EBC-1 (a squamous cell carcinoma cell line), subadditive against PC-3 (an adenocarcinoma cell line) and from subadditive to supraadditive against RERF-LC-MS (an adenocarcinoma cell line). Conclusions: Our findings suggest that the effects of cisplatin, vinorelbine and irinotecan incombination are additive against NSCLC in vitro. These results encourage clinical trials of the three agents in combination chemotherapy for the treatment of NSCLC. Received: 8 December 1995/Accepted: 18 May 1996     

Association of cisplatin nephrotoxicity with patient characteristics and cisplatin administration methods

Objective: To assess factors that affect cisplatin nephrotoxicity. Methods: In 425 patients treated with cisplatin, we assessed the effect of pretreatment factors and treatment conditions on the rise in serum creatinine with the first course of cisplatin, on the maximum rise in serum creatinine over the entire course of the cisplatin therapy, and on residual nephrotoxicity after the last cisplatin treatment ended. (Because of the nature of the relationship between serum creatinine and creatinine clearance, rise in serum creatinine was divided by pretreatment creatinine squared.) Patients were dichotomized into the upper quartile versus the lower three quartiles of degree of nephrotoxicity. Multivariate analyses were based on logistic regression, controlling for cisplatin dose per course. Results: Controlling for cisplatin dose per course, factors most closely associated with nephrotoxicity during the first course of cisplatin were: serum albumin and potassium, body surface area, and administration of cisplatin over 2–5 days per course vs 1 day (negative associations). Controlling for cisplatin dose per course, the single factor most closely associated with maximum life-time cisplatin nephrotoxicity was concurrent use of a vinca alkaloid (negative association). Controlling for cisplatin dose per course, factors most closely associated with residual nephrotoxicity after the end of cisplatin therapy were cumulative dose of cisplatin, concurrent use of metoclopramide (positive associations), uric acid and concurrent use of phenytoin and a vinca alkaloid (negative associations). The association of nephrotoxicity with uric acid and with body surface area was felt to be an artifact resulting from its positive association with pretreatment serum creatinine. Nephrotoxicity during the first course of cisplatin also correlated significantly with autopsy kidney cortex platinum concentrations in 77 evaluable patients. Conclusions: (1) While several factors correlated with cisplatin nephrotoxicity, most of the observed nephrotoxicity was not explained by the variables identified. (2) While most patients received intravenous hydration, patients receiving high hydration volumes did not have significantly less nephrotoxicity than patients receiving lower hydration volumes. (3) Of the variables identified, serum albumin, metoclopramide and phenytoin may have affected nephrotoxicity by altering cisplatin uptake into or distribution within the kidney. Received: 28 February 1996 / Accepted: 17 December 1996     

小细胞肺癌组织显著低表达的SPIDR通过降低血清依赖促进NCI-H446细胞增殖

目的：探讨同源重组修复途径关键调控蛋白SPIDR在小细胞肺癌（small cell lung cancer,SCLC)中的作用与机制。方法：收集2013 年1 月至2015 年1 月上海肺科医院进行肿瘤手术切除、气管镜穿刺的SCLC患者癌组织标本60 例及正常人群肺组织标本44 例,qRT-PCR检测临床组织样本SPIDR mRNA表达水平;经稳定过表达SPIDR 改变NCI-H446 细胞表达水平后,采用MTT、小鼠荷瘤实验等实验方法,在体内、体外探究SPIDR 表达水平对SCLC细胞增殖等影响。结果：吸烟与患者SCLC的发生显著有关（P<0.01）;SPIDR mRNA在SCLC 组织样本中的表达显著低于正常肺组织（P<0.01）。人肺胚成纤维细胞株MRC-5 中SPIDR mRNA和蛋白表达水平明显高于SCLC细胞株NCI-H446（均P<0.05）。在10%胎牛血清常规培养体系中,过表达SPIDR对NCI-H446 细胞增值和化疗药物敏感性无明显影响（均P>0.05）,但小鼠荷瘤实验从第9 天开始,过表达SPIDR组（pMSCV-SPIDR）的瘤体积与原始NCI-H446 组和空载体组（pMSCV）开始出现明显差异,第27 天pMSCV-SPIDR 组移植瘤平均体积分别比原始NCI-H446 组与空载体组缩小58.99%和61.84%（均P<0.01）。在含1%~3%胎牛血清的非常规培养体系中,过表达SPIDR 的NCI-H446 细胞增殖速度显著低于原始NCI-H446 组和空载体组（P<0.05 或P<0.01）。结论：SCLC组织中SPIDR表达水平明显低于正常肺组织,过表达SPIDR的NCI-H446 细胞体内及体外低血清含量培养（<3%）生长速度显著低于对照组,表明SPIDR以低血清浓度依赖方式影响SCLC细胞增殖。     

Combination of arsenic trioxide and chemotherapy in small cell lung cancer

Introduction

Small cell lung cancer (SCLC) carries high mortality despite standard chemotherapy. Arsenic trioxide (ATO) has demonstrated clinical efficacy in leukemia and in vitro activity in various solid tumors. This study was conducted to determine the in vitro and in vivo combination effects of ATO and chemotherapy in SCLC.

Materials and methods

The in vitro model consisted of 5 SCLC cell lines (H187, H526, H69, H841 and DMS79) and the anti-proliferative effects of ATO, cisplatin, etoposide or combinations thereof were measured. Synergism was determined by calculation of the combination index (CI) according to Chou and Talalay. Assays for apoptosis, intracellular glutathione (GSH) content, and mitochondrial membrane depolarization (MMD) were performed. Arsenic content was measured by inductively coupled plasma-mass spectrometry. Expression level of MRP1, MRP2 and pH2AX was detected by Western blot while cellular pH2AX level was monitored by immunofluorescent staining. An in vivo xenograft model in nude mice was established with a H841 cell line to test the effects of drug combinations.

Results

All 5 SCLC cell lines were sensitive to ATO, with IC₅₀ values (48 h) 1.6–8 μM. Synergistic or additive effects were obtained by combining cisplatin with ATO in all 5 cell lines. Combination of etoposide with ATO resulted in antagonistic or barely additive effects. Apoptotic assays and pH2AX immunofluorescent staining corroborated the synergistic combination of ATO and cisplatin. In addition, the ATO/cisplatin combination enhanced MMD, depleted GSH, downregulated MRP2 and elevated intracellular ATO content compared with either ATO or cisplatin alone. In vivo combination of ATO and cisplatin also demonstrated synergism in the H841 xenograft model.

Conclusions

There was clinically relevant in vitro activity of ATO in a panel of 5 SCLC cell lines. Significant synergism was demonstrated with the ATO/cisplatin combination, while antagonism was noted with the ATO/etoposide combination in both in vitro and in vivo models.     

A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.     

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity

Purpose: Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185^erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. Methods: We performed Western blot analysis of Raf-1, p185^erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. Results: We found that downregulation of Raf-1, p185^erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 μM). These inactive drugs also had no or minimal antiproliferative activity (IC₅₀ > 3 μM). All other analogs (at a concentration of 2 μM) downregulated p53, p185^erbB2, and Raf-1, and also displayed cytotoxicity (IC₅₀ in the range 6–600␣nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC₅₀ for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185^erbB2 and Raf-1 were observed at doses that were 4–5 times greater than the cytotoxic IC₅₀. Conclusions: These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185^erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism. Received: 6 June 1996 / Accepted: 28 September 1996     

Combination chemotherapy involving orally administered etoposide and JM-216 in murine tumor models

Purpose: Orally administered VP-16 (etoposide) was evaluated in combination with an orally administered platinum analog, JM-216 [ammine/cyclohexylamine diacetatodichloride Pt(IV)], in mice bearing murine tumors, for therapeutic synergy. Methods: The treatment schedules used involved two courses of therapy, each course consisting of administration every day for 5 days beginning on either day 4 or day 5 posttumor implantation, and again on day 11 or day 12 postimplantation. Result: The amounts of each drug tolerated in the combination treatment setting were much less than their individual maximum tolerated doses (MTDs). Thus, to be used safely, each drug's dose had to be greatly reduced from the amount tolerated when the drugs were given individually. Multiple experiments using a staged P388 leukemia model implanted intravenously yielded confirmatory data supporting the existence of a therapeutic synergy for the drug combination. Identical regimens applied in the staged M5076 sarcoma model implanted subcutaneously, however, were not considered to have yielded data indicative of therapeutic synergy. Conclusions: A clinical phase I study using this combination chemotherapy can be recommended on the basis of the results obtained in the leukemia model. Received: 25 May 1996 / Accepted: 29 October 1996     

In vitro antitumor activity of 3′-desamino-3′(2-methoxy-4-morpholinyl) doxorubicin on human melanoma cells sensitive or resistant to triazene compounds

A new methoxymorpholinyl derivative of Adriamycin (ADR), FCE 23762 (MRD), has recently been selected for phase I clinical trials for its reduced cardiotoxicity and for its cytotoxic activity against a broad spectrum of solid tumors and leukemias that are sensitive or resistant to ADR. The purpose of the present study was to compare the in vitro antitumor activity of MRD and ADR on human melanoma lines with different chemosensitivity to triazene compounds, among which dacarbazine remains a reference drug in the treatment of melanoma. Both MRD and ADR were tested in vitro on three melanoma lines, MI13443-MEL, SK-MEL-28, and M14, previously screened for their chemosensitivity to the triazene compound p-(3-methyl-1-triazeno) benzoic acid, potassium salt (MTBA). The three lines were also analyzed for P-170 expression, total glutathione (GSH) content, and GSH-related enzyme activity. All melanomas, whether sensitive or resistant to MTBA, were susceptible to anthracycline treatment. The cytotoxic activity of MRD was comparable with that of ADR, and no substantial difference was found in cell growth inhibition between the two drugs. When the relative chemosensitivity of the three lines was considered, SK-MEL-28 was found to be slightly less sensitive to MRD treatment than the other tumors. This finding seems to correlate with the higher GSH-peroxidase activity of this melanoma relative to that of the MI13443 and M14 lines. These results show a homogeneous response of melanoma lines to MRD treatment in vitro, suggesting that phase I clinical trials concerning this drug, which in vivo appears to be activated to a more cytotoxic metabolite, could be extended to metastatic melanomas, including those completely resistant to triazene compounds. Received: 11 August 1996 / Accepted: 20 January 1997     

Anti-tumor Efficacy of Paclitaxel against Human Lung Cancer Xenografts

We examined paclitaxel for anti-tumor activity against human lung cancer xenografts in nude mice and compared its efficacy with that of cisplatin, currently a key drug for lung cancer chemotherapy. Five non-small cell lung cancers (A549, NCI-H23, NCI-H226, NCI-H460 and NCI-H522) and 2 small cell lung cancers (DMS114 and DMS273) were chosen for this study, since these cell lines have been well characterized as regards in vitro and in vivo drug sensitivity. These cells were exposed to graded concentrations of paclitaxel (0.1 to 1000 nM ) for 48 h. The 50% growth-inhibitory concentrations (GI₅₀) for the cell lines ranged from 4 to 24 n M , which are much lower than the achievable peak plasma concentration of paclitaxel. In the in vivo study, 4 cell lines (A549, NCI-H23, NCI-H460, DMS-273) were grown as subcutaneous tumor xenografts in nude mice. Paclitaxel was given intravenously as consecutive daily injections for 5 days at the doses of 24 and 12 mg/kg/day. Against every xenograft, paclitaxel produced a statistically significant tumor growth inhibition compared to the saline control. Paclitaxel at 24 mg/kg/day was more effective than cisplatin at 3 mg/kg/day with the same dosing schedule as above, although the toxicity of paclitaxel was similar to or rather lower than that of cisplatin, in terms of body weight loss. In addition, paclitaxel showed potent activity against 2 other lung cancer xenografts (NCI-H226 and DMS114). Therefore, paclitaxel showed more effective, wider-spectrum anti-tumor activity than cisplatin in this panel of 6 lung cancer xenografts. These findings support the potential utility of paclitaxel in the treatment of human lung cancer     

不同铂类为主联合化疗抗人肺癌细胞株的药效学研究*

探讨洛铂（ＬＢＰ）、顺铂（ＤＤＰ）和卡铂（Ｃａｂ）单药或联合紫杉醇（ＰＴＸ）、多西他赛（ＤＯＣ）和长春瑞滨（ＮＶＢ）抗人肺癌细胞株的作用和活性。方法　选用４种人非小细胞肺癌（ＮＳＣＬＣ）细胞株Ａ５４９、ＮＣＩ-Ｈ４６０、Ｃａｌｕ-３、ＮＣＩ Ｈ２３和小细胞肺癌（ＳＣＬＣ）细胞株ＮＣＩ-Ｈ５２６,用不同浓度的ＬＢＰ、ＤＤＰ和Ｃａｂ单独或与ＰＴＸ、ＤＯＣ和ＮＶＢ联合处理上述肺癌细胞株７２ｈ,应用磺酰罗丹明染色法评价细胞增殖,计算半数抑制浓度（ＩＣ_５０）。结果　ＬＢＰ、ＤＤＰ和Ｃａｂ单药均可抑制体外培养的肺癌细胞株生长,其中ＬＢＰ对４种ＮＳＣＬＣ细胞株的ＩＣ５０为１.７～２.４μｍｏｌ／Ｌ,ＤＤＰ为１.８～５.２μｍｏｌ／Ｌ,Ｃａｂ为２２.８～１００.８μｍｏｌ／Ｌ;ＬＢＰ、ＤＤＰ和Ｃａｂ对ＳＣＬＣ细胞株的ＩＣ５０分别为０.３μｍｏｌ／Ｌ、０.６μｍｏｌ／Ｌ和９.８μｍｏｌ／Ｌ。ＬＢＰ分别与ＰＴＸ、ＤＯＣ和ＮＶＢ联合抗ＮＳＣＬＣ细胞株有明显增效作用,其中与ＰＴＸ合用的协同作用最强。ＤＤＰ或Ｃａｂ分别与ＰＴＸ、ＤＯＣ和ＮＶＢ联合的抑制作用与细胞株的类型有关。结论　在上述３种铂类药物中,ＬＢＰ具有明确的抗ＮＳＣＬＣ细胞作用,与ＤＤＰ相似,而强于Ｃａｂ,其抗癌活性与细胞株的类型无关。ＬＢＰ分别联合ＰＴＸ、ＤＯＣ和ＮＶＢ均有协同作用,其中与ＰＴＸ的联合作用最强。     

Effect of single and multiple administration of an O 6-benzylguanine/temozolomide combination: an evaluation in a human melanoma xenograft model

The purpose of the present study was to examine the effect of O ⁶-benzylguanine (O ⁶-BG) on the antitumour activity and toxicity of 8-carbamoyl-3-methylimidazo [5, 1-d ] -1,2,3,5-tetrazine-4(3H)-one (temo-zolomide) in a human malignant melanoma xenograft model following single and multiple administration of the combination. O ⁶-BG irreversibly inactivates the DNA-repair protein O ⁶-alkylguanine-DNA alkyltransferase (AGT), which confers resistance to temozolomide. Preadministration of O ⁶-BG (35 mg/kg, i.p.) 1 h prior to temozolomide (i.p.) was examined using single and daily × 5 dosing regimens in athymic mice bearing subcutaneous A375P xenografts. The AGT activity of A375P tumors was 95 ± 8 fmol/mg protein (mean ± SE, n = 4). O ⁶-BG alone completely suppressed xenograft AGT activity within 1 h of administration but had no effect upon tumor growth. O ⁶-BG did not significantly increase the tumor growth delay induced by a single 200-mg/kg dose of temozolomide (P>0.05, two-tailed Mann-Whitney test) but did increase the associated mean body weight loss (P<0.025). In contrast, when the same dose of temozolomide was divided into five equal fractions (40 mg/kg) and given with O ⁶-BG on 5 consecutive days, a comparable increase in toxicity was accompanied by a very significant increase in tumor growth delay (P<0.0025), equivalent to that produced by a 3-fold greater dose of temozolomide alone. O ⁶-BG with temozolomide also produced a greater antitumour effect than an equitoxic dose of temozolomide alone on this schedule (P<0.005). These data indicate that the enhancement of temozolomide antitumour activity by O ⁶-BG preadministration is dependent upon the schedule of drug administration, with multiple dosing of O ⁶-BG + temozolomide producing the greatest effect. The results also suggest that prolonged administration of the combination can lead to an increase in the therapeutic index of temozolomide. Received: 8 September 1996 / Accepted: 8 February 1997     

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