In addition, IHC showed over-expression of CTSB was more likely to be present with poor differentiation of lung SCC. invasive activity of lung SCC. Conclusions These data suggested that CTSB might serve as a prognostic and therapeutic marker for lung SCC. by PCNA IHC analysis and TUNEL assay (Physique?4C). CTSB-shRNA treated tumor did not show significantly higher percentage of TUNEL-positive nuclei than tumors treated with PBS, Lipo, or NC group (6??1.3 versus 6??1, 7??1.5, or 7.5??1.6, 6??1.6, P? ?0.05). The rate of PCNA-positive nuclei in the four groups reached 93.4??6.42, 89.6??7.09, 85.6??9.73, 82.0??7.13 for PBS, Lipo, NC group and CTSB-shRNA, respectively (Determine?4D). Thus, no statistically significant difference between CTSB-shRNA treated group and the controls was observed in PCNA IHC and TUNEL assay. Reduced metastatic nodules and prolonged survival in mice bearing experimental lung metastatic tumors by CTSB-shRNA The migratory and invasive properties of cancer cells are crucial to tumor progression. We next investigated whether CTSB-shRNA could inhibit metastatic tumors in the lungs. As shown in Physique?5A, B, Mouse monoclonal to PRKDC metastatic nodules and lung weight were obviously reduced in CTSB-shRNA treated mice. The lung weight reached 0.6??0.158, 0.56??0.114, 0.56??0.152, 0.24??0.114 for PBS, Lipo, NC and CTSB-shRNA, respectively (P? ?0.05). Meanwhile, the treatment of CTSB-shRNA prolonged the survival of mice with lung metastasis (P? ?0.01) (Physique?5C). The results above exhibited CTSB influenced the metastatic capacity of lung cancer cells. Open in a separate window Physique 5 CTSB inhibited lung metastases em in vitro and in vivo /em . (A) The number of lung metastatic nodules was dramatically reduced in CTSB-shRNA-treated mice compared with Deruxtecan controls (a: PBS; b: Deruxtecan Lipo; c: NC; d: CTSB-shRNA). (B) The lung weight of mice reached 0.6??0.158, 0.56??0.114, 0.56??0.152, 0.24??0.114 for PBS, Lipo, NC, and CTSB-shRNA, respectively (P? ?0.05). The animal experiment was repeated three times. (C) Kaplan-Meier survival curves of tumor-bearing mice exhibited the treatment of CTSB-shRNA prolonged the survival of mice with lung metastasis (P? ?0.01). (D &E) CTSB-shRNA was effective in decreasing the invasive capacity of lung cancer cells (a: PBS; b: Lipo; c: NC; d: CTSB-shRNA). The invasive capacity of lung cancer cells decreased nearly 80% after treatment with CTSB-shRNA by quantitative analysis (P? ?0.05). Suppression of CTSB remarkably decreased the invasive capacity of lung cancer cell em in vitro /em After treated with PBS, Lipo, NC and CTSB-shRNA, the invasive capacity of A549 cells was determined by the matrigel invasion assay. The results showed that this invasive capacity of lung cancer cells decreased nearly 80% after treatment with CTSB-shRNA by quantitative analysis (Physique?5D, E). Up-regulation of CTSB, Shh and Ptch in metastatic lung SCC The metastatic lung SCC specimens were diagnosed histological after staining with H&E, and the clinical stage was decided according to the TNM classification system of the International Union against Cancer. Detailed information of the patients was shown in Physique?6A. Real-time quantitative RT-PCR and western blotting analysis were conducted to examine the expression level of CTSB, Shh and Ptch. As shown in Physique?6B, the mRNA expression level of CTSB, Shh and Ptch in metastatic lung SCC were significantly higher compared with non-metastatic lung SCC and adjacent normal tissues (p? ?0.05). Furthermore, the protein expression of CTSB, Shh and Ptch in metastatic lung SCC were significantly higher compared with non-metastatic lung SCC and adjacent normal tissues (p? ?0.05) (Figure?6C, D). Deruxtecan This data suggested that hedgehog signaling might be activated in metastatic lung SCC, which could affect expression of CTSB that could promote cancer cell invasion. Open in a separate window Physique 6 Up-regulation of CTSB, Shh and Ptch in metastatic lung SCC. (A) Detailed information of the patients with metastatic lung SCC. (B).