Hemiasterlin衍生物BF65在血癌的抗癌機轉探討

Abstract

BF65為全合成之hemiasterlin衍生物，並已知具有抗微管之作用。典型的hemiasterlins是萃取自海棉的天然化合物，具有三肽(tripeptide)結構，並可與α型微管結合，其結合位點鄰近於長春花生物鹼類(vinca-alkaloids)在β型微管的結合位點，能抑制微管的聚合。BF65除了可影響參與調控細胞週期蛋白質表現，並同時可引起細胞死亡的訊息傳遞，與長春花生物鹼類藥物如vincristine相似。我們利用人類急性前骨髓性細胞白血病(acute promyelocytic leukemia, APL)細胞株HL-60測試BF65的抗癌活性。APL屬於急性骨髓性細胞白血病(acute myelocytic leukemia, AML)下的第三亞型，目前臨床上的治療選擇包含許多化療藥物，如vincristine。已知的抗微管藥物大部分造成細胞週期停滯於有絲分裂階段(mitosis phase)，最終促使細胞凋亡(apoptosis)。然而我們發現HL-60受BF65處理過後也會造成磷酸化組織蛋白H2A.X(phospho-histone H2A.X, γ-H2AX)的蛋白質表現增加及進入S期的細胞數有微幅上升，前述均為DNA受損的表徵，且作用發生的時間點早於有絲分裂期的停滯與細胞凋亡。BF65除了造成γ-H2AX的增加之外，同時降低細胞內的轉錄訊息傳遞及活化子蛋白3(Stat3)與B細胞白血病淋巴瘤蛋白2(Bcl-2)的表現，BF65所引起的這些現象能受到c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)抑制劑SP600125所阻隔。此外我們也發現SP600125同時也能逆轉BF65所引發的粒線體膜電位喪失，但卻不影響細胞週期的改變。我們也注意到BF65能抑制細胞自噬作用(autophagy)。因此，我們推論BF65在抑制微管作用的同時，也會透過JNK的訊息傳遞途徑造成DNA損傷。而BF65抑制自噬和Stat3的能力可能有利於更進一步的抗癌應用。

BF65, a synthetic hemiasterlin, has been known for its anti-microtubule effect. Classic hemiasterlins are natural products extracted from marine sponges. With tripeptide-like structure, hemiasterlins bind to α-tubulin, but the binding site is close to the vinca alkaloid-binding site on β-tubulin, and inhibit tubulin polymerization. BF65 affects proteins involved in cell cycle regulation and signal transduction of cell death in a way similar to vinca alkaloids, such as vincristine. We test the anticancer activity of BF65 in HL-60, a human promyelocytic leukemia cell line. Acute promylocytic leukemia (APL) is a subtype (M3) of acute myeloid leukemia (AML) and several chemotherapy drugs have been approved for AML, including vincristine. Usual anti-microtubule agents cause cell cycle arrest in mitosis, eventually leading cells to apoptosis. However, we find that BF65 also induces increased level of phospho-histone H2A.X (γ-H2AX) and slightly elevates cell population at S-phase, both of which are markers for DNA damage, prior to M-phase arrest in HL-60. Interestingly, BF65-induced γ-H2AX can be partially reduced when c-Jun N-terminal kinase (JNK) is inhibited by SP600125. Moreover, BF65 downregulates signal transducer and activator of transcription 3 (Stat3) and B-cell leukemia lymphoma-2 (Bcl-2), which can be blocked by SP600125 without altering BF65-induced cell cycle arrest. Surprisingly, SP600125 is also able to reverse the mitochondrial membrane potential loss resulted from BF65 treatment. In addition, we notice that BF65 seems to inhibit autophagy. Thus, we propose that BF65 may also cause DNA damage through JNK-mediated pathway while inhibiting normal microtubule function. Besides, BF65-induced decrease in autophagic activity and Stat3 expression may be beneficial for further anticancer applications.