JP2017071592A - 癌幹細胞抑制剤、癌の転移又は再発の抑制剤並びに癌細胞の未分化マーカー発現抑制剤 - Google Patents
癌幹細胞抑制剤、癌の転移又は再発の抑制剤並びに癌細胞の未分化マーカー発現抑制剤 Download PDFInfo
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Abstract
Description
項1. 鉄キレート剤を含む癌幹細胞抑制剤。
項2. 癌幹細胞の増殖を抑制する、項1に記載の癌幹細胞抑制剤。
項3. 癌幹細胞の機能を抑制する、項1に記載の癌幹細胞抑制剤。
項4. 鉄キレート剤がデフェラシロクス又はデフェロキサミンである、項1〜3のいずれか1項に記載の癌幹細胞抑制剤。
項5. 鉄キレート剤を含む癌の転移又は再発の抑制剤。
項6. 鉄キレート剤を含む、癌細胞の未分化マーカー発現抑制剤。
マウスiPS細胞(miPS)を既知の方法によって、肝細胞へと分化誘導した。本製造例ではIwamuroら(Cell Transplant. 2010;19(6):841-7)に準じた方法で以下の手順でEB(Embryonic Body)形成、DE(Definitive Endoderm)への誘導、Heps(Hepatocytes)の作成を行った。
製造例1で得られた癌幹細胞モデル(miPS-LLCcm)を用い、鉄キレート剤(デフェラシロクス、デスフェラール)を用いて癌幹細胞マーカーの発現について検討を行った。この癌幹細胞モデルは、iPS細胞から作成された癌幹細胞モデルのため、Naongと山中因子(c-Myc、Oct3/4、SOX2、Klf4)を発現している。
次に癌幹細胞(miPS-LLCcm)に特異的に発現されている癌幹細胞マーカーの発現に対するデフェラシロクスの効果を、デフェラシロクスを培地に添加して48時間後に蛋白質を回収し、ウエスタンブロット法で検証した(図2)。デフェラシロクスを投与すると癌幹細胞マーカーであるNanogと山中因子(c-Myc、Oct3/4、Sox2、Klf4)の発現が、ある一定の濃度以上になると発現が抑制されることが判明した。
癌幹細胞(miPS-LLCcm)の培地に対して鉄キレート剤のデスフェラールを投与し48時間後に蛋白質を回収し、幹細胞性マーカーのNanogの発現についてウエスタンブロット法で検証した(図3A)。この実験ではデスフェラールが50μM以上となるとNanogの発現が抑制された。
癌幹細胞(miPS-LLCcm)以外に幹細胞性マーカーのNanogを発現しているヒト乳癌細胞株MCF-7についてデフェラシロクスを培地に投与し、48時間後に蛋白質を回収し、ウエスタンブロット法で検証した(図3B)。この実験でもNanogの発現は抑制され、鉄キレート剤を投与すると未分化性マーカーであるNanogを発現抑制する事を示している。
miPS-LLCcm細胞株を用いてヌードマウスで皮下腫瘍を作成し(n=4)、鉄キレート剤であるデフェラシロクスを腫瘍作成1週間後から30mg/Kg、或いは、デスフェラールを腫瘍作成1週間後から30mg/Kgで週5回腫瘍に局所注射で投与すると、各々腫瘍の増殖が抑制された(図4A)。腫瘍作成後19日目に腫瘍を回収し、重量を測定し、免疫組織で幹細胞性マーカーのひとつであるSOX2で染色を行うと、コントロール(鉄キレート剤非投与)では腫瘍全体がSOX2で染色されるのに対してデフェラシロクス、デスフェラール投与群では、腫瘍内部に非染色部位が認められる(図4B)。in vivoにおいてもこれらの除鉄剤は幹細胞性マーカーを抑制する事が可能と考えられる。
マウス繊維芽細胞 :NIH 3T3(図5A)とマウス胚性線維芽細胞:MEF(図5B)に対するデフェラシロクスの投与データを示す。miPS-LLCcmと同様にデフェラシロクス投与48時間後のcell viabilityについて検討を行った。正常細胞に対しても増殖はやや抑制される傾向にあるが、癌幹細胞モデルと比較すると明らかにその効果は弱い。
癌幹細胞モデル(miPS-LLCcm)の鉄依存性について、トランスフェリン鉄を用いて鉄の依存性についても下記の通りcell viabilityを測定して検討を行った(図6)。通常のFBS(牛血清)15%の状態ではトランスフェリン鉄を投与しても48時間後の増殖は刺激されないが、FBS1%にしてトランスフェリン鉄を投与すると約3倍に増殖が刺激される。この事は癌幹細胞モデル(miPS-LLCcm)が高い鉄依存性を有する事を示している。
癌幹細胞(miPS-LLCcm)の培地に対して鉄キレート剤のデスフェラール(デフェロキサミンメシル酸塩)を添加し48時間後に蛋白質を回収し、幹細胞性マーカーのOct3/4, c-Myc, GFPの発現についてウエスタンブロット法で検証した(図7)。この実験ではデスフェラール濃度依存的にOct3/4, c-Myc, GFPの発現が抑制された。
癌幹細胞(miPS-LLCcm)に既存の代表的な抗癌剤である5-FU又はCDDPを投与し、48時間後に細胞生存率をXTT法で計測し、蛋白を回収した。増殖抑制効果は認められたが(図8)、ウエスタンブロット法では未分化マーカーの抑制は認められず(図9)、未分化マーカーの抑制は除鉄剤特有の効果である事を示している。除鉄剤が従来の抗癌剤にはない新しい効能により癌幹細胞治療法になり得る事を示している。
Claims (6)
- 鉄キレート剤を含む癌幹細胞抑制剤。
- 癌幹細胞の増殖を抑制する、請求項1に記載の癌幹細胞抑制剤。
- 癌幹細胞の機能を抑制する、請求項1に記載の癌幹細胞抑制剤。
- 鉄キレート剤がデフェラシロクス又はデフェロキサミンである、請求項1〜3のいずれか1項に記載の癌幹細胞抑制剤。
- 鉄キレート剤を含む癌の転移又は再発の抑制剤。
- 鉄キレート剤を含む、癌細胞の未分化マーカー発現抑制剤。
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CN111363034A (zh) * | 2018-12-25 | 2020-07-03 | 上海市第十人民医院 | 一种NANOG Ser68的抗体,以及抑制剂和应用 |
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WO2011115062A1 (ja) * | 2010-03-15 | 2011-09-22 | 国立大学法人 岡山大学 | 抗腫瘍作用補助剤 |
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WO2011115062A1 (ja) * | 2010-03-15 | 2011-09-22 | 国立大学法人 岡山大学 | 抗腫瘍作用補助剤 |
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CN109745314A (zh) * | 2019-01-30 | 2019-05-14 | 河北师范大学 | 铁螯合剂Deferasirox(DFX)在治疗宫颈癌的药物中的应用 |
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