丹皮酚及雷公藤内酯醇的抗黑素瘤作用及其机制的研究

发布时间：2018-06-25 17:14

本文选题：丹皮酚 + 雷公藤内酯醇　；参考：《北京协和医学院》2012年博士论文

【摘要】：皮肤恶性黑素瘤(CMM)因其易转移、对细胞毒药物耐受等特点而被认为是最具侵袭性和致死率最高的肿瘤之一。近年来该病的发病率和死亡率持续增高,但仍没有理想的治疗药物。因此,寻求治疗黑素瘤的新型药物十分必要。雷公藤内酯醇及丹皮酚作为具有多种药理活性的中药单体,已在多项研究中被证实具有抗肿瘤作用,但抗黑素瘤的作用尚未见报道。本研究旨在探讨此两种中药单体在抗黑素瘤方面发挥的作用及其机制。 (1)丹皮酚和雷公藤内酯醇对黑素瘤细胞增殖的影响利用CCK8方法检测了药物作用后对黑素瘤A375和M14细胞增殖抑制率的影响,并用PI染色-流式细胞术检测了药物对两种黑素瘤细胞细胞周期的影响。结果显示,0.5mM,1mM,2mM,4mM,8mM丹皮酚作用24,48,72h均对A375细胞有抑制作用,各时间点的半数抑制浓度(IC50)分别约为5.19mmM,1.50mM和1.09mM；丹皮酚对M14细胞24,48和72h的半数抑制浓度(IC50)分别约为4.11mM,1.60mM和1.17mM；且可以将细胞阻滞在G2/M期。12.5nM,25nM,50nM,100nM雷公藤内酯醇作用24,48,72h对A375细胞也均有抑制作用,其IC50分别约为84.46nM,33.00nM和8.53nM；雷公藤内酯醇对M14细胞24、48和72h的半数抑制浓度(IC50)分别约为130.90nM,35.90nM和6.50nM；而雷公藤内酯醇是将细胞阻滞在S期。 (2)丹皮酚和雷公藤内酯醇对黑素瘤细胞凋亡的影响利用Hoechst33258染色,荧光显微镜下观察细胞的形态,以流式细胞术检测药物作用下细胞的凋亡率变化。结果表明,分别以5mM丹皮酚作用24h,或30nM雷公藤内酯醇作用48h后,黑素瘤A375和M14细胞均出现凋亡的形态学变化。以0,1.25,2.5,5mM丹皮酚作用24h后,A375细胞的早期凋亡率分别为3.11%±0.53%,13.74%士1.73%,25.95%±0.57%和46.44%±0.81%；M14细胞的早期凋亡率则分别为1.00%±0.08%,2.00%±0.01%,2.99%±0.29%和14.73%±0.94%,各作用组与对照组相比均有统计学意义(P0.05)。0nM,10nM,20nM,30nM雷公藤内酯醇作用48h后,A375细胞的凋亡率分别为4.20%±0.09%,19.50%±0.32%,36.57%±3.85%和58.68%±2.03%；M14细胞的早期凋亡率则分别为2.92%±0.17%,20.99%±0.40%,34.28%±2.04%和63.38%-±0.71%,各作用组与对照组相比均有统计学意义(P0.05)。两种化合物诱导凋亡作用均呈剂量依赖关系。 (3)丹皮酚和雷公藤内酯醇对黑素瘤细胞凋亡机制的研究在上述(1)和(2)中,观察到A375细胞对丹皮酚和雷公藤内酯醇的诱导凋亡作用较M14细胞敏感,为此在本部分实验中,选择A375细胞进行药物诱导黑素瘤细胞凋亡机制的相关研究。本部分实验中检测了caspase3, caspase8和caspase9活性的变化,并利用Western Blot测定p53和NF-κB及其相关蛋白水平的变化。实验结果显示,1.25mM,2.5mM,5mM丹皮酚作用于黑素瘤细胞株A375细胞24h后,caspase3、caspase8及caspase9的活性均发生具有统计学意义的升高；p53及Bax的蛋白水平随药物浓度的增加而升高,而NF-κB、Bcl-2、Bcl-XL的蛋白水平随药物浓度增加而降低,表明丹皮酚对黑素瘤A375细胞的促凋亡作用不仅通过caspase和NF-κB通路,还通过激活p53通路诱导凋亡。以10nM,20nM,30nM雷公藤内酯醇作用于黑素瘤A375细胞株48h后,caspase3和caspase9的活性随浓度的升高而增加,而caspase8的活性则随浓度的升高而略有下降；NF-κB、Bcl-2、 Bcl-XL的蛋白水平随药物浓度增加而降低,表明雷公藤内酯醇仅通过caspase和NF-κB通路诱导黑素瘤A375细胞凋亡。 (4)丹皮酚和雷公藤内酯醇对黑素瘤细胞侵袭的影响及其机制的研究侵袭、转移是恶性肿瘤最本质的特性。本研究采用细胞划痕实验及transwell实验,观察药物对A375细胞侵袭性和迁移能力的影响,结果表明,两种中药单体均对黑素瘤A375和M14细胞具有抑制侵袭的作用。以1.25mM丹皮酚作用24h后,A375细胞的迁移距离由对照组的197.00±11.14缩短至46.67±5.69,M14细胞的迁移距离则由对照组的172.67±9.29缩短至33.00±6.24,药物作用组与对照组结果比较差异均具显著统计学意义(P0.01)；分别以1.25mM,2.5mM,5mmM丹皮酚作用24h后,穿过人工基底膜的A375细胞数由对照组的180.67±13.65分别减少至131.67±10.41,74.33±3.21和33.33±4.16；穿膜的M14细胞数也由对照组的306.00±7.55依次减少至253.67±14.01,150.33±9.6和55.33±5.69,各组与相应对照组比均有明显统计学差异(P0.01)。10nM雷公藤内酯醇作用48h后,A375胞的迁移距离由对照组的286.67±17.62缩短至185.67±7.09,M14细胞的迁移距离则由对照组的415.67±19.76缩短至213.33±10.02,药物作用组与对照组结果比较差异均具显著统计学意义(P0.01)；分别以10nM,20nM,30nM雷公藤内酯醇作用48h后,穿过人工基底膜的A375细胞数由对照组的183.00±10.82分别减少为143.67±7.77,60.67±4.04和27.33±2.52；穿膜的M14细胞数也由对照组的129.67±7.23减少至80.33±2.51,65.00±5.00和20.33±1.53,各组与相应对照组比均有明显统计学差异(P0.01)。本研究以Western Blot、Real-time PCR检测uPA和VEGF的基因表达和蛋白水平的变化,以探讨这两种药物影响人黑素瘤肿瘤细胞侵袭性和迁移能力的可能的机制,结果表明,丹皮酚对uPA和VEGF的基因表达量随浓度增加逐渐升高：0mM(对照组),1.25mM,2.5mM和5mM的结果分别为uPA：0.20±0.02,0.28±0.01,0.38±0.01和1.97±0.21；VEGF:0.59±0.06,0.79±0.04,2.27±0.02和3.61±0.21,而uPA和VEGF的蛋白水平明显降低,0mM(对照组),1.25mM,2.5mM和5mM组的uPA表达与β-actin的灰度比值分别为1.01、0.89、0.68、和0.46,其VEGF表达与β-actin的灰度比值分别为1.13、1.00、0.92和0.52。雷公藤内酯醇对uPA和VEGF的基因表达量随浓度增加逐渐升高：0nM(对照组),10nM,20nM和30nM的结果分别为uPA：0.20±0.02,0.41±0.00,0.95±0.05和5.84±0.16；VEGF:0.59±0.06,0.45±0.11,0.89±0.08和1.00±0.03,而uPA和VEGF的蛋白水平明显降低,0nM(对照组),10nM,20nM和30nM组的uPA表达与β-actin的灰度比值分别为1.01、0.81、0.44和0.15,其VEGF表达与β-actin的灰度比值分别为1.13、1.46、1.34和0.96。表明丹皮酚和雷公藤内酯醇可能通过降低VEGF和uPA的蛋白水平而抑制黑素瘤细胞的侵袭。结论丹皮酚和雷公藤内酯醇均可以抑制人黑素瘤细胞A375和M14增殖,其中丹皮酚的抑制机制可能与抑制黑素瘤细胞在G2/M周期阻止细胞有丝分裂有关,而雷公藤内酯醇的作用机制可能与抑制细胞在S期抑制细胞DNA复制有关。丹皮酚和雷公藤内酯醇能剂量依赖性地诱导人黑素瘤A375和M14细胞凋亡。其中雷公藤内酯醇通过caspase和NF-κB通路诱导人黑素瘤细胞凋亡；而丹皮酚不仅通过caspase和NF-κB通路,还通过激活p53通路诱导黑素瘤A375细胞凋亡。丹皮酚和雷公藤内酯醇均可通过抑制VEGF和uPA的蛋白表达抑制黑素瘤细胞的侵袭和迁移。丹皮酚和雷公藤内酯醇均具有抗黑素瘤的作用,具有皮肤黑素瘤治疗新药的研发前景。
[Abstract]:Cutaneous malignant melanoma (CMM) is considered to be one of the most invasive and fatal tumors because of its easy metastasis and tolerance to cytotoxic drugs. In recent years, the incidence and mortality of the disease have continued to increase, but there is still no ideal treatment. Therefore, it is necessary to seek new drugs for the treatment of melanoma. And paeonol, a Chinese medicine monomer with a variety of pharmacological activities, has been proved to have antitumor effect in a number of studies, but the role of anti melanoma has not yet been reported. The purpose of this study is to explore the role and mechanism of these two kinds of monomers in the anti melanoma.
(1) effects of Paeonol and triptolide on the proliferation of melanoma cells
The effect of drug effect on the proliferation inhibition of melanoma A375 and M14 cells was detected by CCK8, and the effect of drugs on the cell cycle of two melanoma cells was detected by PI staining flow cytometry. The results showed that 0.5mM, 1mM, 2mM, 4mM, and 8mM paeonol had inhibitory effect on A375 cells, and half of the time points were inhibited. The concentration (IC50) was about 5.19mmM, 1.50mM and 1.09mM, and the inhibitory concentration (IC50) of Paeonol on M14 cells 24,48 and 72h (IC50) was about 4.11mM, 1.60mM and 1.17mM, respectively. M, 33.00nM and 8.53nM; the median inhibitory concentration (IC50) of triptolide to M14 cells 24,48 and 72h (IC50) was approximately 130.90nM, 35.90nM and 6.50nM, while triptolide was blocked in the S phase.
(2) effects of Paeonol and triptolide on the apoptosis of melanoma cells
Using Hoechst33258 staining, the morphology of cells was observed under the fluorescence microscope, and the apoptosis rate of cells under the action of drug was detected by flow cytometry. The results showed that the apoptosis of melanoma A375 and M14 cells was changed after the action of 5mM paeonol acting 24h or 30nM triptolide acting as 48h. The early apoptosis rates of A375 cells were 3.11% + 0.53%, 13.74% 1.73%, 25.95% + 0.57% and 46.44% + 0.81%, respectively, and the early apoptosis rates of M14 cells were 1% + 0.08%, 2% + 0.01%, 2.99% + 13.74%, respectively, and all the groups were statistically significant (P0.05).0nM, 10nM, 20nM, and 30nM After 48h, the apoptosis rates of A375 cells were 4.20% + 0.09%, 19.50% + 0.32%, 36.57% + 3.85% and 58.68% + 2.03% respectively. The early apoptosis rate of M14 cells was 2.92% + 0.17%, 20.99% + 0.40%, 34.28% + 2.04% and 63.38%- +, respectively. All the groups were statistically significant compared with the control group (P0.05). Quantity dependence.
(3) the mechanism of Paeonol and triptolide on the apoptosis of melanoma cells
In the above (1) and (2), A375 cells were observed to be sensitive to the apoptosis of Paeonol and triptolide than M14 cells. Therefore, in this part of the experiment, the mechanism of apoptosis induced by A375 cells was selected to induce the apoptosis of melanoma cells. In this part, the changes in the activity of Caspase3, caspase8 and caspase9 were detected, and the benefits were detected in this part of the experiment. The changes in the levels of p53 and NF- kappa B and their related proteins were measured by Western Blot. The experimental results showed that 1.25mM, 2.5mM, and 5mM paeonol acted on the A375 cell 24h of the melanoma cell line, Caspase3, caspase8 and its activity increased statistically, and the level of protein and protein increased with the increase of drug concentration. The protein levels of kappa B, Bcl-2, and Bcl-XL decrease with the increase of drug concentration, indicating that the apoptosis promoting effect of Paeonol on melanoma A375 cells not only passes through the caspase and NF- kappa B pathway, but also induces apoptosis by activating p53 pathway. The activity of caspase8 decreased slightly with the increase of concentration, and the protein level of NF- kappa B, Bcl-2, Bcl-XL decreased with the increase of drug concentration, indicating that triptolide induced the apoptosis of melanoma A375 cells only by caspase and NF- kappa B pathway.
(4) effects of Paeonol and triptolide on the invasion of melanoma cells and its mechanism
Invasion and metastasis are the most essential characteristics of malignant tumor. In this study, the effects of drugs on the invasiveness and migration of A375 cells were observed by cell scratch test and Transwell experiment. The results showed that all two kinds of Chinese medicine monomers had inhibitory effects on melanoma A375 and M14 cells. A375 cell migration after 1.25mM paeonol acted as 24h. The distance from 197 + 11.14 of the control group was shortened to 46.67 + 5.69, and the migration distance of M14 cells was shortened from 172.67 + 9.29 to 33 + 6.24 in the control group. The difference between the drug action group and the control group was statistically significant (P0.01). The number of A375 cells passing through the artificial basement membrane after the action of 1.25mM, 2.5mM, 5mmM with 24h was the number of A375 cells. The 180.67 + 13.65 of the control group decreased to 131.67 + 10.41,74.33 + 3.21 and 33.33 + 4.16, and the number of M14 cells in the membrane also decreased to 253.67 + 14.01150.33 + 9.6 and 55.33 + 5.69 in the control group, and the ratio of each group to the corresponding control group was significantly different (P0.01).10nM after 48h of triptolide, A375 cell The migration distance was shortened from 286.67 + 17.62 to 185.67 + 7.09 in the control group. The migration distance of M14 cells was shortened from 415.67 + 19.76 to 213.33 + 10.02 in the control group. The difference between the drug group and the control group was statistically significant (P0.01). 10nM, 20nM, 30nM triptolide acted after 48h and passed through the artificial basement membrane, respectively. The number of A375 cells decreased from 183 + 10.82 of the control group to 143.67 + 7.77,60.67 + 4.04 and 27.33 + 2.52, and the number of M14 cells in the membrane also decreased from 129.67 + 7.23 to 80.33 + 2.51,65.00 + 5 and 20.33 + 1.53 in the control group, and there were significant differences in the ratio of each group to the corresponding control group (P0.01).
In this study, Western Blot and Real-time PCR were used to detect the gene expression and protein levels of uPA and VEGF, to explore the possible mechanisms of these two drugs to affect the invasiveness and migration of human melanoma tumor cells. The results showed that the gene expression of paeonol to uPA and VEGF increased gradually with the concentration of uPA and VEGF: 0mM (control group), 1.25mM, 2.5. The results of mM and 5mM were uPA:0.20 + 0.02,0.28 + 0.01,0.38 + 0.01 and 1.97 + 0.21 respectively, VEGF:0.59 + 0.06,0.79 + 0.04,2.27 + 0.02 and 3.61 + 0.21, while the protein levels of uPA and VEGF decreased obviously, 0mM (control group). The ratio of tin to 1.13,1.00,0.92 and 0.52. increased with the concentration of uPA and VEGF, respectively: 0nM (control group), the results of 10nM, 20nM and 30nM were uPA:0.20 + 0.02,0.41 + 0.00,0.95 + 0.05 and 5.84 + 0.16, respectively. The protein levels of 0nM (control group), 0nM (control group), 10nM, 20nM and 30nM were 1.01,0.81,0.44 and 0.15, respectively. The ratio of VEGF expression to beta -actin was 1.13,1.46,1.34 and 0.96., indicating that paeonol and triptolide may pass down the level of VEGF and protein and inhibit melanoma. The invasion of the cell.
conclusion
Both paeonol and triptolide can inhibit the proliferation of A375 and M14 in human melanoma cells. The inhibition mechanism of paeonol may be related to inhibiting the mitosis of melanoma cells in the G2/M cycle, and the mechanism of triptolide may be related to the inhibition of cell DNA replication in S cells.
Paeonol and triptolide can induce apoptosis of human melanoma A375 and M14 cells in a dose-dependent manner. In which triptolide induces apoptosis of human melanoma cells through caspase and NF- kappa B pathway; and paeonol not only induces the apoptosis of melanoma A375 cells by activating the caspase and NF- kappa B pathway.
Paeonol and triptolide can inhibit the invasion and migration of melanoma cells by inhibiting the expression of VEGF and uPA protein.
Paeonol and triptolide both have the effect of anti melanoma, and have the research and development prospect of new drugs for the treatment of cutaneous melanoma.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R285.5;R739.5

【参考文献】