Quantitative evaluation of cellular uptake, DNA incorporation and adduct formation in cisplatin sensitive and resistant cell lines: Comparison of different Pt-containing drugs.

The use of Pt-containing compounds as chemotherapeutic agents facilitates drug monitoring by using highly sensitive elemental techniques like inductively coupled plasma mass spectrometry (ICP-MS). However, methodological problems arise when trying to compare different experiments due to the high variability of biological parameters. In this work we have attempted to identify and correct such variations in order to compare the biological behavior of cisplatin, oxaliplatin and pyrodach-2 (a novel platinum-containing agent). A detailed study to address differential cellular uptake has been conducted in three different cell lines: lung adenocarcinoma (A549); cisplatin-sensitive ovarian carcinoma (A2780); and cisplatin-resistant ovarian carcinoma (A2780cis). The normalization of Pt results to cell mass, after freeze-drying, has been used to minimize the errors associated with cell counting. Similarly, Pt accumulation in DNA has been evaluated by referencing the Pt results to the DNA concentration, as measured by (31)P monitoring using flow-injection and ICP-MS detection. These strategies have permitted to address significantly lower Pt levels in the resistant cells when treated with cisplatin or oxaliplatin as well as an independent behaviour from the cell type (sensitive or resistant) for pyrodach-2. Similarly, different levels of incorporation in DNA have been found for the three drugs depending on the cell model revealing a different behavior regarding cell cisplatin resistance. Further speciation experiments (by using complementary HPLC-ICP-MS and HPLC-ESI-Q-TOF MS) have shown that the main target in DNA is still the N7 of the guanine but with different kinetics of the ligand exchange mechanism for each of the compounds under evaluation.