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. 2021 Jul 7;11:14002. doi: 10.1038/s41598-021-93270-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Effect of combination metformin/apigenin on cell growth, cell cycle, cellular ROS and mitochondrial membrane potential in AsPC-1 and HDF cells. (A) The effect of combination of metformin/apigenin on cell proliferation/viability in AsPC-1 and HDF cells. The cells were treated with metformin (5 mM) alone, apigenin (1 or 20 µM) alone or metformin/apigenin combination for 24 to 120 h and the cell proliferation/viability was assessed by MTT assay. (B) Cell cycle was analyzed with PI staining of the cells (AsPC-1 and HDF) treated with metformin (0.05, 0.5 or 5 mM) alone, apigenin (20 µM) alone or metformin/apigenin combination. (C) Cellular ROS levels of metformin-treated (for 24 h) in AsPC-1 and HDF cells were measured by CellROX Green staining when cells (AsPC-1 and HDF) were treated with metformin (0.05, 0.5 or 5 mM), apigenin (20 µM) or combination of metformin and apigenin for 24 h. (D) Mitochondrial membrane potentials were measured when cells (AsPC-1 and HDF) were treated with metformin (0.5 or 5 mM), apigenin (20 µM) or combination of metformin/apigenin for 24 h. Effect of NAC (N-acetyl cysteine, 10 µM) pretreatment on ROS production (A) and cell death (B) induced by combination of metformin/apigenin was examined. The effects of combination of metformin (5 mM)/apigenin (1 and 20 μM) on cell proliferation/viability (E) and cellular ROS level (F) in MIAPaCa-2, DU145, LNCaP and HCC1195 cells. Three different measurements were performed for each sample. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ****p < 0.0001 compared with control group. ^####p < 0.0001 compared with metformin (0.05, 0.5 or 5 mM)/apigenin (20 µM) treated group. ⁺p < 0.05, ⁺⁺⁺p < 0.01, and ⁺⁺⁺⁺p < 0.0001 compared with ASPC-1 control group. ^^p < 0.01, ^^^p < 0.001 and ^^^^p < 0.0001 compared with HDF control group.