Evaluation of the efficacy of selected anticancer compounds in multidrug resistant cell culture models

Abstract

Small cell lung cancer (SCLC) has an aggressive disseminating nature and the relapse of cancer patients after initial treatment is common. Acquired multidrug resistance (MDR) can often be attributed to over-expression of ATP-binding cassette efflux transporters on the cell surface of SCLC cells. In the current study, three SCLC cell lines were evaluated as potential anticancer research models, specifically for investigations to overcome efflux-based MDR in cancer. These cell lines included the drug-sensitive H69V cell line, the multidrug resistance-associated protein 1 (MRP1) hyperexpressing H69AR cell line and the multidrug resistant NCI-H69/LX4 cell line that over-expresses P-glycoprotein (P-gp). A non-cancerous porcine embryonic kidney cell line (LLC-PK1) was included to evaluate the cytotoxic effects of each anticancer drug and to serve as a chemotherapeutic selectivity control. The 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used as a preliminary screening tool to determine the relative cell viability inhibitory concentration (IC) of selected chemotherapeutic drugs (i.e. paclitaxel, cisplatin and irinotecan) on the SCLC cell lines. Following the MTT assay, each cell line was treated with selected concentrations of the anticancer drugs based on the MTT results, which were adjusted daily relative to the wet biomass of the cells. This follow-up advanced cell viability assessment included quantification of intracellular adenosine triphosphate (ATP) and extracellular adenylate kinase (AK) of the different cell lines treated with the selected drugs. The ATP and AK cell viability assessment accurately indicated chemosensitivity of the H69V cell line, as well as resistance to treatment with drugs known to be substrates for the hyperexpressed efflux transporters. The P-gp over-expressing NCI-H69/LX4 cell line exhibited resistance to treatment with the P-gp substrate, paclitaxel (resistance ratio of 215.90; IC50 613.05 nM), while the H69AR cell line (hyperexpressing MRP1) was resistant to irinotecan treatment (resistance ratio of 16.50; IC50 7 716.70 nM), which is known to be a substrate for MRP1-mediated efflux. Cisplatin (ATP, 0.90; AK, 1.21), irinotecan (ATP, 0.94; AK, 1.80) and paclitaxel (ATP, 0.88; AK, 1.94) were cytotoxic to varying degrees in the non-cancerous LLC-PK1 cell line at drug sensitive line (H69V) IC50 concentrations (170.75 nM; 467.01 nM; 2.84 nM), but significantly higher concentrations than those shown to be effective in the SCLC cell lines were needed to determine LLC-PK1 IC50 values (2 619.60 nM; 15 534.00 nM; 29.73 nM). The H69AR cell line also showed resistance to cisplatin (resistance ratio of 2.50; IC50 427.34 nM), an MRP2 substrate, indicating cross-resistance potential between MRP1 and MRP2 transporters (transporter familial resistance). To confirm reports of transporter hyperexpression, a quantitative reverse transcription polymerase chain reaction was completed on all of the cell lines. When normalised to the NCI-H69/LX4 cell line (presented as 0), MRP1 expression was found to be present in the H69V (0.106) and LLC-PK1 (-0.076) cell lines in varying degrees, but as literature reported in the H69AR cell line MRP1 was hyperexpressed (2.037). P-gp expression quantification, normalised to the H69AR cell line (presented as 0), confirmed published reports of hyperexpression in the NCI-H69/LX4 cell line (5.95). No detectable P-gp levels were found to be present in the H69V and LLC-PK1 cell lines. The anticancer results of the selected chemotherapeutic agents were comparable to published in vivo and clinical trial conclusions, confirming the predictive potential of the selected cell lines for cancer treatment screening, including the effectiveness of compounds against MDR in lung cancer. Therefore, the presented SCLC cell model platform can be used as an effective in vitro tool to screen the efficacy of new compounds or to study efflux-based MDR in lung cancer