Figure 1. SPINK1 is identified as a candidate plasma marker for tumor hypoxia and potential therapeutic target for radiosensitization.

(A) HeLa cells were cultured under the indicated oxygen conditions for 24 hours and subjected to DNA microarray analysis. Of 34 genes that exhibited more than 10-fold induction upon hypoxia, the top 4 genes harboring the N-terminus signal peptide are listed. (B and C) HeLa cells were cultured under the indicated oxygen conditions for the indicated periods, and subjected to qPCR for the indicated genes. (D) After being cultured under the indicated oxygen conditions for 48 hours, cell lysates were subjected to qPCR. (E) Changes in the SPINK1 mRNA levels in HeLa cells were quantified at the indicated time points during (prehypoxia) and after (reoxygenation) the severe hypoxic treatment and represented as mean ± SD. (F) After the same treatment as in D, culture media were subjected to the ELISA assay. (G) Scatter plot for correlation analysis between SPINK1 mRNA levels and secreted SPINK1 protein levels in cells cultured under the indicated oxygen conditions for the indicated periods. (H–K) The indicated cells were transfected with either pcDNA4/SPINK1 (SPINK1) or its EV and cultured for 48 hours. Then, both culture media and cell lysates were subjected to Western blotting using the indicated antibodies (H and J), and then, cells were irradiated with the indicated doses of γ-rays and subjected to the clonogenic survival assay (I and K). The exogenously expressed SPINK1 was detected using anti-myc tag Ab (H and J). Data are represented as mean ± SD (B, D, F, I, and K; n = 3 in B–G, n = 6 in I and K). Two-tailed Student’s t test. *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.