Ketamine, an anesthetic, analgesic, or sedative, is widely used for the treatment of cancer pain. presented as the mean??SD (* em P /em ? ?0.05, ** em P /em ? ?0.01, *** em P /em ? ?0.001). GraphPad FK866 irreversible inhibition Prism was used to analyze experiment data (GraphPad Software, USA). Results Ketamine induces A549 cells apoptosis in a dose\dependent manner To evaluate whether ketamine induces apoptosis in A549 cells, the cells were exposed to ketamine (0, 1, 10, and 100? em /em mol/L) for 24?h. We found that 10 and 100? em /em mol/L ketamine induced a significant degree of apoptosis in A549 cells compared with the control group (Fig.?1A). FACS analysis confirmed that ketamine significantly induced the apoptosis of A549 cells at FK866 irreversible inhibition concentrations of 10 and 100? em /em mol/L (Fig.?1B and C). Assessing cell viability by trypan staining showed that 10 and 100? em /em mol/L ketamine induced more than 10C20% of the cells to die (Fig.?1D). Open in FK866 irreversible inhibition a separate window Figure 1 Ketamine induces apoptosis of A549 cells in a dose\dependent manner. (A) Morphology of cells treated with increasing concentration of ketamine. Scale bar represents 100? em /em m. (B) FACS analysis for detecting the increasing apoptosis proportion of cells treated by increasing concentration of ketamine. (C) Statistics of apoptosis proportion of cells treated by ketamine. (D) Trypan blue staining indicated the repression of cell viability in cells treated by ketamine. Data represent mean??SD of three independent experiments ( em n? /em = em ? /em 3, * em P? /em em ? /em 0.05, ** em P? /em em ? /em 0.01, *** em P? /em em ? /em 0.001). Ketamine upregulates the expression of CD69 in A549 cells We further found that CD69 could be upregulated by ketamine in A549 cells at both the mRNA (Fig.?2A) and protein levels (Fig.?2B). The expression of CD69 was FK866 irreversible inhibition clearly augmented as the concentration of ketamine was increased (Fig.?2A and B). We therefore analyzed data from lung cancer patient samples in The Cancer Genome Atlas (TCGA) database and found that CD69 was significantly decreased in cancer samples compared with normal tissues (Fig.?2C). Paired comparison of the normal and cancer samples also indicated the downregulation of CD69 in the cancers (Fig.?2D). The tumor, node, metastases (TNM) stage system is used to classify patients for clinical treatment or participation in clinical trials. We found that the expression level of CD69 was associated with the T stage, especially the T1, T2, and T3 stages, which indicated that low expression of CD69 was associated with an increase in tumors (Fig.?2E). Open in a separate window Figure 2 Effects of ketamine on cell growth inhibition of A549 cells. (A) Detection of CD69 mRNA level in A549 cells treated with 0, 1, 10, and 100? em /em m ketamine for 24?h, respectively. Data represent mean??SD of three independent experiments ( em n? /em = em ? /em 4, * em P? /em em ? /em 0.05, ** em P? /em em ? /em Isl1 0.01, *** em P? /em em ? /em 0.001). (B) The CD69 level detected by western blot analysis. (C) Expression of CD69 in the normal ( em n /em ?=?51) and cancer ( em n /em ?=?501) tissues. em P /em ?=?1.31eC29. (D) CD69 expression was lower in the cancer tissues compared with the normal tissues in same patients. em P /em ?=?7.90e?17. (E) RSEM analysis showed downregulation of the expression level of CD69 in cancer tissues compared with normal tissues and associated with the T stage. Overexpression of CD69 induces apoptosis of A549 cells We then focused our attention on the apoptosis\regulatory function of CD69. To confirm that CD69 crucially contributed to apoptosis, we transfected A549 cells with Fugw\CD69 to overexpress CD69. CD69 levels were significantly higher in transfected cells than in the control group (Fig.?3A). There were more apoptotic cells, which were small and showed shrinkage, in the CD69 overexpressing group (Fig.?3B). As shown in Figure?3C, overexpression of CD69 significantly induced apoptosis compared with the control group, as assessed by FACS. The regulatory effect of CD69 overexpression on the apoptosis rate and viability of A549 cells is shown in Figure? 3D and E. Open in a separate window Figure 3 CD69 induces the cell apoptosis in A549 cells. (A) Detection of CD69 mRNA level in control group and CD69 overexpression group by qPCR. (B) The morphology of cells observed by inverted phase\contrast microscope. Scale bar represents 100? em /em m. (C) FACS analysis of cell apoptosis and (D) statistics of cell apoptosis rate in CD69 overexpression group. (E). Cell viability of the.