EFFECT OF CRYPTOBRACHITONE C ON LDOC1, NFKBIZ, AND HRK GENE EXPRESSION IN MCF-7 CANCER CELL LINE

• Main Author: Kafa Bihi, Abdulloh
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/51028
• Institution: Institut Teknologi Bandung

Breast cancer is the leading cause of cancer death in women. According to WHO's data in 2018, as much as 15% (630,000) of women with cancer who died suffered from breast cancer. The most common therapy for breast cancer is surgery, radiotherapy and chemotherapy using cytotoxic substances like Doxorubicin. However, these therapies have several negative effects. Surgery can cause lymphedema and Doxorubicin have cardiotoxicity effects. Due to the negative effect of these common anticancer therapies, a concerted effort is currently performed to find a novel and safer method to treat breast cancer. One such alternative method is by using natural compounds as a cytotoxic agent in cancer treatment. Cryptocarya is a genus of plant that is widely used as a traditional medicinal plant. Cryptocarya extract is known to possess several biological effects, such as anti-inflammation, antimicrobial properties, antituberculosis, and cytotoxic activity. Several studies have shown that Cryptobrachytone C, a substance isolated from Cryptocarya pulchinervia, has cytotoxic activity on MDA-MB-231 breast cancer cell line and five other cancer cell lines. To determine the mechanism involved in cytotoxic and anti-proliferative effects of Cryptobrachytone C on MCF-7 breast cancer cell line, RNAseq analysis was done. Two samples are used in the transcriptomic analysis: MCF-7 cells without treatment and MCF-7 cells with Cryptobrachytone C 12,94 ?M (IC50) treatment. Treatment was conducted for 24 hours. The objectives of this study are to analyze RNAseq data and verify RNA-seq analysis results using RT-qPCR. Tuxedo protocol was used in RNA-seq data analysis. Gene expression results from RNA-seq analysis were selected by its role in apoptosis, cell proliferation, and cancer progression. LDOC1, NFKBIZ, and HRK are the three genes of interest in this study. RT-qPCR with 18S rRNA as an internal control gene is used to determine the relative expression of LDOC1, NFKBIZ, and HRK at mRNA level on MCF-7 cell line. Five treatment of the qPCR test used in this study are Cryptobrachytone C with three different concentration 4,31 ?M (IC25), 12,94 ?M (IC50), and 38,89 ?M (IC75); Doxorubicin 0,5 ?g/mL; and negative control. To determine the interaction between Cryptobrachytone C and several transcription factors of LDOC1 and HRK, molecular docking using AutoDock Vina are used in this study as further in-silico analysis. Based on the RNA-seq analysis, LDOC1, NFKBIZ, and HRK genes expression levels are higher in the Cryptobrachytone C treatment group rather than the control group. Relative expression of LDOC1 in all treatment groups (IC25, IC50, IC75, and Doxorubicin) was lower compared to the negative control. Relative expression of NFKBIZ in IC50, IC75, and Doxorubicin group was lower compared to the negative control, whereas the IC25 group is not significantly different from negative control. Relative expression of HRK in the IC75 group was lower compared to negative control, whereas IC25 and IC50 groups are not significantly different from negative control. Relative expression of HRK in Doxorubicin was higher compared to negative control. LDOC1, HRK, and NFKBIZ relative expression using qPCR test with Cryptobrachytone C treatment in contrast to the results from RNAseq analysis. Since Cryptobrachytone C has a cytotoxic effect on cancer cell lines according to previous research, qPCR test results of NFKBIZ were in accordance with the role of I?B? protein that can inhibit apoptosis and induce cell proliferation. On the other hand, qPCR test results of LDOC1 and HRK were antagonistic with its role as pro-apoptotic genes. Molecular docking analysis shows that Cryptobrachytone C could bind in the binding site of SAH (AdoHcy), an inhibitor of DNMT1 protein. DNMT1 protein can repress the expression of LDOC1 and HRK. Therefore, it can be predicted that Cryptobrachytone C competes with SAH so the inhibitory effect of SAH is decreasing. It can be concluded that Cryptobrachytone C decreased the relative expression of LDOC1, NFKBIZ, and HRK genes in MCF-7 cells .