Hypoxia induces tumor cell growth and angiogenesis in non-small cell lung carcinoma via the Akt-PDK1-HIF1α-YKL-40 pathway

Figures

• 

  Figure 1. Changes in protein expression in CL1-5 cells under hypoxia. (A) Western blots showing the expression of HIF1α changed over time under hypoxic or normoxic exposure. Β-actin set as internal reference. (B) Representative Western blots pictures; total Akt1, Akt2, and PDK1 showed little difference in a hypoxic or normoxic environment. Cox4 set as internal reference of mitochondrial proteins. (C) A representative Western blot picture of cytoplasm proteins setting β-tubulin as reference. (D) ELISA’s data of mass of TGF-β, YKL-40, and VEGF are depicted as the mean of three independent experiments measured in triplicate ± SEM (**, P<0.01).

• 

  Figure 2. The accumulation of Akt-PDK1 promoted HIF1α expression. (A) The Western blot test of mitochondrial proteins showing that the levels of Akt1, Akt2, and PDK1 phosphorylation increased in a hypoxic environment, and Akt1 siRNA inhibited the expression of Akt1 and its phosphorylation and PDK1 phosphorylation, as did Akt2. Cox4 set as internal reference of mitochondrial proteins. (B) Band map of CL1-5 cells protein expression in each group; control, transfected by blank vector + normoxic culture; PDK1 OE, transfected by PDK1 overexpression vector + normoxic culture; control + hypoxia, transfected by blank vector overexpress + hypoxia culture; PDK1 OE + hypoxia, transfected by PDK1 overexpression vector + hypoxia culture. (C) Band map of cell protein expression under hypoxia; control, CL1-5 cells+ transfected by blank vector; PDK1 OE, CL1-5 cells + transfected by PDK1 ion vector. **, P<0.01. (D) Β-actin set as cytoplasmic internal reference. The levels of HIF1α, YKL-40, and p-Smad3 markedly increased under hypoxia, and Akt1/Akt2 siRNAs inhibited the expression of downstream proteins PDK1, HIF1α, YKL-40, and p-Smad3. (E) ELISA assays of TGF-β/YKL-40/VEGF mass in liquid supernatant. The…

• 

  Figure 3. The accumulation of Akt-PDK1 induced malignant phenotype of CL1-5 cells under hypoxia. (A) CCK-8 assay result shows viability of CL1-5 cells. Representative images of (B) migration, (C) invasion, (D) tube-formation assays showing the positive effects of hypoxia and the negative effects of Akt1/Akt2 siRNA. Scale bars =100 µm (B,C), 200µm (D) Quantitative results for migration (B), invasion (C) data have been shown. Dates are depicted as the mean of three independent experiments measured in triplicate ± SEM (**, P<0.01).

• 

  Figure 4. HIF1α is responsible for expression and secretion of YKL-40 in CL1-5 cells. (A) Band map of CL1-5 cells protein expression in each group; control, transfected by blank vector + normoxic culture; HIF1α OE, transfected by HIF1α overexpression vector + normoxic culture; control + hypoxia, transfected by blank vector + hypoxia culture; HIF1α OE + hypoxia, transfected by HIF1α overexpression vector + hypoxia culture. (B) Band map of cell protein expression. (C) Band map of CL1-5 cells protein expression under hypoxia in each group. (D) ELISA assays of TGF-β/YKL-40/VEGF mass in liquid supernatant of each group. (E) ELISA assays of TGF-β/YKL-40/VEGF mass in liquid supernatant of each group. **, P<0.01.

• 

  Figure 5. YKL40 is a key mediator of the Akt-PDK1-HIF1α signaling pathway. (A) Western blot band map of CL1-5 cells proteins expression under hypoxia in each group. (B) ELISA assays of YKL-40 and VEGF mass in liquid supernatant of each group. (C) CCK-8 kit assay result shows viability of CL1-5 cells. Representative images of (D) migration, (E) invasion, (F) tube-formation assays. Scale bars =100 µm (D,E), 200 µm (F) Quantitative results for migration (D), invasion (E) data have been shown. Dates are depicted as the mean of three independent experiments measured in triplicate ± SEM (**, P<0.01).

• 

  Figure 6. Growth of xenograft of NSCLC could be targeted by Akt inhibition but rescued by YKL-40 expression. (A) Tumor volume on day 32 after subcutaneous transplant of cells in mice; (B) tumor volume increased with time; one-way analysis of variance analysis is used in data statistics, and the error line denotes SD; (C) tumor weight on day 32 in mice after tumor cell transplant. **, P<0.01.

• 

  Figure 7. YKL-40-induced angiogenesis and tumor proliferation in vivo. (A) WB band map of several total protein factors in different groups of mice tumor cells. (B) Representative Ki67 immunostained tumor tissue slice; Ki67 positive cell ratio of tumor cells. (C) Representative vWF immunostained tumor tissue slice. Scale bars =50 µm (B,C). **, P<0.01.

• 

  Figure 8. Model illustrating hypoxia-induced tumor malignancy and angiogenesis that is mediated by Akt-PDK1-HIF1α pathway.

• 

  Figure S1. Relative Akt1/Akt2 mRNA expression in CL1-5 cells that were interfered by 3 different artificial siRNAs or the control siRNA (control siRNA =1.0); employing qRT-PCR to verify the effects. Akt1-siRNA2 and Akt2-siRNA3 were chosen as Akt1-siRNA and Akt2-siRNA to participate in the following experiments. **, P<0.01 vs. NC.

• 

  Figure S2. Akt2 shRNA and PDK1 OE mRNA expression verified by qRT-PCR. (A) Relative Akt1/Akt2 mRNA expression in Akt1/Akt2 shRNA lentiviral vector stably transfected CL1-5 cells; control, without any treatment; scramble shRNA, transfected by scramble shRNA interfering plasmid; Akt1/Akt2 shRNA, transfected by Akt1/Akt2 shRNA interfering plasmid (scramble shRNA =1.0) Akt2 shRNA stably transfected CL1-5 cell was effective (P<0.01) (B) Relative PDK1 mRNA expression in CL1-5 cells. It was confirmed that PDK1 OE transfection succeeded; blank, without any treatment; NC, negative control, transfected by blank vector; PDK1 OE, transfected by PDK1 overexpression vector. **, P<0.01 vs. NC.

• 

  Figure S3. HIF1α and YKL-40 OE mRNA expression verified by qRT-PCR. (A) Relative HIF1α mRNA expression in CL1-5 cells; blank, without any treatment; NC, negative control, transfected by blank vector; HIF1α OE, transfected by HIF1α overexpression vector. (B) Relative YKL-40 mRNA expression in CL1-5 cells confirmed by qRT-PCR; YKL-40 OE, transfected by YKL-40 overexpression vector. **, P<0.01 vs. NC.