Chemopreventive Effect of Stoechospermum marginatum against Experimental Colon Carcinogenesis Induced by 1, 2-Dimethyl Hydrazine

Colorectal cancer is one of the most common malignant cancers of the gastrointestinal tract, representing 13% of all malignant tumours (Granados-Romero et al., 2017). An effective treatment for cancer is still lacking due to development of resistance, side effects and the availability of safe and specific anticancer drug. Natural products have received great attention in the recent years for owing to its reduced toxicity and side effects. 
       
Macroalgae, commonly known as seaweeds, are excellent sources of terpenoids, steroids, pholorotannins, polyketides, alkaloids, flavonoids, polysaccharides and were reported to contain various therapeutic properties including antioxidant, anti-inflammatory, anti-diabetic and anticancer activities (Sharif et al., 2014).
       
Stoechospermum marginatum, brown macroalgae of class phaeophyceae (Family Dictyotaceae) is widely distributed in eastern coastal regions of Tamil Nadu. Phytochemicals such as triterpenoids, sulfated polysaccharides, phenols, flavonoid, tannin, saponin, sterols, steroids, glycosides were reported in S. marginatum (Anbu et al., 2017). S. marginatum was reported to have antioxidant, antiviral, antifungal, antibacterial, cytotoxic and antiproliferative activities (Adhikari et al., 2006; Esmaeili and Khakpoor, 2012; Velatooru et al., 2016). Keeping the fact in view, the present study was undertaken to explore the chemopreventive potential of Stoechospermum marginatum.

The study was conducted at the Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai during January 2019.
 
Preparation of seaweed extract
 
Fresh thallus of Stoechospermum marginatum (Fig 1) was collected from the Gulf of Mannar Region of Mandapam Coast, Tamil Nadu, India and were authenticated. They were washed, air dried and powdered. The ethanolic extract was prepared by continuous hot percolation at 55°C in soxhlet apparatus. The extracts were then vacuum concentrated, air dried and stored at 4°C.
 

 
Experimental animals
 
Healthy male wistar rats weighing around 120-140 g were used. The rats were housed in groups in polypropylene cages and fed with ad libitum pellet feed.  The experimental protocol was approved by the Institutional Animal Ethical Committee, Madras Veterinary College (Approval No. 1467/DFBS/IAEC/2018).
 
In vivo anticancer activity
 
Forty eight healthy male wistar rats were randomly divided into six groups with eight animals in each group. The experimental design is given below.
       
 
 
Colon tumour was induced in male wistar rats by following the protocol of De Jong et al., (2000) with slight modifications. DMH was dissolved in 1 mM EDTA in normal saline (pH adjusted to 6.5 with1 mM NaOH). Colon tumour was induced in group III, IV, V and VI by intraperitoneal injection of DMH at 25 mg/kg b. wt. once a week for initial six weeks.
       
Group I rats (normal control) received 0.1% CMC orally daily for 15 weeks. Group II rats were treated with S. marginatum extract @ 200 mg/kg b. wt. in 0.1% CMC orally for 16 weeks. Group III rats served as tumour control. Group IV rats were treated with standard drug 5-Fluorouracil @ 25 mg/kg b.wt. intraperitoneally once a week starting from week 7. Group V and VI rats were treated with S. marginatum extract at 100 and 200 mg/kg b. wt. in 0.1% CMC orally once daily starting one week before tumour induction. At the end of the experimental period, the rats were sacrificed. Colon tissue were collected and measured for its length and weight. The colon index and colon length to weight ratio were calculated (Chari et al., 2018).
       
Total protein, lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidise (GPx) levels were estimated in colon homogenate according to the methods described by Lowry et al., (1951), Placer et al., (1966), Moron et al., (1979), Marklund and Marklund (1974), Caliborne (1985) and Rotruck et al., (1973), respectively.
       
A piece of colon tissue was cut open longitudinally, fixed in 10% neutral buffered formalin, stained with 0.2% methylene and examined under light microscope for aberrant crypt foci (ACF). ACF were identified by elongated, slit-shaped lumens surrounded by thickened epithelium that stains more intensely than the surrounding normal crypts (Fragoso et al., 2013).
       
Colon tissues were fixed in neutral buffered formalin and processed for histopathology.
 
Quantitative real time polymerase chain reaction analysis
 
RNA was isolated from colon tissue samples using commercially available kit (GETTm Total RNA Kit, USA). The concentration and purity of RNA were determined by measuring optical density at 260 nm/280 nm using spectrophotometer Nanodrop 2000. cDNA was synthesized using commercially available kit (PrimeScript^™ RT reagent Kit, Biorad, Dublin). Real Time PCR was performed using commercially synthesized primers and kits (SYBR® Premix Ex TaqTM II, Tli RNaseH Plus, Cat. No. RR820A).
       
PCR were performed as per the following conditions: 95°C for 3 min for initial denaturation, then 40 cycles of denaturation at 95°C for 30 seconds and annealing at 54°C for β -actin, 53°C for Bcl-2 and COX-2, 50°C for Bax and 61°C for caspase-3 and caspase-9 for 30 seconds and then final extension at extension at 72°C for 45 seconds. The Ct values were recorded. The gene expressions levels were calculated as described by Alshatwi et al., (2012).


 
Statistical analysis
 
The data were analyzed by one way ANOVA with Duncan’s posthoc analysis using SPSS software 20.0.

DMH-induced colon carcinogenesis is widely used model for the evaluation of chemopreventive agents in the colon cancer and also to study morphologic and molecular mechanisms of the multistage development of colon cancer in order to elucidate new targets for chemoprevention (Perse and Cerar, 2011).
       
In the present study, no gross pathological changes were observed in control rats and rats treated with S. marginatum whereas corrugations and thickening of colonic mucosa with small nodular growth was observed in tumour control rats (Fig 2).
 

       
The colon index of DMH-induced tumour rats were significantly higher compared to control rats and rats treated with 5-FU and S. marginatum at both dose levels (Table 1). This may be due to preneoplastic changes like thickening and corrugation of colon mucosa caused by DMH (Chari et al., 2018). Colon length to weight ratio of tumor control rats was significantly lower than control rats. Treatment with 5-FU, S. marginatum at both dose levels improved the colon length to weight ratio similar to control group. The formation of microadenoma, adenoma and carcinonomas would decrease colon length to weight ratio (Chari et al., 2018). Similar to the results of the present study, Chari et al., (2018) reported significantly decreased colon length to weight ratio in DMH treated rats compared to control rats and pretreatment with pumpkin seed extract significantly increased the ratios compared to DMH treated rats.
 

 
Oxidative stress plays an important role in all stages of carcinogenesis and the reduction of antioxidants is one of the cancer self-defense mechanism (Ghareeb et al., 2018). Enhanced LPO is associated with depletion in detoxifying (GPx) and antioxidant (CAT) enzymes in the colon and liver homogenate were the characteristic findings in malignant transformation (Rai et al., 2015).
       
In the present study, SOD, GSH, CAT, GPx activities were significantly decreased in tumour control rats (Fig 3). Treatment with S. marginatum at 200 mg/kg significantly increased and restored the values of these parameters to near normal parameters.
 

       
In the present study, the levels of malondialdehyde (MDA), anend-product of peroxidation of polyunsaturated fatty acids, was found to be significantly increased in the DMH-induced tumour rats. The observed increase in the lipid peroxidation of DMH-induced rats correlates with decline in antioxidant enzymes, SOD and GPx. It may be due to excess utilization to scavenge the products of lipid peroxidation as well as sequestration by tumour cells (Aziza et al., 2014). Treatment with 5-FU and S. marginatum extract significantly decreased the levels of MDA compared to tumour control rats. S. marginatum extract reduces the oxidative injury in DMH-induced tumour rats probably by stimulating endogenous antioxidant defense enzymes and alleviates preneoplastic lesions.
       
ACF are specific preneoplastic lesions and reliable biomarker for diagnosis of early stages of colon cancer and to assess chemopreventive potential of natural products (Fragoso et al., 2013). Tumour control rats showed more ACF that stained intensely compared to normal control rats (Fig 4). Rats treated with 5-FU and S. marginatum extract at 200 mg/kg showed very few initial ACF that stained very lightly compared to tumour control groups. Minimal ACF observed in the colon of rats treated with S. marginatum suggest chemopreventive potential of S. marginatum in alleviating development of preneoplastic lesions.
 

       
Colon of control rats showed normal histology with no signs of apparent abnormality. No histological evidence of toxicity was observed in rats treated with S. marginatum extract alone.
              
DMH-induced tumour control rats showed preneoplastic lesions such as ACF, hyperchromatic nuclei with stratification, mucin depletion, ACF with elongated crypts and branching, cryptic dilatation and dysplastic ACF.

Increased infiltration of mononuclear cells and eosinophils with destruction of crypts of Lieburkuhn was observed. Tumour abscess, vesicular nuclei and goblet cell reduction was observed in one case (Fig 5). Rats treated with S. marginatum extract at 200 mg/kg showed intact colonic mucosal surface, very few initial ACF with minimal mononuclear cells and eosinophilic infiltration. Colon tissue of rats treated with S. marginatum extract at 100 mg/kg showed few ACF with mononuclear cells and eosinophil infiltration in between the crypts (Fig 6).
 

 

       
Histology of colon of tumour control rats showed hyperplastic intraepithelial lesions to intraepithelial neoplasia / dysplasia characterized by hypercellularity with enlarged hyperchromatic nuclei, degree of nuclear stratification, loss of polarity, high nuclear/cytoplasmic ratio, nuclear crowding and increase in mitotic index and decrease in mucin secretion (Perse and Cerar, 2011). These histopathological abnormalities were reduced in rats treated S. marginatum extract suggesting its protective role in development of cancer.
       
Apoptosis is tightly regulated by apoptosis-promoting factors such as p53, Bax and caspases and antiapoptotic factors such as Bcl-2 and survivin. Most colorectal adenomas express Bcl-2 at higher levels and an inverse correlation has been reported between Bcl-2 expression and apoptotic index of colon tissues (Bendardaf et al., 2004). In the present study, the expression of Bcl-2, Bax, caspase 3, caspase 9 and COX-2 were studied by RT-PCR and the results are presented in Table 2. The expression of antiapoptotic gene Bcl-2 was significantly increased while the expression of apoptotic gene Bax was significantly reducedin tumour control rats compared to control rats. Treatment with 5-FU and S. marginatum extract at both dose levels significantly reduced Bcl-2 expression and significantly increased expression of Bax compared to tumour control rats. Thus, the results of the present study suggested that an up regulation of Bax and the corresponding down regulation of Bcl-2 observed in the colon tissue of S. marginatum extract treated rats is one of the critical mechanisms involved in apoptosis.
 

       
The fold change in expression of caspase-3 and caspase-9 in tumour control rats was significantly reduced compared to normal control rats. Treatment with 5-FU and S. marginatum extract significantly increased fold change in expression of caspase 3 and caspase-9 which differed significantly from tumour control and control rats. The induction of apoptosis is almost always associated with the activation of caspase and in accordance with this, the expression of caspase-3 and caspase-9 were significantly enhanced by treatment with S. marginatum. The intrinsic pathway of apoptosis is associated with the disruption of mitochondrial membrane potential and the release of mitochondrial contents resulting in activation of caspase-9 and caspase-3 with decreased levels of Bcl-2 and increased levels of Bax and it is confirmed by the results of the present study. The present study clearly demonstrated that S. marginatum extract induces apoptosis in DMH-induced tumour rats probably through intrinsic pathway and thereby has protective role in colon carcinogenesis.
       
The expression of COX-2, an inflammatory marker was significantly increased by 6.10±0.70 fold relative to control rats. Treatment with 5-FU and S. marginatum extract at both dose levels significantly decreased fold change in expression of COX-2, which differed significantly from tumour control.
       
COX-2 is involved in the synthesis of prostaglandins and thromboxanes, which plays an important role in various biological processes such as cell proliferation, angiogenesis and inflammation and all of which plays a crucial role in the development and progression of tumour (Roelofs et al., 2014). The elevation of COX-2 level reflected the elevation of prostaglandins production which precedes the upregulation of Bcl-2 or inhibition of p53 (Ghareeb et al., 2018).  Overexpression of COX-2 was reported in malignant and premalignant tumours (Roelofs et al., 2014). In agreement with this, increased expression of COX-2 in DMH-induced colon tumour rats was significantly attenuated by S. marginatum treatment suggesting the protective role of S. marginatum extract in colon carcinogenesis by suppressing inflammatory signals.

It can be concluded from the findings of this study that S. marginatum extract exerts chemopreventive action against DMH-induced colon carcinogenesis probably through the attenuation of hyperproliferative responses, inflammatory markers and apoptotic responses in the colon. Since the studies were conducted with ethanolic extracts, it is difficult to attribute the chemopreventive effect of S. marginatum to a single component. The activity could be due either to individual compound or to a combination of polysaccharides, phenols, flavonoids and terpenoids present in the extract. Further studies are warranted to isolate the bioactive compound/fraction and to pinpoint the exact mechanism of action of S. marginatum.

This study was funded by Tamil Nadu Veterinary and Animal Sciences University, Chennai.

The authors declare that they have no conflict of interest.