Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro suppresses NSCLC cell proliferation. (A) Andro inhibited H1975 cell survival in a dose-time-dependent manner, as measured by MTT assessment; (B) Andro inhibited H1975 cell clone formation in a dose-dependent manner, as measured through the colony-formation assay. Colony formation capacities were determined by crystal violet staining and the magnification was 40× under optical microscope. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Colony Assay, Staining, Microscopy

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro exposure promotes NSCLC cell apoptosis. (A) Andro facilitated H1975 cell apoptosis in a dose-dependent manner, as measured through flow cytometry; (B) Andro enhanced cleaved caspase 9, cleaved caspase 8 , and c leaved caspase 3 expression in H1975 cells in a dose-dependent manner, as measured by WB. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; WB, western blotting.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro inhibits NSCLC cell proliferation by activating the mitochondrial apoptosis pathway. (A) Andro weakened ΔΨm in H1975 cells, as measured by JC-1 fluorescence. Scale bar: 20 µm; (B) Andro inhibited mitochondrial cyto C and cytoplasmic Bak expressions, and promoted cytoplasmic cyto C and mitochondrial Bak expressions in H1975 cells in a time-dependent manner, as evaluated through WB; (C) Andro enhanced Bax and Bak and inhibited Bcl-2 expression in H1975 cells in a dose-dependent manner, as evaluated through WB; (D) Transfection with Bak-siRNA significantly inhibited Bak mRNA and protein expressions in H1975 cells, as determined by qRT-PCR and WB, respectively; (E) Bak downregulation promoted H1975 cell proliferation, as measured through CCK-8 assay; (F) Bak downregulation enhanced ΔΨm in H1975 cells, as determined by the JC-1 fluorescence experiment. Scale bar: 20 µm; (G) Bak downregulation markedly inhibited apoptotic executive proteins (cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3) expression in H1975 cells, as measured by WB. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; ΔΨm, mitochondrial membrane potential; JC-1: mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide; cyto C, cytochrome C; Bcl-2, B-cell leukemia/lymphoma 2; Bak, Bcl-2-antagonist/killer (Bak); Bax, Bcl2-associated X; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mrna, messenger RNA; WB, western blotting; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; OD, optical density; NC, negative control; si, small interfering.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Fluorescence, Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Membrane, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Cell Counting, Negative Control

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro suppresses NSCLC cell proliferation by inducing the reprogramming of glucose metabolism. (A) Andro reduced the expression of PKM2, LDHA , and GLUT1 genes, and stimulated the expression of PEPCK1, FBP1 , and PFK genes in H1975 cells, as measured by qRT-PCR; (B) Andro inhibited glucose uptake, lactate production, and intracellular ATP synthesis in H1975 cells; (C) FBP1 -siRNA significantly inhibited FBP1 expression at both the mRNA and protein levels in H1975 cells, as measured by qRT-PCR and WB; (D) in H1975 cells, FBP1 downregulation promoted glucose uptake, lactate production, intracellular ATP synthesis; (E) FBP1 downregulation promoted H1975 cell proliferation, as evaluated through the CCK-8 assay; (F) FBP1 downregulation inhibited cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3 gene expression, as evaluated through WB. *, P<0.05; **, P<0.01. Andro, andrographolide; NSCLC, non-small cell lung cancer; PKM2 , pyruvate kinase M2; LDHA , lactate dehydrogenase A; GLUT1 , glucose transporter 1; PEPCK1 , phosphoenolpyruvate carboxykinase 1; FBP1 , fructose-1,6-bisphosphatase 1; PFK , phosphofructokinase; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; ATP, adenosine triphosphate; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; Mrna, messenger RNA; WB, western blotting; CCK-8, cell counting kit-8; OD, optical density; NC, negative control; si, small interfering.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Cell Counting, Negative Control