臺灣博碩士論文加值系統

肝癌(Hepatocellular carcinoma,HCC)在人類的流行病學當中是一種極為廣泛且致命的癌症類別；在藥物的選擇上，四氫薑黃素(Tetrahydrocurcumin)已證實是薑黃素的主要代謝產物，而薑黃素是屬於天然的多酚類化合物，是一種從薑黃(Curcuma longa)根莖中萃取得到的黃色色素，根據先前研究發現薑黃素的藥理活性較不穩定且不易被人體腸胃道所吸收，於是作用於血液中的濃度並不高因而限制了其療效。近幾年研究指出四氫薑黃素與薑黃素間具有類似的生物活性並且比薑黃素本身還要穩定；在廣泛的研究中指出其具有抗氧化、抗發炎、抗癌及保肝等作用，然而其中的作用機制及如何誘導肝癌細胞死亡的原因至今仍無文獻顯示。
從我們的研究中證實四氫薑黃素能夠明顯抑制肝癌細胞株的生長，在特定濃度處理下發現四氫薑黃素對於肝癌細胞並非顯示典型的細胞凋亡特徵，也非藉由細胞週期性停滯以抑制肝癌細胞的生長。於是我們根據細胞自噬的特性，證實在處理以四氫薑黃素的肝癌細胞明顯地促進酸性自噬囊狀胞器及EGFP-LC3 punctas的形成。而在西方墨點法也證實了四氫薑黃素的確能夠增加細胞自噬相關蛋白表現量，且可能透過下調控PI3K/Akt-mTOR訊息傳遞路徑來達到活化細胞自噬的作用以利肝癌細胞死亡。接著我們以細胞自噬抑制劑去抑制四氫薑黃素所誘導細胞自噬的過程，發現肝癌細胞並無復甦的現象反而加速四氫薑黃素轉而啟動肝癌細胞選擇走向另一途徑之凋亡型式作用的死亡方式。
化療藥物艾黴素(Doxorubicin)對於肝癌病患在臨床治療時所帶來的不良影響及抗藥性至今仍是相當棘手的問題，於是我們透過四氫薑黃素合併低劑量艾黴素的策略，經藥物等效線圖及合併指數分析兩藥物間的合併作用對於肝癌細胞屬於協同效應關係；發現其藥物合併效果有效提升HepG2肝癌細胞凋亡的作用。
總結上述結果，四氫薑黃素確實能夠透過誘導細胞自噬的作用以抑制肝癌細胞的生長，並且我們也首度證實四氫薑黃素能夠提升艾黴素抑制腫瘤生長的活性，但其機制仍需更進一步的探討和評估，對於日後在肝癌標靶藥物或是臨床輔助用藥的選擇上其非常具參考價值及開發的潛力。

Hepatocellular carcinoma(HCC) is among the most lethal and prevalent cancers in human epidemiology. On the choice of drugs, Tetrahydrocurcumin has been demonstrated to be the major metabolite of curcumin.Curcumin is a natural polyphenol compound of yellow pigment, derived from the rhizome of the Curcuma longa plant. According to previous studies found that the pharmacological activity of curcumin is not stable in vivo. Furthermore,it is difficulty absorbed through the gastrointestinal tract. Acting on the blood concentration is not high thus may result in limited its efficacy. Recently, previous studies showed that tetrahydrocurcumin has similar same physiological and pharmacological properties as the active form of curcumin in vivo and it’s more stable than curcumin. Tetrahydrocurcumin has been widely studied due to its potential antioxidant, anti-inflammatory, and anticarcinogenic activities as well as hepatoprotective. However, the mechanism of action and how it causes hepatocellular carcinoma cell death still no literature shows.
The present studies demonstrate that Tetrahydrocurcumin significantly inhibits hepatocellular carcinoma cell proliferation.When hepatocellular carcinoma cell were treated with Tetrahydrocurcumin under the specific concentration,it neither displays features typical apoptosis nor cell cycle arrest to inhibit cancer cell growth.So we based on the characteristics of autophagy,our studies demonstrate that Tetrahydrocurcumin significantly promote autophage marker acidic vascular organelles and EGFP-LC3 punctas formation. At the molecular levels, the results showed that Tetrahydrocurcumin significantly increased autophagy-related protein expression.In addition,Tetrahydrocurcumin may induced autophagic cell death through modulation of PI3K/Akt-mTOR signal transduction pathyway in human hepatocellular carcinoma cells.Then we further suppressed tetrahydrocurcumin -induced autophagy process by autophagy inhibitors.The results showed that autophagy inhibitor didn’t make hepatocellular carcinoma cells revive.On the contrary,it accelerated tetrahydrocurcumin choose to switch another way of apoptosis.
In clinical,Doxorubicin has been commonly used as a chemotherapeutic drug in the treatment of hepatocellular carcinoma patients.But it tend to cause some adverse effects and drug resistance for patients ,this is still thorny problem so far. Accordingly, we aim to investigate the effects of tetrahydrocurcumin combined with doxorubicin for treatment strategy. The results from Isobologram assay and drugs combination index analysis showed that the effects is belong to the synergy in hepatocellular carcinoma cells between the two drugs.We found that the combined effect of drugs effectively enhanced apoptosis in HepG2 cells.
Taken toghter, our present studies demonstrated tetrahydrocurcumin could induce autophagy to inhibit the growth of human hepatocellular carcinoma cells in vitro.And we also have shown for the first time that tetrahydrocurcumin could augment antitumor activity of doxorubicin in vivo.But the mechanism of tetrahydrocurcumin still needs further discussion and evaluation in hepatocellular carcinoma.For future,we provided a choice of targeted drugs and adjuvant drugs for clinical treatment.This is a reference value and has a potential application for hepatocellular carcinoma.

中文摘要....................................................i
英文摘要...................................................ii
目 錄..................................................iii

第 一 章 緒論..............................................1

第 一 節...................................................1

一、肝臟的構造與生理功能.......................................1
二、肝癌....................................................3
三、肝癌的成因及臨床診斷治療....................................3

第 二 節...................................................10

一、四氫薑黃素..............................................10
二、艾黴素.................................................11
三、中西藥物合併艾黴素之策略於肝癌的研究.........................12

第 三 節...................................................13

一、細胞週期................................................13
二、細胞凋亡................................................15
三、細胞自噬................................................17
四、細胞自噬與細胞凋亡間的關聯性................................19

第 二 章 研究目的..........................................20

第 三 章 研究材料與方法.....................................21

一、細胞株培養..............................................21
二、人類周邊血液單核細胞分離...................................24
三、藥物製備................................................25
四、細胞存活率檢測I-MTT assay................................26
五、細胞存活率檢測II-CCK-8 assay.............................27
六、細胞型態觀察.............................................27
七、細胞週期分佈檢測.........................................28
八、細胞凋亡檢測.............................................29
九、細胞自噬之酸性囊狀胞器檢測.................................31
十、EGFP-LC3 punctas螢光影像檢測.............................33
十一、粒線體膜電位測定........................................38
十二、細胞內氧化自由基濃度檢測.................................39
十三、西方墨點法.............................................41
十四、細胞自噬抑制劑之於細胞功能分析............................49
十五、藥物協同作用分析........................................50
十六、異種移植動物模式實驗....................................52
十七、分析與統計方法.........................................54

第 四 章 研究結果..........................................55

一、Tetrahydrocurcumin能夠有效抑制肝癌細胞的生長且對於人類周邊血液單
核細胞毒殺性作用並不會造成太大的影響.........................55
二、Tetrahydrocurcumin對於肝癌細胞HepG2及Hep3B具有抑制其生長的能力
且促成癌細胞型態上的變化...................................55
三、Tetrahydrocurcumin並非促使HepG2及Hep3B肝癌細胞走向細胞凋亡主導
之型式..................................................56
四、Tetrahydrocurcumin並非主要藉由細胞週期性停滯來抑制HepG2及Hep3B
肝癌細胞生長的能力........................................57
五、Tetrahydrocurcumin並無明顯誘導HepG2及Hep3B肝癌細胞之細胞凋亡相
關蛋白的表現量...........................................58
六、Tetrahydrocurcumin能夠誘導HepG2及Hep3B肝癌細胞之酸性囊狀胞器
(Acidic vesicular organelles,AVOs)的形成................58
七、Tetrahydrocurcumin明顯誘導HepG2及Hep3B肝癌細胞之細胞自噬相關蛋
白的表現量..............................................59
八、Tetrahydrocurcumin能夠誘導HepG2及Hep3B肝癌細胞中EGFP-LC3
punctas的形成...........................................60
九、Tetrahydrocurcumin並非藉由增加HepG2及Hep3B肝癌細胞之活性氧物質
(Reactive oxygen species,ROS)的累積來誘導細胞自噬的作用....60
十、Tetrahydrocurcumin並無造成HepG2及Hep3B肝癌細胞之胞內粒腺體膜電
位(Mitochondrial membrane potential, ΔΨm)的去極化及損傷..61
十一、Tetrahydrocurcumin並無明顯誘導HepG2及Hep3B肝癌細胞之內質網逆
境標誌蛋白的表現量......................................62
十二、Tetrahydrocurcumin透過調控PI3K/Akt-mTOR訊息傳遞路徑以誘導肝
癌細胞之細胞自噬作用....................................63
十三、藉由細胞自噬抑制劑(Autophagy inhibitors)啟動
Tetrahydrocurcumin誘導肝癌細胞之細胞凋亡作用的進行.........64
十四、Tetrahydrocurcumin合併化療藥物Doxorubicin後有效提升抑制肝癌
細胞HepG2和Hep3B的生長能力及藥物協同效應..................66
十五、Tetrahydrocurcumin合併化療藥物Doxorubicin後明顯促進肝癌細胞
HepG2細胞凋亡的作用並且降低細胞自噬的能力而Hep3B則不明顯.....67
十六、Tetrahydrocurcumin合併化療藥物Doxorubicin後明顯促使肝癌細胞
HepG2之DNA碎裂的情形發生及誘導Hep3B細胞週期性由G2/M期往前一階段
S期停滯累積的現象.......................................69
十七、Tetrahydrocurcumin合併化療藥物Doxorubicin後明顯抑制HepG2腫瘤
的生長................................................69

第 五 章 研究討論..........................................71

第 六 章 參考文獻..........................................79

第 七 章 實驗結果圖.........................................90

圖一、Tetrahydrocurcumin對於肝癌細胞株及正常人類周邊血液單核細胞之增
生作用的影響...........................................90
圖二、Tetrahydrocurcumin對於肝癌細胞HepG2及Hep3B之細胞型態的影響
.....................................................91
圖三、Tetrahydrocurcumin對於肝癌細胞HepG2之細胞凋亡特性的影響....92
圖四、Tetrahydrocurcumin對於肝癌細胞Hep3B之細胞凋亡特性的影響....93
圖五、Tetrahydrocurcumin對於肝癌細胞HepG2之細胞週期性分佈的影響..94
圖六、Tetrahydrocurcumin對於肝癌細胞Hep3B之細胞週期性分佈的影響..95
圖七、Tetrahydrocurcumin對於肝癌細胞HepG2及Hep3B之細胞凋亡與相關蛋
白變化的影響...........................................96
圖八、Tetrahydrocurcumin對於肝癌細胞HepG2之酸性自噬溶酶體囊狀胞器的
生成作用..............................................97
圖九、Tetrahydrocurcumin對於肝癌細胞Hep3B之酸性自噬溶酶體囊狀胞器的
生成作用..............................................98
圖十、Tetrahydrocurcumin對於肝癌細胞HepG2之細胞自噬相關蛋白變化的影
響...................................................99
圖十一、Tetrahydrocurcumin對於肝癌細胞Hep3B之細胞自噬相關蛋白變化的
影響..............................................100
圖十二、Tetrahydrocurcumin對於誘導肝癌細胞HepG2及Hep3B之細胞內EGFP
-LC3 punctas的形成作用.............................101
圖十三、Tetrahydrocurcumin對於肝癌細胞HepG2之細胞內活性氧自由基累積
的影響.............................................102
圖十四、Tetrahydrocurcumin對於肝癌細胞Hep3B之細胞內活性氧自由基累積
的影響.............................................103
圖十五、Tetrahydrocurcumin對於肝癌細胞HepG2之細胞內粒線體膜電位的影
響................................................104
圖十六、Tetrahydrocurcumin對於肝癌細胞Hep3B之細胞內粒線體膜電位的影
響................................................105
圖十七、Tetrahydrocurcumin對於肝癌細胞HepG2及Hep3B之內質網壓力標誌
蛋白變化的影響......................................106
圖十八、Tetrahydrocurcumin對於調控肝癌細胞HepG2之PI3K/Akt-mTOR訊
息傳遞路徑的影響.....................................107
圖十九、Tetrahydrocurcumin對於調控肝癌細胞Hep3B之PI3K/Akt-mTOR訊
息傳遞路徑的影響.....................................108
圖二十、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞HepG2之細胞
增生作用及LC3蛋白表現量的影響.........................109
圖二十一、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞Hep3B之細
胞增生作用及LC3蛋白表現量的影響.......................110
圖二十二、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞HepG2之細
胞凋亡作用的影響....................................111
圖二十三、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞Hep3B之細
胞凋亡作用的影響....................................112
圖二十四、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞HepG2之細
胞週期性分佈的影響..................................113
圖二十五、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞Hep3B之細
胞週期性分佈的影響..................................114
圖二十六、細胞自噬抑制劑對於Tetrahydrocurcumin處理肝癌細胞HepG2及
Hep3B之細胞凋亡相關蛋白表現量的影響...................115
圖二十七、Tetrahydrocurcumin合併化療藥物Doxorubicin處理於肝癌細胞
HepG2之細胞增生的影響及藥物合併作用之等效線圖分析........116
圖二十八、Tetrahydrocurcumin合併化療藥物Doxorubicin處理於肝癌細胞
Hep3B之細胞增生的影響及藥物合併作用之等效線圖分析........117
圖二十九、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
HepG2之細胞凋亡作用的影響...........................118
圖三十 、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
Hep3B之細胞凋亡作用的影響...........................119
圖三十一、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
HepG2及Hep3B之細胞自噬作用的影響.....................120
圖三十二、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
HepG2之細胞週期性分佈的影響..........................121
圖三十三、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
Hep3B之細胞週期性分佈的影響..........................122
圖三十四、Tetrahydrocurcumin合併化療藥物Doxorubicin對於肝癌細胞
HepG2及Hep3B之細胞自噬與細胞凋亡相關標誌蛋白的影響......123
圖三十五、Tetrahydrocurcumin與化療藥物Doxorubicin單一和合併處理於
HepG2肝癌細胞異種移植動物模式之腫瘤生長的影響...........124
圖三十六、本研究的實驗結果總結示意圖...........................125

第 八 章 附圖與附表........................................126

附圖一、美國癌症協會於西元2013年調查統計當地民眾罹患十大癌症之診斷案例與
死亡率排名統計表.....................................126
附圖二、薑黃素與四氫薑黃素以及艾黴素之化學結構示意圖..............127
附圖三、細胞週期之週期蛋白與其激酶調控示意圖.....................128
附圖四、細胞凋亡之內源性途徑與外源性途徑調控示意圖................129
附圖五、細胞自噬種類與酸性囊狀胞器形成調控示意圖..................130
附圖六、啟動細胞自噬之訊息傳遞路徑調控示意圖.....................131
附圖七、細胞自噬與細胞凋亡間的差異性及相互調控示意圖..............132
附圖八、探討Tetrahydrocurcumin與Doxorubicin單一或合併處理於異種移植
肝癌動物模式之試驗設計示意圖...........................133
附表一、行政院衛生署國民健康局於民國98年調查統計國人男性罹患十大癌症之發
生率與死亡率排名統計表................................134
附表二、行政院衛生署國民健康局於民國98年調查統計國人女性罹患十大癌症之發
生率與死亡率排名統計表................................135
附表三、Tetrahydrocurcumin對於肝癌細胞及人類周邊血液單核細胞之半數
抑制濃度(IC50)換算..................................136
附表四、Tetrahydrocurcumin與Doxorrubicin單獨及合併處理於HepG2和
Hep3B肝癌細胞24小時後，其半數抑制濃度(IC50)換算及藥物協同效應
分析..............................................137
附表五、Tetrahydrocurcumin與Doxorrubicin單獨及合併處理於HepG2和
Hep3B肝癌細胞48小時後，其半數抑制濃度(IC50)換算及藥物協同效應
分析..............................................138

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