Aim To investigate the effects of classic calcium antagonists verapamil(Ver),nifedipine(Nif),diltiazem(Dil)and the novel calcium antagonist N-n-butyl haloperidol iodide(F2)which was synthesized by our lab by regulating Ca2+-independent phospholipase A2(iPLA2)on hypoxia/reoxygenation(H/R)injury of cardiac microvascular endothelial cells(CMECs)and the mechanisms.Methods The CMECs were isolated from SD neonatal rats.The H/R model was established,then cells were treated with different concentrations of calcium antagonists and F2.The content of LDH in the cell supernatant was measured by colorimetric method.The levels of IL-6 and AA in cell supernatant were measured by ELISA;and late-stage apoptosis was measured by TUNEL.The mRNA and protein expression levels of iPLA2 in CMECs were examined by real time-PCR and Western blot analysis.Results Calcium antagonists except Dil decreased the generation of LDH,IL-6 and AA in a dose-dependent manner(P<0.05),and reduced the apoptosis(P<0.05).F2 and Ver decreased the mRNA and protein expression of iPLA2 in a dose-dependent manner,while there were no such effects for Nif and Dil.Conclusions Calcium antagonists except Dil have protective effects against H/R injury.F2 and Ver protect CMECs against H/R injury partly through iPLA2.

Aim To investigate the effect of N-n-butyl haloperidol iodide(F2) on mitochondria-dependent apoptotic pathway of H9c2 cardiac myocytes during hypoxia/reoxygenation(H/R) injury.Methods The H/R models of H9c2 cardiac myocytes were established.The H9c2 cardiac myocytes were randomly divided into five groups: control group(C group), hypoxia/reoxygenation group(H/R group), F2 low concentration group(L), F2 medium concentration group(M), F2 high concentration group(H).Apoptotic rate was evaluated by flow cytometry(FCM).The levels of Cyto C, Bcl-2, Bax were observed by Western blot.Caspase-3 activity was measured with colorimetry.Results Compared with H/R group, F2 low, medium and high concentrations group could significantly decrease apoptosis rate and increase the ratio of Bcl-2 to Bax proteins and inhibit the release of Cyto C into the cytosolic fraction, and decrease caspase-3 activity.Conclusion F2 can protect H9c2 cardiac myocytes against H/R-induced injury through interfering in mitochondria-dependent pathway.

BACKGROUNDIt has been known that Kai1 is one of transmembrane 4 superfamily regulating cell proliferation, adhesion and mobility and its down-regulated expression is closely correlated with progression and prognosis of tumor. Fas ligand (FasL) is one of tumor necrosis factor/nerve growth factor family activating caspase-3, a key proteinase in cell apoptosis and leading immune escape in tumor cells. The aim of this study is to investigate the expression of Kai1 and FasL in non-small cell lung cancer (NSCLC) and to explore their roles and relationship in carcinogenesis and progression of NSCLC.

METHODSKai1 and FasL expressions were examined in 79 NSCLC tissues and their adjacent normal lung tissues by SP immunohistochemistry. Their expressions were compared with clinicopathological features of NSCLC. The relationship between Kai1 and FasL expressions was also analyzed in NSCLC.

RESULTSKai1 expression in NSCLC tissue was remarkably lower than that in their adjacent tissue (55.7% vs 82.6%, P < 0.05). However, it was the converse for FasL (73.4% vs 52.2%, P < 0.05). Kai1 expression was not correlated with age and gender of NSCLC patients, tumor location or histological classification (P > 0.05), but negatively with lymph node metastasis and clinicopathological staging and positively with differentiation degree (P < 0.05). FasL expression was not correlated with age and gender of the patients, tumor location or histological classification (P > 0.05), but positively with lymph node metastasis and negatively with differentiation degree (P < 0.05). Kai1 expression was closely correlated with FasL expression in NSCLC (P < 0.05).

CONCLUSIONSDown-regulation of Kai1 expression and up-regulation of FasL may play important roles in carcinogenesis and progression of NSCLC. Kai1 and FasL could be considered as molecular markers to reflect pathobiological behavior of NSCLC. Additionally, close correlation of FasL expression with Kai1 expression in NSCLC provides a novel insight into the regulatory effects of FasL expression on Kai1 expression.

BACKGROUND: ANXA2 plays a very important role in cancer progression. chemokine ligand 18 (CCL18) is associated with the invasion, migration, metastasis and poor prognosis of lung adenocarcinoma (LUAD). In this study, we aimed to explore whether CCL18 promotes LUAD invasion through ANXA2, and its role and molecular mechanism in LUAD invasion. METHODS: Western blot was used to detect ANXA2 expression in LUAD tissues and adjacent non-tumor tissues, the transfection efficiency of SiANXA2#2 in cells and the role of ANXA2 as an upstream regulator in the AKT/cofilin signaling pathway. In vitro cytological experiments such as chemotaxis experiment and transwell invasion test was used to explore the mechanism of ANXA2 on LUAD metastasis. F-actin polymerization experiment and Western blot were used to detect whether invasion ability alteration of SiANXA2#2 A549 cells are related to F-actin. RESULTS: Western blot analysis showed that compared with adjacent non-tumor tissues, the protein expression level of ANXA2 in cancer tissues increased (P<0.05). In the chemotaxis experiment and invasion experiment, the chemotaxis and invasion ability induced by CCL18 decreased when ANXA2 knockdowned (P<0.05). Compared with the control group, F-actin polymerization was significantly lower in ANXA2 knockdown group, while phosphorylation of AKT at Ser473 and Thr308 and phosphorylation of Cofilin and LIMK were reduced in ANXA2 knockdown group (P<0.05). CONCLUSIONS ANXA2 knockdown can reduce the invasive effect of CCL18 on LUAD cells by reducing phosphorylation of AKT and downstream pathways.

BACKGROUND: Fibroblast activation protein (FAP) is one of the surface markers of cancer-associated fibroblasts (CAFs) and is closely related to the malignant characterization of CAFs. SP13786 is a specific micromolecule inhibitor of FAP and this study is to investigate the effects and mechanism of SP13786 on the migration and invasion of A549 cells through regulating exosomes of CAFs. METHODS: CAFs and paracancerous fibroblasts (PTFs) were isolated and subcultured from freshly resected lung adenocarcinoma tissues and paracancerous normal tissues separately. MTT assay was used to detect the proliferation of CAFs incubated by different concentrations of SP13786; PTFs-exo, CAFs-exo and CAFs+SP13786-exo were extracted by polymer precipitation method. The A549 cells were divided into Ctrl group, PTFs group, CAFs group and SP13786 group and each group was incubated with DMEM, PTFs-exo, CAFs-exo and CAFs+SP13786-exo separately. Laser confocal microscope was used to observe the endocytoses of exosomes by A549 cells. The expression of alpha-smooth muscle actin (α-SMA) and FAP in PTFs and CAFs and the expression of E-cadherin, N-cadherin, Slug, Stat3 and P-Stat3 in A549 cells were detected by immunofluorescence, immunohistochemistry and Western blot. The migration and invasion ability of A549 cells were detected by cell scratch and transwell methods. RESULTS: α-SMA and FAP were expressed much higher in CAFs than that in PTFs which indicate that CAFs and PTFs were successfully obtained from lung adenocarcinoma and paracancerous tissues (P<0.05). MTT showed that the 50% inhibitory concentration (IC50) of SP13786 for CAFs was about 3.3 nmol/L. In addition, SP13786 can significantly decrease the expression of α-SMA and FAP in CAFs which means that targeted inhibition of FAP could reduce the malignant characteristics of CAFs (P<0.05). Laser confocal microscope found that exosomes from CAFs could be taken up by A549 cells and scratch and transwell tests showed that the endocytosed CAFs-exo could promote the migration and invasion of A549 cells (P<0.001), while FAP inhibitor SP13786 could inhibit the effects of CAFs-exo on A549 cells (P<0.05). Furthermore, Immunofluorescence and Western blot showed that CAFs-exo could promote EMT by decreasing E-cadherin expression and increasing N-cadherin, Slug expression in A549 cells while FAP inhibitor SP13786 could significantly supress CAFs-exo-induced epithelial-mesenchymal transition (EMT) of A549 cells (P<0.05). Moreover, the expression of P-Stat3 was obviously increased in A549 cells of CAFs group and significantly down-regulated in SP13786 group (P<0.05) whereas there was no significant difference in total Stat3 between CAFs and SP13786 groups (P>0.05). Finally, WP1066 (a specific inhibitor of Stat3) was used to comfirm whether SP13786 could influence EMT of A549 cells by inhibiting Stat3 phosphorylation via CAFs-Exo. The results showed that when the phosphorylation of Stat3 in CAFs group was inhibited by WP1066, SP13786 could not influence the P-Stat3 expression and EMT of A549 cells anymore (P>0.05). CONCLUSIONS As a specific micromolecule inhibitor of FAP, SP13786 indirectly inhibits the migration and invasion of A549 cells by affecting exosomes of CAFs. The possible mechanism is to inhibit the phosphorylation of Stat3 and thus affect the EMT of A549 cells.