protein levels of PI3K/Akt/ HIF-1 pathway and key enzymes involved in glycolysis

 
X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig. 5. SSL6 suppresses glycolysisvia blocking CD47/PI3K/Akt/HIF-1 signaling pathway. (A) After 72 h of SSL6 treatment (10 μg/mL), protein levels of PI3K/Akt/ HIF-1 pathway and key enzymes involved in glycolysis in Huh-7 were determined. (B) After 72 h of SSL6 treatment (10 μg/mL), protein levels of PLCγ and phosphorylated-PLCγ were assessed. (C) Huh-7 cells were treated with SFN (5 μM) and/or SSL6 (10 μg/mL) for 72 h, the expression of PKA, AMPK and phos- phorylated-AMPK were examined. 

(D,E) After knockdown of CD47, protein levels of PI3K/Akt/HIF-1 pathway and key enzymes in glycolysis in Huh-7 (D) and MHCC97H (E) cells. (F) Flow cytometry detection of cell death after Huh-7 cells were pre-treated with LY294002 (20 μM) or 2-DG (2.5 mM) for 12 h, SFN (5 μM) and/orSSL6(10μg/mL)wereadministratedfor72h.(G,H)Huh-7cellswerepre-treatedwithLY294002(20μM),theintracellularlactatelevels(G)andthemRNA expression of CD47,HIF1A and key enzymes involved in glycolysis (H) were determined. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.NS denotes no significance. for SFN-induced CD47 but SFN might also inhibit CD47 via a PI3K- 3.6. SSL6reversesthesensitivityofSFN-resistant(SFN-R)HCCcells independent pathway when PI3K was blocked. Together these results illustrate that SSL6 down-regulates glycolysis and sensitizes HCC cells We next wondered whether SSL6 could re-sensitize SFN-R HCC to SFNvia CD47/PI3K/Akt/HIF-1 signaling pathway. cells. We established SFN-R Huh-7 and MHCC97H cells by continuous induction with low concentration of SFN for more than 1 month (The 39
X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig. 6. SSL6 re-sensitizes SFN-resistant (SFN-R) HCC cells to SFN.

 (A) Schematic models of SFN-R HCC cell induction. (B) IC50 of control and SFN-R Huh-7 and MHCC97H cells. (C)CD47 mRNA expression in control and SFN-R HCC cells (Huh-7 and MHCC97H). (D) Flow cytometry detection of CD47 expression on control and SFN-R HCC (Huh-7 and MHCC97H). (E) Western-blot of CD47 with control and SFN-R MHCC97H cells. (F) Lactate levels in control and SFN-R HCC cells (Huh-7 and MHCC97H). (G) SFN-R Huh-7 cells were treated with SFN (7.5 μM) and/or SSL6 (10 μg/mL) for 72 h, the cell death were de###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-4.png####termined with flow cytometry. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. IC50 values for SFN in Huh-7 and Huh-7 SFN-R cells were 4.720 and (Fig. 6C Huh-7 cells: the increase percentage of SFN-Rvs. control was 7.060 μM, respectively; in MHCC97H and MHCC97H SFN-R cells were 62 Cell Counting Kit-8 glpbio.64%,p=0.0092;MHCC97Hcells:theincreasepercentageofSFN-R 8.31 and 16.28 μM, respectively) (Fig. 6A and B). These SFN-R cells vs. control was 34.39%, p = 0.0006. Fig. 6D Huh-7 cells: the increase showed increased CD47 expression and elevated glycolysis levels percentage of SFN-R vs. control was 88.99%, p < 0.0001; MHCC97H 40
X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig. 7. SSL6 and SFN synergistically attenuate xenograft HCC growth in vivo. 

(A) Protocol of in vivo experiment model BCI-121. (B) Curves of tumor growth in groups of control (PBS), SSL6 (10 mg/kg), SFN (2 mg/kg), and SFN (2 mg/kg) plus SSL6 (10 mg/kg). (C) Tumor entity view from groups of control (PBS), SSL6 (10 mg/kg), SFN(2mg/kg),andSFN(2mg/kg)plusSSL6(10mg/kg).(D)IHCforCD47expressionintumorscontrol(PBS),SSL6(10mg/kg),SFN(2mg/kg),andSFN(2mg/kg) plus SSL6 (10 mg/kg) 3x flag peptide. cells: the increase percentage of SFN-R vs. control was 35.04%, percentage of SFN vs. control was 177.85%, p = 0.0018; the increase p = 0.0008) (Fig. 6C–F and Supplementary Fig. 4A). In addition, SSL6 percentage of SSL6vs. control was 108.27%, p = 0.0191; the increase promoted the death of SFN-R Huh-7 and MHCC97H cells, as well as percentage of SFN + SSL6vs. SFN was 55.65%, p = 0.0054) (Fig. 6G enhanced SFN-induced cell death (Fig. 6G Huh-7 SFN-R: the increase and Supplementary Fig. 4B). 41

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