Objective To explore the effect of dendritic cells (DCs) allergized by K-ras mutant peptide on expressions of chemokines CCL19, CCL22, and cytoskeletal protein fascin-1. Methods DCs were derived from peripheral blood in the presence of granuloceyte/macrophage-colony stimulating factor, interleukin (IL) -4 in vitro. The DCs were collected on day 7 after culture, and were divided into non-K-ras mutant peptide group (addition of RPMI 1604 culture solution 50 μg/ml) and K-ras mutant peptide group (addition of K-ras mutant peptide 50 μg/ml). Phenotype was identified by flow cytometry. The morphological structure was observed by scanning and transmission electron microscopies, respectively. The expressions of IL-12, CCL19, and CCL22 were tested continuously by enzyme-linked immunosorbent assay (ELISA). The expression of cytoskeletal protein fascin-1 was determined by Western blot. Results ①The expressions of CD1a, CD80, and CD86 after loading K-ras mutant peptide were higher than that before loading K-ras mutant peptide (Plt;0.01). ②The DCs with petal-like and branch-like profections after loading were observed under scanning electron microscopy; The DCs with irregular shapes, branch-like or burr-like were showed under transmission electron microscopy. ③The expressions of IL-12, CCL19, and CCL22 in the Kras mutant peptide group were higher than those in the non-K-ras mutant peptide group at different times (6, 12, 24, and 48 h) after loading Kras mutant peptide (Plt;0.01). ④The expression of fascin-1 in the K-ras mutant peptide group was also higher than that in the non-K-ras mutant peptide group (Plt;0.01). Conclusion K-ras mutant peptide can promote DC to mature and improve the expression of chemokines and cytoskeletal protein which will strengthen DC migration.
To study the effect of the proliferation and apoptosis of Survivin-T34A mutant on breast cancer MCF-7 cell, we adopted the method of cell culture in vitro to observe the proliferation and apoptosis of the cell. In the experiment, MCF-7 cells were randomly divided into three groups and transfected with normal saline, PORF-9-null and Survivin-T34A, respectively. Breast cancer nude mouse models were established to study anti-tumor effect of Survivin-T34A in vivo. The activity of the cells in the Survivin-T34A-transfected group was lower than that in PORF-9-null group. The increase of cell apoptosis was observed under electron microscopy, meanwhile the apoptotic rate was obviously higher than that in PORF-9-null control by flow cytometry. Tumor inhibition effects of the mouse which received the injection of Survivin-T34A intratumoral injection were apparent, and the inhibition ratio was as high as 47.1%. In conclusion, Survivin-T34A mutant has anti-tumor effect through efficiently inhibiting the growth of breast cancer MCF-7 cell and actively promoting apoptosis of cancer cells.
Survivin-D53A (SVV-D53A) is a dominant-negative mutant survivin, which represents a potential promising target for cancer gene therapy. The present study was designed to determine whether SVV-D53A plasmid encapsuled by DOTAP: Chol liposome would have the anti-tumor activity against SPC-A1 lung adenocarcinoma, and to detect the possible mechanisms. In our experiment, SPC-A1 cells were transfected in vitro with SVV-D53A plasmid and examined for protein expression by Western blot, then flow cytometric analysis was used to detect apoptosis. SPC-A1 lung adenocarcinoma xenografts were established in vivo in the nude mice, which received the i.v. administrations of SVV-D53A plasmid/liposome complexes. After mice were sacrificed, the paraffin-embedded tumor tissue sections were used for proliferating cell nuclear antigen (PCNA) expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)assay. Compared with the control group, the mice treated with SVV-D53A plasmid had an obviously reduced tumor volume, with high level of apoptosis and decreased cell proliferation in tumor tissue. The research results proved that the administration of SVV-D53A plasmid resulted in significant inhibition of SPC-A1 cells both in vitro and in vivo. The functional mechanism is that the anti-tumor response causes and induces tumor cell apoptosis.
Objective To investigate the regulatory effects of SHP2 inhibition on the secretion of macrophage-associated inflammatory factors in KRAS-mutant lung cancer cells and to elucidate the underlying mechanisms by which this inhibition remodels the tumor immune microenvironment. Methods Three KRAS-mutant lung cancer cell lines were treated with the SHP2 inhibitor SHP099. The levels of phosphorylated SHP2 and ERK were assessed by Western blot. The expression levels of related inflammatory factors were analyzed using Luminex assay and qRT-PCR assay. Transcriptome sequencing was performed to identify differentially expressed genes and conduct KEGG pathway enrichment analysis. The expression of CXCL8 was validated by flow cytometry and Western blot. Survival analysis and gene set correlation analysis were conducted based on the TCGA database. Results SHP099 significantly inhibited the expression of p-SHP2 and p-ERK proteins, and reduced the secretion of multiple macrophage-related inflammatory factors. qRT-PCR confirmed a decrease in CXCL8 mRNA levels. Transcriptome analysis revealed significant enrichment of the rheumatoid arthritis pathway. Flow cytometry and Western blot validated a significant reduction in CXCL8 protein expression. Survival analysis showed that patients with KRAS-mutant lung adenocarcinoma and high CXCL8 expression had a shorter overall survival, and CXCL8 was positively correlated with M2 macrophage marker genes. Conclusion Targeted inhibition of SHP2 can suppress the expression of some macrophage-related inflammatory factors in KRAS-mutant lung cancer cells, with the most significant inhibition of CXCL8 expression. The mechanism may involve SHP2 regulating the transcription factor AP-1.