THE EFFECT ADMINISTRATION OF PHALERIA MACROCARPA TO CELL PROLIFERATION (AgNOR) AS ADJUVAN OF CHEMOTHERAPY IN COLON

CANCER

Soesilo YR*, Budijitno S**

*) General Surgery Resident of Diponegoro University / RSUP Dr. Kariadi, Semarang**) Staff on Oncology Surgery of Diponegoro University / RSUP Dr. Kariadi, Semarang

Abstract

IntroductionColon cancer cause high mortality both in the world and in Indonesia. Colon cancer treatment modality is currently developed in the form of immunotherapy such as P macrocarpa (Mahkota Dewa) as a companion Surgery. Researchers try to determine the effects of P macrocarpa against colon cancer, especially in terms of cell proliferation.

MethodLaboratory experimental research with post test only group design. Research using Sprague dawley rats strain were divided into 3 groups : group K, P1 (chemotherapy group), and P2 (combination group). Colon cancer derived from 1,2-DMH induction subcutaneously. 5FU-leucovorin chemotherapy is given for 6 cycles according Rosswell Park Regiment. P macrocarpa given at a dose of 0,495 mg / day (0.99 mL / day) orally. Cell proliferation was assessed by histochemical AgNOR staining.

ResultThe research sample as many as 6 tails of each group, dropped out is not found in all treatment groups. Calculation of AgNOR cell proliferation obtained mean in the group K, P1, P2 respectively 9.067 ± 0.432, 8.667 ± 0.350, 8.100 ± 0.374. Test for normality using Sapphiro-Wilk test data obtained normal, followed by statistical analysis using parametric different test One Way ANOVA showed that there were significant differences in AgNOR cell proliferation between groups K vs P2 (p = 0.002)

ConclusionP macrocarpa has immunostimulatory potential as a companion chemotherapy in colon cancer Sprague dawley rats that can increase cell proliferation seen from histochemical AgNOR staining.

Keywords : P macrocarpa, colon cancer, cell proliferation (AgNOR)

Background

The incidence of colon cancer worldwide in 2008 as many as 1,233,700 inhabitants,

the third most widely diagnosed in the male gender patients and the second highest cases in

the female gender patients with mortality rates as much as 608,700 inhabitants, the fourth

highest cause of death in men and third in women. Based on data from the United States

Cancer Statistics as many as 142,950 people in the US were diagnosed with colon cancer in

both men and women, and as many as 52,857 deaths caused by cancer kolon.1 Based on the

reports result from hospitals and health centers in the city of Semarang in 2009, cases of the

disease cancer is found as many as 11,862 cases and become the second largest cause of

death after heart disease and blood vessel disease in the category of non-contagious diseases.2

Increase in the incidence of colorectal cancer is correlated with in creasing consumption of

animal fat and some risk factors such as physical inactivity, obesity, tobacco use, daily diet

that contain high fat and carcinogenic substances.

Surgery remains the primary choice for treatment of localized colon cancer. Other

modalities such as adjuvant therapy in the form of chemotherapy, and radiation, especially

when there is inadequate resection and metastases. Based on the recommendation of the

National Comprehensive Cancer Network (NCCN) in 2013 regimen of chemotherapy in

colon cancer can often be used in combination of mFOLFOX 6, FLOX, CapeOx, and 5FU-

leucovorin (ROSSWELL Park Regiment) or in the form of Capecitabine monotherapy,

Bevacizumab, Cetuximab , Panitumubab, and Irinotecan.3,4

5FU is a fluoropyrimidine analog that works specifically on the S phase of the cell

cycle, and induce cell death. Leucovorin is a reduced folate that can enhance the antitumor

activity of 5FU by strengthening the inhibition of the enzyme thymidylate synthase. Various

studies of clinical trials to prove the effectiveness of 5FU-leucovorin combination in Disease

Free Survival and Overall Survival, and reduce mortality and rekurensi.5 Other colon cancer

therapy modalities that are being developed today is immunotherapy is to modulate the

immune system against the tumor, which is expected to kill cancer cells that spread

systemically after definitive local therapy is done. In Indonesia, one of the traditional

medicinal plants that have been widely used as an anti-cancer drug plants / sitostatika namely

Phaleria macrocarpa (Mahkota Dewa) which have the effect to inhibit the growth of tumor

cells. Phaleria macrocarpa has been widely used and sold in the market as an anti-cancer

treatment at a dose of 5 grams a day in the form of dried and meat of the fruit.6 In this study

we used a crude extract of the fruit Phaleria macrocarpa.

Research on preliminary analysis of secondary metabolites of the mahkota dewa prove

that the leaves and callus of the mahkota dewa contain the same secondary metabolites are

alkaloids, flavonoids, saponins, tannins, and steroids / triterpenoids, and there are active

polyphenolic compounds such as gallic acid (GA: 3.4 , 5-trihydroxybenzoic acid) .7,8

Polyphenol compounds contained in herbal medicine has the effect of blocking the growth

factor receptor, and inhibits mitogen-Activated Protein Kinase (MAPK), the signal path

Receptor Tyrosine Kinases (RTKs) and will block a wide range of Growth Hormone such as

Epidermal Growth Factor Receptor (EGFR), Platelet-Derived Growth Factor Receptor

(PDGF), Fibroblast Growth Factor Receptor (FGR.) who was instrumental in mitosis

sel.9,10,11

Gold standard for the diagnostic assessment of cancer cells, therapeutic and prognostic

evaluation of patients is by histopathology. In the assessment of histopathology of cancer

cells, cell proliferation is a primary assessment factors. Histopathologic assessment which

can simultaneously assess changes in gene and cell proliferation of cancer cells can be shown

by looking at the quantity of nuclear organization region (NOR). NOR can be seen with a

light microscope after staining with silver (Ag). Silver staining to see NOR called

AgNOR.12

Studies in vivo effect of fruit extracts in the form of Phaleria macrocarpa against colon

cancer in particular its effect on cell proliferation was done by the method of IHC staining of

Ki-67. Researchers are trying to prove the effect of Phaleria macrocarpa fruit extracts on cell

proliferation in colon cancer tumor Sprague Dawley rats were given chemotherapy using

histochemical AgNOR staining method.

Methods/ Design

Research Design

This study is a laboratory experimental design with "Post test only control group

design". The research group was divided into 3 groups: control (K), treatment groups 1 and 2

(P1 and P2), with the design of the study as follows:

Description:

K : control group, rats were induced carcinogens, after the onset of bumps, received a

standard feed

P1 : Treatment Group 1 carcinogen-induced rat, after the onset of bumps, received

chemotherapy 5FU-leucovorin

P2 : Treatment Group 2, rats were induced carcinogens, after raised bumps got Phaleria

macrocarpa extract a combination of 0,495 mg / day and chemotherapy 5FU-

leucovorin.

Population and Sample

Population

P2

P1

EksklusiPx

-

Px

PxK+

The population was white strain Sprague Dawley rats were obtained from Integrated

Research and Testing Laboratory 4 (LPPT4), Gadjah Mada University, Yogyakarta.

Determination of female gender is used in this research to facilitate maintenance because the

female sex is not so aggressive so as to avoid injuries due to fights that could cause confusion

in the assessment.

Sample

The samples used were taken at random from the population that is affordable

Sprague Dawley rat strain white female weighing 200 grams with appropriate terms of

inclusion and exclusion criteria.

Inclusion Criteria :

1. Female mice

2. Strain Sprague Dawley

3. Weight 200 grams

4. Arising tumors in the colon

Exclusion criteria :

1. Body weight of less than 200 grams

2. During the 7-day observation seemed ill

3. There are anatomical abnormalities

Each of the samples according to WHO treatment groups of at least 5 ekor. 43 In this study

the number of samples used for each group of 6 rats.

Time and Location Research

Maintenance and animal interventions carried out in the Laboratory of Research and

Testing Integrated 4 (LPPT4), Gadjah Mada University, Yogyakarta. Maintenance since the

time of the selection until the treatment takes place within 5 months. Histopathological

examination ranging from the manufacture of paraffin blocks until histochemical AgNOR

staining performed in the laboratory of PA UGM Faculty.

Variabel Research

Independent Variabel

The independent variable is the provision of 5FU-leucovorin and extract Phaleria

macrocarpa.

Dependent variables

The dependent variable is the proliferation of cells (AgNOR)

Operational definitions

Phaleria macrocarpa is the crude extract that is extracted with ethanol by using

soxhletation method, with the results of the extract concentration of 0.5 mg / mL, containing

3,4,5-trihydroxybenzoic acid 10%, given at a dose of 0,495 mg / day (0.99 mL / day) orally.

5FU was given intravenous doses of 0.27 mg and 0.27 mg intravenous dose leucovorin

according ROSSWELL Park Regiment.4

Scale variables: Nominal

Proliferation of cells (AgNOR) is the average percentage of the number of black spots

located at 100 malignant cell nuclei are counted under a light microscope with a

magnification of 1000x at 5 field of view. AgNOR count measurement scale is a numerical

scale of 1-10 numeric. Calculation of AgNOR count conducted by the pathologist.

Scale variables: Ratio

Materials and Devices Research

Ingredients For Treatment

1. Female rats Sprague Dawley strain weighing 200 grams.

2. Feed the standards provided in the form of feed pellets shaped AD type II (production PT.

Japfa Comfeed Indonesia Tbk) provided ad libitum.

3. Colon cancer is obtained by means of a 1.2-dimethylhidrazine induction dose of 30 mg / kg

/ week for 15 weeks after administration of the first induction. At week 13 and 16 induced

rats were sacrificed each two rats to detect the growth of colorectal cancer.

4. 5FU-leucovorin with Fluorouracyl and Rescuvolin trademark of Kalbe Farma.

5. Phaleria macrocarpa used is crude Phaleria macrocarpa extract, obtained by:

a. 1 kg of fruit Phaleria macrocarpa dried finely ground, then the powder is inserted into the

tool soklet (capacity 50 g) and extraction by means soxhletation using ethanol with cycles 8-

10 times.

b. Results extracts included in the rotary evaporator flask and made up to be concentrated

vacuum distillation (temperature 40ºC).

c. Extract is dried in an oven with a temperature of 40 ° C for 1 hour to evaporate ethanol.

d. Showed 5.5 mg per 1 kg of extract the ingredient (0.55%), containing 3,4,5-

trihydroxybenzoic acid 10%, and the extract was diluted with aquabidest to achieve a

concentration of 0.5 mg / ml.

The dose used was equivalent to the dose used in humans is from the flesh of the fruit powder

5 grams / day, 9 multiplied constant therapy trials in experimental animals (mice) is 0.018

multiplied by the constant 0.0055 extracted, so that the dose administered is 5,000 x 0,018 x

0.0055 = 0,495 mg / day (0.99 ml / day).

Materials for routine histopathological examination of H & E

a. Phosphate Buffered formalin 10%

b. Alcohol 50%, 70%, 80%, 96%, absolute

c. Xylol

d. Liquid paraffin (Histoplast)

e. Albumin and poly-L-lysine

f. Painting materials H & E

g. Canada balsam and entelan

h. Universal Streptavidin-Biotin kit (enVision DakoCytomation®)

Tools for observation and documentation preparation

a. 1 unit of multi-head microscope OlympusR

b. CybershootR Sony digital camera + SD Card

c. 1 unit AtomR netbook Intel Processor

Data collection procedures

A total of 22 rats Sprague Dawley strain of age with a body weight of 200 grams

acclimatized in the laboratory for one week. It is expected that the adjustment of

experimental animals to the environmental conditions that exist so there is no drop outs.

Twenty-two mice were then induced carcinogens for 15 weeks. At week 13 and 16 induced

rats were sacrificed each two rats to detect the growth of colorectal cancer. After growing

proven colorectal cancer histopathologically, randomization, and then divided into three

groups determined by simple random sampling so that each group consisted of 6 rats. Each

group housed individually and get the same standard feed and drink ad libitum. Appropriate

treatment given workflow. After 16 weeks the rats performed the termination of anesthesia

with ether and then taken tumor tissue. Tumor tissue is then processed into paraffin block

preparations. H & E staining is done to confirm the tumor that grows in the form of

adenocarcinoma by 2 pathologists, after unconfirmed performed histochemical AgNOR

staining for assessment of cell proliferation.

Research Procedure

The procedure of making preparations paraffin block

a. Fixation

Pieces of colon cancer included in the solution buffered formalin (10% formalin solution in

Sodium Phosphate buffer to achieve a pH of 7.0). Tissue fixation time of 18-24 hours. Then

the network incorporated in distilled water solution for 1 hour for removal process fixative.

b. Dehydration

Pieces of colon cancer included in the alcohol concentration of multi-storey. Networks

become more clear and transparent. The tissue was then put in a solution of alcohol-xylol for

1 hour and then a solution of pure xylol for 2 x 2 hours.

c. Impregnation

Network incorporated in liquid paraffin for 2 x 2 hours.

d. Embedding

Tissue grown in a solid paraffin having a melting point of 56-58 ° C, wait until the solid

paraffin. Tissue in paraffin cut thickness of 4 microns with a microtome. Tissue sections

mounted on glass objects that have previously been smeared poly- Lysine as an adhesive.

Networking on the slide is heated in an incubator temperature of 56-58 ° C until the paraffin

melts.

Tissue staining procedures with H & E

Sequentially network on the slide included in:

1. Water 11. Xylol 1 minute 15 seconds

2. Xylol 2 minutes 12. Alcohol 80% 15 seconds

3. Xylol 2 minutes 13. Alcohol 96% 30 seconds

4. Alcohol 100% 2 100% Alcohol 14 minutes 45 seconds

5. Alcohol 96% 2 minutes 15. Xylol 1 minute

6. Alcohol 80% 2 minutes 16. Xylol 1 minute

7. Water 1 minute

8. HE Mayer 7.5 minutes

9. Air 7.5 minutes

10. eosin (0.5%) - alcohol-acetic acid 1 minute

AgNOR staining procedure:

1. The paraffin block containing 4 micron thick tissue is cut and attached to the slide.

2. Pieces performed deparafinisasi network and rehydration with distilled water

3. The network Pieces included in the darkroom

4. Incubate tissue sections with AgNOR solution for 45-60 minutes.

5. Dip the pieces of the network as much as 3x in distilled water.

6. Pieces network carried dehydrated with alcohol-rise 50%

until absolute.

7. Soak in xylol for 30 minutes

8. Mounting the balsam Canada

9. Ready to be seen under a light microscope

10. AgNOR solution: mix 2 parts AgNO3 50% and 1 part (sour

format + gelatin

Data analysis

After the data collected performed data cleaning, coding and tabulation. Analysis of the

data include descriptive analysis and hypothesis testing. In the descriptive analysis of the

colon cancer cell proliferation activity presented in tables mean, SD, medians and box plots.

The independent variable using a nominal scale and dependent variables in this study using a

ratio scale form AgNOR cell proliferative activity. The data obtained were tested for

normality of data, if the data obtained resumed normal parametric tests using One-way

ANOVA followed by Post Hoc Test using LSD, when abnormal data obtained nonparametric

test continued using Kruskal-Wallis followed by Mann Whitney. Limit the degree of

significance p 0.05 with 95% confidence intervals. The data were analyzed with SPSS

software Ver. 17.0 for Windows.

RESEARCH RESULT

Gambar 7. Consolidated experiment report

This study used 22 Sprague Dawley strain of rats with standard feed acclimatized for

1 week, then induced a 1.2-dimethylhidrazine with a dose of 30 mg / kg / week sc for

7 weeks. At week 2 5 terminated obtained tumor rats with severe dysplasia results PA,

at week 7 was terminated 2 mice showed PA adenocarcinoma. Then do the random

allocation with simple random sampling method are classified into 3 groups. Group I

without special treatment, the second group was given chemotherapy 5FU-leucovorin,

and the third group was given chemotherapy 5FU-leucovorin and Phaleria macrocarpa

extract at a dose of 0,495 mg / day (0.99 ml / day). At week 16, do termination,

continued isolation of colon cancer tissue, then made into a paraffin block preparation

in Anatomical Pathology laboratory. Each of these preparations was cut as thick as 4

Cell prolifration (AgNOR)

H&EStaining

5FU-LV 5FU-LV + PM

Paraffin Block

P1(6)

P2(6)

K(6)

Exclusion

Randomisation

Induction 1,2 DMH

Adaptation period

22 Sprague dawley mice

1 minggu

7 minggu

7 weeks7 weeks7 weeks

microns and confirmed by pathology by HE staining 2 pathologist. Preparations

paraffin blocks are then taken back thickness of 4 microns to do pegecatan AgNOR

for examination of cell proliferation activity.

Descriptive Analysis

AgNOR cell proliferation measurements by calculating the average percentage

of the number of black spots located at 100 malignant cell nuclei are counted under a

light microscope with a magnification of 1000x. AgNOR count measurement scale is

a numerical scale of 1-10 numeric.

Table 3. Characteristics of data AgNOR cell proliferation

Group Min Max Average ± SD Median

Control 8.4 9.6 9.067 ± 0.432 9.100

P1 8.2 9.2 8.667 ± 0.350 8.700

P2 7.6 8.6 8.100 ± 0.374 8.100

LabelP2P1K

Prol

ifera

si S

el (A

gNO

R)

10.00

9.50

9.00

8.50

8.00

7.50

Figure 8. Graph box plot AgNOR cell proliferation

The mean of cell proliferation was highest in the control group 9067 ± 0432, while the

average of the lowest cell proliferation found in the group P2 is 8100 ± 0374. Similarly, the

median highest and the lowest was found in the control group and the group P2 respectively

in the amount of 9,100 and 8,100.

Data Distribution

Test for normality and homogeneity of cell proliferation of data each group using the

Shapiro-Wilk test and levene's test. Obtained data were normally distributed and

homogeneous in all groups. Exploration of data for each variable of each group can be seen

in the table below.

Table 4. Test of normality and homogeneity of data AgNOR cell proliferation

Variabel Kelompok Mean ± SD Shapiro-Wilk Levene’s test

Cell proliferationAgNOR

K 9.067 ± 0.432 p=0.964

P=0.851P1 8.667 ± 0.350 p=0.918

P2 8.100 ± 0.374 p=0.961

Test Statistics

Shapiro-Wilk test and Levene's test showed that the data of normal cell proliferation

and homogeneous, so proceed One Way ANOVA different test to determine the presence of

tumor cell proliferation AgNOR differences between treatment groups, with the results as

follows

   Table 5. Analysis of cell proliferation AgNOR differences between groups

GroupCell proliferation AgNOR

(Mean ± SD)p

K 9.067 ± 0.432

0.002*P1 8.667 ± 0.350

P2 8.100 ± 0.374

* Tested with One Way ANOVA (significant p <0.05)

    Table 6. A post hoc analysis of cell proliferation AgNOR between groups

Grou

pP1 P2

K 0.281 0.002*

P1 – 0.068

* Tested with Bonferroni (significant p <0.05)

Statistical test results using One Way ANOVA AgNOR cell proliferation rate

differences were significant between groups (p = 0.002) so proceed with using the Bonferroni

post hoc test with significant value p <0.05. Results of post hoc test found significant

differences between the groups K with P2 group (p = 0.002). There were no significant

differences between the groups K with P1 (p = 0281), and P1 to P2 group (p = 0.068).

DISCUSSION

Calculation of cell proliferation using histochemical AgNOR staining techniques

obtained average is the highest in the control group and the mean of the lowest in the group

receiving the combination of 5FU-leucovorin + P macrocarpa. Analysis of cell proliferation

differences among treatment groups obtained a significant difference between the control

group and the group given the combination of 5FU-leucovorin + P macrocarpa. In the group

given chemotherapy 5FU-leucovorin may reduce cell proliferation as in the combination

group 5FU-leucovorin + P macrocarpa but not significantly.

5FU-leucovorin work on the S phase of the cell cycle by inhibiting the enzyme targets

Thymidylate synthase inhibition with the end result of DNA synthesis and function. While

Phaleria macrocarpa through active substances polyphenols such as gallic acid (GA: 3,4,5-

trihydroxybenzoic acid) selectively induce cancer cell death by inhibiting cell proliferation

kanker.10 polyphenolic compounds also inhibit mitogen-Activated Protein Kinase (MAPK),

the signal path Receptor Tyrosine Kinase (RTKs) such as Epidermal Growth Factor Receptor

(EGFR), Platelet-Derived Growth Factor Receptor (PDGF), Fibroblast Growth Factor

Receptor (FGR.) who was instrumental in mitosis sel.9,10 other active substances, namely

flavonoid is a specific inhibitor of the CDKs (Cyclin-dependent kinases), induces arrest in G2

/ M phase, upregulation of p53 and p21 genes, downregulation of cyclin B1 and Cdk1

activity that can alter cell proliferation. In this study, administration of the combination of

5FU-leucovorin and Phaleria macrocarpa working mechanisms reinforce one another

decrease in cell proliferation. In the study conducted Alwi et al, using IHC examination of

cell proliferation Ki-67 in rat colon adenocarcinoma there are significant differences in the

group given the combination of 5FU-leucovorin and Phaleria macrocarpa than other groups,

the research is in line with the results of our research lakukan.44 proliferation Inspection cells

using other techniques such as IHC PCNA in further research is needed as a comparison to

the results of previous studies.

This study uses a single 5-Fluorouracil chemotherapy based protocol "ROSSWELL

Park" which is currently more recommended the use of combination chemotherapy such as

mFOLFOX 6, FLOX, CapeOx based on National Comprehensive Cancer Network (NCCN)

in 2013.3 The results of the data obtained cell proliferation that group K vs P1 and P1 vs P2

is not significant, it does not mean chemotherapy agents failed to lower the cell proliferation,

it is because the current single agent chemotherapies have been abandoned by more recent

recommendations suggest the use of a combination of chemotherapy agents. P macrocarpa is

used instead of pure extract of crude extract but so to do the purification of active substance

in it to find out the more obvious effects against tumor cells, sehinnga it is becoming a

limitation of the study that further research is needed in the form of the use of combinations

of chemotherapeutic agents and purification of the active substance P macrocarpa be studied

benefits.

Phaleria macrocarpa extract has potential as an immunostimulatory whose use is

suplementatif for primary therapy, as an alternative source of traditional medicine use.

Researchers also realize there are still many limitations of the study should be improved to

complement and enhance this research. Where possible this research could be increased to

human clinical trials.

CONCLUSIONS AND RECOMMENDATIONS

Conclusions

1. There is a decrease in cell proliferation of colon cancer tumor tissue Sprague Dawley rats

were given chemotherapy 5FU-leucovorin

2. There is a decrease in cell proliferation of colon cancer tumor tissue Sprague Dawley rats

were given a combination of Phaleria macrocarpa and chemotherapy 5FU-leucovorin

3. There was no significant difference in cell proliferation of colon cancer tumor tissue

Sprague Dawley rats were given chemotherapy 5FU-leucovorin with a combination

Phaleria macrocarpa and chemotherapy 5FU-leucovorin

4.

Suggestions

Keep in consideration of the provision of a combination of chemotherapeutic agents other

groups and purification of active substances to be examined P macrocarpa benefits mainly

active substances gallic acid to enhance the concept of thinking of this research. In addition

to the necessary assessment tiu histopathological grading, lymph node metastasis, and further

investigation of the levels of other cytokines such as CDKs, p53 and p21 genes, VEGF, as

well as toxic effects on vital organs functionally and histologically. The study will be the

basis for the possibility of clinical testing on humans.

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