| BackgroundEndometrial cancer is one of the most prevalent gynaecological cancer, accounting for about 20 ~ 30% of all malignancy of the female genital tract. The incidence of endometrial cancer increased in the recent years, already be close to or even exceed that of cervical carcinoma. The precancerous disease included endometrial hyperplasia.Cyclooxygenase (COX) is the key enzyme in the biosynthesis of prostaglan-din. COX -2 is the inducible form of cyclooxygenase. The expression of COX -2 is up — regulated in various tumor tissues and cell lines. Multiple reports indicated that COX - 2 was closely related to the tumor genesis, progression, invasion, metastasis, prognosis and the sensitivity to radiochemical therapy.A number of epidemiological studies have found associations between long term non - steroidal anti - inflammatory drugs (NSAIDs) use and a lower incidence and mortality from cancers of the esophagus, stomach, breast, lung, prostate, urinary bladder and ovary. The usage of selective COX - 2 inhibitors in the prevention and therapy of tumor has become a research hotspot in the recent years because of the little side — effect of the medicines.ObjectiveTo investigate the role of COX - 2 in the tumorigenesis and maintenance of malignant characteristics of endometrial cancer, we added serial concentrations of celecoxib, a selective COX - 2 inhibitor into the cultured endometrial adeno-carcinoma cell line HEC - 1 - B and observed its effects on cell proliferation andapoptosis.Material and methodsHEC - 1 - B cells were cultured in vitro and exposed to different concentrations of celecoxib (6. 25 ,12. 5 ,25,50, lOOjxmol/L) . The morphologic characters were viewed by inverted phase contrast microscope. Cells were then dyed by Giemsa methods to find out the morphological changes of nuclus and apoptosis. Cell growth curve was made by cell count. MTT assay was used to calculate the inhibiting rate of celecoxib on cell proliferation. Apoptosis induced by celecoxib was then confirmed by DNA Ladder. Flow cytometric analysis was used to measure the percentage of cells in different cell cycle phases, and with a subdiploid DNA content, the hallmark of apoptosis. RT - PCR was applied to detect the degree of COX - 2 mRNA expression.ResultsCelecoxib could inhibit the COX - 2 expression of endometrial cancer cell HEC - 1 - B. COX - 2 mRNA expression was down - regulated after the addition of celecoxib, 50 jxmol/L celecoxib had stronger effect than 20 |xmol/L celecoxib. The inhibiting rate of 20 and 50 |xmol/L celecoxib on COX - 2 mRNA expression was 25. 92% and 50. 81% , respectively. The results showed that celecoxib exerted more inhibitive effect with higher concentration.Celecoxib could inhibit HEC -1 - B cell proliferation in a dose - and time - dependent manner. 25 jxmol/L celecoxib admitted once could markedly inhibit HEC - 1 - B cell proliferation, with about 48% cell proliferation inhibited at day 3. Celecoxib could block the cell cycle process with G2 - M phase cell accumulation.Celecoxib had a dose - dependent induction of apoptosis. Apoptosis was observed when 20 p^mol/L celecoxib was admitted and apoptosis increased with the celecoxib concentration.DiscussionWe added serial concentration of celecoxib(6. 25,12. 5 ,25 ,50, lOOjjumol/ L)to cultured endometrial cancer cell line HEC - 1 - B and found that celecoxib could inhibit HEC - 1 - B cell proliferation, with faster inhibition and longer effect time of the higher celecoxib concentration. With any concentration of celecoxib , the inhibitory effect gradually increase with the extention of time, to be near, celecoxib could inhibit HEC - 1 - B cell proliferation in a dose and time -dependent manner.Cell cycle checkpoints have an vital role in the control of cell cycle. A serial studies in the recent years revealed that many anti - cancer drugs destroied G2 phase checkpoint and induced tumor cell death. In this research, we found with FCM analysis that celecoxib had little effect on Gl - Go phase of HEC - 1 - B cell, it mainly effected G2 - M phase. The proportion of G2 - M phase HEC - 1 - B cell increased from 6. 2% of the control group to 7. 1% and 8. 2% of the 20 and 50|xmol/L celecoxib groups. The results indicated that celecoxib could induce G2 - M phase cell cycle blockage, hinted the possibility of celecoxib as an anti - cancer drug.Apoptosis is also called programmed cell death, to be near, cell died in a gene — controlled, regulated fashion aimed to maintain physiological homeosta-sis. In our study, when observed by inverted phase contrast microscope and Gi-emsa dye methods, HEC - 1 - B cell with celecoxib showed characteristic morphological changes, such as cellular shrinkage, nuclear chromatin condensation , margination and fragmentation, cytoplasmic shrinking, dilated endoplasmic reticulum, membrane blebbing, and apoptotic body formation. The proportion of apoptotic cells increased with the increase of celecoxib concentration.Flow cytometry analysis showed sub - Gx peak, named apoptotic peak in celecoxib group. The apoptotic rate of 50|j,mol/L celecoxib group may reach 22. 5% , significantly higher than 20(xmol/L celecoxib (10. 2% ) and control group (8. 11%). These results indicated that celecoxib could induce apoptosis of HEC - 1 - B cell.We also isolated the HEC -1 - B cell DNA and applied DNA ladder analysis. Characteristic DNA ladder in 50|Jimol/L celecoxib group approved DNA fragmentation and cell apoptosis happened.In summary, the number of HEC - 1 - B cell with celecoxib decreased markedly in a dose - and time - dependent manner. The mechanism may be inhibition of cell proliferation and induction of apoptosis. Combined with results of cell cycle analysis, we concluded that celecoxib might have more effection apoptosis induction than cell proliferation inhibition.Celecoxib could selectively inhibit COX - 2, and inhibit proliferation and induce apoptosis of endometrial cancer cell. However, the underlying molecular mechanism and pathway and the clinical value of celecoxib as an anti - cancer drugs are still unclear and need more research.ConclusionCOX -2 was overexpression in human endometrial cancer cell HEC — 1 — B. Celecoxib could inhibit COX - 2 mRNA expression of HEC - 1 - B cell. Celecoxib inhibited cell proliferation of endometrial cancer in dose and time -dependent manner and induce G2 - M phase cell cycle blockage. Celecoxib could induce HEC -1 - B cell apoptosis, and the apoptosis rate increased with the celecoxib concentration. The study provided experimental evidence for celecoxib usage as an accessorial therapy drug of endometrial cancer.
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