Abstract
We examined whether the increased expression of P-glycoprotein (P-gp) encoded by the human multidrug resistance gene MDR1 is related to the acquired multidrug resistance of lung cancer in vivo. We estimated the chemosensitivity of lung cancer xenografts (LC-6, adenocarcinoma; Lu-24, small-cell cancer) by calculation of relative tumour growth (T/C%, treated/control) in vivo, based on statistical significance determined by the Mann-Whitney U test (P < 0.01, one-sided). MDR1 gene expression levels were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assay. P-gp production and P-gp localisation were examined by Western blotting and by immunohistochemical analysis respectively. LC-6 and Lu-24 were initially sensitive to both vincristine (VCR, 1.6 mg kg-1: LC-6, 45%; Lu-24, 39%) and doxorubicin (DOX, 12 mg kg-1: LC-6, 26%; Lu-24, 27%) in vivo. VCR-resistant variants (LC-6R, 66% and Lu-24R, 68%) selected with VCR (0.4 mg kg-1, x 9) significantly acquired cross-resistance to DOX (LC-6R, 55% and Lu-24R, 55% respectively). RT-PCR assay showed increased levels of MDR1 expression in LC-6R and Lu-24R with stable MDR1 expression levels. P-gp expression levels were elevated, and the percentage of P-gp-positive tumour cells increased in both LC-6R and Lu-24R. These results suggest that P-gp/MDR1 overexpression is related to acquired multidrug resistance in lung cancer in vivo.
Highlights
We examined the gene expression levels of miscellaneous factors associated with multidrug resistance including multidrug resistance-associated protein (MRP), topoisomerase IIcx (Topo Ila) and glutathione-S-transferase-ir (GST-7t) in the xenografts
The growth rates of human lung cancer xenografts in the chemosensitivity tests are shown with relative tumour volume (Figures 1-3)
LC-6 was sensitive to the maximum tolerated doses (MTDs) of both VCR and DOX (Figure la), and LC-6R selected in vivo by VCR was resistant to VCR and acquired cross-resistance to DOX (Figure la)
Summary
Two human xenografts (LC-6, NSCLC, adenocarcinoma; Lu24, SCLC, oat-cell type) were originally established at the Central Institute for Experimental Animals (Kanagawa, Japan) from primary lung cancer materials from patients who had received no anti-cancer chemotherapy. The tumour xenografts were maintained by serial subcutaneous transplantation in nude mice (BALB/c-nu/nu, Clea Japan, Tokyo), and used at 10-20 passages in this study. Xenograft specimens obtained from mice sacrificed under deep anaesthesia were frozen and stored at -80°C until analysed. Tumour xenografts were prepared for routine histopathological values. The drug-sensitive epidermoid carcinoma cell line, KB3-1, and its resistant derivative, KB8-5, were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 5% fetal bovine serum (FBS) at 37°C in a fully humidified 95% air, 5% carbon dioxide atmosphere. P-gp-mediated MDR in lung cancer xenograft Y Abe et al Establishment of VCR-resistant xenografts in vivo
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