Schedule-dependent Interactions between Raltitrexed and Cisplatin in Human Carcinoma Cell Lines in vitro

Raltitrexed ('Tomudex") is a new anticancer agent which inhibits thymidylate synthase. To provide a rational basis for clinical trial design of the combination of raltitrexed and cisplatin, we studied the cytotoxic effects of this combination using various schedules in vitro and four human colon cancer cell lines, Colo201, Colo320, LoVo, and WiDr. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the concentration producing 80% cell growth inhibition (IC₈₀) level were analyzed by the isobologram method. Simultaneous exposure to raltitrexed and cisplatin for 24 h, and sequential exposure to raltitrexed followed by cisplatin produced additive effects in the Colo201, Colo320, and LoVo cells, and additive and synergistic effects in WiDr cells. Sequential exposure to cisplatin followed by raltitrexed produced additive effects in the Colo201 cells and antagonistic effects in other three cell lines. Simultaneous and continuous exposure to both agents for 5 days produced additive effects in all four cell lines. These findings suggest that the simultaneous administration of raltitrexed and cisplatin, or the sequential administration of raltitrexed followed by cisplatin, generally produce the expected cytotoxicity at the cellular level and are optimal schedules, while the sequential administration of cisplatin followed by raltitrexed produces antagonistic effects and is inappropriate for this combination. Further in vivo and clinical studies will be necessary to determine the toxicity and antitumor effects of this schedule.     

Schedule-dependent interactions between raltitrexed and cisplatin in human carcinoma cell lines in vitro.

Raltitrexed ('Tomudex') is a new anticancer agent which inhibits thymidylate synthase. To provide a rational basis for clinical trial design of the combination of raltitrexed and cisplatin, we studied the cytotoxic effects of this combination using various schedules in vitro and four human colon cancer cell lines, Colo201, Colo320, LoVo, and WiDr. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the concentration producing 80% cell growth inhibition (IC(80)) level were analyzed by the isobologram method. Simultaneous exposure to raltitrexed and cisplatin for 24 h, and sequential exposure to raltitrexed followed by cisplatin produced additive effects in the Colo201, Colo320, and LoVo cells, and additive and synergistic effects in WiDr cells. Sequential exposure to cisplatin followed by raltitrexed produced additive effects in the Colo201 cells and antagonistic effects in other three cell lines. Simultaneous and continuous exposure to both agents for 5 days produced additive effects in all four cell lines. These findings suggest that the simultaneous administration of raltitrexed and cisplatin, or the sequential administration of raltitrexed followed by cisplatin, generally produce the expected cytotoxicity at the cellular level and are optimal schedules, while the sequential administration of cisplatin followed by raltitrexed produces antagonistic effects and is inappropriate for this combination. Further in vivo and clinical studies will be necessary to determine the toxicity and antitumor effects of this schedule.     

Schedule-dependent Synergism and Antagonism between Raltitrexed ("Tomudex") and Methotrexate in Human Colon Cancer Cell Lines in vitro

The folate-dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed ("Tomudex"), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (Icg₈₀ and IC₅₀) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule-dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.     

Schedule-dependent synergism and antagonism between raltitrexed ("Tomudex") and methotrexate in human colon cancer cell lines in vitro.

The folate-dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed ("Tomudex") ), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (IC(80) and IC(50)) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule-dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.     

Schedule-dependent synergism and antagonism between pemetrexed and paclitaxel in human carcinoma cell lines in vitro

Pemetrexed is a novel multitargeted antifolate with significant clinical activity against a variety of tumors. We studied the schedule-dependent cytotoxic effects of pemetrexed in combination with paclitaxel in vitro to improve our understanding of how this combination might be used clinically. Human lung cancer A549 cells, breast cancer MCF7, ovarian cancer PA1, and colon cancer WiDr cells were exposed to both pemetrexed and paclitaxel in vitro. Cell growth inhibition after 5 days was determined and the effects of drug combinations were analyzed by the isobologram method (Steel and Peckham). Simultaneous exposure to pemetrexed and paclitaxel for 24 h produced antagonistic effects in A549 and PA1 cells, additive/antagonistic effects in MCF7 cells, and additive effects in WiDr cells. Pemetrexed for 24 h followed by paclitaxel for 24 h produced synergistic effects in A549 and MCF7 cells and additive effects in PA1 and WiDr cells, while the reverse sequence produced additive effects in all four cell lines. Cell cycle analysis supported these observations. Our findings suggest that the simultaneous administration of pemetrexed and paclitaxel is suboptimal. The optimal schedule of pemetrexed in combination with paclitaxel is the sequential administration of pemetrexed followed by paclitaxel, and this schedule should be assessed in clinical trials for the treatment of solid tumors.     

Schedule-dependent synergism and antagonism between paclitaxel and methotrexate in human carcinoma cell lines

Paclitaxel and methotrexate are active against a variety of solid tumors. Because of differences in their mechanisms of action and toxicity profiles, the combination of these two agents has clinical potential. Clinical studies of this combination are in progress. We studied the optimal schedule of paclitaxel and methotrexate in combination at various schedules in vitro using human lung cancer A549, breast cancer MCF7, ovarian cancer PA1, and colon cancer WiDr cells. Cells were simultaneously exposed to paclitaxel and methotrexate for 24 h and sequentially exposed to paclitaxel for 24 h followed by methotrexate for 24 h or vice versa. Cell growth inhibition after 5 days was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentration of drug that produced 80% cell growth inhibition (the IC80 level) were analyzed by the isobologram method. The simultaneous exposure to paclitaxel and methotrexate produced additive to antagonistic effects in the A549 and PA1 cells, and antagonistic effects in the MCF7 and WiDr cells. The sequential exposure to paclitaxel followed by methotrexate produced additive effects in all four cell lines. The reverse sequence produced synergistic effects in the A549, MCF7, and WiDr cells, and additive effects in the PA1 cells. These findings suggest that a sequential administration of methotrexate followed by paclitaxel may be the appropriate schedule for this combination. On the basis of the observed in vitro synergism, further in vivo and clinical studies are necessary to clarify the toxicity and proposed antitumor effects of this schedule.     

Schedule-dependent interaction between paclitaxel and 5-fluorouracil in human carcinoma cell lines in vitro.

We assessed the cytotoxic interaction between paclitaxel and 5-fluorouracil administered at various schedules against four human carcinoma cell lines, A549, MCF7, PA1 and WiDr. The cells were exposed simultaneously to paclitaxel and to 5-fluorouracil for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days respectively. In another experiment, the cells were exposed simultaneously to both agents for 5 days. Cell growth inhibition was determined by MTT reduction assay. The effects of drug combinations at IC80 were analysed by the isobologram. The cytotoxic interaction of paclitaxel and 5-fluorouracil was definitely schedule dependent. Simultaneous exposure to paclitaxel and 5-fluorouracil for 24 h showed mainly subadditive effects in A549, MCF7 and WiDr cell lines, whereas it showed additive effects in PA1 cells. Sequential exposure to paclitaxel followed by 5-fluorouracil showed additive effects in all cell lines. Sequential exposure to 5-fluorouracil followed by paclitaxel showed subadditive effects in A549, MCF7 and PA1 cells. Whereas it showed additive effects in WiDr cells. These findings suggest that maximum cytotoxic effects can be obtained when paclitaxel precedes 5-fluorouracil. Interestingly, the continuous (5-day) exposure to paclitaxel and 5-fluorouracil had additive effects in A549, PA1 and WiDr cells, indicating that the prolonged simultaneous administration of these agents may circumvent the antagonistic interaction produced by short-term simultaneous administration. These findings may be useful in clinical trials of combination chemotherapy with paclitaxel and 5-fluorouracil.     

Schedule-dependent interactions between pemetrexed and cisplatin in human carcinoma cell lines in vitro

The combination of pemetrexed and cisplatin shows good clinical activity against mesothelioma and lung cancer. In order to study the potential cellular basis for this, and provide leads as to how to optimize the combination, we studied the schedule-dependent cytotoxic effects of pemetrexed and cisplatin against four human cancer cell lines in vitro. Tumor cells were incubated with pemetrexed and cisplatin for 24 h at various schedules. The combination effects after 5 days were analyzed by the isobologram method. Both simultaneous exposure to pemetrexed and cisplatin for 24 h and sequential exposure to cisplatin for 24 h followed by pemetrexed for 24 h produced antagonistic effects in human lung cancer A549, breast cancer MCF7, and ovarian cancer PA1 cells and additive effects in colon cancer WiDr cells. Pemetrexed for 24 h followed by cisplatin for 24 h produced synergistic effects in MCF7 cells, additive/synergistic effects in A549 and PA1 cells, and additive effects in WiDr cells. Cell cycle analysis of MCF7 and PA1 cells supported these findings. Our results suggest that the simultaneous clinical administration of pemetrexed and cisplatin may be suboptimal. The optimal schedule of pemetrexed in combination with cisplatin at the cellular level is the sequential administration of pemetrexed followed by cisplatin and this schedule is worthy of clinical investigations.     

Schedule-dependent synergism and antagonism between methotrexate and cytarabine against human leukemia cell lines in vitro.

Methotrexate (MTX) and cytarabine have been widely used for the treatment of acute leukemias and lymphomas for over 30 years. However, the optimal schedule of this combination is yet to be determined and a variety of schedules of the combination has been used. We studied the cytotoxic effects of MTX and cytarabine in combination against human leukemia cell lines at various schedules in vitro. The effects of the combinations at the concentration of drug that produced 80% cell growth inhibition (IC(80)) were analyzed using the isobologram method of Steel and Peckham. Simultaneous exposure to MTX and cytarabine for 3 days produced antagonistic effects in human T cell leukemia, MOLT-3 and CCRF-CEM, B cell leukemia, BALL-1, Burkitt's lymphoma, Daudi, promyelocytic leukemia, HL-60 and Philadelphia chromosome-positive leukemia, K-562 cells. Simultaneous exposure to MTX and cytarabine for 24 h produced antagonistic effects, sequential exposure to MTX for 24 h followed by cytarabine for 24 h produced synergistic effects, and the reverse sequence produced additive effects in both CCRF-CEM and HL-60 cells. Sequential exposure to MTX for 24 h followed by cytarabine for 3 days also produced synergistic effects in MOLT-3 cells. Cell cycle analysis supported these observations. Our findings suggest that the simultaneous administration of MTX and cytarabine is not appropriate and the sequential administration of MTX followed by cytarabine may be the optimal schedule of this combination.     

Schedule‐dependent interactions between vinorelbine and paclitaxel in human carcinoma cell lines in vitro

Vinorelbine and paclitaxel are new anticancer agents that bind to distinct sites on tubulin and affect microtubules in opposite ways. Clinical studies of combinations of these agents have been in progress against breast cancer and some solid tumors. To clarify the optimal schedule for this combination, we studied the scheduledependent cytotoxic effects of vinorelbine and paclitaxel against the human lung carcinoma cell line A549, the breast carcinoma cell line MCF7, the ovarian carcinoma cell line PAl, and the colon carcinoma cell line WiDr in vitro. Tumor cells were incubated with vinorelbine and paclitaxel simultaneously for both 24h and 5 days. Cells were also incubated with vinorelbine for 24h, followed by a 24h exposure to paclitaxel and vice versa. Cell growth inhibition after 5 days was determined by MTT assay. The effects of drug combinations at the concentration producing 80% cell growth inhibition (IC₈₀) were analyzed by the isobologram method (Steel and Peckham).The simultaneous exposures to vinorelbine and paclitaxel for both 24h and 5 days produced additive effects for all four cell lines. The sequential exposure to vinorelbine followed by paclitaxel produced additive effects for the PAl and WiDr cells, additive and antagonistic effects for the A549 cells, and antagonistic effects for the MCF7 cells. The sequential exposure to paclitaxel followed by vinorelbine produced additive effects for the A549, and PAl cells, additive and antagonistic effects for the MCF7 cells, and antagonistic effects for the WiDr cells.Our findings suggest that the simultaneous but not the sequential administration of vinorelbine and paclitaxel may be optimal schedule for this combination of these two agents. Applications of this schedule dependency may be beneficial for the treatment of breast cancer and other solid tumors.     

Schedule-dependent interaction between paclitaxel and doxorubicin in human cancer cell lines in vitro

The schedule-dependent interaction of paclitaxel and doxorubicin was evaluated in four human cancer cell lines. The cells were exposed simultaneously or sequentially to the two agents for 24 h, and were then incubated in drug-free medium for 4 and 3 days, respectively. The cell growth inhibitions were determined by the MTT assay. The cytotoxic interactions at the ₈₀ level were evaluated by the isobologram method of Steel and Peckham. In non-small cell lung cancer A549, breast cancer MCF7 and colon cancer WiDr cells, antagonistic effects were observed for the paclitaxel and doxorubicin combination on simultaneous exposure to the two agents and on sequential exposure to doxorubicin followed by paclitaxel, while additive effects were observed for the combination on sequential exposure to paclitaxel followed by doxorubicin. In ovarian cancer PA1 cells, additive effects were observed for all schedules. These findings suggest that sequential administration of paclitaxel followed by doxorubicin may be the most suitable sequence, while the simultaneous administration of the two agents and the sequential administration of doxorubicin followed by paclitaxel may result in less tumour cell kill than anticipated. Further preclinical and clinical studies are required to elucidate the relationship between paclitaxel and doxorubicin with regard to both antitumour activity and toxicity.     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

Paclitaxel and irinotecan are important new anticancer agents. The combination of these two agents has been considered for use against a variety of advanced solid tumors. Since the schedule-dependent effects of this combination may be crucial to its use, we studied the interaction of paclitaxel and SN-38 (the active metabolite of irinotecan) in various schedules in four human cancer cell lines in culture. Cell growth inhibition after 5 days was determined using an MTT assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. Simultaneous exposure to paclitaxel and SN-38 for 24 h produced antagonistic (subadditive and protective) effects in the human lung cancer cell line A549, the breast cancer cell line MCF7, and the colon cancer cell line WiDr, and produced additive effects in the ovarian cancer cell line PA1. Sequential exposure to paclitaxel for 24 h followed by SN-38 for 24 h, and the reverse sequence, produced additive effects in all four cell lines. These findings suggest that sequential administration, not simultaneous administration, may be the appropriate schedule for the therapeutic combination of paclitaxel and irinotecan. Continued preclinical and clinical studies should provide further insights and assist in determining the optimal schedule for this combination in clinical use. Received: 25 February 1997 / Accepted: 6 November 1997     

In vitro schedule-dependent interaction between paclitaxel and cisplatin in human carcinoma cell lines

 The schedule-dependent interaction of paclitaxel and cisplatin was studied in four human carcinoma cell lines: non-small cell lung cancer, A549; breast cancer, MCF7; ovarian cancer, PA1; and colon cancer, WiDr cells. The cells were exposed simultaneously to the drugs for 24 h and sequentially to paclitaxel first for 24 h followed by cisplatin for 24 h, or vice versa, and then incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was then determined by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. On simultaneous exposure to paclitaxel and cisplatin, additive and sub-additive (slight antagonistic) effects were observed in A549, MCF7, and PA1 cells, while sub-additive and protective (antagonistic) effects were observed in WiDr cells. On sequential exposure to paclitaxel first, followed by cisplatin, additive effects were observed in all cell lines. On sequential exposure to cisplatin first, followed by paclitaxel, additive effects were observed in PA1 cells, while additive, sub-additive, and protective effects were observed in A549, MCF7, and WiDr cells. These findings suggest that the interaction of paclitaxel and cisplatin is schedule- and cell line-dependent. The optimal schedule of this combination may be paclitaxel first followed by cisplatin. Received: 15 November 1994/Accepted: 21 June 1995     

Enhanced antitumor activity of irofulven in combination with 5-fluorouracil and cisplatin in human colon and ovarian carcinoma cells

Irofulven (6-hydroxymethylacylfulvene, MGI-114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product obtained from the Omphalotus mushroom. Irofulven has demonstrated potent activity against a broad range of solid tumors in both cellular and xenograft models and has shown promising activity in clinical trials. To guide the clinical use of irofulven, the present study used the MTT viability assay to examine the cytotoxic effects obtained by combining irofulven with two other anticancer agents: cisplatin and 5-fluorouracil (5-FU). The study was carried out with HT-29 and HCT-116 colorectal and A2780 ovarian carcinoma cells as well as with their irofulven- (HT-29/IF2, HCT-116/IF27) or cisplatin-resistant (A2780/CP70) variants. The combinations showed strong sequence specificity. Simultaneous exposure to cisplatin and irofulven was at least additive for four cell lines including the cisplatin-resistant A2780/CP70 ovarian cells which exhibit a multifactorial resistance phenotype. Cisplatin followed by irofulven was additive for parental HCT-116 and A2780 cells whereas irofulven followed by cisplatin was antagonistic in all cellular models. Simultaneous exposure to 5-FU and irofulven was at least additive for all six cell lines. 5-FU followed by irofulven was additive for the parental HT-29 and A2780 cells and synergistic for the irofulven-resistant HCT-116 cell line. Irofulven followed by 5-FU was synergistic for the two ovarian cell lines and additive for the two parental colon cell lines. These studies demonstrate that simultaneous exposure to irofulven and cisplatin is at least additive for most cell lines whereas simultaneous exposure to irofulven and 5-FU is additive to synergistic for all the cell lines tested, including the irofulven- and cisplatin-resistant variants. The enhanced cytotoxicity of irofulven in combination with cisplatin and 5-FU support the clinical application of these regimens.     

Combination of raltitrexed with other cytotoxic agents: rationale and preclinical observations

Agents for use in combination therapy should be effective as monotherapy in the tumour type of interest, have different mechanisms of action or pharmacology, and preferably non-overlapping toxicity profiles. Raltitrexed is effective as monotherapy in a number of tumour types, but it is hoped that combining it with other cytotoxic agents will lead to enhanced efficacy. Raltitrexed and 5-fluorouracil (5-FU) are specific and non-specific inhibitors, respectively, of thymidylate synthase, a critical enzyme in the de novo synthesis of DNA. Preclinical studies have indicated that raltitrexed and 5-FU have an incompletely overlapping spectrum of antitumour activity and may have additive or synergistic effects on colon carcinoma cells. These interactions are schedule-dependent (raltitrexed should precede 5-FU). Pre-treatment of colon carcinoma cells with raltitrexed has also been shown to increase intracellular levels of phosphoribosyl pyrophosphate resulting in increased incorporation of 5-FU nucleotides into RNA. Raltitrexed has a different mechanism of action from two other new agents active in colorectal cancer, irinotecan and oxaliplatin, and tumours are therefore not necessarily cross-resistant. Short pre-exposure of colon carcinoma cells to the irinotecan active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), prior to exposure to raltitrexed has consistently resulted in synergistic cell kill, whereas the reverse sequence is antagonistic. Preliminary results indicate that equitoxic doses of raltitrexed and cisplatin, or oxaliplatin, are antagonistic in two colon carcinoma cell lines. However, because there are major difficulties in translating preclinical drug combination results to the clinical settings, these results should be interpreted with caution.     

in vitro synergistic antitumor activity of a combination of 5-fluorouracil and irinotecan in human colon cancer

The combination of irinotecan and a fluoro-pyrimidine is widely accepted as a treatment for advanced colorectal cancer. However, evaluable data on the feasibility of these combinations has not been presented, and an optimal sequence for administration has not been experimentally and clinically determined. The sequential effect of a combination of 5-FU and CPT-11 in the human colon cancer cell line LoVo was evaluated by WST-8 colorimetric assay. The cytotoxicity and cell cycle distributions of each drug were analyzed by apoptosis assay and flow cytometry. Further, the potential mechanisms of the sequence-dependent effects were investigated by a microarray technique, and confirmed by Western blot analysis. The cytotoxicity of 5-FU (10, 100, 1000 microM) followed by CPT-11 (1 microM) was significantly greater than that of CPT-11 (1 microM) followed by 5-FU (10, 100, 1000 microM) (p<0.05). In cell cycle distribution, 5-FU exposure for 24 h increased the S phase fraction in a dose-dependent manner; though there was no significant difference in cell cycle distribution in 24 h CPT-11 (0.01-1 microM) exposure. Microarray analysis revealed that expressions of some apoptosis related genes such as Bcl-2 changed, and were correlated with sequence-dependent cytotoxicity of the 5-FU --> CPT-11 sequence. Western blot analysis confirmed that the Bcl-2/Bax ratio was lower after 5-FU --> CPT-11 sequence than before. The sequence-dependent cytotoxic effect may depend on the sensitizing effect of 5-FU pretreatment on CPT-11 cytotoxicity. 5-FU followed by CPT-11 administration may be an optimal sequence for IFL treatment of advanced colon cancer.     

Schedule-dependent synergism and antagonism between pemetrexed and docetaxel in human lung cancer cell lines in vitro

Background  Pemetrexed and docetaxel show clinical activities against a variety of solid tumors including lung cancers. To identify the optimal schedule for combination, cytotoxic interactions between pemetrexed and docetaxel were studied at various schedules using three human lung cancer cell lines A-549, Lu-99, and SBC-5 in vitro. Methods  Cells were incubated with pemetrexed and docetaxel simultaneously for 24 or 120 h. Cells were also incubated with pemetrexed for 24 h, followed by a 24 h exposure to docetaxel, and vice versa. Growth inhibition was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis. Cytotoxic interactions were evaluated by the isobologram method. Results  Simultaneous exposure to pemetrexed and docetaxel for 24 and 120 h produced antagonistic effects in all three cell lines. Pemetrexed (24 h) followed by docetaxel (24 h) produced additive effects in A-549 cells and synergistic effects in Lu-99 and SBC-5 cells. Docetaxel followed by pemetrexed produced additive effects in A-549 and Lu-99 cells and antagonistic effects in SBC-5 cells. The results of cell cycle analysis were fully consistent with those of isobologram analysis, and provide the molecular basis of the sequence-dependent difference in cytotoxic interactions between the two agents. Conclusions  Sequential administration of pemetrexed followed by docetaxel may provide the greatest anti-tumor effects for this combination in the treatment of lung cancer.     

In vitro schedule-dependent interaction between paclitaxel and oxaliplatin in human cancer cell lines

Purpose In order to define the most effective administration schedule of the combination of paclitaxel and oxaliplatin, we investigated the in vitro interaction between these drugs in a panel of three human cancer cell lines (AZ-521 gastric adenocarcinoma cell line, HST-1 tongue squamous carcinoma cell line, and KSE-1 esophageal squamous carcinoma cell line).Materials and methods Cytotoxic activity was determined by the WST-1 assay. Different administration schedules of the two drugs were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay. Cell cycle perturbation and apoptosis were evaluated by flow cytometry.Results Simultaneous treatment of cells with paclitaxel and oxaliplatin showed greater than additive effects. Upon 24-h sequential exposure, the sequence of paclitaxel followed by oxaliplatin showed synergistic effects in AZ-521 and HST-1 cells, and greater than additive effects in KSE-1 cells, while the opposite sequence yielded marked antagonistic effects in all three cell lines. Flow cytometric analysis indicated that paclitaxel induced G₂/M arrest with subsequent induction of apoptosis in the sub-G₁ phase. Apoptosis was most prominent when paclitaxel preceded oxaliplatin, which produced apoptosis in the majority of treated cells (75%). By contrast, the reverse sequence yielded only 39% induction of apoptotic cells, the rate being not different from those induced by each drug singly.Conclusions Our findings suggest that the interaction of paclitaxel and oxaliplatin is highly schedule-dependent and that the sequential administration of paclitaxel followed by oxaliplatin should thus be incorporated into the design of a clinical trial.     

Irinotecan-induced cytotoxicity to colon cancer cells in vitro is stimulated by pre-incubation with trifluorothymidine

SN38 is the active metabolite of the anti-cancer agent irinotecan (CPT-11) and is a potent inhibitor of topoisomerase-I (topo-I), leading to DNA strand breaks and eventually cell death. The pyrimidine analog trifluorothymidine (TFT) is part of the anti-cancer drug formulation TAS-102, which was developed to enhance the bioavailability of TFT in vivo, and is currently being evaluated as an oral chemotherapeutic agent in phase I clinical studies. In this study, the combined cytotoxic effects of dual-targeted TFT with SN38 were investigated in a panel of human colon cancer cell lines (WiDr, H630, Colo320, SNU-C4, SW1116). We used different drug combination treatment schedules of SN38 with TFT, and possible synergism was evaluated using median drug effect analysis resulting in combination indexes (CI), in which CI<0.9 indicates synergism, CI=0.9-1.1 indicates additivity and CI>1.1 indicates antagonism. Drug target analysis was performed to investigate the effect of TFT on SN38-induced DNA damage, cell cycle delay and apoptosis. Simultaneous exposure to SN38 in combination with TFT was not more than additive, whereas pre-incubation with TFT resulted in synergism with SN38 (CI=0.3-0.6). Only for Colo320 synergism could be induced for both simultaneous and sequential drug combinations. SN38 and TFT induced most DNA damage in H630 and Colo320 cells, which was increased in combination. TFT pre-incubation further enhanced SN38-induced DNA strand breaks in H630 and Colo320 (>20%), which was most pronounced in H630 cells (p<0.01). Exposure to SN38 alone induced a clear cell cycle G2M-phase arrest and pre-incubation with TFT enhanced this effect in WiDr and H630 (p<0.05). Both drugs induced significant apoptosis; SN38-induced apoptosis increased significantly in the presence of TFT (p<0.01), either when added simultaneously (about 3-fold) or at pre-incubation (about 2-fold). Topo-I protein levels varied among the cell lines and TFT hardly affected these. In conclusion, TFT pre-incubation can enhance SN38-induced cytotoxicity to colon cancer cells resulting in synergism between the drugs, thereby increasing DNA damage and apoptosis induction.     

Supraadditive effect of 2′,2′-difluorodeoxycytidine (gemcitabine) in combination with oxaliplatin in human cancer cell lines

PURPOSE: This study assessed the cytotoxic effects of the nucleoside analog gemcitabine in combination with the diaminocyclohexane platinum compound oxaliplatin. METHODS: Growth inhibition studies were performed using the human CEM leukemia cell line and the colon-cancer cell lines HCT 116 and Colo 320 DM. Gemcitabine-oxaliplatin combinations were compared with gemcitabine-cisplatin combinations in the same cell lines using similar experimental settings. Cells were exposed for 2 h to gemcitabine and then for 24 h to oxaliplatin or cisplatin, and vice versa. RESULTS: The 50% inhibitory concentrations (IC50 values) in single-drug experiments using 2 h of exposure to gemcitabine and 24 h of exposure to oxaliplatin or cisplatin were, respectively, 89 pM, 11.1 microM, and 10.3 microM for CEM cells; 46 pM, 10.2 microM, and 2.7 microM for HCT 116 cells; and 102 pM, 4.6 microM, and 8.6 microM for Colo 320 DM cells. Gemcitabine-oxaliplatin combinations displayed supraadditive effects in human leukemia and colon-cancer cell lines. The sequence of gemcitabine followed by oxaliplatin was more effective than the opposite sequence in HCT 116 and Colo 320 DM colon-cancer cell lines, whereas the sequence of oxaliplatin followed by gemcitabine yielded to synergistic effects in CEM cells. The cytotoxic effects of gemcitabine-oxaliplatin combinations were better than (HCT 116 cells) or equal to (CEM and Colo 320 DM cells) those of gemcitabine-cisplatin combinations. CONCLUSION: Our data show that the combination of gemcitabine with oxaliplatin exerts potent antiproliferative effects in human leukemia and colon cancer cells, warranting further investigations in the framework of phase I-II trials as an alternative for the treatment of solid malignancies.