Identification of candidate biomarkers of therapeutic response to docetaxel by proteomic profiling.

Docetaxel chemotherapy improves symptoms and survival in men with metastatic hormone-refractory prostate cancer (HRPC). However, approximately 50% of patients do not respond to Docetaxel and are exposed to significant toxicity without direct benefit. This study aimed to identify novel therapeutic targets and predictive biomarkers of Docetaxel resistance in HRPC. We used iTRAQ-mass spectrometry analysis to identify proteins associated with the development of Docetaxel resistance using Docetaxel-sensitive PC3 cells and Docetaxel-resistant PC3-Rx cells developed by Docetaxel dose escalation. Functional validation experiments were performed using recombinant protein treatment and siRNA knockdown experiments. Serum/plasma levels of the targets in patient samples were measured by ELISA. The IC(50) for Docetaxel in the PC3-Rx cells was 13-fold greater than the parent PC-3 cell line (P = 0.004). Protein profiling identified MIC-1 and AGR2 as respectively up-regulated and down-regulated in Docetaxel-resistant cells. PC-3 cells treated with recombinant MIC-1 also became resistant to Docetaxel (P = 0.03). Conversely, treating PC3-Rx cells with MIC-1 siRNA restored sensitivity to Docetaxel (P = 0.02). Knockdown of AGR2 expression in PC3 cells resulted in Docetaxel resistance (P = 0.007). Furthermore, increased serum/plasma levels of MIC-1 after cycle one of chemotherapy were associated with progression of the cancer (P = 0.006) and shorter survival after treatment (P = 0.002). These results suggest that both AGR2 and MIC-1 play a role in Docetaxel resistance in HRPC. In addition, an increase in serum/plasma MIC-1 level after cycle one of Docetaxel may be an indication to abandon further treatment. Further investigation of MIC-1 as a biomarker and therapeutic target for Docetaxel resistance in HRPC is warranted.

Evidence for the involvement of p38 MAP kinase in the action of the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

AIMS: DMXAA (AS1404), a small-molecule vascular disrupting agent that has now completed Phase II clinical trial, induces endothelial cell apoptosis, increased vascular permeability and decreased tumour blood flow in vivo. Its action is incompletely understood and we wished to develop an in vitro system to study its effects. METHODS: Human tumour cell lines developed from aggressive tumours were grown on Matrigel to simulate a tumour microenvironment. Cells were analysed by light microscopy and by gene expression profiling. RESULTS: Several cell lines formed networks when grown on Matrigel and the NZM7 melanoma cell line was chosen for further study. Addition of DMXAA at a clinically achievable concentration (30 microg/mL) prevented network formation, but co-addition of SB203580 (10 microM), a selective inhibitor of p38 MAP kinase, reversed the effect of DMXAA and restored network formation. Analysis of expression genes for endothelial and related functions showed that cells growing on Matrigel expressed a pattern similar to that of NZM7 cells growing as xenografts in vivo but different from that of cells grown on standard tissue culture plates. Addition of DMXAA resulted in the inhibition of expression of several genes including the transcriptional activator Ets1 and matrix metalloproteinase-2 (MMP2), but co-addition of SB203580 did not reverse these effects of DMXAA on gene expression. CONCLUSION: The results suggest that p38 MAP kinase plays an important role in the action of DMXAA and that growth of tumour cells on Matrigel provides a promising model for further studies on the action of this drug.

Mechanisms of tumor vascular shutdown induced by 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Increased tumor vascular permeability.

The novel vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has completed phase 1 clinical trial and has shown tumor antivascular activity in both mice and humans. We have investigated its ability to change tumor vascular permeability, relating it to tumor vascular perfusion and other responses. The murine colon 38 adenocarcinoma was grown in C57Bl wild-type mice and mice lacking expression of either tumor necrosis factor receptor-1 (TNFR1(-/-)) or TNF (TNF-/-). Tumor vascular permeability, as measured by extravasation of albumin-Evans Blue complexes 4 hr after DMXAA treatment, was significantly increased in tumor tissue in C57Bl, TNFR1-/- and TNF-/- mice but not in normal (skin) tissue. Significant linear relationships were found between increased tumor vascular permeability, decreased functioning tumor blood vessels (measured by Hoechst 33342 staining at 4 hr), increased plasma 5-hydroxyindole-3-acetic acid concentrations (as a measure of serotonin release by platelets) and the degree of induced tumor hemorrhagic necrosis. The results support the hypothesis that DMXAA increases tumor vascular permeability both directly and through the induction of other vasoactive mediators, including TNF. DMXAA might be useful clinically to potentiate the vascular permeability of other anticancer modalities such as cytotoxic drugs, antibodies, drug conjugates and gene therapy.

Improvement of the antitumor activity of intraperitoneally and orally administered 5,6-dimethylxanthenone-4-acetic acid by optimal scheduling.

PURPOSE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer drug that has recently completed Phase I clinical trial, is effective against transplantable murine tumors with established vasculature. We wished to determine the relationship between administration schedule and antitumor activity. EXPERIMENTAL DESIGN: C57Bl/6 mice with s.c. implanted Colon 38 tumors were used for determination of maximal tolerated doses and tumor growth delay. Plasma and tissue DMXAA concentrations were measured by high-performance liquid chromatography. RESULTS: Continuous infusion (30 mg/kg/day for 3 days) and daily i.p. administration schedules (7.5 mg/kg) were ineffective. A pharmacokinetically guided schedule was developed to increase tumor tissue drug concentrations without increasing the maximal plasma concentration. A schedule comprising a loading dose (25 mg/kg, i.p.) followed by supplementary doses (5 mg/kg after 4 and 8 h) provided a 1.6-fold increase in tumor tissue area under the concentration-time curve, no increased toxicity, and superior antitumor activity (100% cure rate, as compared with 55% for a single i.p. dose of 25 mg/kg). A similar strategy was developed for oral administration with a loading dose (30 mg/kg) and supplementary doses (15 mg/kg after 4 and 8 h). It provided a 90% cure rate, in contrast to a single oral dose (0% cure rate). CONCLUSIONS: The antitumor action of DMXAA is schedule dependent, and the achievement of an adequate tumor tissue DMXAA concentration above a threshold value appears to be critical for activity. The use of a pharmacokinetically guided schedule provides excellent oral activity against Colon 38 tumors and provides a basis for developing more effective administration schedules in clinical trials.

Oral activity and pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice.

PURPOSE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an anticancer drug with an antivascular action, has recently completed phase I clinical trials. Since oral administration has many advantages, we compared the biological activity and pharmacokinetics of DMXAA in mice following oral and intraperitoneal (i.p.) administration. METHODS: Growth delays of Colon 38 tumours were measured in C57Bl/6 mice. Plasma concentrations of DMXAA, 5-hydroxyindole-3-acetic acid (5HIAA) as a measure of serotonin production, and nitrate as a measure of nitric oxide production, were determined by high-performance liquid chromatography. Tumour necrosis factor (TNF) concentrations in serum and tumour tissues were measured by ELISA. RESULTS: The antitumour activity of DMXAA at the maximum tolerated oral dose (32.5 mg/kg) was low (4-day growth delay, no cures) compared to that (19-day growth delay, 40% cures) at the maximum tolerated i.p. dose (27.5 mg/kg). The pharmacokinetics of DMXAA in plasma, liver and tumour tissue indicated a bioavailability of 73%. Elevation of plasma 5HIAA, measured 4 h following i.p. administration of DMXAA, was linear with DMXAA dose, and the 5HIAA response to oral administration was consistent with its bioavailability. TNF concentrations increased following oral administration (30 mg/kg) and were particularly evident in tumour tissue, but were lower and less prolonged than those in response to i.p. administration at 25 mg/kg. Plasma nitrate levels were not increased following oral administration (30 mg/kg). CONCLUSIONS: DMXAA exhibits good bioavailability, and changes in serum TNF, tissue TNF, plasma 5HIAA and plasma nitrate, as markers of biological response, are consistent with this bioavailability. The low maximal plasma DMXAA concentration following oral administration, resulting in reduced retention of intratumoral TNF, may be responsible for the low antitumour activity.