Effect of Artemisia vulgaris Extract on P53 Expression and Caspase-8 Expression (Study on Adenocarcinoma Mammae C3H Mice Given Adriamycin- Cyclophosphamide Chemotherapy Regimen)

All authors have reviewed and approve d the final version of the manuscri pt. https://doi.org/10.32539/BJI.v7i2.300 A B S T R A C T Background : The incidence of breast cancer worldwide is still high. Surgery remains the top choice with other modalities of chemotherapy. radiation. and immunotherapy such as Artemisia vulgaris (AV). Purpose : The study was aimed to demonstrate that administration of AV extract increased the levels of p53 and Caspase-8 in adenocarcinoma mammae. Methods : This study used "Post test only control group design" on 24 females C3H mice that were randomly selected and divided into four groups : group K (control). P1 (chemotherapy). P2 (extract). and P3 (combination). Adenocarcinoma mammae comes from the inoculation of donor mice. Chemotherapy of Adriamycin 0.18 mg and Cyclophosphamide 1.8 mg were given in 2 cycles. AV 13 mg (0.2 ml) was given once daily orally. P53 and Caspase-8 levels were evaluated by imunohistochemical staining. Results : Mean of p53 and Caspase-8 levels were found in groups K. P1. P2. P3 were 22.06+1.73. 37.16+1.20. 24.60+1.08. 39.78+1.19 dan 17.16+1.28. 26.20+1.11. 24.60+1.08. 39.78+1.19. The statistical analysis showed that there were significant differences in the levels of p53 between groups of K vs P1. P3 (p=0.001). K vs P2 (p=0.048). P1 vs P2 (p=0.001). P1 vs P3 (p=0.039). P2 vs P3 (p=0.001). and in Caspase-8 between groups of K vs P1. P3 (p=0.001). K vs P2 (p=0.048). P1 vs P2 (p=0.001). P1 vs P3 (p=0.039). P2 vs P3 (p=0.001). Correlation analysis between p53 and Caspase-8 showed significant correlation (p=0.047 dan r=0.883). Conclusion : Artemisia vulgaris can improve the effectivity of AdriamycinCyclophosphamide chemotherapy on C3H mice with adenocarcinoma mammae in terms of elevated levels of P53 and Caspase-8.


Introduction
Breast cancer is one of main health concerns for carcinogens. cytotoxic chemotherapy treatment). and also non-genotoxic effects (hypoxia. oncogen activation. nucleotide repression. microtubule damage. and loss of intercellular contact). 8 Some of its functions includes inducing apoptosis. controlling and shutting down cell cycle (cell cycle arrest). helping DNA repair. and inhibiting angiogenesis. 8 The specific qualitative and quantitative activities of P53 depend on its integrity (mutation status). number. post translation specific modification in response to environmental stress and interactions with a number of its cofactors. 9 The function of P53 in regulating cellular response to environmental stress contributes greatly in preventing tumors. which makes P53 known as one of Tumor Suppressor Genes. The evidence of P53 gene functioning as tumor suppressor can be seen from mice which phenotype modified to mutate or lose one or two of P53 allelgen developed tumor and die earlier than mice with normal two alleles of P53 gene. 8 radiotherapy. chemotherapy. and hormonal therapy. 5.8 Chemotherapy is a process of treatment using drugs to destroy cancer cells of slow down their growth. Earlier study on mice with hepatic carcinoma given artemisinin with dose of 100mg/kg per day showed anticancer activity. 7

Research design
This research is an experimental laboratory study with post-test only control group design. The research group was divided into 4. namely the control group (K). treatment 1 (P1). treatment 2 (P2). and treatment 3 (P3). Hospital Yogyakarta.

Research variable
The independent variables in this study were administration of Artemisia vulgaris extract.

administration of chemotherapy Adriamycin and
Cyclophosphamide. and administration of Artemisia vulgaris extract with a combination of chemotherapy Adriamycin and Cyclophosphamide.
The dependent variables in this study were expression of P53 and Caspase-8.

Operational definition
Administration

Data collection
Six micron wide specimen was made from each group. stained with HE and examined for lymphocyte spread and histology degree. Another specimen was also made from each group to evaluate perforin expression using anti-P53 monoclonal antibody staining and Caspase-8 and examined for number of lymphocytes near the cancer cell that produce perforin.

Ethical requirements
This study applies animal ethics in managing sample animals. This study has been approved by Ethics Commission for Health Research. Faculty of Medicine. Diponegoro University. All sample animals were treated and cared for according to standard animal caring procedure.
Difference of P53 level analysis using One Way ANOVA test. followed with Post Hoc test showed a significant difference and thus the first hypothesis may be accepted.

Bonferroni
Post-Hoc test (Table.5   Caspase-8 expression data characteristics presented in percentage.
One way anova test showed p < 0.001

Discussion
This study aimed to prove the influence of Artemisia vulgaris extract administration to C3H mice with mammae adenocarcinoma that received Adriamycin-Cyclophosphamide chemotherapy in terms of P53 and Caspase-8 levels. Result discussion will be explained orderly starting from P53 level. Caspase-8 level.
followed with witnessing the correlation between said two variables.
Average P53 level was found higher in the group that received combination of chemotherapy and Artemisia vulgaris extract than the group that received only chemotherapy. This corresponds to the study by This study was able to prove correlation between P53 level and Caspase -8 level. but there are various other pathways that can cause the apoptosis process.

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Artemisa vulgaris extract has the potential to be used as immunomodulator that supplements a primary therapy. as an alternative use of traditional medicine.
We realized this study contained numerous flaws that needed to be countered in order to complete and perfect the study. To emphasize the concept of this study. there is a need for a follow-up research to determine other factors contributing to the increase of P53 and Caspase-8 levels. A new clinical trial on human subjects may be considered to be carried out.

Conclusion
Artemisia vulgaris suppresses cell proliferation in mammary adenocarcinoma inoculated C3H mice

Conflict of interest and funding
Authors did not received funding or profits from the industry or elsewhere for conducting this research.