Several earlier studies reported that UDCA could protect cells from the pro-apototic effects of a variety of reagents including more hydrophobic bile acids such as DCA (Botla et al., 1995; Cecilia et al., 1998). For the Alzheimer's disease drug, acetylcholinesterase inhibitor-tacrine has a side effect to induce hepatotoxicity, this study shows that the hepatoprotective effect of UDCA on tacrine-induced HepG2 cells. Better understanding of the mechanism underlying UDCA's protection on tacrine-induced hepatotoxicity may provide new concepts for the medical treatment of hepatotoxicity.In this study, we have shown that the hydrophobic bile acid UDCA has no toxic effect on HepG2 cells even up to 200 μM, so 25, 50, and 100 μM were selected to use in the experiment. Tacrine induces apoptotic cell death in HepG2 cells, and the appropriate concentration of THA treated to cells, 0.2 mM, which corresponded with Galisteo et al. (2000). A significantly increase of the proliferation of tacrine-treated HepG2 cells was performed by UDCA supplement. Based on the results, we expect that UDCA is a potential agent in the treatment of tacrine caused human hepatocyte injury.Here, we observed a DNA fragmentation occurred in tacrine-treated HepG2 cells. The cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleasome length (180-200 bp) (Huang et al., 1997) Their characteristic patterns revealed by agarose gel electrophoresis are widely used as one of the biomarkers of apoptosis. The other biochemical markers, a characteristic ultrastructural appearance and numerous fragmented nuclei observed under an electron microscope were displayed in tacrine-evoked HepG2 cells, but UDCA prevented these phenomena in dose-response manner. With these results we predict that exposure of HepG2 to tacrine induces apoptotic cell death.During apoptosis, PARP is selectively cleaved to 24 KDa and 85 KDa fragments representing the N-terminal DNA-binding domain and the C-terminal catalytic subunit, respectively, by several caspases, especially by caspase 3 (Zhao et al., 2006). In this experiment, following the degradation of procaspase 3, the caspase 3 substrate PARP minor 85 KDa fragment was detected after tacrine administration. Again, UDCA inhibited the cleavage of these proteins. Generally, once activated caspase 9 goes on to cleavage of caspase 3 and caspase 7 which cleave several cellular targets, including poly (ADP-ribose) polymerase. Therefore, consistent with these data, the expression of caspase 9 appeared the same tendency with caspase 3. Taken together, these results suggest that suppression of tacrine-induced hepatotoxicity by UDCA treatment in HepG2 cells was mainly caused by regulation of caspase activation through down-expression of cleaved caspase 3 and caspse 9.UDCA has been described as decreasing apoptosis via modulation of mitochondrial function by inhibiting mitochondrial membrane depolarization and cytochrome c release in hepatic and nonhepatic cells (Rodrigues et al., 1999, 1998). Since the activation of pro-caspase 9 associated with the release of cytochrome c from mitochondria to the cytosol-an indicator of apoptosis, we determined the expression of cytochrome c release into cytosolic fraction. Following the down-expression of cytochrome c release in cytoplasm, we obtained down-regulated cytosolic Bcl-2, Bcl-x_(L) in UDCA plus tacrine treated HepG2 cells. The cell death regulator molecules, Bcl-2 family regulates mitochondrial integrity and apoptosis, which include the pro-apoptotic Bax and the anti-apoptotic Bcl-2, Bcl-x_(L). Mechanistically, our data support the suppression of tacrine-induced apoptosis by UDCA through an inhibition of the mitochondria pathway: increased mitochondrial Bcl-2 and Bcl-x_(L), decreased mitochondrial Bax, release of cytochrome c, augmented pro-caspase 9 and pro-caspase 3 (through inhibition of cleavage) and suppressed PARP cleavage and DNA fragmentation.The detail mechanisms for how UDCA affects the mitochondria to inhibit apoptosis signaling as well as a possible involvement of the MAPK (ERK, JNK, p38MAPK) or the PI3K/AKT survival pathway is quite necessary. Our experiment revealed that UDCA treatment didn't lead to any alteration on the expression of phospho-ERK (data not shown). Conversely, we found that the magnitude of the PI3K activity was significantly increased by UDCA supplement in tacrine-induced HepG2 cells. In line with previous result, the level of the PI3K downstream effector, phospho-Akt was enhanced by UDCA in dose-dependent pattern. Next, we determined the Akt downstream protein, Bad. When another pro-apoptotic protein of Bcl-2 family Bad active, Akt phosphorylates Bad on Ser136, causing Bad to dissociate from the Bcl-2/Bcl-x_(L) complex and lose its proapoptotic function (Marlene et al., 2008). In agreement with previously published data, in our study, marked activation of Akt clearly activated Bad by UDCA in HepG2 cells. From these observations, we found that UDCA protect HepG2 from tacrine-induced hepatotoxicity by activation of Bad through the PI3K/Akt cell survival signal pathway.In the result of Akt activation, certain proteins are phosphorylated and lead to cell survival. For example, phosphorylation of IκB by Akt leads to activation of NFκB that promotes IAP family proteins. This study revealed that UDCA up-regulated NFκB-dependent gene activation and induced XIAP expression, possibly suggesting that UDCA protective effect on tacrine-induced apoptosis in HepG2 cells occurred partially via suppressing NFκB translocation from cytosol to nuclei and induction of XIAP expression.We further reported that the PI3K inhibitor, LY294002, can efficiently block the UDCA-induced activation of Akt, Bad, NFκB and XIAP. Furthermore, in contrast to UDCA effect, it increased the activation caspase 3, the cleavage of PARP and release of cytochrome c into cysolic fraction. This strongly suggests that PI3K/Akt dependent pathway displays an important role in UDCA protective effect on tacrine-induced apoptosis in HepG2 cells.Our observations presented in this study recommended that UDCA carried out its protective work on tacrine-treated HepG2 cells by activating a PI3K/Akt dependent survival signal that is mediated by NFκB and IAP family. This concept indicated that the net effect of UDCA in mediating hepatoprotection reflects a balance between pro- and anti-apoptotic processes. Therefore, the investigation of this experiment is novel in demonstrating alteration of mitochondrial function and inhibition of apoptosis by UDCA at physiologically relevant concentrations in tacrine-induced human hepatoma cell line (HepG2).
친수성 담즙산의 일종인 UDCA는 여러 가지 간의 질병, 말하자면 원발성 담즙성 간경변증 (PBC), 일차성 경화성 담관염 (PSC), 만성 C형 간염 등에 치료제로 널리 사용되고 있다. 콜린에스테라제의 길항적 저해제의 하나인 타크린은 치매치료제로써 간 세포독성을 지니고 있는 것으로 알려져 있다. UDCA를 간암세포 HepG2에 대한 독성확인을 위해 25μM, 50μM, 100μM의 농도로 UDCA를 처리한 결과 독성이 없음을 확인하였다. 타크린 0.2 mM을 UDCA 25μM, 50μM, 100μM와 동시에 처리한 결과 UDCA가 농도 의존적으로 간 세포보호 효과가 나타났음을 관찰할 수 있었다. 또 UDCA의 처리에 의하여 타크린이 일으킨 PARP, caspase 3와 caspase 9의 쪼개짐이 농도 의존적으로 억제 되였다는 것을 알 수 있었다. UDCA는 항암작용을 가지고 있는 PI3K의 발현을 증가시킴으로써 pAkt도 증가하는 것을 관찰할 수 있었고 또 다른 cell survival signal pathway인 NFκB를 활성화시킴으로써 apoptosis저해 단백질 가족인 IAP family의 발현을 증가시켰다. 나아가서는 Bcl-2 family인 Bad, Bax의 발현을 효과적으로 억제하고 Bcl-xL, Bcl-2의 발현양을 증가시킴으로써 미토콘드리아의 구멍의 뚫림에 의하여 생긴 Cytochrome c가 cytosol내로의 방출을 막아준다. 따라서 타크린이 HepG2에 일으킨 간 독성에 대한 UDCA의 간 세포보호 효과는 PI3K/pAkt의 신호전달 경로를 통한 전사인자의 발현과 이들 전사인자의 downstream에 존재하는 Bcl-2 family인 Bad, Bax의 발현억제와 Bcl-x_(L), Bcl-2의 발현증가를 통하여 결과적으로 DNA repair작용을 하는PARP의 쪼개짐을 억제하는 것으로 판단된다.