The Effects Of DHA On Cell Apoptosis,Invasion And Migration Of Cervical Cancer Cell Lines

[Background]With the aging of the population gradually accelerated and the quality of living environment declined, global cancer pressure is increasing. According to GLOBOCAN statistics, in 2008, there were about 1.27 million new cancer patients and about 7.6 million people died of cancer. Cervical cancer is the second malignant tumor in women worldwide and is the most common cancer of the female reproductive tract. Since the 1950 s, as a result of cervical cytology screening and virus detection guide constantly updated, cervical cancer incidence and mortality decreased obviously. But in 2011, there were still about 529000 new cases of cervical cancer worldwide, about 275000 deaths,80% of new cases and 85% of deaths occur in developing countries. And the morbidity of our country is as high as 28.8%, the onset age at 40～50 years old,60～70 years old have an anther peak. The morbidity trends not only seriously threat the women’s physical and mental health, but become a focus of attention of the whole society. It is the key of the in-depth discussion in the medical community.Cervical cancer is the most common female reproductive system cancer, the incidence rate increased year by year, and getting younger and younger, so looking for effective mode of treatment for cervical cancer patients is important.The main treatment of cervical cancer are surgery, radiation and chemotherapy. Chemotherapy, as an important part of comprehensive treatment, get more and more attention of scholars both at home and abroad in recent years.The drug resistance of cancer cell limits the application of chemotherapy drugs in clinical.What’s more,drug toxicity of traditional chemotherapy makes a certain contradiction between the does of chemotherapy and effect. So we focus on finding an efficient chemotherapy sensitizer or some drug which have anti-tumor activity.To make cancer patients have a better prognosis with low relapse rate, less chemotherapy side effects and high quality of life, people are constantly updated treatment modalities for cervical cancer and put more concern about the impact on the patient’s own internal environment.At the same time, nutritional oncology were gradually proposed. Many epidemiological studies including in vivo experiments and in vitro experiments showed dietary supplement with ω-3 polyunsaturated fatty acids (PUFAs) could prevent a variety of inflammatory and neoplastic diseases.In 2009, United States Parenteral Nutrition Society(ASPEN) released a new guideline which is about some support treatment for cancer patients indicated that it can help to improve the immune function,correct organ dysfunction and the nutritional status of patients by encouraging the patients to eat normal and accept oral assist nutrition,artificial EN or PN depending on the disease state.It also conformed that nutrition support can be an important part of comprehensive treatment in tumor patient.The guidelines cited some correlation research about cancer patients intook omega-3 polyunsaturated fatty acids in recent years,and pointed out that PUFAs can meintain weight and enhanced immunity of cancer patients.Many clinical studies and epidemiological studies have showed that n-3 fatty acids have a positive effect on infant development, prevention and treatment of cancer, cardiovascular disease.What’s more,some study also suggested that there was a positive role on depression and dementia recently. These unsaturated fatty acid have multiple effects including anti-inflammatory, antiplatelet aggregation, anti-hypertensive and lipid-lowering. These effects may occur through a variety of different mechanisms, including the impact of cell membrane composition and function, gene expression or produce a class Ⅻ acid. Oncology nutrition are concerned about the impact of nutrients on cancer patient body weight, and other aspects of immunity, but gradually begin to focus on nutrients to tumor cell proliferation, metastasis and related substances sensitivity of radiotherapy and chemotherapy.In the beginning of these studies, the scholars mainly gived patients PUFAs by oral or intravenous who would receive elective surgery PUFAs to investigate their effects on postoperative infection rates, length of stay and other related indicators.In some early studies,researchers administered by elective surgery patients oral of intravenous administration of n-3 polyunsaturated fatty acids to explore the clinical effect of surgery.Lone S. Sorensen found that there no obvious clinical active role on patients who were accepted unsaturated fatty acids preoperative and postoperative.But Wachtler study found that preoperative administration of unsaturated fatty acids may reduce postoperative complications.These inconsistent findings may be related to the intake time and acceptable daily intakeof unsaturated fatty acids of patients,but also related to different research methods.It need for more research to find that whather unsaturated fatty acids affect on the prognosis of patients undergoing surgery.These two decades, researchers are more concerned about DHA and EPA for prevention and treatment of tumors.Researchers found that ω-3 polyunsaturated fatty acids(EPA and DHA) have positive biological effects on prostate cancer,breast cancer,coloretal cancer,bladder cancer and other tumor cells in vivo or in vitro. And some prospective studies or cohort studies in Europe found that the intake of w-3 polyunsaturated fatty acids may reduce the risk of colorectal cancer and prostate cancer. Abdi, J found DHA and EPA can induce the apoptosis of MM cells and enhance the chemosensitivity but not normal cells. ALICJA ZAJDEL et al found that these fatty acids, anti-cell proliferation depending on the type of fatty acid types and different cell lines by a study which cultured melanoma cell lines and used AA,EPA,DHA to stimulated the different cell lines. Belmiro Parada found thatω-3 polyunsaturated fatty acids can inhibit the development of precancerous lesions and malignant tissue by establishing an animal model of bladder cancer.And they also discovered that the mechaninsm may be related to its properties of anti-inflammatory, anti-oxidation, anti-proliferative and anti-angiogenic.The researchers found that PUFAs not only could promoted tumor cell apoptosis, also could enhance the sensitivity of tumor cells to chemotherapy, these findings could improve the sensitivity of resistant cells to chemotherapeutic drugs, reduce the chemotherapy drugs dose and the incidence of side effects of chemotherapy drugs.Gaelle Benais-Pont studied the rectal adenocarcinoma cells in vitro found thatω-3 polyunsaturated fatty acids may be used as a nutritional adjuvant to enhance the role of human rectal adenocarcinoma radiotherapy.Through the study between the colorectal cancer stem-like cells and EPA,Flavia De Carlo pointed out that EPA can enhace the coloractal cancer 320D cell line to be more sensitive to standard chemotherapy (5-fluorouracil and oxaliplatin. Anita Vasudevan et also found that EPA can be as an effective prevention strategy of recurrent colorectal cancer by itself or combined with fluorouracil.Although PUFAs have multiple positive biological effects in preventing cancer, the mechanism of its affects was not clear. Study found that DHA act its pro-apoptotic and anti-tumor effects on colorectal cancer by reducing the GRP78 protein expression (one protein component of endoplasmic reticulum, which may be a molecular target of DHA) Jeffrey D.Altenburg etc. discover that EPA and DHA can reduced metastasis, mediated by CXCR4,by down regulating the expression of CXCR4,which is a receptor of cell surface in MDA-MB-231 breast cancer cell lines. Arun Sharma, etc. Through the study of epithelial ovarian carcinoma SKOV-3 and OVCAR-3 cell line,Arun Sharma found that anti-proliferative effect on ovarian cancer cell lines of ω-3 polyunsaturated fatty acids are diverse. Under some certain conditions, anti-proliferative effect of ω-3 polyunsaturated fatty acids may be caused by the TGF-β pathway,or may be related to the regulation of P21 protein.Masayuki Fukui, etc found that EPA can induce autophagy to exert its anti-tumor effect in pancreatic cancer cells by study in vivo or in vitro.But the specific mechanism was not clear,which may related to the level of ROS and the caspase protein expression.In short, the mechanism of PUFAs how to played on the tumor cells were unclear. DHA (Docosahexaenoic acid, docosahexaenoic acid), one of the most important nutritional ingredients, it can anti-inflammatory, anti-atherosclerosis and promote the development of the fetal nervous system. What’s more, there were more and more evidences suggested that it could inhibit the proliferation of tumor cells, but its complex pharmacological effects, antitumor effect mechanism is not clear.The domestic studies have shown that DHA may by mTORCl/2 signaling pathway to inhibit the growth of endometrial cancer, while the research of PUFAs on gynecologic tumor cells was still rarely reported.[Objective]1. To preliminarily investigate the effect of unsaturated fatty acids, Docosahexaenoic acid(DHA), on apotosis of cervical cancer cell lines(HeLa and Siha).2. To preliminarily investigate the effect of unsaturated fatty acids, Docosahexaenoic acid(DHA), on migration and invasion of cervical cancer cell lines(HeLa and Siha).[Contents and methods]Logarithmic phase of Hela, Siha cells were assigned to control group and different concentrations of DHA stimulation groups.1. Docosahexaenoic acid(DHA) on proliferation of cervical cancer cell lines(HeLa and Siha) in vitro.(1) MTT assay was performed to evaluate the repression effect of different concentration of DHA (Oμmol/L、20μmol/L、40μmol/L、60μmol/L、80μmol/L、 100μmol/L) on cell viability after DHA stimulated 24、48、72h;(2) The cervical cancer cell morphological changes after different concentrations of DHA (Oμmol/L、IC25、IC50、IC75) sitmulated 48h were observed under inverted microscope;(3) Hoechst33258 stain was used to detected the cervical cancer cell apoptosis after different concentrations of DHA (Oμmol/L、IC25、IC50、IC75) stimulated 48h;(4) Flow cytometry detection (FCM) was used to detected the apoptosis rate of cervical cancer cells after different concentrations DHA (Oμmol/L、IC25、IC50、 IC75) stimulated for 48h;(5) The expression levels of apoptosis-related protein Bax, Bcl-2 cleaved-caspase3 after different concentration of DHA (Oμmol/L、IC25、IC50、IC75) stimulated were detected by Western Blot.2. Docosahexaenoic acid (DHA) on migration and invasion of cervical cancer cell lines (HeLa and Siha) in vitro.(1) Wound healing assay was observed after stimulation of the cells DHA, changes in cell migration ability in vitro;(2) Transwell assay was used to observed the changes of migration and invasion after different concentrations of DHA stimulated the both cell lines;(3) The expression levels of invasion and metastasis-associated proteins (VEGF-. MMP-9) were detected by Western Blot.3. Statistical Analysis:Data were processed with SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA) and expressed as means±standard deviation. Multiple comparisons of sample means were analyzed by one-way analysis of variance and P<0.05 was considered to indicate a statistically significant difference[Result]1..The cell viability were detected by MTT assay.DHA could significantly inhibit proliferation and induce apotosisof cervical cancer cell lines (HeLa cell and Siha cell) in a concentration-dependent manner as compared with control group (0μmol/LDHA),P<0.05.2. Under inverted microscope, the cell morphology change of two cell lines were obviously after stimulation with different concentrations of DHA for 48 hours,which were compared with the control group (0 μmol/L DHA).In the control group, Siha cells were elongated strip spindle and single cell growth; but HeLa cells were grow like a spindle-shaped nests agglomerate and irregular.In the IC25 group,connections of intercellular were looser than normal group and the cells were began to be rounding.The cell stopped prolife and most cytoplasm becamce translucent.What’s more,there were small amount of cell debris in the culture hole.In IC50 group,floating translucent dead cells could be seen in the culture supernatant.There were further widened between the cells which volume were reduction.We also could see there were more translucent particles aroud the cytoplasm and nucleus,while part of cells were became cell debris.In IC75 group,the cell refraction were further enhanced and the cell density sparser than the other experimental group.There were less normal cells and more debris in the culture hole.Lipid-like particles could be seen in the cell cytoplasm.3. Hoechst 33258 staining was used to observed the apoptotic bodies and other changes.In the control group((0 μmol/L DHA),the nucleus of the cells of HeLa and Siha were round,stained unifore and sharp edges. We could saw that HeLa cells were agglomerate-like growth while Siha cells were single cell growth.In IC25 group,the nucleus of both cell lines were began to shrunken and be small,in the same time,the boundaries of nuclear became blurred and irregular.Further,in the IC50 group,The cell density was significantly decreased.There were less more normal nucleus due to the nucleus were shrinkaged or deformed.The nuclear chromatin accumulated and fractured.Apoptotic bodies could be seen,which were a crescent-shaped or granular bright blu fluorescence.In the IC75 group,the cell density were further reduced.There were apoptotic bodies within the field of view and more cell debris.Sometimes,we could saw a single normal nucleus.4. The AnnexinV-FITC/PI double staining and flow cytometry revealed that in IC25 group,the apoptosis rate of HeLa and Siha cell were 38.43±8.65% and 22.03±2.47% respectively; In IC50 group,the apoptosis rate of HeLa and Siha cell were 47.53±6.33% and 31.50±5.80% respectively; In IC75 group,the apoptosis rate of HeLa and Siha cell were 77.83±2.74% and 55.97±5.35% respectively;The apoptosis rate of control group were 13.03±2.75% and 11.93±2.29%, respectively. The two cell lines were compared with the control group respectively and each grou differences were statistically significant. (P<0.05)。5. In wound healing, cell repaired the scratches area was significantly weaker tha the control group, especially is Siha cell line.The wound healing assay revealed that DHA can effectively inhibit cell migration and invasion of the cancer cells. In the transwell chamber migration assay, with the increasing concentrations of DHA, the number of cells throughing the membrane into the bottom chamber was lower in both cell lines. These results prompted that DHA can inhibit the migration of HeLa cells and Siha cells in vitro.The results of wound healing assay and transwell cell migration assay suggested that different concentrations of DHA can inhibit the migration of HeLa cells and Siha cells in a concentration-dependent manner.6. Transwell chamber assay was used to detect the changes of invasion of HeLa cells and Siha cells after different concentrations of DHA stimulated for 48h in this study. The results revealed that the ability of invasion of HeLa cells and Siha cells were decreased in a concentration-dependent manner after treated with different concentrations of DHA.7. Western blotting prompted that the expression of protein Bcl-2, VEGFand MMP-9 were significantly lower in the experiment group than that in the control group. While the expression of protein Bax and cleaved-caspase 3 were significantly higher in the DHA group than that in the control group. The expression of Bax, Bcl-2 and cleaved-caspase were consistant with the result of MTT assay, Hoechst 33258 staining and flow cytometry.And the expression of VEGF and MMP-9 were consistant with result of wound healing assay.[Conclusion]The research paved a path in the molecular mechanism of DHA revealing its antitumoral property in cervical cancer cells. We found that DHA can promote cervical carcinoma cell apoptosis by down regulating the anti apoptotic proteins (Bax,Bcl-2 cleaved-caspase3) and inhibit cell invasion by decreasing MMP-9, VEGF expression. Bax, Bcl-2 are the specifice protein of mitochondrial membrane.We speculated that DHA may promote the apoptosis of cervical cancer cells through the mitochondrial pathway.