Anti-cell Proliferative and Anti-angiogenic Potential of Andrographolide During 7 , 12-Dimethylbenz ( a ) anthracene Induced Hamster Buccal Pouch Carcinogenesis

Oral cancer, a highly complex multistep process that occurs when squamous epithelium is affected by multiple genetic alterations, is the fifth most common cancer worldwide and 90% of these forms of cancers are squamous cell carcinoma (SCC) (Jemal et al., 2009). While oral carcinoma comprises around 40% of total malignancies in India, in western countries it accounts for only 3-5% of all cancers. High incidence of oral cancer is attributable to the indigenous habit of chewing a mixture of tobacco, areca nut, lime, betel leaf, and spices in several combinations (Chiba, 2001). The status of tumor suppressor genes, oncogenes, cell proliferation markers, angiogenic markers and cell adhesion molecules are utilized as a potential tool to predict the prognosis of patients with SCC (Massano et al., 2006). Golden Syrian hamsters are commonly utilized as an experimental model of oral carcinogenesis due to their pocket (pouch) like anatomy in the mouth, which retains the carcinogen for a longer time (Manoharan et al., 2012; Singh et al., 2012). DMBA, a polycyclic aromatic hydrocarbon, is widely employed to induce oral carcinoma in experimental animals. DMBA induced experimental oral carcinogenesis in the hamster cheek


Introduction
Oral cancer, a highly complex multistep process that occurs when squamous epithelium is affected by multiple genetic alterations, is the fifth most common cancer worldwide and 90% of these forms of cancers are squamous cell carcinoma (SCC) (Jemal et al., 2009).While oral carcinoma comprises around 40% of total malignancies in India, in western countries it accounts for only 3-5% of all cancers.High incidence of oral cancer is attributable to the indigenous habit of chewing a mixture of tobacco, areca nut, lime, betel leaf, and spices in several combinations (Chiba, 2001).The status of tumor suppressor genes, oncogenes, cell proliferation markers, angiogenic markers and cell adhesion molecules are utilized as a potential tool to predict the prognosis of patients with SCC (Massano et al., 2006).
Golden Syrian hamsters are commonly utilized as an experimental model of oral carcinogenesis due to their pocket (pouch) like anatomy in the mouth, which retains the carcinogen for a longer time (Manoharan et al., 2012;Singh et al., 2012).DMBA, a polycyclic aromatic hydrocarbon, is widely employed to induce oral carcinoma in experimental animals.DMBA induced experimental oral carcinogenesis in the hamster cheek pouch produces premalignant and malignant changes that resemble premalignancy and malignancy of human oral mucosa (Morris, 1961).Also DMBA induced oral tumors expressed biochemical and molecular characteristics similar to that of human oral tumors (Shklar et al., 2009).
PCNA, a molecular coordinator for DNA replication, is involved in maintaining genome integrity and serve as a molecular platform to recruit proteins involved in DNA synthesis, cell cycle control and DNA damage repair (Prelich et al., 1987).PCNA, a sliding clamp for DNA polymerase delta, encircles DNA and coordinates multiple genetic functions during DNA replication and repair (McAlear et al., 1994).PCNA has been used as a molecular marker for assessing tumor progression and prognosis (Gulbis et al., 1996;Sakurai et al., 2005).Cyclin D1 is involved in promoting cell progression from G1 to S phase (Lu et al., 2009).Cyclin D1 plays crucial role in cell proliferation and differentiation (Jiao et al., 2013).Cyclin D1 overexpression mediates tumor proliferation, lymph node metastasis and prognosis (Kunisaki et al., 2004).Cyclin D1 is over expressed in a large spectrum of human cancers (Bosch et al., 1994).
Angiogenesis, the growth of new blood vessels from pre-existing ones, is a basic requirement for sustained tumor growth (Lingen, 1999).Angiogenesis is essential to supply the nutrients and oxygen, which are necessary for tumor growth and metastasis (Pratheeshkumar et al., 2012).Under cancerous conditions, an imbalance occurs between pro-angiogenic and anti-angiogenic factors that favor the promotion of tumor angiogenesis.

Golden syrian hamsters
Male golden Syrian hamsters purchased from National Institute of Nutrition, Hyderabad were maintained in the Central Animal House of Annamalai University.The hamsters were housed in polypropylene cages and were maintained under temperature (27±2°C) and humidity (55±5%) with a 12h light/dark cycle.The experimental work was approved by Institutional Animal Ethics Committee (Reg.No 160/1999/CPCSEA).

Experimental design
A total number of 40 hamsters were divided into four categories of 10 hamsters in each.Group 1 hamsters were treated with liquid paraffin alone three times a week for 14 weeks on their left buccal pouches.Hamsters in groups 2 and 3 were treated with 0.5% DMBA in liquid paraffin three times per week for 14 weeks.Hamsters in group 2 received no other treatment.Group 3 hamsters were orally administered with andrographolide (50 mg/ kg bw) three times per week on days alternate to DMBA application, starting 1 week before the exposure to DMBA and continued until one week after the final exposure of the DMBA.Group 4 hamsters were orally administered with andrographolide alone throughout the experimental period.All hamsters were sacrificed by cervical dislocation at the end of experimental period.

Immunoexpression of PCNA and VEGF
The antigen retrieved tissue sections were incubated with respective primary antibodies (PCNA and VEGF) and subsequently treated with secondary antibodies conjugated with horseradish peroxidase.The antigen-antibody complex was then detected using 3,3'-diaminobenzidine.The percentage of positive cells was scored after the slides were counter stained with hematoxylin.

Expression of cyclin D1 using real time PCR
Isolated RNA from the buccal mucosa was reverse transcribed to cDNA with random primers (Table 1) using

Statistical analysis
The data for Cyclin D1 is expressed as mean ± standard deviation (SD).Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT).The statistical evaluation for the score of positively stained cells of PCNA and VEGF was analyzed using Chi-square (χ 2 ) test.The results were considered statistically significant if the p values were 0.05 or less than 0.05.

Results
I m m u n o h i s t o c h e m i c a l a n a l y s i s r e v e a l e d overexpression of PCNA and VEGF in hamsters treated with DMBA (Figures 1 and 2).Oral administration of andrographolide to hamsters treated with DMBA prevented the abnormal expression of PCNA and VEGF during DMBA induced oral carcinogenesis.The score of positively stained cells for PCNA and VEGF expression in control and experimental hamsters are also given in (Table 2).
The primer melting curve and the fold increase in the Cyclin D1 mRNA expression pattern of control and experimental hamsters in each group is shown in Figures 3 and 4

Discussion
Deregulation of cell proliferation and angiogenesis are two important scenarios occurring in oral carcinogenesis (Johnstone et al., 2006).It has been reported that analysis of PCNA protein expression could play an important role in the prediction of long term survival and prognosis of patients.PCNA has been reported as the ring master of the genome due to active participation in several molecular pathways responsible for the survival and death of mammalian cells (Paunesku et al., 2001).Overexpression of PCNA has been reported in several cancers including oral cancer (Manojprabhakar et al., 2012).Our study supports these finding.
Tumour specific alterations noted in the cyclin D1 gene product indicate the importance of cyclin D1 as a driver of the neoplastic process (Gautschi et al., 2007).Cyclin D1 is also involved in the regulation of apoptosis and it may act as pro-apoptotic or anti-apoptotic factor depending on the proliferative and differentiated state of the cell (Han et al., 1999).Profound studies reported that transcriptional up regulation of endogenous cyclin D1 inhibited apoptotic machinery in human carcinoma cells (Fu et al., 2004;Sun et al., 2012).It has been reported that cyclin D1 amplification increased VEGF production and decreased Fas expression in esophageal tumor cells (Tashiro et al., 2007).Over expression of cyclins is one of the common phenomenons of oral cancer (Miyamoto et al., 2003).Overexpression of cyclin D1 was reported in 30-35% of oral carcinogenesis (Sauter et al., 1999;Silvan et al., 2013).
VEGF is a multifunctional cytokine and is a major angiogenic factor whose biological activity is primarily associated with endothelial cells (Bancroft et al., 2001).VEGF, endothelial specific mitogen, determines the fate of an endothelial cell for the angiogenic process.VEGF regulates the proliferation, migration, differentiation of endothelial cells (Li et al., 2009).Investigation of angiogenic inhibitors could thus serve as a new clinical class of drugs for the treatment of cancers.Although angiogenesis is a common phenomenon in physiological conditions, it is also crucial in pathological conditions such as tumor progression and metastasis.Extensive studies reported overexpression of VEGF in oral carcinogenesis (Silvan et al., 2013).Our results are in line with these findings.
Manoharan et al. ( 2012) demonstrated the anti-tumor initiating potential of andrographolide in DMBA induced hamster buccal pouch carcinogenesis.This antitumor initiating potential is probably due to its antioxidant potential as well as modulating effect on xenobiotic metabolizing enzymes during DMBA induced oral carcinogenesis.It has been reported that andrographolide has the potential to protect cell surface abnormalities during DMBA induced hamster buccal pouch carcinogenesis this is probably due to its inhibitory effect on enzymes involved in the glycosylation, silalylation and fucosylation process (Singh et al., 2012).Shanmugam et al. (2012) reported the proapoptotic and anti-inflammatory potential of andrographolide during DMBA induced hamster buccal pouch carcinogenesis.They suggested that the anti-tumor  The percentage positive cells were scored as: 3 + = strong staining, more than 50% of cells were stained, 2 + =moderate staining, between 20 and 50% of cells were stained 1 + = week staining, between 1 and 20% of cells were stained, 0 = negative, less than 1% of cell staining effect of andrographolide could partly be attributed to its apoptotic and anti-inflammatory potential during DMBA induced hamster buccal pouch carcinogenesis.Also, previous studies demonstrated the apoptotic and anti-inflammatory potential of andrographolide in various cancer cells (Cheung et al., 2005;Lee et al., 2011;Pratheeshkumar et al., 2012).It has been demonstrated that andrographolide suppressed VEGF expression in prostate cancer and pulmonary tumors (Zhao et al., 2008).It has been reported that the anti-angiogenic role of VEGF by arresting the cell cycle could play a role in the prevention of pulmonary tumor and its metastasis (Tung et al., 2013).Andrographolide down regulated the expression of cyclin D1 in prostrate cancer (Wang et al., 2011).Also, andrographolide down regulated the expression of PCNA in human skin carcinoma A431 cells (Jing et al., 2013).
In the present study, oral administration of andrographolide at a dose of 50 mg/kg bw down regulated the expression of PCNA, cyclin D1 and VEGF during DMBA induced hamster buccal pouch carcinogenesis.The results of the present study thus revealed the anti-cell proliferative and anti-angiogenic potential of andrographolide during oral carcinogenesis.To conclude, andrographolide could be used as a potent drug to suppress abnormal cell proliferation and angiogenesis occurring in oral carcinogenesis along with the current chemotherapeutic drugs.DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.6001 Andrographolide Protection against 7,12-DMBA-induced Hamster Buccal

Figure 1 .
Figure 1.Immunoexpression Pattern of PCNA Protein in the Buccal Mucosa of Control and Experimental Hamsters in Each Group (405).A and D-Control and Andrographolide alone (expression not detectable); B-DMBA alone (over expressed); C-DMBA+Andrographolide (down regulated respectively.Overexpression expression of cyclin D1 mRNA was noticed in hamsters treated with DMBA alone.Oral administration of andrographolide to hamsters treated with DMBA suppressed the expression of Cyclin D1.Similar Cyclin D1 mRNA expression pattern was noticed in control hamsters and hamsters treated with andrographolide alone.DOI:http://dx.doi.org/10.7314/APJCP.2013.14.10.6001Andrographolide Protection against 7,12-DMBA-induced Hamster Buccal Pouch Carcinogenesis

Figure 3 .Figure 4 .
Figure 3. mRNA Expression Pattern of Cyclin D1 in the Buccal Mucosa of Control and Experimental Hamsters in Each Group