Bone Marrow Cell Therapy on 1,2-Dimethylhydrazine (DMH)-Induced Colon Cancer in Rats

Colon cancer is the foremost cause of malignancy-related mortality in the developed countries. Global, about two-third of newly diagnosed cases occur in the western world [1]. Several risk factors for colorectal cancer including life-style, consumption of red or processed meat but not white, age and family history are important in the induction and progression of this tumor [2]. The early detection of cancer can save the life of patients, however, colon cancer is often detected in the late stages when the symptoms become obvious [3].

Among chemically induced colon cancer in animal models, 1, 2-dimethylhydrazine (DMH) is the most frequently used [4]. DMH is a potent colon carcinogen inducing colorectal tumors shares many resemblances to human colorectal cancer, including resemblance in the response to some promotional and preventive agents [5]. DMH-induced colon cancer is a multistep process comprising a series of pathological changes, such as aberrant cryptic foci formation [6].

Bone marrow cell (BMC) transplantation has been considered as a good candidate for a therapeutic approach for patients with solid tumor as these cells are a good source for hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs). HSCs transplantation was used over several years for breast cancer and other tumors alike [7], although MSCs have received renewed interest in the last years, due to their capability to enhance the immune response as immunomodulators [8].

Tumor cells rapidly develop chemoresistance to anticancer agents because of their genomic instability; therefore, there is an urgent need to design new drugs with a new mechanism to improve therapeutic efficacy [9]. Therefore, we hypothesize that colorectal delivery of bone marrow tissue could be more efficient than systemic administration to minimize the inflammatory and remodeling processes and enhance mucosal epithelial repair in experimental colon cancer. To test our hypothesis, DMH was used to induce colon cancer and colon histology findings, the expressions of multidrug resistance-1 (MDR1), β-catenin and cytokeratin 20 (CK20), as well as, oxidative stress markers were investigated in the current study.

The present experimental study was conducted on the adult male Wistar albino rats; weighing 140– 200 g. The animals were purchased from the National Research center (NRC, Cairo, Egypt). The male rats were housed in metal cages with wire-grid floors. The animals were kept at standard housing facilities (25±2 ºC, 45 ± 5% humidity and 12 hrs. light/dark cycles). They were fed on a standard laboratory chow and water ad libitum. The standard guidelines of National research center were used in handling animals.

Bone marrow cells were collected from male Swiss mice (weight 20–25 g, n = 10) and administered on the same day of collection. Briefly, bone marrow cells (BMCs) were aspirated from the femur and tibia by flushing the bone marrow cavity with saline (0.9% sodium chloride). Cells were centrifuged (500 × g for 10 min) and re-suspended in phosphate-buffered saline. Before the administration of BMCs, cells were counted in a Neubauer chamber with Trypan Blue for evaluation of viability and it was localized injected in the intracolorectum pathway. First, colon of rat was lavage with 2 ml of saline followed by handling palpation of the lower abdomen to expel any remaining feces. 1 ml of BMCs (6 × 10⁶ cells/ml 0.9% saline solution) was injected intra-rectal for 30 seconds using a polyurethane cannula (2 mm in diameter) inserted through the anus up to a distance of 6 cm. The rats were hold in a head-down position for 2 min to prevent the intra-colonic BMCs leakage. In the rats those not treated with BMCs, rats were treated with saline (1 ml) instead of BMCs.

DMH was dissolved in 1 mM ethylenediaminetetraacetic acid–containing 1 mM sodium bicarbonate (pH 6.5). Animals were given a weekly subcutaneous injection of DMH in the groin at a dose of 20 mg/kg body weight for 15 weeks.

To study the effects of BMCs on DMH-induced colon cancer, the rats were randomly divided into two groups, control group (n=7) and experimental group (n=21). The rats in the experimental group were divided into three groups: BMCs alone, DMH alone and BMCs with DMH, each group consists of 7 rats. The control rats were subcutaneously injected with 0.2 ml/week saline for 15 weeks. Prior the weekly BMCs injection, the animals were kept on fast overnight. BMCs were injected intrarectally in saline and DMH was injected subcutaneously starting from zero-day and continued for 15 weeks. The common dose of the BMCs per rat was 6 × 10⁶ cells both in the BMCs alone and BMCs with DMH groups.

Following the last administration by one week, the animals were killed under mild ether anesthesia. Blood was collected from the abdominal aorta using a syringe puncture, and the serum was separated. Colons were excised quickly, cleaned free of irrelevant material and immediately perfused with ice-cold saline.

The excised organ was fixed in 10% neutral buffered formalin solution for about 24 hrs, washed in running water, dehydrated, cleared in xylene and impregnated in parablast for blocking. Serial sections of 5 μm thick were prepared and stained with: Hematoxylin and Eosin, Periodic acid Schiff’s (PAS) and Masson’s trichrome.

Five-micron sections of colon fixed in 10% neutral buffered formalin fixative immunostained using anti-cytokeratin 20 primary antibody (Labvision, Neomarkers, USA) for 90 minutes. This was followed by the secondary antibody application using the immunoperoxidase technique (Vectastain ABC kit; Vector Laboratories, Burlingame, CA).

Colon was weighed and immediately homogenized in an ice-cold medium of 50 mM Tris–HCl (pH 7.4) to give a 10 % (w/v) homogenate. The homogenates were centrifuged at 1000 g for 10 minutes at 4°C. The collected supernatants were used for the various biochemical determinations. The total protein content of the homogenized colon was determined by the method of Lowry et al [10]. using bovine serum albumin as a standard.

Homogenates of colon or serum was used to determine malondialdehyde (MDA) as an indicator of lipid peroxidation (LPO) by reaction of thiobarbituric acid according to the method of Ohkawa et al. [11]. Nitrite/nitrate (nitric oxide; NO) [12] and glutathione (GSH) [13] were also chemically measured. For the enzymatic antioxidant status, colon supernatants were subjected to determine superoxide dismutase (SOD) activity according to Nishikimi et al [14]. and catalase (CAT) activity as described by Aebi [15].

Total RNA was purified from colon tissue using an RNeasy Plus Minikit (Qiagen, Valencia, CA). cDNA synthesis was undertaken using the RevertAid^TM H Minus Reverse Transcriptase (Fermentas, Thermo Fisher Scientific Inc., Canada). The cDNA samples were run in triplicate for real-time PCR analysis. Real-time PCR reactions were performed using Power SYBR® Green (Life Technologies, CA) on the Applied Biosystems 7500 system. Relative values of gene expression were normalized to β-actin. Primer sequences and accession number of the genes are provided in Table 1.

All results were expressed as the mean ± standard error of the mean (SEM). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA). For the comparison of significance between groups, Duncan’s test was used as a post hoc test and the probability level of less than p˂0.05 was considered significant. The data analysis was performed using the SPSS 20 (SPSS Software, SPSS Inc., Chicago, Illinois, USA).

Colon sections of the control group showed a normal histological futures of the mucosa, submucosa and muscularis layers with an intact mucosal gland with lined epithelial cells and goblet cells were noticed in mucosa layer (Fig. 1a). Cancer-induced group showed an erotic epithelial cells in covering layer of the mucosa, an acute infiltration of inflammatory cells was also observed in mucosa and lamina propria, moreover abnormal structure in mucosal glands was noticed represented in dysplasia, anaplasis and hyperchromasia (Fig. 1b). Colon cancer-induced rats treated with BMCs showed a clear improvement in mucosal layer, few inflammatory cells infiltration periglandular and in the lamina propria (Figs 1c). Colon treated with BMCs alone showed a normal structure similar to control one (Fig. 1d).

Histological examination of colon section stained with PAS reaction of the control group showed normal PAS reaction located markedly in goblet cells which secrets mucous (Fig. 2a). While in cancer-induced group faint or weak PAS reaction was located in the goblet cells of mucosal glands (Fig. 2b). A marked improvement was observed in PAS reaction in cancer-induced group treated with BMCs (Fig. 2c).

Histological examination of control colon sections stained with Masson’s trichrome showed normal collagenous content in mucosa, submucosa, and musculosa (Fig. 3a). DMH-induced group showed a mild increase in collagenous content represented in highly compact collagen bundles in submucosa and lamina propria, also in mucosa periglandularly. Dilatation and congestion in blood vessels of submucosa were seen (Fig. 3b). A clear improvement in collagenous content in all layers was showed in cancer-induced group treated with BMCs (Fig. 3c). However, in the group treated alone with BMCs, a moderate increase in collagen content was observed in the submucosa (Fig. 3d).

Immunohistochemical examination of control colon showed a moderate positive reaction in cytokeratin 20 immunostaining located in glandular cells of mucosa layer (Fig. 4a). While in cancer-induced group, sever increase in cytokeratin 20 reaction was clearly shown in mucosal glands (Fig. 4b). After treatment with BMCs to cancer induced group, a moderate positive reaction in cytokeratin 20 in the glandular cells of mucosa layer was observed (Fig. 4c).

Fig. 5 illustrated the oxidative stress markers in the serum and colon of rats. The higher levels of LPO and NO in serum and colon homogenates of rats treated with DMH indicated the induction of oxidative stress. These findings were supported with the depletion of GSH. However, a post hoc comparison of means in DMH+BMCs group showed significant (p<0.05) decrease in lipid peroxidation and NO generation in rats treated with BMCs compared with the rats treated alone with DMH. Furthermore, BMCs injection prevented the depletion of GSH content induced by DMH injection in the serum and colon homogenates (p<0.05). Furthermore, the results also illustrated that DMH injection inhibited the activities of SOD and CAT (p<0.05). Interestingly, treatment with BMCs significantly elevated SOD and CAT activities (Fig. 6).

In order to elucidate the mechanisms of how BMCs exerts its anti-colon carcinogenesis effect, we evaluated the expression of survivin, MDR-1, and β-catenin in the colon tissues. The RT-PCR findings showed that DMH injected induced a significant (p<0.05) up-regulation in the expression of survivin, MDR-1 and β-catenin in the colon tissue compared to the control group. However, BMCs treatment induced a marked (p<0.05) decrease in the expression of survivin, MDR-1 and β-catenin in the rats compared to the DMH group (Fig. 7).

The main objective of the current study was to determine whether BMCs could decrease the risk of DMH-induced colorectal carcinogenesis rats. DMH is a carcinogenic and the mechanism underlying DMH-induced colon cancer is well reported and reactive oxygen species (ROS) production is recognized as a key player leading to the condition of oxidant/antioxidant imbalance. DMH-induced colon toxicity is mainly dependent on biotransformation to form more reactive intermediates, which occurs by two pathways, cytochrome P450 monooxygenases (CYPs) dependent oxidation (Phase I) and glutathione (GSH) conjugation (Phase II) [16-17]. In the current study, the major markers of oxidative stress response were found to be increased including LPO and NO, concurrently with the depletion of glutathione (GSH) and related redox cycle enzymes like SOD and CAT. Lipid peroxidation is one of the important markers of oxidative damage and high level of MDA, the end products of lipid peroxidation, has been found after DMH treatment [18-19]. Consistent with previous studies, our findings also showed noteworthy elevation in the level of LPO after DMH injection.

Inflammation plays an important role and is documented to influence cancer initiation and promotion [6]. NO modifies different cancer-related events including angiogenesis, apoptosis, cell cycle promotion, invasion and metastasis [20]. NO may mediate nucleic acid lesions by formation of toxic and mutagenic substances, by direct modification of DNA, or by suppressions of DNA repair mechanisms [21]. NO reinforces angiogenesis by stimulating of cyclooxygenase which promotes the production of proangiogenic factors and prostaglandins. Femia et al. [22] reported that DMH-induced colorectal cancer has high pro-inflammatory enzyme inducible nitric oxide synthase (iNOS) expression. Many researches report high iNOS activity in colon cancer [23-24] and support our findings.

We have also found the enhanced expression of β-catenin in the colon of rats treated with DMH. Our findings are in accordance with previous studies by Femia et al. [22] and Kansal et al. [25]. The nuclear translocation of β-catenin induces the target gene expression involved in cellular proliferation, such as c-myc, cyclin D1, multidrug resistance protein 1 and PPARγ (peroxisome proliferator-activated receptors γ) [25]. Therefore, blocking β-catenin signaling for cancer therapy has thus gained significant interests [26].

The results demonstrated that DMH repressing the expression survivin (baculoviral inhibitor of apoptosis repeat-containing 5; BIRC5). During colorectal cancer progression, survivin obviously has the dual function of preventing tumor cell apoptosis and controlling the cell-cycle proliferation. This peculiarity concurs to both accelerate proliferative activity and suppress apoptosis [27-28]. High survivin expression has been observed in various types of tumors and therefore may represent a promising drug target for tumor treatment [29].

MDR is the major cause for the aquire resistance to chemotherapy on cancers. The mechanism of MDR function is complex. P-glycoprotein (P-gp) mediated MDR is considered to be the classic mechanism of resistance. P-gp is an ATP-dependent membrane transport protein encoded by the MDR1 gene, and it pumps exogenous cell substances from within cells to protect cells from chemotherapeutic agents [30]. Pgp also plays important roles in cell cycle, differentiation, and apoptosis [31]. MDR mRNA levels were found to be highest in rat colon treated with DMH. The present data is in accordance with the previous study of Kankesan et al [31].

Cytokeratins (CKs) represent the epithelial class of intermediate-sized filaments of the cytoskeleton. To date 20 subtypes of CK intermediate filaments were discovered. Among the most useful cytokeratins is cytokeratin 20 (CK20) [32]. Recently, CK20 protein has gained growing interest due to its highly specific expression in both colorectal and gastric cancers [33]. In the current study, we observed strong CK20 immunostaining in colorectal tissues of rats treated with DMH.

In this study, we observed a complete regeneration of the mucosal epithelium, a marked improvement in the collagen fiber accumulation also a well-seen mucus secretion in the goblet cells were observed after BMCs treatment in MDH-induced colon cancer in rats. Therefore, administration of BMCs may potentiate mucosal epithelial cell repair. BMCs appeared to repair mucosal epithelial cells associated with several features of regeneration, such as enriched lamina propria with clearly seen blood vessels and connective tissue, well improved muscular layer with normal collagenous fiber content. Coinciding with our findings, MSCs accumulate in inflamed areas of the colon and contribute to tissue repair by a differentiation into vascular smooth muscle cells, endothelial cells, pericytes or epithelial cells. In addition, BMCs therapy may be useful antifibrotic treatment by producing the basic fibroblast growth factor (bFGF) that involved in the prevention of fibrosis [34].

Many studies demonstrate that MSCs could be autologously, allogeinic and even xenogeinic transplanted since the infusion of MSCs in animal models did not lead to rejection of the same and provided good therapeutic results [35]. Therefore, use of homograft or xenograft stem cells does not seem to affect the experiment, at least not in our models. Furthermore, MSCs cannot be considered truly immune privileged, rejection of allo-MSCs occurs more slowly than rejection of other allogeneic cell types [36]. Numerous studies have demonstrated that MSCs possess unique immunologic characters. Transplantation of allogeneic mismatched MSCs to adult animals has shown engraftment in murine and baboon experimental models [37-38]. Unlike higher mammals, mouse-to-rats BMCs transplantation didn’t elicit a xenoimmune response that allows researchers to perform multiple repeats, expand time schedules. Rats engrafted with mouse bone marrow stem cells exhibited excellent survival and accepted BMCs xenografts donation.

Many mechanisms have been suggested the epithelial cells regeneration occurs after BMCs treatment, one of this, the fusion between epithelial cells and transplanted BMCs, interestingly, the fusion of bone marrow-derived cells with neoplastic epithelium did not result in tumor initiation [39]. Another mechanism may be that the BMCs provide cytokines and growth factors in their microenvironment that enhance epithelial cells functions by paracrine mechanisms [40]. BMCs promote tissue renewing and repairing through synergistic down regulation of proinflammatory cytokines and increased production of soluble factors with antioxidant property. Furthermore, BMCs enhance cell proliferation or inhibit the epithelial cell apoptosis, or by a combination of both [41]. A third mechanism may be transdifferentiation of BMCs after they are reprogrammed by the microenvironment in the injured colon. So far, no data have been published that directly support this hypothesis. However, in accordance with this hypothesis, Krause et al [42]. published compelling evidence on the fact that a rare population of single BMCs was able to repopulate the hematopoietic system and generate nonhematopoietic cell types in multiple tissues including epithelial cells of the liver and gastrointestinal tract.

Interestingly, many studies reported that stem/progenitor cells suppressed tumor growth. Livraghi et al [43]. reported that the fundamental role of stem cell microenvironment in inhibiting carcinogenesis is by providing signals to suppress proliferation and to enhance differentiation. Additionally, it was suggested that engrafted MSCs also differentiated into colonic interstitial lineage cells and secreted vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-β1) those playing an important role in healing of injured colonic mucosa [41]. Supporting this observation, BMCs allograft to sites of Kaposi’s sarcoma potently inhibits tumor growth by down-regulating protein kinase B (AKT) activity in tumor cells [44]. Furthermore, cancer cells may produce proteins that can activate signaling pathways that facilitate and attract BMCs migration to the tumor site. As a result of injury, tissues produce chemokines attracting the circulated BMCs, and can produce circulating microvesicles including RNA, proteins and a variety of signals argument the tumor growth [40].

In the current study, we observed that BMCs administration to rats treated with DMH-induced oxidative stress in colon tissue enhanced the antioxidant capacity. Supporting this finding, BMCs transplantation to diabetic mice increased the antioxidant capacity by up-regulating heme oxygenase (HO)-1 expression and down-regulating the expression of iNOS and AKT genes [45]. Furthermore, Calió et al [46]. demonstrated that the level of superoxide anion and lipid peroxidation were decreased in the brain of a spontaneous stroke model, indicating that BMCs could have antioxidant potential.

As regards genetic analysis of the current study, our findings showed that BMCs down regulated expression of oncogenes (survivin, β-catenin, and MDR) which are all involved in Wnt/β-catenin signaling that plays major roles in the initial and development of colorectal cancer [47]. These findings provide evidence to support that the Wnt signaling pathway is likely to regulate the suppressive role of BMCs. Djouad et al [48]. reported that BMCs could up-regulate the mRNA expression of cyclin-dependent kinase inhibitor 1, p21; a cell-cycle negative regulator, and apoptosis-associated protease caspase-3 resulting in cell-cycle arrest at the G1/S phase and apoptotic cell death of tumor cells. Furthermore, BMCs also produce Dickkopf-1 to block the Wnt/β-catenin signaling pathway, mitigating the malignant phenotype of tumor cells [49]. Moreover, BMCs are potentially cytotoxic when it injected locally in tumor tissue which they might be effective antiangiogenesis candidates suitable for cancer treatment [50].

Our findings demonstrated that BMCs have tumor suppressive effects in DMH-induced colon cancer as evidenced by down regulation of survivin, β-catenin, and MDR-1 genes and enhancing the antioxidant activity. However, further studies are needed regarding BMCs potential risks versus benefits in colon cancer, the route of delivery, the amount of infused cells and the timing of transplantation.

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research through Research Group Project No. RG-1435-016.

The authors declare that there is no conflict of interests regarding the publication of this article.