| 1.Background and 0bjectiveGallbladder cancer incidence of gastrointestinal malignancies in the first five,is the most common biliary tract cancer,high incidence of 50 to 70 years old,female patients about 2 to 3 times the male patients.Chronic cholecystitis and gallstones are risk factors for gallbladder cancer.Gallbladder cancer early symptoms hidden,rapid disease progression,high degree of malignancy,early diagnosis is more difficult,some cases are laparotomy accidental discovery.When patients with abdominal pain,jaundice and other symptoms,often in the late stages of cancer,missed the chance of radical resection.Local radiotherapy can prolong the median survival time of patients with gallbladder cancer,but the probability of distant metastasis of gallbladder cancer is high,resulting in radiotherapy effect greatly reduced.There is no unified and effective gallbladder cancer chemotherapy,mostly 5-Fu,platinum,gemcitabine,capecitabine,doxorubicin and other drugs,the effect has not been reliable research confirmed.CD147 is an important molecular molecule in the development and progression of malignant tumor,which can induce the production of extracellular matrix metalloproteinases(MMPs),thereby promoting tumor invasion and metastasis;he can upregulate the expression of vascular endothelial growth factor(VEGF)Tumor angiogenesis;can ensure that the high tumor cells anchored non-dependent growth of the ability to antagonize tumor cells inoculation;promote tumor cell multi-drug resistance.MMPs are extracellular matrix metalloproteinases,MMP-9 is a member of the MMPs family,also known as gelatinase B.It can degrade the matrix and basement membrane,promote the spread and metastasis of tumor cells,but also can promote tumor proliferation,regulate the extracellular matrix,adjust a variety of growth factors and promote angiogenesis.Compared with chronic cholecystitis,mild atypical hyperplasia,gallbladder adenoma tissue,most of the gallbladder cancer tissue expression of CD147 and MMP-9,and its expression and gallbladder cancer differentiation,clinical stage,lymph node metastasis and median Survival time was positively correlated,so CD147 and MMP-9 may be a potential target for gallbladder cancer treatment.CD147 antagonist peptide 9(AP-9)is a 12 peptide that can specifically bind to CD147 molecules by random phage peptide libraries that antagonize CD147 and play a desired role,such as reducing the activity of MMP-9.(Doxycycline,DOX)belongs to the third generation of semi-synthetic tetracycline broad-spectrum antibiotics,which can inhibit the role of metalloproteinases,promote tumor cell apoptosis,inhibit tumor cell proliferation and other aspects of anti-tumor effect.To research the effect of AP-9 and DOX on GBC-SD,we detected the proliferation,apoptosis,expression of CD147 and activity of MMP-9 in gallbladder carcinoma cell,with respectively and combination therapy of AP-9 and DOX.2.MethodsGallbladder carcinoma cells were treated with AP-9(50,100,200,400?g/ml),DOX(5,10,20,40?g/ml)and combination therapy(AP-9 200?g/ml + DOX 20?g/ml)for 12,24,48 h,then the inhibition rate of proliferation was detected by CCK-8,expression of CD147 was detected by western blot,the activity of MMP-9 was detected by gelatin zymography,and the early apoptosis rate was detected by flow cytometry with Annexin V/PI staining.3.ResultsAfter treated with several concentrations of AP-9,the apoptotic rate(2.13 ± 0.10)%,(2.92 ± 0.19)% were higher(p=0.002,p<0.001)than control group(1.58±0.08)%;the activity of MMP-9(320.08 ± 33.90,249.59 ± 33.63,259.63 ± 34.08,172.31 ± 35.20)were reduced(p=0.013,0.002,0.003,0.001)than control group;cell proliferation was inhibited than control group(p<0.01).After treated with several concentrations of doxycycline,the apoptotic rate(3.25±0.12)%,(3.63±0.24)% were higher(p<0.001)than control group(1.58±0.08)%;The activies of MMP-9(203.52±18.25,175.22±23.30,147.86±22.83,109.99±29.22)were reduced(p=0.001,p<0.001,p<0.001,p<0.001)than control group(446.08±42.42);cell proliferation was inhibited tha control group(p<0.001);the expression level of CD147(0.97±0.12,0.80±0.10,0.69±0.11,0.51±0.12)were reduced(p<0.001)than cotrol group(2.04±0.15).Compared with single treating.After treated with combination,cell proliferation(38.78±0.39)%,(43.76±0.71)%,(52±0.91)% was higher(p<0.05),the activity of MMP-9(110.19±24.74)was reduced(p<0.05),the apoptotic rate(3.73±0.29)% was higher(p<0.05).4.ConclusionsDOX can inhibit reduce the expression of CD147,AP-9 and DOX alone or in combination can inhibit the proliferation of gallbladder cancer cells,inhibit the activity of MMP-9,and promote apoptosis,and the the inhibitory effect was enhanced after combination therapy... |
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