Jyoti Parmar, Priyanka Sharma, and Pradeep K Goyal*
Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur-302 004, Rajasthan, India.
Received date: 26/03/2013; Accepted date: 07/04/2013; Published date: 25/05/2013
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Investigation of cancer chemopreventive and anti-oxidative property of S.cumini seed extract (SCE) on 7,12-dimethyl benz(a)anthracene (DMBA) induced skin carcinogenesis in mice. Animals were divided into four groups on the basis of their respective treatments wherein mice of Group I & II served as vehicle treated and SCE treated controls respectively. For induction of skin tumors, mice of Group III and IV were applied topically with 7,12-dimethylbenz(a)anthracene (DMBA) followed 2 weeks later by repeated application of croton oil (1% in acetone three times a week) and continued till the end of the experiment (i.e. 16 weeks). Mice of Group IV were administered S.cumini seed extract (SCE) at peri- & post-initiational stage. The results of the study revealed a significant decrease in incidence, cumulative number of papillomas, tumor yield and tumor burden in mice of Group IV as compared with DMBA alone at the end of experiment. A significant reduction in tumor weight and tumor volume was also observed. Reduction in the incidence and number of papilloma, the preneoplastic lesions, was considered to be the mean of assessment. Significant (p<0.01) decrease in the level of lipid peroxidation and significant (p<0.01-p<0.001) enhancement in the activity of antioxidants (GSH, SOD, Vitamin-C) and total proteins levels were recorded in Group IV. SCE was also found potential in reducing the histopathological lesions induced in tumors by DMBA. From the present study, it can be inferred that Syzygium cumini possesses has chemopreventive and anti-oxidative potential for chemical induced carcinogenesis. Such chemopreventive activity may be linked with the antioxidant/free radical-scavenging constituents present in the extract.
Despite the efforts of innumerable researchers worldwide to ameliorate the dismal outcomes of cancer, it stills continues to be a huge burden on mankind. Cancer originates in our own cells, but several factors, both intrinsic and external to the body, which influence our daily life, can add to the life time cancer risk. While cancer, as such, is not infectious, some infections can act as a stimulus to induce and promote cancer development. In addition, environmental pollutants like many chemicals, industrial effluents, some therapeutic drugs, and mutagenic agents, including ionizing radiation, can increase the incidence of cancer. About 50% of all cancers are attributed to life style, eg. diet, tobacco habits and alcohol consumption, and exposure to industrial toxins.
Chemical and UV radiation-induced carcinogenesis in murine skin and possibly human skin is a stepwise process of at least three distinct stages: initiation, promotion, and progression . Tumors can be initiated by a single dose of carcinogen such as 7, 12-dimethylbenz (a) anthracene (DMBA) followed by a repetitive application of a tumor promoter such as TPA . Increasing skin cancer morbidity and mortality is alarming and expensive, in both human and economic terms. New strategies are needed to combat this disease. The goals of cancer chemo-prevention are to reduce the incidence, morbidity and mortality due to cancer through the identification and elimination of precancerous lesions termed intraepithelial neoplasias and/or the early detection of minimally invasive cancers.
Herbal medicines are in great demand in the developed medicines offers a host of problems. To solve this as well as developing countries for primary healthcare problem, first and foremost task is the selection of the because of their wide biological activities, higher safety right kind of plant material which is therapeutically margins and lesser costs. Ayurveda, a traditional Indian medicine of plant drugs has been successful from very early times in using these natural drugs and preventing or suppressing various tumors using various lines of treatment. Recently, phytochemicals and their effects on human health have been intensively studied. In particular, a search for antioxidants, hypoglycemic agents, and anticancer agents in vegetables, fruits, teas, spices and medicinal herbs has attracted great attention.
Some of the useful plant drugs with medicinal properties include vinblastine, vincristine, taxol, podophyllotoxin, camptothecin, digitoxigenin, gitoxigenin, digoxigenin, tubocurarine, morphine, codeine, aspirin, atropine, pilocarpine, capscicine, allicin, curcumin, artemesinin and ephedrine among others.  In some cases, the crude extract of medicinal plants may be used as medicaments. On the other hand, the isolation and identification of the active principles and elucidation of the mechanism of action of a drug is of paramount importance. Hence, investigations on both mixture of traditional medicine and single active compounds are very important .
S. cumini (L.) Skeels has been attributed in the Indian folklore medicine system to possess several medicinal properties . The bark of the plant is astringent, sweet, refrigerant, carminative, diuretic, digestive, anti-helminthic, febrifuge, constipating, stomachic and antibacterial. The fruits and seeds are used to treat diabetes, pharyngitis, spleenopathy, urethrorrhea and ringworm infection. The leaves have been extensively used to treat diabetes, constipation , leucorrhoea, stomachalgia, fever, gastropathy, strangury and dermopathy , and to inhibit blood discharges in the faeces .
Hence, the present study is designed to evaluate the effectiveness of seed extract of Syzygium cuminiin modifying the carcinogenic process, as well as oxidative stress in DMBA- induced skin carcinogenesis.
The animal care and handling was approved by ethical committee of our institution and was done according to guidelines set by the World Health Organization, Geneva, Switzerland, and the Indian National Science Academy, New Delhi, India. The inhibition of tumor incidence by Syzygium cumini seed extract was evaluated on two-stage skin carcinogenesis, induced by a single application of DMBA (initiator), and two weeks later, promoted by repeated application of croton oil (promoter) thrice per week, following the protocol for 16 weeks .Animals
The study was conducted on random-breed male Swiss albino mice of 7-8 weeks old with 24 ± 2 g body weight. These animals were housed in polypropylene cages in the animal house under controlled conditions of temperature (25°C ± 2°C) and light (14 light:10 dark). The animals were fed a standard mouse feed procured from Aashirwad Industries, Chandigarh (India), and water ad libitum. Eight animals were housed in one polypropylene plastic cage containing saw dust (procured locally) as bedding material. For precaution against infections, tetracycline hydrochloride water was given to these animals once each fortnight.
7, 12-Dimethyl Benz (a) anthracene (DMBA) and croton oil was procured from Sigma Chemical Co., USA. DMBA was dissolved at a concentration of 100 μg/ 100μl in acetone. Croton oil was mixed in acetone to give a solution of 1% dilution.
The fruit of Syzygium cumini L. was collected locally after proper identification. The identification of the plant Syzygium cumini L. (Family: Myrtaceae) was done by a botanist (Voucher Specimen No: RUBL- 20425) from Herbarium, Department of Botany, University of Rajasthan, Jaipur, Rajasthan (India). The pulp was removed from the fruit and the seed were washed properly and shade dried, then after fruit was powdered in a mixture and the hydro-alcoholic extract was prepared by refluxing with the double distilled water (DDW) and alcohol (3:1) for 36 (12 x 3) hrs at 40ºC. The extract was cooled and concentrated by evaporating its liquid contents. The prepared Syzygium cumini extract (SCE) was stored at low temperature until its further use and it was redissolved in DDW prior for the oral administration in mice.
The dorsal skin (2 cm diameter) of Swiss albino mice was shaved 2 days before chemical treatment and animals in the resting phase of growth cycle were selected for the experiment. Mice selected from inbreed colony were grouped into following five groups:
Group I: Vehicle treated control
Animals of this group received topical application of acetone (100 μl/ mouse) on the shaven dorsal skin and double distilled water
equivalent to SCE (100 μl/ mouse/ day) by orally for 16 weeks.
Group II: Drug (SCE) treated control
These animals received SCE (125 mg/kg b. wt./animal/day) alone orally during the entire experimental period (i.e., 16 weeks).
Group III: Carcinogen treated control
A single dose of 100 μg of DMBA in 100 μl of acetone was applied topically over the shaven area of the skin of the mice. Two weeks
later croton oil (1% croton oil in acetone) was applied three times per week until the end of the experiment (i.e., 16 weeks).
Group –IV: SCE treated experimental
Animals of this group were administered SCE (125 mg/kg b. wt./animal/day) orally starting from 7 days before of DMBA application
and continued until the end of experiment (i.e., 16 weeks) and served as peri- and post- initiational group.
The Following Morphological Parameters Were Studied
Tumor incidence: The number of mice carrying at least one tumor expressed as a percentage incidence.
Tumor yield: The average number of papillomas per mouse.
Tumor burden: The average number of tumors per tumor bearing mouse.
Diameter: The diameter of each tumor was measured.
Weight: The weight of the tumors of each animal at the termination of each experiment was measured.
Body weight: The weight of the mice was measured weekly.
Average latent period: The time lag between the application of the promoting agent and the appearance of 50% of tumors was determined. The average latent period was calculated by multiplying the number of tumors appearing each week by the time in weeks after the application of the promoting agent and dividing the sum by total number of tumors.
Where F is the number of tumors appearing each week, X is the numbers of weeks, and n is the total number of tumors.
The following biochemical parameters were estimated in the liver and skin of mice.
Lipid peroxidation (LPO)
The level of LPO was estimated spectrophotometrecally by thiobarbituric acid reactive substances (TBARS) method as described by Ohkhawa et al. (1979) . Briefly, thiobarbituric acid (0.6%), sodium dodecyl sulphate (0.1%), and trichloroacetic acid (20%) were added to 200 μl of the tissue homogenate (10%) prepared as described above. This mixture was heated for 60 minutes, cooled, and extracted with N butanol- pyridine (15:1), the optical density (OD) was recorded at 532 nm and the contents were expressed as nmol/mg of tissue.
Reduced glutathione (GSH)
The level of GSH was estimated as total nonprotein sulphahydryl group by the method of Moron et al. (1979) . The homogenate was immediately precipitated with 100 μl of 25% trichloroacetic acid (TCA) and the precipitate was removed after centrifugation. Free endogenous-SH was assayed in a total volume of 3 ml by the addition of 200 μl of 0.6 mM 5, 5′ dithio-bis (2- nitrobenzoic acid) dissolved in 0.2 M phosphate buffer (pH 8.0) to 100 μL of the supernatant and the absorbance was recorded at 412 nm using a UV-VIS Systronics spectrophotometer. Reduced GSH was used as a standard and the levels of GSH were expressed as μmol/gm of tissue.
The catalase activity was assayed by the method of Aebi . The change in absorbance was followed spectrophotometrically at 240 nm after the addition of H2O2 (30 mM) to 100 μL of the supernatant (10% of skin homogenate prepared in 50 mM phosphate buffer and centrifuged for 10 min.) in 50 mM phosphate buffer (pH 7).The activity of the enzyme is expressed as U/mg of tissue, where U is μmol of H2O2 disappearance/min.
Total Proteins were estimated by the method of Lowery et al.,  using bovine serum albumin as a standard and the level was expressed as mg/ gm.
SOD was determined by the method of Marklund and Marklund  by quantification of pyrogallol auto oxidation inhibition and the results are expressed as units/mg protein. Auto oxidation of pyrogallol in Tris-HCL buffer (50 mM, pH-7.5) was measured by increase in absorbance at 420 nm.
For this, tissue, the fresh organs were weighed, homogenized in acetate buffer (20 mg/ ml) extracted with cold 4 per cent trichloroacetic acid, centrifuged, and filtered. Ascorbic acid was determined by the method of Roe and Kuether .
Tumors and normal skin were removed from the sacrificed animals and immediately fixed in 10% formalin fixative for 24h. The tissues were then dehydrated in ascending series of alcohol, embedded in paraffin wax and 5 micron thick sections were cut and these were deparaffinized in xylene, rehydrated in descending concentrations of ethanol, stained with hematoxylin and eosin. and viewed under light microscope.
Data from different experimental groups were analyzed and expressed as mean + SD. The significant level of difference between Carcinogen treated control and SCE treated experimental groups were statistically analyzed using Student’s t-test.
No tumor development was recorded in vehicle treated and SCE treated control (Group I and II) respectively. Papillomas appeared on skin of all the animals of carcinogen treated control (Group-III), where a single topical application of DMBA followed 2 weeks later by repeated application of croton oil, i.e. 100 % incidence was recorded in the Group III (Figure.1). The cumulative number of papillomas as induced during the entire experimental period was 68 (Figure. 2). The average number of tumors per tumor bearing mouse (tumor burden) and tumor yield was found to be as 8.5 (Figure. 3 and 4), and the average latent period was 7.88 weeks (Figure. 5).
Mice of Group IV that received a continuous treatment of S.cumini extract orally at peri- and post- initiational phase, showed a significant reduction in the tumor incidence (62.5%) as compared with the carcinogen treated control (Group I) (Figure.: 1). The cumulative number of papillomas produced during the observation period (i.e., 16 weeks) were considerably lesser (76.47%) than the carcinogen treated control (Figure. 2).
Oral administration of SCE during peri- and post- initational stage (Group IV) of DMBA-induced papillomagenesis reduced the tumor burden and tumor yield to 3.2 (62.35%) and 2(76.47%) respectively (Figures. 3 and 4) The time lag between the application of the promoting agent and the appearance of 50% of tumors i.e., average latent period was documented as 11.75 (49.11%) weeks in Group IV where SCE was administered 7 days before DMBA application and continued throughout the experimental period (Figure. 5). The average weight of tumors was also significantly reduced to 45.5 mg (67.23%) in SCE treated animals than the carcinogen treated control (138.87 mg) (Table 1).
The levels of TBARS, total proteins and antioxidants (GSH, SOD, CAT and vitamin- C), in liver and skin of animals of each group are shown in Figures. 7-12. The concentration of TBARS was significantly (p<0.001) increased, whereas the status of antioxidants and the total proteins was significantly (p<0.001) decreased in animals of carcinogen treated control (Group III), as compared to vehicle treated control (Group I).
Lipid peroxidation in liver and skin, expressed in terms of TBARS, was found to be significantly (p<0.001) reduced at the peri- and post- initiational stage (Group IV) to that measured in the carcinogen treated Control (Group III). Oral administration of SCE to DMBA-painted animals (Group IV) at the peri- and post- initiational stage significantly revert back the status to near normal concentration of TBARS.
SCE treatment during the peri- and post- initiational stage (Group IV) significantly (p<0.01-p<0.001) elevated the hepatic as well as skin antioxidants and total proteins levels when compared with carcinogen treated control (Group III).
Mice treated with S.cumini extract alone (Group II) showed no significant difference in TBARS, proteins and antioxidants status when compared to vehicle treated control animals (Group I).
E- epidermis, D- dermis, B- basal Lamina, H.K- Hyperkeratosis E.H- epidermal hyperplasia, D.I.- Dermal invasion, S.G- sebaceous gland, Ac- Acnathosis, H.C- Horny cyst, P.H.C. Pseudo horny cyst, Rd- reduced stroma with lymphocytes, T.N.-Tumor Nest
The histopathological examination of the skin of carcinogen treated control animals exhibited at the cells with atypical (enlarged and hyperchromatic) nuclei at all the strata of the epidermis, severe epidermal hyperplasia and dermal invasion of epidermis. All of these symptoms were found to be minimal in SCE treated experimental animals (Group IV) (Figure. 13. a–e). Tumor of carcinogen treated control (Group III) showed well differentiated squamous cell carcinoma with severe acanthosis, reduced stroma with lymphocytes and an obvious number of horny pearls, which were larger in size, whereas S.cumini extract administration at the peri- and post- initiational stage (Group IV) showed poorly differentiated papilloma with lesser number and small sized horny pearls.
Figure 13: (a) Photomicrograph showing histological section of skin of normal mice (Group I).
(b) Photomicrograph showing histological section of DMBA/TPA induced mice skin (Group II)
(c) Photomicrograph showing histological section of DMBA/TPA induced skin tumor in mice (Group III)
(d) Photomicrograph showing histological section of DMBA/TPA induced skin in mice administered with SCE (Group IV).
(e) Photomicrograph showing histological section of DMBA/TPA induced skin tumor in mice administered with SCE (Group IV).
7,12-Dimethylbenz[a]anthracene (DMBA) is the best studied polycyclic aromatic hydrocarbon (PAH) and widely used in experimental carcinogenesis [12,13]. Metabolic activation by P450 through AhR is the first step in the long road of PAH carcinogenesis. The metabolism of DMBA is well characterized [14,15,16]. Briefly, P450 oxidizes DMBA to the 3,4-epoxide followed by hydrolysis by mEH to the proximate form, 3,4-diol. This metabolite is again oxidized by P450 to the ultimate form, 3,4-diol-1,2-epoxide that is capable of binding covalently to DNA and causing gene mutation.
The role of polycyclic aromatic hydrocarbons (PAH) is clearly implicated in the process of carcinogenesis especially 7,12-dimethylbenz[a]anthracene (DMBA), which is one of the most potent skin and breast carcinogens known .
In the present investigation, of toxic effects of DMBA there was a significant decrease in the body weight and increase in tumor size of carcinogen treated control mice. Such weight loss and tumor size was rectified by the administration of SCE at the peri- and post- initiational stage. Topical application of DMBA considerably increased the incidence, cumulative number of papillomas, tumor yield and tumor burden whereas it decreased average latent period in animals of carcinogen treated control (Group III) because DMBA, after metabolic activation, has been found to induce cancer through an oxidatively mediated genotoxicity by incorporating diolepoxide and other ROS into DNA [18,19].
Oxidative damage to cellular macromolecules may occur through the overproduction of ROS and faulty antioxidants and/or DNA repair mechanisms that result in cancer . Several transcriptional factors such as nuclear factor kappa B (NFκB) and AP-1 are induced by ROS and are known to have a direct impact on inflammation, cellular proliferation, and apoptosis, thus making ROS instrumental in tumor promotion . Therefore, administration of antioxidants may retard this process.
Oral administration of S.cumini extarct at peri- and post-initiational stage (Group IV) significantly reduced the occurrence of skin papilloma, induced by DMBA–croton oil after 16 weeks of experimental period. Moreover, during the entire experimental period, the cumulative number of papilloma, tumor yield and tumor burden were found to be considerably decreased in Group IV than the carcinogen treated control (Group III).
The inhibition in tumor genesis and reduction in oxidative stress during present experimentation might be because of the presence of several phytochemicals in this plant extract such as acetyl oleanolic acid, triterpenoids, ellagic acid, isoquercitin, quercetin, kaempferol and myricetin [22,23]. Most of these compounds have been reported to possess antioxidant and free radical scavenging activities .
These results suggests an anti-tumorigenic potential of SCE against DMBA–croton oil induced skin carcinogenesis, which might be due to the phyto-constituents (i.e., polyphenols, flavanoids (quercetin, myrecetin), saponins and tannins) present in the seed of S.cumini. Myrecetin inhibits the phorbol ester-induced upregulation of COX-2 protein . As reported earlier that chronic inflammation, predisposes to malignancy  and curumin exerts it’s both anti-inflammatory and chemopreventive effects by inhibiting the phorbol ester-induced expression of COX-2 protein . Quercetin is one of the most abundant dietary flavonoids that arrest the cell cycle at the point where damage occurs and thereby inhibits the initiation of carcinogenesis which clearly reflects in the present study.
These findings were supported by several other finding also. Several studies have indicated that Chinese medicinal plants contain a wide variety of natural antioxidants such as phenolic acids, flavonoids and tannins, which possess more potent antioxidant activity than common dietary plants [28,29]. In recent years, a group of plants under the ‘heat-clearing’ category according to the classification of Chinese medicinal plants have attracted much attention because they have demonstrated significant activities in anti-inflammatory, anti-tumor, anti-allergic, anti-viral and antibacterial assays [30,31].
Histopathological investigations showed a normal architecture of skin in vehicle treated (Group I) and SCE treated control (Group II). DNA is a target molecule of DMBA, and metabolically activated DMBA can bind DNA to form adducts , cause mutations , and then induce tumor formation . Transversion from A to T on codon 61 of the H-ras oncogene is a highly consistent finding in DMBA/ TPA-induced mouse skin papilloma .
Skin of mice of carcinogen treated control (Group III) showed severe epidermal hyperplasia, dermal invasion and hyperkeratosis and tumor histology exhibited well differentiated squamous cell papilloma with hyper chromatic nuclei, reduced stroma with large number of lymphocytes. On the other hand, poorly differentiated papillomas and the extent of all these lesions were observed to be of relatively lesser in the tumors and skin of mice that received SCE treatment at the peri- and post- initiational stage (Group IV).
Increase in epidermal proliferation can lead to the establishment and development of a mutated clone which ultimately can result in papillomas. Cell death is a part of normal physiology for most metazoan species. During development, unwanted cells are removed through programmed cell death, making important contributions to morphogenesis, organogenesis, and other processes.  Defects in the cell death machinery, which prevents the programmed turnover of cells, can increase undesired cell accumulation, genetic instability, enhance cell longevity and permit cells to survive in a suspended state. Thus, defective apoptosis may indirectly promote cancer. Reduction in all the DMBA induced lesions in Group IV by S.cumini seed extract might be because of inhibition of cell proliferation and induction of apoptosis at the target site which is initiated by DMBA.
The formation of malondialdehyde is considered as an index of lipid peroxidation that causes cell injury. Elevation of lipid peroxides, as indicated by increased MDA and decrease in total proteins were observed in liver and skin of tumor bearing animals of carcinogen treated control (Group III). DMBA induces critical oxidative damage in the liver in vivo [37,38]. Significant increase in LPO in carcinogenic process may be due to abnormal levels of reactive oxygen species (ROS). ROS production in excess of cellular antioxidant capacity, may result in damage to lipids, proteins, RNA and DNA or other effects .
A significant decrease in the activity of antioxidants i.e., SOD, CAT and vitamin-C were documented in liver and skin of carcinogen treated control mice when compared to vehicle treated control. These results are in collaboration with the findings of others [40,41].
SOD acts as an anti-carcinogen inhibitor during initiation and promotion/transformation stages of carcinogenesis . Superoxide radicals may be reduced by the enzyme superoxide dismutase to form H2O2 and oxygen. Catalase is an enzyme which converts H2O2 to neutral products like O2 and H2O2. The antioxidant effect of SCE extract was observed by significant increase in the antioxidant activity of SOD, CAT and vitamin-c and by significant decrease in lipid peroxidation in liver and skin of animals of Group IV when compared to carcinogen treated control (Group III).
The antioxidant activity of Eugenia jambolana could be attributed due to the presence of phenolic compounds as they are believed to be the major phytochemicals responsible for antioxidant activity of plant materials [43,44]. Phenolic compounds such as flavonoids, phenolic acid and tannins possess diverse biological activities such as anti-inflammatory and anti-atherosclerotic activities, which might be related to their antioxidant potential .
A significant decreased level of vitamin C was observed in liver and skin of carcinogen treated control. This decreased level of vitamin C may be due to increased utilization in trapping the oxyradicals which were generated by DMBA. Vitamin C is a key antioxidant which particularly protects lipids from per-oxidative damage by aqueous solution, thereby blocking the initiation of carcinogenesis . S.cumini extract treatment boost up the level of vitamin-c in liver as well as in skin of SCE treated animals (Group IV), which might be helpful in neutralizing the DMBA induced free radical and protect the cells from oxidative damage. Sharma et al. have proposed that several medicinal plants and their constituents probably exert their chemopreventive effect, by scavenging reactive oxygen species and improving the antioxidant defence systems.
From the present study, it can be inferred that Syzygium cumini possesses anti-carcinogenic and anti-oxidative potential against chemical induced carcinogenesis in mammals. Such chemopreventive activity may be linked with the antioxidant/free radical-scavenging constituents present in the extract.