Effect of apigenin in combination with oxymatrine on non-small cell lung cancer and mechanism / 中国中药杂志

This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 μmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.

Active fractions of Camellia nitidissima inhibit non-small cell lung cancer via suppressing epidermal growth factor receptor / 中国中药杂志

The present study explored the effects and its underlying mechanisms of four active fractions of Camellia nitidissima(leaf polyphenols, leaf saponins, flower polyphenols, and flower saponins in C. nitidissima) in inhibiting the proliferation and migration of non-small cell lung cancer(NSCLC) by suppressing the epidermal growth factor receptor(EGFR). MTT assay was used to detect the effect of four active fractions on the proliferation of NCI-H1975 and HCC827 cells. Wound healing assay and Transwell assay were adopted to evaluate the effect of four active fractions on the migration of NSCLC. The effect of four active fractions on the enzyme activity of EGFR was detected. Molecular docking was carried out to explore the direct action capacity and action sites between representative components of the four active fractions and EGPR. Western blot assay was employed to investigate the effect of four active fractions on the protein expression in EGFR downstream signaling pathways. The results of the MTT assay indicated that the cell viability of NCI-H1975 and HCC827 cells was significantly inhibited by four active fractions at 50, 100, 150, and 200 μg·mL~(-1) in a dose-dependent manner. Wound healing assay and Transwell assay revealed that the migration of NCI-H1975 and HCC827 cells was significantly suppressed by four active fractions. In addition, the results of the protein activity assay showed that the enzyme activity of EGFR was significantly inhibited by four active fractions. The molecular docking results confirmed that various components in four active fractions possessed strong binding activity to EGFR enzymes. Western blot assay revealed that four active fractions down-regulated the protein expression of EGFR and its downstream signaling pathways. It is concluded that the four active fractions of C. nitidissima can inhibit NSCLC. The mechanism may be related to EGFR and its downstream signaling pathways. This study provides a new scientific basis for the clinical treatment of NSCLC with active fractions of C. nitidissima, which is of reference significance for further research on the anti-tumor mechanism of C. nitidissima.

Construction and verification of anti-MM scFv-tP fusion protein expression vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an expression vector of anti-MM scFv-tP fusion protein and test its expression efficiency and function.</p><p><b>METHODS</b>The truncated protamine (tP) gene sequence was added to the gene of single chain antibody against the specific antigen on the surface of malignant melanoma tumor cells using PCR. A GST-fusion expression vector was constructed and the soluable protein was expressed in the E.coli system. After cleavage and purification, the purified fusion protein was obtained. The binding activity of Anti-MM scFv-tP and siRNA was detected by EMSA. Flow cytometry and confocal microscopy were used to detect the cell surface antigen binding activity of the fusion protein.</p><p><b>RESULTS</b>The expression vector of Anti-MM scFv-tP fusion protein was successfully constructed. The soluable protein could be expressed in the E.coli system, and the purified fusion protein was obtained. The anti-MM scFv-tP fusion protein retained siRNA binding ability and could directly target malignant melanoma (MM) LiBr cells.</p><p><b>CONCLUSION</b>The recombinant GST- Anti-MM-scFv-tp expression vector was successfully constructed. The fusion protein retains siRNA binding ability and can directly target LiBr cells to provide a reliable tool for further study.</p>

5-Fu activates NKG2D ligands MICA/B promoter in transiently transfected A549 cell line / 军事医学科学院院刊

Objective:To analyze the activities of human NKG2D ligand MICA/B promoter induced by 5-Fu.Methods:The 5'-end flanking regions of MICA /B promoter and their different truncated fragments were amplified from A549 genome by PCR. The resulting amplicons were cloned into pGL3-Basic vector to generate the MICA/B luciferase reporter plasmids. All the constructs were transiently transfected A549 cells. The promoter region activities were determined by dual-luciferase reporter assays. The effect of 5-Fu on the promoter activities of MICA/B was also tested.Results and Conclusion:The 5'-end flanking regions of MICA /B promoter and five of their different truncated fragments were successfully obtained. The normalized luciferase reporter gene activities driven by the above promoters and fragments were 3.61,2.26,1.63,0.313,0.711 and 0.663 for MICA and 17.49,10.11,7.398,0.822,0.997 and 0.49 for MICB,respectively. Promoter activities in transiently transfected A549 cells treated by 20,40,80,160 and 320 μg/m of 5-Fu increased 1.69,1.48,1.62,1.55 and 1.78 fold for MICA and 1.44,1.87,1.38,1.19 and 1.25 fold for MICB. Our results suggest that 5-FU can significantly up-regulate the promoter activity of both MICA and MICB.

CD147 increases invasiveness of U937 cells through regulation of matrix metalloproteinase activity / 中国实验血液学杂志

This study was purpose to investigate the effects of CD147 on the invasiveness of leukemia cells U937. The experiments were divided into 4 groups: control group, LPS group, CD147mAb group and LPS+CD147 mAb group. Cells were treated by lipopolysaccharide (LPS) or anti-CD147 monoclonal antibody, and the expression of CD147 and MMP-2, -9, the invasive potential of the cells in vitro and ex vivo, as well as the invasion of the implanted tumors in SCID mice were analysed by RT-PCR, FCM, gel zymography and invasion test in vitro respectively. The results showed that the expression of CD147 was elevated by the induction of LPS, and the enhanced expression of CD147 on U937 cells increased the production and secretion of MMP-2 and MMP-9 as measured by reverse transcription-PCR and gel zymography. An increased number of LPS-induced cells invading through a reconstituted basement membrane were observed by invasion assays. These responses were down-regulated after blocking CD147 with anti-CD147 antibody. At 30 days after intravenous injection of LPS pretreated U937 cells to SCID mice human U937 cells were found in the bone marrow and lung of the mice, indicating the invasion of the tumor cells. And overexpressions of CD147, MMP-2 and MMP-9 were found in the lung tissue of the mice injected with LPS-treated but not anti-CD147 antibody treated tumor cells. It is concluded that overexpression of CD147 on U937 cells may increase the secretion and activation of MMP-2 and MMP-9 and thus promote the invasiveness of the tumor cells.

Protective effect of sodium pyruvate on ischemia/reperfusion injury of rats subjected to hemorrhagic shock / 中国应用生理学杂志

<p><b>AIM</b>To study the protective effect of sodium pyruvate on ischemia/reperfusion injury following hemorrhagic shock.</p><p><b>METHODS</b>Rat models of hemorrhagic shock were built up. When the shed blood was infused, the rats were also randomly provided by one of normal saline, glutathione and sodium pyruvate. Rats were killed 3 hours after the reperfusion, the activity of plasma lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT), the level of tissue malondialdehyde (MDA) and the activity of tissue myeloperoxidase (MPO) were detected. Biopsy specimens were obtained to investigate morphological changes of the myocardial, hepatic, lung and renal tissue.</p><p><b>RESULTS</b>The activity of plasma LDH and GOT, the level of MDA of hepatic, lung and renal tissue and the activity of MPO of myocardial, lung and renal tissue decreased remarkably in group given sodium pyruvate compared with group given normal saline, and the effect of group given sodium pyruvate was more remarkable than group given glutathione.</p><p><b>CONCLUSION</b>These data support the view that sodium pyruvate shows protective effect on ischemia/reperfusion injury following hemorrhagic shock. It is possibly relevant to scavenging of oxygen free radicals, reduction of neutrophil, and anti-inflammatory response.</p>

Analysis of Human Platelet Antigen Genotypic Frequencies in Chinese Population by PCR Amplification with Sequence Specific Primers / 中国实验血液学杂志

In present study, a method for genotyping for human platelet antigen (HPA) systems by means of the polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) technique was developed and employed to determine the human platelet antigen frequencies in the Chinese population. Primers sets were designed to allow PCR amplification for five systems using the same assay conditions. Platelets from 110 random Chinese blood donors were typed for human platelet alloantigens HPA-1 to -5 by the method. The results showed that the HPA genotypic frequencies observed in the 110 donors were 0.91 and 0.09 for HPA-1a and HPA-1b, 0.86 and 0.14 for HPA-2a and HPA-2b, 0.60 and 0.40 for HPA-3a and HPA-3b, 0.92 and 0.08 for HPA-4a and HPA-4b, and 0.85 and 0.15 for HPA-5a and HPA-5b, respectively. In conclusion the method is feasible and practical and may be available to typing for HPA in the clinical laboratories.