Direct and Indirect Effect of TNFα and IFNγ Toward Apoptosis in Breast Cancer Cells

Abstract

Background: Breast cancer (BC) is the leading cause of death cancer in women. Cancer therapies using TNFα and IFNγ have been recently developed by direct effects and activation of immune responses. This study was performed to evaluate the effects of TNFα and IFNγ directly, and TNFα and IFNγ secreted by Conditioned Medium-human Wharton’s Jelly Mesenchymal Stem Cells (CM-hWJMSCs) toward apoptosis of BC cells (MCF7).Materials and Methods: BC cells were induced by TNFα and IFNγ in 175 and 350ng/mL, respectively. CM-hWJMSCs were produced by co-culture hWJMSCs and NK cells that secreted TNFα, IFNγ, perforin (Prf1), granzyme B (GzmB) for treating BC cells. The BC cells were treated with CM-hWJMSCs in 50%. The expression of apoptotic genes Bax, p53, and the antiapoptotic gene Bcl-2 were determined using RT-PCR.Results: TNFα and IFNγ at concentration of 350 ng/mL induced higher Bax expression compared to 175 ng/mL. TNFα and IFNγ 350 ng/mL, 175 ng/mL induced p53 expression, whilst TNFα and IFNγ at 350 ng/mL decreased Bcl-2 expression. Perf1, GzmB, TNFα and IFNγ-containing CM-hWJMSCs induced significantly apoptosis percentage, induced Bax expression, but did not effect p53, Bcl-2 expression.Conclusion: TNFα and IFNγ directly induce Bax, p53, decrease Bcl-2 gene expression. The Prf1, GzmB, TNFα, IFNγ-containing CM-hWJMSCs induce apoptosis and Bax expression.Keywords: breast cancer, Wharton’s Jelly mesenchymal stem cells, TNFα, IFNγ

Hepatoprotective properties of red betel (Piper crocatum Ruiz and Pav) leaves extract towards H2O2-induced HepG2 cells via anti-inflammatory, antinecrotic, antioxidant potency

“…TNF-α level quantification was performed using ELISA assay according to the manufacturer’s protocol (430204, BioLegend, California, US). Optical density (OD) were measured at 450 nm and 570 nm using spectrophotometer ( Laksmitawati et al, 2016 ; Widowati et al, 2018a ).…”

“…To 5 × 10 5 accounted cells, Annexin V binding buffer (130-092-820, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 500 μL, Anti-FITC (130-048-701, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 5 μL, and Propidium Iodide (130-093-233, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 5 μL were added and continued by incubation in the dark at 4 °C. The HepG2 cells apoptotic, necrotic, death, and live cells percentage were measured by MACSquant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) ( Widowati et al, 2018a ; Widowati et al, 2020 ).…”

“…The qPCR process were carried out through 7 min pre-denaturation at 95 °C; continued by 40 cycles of 30 sec of denaturation at 95 °C, 40 sec of annealing at 60 °C, and 60 sec of elongation at 72 °C. Gene expression was measured as relative copy number compared to control using ΔΔCT method ( Widowati et al, 2018a , Widowati et al, 2018b , Afifah et al, 2019 ). The primer sequences of GPX and β-actin gene used in this study can be seen at Table 1 also the concentration and purity of RNA can be seen at Table 2 .…”

Apoptotic Potential of Secretome from Interleukin-Induced Natural Killer Cells toward Breast Cancer Cell Line by Transwell Assay

“…The concentration and purity of RNA of each sample was determined at 260/280 nm. The cDNA synthesis was performed using cDNA synthesis kit (Bio-rad, 170-8841) (Widowati et al 2018;Afifah et al 2019;Widowati et al 2019). Primer sequences were used can be seen at Table 1.…”

“…Cell cultures were maintained in T-75 flasks, at 37°C, 5% CO 2 . Culture mediums were changed every three days and sub-cultured at 80% confluence (Widowati et al 2018;Widowati et al 2019).…”