The in vivo effect of bryostatin-1 on paclitaxel-induced tumor growth, mitotic entry, and blood flow

Pretreatment of tumor cells with the protein kinase C (PKC) inhibitor bryos tatin-1 enhances the cytotoxicity of most chemotherapeutic agents, However, in the case of paclitaxel, this effect has been shown in vitro to be best achieved when bryostatin-1 follows (rather than precedes) paclitaxel treatm ent. With combination trials of bryostatin-1 and paclitaxel planned for cli nical trials and with only in vitro data available regarding drug sequence, me elected to undertake an in vivo study evaluating the effect of sequenti al bryostatin-1 and paclitaxel in a tumor-bearing mouse model and to correl ate this effect to cell cycle events, tumor metabolism, and tumor blood pow , At the maximum tolerated i.p. dose, bryostatin-1 at 80 mu g/kg resulted i n a small but significant increase in tumor doubling time (4.2 +/- 0.3 days ) compared with control tumors (3.0 +/- 0.3 days; P < 0.01). Mice treated w ith i.v. paclitaxel, administered at a dose of 12 mg/kg every 12 h for thre e doses, weekly for 3 weeks, had a tumor doubling time of 23.4 +/- 1.7 days . Mice pretreated with i.p. bryostatin-1 (80 mu g/kg) followed 12 h later b y i.v. paclitaxel (12 mg/kg every 12 h for three doses) weekly for 3 weeks had a tumor doubling time of 9.7 +/- 1.1 days. This was significantly less (P < .001) than paclitaxel alone, which indicated an inhibitory effect by b ryostatin-1 on paclitaxel therapy. In comparison, tumor-bearing mice that w ere treated with the same dose but with the sequence of paclitaxel followed by bryostatin-1 had a tumor doubling time of 29.6 +/- 0.6 days. This was s ignificantly greater than the tumor doubling times for any condition tested (P < 0.01), demonstrating the sequence dependence of this combination. The efficacy of paclitaxel is dependent on mitotic entry a step that requires activation of p34(cdc2) kinase activity. Treatment with paclitaxel in vivo increased p34(cdc2) kinase activity in the mouse mammary tumors, whereas ad ministration of bryostatin-1 before paclitaxel prevented the p34(cdc2) kina se activation by paclitaxel. This was further evaluated in vitro by flow cy tometry in MKN-74 human gastric cancer cells, As determined by MPM-2 labeli ng, which identifies cells in mitosis, pretreatment with bryostatin-1 preve nted paclitaxel-treated cells from entering mitosis. Bryostatin-1 has been reported to induce changes in muscle metabolism and to decrease muscle bloo d flow. These events could impact on the interaction of bryostatin-1 with p aclitaxel. Using proton-decoupled phosphorus nuclear magnetic resonance (P- 31-NMR) spectroscopy in vivo, bryostatin-1 at 80 mu g/kg induced a decrease in both intratumoral pH and high-energy phosphates, In vivo perfusion stud ies, using dynamic enhanced NMR imaging with gadolinium diethylenetriamine pentaacetic acid, also demonstrated decreased tumor blood flow. These studi es suggest that the inhibition of tumor response to paclitaxel by bryostati n-1 is multifactorial and includes such diverse factors as inhibition of ce ll entry into mitosis, a decrease in pH and energy metabolism, and a decrea se in tumor blood flow, These results indicate that, as this combination en ters Phase I clinical trials, the sequence of paclitaxel followed by bryost atin-1 will be critical in the clinical trial design.