Increased sensitivity to platinating agents and arsenite in human ovarian cancer by downregulation of ASNA1.

Platinating agents constitute the first line treatment for ovarian cancer but treatment failure is common because of intrinsic and acquired resistance. Cancer cells develop the RASP-phenotype (cross resistance against arsenite, antimonite and platinum) associated with decreased accumulation of cisplatin and arsenite. ASNA1 is a possible subunit of a transport system for cisplatin and arsenite due to homology to arsA, an ATPase in the E. coli ars-complex responsible for efflux of arsenite and antimonite. Eukaryotic ASNA1 is a targeting factor for membrane insertion of tail-anchored proteins involved in the secretory pathway and cellular stress responses. The purpose with this study was to evaluate if ASNA1 expression influenced cisplatin, carboplatin, oxaliplatin or arsenite sensitivity in ovarian cancer. Human ovarian cancer cell line 2008 was transfected with a sense or an antisense ASNA1 construct. ASNA1 downregulated and overexpressing clones were identified by Western blots. Cell growth and chemosensitivity was determined by the MTT assay. Down-regulated ASNA1 expression was associated with retarded growth and increased sensitivity to cisplatin, carboplatin, oxaliplatin and arsenite whereas the cisplatin resistant 2008/A overexpresses ASNA1. These observations support the hypothesis that ASNA1 is a target to overcome platinum resistance in ovarian cancer.

Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry.

A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines were used. To keep the sample consumption to approximately 10 microl and obtain a high robustness of the system, a flow injection sample introduction system with a 4.6-microl sample loop was used in combination with a conventional pneumatic nebulizer and a spray chamber. The system was optimized with respect to signal/noise ratio using a multivariate experimental design. The system proved to be well suited for routine analysis of large sample series, and several hundreds of samples could be analyzed without maintenance or downtime. The detection limit of the method was 0.12 pg (26 pg/g) platinum. To avoid systematic errors from nonspectral interferences, it was necessary to use reagent matched calibration standards or isotope dilution analysis. An uncertainty budget was constructed to estimate the total expanded uncertainty of the method, giving a quantification limit of 2.3 pg (0.5 ng/g) platinum in DNA samples. The uncertainty was sufficiently low to study quantitative differences in the formation of Pt-DNA adducts after treatment with cisplatin using different exposure times and concentrations.