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P-glycoprotein activity in renal clear cell carcinoma
Urologic Oncology: Seminars and Original Investigations 27 (2009) 363–366
Original article
P-glycoprotein activity in renal clear cell carcinoma Elena Soto-Vega, Ph.D.a,1, Carlos Arroyo, M.D., Ph.D.b,1, Yvonne Richaud-Patin, B.S.c, Mario García-Carrasco, M.D.a, Luis G. Vázquez-Lavista, M.D.d, Luis Llorente, M.D.c,* a Unit of Research of Autoimmune Diseases, Instituto Mexicano del Seguro Social, Puebla, Mexico Department of Surgery, Hospital Universitario de Puebla, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico c Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico DF, Mexico d Department of Urology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico DF, Mexico b
Received 22 November 2007; received in revised form 24 January 2008; accepted 25 January 2008
Abstract Background: The mechanism by which renal cancer patients show poor response to chemotherapy has not been well understood. The aim of this study was to evaluate the functional activity of P-glycoprotein (P-gp) in renal clear cell carcinoma (RCCC) and its possible role in chemotherapy resistance. Methods: We studied 11 patients who underwent radical nephrectomy due to RCCC; from each patient we obtained a sample from the cancer tissue, and another from normal renal tissue. These biopsies were mechanically disaggregated to allow individual cells analysis. Cells were incubated with daunorubicin (a fluorescent drug extruded by P-gp) at 37oC and 4oC for 30 min. P-gp activity was analyzed using flow cytometry. Results were expressed as the percentage of cells with P-gp activity (i.e., low fluorescence). Results: The analysis of renal cells showed that there was no significant difference in size between normal and cancer cells; however there were clusters of cells with different granularities. We divided the cells according to their granularity. The proportion of cells capable of extruding daunorubicin was significantly higher on tumor cells than in normal renal cells independently of the cell granularity. Our results are congruent with those obtained when mRNA or immunohistochemical test were used. This is the first report quantifying the P-gp activity from fresh samples obtained from kidney cancer in humans. Conclusions: Percentage of cells extruding daunorubicin in RCCC is elevated, indicating that P-gp activity may contribute to multidrug resistance in RCCC. © 2009 Elsevier Inc. All rights reserved. Keywords: Renal cancer; P-glycoprotein; Chemoresistance; Multidrug resistance; Daunorubicin
1. Introduction Kidney cancer is the seventh leading malignant condition among men and the twelfth among women, accounting for 2.6% of all cancers in the United States [1]. The most frequent histology is renal clear cell carcinoma (RCCC) [2]. When RCCC is detected in an organ confined stage, it is surgically treated with a good overall survival rate. However, 30% of RCCC are detected in an advanced stage, with a high mortality rate. One-third of the patients with RCCC have metastatic disease at diag-
* Corresponding author. Tel.: ⫹(52) 55 56 55-5954; fax: ⫹(52) 55 55172096. E-mail address: [email protected] (L. Llorente). 1 These authors collaborated equally to this work. 1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2008.01.011
nosis and 50% will progress post-nephrectomy [3]. RCCC shows a poor response to chemotherapy, making it an inappropriate treatment. Current FDA approved treatment strategies for metastatic stages are immunotherapy with cytokines [4] (interferon ␣ and interleukin 2), and the use of antibodies against specific proteins (vascular endothelial growth factor, platelet derived growth factor, and tumor growth factor ␣), such as Bevacizumab [5] and Sunitinib [6]. The RCCC is derived from renal proximal tubular epithelial cells. These cells express several membrane transporters for reabsorption and excretion of various metabolites, including those for diverse drugs such as those employed for cancer chemotherapy [7]. Unresponsiveness to chemotherapy in RCCC may be caused by acquired drug resistance mechanisms. Among these, one of the most extensively studied is the so-called multidrug resistance-1 (MDR-1), which is a 170 kDa
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P-glycoprotein (P-gp) [8,9]. This molecule belongs to a superfamily of the ATP binding cassette (ABC) transporters. Although the physiological role of P-gp is not yet completely understood, it has been implicated in bacterial product detoxification and in hormone as well as metabolite secretion. The P-gp activity involves a decreased sensitivity to several agents, including vinca alkaloids, anthracyclines, and glucocorticoids among others [10,11]. The drug resistance has been attributed to an augmented drug extrusion by P-gp, which diminishes the intracellular drug concentration, thus reducing its therapeutic action. MDR-1 gene expression and protein membrane density have been studied in normal and malignant cells using quantification of P-gp mRNA and immunohistochemical techniques. These methods measure any P-gp detected (protein or mRNA) independently of its functional incorporation in the cell membrane machinery, however protein regulation may occur at the transcriptional, translational, and/or post-translational levels modifying its functionality [12– 15]. An alternative approach that we have taken is the study of the extrusion of fluorescent dyes from individual cells, allowing evaluation of the functional activity of P-gp. Such functional efflux assays have been successfully used to investigate P-gp status and the effects of P-gp modulators in different cells [16 –19]. In the present study, we evaluated the function of P-gp using a functional efflux assay, in cells obtained from a group of patients with RCCC.
2.1. Flow cytometric detection of daunorubicin efflux All surgical specimens were washed twice in phosphate buffer saline (PBS) to eliminate blood component from renal tissue. Then, tissues were mechanically treated with a syringe, passed thorough the syringe until isolated cells were obtained, to allow individual cell analysis. Cells were counted and adjusted to 1 ⫻ 106 cells /ml in PBS. The extrusion of the P-gp substrate daunorubicin from the cells was studied by incubating 2 ⫻ 106 cells with 40 l of 400 M daunorubicin. The cells were then divided into two aliquots. One was placed for 30 min in an ice bath at 4oC in order to inhibit cell metabolism, to allow measurement of basal daunorubicin uptake. The other was incubated for 30 min in a 37oC water bath so that the efflux could take place. The cells were then washed twice with ice-cold medium and kept at 4oC for immediate flow cytometry analysis. All analyses were performed using a FACScan flow cytometer with CellQuest software (Becton Dickinson, San Jose, CA). Electronic gates were set to allow separate analysis of all the cells based on side scatter and forward scatter characteristics (to avoid differences in size or granularity among cell populations, we set narrow gates in the middle of the cells clusters). All analyses were done within these gates. The results were expressed as the percentage of cells capable of extruding daunorubicin. Fluorescence intensity was recorded in each subject and in each cell subpopulation to determine whether effluxing and noneffluxing cells were comparable in tumor and normal tissue.
2. Methods
2.2. Statistical analysis
We obtained 15 samples of patients who underwent radical nephrectomy because of kidney cancer in an organconfined stage as a curative intent; 4 of these samples were not RCCC, so we included only 11 patients (6 females and 5 males) with a mean age of 54.7 years (range 43 to 68 years) with RCCC histopathology. The histopathological results showed that the Fuhrman grade was 2 in seven patients, 3 in three patients, and 4 in one patient, with 9 patients in a stage T3 and two in a stage T2 of the 2002 tumor nodule metastasis classification. None of the patients had any proven metastasis site and no one had received any previous chemotherapy, immunotherapy, or tyrosine kinase inhibitors. From each patient, we obtained a sample of approximately 2 cm3 from the cancer tissue, and another of 2 cm3 from the normal renal tissue. Samples were processed immediately and the rest of the surgical specimens were analyzed histologically by the pathology department. The 11 tissue samples were confirmed as a renal clear cell carcinoma and the normal renal tissue examination revelead no sign of neoplastic cell contamination. The study was approved by our institutional ethics committee, and all subjects gave their written informed consent.
Data were evaluated using the Mann Whitney U-test. P-values less than 0.05 were considered statistically significant.
3. Results The analysis of the normal renal tissue histopathology revealed no sign of neoplastic cell infiltrates. Initially, all the cells were quantified with the flow cytometer without taking into account the size or granularity; the results showed that the cells obtained from RCCC tissue had greater P-gp activity. The percentage of cells extruding daunorubicin was of 77.7% ⫾ 16.7% in cancer cells compared with 53.0% ⫾ 18.8% in normal cells (P ⫽ 0.02). We then analyzed the size and granularity from both normal and RCCC cell to determine if they were comparable. The analysis of both renal tissues showed that there was no difference in size between them; however there were different granularity clusters in both tissues, so, we decided to divide the cells in 3 different cell clusters according to their mean side scatter values; each cell cluster was desig-
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Table 1 The percentage of cell extruding daunorubicin according to the different granularities clusters in normal and tumor cells Cell granularity
Normal renal cells (n ⫽ 11)
RCCC (n ⫽ 11)
P
1 Low 2 Medium 3 High
73 ⫾ 13 46 ⫾ 14 40 ⫾ 14
92 ⫾ 13 74 ⫾ 12 68⫾15
0.01 0.0008 0.0005
nated as 1, 2, or 3 according to its granularity (1 low, 3 high). The analysis of the forward scatter channel in all the samples did not prove any significant variation; the mean size of all the cells was of 157 ⫾ 53. The study of the granularity showed data dispersion but it was possible to differentiate three clusters of cells, and among them the side scatter values for each group were 50 to 200, 240 to 420, and 600 to 810. The result of each cluster obtained was compared with its similar cluster between normal and RCCC. The flow cytometric analysis of P-gp activity showed that in all cell clusters, the proportion of cells capable of extruding daunorubicin was significantly higher on tumor cells than in those obtained from normal renal tissue at 37oC (Table 1). It is important to observe that in both tissues, the cells with the lowest granularity had the greatest P-gp activity, and this percentage of activity was significant when comparing P-gp activity between granularity clusters in the same tissue (Table 2). As a control group, the activity of P-gp at 4oC was examined because at this temperature the cellular metabolism is inhibited, this basal activity at 4oC was compared with the activity obtained at 37oC. As expected, the activity of P-gp at 4oC was always significantly lower than in those samples incubated at 37oC (data not shown). When normal and cancer cells groups were compared according to granularity of the cells, a significant difference between normal renal tissue and RCCC in P-gp activity was found in the three granularity groups at 37°C (Fig. 1). As a control of the fluorescence channel intensity, for each subject, the fluorescence for P-gp positive and P-gp negative cells was recorded. We found no difference in
Table 2 Comparisons of P-gp activity between the different granularity clusters in the same tissue Cell P-gp activity granularity Normal renal cells (n ⫽ 11) 1 vs. 2 1 vs. 3 2 vs. 3
RCCC (n ⫽ 11)
79.9 ⫾ 20 vs. 46.2 ⫾ 13.7* 91.5 ⫾ 13.1 vs. 73.9 ⫾ 12.1* 72.9 ⫾ 20 vs. 39.9 ⫾ 14.2a 91.5 ⫾ 13.1 vs. 67.9 ⫾ 14.6a 46.2 ⫾ 13.7 vs. 39.9 ⫾ 14.2a 73.9 ⫾ 12.1 vs. 67.9 ⫾ 14.6a
* P ⬍ 0.01. a Not significant.
Fig. 1. P-gp activity in normal renal cells and RCCC. The P-gp activity was measured as the percentage of cells capable of extruding daunorubicin in normal renal tissue and RCCC according to their granularity (1 low, 2 medium, 3 high). The bars represent the mean ⫾ standard deviation of the percentage of cells extruding daunorubicin. Black and gray bars represent normal renal cells and RCCC cells at 37°C, respectively (n ⫽ 11 for all bars).
fluorescence intensity in both cell subpopulations, either from normal or malignant renal tissue.
4. Discussion Incidence rates for RCCC have been increasing steadily each year in Europe and the United States in the last three decades [20], with a decrease in tumor size at diagnosis, which should have a relative survival advantage [21]. The only established causal risk factor is cigarette smoking [22] and surgery the only curative procedure in early stages. RCCC has been classified as a marginal poorly responsive tumor in terms of the chemotherapeutic effect (⬍5%), because of the natural resistance of the tumor cells. The function of P-gp in normal renal tissue is mainly the excretion of hydrophobic cationic compounds; studies in malignant cells had found a significant correlation between increased levels of P-gp mRNA and multidrug resistance [23,24]. However it is possible that P-gp detected (protein or mRNA) is not functional, and regulation may occur at the transcriptional, translational, and/or post-translational levels determining the protein function. In cell culture models and in patients with drug refractory leukemia, it has been demonstrated that a rearrangement of the MDR-1 gene takes place [25,26]. An important goal in this paper was the determination of the presence of P-gp by cytometry, and mainly the quantification of the P-gp function directly by daunorubicin extrusion in RCCC cells obtained from fresh human tumors. Previous works have quantified the mRNA and the P-gp histochemistry in cultured cells [12–15].
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A significant increase of P-gp activity in malignant tissue compared with healthy renal tissue was found. However, the small number of cases upon which this results are based is a definite limitation, and our conclusions should be considered with the above in mind. The mechanisms that determine an increase in the activity of P-gp during a cellular transformation into a neoplasia is yet to be understood. P-gp is one of the probable causes of chemotherapy resistance, however it is important to mention that drug sensitivity in kidney cancer is a complex process that most probably involves other factors. In 3 different cells lines of renal carcinoma (VMRC-RCW, OS-RC-2, and TUHR14TKB) it has been shown that drug resistance varies with the expression levels of different factors, including P-gp, resistance-related gene-9, ␥-glutamyl cysteine synthetase, and cis-diamminedichloroplatinum [27]. Although it has been reported that the expression of P-gp is associated with a worse prognosis [28], it is necessary to determine and measure the activity of each resistance factor in tumor tissue in order to better define its participation in the complex chemoresistant process. Inhibitors of P-gp such as PSC833 and VX-710 have been proven useful in some diseases such as lupus erythematosus, in which resistance to steroid therapy develops in parallel with the expression of P-gp [29]. However, for cancer therapy, the concentrations of P-gp inhibitors needed to be effective are toxic for humans, although there are some clinical Phase III trials with PSC 833 in tumors, and the preliminary results showed that it is possible to reduce the concentration of anticancer drugs used in these patients when it is combined with the PSC 833 [30]. Much remains to be elucidated concerning the role of P-gp and drug resistance. Our findings may help to characterize pathologic mechanisms and to identify potential therapeutic strategies for RCCC that deserves to be explored in depth.
References [1] Jemal A, Murray T, Ward E. Cancer statistics. CA Cancer J Clin 2005;55:10 –30. [2] Vogelzang NJ, Stadler WM. Kidney cancer. Lancet 1998;352: 1691– 6. [3] Hock LM, Lynch J, Balaji KC. Increasing incidence of all stages of kidney cancer in the last two decades in the United States: An analysis of surveillance, epidemiology and end results program data. J Urol 2002;167:57– 60. [4] Ramsey S, Aitchinson M. Treatment for renal cell: Are we beyond the cytokine era? Nat Clin Pract Urol 2006;3:478 – 84. [5] Yang JC, Haworth L, Sherry RM, et al. A randomized trial of bevacizumab an anti-vascular endothelial growth factor antibody for metastatic renal cancer. N Engl J Med 2003;349:427–34. [6] Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 2007; 356:115–24. [7] Inui K, Masuda S, Satio H. Cellular and molecular aspects of drug transport in the kidney. Kidney Int 2000;58:944 –58. [8] Chang G. Multidrug resistance ABC transporters. FEBS Lett 2003; 27:102–5.
[9] Liang XJ, Aszalos A. Multidrug transporters as drug targets. Curr Drug Targets 2006;7:911–21. [10] Tsuruo T. Reversal of acquired resistance to vinca alkaloids and anthracycline antibiotics. Cancer Treat Rep 1983;67:889 –94. [11] Ueda K, Cardarelli C, Gottesman MM, et al. Expression of a full length cDNA from the human MDR-1 gene confers resistance to colchicines, doxorubicin, and vinblastine. Proc Natl Acad Sci USA 1987;84:3004 – 8. [12] Juranka PF, Zastawny RL, Ling V. P-glycoprotein: Multidrug resistance and a superfamily of membrane-associated transport proteins. FASEB J 1989;324:485–9. [13] Bradley G, Naik M, Ling V. P-glycoprotein expression in multidrugresistant human ovarian carcinoma cell lines. Cancer Res 1989;49: 2790 – 6. [14] Hill BT, Deuchars K, Hosking LK. Overexpression of P-glycoprotein in mammalian tumor cell lines after fractioned X irradiation in vitro. J Natl Cancer Inst 1990;82:607–12. [15] McClean S, Hill BT. Evidence of post-translational regulation of P-glycoprotein associated with expression of a distinctive multiple drug-resistant phenotype in Chinese hamster ovary cells. Eur J Cancer 1993;29A:2243– 8. [16] Cornwell MM, Pastan I, Gottesman MM. Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane besides and inhibit drug binding to P-glycoprotein. J Biol Chem 1987;262:2166 –70. [17] Safa AR. Photoaffinity labeling of the multidrug resistance-related P-glycoprotein with photoactive analogs of verapamil. Proc Natl Acad Sci 1998;85:7187–91. [18] Safa AR, Glover CJ, Sewell JL, et al. Identification of the multidrug resistance-related membrane glycoprotein as acceptor for calcium channel blockers. J Biol Chem 1987;262:7884 – 8. [19] McCoy JP, Keren DF. Current practices in clinical flow cytometry. A practice survey by the American Society of clinical pathologist. Am J Pathol 1999;111:161– 8. [20] Mathew A, Devesa SS, Fraumeni JF, et al. Global increases in kidney cancer incidence, 1973–1992. Eur J Cancer Prev 2002;11:171– 8. [21] Nguyen MM, Gill IS, Ellison LM. The evolving presentation of renal carcinoma in the United States: Trends from the surveillance, epidemiology and end results program. J Urol 2006;176:2397–2400. [22] Lipworth L, Tarone RE, McLaughlin JK. The epidemiology of renal cell carcinoma. J Urol 2006;176:2353– 8. [23] Fojo T. Multiple paths to a drug resistance phenotype mutations, translocations, deletions and amplifications of coding genes or promoter regions, epigenetic changes and micro RNAs. Drug Resist Update 2007;10:59 – 67. [24] Higgins CF. Multiple molecular mechanisms for multidrug resistance transporters. Nature 2007;446:749 –57. [25] Huff LM, Lee JS, Robey RW, et al. Characterization of gene rearrangements leading to activation of MDR-1. J Biol Chem 2006;281: 36501–9. [26] Harada T, Nagayama J, Kohno K, et al. Alu-associated interstitial deletions and chromosomal rearrangements in two human multidrugresistant cell lines. Int J Cancer 2000;86:506 –11. [27] Asakura T, Imai A, Ohkubo-Uraoka N, et al. Relationship between expression of drug-resistance factors and drug sensitivity in normal human renal proximal tubular epithelial cells in comparison with renal cell carcinoma. Oncol Rep 2005;14:601–7. [28] Mignogna C, Staibano S, Altieri V, et al. Prognostic significance of multidrug-resistance protein (MDR-1) in renal clear cell carcinomas: A five-year follow-up analysis. BMC Cancer 2006;6:293–303. [29] Aouali N, Eddabra L, Macadré J, et al. Immunosuppressors and reversion of multidrug-resistance. Crit Rev Oncol Hematol 2005;56: 61–70. [30] Bates SE. Solving the problems of multidrug resistance: ABC transporters in clinical oncology. In: Holland IB, Cole SP, Kuchler K, et al, editors. ABC proteins: From bacteria to man. London: Elsevier Science, 2002; p. 670 – 8.
Original article
P-glycoprotein activity in renal clear cell carcinoma Elena Soto-Vega, Ph.D.a,1, Carlos Arroyo, M.D., Ph.D.b,1, Yvonne Richaud-Patin, B.S.c, Mario García-Carrasco, M.D.a, Luis G. Vázquez-Lavista, M.D.d, Luis Llorente, M.D.c,* a Unit of Research of Autoimmune Diseases, Instituto Mexicano del Seguro Social, Puebla, Mexico Department of Surgery, Hospital Universitario de Puebla, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico c Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico DF, Mexico d Department of Urology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico DF, Mexico b
Received 22 November 2007; received in revised form 24 January 2008; accepted 25 January 2008
Abstract Background: The mechanism by which renal cancer patients show poor response to chemotherapy has not been well understood. The aim of this study was to evaluate the functional activity of P-glycoprotein (P-gp) in renal clear cell carcinoma (RCCC) and its possible role in chemotherapy resistance. Methods: We studied 11 patients who underwent radical nephrectomy due to RCCC; from each patient we obtained a sample from the cancer tissue, and another from normal renal tissue. These biopsies were mechanically disaggregated to allow individual cells analysis. Cells were incubated with daunorubicin (a fluorescent drug extruded by P-gp) at 37oC and 4oC for 30 min. P-gp activity was analyzed using flow cytometry. Results were expressed as the percentage of cells with P-gp activity (i.e., low fluorescence). Results: The analysis of renal cells showed that there was no significant difference in size between normal and cancer cells; however there were clusters of cells with different granularities. We divided the cells according to their granularity. The proportion of cells capable of extruding daunorubicin was significantly higher on tumor cells than in normal renal cells independently of the cell granularity. Our results are congruent with those obtained when mRNA or immunohistochemical test were used. This is the first report quantifying the P-gp activity from fresh samples obtained from kidney cancer in humans. Conclusions: Percentage of cells extruding daunorubicin in RCCC is elevated, indicating that P-gp activity may contribute to multidrug resistance in RCCC. © 2009 Elsevier Inc. All rights reserved. Keywords: Renal cancer; P-glycoprotein; Chemoresistance; Multidrug resistance; Daunorubicin
1. Introduction Kidney cancer is the seventh leading malignant condition among men and the twelfth among women, accounting for 2.6% of all cancers in the United States [1]. The most frequent histology is renal clear cell carcinoma (RCCC) [2]. When RCCC is detected in an organ confined stage, it is surgically treated with a good overall survival rate. However, 30% of RCCC are detected in an advanced stage, with a high mortality rate. One-third of the patients with RCCC have metastatic disease at diag-
* Corresponding author. Tel.: ⫹(52) 55 56 55-5954; fax: ⫹(52) 55 55172096. E-mail address: [email protected] (L. Llorente). 1 These authors collaborated equally to this work. 1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2008.01.011
nosis and 50% will progress post-nephrectomy [3]. RCCC shows a poor response to chemotherapy, making it an inappropriate treatment. Current FDA approved treatment strategies for metastatic stages are immunotherapy with cytokines [4] (interferon ␣ and interleukin 2), and the use of antibodies against specific proteins (vascular endothelial growth factor, platelet derived growth factor, and tumor growth factor ␣), such as Bevacizumab [5] and Sunitinib [6]. The RCCC is derived from renal proximal tubular epithelial cells. These cells express several membrane transporters for reabsorption and excretion of various metabolites, including those for diverse drugs such as those employed for cancer chemotherapy [7]. Unresponsiveness to chemotherapy in RCCC may be caused by acquired drug resistance mechanisms. Among these, one of the most extensively studied is the so-called multidrug resistance-1 (MDR-1), which is a 170 kDa
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P-glycoprotein (P-gp) [8,9]. This molecule belongs to a superfamily of the ATP binding cassette (ABC) transporters. Although the physiological role of P-gp is not yet completely understood, it has been implicated in bacterial product detoxification and in hormone as well as metabolite secretion. The P-gp activity involves a decreased sensitivity to several agents, including vinca alkaloids, anthracyclines, and glucocorticoids among others [10,11]. The drug resistance has been attributed to an augmented drug extrusion by P-gp, which diminishes the intracellular drug concentration, thus reducing its therapeutic action. MDR-1 gene expression and protein membrane density have been studied in normal and malignant cells using quantification of P-gp mRNA and immunohistochemical techniques. These methods measure any P-gp detected (protein or mRNA) independently of its functional incorporation in the cell membrane machinery, however protein regulation may occur at the transcriptional, translational, and/or post-translational levels modifying its functionality [12– 15]. An alternative approach that we have taken is the study of the extrusion of fluorescent dyes from individual cells, allowing evaluation of the functional activity of P-gp. Such functional efflux assays have been successfully used to investigate P-gp status and the effects of P-gp modulators in different cells [16 –19]. In the present study, we evaluated the function of P-gp using a functional efflux assay, in cells obtained from a group of patients with RCCC.
2.1. Flow cytometric detection of daunorubicin efflux All surgical specimens were washed twice in phosphate buffer saline (PBS) to eliminate blood component from renal tissue. Then, tissues were mechanically treated with a syringe, passed thorough the syringe until isolated cells were obtained, to allow individual cell analysis. Cells were counted and adjusted to 1 ⫻ 106 cells /ml in PBS. The extrusion of the P-gp substrate daunorubicin from the cells was studied by incubating 2 ⫻ 106 cells with 40 l of 400 M daunorubicin. The cells were then divided into two aliquots. One was placed for 30 min in an ice bath at 4oC in order to inhibit cell metabolism, to allow measurement of basal daunorubicin uptake. The other was incubated for 30 min in a 37oC water bath so that the efflux could take place. The cells were then washed twice with ice-cold medium and kept at 4oC for immediate flow cytometry analysis. All analyses were performed using a FACScan flow cytometer with CellQuest software (Becton Dickinson, San Jose, CA). Electronic gates were set to allow separate analysis of all the cells based on side scatter and forward scatter characteristics (to avoid differences in size or granularity among cell populations, we set narrow gates in the middle of the cells clusters). All analyses were done within these gates. The results were expressed as the percentage of cells capable of extruding daunorubicin. Fluorescence intensity was recorded in each subject and in each cell subpopulation to determine whether effluxing and noneffluxing cells were comparable in tumor and normal tissue.
2. Methods
2.2. Statistical analysis
We obtained 15 samples of patients who underwent radical nephrectomy because of kidney cancer in an organconfined stage as a curative intent; 4 of these samples were not RCCC, so we included only 11 patients (6 females and 5 males) with a mean age of 54.7 years (range 43 to 68 years) with RCCC histopathology. The histopathological results showed that the Fuhrman grade was 2 in seven patients, 3 in three patients, and 4 in one patient, with 9 patients in a stage T3 and two in a stage T2 of the 2002 tumor nodule metastasis classification. None of the patients had any proven metastasis site and no one had received any previous chemotherapy, immunotherapy, or tyrosine kinase inhibitors. From each patient, we obtained a sample of approximately 2 cm3 from the cancer tissue, and another of 2 cm3 from the normal renal tissue. Samples were processed immediately and the rest of the surgical specimens were analyzed histologically by the pathology department. The 11 tissue samples were confirmed as a renal clear cell carcinoma and the normal renal tissue examination revelead no sign of neoplastic cell contamination. The study was approved by our institutional ethics committee, and all subjects gave their written informed consent.
Data were evaluated using the Mann Whitney U-test. P-values less than 0.05 were considered statistically significant.
3. Results The analysis of the normal renal tissue histopathology revealed no sign of neoplastic cell infiltrates. Initially, all the cells were quantified with the flow cytometer without taking into account the size or granularity; the results showed that the cells obtained from RCCC tissue had greater P-gp activity. The percentage of cells extruding daunorubicin was of 77.7% ⫾ 16.7% in cancer cells compared with 53.0% ⫾ 18.8% in normal cells (P ⫽ 0.02). We then analyzed the size and granularity from both normal and RCCC cell to determine if they were comparable. The analysis of both renal tissues showed that there was no difference in size between them; however there were different granularity clusters in both tissues, so, we decided to divide the cells in 3 different cell clusters according to their mean side scatter values; each cell cluster was desig-
E. Soto-Vega et al. / Urologic Oncology: Seminars and Original Investigations 27 (2009) 363–366
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Table 1 The percentage of cell extruding daunorubicin according to the different granularities clusters in normal and tumor cells Cell granularity
Normal renal cells (n ⫽ 11)
RCCC (n ⫽ 11)
P
1 Low 2 Medium 3 High
73 ⫾ 13 46 ⫾ 14 40 ⫾ 14
92 ⫾ 13 74 ⫾ 12 68⫾15
0.01 0.0008 0.0005
nated as 1, 2, or 3 according to its granularity (1 low, 3 high). The analysis of the forward scatter channel in all the samples did not prove any significant variation; the mean size of all the cells was of 157 ⫾ 53. The study of the granularity showed data dispersion but it was possible to differentiate three clusters of cells, and among them the side scatter values for each group were 50 to 200, 240 to 420, and 600 to 810. The result of each cluster obtained was compared with its similar cluster between normal and RCCC. The flow cytometric analysis of P-gp activity showed that in all cell clusters, the proportion of cells capable of extruding daunorubicin was significantly higher on tumor cells than in those obtained from normal renal tissue at 37oC (Table 1). It is important to observe that in both tissues, the cells with the lowest granularity had the greatest P-gp activity, and this percentage of activity was significant when comparing P-gp activity between granularity clusters in the same tissue (Table 2). As a control group, the activity of P-gp at 4oC was examined because at this temperature the cellular metabolism is inhibited, this basal activity at 4oC was compared with the activity obtained at 37oC. As expected, the activity of P-gp at 4oC was always significantly lower than in those samples incubated at 37oC (data not shown). When normal and cancer cells groups were compared according to granularity of the cells, a significant difference between normal renal tissue and RCCC in P-gp activity was found in the three granularity groups at 37°C (Fig. 1). As a control of the fluorescence channel intensity, for each subject, the fluorescence for P-gp positive and P-gp negative cells was recorded. We found no difference in
Table 2 Comparisons of P-gp activity between the different granularity clusters in the same tissue Cell P-gp activity granularity Normal renal cells (n ⫽ 11) 1 vs. 2 1 vs. 3 2 vs. 3
RCCC (n ⫽ 11)
79.9 ⫾ 20 vs. 46.2 ⫾ 13.7* 91.5 ⫾ 13.1 vs. 73.9 ⫾ 12.1* 72.9 ⫾ 20 vs. 39.9 ⫾ 14.2a 91.5 ⫾ 13.1 vs. 67.9 ⫾ 14.6a 46.2 ⫾ 13.7 vs. 39.9 ⫾ 14.2a 73.9 ⫾ 12.1 vs. 67.9 ⫾ 14.6a
* P ⬍ 0.01. a Not significant.
Fig. 1. P-gp activity in normal renal cells and RCCC. The P-gp activity was measured as the percentage of cells capable of extruding daunorubicin in normal renal tissue and RCCC according to their granularity (1 low, 2 medium, 3 high). The bars represent the mean ⫾ standard deviation of the percentage of cells extruding daunorubicin. Black and gray bars represent normal renal cells and RCCC cells at 37°C, respectively (n ⫽ 11 for all bars).
fluorescence intensity in both cell subpopulations, either from normal or malignant renal tissue.
4. Discussion Incidence rates for RCCC have been increasing steadily each year in Europe and the United States in the last three decades [20], with a decrease in tumor size at diagnosis, which should have a relative survival advantage [21]. The only established causal risk factor is cigarette smoking [22] and surgery the only curative procedure in early stages. RCCC has been classified as a marginal poorly responsive tumor in terms of the chemotherapeutic effect (⬍5%), because of the natural resistance of the tumor cells. The function of P-gp in normal renal tissue is mainly the excretion of hydrophobic cationic compounds; studies in malignant cells had found a significant correlation between increased levels of P-gp mRNA and multidrug resistance [23,24]. However it is possible that P-gp detected (protein or mRNA) is not functional, and regulation may occur at the transcriptional, translational, and/or post-translational levels determining the protein function. In cell culture models and in patients with drug refractory leukemia, it has been demonstrated that a rearrangement of the MDR-1 gene takes place [25,26]. An important goal in this paper was the determination of the presence of P-gp by cytometry, and mainly the quantification of the P-gp function directly by daunorubicin extrusion in RCCC cells obtained from fresh human tumors. Previous works have quantified the mRNA and the P-gp histochemistry in cultured cells [12–15].
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A significant increase of P-gp activity in malignant tissue compared with healthy renal tissue was found. However, the small number of cases upon which this results are based is a definite limitation, and our conclusions should be considered with the above in mind. The mechanisms that determine an increase in the activity of P-gp during a cellular transformation into a neoplasia is yet to be understood. P-gp is one of the probable causes of chemotherapy resistance, however it is important to mention that drug sensitivity in kidney cancer is a complex process that most probably involves other factors. In 3 different cells lines of renal carcinoma (VMRC-RCW, OS-RC-2, and TUHR14TKB) it has been shown that drug resistance varies with the expression levels of different factors, including P-gp, resistance-related gene-9, ␥-glutamyl cysteine synthetase, and cis-diamminedichloroplatinum [27]. Although it has been reported that the expression of P-gp is associated with a worse prognosis [28], it is necessary to determine and measure the activity of each resistance factor in tumor tissue in order to better define its participation in the complex chemoresistant process. Inhibitors of P-gp such as PSC833 and VX-710 have been proven useful in some diseases such as lupus erythematosus, in which resistance to steroid therapy develops in parallel with the expression of P-gp [29]. However, for cancer therapy, the concentrations of P-gp inhibitors needed to be effective are toxic for humans, although there are some clinical Phase III trials with PSC 833 in tumors, and the preliminary results showed that it is possible to reduce the concentration of anticancer drugs used in these patients when it is combined with the PSC 833 [30]. Much remains to be elucidated concerning the role of P-gp and drug resistance. Our findings may help to characterize pathologic mechanisms and to identify potential therapeutic strategies for RCCC that deserves to be explored in depth.
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