The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model
碩士 === 國立東華大學 === 生命科學系 === 102 === Prostate cancer is one of the susceptible diseases observed among older men. Due to the gradual westernization of dietary and lifestyle habits, leading to prostate cancer incidence and mortality rates increased year by year. Antrodia cinnamomea (AC) is an unique m...
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ndltd-TW-102NDHU51080052019-07-16T03:44:59Z http://ndltd.ncl.edu.tw/handle/p7h8yz The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model 特定牛樟芝子實體純化物對人類前列腺癌細胞在動物實驗內之細胞凋亡機制探討 YU-HAO LUO 羅宇浩 碩士 國立東華大學 生命科學系 102 Prostate cancer is one of the susceptible diseases observed among older men. Due to the gradual westernization of dietary and lifestyle habits, leading to prostate cancer incidence and mortality rates increased year by year. Antrodia cinnamomea (AC) is an unique mushroom that grows in dark and humid conditions, and it specifically prefers to grow on the inner caving of the endemic species Cinnamomum kanehirae ( Bull camphor tree ), which is a very rare and one of conservation plants in Taiwan. AC contains abundant triterpenoids and polysaccharides, lot of previous researches have shown that AC has many pharmacological effects such as anti-cancer, hepatoprotective, and anti-oxidant, etc. The purpose of this study was to investigate the cytotoxic effect and molecular mechanism of AC’s bioactive components in prostate cancer cell lines both in vitro and in vivo with xenograft mode. This study was mainly divided into three parts: first, the main aim is to screen out the target compound from AC's fruiting body which can effectively inhibit the growth of prostate cancer cells. The results of MTT assay showed that FK5 expressed best inhibitory effect (IC50 70.36 μg / ml and 52.95 μg / ml in PC-3 and LNCaP, respectively) among all test fractions (FK1 to FK6). After that, K5-2 was further purified from FK5 by using semi-preparative HPLC column, which inhibits both PC-3 and LNCaP with IC50 were approximately 2.5 μg / ml but no toxicity to the normal cell CCD-966SK up to the concentration of 10 μg / ml. Microscopic examination found that many changes in cell morphology such as shrinkage and fragmentation after K5-2 treatment. Flow cytometry analysis showed that LNCaP’s after K5-2 treatment resulted in cell cycle arrested at SubG1 phase, but no significant change in PC-3. However, Annexin-V FITC & PI double staining showed that the percentage of apoptotic cells of PC-3 and LNCaP were increased with the concentration of K5-2 and reached a maximum at 5 μg / ml. The second part of this study was to investigate the effect of K5-2 on PC-3 cells in vivo by using xenograft in NOD-SCID mice to induce prostate tumors grow. Intraperitoneal injected series dosages of K5-2 in NOD-SCID mice; the results showed that K5-2 could effectively suppress the tumor volume and mass at concentration level of 10 mg/kg. Subsequently, TUNEL Assay showed that the proportion of apoptotic cells increased with K5-2 in dose-dependent manner. Finally, to elucidate the apoptotic pathway by using protein extracts from homogenized tumor tissue, Western blot results showed that K5-2 induced apoptosis both associated with intrinsic and extrinsic pathway. The results were summary as followings: both DR4 / DR5 and Fas death receptors were triggered and Caspase-8, -3 were activated after K5-2 treated. Down-regulation of Bcl-2 and up-regulation of Bax, Bad were observed along with the release of Cytochrome C. Subsequently, the level of Caspase-9, -3 were activated which induced apoptosis. The activation of Caspase-3 suppressed the PARP level which could repair DNA. Furthermore, the up-regulation of p53 was observed, which indirectly impacted apoptosis. Therefore, our results suggest that K5-2 may have a great potential to develop as a drug originating from medicinal fungus AC for prostate cancer treatment and will not damage normal. Tzong-Zeng Wu 吳宗正 2014 學位論文 ; thesis 134 |
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碩士 === 國立東華大學 === 生命科學系 === 102 === Prostate cancer is one of the susceptible diseases observed among older men. Due to the gradual westernization of dietary and lifestyle habits, leading to prostate cancer incidence and mortality rates increased year by year. Antrodia cinnamomea (AC) is an unique mushroom that grows in dark and humid conditions, and it specifically prefers to grow on the inner caving of the endemic species Cinnamomum kanehirae ( Bull camphor tree ), which is a very rare and one of conservation plants in Taiwan. AC contains abundant triterpenoids and polysaccharides, lot of previous researches have shown that AC has many pharmacological effects such as anti-cancer, hepatoprotective, and anti-oxidant, etc. The purpose of this study was to investigate the cytotoxic effect and molecular mechanism of AC’s bioactive components in prostate cancer cell lines both in vitro and in vivo with xenograft mode. This study was mainly divided into three parts: first, the main aim is to screen out the target compound from AC's fruiting body which can effectively inhibit the growth of prostate cancer cells. The results of MTT assay showed that FK5 expressed best inhibitory effect (IC50 70.36 μg / ml and 52.95 μg / ml in PC-3 and LNCaP, respectively) among all test fractions (FK1 to FK6). After that, K5-2 was further purified from FK5 by using semi-preparative HPLC column, which inhibits both PC-3 and LNCaP with IC50 were approximately 2.5 μg / ml but no toxicity to the normal cell CCD-966SK up to the concentration of 10 μg / ml. Microscopic examination found that many changes in cell morphology such as shrinkage and fragmentation after K5-2 treatment. Flow cytometry analysis showed that LNCaP’s after K5-2 treatment resulted in cell cycle arrested at SubG1 phase, but no significant change in PC-3. However, Annexin-V FITC & PI double staining showed that the percentage of apoptotic cells of PC-3 and LNCaP were increased with the concentration of K5-2 and reached a maximum at 5 μg / ml. The second part of this study was to investigate the effect of K5-2 on PC-3 cells in vivo by using xenograft in NOD-SCID mice to induce prostate tumors grow. Intraperitoneal injected series dosages of K5-2 in NOD-SCID mice; the results showed that K5-2 could effectively suppress the tumor volume and mass at concentration level of 10 mg/kg. Subsequently, TUNEL Assay showed that the proportion of apoptotic cells increased with K5-2 in dose-dependent manner. Finally, to elucidate the apoptotic pathway by using protein extracts from homogenized tumor tissue, Western blot results showed that K5-2 induced apoptosis both associated with intrinsic and extrinsic pathway. The results were summary as followings: both DR4 / DR5 and Fas death receptors were triggered and Caspase-8, -3 were activated after K5-2 treated. Down-regulation of Bcl-2 and up-regulation of Bax, Bad were observed along with the release of Cytochrome C. Subsequently, the level of Caspase-9, -3 were activated which induced apoptosis. The activation of Caspase-3 suppressed the PARP level which could repair DNA. Furthermore, the up-regulation of p53 was observed, which indirectly impacted apoptosis. Therefore, our results suggest that K5-2 may have a great potential to develop as a drug originating from medicinal fungus AC for prostate cancer treatment and will not damage normal.
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author2 |
Tzong-Zeng Wu |
author_facet |
Tzong-Zeng Wu YU-HAO LUO 羅宇浩 |
author |
YU-HAO LUO 羅宇浩 |
spellingShingle |
YU-HAO LUO 羅宇浩 The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
author_sort |
YU-HAO LUO |
title |
The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
title_short |
The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
title_full |
The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
title_fullStr |
The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
title_full_unstemmed |
The apoptotic mechanism of specific compound purified from Antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
title_sort |
apoptotic mechanism of specific compound purified from antrodia cinnamomea’s fruiting body in prostate carcinoma xenograft model |
publishDate |
2014 |
url |
http://ndltd.ncl.edu.tw/handle/p7h8yz |
work_keys_str_mv |
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