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All Journal Jurnal Teknosains Indonesian Journal of Cancer Chemoprevention
Kumara, Dennaya
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Biochemistry, Genetics & Molecular Biology Civil Engineering, Building, Construction & Architecture Industrial & Manufacturing Engineering Mechanical Engineering Medicine & Pharmacology

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Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging Kumara, Dennaya; Harsan, Hayfa Salsabila; Novianti, Metta; Lestari, Dinda; Septisetyani, Endah Puji; Prasetyaningrum, Pekik Wiji; Paramitasari, Komang Alit; Meiyanto, Edy
Jurnal Teknosains Vol 13, No 1 (2023): December
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/teknosains.89479

Abstract

The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.
Citrus Flavonoids from Citrus reticulata Peels Potentially Target an Autophagy Modulator, MAP1LC3A, in Breast Cancer Anggoro, Bayu; Kumara, Dennaya; Angelina, Dhella; Ikawati, Muthi
Indonesian Journal of Cancer Chemoprevention Vol 12, No 3 (2021)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev12iss3pp114-122

Abstract

Citrus flavonoids have been known for their vast biological activities including chemoprevention activities. However, the organic solvent extraction system limits its potential utilization. We recently adopted a hydrodynamic-cavitation method to extract citrus flavonoids from citrus peels. In this study we verified the high flavonoid content of the hydrodynamic-cavitation extract from Citrus reticulata peels and explore the potency of its citrus flavonoid contents as targeted chemoprevention agent for breast cancer by using bioinformatics. Based on a thin layer chromatography, the extract positively yielded high content of citrus flavonoids represented by hesperidin. The toxicity analysis by Protox II Online Tool revealed that hesperidin as the major citrus flavonoid in the extract was considered safe with a predicted LD50 of 12,000 mg/kg. We then further exploring citrus flavonoids’ capacity in targeting MAP1LC3A, a key protein in autophagy. UALCAN analysis validated that low expression of MAP1LC3A is associated with low survival rates in breast cancer patients. Limonin, hesperidin, narirutin, neohesperidine, and naringin are flavonoids from citrus peels that predicted to have inhibitory activity against Protein Kinase A (PKA), a negative upstream of MAP1LC3A, calculated by KNIME. Citrus flavonoids scoparone, cirsimaritin, 4',5,7-trimethoxyflavone, eupatorine, and hesperidin were also exhibit similar structure to an agonist of ATG4B, a protein that plays a role in MAP1LC3A activation. Furthermore, eupatorine, hesperidin, and cirsimaritin displayed a high affinity to ATG4B based on a molecular docking. We concluded that citrus flavonoids from citrus peels are safe to normal cells, and the citrus flavonoids potentially targets MAP1LC3A by inhibiting PKA and acting as ATG4B agonists. Thus, this extract-contained flavonoids from citrus peels is potential to be investigated further as a chemoprevention agent by inducing autophagy, especially for breast cancer.Keywords: Citrus reticulata, citrus flavonoid, autophagy, MAP1LC3A, breast cancer.
Co-Authors Angelina, Dhella Anggoro, Bayu Edy Meiyanto Endah Puji Septisetyani, Endah Puji Harsan, Hayfa Salsabila Komang Alit Paramitasari Lestari, Dinda Muthi Ikawati Novianti, Metta Prasetyaningrum, Pekik Wiji