Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro

The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C → T and C → S), and (3) TPT or SN-38 followed by cisplatin (T → C and S → C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C → T (or C → S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROV_CDDP was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROV_CDDP lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G₂/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors. Received: 20 April 1997 / Accepted: 28 August 1997     

Abrogated energy-dependent uptake of cisplatin in a cisplatin-resistant subline of the human ovarian cancer cell line IGROV-1

The parental IGROV-1 human ovarian adenocarcinoma cell line was intermittently exposed to increasing concentrations of cisplatin to obtain resistant sublines. A stable resistant subline with a resistance factor of 8.4 had been developed after 9 months and 28 passages, which was denoted IGROV_CDDP. A high correlation coefficient of 0.97 was found between the log cell survival and the DNA-adduct peak level during the process of resistance development. IGROV_CDDP was strongly cross-resistant to carboplatin and doxorubicin and moderately cross-resistant to etoposide, docetaxel, and topotecan. Only minor resistance against 5-fluorouracil was observed, whereas IGROV_CDDP was not cross-resistant to methotrexate. Intracellular accumulation of cisplatin was 65% lower in IGROV_CDDP as compared with parental IGROV-1 at 37  °C under normal conditions. Coincubation of cisplatin with the Na⁺/K⁺-ATPase inhibitor ouabain resulted in a more pronounced decrease in platinum accumulation in IGROV-1 (44% decrease) than in IGROV_CDDP (26% decrease). Under energy-depleting conditions the accumulation of cisplatin in the parental cell line was approximately 60% lower than that observed under normal (energy [i.e., ATP] rich) culture conditions. In contrast, the accumulation in IGROV_CDDP was not affected by ATP-depletion. There appeared to be no significant difference between the intracellular accumulation of platinum in the resistant and sensitive cells under conditions of energy deprivation or when the uptake was studied at 0  °C. In conclusion, abrogation of energy-dependent accumulation in IGROV_CDDP seems to be a major mechanism of resistance to cisplatin in this cell line. Received: 21 January 1997 / Accepted: 22 July 1997     

The significance of the sequence of administration of topotecan and etoposide

 Purpose: Often the best method of integrating chemotherapeutic agents is unknown. Recently there has been interest in the use of combinations of the topoisomerase II inhibitors and the topoisomerase I inhibitors as these agents have shown individual activity in malignancies such as non-small-cell lung cancer. This study examined the interaction of the topoisomerase II inhibitor etoposide with the topoisomerase I inhibitor topotecan (Tpt) in V79 cells (hamster lung fibroblast cells) to determine the optimal method of delivering these agents. Methods and results: Cell survival was assessed by colony formation. Synergistic interactions were assessed by the median effect principle in which a combination index (CI) of less than one suggests a synergistic interaction. The V79 cells were exposed to sequential 24-h incubations with the two chemotherapeutic agents. Initially, equitoxic doses of the two agents were delivered (i.e. 0.0275 μg/ml of topotecan alone or 0.089 μg/ml of etoposide alone resulting in a surviving fraction of 70%; Tpt : etoposide ratio 1 : 3.2). It was determined that a sequence-dependent synergistic interaction (CI<1) resulted at a lower level of cytotoxicity if the etoposide exposure followed the Tpt exposure compared to the opposite sequence. This same effect was seen after treatment of cells with various concentration (μg/ml) ratios of Tpt : etoposide (1 : 4.0, 1 : 1, 2.5 : 1). Conclusions: These results suggest that maximum synergy occurs for the delivery of etoposide following Tpt exposure (compared to the opposite sequence) and these findings may have important clinical implications. Received: 29 September 1995/Accepted: 25 March 1996     

Enhancement of cisplatin cytotoxicity by terbium in cisplatin-resistant MDA/CH human breast cancer cells

Purpose: The development of cisplatin resistance is a major problem in the treatment of cancer patients with cisplatin chemotherapy. The membrane binding of terbium (Tb³⁺) has been shown to increase the cellular accumulation of cisplatin in breast cancer cells. Therefore, the ability of Tb³⁺ to modulate the cytotoxicity of cisplatin was investigated in cisplatin-sensitive (MDA) and cisplatin-resistant (MDA/CH) MDA-MB-231 human breast cancer cells. Methods: The cytotoxic parameters of cisplatin were determined using live cell microfluorometry and median effect analysis. Results: MDA/CH cells (IC₅₀ = 142 ± 9 μM) were found to be approximately 3.3-fold more resistant to cisplatin than MDA cells (IC₅₀ = 43.5 ± 3.0 μM). In both cell lines, the IC₅₀ value for cisplatin was reduced two-fold in the presence of 80 μM Tb³⁺, thus indicating that the cytotoxicity of cisplatin is increased by Tb³⁺. The cytotoxic activity of cisplatin alone was observed to be 5.7 and 1.6 times more potent than that of Tb³⁺ alone in MDA and MDA/CH cells, respectively. Combination index analyses revealed that the interaction between cisplatin and Tb³⁺ was only synergistic at very low indices of cell death in MDA cells. However, in MDA/CH cells, the two drugs were synergistic up to intermediate levels of cell death. Conclusions: Our results suggest that the enhancement of cisplatin cytotoxicity by Tb³⁺ is more effective in cisplatin-resistant MDA/CH cells than in cisplatin-sensitive MDA cells. Therefore, terbium is potentially useful in cisplatin combination therapy for breast cancer patients, especially for those patients who have developed resistance to the drug. Received: 16 December 1998 / Accepted: 19 January 1999     

Therapeutic effects of the combination of fenretinide and all-trans-retinoic acid and of the two retinoids with cisplatin in a human ovarian carcinoma xenograft and in a cisplatin-resistant sub-line

We previously showed that fenretinide (4-HPR), a synthetic derivative of all-trans retinoic acid (RA), is effective in mice bearing the human ovarian carcinoma IGROV-1 and it significantly enhances the antitumour activity of cisplatin on the same tumour. The present study examined the therapeutic effects of the combination of 4-HPR and RA and of the two retinoids with cisplatin as intracavitary treatments of mice bearing IGROV-1 and IGROV-1/cisplatin tumours, the latter derived from a sub-line with an in vivo reduced sensitivity to cisplatin. 4-HPR, as a single agent, was effective against both tumours, whereas RA had no effect. In IGROV-1 tumour-bearing mice, the combination of RA and 4-HPR significantly improved the efficacy of 4-HPR, resulting in an antitumour activity similar to that obtained with cisplatin alone. N-(4-methoxyphenylretinamide), the main metabolite of 4-HPR, had no antitumour effect and it did not increase 4-HPR activity in IGROV-1 tumour-bearing mice. In the same tumour model, 4-HPR and RA separately increased cisplatin activity, even though for RA the increase was not statistically significant. In contrast, the association of the two retinoids together with cisplatin did not produce any benefit and resulted in increased toxicity. In IGROV-1/cisplatin tumour-bearing mice, the association of 4-HPR (but not of RA) to cisplatin significantly increased cisplatin activity, resulting in the reversal of cisplatin resistance. These findings demonstrate that 4-HPR may be effective and enhance cisplatin sensitivity in cisplatin-sensitive and -resistant ovarian tumours and that the association of RA and 4-HPR may result in increased 4-HPR antitumour activity.     

Therapeutic uses of topotecan for thoracic malignancies

Topotecan, a topoisomerase 1 inhibitor (topo1-i), is a semi-synthetic derivative of camptothecan with well-established cytotoxic properties. It is licensed for the treatment of relapsed, sensitive small cell lung cancer (SCLC) and for second line treatment in ovarian cancer. Studies have also shown this drug to be an effective first line treatment for SCLC with either cisplatin or in combination with a topoisomerase 2 inhibitor (topo2-i), etoposide; both combinations have shown similar efficacy. Oral and parenteral formulations of topotecan have proven to be equivocal in relapsed SCLC patients. The parenteral form showed reduced rates of severe haematological toxicities and better symptom control than an anthracycline combination (CAV). In relapsed SCLC, a randomised phase III trial of oral topotecan versus best supportive care showed a statistically significant increase in overall survival. In the second line treatment of non-small cell lung cancer (NSCLC), oral topotecan was not inferior to the current standard iv docetaxel for 1 year survival rates. Worthwhile potential investigations include combining oral topotecan with other oral agents to design treatments for different lines of therapy. The new oral formulation of topotecan also lends itself to testing with small molecule tyrosine kinase inhibitors. The myelosuppressive toxicity profile needs to be vigorously monitored and managed. The trials with oral topotecan have also highlighted the need to reassess the utility of the terms ‘sensitive’ and ‘resistant’ in the selection of patients with relapsed small cell lung cancer.     

Cross-resistance of topoisomerase I and II inhibitors in neuroblastoma cell lines

Purpose: We have previously shown that neuroblastoma cell lines established from patients after intensive chemotherapy show sustained resistance to various drugs and especially high resistance to etoposide (up to 51 times higher than a clinically achievable level). To determine whether topoisomerase I inhibitors (topotecan and CPT-11) are effective against etoposide-resistant neuroblastomas, we studied the response to topotecan and the active metabolite of CPT-11 (SN-38) in 19 cell lines with a spectrum of sensitivities to etoposide. Materials and methods: The panel included cell lines established at diagnosis and after disease progression either during induction chemotherapy or after myeloablative therapy supported with bone marrow transplantation. Cytotoxicities of topotecan, SN-38, and etoposide were determined using a microplate digital image microscopy (DIMSCAN) assay with a 4-log dynamic range. Results: All six etoposide-resistant cell lines were resistant to topotecan and SN-38 (resistance defined as LC₉₀ higher then clinically achievable levels for the drug). Significant cross-resistance by Pearson's correlation analysis (r ≥ 0.6) occurred between topotecan + etoposide, topotecan + SN-38, and etoposide + SN-38. Conclusions: Topotecan and CPT-11 do not have significant activity against most etoposide-resistant neuroblastoma cell lines and this suggests that agents other than topoisomerase inhibitors should be explored for the treatment of recurrent neuroblastomas. Received: 19 January 1999 / Accepted: 24 May 1999     

Phase II and pharmacokinetic/pharmacodynamic trial of sequential topoisomerase I and II inhibition with topotecan and etoposide in advanced non-small-cell lung cancer

PURPOSE: In vitro and in vivo preclinical models have demonstrated synergistic activity when topoisomerase I and II inhibitors are administered sequentially. Topoisomerase I inhibitors increase topoisomerase II levels and increase cell kill induced by topoisomerase II poisons. We evaluated this hypothesis in a cohort of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: A group of 19 patients with advanced NSCLC (70% adenocarcinoma) received topotecan at a dose of 0.85 mg/m2 per day as a continuous 72-h infusion from days 1 to 3. Etoposide was administered orally at a dose of 100 mg twice daily for 3 days on days 7-9 (schedule and dose derived from prior phase I trials). Total and lactone topotecan concentrations were measured at the end of the 72-h infusion. Blood samples were obtained immediately after each 72-h topotecan infusion in order to measure the mutational frequency at the hypoxanthine phosphoribosyl transferase (HPRT) locus in peripheral lymphocytes. RESULTS: A total of 55 cycles were administered. Toxicity was mainly hematologic with grade 4 neutropenia occurring in 7% of courses. Only one partial response and two stable diseases were observed. The 1-year survival rate was 33%. There was a statistically significant difference between steady-state lactone concentrations between cycle 1 and cycle 2 with decreasing concentrations with cycle 2 (P = 0.02). This was explained by a statistically significant increase in the clearance of topotecan lactone during cycle 2 (P = 0.03). Total but not lactone concentrations correlated with nadir WBC, ANC and platelet levels. Steady-state plasma lactone levels correlated with the mutational frequency at the HPRT locus (P = 0.06). In the one patient with a partial response a sixfold increase in HPRT mutational frequency was observed, which was not seen in patients with progressive disease. CONCLUSION: The combination of topotecan and etoposide in this schedule of administration has minimal activity in adenocarcinoma of the lung. This lack of activity may be due to the delay in administration of etoposide after the topotecan as studies have shown that the compensatory increase in topoisomerase II levels after treatment with topoisomerase I inhibitors is shortlived (<24 h). The HPRT mutational frequency results suggest that the lack of clinical response may be associated with failure to achieve sufficient cytotoxic dose as indicated by a lack of increase in mutational frequency in those patients with progressive disease. HPRT mutational frequency may correlate with plasma steady-state topotecan lactone levels. Future studies should be directed toward earlier administration of topoisomerase II inhibitors after topoisomerase I inhibition.     

Glucocorticoid pretreatment reduces the cytotoxic effects of a variety of DNA-damaging agents on rat tibial growth-plate chondrocytes in vitro

It is apparent that cytotoxic chemotherapy used to treat childhood malignancies has a major impact on subsequent growth. Our initial studies have demonstrated a direct adverse effect of individual glucocorticoids and cytotoxic agents on the proliferative capacity of rat tibial growth-plate chondrocytes in vitro. In the present study we investigated the interaction between these classes of agents using in vitro cultures of chondrocytes and examined the potential of these cells to recover from the adverse effects of the drugs as applied either alone or in combination. The glucocorticoids prednisolone and dexamethasone significantly reduced the growth rate of chondrocytes when present in cultures for 3 days. The growth rate increased following the removal of prednisolone and dexamethasone from cultures and reached 83.9 ± 0.8% and 62.4 ± 4.0%, respectively, of the control values after 11 days of culture. In contrast, cell numbers were significantly reduced when the DNA-damaging agents cisplatin, carboplatin, etoposide or actinomycin-D were present in cultures for 3 days. Very little recovery of cell growth was observed after removal of the drugs from cultures, with cell loss occurring in the cisplatin- and actinomycin-D-treated cultures. However, pretreatment of chondrocytes with either of the glucocorticoids completely ameliorated the cytotoxic effects of etoposide and carboplatin and significantly reduced those of cisplatin and actinomycin-D. Recovery of the cells treated with a combination of glucocorticoid and DNA-damaging agent was demonstrated by a significant increase in their ability to form colonies in suspension culture. Colony numbers were increased by a factor of between 5 and 80 as compared with the cells receiving medium alone followed by DNA-damaging agent. The glucocorticoids offer a protective effect in terms of the reduced cytotoxicity of DNA-damaging agents and improve the subsequent clonogenicity and recovery of growth-plate chondrocytes. This has important implications for treatment schedules involving both cytotoxic agents and glucocorticoids in childhood malignancies. Received: 21 October 1997 / Accepted: 20 January 1998     

Successful local regional therapy with topotecan of intraperitoneally growing human ovarian carcinoma xenografts.

The therapeutic effects of intraperitoneal topotecan, a water-soluble camptothecin analogue, were investigated in two models of human ovarian carcinoma xenografted intraperitoneally into nude mice: the IGROV-1 tumour, which originated from an untreated patient, and the A2780 tumour, selected for resistance in vitro to cisplatin (A2780DDP). In IGROV-1 tumour-bearing mice, the optimal dose (10 mg kg-1) of topotecan, given intraperitioneally every 4 days for four occasions markedly increased survival time over control mice (300 T/C%) and cured 4/9 mice, and such effects were not achieved by any of the clinically available drugs tested, i.e. cisplatin carboplatin and doxorubicin delivered intraperitonally according to their optimal doses and schedules. In the treatment of A2780DDP tumour-bearing mice, topotecan was very effective since, at dose levels of 6.6 and 10 mg kg-1 every 4 days for four occasions, 15/18 mice survived more than 100 days, and most of them (12/15) were found to be tumour free. The high responsiveness of this tumour to topotecan might be related to the elevated expression of the target enzyme topoisomerase I. From these results, intraperitoneal treatment with topotecan appears to be a promising approach in the therapy of refractory ovarian cancer confined to the peritoneal cavity.     

Stimulation of the apoptotic response as a basis for the therapeutic synergism of lonidamine and cisplatin in combination in human tumour xenografts.

The pharmacological interest in lonidamine is related to its ability to enhance the cytotoxic effects of several DNA-damaging anti-tumour agents. This study was undertaken to better understand the in vivo interaction between lonidamine and cisplatin in the treatment of human tumour xenografts, including three carcinoma models characterized by a different responsiveness to cisplatin, in spite of the presence of a wild-type p53 gene in all tumours. The drug combination was more effective in tumour growth inhibition than cisplatin alone against MX-1 breast carcinoma and A2780 ovarian carcinoma, both highly responsive to cisplatin, whereas no influence of ionidamine was observed on anti-tumour activity of cisplatin in the treatment of the relatively resistant IGROV-1 ovarian carcinoma. As cisplatin activity is related to induction of apoptosis, the modulation of drug-induced apoptosis by lonidamine was investigated. Under conditions in which lonidamine itself had negligible effects on tumour growth and apoptosis, the modulating agent stimulated the apoptotic response induced by cisplatin in the responsive but not in the resistant tumours. Tumour response was dependent not only on the drug activation of apoptosis, but mainly on the persistence over time of the event. In the breast carcinoma MX-1, hypersensitive to cisplatin and to the lonidamine+cisplatin combination, the efficacy of drug treatment was associated with phosphorylation of bcl-2 followed by down-regulation of the protein. Lonidamine itself caused a delayed phosphorylation of bcl-2. These results are consistent with the interpretation that lonidamine is effective in modulating biochemical factors involved in regulation of apoptosis.     

Genetic basis of drug sensitivity in human testis tumour cells

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98 or CI). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning. © 1996 Wiley-Liss, Inc.     

Reduced cellular accumulation of topotecan: a novel mechanism of resistance in a human ovarian cancer cell line.

In order to unravel possible mechanisms of clinical resistance to topoisomerase I inhibitors, we developed a topotecan-resistant human IGROV-1 ovarian cancer cell line, denoted IGROV(T100r), by stepwise increased exposure to topotecan (TPT). The IGROV(T100r) cell line was 29-fold resistant to TPT and strongly cross-resistant to SN-38 (51-fold). However, the IGROV(T100r) showed only threefold resistance to camptothecin (CPT). Remarkably, this cell line was 32-fold resistant to mitoxantrone, whereas no significant cross-resistance against other cytostatic drugs was observed. No differences in topoisomerase I protein levels and catalytic activity as well as topoisomerase I cleavable complex stabilization by CPT in the IGROV-1 and IGROV(T100r) cell lines were observed, indicating that resistance in the IGROV(T100r) cell line was not related to topoisomerase I-related changes. However, resistance in the resistant IGROV(T100r) cell line to TPT and SN-38 was accompanied by decreased accumulation of the drugs to approximately 15% and 36% of that obtained in IGROV-1 respectively. No reduced accumulation was observed for CPT. Notably, accumulation of TPT in the IGROV-1 cell line decreased under energy-deprived conditions, whereas the accumulation in the IGROV(T100r) cell line increased under these energy-deprived conditions. The efflux of TPT at 37 degrees C was very rapid in the IGROV-1 as well as the IGROV(T100r) cell line, resulting in 90% efflux within 20 min. Importantly, the efflux rates of TPT in the IGROV-1 and IGROV(T100r) cell lines were not significantly different and were shown to be independent on P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP). These results strongly suggest that the resistance of the IGROV(T100r) cell line to TPT and SN-38 is mainly caused by reduced accumulation. The reduced accumulation appears to be mediated by a novel mechanism, probably related to impaired energy-dependent uptake of these topoisomerase I drugs.     

Effect of topoisomerase modulators on cisplatin cytotoxicity in human ovarian carcinoma cells

The in vitro interaction of modulators of topoisomerase I and II with cisplatin in human ovarian carcinoma cells might be synergistic. The interactions were evaluated by median effect analysis of survival data derived from continuous exposure to drug combinations for 10 days in colony-forming assays. The interaction between cisplatin and the topoisomerase I inhibitor camptothecin and the topoisomerase I activator β-lapachone was additive, as was that between cisplatin and the topoisomerase II inhibitor novobiocin. Despite the clinical efficacy of the combination of etoposide (a topoisomerase II inhibitor) and cisplatin, the combination index at 50% cell kill indicated antagonism between these two drugs. Thus, biochemical synergism at the cellular level is not a prerequisite of improved therapeutic efficacy.     

131I]meta-iodobenzylguanidine and topotecan combination treatment of tumors expressing the noradrenaline transporter.

PURPOSE: Both [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [(131)I]MIBG in combination with topotecan in vitro and in vivo. EXPERIMENTAL DESIGN: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [(131)I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice. RESULTS: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([(131)I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3. CONCLUSIONS: The combination of topotecan and [(131)I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.     

Interaction of ionizing radiation with topotecan in two human tumor cell lines

The effect of topotecan, a topoisomerase I inhibitor, on ionizing radiation-induced cytotoxicity was studied in 2 human tumor cell lines characterized by a different expression of the target enzyme. The cytotoxicity of topotecan alone or in combination with radiation was assessed in exponentially growing non-small-cell lung cancer (H460) and glioblastoma (GBM) cells using the colony-forming assay. An isobologram method was used to evaluate the treatment interaction. An apparent supra-additive effect in cell killing following drug-radiation-combined treatment was observed only in GBM cells exposed to topotecan for 24 hr. In the case of H460 cells, interaction varied from a strong intra-additive effect at low radiation doses to a slight supra-additive effect when cells were exposed to radiation doses greater than 3 Gy. Northern blot analysis indicated that topoisomerase I expression in H460 cells was 8-fold higher than that of GBM cells. Although the H460 cell line exhibited an increased sensitivity to topotecan, only in the GBM cell line (which expressed a lower level of topoisomerase I) did the drug potentiate the radiation cytotoxicity. The observation that the radiosensitization by topotecan was related to topoisomerase I level is consistent with a putative role of the enzyme in processes involved in the repair of radiation damage. It is conceivable that the modulation of enzyme function results in an effective reduction of cellular capability for repair of radiation damage only if the enzyme is not over-expressed. Although a precise role of topoisomerase I in the cellular response to ionizing radiations (in particular, in DNA repair) remains to be documented, such results suggest the potential interest of topoisomerase I inhibitors in combination with radiation therapy for tumors expressing low topoisomerase I levels. © 1996 Wiley-Liss, Inc.     

Cell cycle perturbations in cisplatin-sensitive and resistant human ovarian carcinoma cells following treatment with cisplatin and low dose rate irradiation

Purpose: To investigate cell cycle pertubations in plateau-phase human ovarian carcinoma cells following treatment with cisplatin, low dose-rate irradiation (LDRI), or combined cisplatin and LDRI, in order to understand cell cycle mechanisms by which these two treatment modalities interact. Methods: Human ovarian carcinoma cells sensitive (A2780) and resistant (2780^CP) to cisplatin were grown to plateau phase and given protracted cisplatin treatments (A2780 0.7 and 2 μg/ml; 2780^CP 5 and 15 μg/ml) and/or LDRI (0.41 cGy/min). Cell cycle distribution following treatment was determined by two-parameter flow cytometry, based on bromodeoxyuridine (BrdU) uptake and DNA content using propidium iodide staining.Results: The cisplatin-sensitive A2780 cells exposed to cisplatin alone for up to 28 h showed depletion of the G1 fraction and accumulation in S-phase, although the percentage of S-phase cells actively incorporating BrdU dropped to almost zero. The cisplatin-resistant 2780^CP cells exposed to cisplatin alone showed a G1 arrest when exposed to 15 μg/ml, but not when exposed to 5 μg/ml. LDRI alone caused little cell cycle redistribution different from controls in either cell line. When LDRI was combined with cisplatin, no significant cell cycle redistribution was observed, apart from a decline in the actively incorporating S-phase fraction. Conclusions: Cisplatin caused A2780 cells to accumulate in nonincorporating S-phase, with no evidence of G1 arrest. Cisplatin-resistant 2780^CP cells showed a G1 block when exposed to a high enough cisplatin concentration. This could indicate a mechanism of cisplatin resistance in these cells. LDRI alone or in combination with cisplatin did not result in significant cell cycle redistribution. Received: 22 December 1995 / Accepted: 28 September 1996     

Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells

Purpose: The combination of paclitaxel (PTX) and cisplatin (DDP) shows good clinical efficacy against ovarian cancer. In order to examine the potential cellular basis for this, and provide leads as to how to optimize the combination, we examined the role of sequence of exposure to PTX and DDP on cell growth in vitro. Methods: Four human ovarian carcinoma cell lines, A121, A2780/WT, A2780/DX5B and A2780/CP3, two human head and neck carcinoma cell lines, A253 and FaDu, and the human ileocecal carcinoma cell line, HCT-8, were treated with PTX + DDP with seven schedules: (A) 96 h exposure to PTX + DDP; (B) 24 h PTX alone, then 72 h PTX + DDP; (C) 4 h DDP alone, then 92 h PTX + DDP; (D) 24 h PTX alone, 4 h DDP alone, then 68 h drug-free; (E) 4 h DDP alone, 24 h PTX alone, then 68 h drug-free; (F) 3 h PTX alone, 1 h DDP alone, then 92 h drug-free; and (G) 1 h DDP alone, 3 h PTX alone, then 92 h drug-free. Each of 66 two-drug experiments included five plates (440 randomly treated wells per experiment). Cell growth was measured by the sulforhodamine B assay. The nature and the intensity of the drug interactions were assessed by fitting a seven-parameter model to data with weighted nonlinear regression, enabling the estimation of an interaction parameter, α, with its standard error. Results: Overall there was very little departure from Loewe additivity: 43 experiments showed Loewe additivity, 10 showed Loewe antagonism, and 13 showed slight Loewe synergy. In vitro Loewe synergy was rare, was small when present, and reproducible only for the A121 and HCT-8 cells exposed to schedule D (24 h PTX prior to 4 h DDP). Isobolographic analysis showed complex combined-action surfaces with regions of local Loewe synergy and antagonism. Conclusion: It appears unlikely that the good clinical efficacy of the combination is primarily caused by a synergistic interaction at the cellular level. Received: 24 October 1996 / Accepted: 5 March 1997     

Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225.

Epidermal growth factor (EGF)-related proteins such as transforming growth factor alpha (TGF-alpha) control cancer cell growth through autocrine and paracrine pathways. Overexpression of TGF-alpha and/or its receptor (EGFR) has been associated with a more aggressive disease and a poor prognosis. The blockade of EGFR activation has been proposed as a target for anticancer therapy. Monoclonal antibody (MAb) C225 is an anti-EGFR humanized chimeric mouse MAb that is presently in Phase II clinical trials in cancer patients. Previous studies have suggested the potentiation of the antitumor activity of certain cytotoxic drugs, such as cisplatin and doxorubicin, in human cancer cell lines by treatment with anti-EGFR antibodies. We have evaluated in human ovarian, breast, and colon cancer cell lines, which express functional EGFR, the antiproliferative activity of MAb C225 in combination with topotecan, a cytotoxic drug that specifically inhibits topoisomerase I and that has shown antitumor activity in these malignancies. A dose-dependent supraadditive increase of growth inhibition in vitro was observed when cancer cells were treated with topotecan and MAb C225 in a sequential schedule. In this respect, the cooperativity quotient, defined as the ratio between the actual growth inhibition obtained by treatment with topotecan followed by MAb C225 and the sum of the growth inhibition achieved by each agent, ranged from 1.2 to 3, depending on drug concentration and cancer cell line. Treatment with MAb C225 also markedly enhanced apoptotic cell death induced by topotecan. For example, in GEO colon cancer cells, 5 nM topotecan, followed by 0.5 microg/ml MAb C225, induced apoptosis in 45% cells as compared with untreated cells (6%) or to 5 nM topotecan-treated cells (22%). Treatment of mice bearing established human GEO colon cancer xenografts with topotecan or with MAb C225 determined a transient inhibition of tumor growth because GEO tumors resumed the growth rate of untreated tumors at the end of the treatment period. In contrast, an almost complete tumor regression was observed in all mice treated with the two agents in combination. This determined a prolonged life span of the mice that was significantly different as compared with controls (P < 0.001), to MAb C225-treated group (P < 0.001), or to the topotecan-treated group (P < 0.001). All mice of the topotecan plus MAb C225 group were the only animals alive 14 weeks after tumor cell injection. Furthermore, 20% of mice in this group were still alive after 19 weeks. The combined treatment with MAb C225 and topotecan was well tolerated by mice with no signs of acute or delayed toxicity. These results provide a rationale for the evaluation of the anticancer activity of the combination of topoisomerase I inhibitors and anti-EGFR blocking MAbs in clinical trials.