< Abstract >

Management strategies of chronic phase chronic myelogenous leukemia (CML) have drastically improved due to the development of selective tyrosine kinase inhibitor, imatinib mesylate (Glivec). However, there is the continuously arising problem of imatinib resistance mainly due to certain point mutations within the ATP-binding pocket of BCR/ABL suggesting for the necessity of alternative drugs.

Epigallocatechin-gallate (EGCG) is the major catechin component and chemopreventive polyphenol that is found in green tea, one of the most consumed beverages in the world. Anti-tumour activity of EGCG has been gaining much attention due to their selective cytotoxic effects towards various tumour cell lines and not to their normal counterparts. Anti-tumour activity of EGCG has been reported to result from inhibition of multiple signaling pathways such as cell cycle arrest, inhibition of telomerase activity, and inhibition of metastasis via binding to laminin receptor, LR67. Although several mechanisms for EGCG-induced anti-tumour activity have been proposed, they generally shared a common final phase of apoptosis. However, our preliminary data on Wright-Giemsa-stained CML cells after EGCG treatment showed atypical cell death morphology where apoptotic morphology was absent. This prompted us to specify the mode of EGCG-induced cell death in CML cell lines based on biochemical and morphological approaches not only to clarify its anti-tumour activity, but also on the hypothesis that if EGCG induced nonapoptotic cell death, it may be clinically beneficial in overcoming both imatinib-sensitive and -resistant CML cells, often with apoptosis-resistant characteristics due to constitutively active BCR/ABL suppressing the apoptotic pathway.

< Methods and Results >

EGCG induces cell death distinct from imatinib-induced apoptosis

In order to verify the effect of EGCG and imatinib on the mitochondrial transmembrane potential (MMP) and plasma membrane, K562 and C2F8 CML cell lines were stained with DiOC(6), mitochondria-specific marker, and PI, after treatment with increasing concentrations of EGCG and imatinib. As a result, both EGCG and imatinib induced the reduction of MMP and increasing of PI-positive cells (Fig 1), however, distinct cell death population pattern resulted between EGCG and imatinib treatments, suggesting for their distinct modes of cell death.

Caspase-independent necrosis-like cell death in EGCG-induced cell death

To ascertain whether EGCG induced a non-apoptotic cell death, activation of caspases, key executioners in apoptotic pathway, were examined in EGCG-induced cell death by Western blotting. Although imatinib-treated K562 and C2F8 cells showed activation of all caspases tested and PARP cleavage at 24 h (Fig 2A), EGCG-treated K562 and C2F8 cells only showed a partial activation of caspases-9 and caspase-8, but

caspase-3 and PARP were not activated with exposure to EGCG at 24 h (Fig 2A). Also, to confirm this result, K562 cells were treated with either EGCG or imatinib alone or after pretreatment with Z-VAD-FMK, broad caspase inhibitor, for 48h. Imatinib-induced apoptosis was prevented by Z-VAD-FMK dose-dependently (p < 0.01) (Fig 2B), however, EGCG-induced cell death was not prevented at all by any concentrations of Z-VAD-FMK (p < 0.01) (Fig 2B), suggesting that EGCG induced predominantly caspase-independent cell death. Also, reduction of anti-apoptosis factors such as inhibitor of apoptosis proteins (IAP) and Bcl-2 could not be observed in EGCG-induced cell death, suggesting that EGCG did not interfere either with the safeguard apoptotic pathway (Data not shown).

Since a clearer delineation of the mode of cell death was based on morphological studies, we next examined the morphological differences in EGCG-treated and imatinib-treated cell deaths in K562 and C2F8 cells using an electron microscope. As a result, imatinib-induced cell death exhibited a typical apoptotic feature with smoothing of cell surface, nuclear fragmentation with compaction of chromatin to the crescents adjacent to the nuclear envelope and rather structurally intact mitochondria (Fig 3A). EGCG-induced cell death resembled significantly the necrotic morphology induced by ATP-depletion without nuclear fragmentation but with significant cytoplasmic vacuolations with severe disruption of cell plasma membrane, mitochondrial membrane, and even detachment of nuclear envelope developing a large vacuole (Fig 3A).

Quantification analysis of these distinct types of cell death by counting toluidine blue stained cells showed that EGCG predominantly induced cell death with necrotic characteristics such as cytoplasmic vacuolated cells (65 ± 5%) with lumpy chromatin with almost no apoptptic bodies (0.6 ± 5%) (Fig 3B).

EGCG effect on imatinib-resistant CML cells

In addition, we evaluated the effectiveness of EGCG towards imatinib-resistant CML cells, K562/sti, and EGCG effectively induced cell death in imatnib-resistant CML cell lines with similar IC50 value as for imatinib-sensitive cell line, whereas this cell line showed strong resistance to imatinib (Fig 4). Moreover, combination treatment of EGCG and imatinib induced enhanced (additive) cell death in K562 cells (Data not shown), which probably reflects the fact that EGCG and imatinib trigger distinct cell death pathways. Meanwhile, EGCG is expected to have minimal side effects to the patients as the anti-cancer drug, because EGCG did not show significant cytotoxicity to healthy peripheral blood mononuclear (PBMC) cells. These results suggest for the possibility of EGCG as a potential anticancer agent that may overcome the imatinib-resistance, and worthy of further pre-clinical experiments using mouse models.

< Summary >

We have demonstrated for the first time that EGCG predominantly induced necrosis-like cell death in CML cell lines via a caspase-independent mechanism, distinct from imatinib-induced typical apoptosis. We have also

shown that activating this alternative pathway has been advantageous in overcoming the imatinib-resistant CML cells. Since failure in apoptotic machinery is one of the features of chemoresistant tumors, this pro-necrotic effect of EGCG can possibly be extrapolated to other multidrug resistant leukemia or other types of tumors. It should also be of interests to oncologists and immunologists to further investigate the impact of the necrotic cell death in surrounding tissues and our immune response against cancer.

Fig 1. EGCG and imatinib showed distinct cell death patterns.

K562 cells were treated with 0.1% ethanol (control), EGCG or imatinib for 48 h, harvested and incubated with DiOC6(3) and PI. Mitochondrial transmembrane potential (Δψm) and cell death were determined by dual-parameter flow cytometry. Results are representative of three independent experiments.

Fig 2. (A) EGCG-induced cell death is caspase-independent.

K562 cells were treated with 0.1% ethanol (control), EGCG or imatinib for 24 h, and subjected to Western blotting. (B) Z-VAD-FMK fails to prevent EGCG-induced cell death. K562 cells were preincubated with 0.1% ethanol (control) or increasing doses of Z-VAD-FMK for 1 h and subsequently treated with or without 200 μM EGCG or 1 μM imatinib for 48 h. The cell viability was measured based on intracellular ATP content. Asterisks indicate significant difference from single imatinib treatment and significant indifference for EGCG-treated cells (P < 0.01).

Fig 3. (A) EGCG treatment caused necrotic cell death morphology resembling ATP-depleted condition.

K562 cells were grown in glucose-free medium with 5 μg oligomycin for ATP-depleted condition for 12 h; treated with 1 μM imatinib or 200 μM EGCG for 48 h and compared with the 0.1% ethanol-treated control K562. (B) Quantification analysis of necrotic (cytoplasmic vacuolation) and apoptotic cells induced by EGCG, imatinib or ATP-depletion.

Fig 4. EGCG was equally effective to induce cell death in imatinib-resistant CML cells.

Imatinib-resistant K562 (K562/sti) cells were seeded at a density of 5 x 103 cells per well in 96-well plates, and treated with increasing concentrations of EGCG or imatinib for 48 h, and the number of viable cells were measured based on intracellular ATP content.

本論文は緑茶成分カテキンの慢性骨髄性白血病細胞に対する細胞障害活性を検討した論文である。

慢性骨髄性白血病(CML)の治療は、CMLの癌遺伝子であるBcr-Ablを分子標的としたチロシンキナーゼ阻害剤imatinib mesylate(商品名Glivec)の開発により、劇的な変化を示した。本薬剤の開発以前は、大多数の患者が5から7年の間に急性転化を示し、種々の治療に抵抗性を示して死に至っていた。血液幹細胞移植が唯一治癒を望める治療法であったが、ドナーの問題と患者の年齢等で、適応に限界があった。しかし、現在では、大部分の患者でGlivec服用によりBcr-Abl陽性腫瘍細胞は検出不可能なレベルまで抑制され、「治癒」と言う概念が議論されるまでに至っている。一方、現在ではBcr-Abl kinaseのATP結合ポケット領域の点突然変異に起因するGlivecに対する薬剤耐性が新たな問題となっている。薬剤耐性克服のために、新たに様々のBcr-Abl阻害剤の開発が進められているが、耐性克服には全く新たな視点での薬剤の開発も重要な視点である。

Epigallocatechin-gallate (EGCG)は緑茶成分に含まれる主要なカテキンの成分であり、がんの化学予防効果が注目されるpolyphenolである。近年、EGCGの抗腫瘍効果が注目されている.理由は、EGCGが種々の腫瘍細胞に対して選択的な細胞毒性を示す事であり、腫瘍細胞に対応する正常細胞に対しては細胞毒性を示さないとされている。従来、EGCGの抗腫瘍効果は、細胞周期停止、telomerase活性の阻害、あるいはラミニン受容体LR67への結合を介する転移の抑制等によるものと報告されている。これら種々の抗腫瘍機構は最終的には共通のアポトーシスへとつながっている。しかしながら、筆者らの予備実験の結果では、形態学的にCML細胞はアポトーシスとは異なる非典型的な形態を示した。これに基づいて、筆者らは、EGCGによるCML細胞の細胞死誘導のメカニズムを明らかにすることにした。さらに、恒常的活性化Bcr-Ablキナーゼによる抗アポトーシス作用を示すと考えられる薬剤抵抗性CML細胞に対する有効性を含めて解析し、細胞死誘導の分子機構と薬剤耐性CML細胞への有効性を検討した。

解析の結果、以下の事が明らかになった。1)CML細胞株K562をEGCGで処理し、ミトコンドリア膜電位のDiOC6での検出とPropidium iodideの染色をdual parameter flow cytometryで解析した結果、K562をimatinibで処理した場合とは異なった細胞死が誘導されること。2)この細胞死に際してはcaspaseの活性化を示す分解産物は検出されないこと。3)この細胞死を、caspase阻害剤Z-VAD-FMKが抑制しないこと。4)EGCGで処理されたK562細胞を電子顕微鏡で観察すると、細胞質の空泡化等を伴うATP除去によって誘導されるネクローシスと同様の形態を示したこと。5)Glivec耐性K562細胞はEGCGにたいして、親株と同様の感受性を示したこと。以上の結果から、EGCGはCML細胞に、アポトーシスとは明確に異なる、caspase非依存的なネクローシス様の細胞死を誘導すること、さらに、この細胞死経路の活性化がimatinib耐性を示すCML細胞に対しても有効であることを示した。多くの薬剤耐性腫瘍細胞においてはアポトーシス経路の機能不全が特徴である事から、EGCGのネクローシス様細胞死誘導機能は、多剤耐性白血病細胞や他の薬剤耐性腫瘍細胞における耐性克服の新たな可能性を示すものである。また、ネクローシス様の細胞死が周囲の組織や免疫反応へ与えるインパクトは、腫瘍学にとって興味深い検討課題であると考えられる。

なお、本論文の主要部分は、伊藤金次、石田 尚臣、浜之上 誠、 足立 壮一、 渡邉俊樹、佐藤 裕子との共同研究であるが、論文提出者が主体となって分析及び検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学位を授与できると認める。