鋅手指樣蛋白 (Zinc finger-like protein that regulates apoptosis, Zfra)是一個僅含31個胺基酸的小型蛋白質。在先前的研究中已經證實Zfra能夠預防癌細胞的生長，預先於小鼠尾靜脈注射全長的Zfra1-31或截斷型Zfra4-10將會活化小鼠脾臟內一種特殊的免疫細胞¬¬－Hyal-2+ CD3- CD19- Z細胞。當Z細胞活化後將能夠預防癌細胞的生長，但在先前的研究中顯示，Zfra對於癌症的治療效果並不理想，推測與Zfra的溶劑有著密切的關係。在這份研究中，我們發現當Zfra (4-10)溶於二甲基亞碸 (Dimethyl sulfoxide, DMSO)能夠有效的治療癌症。在已生長腫瘤的BALB/c小鼠以及NOD-SCID小鼠身上以尾靜脈注射Zfra4-10後，腫瘤的生長相對於控制組受到明顯的抑制，並且Zfra的抑癌效果與市面上現行之抗癌藥物Ceritinib以及Docetaxel並沒有產生顯著差異。同時，癌細胞的肺轉移也明顯的受到抑制。另外，我們發現帶有Zfra基因的質體DNA同樣具有抑癌效果，注射了帶有Zfra cDNA的質體後，小鼠身上的4T1與B16F10癌細胞生長明顯受到抑制。我們推測，Zfra除了能活化Z細胞引起抗癌免疫反應之外，癌細胞可能吸收了Zfra基因並因過度表現Zfra而死亡。ANR40與Zfra同屬於會和抗癌蛋白WWOX結合的蛋白質。在類似的動物實驗中，我們也發現過度表現ANR40基因與Wwox基因能抑制癌細胞的生長。藉由免疫共沉澱與以及螢光共振能量轉移實驗我們驗證了Zfra會與ANR40直接結合。同時過度表現ANR40以及Zfra能使B16F10黑色素瘤細胞在細胞週期中的G2/M期被遏止，G2/M期的比例從20.3%提升至37.6%。ER促進劑能抑制ANR40以及Zfra所引起的G2/M期上升，並且我們透過免疫共沉澱也證實ANR40與pERβ能形成複合物。過度表現Zfra與 ANR40抑制了ERβ下游的AKT及MAPK訊息傳遞。此外，過度表現ANR40以及Zfra會降低黑色素瘤癌症幹細胞標記CD133的表現與B16F10的致瘤性。Zfra能活化ANR40的作用去抑制癌細胞生長。總而言之，我們的數據顯示ANR40與Zfra能共同抑制黑色素瘤的生長。

Zfra (Zinc finger-like protein that regulates apoptosis) is a 31-amino-acid zinc finger-like protein which prevents many types of cancer growth in mice. Post tail vein injection, full length Zfra1-31 or truncated Zfra4-10 peptide is deposited mainly in mouse spleen. Zfra activates novel type of splenocytes which are identified to be Hyal-2+ CD3- CD19- Z cells. Despite its role in cancer suppression, Zfra antitumor function has not been maximized. Phosphate-buffered saline induces time-related overpolymerization of Zfra and leads to its functional inactivation. Here, Zfra, dissolved in dimethyl sulfoxide (DMSO) effectively blocks the growth of breast 4T1 cancer cells and B16F10 melanoma cells in BALB/c mice and NOD-SCID mice, which are comparable to those effects of anticancer drugs ceritinib and docetaxel. In parallel, injected Zfra cDNA in mammalian expression vector exhibited similar results in vivo. Both Zfra and ankyrin repeat domain 40 (ANKRD40 or designated as ANR40) are binding proteins of tumor suppressor WW Domain-Containing Oxidoreductase (WWOX). Transiently overexpressed ANR40 effectively suppressed B16F10 cells growth in vivo and in vitro. ANR40 physically interacts with Zfra, as determined by co-immunoprecipitation and Förster resonance energy transfer. Ectopic expression of ANR40 and Zfra arrests cell cycle progression at the G2/M phase. Estrogen receptor β agonist ERB-041 blocked the G2/M arrest. The ANR40/ER complex was verified by immunoprecipitation, suggesting that ANR40 directly inhibited ER. The ANR40/ER complex was dissociated by 17beta-Estradiol. Zfra may covalently conjugate with proteins. Ectopic expression of ANR40 and Zfra reduced the underlying pro-survival AKT and MAPK pathway. In addition, ectopic expression of ANR40 and Zfra reduced transforming growth of B16F10, as well as its stem cell marker CD133. After binding with Zfra, ANR40 becomes active to suppress cancer growth. Together, our data suggest that ANR40 and Zfra synergistically act in melanoma suppression in vivo.

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