Table of Contents 
ORIGINAL ARTICLE
Year : 2015  |  Volume : 22  |  Issue : 1  |  Page : 21-29  

Terminalia chebula attenuates DMBA/Croton Oil-Induced oxidative stress and inflammation in Swiss albino mouse skin


Department of Medical Elementology and Toxicology, Faculty of Science, Section of Molecular Carcinogenesis and Chemoprevention, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, India

Date of Web Publication21-Dec-2015

Correspondence Address:
Sarwat Sultana
Department of Medical Elementology and Toxicology, Faculty of Science, Section of Molecular Carcinogenesis and Chemoprevention, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi- 110 062
India
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DOI: 10.4103/0971-6580.172252

PMID: 26862256

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   Abstract 


Objective: The present study was designed to investigate underlying molecular mechanism for antitumorigenic potential of Terminalia chebula (TC) against chemically-induced skin tumorigenesis in Swiss albino mice. It is used as herbal medicine because it exhibits antioxidant, anti-inflammatory, and anticarcinogenic activity. However, the précised underlying mechanism remains to be elucidated. Materials and Methods: In light of the important role of nuclear factor-kappaB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (i-NOS), ornithine decarboxylase (ODC), proinflammatory cytokines, oxidative stress in carcinogenesis, chemopreventive efficacy of TC against 7,12-dimethylbenz[a] anthracene (DMBA), and croton oil-induced 2-stage skin carcinogenesis was studied in terms of cytoprotective antioxidant enzymes activity, lipid peroxidation (LPO), inflammatory responses, and expression of various molecular markers in skin tissues. Results: We found that topical application of TC at dose of 30 mg/kg b. wt. mouse effectively suppressed oxidative stress and deregulated activation of inflammatory mediators and tumorigenesis. Histological findings further supported the protective effects of TC against DMBA/croton oil-induced cutaneous damage. Conclusion: The findings of the present study suggest that the chemopreventive effect of TC is associated with upregulation of endogenous cytoprotective machinery and downregulation of inflammatory mediators (interleukin (IL)-6, COX-2, i-NOS, ODC, and NF-κB).

Keywords: Cytokines, inflammation, proinflammatory markers, Terminalia chebula, Skin carcinogenesis


How to cite this article:
Majed F, Nafees S, Rashid S, Ali N, Hasan SK, Ali R, Shahid A, Sultana S. Terminalia chebula attenuates DMBA/Croton Oil-Induced oxidative stress and inflammation in Swiss albino mouse skin. Toxicol Int 2015;22:21-9

How to cite this URL:
Majed F, Nafees S, Rashid S, Ali N, Hasan SK, Ali R, Shahid A, Sultana S. Terminalia chebula attenuates DMBA/Croton Oil-Induced oxidative stress and inflammation in Swiss albino mouse skin. Toxicol Int [serial online] 2015 [cited 2017 Mar 28];22:21-9. Available from: http://www.toxicologyinternational.com/text.asp?2015/22/1/21/172252




   Introduction Top


Skin cancer is mainly caused by various environmental carcinogens, inflammatory agents, ultraviolet (UV) irradiation and/or tumor promoters. Murine skin tumorigenesis is an excellent in vivo model to study the chemopreventive activity of natural/synthetic agents.[1] Tumor development can also be caused by multiple applications of croton oil, a tumor promoter, which is a slow and reversible process and leads to skin tumors in mouse by inducing hyperplasia and inflammatory responses.[2],[3] 7,12-dimethylbenz[a] anthracene (DMBA), a carcinogen, initiates skin tumorigenesis which is a rapid and irreversible process. The croton oil is the most widely used distinguished promoting agent to understand the cellular and molecular alterations associated with promotion stage and also a well-known model to understand the role of inflammation, generation of reactive oxygen species (ROS), and hyperplasia in cancer promotion.[4],[5]

Inflammatory responses play an important role in all the stages of carcinogenesis, including; initiation, promotion, malignant conversion, invasion, and metastasis.[6] Various observations reveal that inflammation is one of the key events inducing cancer development by various mechanisms, such as production of ROS. ROS and free radicals play a significant role in induction of many diseases such as cancer, ageing, atherosclerosis, asthma, and diabetes.[7],[8] However, uncontrolled production of ROS or change in intracellular antioxidants level causes damage or modification of cellular macromolecules,[9] which on activation produce these kinds of free radicals to kill the pathogens.

Aberrant production of inflammatory mediators like proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and NO have critical role in multistage cancer development. Cytokines, chemokines, and eicosanoids are the inflammatory mediators known to have important role at promotion phase of carcinogenesis inflammatory responses including expression of cyclooxygenase-2 (COX-2) enzyme and ROS. These factors play a critical role in carcinogenesis stage.[6],[10] Ornithine decarboxylase (ODC) enzyme activity and thymidine incorporation into DNA synthesis can be used as biomarkers of skin tumorigenesis.[11] Nuclear factor-kappa B (NF-κB) is one of the most important transcription factor known to regulate the expression of genes involved in inflammation, cell proliferation, and survival. One of the downstream targets of NF-κB is COX-2. Uncontrolled expression of COX-2 has been observed in different premalignant as well as in malignant stage.[12] TC, commonly known as the king of medicine, is a plant of Combretaceae family which is used in number for ayurvedic productions.[13] Naturally occurring phenolics are richly found in edible vegetables, nuts, and fruits and constitute an important part of the human diet. TC has been reported to exhibit a variety of biological effects, for example, antioxidant, anticancer, and antimutagenic.[14] In antioxidant patterns, the active constituents of TC are gallic acid, ellagic acid, tannic acid, ethyl gallate, chebulagic acid, chebulinic acid, etc.

The present study was designed to investigate the preventive effects of TC on DMBA/croton oil-induced initial and promotional changes in mouse skin and explore the underlying molecular mechanism. To test this possibility, we have studied the preventive effects of TC on the DMBA/croton oil-induced activation of NF-κB and its downstream target COX-2, ODC, and inducible nitric oxide synthase (i-NOS) via cutaneous oxidative stress, inflammation, and IL-6 production.


   Materials and Methods Top


Chemicals

Reduced glutathione (GSH), glutathione reductase (GR), oxidized glutathione (GSSG), 1-chloro-2,4-dinitrobenzene (CDNB), 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), ethylenediamine tetraacetic acid (EDTA), sulfosalicylic acid, horseradish peroxidase (HRP), hydrogen peroxide, xanthine, xanthine oxidase (XO), acetone, DMBA, Terminalia chebula (TC), and croton oil were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). All the other reagents used were of analytical grade and highest purity.

Animals

Female Swiss albino mice 6–8-weeks-old, weighing 20-30 g, were obtained from the Central Animal House Facility of Hamdard University. Animals received humane care in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, and prior permission was sought from the Institutional Animal Ethics Committee (IAEC) of the university.

Treatment regimen

To observe the effect of TC on DMBA/croton oil-induced cutaneous oxidative and inflammatory responses, animals were divided into four groups (I–IV) of 10 animals (n = 10) each. Dorsal skin of all the animals was shaved with the electric clipper 2 days prior to the start of the experiment. Only mice that showed no signs of hair regrowth were included in the experiments. All the treatments were done topically onto the shaved area of dorsal skin.

Group I: Animals of this group served as control and were given topical application of vehicle (0.2 ml acetone).

Group II: Animals were given a single topical application of DMBA (50 µg) in 0.2 ml acetone. One week after the initiation, croton oil (1%) in 0.2ml acetone was applied twice a week for 20 consecutive weeks.

Group III: Animals were given a single topical application of DMBA (50 µg) in 0.2 ml acetone. After 1 week of pretreatment of TC at the dose 30 mg/kg b. wt., in 0.2 ml acetone 30 min before croton oil (1% in 0.2 ml acetone) was given topically twice a week until the termination of the experiment at 20 weeks.

Group IV: Animals were given topical application of TC (30 mg/Kg b. wt.) in 0.2 ml acetone up to the termination of the experimentat 20 weeks.

At the end of 20 weeks, all the animals were euthanized by light ether anesthesia and exsanguinations. Their skin was quickly removed and processed for various molecular, histopathological, and immunohistochemical studies.

Biochemical studies

Assay for lipid peroxidation (LPO), catalase (CAT), GSH, glutathione peroxidase (GPx), GR, glutathione-S-transferase (GST), XO, hydrogen peroxide (H2O2), and protein estimation was done by the method as described by Khan et al.[15]

Assay for IL-6 level

Level of all the proinflammatory cytokines was measured with commercially available kits. Tissue was homogenized and measured by enzyme-linked immunosorbentassay (ELISA) kit (eBioscience Inc, San Diego, USA). Analysis was performed according to the manufacturer's instruction.

Immunohistochemistry

Skin sections on polylysine coated slides obtained were fixed in neutral buffered formalin, embedded in paraffin, and was treated for NF-κB, COX-2, ODC, and iNOS antibody for immunohistochemical analysis. The procedure was processed according to the manufacturer's protocol with slight modifications.

Histological investigation

The skin were quickly removed after sacrifice and preserved in 10% neutral buffered formalin for histopathological processing. The skin were embedded in paraffin wax and longitudinally sectioned with a microtome. Hematoxylin and eosin staining of the sections was observed under an Olympus microscope.

Morphological assessment

Observed and measured at weekly interval percentage of tumor incidence, tumor bearing mouse, body weight, and cumulative number of papillomas. Only tumors, those persisted more than 2 week with diameter greater than 1 mm were taken into consideration for evaluation of the data. Percentage of tumor incidence was calculated by dividing the number of tumor-bearing mice with the total number of mice in a particular group and multiplied with 100%.

Statistical analysis

The data from individual groups were presented as the mean ± standard error of the mean (SEM). Differences between groups were analyzed using analysis of variance (ANOVA) and minimum criterion for statistical significance was set at P < 0.05.


   Results Top


Effect of TC on DMBA/croton oil-induced oxidative stress in mouse skin

Exposure of DMBA and croton oil resulted in significant increase in the level of MDA in Group II as compared Group I (P < 0.001). In Group III, it significantly (P < 0.001) inhibited the formation of MDA as compared to Group II. There was no significant change observed in the level of MDA between Groups I and IV [Figure 1]a. Skin H2O2 content was measured and the results were shown in [Figure 1]b. There was significant increase between Groups I and II (P < 0.001). We have observed that in Group III, it significantly inhibited H2O2 formation (P < 0.001) as compared to Group II (P < 0.001). There is no significant difference between Groups I and IV. Also, elevation of XO activity was observed in Group II when compared with Group I (P < 0.001). We have observed that topical application of TC was significantly effective in the reduction of XO activity in Group III when compared with Group II (P < 0.001). There was no significant difference observed betweenGroups I and IV as far as XO activity is concerned [Figure 1]c. It was found effective in restoration of endogenous antioxidant CAT activity. There was significant depletion in the level of CAT activity in Group II as compared to Group I (P < 0.001). Group III showed significant increase in the level of CAT (P < 0.001) as compared to Group II. There was no significant difference in CAT level between Groups I and IV [Figure 2]a and [Table 1]. It has been observed that the level of educed GSH was significantly depleted in Group II as compared with Group I (P < 0.001). Group III showed significant increase in the level of GSH (P < 0.001) as compared to Group II. There was no significant difference in GSH level between Groups I and IV [Figure 2]b. Also, depletion in the activities of different antioxidant enzymes was examined and the results are shown in [Table 1]. We have observed that there was a significant (P < 0.001) decrease in the activity of different antioxidant enzymes in Group II as compared to Group I. However, Group III significantly (P < 0.001) restored the activity of antioxidant enzymes when compared with Group II. There was no significant difference observed between Groups I and IV.
Figure 1: Effect of Terminalia chebula (TC) on DMBA/croton oil-induced (a) lipid peroxidation, (b) H2O2production, and (c) xanthine oxidase activity in skin of mice from different treatment groups. Values are expressed as means±SEM (n=10) of nmol MDA formed/h/g of tissue, nmol H2O2/h/g of tissue, and μg uric acid/mg protein, respectively. ***P<0.001 shows significant difference in Group II when compared with Group I. ###P<0.001, ###P<0.001 shows significant difference in the Group III when compared with Group II. SEM=Standard error of the mean, DMBA = 7, 12-dimethylbenz[a]anthracene

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Figure 2: Effect of TC pretreatment on DMBA/croton oil-induced cutaneous (a) CAT and (b) GSH level. Values are expressed as mean±SEM (n=10). ***P<0.001 shows significant difference in Group II (DMBA/croton oil (1%) in 200 μl acetone) when compared with Group I. ###P<0.001 shows significant difference in Group III (TC (30 mg) + DMBA/croton oil (1%)) when compared with DMBA/croton oil-treated Group II. CAT = catalase, GSH = reduced glutathione

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Table 1: Effect of TC on the antioxidant enzymes

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Effect of TC on IL-6

IL-6 levels in the skin tissue of Group II was significantly elevated (P < 0.001) as compared to Group I. TC administration showed a significant reduction of IL-6 levels production in Group III when compared with Group II (P < 0.001). There was no significant difference found between Groups I and IV as far as release of IL-6 is concerned. There was no significant difference found between Groups I and IV as far as release of IL-6 is concerned [Figure 3].
Figure 3: Effect of TC pretreatment on DMBA/croton oil-induced cutaneous IL-6 cytokines. Values are expressed as mean±SEM (n=10). ***P<0.001 shows significant difference in Group II (DMBA/croton oil (1%) in 200 ml acetone) when compared with Group I (vehicle alone), ###P<0.001 shows significant difference in the Group III (TC (30 mg) + DMBA/croton oil (1%)) when compared with DMBA/croton oil-treated Group II

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Effect of TC on tumor incidence and histopathological observations in tumor study

Topical application of TC was further observed for cutaneous histological alteration caused by DMBA + croton oil application with reference to neutrophil infiltration and epidermal hyperplasia as shown in [Figure 4]. We found an increase in the leukocyte infiltration and epidermal thickening in Group II [Figure 4]b as compared to Group I [Figure 4]a. In Group III, diminished leukocyte infiltration as well as hyperplasia was observed [Figure 4]c. There was no distinguished change observed between Groups I and IV [Figure 4]d as far as leukocyte infiltration and hyperplasia is concerned. Tumors begin to appear on the skin from week 9 to 20, during the promotion period. Protective effects of TC on skin carcinogenesis are shown in [Table 2]. Animals of Groups I and IV did not show any significant variation in body weight during the entire experiment and there was no mortality. No noticeable signs of weight loss and papilloma were detected in the mice of Groups IV and I, but was found significant in Groups II and III. The incidence of papilloma was different in Group II (100%) as compared to Group III (40%). Cumulative number of papillomas was 118 in Group II, which was significantly reduced to 21 in Group III.
Figure 4: Effect of TC pretreatment on skin histological alterations caused by DMBA/croton oil application. (a) Skin of the acetone vehicle control group reflects normal skin architecture with no change in epidermis and dermis region. (b) Skin of the DMBA/croton oil-treated animals. There is marked swelling and thickening of the epidermis as compared to the control. (c) Skin of the animals pretreated with TC before DMBA/croton oil shows improved skin architecture as far as neutrophil infiltration and epidermal thickness is concerned. (d) Shows the skin of the group treated with dose of TC only with no significant change as compared to the control. Original magnification, ×10

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Table 2: Effect of TC on DMBA/croton oil-induced carcinogenesis in mice

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Effect of TC on expression of i-NOS, COX-2, ODC, and NF-κBof skin

Cutaneous expression of the above mentioned proteins are shown in [Figure 5], [Figure 6], [Figure 7], [Figure 8], respectively. They show the epidermal cells of the papilloma with very good positive staining for havingCOX-2, ODC, i-NOS, and NF-κ B expression in Group II as compared to that of Group I. In Group III, showing the epidermal cells of the papilloma with very weak positive staining for having expression of NF-κB, COX-2, and i-NOS and has less immunopositive cells with weak staining as compared to Group II.
Figure 5: Effect of TC pretreatment on COX-2 expression. DMBA/croton oil-treated Group II has more COX-2 positive cells as compared to acetone-treated Group I (a and b). Pretreatment of TC in the Group III (DMBA/croton oil + TC; C) and TC-treated Group IV, (c and d) have less staining as compared to Group II. COX = Cyclooxygenase

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Figure 6: Effect of TC pretreatment on i-NOS expression. DMBA/croton oil-treated group II has more i-NOS positive cells as compared to acetone-treated Group I (a and b). Pretreatment of TC in Group III (DMBA/croton oil + TC; C) and TC-treated group IV (c and d) have less staining as compared to Group II. However, there was no significant difference in Groups I and IV (a and d) as far as staining is concerned. i-NOS = Inducible nitric oxide synthase

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Figure 7: Effect of TC pretreatment on DMBA/ croton oil-induced cutaneous NF-κB expression. Representative photomicrographs (magnification: × 40), (a) only vehicle (Group I), (b) DMBA/croton oil (Group II), (c) TC + DMBA/croton oil (Group III), and (d) only TC Group IV). DMBA/croton oil-treated group (Group II) shows more NF-κB immunopositive cells as compared to vehicle-treated group (Group I). TC pretreatment (Group III) reduces NF-κB immunopositive cells as compared to Group II. However, there was no significant difference in the NF-κB immunostaining in Group IV as compared to Group I. NF-κB = nuclear factor-kappa B

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Figure 8: Effect of TC pretreatment on DMBA/croton oil-induced cutaneous ODC expression. Representative photomicrographs (magnification: ×40), (a) only vehicle (Group I), (b) DMBA/croton oil (Group II), (c) TC + DMBA/croton oil (Group III), and (d) only TC (Group IV). DMBA/croton oil-treated group (Group II) shows more ODC immunopositive cells as compared to vehicle-treated group (Group I). TC pretreatment (Group III) reduces ODC immunopositive cells as compared to Group II. However, there was no significant difference in the ODC immunostaining in Group IV as compared to Group I. ODC = Ornithine decarboxylase

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   Discussion Top


The mice model of skin carcinogenesis represents one of the best means to study malignant transformation, including the multistage nature of tumor development and the molecular mechanism that contribute to the development of human epithelial cancers. It has been found to be a very good test system for investigating the influence of dietary and chemoprevention, both mechanistically and operationally.[16]

TC has high phenolic content and strong biological activities, including anticancer, anti-LPO, and antioxidant activity.[17],[18] Further, evaluation of antitumorigenic potential of TC on DMBA-initiated and croton oil-promoted mouse skin carcinogenesis showed that pretreatment with TC significantly prolongs the formation of tumors in skin and reduced the tumor incidence and cumulative number of papillomas. In this study, it has been reported that the attenuative effects of TC with reference to croton oil-induced inflammatory responses, which is critically associated with the promotion phase of cancer development and their mediators, are recognized as an important regulator of tumor promotion.

The results of the present study showed that TC inhibits DMBA and croton oil-induced cutaneous LPO. It is a major cellular macromolecules which leads to improper functioning of various inter- and intracellular signaling pathways and has critical association with the promotion stage of cancer development.[19] The level of MDA had elevated after DMBA and croton oil topical administration. Pretreatment with TC reduced MDA formation which suggests the strong antitumor promotional potential of TC. Further, it was observed that TC also upregulates the levels and activities of the major endogenous antioxidants viz., GSH, catalase, GR, and GPx; and therefore, confirms its antioxidant potential.

The data of present study suggested that antioxidants and detoxifying enzymes play critical role in croton oil-induced early tumor promotional events and pretreatment with TC repressed the oxidative damages possibly by supporting endogenous antioxidant defense machinery via triggering their transcriptional upregulation, which is in accordance with previous findings. TC indirectly scavenges the oxygen free radicals, as evidenced by the activities of CAT, GPx, GR, GST, and levels of GSH and LPO.[20] DMBA and croton oil-induced inflammatory responses (leukocytes infiltration, increase production of ROS, and hyperplasia) increased proinflammatory cytokines production and over expression of COX-2 and i-NOS, which are closely related with skin tumor promotion.[21]

NF-κB plays a critical role in regulation of cell survival genes and coordinates the expressions of proinflammatory enzymes and cytokines such as i-NOS, COX-2, ODC, and IL-6. Since the expressions of these proinflammatory mediators are modulated by NF-κB. The chief molecular factors involved in inflammation are prostaglandins, cytokines, NF-κB, chemokines, and angiogenic factors.[22] These cytokinesplaycritical roles in tumor growth and progression of tumor development. Chebulagic acid from TC showed potent inhibition activity against COX-2.[23] The expression of i-NOS and COX-2 appear to be regulated by NF-κB in mouse skin with DMBA and croton oil-induced cutaneous inflammation and observed several chemopreventive phytochemicals have been shown to inhibit COX-2 and i-NOS expression by blocking improper NF-κB activation; and they are associated with cutaneous inflammation, cell proliferation, and skin tumor promotion.[24]

Present study demonstrates that TC strongly suppress the activation of NF-κB that might be mediated via inhibition of the phosphorylation or degradation of IκB, as previous findings implicate that natural antioxidants inhibit NF-κB activation by blocking modification of IκB.[25] NO and i-NOS show overexpression during inflammatory responses and are believed to be responsible for enhancing carcinogenesis and tumor progression, stimulation of angiogenesis, and promotion of metastasis.[26] Thus, inhibition of i-NOS and NO may serve as anticancer target. TC inhibited DMBA/croton oil-induced inflammatory responses, such as leukocyte infiltration; proinflammatory cytokines production expression of COX-2, i-NOS, and ODC; and activation of NF-κB.[18]

IL-6isan important proinflammatory agent and signaling messenger which plays the key role in promotion stage of carcinogenesis. Their role in production of growth factors, eicosanoids, NO, and activation of the NF-κB is also well established.[27] Increased production of IL-6 have been associated with promotion phase of skin carcinogenesis and was confirmed by the previous observation that mouse deficient in IL-6 is resistant to skin tumor formation.[28] ROS play an important role in tumor initiation by enhancing or facilitating the metabolic activation and/or initiating effects of carcinogens and induction of epidermal ODC.[17],[29] Topically treated croton oil animals show enhanced ODC, a key enzyme in the polyamine synthesis pathway and is overexpressed in a variety of cancers.[11] The present study showed that TC reduced ODC expression in DMBA and croton oil-treated mouse skin. In a recent study, topical exposure of TC prior to that of DMBA and croton oil resulted in a significant inhibition of DMBA and croton oil-mediated, and pretreatment with TC on ODC expression shows that it inhibits DMBA and croton oil-mediated induction of epidermal ODC.[30] The ODC, i-NOS, and COX-2 proteins were highly expressed in mouse skin with hyperplasia, compared to mice treated with vehicle alone. The increased expression of these biomarkers was maintained during the development of papilloma with dysplasia. Histological examination of mouse skin after DMBA and croton oil application showed marked cellular anomalies like epidermal thickness, disorganization of epithelium, presence of necrotic cells, and focal proliferative area. Inflammatory cell infiltration of the dermis and intercellular edema in the epidermis were also observed. Similarly, hematoxylin and eosin stained sections showed pronounced preventive effects of TC on DMBA and croton oil-induced changes in animal's skin. They also found a decrease in tumor incidence, cumulative number of papillomas, and body weight. Further, the findings of the present study demonstrate that TC has strong antitumorigenic potential against DMBA-initiated and croton oil-promoted mouse skin tumorigenesis; and the underlying mechanism may involve inhibition of croton oil-induced inflammatory responses, hyperproliferation, oxidative stress, and endogenous proinflammatory cytokines. Induction of various antioxidant enzymes also provides substantial evidence for the antitumorigenic potential of TC.


   Conclusion Top


The findings of the present study showed that TC strongly suppressed cutaneous tumorigenesis via boosting of endogenous antioxidant defense system, inhibition of oxidative stress, and inflammation. Inhibition of COX-2 expression, i-NOS expression, and proinflammatory cytokine production activation of NF-κB and ODC by TC provide the molecular basis of the antitumor promotional potential of TC. These findings suggest the strong anticarcinogenic potential of TC, and hence may serve as one of the important strategies for the prevention of skin cancer.

Acknowledgment

Dr. Sarwat Sultana is thankful to Ministry of Higher Education Republic of Iraq to provide fellowship to one of the authors (Ms. Ferial Majed) to carry out this research work at Jamia Hamdard (Hamdard University), New Delhi, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2]



 

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