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[Cancer Research 66, 9682-9690, October 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

The 3p21.3 Tumor Suppressor NPRL2 Plays an Important Role in Cisplatin-Induced Resistance in Human Non–Small-Cell Lung Cancer Cells

Kentaro Ueda1, Hiroyuki Kawashima1, Shoichiro Ohtani1, Wu-Guo Deng1, Murali Ravoori2, Jim Bankson3, Boning Gao4, Luc Girard4, John D. Minna4, Jack A. Roth1, Vikas Kundra2 and Lin Ji1

1 Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, 2 Department of Diagnostic Radiology, and 3 Department of Imaging Physics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; and 4 Department of Internal Medicine and Pharmacology, Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Lin Ji, Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-745-4530; Fax: 713-794-4901; E-mail: lji{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non–small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of –0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle–mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment. (Cancer Res 2006; 66(19): 9682-90)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NPRL2 (GenBank accession no. AF040707) is one of the candidate tumor suppressor genes identified in the human chromosome 3p21.3 region, in which genomic abnormalities such as a loss of heterozygosity and homozygous deletion are frequently found in the early stages of the development of various human cancers, including lung cancer (1–3). Cancer-associated inactivation of the NPRL2 gene, via aberrant splicing transcripts, multiple exon deletions, or intragenic homozygous deletions, has been found in renal cell carcinoma, lung carcinoma, and other human cancers and cancer-derived cell lines, suggesting that NPRL2 may be a tumor suppressor and its inactivation may promote tumorigenesis (1, 4). We previously showed that reactivation of the wild-type (wt) NPRL2 by recombinant adenoviral vector–mediated transfer in lung cancer cells with abnormal 3p21.3 genes inhibited tumor cell growth by inducing apoptosis and altering cell kinetics in vitro and significantly suppressed primary tumor growth and inhibited tumor progression and metastasis by either intratumoral injection or systemic administration of protamine-complexed recombinant adenoviral vector of NPRL2 in various human lung cancer mouse models (5). Similarly, a NPRL2-mediated tumor suppression activity has also recently been shown in NPRL2-deficient renal cell carcinoma KRC/Y cells, small-cell lung carcinoma U2020 cells, and non–small-cell lung cancer (NSCLC) A549 cells when NPRL2 expression was induced at a physiologic level (4). Although the mechanism of NPRL2-mediated tumor suppression activity remains unknown, recent studies have suggested that NPRL2 is involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway (4, 6).

Cisplatin can form platinum-DNA adducts and induce cytotoxicity by interfering with the DNA-damage repair in various cancer cells (7). It is among the most effective cytotoxic agents for advanced cancer treatments including NSCLC (8). Unfortunately, intrinsic or acquired resistance to cisplatin (cis-diammine-dichloroplatinum) is frequently encountered and severely limits its therapeutic potential (8, 9). Although the exact molecular mechanisms that control cellular sensitivity to cisplatin have yet to be elucidated, some molecular genetic and cytogenetic studies of gene knockouts in yeast and in mammalian cells have unambiguously revealed genes and gene products that are involved in the modulation of cisplatin-induced cytotoxicity and resistance (8, 9). Disruption of the nitrogen permease regulator protein gene (NPR2) in yeast cells has been shown to contribute to cellular resistance to cisplatin- and doxorubicin-mediated cell killing (4, 6). The full-length cDNA of the human NPRL2 gene consists of 1,351 bp and encodes a protein of 380 amino acid residues. The NPRL2 protein sequence has a high degree of homology to that of the yeast NPR2 protein with a 32% identity (53% similarity) over 152 amino acids in a consensus region of the NPR2 protein family (4, 5, 10, 11). Because of the high degree of structural similarity between the human NPRL2 and the yeast NPR2 proteins, we hypothesized that NPRL2 has a similar biological function in mediating cellular responses to treatment with chemotherapeutic drugs such as cisplatin and that restoring normal NPRL2 function in cisplatin-resistant tumor cells may resensitize or synergize their response to treatment.

To test these hypotheses, we quantitatively analyzed expression of the NPRL2 protein and determined IC50 values of cisplatin in 40 NSCLC cell lines to evaluate the potential correlation between NPRL2 protein expression and cisplatin sensitivity. Next, we studied the effect of exogenous expression of NPRL2 induced by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate (DOTAP):cholesterol (DC) nanoparticle–mediated gene transfer on the growth and apoptosis of cisplatin-sensitive and cisplatin-resistant NSCLC cells in vitro. Finally, we explored the potential of treatment with a combination of systemic injection of DC-NPRL2 nanoparticles and i.p. injection of cisplatin for enhancing antitumor efficacy and overcoming drug resistance in mouse models of human lung cancer. Our results show for the first time, to our knowledge, a significant correlation between the level of NPRL2 protein expression and sensitivity to cisplatin treatment and show that cisplatin-induced cytotoxicity in NPRL2-deficient and cisplatin-resistant NSCLC cells can be modulated in vitro and in vivo by reactivating NPRL2. These findings suggest that NPRL2 may serve as a biomarker for the prediction of cisplatin response and a prognositic indicator in lung cancer patients, as well as a molecular therapeutic agent for enhancing and resensitizing nonresponders to cisplatin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell cultures. Human NSCLC cell lines (n = 40) were either obtained from American Type Culture Collection (Manassas, VA) or established from primary lung tumors through University of Texas Specialized Program of Research Excellence in Lung Cancer. Cells were maintained in either Ham's F12 medium supplemented with 10% FCS or RPMI 1640 supplemented with 10% FCS and 5% glutamine. The cisplatin-sensitive H1437S cell line was directly established from a primary lung tumor that responded favorably to cisplatin treatment, and the cisplatin-resistant H1437R cell line was established from the parental H1437S cells by selection against cisplatin in the laboratory. The genomic and genetic status of the 3p21.3 region and genes in the region in these NSCLC cell lines has been described previously (1, 2, 3, 12).

Reagents and antibodies. Cisplatin was purchased from Bristol-Myers Squibb Company (Princeton, NJ). DC-encapsulated plasmid DNA nanoparticles were produced in our laboratory as previously described (13–15) and used both as a transfection agent in vitro and as a plasmid DNA delivery vehicle in vivo. Anti-NPRL2 (Abcam, Cambridge, MA), anti–caspase-2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti–caspase-3 (Cell Signaling Technology, Beverly, MA), anti–caspase-8 (BD Biosciences, San Jose, CA), anti–caspase-9 (Cell Signaling Technology), anti–poly(ADP-ribose) polymerase (BD Biosciences), and anti–ß-actin (A1978; Sigma, St. Louis, MO) were purchased.

Construction of NPRL2-expressing plasmid vectors. The wt NPRL2 cDNA was inserted into an expression cassette driven by a cytomegalovirus promoter and tailed with a bovine growth hormone polyadenylation signal sequence using a plasmid vector backbone that has been approved by the U.S. Food and Drug Administration for human clinical application (13–15). A Myc-NPRL2 fusion protein–expressing vector was also constructed to facilitate biochemical characterization of the NPRL2 protein. An empty vector without a transgene insert, a plasmid vector expressing the LacZ gene, and a plasmid vector expressing the wt p53 gene were used as negative, nonspecific, and positive controls, respectively.

Western blot analysis. Cell lysates were prepared from various human NSCLC cells, normal human bronchial epithelial cells, and WI-38 lung fibroblasts in Laemmli/urea buffer [125 mmol/L Tris (pH 6.8), 4% SDS, 10% glycerol, and 6 mol/L urea] with proteinase inhibitor (Roche Molecular Biochemicals, Mannheim, Germany). Protein concentrations were assayed with bicinchoninic acid protein assay reagent (Pierce Biotechnology, Rockford, IL). The cell lysates (~50 µg) were separated by standard SDS-PAGE and detected by Western blotting. The densities of NPRL2 and ß-actin immunoblot bands on the gel from each cell type were measured by an automated digitizing system and quantitatively analyzed with the system software (Silk Scientific, Orem, UT). The level of NPRL2 protein expression in each cell line was normalized to that of the ß-actin band.

Determination of cisplatin sensitivity in NSCLC cells. To determine the IC50 and IC20 values of cisplatin in 40 NSCLC cell lines, a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Cell Proliferation Kit II, Roche Molecular Biochemicals) was used according to the instructions of the manufacturer. Briefly, 2 x 103 cells were plated onto each well of a 96-well plate for 24 hours and then treated with various concentrations of cisplatin for 72 hours. The IC50 and IC20 values of cisplatin were calculated using a curve-fitting software previously described (16).

Immunocytochemical analysis of NPRL2 expression. The level and localization of NPRL2 protein expression in cells were also analyzed by immunocytochemical staining with anti-NPRL2 goat polyclonal antibodies. The 10% formalin-fixed cell lines were incubated with anti-NPRL2 antibodies (2.5 µg/mL in PBS) and immunostaining was done with a Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) according to the instructions of the manufacturer. Subsequently, the samples were counterstained with hematoxylin (Sigma) and examined under a light microscope equipped with a digital camera and imaging software (Nikon, Tokyo, Japan).

Tumor cell–induced clonogenicity assay. To analyze the effect of NPRL2 protein on tumor cell–derived clonogenicity in vitro, we transfected H1299 cells (2 x 105) on six-well plates with a vector expressing NPRL2 or Myc-NPRL2 using DOTAP. H1299 cells were cotransfected with 2 µg of plasmid DNA and 1 µg of pcDNA3.1 vector (Invitrogen, Carlsbad, CA) containing a neomycin-resistant gene. Untransfected cells and cells transfected with the pcDNA3.1 vector alone (1 µg) or with 2 µg of empty vector or p53 plasmid DNA served as controls. Twenty-four hours after transfection, cells were harvested, stained with trypan blue, counted, and replated onto a 100-mm dish (1 x 104 per dish) in triplicate. Cells were grown in RPMI 1640, supplemented with 10% FCS and containing 400 µg/mL G418, for 2 weeks. The G418-resistant colonies were counted after staining with crystal violet.

Cell viability assay. Inhibition of tumor cell growth by treatment with NPRL2-expressing vector and cisplatin was analyzed by viable cell counting with a trypan blue exclusion assay. H1437S, H1437R, H1299, and H322 cells were seeded in six-well plates at a density of 2 x 105 per well. The next day, the cells were transfected with 2 µg of NPRL2 vector or empty vector using DOTAP reagent in serum-free medium. Two hours later, the cells were replenished with appropriate medium for each cell line containing various concentrations of cisplatin. Cells were harvested 72 hours after transfection and cell viability was determined by counting under a light microscope. The transfection efficiency was assessed by a parallel transfection with an equal amount of enhanced green fluorescent protein–expressing plasmid vector with a similar backbone in each cell lines. The transfection efficiency was in the range of 30% to 45% in those cell lines.

Analysis of apoptosis in vitro. Induction of apoptosis in tumor cells treated with NPRL2 vector and cisplatin was analyzed with Apo-BrdU Kit (BD Biosciences PharMingen) by flow cytometry [fluorescence-activated cell sorting (FACS)]. Briefly, cells were plated on 60-mm dishes at 4 x 105 per dish for 24 hours and then treated with 4 µg of empty vector or NPRL2-expressing vector in the absence or presence of cisplatin at the IC20 dose determined earlier. After 72 hours, cells were harvested and fixed in 1% paraformaldehyde. Bromodeoxyuridine triphosphate incorporated into DNA was detected with FITC-labeled anti-bromodeoxyuridine antibody and analyzed by FACS. The same cell samples were also stained with propidium iodide/RNase buffer and analyzed by FACS for determination of cell cycle kinetics.

Caspase activity assay. Induction of caspase activity in H1299 cells treated with NPRL2 vector and cisplatin was determined with the ApoAlert caspase profiling assay (BD Biosciences). Briefly, H1299 cells were plated in 60-mm dishes at a density of 4 x 105 per dish for 24 hours and then treated with 4 µg of plasmid vector in the absence or presence of 3.0 µmol/L cisplatin. After 72 hours, cells were harvested, cell lysates were prepared, and caspase activity was analyzed using the ApoAlert assay according to the instructions of the manufacturer.

Animal studies. All mice were maintained and animal experiments were done at the Animal Core Facility at The University of Texas M.D. Anderson Cancer Center with institutionally approved animal protocols following the NIH guidelines. An orthotopic mouse model of human NSCLC H322 was used to evaluate the therapeutic efficacy of systemic administration of the DC-NPRL2 nanoparticles and cisplatin in vivo. The animals used in this study were female nu/nu mice (4-6 weeks old) purchased from Charles River Laboratories (Wilmington, MA). Before tumor cell inoculation, mice were subjected to 3.5-Gy total body irradiation with an external 137Cs source. To establish orthotopic pleural tumors, the mice were inoculated with 2 x 106 H322 cells in 100 µL of PBS by intrathoracic injection with a 27-gauge needle. These mice were randomly divided into six groups (eight mice per group): (i) PBS; (ii) PBS plus cisplatin; (iii) LacZ; (iv) LacZ plus cisplatin; (v) NPRL2; and (vi) NPRL2 plus cisplatin. Orthotopic tumors are usually established on the lung or on the inner thoracic membrane 10 to 14 days after tumor cell inoculation. Before the treatment, four mice from each group were subjected to magnetic resonance imaging (MRI) analysis to establish baseline tumor volumes. On days 10, 13, and 16 after tumor inoculation, the mice were given systemic DC-DNA nanoparticles by tail-vein injection at a dose of 25 µg of plasmid DNA and 10 nmol DC in 100 µL of 5% dextrose in water per mouse, alone or in combination with i.p. injection of cisplatin at a dose of 2.5 mg/kg. Twelve days after the last treatment, a second MRI was done on the previously imaged mice from each group. All mice were then killed and the total number and total weight of intrathoracic and pleural tumors of each mouse were examined in each animal.

To determine NPRL2 expression and apoptosis in tumors, two mice in each group were treated with the designated agents and killed 48 hours later, and tumors >5 mm were harvested and freshly frozen. NPRL2 protein expression in frozen tumor sections was detected by anti-NPRL2 antibodies (1:100) with the Vectastain kit and examined under a light microscope. Induction of apoptosis was analyzed with an in situ cell death detection kit and terminal dUTP nick-end labeling (TUNEL) staining with FITC-labeled dUTP (Roche Molecular Biochemicals) according to the instructions of the manufacturer. The images were examined under a Nikon TC200 fluorescence microscope equipped with a digital camera. Percentage of TUNEL-staining positive cells in each treatment was determined by counting the numbers of cells with nuclear 4',6-diamidino-2-phenylindole (DAPI) staining (blue) and TUNEL staining (green) in at least three fields in each section using the ImagePro image analysis software (Media Cybernetics, Silver Spring, MD) and three TUNEL-stained frozen tissue sections were examined.

Evaluation of therapeutic efficacy by a noninvasive MRI. Mice were imaged using a 4.7-T small animal MRI scanner (Biospec, Bruker Biospin MRI, Billerica, MA). Mice anesthetized with 1% to 3% isofluorane were placed prone and head first on a positioning sled. Electrocardiography leads were positioned on the forepaws and tail and respiratory bellows were positioned over the abdomen. Respiratory and cardiac gated axial imaging was done on the thorax using a fat-suppressed T2 weighted echo planar sequence (TE, 40 ms; TR, ~16 seconds; 3 x 3-cm field of view; 1.25-mm slice thickness; 128 x 128 image matrix; 8 shots) and using a T1-weighted spoiled gradient echo sequence (TE, 1.4 ms; TR, ~200 ms, 3 x 3-cm field of view; 1.25-mm slice thickness; 128 x 128 image matrix; 65-degree flip angle) with or without intravenous contrast. The fat suppression pulse was manually adjusted for each mouse using a one-pulse spectroscopic sequence.

Tumor measurements were done as described (17) with Image J software (NIH, Bethesda, MD). In each echo planar image containing a tumor, the periphery of the mass was traced and the area of the drawn region was calculated. The areas were then multiplied by the slice thickness plus the skip distance to obtain the volume of each slice containing the object of interest. The slice volumes were then summed. To control for volume averaging, half of the volume containing tumor in the most superior or inferior slice was included in the final volume.

Statistical analysis. All in vitro experiments were done at least twice with duplicates or triplicates of samples. ANOVA and Fisher's test or t test were used to compare the values of the test and control samples in vitro and in vivo. Correlation of endogenous NPRL2 protein with cisplatin sensitivity was analyzed with the nonparametric Spearman rank order correlation (Spearman R). P < 0.05 was considered statistically significant. STATISTICA 6.0 software (StatSoft, Tulsa, OK) was used for all statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Correlation of endogenous NPRL2 protein expression with cisplatin sensitivity in NSCLC cell lines. To evaluate the potential correlation between NPRL2 protein expression and cisplatin sensitivity in lung cancer cells, we first examined the expression of endogenous NPRL2 protein in 40 NSCLC cell lines by Western blot analysis (Fig. 1A ). Moreover, we quantitatively measured the density of the NPRL2 protein and ß-actin bands on Western blots of each cell line and normalized the level of NPRL2 protein expression in each cell line to that of ß-actin. Various levels of NPRL2 expression were detected in the following 21 cell lines: A549, H358, H441, H1437S, H125, H596, H292, H460, H226B, H226Br, CALU-1, CALU-3, H2887, H3255, H820, H1650, H1819, HCC366, HCC44, HCC78, and HCC9. The other 19 NSCLC cell lines had little or no NPRL2 protein expression. A moderate level of endogenous NPRL2 expression was detected in WI-38 fibroblasts and human bronchial epithelial cells (data not shown). Interestingly, a high level of NPRL2 protein was detected in the NSCLC cell line H1437S that was established from tumors sensitive to cisplatin, but no NPRL2 protein expression was detected in the cisplatin-resistant H1437R cell line that was selected against cisplatin from the parent H1437S cells as shown by a >15-fold increase in the IC50, suggesting that NPRL2 is involved in cisplatin-induced resistance in vitro.


Figure 1
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  		Figure 1. Correlation of expression of endogenous NPRL2 proteins and cisplatin (CDDP) sensitivity in NSCLC cell lines. A, expression of endogenous NPRL2 protein was analyzed by Western blotting in 40 NSCLC cell lines. The expression was determined by the intensity of the immunoblot normalized to that of ß-actin in each cell line. IC50 values of cisplatin in these NSCLC cells were determined by an XTT assay. Intensity index and IC50 values are indicated at the bottom of the Western blot images. H1437S, cisplatin-sensitive cells; H1437R, cisplatin-resistant cels. B, nonparametric Spearman rank order correlation analysis of NPRL2 protein expression with cisplatin sensitivity. The Spearman correlation coefficient R is indicated in the graph and the significance of the correlation was evaluated by paired t test.

 
To evaluate the sensitivity of tumor cells to cisplatin treatment, we determined the IC50 values of cisplatin in the 40 NSCLC cell lines. As shown in Fig. 1A (bottom line under each Western blot), 15 of the 21 (71%) NPRL2+ cell lines were highly sensitive to cisplatin (IC50 < 5 µmol/L) and 15 of the 19 (79%) NPRL2– cell lines were cisplatin resistant (IC50 > 5 µmol/L). Five of 6 (83%) cell lines that were rated as highly cisplatin resistant (IC50 > 10 µmol/L) were NPRL2–. Furthermore, we did a nonparametric Spearman rank order correlation analysis on NPRL2 protein expression and cisplatin sensitivity in these NSCLC cell lines and found that expression of NPRL2 significantly and reciprocally correlated with cisplatin sensitivity, with a Spearman coefficient of –0.676582 (P < 0.00001; Fig. 1B).

Inhibition of tumor cell clonogenicity and growth by forced expression of NPRL2 and cisplatin. To test whether reactivation of NPRL2 could reverse the tumor clonogenic phenotype in NPRL2-deficient NSCLC cells, we first analyzed the effect of exogenous expression of NPRL2 on tumor cell–induced clonogenicity in deficient H1299 cells (NPRL2– and cisplatin-resistant; IC50 = 7.6 µmol/L) transfected with wt NPRL– or Myc-NPRL2–expressing plasmid vector, with empty vector and a wt p53–expressing vector as negative and positive controls, respectively (Fig. 2 ). Colony formation was significantly inhibited in both NPRL2- and Myc-NPRL2–transfected H1299 cells compared with empty vector–transfected cells (P < 0.0001). Similar inhibition of H1299-induced clonogenicity was also observed in p53-transfected cells. The expression of endogenous NPRL2 or Myc-NPRL2 proteins in transfectants was confirmed by Western blotting. These results suggest that, like p53, NPRL2 is a potent suppressor of the development of lung cancer.


Figure 2
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  		Figure 2. Effect of exogenous expression of NPRL2 on tumor cell–induced clonogenicity in NPRL2– H1299 cells. A, for the clonogenicity assay, an NPRL2- or Myc-tagged NPRL2 (Myc-NPRL2)–expressing plasmid vector was cotransfected into H1299 cells with a neomycin-resistant gene–containing pcDNA3.1 vector. Empty vector (EV) and a wt p53–expressing vector were used as negative and positive controls, respectively. After 24 hours, an equal number of viable cells from each group was replated onto a soft agar plate and grown for 2 weeks in the presence of G418. B, the number of G418-resistant colonies was counted after staining with crystal violet. Columns, mean of three repeated experiments; bars, SD. C, expression of NPRL2 was confirmed by Western blotting. D, effects of exogenous expression of NPRL2 and cisplatin treatment on tumor cell growth in human NSCLC cells. NSCLC cell lines H1437S, H1437R, H1299, and H322 that are cisplatin sensitive (S) or cisplatin resistant (R) with or without endogenous NPRL2 expression (NPRL2– or NPRL2+), as indicated on the graph, were transfected with an NPRL2-expressing plasmid vector in the absence or presence of various concentrations of cisplatin. The last two concentrations of cisplatin represent the IC20 and IC50 doses for each cell line. Cells treated with empty vector and cisplatin were used as negative controls. The percent cell viability in each treatment group was calculated relative to untreated cells. Columns, mean of three individual experiments; bars, SD. *, P < 0.05; **, P < 0.005, significant differences between treatment groups (ANOVA and Fisher's test).

 
To further assess the biological involvement of NPRL2 activity in cisplatin-mediated cytotoxicity and treatment sensitivity in tumor cells and to test whether restoring NPRL2 function could enhance cisplatin-mediated anticancer cytotoxicity and resensitize cisplatin-resistant tumor cells to treatment, we evaluated the effect of forced expression of NPRL2 on tumor cell growth by DC nanoparticle–mediated gene transfer in the absence and presence of cisplatin in various cisplatin-sensitive and cisplatin-resistant NSCLC cells (Fig. 2D). Inhibition of tumor cell growth was more significantly enhanced in cisplatin-sensitive H1437S cells transfected with NPRL2 and treated with cisplatin at both the IC20 (0.8 µmol/L) and IC50 (1.2 µmol/L) levels, with >50% (P < 0.025) and 80% (P < 0.005) reduction in cell viability, respectively, than in cells treated with either NPRL2 (30% reduction) or cisplatin alone at IC20 and IC50 levels, or with empty vector plus cisplatin at the same dose levels; cells treated with empty vector alone was used as the referent group with cell viability set at 100%. The enhancement in growth inhibition by treatment with a combination of NPRL2 and cisplatin was more dramatic in cisplatin-resistant H1437R cells than in H1437S cells. A significant synergistic inhibition of tumor cell growth was detected in all cisplatin-resistant cells (H1437R, H1299, and H322) treated with NPRL2 and cisplatin at both IC20 and IC50 levels. These results provide biological evidence that NPRL2 is involved in cisplatin-mediated cytotoxicity and modulates tumor cell sensitivity to cisplatin.

Induction of apoptosis by treatment with a combination of NPRL2 and cisplatin. One of the key pathways in NPRL2- or cisplatin-mediated tumor cell killing is by inducing apoptosis. To better understand the mechanism behind the observed synergism in tumor growth inhibition by NPRL2 and cisplatin, we evaluated the effects of NPRL2 activation and cisplatin treatment on the induction of apoptosis in both cisplatin-sensitive and cisplatin-resistant NSCLC cells by FACS analysis with a TUNEL reaction (Fig. 3A ). In cisplatin-sensitive (NPRL2+) H1437S cells, the exogenous expression of NPRL2 caused very little apoptosis (~4%), a level slightly lower than that of cisplatin treatment alone (7%) at IC20 after 72 hours. However, the induction of apoptosis was dramatically increased in NPRL2-transfected, cisplatin-resistant H1437R cells, in which the expression of NPRL2 was lost under the negative pressure of cisplatin selection (Fig. 1A), with a level of apoptosis induction (>15%) slightly higher than that of cisplatin treatment at IC20 in the same cells. Furthermore, a significant and synergistic induction of apoptosis was detected in all three NPRL2-, cisplatin-resistant cell lines treated with a combination of DC-NPRL2 nanoparticles with cisplatin at the same IC20 dose level, with a 33.4% induction of apoptosis in H1437R cells, 42.0% in H1299 cells, and 21.8% in H322 cells, compared with those treated with either agent alone or the control cells that were untreated (PBS) or treated with empty vector or empty vector plus cisplatin. These results suggest that the synergism in tumor cell growth inhibition and killing by a combination treatment with NPRL2 and cisplatin is mediated by a synergistic induction of apoptosis in these cisplatin-resistant cells.


Figure 3
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  		Figure 3. Activation of apoptosis by exogenous expression of NPRL2 and cisplatin in NSCLC cells. A, induction of apoptosis by NPRL2 and cisplatin. NSCLC cells were transfected with an NPRL2-expressing vector in the absence or presence of cisplatin at an IC20 dose level for each cell line as indicated in Fig. 2D; untreated or empty vector–transfected cells were used as negative controls. After 72 hours, the percentage of apoptotic cells was determined by FACS analysis with TUNEL staining. Columns, mean of three individual experiments; bars, SD. *, P < 0.05; **, P < 0.005, significant differences between treatment groups (ANOVA with Fisher's test). B, activation of caspases in lung cancer cells by exogenous expression of NPRL2 and cisplatin treatment shown by Western blotting. Cell lysates were prepared from H1299 cells transfected with NPRL2 in the absence or presence of cisplatin at an IC20 dose level (3.0 µmol/L) for 72 hours, with empty vector as a negative control. The activation of caspases and protease was detected by the cleaved fragment bands from the pro-proteins (arrows). C, effect of exogenous NPRL2 expression and cisplatin treatment on caspase activity in H322 cells as shown by a caspase activity profiling assay. Cell lysates from H322 cells transfected with NPRL2 in the absence or presence of cisplatin at an IC20 dose level (5.0 µmol/L) were used for the assay. Columns, mean of three individual experiments; bars, SD. The significance of differences in caspase activity between treatment groups was analyzed by ANOVA and Fisher's test.

 
Next, we analyzed the effects of exogenous expression of NPRL2 and cisplatin treatment on some key caspases and proteases that are involved in signal transduction of apoptotic stimuli. We used both Western blotting and a caspase activity profiling assay in H1299 and H322 cells transfected with NPRL2 vector with or without cisplatin treatment at a dose level of IC20. Untreated cells (PBS) and cells treated with empty vector or a combination of empty vector and cisplatin were used as negative controls (Fig. 3B and C). Activation of the caspase cascade including caspase-2, caspase-3, caspase-8, caspase-9, and the protease poly(ADP-ribose) polymerase were clearly increased in H1299 cells treated with both NPRL2 and cisplatin compared with cells treated with either agent alone or negative controls, as indicated by the increased intensity in the cleaved bands from pro-proteins on the Western blot. Similarly, the activity of caspase-3 increased by 23.7-fold (P < 0.0001), caspase-8 by 10.4-fold (P < 0.002), and caspase-2 by 17.2-fold (P < 0.001) in H322 cells transfected with NPRL2 and treated with cisplatin at an IC20 dose compared with those treated with either agent alone or the negative controls, as shown by the ApoAlert assay. These results are consistent with our observations of apoptosis induction and suggest that the synergistic induction of apoptosis in cells treated with both NPRL2 and cisplatin is mediated by concurrent activation of a caspase cascade in cisplatin-resistant NSCLC cells.

Enhanced tumor growth inhibition and apoptosis induction by systemic treatment with NPRL2 nanoparticles and cisplatin in vivo. Based on the results of our in vitro studies, we explored the potential of NPRL2 as a novel molecular therapeutic agent for more effectively suppressing tumor growth and modulating tumor cell response to chemotherapeutic drugs in mice with human lung cancer xenografts. Using a quantitative, noninvasive MRI analysis, we evaluated the therapeutic efficacy of systemic treatment with i.v. injection of DC-NPRL2 nanoparticles and i.p. injection of cisplatin in an orthotopic mouse model of human H322 NSCLC (Fig. 4 ). MRI analysis was done before treatment to establish baseline tumor loads and volumes and after treatment to assess therapeutic efficacy. The mice were killed after the second MRI, and tumors from each animal were dissected, counted, and measured. The MRI findings of these tumors were confirmed by autopsy (Fig. 3A). Systemic administration of DC-NPRL2 nanoparticles alone for a total dose of 75 µg of DNA significantly inhibited tumor growth, yielding a reduction of >40% (P < 0.005) in total tumor volumes after 14 days. In contrast, treatment with three i.p. injections of cisplatin alone at a standard dose of 2.5 mg/kg body weight did not inhibit tumor growth in this cisplatin-resistant lung tumor mouse model (Fig. 3B and C). Moreover, tumor growth was significantly and synergistically inhibited, with total tumor volumes reduced by >90%, in mice treated with three injections of NPRL2 nanoparticles and cisplatin together at same doses as each agent given alone, compared with treatment with PBS, PBS and cisplatin, LacZ, or LacZ and cisplatin (P < 0.005), or with NPRL2 alone (P = 0.03; Fig. 4B and C). Similarly, a significant reduction in both the total tumor numbers (Fig. 4D) and the total fresh tumor weights was seen in mice treated with both NPRL2 and cisplatin compared with treatment with PBS, PBS and cisplatin, LacZ, or LacZ and cisplatin (P < 0.02), as shown by autopsy examination of the tumors in each treatment group. These findings were consistent with those obtained by MRI analysis.


Figure 4
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  		Figure 4. Effect of treatment with a combination of systemic administration of DC-NPRL2 nanoparticles with cisplatin on tumor growth in human H322 orthotopic lung cancer mouse modes using quantitative MRI analysis. A, orthotopic pulmonary tumors were established by intrathoracic injection of 2 x 106 H322 cells in 100 µL of PBS in nude mice. The first MRI was done 10 to 14 days after tumor cell inoculation and before treatment to identify tumor-bearing mice and to establish baseline tumor volume. A second MRI was done 12 to 14 days after the last treatment to determine therapeutic efficacy. A, representative magnetic resonance images before and after treatment along with necropsy photos of the thoracic cavity. Arrows, tumors. B, analysis of orthotopic tumor growth. The total tumor volumes in each treatment group were determined by serial MRI analysis and the absolute growth represented changes in the total tumor volume after treatment divided by the volume before treatment. C, relative growth was calculated using the PBS-treated cells as 100%. At the end of the experiments, the mice were killed and the remaining tumors were dissected. D, the total number of tumors in each mouse were counted. E, the total fresh tumor weights were measured. Bars, SDs of the total tumor volumes (n = 4) or numbers (n = 8) or weights (n = 8) in each treatment group. ANOVA and Fisher's test were used to determine the statistical significance between treatment groups. F, induction of apoptosis by systemic treatment with NPRL2 nanoparticles and cisplatin in vivo. Two H322 tumor–bearing nude mice were treated with agents described above. After 48 hours, the mice were killed, and tumors >5 mm in diameter were harvested and freshly frozen. Apoptosis in frozen tumor tissue sections was detected with an in situ cell death detection kit and TUNEL staining (bright green). The samples were also examined under a fluorescence microscope equipped with a digital camera for nuclear staining by DAPI (blue). Percentage of TUNEL-staining positive cells was determined by counting the numbers of DAPI- and TUNEL-stained cells in at least three fields in each section with an ImagePro image analysis software (Media Cybernetics) and three TUNEL-stained frozen tissue sections were examined. G, columns, percentage of TUNEL+ cells; bars, SD. The significance of differences in induction of apoptosis between treatment groups was analyzed by ANOVA and Fisher's test. NPRL2 protein expression was analyzed by immunohistochemical staining with anti-NPRL2 antibody. Magnification, x400.

 
To evaluate the biological action of systemic NPRL2 nanoparticle and cisplatin treatments in tumors in mice, we examined the expression of NPRL2 protein by immunohistochemical staining with anti-NPRL2 antibodies. We also analyzed the induction of apoptosis by TUNEL staining with an in situ cell death detection kit in flash-frozen tumor tissue samples prepared from H322 tumor–bearing mice 48 hours after treatment (Fig. 4F and G). Induction of apoptosis in the tumors was detected in mice treated with cisplatin or NPRL2 nanoparticles, either alone or together, and in those treated with combined LacZ and cisplatin, but not in those treated with PBS or LacZ alone. Much higher apoptosis induction was detected in tumors treated with NPRL2 nanoparticles (24.4%) than in those treated with cisplatin alone (4.8%), and a significantly and synergistically increased apoptosis (>40%) was registered in tumors treated with both NPRL2 nanoparticles and cisplatin compared with that treated with either agent alone (P = 0.0013), as indicated by extended and intensified TUNEL staining. The expression of NPRL2 proteins in those NPRL2-transfected tumor cells was confirmed by immunohistochemical staining, indicating the capability and efficiency of DC nanoparticle–mediated gene delivery for systemic cancer treatment. These in vivo results were consistent with those observed in vitro, suggesting that treatment with systemic administration of NPRL2 nanoparticles and cisplatin could be a useful therapeutic strategy for enhancing antitumor efficacy and overcoming drug-induced resistance for patients with lung cancer and potentially other cancers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that endogenous NPRL2 protein expression was significantly and reciprocally correlated with cisplatin sensitivity in NSCLC cells. Interestingly, we also noticed that the cisplatin-sensitive H1437 cells that were derived from a patient with cisplatin-responsive lung cancer produced a fairly high level of NPRL2 protein, but that expression was completely lost in the cisplatin-resistant H1437R cells that were selected against cisplatin from the parental H1437 cells. These results suggest that NPRL2 activity plays a role in cytotoxicity mediated by DNA-damaging agents such as cisplatin or {gamma}-irradiation and could be involved in drug resistance. On the other hand, the activity of NPRL2 itself may also be affected by these agents. NPRL2 is a newly identified gene, and the structure and function of its protein are largely unknown. A further study of NPRL2 expression in primary human lung tumors will provide a valuable validation of the findings observed in these cell lines.

Although the exact mechanism involved in the inactivation of NPRL2 in human cancers remains unknown, dysfunctional alterations of the NPRL2 gene and its products (e.g., aberrant splicing transcripts and intragenic homozygous deletions) have been found in various human cancers and cancer cell lines (1, 4). Nonsense and missense mutations in NPRL2 gene clones generated from various primary human tumors (4) and differences in NPRL2 expression between various human cancers and normal counterpart cells (data not shown) have also been revealed by a computer-aided analysis of expressed sequence tag and serial analysis of gene expression databases. The extensive loss of protein expression or alterations in gene products have also been found in several other candidate 3p21.3 tumor suppressors, such as RASSF1A and FUS1, in primary human lung cancers and cancer cell lines (10, 18, 19), suggesting that similar mechanisms such as chromosome instability, aneuploidy, promoter methylation, haploinsufficiency, and altered RNA splicing, as well as defects in transcriptional, translational, and posttranslational processes that are frequently found in the 3p region and in 3p21.3 genes, may play a role in the ultimate inactivation of these 3p21.3 tumor suppressor genes in lung cancers.

The mechanisms underlying cisplatin-induced resistance in lung cancer are also poorly understood, but several in vitro studies with cisplatin-resistant lung cancer cell lines and epigenetic studies in cisplatin-treated cancer patients have revealed some molecular profiles and interrelated mechanisms involved in the drug-resistant phenotype (20–25). One possible mechanism of cisplatin resistance is through alterations in cellular proteins involved in DNA-damage repair and in growth and apoptosis signaling (22–27). Preliminary observations showed microsatellite instability and inactivating homozygous deletions of the 3' coding region of NPRL2 in various human primary tumors and suggested that NPRL2 may be a novel 3p mismatch repair gene (4, 6). Our finding of a significant correlation between endogenous NPRL2 protein expression and cisplatin sensitivity in lung cancer cells is consistent with the suggestion that NPRL2 plays a role in regulating the DNA-damage repair pathway and that inactivation of NPRL2 in tumor cells may promote drug resistance, probably by interrupting DNA-damage repair and apoptotic signaling. In yeast, the NPR2-defective strain did not reduce platinum accumulation but displayed an enhanced rate of spontaneous mutation compared with the isogenic parent cells, suggesting that the NPR2 may be a selective target of the drug persistent stress (8, 9). Our observation of the loss of human NPRL2 expression in the cisplatin-resistant cells is consistent with similar findings in yeast. However, more detailed studies of the potential genetic alterations and the expression profiles of the NPRL2 gene and gene products in association with anticancer agents in human primary cancers and cancer cell lines are needed to confirm the involvement of NPRL2 gene and gene product in cisplatin resistance.

We biologically confirmed that restoring NPRL2 function in NPRL2-deficient and cisplatin-resistant NSCLC cells by DC-NPRL2 nanoparticle–mediated gene transfer resensitized the cell response to cisplatin treatment and synergistically promoted tumor suppression activity in vitro and in mouse models. We also showed that the enhanced tumor growth inhibition by treatment with a combination of NPRL2 and cisplatin was mediated by enhanced activation of caspase cascades and synergistically accelerated induction of apoptosis in vitro and in vivo. The synergistic effect of NPRL2 and cisplatin on tumor growth inhibition and apoptosis is much stronger in NPRL2– and cisplatin-resistant cells than in NPRL2+ and cisplatin-sensitive cells, suggesting that activation of NPRL2 could be effective in overcoming drug-induced resistance in the treatment of lung cancer and other cancers. Moreover, these cisplatin-resistant NSCLC cell lines present a varied status of p53 (e.g., H322 cells contain a R248L inactive mutation, H1299 a homozygous deletion, and A549 a wild-type p53 gene), suggesting that the NPRL2-mediated growth inhibition and apoptosis induction are p53 independent and their association with resistance to chemotherapy may involve different pathways. A detailed study of the mechanisms involved in NPRL2-facilitated activation of cisplatin-mediated cell death and apoptotic signaling pathways is under way in our laboratory and will be presented in a follow-up report. The significant synergism in tumor growth inhibition was also observed in treatment with a combination of NPRL2 nanoparticles and cisplatin at an IC20 dose level, suggesting that a much lower dose of chemotherapeutic agents could be used in a systemic combined treatment with NPRL2 nanoparticles to enhance drug-mediated anticancer efficacy and reduce high-dose drug-induced (toxicity) adverse effects.

In summary, we have shown for the first time that the expression of endogenous NPRL2 significantly and reciprocally correlated with cisplatin sensitivity in NSCLC cells. Reactivation of NPRL2 in NPRL2-deficient and cisplatin-resistant NSCLC cells by NPRL2 nanoparticle–mediated gene transfer synergistically enhanced cisplatin-mediated cytotoxicity by facilitating apoptosis induction in vitro and in vivo. Our results suggest that NPRL2 has potential as a biomarker for the prediction of cisplatin response and prognosis in lung cancer patients and other human cancers and as a molecular therapeutic agent for enhancing and resensitizing the response of nonresponders to cisplatin treatment.


   Acknowledgments
 
Grant support: National Cancer Institute, NIH, Specialized Program of Research Excellence grants CA70970, CA71618, and MMHCC U01CA10535201; Department of Defense TARGET Lung Cancer Programs grant DAMD17-02-1-070; a W.M. Keck Gene Therapy Career Development grant; M.D. Anderson Cancer Center Support Core grant CA16672; and a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/26/06. Revised 7/13/06. Accepted 8/ 7/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 2000;60:6116–33.[Abstract/Free Full Text]
  2. Wistuba II, Behrens C, Virmani AK, et al. High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 2000;60:1949–60.[Abstract/Free Full Text]
  3. Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene 2002;21:6915–35.[CrossRef][Medline]
  4. Li J, Wang F, Haraldson K, et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C. Cancer Res 2004;64:6438–43.[Abstract/Free Full Text]
  5. Ji L, Nishizaki M, Gao B, et al. Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer Res 2002;62:2715–20.[Abstract/Free Full Text]
  6. Schenk PW, Brok M, Boersma AW, et al. Anticancer drug resistance induced by disruption of the Saccharomyces cerevisiae NPR2 gene: a novel component involved in cisplatin- and doxorubicin-provoked cell kill. Mol Pharmacol 2003;64:259–68.[Abstract/Free Full Text]
  7. Baruah H, Barry CG, Bierbach U. Platinum-intercalator conjugates: from DNA-targeted cisplatin derivatives to adenine binding complexes as potential modulators of gene regulation. Curr Top Med Chem 2004;4:1537–49.[CrossRef][Medline]
  8. Perez RP, Perez RP. Cellular and molecular determinants of cisplatin resistance. Eur J Cancer 1998;34:1535–42.[CrossRef][Medline]
  9. Niedner H, Christen R, Lin X, et al. Identification of genes that mediate sensitivity to cisplatin. Mol Pharmacol 2001;60:1153–60.[Abstract/Free Full Text]
  10. Ji L, Minna JD, Roth JA. 3p21.3 tumor suppressor cluster: prospects for translational applications. Future Oncol 2005;1:79–92.[CrossRef][Medline]
  11. Wistuba I, Montellano FD, Milchgrub S, et al. Deletions of chromosome 3p are frequent and early events in the pathogenesis of uterine cervical carcinoma. Cancer Res 1997;57:3154–8.[Abstract/Free Full Text]
  12. Fondon JW, Mele GM, Brezinschek RI, et al. Computerized polymorphic marker identification: experimental validation and a predicted human polymorphism catalog. Proc Natl Acad Sci U S A 1998;95:7514–9.[Abstract/Free Full Text]
  13. Ito S, Ji L, Tanaka F, et al. Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo. Cancer Gene Ther 2004;11:733–9.[CrossRef][Medline]
  14. Ramesh R, Saeki T, Templeton NS, et al. Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol Ther 2001;3:337–50.[CrossRef][Medline]
  15. Templeton NS, Lasic DD, Frederik PM, et al. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 1997;15:647–52.[CrossRef][Medline]
  16. Nishizaki M, Meyn RE, Levy LB, et al. Synergistic inhibition of human lung cancer cell growth by adenovirus-mediated wild-type p53 gene transfer in combination with docetaxel and radiation therapeutics in vitro and in vivo. Clin Cancer Res 2001;7:2887–97.[Abstract/Free Full Text]
  17. Yang D, Han L, Kundra V. Exogenous gene expression in tumors: noninvasive quantification with functional and anatomic imaging in a mouse model. Radiology 2005;235:950–8.[Abstract/Free Full Text]
  18. Burbee DG, Forgacs E, Zochbauer-Muller S, et al. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst 2001;93:691–9.[Abstract/Free Full Text]
  19. Uno F, Sasaki J, Nishizaki M, et al. Myristoylation of the FUS1 protein is required for tumor suppression in human lung cancer cells. Cancer Res 2004;64:2969–76.[Abstract/Free Full Text]
  20. Whiteside MA, Chen DT, Desmond RA, et al. A novel time-course cDNA microarray analysis method identifies genes associated with the development of cisplatin resistance. Oncogene 2004;23:744–52.[CrossRef][Medline]
  21. Rosell R, Taron M, Barnadas A, et al. Nucleotide excision repair pathways involved in Cisplatin resistance in non-small-cell lung cancer. Cancer Control 2003;10:297–305.[Medline]
  22. Rosell R, Lord RV, Taron M, et al. DNA repair and cisplatin resistance in non-small-cell lung cancer. Lung Cancer 2002;38:217–27.[CrossRef][Medline]
  23. Schiller JH. Small cell lung cancer: defining a role for emerging platinum drugs. Oncology 2002;63:105–14.[CrossRef][Medline]
  24. Rosell R, Taron M, O'Brate A. Predictive molecular markers in non-small cell lung cancer. Curr Opin Oncol 2001;13:101–9.[CrossRef][Medline]
  25. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003;22:7265–79.[CrossRef][Medline]
  26. Tsai CM, Chang KT, Li L, et al. Interrelationships between cellular nucleotide excision repair, cisplatin cytotoxicity, HER-2/neu gene expression, and epidermal growth factor receptor level in non-small cell lung cancer cells. Jpn J Cancer Res 2000;91:213–22.[CrossRef][Medline]
  27. Siddik ZH. Biochemical and molecular mechanisms of cisplatin resistance. Cancer Treat Res 2002;112:263–84.[Medline]



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