類黃酮素及棉子酚抗腫瘤分子機轉之探討
Anti-tumor Molecular Mechanism of Flavonoids and Gossypol
中文摘要
有許多類黃酮素化合物的生理活性已經廣泛的被報導，然而化學結構特徵對其 抗腫瘤活性效果之重要性仍尚未釐清。在本研究中，針對類黃酮素化合物化學結 構上的特徵，與其誘導腫瘤細胞凋亡及抑制腫瘤細胞轉移兩種生理活性進行分 析；實驗結果發現flavanone 結構上 C4’及 C6 上的氫氧基化顯現出有效誘導血癌 HL-60 細胞凋亡之毒性，而且 4’-OH flavanone 及 6-OH flavanone 均能誘導大量 內生性活化態氧的產生；抗氧化劑N-acetyl-cysteine (NAC)、catalase
(CAT) 、superoxide dismutase (SOD)及 allopurinol (ALL)，而非 pyrrolidine dithiocarbamate (PDTC)或 diphenylene iodonium (DPI)的前處理下，除了能抑制 上述兩化合物所誘導之活化態氧產生外，並同時可以抑制其所誘導之細胞凋亡 進行。而4’-OH flavanone 及 6-OH flavanone 所誘導之細胞凋亡過程，乃降低了 Mcl-1 蛋白的表現，並增加 Bax 及 Bad 的蛋白表現，透過 caspase-9 的活化 、 cytochrome c 的釋出，進而活化胞內 caspase-3 促使細胞凋亡的進行。除此之外，
另一類黃酮素化合物 myricetin (ME)，亦發現具有極明顯之抗腫瘤活性；ME 而 非其醣基化合物myricitrin (MI)，能有效造成粒線體功能的喪失、降低 Bcl-2/Bax 蛋白表現的比例、cytochrome c 的釋出，進而誘導 HL-60 細胞凋亡。而利用 ethidium bromide (EtBr)造成 HL-60 細胞粒線體的功能喪失，能顯著降低 ME 所 造成之細胞毒性，然而此一誘導粒線體功能喪失之毒性，非與活化態氧的產生 有關，因藉由DCHF-DA、DPPH assay 及 plasmid digestion 分析，除無法偵測到
自由基的產生及胞內氧化壓力的產生外，抗氧化劑NAC、CAT、SOD 及 TIR 的
前處理，亦無法抑制ME 所誘導的 HL-60 細胞死亡；而此 ME 誘導的 HL-60 細 胞凋亡過程可藉由PKC 活化劑 12-O-tetradecaoylphorbol-13-acetate (TPA)的前處 理，阻斷cytochrome c 的釋出、維持粒線體膜電位及抑制 Bcl-2/Bax 蛋白表現比
例的降低，進而抑制ME 所誘導的細胞凋亡進行；而 PKC 抑制劑，GF-
109203X、H7、 staurosporine 的前處理，可以阻斷 TPA 所提供的細胞保護作用，
證明PKC 的活化可以保護 ME 所誘導之細胞凋亡。而在其他血球細胞毒性分析
上，4’-OH flavanone、6-OH flavanone 及 ME 對另一血癌 Jurkat 細胞具細胞凋亡 毒性，而對成熟THP-1 血球細胞、正常人類 polymorphonuclear neutrophils (PMN)細胞及大鼠 peritoneal macrophages (PM)細胞則無觀察到任何明顯之毒性。
此外在抗腫瘤轉移方面，ME 而非 MI，能有效抑制腸癌細胞
COLO205、COLO320HSR、COLO320DM、HT29 及 COLO205-X 所產生之 MMP-2 活性，IC50 分別為 7.82、11.18、11.56、13.25 及 23.51M；ME 能直接抑制 MMP- 2 酵素活性並降低其蛋白表現，並可抑制 TPA 所誘導 PKC位移、ERK 磷酸化及 c-Jun 表現，達致抑制 MMP-2 活性表現。此外，利用體外 EHS matri-gel transwell 分析，ME 亦能有效抑制 TPA 所誘導之 COLO205 細胞轉移。總結本研究發現 4’-
OH flavanone、6-OH flavanone 及 ME 分別透過活化態氧或非活化態氧途徑，造
成粒線體功能喪失，致使HL60 細胞凋亡；另 ME 可以透過直接抑制內生性蛋
白生成、酵素活性或阻斷TPA 誘導 MMP-2 活化，而抑制 COLO205 細胞轉移。
(Gossypol)
Gossypol (GOS)是一存於棉花種子中多酚類化合物，在許多研究中指出能誘導 多種腫瘤細胞凋亡，然而其於體內之效應為何仍尚未釐清。本實驗中針對十三種
不同之動物細胞，觀察GOS 之細胞毒性；經由 MTT assay 實驗結果顯示，GOS
對人類直腸癌細胞包含HT29、COLO 205、COLO 320HSR 及 COLO 320DM 等細
胞，表現出特別顯著之細胞毒性，而此GOS 所造成之細胞毒性為誘導常癌細胞
凋亡所致，包含造成DNA 階梯狀斷裂、凋亡小體的產生以及染色體的皺縮等細
胞凋亡特徵。此外，GOS 誘導腸癌細胞 caspase 3, 6, 8, 9 而非 caspase 1 的活化，
造成PARP 蛋白(85kDa)的切割及 caspase 3 活化態(p17/p15)的產生，並造成抗凋 亡Bcl-xL 蛋白的降低以及促凋亡 Bad 蛋白的磷酸化；進一步利用 DCHF-DA assay 及 plasmid digestion 分析，GOS 除能產生自由基外，並會造成胞內氧化壓 力增加，致使大量活化態氧產生；而抗氧化劑N-acetyl-cysteine (NAC)、catalase (CAT)、tempol (TEM)及 melatonin (MEL)，而非 allopurinol (ALL)、pyrrolidine dithiocarbamate (PDTC)或 diphenylene iodonium (DPI)的投予，能顯著的抑制 GOS 所誘發的活化態氧產生，並同時抑制了 GOS 所誘導腸癌細胞凋亡之毒性。
而藉由誘導活化態氧產生，更促使細胞粒線體膜電位的流失、粒線體cytochrome
c (cyt c)及 apoptosis-inducing factor (AIF)的釋出，造成粒線體功能的喪失，引發 細胞凋亡；進一步利用ethidium bromide (EtBr)破壞細胞粒線體功能，結果發現
大幅降低GOS 所誘導之細胞死亡，顯示出粒線體於 GOS 誘導細胞凋亡作用中
之重要性。在裸鼠體內實驗方面，GOS 或 GAA (gossypol acetic acid)的投予，均 能有效抑制COLO205 細胞於皮下腫瘤的生長；在 ex vivo 實驗方面，GOS 亦能
對取自裸鼠初代培養之COLO205-X 細胞誘導細胞凋亡作用的進行，包含 DNA
階梯狀斷裂及caspaes 瀑布效應的活化。本研究首度提供了體內實驗數據證實
GOS 於活體的抗腫瘤效果，並歸納出一活化態氧依循性之粒線體必需之細胞凋 亡途徑。
英文摘要
Various biological activities of flavonoids had been reported, however the importance of structural characteristics in their anti-tumor effects is still undefined. In the present study, apoptosis-inducing and anti-metastasis activities of flavonoid derivatives with different functional groups substitution were evaluated. Hydroxylation, but not methoxylation, at C4′ and C6 of flavanone showed significant cytotoxic effect in human leukemia HL-60 cells accompanied by the characteristics of apoptosis.
Induction of endogenous ROS production was detected in 4′-OH or 6-OH flavanone- treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetyl-cysteine
(NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4′-OH or 6-OH flavanone-induced ROS production and apoptosis. A upstream caspase 9 activation, mitochondrial cytochrome c release, then caspase 3 cascade induction by 4′-OH or 6-OH flavanone, accompanied by a decrease in the anti-apoptotic Mcl-1 protein and increases in the pro-apoptotic proteins, Bax and Bad, were identified in apoptosis process. In addition, myricetin (ME) but not its respective glycoside myricitrin (MI; Myricetin-3-O-rhamnose) also reduces the viability of HL60 cells via apoptosis through mitochondrial dysfunction characterized by a decrease in Bcl-2/Bax protein ratio, and translocation of cytochrome c from mitochondria to cytosol. Removing mitochondria by ethidium bromide (EtBr) treatment reduces the apoptotic effect of ME. However, no significant induction of intracellular ROS level by ME was observed by DCHF-DA assay, DPPH assay, and plasmid digestion assay, and antioxidants including NAC, CAT, SOD, and tiron (TIR) show no protective effects on ME-induced apoptosis. A PKC activator, 12-O-
tetradecaoylphorbol-13-acetate (TPA) significantly attenuates ME-induced apoptosis via preventing cytochrome c release to cytosol and maintaining mitochondria
membrane potential with inhibiting a decrease in Bcl-2/Bax protein ratio, those effects were blocked by PKC inhibitors including GF-109203X, H7, and staurosporine.
Moreover, apoptosis-inducing activity of ME, 4′-OH or 6-OH flavanone was also observed in another leukemia cell line (Jurkat), but not found in mature monocytic cells (THP-1), normal human polymorphonuclear neutrophils (PMN), and murine peritoneal macrophages (PM). Furthermore, among thirty-six flavonoids examined in this study, ME was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells with an IC50 value of 7.82 M. And the inhibition of MMP-2 enzyme activity was also found in other colorectal carcinoma cells COLO 320HSR, COLO 320DM, HT 29 and COLO 205-X with IC50 values of 11.18, 11.56, 13.25 and 23.51M, respectively. ME, but not MI inhibition of MMP-2 enzyme activity was due to directly suppress MMP-2 enzyme activity with reduced MMP-2 protein expression.
In the TPA stimulation condition, an increase in MMP-2 protein expression and enzyme activity was observed in according with inducing PKC protein translocation, ERK1/2 protein phosphorylation, and c-Jun protein expression in COLO 205 cells.
ERKs inhibitor (PD98059) and PKC inhibitors (GF and H7), but not p38 inhibitor (SB203580) or JNK inhibitor (SP600125), significantly inhibited TPA-induced MMP-2 protein expression with reduced ERKs phosphorylation and c-Jun protein expression. Addition of ME but not MI suppressed TPA-induced MMP-2 protein expression with blocking the TPA-induced events including translocation of PKC
from cytosol to membrane, phosphorylation of ERK1/2 protein, and induction of c-
Jun protein expression in COLO 205 cells. Addition of PD98059 or GF significantly enhances the inhibitory effect of ME on MMP-2 enzyme activity induced by TPA.
Furthermore, ME, but not MI, suppressed TPA-induced invasion of COLO205 cells in in vitro invasion assay using EHS matrigel-coated transwells. Results of the present study indicated that 4′-OH or 6-OH flavanone induced apoptosis in HL-60 cells through a ROS-dependent mitochondrial pathway. In the contrast, apoptosis induced by ME is throguh a novel mitochondria-dependent, ROS-independent, pathway; and TPA protection of cells from ME-induced apoptosis via PKC activation avoid mitochondria destruction. ME also significantly blocked both endogenous and TPA- induced MMP-2 enzyme activity via inhibition of its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERKs
phosphorylation, and c-Jun protein expression. In structure activity relationship (SAR) discussion, hydroxylation at C4′ or C6 of flavanone, OH at C3’, C4’, and C5’
of ME is important for the apoptosis-inducing activities. Besides, glycosylation is negtive moiety in ME mediated anti-tumor effects.
(Gossypol)
Gossypol (GOS), a polyphenolic compound in cotton seeds, has been shown to induce apoptosis in several cancer cells, however the in vivo effect is unclear. In thirteen tested cells, GOS showed the most significant cytotoxic effect in human colorectal carcinoma cells including HT29, COLO205, COLO320HSR, COLO320DM by MTT assay. The cytotoxic effect of GOS was mediated by inducing the occurrence of apoptosis characterized by the occurrence of DNA ladders, apoptotic bodies, and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS-treated cells. A decrease in Bcl-xL and phosphorylated Bad proteins were found in cells under GOS treatment. GOS induction of ROS production was detected by in vitro plasmid digestion and an increase in intracellular peroxide level was observed in GOS-treated COLO 205 cells by DCHF-DA assay. Antioxidants including N-acetyl-cysteine (NAC), catalase (CAT), tempol (TEM), and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI),
significantly inhibited GOS-induced ROS production with blocking the occurrence of apoptosis. GOS induces the dysfunction of mitochondria characterized by loss of mitochondria membrane potential accompanied by a release of cytocrome c (Cyt C) and apoptosis-inducing factor (AIF) from mitochondria to cytoplasm. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO 205 cells. Furhtermore, i.p injection of GOS or GAA (gossypol acetic acid) significantly reduced the tumor growth of COLO205
cells injected subcutaneously in nude mice. A primary tumor cells (COLO205-X) derived from tumor specimen elicited by COLO205 were established, and GOS effectively induced apoptosis in COLO 205-X cells with the occurrence of apoptotic events including DNA fragmentations, caspases cascade, and PARP protein cleavage.
Results of the present study provide first evidences to demonstrate the in vivo anti- tumor effects of GOS via apoptosis induction, and apoptotic mechanism was also elucidated.
