Abstract
In this study, we developed a strategy to determine atto- and femtomolar amounts of metal ions in lysates and mineralizates of cells (human non-small-cell lung carcinoma (NSCLC, A549) and normal lung (MRC-5)) exposed to cytotoxic metallo-drugs: cisplatin and auranofin at concentrations close to the half-maximal inhibitory drug concentrations (IC50). The developed strategy combines data obtained using biological and chemical approaches. Cell density was determined using two independent cell staining assays using trypan blue, calcein AM/propidium iodide. Metal concentrations in lysed and mineralized cells were established employing a mass spectrometer with inductively coupled plasma (ICP-MS) and equipped with a cross-flow nebulizer working in aspiration mode. It allowed for detecting of less than 1 fg of metal per cell. To decrease the required amount of sample material (from 1.5 mL to ~100 µL) without loss of sensitivity, the sample was introduced as a narrow band into a constant stream of liquid (flow-injection analysis). It was noticed that the selectivity of cisplatin accumulation by cells depends on the incubation time. This complex is accumulated by cells at a lower efficiency than auranofin and is found primarily in the lysate representing the cytosol. In contrast, auranofin interacts with water-insoluble compounds. Despite their different mechanism of action, both metallo-drugs increased the accumulation of transition metal ions responsible for oxidative stress.
Highlights
Lung cancer is the most common type of cancer and causes many deaths (1.6 million) worldwide annually [1]
Comparing the total metal contents in the cells with those determined in the cell lysates, more than 80% of the platinum remains in the lysate for normal cells (MRC-5), and less than 10% for cancer cells
Bioanalytical methods provide the key information necessary to design an appropriate procedure for studying the degree of accumulation of a cytostatic compound by cells: determination of the IC50 parameter helps to select the concentration of the compound in the medium, imaging of stained cells allows for the determination of their quantity in a defined volume cell density or to confirm changes of metal amounts in cells established by inductively coupled plasma mass spectrometer (ICP-MS)
Summary
Lung cancer is the most common type of cancer and causes many deaths (1.6 million) worldwide annually [1]. Since the hydrolysis process of cisplatin is very slow compared to auranofin (minimum 48 h to reach a reaction efficiency of up to 50% [37,38], while for auranofin the reaction can be completed within few hours) and that only the formed derivatives of prodrugs are biologically active, it is possible to observe a significant change in the cytotoxicity of cisplatin (normal vs cancer cells) given such long exposure times (24, 48 and 72 h) of cells to the drug Since drugs such as cisplatin can be present in the human body for up to 20 months after treatment [49], and the time required for cisplatin transformation can exceed 48 h [35,36], cells exposed to 72 h incubation were selected for further testing by ICP-MS. Metallodrugs (auranofin and cisplatin) concentration required for 50% inhibition of cell biochemical function Figu(IrCe502).dMeteetramllionderdufgosr A(a-u5r4a9ncoafinncearncedllcsiaspndlantionr)mcoalnMceRnCtr-a5ticoenllsreafqtuerirdeidffeforern5t0i%ncuinbhaitbioitniotinmoefs caenldl bcioorcrheleamtioicnaclofeufnficctiieonnt (IC5(0r)2)d. eICte5r0mwinasedesftoarbAlis-h54ed cuasnincegracuetlolsmaantdicanlolyrmfiattleMd RcuCr-v5ecsedllessacfrtiebreddifbfyerHeniltlienqcuuabtaitoinontotimreleastiavnedcchoarnrgeelastioofnacboseofrfibcainecnet (r2).oIbCta5i0nwedasvieasMtabTlTistheesdt (uDsrinFigt saoufttowmaraet)icfaolrlycafintcterdlucunrgvceeslldseAs5cr4i9baenddbnyoHrmillalelquunagticoenllstoMrRelCat-i5veexcphoasnedgetso oafuarabnsofribnaanncde obtacinspeldatviniafoMrT7T2 thes(ta)(.DMr iFcirtosocoftpwicarime)afgoerscwanecrerolbutnaginecedllfsorAl5u4n9gacnadncneorr(mAa5l4l9u)nagndcenllosrMmaRlC(M-5ReCxp-5o)sceedlltsoeaxuproasnedofitno andacuirsapnlaotfiinn faonrd7c2ishp(laat)i.nMstiacrinoesdcowpiicthimcaalcgeeisnw(CeAreMo,bgtareineend), fporrolpuindgiucmaniocedrid(Ae (5P4I9,)readn)d, annodrmtraylp(aMnRbClu-e5)(gcreelyls) e(bxp).osed to auranofin and cisplatin stained with calcein (CAM, green), propidium iodide (PI, red), and trypan blue (grey) (b)
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