CHEMOPREVENTION OF LUNG CARCINOGENESIS IN STRAIN BALB/C MICE WITH POLYPHENOLS OF MAHKOTA DEWA ( Phaleria macrocarpa ) FROM INDONESIAN HERBAL.

Backgroud and objective: Polyphenols of Mahkota Dewa ( Phaleria macrocarpa ) has potential as anticancer that is expected to inhibit lung carcinogenesis, but it has not been widely studied. This study aims to prove the role of Mahkota Dewa polyphenols to inhibit the progression of lung carcinogenesis development in mice. Methods: The design used in this study is posttest serial control group design with 40 strain Balb/c mice as samples, aged 1-2 weeks, weighted 20-30 g, and in healthy condition. All mice were induced using B(a)P then were randomized into 2 groups: the control group and the 50 mg polyphenols treatment group. The development of lung tumor was observed with tissue surgery at week-8, -17, and -26. The examination of lung carcinogenesis incidence was done using H&E staining. Results: The data were analyzed using Kruskal-Wallis, Mann-Whitney, with significance level of p<α (0.05). Oral administration of Mahkota Dewa’s polyphenols significantly showed the inhibition of lung carcinogenesis incidence in the treatment group at week-8, -17, and -26 (p=0.000). The incidence of lung carcinogenesis in the control group at week-8, -17, and -26 were 2.32±0.26 and 3.93±0.46; while in the treatment group were 1.88±0.38 and 0.88±0.22 (p=0,000). Conclusion: The administration of Mahkota Dewa’s polyphenols was effective to inhibit lung carcinogenesis in Balb/c strain mice. study using B(a)P as carcinogen, capable of causing the development of hyperplasia in strain A/J mice at week-3, adenoma appeared at week-12, and carcinoma appeared at week-26 post-administration of B(a)P. While research conducted by Yang Y, et al (2007) showed that the lung carcinogenesis in strain A/J mice in the control group developed until at week-18 before it was terminated. hyperplasia the there was an of hyperplasia to developed into after Mahkota Dewa’s polyphenols. The results of histopathological examination using H&E staining method, showed visible changes in the bronchiole epithelial. These changes included proliferation of bronchiole epithelial, hyperchromatic nucleus, enlarged nucleus, and cytoplasm becomes narrower and there was found an image of mitosis. were found changes in the cells such change the chromatin becomes more dense and darker (hyperchromatic), abnormal image of mitosis, narrower cytoplasm, enlarged nucleus, and anaplasia cell form becomes larger and irregular).


ISSN: 2320-5407
Int. J. Adv. Res. 6(5), 580-590 581 Polyphenols of Mahkota Dewa is expected as potential chemopreventive to disrupt or reversing the carcinogenesis process by acting on intracellular signalling of molecular tissue that is involved in iniation and/or promotion stage, and also can stop or reversing the developmental stage of cancer (Manson M, 2003;Surh YJ, 2003). The use, effectivity, and the utilization of Mahkota Dewa had been demonstrated as do not have any side effects, both empirically and in pre-clinical studies (Soeksmanto A, 2006 a,b ). Until recently, polyphenols of Mahkota Dewa has never been reported as to its inhibition of lung carcinogenesis in experimental animals through the process of cell proliferation and apoptosis that are considered as targets in the prevention of carcinogenesis; so it is necessary to do further research in-vivo.
Various researchs had began to studied about the possible involvement of molecular mechanisms such as cell cycle, cell proliferation, regulation of apoptosis, and tumor suppressor genes in the pathogenesis and progession of lung cancer (Esposito et al, 2002;, 2004dan Caputi M, et al, 2005. The disruption of homeostatic which regulate cell proliferation and cell death (apoptosis) may contribute to the development and growth rate of tumor. This suggests that the ability to induce apoptosis and inhibit proliferation are considered as the primary factor in determining the success of chemopreventive agents (Benerjee S, et al, 2005).
Chemotherapy and radiotherapy programs are the main therapy for most patients with lung cancer, but the expected results with such therapy have not been helpful and the result itself is still limited because most patients often experiencing a relapse (Zochbauer-Muller S, et al, 2002;Benerjee S, et al, 2005). The lots of failure in chemotherapy and radiotherapy of lung cancer usually associated with the failure of anticancer agents in inducing the programmed cell death (apoptosis) of cancer (Fisher DE, 2004). In addition to chemotherapy and radiotherapy, the management of lung cancer can be done with chemopreventive measures (prevention). The aim of the chemopreventive measures are to intervent the cancer by administering natural ingredients that can inhibit, delaying, and blocking or reversing the carcinogenesis process, which can be used as protection from carcinogenic exposure and reduce the risk of lung cancer (Benerjee S, et al, 2006b).

Methods:-
Research Samples:-A total of 40 of strain Balb/c mice aged 1-2 weeks were adapted for a week in laboratorium of experimental animals, which the animals were caged in a sterile manner with adequate lighting.

Induction with B(a)P:-
The lung cancer was induced according to the carcinogen test conducted by Murwanti R, et al, 2004; Benerjee S, et al, 2006 a,b . The first induction was done on day-1 with a dose of 0.2 mL/0.025 cc. The second induction was done on day-8 with a dose of 0.4 mL/0.025 cc and the third induction was done on day-15 with a dose of 0.8 mL/0.025 cc. Each young mice got (received) an induced lung tumor subcutaneously in the sub-scapular region with 0.1 mL of B(a)P suspension with a concentration of 0.2 mg which was already diluted into DMSO (single dose). Carcinogen was used within 1 hour after emulsification. Once injected, the young mice were allowed to live with their mother, were given water and food ad libitum during the weaning process. DMSO was not toxic so it was safe to used as diluent solution and was diluted with B(a)P (inductor).

Treatment in Strain Balb/c Mice:-
As much as 8 mice were sacrificed to determine the growth of tumor cells in a row at week-4 and week-5 to observed the carcinogenesis development into hyperplasia and dysplasia stadium in lung epithelial cells of mice.
Entering week-5, the strain Balb/c mice that were already into hyperplasia stadium were divided into 2 groups by random allocation and were treated as follows: -Group C : a total of 15 mice in the control group, untreated with polyphenols of Mahkota Dewa until week-26. -Group X : a total of 15 mice in the treatment group were administered orally with polyphenols of Mahkota Dewa at a dose of 50 mg/kg/mice/day from week-5 and continued until week-26.
The mice from 2 groups (control and treatment) were terminated at week-8, -17, and -26 to study the development of stadium with different time intervals. All of lung organs were excised and washed with 0.9% NaCl solution, were dried and fixated with 10% of formaldehyde buffer for 24 hours, and then the paraffin blocks were ready to made.
As much as 5 lung lobes were cut with each slide has 4 microns sized with 4 pieces of tissues, then the observations were made.

H&E Staining:-
The next process included the processing of histological preparation through the stages of fixation, dehydration, impregnation, and embedding. The tissues of 2 groups that had been made into paraffin blocks were cut to 4 microns thick and then were stained using Hematoxylin Eosin (H&E) to calculate the incidence of hyperplasia and dysplasia in the bronchiole epithelium of lung tissue with light microscope using 400X magnification.
After being stained with H&E, the slides were observed for hyperplasia and dysplasia incidence, also the inhibition in bronchiole epithelial at week-8, -17, and -26 both in the control group and treatment group. The calculation of changes in hyperplasia and dysplasia also the inhibition were in accordance with calculation method by Benerjee S, et al (2004) i.e. % inhibition= (1-T/C) x 100.

Observations to Hyperplasia-Dysplasia:-
The number of bronchial epithelial undergo hyperplasia and dysplasia which is observed with H & E staining. Excessive cell growth which occurs does not indicate malignancy which showed hyperplasia. On epithelium, it is shown that cell core grows more, but the cell shape still looks the same. Dysplasia Development showed growth abnormalities with the changes in the structure and morphology of the cells. The size of cell core is large/small, cell shape is varied, the cell is not uniform, the color is darker, the structure of the cell core is irregular and the ratio of cell core and cytoplasm are rising.

Data Analysis Technique:-
Incidence data of lung carcinogenesis is a ratio scale. The data were descriptively analyzed and were presented in the form of mean, median, mode, and standard deviation; then considered to have normal distribution or not by assessing the histograms and boxplot. Before the data were analyzed, the normality test and homogeneity test were performed first. Data analysis included non-parametric test of Kruskall-Wallis then followed with Mann-Whitney test to determine the incidences of lung cancer, with significant level of p<0.05 and confidence interval of 95%. The data obtained were statistically analyzed using SPSS ver.19.

Results and Discussion:-
Result:-The incidence of hyperplasia and dysplasia:-After the mice were overall terminated at week-26, the lung organs ( Figure 1) were isolated and were made into paraffin blocks. H&E staining were done to observed the hyperplasia and dysplasia of bronchiole epithelial. Figure  1A is the result of this study. The inhibition and incidence of hyperplasia and dysplasia in bronchiole epithelial at week-8, -17, and -26 both in the control group and the treatment group can be seen in Table 1 below.   Table 1 shows the protective effects of Mahkota Dewa polyphenols throughout the lung carcinogenesis that was induced by B(a)P. In this study the hyperplasia incidence were 58% in the control group and then fell to 47% in the treatment group, so the inhibition percentage by polyphenols of Mahkota Dewa was 18.96%. At week-17 it was 68% in the group exposed by B(a)P, 31% in the treatment group, thus the inhibition percentage recorded was 54.41% after administration of Mahkota Dewa polyphenols. At week-26 it was 96% in the control group, and there was a decrease in the hyperplasia incidence to 21% in the treatment group, which showed 78.12% after the given treatment.
Based on this study results, the histopathological changes in the experimental animals may occur but the changes occur until the development of dysplasia in the control group. While in the treatment group, the exposure of Mahkota Dewa polyphenols could delay the formation and development of hyperplasia into dysplasia at the postinduction of carcinogens.
In the control group, the tumor incidence entered the development stage of hyperplasia and progressed into dysplasia. While in the treatment group the inhibition of tumor development occurred after exposure of Mahkota Dewa polyphenols so it did not enter into dysplasia development stage. The results of histopathological examination using H&E staining method showed visible changes in the bronchiole epithelial. That changes included the proliferation of bronchiole epithelial, hyperchromatic nucleus, enlarged nucleus, and cytoplasm becomes narrower and there was found an image of mitosis.
Based on Figure 2, at week-8 there were noticeable changes of hyperplasia in bronchiole epithelial cells in the control group, while there were no changes of hyperplasia in bronchiole epithelial cells in the treatment group. In Figure 3, at week-17 the development of hyperplasia in the control group remained the same, while the administered Mahkota Dewa polyphenols in the treatment group has affected the majority of epithelial cells so that it looks normal. At week-26, there were histopathologic changes towards dysplasia in the control group, but in the treatment group there were no changes towards dysplasia and still showed hyperplasia.
In the control group at week-8 and week-17, there were excessively cell growth in the bronchiole epithelial that showed no malignancy and showed hyperplasia, in the epithelial cells showed a lot of cell nucleus growth ( Figure  2A,B). The histopathological observation on bronchiole epithelial after 26 weeks showed dysplasia, in which there were growth abnormalities that lead to changes in the structure and morphology of the cells. The size of the cell nucleus was bigger/smaller, the cell structure had been varied and the shape was not uniform, the color looks darker and the cell nucleus had been irregularly arranged (polarization), also the ratio of cell nucleus and cytoplasm were increased and the chromatin was clumped (darker) ( Figure 2C).  These results indicate that, in the control group of carcinogens at week-8, there was an increase of lung carcinogenesis incidence, and continued to increase until week-17 and 26. While in the group that was exposed to polyphenols of Mahkota Dewa, there was a decrease of lung carcinogenesis incidence at week-8, -17, and -26. Statistical test using Kruskall-Wallis test showed significant improvement (p=0.000) (see appendix 12). Then, the further differences between the experimental groups were analyzed using Mann-Whitney test.

Discussion:-
To determine the role of antitumor compounds that precisely inhibit the growth of tumor cells, it would require an in-vivo study using experimental animals. Various methods of tumor inductions require considerable time with large doses of carcinogens to grow the tumor as well as the necessary of certain strains of experimental animals with high sensitivity level. Sensitivity towards inductor is affected by species, strain, age, gender, and target organs. In this study, the use of experimental animals with young age is expected to have high sensitivity towards B(a)P inductor than the old age. The use of young mice is expected to have high sensitivity when the test takes place.
This study uses young age of strain Balb/c mice with the consideration that this strain has the highest incidence of spontaneous tumor/cancer and also highly responsive to chemical induction of lung cancer ( In the control group induced by B(a)P, although the induction was discontinued at week-4, but development of lung cancer continued until week-26. This was caused by the ability of B(a)P in inducing glutathione-S-transferase (GST) enzyme, which is a phase II metabolic enzyme. Additionally, B(a)P can induce cell proliferation caused by overexpression of enzyme involved in synthesis of nucleotide and DNA, also it regulates the differentiation that causes cells to proliferate uncontrollably (Giovannini C, et al, 2007). The results of this study are supported by a research conducted by Benerjee S, et al (2005), which stated that during the lung carcinogenesis in strain A mice caused by B(a)P there were changes in cellular and histopathological cells identified as hyperplasia, dysplasia, insitu carcinoma, and invasive carcinoma. Another research by Estensen RD, et al (2004) showed the results of the

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Inhibition Hyperplasia and Dysplasia Group C = Control; X = Treatmen study using B(a)P as carcinogen, capable of causing the development of hyperplasia in strain A/J mice at week-3, adenoma appeared at week-12, and carcinoma appeared at week-26 post-administration of B(a)P. While research conducted by Yang Y, et al (2007) showed that the lung carcinogenesis in strain A/J mice in the control group developed until at week-18 before it was terminated.
The observation results based on histopathological image of lung tissue of strain Balb/c mice showed that thre were specific changes in lung cells, the bronchiole epithelial cells were seen arranged into two layers although some of the epithelial cells were still arranged into one layer. In the control group, the bronchiole epithelial cells entered the stage of hyperplasia and developed into dysplasia. While in the treatment group there was an inhibition of hyperplasia to developed into dysplasia after administration of Mahkota Dewa's polyphenols. The results of histopathological examination using H&E staining method, showed visible changes in the bronchiole epithelial.   , et al, 2008). The delayed progression on histopathological changes of hyperplasia into dysplasia, in the treatment group may be associated with the increase of stable apoptosis, will provide the important protective activity on various chemopreventive agents.
The growth of lung cancer is the same as other types of cancer through 3 phases: initiation phase, promotive phase, and progressive phase. The process of carcinogenesis need to be control through physiologic as well as pharmacologic process in the stage of preneoplastic so that the further development of promotive phase can be hold and prevented. This control can be done through preventive measures such as avoidance to various agents known to induce cancer, improve the resistance to cancer, changing lifestyles and the use of chemopreventive agents (chemoprevention). Chemoprevention involve substances that can delaying or stopping the process of carcinogenesis resulting in lower risk of cancer invasion process. By studying various pathways such as cell proliferation and apoptosis, it was to be expected the use of chemoprevention to inhibit the cell proliferation and induce apoptosis (Sun S-Y, et al, 2004). Increased or uncontrolled proliferation and impaired apoptosis play a role in determining the accumulation of malignant cells and cause the formation of multistage carcinogenesis.
Thus, it can be concluded that the polyphenols of Mahkota Dewa has anticancer activity which plays a role in the inhibition of lung carcinogenesis in experimental animals through the mechanism of lung carcinogenesis inhibition.

Conclusion:-
Based on the results of analysis and the discussion of this research, it can be concluded that polyphenols of Mahkota Dewa proved to be effective to inhibit lung carcinogenesis which is marked by the inhibition of hyperplasia changes into dysplasia in the bronchiole epithelial in the strain Balb/c mice. It is recommended to do further research on the role of proteins and enzymes that cause damage to the lung tissue of strain Balb/c mice.

Limitation:-
This research in limited onlu on patologic parameter. For other parameter like imunology parameter do not yet. For furture research adding variable in relation with in this study is needed.