||Study of the therapeutic efficacy and mechanism of action of a novel HDAC inhibitor, MPT0B390, against androgen-dependent and -independent prostate cancer
||Institute of Molecular Medicine
攝護腺癌是一種在西方國家的成年男性常見的癌症。而常見的治療方式為手術切除合併抗雄性素的治療為主，又稱荷爾蒙療法。但是根據治療後病患追蹤統計，有八成至九成的病患可能在兩年內復發成為對賀爾蒙療法有抗性的攝護腺癌，而目前對於這類型的癌症尚未發展出有效的治療方法。組蛋白去乙醯酶為一種對組蛋白進行去乙醯化修飾的酵素，可以進而去調控基因的表現。先前的研究中指出，此類酵素在許多癌症中，包括攝護腺癌等，都有大量表現的情形。因此，此類酵素的抑制劑被認為是具有潛力的抗癌藥物，全世界許多的藥廠及實驗室也致力於相關抑制劑之研發。MPT0B390是本實驗室所開發對組蛋白去乙醯酶有抑制作用的化合物，對於很多癌細胞株都具有很好的毒殺效果。在本研究中，我們選擇攝護腺癌為研究模式，探討這個化合物在治療攝護腺癌的療效以及作用的機制。我們利用三種不同類型的攝護腺癌細胞，分別為一株對雄性激素依賴性細胞—LNCap 104-S，2株為對對雄性激素非依賴性細胞LNCap 104-R1和PC3，探討MPT0B390的細胞毒性以及相關作用機制。因為MPT0B390其化學結構類似於目前臨床上使用之組蛋白去乙醯酶抑制劑-SAHA，因此本研究以SAHA做為正對照組。酵素活性抑制性分析顯示，相較於SAHA，MPT0B390傾向於抑制第一類的組蛋白去乙醯酵素，如HDAC1及HDAC2。細胞毒性分析結果顯示，MPT0B390的毒殺效果明顯比其他2種常見的組蛋白去乙醯酶抑制劑 (SAHA與MS275) 要好。我們進一步分析MPT0B390造成細胞死亡的機制，發現在MPT0B390作用下會影響攝護腺癌細胞之細胞週期的分佈，並產生sub-G1的堆積，推測可能有細胞凋亡的現象發生。進一步使用西方墨點法確認細胞凋亡的相關調控因子，如凋亡蛋白酶(caspase)-3、-8、和-9 有被活化的現象，而多聚ADP 核糖聚合酶 (PARP) 有85 kDa片斷的產生，顯示MPT0B390誘發細胞的凋亡。已知雄性素接受器(androgen receptor)在荷爾蒙治療後復發的攝護腺癌癌化進展中扮演重要的腳色，並且也被證實為HDAC1的非組蛋白受質，因此，我們假設MPT0B390可能會藉由去抑制雄性素接受器的表現，進而影響攝護腺癌細胞的生長。利用西方墨點法分析，雄性素接受器蛋白質表現確實受到MPT0B390的影響而表現量降低。另外，使用反轉錄聚合酶連鎖反應分析雄性素接受器訊息核糖核甘酸(mRNA)，也發現隨著化合物濃度的增加而表現量下降。MPT0B390降低雄性素接受器訊息傳遞路徑的下游蛋白—攝護腺特異性抗原的表現，結果顯示化合物確實透過抑制了雄性素接受器的表現而達到抑制傳導路徑活性。最後，我們使用PC3細胞異體移植所形成的腫瘤作為臨床前活體動物抗癌活性之評估。利用口服餵食的方式將50 mg/kg5 SAHA及MPT0B390給予裸鼠(給藥時程：連續餵食5天，停藥2天，再連續餵食5天)，結果顯示，MPT0B390處理的組別所形成的腫瘤比起SAHA有明顯縮小的現象。綜合上述的結果，MPT0B390 不論是在活體外或是活體內的實驗中，皆顯示對於治療不同種類的攝護腺癌都有很好的療效，顯示其為未來值得發展的抗癌藥物。
Prostate cancer is a common male malignancy and a frequent cause of mortality worldwide, especially in western countries. The common treatment is bilateral orchiectomy combined with antiandrgen, also called hormonal therapy. According to the statistics of tracked patients receiving hormonal treatment, most patients relapsed to the hormone refractory prostate cancer within two years. Nowadays, there is no effective treatment to cure the malignant prostate cancer. Histone deacetylase is one of enzymes to regulate gene expression by removing the acetyl group of lysine on histone. Previous studies indicated that HDACs were overexpressed on various cancers, including prostate cancer. Thus, HDAC inhibitor is considered to be the potential anti-cancer candidate. Nowadays, more and more pharmaceutical companies and laboratories have developed novel HDAC inhibitors. Recently, our laboratory aimed to develop potential HDAC-inhibitory compounds for cancer treatment. Among them MPT0B390, a novel HDAC inhibitor, were identified to possess potent cytotoxic effect in many cancer cell lines. In this study, we investigated the therapeutic efficacy and mechanism of action of MPT0B390 against androgen-dependent and –independent prostate cancer. To this end, we selected three prostate cancer cell lines, one was androgen-dependent (LNCap 104-S), and another ones were androgen independent (LNCap 104-R1, and PC3) prostate cancer cells, to investigate the cytotoxicity and mechanism of action of MPT0B390. Because MPT0B390 and current clinical use HDAC inhibitor SAHA belong to hydroximate-based compound, we therefore chose SAHA as control compound in this study. The result demonstrated that MPT0B390 significantly inhibited HDAC1 and HDAC2 from class I as well as HDAC6 form class IIb at nanomolar concentrations. While MPT0B390 was more potent than SAHA against recombinant HDAC1 and HDAC2. Comparing the cytotoxicity against a panel of prostate cancer (PCa) cells with SAHA, MPT0B390 is much more potent than that in SAHA and MS275. We further found that MPT0B390 affected the cell cycle distribution and produced the accumulation of sub-G1 cell in PCa cells. After MPT0B390 treatment, it revealed an activation of caspase-3,-8, -9 and production of 85kDa fragments of the poly(ADP-ribose) polymerase (PARP) in PCa cells. These evidences indicated that MPT0B390 induced PCa cells apoptosis. On the other hand, it was well-known that androgen receptor (AR) plays an important role in the prostate cancer progression, and the protein also convinced the non-histone substrate of HDAC1. Thus, we presumed that MPT0B390 suppressed the AR expression to affect the cell growth of PCa cells. Our data showed that the expression levels of both AR protein and mRNA were down-regulated after MPT0B390 treatment in a concentration-dependent manner. MPT0B390 also reduced the levels of PSA, the downstream protein of AR signaling pathway, and it showed that MPT0B390 block the activity of AR signaling pathway. Finally, we use PC3 xenograft animal model to evaluate anticancer activity of MPT0B390. The nude mice were received 50 mg/kg of SAHA or MPT0B390 5 days/week for 2 consecutive weeks. The result indicated that MPT0B390 treatment resulted in shrinkage of PC3 cells tumor growth in xenografted mouse model and was superior to the therapeutic effect of SAHA. Taken together, whether in vitro or in vivo experiments, MPT0B390 shows significant efficacy for different types of PCa cells. It is worthwhile for further development of this compound.
Chinese Abstract I
English Abstract III
Table Contents IX
Figure Contents X
1. Prostate cancer 1
1-1. Epidemiology 1
1-2. The therapies for prostate cancer 2
1-3. The therapy targets for HRPC 2
1-3-1. Inhibitors of the androgen synthesis and androgen receptor signaling 2
1-3-2. Inhibitors of growth factor receptors signaling 3
1-3-3. Anti-angiogenesis targeting therapy 3
1-3-4. Other molecular targets for prostate cancer therapy 4
2. Epigenetic modification 4
2-1. HDAC classification and biological function 5
2-2. HDAC inhibitors 6
2-3. The role of HDAC in prostate cancer 7
2-4. HADC inhibitor used in prostate cancer therapy 7
Specific aims 9
Experimental Design 11
Materials and Methods 12
1. Chemicals. 12
2. Cell lines and culture conditions. 12
3. HDAC Activity Assays. 12
4. Cell Growth Inhibitory Assay. 13
5. Cell cycle analysis. 14
6. Western blot analysis and primary antibodies. 14
7. RT-PCR. 15
8. In vivo experiment. 15
9. Statistical Analysis. 16
Results and Discussion 17
1. Inhibitory effect of MPT0B390 and SAHA on histone deacetylase (HDAC) 1, 2, and 6 enzymatic activity. 17
2. Anti-proliferative activity of HDAC inhibitors toward prostate cancer cells 17
3. Dose effect of MPT0B390 on cell cycle distribution in PCa cells 18
4. The effect of MPT0B390 on apoptosis 20
5. Down-regulation of the protein and mRNA levels of androgen receptor in androgen-dependent and -independent PCa cells 21
6. Down-regulation of androgen receptor signaling pathway 22
7. Investigation of in vivo antitumor efficacy of MPT0B390 in PC3 xenograft mouse model 22
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