Anticancer effect of Sargassum oligocystom hydroalcoholic extract against SW742, HT-29, WiDr and CT-26 colorectal cancer cell lines and expression of P53 and APC genes

Colorectal cancer (CRC) is the third most common cancer in the world, with enhancing morbidity and mortality each year. Due to the drug resistance against CRC, the use of novel compounds besides chemotherapy is required. Natural seafood contains large amounts of biologically active substances with new chemical structures and new medicinal activities. The aim of this study was to evaluate the effects of hydroalcoholic extract of Sargassum oligocystom algae on SW742, HT-29, WiDr and CT-26 CRC cell lines and to evaluate the expression of P53 and APC genes using quantitative real time PCR (RT-qPCR). The cytotoxicity of oligocystom was by and trypan blue in six different concentrations including and / on various cell lines and a control group. The expression of P53 and APC genes in exposure to 2mg/mL of the extract was also evaluated using RT-qPCR. had anticancer effect via increase in the APC gene expression.


Introduction
One of the most common problems in the medical world has been the resistance of cancer cells to antitumor drugs, hence nding novel anti-cancer compounds with minimal side effects seems necessary with this regard. Colorectal cancer (CRC) mainly originates from adenomatous polyps, some of which are premalignant and develop into cancer [1,2]. CRC generally occurs among people > 50 years of age and older when abnormal cells divide in the large intestinal epithelium. The mortality rate from CRC is about 40%.
Genetic factors and in ammation of colon epithelial cells, epigenetics, and individual behavior and nutrition are important in the onset and progression of CRC [3,4]. Approximately 30% of cases are inherited and people who consume more calories, protein and fat are at greater risk. The apoptosis in cancer cells inhibits cancer progression [5,6]. The development of anticancer drug resistance is also a dilemma. Anticancer drugs should act exclusively on cancer cells, while some of the chemotherapy drugs currently used in cancer patients have many side effects on the human body [7,8]. These effects include bleeding, hair loss, diarrhea, and device suppression, so research is needed to nd a compound with especial targeting. Antitumor properties that have the ability to prevent the spread and growth of cancer cells have made signi cant progress in recent years due to the vital biological role of seaweed in the safety and improvement of life of cancer patients [7,8]. Extensive studies in medical-industrial applications of these products have been developed and their anti-tumor effects have led to the pursuit of wider studies by researchers. The cytotoxic effects of extracts of some green and brown algae in a dosedependent response against leukemia in mice were subsequently studied, assessing the effect of Spirulina blue algae polysaccharide inhibitory effects against several tumors [10,11]. The cytotoxic effects of red seaweed Sargassum crispum and Sargassum oligocystom has revealed promising results when determined with IC50 in vitro [12][13][14]. The apoptotic effects of brown algae have also been con rmed by microscopic observations and analytical methods by MTT assay and enzyme-linked immunosorbent assay (ELISA). Bioactive compounds that induce apoptosis in cancer cells can be considered as an effective therapeutic agent. Aqueous algae compounds mostly include polysaccharides, ortanins, carotenoids, minerals, peptides and sulfo-peptides [10,11]. Among brown algae, Sargassum spp. contain a glycoprotein with anti-cancer effects against the human CRC [13,14]. The anticancer effects of several algal genera, particularly Sargassum spp against human leukemia cells (MOLT), K562, mouse lymphocytic leukemia cells (p.388), 180-sarcoma cells have been revealed. Our aim was assessment of anticancer effect of S. oligocystom hydroalcoholic extract against SW742, HT-29, WiDr and CT-26 CRC cell lines and expression of P53 and APC genes.

Materials And Methods
Algae collection and extract preparation S. oligocystom was collected from the Persian Gulf. After washing the algae, it was dried at room temperature for two weeks and after cleaning the obtained ne powder, it was combined with 300 mL of deionized water and the obtained suspension was boiled for 3 hours and then the suspension was passed through paper. The ltered hydroalcoholic extract was lyophilized into the powder and stored at 4°C until use [15].
Preparation and culture of cell lines Various CRC cell lines including SW742, HT-29, WiDr and CT-26 cells were purchased from Pasteur Institute of Iran. The cell lines were placed in DMEM medium with 10% fetal bovine serum (FBS) and 100 μg / mL of penicillin and streptomycin and incubated at 37 ° C containing 5% CO2 and 90% of humidity.

Preparation of the algae extract
Firstly, 100 mg of lyophilized S. oligocystom powder was weighed and 1 mL of phosphate buffered saline (PBS) was added to the powder, and after vortex, 9 mL of medium was added and extract was reached to a volume of 10 mL and then ltered using a 0.2 µL lter and completely puri ed and homogenized. The prepared extract was stored at -20°C until use [15,16].

The MTT assay
In this method, cells (100µL) were cultured in 96-well plates. Then, concentrations 0.1, 0.2, 0.5, 1 , 2 and 4mg / mL of the extract were prepared and each concentration was exposed to each 96-well plate containing each cell line as an independent group. The plates were incubated in 5% CO2 and 90% humidity for 24 and 48 hrs separately. Then each supernatant was taken and MTT dye was added to wells and the plates were wrapped in aluminum foil and incubated for 4 hours. Then the MTT dye was taken and DMSO was added to each well and placed in the shaker for 20 minutes to make it completely uniform and then light absorbance rate of each well was measured at 570 nm wavelength [16,17].
Cells viability using trypan blue dye Brie y, the cells were cultured in 96-well plates containing the DMEM medium and various concentrations of S. oligocystom was added and incubated for 72h at 37°C and supplementation of 5% CO2. Next 20µL of trypan blue was mixed with 20µL of culture cell and the number of cells were counted using hemocytometer neobar lamella. The percentage of living cells was measure using the following formula: Cells viability percentage= 1-(living cells/total cells)×100

Expression of APC and P53 genes
The cell lines were exposed to 2mg/mL of S. oligocystom for 24h. Next, RNA extraction from each cell line was conducted using Gen-All kit according to protocol of the manufacturer. The real-time PCR reaction was performed at a nal volume of 20λ and repeated twice for each group. The concentration of primers was 150 nM. The quanti cation and analysis of gene expression was performed using semiquantitative method considering ΔΔCT formula and RealTime PCR ABI software [].

Data analysis
Results analysis was performed using SPSS software version 21 and One-way ANOVA statistical test.
Quantity difference was de ned at level of 0.05.
According to the MTT assay, S. oligocystom exerted a substantial anticancer effect at 4mg/mL, however, this concentration was also toxic against normal cells. Therefore, the concentration of 2mg/mL was e cient against all cell lines. Moreover, these effects were time-dependent.

Trypan blue test
In the concentration of 4mg/mL of S. oligocystom, 96% of cells were killed, and at concentrations 2, 1, 0.5, 0.2 and 0.1 mg/mL 91%, 81%, 56%, 31 and 11% of them were killed, respectively (table1). There was no signi cant difference among various cell lines, but a signi cant viability decrease was observed at concentrations ≥1mg/mL. Gene expression The effect of 2 mg/mL of S. oligocystom on the expression of APC and P53 genes after 24h included 2.2 fold (p<0.001) increase in the former and 0.66 fold (p=0.323) increase in the latter genes. Therefore, the S. oligocystom had anticancer effect via increase in the APC gene.

Discussion
The cancer has an increasing trend around the world. Physicians and researchers have been trying to improve the general condition of cancer patients using different methods of chemotherapy, radiation therapy and surgery [1][2][3]. However, despite the development of therapeutic interventions, development of novel chemotherapeutics, the mortality rate of patients with CRC is still high [18]. Therefore, the application of novel alternative compounds in various extracts will be promising for inducing cell death (apoptosis) in cancer cells. It has been shown that hydroalcoholic extract of some algal species had signi cantly higher anticancer effects than other extracts against cancer cells [19][20][21]. In another study, alcoholic and chloroform extracts of Polysiphonia lanosa were signi cantly more effective against DLD-1 and HTC-116 CRC cell lines [22]. Moreover, Gracilaria edulis methanolic extract had signi cantly higher effect against HT-29 CRC cells [23].
In this study, the effect of hydroalcoholic extract of S. oligocystom was evaluated against several CRC cell lines. The 50% cell cytotoxicity (LD50) of S. oligocystom against SW742, HT-29, WiDr and CT-26 cell lines after 24h included 0.5, 1, 1 and 0.5mg/mL, respectively (Fig. 1). After 48h, the LD50 of this algae included 0.2, 0.5, 0.5 and 0.2 mg/mL, respectively. Moreover, LD90 of this algae was > 2mg/mL after 24h and > 1mg/mL after 48h for all cell lines. The results exhibited that concentration of ≥ 0.5mg/mL of S. oligocystom can be considered for anticancer therapies. It has been veri ed that Sargassum spp have antioxidant and anticancer effects against some cancer cell lines such as HepG2, Hela, MDA-MB-231, MCF-7, HT-29 and LNCap in vitro. We also did not assess the in vivo results. In previous studies, Sargassum spp has conferred anticancer effects at higher concentrations [21][22][23][24][25][26].
In the trypan blue test, in the concentration of 4mg/mL of S. oligocystom, a mean of 96% of cells were killed, and at concentrations 2, 1, 0.5, 0.2 and 0.1 mg/mL, 91%, 81%, 56%, 31 and 11% of them were killed, respectively. There was no signi cant difference among various cell lines, but a signi cant viability decrease was observed at concentrations ≥ 1mg/mL. It is crucial to determine a special dose for anticancer treatment using more exact veri cation of anticancer effects of S. oligocystom because of potential effects in this study.
We also observed that S. oligocystom can increased the expression of APC gene, a regulatory gene necessary for the control of cell division. One of the most common mutations in the CRC development includes the inactivation of the APC gene which results in uncontrolled cells proliferation and polyp development. However, patients with APC mutations have the risk of developing CRC approximately at the age of 40 [27]. The APC protein from mutation is truncated, abnormal, and dysfunctional. This short protein cannot prevent cell overgrowth, thus leading to the formation of polyps that can become cancerous. APC is also involved in the demonstration of microtubules by binding to the PD2 domain. APC inactivation can be initiated after speci c chain reactions in the cytoplasm [28]. Mutations in the APC gene mostly occur early in cancers, such as CRC. Human develop the CRC due to mutations in the APC gene.
In addition, P53 acts as a guardian of the genome to maintain genome stability by preventing incidence of mutations. This suggests that the TP53 gene plays an important role in preventing cancer formation, with proteins encoded by TP53 binding to DNA and regulating gene expression to prevent genome mutation (in normal cells P53 binds to its negative regulator, MDM2 complex). Following DNA damage or

Conclusion
Herein, S. oligocystom was rstly studied to effect on SW742, HT-29, WiDr and CT-26 CRC cell lines and it was con rmed that the algae hydroalcoholic extract conferred toxic activity and growth inhibition against CRC cells. Gene expression analysis exhibited cell death inducing by the extract of the S. oligocystom through activating and increasing the expression of the APC gene, which is a tumor suppressor gene, especially in CRC cells. According to our results, S. oligocystom exerted a substantial anticancer effect at 4mg/mL, however, this concentration was also toxic against normal cells. Therefore, the concentration of 2mg/mL was safe and e cient against all cell lines. Moreover, these effects were time-dependent.

Declarations
Funding This study was supported by Baghdad University.  Figure 1 The MTT assay of the S. oligocystom against SW742, HT-29, WiDr and CT-26 cell lines after 48h (viability percentage)