類黃酮素及棉子酚抗腫瘤分子機轉之探討
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) 、 catal ase (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 digest ion 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 flavanon e 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 macro phages (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 23.51 M ； 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 COL O 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 (p 17/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 fa ctor (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 casp aes 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。
Anti-tumor Molecular Mechanism of Flavonoids and Gossypol
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, apopto sis-inducing and anti-metastasis activities of flavonoid derivatives with different functional groups substitution were evaluated. Hydroxylation, but not methoxylation, at C4′ and C6 of fla vanone 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 incre ases 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-rhamnos e) 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-indu ced apoptosis. A PKC activator, 12-O-tetradecaoylphorbol-13-acetate (TPA) significantly attenuates ME-induced apoptosis via preventing cytochrome c release to cytosol and maintainin g 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. M oreover, 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 h uman polymorphonuclear neutrophils (PMN), and murine peritoneal macrophages (PM). Furthermore, among thirty-six flavonoids examined in this study, ME was found to be the most p otent 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 23.51 M, respectively. ME, but not MI inhibition of MMP-2 enzyme activit
y 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 (PD980 59) 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 transloca tion 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 enh ances 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 ass ay 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 P KC activation avoid mitochondria destruction. ME also significantly blocked both endogenous and TPA-induced MMP-2 enzyme activity via inhibition of its protein expression and enzy me activity. The blockade involved suppression of PKC translocation, ERKs phosphorylation, and c-Jun protein expression. In structure activity relationship (SAR) discussion, hydroxyla tion 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 effe cts.
(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 cell s. 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 (T EM), and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS-induced ROS production with bl ocking the occurrence of apoptosis. GOS induces the dysfunction of mitochondria characterized by loss of mitochondria membrane potential accompanied by a release of cytocrome c (C yt C) and apoptosis-inducing factor (AIF) from mitochondria to cytoplasm. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of G OS 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 occurre nce 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-t umor effects of GOS via apoptosis induction, and apoptotic mechanism was also elucidated.
