Dihydrodenbinobin在人類賀爾蒙不依賴型前列腺癌細胞之抗癌作用機轉探討

Abstract

Dihydrodenbinobin為對天然抗癌活性物質denbinobin做結構修飾之全合成衍生物，能夠抑制賀爾蒙不依賴型前列腺癌PC-3細胞株的生長，利用SRB assay測定其GI50濃度為3.45 μM。使用流式細胞儀分析細胞內DNA含量，發現dihydrodenbinobin隨時間與濃度增加可顯著的增加sub-G1細胞的數量，但沒有觀察到細胞週期停滯。透過JC-1染劑測量粒線體之膜電位，發現JC-1之綠色螢光單體形式增加，表示有粒線體損傷之情形。接著利用西方墨點法觀察細胞內蛋白質的表現情形，發現AMPK在Thr172的磷酸化增加、其下游之蛋白如p70S6KThr389、p70S6KThr421/Ser424、4E-BP1Thr37/46磷酸化降低以及eIF4ESer209之磷酸化增加，mTOR之磷酸化表現則不受影響。值得注意的是，dihydrodenbinobin引起之AMPK活化，並不是透過上游激酶LKB1及CaMkkβ所影響，有可能是直接透過ATP的減少所調控。另外，加藥處理所造成之細胞死亡可以被p38 MAPK抑制劑SB203580所抑制，表示p38 MAPK在dihydrodenbinobin造成的細胞死亡中扮演了重要的角色。使用DCFH-DA染劑發現dihydrodenbinobin可在短時間內使活性氧物質 (ROS) 增加；抗氧化劑N-acetyl-L-cysteine亦可有效抑制dihydrodenbinobin引起之細胞死亡與蛋白質表現情形。利用kinase assay與西方墨點法皆可發現dihydrodenbinobin具有抑制Aurora A kinase之活性。總結來說，dihydrodenbinobin可透過活化AMPK、p38 MAPK與抑制Aurora A的活性，導致PC-3前列腺癌細胞的死亡。

Dihydrodenbinobin, a total synthetic compound through structural modification of the naturally occurring anti-cancer agent denbinobin, was effective against the androgen-independent prostate cancer PC-3 cells, with a concentration at 50% inhibition of proliferation (GI50) value of 3.45 μM measured by sulforhodamine B assay. The flow cytometric analysis of DNA content revealed that dihydrodenbinobin significantly increased the sub-G1 population of PC-3 cells in a time- and concentration-dependent manner without any effect on phase arrest of the cell cycle. JC-1 dye was used to measure mitochondrial membrane potential in this study. The data demonstrated that an increase of JC-1 monomers (green fluorescence) was detected indicating mitochondrial damages. Dihydrodenbinobin induced the phosphorylation of AMP-activated protein kinase (AMPK) at Thr172, and the downstream proteins were also affected, including decreased p70S6K phosphorylation at Thr389 and Thr421/Ser424, 4E-BP1 phosphorylation at Thr37/46 and increased eIF4E phosphorylation at Ser209, though mTOR activity was not modified after digydrodenbinobin treatment. Notably, neither the AMPK upstream kinase LKB1 was affected by dihydrodenbinobin nor CaMkkβ inhibitor STO609 rescued the modified downstream signals. The activation of AMPK might result from the reduction of ATP level in cell. In addition, the cell death caused by dihydrodenbinobin was completely blocked by SB203580, an inhibitor of the p38 MAPK, indicating the important role of p38 MAPK. A short-term increase of reactive oxygen species (ROS) generation was also detected in the exposure to dihydrodenbinobin, while antioxidant N-acetyl-L-cysteine can reverse cell death as well as protein expressions. Moreover, both kinase assay and Western blotting showed that dihydrodenbinobin displayed an inhibitory activity against Aurora A. In summary, the data suggest that dihydrodenbinobin induces anticancer activity through the activation of AMPK and p38 MAPK. The anti-Aurora A activity may also contribute to dihydrodenbinobin -mediated apoptotic mechanism in prostate cancer cells