Journal Article 口腔癌細胞のシスプラチン耐性化に伴うNa, K-ATPase及びouabain感受性の変化

平川, 直  ,  出山, 義昭  ,  吉村, 善隆  ,  鈴木, 邦明  ,  鄭, 漢忠

38 ( 1 )  , pp.40 - 47 , 2017-09 , 北海道歯学会
ISSN:0914-7063
Description
cis-diamminedichloro-platinum (II) (シスプラチン,CDDP)による癌の化学療法においては癌細胞の耐性化が障害となるが,その機序には不明な点も多い。そこで,口腔癌細胞株(Sa3,H1,KB)及びそれらのCDDP耐性株(Sa3R,H1R,KBR)と正常歯肉から採取した細胞を用いて,口腔癌細胞のCDDP耐性化に伴うNa,KATPaseの変化を中心として検討した.  CDDPによる 50%細胞生存濃度(IC50)は,H1とH1Rではそれぞれ1.5×10-4及び7.5×10-4 M,Sa3とSa3Rでは4.5×10-3及び5.5×10-3 M,KBとKBRでは1.0×10-4及び7.0×10-4 Mであり,正常歯肉細胞は5.0×10-5 Mであった.癌細胞は正常歯肉細胞に比べてCDDP感受性が低く、耐性株はそれぞれの感受性株より低い感受性を示した.Na,K-ATPase活性は,Sa3よりSa3Rが高く,H1よりH1Rがわずかに高値を示したが,KBとKBRで差はなかった. Ouabain存在下での各細胞のIC50は、H1とH1Rでは5及び20 nM,Sa3とSa3Rでは80及び180 nM,KBとKBRでは40及び130 nMであり,正常歯肉細胞は5 nMであった.この結果から,CDDP耐性化はouabainに対する耐性化も伴うことが示唆された.各親株と耐性株間のNa,K-ATPase活性阻害に対するouabain濃度依存性は細胞によって異なり,ouabainに対する耐性化はNa, K-ATPaseの変化によるものではないと示唆された.各細胞のP-糖タンパク質とATP7Aの発現を親株と耐性株間で比較したところ,発現量は細胞によって異なった.以上の結果から,CDDP耐性化のメカニズムは細胞の種類によって異なることと,CDDP耐性化はouabain耐性化を伴うことが示唆された.
Purpose : Resistance of cancer cells is an obstacle in chemotherapy for cancer by cisplatin (CDDP). But it is not well elucidated in the mechanism. We examined the change of Na, K-ATPase accompanied by the acquisition of the CDDP resistance of oral cancer cells. Methods : Oral cancer cell lines (Sa3, H1, KB) and CDDP-resistant lines of (Sa3R, H1R, KBR) provided from Chiba University, Faculty of Medicine and cells collected from normal gingiva, were used. Na, K-ATPase activity and cell viability under treatment ouabain and CDDP for each cell was measured. Assessment of Na, K-ATPase α subunits, ATP7A and P-glycoprotein (P-gp) was done by western blotting. Results : According to CDDP, 50% inhibitory concentration (IC50) was, 7.5 × 10-4 M and 1.5 × 10-4 M respectively in H1R and H1, 6.0×10-3 M and 4.5×10-3 M respectively in Sa3R and Sa3, 7.0×10-4 M and 1.0×10-4 M respectively in KBR and KB. CDDP sensitivity was lower than the normal gingival cells to cancer cells. Resistant strains showed a lower sensitivity than the more sensitive strains of each.  Measurement of Na, K-ATPase activity, Sa3R is higher than Sa3, H1R was higher than H1 slightly, but there was no significant difference in the KBR and KB. Activity measurements were consistent in Na, K-ATPase and protein expression levels. IC50 was examined for each cell in the presence of ouabain and were 80 nM and 60 nM in the H1R and H1, 180 nM and 80 nM in the Sa3R and Sa3, 130 nM and 40 nM in the KBR and KB. The normal gingival cells was 5 nM.  From the results of these examinations, it was suggested the cancer cells resistant to CDDP get resistant to ouabain at the same time. As for the ouabain concentration dependence of Na, K-ATPase inhibition of the cells, there is no significant difference in the 50% inhibitory concentration between resistant and parent lines. Therefore, change of Na,KATPase sensitivity to ouabain was not related to resistance of the cells against ouabain. It was suggested that P-gp and Na, K-ATPase, ATP7A is involved in CDDP resistance. The mechanism depends on the type of cells, although we found no features that are common in the between the parent and resistant lines for the expression of ATP7A and P-gp.
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