The Citrus sinensis Peel Extract Elevates the Cytotoxicity Effect of Doxorubicin and Inhibits 4T1 Cell Migration

Shofa Khamdanatuz  Zufairo'; Desty Restia Rahmawati; Amaliya Permata Putri; Anif Nur Artanti; Faaza Aulia Rahman; Edy Meiyanto; Ratna Asmah Susidarti

doi:10.22146/jtbb.18058

Shofa Khamdanatuz Zufairo' Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281; Master Student of Biotechnology Study Program, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia, 55284 https://orcid.org/0009-0004-4194-6713
Desty Restia Rahmawati Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281
Amaliya Permata Putri Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281
Anif Nur Artanti Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281; Departement of Pharmacy, Vocational College, Universitas Sebelas Maret, Surakarta, 57126, Indonesia https://orcid.org/0009-0001-8015-2850
Faaza Aulia Rahman Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281
Edy Meiyanto Laboratory of Macromolecular Engineering, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281 https://orcid.org/0000-0002-0886-6322
Ratna Asmah Susidarti Cancer Chemoprevention Research Center (CCRC), Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia, 55281; Laboratory of Medicinal Chemistry, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia, 55281 https://orcid.org/0000-0002-0973-441X

DOI: https://doi.org/10.22146/jtbb.18058

Keywords: Citrus sinensis, Co-chemotherapy, Doxorubicin, Sinensetin, TNBC

Abstract

Combination therapy using natural-based substances is an effective approach to increase the efficacy of doxorubicin (Dox) while reducing its toxicity towards normal cells. This general chemotherapeutic agent treats various cancer cells, including triple-negative breast cancer (TNBC). Citrus sinensis peel extract (CPE) contains sinensetin (Sin), which exhibits anti-cancer effects, offering good prospects as a co-chemotherapy agent. This research aims to evaluate the co-chemotherapeutic potency of CPE-contained Sin when combined with Dox and also study its influence on 4T1 cell migration. Assays performed in this experiment included cell viability assay using MTT in individual and combination manner, clonogenic assay to analyse the proliferation inhibition effects of the treatments, scratch wound healing assay to assess the cells’ migration status, and gelatine zymography to evaluate cells’ matrix metalloproteinase-9 (MMP-9) activity.. This experiment confirmed that CPE and Sin had cytotoxic effects on 4T1 cells with the IC₅₀ of 536 μg mL^-1 and 120 μM, respectively, while Dox performed cytotoxicity on 4T1 cells with an IC₅₀ of 2 μM. The combination of CPE and Dox showed strong synergistic effects with the combination index (CI) <1.0. CPE and its co-administration with Dox permanently suppressed the colony formation of 4T1 cells after 10 days of treatment removal. CPE and Sin inhibited the 4T1 cell migration strongly compared to Dox. CPE in its single and mixture form with Dox inhibited the MMP-9 activity. These results suggest that CPE, when combined with Dox, could be a promising co-chemotherapy regimen for TNBC treatment, potentially reducing the risk of metastasis and relapse.

References

Amalina, N.D. et al., 2023. In vitro synergistic effect of hesperidin and doxorubicin downregulates epithelial-mesenchymal transition in highly metastatic breast cancer cells. Journal of the Egyptian National Cancer Institute, 35(1), 6. doi: 10.1186/s43046-023-00166-3.

Artanti, A.N. et al., 2023. Cytotoxic Activity and Senescence Modulatory Effect of Hesperetin on Triple-Negative Breast Cancer Cells and Kidney Cells Co-Treatment with Cisplatin. Indonesian Journal of Cancer Chemoprevention, 14(3), pp.181-188. doi: 10.14499/indonesianjcanchemoprev14iss3pp181-188.

Chen, Z.T. et al., 2012. Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress. Food chemistry, 135(4), pp.2119-2127. doi: 10.1016/j.foodchem.2012.07.041.

Chu, C.C. et al., 2017. Antiproliferative effect of sweet orange peel and its bioactive compounds against human hepatoma cells, in vitro and in vivo. Journal of Functional Foods, 33, pp.363-375. doi: 10.1016/j.jff.2017.03.051.

Haryanti, S. et al., 2022. The cytotoxic and anti-migratory properties of Caesalpinia sappan and Ficus septica, in combination with doxorubicin on 4T1 TNBC cells with nephroprotective potential. Asian Pacific Journal of Cancer Prevention: APJCP, 23(2), pp.743-753. doi: 10.31557/APJCP.2022.23.2.743.

Hermawan, A. et al., 2021. Bioinformatics and in vitro studies reveal the importance of p53, PPARG and notch signaling pathway in inhibition of breast cancer stem cells by hesperetin. Advanced Pharmaceutical Bulletin, 11(2), pp.351-360. doi: 10.34172/apb.2021.033.

Ikawati, M. et al., 2023. The Synergistic Effect of Combination of Pentagamavunone-1 with Diosmin, Galangin, and Piperine in WiDr Colon Cancer Cells: In vitro and Target Protein Prediction. Journal of Tropical Biodiversity & Biotechnology, 8(2), jtbb80975. doi: 10.22146/jtbb.80975.

Indriyanti, R.A. et al., 2023. The effect of Sinensetin and Imperatorin on A-549 lung cancer cell viability in vitro. Pharmacognosy Journal, 15(1), pp.38-46. doi: 10.5530/pj.2023.15.6.

Kciuk, M. et al., 2023. Doxorubicin—an agent with multiple mechanisms of anticancer activity. Cells, 12(4), pp.26-32. doi: 10.3390/cells12040659.

Li, Y. et al., 2023. Natural flavonoid sinensetin inhibits cisplatin-induced pyroptosis and attenuates intestinal injury. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1869(3), 166637. doi: 10.1016/j.bbadis.2023.166637.

Manjunath, M. & Choudhary, B., 2021. Triple-negative breast cancer: A run-through of features, classification and current therapies. Oncology Letters, 22(1), 512. doi: 10.3892/ol.2021.12773.

Meiyanto, E., Hermawan, A. & Anindyajati, A., 2012. Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents. Asian Pacific Journal of Cancer Prevention, 13(2), pp.427-436. doi: 10.7314/APJCP.2012.13.2.427.

Nurhayati, I.P. et al., 2020. Cytotoxic and antimetastatic activity of hesperetin and doxorubicin combination toward Her2 expressing breast cancer cells. Asian Pacific Journal of Cancer Prevention: APJCP, 21(5), pp.1259-1267. doi: 10.31557/APJCP.2020.21.5.1259.

Reynolds, C.P. & Maurer, B.J., 2005. Evaluating response to antineoplastic drug combinations in tissue culture models. Chemosensitivity: Volume 1 In Vitro Assays, 110, pp.173-183. doi: 10.1385/1-59259-869-2:173.

Rosińska, M. et al., 2024. Retrospective Observational Study to Determine the Epidemiology and Treatment Patterns of Patients with Triple-Negative Breast Cancer. Cancers, 16(6), 1087. doi: 10.3390/cancers16061087.

Samidurai, D. et al., 2020. Sinensetin isolated from Orthosiphon aristatus inhibits cell proliferation and induces apoptosis in hepatocellular carcinoma cells. Process Biochemistry, 88, pp.213-221. doi: 10.1016/j.procbio.2019.09.031.

Schrörs, B. et al., 2020. Multi-omics characterization of the 4T1 murine mammary gland tumor model. Frontiers in oncology, 10(6), 1195. doi: 10.3389/fonc.2020.01195.

Shi, M.D. et al., 2013. Nobiletin attenuates metastasis via both ERK and PI3K/Akt pathways in HGF-treated liver cancer HepG2 cells. Phytomedicine, 20(8-9), pp.743-752. doi: 10.1016/j.phymed.2013.02.004.

Tafrihani, A.S. et al., 2023. Cardamom essential oil extract suppress the progression of triple-negative breast cancer 4T1 cell line. The Indonesian Biomedical Journal, 15(2), pp.150-156. doi: 10.18585/inabj.v15i2.2140.

Vuger, A. T. et al., 2020. Characteristics and prognosis of triple-negative breast cancer patients: a Croatian single institution retrospective cohort study. Acta clinica Croatica, 59(1), pp.97-107. doi: 10.20471/acc.2020.59.01.12.

Wardhani, B.W. et al., 2021. TGF-β-induced TMEPAI promotes epithelial–mesenchymal transition in doxorubicin-treated triple-negative breast cancer cells via SMAD3 and PI3K/AKT pathway alteration. Breast Cancer: Targets and Therapy, 13, pp.529-538. doi: 10.2147/BCTT.S325429.

Xu, Y. et al., 2019. Simultaneous separation of six pure polymethoxyflavones from sweet orange peel extract by high performance counter current chromatography. Food Chemistry, 292, pp.160-165. doi: 10.1016/j.foodchem.2019.04.031.

Zhu, S. et al., 2024. Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition. Translational Cancer Research, 13(1), pp.348-362. doi: 10.21037/tcr-23-1317.

Zufairo’, S.K. et al., 2023. Citrus sinensis Peel Extract Synergistically Enhances the Cytotoxic Effect of Chemotherapeutic Agents on HepG2 Cells. Indonesian Journal of Cancer Chemoprevention, 14(3), pp.151-159. doi: 10.14499/indonesianjcanchemoprev14iss3pp151-159.