The Anti-proliferative Effect Of CPT21, A Novel Semi-sythethized Camptothecin Analogue, Agaisnt Gastric Cancer SGC-7901

Camptothecin(CPT)is an alkaloid extracted from Camptotheca acuminata.CPT binds to topoisomerase I,an enzyme that relaxes the supercoiled DNA duplex during replication and transcription.Because tumor cells have been shown to contain more topoisomerase I compared with normal tissue,CPT and its analogs are good candidates for specific cancer treatment.These CPTs contain a terminal lactone ring that makes them unstable in aqueous solutions by undergoing a rapid,pH-dependent, nonenzymatic hydrolysis to form an open-ring hydroxy carboxylic acid.This hydrolysis makes them much less potent inhibitors of topoisomerase I. Recently,SAR data suggested that 7 position-modified CPT analogues might dramatically enhance the biological profiles.Thus,a series of 7-ankyl CPTs was synthesized via Sonogashira coupling reaction.Among them, 7-[(3-piperidyl)-1-propinyl]-camptothecin(CPT21),a novel semi-synthesized compound,showed preferred stability in water,belonging to the family of camptothecin,and is able to stabilize DNA-topoisomerase I complexes.Purpose:In the present study,we determined the potent anti-proliferative effect of CPT21 against cancer cells both in vivo and in vitro.Methods:IC₅₀values were determined by MTT method. Topoisemerase I catalytic activity assay was used to evaluate the inhibitory effect of CPT21 on the normal function of Topoisemerase I.SGC-7901 xenografted athymic mice model was utilized to evaluate the anti-tumor activity of CPT21 in vivo.Apoptosis was analyzed by flow cytometry. Mitochondria membrane potential(△ψm)was evaluated by JC-1 staining. Expression of proteins was determined by Western Blotting method.Chick embryo chorioallantoic membrane(CAM)assay was utilized to evaluate the anti-angiogenesis activity of CPT21.Results:1.CPT21 effectively inhibited ten cancer cell lines in vitro.The IC₅₀ values of hepatoma Bel-7402 cells,chronic myelocytic lecukemia K562 cells, acute promyelocytic leukemia HL-60 cells,androgen-independent prostate cancer PC-3 cells,oral squamous carcinoma KB cells,esophageal cancer ECA-109 cells,non-small cell lung cancer A549 cells,breast cancer MCF-7 cells,glioblastoma U251 cells and gastric cancer SGC-7901 cells were 3.0±1.7μM,0.13±0.04μM,0.10±0.05μM,0.10±0.08μM,5.0±2.1μM,7.9±3.5μM,7.3±3.5μM,1.3±1.1μM,12.0±4.1μM and 6.3±3.1μM respectively.2.CPT21 was able to inhibit Topo I-catalyzed relaxation of supercoiled DNA in a dose-dependent manner,and its ability was more potent than SN38 at 200μM concentration.3.CPT21 effectively delayed tumor progression in vivo.Compared with the control group,the tumor growth was significantly inhibited(p＜0.05-0.001)in the groups treated with CPT21(5.0 and 10.0 mg/kg)with the corresponding tumor inhibition rate at 45.2%and 75.1%,compared with Irinotecan 10.0 mg/kg,which was 65.1%.4.CPT21 induced apoptosis of SGC-7901 cells.The percentages of apoptotic SGC-7901 which were treated with 5μM CPT21 for 12,24 and 48h were 10.2,13.5 and 69.7%respectively in a time-dependent manner.In addition,CPT21 also induced a concentration-dependent apoptosis.The results showed that after incubated with CPT21(2.5,5.0 and 10.0 pM)for 24h,the corresponding apoptotic rates were 6.2%,13.5%and 34.5%.5.CPT21 caused decrease of△ψm in SGC-7901 cells.After treated with 5.0μM CPT21 for 12,24 and 48 h,the ratio of green to red fluorescence rose from 2.9 to 12.3.While treated with various concentrations of CPT21 (2.5,5.0 and 10.0μM)for 24h the ratio of green to red fluorescence was elevated from 2.9 to 45.0.6.The protein expression of p53 and p21 increased visibly in accordance with the treatment time,accompanied with the decrease of Bcl-2/Bax.As a proapoptotic signal,the expression level of phosphate-JNK was elevated after treatment with CPT21.In addition,CPT21 also stirred the caspases cascade,the cleaved fragment of Poly(ADP-ribose) polymerase(PARP)appeared and the expression of caspase-9 and XIAP decreased as the treatment time prolonged,meanwhile,the activity of caspase-3 was raised after cells were incubated with CPT21.7.CPT21 remarkably showed anti-angiogenesis ability.5.0μg/egg and 1.0μg/egg CPT21 significantly inhibit the growth of novel vessels by using the CAM assay,and its anti-angiogenesis activity is similar to topotecan at the concentration of 5.0μg/egg.Conclusion:Altogether,the present data demonstrated that CPT21 was a potent anti-tumor compound which was able to induce the apoptosis of SGC-7901 cells,and the mechanism may include:1)Mediate Bcl-2 family to affect the normal function of mitochondrion and further stirring the downstream caspase family;2)Activate the p53 pathway to induce apoptosis.