Sirolimus increases the anti-cancer effect of Huai Er by regulating hypoxia inducible factor-1α-mediated glycolysis in hepatocellular carcinoma

Figure 1 Expression of hypoxia inducible factor-1α, lactate dehydrogenase A, and glucose transporter 1 in hepatocellular carcinoma. A and B: Expression of hypoxia inducible factor-1 (HIF-1α) in poorly (A) and highly-moderately differentiated hepatocellular carcinoma (HCC) (B); C and D: Expression of lactate dehydrogenase A (LDHA) in poorly (C) and highly-moderately differentiated HCC (D); E and F: Expression of glucose transporter 1 (GLUT1) in poorly (E) and highly-moderately differentiated HCC (F); F: Expression of GLUT1 in high-moderately HCC, (Magnification: 200 ×, scale bar 10 µm); G-I: Comparison of HIF-1α (G), LDHA (H), and GLUT1 (I) in different groups by t test. Magnification: 200 ×, scale bar = 10 µm. ^aP < 0.001 vs poorly differentiated HCC; ^bP < 0.001 vs cancer; ^cP < 0.05 vs cancer.

Figure 2 Inhibitory effect of different drugs on HepG2 and Huh 7 cell invasion and proliferation as demonstrated by scratch and plate clone formation assays. A and B: Scratch images of HepG2 (A) and Huh7 cells (B) treated with different drugs; C and D: Effect of single drug treatment (C) and combination treatment (D) on HepG2 invasion; E and F: Effect of single drug treatment (E) and combination treatment (F) on Huh 7 invasion; G and H: Clonal size of HepG2 (G) and Huh 7 cells (H) treated with different drugs. ^aP < 0.05 vs control; ^bP < 0.01 vs control; ^cP < 0.001 vs control. H8: 8 mg/mL for Huai Er; S50: 50 nM for sirolimus; Ly25: Ly294002; KC7: 20 μM for KC7F2.

Figure 3 Effects of sirolimus and Huai Er on apoptosis of HepG2 and Huh 7 cells under normoxic and hypoxic conditions. A-F: Scatter diagrams of apoptosis of HepG2 cells intervened with sirolimus (SRL) (A), with Huai Er (B), with SRL under hypoxia (C), with Huai Er under hypoxia (D), with S50 + H8 under normoxia (E), and with S50 + H8 under hypoxia (F); G and H: Apoptosis rates of HepG2 cell at 24 h and 48 h under normoxia (G) and under hypoxia (H); I-N: Scatter diagrams of apoptosis of Huh 7 cells intervened with SRL (I), with Huai Er (J), with SRL under hypoxia (K), with Huai Er under hypoxia (L), with S50 + H8 under normoxia (M), and with S50 + H8 under hypoxia (N); O and P: Apoptosis rates of Huh 7 cells at 24 h and 48 h under normoxia (O) and under hypoxia (P). ^aP < 0.01 vs H8 + S50/hypoxia; ^bP < 0.001 vs H8 + S50/hypoxia; ^cP < 0.05 vs H8 + S50/hypoxia. H8: 8 mg/mL for Huai Er; S50: 50 nM for SRL.

Figure 4 Effects of sirolimus and Huai Er on cell cycle progression of HepG2 and Huh 7 cells under normoxic and hypoxic conditions. A-C: Peak graphs of apoptosis of control HepG2 cells (A) and those treated with sirolimus (SRL) (B) and Huai Er (C) under normoxia; D: HepG2 cell cycle distribution at 24 h and 48 h under normoxia; E-G: Peak graphs of apoptosis of control HepG2 cells (E) and those treated with SRL (F) and Huai Er (G) under hypoxia; H: HepG2 cell cycle distribution at 24 h and 48 h under hypoxia; I-K: Peak graphs of apoptosis of control Huh 7 cells (I) and those treated with SRL (J) and Huai Er (K) under normoxia; L: Huh 7 cell cycle distribution at 24 h and 48 h under normoxia; M-O: Peak graphs of apoptosis of control Huh 7 cells (M) and those treated with SRL (N) and Huai Er (O) under hypoxia; P: Huh 7 cell cycle distribution at 24 h and 48 h under hypoxia. ^aP < 0.01 vs control; ^bP < 0.001 vs control; ^cP < 0.01 vs hypoxia; ^dP < 0.001 vs hypoxia; ^eP < 0.05 vs control; ^fP < 0.05 vs hypoxia. H8: 8 mg/mL for Huai Er; S50: 50 nM for SRL.

Figure 5 Effects of sirolimus & Huai Er on expression of target genes under under normoxic and hypoxic conditions. A: The mRNA levels of Akt, p27, PTEN, FOXP3, and VEGF in cells treated with sirolimus (SRL) under normoxic condiction at 24 h and 48 h; B: The mRNA levels of Akt, mTOR, FOXP3, and VEGF in cells treated with Huai Er under normoxic condiction at 24 h and 48 h; C: The mRNA level of Akt, mTOR, VEGF, HIF-1α, LDHA, GLUT-1, FOXP3, p27, and PTEN in cells treated with SRL and Huai Er under hypoxia at 24 h and 48 h. ^aP < 0.05 vs control; ^bP < 0.05 vs control; ^cP < 0.01 vs hypoxia; ^dP < 0.001 vs hypoxia. PTEN: Phosphatase and tensin homolog deleted on chromosome ten; FoxP3: Forkhead box P3; VEGF: Vascular endothlial growth factor; mTOR: Mammalian target of rapamycin; HIF-1α: Hypoxia inducible factor-1α; LDHA: Lactate dehydrogenase A; GLUT-1: Glucose transporter 1.

Figure 6 Effect of intervention with LY49002 and KC7F2 on mRNA expression of target genes. A: Effect of LY294002 intervention on expression of different genes under normoxic condition; B: Effect of KC7F2 intervention on expression of different genes under normoxic condition. ^aP < 0.05 vs control; ^bP < 0.05 vs 24 h; ^cP < 0.05 vs control; ^dP < 0.01 vs control. mTOR: Mammalian target of rapamycin; VEGF: Vascular endothlial growth factor; FoxP3: Forkhead box P3; HIF-1α: Hypoxia inducible factor-1α; LDHA: Lactate dehydrogenase A; GLUT-1: Glucose transporter 1; PTEN: Phosphatase and tensin homolog deleted on chromosome ten.

Figure 7 Effect of single or combined drug intervention on target gene mRNA expression. A: Effects of LY294002 and KC7F2 on expression of different genes under hypoxia; B: Effect of S50 combined with Ly25 on expression of different genes. ^aP < 0.05 vs hypoxia; ^bP < 0.05 vs hypoxia; ^cP < 0.05 vs S50 + Ly25; ^dP < 0.01 vs S50 + Ly25. mTOR: Mammalian target of rapamycin; VEGF: Vascular endothlial growth factor; FoxP3: Forkhead box P3; HIF-1α: Hypoxia inducible factor-1α; LDHA: Lactate dehydrogenase A; GLUT-1: Glucose transporter 1; PTEN: Phosphatase and tensin homolog deleted on chromosome ten.

Figure 8 Effect of S50 combined H8 or KC7 intervention on target gene mRNA expression. A: Regulatory effect of S50 + H8 on expression of different genes under hypoxia condition; B: Effect of S50 + KC7 on expression of different genes. ^aP < 0.05 vs S50 + H8; ^bP < 0.01 vs S50 + H8; ^cP < 0.05 vs S50 + KC7; ^dP < 0.01 vs S50 + KC7. mTOR: Mammalian target of rapamycin; VEGF: Vascular endothlial growth factor; FoxP3: Forkhead box P3; PTEN: Phosphatase and tensin homolog deleted on chromosome ten.