第二型拓樸異構酶IIβ抑制藥物用以減少化療副作用之開發

Abstract

人類第二型拓樸異構酶（topoisomerase II, Top2）是癌症治療的良好標的目標，而抗第二型拓樸異構酶的藥物可依據藥物作用機轉分為兩類：（一）第二型拓樸異構酶的可切性複合體（Top2 cleavable complex, Top2cc）產生藥物：現行癌症化療藥物，除了抑制第二型拓樸異構酶的酵素活性，由於停留於特別的步驟，造成第二型拓樸異構酶與斷裂DNA共價結構之可切性複合體並且造成短暫DNA雙股斷裂（稱為 Top2 poisoning activity），並主動引發細胞死亡，為腫瘤治療主要療效來源，例：多柔比星（小紅莓，doxorubicin）；（二）第二型拓樸異構酶的酵素拮抗劑（Top2-antagonizing inhibitors）：其他第二型拓樸異構酶的酵素活性抑制劑，則只抑制酵素活性，其中過程並不產生第二型拓樸異構酶可切性複合體，但可以拮抗可切割性複合體的產生，減低癌症化療藥物的細胞毒殺性，目前有合併治療，使用於減低小紅莓引起的心臟毒性等臨床副作用，例：右雷佐生（dexrazoxane, ICRF-187）。近年研究闡明人類第二型拓樸異構酶包含兩種亞型（α and β isozymes）。先前我們與其他文獻指出在化療中，Top2α亞型主要是抗癌藥物的標的目標並為影響化療的療效的主因與的作用機制（mechanism of action, MOA），而以Top2β亞型的可切性複合體則是造成二度癌化與心臟毒性等副作用的潛在的作用機制。因此，開發β亞型專一性的Top2β酵素拮抗劑，應可以有效減少第二型拓樸異構酶可切性複合體產生藥物所引發的臨床副作用與毒性，但不影響其抗腫瘤療效，有效提升現行藥物的治療指數（therapeutic index, TI），這也是本論文的研究目標。另外，實驗室先前對蒽醌（mitoxantrone）衍生藥物有相當的研究，所以本論文首先聚焦於的蒽醌衍生物上，利用第二型拓樸異構酶的DNA解螺旋（relaxation）和切割（cleavage）生化實驗，我們首先篩選出蒽醌衍生物CL-01和CL-14具有較專一性抑制Top2β第二型拓樸異構酶β亞型解DNA螺旋的能力。不同於蒽醌，CL-01和CL-14只具有很弱的DNA結合力。另外，和Top2可切性複合體產生藥物小紅莓或依托泊苷（etoposide, VP-16）共同作用時，能減低其所造成的 DNA斷裂。為了進一步研究心臟毒性，我們使用HL-1心肌細胞。我們發現在彗星（comet）實驗中，CL-01和CL-14均能夠減少血癌與心肌細胞中被小紅莓或依托泊苷所造成的DNA損傷。CL-01能夠有效的拮抗第二型拓樸異構酶Top2α與Top2β亞型表現的細胞中的DNA損傷；相較於CL-01，CL-14更具有抑制第二型拓樸異構酶Top2β亞型切割複合體產生的能力。最後在動物實驗中，CL-01和CL-14可以防止小紅莓所引起的乳酸去氫酶（LDH, 心毒性指標之一）、天冬氨酸氨基轉移酶（AST, 心毒性指標之一）和丙氨酸基轉移酶（ALT, 心毒性指標之一）上升；同樣地，CL-01和CL-14亦可以防止小紅莓所引起的心肌纖維化（fibrosis, 心毒性指標之一）。而在少量動物實驗結果中，CL-01也有抑制小紅莓所造成的其他副作用，例：骨髓抑制/毒性（myelosuppression/bone marrow toxicity）。結論上，許多蒽醌衍生物，尤其CL-14，具有較偏第二型拓樸異構酶β亞型專一性的抑制能力，能夠減少臨床上使用的第二型拓樸異構酶可切性複合體產生藥物所引起之化療副作用，尤其是心臟與肝臟毒性。

Human type II topoisomerases (topoisomerase II, hTop2) have been proven to be an excellent therapeutic target for anti-cancer drugs. Based on underlying mechanism of action (MOA), Top2-targeting agents are classified into two classes: the Top2 cleavable complex (Top2cc)-producing drugs, such as doxorubicin and etoposide, and the Top2-antagonizing inhibitors, such as dexrazoxane (ICRF-187) and aclarubicin. In this regard, the Top2cc-producing drugs inhibit a specific intermediate stage of Top2 catalytic cycle and stabilize transient Top2-linked double-stranded DNA break (DSB) structure termed Top2cc and leading to cell death and anticancer efficacy; while the Top2-antagonizing inhibitors not only interfere Top2 catalytic activity without inducing Top2cc but also antagonize the Top2cc-mediated cytotoxicity caused by Top2cc-producing drugs. It is interesting to note that there are two hTop2 (α and β) isozymes which exhibit distinct functions and properties in mammalian cells. Our and other recent studies suggest that hTop2α is the main MOA target for anti-cancer efficacy, whereas the MOA hTop2β-targeting leads to clinical adverse effects such as cardiotoxicity and 2nd malignancy, that is, hTop2α and hTop2β are MOAs responsible respectively for the anti-cancer efficacy and clinical adverse effects. Therefore, identifications of hTop2β-specific antagonizing inhibitors might create a new revenue for reducing unwanted clinical side effect of the Top2cc-based anti-cancer therapy and improving their therapeutic index, and this is also the specific aim for my dissertation study. Through performing in vitro Top2 relaxation and cleavage assays using purified recombinant hTop2 isozymes, we have screened and found that anthracenediones, such as CL-01 and CL-14, were shown to inhibit hTop2β relaxation activity preferentially and could reduce the hTop2β-mediated DNA breaks when co-treated with two Top2cc-producing drugs, doxorubicin and etoposide. In order to further investigate the adverse effect of cardiotoxicity during doxorubicin therapy, we took advantage of the HL-1 cardiomyocyte. We showed that, in both leukemia cells and cardiomyocytes, CL-01 or CL-14 co-treatments reduced of the Top2cc-mediated DNA damage induced by etoposide and doxorubicin. In addition, compared to CL-01, CL-14 has a better ability to preferentially antagonize Top2βcc formation by etoposide. To study further the isozyme preference, we then employed the RNAi knockdown approach to generate the individual Top2 isozyme-deficient cell lines. Unlike a control ICRF-193 and CL-01, CL-14 was able to antagonize the etoposide-induced and Top2βcc-mediated DNA damage in the Top2α-specific knockdown cells better than the etoposide-induced and Top2αcc-mediated in the Top2β-specific knockdown cells. Lastly, using the animal experimental models, pretreatment of CL-14 effectively relieved the elevated levels of LDH (a serum biomarker for cardiotoxicity), AST, ALT (two serum biomarkers for hepatotoxicity), and also reduce myelosuppression (i.e. bone marrow toxicity), those adverse effects caused by doxorubicin. With the preliminary and limited data, we found that CL-01 also showed abilities to relieve doxorubicin adverse effects. Consistent with the notion that CL-01 and CL-14 can reduce the cardiotoxicity adverse effect induced by doxorubicin, pretreatments of CL-01 and CL-14 could both reduce the doxorubicin-mediated heart tissue fibrosis using the Masson trichrome staining. Taken together, anthracenediones, especially CL-14, may be a good potential as the hTop2βcc-specific antagonizing inhibitor to reduce adverse effects caused by the clinically useful Top2cc-producing anticancer drugs.