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Recent research has shown that lactoferrin indirectly suppresses osteoclastogenesis by affecting osteoblasts and periodontal ligament fibroblasts. However, the mechanism by which lactoferrin directly affects osteoclastogenesis is yet to be reported. Therefore, this study examined the direct effects of lactoferrin on RANKL-induced osteoclast differentiation of murine osteoclastic RAW 264.7 cells. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. The effect of lactoferrin on the number of osteoclasts was measured, and the effect on the mRNA expression of osteoclast differentiation markers was assayed using real-time PCR. Lactoferrin decreased the number of osteoclasts (_2 nuclei) and large osteoclasts (_8 nuclei) in a dose-dependent manner without affecting the viability of RAW 264.7 cells. Additionally, it only mediated these effects within 48 h of culturing the RAW 264.7 cells with RANKL. Lactoferrin also significantly inhibited RANKL-induced mRNA expressions of osteoclastic differentiation genes, such as NFATc1, RANK, DC-STAMP, and OC-STAMP. Thus, these findings suggest that lactoferrin directly suppresses osteoclastogenesis within 48 h of culturing the RAW 264.7 cells with RANKL. Therefore, lactoferrin may be a novel and innovative therapy for treating bone diseases.

【目的】白金系抗がん剤による化学療法において耐性化が障害となるが，その機序には不明な点も多い．そこで耐性化の機構の検討を目的に，シスプラチン耐性化に伴う白金系抗がん剤及び抗がん活性を示す強心配糖体のウアバインに対する感受性の変化を検討した． 【材料と方法】口腔がん細胞株（親株：H1, KB）及びそれらのシスプラチン耐性株（耐性株：H1R, KBR）のシスプラチン，カルボプラチン及びネダプラチン（抗がん剤と総称）とウアバインに対する感受性の変化を検討した．細胞生存率を測定するために細胞内ATP量を測定した． 【結果と考察】1．H1とKB，H1RとKBRはそれぞれ親株と耐性株として類似した結果を示した．2．親株，耐性株とも抗がん剤の濃度及び作用時間に依存して細胞数は減少した．3．耐性株は，低濃度の抗がん剤に対して親株と比較して耐性を示した．4．親株，耐性株とも50％細胞数を減少するのに必要な抗がん剤の濃度はシスプラチン，ネダプラチン，カルボプラチンの順に増加した．5．親株は低濃度の抗がん剤存在下で細胞数が減少したが，さらに濃度が上がると細胞数の減少が抑制される濃度域が存在した．一方，耐性株は抗がん剤の濃度依存性に細胞数が減少するのみであった．6．親株及び耐性株はウアバインの濃度及び作用時間に依存して細胞数が減少した．7．適当な濃度の抗がん剤はウアバイン存在下での細胞数の減少を保護した．以上の結果は，口腔がん細胞及びそのシスプラチン耐性株に対する白金系抗がん剤の作用はその濃度によって異なることと，シスプラチン耐性化に伴いウアバインの作用も変化することを示唆する． 【結論】口腔がん細胞株であるH1及びKBのシスプラチン耐性化によりカルボプラチン及びネダプラチンに対する交叉耐性を示す．また，耐性化に伴い，白金系抗がん剤及び強心配糖体の作用が変化する．, Purpose: Cancer chemotherapy using platinum-based anticancer agents is inhibited by cancer cell cisplatin resistance, but the mechanism is unknown. Hence, we examined how cisplatin-resi stant oral cancer cells changed their sensitivity toplatinum coordination complex and cardiac glycosides. Materials and methods: We used oral cancer cells (parent cell line: H1, KB) and cisplatin-resistant lines (resistant cell line: H1R, KBR). The cells were dispersed in a 96-wells plate and inc ubated with 8, 16, 31, 63, 125, 250, and 500 μM cisplatin, carboplatin, or nedaplatin (anticancer agent) for 6, 12, 24, 48, and 72 h in the absence or presence of ouabain. The survival rate of the cells was measured by intracellular adenosine triphosphate content using ViaLigh plus kit (LONZA). Results and discussion: 1. In all experimental conditions, H1 and KB, H1R, and KBR showed similar results as parent or resistant cell lines. 2. The viable cell count of a parent or resistant cells decreased in a concentration and time-dependent manner in response to the anticancer agent. 3. When compared to the parent cell line, the resistant cell line showed resistance to not only cisplatin but also carboplatin and nedaplatin. 4. In parent and resistant cell lines, the 50% inhibitory concentrations of the anticancer agent ranged from low to high in the order of cisplatin, nedaplatin, and carboplatin. 5. In the presence of a low concentration anticancer agent, the number of parent cells was reduced, but it was protected at higher concentrations. On the other hand, there was no such concentration range for anticancer agent-resistant cell lines. 6. The viable cell count of a parent or resistant cells decreas ed in a concentration and time-dependent manner in response to ouabain. 7. In presence of ouabain, there was a concentration range of anticancer agents that protected against a decrease in the number of cells. This effect occurred only in the parent cell l ine incubated with cisplatin, but not in the parent or resistant cell line incubated with carboplatin or nedaplatin. Conclusion: Cisplatin resistance in the oral cancer cell lines H1 and KB showed cross-resistance to carboplatin and nedaplatin. The effect of the platinum coordination complex and cardiac glycosides changed as they became cisplatin-resistant.

ヒトには遺伝子型の異なる4 種に加えて糖鎖による修飾が異なる多数のアルカリ性ホスファターゼ（ALP）アイソザイムが存在するが，骨型ALPのピロリン酸及び小腸型ALPのピリドキサルリン酸を除いて生理的な基質と機能に関しては不明な点が多く，また活性の至適pHがアルカリ性である意味も不明である． ヒト胎盤型ALPの結晶構造解析の結果からヒトのALPには1 サブユニットあたり1 Mg及びCaと2 Znの結合部位の存在が示されたが，ALP活性における各イオンの機能については報告が少ない．そこで，ヒトALPアイソザイムに対する各イオンの作用を比較することにより，アイソザイムによる活性の至適pH及び基質への親和性の相違を明らかにすることを目的に研究を行った．市販のヒト骨型，肝臓型，胎盤型及び小腸型ALPを使用した．基質にはパラニトロフェニルリン酸（pNPP），Na-PPi及びATPを使用し以下の結果を得た．1）pNPPを基質として，4 種類のALP活性はMg，Ca及びZn濃度依存性に増加し，50％活性化濃度はALPの種類にはよらず，Zn，Ca，Mgの順に増加した．Znは低濃度では活性を促進したが，高濃度では逆に抑制し， 2個のZn結合部位の性質は異なることを示唆した．また，各ALP活性の至適pHはALP，基質及びイオンの種類により変化した．2）pNPP 及びNa-PPiを基質として，骨型ALP活性はMg，Ca及びZn濃度依存性に増加したが，濃度依存曲線は基質によって異なった．3）MgとCaあるいはMgとZnを組み合わせて添加することにより骨型ALP活性は相加的に増加した．4）イオン及び基質の種類によらず，骨型ALP活性は阻害剤であるlevamisoleあるいはtetramisoleによって濃度依存的に阻害された．以上の結果はin vitroでは4 種類のALPには Mg，Ca及びZn結合部位が存在し独立してALP活性の促進が可能であること，活性の至適pH及び基質に対する親和性は存在するイオンによって変化しうることを示唆した．In vivoでもALPの生体内での基質は存在する2 価金属イオンの種類と周囲のpHによって変化する可能性がある．, Objectives: One Mg, one Ca, and two Zn binding sites per subunit of the human placenta type alkaline phosphatase (ALP) were detected by analyzing its crystalline structure. It was an ticipated that other human ALPs also contain these ion binding sites, as there are few reports about the function of each of these ions on ALP activity. We examined the affinity of ALP isozymes for Mg, Ca, and Zn. Moreover, we also examined the effects of Mg, Ca, and Zn on the optimum pH for ALP activity and substrate specificity. Methods: We utilized commercial human bone, liver, placenta, an d small intestine type ALP, p-nitrophenyl phosphate (pNPP), and Na-PPi as substrates, and assayed the Pi released from the substrates using the Chifflet method. Results and discussion: 1) The ALP activity of all four isozyme s increased in Mg, Ca, and Zn concentration-dependent manner with pNPP as the substrate. Remarkably, the 50% activated concentrations (K0.5) of each ion did not depend on the type of ALP and increased as the concentration of Zn, Ca, a nd Mg increased. Zn promoted the activity with a low concentration, but decreased it with a higher concentration, suggesting that the first Zn binding site stimulates activity but the second one inhibits it. The optimum pH for ALP activity varied according to the ion present. We observed that Zn, Ca, and Mg binding sites are present in all ALP. 2) The concentration dependency of Mg, Ca, and Zn on the bone ALP activity was examined using pNPP and Na-PPi as substrates. Its activity was promoted by Mg, Ca, and Zn in a concentration dependent manner, but the concentration dependency curve was different between pNPP and Na-PPi, suggesting that the affinity for Mg, Ca, and Zn depends on the substrate. 3) The activity increased remarkably when divalent cations were present in combination with Mg, Ca, and Z n, suggesting that each ion promoted ALP activity independently. 4) The ALP activity in the presence of each ion was inhibited in a concentration-dependent manner by specific ALP inhibitors such as levamisole and tetramisole. Moreover, the inhibition did not depend on the substrate used. 5) Substrate concentration-dependency of ALP activity was measured in the presence of Mg, Ca, and Zn. The K0.5 values were 0.73 mM, 0.57 mM, and 0.53 mM for pNPP with Mg, Ca, and Zn, respectively. The K 0.5 values were 1.8 mM, 0.35 mM, and 0.15 mM for Na-PPi with Mg, Ca, and Zn, respectively. The affinity for the substrate of ALP varied according to the type of substrate but not to the type of ion. Conclusion: Mg, Ca, and Zn possibly promote ALP activity independently. Our results suggest that the affinity for the substrate and the optimum pH could change depending on the ions present. It is possible that the function of ALP changes when divalent cations and a certain substrate is present.

【目的】最近のi P S 細胞や腫瘍細胞に関する研究はALPが細胞の分化に関与することを示唆する．そこで，腫瘍細胞と非腫瘍細胞のALPを比較することを目的にALP阻害剤に対する反応性の違いを検討した．【材料及び方法】市販のヒトの骨，小腸，胎盤及び肝臓由来のALP，及びヒト腫瘍細胞であるSaOs，Hela，P19とP19をレチノイン酸（RA）で処理して神経系細胞に分化させたRA-P19のALPを使用しALP活性を測定した．また，ALP阻害剤としてtetramisole，levamisole，L-homoarginine，etidronate及びvanadateに対する反応性を調べた．【結果と考察】ヒト臓器由来のALP活性の各阻害剤に対する反応性はアイソザイムの型によって異なったが，ヒト腫瘍細胞由来のALP活性の反応性には大きな相違はなく，未分化な腫瘍細胞のALPは，臓器の由来にかかわらず類似した性質を示す可能性が示唆された．P19とRA-P19のALPはetidronate以外の阻害剤には類似した反応性を示したが，etidronatに対しては異なった．EDTAでも同様の結果が得られたため，etidronateはキレート作用によりALP活性を阻害すると結論した． ALP活性に必要なZnの両ALP活性に対する作用を調べたところ，低濃度ではALP活性を促進したが，高濃度では逆に阻害し，P19には親和性の異なる2 種のZn結合部位が存在することが示唆された．さらにRA処理によるZnの低親和性部位の変化が，P19とRA-P19のALP活性のetidronateに対する反応性の相違を引き起こすことを示唆する結果を得た．【結論】腫瘍細胞のALPは臓器特異的な阻害剤に対する反応性を喪失する可能性と活性発現に必須のZn結合部位の変化も阻害剤に対する反応性に影響を及ぼす可能性を示した．, [Objectives] In a recent study on iPS and tumor cells, it was suggested that different types of cells have different levels of alkaline phosphatase (ALP) activity. Therefore, we examined differences in sensitivity to ALP inhibitors in both non-tumor cells and tumor cells.[Materials and methods] We measured ALP activity using commercial ALP derived from the small intestine, humanbone, placenta, and liver. ALP activity in human tumor cells: SaOs, Hela, P19, and RA-P19, which was differentiated into neurologic cells with retinoic acid (RA) treatment. Additionally, we examined the sensitivity of ALP activity to inhibitors using tetramisole, levamisole, L-homoarginine, etidronate, and vanadate. [Results and Discussion] The sensitivity to each inhibitor of ALP activity varied depending on the isozyme type and its human organ origin. The sensitivity of ALP activity derived from human tumor cells did not show large differences, suggesting that ALP activity of undifferentiated tumor cells has similar characteristics regardless of the organ origin. ALP activity in P19 and RA-P19 showed similar sensitivity to inhibitors, except for etidronate, which produced different sensitivity. Similar results were obtained with EDTA, thus etidronate inhibited ALP activity by chelation effects was concluded. We examined the effects of Zn, which is necessary for ALP activity, on ALP activity in P19 and RA-P19. Zn promoted ALP activity at a low concentration, but it inhibited ALP activity at a high concentration. It has been proposed that two types of Zn binding sites with either low- or high-affinity are present in P19 cells. Furthermore, our results suggested that changes in the low-affinity site of Zn with RA treatment caused differences in sensitivity of ALP activity to etidronate in P19 and RA-P19.[Conclusion] The changes in the Zn binding site, which are essential for inhibitory activity, changed sensitivity to the inhibitor along with ALP activity in tumor cells that lost organ-specific sensitivity to inhibitors.

骨芽細胞にはCaで活性化されるATPase（Ca－ATPase）が存在し石灰化との関連が推測されるが報告は少ない．そこで，骨芽細胞様細胞（MC3T3－E1）のCa-ATPaseの性質を調べた．E1細胞を培養後，細胞破砕物を作成したのち，ミクロソーム分画とした．さらにドデシル硫酸ナトリウム（SDS）を添加して可溶化した後，グリセロールの密度勾配遠心にかけてATPase活性のある分画を得た．細胞破砕物にはCa，Mg－ATPase以外に，pH 9.6（ Ca－P1）とpH10.8（Ca－P2）で活性が高いCa－ATPase及びpH 9.6（Mg－P1）が主でpH11.1（Mg－P2）にわずかな活性を示すMg－ATPase活性が検出された．Mg－P1の最大活性はCa-P1及びCa-P2の約4.5倍であった．これらの活性はSERCAの特異的阻害剤とされるthapsigarginによって阻害されたが，P型ATPaseを阻害するvanadateによる阻害は認められなかった．また2価金属のキレーターであるEDTA及びEGTAは濃度依存性に両ATPase活性を阻害した．ミクロソーム分画ではCa－P1が顕著に減少し，Ca－P2の割合が増加した．グリセロール密度勾配遠心後には，Mg－P2とその約3倍のCa－P2活性が検出された．両活性はthapsigarginにより阻害された．両活性の石灰化への関与を調べるために，25及び50μMのthapsigargin存在下でのE1細胞の培養を行ったが，thapsigarginのアポトーシス誘導作用のため石灰化の検出まで細胞を培養することはできなかった．本研究ではthapsigarginにより阻害されP型ではないアルカリ性至適pH のCa-ATPaseがE1細胞に存在することを示し，その性質の検討が可能な程度まで分離を行うことができた., Adenosinetriphosphatase (ATPase) activated by calcium ions (Ca-ATPase) exists and plays a role in bone mineralization; however, not enough research has been conducted on it. Therefore, this report studied the properties of Ca-ATPase in osteoblastic cells (MC3T3E-1). Osteoblastic cells were cultured and harvested at 4 weeks after confluence. The cells were homogenized through ultrasonication and separated into the microsome and cytoplasm by centrifugation. Further, they were solubilized using sodium dodecyl sulfate and separated into fractions, including Ca-ATPase activity on the density gradient of glycerol. In addition, the cell homogenate detected high Ca-ATPase activity at a pH of 9.6 (Ca-P1) and 10.8 (Ca-P2), and Mg-ATPase activity at a pH of 9.6 (Mg-P1). Low Mg-ATPase activity at a pH of 11.1 (Mg-P2) was also detected. The maximum activity of Mg-P1 was 4.5 times higher than that of Ca-P1 and Ca-P2. These activities of ATPase were inhibited by thapsigargin, the specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, but not by vanadate, an inhibitor of P-type ATPase. Ethylene glycol-bis (β-aminoethyl ether) -N,N,N′,N′-tetraacetic acid and ethylenediaminetetraacetic acid, chelators of divalent metallic ions, inhibited both ATPases concentration-dependently. In the microsome fractions, the activity of Ca-P1 decreased remarkably. The activities of Ca-P2, Mg-P1, and Mg-P2 remained the same, but the ratio of Ca-P2 increased. Mg-ATPase was inhibited by azide, an inhibitor of mitochondrial ATPase, but Ca-ATPase was not inhibited. After centrifugation of the density gradient of glycerol, the activity of Mg-P1 decreased remarkably, but the activities of Mg-P2 and Ca-P2 were maintained. The activity of Ca-P2 was found to be thrice higher than that of Mg-P2. Both ATPases were inhibited by thapsigargin. To investigate the role of both ATPases in mineralization, MC3T3-E1 cells were cultured with 25 and 50 μM thapsigargin. Three days after culture, the surface layers of cells separated, and the number of cells decreased. As thapsigargin strongly induced cell apoptosis, we could not culture the cell until mineralization was detected. These results suggested that the E-1 cell has Ca-ATPase with optimal pH at the alkaline side which is inhibited by thapsigargin but not by P-type ATPase inhibitor. In addition, we could separate Ca-ATPase to the extent required for use in its characterization

ビスホスホネート（BPs）はアルカリ性ホスファターゼ（ALP）活性の発現に必要な遊離Mg2+（ Mg）をキレ ートしてALP活性を阻害するとされているが，不明な点も多い．また，最近，ALP活性はMg, Ca2+（ Ca）及びZn2+ （Zn）により独立して活性化され（Mg, Ca及びZn-ALPとする），活性化には基質の種類も影響することが報告され たが，それらに対するBPsの作用は明らかではない．そこでヒト骨型ALPのMg, Ca及びZn-ALP活性のエチドロネ ート（etidronate）による阻害機構を，基質としてパラニトロフェニルリン酸（pNPP）またはピロリン酸（PPi）を 使用して検討した．種々濃度のMg，Ca，Zn存在下でpNPPを基質としてALP活性を測定すると， etidronateによる 50 %活性阻害濃度（Ki 0.5）はMg濃度が高いほど増加したが，Ca及びZnでは顕著な変化は認められず，etidronateは Mgとは拮抗するが， Ca及びZnと拮抗しないことが示唆された．一方， PPiが基質の場合は， Mg，Ca，Znとの拮抗 が示唆された．Mg， Ca及びZn-ALP活性のpNPP濃度依存性に対するetidronateの影響を調べると， etidronateの濃 度に依存して各活性は抑制されたが， pNPPの50 %活性化濃度（K 0.5）に対する顕著な影響はなく， etidronateは pNPPとは拮抗しないと示唆された．一方，基質がPPiの場合，各活性はetidronate濃度に依存して抑制され，Ca及 びZn-ALP活性ではetidronateはPPiとは非拮抗的であった．しかし，Mg-ALP活性ではetidronate濃度に依存した PPiに対するK 0.5の減少が観察された．以上の結果は，etidronateによる骨型ALPの阻害様式は基質及び活性化する 2 価金属の種類によって異なることを示唆した．, Bisphosphonates (BPs) inhibited alk aline phosphatase (ALP) activity by chelating Mg2+ (Mg) that i s necessary for ALP activation; however, many points are still unclear. Recent investigations have demonstrated that ALP activity is independently activated by Mg, Ca 2+ (Ca), and Zn 2+ (Zn) (Mg-, Ca-, and Zn-ALP) and affected by various substrates. The effects of etidronate on human bone-type ALP ac tivity activated by Mg, Ca, and Zn (Mg-, Ca-, and Zn-ALP) with p-nitrophenylphosphate (pNPP) or pyrophosphate (Na-PPi) as substrates were tested. The 5 0% activity inhibition concentration (Ki0.5) of Mg-ALP activity inhibition by etidronate increased, wherea s Ki0.5 of Ca- or Zn-ALP activity inhibition by etidronate remained almost unchanged. Etidronate competitively inhibited Mg-ALP activity with Mg but noncompetitively inhibited Ca- and Zn-ALP activities wit h Ca and Zn, with pNPP as a substrate, whereas etidronate competitively inhibited Mg-, Ca-, and Zn-ALP activities with Mg, Ca, and Zn with Na-PPi as a substrate. Furthermore, we examined the effects of etidronate on substrate concentration dependency of ALP activities. Based on etidronate concentration, the ALP activity was inhibited. The 5 0% activation concentration (K 0.5) of pNPP remained unaffected. Etidronate noncompetitively inhibited Mg-, Ca-, and Zn-ALP activities with pNPP based on its concentration. Furthermore, the substrates are hydrolyzed to influence Mg, Ca, and Zn in proximity to the substrate-binding site of ALP. Etidronate also noncompetitively inhibited Ca- and Zn-ALP activities with PPi based on its concentration; however, we observed that K0.5 of Mg-ALP activity decreased based on etidronate concentration as PPi. These results demonstrate that the mechanism of human bone-type ALP activity restraint by etidronate changes by the kind of 2 values metal ions and the substrate binding to the ALP.

骨改造において機械的刺激が重要な役割を果たしていることが明らかとなっているが，破骨細胞の分化誘導系に対し機械的刺激を直接作用させた報告は我々の研究のみである．これまで我々は周期的および持続的な機械的刺激による破骨細胞分化の抑制に関して報告してきた．本研究では，可撤式矯正装置の間歇的な使用による組織変化を想定して，間歇的な機械的刺激を加えた場合の破骨細胞分化への影響について検討した．RAW 264.7 細胞を通法に従い３日間培養した．その後，Flexcell tension system を用い，培養４日目から48時間，伸展率10 %で，合計刺激時間を24時間とし，1，2，3，4，6，12時間毎にそれぞれ持続的機械的刺激と無刺激とを繰り返し，間歇的な機械的刺激とした．48時間無刺激としたものを対照群とした．TRAP 染色にて核数ごとに破骨細胞数を測定し，破骨細胞関連遺伝子の mRNA 発現量をリアルタイム PCR 法にて定量した．全ての実験群では破骨細胞数が有意に抑制された．また，１時間毎の刺激付与群（１時間群）と12時間毎の刺激付与群（12時間群）を比較すると，1時間群において総破骨細胞数は有意に抑制された．特に2，3，4核の破骨細胞数が有意に抑制された．両群のmRNA 発現量の比較では，DC-STAMP および OC-STAMP，CD47 の発現は１時間群において有意に抑制された．12時間群に比べ１時間群では，単核の前破骨細胞同士，および単核の前破骨細胞と２～４核の小数核の破骨細胞との融合に関与している CD47 の発現抑制により，２～４核の破骨細胞数が抑制され，さらに DC-STAMP および OC-STAMP の発現抑制により総破骨細胞数が抑制されることが示唆された．以上から，間歇的な機械的刺激を付与する回数，時間によって細胞融合因子が抑制され，破骨細胞分化を抑制することが示唆された．, Mechanical stress plays an important role in the remodeling of bone. In previous reports by this research team, we showed the suppression of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effects of intermittent continuous mechanical stress on osteoclastogenesis. We cultured RAW264.7 cells stimulated with the receptor activator of nuclear factor-κB ligand (RANKL) using a BioFlex plate. Osteoclasts were stimulated using a Flexcell tension system at elongation rates of 10 % for 2 days from the fourth day of culture. In different treatments, mechanical stress was applied for 24 h, followed by bouts of continuous mechanical stress and no stress alternating at 1, 2, 3, 4, 6, or 12 h. In addition, we assayed osteoclast-related genetic mRNA expression using the real-time PCR method. We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts with 2, 3, 4, 5, 6, or 7 nuclei, and those with more nuclei. The number of osteoclasts was lower after mechanical stress when compared with no mechanical stress. The number of total osteoclasts was significantly suppressed in the group stimulated every hour (1-h group) when compared with the group that was stimulated every 12 h (12-h group). Furthermore, we found that the number of osteoclasts with 2-4 nuclei was significantly reduced by intermittent continuous mechanical stress. The measurement of mRNA expression using real-time PCR showed the suppression of DC-STAMP, OC-STAMP, and CD47, which are fusion factors for osteoclasts. These significantly decreased in the 1-h group compared with that in 12-h group. CD47 participates in cell fusion with the mononuclear preosteoclasts among each other, and among the mononuclear preosteoclasts and those with 2-5 nuclei. Thus, the suppression of CD47 expression could reduce the number of osteoclasts with 2-4 nuclei. Furthermore, the total number of osteoclasts was reduced by the suppression of DC-STAMP and OC-STAMP expression. These results suggest that intermittent continuous mechanical stress is one factor that suppresses the fusion of osteoclasts, and that the cell fusion factor is controlled by the duration and frequency of intermittent stimulation by sustained extension.

第35回宇宙環境利用シンポジウム(2021年1月19日-20日. オンライン開催), Space Utilization Research (January 19-20, 2021. Online Meeting), 著者人数: 17名, 資料番号: SA6000156028, レポート番号: S-04

第35回宇宙環境利用シンポジウム(2021年1月19日-20日. オンライン開催), Space Utilization Research (January 19-20, 2021. Online Meeting), 資料番号: SA6000156029, レポート番号: S-05

第21回宇宙科学シンポジウム (2021年1月6日-7日. オンライン開催), 21st Space Science Symposium (January 6-7, 2021. Online Meeting), 著者人数: 17名, 資料番号: SA6000163180, レポート番号: Pb.06

著者人数: 13名, Accepted: 2018-08-02, 資料番号: SA1180331000

【目的】口腔がん細胞のシスプラチン耐性化に伴うネダプラチンおよびカルボプラチンに対する感受性の変化を検討することを目的に本研究を行った． 【材料と方法】口腔がん細胞である KB 細胞（KB）及びそのシスプラチン耐性株（KB-R）をネダプラチンおよびカルボプラチン存在下で培養し，細胞生存率の指標として細胞内 ATP 量を測定した． 【結果と考察】ネダプラチンおよびカルボプラチン存在下で 72 時間培養した KB および KB-R は濃度に依存して細胞生存率が低下した．KB および KB-R の細胞生存率が 50％低下する濃度は，ネダプラチン存在下では 11 μMおよび 69 μM，カルボプラチン存在下では 86 μM および 334 μM となり，シスプラチン耐性株はネダプラチンおよびカルボプラチンに対しても耐性化していることが示された．また，細胞生存率の低下において，カルボプラチンはネダプラチンと比較してより高濃度を必要とした．KB は 62.5 μM のネダプラチンおよび 125 μM のカルボプラチン存在下で細胞生存率が最低となり，より高濃度のネダプラチンおよびカルボプラチン存在下では細胞生存率が増加した．ネダプラチンおよびカルボプラチンには，KB の細胞死を引き起こす濃度域と，細胞死を抑制する濃度域が存在することが示唆された．KB-R は，低濃度のネダプラチンおよびカルボプラチンに対して KB よりも耐性を示したが，より高濃度のネダプラチンおよびカルボプラチン存在下では，KB で観察される細胞生存率の増加を示さなかった．以上の結果は，KB と KB-R はネダプラチンおよびカルボプラチンに対する反応性が異なること，及び，シスプラチン耐性株はネダプラチンおよびカルボプラチンに対しても耐性を示すことを示唆する． 【結論】口腔がん細胞株である KB がシスプラチンに対して耐性化すると，ネダプラチンおよびカルボプラチンに対する交叉耐性を示す．また，耐性化に伴い，広い濃度範囲でネダプラチンおよびカルボプラチンに対する感受性が変化する．, Purpose : We examined the change of sensitivity for nedaplatin and carboplatin of oral carcinoma cell line, KB, and its cisplatin-resistant cell line, KB-R. Materials and methods : We used oral carcinoma cells (parent cell line: KB) and cisplatin-resistant line of KB (resistant cell line: KB-R). The cells were disseminated at a 96-well plate and incubated with 0, 7.8, 15.6, 31.2, 62.5, 125, 250 and 500μM nedaplatin or carboplatin for 12, 24 or 48 and 72 hours. Then the intracellular ATP content was measured by use of ViaLight? plus kit (LONZA) for the measurement of the survival rate of the cells. Results and discussion : The survival rate of KB and KB-R decreased depending on the concentration of nedaplatin or carboplatin after 72 hours incubation. The 50% inhibitory concentrations of survival rate for KB and KB-R were 11 and 69 μM for nedaplatin and 86 and 334 μM for carboplatin. This suggests that the cisplatin resistant cell line showed resistance to nedaplatin or carboplatin. A higher concentration was required for carboplatin to decrease the survival rate compared to nedaplatin. The survival rate of KB was the lowest at 62.5 μM nedaplatin and 125 μM carboplatin, but the survival rate increased at higher concentrations of nedaplatin or carboplatin. This suggests there are two concentration ranges for nedaplatin or carboplatin, and one is for cell death and the other is for recovery from cell death. KB-R showed resistance to lower concentration of nedaplatin or carboplatin compared to KB, but KB-R did not show an increase of survival rate, which KB showed, at higher concentration of nedaplatin or carboplatin. These results suggest that cisplatin resistant KB-R show resistance to nedaplatin or carboplatin and that KB and KB-R possess different sensitivity to nedaplatin or carboplatin. Conclusion : Cisplatin resistant KB-R showed cross resistance to nedaplatin and carboplatin. Also, the effect of nedaplatin and carboplatin on KB and KB-R changed due to becoming cisplatin resistant.

ビスホスホネート（BP）によるALP活性抑制の報告があるが，その機構は不明な点が多い．そこで，ヒト組織非特異型ALP（骨型，肝臓型），及び組織特異型ALP（胎盤型）とマウス由来骨芽細胞様細胞であるMC3T3-E1細胞のALPに対する，窒素非含有型のclodronate，窒素含有型のrisedronate及びalendronateによるALP活性の阻害機構を検討した．Mg，Zn及びCa存在下の活性を，それぞれ，Mg-ALP，Zn-ALP及びCa-ALPとし，基質としてパラニトロフェニルリン酸（p NPP）あるいは無機ピロリン酸（PPi）を使用した．p NPPを基質とした骨型及び肝臓型の各ALP活性はclodronate，risedronate及びalendronateの濃度に依存して抑制された．Mg-ALPでは，各BPとMgとの拮抗が見られたが，Zn-及びCa-ALP活性におけるBPとZn及びCaとの拮抗は，Mg-ALPほど顕著ではなかった．また，clodronateと比較してrisedronate及びalendronateのほうが強いMg-ALP抑制作用を示し，阻害作用は窒素非含有型と比較して窒素含有型BPのほうが強いことが示唆された． PPiを基質とすると，clodronateはMC3T3-E1及びヒト胎盤型ALPのCa-ALP活性を，Caと拮抗して濃度依存性に抑制した．P-C-P結合を持つBPがP-O-P結合を持つ基質と拮抗する可能性を考えて，骨型及び肝臓型ALPを使用して，Mg-，Zn-及びCa-ALP活性のp NPP濃度依存性に対するclodronate，risedronate及びalendronateの影響を検討した．BPは濃度に依存して最大活性を抑制したがp NPPによる50％活性化濃度には顕著な影響を与えず，BPはALPの基質であるp NPPとは拮抗しないことを示唆した．以上の結果は，BPはALPの種類，基質，活性化する２価金属の種類に関係なく，２価金属イオンとの拮抗によりALP活性を阻害し，その作用は窒素含有型が非含有型と比較して強いことを示唆した．, Recent investigations have demonstrated that bisphosphonate (BP) inhibits ALP activities, but there are still many points that remain uncovered. We tested the inhibitions of human bone, placental, and liver type ALP activity in the presence of Mg, Zn and Ca (as Mg, Zn, and Ca-ALP respectively), with paranitrophenyl phosphate (p NPP) or inorganic pyrophosphate (PPi) as substrates, using various BPs: non- nitrogen-containing BP clodronate and nitrogen-containing BP risedronate and alendronate for inhibitors. Using p NPP as a substrate, the relationship between the effects of clodronate, risedronate or alendronate on bone or liver Mg, Zn and Ca-ALP activities, and the concentrations of coexisting Mg, Zn and Ca were examined. Mg, Zn, and Ca-ALP activities of both isozymes decreased to the concentration dependency of each BP ; this was required as higher concentrations of BPs as the coexisting Mg concentration was higher for the suppression of Mg-ALP activity by BPs. In addition, risedronate and alendronate showed stronger Mg-ALP inhibitory action compared with clodronate. These results suggest that BP inhibits human bone and liver Mg-ALP activity competitively with Mg. Also, the strength of the inhibition by nitrogen-containing BPs are stronger than those of non-nitrogen-containing BPs. For Zn and Ca-ALP activities, BPs also showed a tendency of competition with Zn or Ca, but not as significant as Mg. Clodronate suppressed the Ca-ALP activities of MC3T3-E1 and human placental ALP using PPi as substrates, to the concentration dependency competitively with Ca. Considering the possibility that BP with a P-C-P structure might compete with the substrates containing a P-O-P structure, we measured the effects of clodronate, risedronate, and alendronate on p NPP concentration dependency of human bone and liver Mg, Zn and Ca-ALP activities. BPs inhibited the maximum activity depending on the concentration, but did not significantly affect the 50 % activtity concentration by p NPP. This suggests that BPs do not compete with the substrate of ALP, p NPP. These results suggest that BP inhibits ALP activity by competition with bivalent metal, regardless of the type of ALP, substrate, and the type of bivalent metal for the activation. In addition, its action of nitrogen-containing BP is stronger than non-nitrogen-containing BP.

形態学的な研究から，硬組織形成部位にアルカリ性至適pHのCa-ATPaseの存在が示唆されているが酵素学的な性質の報告は少ない．そこで骨芽細胞様細胞であるMC3T3-E1細胞が保持するCa-ATPase活性に関して研究を行った．細胞を石灰化時期まで培養後回収し，超音波破砕後に遠心分離操作を行って膜分画を得た．ATP加水分解により生じた無機リン酸をChifflet法で定量してATPase活性を測定し，以下の結果を得た．１．膜分画にはカルシウム（Ca）あるいはマグネシウム（Mg）により活性化されるATPaseが存在し両酵素ともthapsigarginによって阻害された．Ca存在下のATPase活性はMgによって拮抗されることと，Mg-ATPaseを阻害するazideによって阻害されないことから両酵素は別の酵素と示唆された．２．Ca-ATPase活性はCa濃度依存性に増加して1 mMの遊離Ca濃度で飽和し，50 ％活性化濃度は0.3 mMであった．３．活性はpH依存性に増加し，pH 9.1でpH 7.5のほぼ３倍の活性を示して最大となりpH 10.0までは同程度の活性を示した．４．活性はATP加水分解の過程においてリン酸化酵素を形成するP型ATPaseの阻害薬であるvanadateとエタクリン酸によっては阻害されなかった．５．活性は２価金属イオンのキレーターであるEGTAおよびEDTAにより濃度依存性に阻害されたが，ビスホスホネートによっては阻害されなかった．６．遊離Ca濃度100 nMでは， Ca-ATPase活性はほぼ検出されないが，カルモジュリンを添加すると濃度に依存して活性は増大し，50 ％活性化濃度は約6 μMであった．７．カルモジュリン非添加における活性は，カルモジュリン拮抗薬であるW7によって濃度依存性に抑制され，50 ％阻害濃度は0.3 mMであった．以上の結果は，E1細胞にはアルカリ性至適pHのP型ではないカルモジュリン依存性Ca-ATPaseが存在することを示唆する．本酵素は，形態学的に存在が示唆されるCa-ATPaseと類似しており，硬組織形成に関与する可能性がある．, Morphological studies suggest the presence of Ca-ATPase at alkaline pH optimum at the site of hard tissue formation, but there are few reports on enzymological properties. Therefore, we investigated Ca-ATPase activity using clonal osteoblastic cells (MC3T3-E1). The cells were cultured until calcification. The cells were then ultrasonically crushed. Further, they were centrifuged to obtain a membrane fraction. The ATPase activity was measured by measuring inorganic phosphate produced by ATP hydrolysis by the Chifflet method. The following results were obtained : 1. Ca-ATPase and Mg-ATPase exist in membrane fractionation. They are inhibited by thapsigargin. Ca-ATPase activity is inhibited by Mg. Mg-ATPase is inhibited by azide, but Ca -ATPase was not inhibited. Both are different enzymes. 2. Ca-ATPase activity increased with Ca concentration dependence, saturated at 1 mM free Ca concentration, and 50% activation was 0.3 mM. 3. Ca-ATPase activity increased in pH dependence and showed maximum activity at pH 9.1. It is nearly three times that of pH 7.5. It is the same value up to pH 10.0. 4. Ca-ATPase activity is not inhibited by vanadate, an inhibitor of P-type ATPase. Its activity is not inhibited by ethacrynic acid, an inhibitor of Mg-ATPase. 5. Ca-ATPase activity is inhibited in a concentration-dependent manner by EGTA and EDTA of bivalent metal chelators. Its activity is not inhibited by bisphosphonates. 6. At free Ca concentration of 100 nM, Ca-ATPase activity in the absence of calmodulin was not detected. Ca-ATPase activity increases with the addition of calmodulin depending on the concentration, and the 50 % activation concentration is about 6 μM. 7. Ca-ATPase activity is inhibited in a concentration-dependent manner by W7 which is a calmodulin antagonist when calmodulin is not added. The 50 % inhibitory concentration is 0.3 mM. The above results suggest that Ca-ATPase in MC3T3-E1 cells is calmodulin-dependent, which is optimal at alkaline pH and not P-type. This enzyme may be identical to Ca-ATPase which is morphologically suggested to be present, and it may be involved in hard tissue formation.

漢方薬のセンソは約100種類のbufadienolidesを含む．Bufadienolidesは心不全治療薬であるジギタリスなどと同様の強心ステロイドであり，Na, K-ATPase活性の阻害作用を示すが，報告は少ない．本研究は，bufadienolidesのNa, K-ATPaseに対する作用とその機構を明らかにすることを目的に行った．Bufadienolidesとしてbufalin，cinobufagin，cinobufotalin，ジギタリス類としてouabainを使用し，ウサギ及びラット脳から精製したNa,K-ATPase活性に対する作用を検討した．Bufadienolidesとouabainは，ウサギ及びラット脳Na,K-ATPase活性をほぼ完全に阻害した．両Na, K-ATPase活性に対する50 ％阻害濃度（IC50）は， ouabainでは290及び260 nM，bufalinでは40及び20 nM，cinobufaginでは230及び90 nM，cinobufotalinでは300及び150 nMであった．ウサギ脳Na, K-ATPaseに対するcinobufotalinを除いて，bufadienolidesはNa, K-ATPaseの特異的阻害薬とされるouabainよりも強い抑制作用を示し，特に，bufalinは強い作用を示した．また，ラット脳Na, K-ATPaseはウサギよりも強心ステロイドに対する感受性が高いことが示唆された．次に，ウサギ及びラット脳Na, K-ATPase活性のNa+，K+，Mg2+，ATP濃度依存性に対するouabain，bufalin，cinobufagin，cinobufotalinの作用を検討した．Ouabainとbufadienolidesはラット及びウサギ脳Na, K-ATPase活性の，Na+，Mg2+，ATPに対する親和性を増大し，K+に対する親和性を低下させた．これらの結果は，ouabainと本研究において使用したbufalin，cinobufagin，cinobufotalinは，同様の機構で，Na, K-ATPaseを抑制することを示唆する．すなわち，センソの強心作用及び利尿作用は，ジギタリス類と同様にNa, K-ATPaseの抑制作用に基づくことを示唆する．, The use of toad venom in Chinese medicine includes approximately 100 kinds of bufadienolides. Bufadienolides are cardiotonic steroids similar to the digitalis which is a heart failure therapeutic drug, and show inhibition of the Na, K-ATPase activity, but there are few reports. This study was carried out for the purpose of determining the effects of bufadienolides on Na, K-ATPase and its mechanism. We examined the effects of bufalin, cinobufagin, cinobufotalin, and ouabain, one of digitalis-like substances, on Na, K-ATPase activity purified from rabbit and rat brains. Bufadienolides and ouabain approximately demonstrated complete inhibition of rabbit and rat brain-derived Na, K-ATPase activity. The 50 percent inhibitory concentrations for both Na, K-ATPase activites were 290 and 260 nM for ouabain, 40 and 20 nM for bufalin, 230 and 90 nM for cinobufagin and 300 and 150 nM for cinobufotalin. Excluding cinobufotalin for rabbit brain Na, K-ATPase, bufadienolides showed stronger inhibitory effect than ouabain, specific inhibitor of Na, K-ATPase, and bufalin showed particularly strong effects. It was suggested that rat brain Na, K-ATPase was more susceptible to the cardiotonic steroid than rabbit brain. We then examined the effects of ouabain, bufalin, cinobufagin, cinobufotalin on Na+, K+, Mg2+ and ATP concentration-dependency of rabbit and rat brain Na, K-ATPase activity. Ouabain and bufadienolides increased the affinity for Na+, Mg2+ and ATP of Na, K-ATPase activity, and reduced affinity for K+. These results suggest that bufalin, cinobufagin, cinobufotalin inhibit Na, K-ATPase by a similar mechanism of ouabain. In other words, cardiotonic action and diuretic effects of toad venom are based on the inhibitory effect of Na, K-ATPase similar to digitalis.

歯科矯正力により歯牙には牽引側では伸展力が，圧迫側では圧縮力が作用する．我々は破骨細胞に直接圧縮力を加えた際の影響について報告しているが，破骨細胞に圧縮力を加えた後，長期的に培養した場合の動態については未だ報告がない．本研究では，破骨細胞に圧縮力を加え，長期的に培養して分化誘導系に与える影響を検討した．RANKL添加培養液を用いてRAW264.7細胞を７日間培養し，破骨細胞数の推移を観察した．培養６日目までは破骨細胞数は増加したが，それ以降は減少傾向を示した．次に培養３日目に圧縮力を破骨細胞に加え24, 48および72時間培養した．圧縮力を加えず培養したものを対照群，圧縮力を24時間加えた後それを解放して培養したものを解放群，圧縮力を加えたまま培養したものを圧縮群とした．培養３日目から24時間圧縮力を加えると，破骨細胞数は増加した．培養５日目の時点では解放群，圧縮群では対照群と比較して増加した．しかし，解放群，圧縮群の間に有意差が認められなかったことから，24時間以上圧縮力を加えても破骨細胞の分化・融合を促進しない可能性が示唆された．培養６日目の時点では，解放群，圧縮群では対照群と比較して破骨細胞数が減少した．また培養５日目から６日目の間において対照群では破骨細胞数が増加したが，他の群では減少した．これらの結果より，圧縮力は培養後期における破骨細胞の分化・融合を抑制する可能性が示唆された．対照群と圧縮群の破骨細胞関連遺伝子であるNFATc1, RANK, TRAP, DC-STAMPおよびOC-STAMPのmRNA発現量を比較すると，培養４日目の時点では有意差が認められなかったが，培養５日目の時点では圧縮群ではそれらすべての発現が有意に抑制されていた．以上より，圧縮力は培養後期において破骨細胞関連遺伝子の発現を抑制することで破骨細胞の分化・融合を抑制することが示唆された．, Orthodontic force consists of tensile and compressive force. On the pressure side, osteoclasts are subjected to compressive force. In contrast, on the tension side, osteoclasts are subjected to tensile force. Here we present our reports about the direct effects of compressive force on osteoclast. We found no other reports that demonstrated the change of the osteoclasts when they are cultured long term after compression. Therefore, we investigated the effects of compressive force on osteoclastogenesis when we cultured osteoclasts long term after application of a compressive force in this study. We cultured RAW264.7 (RAW) cells with a culture medium added receptor activator of nuclear factor-κB ligand (RANKL) for 7 days and observed the change in the number of osteoclasts. Osteoclasts increased up to the 6th day, but decreased after that. To investigate the effects of compressive force, we added weight to osteoclasts at day 3, and cultured them for 24, 48 and 72 h. The control group was cultured without weight. The compressed off (CF→ off) group was cultured for 24 h with weight, and then cultured without it. The compressed (CF) group was cultured with weight. The number of osteoclasts were counted and compared. The number of osteoclasts increased when we applied compressive force for 24 h from the 3rd day. At day 5 , CF→ off group and CF group increased more than the control group. No significant difference was recognized among the former two groups. This suggested the possibility that over 24 h compression did not promote osteoclast differentiation and fusion. On the 6th day, the control group increased more than the other groups. In addition, the control group increased during the culture of the 5th day to the 6th day, but the other groups decreased. These results suggested the possibility that the compressive force suppressed differentiation and fusion of osteoclasts in late stages. Comparing the control group with the CF group about mRNA expression of osteoclastassociated genes, including nuclear factor of activated T cells c1 (NFATc1), TRAP, receptor activator of NF-κB (RANK), dendritic cell specific trans membrane protein (DC-STAMP), and osteoclast stimulatory trans membrane protein (OCSTAMP) on the 4th day, no significant differences were observed. But on the 5th day, the CF group was significantly suppressed. This showed the compressive force suppressed expression of osteoclast-associated genes in the late stages. These results suggested that compressive force suppressed osteoclast differentiation and fusion by suppressing expression of osteoclast-associated genes in late stages of the experiment.