"探討Physalin F, NPRL-Z-1與Heteronemin在人類腎癌細胞株之抗癌機轉"

Abstract

腎細胞癌Renal Cell Carcinoma，簡稱RCC，是成人最常見的腎臟癌，佔了九成以上的病例。在全美的罹癌統計中，腎細胞癌名列男性最常見第七位，與女性最常見第九位的癌症。而男性罹病與死亡的機率將近是女性的兩倍。雖然手術切除在罹病的早期是最有效的方式，但是有近三成的病人在被診斷出患有腎細胞癌的同時卻也發現已經轉移，主要是因為腎細胞癌的臨床症狀不明顯。由於腎細胞癌對於化學治療及放射線療法的反應不佳，因此，目前著重於研發針對腎細胞癌的標靶治療藥物。在本篇論文中，我們探討從天然物中萃取或合成的抗癌化合物，利用其作用機轉以提供發展治療腎細胞癌藥物的優勢。 Physalin F是從苦蘵所萃取出來的天然化合物之一。過去常被用於治療瘧疾、氣喘、肝炎、皮膚炎及風濕。近幾年發現全株藥用或其他化合物也具有抗癌作用。在本篇論文中，深入研究Physalin F在腎癌細胞株引起細胞凋亡的機轉。研究發現，Physalin F可以誘發自由基的產生、p53與p21的表現量增加、粒線體膜電位的減少，釋放cytochrome c，最後造成caspase cascade的活化而引起細胞凋亡。另一方面，Physalin F也能抑制IkBα磷酸化的增加、p65與p50的入核、與NF-ĸB與DNA之間的結合以達到促進細胞凋亡的結果。而抗氧化劑NAC與GSH可以逆轉以上這些現象，表示自由基在Physalin F所引起的細胞凋亡中扮演了重要的角色。 NPRL-Z-1是epipodophyllotoxin的衍生物，藉由將C7接上的較大的取代基以加強etoposide analogues對第二型拓樸異構酶 (topoisomerase) 的抑制作用、克服抗藥性及改善藥物水溶性。本篇研究發現，NPRL-Z-1會與TOP2形成TOP2 cleavage complexes，而造成DNA雙股斷裂，而使ATM活化下游的反應蛋白，最終造成細胞週期停滯與細胞凋亡的現象。同時，NPRL-Z-1也能誘發自由基的產生，促進細胞凋亡。本篇研究證實NPRL-Z-1是新型的第二型拓樸異構酶的毒物，並對於腎癌細胞株有較佳的抗癌效果。 Heteronemin是自海綿所萃取出來的海洋天然化合物。之前的研究顯示其亦具有抗癌作用。本篇研究發現Heteronemin對人類腎癌細胞株有較佳的毒殺作用之外，Heteronemin亦會降低Bcl-2及Bcl-xL的蛋白質表現量，卻增加Bax的蛋白質表現量；因而造成粒線體膜電位的減少，釋放cytochrome c，最後造成caspase cascade的活化引起細胞凋亡。Heteronemin還能活化p38與JNK，並抑制ERK與Akt的磷酸化。當p38被抑制時，可逆轉Heteronemin所引起的細胞凋亡作用。Heteronemin亦能引起腎癌細胞株A98的細胞自噬作用；使用chloroquine (細胞自噬抑制劑) 與SP600125 (JNK抑制劑) 不但能抑制Heteronemin引起的細胞自噬作用，還能促進Heteronemin所造成的細胞凋亡的反應。因此，本篇研究提出結合Heteronemin與細胞自噬抑制劑，做為新的治療策略之方向，以促使腎癌細胞的凋亡。

Renal cell carcinoma (RCC) represents approximately 90% of the adult kidney cancer. In America, it is the seventh most common cancer in men and the ninth most common in women and the incidence and mortality rates in men are almost twice that in women. Although surgical innovation is the most effective treatment in early stage, 30% of patients have metastases when they are diagnosed and do not apply to surgery. Because RCC is resistant to chemotherapy and radiotherapy, scientists are dedicated and focus on the investigation of target therapy of RCC. In this thesis, we explored the anticancer mechanisms of natural or synthetic compounds, and provided the advantages of developing the new treatment strategy of RCC. Physalin F is extracted and purified from Physalis angulata L which has been widely used to treat malaria, asthma, hepatitis, dermatitis, and for rheumatism. The goal of this study, was to investigate the mechanisms of physalin F associated with cell apoptosis in the renal carcinoma cells, A498. The results of this study showed that physalin F induced ROS generation and caused cell apoptosis. Because of the loss of the mitochondria membrane potential, cytochrome c was released into the cytosol and induced caspase activation resulting in apoptosis. Moreover, the phosphorylation of IĸBα was inhibited and prevented NF-ĸB nuclear translocation in physalin F-treated A498 cells. These phenomena were reversed by NAC and GSH. As measured by EMSA, physalin F blocked NF-ĸB activation in A498 cells. These findings suggest that ROS/NF-ĸB is involved in the physalin F-induced A498 cell apoptosis pathway. NPRL-Z-1 is a podophyllotoxin derivative and designed to enhance TOP2 inhibition, overcome drug resistance, and modulate water solubility of etoposide analogues by extending the bulky substituent at C7. In this study, NPRL-Z-1 induced DNA DSBs, TOP2 cleavage complexes formation, and ROS production in A498 cells. When ATM was activated by DSBs, p53 and p21 expression increased and cell cycle was arrested. Ultimately, NPRL-Z-1 induced cell apoptosis. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator, and had better cytotoxicity in RCC cell lines. Heteronemin is a bioactive marine sesterterpene isolated from the sponge Hyrtios sp. Previous reports have shown that heteronemin possesses anticancer activity. Here heteronemin displayed potent cytotoxic effects in A498 human renal carcinoma cells. Heteronemin initiates apoptotic cell death by down-regulating Bcl-2 and Bcl-xL, and up-regulating Bax, leading to the disruption of the mitochondrial membrane potential and the release of cytochrome c from the mitochondria. These effects were associated with the activation of caspase-3/-8/-9, followed by PARP cleavage. Furthermore, heteronemin inhibited the phosphorylation of ERK and Akt signaling pathways and activated p38 and JNK. The specific inhibition of the p38 pathway by SB203580 or p38 siRNA treatment reversed the heteronemin-induced cytotoxicity and apoptotic signaling. Heteronemin also induced autophagy in A498 cells, and treatment with chloroquine (autophagy inhibitor) or SP600125 (JNK inhibitor) inhibited autophagy and increased heteronemin-induced cytotoxicity and apoptotic signaling. Taken together, this study proposes a novel treatment paradigm in which the combination of heteronemin and autophagy inhibitors leads to enhanced RCC cell apoptosis.