Table 1.
Patient characteristics and relative factors in 150 SCLC patients (Univariate Survival Analyses; log-rank test).
Figure 1.
The expression of ADAM-12 in small cell lung cancer tissue samples.
ADAM-12 was detected in small cell lung cancer tissue by immunohistochemistry staining. The dilution ratio of primary antibody was 1:200 for ADAM-12. (A) Normal lung tissue; (B) HE staining in SCLC tissue; (C) Negative control (incubated with PBS instead of primary antibody overnight at 4°C); (D) Staining for ADAM-12 in SCLC tissue.
Figure 2.
Survival curve by univariate survival analysis.
Patient survival time was obtained by follow-up. Univariate survival analysis was performed to assess whether ADAM-8, -10, -11, -12, -15 and -17 could be considered as independent prognostic factors. The result showed ADAM-12 to be an independent and adverse factor (P = 0.022).
Table 2.
Clinical and IHC variables influencing prognosis assessed by Multivariate survival analysis.
Figure 3.
The expression of ADAM-12 in serum and urine.
(A) ADAM-12 staining was strongly positive in mucus secretions (the region is indicated by the arrow). (B, C) An ROC curve was performed to assess the cut-off for patients with SCLC and healthy volunteers; ROC = receiver operating characteristics. (D, E) The expression levels of ADAM-12 in serum and urine from SCLC patients and normal controls were detected using an ELISA kit. ** P<0.01, SCLC patients vs. normal controls, SCLC patients with extensive disease vs. limited disease.
Figure 4.
Analysis of proliferation and metastasis in H1688 cells after interfering with ADAM-12 expression.
(A) The expression of ADAM-12 was higher in H1688 and H446 cells compared with H345 cells by Western blotting. (B) The mRNA and protein expression of ADAM-12 was significantly reduced when ADAM-12 expression was silenced by a specific siRNA targeting ADAM-12 (ADAM-12i) compared with untreated (Mock) and scrambled siRNA (NCi) in H1688 cells, ** P<0.01. (C) The cells induced by the serum filtering chamber membrane were significantly reduced in the ADAM-12i group compared with the Mock and NCi groups in H1688 cells, ** P<0.01. (D) The cell cycle was significantly arrested in the G0-G1 phase in the ADAM-12i group. Mock: untreated cells; NCi: the cells were treated with scrambled siRNA. ADAM-12i: the cells were treated with siRNA targeting ADAM-12.
Figure 5.
The functional analysis of ADAM-12-L and -S in H345 cells.
(A) The mRNA expressions of ADAM-12-L and -S were detected by real time PCR in H345 cells transfected with the expression vector of ADAM-12-L and -S, ** P<0.01. Mock: untreated group; pcDNA3.1: negative group. 12-L: transfected with pcDNA3.1-ADAM-12-L; 12-S: transfected with pcDNA3.1-ADAM-12-S. (B) The cells induced by the filtering chamber membrane were not significantly different in the H345 cells transfected with pcDNA3.1-ADAM-12-L (named H345-L), however the cells were significantly increased in H345 cells transfected with pcDNA3.1-ADAM-12-S (named H345-S), * P<0.05. (C) ADAM-12-L induced cells to go from the G0-G1 phase into the S phase, but ADAM-12-S did not influence cellular proliferation in H345 cells.
Figure 6.
ADAM-12 was up-regulated when the invasion and metastasis of SCLC cells was enhanced.
(A) A highly metastatic animal model was constructed, 1×106 cells were administered subcutaneously and the subcutaneous tumors (parental cells) were injected into nude mice via tail vein to form metastatic tumors (metastatic cells). (B) Morphological differences were observed between parental cells and metastatic cells via hematoxylin and eosin (HE) staining. Compared with parental cells, metastatic cells were smaller, had shoaled cytoplasm staining, deeper nuclear staining, thinner cell density and weaker cell compact. (C) There was no difference in ADAM-12-L mRNA expression between parental and metastatic cells, however ADAM-12-S mRNA was significantly higher in metastatic cells, * P<0.05. (D) ADAM-12 protein expression was significantly increased in metastatic cells by IHC and western blotting. P1-P5 indicates the five subcutaneous tumors and M1-M5 indicates the five metastatic tumors.