The Effects And Mechanisms Of Folate On Cervical Cancer

ObjectiveFolate(FL)is one of the most important micronutrients. It mediates one-carbon transfer and acts as coenzyme in the synthesis of DNA, RNA and proteins for normal cellular differentiation and proliferation. It is only recently that folate has been implicated in the development of cancers,such as colorectal,gastric and breast cancer.But there are few studies about relationship between folate and cervical cancer(CC). Cervical cancer is the most common cancer among women worldwide and is a major public health problem. HPV infection is a necessary cause of CC, but there are cofactors may facilitate development of CC. Epidemiological evidence remains uncertain for the role of folate in the development of CC.Most studies indicate that Folate deficiency(FD)is associated with high risk of CC; Folate supplementation may improve precancerosis of CC. But the mechanisms by which FL might protect against CC have not yet been clearly elucidated. More prospective studies and clinic experiments are needed to resolve the role of the folate in the etiologly of cervical cancer.Population investigation and experiment in vitro were conducted in our study. The possible effects of serum folate and HPV16 on CC were explored by a case-dontrol stuey.Then the relationship between CC and serum folate was analyzed by Meta analysis. And human cervical cancer cell lines, C33A cell with HPV negative and CaSki cell with HPV16 positive, were cultured with different concentration of folate in vitro so as to evaluate FL effected on the cell proliferation and cell apoptosis, and to examine expression mRNA of HPV16 E2 and E6 genes. These studies would offer some new evidences for the etiology, prevention and treatment of cervical cancer.Methods1,Population investigation: A hospital-based case-control study was conducted in Shanxi Tumor Hospital from Jane 2005 to December 2005. The study included 103 women newly diagnosed by pathology of cervical cancer and 103 control of hysteromyoma. A standardized questionnaire was used to get demographic information and other correlated factors of cervical cancer. Blood samples were drawn in before having breakfast.HPV16 was gained by PCR,serum folate was measured using radiobinding assay.2,Meta analysis: The literature about the association of serum folate and cervical cancer from January, 1990 to July, 2007 were searched for according to inclusion and exclusion criteria.The data in the studies selected and our study was analyzed by REVMAN 4.2 software.3,experiments in vitro: The C33A and CaSki cells were cultured routinely in vitro. Cells collected in the logarithm growth stage were treated by groups with the different concentration of folate.Groups were divided as follows:â‘ control group: concentration of FL was 1μg/ml (concentration of normal media);â‘¡FD group: concentration of FL was 0.1μg/ml;â‘¢groups of adding FL: concentration of FL was 10μg/ml, 50μg/ml, 100μg/ml, 500μg/ml, 1000μg/ml and 2000μg/ml, respectively.The effect on the activity and function of two cell lines were evaluated by MTT assay, then calculated relative inhibitive rate of cell growth and IC₅₀ value. Cell growth suppression of two cell lines was assessed by Cell proliferation curve analysis. Changes of cell cycle phase and cell apoptosis of two cell lines were analyzed by FCM when the cells were treated with different concentration of FL for 48 hours and the cells of two groups (the control group of 1μg/ml folate, the experiment group of 1000μg/ml folate) were cultured continuously for 4 days. The mRNA levels of HPV16 E2 and E6 gene were detected by RT-PCR when CaSki cells were cultured with different concentration of folate for 48 hours and with 1000μg/ml concentration of folate for 4 days continuously.Results1,Serum folate and HPV16 were associated with cervical cancer(1)Serum folate level in case(2.10±1.03ng/ml)was significantly lower than it in control(3.04±1.99ng/ml).There was significant difference between two groups(Z=3.98, P<0.001).By Quartile based on serum folate of control, there was a dose-responded relationship between increased risk of cervical cancer and decreased serum folate(χ²_{趋势}=12.42, P=0.000).(2)HPV16 infection rate is significantly higher in case than it in control(55.34% and 31.07%), the odds ratios is 2.75(95%CI: 1.56⁴.86). (3)Folate deficiency and HPV16-infected showed a significant interaction in the development of cervical cancer ,OR=5.79(95%CI: 2.66¹2.63)。2,Meta analysis of the association between serum folate and cervical cancer The cumulative cases and controls were 882 and 1477 in 6 studies collected into Meta ananlysis, respectively. In low serum folate levels(50 value was calculated. The results of cell proliferation curve analysis showed there was no significant difference on the cell proliferation of two cell lines cultured with folate deficiency media and normal media(P>0.05).The adding different concentration of folate inhibited cell proliferation obviously. The effects of folate on the cell growth suppression were increased gradually with the concentration of folate increasing and cultured duration prolonging. And there was a dose-responded relationship between folate's concentration and inhibition rate(P<0.05).It showed there was no significant difference on activity and proliferation of HPV negative C33A cells and HPV positive CaSki cells effected by same concentration of FL(P>0.05).(2)By FCM, it showed there was no significant difference on the changes of cell cycle phase and cell apoptosis of two cell lines cultured with folate deficiency media and normal media(P>0.05). The adding different concentration of folate induced cell apoptosis and changed the percentage of cell cycle phase. With the concentration of folate increasing, the percentage of G0/G1 cells and cell apoptosis ratio increased gradually. 1000μg/ml FL treated groups and control groups of two cell lines were selected to ananlyze cell apoptosis and changed the percentage of cell cycle phase in different time. With cultured duration prolonging, the percentage of G0/G1 cells and cell apoptosis ratio increased which showed statistics significance between two groups(P<0.05). It showed there was no significant difference on cell apoptosis and changes of cell cycle phase of HPV negative C33A cells and HPV positive CaSki cells treated with different concentration of FL and with 1000μg/ml FL for different days(P>0.05).But increased cell apoptosis rate of CaSki was higher slightly than C33A.(3)There was no significant difference on expression of HPV16 E2 and E6 genes by RT-PCR whether CaSki cells cultured with different concentration of FL or with 1000μg/ml FL in different days.Conclusions1,Low serum folate status might be a risk factor for cervical cancer.2,HPV16 infection was the etiology associated with the development of cervical cancer.3,Serum folate had effect with HPV16 infection on the development of cervical cancer.4,The adding different concentration of FL inhibited cell proliferation and enhanced cell apoptosis. And with the concentration of FL increasing and cultured duration prolonging, the effects of folate on the cell growth suppression, the percentage of G0/G1 cells and cell apoptosis rate increased gradually. But FD had no effects on cell proliferation and apoptosis of two cervical cnacer cell lines.5,The effects of folate on expression of mRNA of CaSki HPV16 E2 and E6 genes had no obvious changes, which means effect of FL on transcription of CaSki cells may be slight.6,It was similarly that different concentration of FL inhibited cell proliferation and enhanced cell apoptosis of C33A and CaSki. Folate may be an independent risk factor for CC.