Addition of 5-fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines.

INTRODUCTION: The aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5-fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells. METHODS: The study was performed on MCF-7 and BRC-230 cell lines. The cytotoxic activity was evaluated by sulphorhodamine B assay and the type of drug interaction was assessed by the median effect principle. Cell cycle perturbation and apoptosis were evaluated by flow cytometry, and apoptosis-related marker (p53, bcl-2, bax, p21), caspase and thymidylate synthase (TS) expression were assessed by western blot. RESULTS: 5-FU, used as a single agent, exerted a low cytotoxic activity in both cell lines. The Dox-->Pacl sequence produced a synergistic cytocidal effect and enhanced the efficacy of subsequent exposure to 5-FU in both cell lines. Specifically, the Dox-->Pacl sequence blocked cells in the G2-M phase, and the addition of 5-FU forced the cells to progress through the cell cycle or killed them. Furthermore, Dox-->Pacl pretreatment produced a significant reduction in basal TS expression in both cell lines, probably favoring the increase in 5-FU activity. The sequence Dox-->Pacl-->48-h washout-->5-FU produced a synergistic and highly schedule-dependent interaction (combination index < 1), resulting in an induction of apoptosis in both experimental models regardless of hormonal, p53, bcl-2 or bax status. Apoptosis in MCF-7 cells was induced through caspase-9 activation and anti-apoptosis-inducing factor hyperexpression. In the BRC-230 cell line, the apoptotic process was triggered only by a caspase-dependent mechanism. In particular, at the end of the three-drug treatment, caspase-8 activation triggered downstream executioner caspase-3 and, to a lesser degree, caspase-7. CONCLUSION: In our experimental models, characterized by different biomolecular profiles representing the different biology of human breast cancers, the schedule Dox-->Pacl-->48-h washout-->5-FU was highly active and schedule-dependent and has recently been used to plan a phase I/II clinical protocol.

Cellular basis of antiproliferative and antitumor activity of the novel camptothecin derivative, gimatecan, in bladder carcinoma models.

To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting antiproliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

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

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

NCX 4016, a nitric oxide-releasing aspirin derivative, exhibits a significant antiproliferative effect and alters cell cycle progression in human colon adenocarcinoma cell lines.

Nitric oxide-releasing non-steroidal antiinflammatory drugs (NO-NSAIDs) are safer than NSAIDs due to their ability to reduce gastric toxicity. We assessed the cytotoxic activity of a new aspirin derivative, NCX 4016, after different exposure schedules, in three human colon adenocarcinoma cell lines. All the lines were positive for COX-1 protein and mRNA, as evaluated by Western blot and RT-PCR, respectively, while only one was positive for COX-2. The cytostatic and cytocidal activity was determined by sulforhodamine B assay and evaluated according to Monks' model. Cytostatic activity was observed after a 24-h drug exposure and 50% growth inhibition was reached at concentrations ranging from 165 to 250 micro M in all cell lines, whereas with aspirin the IC50 was never reached, even at the maximum concentration tested (500 micro M), and was independent of COX-1 or COX-2 status. Cytocidal activity was observed only at the highest concentrations and persisted for a long time after drug removal. Flow cytometric analysis showed that the NO-aspirin compound induced a persistent accumulation of cells in G2-M phase in all the cell lines after at least 48 h exposure. Specifically, the block pertained mainly to G2 phase, whereas mitotic index was not affected at all. Our results indicate that NCX 4016 has an in vitro cytostatic activity superior to that of its parental aspirin compound, which makes it a potentially important tumor preventive agent. Furthermore, the cytocidal effect observed at the highest concentrations and the induction of a specific block in G2 phase renders it a promising candidate for drug combination treatments.

In vitro schedule-dependent interaction between docetaxel and gemcitabine in human gastric cancer cell lines.

PURPOSE: The purpose of this study was to assess the activity of clinically used drugs and to define the most effective treatment scheme in human gastric cancer cell lines. EXPERIMENTAL DESIGN: Cytotoxic activity was evaluated by sulforhodamine B assay, potential clinical activity was estimated by relative antitumor activity, and the type of drug interaction was assessed using the method of Chou and Talalay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry, mitotic index by microscopic analysis, bax, bcl-2, and p53 by immunohistochemistry, and cyclin B expression by Western blot. RESULTS: Gemcitabine (GEM) and docetaxel (DOC) were the most potent of the seven drugs tested, with maximum relative antitumor activity values in all of the cell lines. Simultaneous treatment with GEM and DOC, and the sequence GEM-->DOC caused an antagonistic interaction, as shown by the combination index >1, at all levels of killed cell fraction. Conversely, the sequential treatment DOC-->GEM produced a synergistic interaction (combination index < 1). On the basis of cell cycle perturbations, it can be hypothesized that the antimetabolite (GEM) attacks cells recovering rapidly from an M block induced by DOC as they progress to the S phase, producing a powerful cytocidal effect, as shown by the increase from 15 to 75% of apoptotic cells. CONCLUSIONS: Our findings suggest that the interaction of DOC and GEM is highly schedule dependent and has been used recently to plan a Phase I-II clinical protocol.

Docetaxel followed by gemcitabine in the treatment of advanced non-small cell lung cancer: a phase I study.

AIMS AND BACKGROUND: Based on the results of a preclinical study, a phase I trial was conducted to evaluate the feasibility of administering docetaxel followed by gemcitabine in non-small cell lung cancer patients. STUDY DESIGN: Sixteen patients with advanced non-small cell lung cancer (stages III B-IV) were treated on the 1st day with docetaxel and on the 8th day with gemcitabine. Treatment was repeated every three weeks for a maximum of six cycles. Five groups received docetaxel/gemcitabine (mg/ml): 50/800, 60/800, 60/900, 60/1,000, 70/1,000. All patients and 57 cycles were assessed for toxicity. RESULTS: The most important side effects were grade IV neutropenia in 4 patients (2 at the 60/1000 level and 2 at the 70/1000 level) and grade III leukopenia and neutropenia without fever in 4 and 6 patients, respectively. Maximum tolerated dose was not reached. CONCLUSIONS: The sequence docetaxel-gemcitabine appears well tolerated and easy to administer. For this reason, a phase II study is ongoing to fully assess its antitumor activity.

In Vitro schedule-dependent interactions between the multitargeted antifolate LY231514 and gemcitabine in human colon adenocarcinoma cell lines.

PURPOSE: Multitargeted antifolate (MTA) and gemcitabine (GEM) have shown preclinical and clinical activity in tumor histotypes such as colon, renal, small and non-small cell lung cancers, hepatomas and carcinoid tumors. In our study, we investigated the cytotoxic activity of MTA alone or in combination with GEM using different exposure schedules in three different colon cancer cell lines (LoVo, WiDr, and LRWZ). EXPERIMENTAL DESIGN: Cytotoxic activity was evaluated by sulforhodamine B assay, cell cycle perturbations and apoptosis were evaluated by flow cytometry, and thymidylate synthase expression was evaluated by immunohistochemical method. RESULTS: A 48-h exposure to MTA caused a minimal and no-dose-response effect on the three cell lines used. Flow cytometric analysis showed a cell accumulation in S phase that completely resolved in LoVo and LRWZ cell lines and persisted in WiDr cells after a 48-h washout. Moreover, a significant increase in thymidilate synthase expression was observed in all of the cell lines after MTA exposure. Among the different combinations tested, the highest synergistic interaction, assessed using Kern's method and expressed as the synergistic ratio index, was produced by pretreatment with GEM followed by MTA (ratio index: 1.3- 6.7). It is possible that the depletion of nucleotide pools induced by MTA and required for DNA synthesis prevented cells from repairing DNA damage caused by GEM. The type and degree of drug interactions were not paralleled by apoptosis, which was almost always negligible, or by the type and persistency of the cell cycle perturbations. CONCLUSIONS: Our results indicate that the sequential administration of GEM --> MTA provides the greatest benefit in the clinical treatment of colon cancer.