Journal: Cancer Management and Research

Article Title: Allylated Curcumin Analog CA6 Inhibits TrxR1 and Leads to ROS-Dependent Apoptotic Cell Death in Gastric Cancer Through Akt-FoxO3a

doi: 10.2147/CMAR.S227415

Figure Lengend Snippet: CA6 induces ER stress and gastric cancer cell apoptosis. ( A ) BGC-823 and SGC-7901 cells were pretreated with NAC (5 mM) for 2 h before exposure to CA6 for 24 h. Percentage of cell apoptosis was determined by Annexin-V/PI staining. Representative dot plots shown [n = 3]. ( B, C ) BGC-823 (B) and SGC-7901 ( C ) cells were challenged with CA6 (15 µM) for indicated time points. Levels of ER stress-related proteins p-eIF2α, ATF4, and CHOP were determined. Curcumin was used as a positive control at the dose of 20 µM. GAPDH was used as the loading control. Representative blots shown. ( D, E ) BGC-823 ( D ) and SGC-7901 ( E ) cells were exposed to CA6 at the dose of 5, 10 and 15 µM for 6 h. Where indicated, NAC pretreatment (5 mM) was carried out for 2 h. Cells treated with curcumin (20 µM) for 12 h were used as the positive control. Protein levels of p-eIF2α, ATF4 and CHOP were determined. ( F ) BGC-823 cells were transfected with siRNA against ATF4. Cells were then exposed to CA6 (15 μM) for 6 h. Levels of ATF4 were determined. GAPDH was used as loading control. Representative immunoblot (upper panel) and densitometric quantification (lower panel) is shown [Mean ± SEM; n = 3; ** P <0.01]. ( G ) BGC-823 cells were exposed to CA6 (15 μM) for 12 h, with or without pretreatment with NAC (5 mM) for 2 h. The activity of caspase-9 was determined [Mean ± SEM; n = 3; ** P <0.01, *** P < 0.001]. ( H, I ) Flow cytometric analysis of apoptotic cells following knockdown of ATF4 and exposure to CA6. BGC-823 cells transfected with ATF4 siRNA were exposed to CA6 (15 µM) for 24 h. Dot plots ( H ) and quantification of apoptotic cells ( I ) are shown [Mean ± SEM; n=3; * P < 0.05, ** P < 0.01, *** P < 0.001].

Article Snippet: Cell cycle and apoptosis detection kits, Annexin V/Propidium iodide (PI) were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Staining, Positive Control, Transfection, Western Blot, Activity Assay

Journal: Cancer Management and Research

Article Title: Allylated Curcumin Analog CA6 Inhibits TrxR1 and Leads to ROS-Dependent Apoptotic Cell Death in Gastric Cancer Through Akt-FoxO3a

doi: 10.2147/CMAR.S227415

Figure Lengend Snippet: CA6 induces apoptosis in gastric cancer cells through ROS-dependent modulation of AKT and FOXO3a. ( A ) BGC-823 cells were treated with CA6 (15 μM) for indicated time points. Curcumin was used at the dose of 20 µM as positive control. Levels of phosphorylated Akt (Thr308 and Ser473) and PTEN were determined. GAPDH was used as loading control. Representative immunoblots shown [n = 3]. ( B ) Cells treated as indicated in panel A were probed for levels of phosphorylated-FoxO3a and cleaved-PARP. ( C ) BGC-823 cells were exposed to CA6 (15 μM) for 12 h, with or without pretreatment with NAC (5 mM) for 2 h. Levels of p-Akt473, p-Akt308, PTEN, and cleaved-PARP were determined. Representative immunoblots shown [n = 3]. ( D ) Cytosolic and nuclear fractions from BGC-823 cells treated as indicated in panel C. Protein levels of FoxO3a were examined. GAPDH and Lamin B1 were used as loading control. Representative blots shown in the upper panel and densitometric quantification are shown in the lower panel [Mean ± SEM; n = 4; * P < 0.05; *** P < 0.001].

Article Snippet: Cell cycle and apoptosis detection kits, Annexin V/Propidium iodide (PI) were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Positive Control, Western Blot