|Year : 2022 | Volume
| Issue : 3 | Page : 180-185
Curcuma longa (Curcumin) Abrogates Hyperhomocysteinemia and Oxidative Stress in a Rat Model of Colon Cancer
Mostafa I Waly, Lyutha Al Subhi
Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Oman
|Date of Submission||07-May-2022|
|Date of Decision||22-May-2022|
|Date of Acceptance||07-Jun-2022|
|Date of Web Publication||3-Oct-2022|
PhD, MPH, MSc Mostafa I Waly
Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Hyperhomocysteinemia is involved in the pathogenesis of oxidative stress, a well-known etiological factor for different types of cancer, including colon cancer. Although Curcuma longa (curcumin) is a well-known antioxidant shown to prevent oxidative stress in different experimental models, yet its preventive role against hyperhomocysteinemia has not been addressed in experimental model for colon cancer. Objective: This study aimed to assess the protective role of C. longa (curcumin) as a natural antioxidant against the development of hyperhomocysteinemia-mediated oxidative stress and its associated carcinogenesis in rat colon. Methods: Forty-eight adult male Sprague Dawley rats were divided into four groups (12 rats/group): control, curcumin-supplemented group which received a daily dose of 200 mg curcumin/kg body weight, azoxymethane (AOM)-induced colon cancer group, and AOM group + curcumin supplementation. At the end of the experiment, 16 weeks, rats were sacrificed and colon tissues were collected to measure homocysteine level, oxidative stress markers [glutathione (GSH), total antioxidant capacity (TAC), lipid peroxides, and nitric oxide], and antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Colon histological sections were also examined for any histopathological changes. Results: The study results revealed that the colon tissue of the AOM-injected group had higher levels of homocysteine and markers of oxidative stress (GSH depletion, impairment of TAC, and inhibition of antioxidant enzymes) as compared to the control group, P < 0.05. Curcumin supplementation in the AOM + curcumin group significantly alleviated antioxidant enzymes activities as well as hyperhomocysteinemia, P < 0.05. AOM has also caused a significant increase in the size and numbers of aberrant crypt foci, marker lesions of colon tumors. Conclusion: Hyperhomocysteinemia results in the generation of reactive oxygen species, and thereby contributing to the oxidative stress-associated colon cancer pathogenesis. Curcumin as a functional food might be used as a preventative nutritional strategy against colon dysfunction that leads to cancer process.
Keywords: Colon cancer, curcumin, hyperhomocysteinemia, oxidative stress
|How to cite this article:|
Waly MI, Al Subhi L. Curcuma longa (Curcumin) Abrogates Hyperhomocysteinemia and Oxidative Stress in a Rat Model of Colon Cancer. Int J Nutr Pharmacol Neurol Dis 2022;12:180-5
|How to cite this URL:|
Waly MI, Al Subhi L. Curcuma longa (Curcumin) Abrogates Hyperhomocysteinemia and Oxidative Stress in a Rat Model of Colon Cancer. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 Nov 29];12:180-5. Available from: https://www.ijnpnd.com/text.asp?2022/12/3/180/357216
| Introduction|| |
Colorectal cancer (CRC) is the third most common cancer among adults worldwide. CRC pathogenesis is characterized by oxidative stress precipitated by increased production of reactive oxygen species (ROS) and diminished cellular antioxidant defenses. ROS molecules are short-lived and cause oxidative damage of cell membranes and intracellular organelles, including DNA. Azoxymethane (AOM), a specific colon cancer agent in rats, increases the ROS production and causes increase in colonic cellular oxidative activity and decreased activity of antioxidant enzymes.,, Several experimental models suggest that functional foods can prevent early steps of CRC development (i.e., aberrant crypt foci and adenomatous polyp formation); yet no recent studies have examined the biochemical and histopathological effects of curcumin supplementation in relation to CRC development. Curcumin is a polyphenolic chemical constituent that is derived from the rhizome of turmeric (Curcuma longa) plant; it has powerful medicinal applications, based on its biological effects that range from antioxidant, anti-inflammatory to inhibition of angiogenesis. The molecular mechanism of its varied cellular effects has been shown to have multiple targets and interacting macromolecules within cells. Curcumin has been shown to regulate the production of pro-inflammatory cytokines tumor necrosis factor-α, and inhibit the activation of transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1), which regulate the genes for pro-inflammatory mediators and protective antioxidant genes. Curcumin inhibited NF-κB activation by blocking phosphorylation of I-κB, through inactivation of I-κB kinase complex. Suppression of AP-1 was due to a direct interaction of curcumin with AP-1 binding to its DNA binding motif and due to inhibition of c-Jun and c-fos, components of AP-1. It was also reported that curcumin suppresses the activity of a number of enzymes such as cytochrome P450 and COX-2 in animal models with no toxicity. Hence, this study was undertaken to examine the biochemical and cellular role of curcumin as a potential bioactive agent against AOM-induced hyperhomocysteinemia and oxidative stress in an experimental model of colon cancer in rats.
| Methods|| |
Chemicals and supplies
AOM and curcumin were purchased from Sigma Chemical Co (St Louis, MO).
Animal experiments were conducted in compliance with the principles of laboratory animal care guidelines published by the National Institute of Health, and were approved by the animal care committee at Sultan Qaboos University (SQU/AEC/2015-16/8). Forty-eight adult male Sprague Dawley rats weighing 200 ± 5 g were obtained from the animal breed at the animal house facility, Sultan Qaboos University, Muscat, Oman. The animals were housed in individual cages at standard conditions (temperature 22 ± 2°C, relative humidity about 60%, and 12 hours’ light/dark cycles), and all rats were fed a standard chow diet and given water ad libitum. They were acclimatized for a week prior to the experiment then randomly divided into four groups (n = 12 rats/group). The control group was fed a standard chow diet and also received a single intraperitoneal injection of 0.9% physiological saline; AOM-treated group was fed a standard chow diet and got an intraperitoneal dose of AOM (30 mg/kg body weight) dissolved in 0.9% physiological saline. The other two groups were fed a standard chow diet and received intragastric intubation of 1 mL of curcumin mixed with water (200 mg/kg body weight/day) in the presence or absence of AOM injection. Effective doses for curcumin and AOM were based on those reported in a previous study. The trail was carried for 16 weeks.
Body weight, food intake, and tissue harvest
Body weight and food intake were recorded weekly for the entire duration of the experiment. After 16 weeks, the animals were fasted overnight then were anesthetized with a lethal dose of a cocktail containing ketamine (1 mg), xylazine (5 mg), and acepromazine (0.2 mg). The gastric tissues were excised for histopathological examination of any cancer lesions development, and for biochemical measurements of oxidative stress indices. For each group, the colons were excised carefully from each rat and were kept on a glass plate in ice jackets; then they were divided into two parts symmetrically (70 mg each). The first parts were rinsed with ice-cold physiological saline and were processed as follow: 50 mg was immediately homogenized in 100 mM potassium phosphate buffer (pH 7.4) using a Potter–Elvehjem tissue homogenizer (Thomas Scientific, Swedesboro, NJ). Meanwhile, 20 mg was used for DNA extraction as described below. The second parts were fixed flat in 10% formalin solution (Fisher Scientific, Fair Lawn, NJ) overnight and used for histopathological examination and for electron microscopic examination.
Colon histopathological examination
Following tissue processing and paraffin embedding, the fixed colon tissues were sectioned on a microtome to 4 μm thickness and placed on glass slides which were then stained with hematoxylin and eosin (H&E) and examined for architecture histology (dysplasia and carcinoma) under light microscope (Nikon Corporation (Nikon), Tokyo, Japan) examination (magnification 40×).
Measurements of cellular oxidative stress markers
The homogenized colon tissue was centrifuged at 10,000 g at 4°C for 30 minutes. The resulting supernatant was separated into aliquots and used for the determination of reduced glutathione (GSH, Catalog #K464), lipid peroxidation (malondialdehyde, Catalog #K739), total antioxidant capacity (TAC, Catalog #K274), and antioxidant enzymes activities [catalase (CAT, Catalog #K773), glutathione reductase (GR, Catalog #K761), glutathione peroxidase (GPx, Catalog #K762), and superoxide dismutase (SOD, Catalog #K335)]. All assay kits were conducted based on the procedures of the manufacture (Biovision Company, Mountain View, CA).
Protein content analysis
Protein content of colon tissue homogenates was assayed by the method of Lowry et al., using bovine serum albumin as a standard.
Measurements of homocysteine in colon tissue homogenates
The concentrations of total homocysteine in colon tissue homogenate was measured by high performance liquid chromatography (HPLC) following the procedure of Durand et al.
Estimation of DNA oxidative damage
Formation of 8-hydroxydeoxyguanosine (8-OHdG) is a ubiquitous marker of oxidative DNA damage, and was measured in DNA samples extracted from all colon tissues using a commercial Oxidative DNA Damage ELISA assay kit (OxiSelect, Catalog #STA-320, purchased from Cell Biolabs Inc, San Diego, CA). The quantity of 8-OHdG in the extracted DNA of each gastric tissue (ng/mL) was measured at 450 nm and quantified by comparison with an 8-OHdG standard curve.
Analyses were done using GraphPad Prism (version 5.03; GraphPad Software Inc, San Diego, CA). One-way analysis of variance was applied to compare group means of each assay followed by Tukey post hoc test for multiple comparisons within assays. P-value < 0.05 was considered significant.
| Resultsand Discussion|| |
[Figure 1] represents weekly body weight changes in the experimental groups over the duration of the experiment. There was a significant weight reduction in the experimental group that received AOM injection as compared to the control group, P < 0.05. Curcumin showed no effect on the body weight changes throughout the experiment as compared to the control group, P > 0.05. Meanwhile, curcumin supplementation in the AOM injected group resulted in improvement in body weight loss detected at 3 weeks and became significantly higher than AOM group at 6 weeks to the end of the experiment, P < 0.05.
|Figure 1 Changes in body weight of rats supplemented with curcumin in the presence or absence of azoxymethane carcinogen. Animals in the four groups were examined for changes in body weight every week for 16 weeks. Values without superscript are not significantly different as compared to the control group. *Significantly lower as compared to the control group and curcumin supplemented groups, P < 0.05.|
Click here to view
AOM acted as an oxidizing agent in the AOM-injected group and resulted in significant reduction of the intracellular GSH level, impairment of TAC, increase in the lipids peroxides, and release of nitric oxide level as compared to the control group, P < 0.05 [[Figure 2]A–D, respectively]. It has been observed that the curcumin supplementation to the AOM-injected group resulted in combating the observed AOM-induced oxidative stress indices, specifically by restoring the level of depleted GSH to a level that is comparable to that of the control group, P > 0.05 [[Figure 2]A]. The same trend was observed for the protective effects of curcumin supplementation on abrogating the AOM-mediated effect on TAC, lipids peroxides, and nitric oxide levels [[Figure 2]B–D, respectively].
|Figure 2 Measurements of cellular oxidative stress indices in colon tissue homogenates of rats supplemented with curcumin in the presence or absence of azoxymethane carcinogen: (A) glutathione (GSH), (B) total antioxidant capacity (TAC), (C) lipid peroxides, and (D) nitric oxide. Data are expressed as mean ± SD. Values without superscript are not significantly different as compared to the control group, P > 0.05. *Significantly lower as compared to the control group. **Significantly higher than the azoxymethane-injected group. ^Significantly higher as compared to the control group. ^^Significantly lower than the azoxymethane-injected group, P < 0.05.|
Click here to view
As illustrated in [Figure 3] and [Figure 4], AOM caused oxidative damage to the DNA in the colon tissues of rats injected with AOM and the difference was significantly higher than the control group, P < 0.05. Meanwhile, curcumin supplementation showed a significant reduction in the DNA damage in the AOM-injected group, P < 0.05.
|Figure 3 DNA oxidative damage in colon tissues of the experimental groups. Data are expressed as mean ± SD. Values without superscript are not significantly different as compared to control group, P > 0.05. 8-OHdG, 8-hydroxydeoxyguanosine. *Significantly higher than control group. **Significantly lower than azoxymethane-injected group, P < 0.05.|
Click here to view
|Figure 4 Representative section of colon architecture histology under the light microscope after hematoxylin and eosin staining. (A) Control rats with normal colon architecture. (B) azoxymethane-treated rats showed colonic mucosa with preserved architecture and lined by unremarkable epithelium in nearly two-thirds of the examined area. There is an increase in mitotic activity in the crypts compared to the control rats which may suggest regeneration in response to AOM-induced-injury. (C) A representative section for curcumin-treated groups; showed histological appearance similar to the control rats. (D) Colons of azoxymethane + curcumin rats showed dramatic improvement in the histological appearance similar to the control rats.|
Click here to view
As illustrated in [Table 1], curcumin supplementation protected against AOM-induced hyperhomocysteinemia and antioxidant enzymes inhibition in colon tissue homogenates. AOM caused a significant inhibition in the activities of CAT, GPx, GR, and SOD as compared to control group, P < 0.05. AOM caused an elevation in the homocysteine level as compared to the control group, P < 0.05. The concomitant treatment of the AOM-injected groups with curcumin supplementation has significantly alleviated the AOM-associated inhibition of antioxidant enzymes’ activities as well as hyperhomocysteinemia, P < 0.05.
|Table 1 Effect of curcumin supplementation and azoxymethane on hyperhomocysteinemia and antioxidant enzymes in colon tissue homogenates|
Click here to view
The present study elucidated the role of curcumin in alleviating AOM-induced oxidative stress and its associated colon carcinogenesis in an experimental model. It is known that ROS (reactive oxygen species) are the mediators of oxidative stress and cellular insults by inducing lipid peroxidation in the cell-membrane moiety, protein damage, DNA fragmentation, gene mutations, and inhibition of antioxidant enzymes. We reported that rats injected with AOM developed hyperhomocysteinemia, oxidative stress in the colon tissues as evidenced by GSH depletion, increased level of lipids peroxides, nitric oxide release, impairment of total antioxidant capacity, DNA oxidative damage, and inhibition of antioxidant enzymes (CAT, GPx, GR, SOD), as well as histopathological changes which manifested carcinogenic effect in the examined gastric tissues. However, curcumin supplementation has suppressed the oxidative damage associated with AOM injection and mitigated its carcinogenic effect. These findings suggested that curcumin had a gastroprotective effect against AOM-induced oxidative stress and its associated colon carcinogenesis.
Our findings are consistent with the well-documented role of curcumin in the treatment and prevention of chronic noncommunicable diseases, including cancer. Curcumin is found ubiquitously in plants and its regular consumption has wide medicinal applications. Previous studies have long identified that curcumin prevents generation of cellular oxidative stress by acting as upstream therapeutic barrier to oxidative stress and inflammation, hence offering a novel approach to prevent oxidative stress-induced carcinogenesis. It is well-documented that curcumin scavenges the carcinogen-induced oxidative stress in different experimental models, this is consistent with our reported results which address the primary prevention of oxidative stress as a well-known etiological factor for colon cancer. However, it is essential to conduct human-based clinical trials to evaluate the specific effect of curcumin supplementation against hyperhomocysteinemia-mediated oxidative stress.
Financial support and sponsorship
The authors merit Sultan Qaboos University Internal Grant Fund (IG/AGR/FOOD/20/01) awarded to the authors for providing financial support to carry out this research project.Conflicts of interest
There are no conflicts of interest.
| References|| |
Waly MI, Arafa MA, Jriesat SB, Sallam SA, Al-Kafajei A. Folate and vitamin B12 deficiency is associated with colorectal cancer in Jordan. Int J Nutr Pharmacol Neurol Dis 2012;2:57-60. doi: 10.4103/ 2231-0738. 93127 [Full text]
Padmanabhan S, Waly MI, Taranikanti V et al.
Folate/vitamin B12 supplementation combats oxidative stress-associated carcinogenesis in a rat model of colon cancer. Nutr Cancer 2019;71:100-10. doi: 10.1080/01635581.2018.1513047
Pettersen K, Monsen VT, Pettersen CH et al.
DHA-induced stress response in human colon cancer cells − focus on oxidative stress and autophagy. Free Radic Biol Med 2016;90:158-72. doi: 10.1016/j.freeradbiomed.2015.11.018
Waly MI, Al-Ghafri BR, Guizani N, Rahman MS. Phytonutrient effects of date pit extract against azoxymethane-induced oxidative stress in the rat colon. Asian Pac J Cancer Prev 2015;16:3473-7. doi: 10.7314/apjcp.2015.16.8.3473
Waly MI, Al-Rawahi AS, Al Riyami M et al.
Amelioration of azoxymethane induced-carcinogenesis by reducing oxidative stress in rat colon by natural extracts. BMC Complement Altern Med 2014;14:60. doi: 10.1186/1472-6882- 14-60.
Al Hasani S, Al-Attabi ZH, Waly M, Rahman MS, Tamimi Y. Antioxidant and antitumor properties of wild blueberry (sideroxylon mascatense): effects of drying methods. Int J Nutr Pharmacol Neurol Dis 2021;11:71-9. doi: 10.4103/ijnpnd.ijnpnd_76_20
Al-Numair KS, Waly MI, Ali A, Essa MM, Farhat MF, Alsaif MA. Dietary folate protects against azoxymethane-induced aberrant crypt foci development and oxidative stress in rat colon. Exp Biol Med (Maywood) 2011;236:1005-11. doi: 10.1258/ebm.2011.011010
Mullaicharam AR, Maheswaran A. Pharmacological effects of curcumin. Int J Nutr Pharmacol Neurol Dis 2012;2:92-9. doi: 10.4103/ 2231-0738. 95930 [Full text]
Giordano A, Tommonaro G. Curcumin and cancer. Nutrients 2019;11:2376. doi: 10.3390/nu11102376
Deguchi A. Curcumin targets in inflammation and cancer. Endocr Metab Immune Disord Drug Targets 2015;15:88-96. doi: 10.2174/1871530315666150316120458
Castaño PR, Parween S, Pandey AV. Bioactivity of curcumin on the cytochrome P450 enzymes of the steroidogenic pathway. Int J Mol Sci 2019;20:4606. doi: 10.3390/ijms20184606
Hamzehzadeh L, Atkin SL, Majeed M, Butler AE, Sahebkar A. The versatile role of curcumin in cancer prevention and treatment: a focus on PI3K/AKT pathway. J Cell Physiol 2018;233:6530-7. doi: 10.1002/jcp.26620
Waly MI, Al-Bulushi IM, Al-Hinai S, Guizani N, Al-Malki RN, Rahman MS. The protective effect of curcumin against nitrosamine-induced gastric oxidative stress in rats. Prev Nutr Food Sci 2018;23:288-93. doi: 10.3746/pnf.2018.23.4.288
Upreti GC, Ratcliff RA, Riches PC. Protein estimation in tissues containing high levels of lipid: modifications to Lowry’s method of protein determination. Anal Biochem 1988;168:421-7. doi: 10.1016/0003-2697(88) 90339-9.
Durand P, Fortin LJ, Lussier-Cacan S, Davignon J, Blache D. Hyperhomocysteinemia induced by folic acid deficiency and methionine load: applications of a modified HPLC method. Clin Chim Acta 1996;252:83-93. doi: 10.1016/0009-8981(96) 06325-5.
Lee HM, Patel V, Shyur LF, Lee WL. Copper supplementation amplifies the anti-tumor effect of curcumin in oral cancer cells. Phytomedicine 2016;23:1535-44. doi: 10.1016/j.phymed.2016.09.005
Hejazi J, Rastmanesh R, Taleban FA et al.
Effect of curcumin supplementation during radiotherapy on oxidative status of patients with prostate cancer: a double blinded, randomized, placebo-controlled study. Nutr Cancer 2016;68:77-85. doi: 10.1080/01635581.2016.1115527
Doello K, Ortiz R, Alvarez PJ, Melguizo C, Cabeza L, Prados J. Latest in vitro and in vivo assay, clinical trials and patents in cancer treatment using curcumin: a literature review. Nutr Cancer 2018;70:569-78. doi: 10.1080/01635581.2018.1464347
Aroch I, Kraus S, Naumov I et al.
Chemopreventive effects of Coltect, a novel dietary supplement, alone and in combination with 5-aminosalicylic acid in 1,2-dimethylhydrazine-induced colon cancer in rats. Therap Adv Gastroenterol 2010;3:281-9. doi: 10.1177/1756283 ( 10379258
[Figure 1], [Figure 2], [Figure 3], [Figure 4]