In vitro and in vivo effects of cisplatin and etoposide in combination on small cell lung cancer cell lines

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isobologram analysis. Used together, CDDP and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC-3), additive effects on 6 (SBC-2, SBC-5, Lu130, Lu134AH, Lu135T and H69) and an antagonistic effect on 1 (SBC-1). In the in vivo experiment, nude mice were inoculated with SBC-1, SBC-3 and SBC-5 cells. Two or 5 mg/kg CDDP and 10 or 30 mg/kg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may be attributable not to drug interaction at the cellular level but to the feasibility of combined use of them at full doses without overlapping side effects.     

Importance of the irradiation timing within a chemoradiotherapy sequence including cisplatin and 5-FU-folinic acid. Experimental results

The objective of the present in vitro study was to determine an optimal timing of the irradiation in the combination cisplatin (CDDP) and 5-fluorouracil-folinic-acid (5-FU-FA) allowing a maximal cytotoxic effect on a human cell line derived from a head and neck carcinoma (CAL 27 cells). The various tested chemoradiotherapy sequences were applied in parallel to human keratinocytes in culture (SVK 14 cells). This was done in order to define the best sequence allowing the achievement of an optimal selectivity of the cytotoxic effects. The drug sequence was: CDDP over 2 h then fresh medium was added including the tandem 5-FU-d,I FA applied 6 h after CDDP, for 5 days. Irradiation was applied only once and at various times within the drug sequence. The cytotoxicity effects of the different chemoradiotherapy combinations were assessed by the MTT semi-automated test. The part taken by the 5-FU-FA combinations in the overall cytotoxicity was examined; an effect was apparent on CAL 27 cells only. The evolution of the radiation effect (RE = cell survival after drugs/cell survival after drugs plus irradiation) was analysed as a function of the different times of irradiation within the given drug sequence. Clearly, the RE values were dependent upon time at which the radiation dose in the chemoradiotherapy regimen was administered. For CAL 27 cells, irradiation effects were maximal at the first irradiation time tested after the end of the CDDP exposure (i.e. t = 3.5 h). In contrast, this optimal chemoradiotherapy timing for better cytotoxicity on CAL 27 cells did not correspond to that of SVK 14 cells. Consequently, it was possible to establish that the best time for the selectivity index was located shortly after the CDDP exposure.     

Modulation of fluorouracil cytotoxicity by interferon-alpha and -gamma

Because interferons (IFN)-alpha and -gamma individually have increased fluorouracil (FUra) cytotoxicity in several in vitro models, we studied the effects of FUra combined with IFN-alpha + gamma in HT29 colon cancer cells. A 96-hr exposure to IFN-alpha (500 units/ml) plus IFN-gamma (10 units/ml) and a 72-hr exposure to 0. 25-1 microM FUra (hr 24-96) inhibited cell growth and colony formation in an additive or more-than-additive fashion. When cells were exposed to IFN-alpha + gamma and FUra, free FdUMP levels became detectable, whereas [3H]FUra-RNA incorporation decreased. Exposure to IFN-alpha + gamma, FUra, or the combination decreased dTTP pools to 58%, 43%, and 17% of control, respectively. A marked increase in the dATP to dTTP ratio was seen with FUra with or without IFN-alpha + gamma. Thymidylate synthase catalytic activity was reduced to 28% and 24% of control with FUra with or without IFN-alpha + gamma, suggesting that the enhanced dTTP depletion must be due to another mechanism. FUra-mediated thymidylate synthase inhibition was accompanied by a 124-fold increase in total deoxyuridylate immunoreactivity and a 31-fold increase in dUTP pools, but the addition of IFN-alpha + gamma attenuated the accumulation. Treatment with IFN-alpha + gamma and FUra individually interfered with nascent DNA chain elongation, whereas the three-drug combination produced the most striking effects. IFN-alpha + gamma plus FUra produced the greatest amount of single-strand breaks in nascent DNA and dramatically decreased net DNA synthesis. IFN-alpha + gamma with or without FUra produced double-strand breaks in parental DNA. These results suggest that dTTP depletion, dATP/dTTP imbalance, pronounced inhibition of DNA synthesis, and damage to nascent and parental DNA contribute to the enhanced cytotoxicity with the triple combination.     

Cytotoxic effects of topotecan combined with various anticancer agents in human cancer cell lines

BACKGROUND: Topotecan (TPT) is a topoisomerase I poison that exhibits antineoplastic activity. Analysis of the cytotoxic effects of combinations of TPT and other anticancer agents has been limited. PURPOSE: We assessed the cytotoxic effects produced by combinations of TPT and other antineoplastic agents in experiments involving multiple human cancer cell lines of diverse histologic origins. METHODS: The cytotoxic effects of various antimetabolites (fluorouracil, methotrexate, or cytarabine), antimicrotubule agents (vincristine or paclitaxel [Taxol]), DNA alkylating agents (melphalan, bis[chloroethyl]nitrosourea [BCNU], or 4-hydroperoxycyclophosphamide [4HC]), and a DNA-platinating agent (cisplatin), alone and in combination with TPT, were measured in clonogenic (i.e., colony-forming) assays. HCT8 ileocecal adenocarcinoma, A549 non-small-cell lung carcinoma, NCI-H82ras(H) lung cancer, T98G glioblastoma, and MCF-7 breast cancer cell lines were used in these assays. The data were analyzed by the median effect method, primarily under the assumption that drug mechanisms of action were mutually nonexclusive (i.e., completely independent of one another). For each level of cytotoxicity (ranging from 5% to 95%), a drug combination index (CI) was calculated. A CI less than 1 indicated synergy (i.e., the effect of the combination was greater than that expected from the additive effects of the component agents), a CI equal to 1 indicated additivity, and a CI greater than 1 indicated antagonism (the effect of the combination was less than that expected from the additive effects of the component agents). RESULTS: When the mechanisms of drug action were assumed to be mutually nonexclusive, virtually all CIs for combinations of TPT and either antimetabolites or antimicrotubule agents revealed cytotoxic effects that were less than additive. The CIs calculated at low-to-intermediate levels of cytotoxicity for combinations of TPT and the DNA alkylating agents melphalan, BCNU, and 4HC also showed drug effects that were less than additive; in most cases, however, nearly additive or even synergistic effects were observed with these same drug combinations at high levels of cytotoxicity (i.e., at > or = 90% inhibition of colony formation). Results obtained with combinations of TPT and cisplatin varied according to the cell line examined. With A549 cells, less than additive effects were seen at low-to-intermediate levels of cytotoxicity, and more than additive effects were seen at high levels of cytotoxicity. With NCI-H82ras(H) cells, synergy was observed over most of the cytotoxicity range. CONCLUSIONS AND IMPLICATIONS: TPT cytotoxicity appears to be enhanced more by combination with certain DNA-damaging agents than by combination with antimetabolites or antimicrotubule agents. Interactions between TPT and other drugs can vary depending on the cell type examined. Further investigation is required to determine the basis of the observed effects and to determine whether these in vitro findings are predictive of results obtained in vivo.     

Sensitization of human bladder cancer cells to Fas-mediated cytotoxicity by cis-diamminedichloroplatinum (II)

PURPOSE: The resistance of bladder cancer to anticancer chemotherapeutic drugs is a major problem. Several immunotherapeutic approaches have been developed to treat drug-resistant tumor cells. The Fas antigen (Fas)-Fas ligand pathway is involved in cytotoxic T lymphocyte and natural killer cell-mediated cytotoxicity. Like the Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis in tumor cells expressing Fas. Several anticancer drugs also mediate apoptosis and may share with Fas common intracellular pathways leading to cell killing. We reasoned that treatment of drug-resistant cancer cells with a combination of anti-Fas mAb and drugs might overcome their resistance. This study has investigated whether anticancer drugs synergize with anti-Fas mAb in cytotoxicity against bladder cancer cells. MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. RESULTS: Treatment of the T24 human bladder cancer cell line with anti-Fas mAb in combination with 5-fluorouracil, mitomycin C or methotrexate did not overcome resistance to these agents. However, treatment of T24 tumor cells with a combination of anti-Fas mAb and cisdiamminedichloroplatinum (II) (CDDP) resulted in a synergistic cytotoxic effect. In addition, the CDDP-resistant T24 line (T24/CDDP) was sensitive to treatment with a combination of anti-Fas mAb and CDDP. Synergy by combination of anti-Fas mAb and CDDP was also achieved in three other bladder cancer lines and four freshly derived human bladder cancer cells. The combination of anti-Fas mAb and carboplatin also resulted in a synergistic cytotoxic effect on T24 cells; however, the combination of anti-Fas mAb and trans-diamminedichloroplatinum (II) resulted in an additive cytotoxic effect. Treatment with CDDP enhanced the expression of Fas on T24 cells. The synergy achieved in cytotoxicity with anti-Fas mAb and CDDP was also achieved in apoptosis. Incubation of T24 cells with anti-Fas mAb increased the intracellular accumulation of CDDP. Treatment of freshly isolated bladder cancer cells with CDDP enhanced their susceptibility to lysis by autologous lymphocytes. CONCLUSIONS: This study demonstrates that combination treatment of bladder cancer cells with anti-Fas mAb and CDDP overcomes their resistance. Synergy was achieved with established CDDP-resistant bladder cancer cells and freshly isolated bladder cancer cells. In addition, the sensitization required low concentrations of CDDP, thus supporting the potential in vivo application of combination of CDDP and immunotherapy in the treatment of CDDP- and/or immunotherapy-resistant bladder cancer.     

Interleukin 15 protects against toxicity and potentiates antitumor activity of 5-fluorouracil alone and in combination with leucovorin in rats bearing colorectal cancer

5-Fluorouracil (FUra) modulated by leucovorin (LV) is active in the treatment of colorectal cancer. Diarrhea and stomatitis are the most common dose-limiting toxicities. We have developed a model system in rats bearing a transplantable colon carcinoma sensitive to FUra therapy with dose-limiting toxicity profiles similar to what is observed in patients treated with either daily or weekly schedules of FUra plus LV. Interleukin 15 (IL-15), a cytokine that shares many biological activities with IL-2, was used at different doses (25, 100, and 400 microg/kg) and schedules (three doses before a single dose of FUra, FUra/LV daily x 5, or before each week of FUra/LV weekly x 4, or three doses before a single dose of FUra or FUra/LV daily x 5, then twice daily x 5 for a total of 11 doses) to evaluate its role in the modulation of the therapeutic selectivity of FUra alone and modulated by LV. IL-15 induced a dramatic decrease in chemotherapy-induced gastrointestinal toxicities, significant potentiation of antitumor activity, and an increased therapeutic index of FUra administered on single dose, daily x 5 and weekly x 4 schedules. In contrast, IL-2 (400 microg/kg) significantly potentiated the toxicity of FUra administered as a single i.v. push, with minimal potentiation of the antitumor activity. Taken together, the results clearly demonstrated the ability of IL-15, but not IL-2, to provide significant improvement of the therapeutic index of FUra alone and in combination with LV. The clinical relevance of the results obtained in this model system needs to be confirmed.     

Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.     

Synergistic interaction between cisplatin and gemcitabine in vitro

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.     

Sub-additive effect of the combination of radiation and cisplatin in cultured murine and human cell lines

The use of cisplatin (CDDP) as a potential radiosensitizer in tumors is controversial. Reports about CDDP interaction with radiation range from high radiosensitization to a clear sub-additive effect. We examined the effect of the combination of different concentrations of CDDP with radiation in murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cell lines. CDDP was given in the dose range of 0.01-3.0 micrograms/ml and radiation in the dose range of 1-6 Gy. A methylene blue assay of cell density was used for the evaluation of cell survival and rate of proliferation in 96-microwell plates. The validity of this assay for evaluation of cell survival was verified by colony-forming assay and radiolabeled thymidine uptake. The dose response to CDDP for both OV-1063 and EMT-6 cells lines was examined; the ID50 was 0.06 and 0.9 micrograms/ml respectively. A sub-additive effect of the combination of radiation with CDDP was clearly observed in the two cell lines tested; the increase in dose of each modality resulted in a decrease of the relative contribution on the effect of the other. These findings question the rationale of combining CDDP with radiation for the enhancement of tumor response, since with the increase in the dose of either modality the additional effect of the other decreases.     

In vitro synergism between 5-fluorouracil and natural beta interferon in human colon carcinoma cells

The combination of 5-fluorouracil (FUra) plus IFN-beta was studied in vitro using a human colon carcinoma cell line, HCT-8. Continuous exposure to high concentrations of IFN-beta is cytotoxic to these cells (ED50, 600 +/- 50 IU/ml). A strong synergism (P < 0.002) was observed when a short-term (1-h), high-concentration exposure to fluoropyrimidine (300 or 1000 microM) was followed by IFN-beta given continuously. In fact, the mean ratio between the expected (product of the survival of each agent alone) and the observed clonogenic cell survival rates of the combination was 3.4 (range, 2.4-4.9). Longer exposures to the fluoropyrimidine (24 h, 7 days) produced less than additive effects with IFN-beta, indicating strong schedule dependency for this synergism. The mechanism of interaction was studied at four levels. First, thymidylate synthase (TS) activity, inhibition, and recovery were measured by an in situ assay in cells treated with FUra, IFN-beta, and their combination. When the prolonged infusion of IFN-beta followed a 1-h exposure to FUra, the observed TS inhibition and recovery, at each time point, were very similar to the expected values, indicating that the interactions between these drugs at the level of TS are not the determinant of the synergism. Second, cell cycle analysis was done. During the continuous exposure to IFN-beta, a significant accumulation of HCT-8 cells in S-phase was observed at 24, 48, and 72 h compared to untreated controls (P = 0.003); however, under the same experimental conditions producing synergy in the clonogenic assay, no significant cell cycle perturbations were produced by the combination of FUra followed by IFN-beta compared to those caused by each agent alone. Third, using the alkaline elution test, we also demonstrated that the synergism is not due to enhanced FUra-induced DNA single-strand break frequency in high molecular weight DNA. Finally, nucleic acid incorporation experiments, using tritiated FUra, showed that the cytokine, given continuously (300 IU/ml), enhanced the amount of FUra incorporated into nucleic acids 24 h after a 1-h exposure to 300 and 1000 microM of FUra. The median percentage of enhancement values were 31.6 +/- 11.5%,m for the lower drug concentration and 18. 4 +/- 8.1% for the higher drug concentration tested. These results suggest that the mechanism of this synergism may be related to the ability of IFN-beta to promote the incorporation of intracellular FUra metabolites into nucleic acids and/or to inhibit the cleavage of FUra nucleotides from RNA/DNA.     

In vivo modulation of several anticancer agents by beta-carotene

The ability of the collagenase inhibitor minocycline and of beta-carotene to act as positive modulators of cytotoxic anticancer agents was assessed in vitro and in vivo. Cell-culture studies were conducted using the human SCC-25 squamous carcinoma cell line. Simultaneous exposure of the cells to minocycline and beta-carotene or 13-cis-retinoic acid along with cisplatin (CDDP) resulted in a small decrease in the cytotoxicity of the CDDP. The addition of each of the modulator combinations for 1 h or 24 h to treatment with melphalan (L-PAM) or carmustine (BCNU) resulted in greater-than-additive cytotoxicity with each of four regimens. The modulator combinations of minocycline and beta-carotene applied for 1 h or 24 h and the modulator combination of minocycline and 13-cis-retinoic acid produced greater-than-additive cytotoxicity at 50 microM 4-hydroperoxycyclophosphamide (4-HC), whereas minocycline and 13-cis-retinoic acid applied for 1 h was antagonistic with 4-HC and the other modulator treatments at low concentrations of 4-HC resulted in subadditive cytotoxicity. The effect of treatment with beta-carotene alone and in combination with several different anticancer agents was examined in two murine solid tumors, the FSaII fibrosarcoma and the SCC VII carcinoma. Administration of the modulators alone or in combination did not alter the growth of either tumor. Whereas increases in tumor growth delay occurred with the antitumor alkylating agents and beta-carotene and with minocycline and beta-carotene, a diminution in tumor growth delay was produced by 5-fluorouracil in the presence of these modulators. The modulator combination also resulted in increased tumor growth delay with adriamycin and etoposide. Tumor-cell survival assay showed increased killing of FSaII tumor cells with the modulator combination and melphalan or cyclophosphamide as compared with the drugs alone. These results indicate that further investigation of this modulator strategy is warranted.     

The efficacy of 2',2'-difluorodeoxycytidine (gemcitabine) combined with interferon in human renal cell carcinoma cell lines

The present in vitro study on three human renal cell carcinoma (RCC) cell lines (A-498, ACHN, SN12C) evaluated the efficacy of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine), vinblastine (VBL), rhu-interferon-alpha (IFN-alpha) and rhu-interferon-gamma (IFN-gamma) alone or in combinations. The cytotoxicity was measured by using the sulphorhodamine B colorimetric cytotoxicity assay. Analyses were made from cells being continuously long-term (4 weeks) or short-term (4 h) with IFN-alpha or IFN-gamma with regard to the cytotoxicity of the chemotherapeutic agents. dFdC was more cytotoxic against ACHN and A-498 cells compared to VBL. Pre-treatment with IFN-alpha enhanced growth inhibition caused by dFdC (4/4 cell lines) and VBL (2/3 cell lines), and was more effective than IFN-gamma. Pre-exposure with IFN-alpha sensitized SN12C and ACHN cells for dFdC. A-498 cells achieved a decreased sensitivity to dFdC and VBL after pre-exposure to IFN-gamma. The resistance of newly established dFdC-resistant SN12C cells (23-times) decreased when pre-treated with IFN-alpha. The data demonstrate efficacy of dFdC in human RCC at concentrations below clinically achievable doses. dFdC was more effective compared to VBL. Combined therapy preferentially with IFN-alpha increased cytotoxicity of dFdC in vitro. In vivo studies in nude mice xenografts are under investigation to support these observations.     

5-Fluorouracil prodrug: role of anabolic and catabolic pathway modulation in therapy of colorectal cancer

Following p.o. administration to rats bearing advanced colorectal carcinoma, Ftorafur (FT) is converted to 5-fluorouracil (FUra) by microsomal P450 in the liver. To optimize the therapeutic selectivity of the FUra generated from FT, three approaches were utilized: (a) inhibition of FUra degradation to dihydrofluorouracil by uracil as an alternative substrate for uracil reductase in the molar ratio of 4 uracil:1 FT (UFT); (b) modulation of drug inhibition of thymidylate synthase by leucovorin (LV); and (c) by increasing the level of FUra incorporation into cellular RNA by N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbomylase. The maximum tolerated dose (MTD) of FT and UFT, administered 3 times a day for 28 days, was 150 mg/kg/day and 60 mg/kg/day, respectively. The MTDs were not significantly modified by LV (150 or 600 mg/kg/day), administered by the p.o. route with the drugs, or by PALA (100 mg/kg) administered weekly by the i.v. route. The dose-limiting toxicity of FT alone and in combination with the modulators was stomatitis. The severe alopecia observed with FT alone was reduced significantly by uracil. At the MTD, the antitumor activity of UFT was superior to those of FT and FUra alone and in combination with LV and/or PALA. The 3-month sustained complete tumor regression for UFT, FT, and FUra was 38%, 0%, and 13% (for the weekly schedule), respectively. Although uracil, LV, and PALA individually increased the antitumor activity of FT at its MTD, the combination of the three modulators produced the highest therapeutic efficacy in rats bearing advanced colorectal carcinoma, in which 100% of the treated animals achieved complete and sustained tumor regression. The therapeutic efficacy observed with FT modulation could not be achieved with FUra administered by different schedules, each at its MTD alone or in combination with either LV or PALA. In brief, modulation of FT produced greater therapeutic efficacy and selectivity than FUra. Furthermore, the combined use of modulators capable of inhibiting the degradation pathway of FUra and potentiating the effects of the anabolic metabolites action appears to offer the greatest therapeutic potential.     

Revertant and potentiating activity of lonidamine in patients with ovarian cancer previously treated with platinum

PURPOSE: Lonidamine (LND) is an energolytic derivative of indazol-carboxylic acid that has been shown to enhance cisplatin (CDDP) activity in both sensitive (A2780) and resistant (A2780/Cp8) ovarian cancer cell lines. The aim of this study was to confirm the potentiating or reverting activity of LND on CDDP activity obtained in experimental models in a phase II study of advanced ovarian cancer patients previously treated with platinum-based regimens. PATIENTS AND METHODS: Twenty-seven consecutive women with histologically proven and measurable ovarian cancer previously treated with platinum compounds were treated with CDDP plus LND. CDDP was administered at 1 mg/kg intravenously (IV) once weekly for 6 weeks and every 3 weeks thereafter until disease progression or toxicity. LND was administered at 450 mg daily (1 tablet every 8 hours) for the entire period of therapy starting 3 days before the first CDDP administration. In addition, a higher LND dosage was provided on the day of CDDP administration in an attempt to maximize the synergy of this drug with CDDP. RESULTS: Ten patients achieved a complete response (CR) or partial response (PR) for an overall response rate of 37% (95% confidence interval [CI], 19% to 55%). In particular, responses were observed in five of 18 (28%) refractory or early relapsed patients (one CR and four PRs) and in five of nine patients (55%) in the late-relapsed group (two CRs and three PRs). Grade 3 or 4 anemia, leukopenia, and thrombocytopenia were observed in 19%, 15%, and 11% of patients, respectively, whereas seven of 27 patients (26%) showed LND-related myalgia. Grade 3 renal toxicity was observed in two patients (8%). Neurotoxicity, often concealed by LND-related myalgia, was recorded as grade 1 or 2 in six patients (22%) and as grade 3 in one (4%). CONCLUSION: The 37% response rate observed in this study (28% in refractory or early-relapsed patients), suggests that the synergism between CDDP and LND observed in vitro against ovarian cancer cell lines can be clinically confirmed. However, larger series and randomized studies are needed to assess definitely the revertant activity of LND on CDDP-refractory patients.     

An early phase II trial combining cisplatin and carboplatin in advanced non-small cell lung cancer

We conducted an early phase II trial in advanced non-small cell lung cancer (NSCLC) to evaluate response efficacy of a combination of Cisplatin (CDDP) and Carboplatin (CBDCA). The twenty-six patients in the study had had no previous treatment. They received a sequential administration of 300 mg/m2 CBDCA and 80 mg/m2 CDDP with approximately 3,500 ml of hydration on day 1 every 4 weeks. All patients were evaluable for response and toxicity. Ten (38.5%) of all assessable patients achieved a partial response (95% confidence interval, 19.8-57.2%). Response rates for patients with stage III A, III B and IV- disease were 40.0 (2/5), 70.0 (7/10) and 9.1% (1/11), respectively. Response rates for patients with squamous cell carcinoma, adenocarcinoma and large cell carcinoma were 35.7 (5/14), 45.5 (5/11) and 0.0% (0/1), respectively. The median survival time (MST) of all patients was 11 months. The MST for patients with stage III disease was 14 months; for those with stage IV disease it was 7 months. The MST for responding patients was 15 months and for not responding patients 5 months. Major toxicities were hematologic and gastrointestinal, and the dose-limiting factor was thrombocytopenia. This combination chemotherapy was effective against NSCLC with tolerable toxicities. Further trials are warranted to determine the efficacy of the combination chemotherapy.     

Personalities and alimentary behaviors in obese patients

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of cisplatin (CDDP) and epidoxorubicin (EPI), singly or in combination, was investigated in two human breast cancer cell lines (MCF7 and T47D). A 72-hr post-incubation with a non-cytotoxic concentration of LND (75 microM) increased the activity of a 1-hr CDDP treatment as well as that of a 1 to 16-hr EPI treatment. A different pattern of interaction among the drugs and modulator was observed as a function of the sequence of drug treatment. Specifically, supra-additive or additive effects of the combination were obtained in the two cell lines according to the different treatment schemes. In particular, the maximum potentiation was observed in MCF7 cells simultaneously exposed to CDDP, EPI and LND for 1 hr and then post-incubated with LND for 72 hr, and in T47 first exposed to EPI and LND, then to CDDP and LND, and finally post-incubated with LND. Flow cytometric analysis of MCF7 cell distribution in the different cycle phases showed that combined treatment with EPI/CDDP/LND was able to stabilize cell cycle perturbations (mainly G2M accumulation) induced by individual agents. The ability of LND to potentiate CDDP and EPI cytotoxicity, and the consideration that LND causes side effects different from those caused by alkylating agents and anthracyclines, make this compound an attractive candidate for multidrug combination therapy in breast cancer.     

UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status

Our goal was to determine the cytotoxicity of 7-OH-hydroxystaurosporine (UCN-01) as a single agent and in combination with cis-diamminedichloroplatinum(II) (CDDP) in a panel of ovarian carcinoma cells. We sought to examine the role of p53 gene function and alterations in cell cycle progression or other mechanisms of action of UCN-01 including perturbation of the apoptosis pathway mediated by NF-kappaB and Bcl-2/Bax. Cytotoxicity was determined from dose-response curves established by the Alamar blue vital dye indicator assay. Restoration of wild-type p53 in a p53 null cell line, SKOV 3, was achieved by transfection of a p53 expression vector. Cell cycle distribution was measured by fluorescence-activated cell sorting analysis of ethidium bromide-stained nuclei. Apoptosis was measured by quantitative fluorescence microscopy. NF-kappaB DNA binding activity was measured by electrophoretic mobility shift assay. Bcl-2 and Bax levels were determined by Western immunoblotting. UCN-01 was effective as a cytotoxic agent alone and in combination with CDDP in all cell lines studied, regardless of p53 status. The degree of sensitization to CDDP conferred by UCN-01, however, was found to correlate with p53 gene status. p53 wild-type cells seem to be more sensitive to the cytotoxic effects of the combination of UCN-01 + CDDP than the p53 mutant cells. This was confirmed in cells in which p53 wild-type function was restored by transfection of p53 cDNA, but these cells are also significantly more sensitive to CDDP alone. The effects of UCN-01 on cell cycle progression also appear to be p53 dependent but may not be the primary mechanism of action. The rate of apoptosis is increased 4-fold in UCN-01 + CDDP-treated cells compared to either agent alone. UCN-01 does not effect NF-kappaB DNA binding activity or Bcl-2 and Bax levels. UCN-01 enhances CDDP cytotoxicity and apoptosis in ovary cancer cells. This occurs regardless of p53 status, but wild-type p53 seems to increase the degree of sensitization.     

Synergistic antitumor activity of cisplatin and interleukin 1 in sensitive and resistant solid tumors

The antitumor activity of cis-diamminedichloroplatinum(II) (cP) and human recombinant interleukin-1 alpha (IL-1 alpha) was studied in RIF-1 and SC VII solid tumor models and in a cP-resistant subline of RIF-1 designated RIF-R1cP. In RIF-1 tumors, clonogenic cell survival after cP plus IL-1 alpha combinations was highly schedule and IL-1 alpha dose dependent. More than additive clonogenic cell kill was seen when cP was given 6 h before, but not 8 h before or at 2-6 h after IL-1 alpha. Time course studies indicated that maximal clonogenic cell killing was achieved within 4-6 h after the cP plus IL-1 alpha combination, with little or no recovery for up to 24 h. In vivo dose-response studies indicated that cP plus IL-1 alpha combinations induced more clonogenic cell kill than cP alone in all three tumor models, and analysis by the median effect principle indicated highly synergistic antitumor activity. Dexamethasone but not indomethacin inhibited the synergistic interaction. IL-1 alpha had no effect on the cytotoxicity of cP in SCC VII cells in vitro, and neither in vitro hypoxia nor in vivo ischemia, induced by clamping tumor blood supply, significantly affected cP clonogenic cell killing. Increased clonogenic cell killing was seen, however, after removal of the clamp, implicating reperfusion events, such as oxyradical stress, as a potential mechanism for increased cP cytotoxicity in SCC VII solid tumors. The data from our model systems provide a rationale for additional work to define the mechanisms of the synergistic antitumor activity of the cP plus IL-1 alpha combination and indicate that IL-1 alpha might be a useful adjunct to increase the clinical efficacy of cP-containing strategies for both sensitive and cP-resistant cancers.     

Cell cycle dependency of 67gallium uptake and cytotoxicity in human cell lines of hematological malignancies

67Gallium (67Ga) is a radionuclide which accumulates in hematological malignancies and is used for diagnostic imaging. We investigated in this in vitro study the cell cycle dependency of cellular uptake and cytotoxicity of 67Ga. Cell cycle synchronization of cells was achieved by counterflow centrifugal elutriation and the use of cytostatic drugs. The human lymphoma cell lines U-937 and U-715 were used and in elutriation experiments we also used the leukemic cell line HL-60. The transferrin receptor (CD71) expression, 67Ga uptake and cell proliferation inhibition were the parameters measured. We also studied cytotoxicity in various schedules for combination of 67Ga and drugs and the residual proliferative capacity was measured. The CD71 expression in the three cell lines increased from 106-177% on S phase cells and from 118-233% on G2M cells, as compared to the G0/G1 cell fraction. The 67Ga uptake varied from 108-127% for S cells and 128-139% for G2M cells. The drugs chosen induced cell cycle phase accumulation in S and/or G2M phase during preincubation. 67Ga preincubation induced accumulation in the G2M phase. Almost all combinations of 67Ga and drugs resulted in a non-interactive effect, except for methotrexate which resulted in an antagonistic effect. No preferential effect of any of the incubation schemes was seen. CD71 expression and 67Ga uptake were increased in S and G2M cells. Combination of 67Ga with drugs which arrest cells in these cell cycle phases did not result in a change in cytotoxicity. However, these results implicate that 67Ga and the cytostatic drugs tested except for methotrexate might be used together or sequentially in therapy.     

Combination chemotherapy studies with gemcitabine and etoposide in non-small cell lung and ovarian cancer cell lines

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and etoposide (4'-demethylepipodo-phyllo-toxin-9-4,6-O-ethylidene-beta-D-g lucopyranoside, VP-16) are antineoplastic agents with clinical activity against various types of solid tumors. Because of the low toxicity profile of dFdC and the differences in mechanisms of cytotoxicity, combinations of both drugs were studied in vitro. For this purpose, we used the human ovarian cancer cell line A2780, its cis-diammine-dichloroplatinum-resistant and VP-16 cross-resistant variant ADDP, and two non-small cell lung cancer cell lines, Lewis Lung (LL, murine) and H322 (human). The interaction between the drugs was determined with the multiple drug effect analysis (fixed molar ratio) and with a variable drug ratio. In the LL cell line, the combination of dFdC and VP-16 at a constant molar ratio (dFdC:VP-16 = 1:4 or 1:0.125 after 4- or 24-hr exposure, respectively) was synergistic (combination index [CI], calculated at 50% growth inhibition = 0.7 and 0.8, respectively; CI <1 indicating synergism). After 24- and 72-hr exposure to both drugs at a constant ratio, additivity was found in the A2780, ADDP, and H322 cell lines (dFdC:VP-16 = 1:500 for both exposure times in these cell lines). When cells were exposed to a combination of dFdC and VP-16 for 24 or 72 hr, with VP-16 at its IC25 and dFdC in a concentration range, additivity was found in both the LL and H322 cells; synergism was observed in the A2780 and ADDP cells, which are the least sensitive to VP-16. Schedule dependency was found in the LL cell line; when cells were exposed to dFdC 4 hr prior to VP-16 (constant molar ratio, total exposure 24 hr), synergism was found (CI = 0.5), whereas additivity was found when cells were exposed to VP-16 prior to dFdC (CI = 1.6). The mechanism of interaction between the drugs was studied in more detail in the LL cell line; dFdCTP accumulation was 1.2-fold enhanced by co-incubation with VP-16, and was even more pronounced (1.4-fold) when cells were exposed to VP-16 prior to dFdC. dCTP levels were decreased by VP-16 alone as well as by the combination of both compounds, which may favor phosphorylation of dFdC, thereby increasing dFdCTP accumulation. DNA strand break (DSB) formation was increased for exposure to both compounds together compared to exposure to each compound separately, this effect being most pronounced when cells were exposed to VP-16 prior to dFdC (38% and 0% DSB for dFdC and VP-16 alone, respectively and 97% DSB for the combination). The potentiation in DSB formation might be a result of the inhibition of DNA repair by dFdC. Provided the right schedule is used, VP-16 is certainly a compound eligible for combination with dFdC.