Effects of FUX on gemcitabine sensitivity in lung cancer cells
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Lung cancer has accounted for the most deaths from cancer (19.2% of all cancer deaths) in registered cancer cases in New Zealand. At present lung cancer treatment is inadequate, as patients treated with the front-line drugs, such as gemcitabine, rapidly develop drug resistance by decreasing cellular accumulation and/or avoiding apoptosis. Fucoxanthin (FUX), extracted from edible seaweed such as Undaria pinnatifida, has recently been reported to inhibit membrane drug efflux transporters (ABC transporters) and induce apoptosis in various cancer cell lines. Previous studies in AUT have defined FUX extracted from New Zealand Undaria pinnitifida with anti-cancer properties by using in vitro cell models. FUX has been reported to have few adverse effects in some animal models. We hypothesize that FUX may be a safe sensitizer to reverse gemcitabine resistance in lung cancer cells by increasing cellular accumulation of gemcitabine. The primary objective of this study was to assess the potential effects of FUX to reverse gemcitabine resistance in human lung cancer cell lines. The secondary objective of current study is to investigate the mechanisms of FUX actions if FUX may potentiate gemcitabine sensitivity. The third objective of this study is to evaluate the effects of FUX on modifying gemcitabine toxicity in two typical normal human cell lines. Several types of human cell lines were used in this study including a lung carcinoma cell line A549, and two typical normal human cell lines embryonic kidney cell HEK293 and adult dermal fibroblasts (HDFa). Anti-proliferative effects were determined by 48-hr and 72-hr MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. As a single agent, either gemcitabine or FUX showed concentration-dependant inhibition of lung cancer proliferation in 72-hr MTT assays, with IC50 values of 9nM and 13μM, respectively. FUX increases gemcitabine sensitivity in an NSCLC cell line, A549 cell in a time and concentration dependant manner. Indeed, the 72-hr IC50 value for gemcitabine was only 3.9nM in the presence of 8μM FUX, which was decreased by 59% when comparing with control (P< 0.05). More importantly, FUX has no apparent effects on gemcitabine toxicity in two typical cell lines representing normal human tissues. It would be expected that FUX may represent a unique sensitizer, which may turn a less effective anti-cancer drug into an exceptional one. To elucidate the mechanisms of action of FUX, it is necessary to carry out a mechanistic study to investigate if FUX changes the intracellular gemcitabine accumulation in A549 cells. To determine gemcitabine in A549 cellular homogenates, an HPLC method has been developed and validated. In this study, while gemcitabine cannot be separated sufficiently from the cellular interferences using a conventional C18 column, aphenyl-hexyl column was found to be efficient to achieve better separation for quantitation of gemcitabine. This is because that separation using the phenyl column is conducted via the π electron, which in this case utilizes the π-π interaction between the phenyl group π electron and the analyte's π electron. Validation data indicates that the method is sensitive and reliable, with acceptable accuracy (85-115% of true values) and precision (CV < 15%). The assay specificity was indicated by the absence of interfering chromatographic peaks in cellular homogenates, and the LOQ of the assay was 0.5 μM. Calibration curves for gemcitabine were linear with the mean correlation coefficients > 0.987. This method has the advantage of being relatively rapid and efficient, with the retention time of gemcitabine separated from the substances in cellular homogenates. Therefore, this HPLC method is suitable for gemcitabine measurement in A549 cellular homogenates studies. Cellular accumulation studies suggest uptake of gemcitabine may reach equilibrium after 4-hr in the presence or absence of FUX. FUX (10 μM) shows the potentials to increase the steady-state accumulation of gemcitabine in A549 cells. However, it does not affect the initial cellular uptake of gemcitabine in A549 cells. While this mechanistic research provides some clues to elucidate the effects of FUX on gemcitabine accumulation, more details about the exact mechanisms of its action, are warranted for further studies in the future. However, a major limitation of this HPLC method is a lack of detection of gemcitabine metabolites. The cytotoxic action of gemcitabine has been attributed to inhibition of DNA synthesis by dFdCDP and dFdCTP. The HPLC method described in this study may not be suitable to simultaneously measure these active metabolites. Thus it is worthwhile to determine the cellular pharmacokinetics of gemcitabine and its metabolites in A549 cells and other NSCLC cells simultaneously by using an LC-MS/MS system. In conclusion, fucoxanthin increases gemcitabine sensitivity to A549 cancer cell lines, and more importantly, it has no apparent effects on gemcitabine toxicity in two typical cell lines representing normal human tissues. It would be expected that FUX may represent a unique sensitizer, which may turn a less effective anti-cancer drug into an exceptional one.