| Summary: | 碩士 === 國立臺灣師範大學 === 人類發展與家庭學系 === 101 === Abstract
The 2011 statistics of Department of Health (DOH) shows that human hepatocellular carcinoma (HCC) is the second death of cancer in Taiwan, and is the third death of cancer in the world. Chemotherapy is the traditional choice for advanced HCC, but the FDA-approved targeted therapy Sorafenib limits the therapeutic effect due to its intolerable side effects to patients. Thus, to develop new drugs for HCC is urgent. Justicidin A (JA) is a purified compound isolated from Justicia procumbens. Our previous published report indicates that JA induced apoptosis of human HCC cells in vitro and in vivo. Recently, many reports indicate that there are interactions between autophagy and apoptosis, and the autophagic effect induced by anticancer drugs may inhibit or promote apoptotic cell death. The present study is proposed to investigate if JA can induce autophagy of HCC and to find out how JA-induced autophagy influences the process of JA-induced apoptosis. When autophagy was induced in cells, acidic vescular organelles (AVOs) produced in cells. By staining cells with acridine orange, induction of autophagy can be investigated. In the present study, JA increased the percentages of AVOs-positive cells in a time- and dose-related manners using flow cytometry. The expressions of LC3-II and LAMP2a increased as the incubation time increased, but that of p62 decreased between 24 to 96 h. To confirm the process of autophagy flux, Hep 3B cells were pretreated with or without autophagy inhibitor Bafilomycin A1 (BAF) and then treated with JA. The increase in protein expression of LC3-II, p62 and LAMP2a using Western blot and the decrease in colocalization of LC3 and LAMP2a puncta using confocal microscopy in BAF-pretreated group confirm that JA not only induced autophagy but also completed the autophagy flux. In order to analyze the interaction between autophagy and apoptosis, Hep 3B cells were pretreated with BAF or pan-caspase inhibitor zVAD-fmk to inhibit autophagy and apoptosis, respectively. In BAF-pretreated Hep 3B cells, JA elevated the percentages of cells at sub-G1 phase and raised the expression of cleaved caspase 3. However, in zVAD-fmk-pretreated Hep 3B cells, no changes in the expression of autophagy-related LC3-II, p62 and LAMP2a proteins was observed, suggesting that using autophagy inhibitor promoted JA-induced apoptosis and JA-induced apoptosis didn’t affect JA-induced autophagy. Moreover, to investigate the molecules involved in JA-induced autophagic pathway, we observed that JA-induced autophagy was via activation of Raf/MEK/ERK pathway, and was not associated with inhibition of class I PI3K/Akt/mTOR or induction of class III PI3K/Beclin 1 pathway by western blot. To examine if addition of JA promotes chemosensitivity of Sorafenib, cell growth inhibition was analyzed using MTT assay. Addictive effect was found when cells were treated with 10 M Sorafenib, the dosage uses for patients, in combination with 0.5 or 1 M JA, suggesting the benefit of using JA in addition to Sorafenib. Taken together, our data suggest that the use of autophagy inhibitor should be taken into consideration to enhance the anticancer effect of JA and addition of JA promotes the chemosensitivity of FDA-approved targeted therapy Sorafenib.
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