Upregulation of KCNMA1 facilitates the reversal effect of verapamil on the chemoresistance to cisplatin of esophageal squamous cell carcinoma cells.

OBJECTIVE: This study aimed to investigate the reversal effect of verapamil (VER) on the chemoresistance to cisplatin of esophageal squamous cell carcinoma (ESCC) cells. PATIENTS AND METHODS: The reversal effect of VER on cisplatin resistance in ESCC cells was evaluated via CCK-8 assay, colony formation assessment, and flow cytometry. The key genes that mediate this effect were screened via high-throughput transcriptome se¬quencing. The mRNA and protein expression levels of potassium calcium-activated channel subfamily M alpha 1 (KCNMA1) in ESCC cells were examined via quantitative real-time PCR and Western blot analysis, respectively. The protein expressions of KCNMA1 in tissue samples from patients with either positive or negative responses to the therapeutic regimen of VER were determined via immunohistochemistry assay. Cell models with KCNMA1 knockdown and overexpression were es¬tablished to examine the role of KCNMA1 in mediating the reversal effect of VER on the chemoresistance to cisplatin of ESCC cells. RESULTS: Results revealed that VER significantly decreased the 50% inhibitory concentration of cisplatin, inhibited colony formation, and induced apoptosis in ESCC cells. The curative effects of VER combined with chemotherapeutic drugs in KCNMA1-positive patients were better than those in KCNMA1-negative patients. KCNMA1 upregulation enhanced the reversal effect of VER on the chemoresistance to cisplatin of ESCC cells. CONCLUSIONS: KCNMA1 facilitated the reversal effect of VER on cisplatin resistance in ESCC cells.

Effect of verapamil in the reversal of doxorubicin chemotherapy resistance in advanced gastric cancer.

OBJECTIVE: Gastric carcinoma is one of the most common malignant tumors and one of the most common cancer-related fatal diseases. Chemotherapy is considered as the major therapy for advanced gastric cancer, and the curative effect of chemotherapy directly affects the treatment of advanced gastric cancer. Drug resistance of tumor cells is one of the important causes that induces failure of chemotherapy. Previous studies have demonstrated that verapamil (VER) can reverse drug resistance by inhibiting the P-glycoprotein (P-gp), which is one of the main targets of VER. The present study aimed at investigating the function of glucosylceramide synthase (GCS) in the VER-induced reversal of doxorubicin (ADM) chemotherapy resistance in gastric carcinoma. PATIENTS AND METHODS: In the current study, the 4 GC cell line was selected for investigation. The IC50 values of gastric cancer cells were measured using CCK-8 assay. The expression levels of candidate genes in gastric carcinoma cells were measured by RT-qPCR. The expression levels of candidate protein in gastric carcinoma cells were measured by Western blot. The expression of GCS protein in clinical specimens of GC receiving VER+TACE therapy was measured by immunohistochemistry. The test of gastric carcinoma cell apoptosis was measured by Annexin V-FITC/PI double-staining. RESULTS: We found that the expression levels changes of the GCS gene can influence the effects of ADM+VER on cell apoptosis. The role and mechanism of GCS gene in reversing the chemotherapy resistance of gastric carcinoma cells to ADM were explored. CONCLUSIONS: In future research, we will explore the mechanism of how GCS affects drug resistance in gastric carcinoma and related signal transduction pathway.

The clinical observation of verapamil in combination with interventional chemotherapy in advanced gastric cancer.

OBJECTIVE: We analyzed the clinical observations of target arterial infusion of verapamil combined with chemotherapy as therapy for advanced gastric cancer. PATIENTS AND METHODS: From March 2012 to December 2015, a total of 63 patients with advanced gastric cancer were admitted to our department. The target artery in the control group was perfused with chemotherapy drugs only, and the target artery in the therapy group was injected with verapamil combined with chemotherapy drugs. RESULTS: The therapeutic effect of the therapy group was significantly better than that of the control group in the primary foci of gastric cancer. Liver metastatic lesions: 11 patients in the control group had liver metastases and 25 patients in the therapy group had liver metastases. The effective rate (CR+PR) of the therapy group was significantly better than the control group. Clinical benefit evaluation: in the therapy group of 43 cases, 40 cases presented positive clinical benefit and 38 cases positive clinical weight in KFS scoring system; the clinical benefit of the therapy group was significantly better than control group. Survival analysis: the disease progression-free rate and survival rate of the therapy group were 12 months and 24 months, which were higher than those in the control group. The median PFS and median OS were also significantly longer than those in the control group (p<0.01). In the therapy group, adverse effects of chemotherapy in 43 patients were relieved in a short time. CONCLUSIONS: Target arterial infusion of verapamil combined with chemotherapy drugs for advanced gastric cancer can significantly improve the efficacy of chemotherapy drugs and prolong the survival of patients.

A new automatic algorithm to extract craniofacial measurements from fetal three-dimensional volumes.

OBJECTIVES: Three-dimensional (3D) ultrasound is useful in the prenatal evaluation of fetal craniofacial structures, particularly as it provides a multiplanar view. However, an expert must designate the area of interest and the appropriate view, making measurement of fetal structures using 3D ultrasound both time-consuming and subjective. In this study we propose an image analysis system that measures automatically and precisely the fetal craniofacial structures and evaluate its performance in the second trimester of pregnancy using a new 3D volume analysis algorithm. METHODS: A universal facial surface template model containing the geometric shape information of a fetal craniofacial structure was constructed from a fetal phantom. Using the proposed image analysis system we fitted this stored template model using a model deformation approach to individual fetal 3D facial volumes from 11 mid-trimester fetuses, and extracted automatically the following standard measurements: biparietal diameter (BPD), occipitofrontal diameter (OFD), interorbital diameter (IOD), bilateral orbital diameter (BOD) and distance between vertex and nasion (VN). The same five parameters were measured manually by an expert and the results compared. RESULTS: Comparison of the algorithm-based automatic measurements with manual measurements made by an expert gave correlation coefficients of 0.99 for BPD, 0.98 for OFD, 0.80 for BOD, 0.83 for IOD and 0.99 for VN. There were no significant differences between automatic and manual measurements. CONCLUSION: Our proposed system measures precisely the fetal craniofacial structures using 3D ultrasound, making it potentially useful for clinical service. This system could also be applied to other clinical fields in future testing.

Quantification of Fusarium graminearum in harvested grain by real-time polymerase chain reaction to assess efficacies of fungicides on fusarium head blight, deoxynivalenol contamination, and yield of winter wheat.

We used a real time polymerase chain reaction-based assay and visual disease assessment to evaluate the efficacies of Js399-19, tebuconazole, a mixture of tebuconazole and thiram, azoxystrobin, carbendazim, and thiram on the development of Fusarium head blight (FHB) and deoxynivalenol (DON) contamination and on the yield of winter wheat (cv. Nannong no. 9918) after artificial inoculation under field conditions with Fusarium graminearum. The incidence of infected spikelets (IIS), amount of F. graminearum DNA (Tri5 DNA), total DON (containing DON, 3-acetyl-deoxynivalenol, and 15-acetyl-deoxynivalenol) concentration, and 1,000-grain weight (TGW) were quantified in 2006 and 2007. A strong positive correlation was found between IIS or Log10Tri5 DNA and total DON concentration in the harvested grain. The Js399-19, tebuconazole, and the mixture of tebuconazole and thiram significantly reduced IIS of FHB, amount of Tri5 DNA, and total DON within the grain and increased TGW. Although azoxystrobin, carbendazim, and thiram can increase TGW, they had no effect on the occurrence of F. graminearum compared with those of the untreated controls. Surprisingly, azoxystrobin and carbendazim significantly increased the total DON content in the harvested grain because they might have stimulated the amount of total DON production per Tri5 DNA. The fungicides Js399-19, tebuconazole, and the mixture of tebuconazole and thiram were the most effective in controlling FHB and reducing DON contamination of the wheat.

Effects of ultraviolet B irradiation, proinflammatory cytokines and raised extracellular calcium concentration on the expression of ATP2A2 and ATP2C1.

BACKGROUND: Darier disease (DD) and Hailey-Hailey disease (HHD) are autosomal dominantly inherited skin disorders that histologically share the characteristics of suprabasal separation and acantholysis of epidermal keratinocytes. Various mutations in the DD gene (ATP2A2) and the HHD gene (ATP2C1) (respectively encoding the calcium pumps of the sarco/endoplasmic reticulum and the Golgi apparatus) have recently been described in multiple families with DD and HHD. Mutations in ATP2A2 or ATP2C1 have been suggested as causing the conditions via the mechanism of haploinsufficiency. Ultraviolet (UV) B irradiation is thought to be an aggravating factor in both diseases. OBJECTIVES: To examine the effects of various stimuli on ATP2A2 and ATP2C1 mRNA expression, and to examine the role of calcium pumps during keratinocyte differentiation. METHODS: The effects of UVB irradiation, of UVB-inducible inflammatory cytokines produced by keratinocytes and of high-calcium medium (1.8 mmol L(-1) as opposed to 0.08 mmol L(-1) Ca2+) on ATP2A2 and ATP2C1 mRNA expression were quantified in cultured normal human keratinocytes using reverse transcription-polymerase chain reaction. RESULTS: Expression of ATP2A2 and ATP2C1 mRNA was suppressed immediately after exposure to UVB irradiation, and modulation of mRNA expression was achieved in keratinocytes cultured with proinflammatory cytokines. The mRNA expression of both genes was increased significantly after the shift to high extracellular Ca2+ concentration. CONCLUSIONS: The results suggest that modulation of ATP2A2 and ATP2C1 mRNA expression by UV or cytokines might contribute to the clinical presentations unique to DD and HHD, and that the controlled expression of these genes plays an important role in keratinocyte homeostasis, function and differentiation.

Effect of quercetin on adhesion of platelets to microvascular endothelial cells in vitro.

AIM: To study the effect of quercetin(Que) on the adhesion of platelets to microvascular endothelial cells (MVEC) isolated from human skin. METHODS: [3H]-adenine-labeled platelets were incubated with MVEC. Effect of Que on platelet endothelial cell adhesion molecular (PECAM) expression on MVEC was also evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: Adhesion of platelet to MVEC reached to maximum at about 30 min. Que inhibited the adhesion of platelets to MVEC in a concentration-dependent manner. Que 5 micromol/L did not show any significant inhibition. When the concentration of Que increased to 10, 20, and 40 micromol/L, the inhibition rate increased to 10.5 %, 20.0 %, and 42.2 %, respectively. Pre-incubation of Que (10 - 40 micromol/L) with labeled platelets for 30 min also inhibited the adhesion but Que 5 micromol/L did not. The inhibition rate of Que 10, 20, and 40 micromol/L was 18.2 %, 29.8 %, and 65.3 % respectively. Expression of PECAM on the endothelial cells was decreased in a concentration-dependent manner when MVEC were treated with Que (10 - 40 micromol/L) for 12 h but Que 5 micromol/L did not significantly affect the expression. CONCLUSION: Que could inhibit the adhesion of platelets to MVEC. This effect may be related to decreased expression of PECAM on MVEC.

Initial evidence of endothelial cell apoptosis as a mechanism of systemic capillary leak syndrome.

BACKGROUND: Systemic capillary leak syndrome (SCLS) is a rare disorder of unknown etiology that is characterized by acute recurrent attacks of hypovolemic shock commonly following an inflammatory stimulus such as a viral illness. Prophylactic therapy is generally ineffective, and the outcome is frequently fatal. METHODS: In order to investigate the cellular mechanisms leading to SCLS, we examined the effects of sera from two patients with active SCLS on microvascular endothelial cell apoptosis in vitro. Apoptosis was determined by morphologic criteria, DNA fragmentation, annexin V stain, and by a quantitative photometric assay. The apoptotic pathway was investigated by Western blot of endothelial cells lysate after exposure to SCLS sera. RESULTS: The sera from patients with active SCLS mediated profound apoptosis of microvascular endothelial cells shortly after exposure. The exposed microvascular endothelial cells underwent immediate apoptosis as evidenced by morphologic changes, plasma membrane phosphatidylserine exposure, and by DNA fragmentation. Increased Bax/Bcl-2 ratio in endothelial cells exposed to SCLS sera was observed and suggested an oxidation injury as the possible mechanism for endothelial apoptosis. This potential mechanism was further explored by measuring intracellular reactive oxygen species (ROS) following SCLS serum exposure. Sera from both patients caused marked increases in ROS, initially detectable at 1 h and persisted for at least 12 h, with control serum from healthy subjects showing no effect on basal endothelial cell ROS concentrations. CONCLUSION: Components from the sera of patients with active systemic capillary leak syndrome in contrast to healthy subject sera mediate early and extensive endothelial apoptosis in vitro that is associated with oxidation injury. These data represent compelling initial evidence for oxidation-induced apoptosis as a likely mechanism for endothelial injury leading to SCLS.

Nerve growth factor and neuropeptides circulating levels in systemic sclerosis (scleroderma).

OBJECTIVE: To determine the circulating levels of nerve growth factor (NGF), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP) in systemic sclerosis (SSc), and to correlate these levels with clinical and laboratory features. METHODS: Forty four patients with SSc were evaluated for circulating NGF (immunoenzymatic assay), NPY and VIP (radioimmunoassay), anticentromere and antitopoisomerase I autoantibodies, lung disease (pulmonary function tests with carbon monoxide transfer factor (TLCO), ventilation scintiscan with 99mTc DTPA radioaerosol, high resolution computed tomography (HRCT), pulmonary pressure (echo colour Doppler)), heart disease (standard and 24 ECG, echocardiography), cutaneous involvement (skin score), joint involvement (evidence of tender or swollen joints, or both), peripheral nervous system (PNS) involvement (electromyography), rheumatoid factor, angiotensin converting enzyme (fluorimetric method), von Willebrand factor (ELISA), and erythrocyte sedimentation rate (ESR) (Westergren). RESULTS: Circulating NGF levels in SSc were significantly increased compared with controls (p<0.00001) and significantly higher in the diffuse than in the limited subset of patients (p<0.01). Patients with articular disease had significantly higher levels of NGF. A significant indirect correlation between NGF levels and TLCO was detected (p<0.01), but no correlation was found between NGF and HRCT, DTPA, skin score, PNS involvement and angiotensin converting enzyme and von Willebrand factor levels, antitopoisomerase or anticentromere antibodies, and ESR. NGF levels increased progressively as the disease worsened. Similarly, VIP circulating levels were significantly increased in patients with SSc (p<0.001), whereas the increase of NPY levels did not reach statistical significance. However, both neuropeptides, following the same trend as NGF, increased as the disease worsened (skin score and lung disease). CONCLUSIONS: The increase of NGF and VIP in patients with SSc, the former in the diffuse subset of the disease, and in patients with prominent articular disease, may suggest a link between neurotransmitters and the disease pathogenesis. Neuropeptide circulating levels seem to increase only in patients with the most severe disease.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR.

BACKGROUND: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. METHODS AND RESULTS: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. CONCLUSION: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc.

Mechanism of serum-mediated endothelial injury in scleroderma: identification of a granular enzyme in scleroderma skin and sera.

Circulating endothelial cell growth-inhibitory factor with a molecular weight of 40-60 kDa was described in scleroderma (SSc) sera and shown to have a proteolytic action. In view of the recent demonstration of cellular immune activation in SSc, and because of the description of novel serine proteases in the granules of activated cytolytic T cells (granzymes), we hypothesized that granzymes represent the endothelial inhibitory principal in SSc sera. Granular enzymes were isolated from IL-2-activated nonadherent normal lymphocytes, and a 60-kDa granzyme was isolated using benzamidine-affinity column and molecular sieve column. A polyclonal antiserum was generated by immunizing rabbits with the isolated granzyme. Anti-granzyme antibody abolished SSc serum-mediated EC growth inhibition. Furthermore, a circulating protein similar to isolated granzyme was identified as a 60-kDa band on Western blots of benzamidine column-purified SSc sera. Immunofluorescence studies of SSc skin biopsies using anti-granzyme antibody demonstrated the presence of granzyme reactivity, while healthy control tissues were negative. Moreover, granzyme A gene expression was identified in SSc skin biopsies by a PCR method. The data suggest cytolytic mechanism involvement in the pathogenesis of scleroderma.

Effect of cytokines on the production of endothelin by endothelial cells.

OBJECTIVE: Circulating levels of endothelin (ET), a potent vasoconstrictor peptide and a mitogen for smooth muscle cells and fibroblasts, are reported to be increased in a variety of human diseases characterized by vascular pathology. In view of the probable immune bases for vascular injury in connective tissue disorders, we examined the effect of the cytokines IL-1 alpha, IL-4, IL-6, TNF-alpha and lymphotoxin on the production of ET-1 by cultured vascular endothelial cells. RESULTS: ET levels in endothelial cell conditioned media were measured by radioimmunoassay. IL-4 and lymphotoxin had no effect on ET release by endothelial cells, while IL-6, TNF-alpha and IL-1 alpha stimulated ET mRNA expression and ET release in a dose dependent fashion. IL-6 was the most potent stimulator and IL-1 was the least effective. The addition of neutralizing antibodies to the cytokines inhibited the observed increase in ET release. CONCLUSIONS: These results suggest that cytokines may play a significant role in the control of vascular tone. Furthermore, cytokines may indirectly contribute to the development of proliferative vascular lesions by stimulating smooth muscle and interstitial cell proliferation through their effects on endothelin release by the vascular endothelium.

Effects of quercetin on Na(+)-K(+)-exchanging ATPase and Ca(2+) Mg(2+)-ATPase in rats.

Quercetin (Que) ig 200 mg.kg-1, qd x 14 d decreased activities of the Na(+)-K(+)-exchanging ATPase (I) of rat brain plasma membranes and heart sarcolemmal and Ca(2+) Mg(2+)-ATPase (II) of heart sarcolemmal membrane. Que 100 mg.kg-1 reduced the activity of I from rat heart sarcolemmal preparation, but had no effect on that from rat brain plasma membranes. The result shows that I of myocardium is more sensitive than that of brain in rat. Que also showed a remarkable inhibitory effect in the II of heart sarcolemma.

[Effects of total saponins of Panax notoginseng on increasing PGI2 in carotid artery and decreasing TXA2 in blood platelets].

Total saponins of Panax notoginseng (PNS) were given orally 100 mg/(kg.d) to rabbit for 8 wk. Aortic atherosclerotic plaque formation was restrained as compared to the control group. Radioimmunoassay was used to investigate the effects of PNS on the contents of prostacyclin in carotid artery and thromboxane A2 in blood platelets of rat. Oral administration of PNS 25,50,100 mg/(kg.d) for 10 d, the caused an increase of prostacyclin in carotid artery and a decrease of thromboxane A2 in blood platelets as compared to the control group. These results show that the anti-atherosclerotic action of PNS may be a result of the correction of the unbalance between prostacyclin and thromboxane A2.