| 摘要: | part I
人類骨肉瘤(human osteosarcoma)是一種極惡性的癌症。其中顯示造骨細胞(osteoblastic)的惡性轉化的特性並產生類骨質(osteoid)的疾病。早先的研究已經證實，阿黴素(Doxorubicin)的化學治療當中，可以降低人類骨肉瘤細胞的增生，並可藉此治療過程中，增強與ZAK相關的RNA的表現。然而，在經由阿黴素抑制人類骨肉瘤細胞的增生中，過度表現ZAKα的影響與其在這影響之下的分子機制尚未十分明瞭。ZAK是MAPKKK家族中的一個蛋白質激?，在功能上可以促進細胞凋亡(apoptosis)。在我們的研究中，發現在人類骨肉瘤細胞過度表現ZAKα時，將會導致細胞凋亡的效應。並且也發現對人類骨肉瘤細胞使用阿黴素(Doxorubicin)會增強ZAKα的表現，進而減少癌細胞的存活更增加癌細胞的凋亡。然而在研究中，我們發現利用shRNA和抑制劑D1771抑制了ZAKα的表現，進而可以抑制阿黴素對人類骨肉瘤細胞的療效。這些發現都一再顯示出阿黴素在人類骨肉瘤細胞治療上展新的分子機制。於此，我們的研究證明人類骨肉瘤細胞在阿黴素的處理之下，會增強ZAKα的表現近而達到增強細胞凋亡效應，降低癌細胞的存活。
part II
ZAK是一個位在細胞質的mixed lineage類激?蛋白質，其含有leucine-zipper (LZ)和sterile-alpha motif (SAM)當做蛋白質與蛋白質反應的區域。ZAK具有兩種不同的接合型式，被命名為ZAKα與ZAKβ。在之前的研究中，ZAKα已被研究出參與各種不同的細胞分子機制過程，其中包括細胞增生、細胞分化與心機肥大的分子機制，但是ZAKβ的分子機制，至今仍不是非常清楚。在我們最近的研究中，發現ZAKβ可以改善因為ZAKα所誘導的心肌細胞凋亡的情況。於此，我們假設ZAKβ在人類骨肉瘤細胞中，同樣可以改善ZAKα所誘導的細胞凋亡效應。結果顯示，ZAKβ在人類骨肉瘤細胞中，卻扮演著與ZAKα類似的角色，同樣可以誘導細胞凋亡並降低細胞的存活率。有趣的是，在人類骨肉瘤細胞的抑制劑實驗中，ZAKβ的表現呈現對ZAKα表現的高度依賴性。然而ZAKβ的表現又可以有效的誘導ZAKα的表現上升，並導致加成性的細胞凋亡現象。在人類骨肉瘤細胞中，可以更增強經由ZAKα所誘導的細胞凋亡效應。最後，透過co-immunoprecipitation的結果顯示ZAKα可以直接與ZAKβ之間有蛋白質與蛋白質的交互作用，這也許是造成加成性細胞凋亡現象的原因之一。
part III
骨肉瘤 (Osteosarcoma) 是一種會造成癌症相關死亡的腫瘤。過去的研究指出，化學治療不只可以降低人類骨肉瘤細胞的增生也可以增加細胞的凋亡，但是對於骨肉瘤臨床上的預後結果卻比較缺乏。漆黃素 (Fisetin) 是一存在蔬菜與水果中的類黃酮化合物，並且也被研究出可以抑制多數癌細胞的生長。但是對於人類骨肉瘤細胞的分子機制，卻鮮為人知。ZAK是MAPKKK家族中的一個蛋白質激?並涉及了很多細胞上的功能包括：細胞增生、細胞凋亡等等。在我們的研究團隊中，已經證明了過度表現ZAK會誘導人類骨肉瘤細胞的凋亡。然而在過去的研究中，MAP3K的上游MAP4K同樣可以藉由活化MST1/2的活性而扮演誘導Hippo pathway的角色。誘導Hippo pathway可以降低癌細胞的增生並導致癌細胞凋亡的情形增加。在此研究中，我們發現在漆黃素 (Fisetin) 的刺激之下，可以調節ZAK的表現增加進而誘導Hippo pathway開啟，同時可以增加JNK/ERK的活性並且透過AP-1的活化而引起人類骨肉瘤細胞的凋亡產生。這些發現顯示了漆黃素 (fisetin) 在分子機制上對人類骨肉瘤細胞影響的重要性。
part I
Human osteosarcoma is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin chemotherapy decreases human osteosarcoma cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the osteosarcoma cell proliferation that is inhibited by doxorubicin and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human osteosarcoma cells. Treatment of human osteosarcoma cells with doxorubicin enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human osteosarcoma cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the doxorubicin therapeutic effect. These findings reveal a novel molecular mechanism underlying the doxorubicin effect on human osteosarcoma cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in doxorubicin-treated human osteosarcoma cells.
part II
ZAK is a novel mixed lineage kinase-like protein that contains a leucine-zipper (LZ) and a sterile-alpha motif (SAM) as a protein-protein interaction domain, and it is located in the cytoplasm. There are two alternatively spliced forms of ZAK: ZAKα and ZAKβ. Previous studies showed that ZAKα is involved in various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy, but the molecular mechanism of ZAKβ is not yet known. In a recent study in our lab, we found that ZAKβ can ameliorate the apoptotic effect induced by ZAKα in H9c2 cells. We further hypothesized that ZAKβ could also improve the apoptotic effect induced by ZAKα in human osteosarcoma cells. The results of this study show that ZAKβ can induce apoptosis and decrease cell viability similar to the effects of ZAKα. Interestingly, our ZAKα-specific inhibitor assay shows that the expression of ZAKβ is highly dependent on ZAKα expression. However, ZAKβ expression effectively induces ZAKα expression and results in synergistic enhancement of apoptosis in human osteosarcoma cells. Furthermore, co-immunoprecipitation results revealed that ZAKα can directly interact with ZAKβ, and this interaction may contribute to the enhanced apoptotic effects.
part III
Osteosarcoma (OS) is a tumor entity that leads to a substantial number of cancer-associated deaths. Previous studies have indicated that chemotherapy can decrease human OS cell proliferation and increase cell apoptosis, but the outcomes of OS clinical prognosis remain poor. Fisetin is a flavonol found in fruits and vegetables, and this compound can inhibit cell growth in numerous cancer cells. However, the molecular mechanism underlying fisetin in human OS cells is not yet known. Previous studies have shown that ZAK, a kinase in the MAP3K family, is involved in various cell processes, including cell proliferation and cell apoptosis. We have previously demonstrated that ZAK overexpression can induce cell apoptosis in human OS cells. In previous studies, MAP4K, the upstream activator of MAP3K, acts in parallel with MST1/2 to activate LATS1/2 in the Hippo pathway. Turning on the Hippo pathway can decrease cell proliferation and cause cell apoptosis in cancer cells. In the present study, we found that fisetin can up-regulate ZAK expression to induce the Hippo pathway and mediate JNK/ERK activation, which is downstream of ZAK, to trigger cell apoptosis in an AP-1-dependent manner in human OS cells. These findings reveal a novel molecular mechanism underlying the fisetin effect on human OS cells. |
