Downregulation of Smac/DIABLO Expression in Renal Cell Carcinoma and Its Prognostic Significance
http://www.100md.com
《临床肿瘤学》
the Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, CA
ABSTRACT
MATERIALS AND METHODS: The level of Smac/DIABLO expression was quantified by Western blot analysis using nonfixed fresh frozen tissues.
RESULTS: The expression of Smac/DIABLO was lower in RCC compared with the autologous normal kidney. Sixty-four (82%) of 78 of RCC expressed Smac/DIABLO, and 18% were negative, whereas 100% of normal kidney tissues were positive. In stage I/II RCC, 96% expressed Smac/DIABLO, whereas only 50% expressed Smac/DIABLO in stage III/IV. Smac/DIABLO expression inversely correlated with the grade of RCC. Patients with RCC expressing Smac/DIABLO had a longer postoperative disease-specific survival than those without Smac/DIABLO expression in the 5-year follow-up. Transfection with Smac/DIABLO cDNA enhanced tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) –mediated and cisplatin-mediated cytotoxicity in RCC.
CONCLUSION: The present study demonstrates for the first time that Smac/DIABLO expression was downregulated in RCC and that no Smac/DIABLO expression in RCC predicted a worse prognosis. In addition, transfection with Smac/DIABLO sensitized RCC to TRAIL/cisplatin-induced apoptosis. These results suggest that Smac/DIABLO expression in RCC may be used as a prognostic parameter, and that enhancement of Smac/DIABLO expression in RCC may potentiate immunotherapy and chemotherapy.
INTRODUCTION
Recent studies have reported that overexpression of Smac/DIABLO can induce apoptosis and/or sensitize the resistant cancer cells to death receptor- or cytotoxic drug-induced apoptosis.8,9 These findings suggest that Smac/DIABLO plays an important role in the regulation of apoptotic responses in cancer cells to both immune- and drug-mediated therapies. We hypothesized that the levels of Smac/DIABLO expression may decrease as a function of tumor cell progression. The expression of Smac/DIABLO in cancers has not been examined, with the exception of a recent study.10 There have been no reports on the expression of Smac/DIABLO in RCC and its potential clinical significance. This study was designed to test our hypothesis and investigate the level of Smac/DIABLO expression in RCCs compared with its expression in autologous normal kidneys and determine its prognostic significance.
MATERIALS AND METHODS
This study was performed after approval by a local human investigations committee. Informed consent was obtained from each patient.
RCC Cell Lines
NC65, ACHN, and Caki-1 human RCC cell lines11,12 were maintained in monolayers on plastic dishes in RPMI-1640-1640 medium (Gibco Biocult, Glasgow, Scotland) supplemented with 25 mmol/L HEPES (Gibco Biocult); 2 mmol/L L-glutamine (Gibco Biocult); 1% nonessential amino acid (Gibco Biocult); 100 units/mL penicillin (Gibco Biocult); 100 μg/mL streptomycin (Gibco Biocult); and 10% heat-inactivated fetal bovine serum (Gibco Biocult), hereafter referred to as complete medium.
Measurement of Smac/DIABLO Expression in RCC and Normal Kidney by Western Blot Analysis and Definition of Smac/DIABLO Expression
The expression of Smac/DIABLO was determined by Western blot analysis as previously described.13 We electrophoresed 20 μg of the sample proteins on 7.5% polyacrylamide gels in Tris-glycin buffer and transferred it to nitrocellulose membranes. The membrane was blocked for 30 minutes in blocking buffer (5% skim milk in 1% Tween-phosphate-buffered saline) and probed first with the anti–Smac/DIABLO antibody (Imgenex, San Diego, CA) for 1 hour. The membrane was washed and then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) and developed with the use of an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The relative expression of Smac/DIABLO protein was determined with a chemiluminescence imaging system and quantified by image analysis (Gel Doc 2000; BIO-RAD, Osaka, Japan).
The NC65 cell line constitutively expressed Smac/DIABLO and was used as the internal standard to compare assays. All samples were analyzed at the same time. Repeated measurements yielded the same results. When Smac/DIABLO expression was not visually observed by the Western blot analysis, it was regarded as no or negative expression. In contrast, expression of Smac/DIABLO was regarded as positive expression, if a visual band was detected by Western blot analysis regardless of the variation of the levels of expression. Positive expression meant unambiguous visual detection of Smac/DIABLO protein band by chemiluminescence and did not refer to the level of Smac/DIABLO expression.
Transient Transfection of RCC Cells With Smac/DIABLO cDNA
Transient transfection of RCC cells with Smac/DIABLO cDNA was determined as described by us previously.9 The transfection of RCC cell lines was performed with the pcDNA3.1 vector containing full-length Smac/DIABLO or an empty vector using the polycationic liposome reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The transfection was done according to the manufacturer's instructions. Overexpression of Smac/DIABLO was observed by this transfection procedure.9
Reagents
Recombinant human tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was purchased from Peprotech (Rocky Hill, NJ). Cisplatin was supplied by Nippon Kayaku Co Ltd (Tokyo, Japan).
Cytotoxicity Assay
Microculture tetrazolium dye assay was used to determine tumor cell lysis as previously described.14,15 Briefly, 100 μL of target cell suspension (2 x 104 cells) was added to each well of 96 well flat-bottom microtiter plates (Corning Glass Works, Corning, NY), and each plate was incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. After incubation, the supernatants were aspirated, and tumor cells were washed three times with RPMI-1640 medium, and 200 μL of drug solution or complete medium for control were distributed in the 96-well plates. Each plate was incubated for 24 hours at 37°C. Following incubation, 20 μL of microculture tetrazolium dye working solution (5 mg/mL; Sigma Chemical Co, St Louis, MO) was added to each culture well and the cultures were incubated for 4 hours at 37°C in a humidified 5% CO2 atmosphere. The culture medium was removed from the wells and replaced with 100 μL of isopropanol (Sigma Chemical Co) supplemented with 0.05 normal HCl. The absorbance of each well was measured with a microculture plate reader (Immunoreader; Japan Intermed Co Ltd, Tokyo, Japan) at 540 nm. The percent cytotoxicity was calculated by the following formula:
Statistical Analysis
All determinations were made in triplicate. For statistical analysis, Student's t test and a {chi}2 test were used. Postoperative disease-specific survival was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in survival between RCC patients with and without Smac/DIABLO expression. A P value ≤ .05 was considered significant.
RESULTS
Smac/DIABLO expression was determined in 78 normal kidneys and 78 RCCs. The percentages of cases expressing Smac/DIABLO and those not expressing Smac/DIABLO were determined and are summarized in Table 1. Smac/DIABLO expression was detected in all normal kidney specimens. The Smac/DIABLO expression in normal kidneys in patients with RCC was similar to that in patients with renal pelvic cancer or ureteral cancer (data not shown). Overall, 64 (82%) RCCs were positive for Smac/DIABLO and 14 (18%) were negative. The mean ± SE ratio of Smac/DIABLO expression in RCC compared with normal kidney was 0.27 + 0.03. In stage I/II RCC (n = 54), 52 (96%) were positive and two (4%) were negative. However, in stage III/IV RCC (n = 24), 12 (50%) were positive, and 12 (50%) were negative. The ratio of Smac/DIABLO expression in stage I/II RCC compared with normal kidney was 0.37, and that in stage III/IV RCC, was 0.04. These findings were corroborated with grades of RCC. In grade 1/2 RCC (n = 56), 53 (95%) were positive and 3 (5%) were negative. In contrast, in grade 3 RCC (n = 22), 11 (50%) were positive and 11 (50%) were negative. The ratios of Smac/DIABLO expression in grade 1/2 and grade 3 RCCs compared to normal kidney were 0.35 and 0.06, respectively. These data show significant decrease of Smac/DIABLO expression in RCC as compared to normal kidneys. Furthermore, Smac/DIABLO expression inversely correlated with the stage progression and the increase of the histologic grade of RCC.
Representative data of Smac/DIABLO expression of RCC and normal kidneys from the same patients are shown in Figure 1B, and Figures 2A to 2C. The mean level of Smac/DIABLO expression in normal kidneys was approximately fourfold higher than that in RCCs. Smac/DIABLO expression was not seen in 14 (18%) of 78 RCC (cases 2, 6 to 11). Preliminary experiments in three patients with metastatic RCC demonstrated that Smac/DIABLO expression was significantly lower in metastatic RCC than in primary RCC (Fig 2C).
The level of Smac/DIABLO expression in clear-cell RCC was similar to that in papillary RCC (data not shown). In contrast with RCC, Smac/DIABLO expression was upregulated in oncocytoma compared with normal kidney (Fig 3).
These findings demonstrate that Smac/DIABLO expression was downregulated in RCC compared with normal kidneys, and a significant population of patients with disease progression did not show Smac/DIABLO expression.
Correlation Between Smac/DIABLO Expression and Postoperative Disease-Specific Survival in Patients With RCC
RCC patients undergoing radical nephrectomy were evaluated for the postoperative clinical course. Postoperative disease-specific survival was estimated by Kaplan-Meier analysis. Based on this analysis, patients with RCC were divided into two groups; namely, those with positive Smac/DIABLO expression and those with negative expression, as described in Materials and Methods. Patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival compared with those with negative expression in the 5-year follow-up (Fig 4). Moreover, it is noteworthy that only one patient with RCC with positive Smac/DIABLO expression died in this study, and the expression of Smac/DIABLO was very low in the primary tumor and negative in the metastatic tumor (representative case 14, Fig 2C). These findings suggest that the level of Smac/DIABLO expression in RCC may be a prognostic indicator, and that positive Smac/DIABLO expression in RCC may be a good prognostic sign.
Sensitization of RCC Cells to TRAIL/ Cisplatin–Mediated Cytotoxicity by Smac/DIABLO Transfection
Since Smac/DIABLO expression was downregulated in RCC, we then examined the effect of transfection of RCC with Smac/DIABLO cDNA on tumor growth and TRAIL/cisplatin–induced cytotoxicity. We have previously demonstrated that the cells transfected with pcDNA3.1–Smac/DIABLO overexpressed the protein.9 The transfection with Smac/DIABLO cDNA had no effect on the growth of NC65 and Caki-1 RCC cell lines (data not shown). Transfection of NC65 cells with Smac/DIABLO enhanced TRAIL-mediated cytotoxicity (Table 2). In addition, when the Caki-1 cell line that expressed less Smac/DIABLO compared with the NC65 line was used as a target, Smac/DIABLO transfection markedly potentiated TRAIL-induced cytotoxicity. Overexpression of Smac/DIABLO by tranfection also sensitized NC65 cells to cisplatin-mediated cytotoxicity.
These findings suggest that low expression of Smac/DIABLO in RCC may be associated with drug/immune resistance, and that overexpression of Smac/DIABLO may enhance TRAIL/cisplatin-mediated apoptosis in RCC.
DISCUSSION
Patients with RCC respond very poorly to chemotherapy and radiotherapy.16 RCC cell lines have been described to be resistant to apoptosis-inducing stimuli. A set of cell lines derived from human RCC almost completely lacked the expression of caspase 3 and further expressed other caspases at low levels.17 Such alteration might contribute to RCC development. A recent study by Gerhard et al18 examined the functional competence of the apoptosome in RCC cell lines and RCC fresh tissues. They found that the apoptosome is structurally and functionally intact in both RCC cell lines and primary RCC by the criteria of adding exogenous cytochrome c. These findings suggested that the apoptosome may not be directly involved in resistance. Their study, however, did not examine the activation of the apoptosome and apoptosis by intrinsic cytochrome c and the role of Smac/DIABLO in the activation. The interaction of the apoptosome with low expression of cytochrome c or Smac/DIABLO may not be sufficient to trigger the apoptosome. The present study shows that low expression of Smac/DIABLO with a possibly intact apoptosome may be associated with resistance and suggests the therapeutic effect of overexpressing Smac/DIABLO in the reversal of resistance.
The present study has shown that the expression of Smac/DIABLO in RCC was significantly lower than that in the normal kidney, and approximately 20% RCC lacked Smac/DIABLO expression, though all normal kidney specimens expressed Smac/DIABLO. A recent study by Yoo et al10 has reported analysis of archival tissues of carcinoma and sarcoma by immunohistochemical analysis for the expression of Smac/DIABLO. Smac/DIABLO expression was observed in 62% of carcinomas and 22% of sarcomas. The level of Smac/DIABLO expression varied depending on the individual tumor. For instance, two of 10 prostate carcinomas were positive for Smac/DIABLO, whereas the remaining eight were negative. Normal tissues adjacent to the cancer showed various degrees of Smac/DIABLO expression. However, in this report, there were no data on the expression of Smac/DIABLO in RCC. Our studies and those of Yoo et al,10 demonstrating that the expression level of Smac/DIABLO in carcinoma and sarcoma is significantly different from the corresponding noncancerous tissues, suggest strongly that Smac/DIABLO might play a role in the development of cancer.
This study is the first to demonstrate that Smac/DIABLO expression in RCC predicts the clinical outcome. The precise reasons responsible for this relationship remain unclear at present. Since Smac/DIABLO is a proapoptotic regulatory molecule, it is reasonable to assume that despite treatments, clones of cells that do not express Smac/DIABLO will not undergo apoptosis and will be selected to grow more easily and rapidly than clones that overexpress Smac/DIABLO. In addition, the current study has shown that Smac/DIABLO was less expressed in the metastatic RCC than in the primary RCC. These findings suggest that Smac/DIABLO agonists may provide a therapeutic means of preventing metastasis and growth of RCC.
Cytotoxic chemotherapy, an integral part of the therapeutic approach for many solid tumors, has shown little or no antitumor activity against RCC and has played no role in either an adjuvant or a neoadjuvant support therapy.16 Immunotherapy including interleukin-2 and interferon alfa is relatively effective against metastatic RCC, and the overall response rate of immunotherapy and/or chemotherapy has gradually improved. However, the response rate is approximately 20%, and metastasis and recurrence still remain major problems in the therapy for RCC.19 Therefore, new therapeutic approaches are required. The down-regulation of Smac/DIABLO expression in RCC compared with the normal kidney identifies Smac/DIABLO as a molecular therapeutic target. Our observation that overexpression of Smac/DIABLO in RCC by transfection resulted in high sensitivity to TRAIL/cisplatin–mediated killing, may be clinically relevant in the management of patients with RCC. The endogenous low level of Smac/DIABLO in RCC may not be adequate to neutralize the antiapoptotic mechanism regulated by IAPs. Thus, immunotherapy/chemotherapy in combination with Smac/DIABLO agonists may be a promising strategy against RCC. Furthermore, enhancement of Smac/DIABLO expression by gene therapy may also provide a novel therapeutic means of overcoming the resistance of RCC to immunotherapy/chemotherapy.
IAPs such as XIAP are highly expressed in various cancers and are associated with poor prognosis and resistance to apoptosis.20,21 Preliminary experiments demonstrated that the expression of XIAP in RCC was higher than that in the normal kidney. Since XIAP blocks apoptosis at the effector phase, strategies targeting XIAP may be especially effective at overcoming resistance to apoptosis. Smac/DIABLO is able to bind to IAP family members, and XIAP is a predominant Smac/DIABLO binding protein. Smac/DIABLO binds to XIAP, displaces XIAP from caspase-9, promotes cleavage of effector caspases, and induces apoptosis.22,23 Therefore, the measurement of XIAP expression as well as Smac/DIABLO expression may be necessary for the accurate evaluation of the efficacy of therapy with Smac/DIABLO.
Drugs that can antagonize IAPs may have benefits, particularly when combined with chemotherapeutic drugs or TRAIL. For instance, Arnt et al24 found that the first four amino acids of Smac/DIABLO increased apoptosis in cell lines treated with paclitaxel, etoposide, camptothecin, and doxorubicine.
Cancer therapy using TRAIL or anti-DR4/5 monoclonal antibody is currently being investigated in clinical trials due to their low toxicity to normal tissues.25,26 However, not all tumors respond to TRAIL, and resistance to TRAIL has been shown to be overcome by drugs,27,28 by overexpression of Smac/DIABLO, or by Smac/DIABLO peptides.8,9,24 Thus, analysis of the expression of Smac/DIABLO in cancer may be helpful for determining therapeutic modalities such as TRAIL therapy.
The findings of this study showed that patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival than those with negative expression. Fundamentally, patients without metastasis or recurrence received no postoperative treatments. The first- and the second-line treatments for metastasis or recurrence were intramuscular interferon alfa monotherapy and combination therapy with intramuscular interferon alfa and intravenous interleukin-2, respectively. The third-line treatment or surgery was dependent on each patient. Therefore, differing therapies may in part account for the different survival curves.
The dramatic postoperative disease-specific survival advantage for Smac/DIABLO-positive RCCs is the central issue in this study. In stage III/IV RCC patients (n = 24), 10 patients (83%) with negative Smac/DIABLO expression (n = 12) died of RCC. In contrast, only one patient (8%) with positive expression (n = 12) died. However, the numbers do not permit multivariate analysis. Therefore, it is not clarified whether Smac/DIABLO expression adds prognostic importance to tumor stage or not. The potential biologic significance of Smac/DIABLO expression as a component of a more generalized problem of apoptotic resistance warrants further investigation and suggests future clinical study.
In conclusion, the current study demonstrated that Smac/DIABLO expression was downregulated in RCC, and that negative Smac/DIABLO expression was a poor prognostic sign. Furthermore, elevated Smac/DIABLO expression by transfection rendered resistant RCC cells sensitive to TRAIL/cisplatin-mediated cytotoxicity. These findings suggest that the assessment of Smac/DIABLO expression may be useful in the management of RCC. Since Smac/DIABLO expression could be used as a prognostic parameter in patients with RCC, the accurate prediction of prognosis may help select patients for more intensive surgical or immunochemotherapeutic approaches in combination with Smac/DIABLO agonists. However, further studies are needed to determine the regulatory effects of Smac/DIABLO expression in RCC.
Authors' Disclosures of Potential Conflicts of Interest
Acknowledgment
We acknowledge the assistance of Ms Yukako Morioka and Ms Kate Dinh in the preparation of this manuscript.
NOTES
Supported in part by grants in aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 15390496), and from the US Department of Defense (DAMD17-02-1-0023; B.B.).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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10. Yoo NJ, Kim HS, Kim SY, et al: Immunohistochemical analysis of Smac/DIABLO expression in human carcinomas and sarcomas. APMIS 111:382-388, 2003
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12. Mizutani Y, Bonavida B, Koishihara Y, et al: Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum (II) by anti-interleukin-6 monoclonal antibody or anti-interleukin-6-receptor monoclonal antibody. Cancer Res 55:590-596, 1995
13. Mizutani Y, Kamoi K, Ukimura O, et al: Synergistic cytotoxicity and apoptosis of JTE-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer. J Urol 168:2650-2654, 2002
14. Mizutani Y, Wada H, Yoshida O, et al: Significance of thymidylate synthase activity in renal cell carcinoma. Clin Cancer Res 9:1453-1460, 2003
15. Mizutani Y, Wada H, Fukushima M, et al: Prognostic significance of orotate phosphoribosyltransferase activity in bladder cancer. Cancer 100:723-731, 2004
16. Yagoda A: Chemotherapy of renal cell carcinoma: 1983–1989. Semin Urol 7:199-206, 1989
17. Kolenko V, Uzzo RG, Bukowski R, et al: Dead or dying: Necrosis versus apoptosis in caspase-deficient human renal cell carcinoma. Cancer Res 59:2838-2842, 1999
18. Gerhard MC, Zantl N, Weirich G, et al: Functional evaluation of the apoptosome in renal cell carcinoma. Br J Cancer 89:2147-2154, 2003
19. Bukowski RM: Natural history and therapy of metastatic renal cell carcinoma. CANCER 80:1198-1220, 1997
20. Deveraux QL, Reed JC: IAP family proteins-suppressors of apoptosis. Genes Dev 13:239-252, 1999
21. Sasaki H, Sheng Y, Kotsuji F, et al: Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells. Cancer Res 60:5659-5666, 2000
22. Goyal L: Cell death inhibition: Keeping caspases in check. Cell 104:805-808, 2001
23. Srinivasula SM, Hegde R, Saleh A, et al: A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature 410:112-116, 2001
24. Arnt CR, Chiorean MV, Heldebrant MP, et al: Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ. J Biol Chem 277:44236-44243, 2002
25. Ashkenazi A, Dixit VM: Death receptors: Signaling and modulation. Science 281:1305-1308, 1998
26. Walczak H, Miller RE, Ariail K, et al: Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med 5:157-163, 1999
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Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, CA
ABSTRACT
MATERIALS AND METHODS: The level of Smac/DIABLO expression was quantified by Western blot analysis using nonfixed fresh frozen tissues.
RESULTS: The expression of Smac/DIABLO was lower in RCC compared with the autologous normal kidney. Sixty-four (82%) of 78 of RCC expressed Smac/DIABLO, and 18% were negative, whereas 100% of normal kidney tissues were positive. In stage I/II RCC, 96% expressed Smac/DIABLO, whereas only 50% expressed Smac/DIABLO in stage III/IV. Smac/DIABLO expression inversely correlated with the grade of RCC. Patients with RCC expressing Smac/DIABLO had a longer postoperative disease-specific survival than those without Smac/DIABLO expression in the 5-year follow-up. Transfection with Smac/DIABLO cDNA enhanced tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) –mediated and cisplatin-mediated cytotoxicity in RCC.
CONCLUSION: The present study demonstrates for the first time that Smac/DIABLO expression was downregulated in RCC and that no Smac/DIABLO expression in RCC predicted a worse prognosis. In addition, transfection with Smac/DIABLO sensitized RCC to TRAIL/cisplatin-induced apoptosis. These results suggest that Smac/DIABLO expression in RCC may be used as a prognostic parameter, and that enhancement of Smac/DIABLO expression in RCC may potentiate immunotherapy and chemotherapy.
INTRODUCTION
Recent studies have reported that overexpression of Smac/DIABLO can induce apoptosis and/or sensitize the resistant cancer cells to death receptor- or cytotoxic drug-induced apoptosis.8,9 These findings suggest that Smac/DIABLO plays an important role in the regulation of apoptotic responses in cancer cells to both immune- and drug-mediated therapies. We hypothesized that the levels of Smac/DIABLO expression may decrease as a function of tumor cell progression. The expression of Smac/DIABLO in cancers has not been examined, with the exception of a recent study.10 There have been no reports on the expression of Smac/DIABLO in RCC and its potential clinical significance. This study was designed to test our hypothesis and investigate the level of Smac/DIABLO expression in RCCs compared with its expression in autologous normal kidneys and determine its prognostic significance.
MATERIALS AND METHODS
This study was performed after approval by a local human investigations committee. Informed consent was obtained from each patient.
RCC Cell Lines
NC65, ACHN, and Caki-1 human RCC cell lines11,12 were maintained in monolayers on plastic dishes in RPMI-1640-1640 medium (Gibco Biocult, Glasgow, Scotland) supplemented with 25 mmol/L HEPES (Gibco Biocult); 2 mmol/L L-glutamine (Gibco Biocult); 1% nonessential amino acid (Gibco Biocult); 100 units/mL penicillin (Gibco Biocult); 100 μg/mL streptomycin (Gibco Biocult); and 10% heat-inactivated fetal bovine serum (Gibco Biocult), hereafter referred to as complete medium.
Measurement of Smac/DIABLO Expression in RCC and Normal Kidney by Western Blot Analysis and Definition of Smac/DIABLO Expression
The expression of Smac/DIABLO was determined by Western blot analysis as previously described.13 We electrophoresed 20 μg of the sample proteins on 7.5% polyacrylamide gels in Tris-glycin buffer and transferred it to nitrocellulose membranes. The membrane was blocked for 30 minutes in blocking buffer (5% skim milk in 1% Tween-phosphate-buffered saline) and probed first with the anti–Smac/DIABLO antibody (Imgenex, San Diego, CA) for 1 hour. The membrane was washed and then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) and developed with the use of an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The relative expression of Smac/DIABLO protein was determined with a chemiluminescence imaging system and quantified by image analysis (Gel Doc 2000; BIO-RAD, Osaka, Japan).
The NC65 cell line constitutively expressed Smac/DIABLO and was used as the internal standard to compare assays. All samples were analyzed at the same time. Repeated measurements yielded the same results. When Smac/DIABLO expression was not visually observed by the Western blot analysis, it was regarded as no or negative expression. In contrast, expression of Smac/DIABLO was regarded as positive expression, if a visual band was detected by Western blot analysis regardless of the variation of the levels of expression. Positive expression meant unambiguous visual detection of Smac/DIABLO protein band by chemiluminescence and did not refer to the level of Smac/DIABLO expression.
Transient Transfection of RCC Cells With Smac/DIABLO cDNA
Transient transfection of RCC cells with Smac/DIABLO cDNA was determined as described by us previously.9 The transfection of RCC cell lines was performed with the pcDNA3.1 vector containing full-length Smac/DIABLO or an empty vector using the polycationic liposome reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The transfection was done according to the manufacturer's instructions. Overexpression of Smac/DIABLO was observed by this transfection procedure.9
Reagents
Recombinant human tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was purchased from Peprotech (Rocky Hill, NJ). Cisplatin was supplied by Nippon Kayaku Co Ltd (Tokyo, Japan).
Cytotoxicity Assay
Microculture tetrazolium dye assay was used to determine tumor cell lysis as previously described.14,15 Briefly, 100 μL of target cell suspension (2 x 104 cells) was added to each well of 96 well flat-bottom microtiter plates (Corning Glass Works, Corning, NY), and each plate was incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. After incubation, the supernatants were aspirated, and tumor cells were washed three times with RPMI-1640 medium, and 200 μL of drug solution or complete medium for control were distributed in the 96-well plates. Each plate was incubated for 24 hours at 37°C. Following incubation, 20 μL of microculture tetrazolium dye working solution (5 mg/mL; Sigma Chemical Co, St Louis, MO) was added to each culture well and the cultures were incubated for 4 hours at 37°C in a humidified 5% CO2 atmosphere. The culture medium was removed from the wells and replaced with 100 μL of isopropanol (Sigma Chemical Co) supplemented with 0.05 normal HCl. The absorbance of each well was measured with a microculture plate reader (Immunoreader; Japan Intermed Co Ltd, Tokyo, Japan) at 540 nm. The percent cytotoxicity was calculated by the following formula:
Statistical Analysis
All determinations were made in triplicate. For statistical analysis, Student's t test and a {chi}2 test were used. Postoperative disease-specific survival was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in survival between RCC patients with and without Smac/DIABLO expression. A P value ≤ .05 was considered significant.
RESULTS
Smac/DIABLO expression was determined in 78 normal kidneys and 78 RCCs. The percentages of cases expressing Smac/DIABLO and those not expressing Smac/DIABLO were determined and are summarized in Table 1. Smac/DIABLO expression was detected in all normal kidney specimens. The Smac/DIABLO expression in normal kidneys in patients with RCC was similar to that in patients with renal pelvic cancer or ureteral cancer (data not shown). Overall, 64 (82%) RCCs were positive for Smac/DIABLO and 14 (18%) were negative. The mean ± SE ratio of Smac/DIABLO expression in RCC compared with normal kidney was 0.27 + 0.03. In stage I/II RCC (n = 54), 52 (96%) were positive and two (4%) were negative. However, in stage III/IV RCC (n = 24), 12 (50%) were positive, and 12 (50%) were negative. The ratio of Smac/DIABLO expression in stage I/II RCC compared with normal kidney was 0.37, and that in stage III/IV RCC, was 0.04. These findings were corroborated with grades of RCC. In grade 1/2 RCC (n = 56), 53 (95%) were positive and 3 (5%) were negative. In contrast, in grade 3 RCC (n = 22), 11 (50%) were positive and 11 (50%) were negative. The ratios of Smac/DIABLO expression in grade 1/2 and grade 3 RCCs compared to normal kidney were 0.35 and 0.06, respectively. These data show significant decrease of Smac/DIABLO expression in RCC as compared to normal kidneys. Furthermore, Smac/DIABLO expression inversely correlated with the stage progression and the increase of the histologic grade of RCC.
Representative data of Smac/DIABLO expression of RCC and normal kidneys from the same patients are shown in Figure 1B, and Figures 2A to 2C. The mean level of Smac/DIABLO expression in normal kidneys was approximately fourfold higher than that in RCCs. Smac/DIABLO expression was not seen in 14 (18%) of 78 RCC (cases 2, 6 to 11). Preliminary experiments in three patients with metastatic RCC demonstrated that Smac/DIABLO expression was significantly lower in metastatic RCC than in primary RCC (Fig 2C).
The level of Smac/DIABLO expression in clear-cell RCC was similar to that in papillary RCC (data not shown). In contrast with RCC, Smac/DIABLO expression was upregulated in oncocytoma compared with normal kidney (Fig 3).
These findings demonstrate that Smac/DIABLO expression was downregulated in RCC compared with normal kidneys, and a significant population of patients with disease progression did not show Smac/DIABLO expression.
Correlation Between Smac/DIABLO Expression and Postoperative Disease-Specific Survival in Patients With RCC
RCC patients undergoing radical nephrectomy were evaluated for the postoperative clinical course. Postoperative disease-specific survival was estimated by Kaplan-Meier analysis. Based on this analysis, patients with RCC were divided into two groups; namely, those with positive Smac/DIABLO expression and those with negative expression, as described in Materials and Methods. Patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival compared with those with negative expression in the 5-year follow-up (Fig 4). Moreover, it is noteworthy that only one patient with RCC with positive Smac/DIABLO expression died in this study, and the expression of Smac/DIABLO was very low in the primary tumor and negative in the metastatic tumor (representative case 14, Fig 2C). These findings suggest that the level of Smac/DIABLO expression in RCC may be a prognostic indicator, and that positive Smac/DIABLO expression in RCC may be a good prognostic sign.
Sensitization of RCC Cells to TRAIL/ Cisplatin–Mediated Cytotoxicity by Smac/DIABLO Transfection
Since Smac/DIABLO expression was downregulated in RCC, we then examined the effect of transfection of RCC with Smac/DIABLO cDNA on tumor growth and TRAIL/cisplatin–induced cytotoxicity. We have previously demonstrated that the cells transfected with pcDNA3.1–Smac/DIABLO overexpressed the protein.9 The transfection with Smac/DIABLO cDNA had no effect on the growth of NC65 and Caki-1 RCC cell lines (data not shown). Transfection of NC65 cells with Smac/DIABLO enhanced TRAIL-mediated cytotoxicity (Table 2). In addition, when the Caki-1 cell line that expressed less Smac/DIABLO compared with the NC65 line was used as a target, Smac/DIABLO transfection markedly potentiated TRAIL-induced cytotoxicity. Overexpression of Smac/DIABLO by tranfection also sensitized NC65 cells to cisplatin-mediated cytotoxicity.
These findings suggest that low expression of Smac/DIABLO in RCC may be associated with drug/immune resistance, and that overexpression of Smac/DIABLO may enhance TRAIL/cisplatin-mediated apoptosis in RCC.
DISCUSSION
Patients with RCC respond very poorly to chemotherapy and radiotherapy.16 RCC cell lines have been described to be resistant to apoptosis-inducing stimuli. A set of cell lines derived from human RCC almost completely lacked the expression of caspase 3 and further expressed other caspases at low levels.17 Such alteration might contribute to RCC development. A recent study by Gerhard et al18 examined the functional competence of the apoptosome in RCC cell lines and RCC fresh tissues. They found that the apoptosome is structurally and functionally intact in both RCC cell lines and primary RCC by the criteria of adding exogenous cytochrome c. These findings suggested that the apoptosome may not be directly involved in resistance. Their study, however, did not examine the activation of the apoptosome and apoptosis by intrinsic cytochrome c and the role of Smac/DIABLO in the activation. The interaction of the apoptosome with low expression of cytochrome c or Smac/DIABLO may not be sufficient to trigger the apoptosome. The present study shows that low expression of Smac/DIABLO with a possibly intact apoptosome may be associated with resistance and suggests the therapeutic effect of overexpressing Smac/DIABLO in the reversal of resistance.
The present study has shown that the expression of Smac/DIABLO in RCC was significantly lower than that in the normal kidney, and approximately 20% RCC lacked Smac/DIABLO expression, though all normal kidney specimens expressed Smac/DIABLO. A recent study by Yoo et al10 has reported analysis of archival tissues of carcinoma and sarcoma by immunohistochemical analysis for the expression of Smac/DIABLO. Smac/DIABLO expression was observed in 62% of carcinomas and 22% of sarcomas. The level of Smac/DIABLO expression varied depending on the individual tumor. For instance, two of 10 prostate carcinomas were positive for Smac/DIABLO, whereas the remaining eight were negative. Normal tissues adjacent to the cancer showed various degrees of Smac/DIABLO expression. However, in this report, there were no data on the expression of Smac/DIABLO in RCC. Our studies and those of Yoo et al,10 demonstrating that the expression level of Smac/DIABLO in carcinoma and sarcoma is significantly different from the corresponding noncancerous tissues, suggest strongly that Smac/DIABLO might play a role in the development of cancer.
This study is the first to demonstrate that Smac/DIABLO expression in RCC predicts the clinical outcome. The precise reasons responsible for this relationship remain unclear at present. Since Smac/DIABLO is a proapoptotic regulatory molecule, it is reasonable to assume that despite treatments, clones of cells that do not express Smac/DIABLO will not undergo apoptosis and will be selected to grow more easily and rapidly than clones that overexpress Smac/DIABLO. In addition, the current study has shown that Smac/DIABLO was less expressed in the metastatic RCC than in the primary RCC. These findings suggest that Smac/DIABLO agonists may provide a therapeutic means of preventing metastasis and growth of RCC.
Cytotoxic chemotherapy, an integral part of the therapeutic approach for many solid tumors, has shown little or no antitumor activity against RCC and has played no role in either an adjuvant or a neoadjuvant support therapy.16 Immunotherapy including interleukin-2 and interferon alfa is relatively effective against metastatic RCC, and the overall response rate of immunotherapy and/or chemotherapy has gradually improved. However, the response rate is approximately 20%, and metastasis and recurrence still remain major problems in the therapy for RCC.19 Therefore, new therapeutic approaches are required. The down-regulation of Smac/DIABLO expression in RCC compared with the normal kidney identifies Smac/DIABLO as a molecular therapeutic target. Our observation that overexpression of Smac/DIABLO in RCC by transfection resulted in high sensitivity to TRAIL/cisplatin–mediated killing, may be clinically relevant in the management of patients with RCC. The endogenous low level of Smac/DIABLO in RCC may not be adequate to neutralize the antiapoptotic mechanism regulated by IAPs. Thus, immunotherapy/chemotherapy in combination with Smac/DIABLO agonists may be a promising strategy against RCC. Furthermore, enhancement of Smac/DIABLO expression by gene therapy may also provide a novel therapeutic means of overcoming the resistance of RCC to immunotherapy/chemotherapy.
IAPs such as XIAP are highly expressed in various cancers and are associated with poor prognosis and resistance to apoptosis.20,21 Preliminary experiments demonstrated that the expression of XIAP in RCC was higher than that in the normal kidney. Since XIAP blocks apoptosis at the effector phase, strategies targeting XIAP may be especially effective at overcoming resistance to apoptosis. Smac/DIABLO is able to bind to IAP family members, and XIAP is a predominant Smac/DIABLO binding protein. Smac/DIABLO binds to XIAP, displaces XIAP from caspase-9, promotes cleavage of effector caspases, and induces apoptosis.22,23 Therefore, the measurement of XIAP expression as well as Smac/DIABLO expression may be necessary for the accurate evaluation of the efficacy of therapy with Smac/DIABLO.
Drugs that can antagonize IAPs may have benefits, particularly when combined with chemotherapeutic drugs or TRAIL. For instance, Arnt et al24 found that the first four amino acids of Smac/DIABLO increased apoptosis in cell lines treated with paclitaxel, etoposide, camptothecin, and doxorubicine.
Cancer therapy using TRAIL or anti-DR4/5 monoclonal antibody is currently being investigated in clinical trials due to their low toxicity to normal tissues.25,26 However, not all tumors respond to TRAIL, and resistance to TRAIL has been shown to be overcome by drugs,27,28 by overexpression of Smac/DIABLO, or by Smac/DIABLO peptides.8,9,24 Thus, analysis of the expression of Smac/DIABLO in cancer may be helpful for determining therapeutic modalities such as TRAIL therapy.
The findings of this study showed that patients with RCC with positive Smac/DIABLO expression had a longer disease-specific survival than those with negative expression. Fundamentally, patients without metastasis or recurrence received no postoperative treatments. The first- and the second-line treatments for metastasis or recurrence were intramuscular interferon alfa monotherapy and combination therapy with intramuscular interferon alfa and intravenous interleukin-2, respectively. The third-line treatment or surgery was dependent on each patient. Therefore, differing therapies may in part account for the different survival curves.
The dramatic postoperative disease-specific survival advantage for Smac/DIABLO-positive RCCs is the central issue in this study. In stage III/IV RCC patients (n = 24), 10 patients (83%) with negative Smac/DIABLO expression (n = 12) died of RCC. In contrast, only one patient (8%) with positive expression (n = 12) died. However, the numbers do not permit multivariate analysis. Therefore, it is not clarified whether Smac/DIABLO expression adds prognostic importance to tumor stage or not. The potential biologic significance of Smac/DIABLO expression as a component of a more generalized problem of apoptotic resistance warrants further investigation and suggests future clinical study.
In conclusion, the current study demonstrated that Smac/DIABLO expression was downregulated in RCC, and that negative Smac/DIABLO expression was a poor prognostic sign. Furthermore, elevated Smac/DIABLO expression by transfection rendered resistant RCC cells sensitive to TRAIL/cisplatin-mediated cytotoxicity. These findings suggest that the assessment of Smac/DIABLO expression may be useful in the management of RCC. Since Smac/DIABLO expression could be used as a prognostic parameter in patients with RCC, the accurate prediction of prognosis may help select patients for more intensive surgical or immunochemotherapeutic approaches in combination with Smac/DIABLO agonists. However, further studies are needed to determine the regulatory effects of Smac/DIABLO expression in RCC.
Authors' Disclosures of Potential Conflicts of Interest
Acknowledgment
We acknowledge the assistance of Ms Yukako Morioka and Ms Kate Dinh in the preparation of this manuscript.
NOTES
Supported in part by grants in aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 15390496), and from the US Department of Defense (DAMD17-02-1-0023; B.B.).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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