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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 798-804

Treatment of undifferentiated colorectal cancer cells with fish-oil derived docosahexaenoic acid triggers caspase-3 activation and apoptosis


1 Department of Histology and Embryology, Faculty of Science; Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia, Iran
2 Department of Histology and Embryology, Faculty of Science; Department of Cellular and Molecular Biotechnology, Institute of Biotechnology; Royan Stem Cell Technology Company, West Azarbaijan Cord Blood Bank, Urmia, Iran
3 Department of Histology and Embryology, Faculty of Science, Institute of Biotechnology, Urmia, Iran
4 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Date of Web Publication25-Jul-2016

Correspondence Address:
Mohammad Reza Sam
Institute of Biotechnology, Urmia University, P.O.Box: 165, Urmia
Iran
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Source of Support: The sources of financial support were National Cell Bank of Iran (NCBI), Pasture Institute of Iran and Urmia University, Conflict of Interest: None


DOI: 10.4103/0973-1482.157326

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 > Abstract 


Background: The effectiveness of chemotherapy is often limited by the side effects on normal tissues. Consequently, the search for new therapeutic agents with minimal toxicity is of particular interest in cancer management. Many studies have shown that docosahexaenoic acid (DHA) have cytotoxic effects against different kinds of cancer cells. However, little attention has been paid to explore the effect of DHA on undifferentiated colorectal cancer cells. In this study, the effects of DHA on LS174T cells as an early stage of tumor initiation were investigated.
Materials and Methods: Tumor cells were treated to various concentrations of DHA and proliferation, survivin expression, caspase-3 activation, and apoptosis were evaluated by different cellular and molecular techniques.
Results: Following 48 h treatment, proliferation was measured to be 73 ± 4.5% (P = 0.000), 53 ± 5.7% (P = 0.000) and 26.3 ± 3.5% (P = 0.000) for 50, 100, and 150 µM DHA, respectively compared to untreated cells. This molecule induced 63% (P = 0.001) and 46% (P = 0.000) decrease in survivin messenger ribonucleic acid (mRNA) level as well as 1.8 (P = 0.001) and three-fold (P = 0.000) increase in caspase-3 activation for 50 and 100 µM DHA, respectively compared to untreated cells. Our evidence showed that survivin mRNA is expressed at the early stage of colorectal cancer cells and DHA-treated cells expressed markedly a lower survivin mRNA compared to untreated cells.
Conclusions: DHA is an attractive repressor of survivin expression, increases caspase-3 and apoptosis in colorectal cancer cells and may provide a novel approach to the treatment of colorectal cancer at the early stage of tumor initiation.

Keywords: Apoptosis, caspase-3, colorectal cancer, docosahexaenoic acid, proliferation, survivin


How to cite this article:
Ahangar P, Sam MR, Nejati V, Habibian R. Treatment of undifferentiated colorectal cancer cells with fish-oil derived docosahexaenoic acid triggers caspase-3 activation and apoptosis. J Can Res Ther 2016;12:798-804

How to cite this URL:
Ahangar P, Sam MR, Nejati V, Habibian R. Treatment of undifferentiated colorectal cancer cells with fish-oil derived docosahexaenoic acid triggers caspase-3 activation and apoptosis. J Can Res Ther [serial online] 2016 [cited 2019 Dec 13];12:798-804. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/798/157326




 > Introduction Top


Colorectal cancer (CRC) is one of the leading causes of death in the world. In the United States, CRC was the third most common cancer with incidence rate of about 71,830 cases in men and 65,000 cases in women in 2014.[1] It has been also shown that CRC is the second most common cause of death due to cancer causing death in Europe with an incidence rate of 464,000 cases per year.[2],[3] In Europe, Czech Republic has one of the highest rates of colorectal cancer in men with incidence rate of 54 cases per 100,000.[3] In Asia, the incidence rate of CRC among the Chinese was 253,427 new cases in 2012.[4] In Iran, CRC is the most common cancer with 5,000 new cases per year.[5]

In CRC, the main curative strategy is complete surgical resection of the tumor associated with radiotherapy and/or chemotherapy. Unfortunately, CRC remains often refractory to these classic therapies [6] and the effectiveness of chemotherapeutic agents is often limited by the side effects on normal cells. With this in mind, the search for new therapeutic agents with minimal toxicity is of particular interest in colon cancer treatment.

Epidemiological studies have shown an inverse relationship between colorectal cancer incidence and fish or fish polyunsaturated fatty acids (PUFAs) consumption,[7] suggesting that fish-oil derived docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may have a protective effect against colorectal cancer development. In this regard, experimental data have shown that DHA and EPA inhibit the abnormal proliferation of epithelial cells of colon crypts of patients with sporadic colorectal adenomatous at high risk of colon cancer.[8],[9] Data are also available to show the growth inhibitory effects of n-3 PUFAs on colonic cancer cells in vitro and in vivo and on various kinds of cancer cells.[10],[11],[12],[13],[14],[15]

DHA is an omega-3 fatty acid with 22 carbons and six double bonds (22:6n-3). Several molecular mechanisms whereby n-3 fatty acids may modify the neoplastic process have been proposed. This molecule inhibits cyclooxygenase (COX) pathway and influences the neoplastic process through its role in suppressing effect on the biosynthesis of arachidonic acid (AA)-derived eicosanoids and downregulation of prostaglandins and thromboxanes.[16] Compared with AA, n-3 fatty acids is the preferential substrate for lipoxygenase and recently, it has been shown that 15-lipoxygenase metabolites of DHA; 17-hydroperoxy-; 17-hydroxy-; 10,17-dihydroxy-; and 7,17-dihydroxy-DHA inhibit the proliferation of prostate cancer cells.[17]

DHA and other essential fatty acids are known to increase cell membrane fluidity, enhance the activity of protein kinase C and other second messenger systems, as well as increase reactive oxygen species and lipid peroxidation.[18],[19] Furthermore, these fatty acids are also able to induce tumor apoptosis through cell cycle gene modulation or to activate and induce cell death via both a mitochondrial-dependent and a bax-mediated, mitochondrial-independent pathway.[13] DHA has also been shown to increase the therapeutic effects of chemotherapeutic drugs.[20]

Recently, it has been suggested that DHA may exert pro-apoptotic effects in cancer cells via regulation of apoptotic and anti-apoptotic genes.[21] Expression of survivin as an anti-apoptotic gene has been shown to be up-regulated in colorectal cancer.[22] The up-regulation of survivin gene has been reported to be correlated to chemoresistance of colorectal cancer cells and poor prognosis.[23] Survivin is involved in both inhibition of apoptosis and regulation of cell division, expressed in the most human neoplasms but rarely in normal tissues.[24] These facts candidate survivin as ideal marker for cancer directed-therapy. In consequence, the search for new therapeutic agents with property of survivin down-regulation may provide minimal toxicity on normal tissues in cancer therapy.

Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis [25] and the activation of caspase-3 is considered to be the final step in many apoptosis pathways.[26] It has been reported that, this protein was inhibited by survivin,[27] suggesting a relationship between apoptosis and cell cycle.

In the present study, we aim to determine the effects of different concentrations of DHA on cell viability, growth rate of malignant cells on a dose response and time course basis, survivin messenger ribonucleic acid (mRNA) level, caspase-3 activity assay, and apoptosis.


 > Materials and Methods Top


Cell line, culture condition, and primers

LS174T colorectal cancer cells (NCBI C568, National Cell Bank of Iran, Tehran) maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 µg/ml), all from (PAA, Austria); and 20 mM HEPES and 2 mM L-glutamine (Roche, Germany) at 37°C in a humidified incubator. The oligonucleotides for polymerase chain reactions (PCRs) [Table 1] were synthesized by MWG Biotech (Ebersberg, Germany).
Table 1: List of primers used in this study

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Preparation of fish-oil derived DHA and cell treatments

DHA (Sigma, USA) was dissolved in absolute ethanol (Merck, Germany) to prepare a 100 mM stock solution and stored in the dark at -80°C. For each experiment, the DHA was freshly prepared from the stock solution at the concentration ranging from 50 to 150 µM by serially diluting in culture medium. Control cells were cultured in the medium containing same concentration of absolute ethanol (v/v) as the DHA containing medium. The final ethanol concentration never exceeded 0.5% (v/v).

Cell count and viability test

LS174T colorectal cancer cells were seeded at a density of 2.5 × 105 cells/well in 1.5 ml of complete medium in six-well plates and incubated at 37°C for 24 h. After 24 h, the culture medium was replaced with fresh complete medium containing DHA at concentrations of 50, 100, and 150 µM and the cells were incubated for 48 h. After 48 h, the cells were harvested using trypsin-ethylenediaminetetraacetic acid (EDTA; Roche, Germany) and viable cells were counted under a microscope by trypan blue (Sigma, USA) exclusion.

Cell proliferation assay

Malignant cells were seeded into 96-well culture plates at a density of 5 × 103 per well. After 24 h incubation, the cells were treated with DHA at concentration ranging from 50 to 150 µM for 24, 48, and 72 h cell proliferation rates were evaluated by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Atocel, Austria) assay. Following each incubation period, the MTT solution (5 mg/ml in phosphate-buffered saline (PBS)) was added to culture medium and incubated for 4 h at 37°C. The culture medium of wells was removed and replaced with 200 μl of dimethyl sulfoxide (DMSO; Merck, Germany) to dissolve the MTT formazan crystals. The absorbance in each well was then measured with a microplate reader (State fox, USA) at 492 nm and cell proliferation was calculated as (Asample - Ablank)/(Acontrol - Ablank) ×100%. Thereafter, the results were expressed as the percentages of nontreated control cells. All experiments were performed in triplicate on three separate occasions.

Apoptosis Assays

Flow cytometric analysis

Apoptosis in LS174T cells was analyzed by a double staining method using annexin V-FLOUS/propidium iodide (PI) labeling solution (Roche, Germany) according to the manufacturer's instructions. Briefly, cells were culture in the absence or in the presence of 50, 100, and 150 µM DHA for 48 h. The cells were suspended by a trypsinization and washed twice with cold PBS. Cell pellets were then resuspended in 100 μL of × 1 binding buffer at a density of 4 × 105 cells/ml with fluorescein isothiocyanate (FITC)-annexin V. The cells were gently mixed and incubated in the dark at room temperature for 20 min. To differentiate cells with membrane damage, PI solution was added to the cell suspension prior to the flow cytometric analysis using a fluorescence-activated cell sorter (FACScan, USA).

Caspase-3 activity assay

Caspase-3 activation was determined using fluorometric-immunosorbent enzyme assay (Roche, Germany) according to manufacturer's instruction. Briefly, LS174T cells were cultured in the absence or in the presence of 100 and 150 µM DHA for 48 h. After trypsinization, cells were washed twice with ice-cold PBS and suspended in cold lysis buffer for 30 min followed by centrifugation at 12,000g for 10 min at 4°C. Thereafter, clear lysate was used for caspase-3 activation assay. The principle was that caspase-3 derived from cellular lysates is captured by a monoclonal antibody. The amount of activated caspase-3 was cleaved proportionally through the addition of fluorogenic substrate in a reaction buffer. Due to proteolytic cleavage of the substrate, free fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) is generated and measured with excitation at 380 nm and emission at 460 nm using a fluorescence spectrophotometer (Biotec, USA).

RNA preparation and reverse transcription PCR (RT-PCR)

Total cellular RNAs were extracted from treated and untreated cells using RNA preparation kit (Cinaclon, Iran) and used as the template to perform RT-PCR method to generate a first complementary DNA (cDNA) strand (Fermentas, Canada) according to the manufacturer's instruction. The generated fragment was subsequently used as template for the PCR-amplification of the double-stranded cDNA corresponding to a section of the survivin coding sequence, using oligonucleotides Survivin-F and Survivin-R as forward and reverse primers, respectively [Table 1] with the following temperature profile: After an initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 5 min, annealing at 61°C for 1 min, extension at 72°C for 1 min were followed by a final extension at 72°C for 7 min. In parallel, for internal control and normalization, the generated cDNA was used as template for the PCR amplification of a section of the human GAPDH coding sequence, using oligonucleotides GAPDH-F and GAPDH-R as forward and reverse primers, respectively [Table 1]. PCR products were visualized on 1% agarose gel (Roche, Germany) with ethidium bromide (Merck, Germany) staining.

Statistical analysis

The results are expressed as mean ± standard deviation (SD). Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) version 15 software. A value of P < 0.05 was considered to be statistically significant.


 > Results Top


Effect of DHA on cell numbers and viability

LS174T cells at a density of 2.5 × 105 were cultured with or without DHA (0, 50, 100, and 150 µM) for 48 h, to evaluate the antiproliferative effects of DHA on cell numbers and viability. As shown in [Figure 1]a, following 48 h treatment, DHA at 50, 100, and 150 µM concentrations diminished significantly cell numbers by 16.5 × 105 (P = 0.001), 12.5 × 105 (P = 0.000), and 8.1 × 105 (P = 0.000); and decreased viability by 63 ± 4.1% (P = 0.000), 48 ± 6.1% (P = 0.000), and 31.5 ± 5.1% (P = 0.000) at indicated concentrations, respectively compared to the untreated cells [Figure 1]b. As the data indicates, reduction in cell viability correlates directly with the concentration of DHA.
Figure 1: Effects of various concentrations of DHA on cell number (a) and viability (b) after 48 h treatment of 2.5 × 105cells/well. Data represent at least three independent experiments. (c) Untreated control cells. DHA = Docosahexaenoic acid

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Effect of DHA on cell proliferation

The MTT assay was used to determine the effect of DHA on the mitochondrial activity of the LS174T cells as an indicator of viable cells. Treatment of cells with increasing concentrations of DHA significantly diminished cell growth in a dose- and time-dependent manner [Figure 2]a and [Figure 2]b. DHA at 50, 100, and 150 µM concentrations inhibited growth rate by 64.3 ± 4%, (P = 0.000), 44 ± 2.1% (P = 0.000), and 19.7 ± 5.5% (P = 0.000) after 72 h treatment, respectively compared to untreated control cells. Moreover, at the same concentrations, cell proliferation was inhibited in a time-dependent manner [Figure 2]b.
Figure 2: Effect of DHA on the cell growth (5 × 103 cells/well) on a dose response (a) and time course basis (b). Data represent at least three independent experiments. **Sample that is significantly different compared to untreated control cells. Significant difference was not observed between 24h treatment with 50 μM DHA (a and b) and untreated control cells. P values were measured to be 0.000 for all treatments

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Effect of DHA on survivin mRNA level

Survivin is a novel member of the inhibitor of apoptosis and determines the susceptibility of malignant cells to proapoptotic stimuli. As shown in [Figure 3], after 48 h treatment with DHA, survivin mRNA level was downregulated significantly by 63% (P = 0.001) and 46% (P = 0.000) at concentrations 50 and 100 µM DHA, respectively, as compared to the untreated cells. The higher concentrations of DHA induced lower survivin mRNA level and higher apoptosis rate in treated cells.
Figure 3: Effect of DHA on survivin mRNA level. Malignant cells were incubated with DHA at concentrations of 50 and 100 μM and survivin mRNA level was determined by semiquantitative RT-PCR. (c) Untreated control cells. RT-PCR = Reverse transcription polymerase chain reaction

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Effect of DHA on caspase-3 activation

Caspase-3 belongs to a family of cysteine proteases that are involved downstream in the process of apoptosis. Caspases exist as inactive proenzymes in the cells, which undergo proteolytic cleavage to become active enzymes. In order to show that the DHA-induced reduction of cellular proliferation and/or viability was due at least in part to apoptosis, caspase-3 activation was measured in LS174T cells after 48 h treatment with DHA. As the data indicate, under the treatment of DHA, caspase-3 activation was induced in a dose-dependent manner. The effect of DHA was evidenced by 1.8 (P = 0.001) and threefold (P = 0.000) increase in caspase-3 activation for 50 and 100 µM DHA, respectively, as compared to untreated cells. The higher concentration of DHA induced higher caspase-3 activation [Figure 4].
Figure 4: Effect of DHA on caspase-3 activation. LS174T cells were exposed to DHA for 48 h and caspase-3 activation was measured with fluorometric-immunosorbent enzyme assay. Data represent at least two independent experiments. (c) Untreated cells

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Effect of DHA on apoptosis

To evaluate whether the growth-inhibitory effect observed upon treatment of LS17T cells with DHA was due to the induction of apoptosis, cells were treated with DHA at concentration ranging from 50 to 150 µm for 48 h, and subsequently stained with annexin V/propidium iodide (PI) and analyzed by means of flow cytometer. With this method, cells stained single positive for PI were considered mostly necrotic cells, and cells stained single-positive for annexin V were considered mostly early apoptotic cells, but cells that were stained double-positive could be either necrotic or apoptotic cells. As depicted in [Figure 5]a and [Figure 5]b, LS174T cells treated with DHA at various concentrations of 50, 100, and 150 µM displayed approximately 2.7, 18.7, and 29% late apoptotic and necrotic cells as well as 2.1, 13.2, and 17% necrotic cells, respectively, compared to the untreated cells. Based on our data, treatment of cells with DHA increased significantly apoptosis and necrosis rates compared to the untreated cells in a dose-dependent manner.
Figure 5: Effect of DHA on apoptosis. (a) After 48 h treatment, DHA-treated cells stained with annexin V/propidium iodide and analyzed by means of flow cytometer. Q1 = Prenecrotic cells, Q2 = late apoptosis + necrosis, Q3 = living cells, Q4 = early apoptotic cells. (b) Quantification of apoptotic and necrotic cells at different concentrations of DHA

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Relationship between survivin mRNA level and apoptosis rate

In treated cells, expression of survivin was downregulated by DHA. To evaluate the functional role of survivin downregulation in colorectal cancer cells, evaluation of cell proliferation and apoptosis is necessary. In this regard, we found that, there is a strong association between levels of survivin mRNAs and apoptosis rate in DHA-treated cells. The higher concentrations of DHA which is followed by decreasing in cell proliferation rate, induced lower survivin mRNA level and higher apoptosis rate in treated cells as compared to the untreated cells [Figure 6].
Figure 6: Relationship between survivin mRNA level and apoptosis rate. Malignant cells were treated to 50 and 100 μM DHA and survivin mRNA level and apoptosis were measured. The results represent mean and standard deviation

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 > Discussion Top


The epidemiological studies, animal studies, and in vitro experiments show that DHA is a major component of fish oil, possess anticancer properties,[7] and induce programmed cell death in various kinds of cancer cells such as leukemia, prostate, breast, pancreatic, and lung cancer cells.[13], 17, [28],[29],[30]

In vitro studies have shown that treatment of different colorectal cancer cell lines with DHA induce growth-inhibitory effects on these cells.[31],[32] However, little attention has been paid to explore the potential of this fatty acid on proliferation, induction of apoptosis, and survivin gene expression in primitive and undifferentiated colorectal cancer cells.

As LS174T cells represent an early stage of tumor development and has a potent prostaglandin E2 (PGE2)-triggered activation of proliferation,[32] evaluation of the effects of DHA on this cell line could provide invaluable information about apoptosis and cell proliferation at the early stages of tumor initiation and progression.

Based on our data, DHA significantly reduces the viability and proliferation of colorectal cancer cells and also promotes apoptosis compared with untreated cells. In the present study, DHA could inhibit the growth of LS174T cells in a time- and dose-dependent manner, which is in accordance with the results obtained by Dommels et al., showed a dose-dependent decrease in proliferation of Caco-2 cells by treatment with DHA.[33]

In this study, proliferation rates were lowest in cells treated with150 µm DHA after 72 h compared with other treatments and time points. It seems that the DHA requires some time to develop its cytotoxic-enhancing mechanisms. This is possibly due to the time required to form free radicals in the cells that are the most probable potentiating agents.[34]

Our results are in agreement with previous reports showing that PUFAs such as DHA induce apoptosis in colorectal cancer cells.[10],[31],[35] In this study, DHA could induce apoptosis via caspase-3-dependent mitochondrial death pathway which was previously shown in the DHA-induced apoptosis in U937 monocytic leukemia cells.[18] Activation of caspase-3, resulting in the death of LS17T cells, was well suited with the fall in cell count, viability, and proliferation. These results are in accordance with previous findings obtained with DHA using different cellular models.[18],[21]

In the present study, quantification of apoptotic cells revealed that, DHA is a potent inducer of apoptosis in undifferentiated colorectal cancer cells, raising the possibility of its therapeutic use as an antiproliferative candidate drug to enhance the efficacy of colorectal cancer treatment at the early stage of tumor initiation.

The differential survivin expression in tumor cells make it a promising target for cancer directed-therapy.[24] This molecule is a cellular factor involved in colon carcinogenesis and known to be related to cancer cell resistance to apoptosis.[21] Given that survivin is a potent inhibitor of caspase activation and the role of survivin in DHA-induced apoptosis has never been addressed in undifferentiated colorectal cancer cells, we were interested to examine whether DHA could downregulate survivin for apoptosis. Our evidence showed that survivin mRNA is expressed at the early stage of colorectal cancer cells and DHA-treated cells expressed markedly a lower survivin mRNA level and higher caspase-3 activation compared to the untreated cells in a dose-dependent manner. Therefore, the proapoptotic effect of DHA in this study may be related to the DHA-induced downregulation of survivin, which has provided activation of caspase-3 to continue the final step in apoptosis. Based on these results, survivin inhibition clearly validates survivin as a valuable drug target. Survivin is expressed in most of the human neoplasms, but rarely in normal tissues.[24] These facts bring this molecule at the center of attention for cancer directed-therapy. It is possible that by combining safe, readily available dietary fatty acids with standard chemotherapy treatments, the expression of survivin can be inhibited effectively in cancer cells in vivo. This promising strategy may increase sensitivity of cancer cells to some chemotherapeutic agents and may induce lower side effects on normal tissues.


 > Conclusion Top


DHA is an attractive and a newly identified repressor of survivin expression, inhibits cell proliferation, increases caspase-3 activation, and induces apoptosis in undifferentiated colorectal cancer cells. These results suggest that survivin may provide a valuable drug target and may provide a novel therapeutic approach to enhance the efficacy of colorectal cancer treatment at the early stage of tumor initiation.

 
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