Colitis associated Colorectal cancer (CACC) is a very common malignancy and the second leading cause of cancer related mortality in the world. Aspirin therapy is well accepted as an analgesic, antipyretic and for cardiovascular prophylaxis. Several decades of research provided considerable evidence demonstrating its potential for the prevention of cancer, particularly colorectal cancer . Zinc plays a major role in various physiological processes; acts as an intracellular signalling molecule, repairs DNA damage, inhibits NADPH oxidase, participates in the structure and stability of some enzymes, modulates ATP function, and maintains the immune and anti-inflammatory systems . To understand the pathogenesis of colitis associated colorectal cancer and the preventive effects of combined administration of aspirin and zinc, a novel colitis-related mouse CRC model (a two-stage mouse colon carcinogenesis model) initiated with DMH and promoted by DSS was developed . In the present investigation, body weight loss, loose stool, faecal occult bleeding was observed during initial period and DAI, which is an indicator of chronic intestinal inflammation was significantly increased. Later on, rectal swelling, aberrant crypt foci formation, tumor formation, tumor multiplicity were also observed. This inferred that DSS induced chronic inflammatory microenvironment favoured the tumorigenesis in colon leading to CACC progression with time. The milieu of chronic intestinal inflammation plays a vital role in the transformation of colitis into colon carcinogenesis by facilitating cell proliferation, migration and angiogenesis, thereby promoting tumor development, growth and progression. Administration of aspirin along with zinc reduced the DAI and tumor progression in DMH-DSS induced colitis associated colorectal cancer. Histopathological analysis of colon tissue revealed cell infiltration, crypt damage, epithelium loss, goblet cell loss was observed in DMH + DSS group. On the contrary all these lesions were less observed in combination treatment group . From the TUNEL assay analysis, number of TUNEL positive cells was decreased in tumor region whereas surrounding areas around tumor (normal cells) was observed with more number of apoptotic cells. It has been found that imatinib and its combination with 2,5-dimethyl-celecoxib increases apoptosis in human HT-29 colorectal cancer cells by the upregulation of Caspase-3 enzyme activity .
Expression of COX-2 was significantly increased in DMH + DSS group, whereas it was significantly decreased in aspirin and combination treatment group observed by immunohistochemical analysis. COX-2 is least expressed in normal tissues; however, COX-2 expression is increased in inflammatory and tumor tissues and plays vital role in inflammation, cell proliferation, and differentiation. COX-2 can: (i) increased prostaglandins production and modulate the body’s immune response; (ii) inhibit apoptosis in tumor cells and promote cell proliferation; (iii) regulation of cell cycle progression; (iv) promote tumor angiogenesis; (v) increase matrix metalloproteinases expression in tumor cells; and (vi) precursors of carcinogenic substances activation . Various preclinical and clinical studies reported that COX-2 inhibitors exerted the inhibitory activities during the initiation and post-initiation stages of colorectal carcinogenesis . Further, clinical data of colorectal cancer patients reported that COX-2 mRNA expression was higher in colon tumors compared to the corresponding normal colorectal mucosa . In the present investigation, expression of metallothionein was decreased in DMH + DSS group whereas zinc and combination group were observed with increased metallothionein expression. Metallothioneins (MT) are silenced during tumor progression, mainly through epigenetic changes, and this loss is associated with poor survival of colorectal cancer patients . Zinc ions also potently increase metallothionein expression and are cytotoxic to tumor cells. Various studies reported that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents . In the present investigation, PCNA expression was increased in DMH+DSS group indication of cell proliferation as compared to Control group. PCNA is a chief marker reflecting the activity of cell proliferation, which is closely related to invasion and metastasis of malignant neoplasms and their prognosis . Further, present investigation representing the increased expression of NFκBp65 and Caspase-1 in DMH + DSS group as compared to Control group animals. As a molecular hub linking inflammation and cancer, NFκB and Caspase-1 has been established as a crucial contributor in the development of malignant tumors . In earlier reports, it has been found that NF-κB activation supports tumorigenesis by enhancing cell proliferation and angiogenesis, inhibiting apoptosis, and promoting cell invasion and metastasis . Further, activation of NFkB signalling promotes the expression of various pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6, that further leads to colonic inflammation and damage . It has been reported that chrysophanol showed protective effect in DSS induced colitis by suppression of NF-kB/Caspase-1 activation .
The present study reported an increased expression of IL-6 and STAT3 expression in DMH + DSS group whereas combination treatment group significantly decreased the expression of IL-6 and STAT3. Further, Nrf2 and SOD expression has been significantly increased in tumor tissue. It has been reported that IL-6/STAT3 is one of the most critical cellular signal transduction pathways known to malfunction in colorectal cancer (CRC) . IL-6 is the key pro-inflammatory cytokine and its overexpression in colonic mucosa leads to mucosal lesions . The transcription factor STAT3 is an oncogenic protein that is constitutively activated in most tumor cells but not in normal cells. Activation of STAT3 leads to upregulation of the expression of various critical genes involved in cell proliferation and survival, such as the pro-proliferative cyclin D1 and the anti-apoptotic Bcl-2 . It has also been found that aspirin enhanced apoptosis rate by down regulation of IL-6-STAT3 signalling pathway in azoxymethane (AOM) and dextran sodium sulfate (DSS) induced colorectal cancer in BALB/c mice . Under healthy conditions, Nrf2 protects against tumorigenesis and cancer progression by attenuating genotoxic compounds that emerge both intrinsically and extrinsically. However, activation of the Nrf2 defence response can promote the survival of both normal and cancer cells by creating an optimal tumor microenvironment for cell growth . Further, Nrf2 knockout mice were more susceptible to AOM/DSS induced inflammation-associated colorectal cancer with impaired antioxidant/detoxifying mechanisms coupled with increased expressions of COX-2, 5-LOX and proinflammatory metabolites of arachidonic acids . Furthermore, Nrf2 protects tumour cells from oxidative stress, chemotherapeutic agents, and radiotherapy, promoting tumour genesis and progression, and also metabolic reprogramming to anabolic pathways .
From the present study, it can be concluded that combined treatment of low dose aspirin and low dose zinc showed protective effect against DMH + DSS induced colitis associated colorectal cancer. Cell proliferation, inflammation has been increased inside the tumor region as evident from the expression of various proliferation and inflammation markers such as PCNA, IL-6, STAT-3, pNFκBp65, Caspase-1 p10. Further, antioxidant defence system has been generated inside the tumor tissue as observed from the expression Nrf2 and SOD. Combination of aspirin and zinc showed protective effect by targeting inflammation, oxidative stress, cell proliferation and apoptosis. The scientific basis for the combined intervention of aspirin and zinc for the prevention of DMH+DSS induced colorectal cancer needs to further strengthen by further preclinical and clinical studies.