In this study, we investigated the male rats to avoid the effect of hormonal changes of oestrus cycles of females21. Progressive loss of body fat and lean body mass accompanied by profound weakness, anorexia, and anemia accompanying cancer is referred to as cachexia. Unlike starvation, the weight loss seen in cachexia results equally from loss of fat and lean muscle22. There is some correlation between the tumour burden and the severity of the cachexia13. It is suspected that tumour necrosis factor (TNF) produced by macrophages in response to tumour cells or by the tumour cells themselves mediates cachexia23. TNF at high concentrations mobilizes fats from tissue stores and suppress appetite; both activities would contribute to cachexia (or weight loss). Other cytokines, such as IL-1, IL-6 and interferon-γ, synergizes with TNF to cause weight loss (or cachexia)23. Mice bearing ectopically-implanted C26 colon carcinoma are widely used as an experimental model of cancer cachexia24. The weight loss seen in the positive control group may be related to the presence of tumour growth within them. The weight gain observed in the groups given OG was directly proportional to the concentration of OG given. This is similar to a work by Costelli et al., 1993 that showed weight loss in tumour bearing rats25. They also showed that this is mediated by TNF. There was also a weight gain in the group treated with celecoxib. This is similar to the work by Lai et al., 2008 which showed that celecoxib ameliorates the effect of tumour cachexia (weight loss associated with cancer) in patients with cancer of the gastrointestinal tract26. The weight gain seen in the group treated with celecoxib is statistically similar to those given the lowest dose of OG even though the group was characterized by tumour growth suggesting that even though the lowest dose of OG given was not completely effective at preventing tumour growth, it showed similar ameliorating effect on tumour cachexia when compared to celecoxib treated group. Higher doses of OG have a better preventive effect against tumour cachexia than celecoxib as seen in this research work. Yanagihara et al., 2013 reported the protective role of flaviniods one of the compounds in OG on tumour cachexia27. The phytochemical screening in this study confirms the presence of flavonoids.
There was no observable tumour seen in the rats treated with celecoxib which is a selective COX-2 inhibitors as well as higher doses of OG. Non-steroidal anti-inflammatory drugs especially the COX-2 inhibitors have long been known to be protective against the development of colon cancers28. They do so by inhibiting the production of prostaglandin E2 which promotes the growth of clones of malignant cells. The absence of tumours in the groups given higher doses of OG may be due to the protective effect of the phytochemical constituents of OG on cancer. Alkaloids which are present in OG have been shown to be protective against colon cancer development in humans29. Cardiac glycosides which are also present in OG are known to be protective against cancer development30. George et al., 2017 also demonstrated a protective effect of flavonoids against DNA mutation which is an important step in carcinogenesis and as earlier mentioned are present in OG31. Steroids which are also anti-inflammatory will also reduce the concentration of prostaglandin E2 which is associated with colon cancer. Steriods are also present in OG as seen in the phytochemical screening. Tannins which are present in OG are also known to be chemopreventive against the development of colon cancer32. Terpenoids which are also present in OG have been observed to induce colon cancer cell death33. All these show that OG leaves have a preventive effect against colon cancer development and this effect is similar to that obtained with celecoxib. The colon cancer seen in the positive control group shows that the DMH is effective in inducing colon cancer.
Staging of cancer helps to give an idea about tumour progression. A rat in the positive control group had colon cancer at Duke stage D while those seen in group C are in Duke stage A. This showed that OG also has an ameliorating effect on tumour progression. The Duke stage of the rats given celecoxib and those given higher doses of OG was Stage 0. This means that higher doses of OG prevented the development of cancer just like celecoxib as earlier emphasized. The rat with stage D had tumour deposits in the posterior abdominal wall which is similar to the work by Ye et al., 2013 which also documented tumour spread to the posterior abdominal wall34. Apart from posterior abdominal wall, they can also metastasize to the liver and lungs13.
There was a reduction in goblet cells in the colon of the positive control group as well as the group given the lowest dose of OG. This reduction in goblet cells is an evidence of loss of differentiation within malignant colonic glands. Maliganant colonic glands were seen in the positive control group and the group given the lowest dose of OG. This finding is similar to the work carried out by Xu, (1990) where he sterelogically studied goblet cells in the colon. He found out that goblet cell count in the colon reduces as the differentiation of the colonic epithelium reduces35. He noticed that it can be reduced as much as 50% in settings of adenocarcinoma. This may be responsible for some of the symptoms like constipation, obstruction, perforation e.t.c. which patient with colon cancer experience. The groups given celecoxib and higher doses of OG have higher number of goblet cells.
There is reduced collagen staining in the basement membrane, lamina propria and submucosa in the positive control group and the group given the lowest dose of OG. While rats given higher doses of OG as well as celecoxib had higher amount of collagen in their basement membrane, lamina propria and sub-mucosa. Degradation of collagen within the basement membrane, lamina propria and sub-mucosa is an important step in tumour invasion and metastasis36. These are mediated by the proteolytic effect of tumour cells which secrete matrix metalloproteinases on the collagen within the basement membrane, lamina propria and sub-mucosa. These cancer cells generally need to degrade the collagen in basement membrane and lamina propria in other to progress to higher stages of the disease. OG has demonstrated a protective effect in this regard by limiting the degradation of collagen. This is similar to the work done by Bode et al., 2000 which showed that collagen fibers are reduced in the basement membrane in colon cancer37.
Increased nucleo-cytoplasmic ratio was seen in the cells of the malignant glands in positive control group and those given the lowest dose of OG. Those given the standard drug as well as higher dose of OG had a low and normal nucleo-cytoplasmic ratio. This still corresponds to the result on tumour incidence because malignant cells as a rule have increased nucleo-cytoplasmic ratio. It is also a reflection of the possible protective effect of OG on colon cancer development because before cells become malignant they are expected to accumulate ribonucleic acids and nucleotides in the nucleus before cell division can start. This leads to increase in nuclear diameter and subsequently increased nucleocytoplasmic ratio. So based on the above mentioned it can be said celecoxib and higher doses of OG have a preventive effect on colon carcinogenesis.
Nitric oxide (NO) is a ubiquitous, water soluble, free radical gas, which plays key role in various physiological as well as pathological processes. NO has emerged as a molecule of interest in carcinogenesis and tumour growth progression. However, there is considerable controversy and confusion in understanding its role in cancer biology. It is said to have both tumoricidal as well as tumour promoting effects which depend on its timing, location, and concentration38. NO has been suggested to modulate different cancer-related events including angiogenesis, apoptosis, cell cycle, invasion and metastasis38. Nitric oxide delivered by inflammatory cells, in particular, is important in causing changes in intestinal epithelial cells, setting the stage for colon cancer development39. There is up-regulation of nitric oxide synthase in rats who have colonic adenomas (pre-malignant lesion)17. The concentration of nitric oxide is higher in the positive control group and the group given lowest dose of OG. This is because nitric oxide is associated with inflammation and chronic inflammation is a known predisposing factor to development of colon cancer. The concentration of nitric oxide seen in the standard group as well as groups given higher doses of OG are similar which showed that higher doses of OG reduced the concentration of nitric oxide, a gas associated with inflammation in the same way as celecoxib does. By so doing, OG at higher doses reduces the concentration of NO in the colon just like celecoxib thereby preventing colon cancer development. Alkaloids, steroids, anthraquinones, flavonoids, saponins and tannins which are phytochemical constituents of OG are known to have anti-inflammatory properties39.
Cyclooxygenase-2 (COX-2) is highly expressed in 90% of colorectal carcinomas and 40% to 90% of adenomas (premalignant lesion). COX-2 is necessary for production of prostaglandin E2, which promotes epithelial proliferation including clones of malignant epithelial cells13. Prostaglandin E2 (PGE2) induces colon cancer stem cell like expansion in vivo and ex vivo40. Based on this, non-steroidal anti-inflammatory drugs (NSAIDS) especially COX-2 inhibitors are given to patients with colonic adenoma to prevent progression to carcinoma41. The concentration of prostaglangin E2 was higher in the positive control and the group given the lowest concentration of OG. The level of prostaglandin E2 in groups given celecoxib (standard group) as well as groups given higher doses of OG are low and are statistically similar. This shows that higher doses of OG reduce the concentration of PGE2 in the same way as will be expected for celecoxib. Prostaglandin E2 is also a cytokine and a derivative of arachidonic acid which is produced during inflammation. This result is in consonance with that obtained from nitric oxide which shows the anti-inflammatory properties of OG. Just as discussed in the preceeding paragraph, alkaloids, steroids, anthraquinones, flavonoids, saponins and tannins which are phytochemical constituents of OG are known to have anti-inflammatory properties.
In conclusion aqueous extract of OG leaves most likely protects against development of colon cancer and as such might be beneficial in the primary prevention of colon cancer.