The combined effect of fish oil containing Omega‐3 fatty acids and Lactobacillus plantarum on colorectal cancer

Abstract Colorectal cancer (CRC) is one of the deadliest malignancies. Recent attempts have indicated the role of diet in the etiology of CRC. Natural dietary compounds such as probiotics and Omega‐3 fatty acids that act synergistically can be beneficial in finding a tremendous solution against CRC. To date, the combined effect of fish oil containing Omega‐3 fatty acids (Omega‐3) and Lactobacillus plantarum (L. plantarum) on CRC has been left behind. We here evaluated the effects of co‐encapsulation of Omega‐3 and probiotic bacteria on CRC cell lines compared to normal cells. Omega‐3 and L. plantarum bacteria were co‐encapsulated in three ways, including gelatin–gum Arabic, gelatin–chitosan, and chitosan–gum Arabic complex coacervate microcapsules. After treatment of cells (Normal [L929] and colorectal [C26]) by L. plantarum, Omega‐3, and microcapsules, viability and growth capacity of cell lines were measured using the MTT (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide) assay. Isolated total RNA was used to evaluate the expression profile of BCL2‐associated X protein (BAX), B‐cell lymphoma 2 (BCL‐2), and Caspase‐3 (CASP3) genes by real‐time polymerase chain reaction (PCR). Statistical analysis was performed with SPSS 25 software. A value of p < .05 was considered statistically significant. The results indicated a significant reduction in cell viability of C26 in a concentration‐dependent manner in the treated cells with all treatments, except gelatin–gum Arabic microcapsules. The messenger RNA (mRNA) expression level of the BAX and CASP3 genes in C26 cells being treated with all treatments significantly increased than in untreated cells, and the expression level of the anti‐apoptotic factor of the BCL‐2 gene decreased in C26 cells simultaneously (p < .05). Although, the combined effect of Omega‐3 and L. plantarum and microcapsulated treatments had no more effect on viability and apoptosis gene expression of cancer cells compared to Omega‐3 or L. plantarum. In conclusion, combination therapy with fish oil containing Omega‐3 and L. plantarum does not improve the anticancer effect of each alone.

a tremendous solution against CRC. To date, the combined effect of fish oil containing Omega-3 fatty acids (Omega-3) and Lactobacillus plantarum (L. plantarum) on CRC has been left behind. We here evaluated the effects of co-encapsulation of Omega-3 and probiotic bacteria on CRC cell lines compared to normal cells. Omega-3 and L. plantarum bacteria were co-encapsulated in three ways, including gelatin-gum Arabic, gelatin-chitosan, and chitosan-gum Arabic complex coacervate microcapsules. After treatment of cells (Normal [L929] and colorectal [C26]) by L. plantarum, Omega-3, and microcapsules, viability and growth capacity of cell lines were measured using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Isolated total RNA was used to evaluate the expression profile of BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and Caspase-3 (CASP3) genes by real-time polymerase chain reaction (PCR). Statistical analysis was performed with SPSS 25 software. A value of p < .05 was considered statistically significant. The results indicated a significant reduction in cell viability of C26 in a concentration-dependent manner in the treated cells with all treatments, except gelatin-gum Arabic microcapsules. The messenger RNA (mRNA) expression level of the BAX and CASP3 genes in C26 cells being treated with all treatments significantly increased than in untreated cells, and the expression level of the anti-apoptotic factor of the BCL-2 gene decreased in C26 cells simultaneously (p < .05). Although, the combined effect of Omega-3 and L. plantarum and microcapsulated treatments had no more effect on viability and apoptosis gene expression of cancer cells compared to Omega-3 or L. plantarum. In conclusion, combination therapy with fish oil containing Omega-3 and L. plantarum does not improve the anticancer effect of each alone.

| INTRODUC TI ON
Colorectal cancer (CRC) is the third most commonly diagnosed cancer type and the second most common cause of cancer death worldwide, with almost 2 million new cases in 2020 years (Sung et al., 2021). Environmental factors like particular dietary, lifestyle, and genetic factors can facilitate polyp growth and the development of CRC by promoting intestinal inflammation and changing the microbiome of the colorectal (Rafter & Gastroenterology, 2003). Due to the complex relationship between microflora and CRC development, finding the mechanisms by which the bacterial community impacts CRC could introduce a new therapeutic approach. However, detecting a particular bacterial community or even modifying the abundance of one strain responsible for CRC development is not easy (Rafter & Gastroenterology, 2003). Recent studies have revealed that probiotic consumption affects the diversity and richness of microbiota and can reduce chronic inflammation (Liu et al., 2011;Ohara et al., 2010). Among the identified probiotic bacteria strains, Lactobacillus plantarum (L. plantarum) is well documented based on its activity and anti-inflammatory and anticarcinogenic role in cells (Dong et al., 2012;Evrard et al., 2011). It causes apoptosis by inducing reactive oxygen species (ROS), overexpression of pro-apoptotic proteins of BAX, cytochrome C (Cyt C), Caspase-3 (CASP3), Caspase-8 (CASP8), and Caspase-9 (CASP9), and inhibition of antiapoptotic protein of B-cell lymphoma 2 (BCL-2) (Kim et al., 2015;Sun et al., 2021). However, the clinical efficacy of probiotics remains low due to reducing their survival rate during processing, storage, and passage through the gastrointestinal (GI) tract (Razavi et al., 2021).
Exposure to low gastric pH, oxygen, and temperature in the GI tract can influence quantities of probiotic bacteria at the site of action.
Nowadays, microencapsulation of probiotics has been considered an appropriate way to improve the survival rate and protection of probiotics in the GI tract (Razavi et al., 2021). Microencapsulation is a process to protect cells from injury or loss by retaining cells within a specific encapsulating membrane (Krasaekoopt et al., 2003). On the other hand, Omega-3 fatty acids (Omega-3) in fish oil, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have anticancer effects (Gutiérrez et al., 2019). They can decrease cell viability and induce pro-apoptotic pathways in CRC (Cockbain et al., 2012;Volpato & Hull, 2018). Previous investigations have shown a synergetic effect between probiotic bacteria and Omega-3 in the GI tract by enhancing the binding probiotic to the intestinal wall (Eratte et al., 2015). Additionally, a study showed that the viability of bacteria increased when they were co-encapsulated with tuna oil compared to being encapsulated on their own (Eratte et al., 2015).
To date, the combined effect of co-encapsulated Omega-3 and probiotic bacteria on CRC cell lines has not been reported. Therefore, this study aimed to evaluate the effect of Omega-3 and L. plantarum combination therapy on survival and expression of apoptosis-related genes in the C26 colorectal cancer (CRC) cell line compared to that in a normal L929 fibroblast cell line.

| Chemicals and reagents
Lactobacillus plantarum, a gram-positive lactic acid bacterium, can mainly be found in fermented food and the GI tract and nowadays it is utilized in the food industry as a probiotic. L. plantarum bacteria (PTCC 1058) was purchased from the Biotechnology Department in the Iranian Research Organization for Science and Technology (IROST), Iran. Omega-3 dietary supplement (fish oil) was purchased from Golden Seas company (Yazd, Iran). According to data provided by the supplier, the major fatty acids in this fish oil were as follows: 240 mg DHA and 360 mg EPA. Gelatin, chitosan, and gum Arabic, and other materials were purchased from Sigma-Aldrich Ltd., Australia.
Fetal bovine serum (FBS) and trypsin were purchased from Gibco, Germany. Cell culture plates and flasks were purchased from SPL, Korea, and the microtubes were purchased from Ratiolab, Germany.
All primers were designed and purchased from DNA Pioneer, Korea.
RNA extraction kit and complementary DNA (cDNA) synthesis kit were purchased from FAVORGEN, Taiwan, and Addbio, Korea.

| Preparation of L. plantarum culture and L. plantarum extract
The L. plantarum was cultured for 48 h in MRS (Man, Rogosa, and Sharpe) broth at 35°C. All the inoculation works were done under laminar air flow. Finally, the cells were harvested at the stationary growth phase by centrifuging at 1500 rpm for 15 min. The supernatant was separated and considered as the L. plantarum extract. After discarding the supernatant, the precipitated cell mass was washed twice with sterile Normal saline (0.9% NaCl) to obtain L. plantarum suspension.

| Microencapsulation preparation
The complex coacervation and microencapsulation procedures were carried out as detailed in our previous study (In press). Briefly, co-encapsulation, colorectal cancer, Lactobacillus plantarum, omega-3 fatty acids Omega-3 and L. plantarum were co-encapsulated in three ways, including gelatin-gum Arabic, gelatin-chitosan, and chitosan-gum Arabic complex coacervate microcapsules with the wall to a core ratio of 4:1. Microcapsules were prepared with a high-speed homogenizer at 7200 rpm for 10 min and an equal proportion of bacteria and Omega-3 fish oil (1:1).

| Cell culture and viability assay
Normal subcutaneous connective tissue (L929) and colorectal mouse (C26) cell lines were purchased from Pasteur Institute (Tehran, Iran) and cultured in DMEM low glucose media containing 10% FBS and antibiotic (containing 50 U/ml Penicillin and 50 μg/ml of Streptomycin). These cells have fibroblast morphology. The cells were incubated at 37°C in 90% humidity containing 5% CO 2 . After the cells reached 70% confluence, they were dissociated by trypsin 0.25% and centrifuged at 1800 rpm for 5 min (Yazdani et al., 2020).  Table 1). The untreated cells are considered a control group. After 24 h, the cells in each study group were treated with 20 μl of the MTT reagent (5 mg/ml in sterile phosphate-buffered saline [PBS]) and incubated at 37°C for 4 h. Finally, the culture medium was removed, and 200 μl of DMSO was added to dissolve the formazan crystals.
The results were measured at the absorbance of 570 nm in a microplate reader (Bio-Tek Instruments, Inc., Vermont, USA). The percentage of cell viability was calculated following the formula: % viability = absorbance test/absorbance control × 100 (Yazdani et al., 2020).

| Gene expression
The L929 and C26 cell lines were cultured at 2 × 10 6 cells per well in 6-well plates. After 24 h, cells were treated with an IC50 concentration of L. plantarum bacteria, L. plantarum extract, Omega-3, gelatin-gum Arabic microcapsule, gelatin-chitosan microcapsule, chitosan-gum Arabic microcapsule, and combination therapy of Omega-3 and L. plantarum, and untreated cells considered as controls. Total RNA was extracted and cDNA was synthesized using Addbio cDNA Synthesis Kit, Korea, based on the manufacturer's protocols. The synthesized cDNA was directly used as a template for real-time quantitative polymerase chain reaction (RT-qPCR). The primer design was performed by Oligo 7.0 software for BAX, BCL2, and CASP3 genes and glyceraldehyde 3-phosphate dehydogenase (GAPDH) as a reference housekeeping gene (Yazdani et al., 2021).
It is noticed that only a specific reverse primer was used for cDNA synthesis. Therefore, for the TaqMan real-time quantitative PCR (RT-qPCR), forward primers were used and Bio-Rad CFX qPCR Instrument was used to carry out a real-time PCR. Amplification reactions were performed in a 15 μl reaction volume containing 13.5 μl Master Mix, 0.5 μl of specific forward primer (10 μM), 1 μl of cDNA, 2 μl of H 2 O. Each sample was tested in triplicate. The optimized PCR conditions were 95°C for 15 min, followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min. The levels of mRNA expression were normalized to GAPDH and were calculated using the 2 −ΔΔCt method (Yazdani et al., 2021).

| Statistical analysis
All data were presented as mean ± standard error. Statistical significance of differences between mean values was analyzed by oneway analysis of variance (ANOVA) followed by Tukey's test using IBM SPSS Statistics 25 software. A value of p < .05 was considered statistically significant.

| Co-encapsulation efficiency
The complex coacervation and microencapsulation procedures were carried out as detailed in our previous study (In press). Briefly, the viability of L. plantarum in gelatin-gum Arabic, gelatin-chitosan, and chitosan-gum Arabic microcapsules is presented in Table 1.  with fish gelatin biopolymers, chitosan, and gum Arabic as wall materials can produce Omega-3 capsules and L. plantarum in micrometer sizes following the protocol described in our previous study (In press).

| Cytotoxicity test
The cytotoxic effect of different treatments on normal (L929) and cancer (C26) cells was tested using MTT assay. Figure 1 Table 2 shows the IC50 of each treatment on the C26 cell line.

| DISCUSS ION
In the present study, the findings manifested that Omega-3 exhibited potent antitumor effects on C26 cancer cells. Previous studies revealed that both DHA and EPA affect CASP3 and, consequently induction of apoptosis (Sauer et al., 2005;Xue et al., 2017). Also, the BCL-2 family proteins are essential in Omega-3-induced cell death (Çetin et al., 2021;Lessa et al., 2021;Ma et al., 2021). extract. Therefore, the BAX/BCL-2 ratio was significantly increased, which may lead to the collapse of mitochondrial membrane potential, resulting in the release of cytochrome c (Cyt) and consequently causing cell apoptosis (Naseri et al., 2015). Additionally, upregulation of CASP3 gene expression in C26 cells was detected in cells being treated with L. plantarum, suggesting the activation of mitochondrial apoptotic pathways (Brentnall et al., 2013). Our data are consistent with the previous reports (Jiang et al., 2020;Sentürk et al., 2020;Sun et al., 2021;Yue et al., 2020).
The stability and delivery of bacteria extract to the exact site of action is a crucial factor in cancer treatment. Therefore, it is essential to utilize a suitable method of delivery and stability for effective cancer therapeutics. Microencapsulation of probiotics has been con-

| CON CLUS ION
The present study showed that Omega-3 selectively induces cytotoxicity and apoptosis in tumor cells. In addition, the suspension of L. plantarum and bacteria extract induced apoptosis in cancer cells more than in normal cells. The combined effect of L. plantarum and Omega-3 did not improve cytotoxicity and apoptosis in colorectal C26 cancer cells compared with monotherapy with L. plantarum or Omega-3.

ACK N OWLED G M ENTS
The authors thank the Mazandaran University of Medical Sciences, and Sari Agricultural Sciences and Natural Resources University for their assistance.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
The protocol of this study was approved by the Research Ethics