Abstract
The present study attempted to explore the efficacy of curcumin and resveratrol in modulating mitotic catastrophe and apoptosis during lung carcinogenesis. The mice were segregated into five groups, which included normal control, benzo(a)pyrene (BP)-treated, BP + curcumin (C)-treated, BP + resveratrol (R)-treated and BP + C + R-treated groups. The BP treatment resulted in a significant increase in the formation of micronuclei as well as in the protein expression of bcl-2 in the lungs of mice. On the other hand, a significant decrease was observed in the number of apoptotic cells and protein expression of bax in the lungs of BP-treated mice. Supplementation of curcumin and resveratrol individually to BP-treated animals resulted in a decrease in the micronuclei formation; however, it was not statistically significant. Interestingly, combination of curcumin and resveratrol resulted in a statistically significant decrease in micronuclei formation. Moreover, phytochemicals in combination significantly reduced the protein expression of bcl-2 in BP-treated mice. Furthermore, supplementation of phytochemicals in combination brought a noticeable improvement in the number of apoptotic cells as well as in the protein expression of bax. The present study, therefore, concludes that the combined treatment with curcumin and resveratrol modulates mitotic catastrophe by stimulating apoptosis in BP-treated mice.
Introduction
Lung cancer is a major cause of mortality throughout the world and is responsible for the deaths of more than one million people annually.1 –3 According to the estimates, 90% of lung cancers are caused by cigarette smoking. 4 Chemoprevention of cancer is an emerging approach, which involves adequate intake of dietary constituents including phytochemicals to reverse or inhibit the process of carcinogenesis.5,6
Among various chemopreventive agents, phytochemicals have shown great potential in preventing the occurrence of cancer and other chronic diseases that result from oxidative stress induced by free radicals. 7 In the present study, curcumin and resveratrol are the phytochemicals of interest. Curcumin, a polyphenol, has been reported to have antiinvasive property. 8 Furthermore, curcumin has been shown to activate apoptotic machinery in patients with lung cancer. 9 On the other hand, resveratrol (trans-3,4′,5-trihydroxystilbene) is a phytoalexin that is present naturally in grapes as well as in a variety of medicinal plants and has been shown to exhibit antioxidant activity with a potential to induce apoptosis.10,11
Although the phytochemicals present in food and vegetables possess potent and desirable biological activities against cancer, the information is still lacking with regard to combined chemopreventive efficacy of curcumin and resveratrol by delaying the onset of carcinogenesis. So, the present study is focused to evaluate the efficacy of combined chemoprevention approach using curcumin and resveratrol in modulating mitotic catastrophe during lung carcinogenesis. Mitotic catastrophe is a condition of abnormal mitosis that leads to the formation of micronuclei during the process of carcinogenesis, when cell is unable to induce apoptosis. 12 ,13 So, the present study is an attempt to explore the synergistic prophylactic action of these two chemopreventive agents in modulating mitotic catastrophe and key regulatory apoptotic genes during lung carcinogenesis.
Material and methods
Animals
Male Laka mice weighing 18–20 g were procured from the central animal house, Panjab University, Chandigarh, India. The animals were housed in the polypropylene cages under hygienic conditions in the departmental animal house by strictly following the guidelines as outlined by institutional ethical committee.
Chemicals
All the chemicals used in this study were of analytical grade. Benzo(a)pyrene (BP), resveratrol and curcumin were procured from Sigma Aldrich company (USA). Antibodies were procured from Biovision (USA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was procured from Trevigen, USA.
Experimental design
Animals in group I served as normal controls and were also administered with corn oil intraperitoneally (IP), which were used as the vehicle for the BP-treated animals. Animals in group II were given a single IP injection of BP in corn oil at a dose of 100 mg/kg body weight. 14 Group III animals were given curcumin orally in water (using intubation gavage) at a dose of 60 mg/kg body weight, thrice a week. 15 Animals in group IV were given resveratrol orally in water (using intubation gavage) at a dose of 5.7 μg/ml (25 μM), thrice a week. 16 Animals in group V were given a combined treatment of curcumin and resveratrol in a similar manner as was given to groups III and IV animals, respectively. The animals were subjected to treatment with phytochemicals, 10 days prior to single BP injection and continued for a total duration of 22 weeks. All the animals had free access to the diet and water.
Micronucleus assay
The micronucleus assay was carried out according to the method described by Schmid in 1975. 17 The tissue samples were minced and suspended at the rate of 1 mg/ml and homogenized at 1500 r/min. The homogenates were then cold centrifuged at 7000 r/min for 10 min. The supernatants were discarded and fresh homogenizing buffer was poured again for resuspension. A small drop of prepared suspension was put at one end of a precleaned, grease free microscopic slide and was spread using cover slip held at an angle of 45° into a smooth layer. The slides were then air dried in a dust free environment for at least 12 h before staining. The slides were then stained with May Graunwald (MG) stain for 1–2 min followed by staining with Geimsa for 10 min. The slides were then rinsed twice in distilled water, dried and then rinsed in methanol. The slides were then cleaned in xylene, mounted with distyrene plasticizer xylene (DPX) and observed under microscope. Cells were counted for the presence of micronuclei using light microscope at ×40.
TUNEL assay
The assay was carried out according to the instructions given in the Trievigen (USA) TACS-XL-blue label in situ apoptosis detection kit. Hydrated, fixed and immobile sample slides were immersed in phosphate buffer saline (PBS) for 10 min. The slides were covered with 50 μl of protein kinase K solution for 30 min, which was followed by 2 washings for 2 min each with deionized water. The next step involved immersion of slides into quenching solution for 5 min, followed by a wash with PBS for 1 min. The slides were then immersed in terminal deoxynucleotidyl transferase (TdT)-labeling buffer. Slides were then covered by 50 μl of TdT-labeling reaction mixture and were incubated at 37°C for 30 min. The labeling reaction was stopped by putting the slides in TdT stop buffer for 5 min, followed by 2 washings of 2 min each with PBS. Slides were then covered again by 50 μl of biotinylated anti-5-bromo-2-Deoxyuridine (BrdU) antibody and were incubated at 37°C for 30 min. This was then followed by counterstaining with nuclear fast red stain. Finally, slides were then mounted with glass coverslips using mounting solution. The cells stained blue were apoptotic positive cells and were counted for apoptosis analyses.
Immunohistochemical analyses
Seven-micrometer-thick paraffin sections of mice lungs were incubated at 60°C for 30 min and deparaffinized in xylene for 2 h. The sections were then gradually hydrated in descending series of alcohol (100%, 90%, 70%, 50% and 30%). Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 30 min at 37°C. The nonspecific binding was blocked by incubating the sections with 2% bovine serum albumin in PBS. The sections were then incubated with polyclonal antibodies, anti-bcl-2 (1:100) and anti-bax (1:100), separately for 2 h at 37°C. After incubation with primary antibodies, the sections were washed with PBS, PBS-Tween and PBS successively for 5 min each. The sections were then incubated with peroxidase labeled anti-rabbit IgG (1:100) for 2 h at room temperature. Sections were washed again similarly as described above and the reaction product was developed using diaminobenzidine and H2O2 under dark conditions. Reaction was stopped by washing with distilled water, and the sections were mounted with DPX. The slides were then analyzed under microscope. Immunohistochemistry (IHC) slides were analyzed quantitatively by using single channel color analyses using adobe photoshop 7.
Statistical analysis
The statistical significance of the data has been determined using one-way analysis of variance (ANOVA) followed a multiple post hoc least significant difference (LSD) test. The results are represented as means ± SD.
Results
The results of all the experiments conducted during the current study are depicted in various tables and figures. All the results of various treatment groups have been compared with their corresponding normal controls. Furthermore, results obtained from BP + curcumin (C), BP + resveratrol (R) and BP + C + R-treated groups were compared with BP-treated group and the results of BP + C and BP + R-treated groups were also compared with BP + C + R combined treatment group. The statistical significance of the values has been determined using ANOVA, followed by least significant test and the results are represented as mean ± SD.
Micronucleus assay
Micronucleus assay showed induction of mitotic catastrophe as observed by increase in the frequency of micronuclei (Table 1; Figure 1) in the lungs of BP-treated mice. Curcumin and resveratrol individual treatments did not bring statistically significant change. However, curcumin and resveratrol in combination significantly moderated mitotic catastrophe in the BP-treated mice as evidenced by an appreciable decrease in the number of micronuclei.
(a) showing normal cells; (b) showing a cell with mircronuclei.
Effects of curcumin and resveratrol on micronuclei formation in mice subjected to benzo(a)pyrene treatment
Data are expressed in mean ± SD.
a p < 0.05 and c p < 0.001 by least significance difference test when values are compared with normal control group.
x p < 0.05 by least significance difference test when values of groups III, IV and V are compared with group II.
TUNEL assay
TUNEL assay showed a significant decline in the apoptosis as observed (Figure 2; Table 2) by a significant decrease in the number of apoptotic cells (stained blue) in the lungs of BP-treated mice. Treatment with curcumin as well as resveratrol significantly induced apoptosis in the BP-treated mice as evidenced by an appreciable increase in apoptotic cells. Furthermore, induction of apoptosis was significantly higher (80.7% approx.) in the combined treatment group as compared to individual treatment groups of curcumin (70% approx.) and resveratrol (76% approx.).
TUNEL assay in Control (c), Benzo(a)pyrene treated (BP), Benzo(a)pyrene + Curcumin treated(BP+C), Benzo(a)pyrene + Resvertrol treated(BP+R) and Benzo(a)pyrene + Curcumin + Resveratrol treated(BP+C+R) animals.
Effects of curcumin and resveratrol on the number of apoptotic cells in mice subjected to benzo(a)pyrene treatment
Data are expressed in mean ± SD.
a p < 0.05, and c p < 0.001 by least significance difference test when values are compared with normal control group.
z p < 0.001 by least significance difference test when values of groups III, IV and V are compared with group II.
Bcl-2 and bax IHC analyses
There was a significant increase in the protein expression of bcl-2 (Figure 3) in the lungs of mice treated with BP. On the other hand, a significant decrease in the protein expression of bax (Figure 4) was observed in the lungs of mice treated with BP. Supplementation with phytochemicals resulted in a significant decrease in the expression of bcl-2. However, decrease in the expression of bcl-2 was much more in the combined phytochemicals treatment group. Furthermore, statistically significant improvement in the protein expressions of bax was observed in combined treatment group, which was better as compared to single phytochemical supplemented groups.
Protein expression of Bcl2 by immunohistochemical (IHC) analyses in Control (c), Benzo(a)pyrene treated (BP), Benzo(a)pyrene + Curcumin treated(BP+C), Benzo(a)pyrene + Resvertrol treated(BP+R) and Benzo(a)pyrene +Curcumin+Resveratrol treated(BP+C+R) animals.
Protein expression of Bax by immunohistochemical (IHC) analyses in Control (c), Benzo(a)pyrene treated (BP), Benzo(a)pyrene + Curcumin treated(BP+C), Benzo(a)pyrene + Resvertrol treated(BP+R) and Benzo(a)pyrene + Curcumin+Resveratrol treated(BP+C+R) animals.
Discussion
The present study focused on the evaluation of combined prophylactic chemopreventive potential of phytochemicals, namely, curcumin and resveratrol against BP-induced lung carcinogenesis in the mice. The reason being that both these phytochemicals have been reported extensively for their chemopreventive potential against cancer18,19; however, information with regard to synergistic supplementation of curcumin and resveratrol in relation to lung cancer is not available. So, the present study explored the efficacy of combined supplementation of curcumin and resveratrol in modulating BP-initiated genotoxicity in mice. The study clearly demonstrates the synergistic potential of curcumin and resveratrol in modulating mitotic catastrophe and key apoptosis regulatory genes. The TUNEL assay showed a significant decrease in apoptotic cells, which suggests decline in the active tumor suppressor proteins in the lungs of BP-treated mice. Moreover, the protein expression of bcl-2 and bax also showed a significant increase and a significant decrease, respectively, in the lungs of BP-treated mice. Bcl-2 is an antiapoptotic protein, 20 so increase in its expression indicated decline in apoptosis. On the other hand, bax is an apoptosis inducing protein 21 ; therefore, observed decrease in its expression also contributed toward decline in apoptosis in BP-treated mice. Low bcl-2-to-bax ratio is a key factor that is responsible for the induction of apoptosis; however, we have observed an opposite high ratio of bcl-2 to bax in the BP-treated mice, which must have contributed toward decline in apoptosis. Phytochemicals treatments individually and in combination were successful in inducing apoptosis in BP-treated mice by moderating the protein expression of bcl-2 and improving the protein expression of bax, respectively. However, the improvement was much more in the combined treatment group. The logical reason appears to be that the phytochemicals contribute toward the maintenance of genome stability, 22 induction of apoptosis 23 and chromatin remodeling encompassing events.
Genotoxic effects of BP were evaluated by studying the formation of micronuclei in the lung cells. BP treatment resulted in mitotic catastrophe which is basically a significant increase in the formation of micronuclei in lung cells. Mitotic catastrophe is a condition that is opted by cell to counter rapidly proliferating cells when apoptosis is withheld due to altered expressions of key apoptosis regulatory genes like bcl-2 and bax.22,23 Mitotic catastrophe results in the formation of micronuclei due to abnormal mitosis, which was observed in our present study in the BP-treated mice. Mitotic catastrophe moderated significantly upon supplementation with phytochemicals in combination, which could be due to phytochemicals in combination induced appreciable increase in the apoptosis by effective maintenance of low bcl-2-to-bax ratio in BP-treated mice. In conclusion, the present study demonstrates that synergistic supplementation of phytochemicals can effectively induce apoptosis by maintaining low bcl-2-to-bax ratio in rapidly proliferating cells and thus significantly moderates mitotic catastrophe in order to contribute toward chemoprevention of lung carcinogenesis.
Footnotes
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.