Ding-Wu Shen

Cisplatin Resistance: A Cellular Self-Defense Mechanism Resulting from Multiple Epigenetic and Genetic Changes

Cisplatin is one of the most effective broad-spectrum anticancer drugs. Its effectiveness seems to be due to the unique properties of cisplatin, which enters cells via multiple pathways and forms multiple different DNA-platinum adducts while initiating a cellular self-defense system by activating or silencing a variety of different genes, resulting in dramatic epigenetic and/or genetic alternations. As a result, the development of cisplatin resistance in human cancer cells in vivo and in vitro by necessity stems from bewilderingly complex genetic and epigenetic changes in gene expression and alterations in protein localization. Extensive published evidence has demonstrated that pleiotropic alterations are frequently detected during development of resistance to this toxic metal compound. Changes occur in almost every mechanism supporting cell survival, including cell growth-promoting pathways, apoptosis, developmental pathways, DNA damage repair, and endocytosis. In general, dozens of genes are affected in cisplatin-resistant cells, including pathways involved in copper metabolism as well as transcription pathways that alter the cytoskeleton, change cell surface presentation of proteins, and regulate epithelial-to-mesenchymal transition. Decreased accumulation is one of the most common features resulting in cisplatin resistance. This seems to be a consequence of numerous epigenetic and genetic changes leading to the loss of cell-surface binding sites and/or transporters for cisplatin, and decreased fluid phase endocytosis.

Decreased accumulation of [14c]carboplatin in human cisplatin-resistant cells results from reduced energy-dependent uptake†

We have isolated cisplatin‐resistant human liver carcinoma (7404‐CP20) cells with reduced accumulation of cisplatin and other drugs (methotrexate, arsenate, and arsenite) to which these cells are cross‐resistant. To determine whether the reduction of drug accumulation in cisplatin‐resistant cells results from impaired uptake or from active efflux, [14C]carboplatin was used for kinetic analysis of drug uptake and efflux. We demonstrate here that the uptake of [14C]carboplatin in 7404 parental cells is time, temperature, and energy dependent, and that the rate of uptake is reduced in 7404‐CP20 cells. Efflux of [14C]carboplatin in cisplatin‐resistant cells was comparable to efflux in the parental cisplatin‐sensitive cells. There was little effect of temperature (between 37°C and 4°C) on efflux in cisplatin‐resistant cells. Immunoblotting with specific antibodies directed to MRP1 and MRP2 (cMOAT) also showed that expression of these two ABC transporter genes was considerably reduced in 7404‐CP20 cells and another cisplatin‐resistant cell line KB‐CP20, in contradistinction to previous studies suggesting that MRP might be responsible for cisplatin efflux. To rule out a generalized defect in uptake of small molecules, fluorescence‐activated cell sorter (FACS) analysis of rhodamine 123 uptake showed that there was no difference between cisplatin‐sensitive and ‐resistant cells. The presence of a pleiotropic defect in uptake of [14C]carboplatin, [3H]methotrexate, [73As]arsenate, and [73As]arsenite in cisplatin‐resistant cells, in association with reduced expression of related cell surface proteins as demonstrated in our previous work, suggests a novel mechanism for acquisition of resistance to cisplatin associated with reduced activity of many different specific uptake systems. J. Cell. Physiol. 183:108–116, 2000.

Metallofullerene nanoparticles circumvent tumor resistance to cisplatin by reactivating endocytosis

Cisplatin is a chemotherapeutic drug commonly used in clinics. However, acquired resistance confines its application in chemotherapeutics. To overcome the acquired resistance to cisplatin, it is reasoned, based on our previous findings of mediation of cellular responses by [Gd@C82(OH)22]n nanoparticles, that [Gd@C82(OH)22]n may reverse tumor resistance to cisplatin by reactivating the impaired endocytosis of cisplatin-resistant human prostate cancer (CP-r) cells. Here we report that exposure of the CP-r PC-3-luc cells to cisplatin in the presence of nontoxic [Gd@C82(OH)22]n not only decreased the number of surviving CP-r cells but also inhibited growth of the CP-r tumors in athymic nude mice as measured by both optical and MRI. Labeling the CP-r PC-3 cells with transferrin, an endocytotic marker, demonstrated that pretreatment of the CP-r PC-3-luc cells with [Gd@C82(OH)22]n enhanced intracellular accumulation of cisplatin and formation of cisplatin-DNA adducts by restoring the defective endocytosis of the CP-r cancer cells. The results suggest that [Gd@C82(OH)22]n nanoparticles overcome tumor resistance to cisplatin by increasing its intracellular accumulation through the mechanism of restoring defective endocytosis. The technology can be extended to other challenges related to multidrug resistance often found in cancer treatments.

Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.

Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.

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

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 receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40–60% of the 125I-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 125I-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 A1, a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, 125I-EGF, 14C-carboplatin, and release of TCA precipitable 125I-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.

SIRT1 Contributes in Part to Cisplatin Resistance in Cancer Cells by Altering Mitochondrial Metabolism

Tumors frequently develop resistance to cisplatin, a platinum drug used as a cornerstone of present-day chemotherapy regimens, significantly decreasing its usefulness in the clinic. Although it is known that cisplatin-resistant (CP-r) cancer cells commonly grow more slowly and exhibit reduced uptake of various compounds, including nutrients, the effect of tumor metabolism on cisplatin resistance is unclear. It was found that in CP-r cells, uptake of 2-deoxyglucose was reduced due to dysfunction and altered morphology of mitochondria compared with cisplatin-sensitive parental cancer cells. The CP-r cells overexpressed SIRT1, a histone deacetylase that plays a central role in DNA damage response and transcriptional silencing. Incubation of drug-sensitive cells in low glucose medium induced the expression of SIRT1 and increased cellular resistance to cisplatin. Reduced SIRT1 expression by a SIRT1 SMART small interfering RNA duplex sensitized the >20-fold resistant CP-r cells to cisplatin treatment 1.5- to 2-fold, and SIRT1 overexpression by SIRT1 cDNA transfection increased cisplatin resistance in cisplatin-sensitive cells by 2- to 3-fold. Our findings therefore suggest that reduced glucose use and altered mitochondrial metabolism mediated by SIRT1 is one of several alterations that contribute to cellular resistance to cisplatin. (Mol Cancer Res 2008;6(9):1499–506)

Endocytic Recycling Compartments Altered in Cisplatin-Resistant Cancer Cells

The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.

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

Reduced accumulation of cisplatin is the most consistent feature seen in cisplatin-resistant (CP-r) cells that are cross-resistant to other cytotoxic compounds, such as methotrexate. In this report, defective uptake of a broad range of compounds, including [14C]-carboplatin, [3H]MTX, [3H]folic acid (FA), [125I]epidermal growth factor, 59Fe, [3H]glucose, and [3H]proline, as well as 73As5+ and 73As3+, was detected in CP-r human hepatoma and epidermal carcinoma cells that we have previously shown are defective in fluid-phase endocytosis. Downregulation of several small GTPases, such as rab5, rac1, and rhoA, which regulate endocytosis, was found in CP-r cells. However, expression of an early endosomal protein and clathrin heavy chain was not changed, suggesting that the defective endocytic pathway is clathrin independent. Reduced expression of the cell surface protein, folate-binding protein (FBP), which is a carrier for the uptake of MTX, was also observed in the CP-r cells by confocal immunofluorescence microscopy and Real-Time PCR. Reactivation of the silenced FBP gene in the CP-r cells by a DNA demethylation agent, 2-deoxy-5-aza-cytidine (DAC) demonstrates that hypermethylation occurred in the CP-r cells. The uptake of [14C]carboplatin, [3H]FA, and [3H]MTX increased in an early stage CP-r cell line (KB-CP1) after treatment with DAC. Both a defective endocytic pathway and DNA hypermethylation resulting in the downregulation of small regulatory GTPases and cell surface receptors contribute to the reduced accumulation of a broad range of compounds in CP-r cells.

Trafficking and localization of platinum complexes in cisplatin-resistant cell lines monitored by fluorescence-labeled platinum.

Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. Resistance to cisplatin represents a major obstacle to effective cancer therapy because clinically significant levels of resistance quickly emerge after treatment. Based on previous studies indicating abnormal plasma membrane protein trafficking in cisplatin‐resistant (CP‐r) cells, Fluorescence (Alexa Fluor)‐labeled cisplatin was used to determine whether this defect altered the trafficking and localization of cisplatin by comparing drug sensitive KB‐3‐1 and KB‐CP‐r cells. Alexa Fluor–cisplatin was readily internalized and localized throughout the KB‐3‐1 cells, but overall fluorescence decreased in KB‐CP‐r cells, as detected by flow cytometry (FACS) and confocal microscopy. Only punctate cytoplasmic staining was observed in KB‐CP‐r cells with less fluorescence observed in the nucleus. Colocalization experiments with a Golgi‐selective stain indicate the involvement of Golgi‐like vesicles in initial intracellular processing of Alexa Fluor conjugated cisplatin complexes. As detected using an antibody to Alexa Fluor–cisplatin, cisplatin complex‐binding proteins (CCBPs) were reduced in membrane fractions of single‐step cisplatin‐resistant KB‐CP.5 cells, and increased in the cytoplasm of KB‐CP.5 cells compared to KB‐3‐1 cells. CCBPs localized to lower density fractions in KB‐CP.5 cells than in KB‐3‐1 cells as determined by iodixanol gradient centrifugation. In summary, inappropriate trafficking of CCBPs might explain resistance to cisplatin in cultured cancer cells, presumably because membrane binding proteins for cisplatin are not properly located on the cell surface in these cells, but are instead trapped in low density vesicles within the cytoplasm.

Elevated Expression of TMEM205, a Hypothetical Membrane Protein, is Associated with Cisplatin Resistance

Development of cisplatin resistance in cancer cells appears to be a consequence of multiple epigenetic alterations in genes involved in DNA damage repair, proto‐oncogenes, apoptosis, transporters, transcription factors, etc. In this study, we found that expression of the hypothetical transmembrane protein TMEM205 (previously known as MBC3205) is associated with cisplatin resistance. TMEM205 was first detected by functional cloning from a retroviral cDNA library made from human cisplatin‐resistant (CP‐r) cells. TMEM205 is predicted to be a transmembrane protein, but its expression, localization, and function have not previously been investigated. A polyclonal antibody directed to the TMEM205 protein was raised in our laboratory. Using this antibody, it was demonstrated that this protein is located at the cell surface. Its expression is increased in our cisplatin‐selected CP‐r cell lines, as demonstrated by immunoblotting, confocal examination, and immuno‐electron microscopy. Stable transfection of the TMEM205 gene confers resistance to cisplatin by approximately 2.5‐fold. Uptake assays with Alexa Fluor‐cisplatin showed reduced accumulation in CP‐r KB‐CP.3 and KB‐CP.5 cells, and in TMEM205‐transfected cells. Analysis of TMEM205 expression profiles in normal human tissues indicates a differential expression pattern with higher expression levels in the liver, pancreas, and adrenal glands. These results indicate that a novel mechanism for cisplatin resistance is mediated by TMEM205, and also suggest that overexpression of TMEM205 in CP‐r cells may be valuable as a biomarker or target in cancer chemotherapy. J. Cell. Physiol. 225: 822–828, 2010.