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Vol-7, Special Issue-Number4-June, 2016, pp472-478 http://www.bipublication.com

Research Article

A Study of the Effect of Omega-3, -6 and -9 Fatty Acids on Bcl-xL Gene

Expression in the Cancerous Gastric Tissue

Taniya Bagheri and Mohammadreza Mashayekhi

Biology Department, Genetics, East Azarbayjan Science and Reserch branch,

Islamic Azad University, Tabriz, Iran

ABSTRACT

Background and objective: Bcl-xL is an anti-apoptotic protein that its overexpression in cancer patients results in reduced level of apoptosis in cells and resistance to chemotherapy. If they are used as food supplements with chemotherapy, omega-3, -6 and -9 fatty acids can induce apoptosis. In this study, we aim to examine the effect of omega-3, -6 and -9 fatty acids on Bcl-xL gene expression in patients with gastric cancer.

Materials and methods: In the study, which is a clinical trial, 34 patients diagnosed with gastric cancer were taken biopsy samples, and they were then classified into two groups of 17 people. During chemotherapy, in group 1 as the control group, patients used cis-platinum drugs and in group 2, patients used cis-platinum drugs with omega-3, 6 and 9 fatty acids. After chemotherapy, biopsy samples were also taken from patients; Bcl-xL gene expression was determined by Real-time PCR and a quantitative scale of protein Bcl-x was determined by immunohistochemical staining and frozen section.

Findings: Omega-3, -6 and -9 fatty acids significantly reduced the quantitative amount of Bcl-xL and Bcl-xL gene expression in gastric cancer patients in the target group than in the control group (p < 0.05).

Conclusions: Omega-3, -6 and -9 fatty acids negatively affect Bcl-xL gene expression in cancerous stomach cells and likely increase the effectiveness of chemotherapy in these patients through increased apoptosis induction.

Keywords: Gastric cancer, Apoptosis, Fatty acid, Gene expression, Bcl-xL

INTRODUCTION

Gastric cancer is an invasive disease that is always one of concerns about health in the world. In spite of its total reduction over several past decades, gastric cancer is the fourth common cancer in the world (1). According to global estimates, 930000 cases of gastric cancer are diagnosed annually that 700,000 cases cause death (2). To treat gastric cancer is one of the most important challenges in medical studies, which requires further research and examination on prevention, early diagnosis and new treatment options. Adenocarcinoma is the

most common form of gastric cancer;

Helicobacter pylori infections, smoking, salty diet and insufficient antioxidant intake are factors contributing to developing this disease (3).

Because gastric cancer is usually diagnosed in advanced and metastatic stage, chemotherapy is a standard treatment method for treating this disease; resistance to chemotherapy is a major clinical problem that leads to death in advanced and metastatic stage (5, 4). Increased anti-apoptosis gene expression plays an important role in resistance to chemotherapy in this stage (6). Apoptosis or programmed cell death depends on actions and interactions of some gene products activating or inhibiting cell death. More than 100 genes are identified that affect cell survival. The Bcl-2 family and caspases are involved in the apoptotic pathway. The Bcl-2 family has two

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proteins, and inhibiting apoptosis, anti-apoptotic proteins; the average ratio of these proteins determines the fate of a cell (7, 8).

Bcl-xL is an anti-apoptotic protein from the Bcl2 family that its locus is on the chromosome 20q11.21 and its sequence is 2610 bp. Bcl-xL has a role in stimulating mitochondria, releasing cytochrome c out, and activating the intrinsic pathway of apoptosis such that any decrease in its expression results in apoptosis induction (9, 10, 11).

Bcl-xl in stimulating mitochondrial release of cytochrome c and activation of the intrinsic pathway of apoptosis abroad plays a role in a way that leads to the induction of apoptosis is down-regulated. (9, 10, 11) Hence, examining factors contributing to decreased anti-apoptotic Bcl-xL gene expression and the reduced amount of this protein in cancer cells could be a new strategy to reduce resistance to chemotherapy.

Necessary fat acids are unsaturated fatty acids of long chains that are not often built by body and must be supplied by food. Necessary fatty acids are divided into two main families: omega-3 and omga-6. Omega-9 is required but not “necessary” because body is able to build few omega-9 by necessary fatty acids in diet. Omega-3, 6 and 9 fatty acids are derived from linolenic acid, linoleic acid and oleic acid, respectively (12). Omega-3, 6 and 9 fatty acids protect heart, vessels, genitals, defensive system and nervous system in body. Deficiency in essential fatty acids and imbalance in the omega-6 to omega-3 ratio causes serious problems like heart attack, cancer, insulin

resistance, asthma, lupus, schizophrenia,

depression, depression after pregnancy, early aging, arteriosclerosis, obesity, diabetes, arthritis and Alzheimer.

The body needs them to build and repair cell membrane, as well as to detoxify and supply food. Their main work is to produce prostaglandins, which regulate such actions as heart beat, blood pressure, blood clotting, fertility and fertilization, as well as play an important role in the correct performance of immune system by controlling

inflammations and fighting infections.

Inflammation and environmental stress like free radical are among factors activating cell death (12, 13, 14).

We aim to examine the effect of administering omega-3, -6 and -9 on Bcl-xL gene expression and protein expression in patients diagnosed with adenocarcinoma during chemotherapy.

PROCEDURE

The present study is a double blind clinical trial conducted over 2013-2014 at Tabriz University of Medical Sciences. The experiment was conducted as following.

Selection of patients. Patients referring to the Endoscopy Clinic at Tabriz University of Medical Sciences who were with diagnosis of gastric cancer were taken three biopsy samples of stomach tumor tissue, the third distal, and then transferred into a nitrogen tank. Before any sampling of patients, individual people in the study were informed from the objectives of the study and written consent was taken from them. These 34 patients were divided into two groups of 17 people. Then, these patients were referred to an oncology specialist and their chemotherapy was started by the oncology specialist. In the two selected groups, group 1 was treated by platinum drugs without administering any supplements, and group 2 was treated by platinum drugs with the pill Natural Factors Ultimate-Omega Factors 1200 mg, an omega-3, 6 and 9 fatty acid supplement

with formulation of Fish Oil Blend 400 mg ،

Flexseed Oil 400 mg ،Borage Oil 400 mg, in a dose of 3600 mg (three 1200-mg pills) per day over three courses (each course lasting three

weeks). Then, consistent with mandatory

treatment follow-up, endoscopy was performed on these people; it was followed by stomach biopsy to treat tumor. Again, samples taken from patients were transferred to the nitrogen tank.

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the samples were blocked by skim milk, and then they were incubated by primary monoclonal antibodies against the Bcl-xL protein at 4 ° C for 16-18 hours. After being washed by TBST buffer, a secondary antibody was added; they were incubated for one hour. To appear the Bcl-xL protein, DAB was used as a substrate and hematoxylin as a counterstain.

In this test, sample analysis under microscope was graded by conventional numbers from -1 to +4, based on the intensity and spread of red dots. Real-time PCR. First, using the kit purchased from Bio Basic Co. and according to respective directions, RNA from the prepared tissues was

extracted. Then, RNA concentration was

determined by NanoDrop through its light absorption at a wavelength of 260 nm, and RNA quality was confirmed by agarose gel. (18sRNA and 28sRNA were observed in 1.5% agarose gel.) Next, Using the RevertAidTM First strand cDNA synthesis kit (Takara Co.) and according to the

manufacturer’s directions, cDNA was constructed from the resulting RNA. Then, the constructed cDNA was proliferated by the Corbet Rotor gene 6000 apparatus and SYBER-Green (Takara Company) diagnostic mix and the Real time PCR reaction, as well as primer designed for the genes Bcl-xL and GAPDH; the amount of product in each target sample was compared with the control amount. The reference gene GAPDH was used as a normalizer.

Specific primers (BIONEER Co.) used in the research included:

Bcl-xL(Reverse):

5`- CTGAAGAGTGAGTGAGCCCAGCA-3`

Bcl-xL(Forward):

5`-GCCACTTACCTGAATGACCAC-3`

Finally, the data obtained was analyzed by SPSS Version 22, using the ANOVA test. Significance level was considered less than 0.05. The data was presented as SEM+-.

Findings

The tissues tested were stained by the immunohistochemistry method and using anti-Bcl-xL antibodies, examples of which are shown in Figure 1. Yellow parts, which are marked by arrows, qualitatively show the amount of the Bcl-xL protein. The results suggest a considerable difference in the amount of the Bcl-xL protein in the cancerous gastric tissue before and after chemotherapy. Also, the amount of the Bcl-xL protein in the target group (using omega-3, -6 and -9 fat acids with cis-platinum drug during chemotherapy) was reduced than that in the control group (using only cis-platinum drug during chemotherapy).

Figure 1. Images taken from immunohistochemically staining a cancerous gastric tissue by an anti-Bcl-xL antibody before and after chemotherapy in the two groups studied.

The findings related to anti-apoptotic Bcl-xL protein expression in the two groups studied after

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received fat acid supplements with chemotherapy, was significantly lower than that in the control group. Mean Bcl-xL gene expression in the control group was 2.32±1.66 and in the case group 1.05±0.66. The value of p was 0.002 and it indicated a significant difference (P < 0.05).

Chart 1. A comparison of Bcl-xL gene expression in the two groups studied.

As seen in chart 1, which shows Bcl-xL gene expression in the groups studied compared to a standard gene, Bcl-xL gene expression in Group 2 (the target group who received omega-3, -6 and -9

fat acids with cis-platinum drug during

chemotherapy) is significantly lower than that in Group 1 (control group who received only cis-platinum drug during chemotherapy).

DISCUSSION

In this study, we examined the effect of administering omega-3, -6 and -9 fat acids on Bcl-xL gene expression in gastric cancer; it was recognized that using omega-3, -6 and -9 fat acids with cis-platinum drug during chemotherapy after three courses of chemotherapy significantly reduced Bcl-xL gene expression in patients with adenocarcinoma than that in patients using only cis-platinum drug during chemotherapy (P=0.06).

Many studies have indicated the importance of Bcl-xL performance in regulating cell apoptosis as an anti-apoptotic protein (9, 10, 15). It is recognized that Bcl-xL has a central role in the survival of tumor cells (17, 18).

Also, in most studies it is recognized that Bcl-xL expression is increased in most human cancers, preventing programmed death of these cells and resistance to chemotherapy (18, 19, 20, 21). In a study by Gryko et al. in 2012, protein expression involved in apoptosis, including Bcl-xL, in healthy cells in gastric mucosa was compared with that in cancer cells through the

immunohistochemistery method. This study

indicated disordered Bcl-xL expression in cancer cells, which is involved in gastric cancer (22). On the other hand, studies have shown that omega fat acids have toxic effects on cancer cells (23, 24).

Some studies stated the toxic effect of omega fat acids by inducing oxidative stress and reducing cancer cell growth (25, 26).

Most studies, however, believe that the toxic effect of omega fat acids is made by inducing apoptosis in these cells and by regulating gene expression involved in apoptosis, consistent with our study (27, 28).

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confirmed their results. An ambiguous and controversial issue in similar studies is whether the toxic effect of omega fat acids on healthy cells is the same as that on cancer cells.

This could be examined by the effect of different unsaturated fat acids on the survival of normal and tumor cells. In a study by Jingfeng Dia et al. in 2013, the effect of different unsaturated fat acids on normal stomach cells and cancerous stomach cells was examined (33).

In this study, it was recognized that low concentrations of about 120 to 160 micromolar of unsaturated fat acids could inhibit cancerous stomach cells; however, if they could be consumed more than 40 miligrams per liter to every cell, they would be harmful to healthy cells. Therefore, in our study, 3600 miligrams of fat acids per day were administered to every patient. This study, however, was conducted in laboratory conditions without a combination of unsaturated fat acids with other compounds so it is suggested that the study be conducted in clinical conditions.

CONCLUSIONS

The results from the study show that omega-3, -6 and -9 fatty acids as supplements with cis-platinum may be effective to induce apoptosis in cancerous stomach cells by reducing Bcl-xL gene expression. The results may represent an increase in the effectiveness of chemotherapy in patients with adenocarcinoma, which could be useful to respond to treatment in these patients.

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Imagem

Figure 1. Images taken from immunohistochemically staining a cancerous gastric tissue by an anti-Bcl-xL antibody  before and after chemotherapy in the two groups studied

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