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スベロイルビス-ヒドロキサム酸によるカポジ肉腫関連ヘルペスウイルスの再活性化およびリンパ腫細胞へのアポトーシス誘導機構の解明

飯田 俊 東北大学

2022.03.25

概要

要約
カポジ肉腫関連ヘルペスウイルス (KSHV) は,カポジ肉腫や原発性体腔液リンパ腫 (PEL) といった悪性腫瘍の他,多中心性キャッスルマン病 (MCD) や KSHV inflammatory cytokine syndrome (KICS) などのヒト疾患の原因病原体である.KSHV では他のヘルペスウイルスと同様に潜伏感染および溶解感染という二つの感染様式が見られ,前者はカポジ肉腫や PEL と,後者は MCD や KICS の病態形成と密接に関与する.PEL は免疫不全を背景に発生する高悪性度の B 細胞リンパ腫である.予後は不良であり,これまで PEL に対する特異的な治療法は確立されていないことから,新規治療法の開発が望まれている.

本研究では,ヒストン脱アセチル化酵素 (HDAC) 阻害薬であるスベロイルビス-ヒドロキサム酸 (SBHA) が,KSHV 陽性 PEL 細胞株においてウイルスを潜伏感染から溶解感染へと再活性化させるのみならず,細胞の生存を著しく阻害することに着目し,その作用機序の解明を目的に分子生物学的な検討をおこなった.
まず,KSHV 再活性化について,蛍光タンパク質を発現する組換え KSHV を感染させたBurkitt リンパ腫細胞株を用いて評価したところ,SBHA は用量依存的にKSHVを再活性化することが明らかとなり,その 50% 効果濃度 (EC50) は 2.71-2.95×10-5 mol/L であった.また,SBHA は,KSHV の溶解感染への移行を制御する replication and transcription activator (RTA) の発現を誘導し,PEL 細胞株においても KSHV を再活性化した.
KSHV 遺伝子の発現変化について次世代シークエンサーを用いて網羅的解析したところ,ウイルス感染細胞の全転写産物に占める KSHV 由来の転写産物の割合は,SBHA 刺激前では 1%未満であったのに対し,SBHA 刺激後では 40%以上に達していた.KSHV 再活性化の機序についてクロマチン免疫沈降法により解析したところ,SBHA は RTA 遺伝子のプロモーターにおいてヒストン H3 および H4 のアセチル化を誘導しており,ヒストン修飾の変化を通じて KSHV を再活性化していると考えられた.次に,PEL 細胞株の生存や増殖への影響について検討したところ,SBHA は用量依存的に PEL 細胞株の生存および増殖を阻害することが明らかとなった.細胞増殖に対する 50% 阻害濃度 (IC50) は 2.40-5.20×10-6 mol/L であり,KSHV 再活性化における EC50 より低濃度であった.
また,SBHA は PEL 細胞株に対して用量依存的にアポトーシスを誘導することが annexin V アッセイにより明らかとなり,アポトーシスにより細胞の生存,増殖が阻害されていると考えられた.
アポトーシス誘導の機序について検討したところ,SBHA 刺激後に caspase-3 および caspase-7 の活性化,p53 のアセチル化およびリン酸化,Bim や Bax の発現上昇ならびに Bcl-xL の発現低下が認められたことから,ミトコンドリア経路の活性化を介してアポトーシスが誘導されていると考えられた.

本研究で得られた知見は,SBHA が実験室において KSHV を効率良く再活性化させるためのツールとして有用であるのみならず,PEL に対する新規治療薬の候補として有望であることを示すものである.

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