2004 Fiscal Year Final Research Report Summary
The role of Na+-K+-ATPase in urological cancer: calcium signaling and induction of apoptosis.
| Project/Area Number |
15591700
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | Tokyo Medical University |
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Principal Investigator |
OHNO Yoshio Tokyo Medical Unuversity, Medicine, Assistant Professor, 医学部, 講師 (40266482)
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| Co-Investigator(Kenkyū-buntansha) |
TACHIBANA Masaaki Tokyo Medical Unuversity, Medicine, Professor, 医学部, 教授 (70129526)
OGAWA Yoshihide Ryukyu University, Medicine, Professor, 医学部, 教授 (50051719)
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| Project Period (FY) |
2003 – 2004
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| Keywords | prostate cancer / renal cell carcinoma / Na, K-ATPase / apoptosis / digitalis / italis / ouabain |
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| Research Abstract |
We evaluated the effects of cardiac glycoside, ouabain, on the proliferation of various types of cancer cell lines and normal proximal tubular cells, and also studied the distribution of Na^+-K^+-ATPase in surgical specimens of renal cell carcinoma. We used the prostate cancer cell lines (LNCaP, DU145, PC3) and renal cell carcinoma cell lines (Caki-1, KU19-20). Normal renal proximal tubular cells and renal cell carcinoma cells were obtained from normal renal cortex and tumor in a tumor bearing kidney. Seven lines of normal proximal tubular cell and 4 lines of renal cell carcinoma were cryopreserved. To certify the origin of cultured cells, the expression of megalin and cubilin mRNA was examined by reverse transcriptase-polymerase chain reaction. All proximal tubular cells and one renal cell carcinoma expressed megalin and cubilin mRNA. The growth inhibitory effect of ouabain was determined by dye reduction method using Alamar Blue. Ouabain inhibited the proliferation of LNCaP, DU145, P
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C3, Caki-1, and KU19.20 at a dose of 10 to 200 μM. The percent of apoptotic cells after ouabain treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method. The TUNEL method analysis detected apoptotic cell in cancer cells 3 h after ouabain treatment. However, no significant correlation between the growth inhibory effect of ouabain and the percentage of apoptotic cells. The proximal tubular cells retrieved from cryopreservation lost their viability, thus the proliferation assay was failed. Now, we have continued to collect primary culture cells of proximal tubules from surgical specimen of kidney. In order to assess the distribution of Na^+-K^+-ATPase in surgical specimens of renal cell carcinoma, we performed immunohistochemical analysis using paraffin embedded specimens. The immunohistochemical analysis did not show any significant expression of Na^+-K^+-ATPase in both normal renal cortex and renal cell carcinoma. We supposed that there are several problems in paraffin embedded tissue and anti- Na^+-K^+-ATPase antibody. It might be necessary to carry out immunohistochemical analysis using crypreserved surgical specimens for detection of Na^+-K^+-ATPase. Ten nM ouabain, which concentration of digitalis is used in patients for the treatment of congestive heart failure, exhibited the growth inhibitory effect in renal cell carcinoma cells as well as in prostate cancer cells. However, combination of another drug will be needed to enhance the effect of digitalis because the growth inhibitory effect was weaker than expected. Less
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