8-烯丙基山竹醇在口腔鳞癌化学预防中的作用

Role of 8-allyl Garcinol in the Chemoprevention of Oral Squamous Cell Carcinoma

目的评估8-烯丙基山竹醇在口腔鳞癌化学预防中的作用。方法培养人口腔鳞癌细胞系CAL27,以不同浓度的山竹醇和8-烯丙基山竹醇处理细胞,采用MTT实验、克隆形成实验、划痕实验及流式细胞术检测其对CAL27细胞生物学行为的影响。建立对二甲基苯并蒽(DMBA)诱导的地鼠颊囊异常增生模型,阴性对照为不做处理,阳性对照为左侧颊囊涂DMBA 3周,实验组为涂DMBA 3周后分别涂0. 5、1. 0 mmol/L山竹醇或8-烯丙基山竹醇2周,实验结束后处死动物,取左侧颊囊进行组织病理学观察和BrdU免疫组织化学染色。结果 MTT实验结果显示,山竹醇和8-烯丙基山竹醇均可抑制CAL27细胞增殖,且呈一定浓度和时间依赖关系。药物作用72 h,8-烯丙基山竹醇的半数抑制浓度(IC50)为(13. 13±2. 55)μmol/L,明显低于山竹醇的(32. 20±3. 24)μmol/L (t=8. 008,P=0. 001);两种药物同种浓度作用效果相比,8-烯丙基山竹醇对细胞增殖的抑制作用明显强于山竹醇,以10 (24 h:t=8. 012,P=0. 001; 48 h:t=5. 939,P=0. 001; 72 h:t=12. 551,P=0. 001)和20μmol/L (24 h:t=8. 887,P=0. 001; 48 h:t=9. 324,P=0. 002; 72 h:t=5. 361,P=0. 002)浓度时差异最为显著。克隆形成实验结果显示,山竹醇和8-烯丙基山竹醇用药组20μmol/L浓度时的克隆形成率分别为(44. 1±0. 4)%和(23. 6±0. 6)%,明显低于10μmol/L浓度时(55. 6±2. 8)%(t=6. 894,P=0. 019)和(31. 0±0. 6)%(t=15. 556,P=0. 001); 10 (t=14. 682,P=0. 003)和20μmol/L (t=51. 514,P=0. 001)浓度时8-烯丙基山竹醇对CAL27细胞菌落形成的抑制作用明显强于山竹醇。划痕实验结果显示,用药12 h时,10和20μmol/L山竹醇组的细胞相对迁移率分别为(16. 00±4. 55)%(t=3. 139,P=0. 026)和(3. 00±3. 16)%(t=6. 608,P=0. 001),明显低于阴性对照组的(30. 33±7. 64)%; 10和20μmol/L 8-烯丙基山竹醇组的细胞相对迁移率分别为(16. 25±3. 86)%(t=3. 245,P=0. 023)和(6. 00±2. 65)%(t=5. 214,P=0. 006),也均明显低于阴性对照组的(30. 33±7. 64)%。用药24 h时,10和20μmol/L山竹醇组的细胞相对迁移率分别为(23. 75±4. 57)%(t=4. 718,P=0. 005)和(5. 75±1. 50)%(t=10. 432,P=0. 001),均明显低于阴性对照组的(45. 33±7. 64)%; 10和20μmol/L 8-烯丙基山竹醇组的细胞相对迁移率分别为(23. 50±2. 38)%(t=5. 529,P=0. 003)和(11. 67±2. 31)%(t=7. 308,P=0. 002),也均明显低于阴性对照组的(45. 33±7. 64)%。20μmol/L浓度作用24 h时,山竹醇组的细胞相对迁移率明显低于8-烯丙基山竹醇(t=4. 151,P=0. 009)。凋亡实验结果显示,10μmol/L时,山竹醇组的早期凋亡率为(5. 00±0. 10)%,明显高于阴性对照组的(1. 57±0. 21)%(F=70. 950,P=0. 001)。20μmol/L时,山竹醇组的早期和晚期凋亡率分别为(5. 90±0. 78)%(t=39. 384,P=0. 001)和(9. 73±1. 67)%(t=10. 101,P=0. 001),均明显高于阴性对照组; 8-烯丙基山竹醇组的早期凋亡率为(4. 63±1. 16)%,明显高于阴性对照组(t=4. 511,P=0. 041)。同种浓度作用效果相比较,8-烯丙基山竹醇对细胞凋亡的促进作用明显弱于山竹醇(10μmol/L:t=5. 982,P=0. 004; 20μmol/L:t=8. 578,P=0. 001)。组织病理学观察结果显示,0. 5 mmol/L山竹醇处理组(t=2. 546,P=0. 031)、0. 5 mmol/L 8-烯丙基山竹醇处理组(t=3. 485,P=0. 008)、1. 0 mmol/L山竹醇处理组(t=4. 556,P=0. 001)和1. 0 mmol/L 8-烯丙基山竹醇处理组(t=5. 393,P=0. 001)地鼠的口腔黏膜上皮单纯增生面积均明显低于阳性对照组; 0. 5 mmol/L 8-烯丙基山竹醇处理组(t=2. 130,P=0. 046)、1. 0 mmol/L山竹醇处理组(t=3. 434,P=0. 010)和1. 0 mmol/L 8-烯丙基山竹醇处理组(t=4. 518,P=0. 004)地鼠的口腔黏膜上皮异常增生面积也均明显低于阳性对照组,1. 0 mmol/L山竹醇处理组(t=2. 793,P=0. 023)和1. 0 mmol/L 8-烯丙基山竹醇处理组(t=4. 997,P=0. 001)均明显低于0. 5 mmol/L山竹醇处理组。BrdU免疫组织化学染色结果显示,阴性对照组(t=7. 563,P=0. 001)、0. 5 mmol/L山竹醇处理组(t=2. 862,P=0. 029)、0. 5 mmol/L8-烯丙基山竹醇处理组(t=4. 693,P=0. 002)、1. 0 mmol/L山竹醇处理组(t=5. 071,P=0. 002)和1. 0 mmol/L 8-烯丙基山竹醇处理组(t=5. 133,P=0. 001)地鼠的BrdU增殖指数均明显低于阳性对照组,0. 5 mmol/L 8-烯丙基山竹醇处理组(t=3. 724,P=0. 007)、1. 0 mmol/L山竹醇处理组(t=7. 000,P=0. 001)和1. 0 mmol/L 8-烯丙基山竹醇处理组(t=4. 413,P=0. 003)均明显低于0. 5 mmol/L山竹醇处理组。结论 8-烯丙基山竹醇可抑制人口腔鳞癌细胞CAL27的增殖和迁移,促进细胞凋亡;对DMBA诱导的地鼠口腔颊囊异常增生具有显著抑制作用,但作用效果与山竹醇存在一定差异。

Objective To evaluate the chemopreventive effects of 8-allyl garcinol on oral squamous cell carcinoma( OSCC). Methods OSCC cell line CAL27 were cultured and treated with different concentrations of garcinol or 8-allyl garcinol. Their effects on the biological behaviors of OSCC cell line CAL27 were measured by MTT assay,clony formation assay,scratch migration assay,and flow cytometry with Annexin V-FITC/PI staining assay. We established DMBA-induced hamster cheek pouch models of dysplasia. While the negative control group was not treated,the positive group was treated with 0. 5% DMBA solution tropically to the left cheek pouch three times per week for three consecutive weeks. The other four groups received 0. 5 mmol/L or1. 0 mmol/L garcinol or 8-allyl garcinol respectively three times within the following two weeks after DMBA treatment. Hamsters were sacrificed at the fifth week to obtain tissue samples of the left cheek pouch. The samples were examined by histopathology and BrdU immunohistochemisty. Results MTT assay showed that both garcinol and 8-allyl garcinol inhibited the proliferation of CAL27 cells in a concentration-and time-dependent manner. The half maximal inhibitory concentration( IC50) of 8-allyl garcinol [( 13. 13 ± 2. 55) μmol/L]was significantly lower than garcinol [( 32. 20 ± 3. 24) μmol/L;t = 8. 008,P = 0. 001]. Comparing the two grougs of medicine in the same concentration,the inhibiting proliferation effects 8-allyl garcinol had significantly stronger effect in inhibiting proliferation than garcinol when the same dose was applied,and the difference was largest at the concentrations of 10( 24 h: t = 8. 012,P = 0. 001;48 h: t = 5. 939,P = 0. 001;72 h: t = 12. 551,P =0. 001) and 20 μmol/L( 24 h: t = 8. 887,P = 0. 001;48 h: t = 9. 324,P = 0. 002;72 h: t = 5. 361,P =0. 002). The clone formation assay showed the clone formation rates after the treatment with 20 μmol/L garcinol and 20 μmol/L 8-allyl garcinol were( 44. 1 ± 0. 4) % and( 23. 6 ± 0. 6) %,respectively,which were significantly lower than those after treatment with 10 μmol/L garcinol [( 55. 6 ± 2. 8) %;t = 6. 894,P = 0. 019]and10 μmol/L 8-allyl garcinol [( 31. 0 ± 0. 6) %;t = 15. 556,P = 0. 001]. The inhibiting effects of 8-allyl garcinol at the concentrations of 10 μmol/L( t = 14. 682,P = 0. 003) and 20 μmol/L( t = 51. 514,P = 0. 001)were significantly stronger than garcinol. Scratch test showed the relative cell migration rates after treatment with10 and 20 μmol/L garcinol for 12 hours were( 16. 00 ± 4. 55) %( t = 3. 139,P = 0. 026) and( 3. 00 ±3. 16) %( t = 6. 608,P = 0. 001),respectively,which were lower than negative control [( 30. 33 ± 7. 64) %].The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 12 hours were( 16. 25 ±3. 86) %( t = 3. 245,P = 0. 023) and( 6. 00 ± 2. 65) %( t = 5. 214,P = 0. 006),respectively,which were also lower than negative control [( 30. 33 ± 7. 64) %]. In addition,the relative cell migration rates after treatment with 10 and 20 μmol/L garcinol for 24 hours were( 23. 75 ± 4. 57) %( t = 4. 718,P = 0. 005) and( 5. 75 ± 1. 50) %( t = 10. 432,P = 0. 001),respectively,which were lower than negative control [( 45. 33 ±7. 64) %]. The relative cell migration rates after treatment with 10 and 20 μmol/L 8-allyl garcinol for 24 hours were( 23. 50 ± 2. 38) %( t = 5. 529,P = 0. 003) and( 11. 67 ± 2. 31) %( t = 7. 308,P = 0. 002),respectively,which were also lower than negative control [( 45. 33 ± 7. 64) %]. Furthermore,the relative cell migration rate after treatment with 20 μmol/L garcinol for 24 hours was significantly lower than after treatment with8-allyl garcinol( t = 4. 151,P = 0. 009). The apoptosis experiments showed that the early apoptosis rate of CAL27 cells was( 5. 00 ± 0. 10) % after treatment with 10 μmol/L garcinol,which was significantly higher than negative control [( 1. 57 ± 0. 21) %;F = 70. 950,P = 0. 001]. The early and late apoptosis rates of CAL27 cells were( 5. 90 ± 0. 78) %( t = 39. 384,P = 0. 001) and( 9. 73 ± 1. 67) %( t = 10. 101,P = 0. 001),respectively,after treatment with 20 μmol/L garcinol,which were also significantly higher than negative control. The early apoptosis rate of CAL27 cells was( 4. 63 ± 1. 16) % after treatment with 8-allyl garcinol,which was significantly higher than negative control( t = 4. 511,P = 0. 041). The effects of 8-allyl garcinol in promoting cell apoptosis were weaker than garcinol( 10 μmol/L: t = 5. 982,P = 0. 004;20 μmol/L: t = 8. 578,P= 0. 001). The histopathological test also showed that the hyperplastic areas of oral mucosal epithelium in hamsters after treatment with 0. 5 mmol/L garcinol( t = 2. 546,P = 0. 031),0. 5 mmol/L 8-allyl garcinol( t =3. 485,P = 0. 008),1. 0 mmol/L garcinol( t = 4. 556,P = 0. 001),and 1. 0 mmol/L 8-allyl garcinol( t =5. 393,P = 0. 001) were significantly smaller than positive control. The dysplasia areas of oral mucosal epithelium in hamsters after treatment with 0. 5 mmol/L 8-allyl garcinol( t = 2. 130,P = 0. 046),1. 0 mmol/L garcinol( t = 3. 434,P = 0. 010),and 1. 0 mmol/L 8-allyl garcinol( t = 4. 518,P = 0. 004) were also smaller than positive control;1. 0 mmol/L garcinol group( t = 2. 793,P = 0. 023) and 1. 0 mmol/L 8-allyl garcinol group( t = 4. 997,P = 0. 001) were smaller than 0. 5 mmol/L garcinol treatment group. Immunohistochemical staining of BrdU showed that the BrdU-labeled indicators were significantly lower in negative control group( t = 7. 563,P= 0. 001),0. 5 mmol/L garcinol( t = 2. 862,P = 0. 029),0. 5 mmol/L 8-allyl garcinol( t = 4. 693,P =0. 002),1. 0 mmol/L garcinol( t = 5. 071,P = 0. 002),and 1. 0 mmol/L 8-allyl garcinol( t = 5. 133,P =0. 001) when compared with the positive control. The Brd U-labeled indicators in 0. 5 mmol/L 8-allyl garcinol( t =3. 724,P = 0. 007),1. 0 mmol/L garcinol( t = 7. 000,P = 0. 001),and 1. 0 mmol/L 8-allyl garcinol( t =4. 413,P = 0. 003) were also significantly lower than in 0. 5 mmol/L garcinol group. Conclusions 8-allyl garcinol could inhibit the proliferation and migration of OSCC cell line CAL27 and promotes apoptosis. It also has prominent inhibitory effects on DMBA-induced hamster cheek pouch dysplasia. However, the specific effects are slightly different from garcinol.