Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines

Abstract

We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptormediated endocytosis, was similar in sensitive and resistant cells. However, 40 -60% of the 125 I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised 125 I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A 1 , a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, 125 I-EGF, 14 C-carboplatin, and release of TCA precipitable 125 I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance. British Journal of Cancer (2003) Cisplatin (CP) is a component of standard treatment regimens for testicular, ovarian, bladder, cervical, head and neck and small-cell and nonsmall-cell lung cancers (Ozols and Williams, 1989;Perez, 1998). Adducts of DNA with CP induce apoptosis leading to cell death (Minn et al, 1995;Simonian et al, 1997). Intrinsic or acquired resistance of tumour cells to CP undermines its clinical effectiveness (Niedner et al, 2001). Mechanisms of resistance include decreased drug accumulation (Shen et al, 2000), changes in DNA repair proficiency (Fink et al, 1998;Zhen et al, 1992;Chu, 1994;Lai et al, 1995;Johnson et al, 1996), metallothionein (MT II) (Kelly et al, 1988;Kondo et al, 1995), glutathione-related enzymes (Moscow and Cowan, 1988;Godwin et al, 1992;Zaman et al, 1995), stress response proteins (Shen et al, 1995;Hettinga et al, 1997), proto-oncogenes or apoptosis-related genes, and cancer susceptibility genes (Fanidi et al, 1993;Lowe et al, 1993;DeFeudis et al, 1996;Husain et al, 1998;Moorehead and Singh, 2000). Protein kinases (PKs) like PKA and PKC have also been associated with CP-resistance (CP-r) and use of their specific inhibitors has been demonstrated to increase CP cytotoxicity in resistant tumour cells (Gosland et al, 1996). Recently, our laboratory (Shen et al, 2000) reported decreased energy-dependent uptake of 14 C-carboplatin by CP-r cells. Copper transporters have recently been ...

Reduced expression of small GTPases and hypermethylation of the folate binding protein gene in cisplatin-resistant cells

“…In this report, we demonstrate that the KB-CP20 cells have a more global defect in the uptake of a variety of radioactively labelled compounds. As shown in Figure 2, 125 I]EGF is correlated with decreased expression of the epidermal growth factor receptor (EGF-R) in our CP-r cells as described previously (Chauhan et al, 2003).…”

“…A defect in fluid-phase endocytosis was found in higher level CP-r cell lines isolated in multiple steps (Chauhan et al, 2003). To investigate the cellular and molecular genetic events involved in cisplatin resistance, particularly for the reduced uptake of a variety of compounds, reduced expression of several small GTPases involved in the endocytic pathway and FBP was found in the CP-r cells as detected by confocal immunofluorescence microscopy,immunoblotting, and realtime (RT)-PCR.…”

Increased levels and defective glycosylation of MRPs in ovarian carcinoma cells resistant to oxaliplatin

“…Acquired resistance to platinum compounds has been recently related to restoration of BRCA1/2 expression and homologous recombination function in tumors with frame-shift mutations of such genes [14]. Cells that have acquired resistance to cisplatin often have impaired drug uptake [15] and this has been linked to altered expression of genes involving copper metabolism [16][17][18][19][20][21]. Additional studies have implicated members of the ATP binding cassette (ABC) family of transporters such as Multidrug resistance associated protein (MRP) 2 and MRP1 in the accumulation defects found in cisplatin-resistant cells [22,23].…”

Metallofullerene nanoparticles circumvent tumor resistance to cisplatin by reactivating endocytosis

“…The difference in transferrin distribution between the CP-r and CP-s cells may result from defective endocytosis of the CP-r cells as demonstrated by our previous studies (32,35). Cisplatin passes through the plasma membrane and enters cells in part by endocytosis (36)(37)(38). We believe that multihydroxylated metallofullerene [Gd@C 82 (OH) 22 ] n nanoparticles reactivate endocytosis through their unique nanoscale properties.…”