Molecular and cellular effects of AP5346, a novel DACH-platinum linked polymer-compound, compared to oxaliplatin and cisplatin in a panel of human cancer cell lines

C105 Background: AP5346 comprises a novel hydrophilic biocompatible copolymer which acts as a macromolecular carrier, combined with a bioactive DACH-platinum (Pt) complex, and has recently entered clinical trials. The pH-dependent sustained Pt-release from the polymer may improve efficacy and safety as compared to oxaliplatin and cisplatin. Our aim was to determine the mechanism of AP5346 action, its transport, distribution and metabolism in cancer cell lines, in comparison to oxaliplatin and cisplatin.
 Methods : Antiproliferative effects of AP5346, oxaliplatin and cisplatin were evaluated in human colon (HT29, HCT116, Colo205, HCC2998), breast (MCF7, MDA-MB-435, MDA-MB-361, SKBR-3), lung (HOP62, HOP92), ovarian (OVCAR3, IGROV1, CAOV3), prostate (PC3) and leukemia (CCRF-CEM, K562) cancer cell lines, by MTT assay. Apoptosis induction was evaluated by FACS analysis. The rate of Pt release was determined over several incubation times and at several pH levels. Pt levels in solution, supernatant, cell lysates and in DNA were measured by atomic absorption. Expression level of genes was determined using q-RT-PCR.
 Results: AP5346 displayed cytotoxic effects against human cancer cell lines (IC 50 values ranging from 3-85 µM), with HT29 being the most sensitive. At equimolar concentrations, AP5346 and oxaliplatin displayed similar spectra of activity notably different from that of cisplatin. Exposure to IC 50 concentrations of AP5346 led to the accumulation of HT29 and HCT116 cells in the G2/M phase of cell cycle, as well as apoptosis induction. In HCT116 cells, these effects were associated with >5-fold increase of p21 expression and about 10-fold decrease of Ki67 and NEK2 mRNA levels. Exposure to 120 µM AP5346 led to the incorporation of approximately 0.1 µg Pt per mg of DNA. The amount of Pt adducts observed with AP5346 was less than that observed with equiplatinum concentrations of oxaliplatin (4 hours, pH 7) presumably due to the slow release of Pt from the polymer. After 4 hours, 4% of platinum was released at pH 5.4 whereas 70% of platinum was released at pH 3.0 in the same time period. In HCT116 MMR proficiency cells, a 4-hour exposure to 120 µM AP5346 resulted in approximately 0.33% intracellular Pt accumulation, decreasing to approximately 0.07% 24 hours after the removal of the drug from media. In HCT116 cells a 4-hour exposure to AP5346 at pH 3.0 resulted in approximately in two log more Pt adducts per mg of DNA, than at pH 5.4 which, in turn doubled the amount of Pt adducts found at pH 7.0. Expression of MLH1, MDR1 and GSTP1 genes seems to correlate with AP5346 cytotoxicity in our panel of cancer cell lines. The impact of mismatch repair and p53 cell expression on AP5346 cytotoxicity is currently being explored using isogenic cell lines. Conclusions: AP5346 displayed a cytotoxic profile similar to that of oxaliplatin in most human cancer cell lines. The rate of Pt release from the polymer and incorporation into DNA is inversely related to the pH. Some proteins implicated in DNA-repair and metabolism may be important factors of AP5346 cytotoxicity to develop; cellular effects of AP5346 were associated with changes of expression of several cell cycle-related genes. Based on the expected toxicity profile, AP5346 is currently being extensively studied in clinical trials.