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Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human.

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Presentation on theme: "Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human."— Presentation transcript:

1 Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human lung cancer cells  Chih-Jung Yao, Jyh-Ming Chow, Shuang-En Chuang, Chia-Lun Chang, Ming-De Yan, Hsin-Lun Lee, I-Chun Lai, Pei-Chun Lin, Gi-Ming Lai  Journal of Ginseng Research  Volume 41, Issue 3, Pages (July 2017) DOI: /j.jgr Copyright © Terms and Conditions

2 Fig. 1 Effects of KG-135 on the proliferation and cell cycle distribution of A549 human nonsmall cell lung cancer cells. (A) After treatment with KG-135 for 72 h, the cell viability of A549 cells was measured using sulforhodamine B binding assay. (B) The cell cycle distribution was analyzed using flow cytometry. Cell viability data are expressed as mean ± standard error and analyzed as described in Section 2. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

3 Fig. 2 Effects of KG-135 and autophagy inhibitor hydroxychloroquine (HCQ) on the cell viability of normal human fetal lung fibroblast WI-38 cells. (A) After treatment with KG-135 alone or in the presence of HCQ for 72 h, the cell viability was determined using sulforhodamine B binding assay and (B) examined using phase contrast microscopy, scale bar = 100 μm. Cell viability data are expressed as mean ± standard error and analyzed as described in Section 2. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

4 Fig. 3 Effects of KG-135 on the cell cycle regulation proteins and tumor suppressor FOXO3a of A549 human nonsmall cell lung cancer cells. (A) After treatment with KG-135 for 72 h, Western blot was used to analyze the protein levels of cyclin-dependent kinase (CDK)4, CDK6, cyclin D1, Rb, and pRb, (B) p18 and p27, and (C) FOXO3a. FOXO3a, Forkhead Class box O3a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pRb, phospho-retinoblastoma protein. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

5 Fig. 4 Effects of KG-135 and metformin on autophagy related proteins in A549 human nonsmall cell lung cancer cells. (A) After treatment with KG-135 or (B) metformin for 72 h, Western blot was used to analyze the protein levels of LC3-I, LC3-II, Beclin-1, and p62 (sequestosome 1). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

6 Fig. 5 Effects of autophagy inhibitor hydroxychloroquine (HCQ) on KG-135-induced LC3-II accumulation and acidic vesicular organelle (AVO) formation in A549 human nonsmall cell lung cancer cells. (A) After treatment with KG-135 alone or in the presence of HCQ for 72 h, the protein levels of LC3-I and LC3-II were analyzed by Western blot. (B) As described in Section 2, the formation of acridine orange-stained AVOs was examined using fluorescence microscopy, scale bar = 50 μm and (C) quantified using flow cytometry. The AVOs in KG-135-treated cells showed bright red, whereas the cytoplasm and the nucleus showed dominant green fluorescence. The percentage of cells with bright red fluorescence (the upper two FL3-positive quadrants) is indicated in the parentheses of each flow cytometric dot plot. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

7 Fig. 6 Effects of autophagy inhibitor hydroxychloroquine (HCQ) on KG-135-mediated growth inhibition and apoptosis induction in A549 human nonsmall cell lung cancer cells. (A) After treatment with KG-135 alone or in the presence of HCQ for 72 h, the cell viability was determined using sulforhodamine B binding assay and (B) examined using phase contrast microscopy, scale bar = 100 μm. (C) The sub-G1 apoptotic fraction was analyzed using flow cytometry. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

8 Fig. 7 Effects of autophagy inhibitor hydroxychloroquine (HCQ) on KG-135-mediated activation of apoptosis pathways and upregulation of tumor suppressor FOXO3a in A549 human nonsmall cell lung cancer cells. (A) The protein level of FOXO3a was analyzed using Western blot after treatment with KG-135 alone or in the presence of HCQ for 24 h and 72 h. (B) The extrinsic apoptosis-related proteins Fas ligand (FasL) and full-length caspase-8 were analyzed using Western blot after treatment with KG-135 alone or in the presence of HCQ for 48 h. (C) The mitochondrial (intrinsic) apoptosis-related proteins were analyzed using Western blot 48 h (survivin and caspase-9 cleaved at Asp315 and Asp330) and 72 h (Bcl-2) after treatment with KG-135 alone or in the presence of HCQ. FOXO3a, Forkhead Class box O3a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

9 Fig. S1 Effect of autophagy inhibitor hydroxychloroquine (HCQ) 25μM combined with 50 μg/mL of KG-135 on apoptosis induction in A549 human nonsmall cell lung cancer cells. After treatment with KG-135 (50 μg/mL) alone or in the presence of HCQ (25μM) for 72 h, the sub-G1 apoptotic fraction was analyzed with flow cytometry. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

10 Fig. S2 The FOXO3a protein in A549 cells decreases with the culture time. The protein level of FOXO3a was analyzed using Western blot after treatment with KG-135 for 4 h, 24 h, and 72 h. FOXO3a, Forkhead Class box O3a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

11 Fig. S3 Effects of KG-135 in H460 human large-cell lung cancer cells. (A) After treatment with KG-135 for 72 h, Western blot was used to analyze the protein levels of LC3-I, LC3-II, Beclin-1, and p62. (B) The protein level of FOXO3a and (C) sub-G1 apoptotic fraction were analyzed after treatment with KG-135 alone or in the presence of hydroxychloroquine (HCQ) for 72 h. H460 cells were maintained and treated with drugs in the same condition as that for A549 cells. FOXO3a, Forkhead Class box O3a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Ginseng Research  , DOI: ( /j.jgr ) Copyright © Terms and Conditions

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