Effects of polyunsaturated fatty acids on the growth of gastric cancer cells in vitro
- Jinfeng Dai†1,
- Junhui Shen†2,
- Wensheng Pan3,
- Shengrong Shen1Email author and
- Undurti N Das4, 5, 6Email author
© Dai et al.; licensee BioMed Central Ltd. 2013
Received: 30 January 2013
Accepted: 19 April 2013
Published: 10 May 2013
Polyunsaturated fatty acids (PUFAs) have tumoricidal action, though the exact mechanism of their action is not clear. The results of the present study showed that of all the fatty acids tested, linoleic acid (LA) and α-linolenic acid (ALA) were the most effective in suppressing the growth of normal gastric cells (GES1) at 180 and 200 μM, while gastric carcinoma cells (MGC and SGC) were inhibited at 200 μM. Arachidonic acid (AA) suppressed the growth of GES1, MGC and SGC cells and lower concentrations (120 and 160 μM) of AA were more effective against gastric carcinoma (MGC and SGC) cells compared to normal gastric cells (GES1). Paradoxically, both eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids though are more unsaturated than AA, were less effective compared with LA, ALA and AA in suppressing the growth of both normal and cancer cells. At the concentration used, methotrexate showed much less growth suppressive action compared to all the fatty acids tested. PUFAs-treated cells showed accumulation of lipid droplets. A close association was noted between apoptosis and lipid peroxides formed compared to the ability of normal and tumor cells to generate ROS (reactive oxygen species) and induce SOD (superoxide dismutase activity) in response to fatty acids tested and methotrexate. Both normal and tumor cells generated lipoxin A4 (LXA4) in response to supplementation of fatty acids and methotrexate though no significant correlation was noted between their ability to induce apoptosis and LXA4 formed. These results suggest that PUFAs induced apoptosis of normal gastric and gastric carcinoma cells could, partly, be attributed to lipid peroxidation process.
KeywordsPolyunsaturated fatty acids Gastric cancer cells Oxidative stress de novo lipid synthesis Lipid metabolites
Gastric cancer is the fourth most prevalent malignant disease and the second leading cause of cancer death worldwide [1, 2]. Despite significant advances, gastric cancer remains a formidable disease to manage.
There is considerable evidence to suggest that essential fatty acids (EFAs): cis-linoleic acid (LA, 18:2, ω-6) and α-linolenic acid (ALA, 18:3, ω-3) and their metabolites exert significant inhibitory action on the growth of tumor cells both in vitro and in vivo [3–16]. It has been documented that tumor cells have decreased activity of Δ6 and Δ5 desaturases that are essential for the metabolism of LA and ALA to their respective long-chain metabolites [17–19]. The long-chain metabolites of EFAs: arachidonic acid (AA, 20:4 ω-6), eicosapentaenoic acid (EPA, 20:5 ω-3) and docosahexaenoic acid (DHA, 22:6 ω-3) not only give rise to prostaglandins, leukotrienes and thromboxanes but also to anti-inflammatory compounds lipoxins, resolvins, protectins and maresins [20, 21].
Previously, we and others showed that gamma-linolenic acid (GLA, 18:3 ω-6), AA, EPA and DHA could be selectively cytotoxic to various tumor cells in vitro and in vivo [3–16]. Many of these studies were performed without taking into consideration the action(s) of these fatty acids on respective normal cells. Hence, in the present study we examined the effect of various long-chain fatty acids on the growth of gastric carcinoma cells and their respective normal gastric cells. We also studied fatty acid profile of cells supplemented with various fatty acids and their influence on the formation of lipid peroxides and free radical generation.
In addition to the generation of various prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs) from PUFAs, especially from DGLA, AA, and EPA that are pro-inflammatory in nature, AA, EPA and DHA also form precursor to anti-inflammatory compounds such as lipoxins (LXs), resolvins and protectins [20, 21]. There is reasonable evidence to suggest that cancer could be a low-grade systemic inflammatory condition [22, 23] that is supported by the observation that tumor cells produce significant amount of pro-inflammatory eicosanoids [24–26]. But, it is not known whether tumor cells are capable of producing anti-inflammatory compounds such as LXs, resolvins and protectins and, if so, how supplementation of various PUFAs alters their generation and the relationship between the generation of these anti-inflammatory compounds and tumor cell growth. Hence, in the present study, we also measured the amounts of LXA4 generated by normal and gastric cancer cells when supplemented with various PUFAs and the results are reported here.
Materials and methods
LA, ALA, AA, EPA, DHA were obtained from Sigma (St. Louis, MO, USA). The human gastric cancer cell line, MGC (undifferentiated), and normal stomach cell line GES1 were kindly provided by Dr. P. Wensheng (Zhejiang University, Hangzhou, China). Human gastric cancer cell line SGC (semi-differentiated) was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. RPMI medium 1640 was purchased from GIBCO (Grand Island, NY, USA). As a positive control, anticancer drug methotrexate (MTX) was used. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) was purchased from Sigma corporation. All other chemicals were of extra-pure grade or analytical grade.
Gastric cancer cells (MGC and SGC) and normal stomach cell line (GES1) were maintained in RPMI-1640, containing 10% fetal bovine serum and 100 U/ml penicillin-streptomycin at 37°C. LA and ALA were dissolved in 0.1 N NaOH at a concentration of 20 mM. AA, EPA and DHA were dissolved in absolute ethyl alcohol at a concentration of 10 mg/ml. Stock solutions were filter-sterilized and diluted with cell culture media for use. The final concentrations of the solvents were 0.001 M of NaOH and 0.6% of ethyl alcohol that were found to have little effect on the growth of the cells.
Cell viability assay
GES1, MGC and SGC cells were seeded in 96-well plates at a density of 10,000 cells per well and allowed to attach overnight, after which cells were supplemented with different concentrations of LA, ALA, AA, EPA and DHA. The doses of fatty acids tested ranged from 0 to 200 uM. 10 uM of antitumor drug methotrexate (MTX) was used as a positive control. After 48 h of incubation with fatty acids and methotrexate, medium was removed and treated with 20 ul MTT solution (5 mg/ml) at 37°C for 4 hours for assessing cell viability by measuring optical density at 492 nm after dissolving the dye in 150 ul of DMSO. The viability was defined as [OD (cells plate)-OD (medium plate)]/[OD (control cell plate)-OD (control medium plate).
Flow cytometric analysis of apoptosis
The apoptotic rate of cells was detected using FCM with the Annexin V-FITC/PI double labeling method . GES1, MGC and SGC cells in logarithmic growth phase were seeded in 6-well plates (Corning Costar) at a density of 100,000 cells/ml (3 ml per well). After adherence for 24 h, the medium was then replaced with refresh RPMI 1640 medium supplemented with different treatment reagents: without fatty acids (control), with 150 uM LA, 150 uM ALA, 180 uM AA, 180 uM EPA, 180 uM DHA and 10 uM antitumor drug MTX. After 48 h of incubation, the cells were harvested using trypsin, washed twice with cold PBS. The cell suspension (1 mL) was centrifuged at 2000×g for 10 min. After discarding the supernatant, the pellet was re-suspended gently in 400 uL Annexin V-FITC binding buffer and incubated with 5 uL Annexin V-FITC in dark at ambient temperature for 15 min. Subsequently, cells were incubated with 10 uL PI solution for 5 min on an ice bath in the dark. Cell apoptosis was subsequently performed by flow cytometry using Cytomics FC 500 MCL (Beckman Coulter, Inc.USA).
Malondialdehyde (MDA) and superoxide dismutase (SOD) assay
The levels of MDA and the activity of SOD were determined by using commercial MDA and SOD assay reagent kits obtained from Nanjing Kaiji Bioengineering Institute (Nanjing, China) . GES1, MGC and SGC cells supplemented with various fatty acids and methotrexate for 48 h were harvested using trypsin, and washed twice with cold PBS to remove excess trypsin. The cell suspension (1 mL) was centrifuged at 1000×g for 5 min. The supernatant was discarded; the pellet was re-suspended gently in 200ul PBS. After ultrasonication, the MDA content and SOD activity were determined and defined as corresponding value: Corresponding MDA (or SOD) concentrations (%) = C treatment / CCK×100%.
Intracellular reactive oxygen species (ROS) generation assay
The generation of reactive oxygen species (ROS) was monitored using DCFH-DA method  with Reactive Oxygen Species Assay Kit (Beyontime Company, China). After freely passing through membrane into the cell, DCFH-DA without fluoresce itself, can be hydrolyzed by intracellular esterases to generate membrane-impermeable compound DCFH which is oxidized by intracellular ROS to fluorescent compound 2, 7-dichlorofluorescein (DCF). GES1, MGC and SGC cells that were supplemented with various fatty acids and methotrexate, after the treatment period, were centrifuged, cell pellet was collected and the same was re-suspended in serum-free PRMI-1640 medium containing 10uM DCFH-DA and incubated for 30 min in dark. The level of DCFH fluorescence was analyzed (excitation wavelength of 488 nm and emission wavelength of 525 nm) by SpectraMax M5, Molecular Devices.
Determination of LXA4 levels
The LXA4 concentrations in the cell culture medium of gastric normal and cancer cells in vitro were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kit  from BOSTER Company (Wuhan, China) according to the instructions of the manufacturer. The sensitivity by this assay was 1.0 pg/ml of LXA4. All measurements were done in duplicate.
Fatty acid analysis of cells
GES1, MGC and SGC cells supplemented with various fatty acids and methotrexate, after the treatment period, were centrifuged and the cell pellet was collected and re-suspended in 500 μL distilled water and then 1 mL 5% hydrochloric acid-methanol mixed solution (v/v) to extract fatty acids. The collected solution was sealed immediately and then evaporated at 100°C for 3 h. After cooling to room temperature, 500 uL distilled water was added to each glass tube and extracted with 3 ml of hexane thrice by mechanical shaking . The collected supernatant was evaporated to dry under N2 and the dried residue was dissolved in 100 μL of hexane for GC analysis.
For fatty acid analysis, the samples were injected into an Agilent 6980 GC system equipped with a DB23 capillary column (0.25 mm×60 m×0.25 μm), and a FID detector (Agilent Technologies, Palo Alto, CA, USA). Helium was used as the carrier gas with a constant flow rate of 1.0 mL/min. One μL of the sample was injected into the Agilent 6980 GC system. The column temperature was initially kept at 130°C for 1 min, and then elevated to 170°C at an increasing rate of 6.5°C per min, followed by 2.75°C per min to 260°C for10 min. Both of the interface and ion source temperature were 200°C.
Oil red ‘O’ stain
To know whether the cells supplemented with various fatty acids form lipid droplets in the cytoplasm, the cells were fixed in 10% formalin after 48 hours of incubation with 180 μM of various fatty acids. Oil red ‘O’ solution that was prepared by dissolving 0.25 g of oil red O in 100 mL isopropyl alcohol by gentle heat at 56°C for 1 hour. The solution obtained at the end of 1 hour is allowed to cool and the cooled solution is filtered through a coarse filter paper that was used as the stock solution. The working solution was prepared by diluting 3 parts of the stock solution in 2 parts of double distilled water (stock solution: double distilled water = 3:2).
GES1, MGC and SGC cells were seeded in 24-well plates (Corning Costar) at a density of 10,0000 cells per well and allowed to attach overnight, then cells were treated in 1640 medium with 180 uM concentrations of LA, ALA, AA, EPA and DHA and 10 uM methotrexate (MTX). After 48 h incubation, the medium was removed and cells were washed three times with PBS and then fixed in 10% formalin for 10 minutes. The fixed cells were washed three times PBS and air dried for 20 minutes. These fixed cells were immersed in 1 ml Oil Red ‘O’ solution, and allowed to stand for 30 minutes. At the end of 30 minutes of treatment, cells were washed with distilled water three times and observed and photographed.
Data obtained from the present study was expressed as mean ± SD, and were analyzed with SPSS 13.0 software. Significance of differences analyses between different groups were performed using a one-way ANOVA test.
Effect of PUFAs on cell viability
PUFAs induce apoptosis of cells
Effect of PUFAs on morphology of gastric normal and cancer cells
Effect of PUFAs on oxidative stress
On the other hand, ROS generation was significantly enhanced in MGC cells by LA, ALA and DHA and also by MTX. In contrast, AA and EPA did not produce any increase in ROS in MGC cells. In fact, both AA and EPA suppressed ROS production in MGC cells. In contrast, all the fatty acids tested produced a significant increase in ROS generation in SGC cells (Figure 4), whereas MTX reduced ROS production in these cells.
Calculated LP/SOD ratio and ROS generated in response to supplementation of 180 μM of various PUFAs and 10 μM of methotrexate for 48 hours by various cells
LP/SOD ratio in GES1
% of viable GES1 cells at 180 μM
LP/SOD ratio in MGC
% of viable MGC cells at 180 μM
LP/SOD ratio in SGC
% of viable SGC cells at 180 μM
LXA4in the medium expressed in pg/ml GES1
LXA4in the medium in pg/ml MGC
LXA4in the medium in pg/ml SGC
GES% of C
MGC% of C
SGC% of C
trexate 10 μM
Effect of PUFAs on the generation of LXA4
AA (EPA > ALA > LA > MTX > DHA > Control > AA). The decrease in the production of LXA4 by GES1 cells in the presence of AA is rather surprising since AA forms the precursor of LXA4. In contrast, MGC cells produced significantly large amounts of LXA4 when supplemented with AA. Though other fatty acids were also effective in increasing LXA4 production in MGC cells, they were much less effective compared to AA (AA > EPA > Control > DHA > LA > ALA > MTX). Thus, in MGC cells only AA and EPA were able to enhance LXA4 production compared to control, while all other fatty acids decreased its synthesis. In SGC cells, ALA was the only fatty acid that enhanced LXA4 synthesis while all other fatty acids inhibited its production (ALA > Control > AA > LA = MTX > DHA > EPA). Thus, there was no consistent pattern with regard to the effect of various PUFAs on the production of LXA4 by GEs1, MGC and SGC cells. Nor we could find any correlation among formation of lipid peroxides, SOD, LXA4 formation and relative viability of cells when were supplemented with various PUFAs (see Table 1).
Changes in the fatty acid composition of cells
Changes in the plasma PL fatty acids in GES1, MGC and SGC cells in response to supplementation of various PUFAs (180 μM) and methotrexate (10 μM) and after 48 hours of incubation
GES1 cell line
Plasma PL fatty acid profile of GES1, MGC and SGC cells supplemented with various fatty acids (180 μM) and methotrexate for 48 hours
Fatty Acid supplementation or Methotrexate supplementation
GES1 MGC SGC
GES1 MGC SGC
GES1 MGC SGC
GES1 MGC SGC
GES1 MGC SGC
GES1 MGC SGC
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Supplementation of methotrexate to GES1, MGC and SGC cells produced few changes in the fatty acid composition of GES1 cells while MGC cells showed significant elevation in the PL content of LA, GLA, DGLA, AA, EPA and DHA, whereas similar increase in the content of these fatty acids was seen in SGC cells also but to a much less significant extent.
Tumor cells are known to have low activity of Δ6 and Δ5 desaturases [17–19]. Hence, we calculated the activity of these enzymes based on the levels of LA and AA and ALA and EPA seen in the three cell lines studied. The ratio between AA and LA (that is a reflection of the activities of Δ6 and Δ5 desaturases) was found to be 1.13 in GES1 cells, while the same was 0.05 in MGC cells and 2.97 in SGC cells (GES1 vs MGC vs SGC = 1.13 vs 0.05 vs 2.97 respectively). On the other hand, the ratio between EPA and ALA (that is a reflection of the activities of Δ6 and Δ5 desaturases) in GES1 cells was 1.13, in MGC cells 0.01 and in SGC cells 0.18. These results suggest that the activity of Δ6 and Δ5 desaturases is very low in MGC and SGC cells in comparison with GES1 cells (GES1 vs MGC vs SGC = 1.13 vs 0.01 vs 0.18 respectively).
Formation of lipid droplets in fatty acid supplemented cells
Previously, we and others showed that PUFAs have cytotoxic action on tumor cells [3–16]. The mechanisms(s) by which PUFAs induce tumor cell death has been controversial. The suggested mechanism(s) of the tumoricidal action of PUFAs include: (a) increased generation of ROS; (b) enhanced lipid peroxidation resulting in accumulation of toxic lipid peroxide products in the cells that ultimately results in cell death; (c) activation of caspases; (d) activation of PPARs; (e) modulating gene/anti-oncogene expression, and (f) induction of chromosomal damage [1–16, 31–37]. Though majority of evidences have been documented employing in vitro studies, some of these evidences have also been obtained using experimental animals [5–8, 10, 11]. Despite these evidences, it is still not clear as to the exact mechanism(s) of the tumoricidal action(s) of various PUFAs. For instance, majority of the studies employed cell culture techniques raising the question of their relevance to an in vivo situation. Furthermore, most of the in vitro studies were performed using only tumor cells without a simultaneous comparison or use of relevant normal cells. Thus, it is doubtful whether PUFAs are toxic only to tumor cells without being cytotoxic to relevant normal cells. This controversy could be answered by studying the effect of various PUFAs on the survival of normal and tumor cells employing similar, if not, identical cell culture conditions. In a previous study [1, 2], we did show that some of the PUFAs especially γ-linolenic acid (GLA, 18:3 n-6), arachidonic acid (AA, 20: 4 n-6), eicosapentaneoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) does possess such selective tumoricidal action with little or no effect on normal cells. In these studies [1, 2], it was noted that at higher concentrations (> 40 μg/ml/0.5 × 105 cells) AA, EPA and DHA were toxic to normal cells (41-SK: human skin fibroblasts) in comparison to human breast cancer, prostate cancer and lung carcinoma cells. Of all the fatty acids tested, only GLA showed selective tumoricidal action. Despite this evidence, human skin fibroblasts cannot be taken as the normal counterpart of human breast cancer cells to conclude that GLA and other fatty acids are selectively toxic to tumor cells. In view of this uncertainty, in the present study we studied the effect of various PUFAs on normal gastric cells (GES1) and corresponding gastric carcinoma cells (MGC and SGC) and explored the possible mechanisms of their tumoricidal action.
It is evident from the results of the present study that all the PUFAs tested (LA, AA, ALA, EPA and DHA) and methotrexate were able to induce apoptosis of the three types of cells tested (both normal and gastric cancer cells) and showed very little differential action on normal and tumor cells (see Figure 1). Cell viability was affected only at higher concentrations (180 and 200 μM) of various PUFAs suggesting that both normal (GES1) and gastric tumor (MGC and SGC) cells are relatively resistant to the cytotoxic action of fatty acids tested. Such a relatively equal sensitivity of both normal and gastric tumor cells to the cytotoxic action of various PUFAs and methotrexate may explain why it is hard to treat gastric cancer since at doses at which anti-cancer drugs are able to kill tumor cells; perhaps, even normal gastric cells will also be affected. Such an equal sensitivity of both normal and tumor gastric cancer cells to the cytotoxic action of anti-cancer drugs could be the reason for various gastrointestinal side-effects observed during the treatment of gastric cancer. In the absence of a selective cytotoxic action of anti-cancer drugs on gastric cancer cells, normal gastric cells also bear the brunt of the actions of chemotherapy and lead to significant side-effects and complications in the management of gastric cancer.
One distinct observation that was made in the present study was the accumulation of lipid droplets in both normal GES1 and gastric tumor cells MGC and SGC that were supplemented with 180 μM of various fatty acids for 48 hours when stained with oil red “O” (see Figure7 which shows lipid droplets in fatty acid supplemented GES1, MGC, and SGC cells and the corresponding control). In general, ALA, AA, EPA and DHA supplemented cells showed more number of lipid droplets compared to the control and methotrexate treated cells.
Studies into the mechanism(s) of cytotoxic action of PUFAs and methotrexate showed little correlation among cytotoxic action of PUFAs and methotrexate on GES1, MGC and SGC cells; production of ROS, formation of lipid peroxides, changes in the levels of SOD and LXA4 in these cells, suggesting that none of these mechanisms seem to be solely responsible for the cytotoxic action of fatty acids and methotrexate tested. Of all, only accumulation lipid peroxides seems to show the most correlation between the cytotoxic action of PUFAs and methotrexate on GES1, MGC and SGC cells and apoptosis. This indicates that formation of lipid peroxides in the normal and cancer cells on supplementation with various PUFAs and anti-cancer drug (methotrexate in the present instance) are the best predictors of their cytotoxic action. In the present study, we have not studied the effect of GLA and DGLA nor did we evaluate the affect of PUFAs on gene/oncogene expression. Such studies may give further insight into the mechanism(s) of cytotoxic action of PUFAs on normal and tumor cells. It is also important to evaluate the affect of combined action of PUFAs and various anti-cancer drugs on the survival of normal and tumor cells. Some of these studies are planned in the near future.
UND is in receipt of Ramalingaswami Fellowship of the Department of Biotechnology, New Delhi during the tenure of this study. This study was funded, in part, by a grant from the Department of Science and Technology to UND (No. IR/SO/LU/03/2008/1) under Intensification of Research in High Priority Areas (IRPHA).
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