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Studies on Cytotoxic Effects of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) to Canine Cell Lines Derived from Hemangiosarcoma and Mammary Epithelial Tumor

後藤, みなみ 岐阜大学

2020.03.13

概要

血管肉腫は,人および犬ともに悪性度が高く,予後不良であり,未だに有効な新規治療法は確立されていない。犬の血管肉腫については,その特徴が明らかになりつつあるが,不明な点も多く,新規治療法開発の妨げとなっている。また,犬の乳腺腫瘍は,良性のものから悪性のものまで,様々な病態を呈し,その中でも脈管浸潤を伴う悪性度の高いものは,非常に予後が悪く,有効な治療法も存在せず,新規治療法の開発が望まれている。これらの犬の悪性腫瘍は,獣医学分野における治療の観点の重要とともに,腫瘍発生のリスク因子や関連遺伝子,組織学的特徴などを比較腫瘍学的に解析できる人の腫瘍の自然発生モデルとして期待されている。

医学領域では抗腫瘍サイトカインの 1 つとして注目されている Tumor necrosis factor related apoptosis inducing ligand (TRAIL)は,腫瘍細胞に選択的にアポトーシスを誘導し,全身的な副作用も少ないため,幅広い腫瘍への適用が期待されている。しかし,獣医学領域においては腫瘍細胞に対する TRAIL の効果についての報告は乏しく,伴侶動物における TRAIL の作用機序に関する基盤的知見も乏しいのが現状である。

上記の観点から,本研究では,医学領域でも検証されていない血管肉腫に対する TRAILの効果を,犬血管肉腫細胞株を用いて検証し,さらに獣医領域における腫瘍に対する TRAIL研究の基礎となる情報を蓄積することを目的として,犬乳腺腫瘍由来の細胞株に対する TRAIL のアポトーシス誘導作用を評価した。

第 1 章では,犬血管肉腫細胞株に対する TRAIL の細胞傷害性の評価を目的とし,三量体形成しやすくした isoleucine-zipper TRAIL(izTRAIL)を含む,3 種類のヒト組み換え TRAILの犬血管肉腫細胞株に対する TRAIL の細胞傷害性を比較し,izTRAIL が最も効果的に犬血管肉腫細胞株の細胞生存率を減少させることを見出した。一方,最も効果的な izTRAIL であっても,正常な血管内皮細胞の細胞生存率には影響せず,izTRAIL は腫瘍細胞に対して選択性な細胞傷害効果を示すことを明らかにした。また,犬血管肉腫細胞は,izTRAIL の添加によって Annexin V+/ Propidium Iodide(PI)-を示す初期アポトーシスの細胞と,PI染色により核の断片化を示すSubG-1 相細胞の増加が確認され,izTRAIL の添加後にcaspase(Casp)-8 および Casp-3 が活性化し,Casp-3 の基質である polyclonal anti-Poly (ADP-ribose) polymerase (PARP)の分解も見られた。これらの変化は Casp-8 および Casp-3の阻害剤を加えることで抑制された。以上より,第 1 章では,izTRAIL が犬血管肉腫細胞株に Casp-8 を介したアポトーシスによる細胞傷害作用を示すことが明らかとなった。

第 2 章では,犬の乳腺腫瘍細胞に対する izTRAIL のアポトーシス誘導作用について評価した。まず,乳腺由来腫瘍細胞株の作製のために,良性の乳腺腫瘍 1 例,悪性の乳腺腫瘍2 例について,腫瘍組織から細胞を分離・採取し,60 代以上継代し,3つの細胞株を樹立した。これらの培養細胞は,細胞形態と免疫蛍光染色の結果から,上皮性腫瘍の特性を有していることが明らかとなった。これらの 3 種類の乳腺由来腫瘍細胞株では,izTRAIL の添加によって細胞生存率が低下し,izTRAIL 添加後には,Annexin V+/PI-を示す初期アポトーシス細胞の増加がみられ,PI 染色によって SubG-1 相の細胞の増加も確認された。また,izTRAIL 添加によって Casp-8 および Casp-3 が活性化し,PARP の分解も見られた。これらの変化は Casp-8 および Casp-3 の阻害剤により抑制された。以上より,第 2 章では, izTRAIL は,犬血管肉腫細胞株と同様に,犬乳腺上皮性腫瘍由来株に対しても Casp-8 を介したアポトーシスによる細胞傷害作用を示すことが明らかとなった。

TRAIL は Casp-8 を活性化させ,p53 の関与しない外因性のアポトーシスを誘導するので, p53 の異常による内因性アポトーシス経路が不活性化した腫瘍にも効果を発揮することが 期待されている。実際,本研究に用いた犬血管肉腫細胞株は,p53 の不活性化した細胞株 であり,それらに対して izTRAIL によってアポトーシス誘導が可能であった点から,p53 の異常に関与しない抗腫瘍効果が期待できるものである。本研究で得られた血管肉腫に対 する TRAIL の抗腫瘍効果は,人の血管肉腫においても報告されておらず,人の血管肉腫の 新しい治療法開発の知見の一つともなりうる。さらに,犬乳腺上皮性腫瘍由来細胞株に対 しても同様のアポトーシス誘導作用による抗腫瘍効果が明らかとなり,本研究の結果は, 獣医領域における腫瘍性疾患の治療法としての TRAIL の広い適用性を示唆するものである。

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