An orally administered DNA vaccine targeting vascular endothelial growth factor receptor-3 inhibits lung carcinoma growth.

Lung cancer is the leading cause of mortality and 5-year survival rate is very low worldwide. Recent studies show that vascular endothelial growth factor receptor-3 (VEGFR-3) signaling pathway contributes to lung cancer progression. So we hypothesize that an oral DNA vaccine that targets VEGFR-3 carried by attenuated Salmonella enterica serovar typhimurium strain SL3261 has impacts on lung cancer progression. In this study, the oral VEGFR-3-based vaccine-immunized mice showed appreciable inhibition of tumor growth and tumor lymphatic microvessels in lung cancer mice model. Moreover, the oral VEGFR-3-based vaccine-immunized mice showed remarkable increases in both VEGFR-3-specific antibody levels and cytotoxic activity. Furthermore, the oral VEGFR-3-based vaccine-immunized mice showed a significant increase in the levels of T helper type 1 (Th1) cell intracellular cytokine expression (IL-2, IFN-γ, and TNF-α). After inoculation with murine Lewis lung carcinoma (LLC) cells, CD4(+) or CD8(+) T cell numbers obviously declined in control groups whereas high levels were maintained in the oral VEGFR-3-based vaccine group. These results demonstrated that the oral VEGFR-3-based vaccine could induce specific humoral and cellular immune responses and then significantly inhibit lung carcinoma growth via suppressing lymphangiogenesis.

Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and ß-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and ß-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in colon carcinomas.

[Expression of transcription factor SOX4 and its clinical significance in female lung cancer patients in Xuanwei area, Yunnan Province].

OBJECTIVE: To investigate the expression of transcription factor SOX4 in lung cancer tissues of female patients in Xuanwei area, Yunnan Province, and explore its correlation with clinicopathological characteristics and prognosis of the female patients. METHODS: Real-time PCR was applied on lung cancer specimens and their corresponding normal lung tissues from 96 female cases of Xuanwei area to assess the expression of SOX4 mRNA. Immunohistochemical staining was performed to investigate the SOX4 protein expression, and further to elucidate its correlation with clinicopathological characteristics and prognosis. RESULTS: The expression level of SOX4 mRNA in the cancer tissues (2.53 ± 1.65) was significantly higher than that of matched normal tissues (1.43 ± 1.14, P = 0.003). Immunohistochemical staining showed that there were 53.1% (51/96) positive expression of SOX4 protein in the cancer tissue and only 26.0% (25/96) in matched normal tissue (P < 0.001). The expression of SOX4 protein had a significant correlation with clinical stage, lymph node metastasis and differentiation of tumor (P < 0.05). The survival analysis by Kaplan-Meier method showed that patients with positive expression of SOX4 protein, lymph node metastasis and advanced tumor stage had a significantly shorter median survival time (P < 0.05). Cox regression survival analysis showed that pathological grade was a significant independent factor affecting prognosis. CONCLUSIONS: The expressions of SOX4 mRNA and protein are significantly up-regulated in Xuanwei female lung cancer patients. Patients with positive SOX4 expression have a shorter median survival time. SOX4 protein expression level combined with pathological grade can be used as a prognostic indicator of female lung cancer patients in Xuanwei area, Yunnan Province.

Detection of cytokeratin 19, human mammaglobin, and carcinoembryonic antigen-positive circulating tumor cells by three-marker reverse transcription-PCR assay and its relation to clinical outcome in early breast cancer.

AIMS: To investigate the diagnostic, predictive, and prognostic value of the detection of circulating tumor cells (CTCs) using a three-marker (CK19, hMAM and CEA) RT-PCR assay in patients with early breast cancer. PATIENTS AND METHODS: Peripheral blood was obtained from 50 patients with early-stage breast cancer before any systemic adjuvant therapy and analyzed for the presence of CK-19, hMAM and CEA mRNA-positive CTCs using an RT-PCR assay. The specificity of the primers used was evaluated in 20 healthy individuals, 24 patients with benign breast disease, and 30 patients with metastatic breast cancer. The detection of CTCs was correlated with clinical outcome. RESULTS: The detection rate of three-marker-positive CTCs in the blood of patients with early breast cancer was 54.0%, significantly higher than in patients with benign breast disease and healthy blood donors (p=0.002 and p=0.000, respectively). The three-marker RT-PCR assay had 58.8% sensitivity in the parallel test and 100% specificity for CTC detection in the serial test, which was higher than the sensitivity and specificity of single-marker assays. For early breast cancer, correlation analysis between detection of three-marker-positive CTCs and clinicopathological characteristics indicated that detection of threemarker-positive CTCs was significantly correlated with elevated serum CEA levels (p=0.001). After three years of follow-up, 13 of the 27 patients with three-marker-positive CTCs in their blood had relapsed and detection of three-marker-positive CTCs was significantly associated with locoregional recurrence and/or distant metastasis (p=0.002). Detection of three-marker-positive CTCs in peripheral blood was an independent risk factor for reduced median relapse-free interval (p=0.000). CONCLUSION: The three-marker RT-PCR assay can enhance the sensitivity and specificity of CTC detection compared to singlemarker assay. Detection of three-marker-positive CTCs was associated with relapse and might have important predictive and prognostic implications in early breast cancer.

Orally administered DNA vaccine delivery by attenuated Salmonella typhimurium targeting fetal liver kinase 1 inhibits murine Lewis lung carcinoma growth and metastasis.

The vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2), also called fetal liver kinase 1 (FLK1) in mice and kinase insert domain receptor (KDR) in humans, is an endothelial cell specific receptor tyrosine kinase that mediates lung cancer angiogenesis. We hypothesized that an active immunotherapy approach targeting FLK1 may inhibit lung cancer growth and metastasis. To test this hypothesis, we evaluated whether immune responses to FLK1 could be elicited in mice by immunization with an orally administered DNA vaccine encoding the extracellular domain (ECD) of FLK1 (pcDNA3.1-FLK1(ECD)) carried by attenuated Salmonella typhimurium. We found that the vaccine was effective at protective antitumor immunity in Lewis lung carcinoma models in mice by breaking immune tolerance to FLK1 self-antigen. Both FLK1-specific humoral and cellular immune responses against endothelial cells can be induced in mice by immunization with pcDNA3.1-FLK1(ECD). Immunization with pcDNA3.1-FLK1(ECD) resulted in tumor suppression and prolonged survival in mice challenged with Lewis lung carcinomas cells. Experimental pulmonary metastases were strongly inhibited in pcDNA3.1-FLK1(ECD) immunized mice challenged with Lewis lung carcinoma cells. Thus, we conclude that the plasmid DNA vaccine encoding the extracellular domain of FLK1 could be an important component of FLK1 DNA vaccine to prevent lung carcinoma recurrence and metastasis after surgery.

Nomogram For The Prediction Of Malignancy In Small (8-20 mm) Indeterminate Solid Solitary Pulmonary Nodules In Chinese Populations.

PURPOSE: This study aimed to develop and validate a nomogram for predicting the malignancy of small (8-20 mm) solid indeterminate solitary pulmonary nodules (SPNs) in a Chinese population by using routine clinical and computed tomography data. METHODS: The prediction model was developed using a retrospective cohort that comprised 493 consecutive patients with small indeterminate SPNs who were treated between December 2012 and December 2016. The model was independently validated using a second retrospective cohort comprising 216 consecutive patients treated between January 2017 and May 2018. The investigated variables included patient characteristics (e.g., age and smoking history), nodule parameters (e.g., marginal spiculation and significant enhancement), and tumor biomarker levels (e.g., carcinoembryonic antigen). A prediction model was developed by using multivariable logistic regression analysis, and the model's performance was presented as a nomogram. The model was evaluated based on its discriminative ability, calibration, and clinical usefulness. RESULTS: The developed nomogram was ultimately based on age, marginal spiculation, significant enhancement, and pleural indentation. The Harrell concordance index values were 0.869 in the training cohort (95% confidence interval: 0.837-0.901) and 0.847 in the validation cohort (95% confidence interval: 0.792-0.902). The Hosmer-Lemeshow test revealed good calibration in each of the training and validation cohorts. Decision curve analysis confirmed that the nomogram was clinically useful (risk threshold from 0.10 to 0.85). CONCLUSION: Patient age, marginal spiculation, significant enhancement, and pleural indentation are independent predictors of malignancy in small indeterminate solid SPNs. The developed nomogram is easy-to-use and may allow the accurate prediction of malignancy in small indeterminate solid SPNs among Chinese patients.

The Correlation of Visfatin and Its Gene Polymorphism with Non-Small Cell Lung Cancer.

Objective: To investigate the protein expression of visfatin and its gene polymorphism in non-small cell lung cancer (NSCLC) patients. Methods: The plasma level of visfatin was detected by enzyme-linked immunosorbent assay, and the genotypes rs59744560, rs9770242, and rs61330082 in the visfatin gene were detected by gene sequencing. Result: This study revealed that plasma levels of visfatin in NSCLC patients were significantly higher than the levels in healthy people (p < 0.01). The high level of plasma visfatin was found to be significantly correlated with TNM stage (p < 0.05). No mutations were detected in rs59744560 and rs9770242 loci. Three genotypes (CC, CT, and TT) were detected in rs61330082 locus, and the differences in the frequency distribution of these genotypes were significant in the two groups (p < 0.05). Central obesity and the CC genotype were independent risk factors in the pathogenesis of NSCLC (p < 0.05). Conclusion: The plasma visfatin level in NSCLC patients significantly increased, and high plasma visfatin levels were correlated with tumor stage. Gene polymorphism was found in the visfatin gene rs61330082 locus. The CC genotype might increase the risk for patients suffering from NSCLC, while the CT genotype, TT genotype, and T allele may reduce the risk of NSCLC. The rs61330082 locus can be used as genetic markers of high-risk populations.

MicroRNA-370 inhibits the growth and metastasis of lung cancer by down-regulating epidermal growth factor receptor expression.

Abnormal microRNA-370 (miR-370) expression has been frequently reported in several types of cancers, including lung cancer. However, the role and molecular mechanisms of miR-370 in regulating the growth and metastasis of lung cancer have not been clarified. Here, we show higher levels of epidermal growth factor receptor (EGFR), but lower levels of miR-370 expression in most human lung cancer cells and non-tumor cells. Induction of miR-370 over-expression significantly reduced the levels of EGFR expression and the EGFR 3'untranslated region (UTR)-regulated luciferase activity in XWLC-05 and H157 cells, suggesting that miR-370 may bind to the 3'UTR of EGFR mRNA. Compared with the control cells, induction of miR370 overexpression significantly inhibited the proliferation, clone formation capacity, migration and invasion of XWLC-05 and H157 cells while miR-370 inhibitor over-expression enhanced their tumor behaviors in vitro. Furthermore, miR-370 over-expression down-regulated the EGFR and hypoxia-inducible factor (HIF)-1α expression, and attenuated the extracellular single-regulated kinase (ERK)1/2 and AKT phosphorylation in XWLC-05 and H157 cells. In contrast, miR370 inhibitor over-expression increased the EGFR and HIF-1α expression as well as the ERK1/2 and AKT phosphorylation in XWLC-05 and H157 cells. Moreover, miR-370 over-expression significantly reduced the levels of EGFR and CD31 expression and inhibited the growth and lung metastasis of xenograft NSCLC tumors in mice. Our study indicates that miR-370 may bind to the 3'UTR of EGFR to inhibit EGFR expression and the growth, angiogenesis and metastasis of non-small cell lung cancer by down-regulating the ERK1/2 and AKT signaling.

[Evaluation of effects of quality scale for removable partial dentures in clinical application].

PURPOSE: To evaluate the effects of quality scale for removable partial dentures (RPD)in clinical application. METHODS: Quality scale for removable partial dentures was designed. Twelve items were devised for visual survey and try-in in base, artificial teeth, clasp, rest, connector and adjustment. The assessments were divided into 3 grades A, B and C. Four commercial dental laboratories were divided into experimental group and control group randomly. All RPD made in two groups were given score with the quality scale by single-blind method. In the experimental group,the technicians were familiar with the quality scale. The assessments were periodically feedbacked to administrative staffs and exchanges were carried out between doctors and technicians by telephone. No feedback information was provided in the control group. The assessments were compared between the two groups. The data was analyzed with SPSS17.0 software package. RESULTS: The scores of assessments for base, artificial teeth, clasp, rest, connector and adjustment in the experimental group were greater than that in the control group. The difference was significant between the two groups by analysis of variance (P<0.01). The grade A and C for RPD used acrylic resin, flexible resin and cast framework in the experimental group was 27.2%,39.5%,40.6% and 9.2%, 7.9%,7.2%, respectively. The grade B was in the majority. In the control group, the grade A and C was 9.4%,15.6%,15% and 40.6%,23.6%,25%,respectively. The majority was grade B and the grade C was significantly higher than the experimental group(P<0.05). CONCLUSION: Applying the quality scale of RPD can improve the fabricating quality of prosthesis.

Moderating effects and maintenance of lung cancer cellular immune functions by CIK cell therapy.

AIMS: To study the CIK cell treatment effects on regulation of cellular immune function disorders in patients with lung cancer, and to analyze the time characteristics. METHODS: Cellular immune function was assessed by FCM, and patients with functional disorders were randomly divided into two groups, one given CIK cell therapy within 18 months (5 courses) and the other the controls, which were followed up for 1 year with cellular immune functions tested once a month. RESULTS: There were 5 types of cellular immunity, 4 of which are disorders; after CIK treatment, the improvement rate of the 4 groups were 79.1%, 70.8%, 76.0% and 70.0%, intergroup differences not being statistically significant (P=0.675), all significantly higher than in the control group (P=0.000). The median maintenance times for the 4 groups were 10.4 months (9.76-11.04), 8.4 months (7.86-8.94), 9.8 months (9.20-10.4) and 7.9 months (6.25-9.55), respectively. CONCLUSIONS: CIK cells were able to improve the immune functions of patients with lung cancer, the rate of improvement and maintenance time being related to the immune function before the treatment and CIK-cell-therapy courses.

Thyroid transcription factor 1 represses the expression of Ki-67 and induces apoptosis in non-small cell lung cancer.

The thyroid transcription factor 1 (TTF-1) gene is associated with the differentiation of lung epithelial cells and has been reported to be an independent prognostic factor for lung adenocarcinoma patients. The aim of the present study was to detect the expression of TTF-1 in human lung cancer cell lines and to evaluate the association of overexpressed TTF-1 with Ki-67 and apoptosis in the A549 cell line. We also investigated the expression of TTF-1 and Ki-67 in Xuanwei lung adenocarcinoma. TTF-1 mRNA expression was evaluated in 10 non-small cell lung cancer (NSCLC) cell lines by quantitative real-time RT-PCR (qRT-PCR). Overexpression of TTF-1 in A549 cells was achieved by transient transfection. The TTF-1 and Ki-67 proteins were detected by immunohistochemistry and apoptosis was detected by flow cytometry. We also investigated immunohistochemically the expression of TTF-1 and Ki-67 in 62 resected cases of Xuanwei lung adenocarcinoma. Overall the expression of TTF-1 mRNA in the 10 cell lines was low. Overexpression of TTF-1 mRNA was found only in 3 (30%) of 10 NSCLC cell lines, including 1 (25%) of 4 adenocarcinoma cell lines. A549 cells overexpressing TTF-1 were found to have repressed expression of Ki-67 (P=0.012) and increased apoptosis (P=0.000). Immunohistochemical analysis of resected cases of Xuanwei lung adenocarcinoma (n=62) showed the expression of TTF-1 in 58 (93%) of 62 and Ki-67 in 22 (35%) of 62. Patients with strong immunohistochemical expression TTF-1 were statistically associated with well-differentiated phenotype (P=0.006) and inverse correlation with Ki-67 expression (P=0.016). These data suggest that TTF-1 may serve as a tumor suppressor gene based on its inverse correlation with Ki-67 proliferative activity and increase of cellular apoptosis.

Characterization of two human lung adenocarcinoma cell lines by reciprocal chromosome painting.

Lung cancer is a leading cause of cancer death worldwide. Some lung cancer patients correlate with a gas of radon besides smoking. To search for common chromosomal aberrations in lung cancer cell lines established from patients induced by different factors, a combined approach of chromosome sorting, forward and reverse chromosome painting was used to characterize karyotypes of two lung adenocarcinoma cell lines: A549 and GLC-82 with the latter line derived from a patient who has suffered long-term exposure to environmental radon gas pollution. The chromosome painting results revealed that complex chromosomal rearrangements occurred in these two lung adenocarcinoma cell lines. Thirteen and twenty-four abnormal chromosomes were identified in A549 and GLC-82 cell lines, respectively. Almost half of abnormal chromosomes in these two cell lines were formed by non-reciprocal translocations, the others were derived from deletions and duplication/or amplification in some chromosomal regions. Furthermore, two apparently common breakpoints, HSA8q24 and 12q14 were found in these two lung cancer cell lines.

A comparative study of gene vaccines encoding different extracellular domains of the vascular endothelial growth factor receptor 2 in the mouse model of colon adenocarcinoma CT-26.

AIMS: To compare the immunogenicity and anti-tumor effects of gene vaccines encoding domains 1-4 with full length of extracellular region of VEGFR2. RESULTS: Both DNA vaccines decreased VEGF levels and rose specific antibodies; the lymphocyte subsets of vaccinated mice maintained high; tumor latency period and survival time of immunized mice were prolonged; tumor size, weight, MVD and liver metastases were significantly less than the control groups. METHODS: Mouse model of CT-26 adenocarcinoma of colon were treated with orally immunized gene vaccine encoding extracellular 1-4 and full length of VEGFR2 respectively. The effect of anti-tumor was evaluated by detecting the tumor volume, mice survival time, intratumoral microvessel density (MVD) and liver metastases. To explore the reasonable mechanism of the oral gene vaccines, the levels of VEGF and anti-VEGFR2 antibody in serum were detected by ELISA, CD4+ and CD8+ T cells in peripheral blood and subcutaneous tumors were analyzed by flow cytometer and immunohischemistry respectively. CONCLUSION: Compared with full length of extracellular domain of VEGFR2, extracellular regions 1-4 of VEGFR2 has been sufficient to decrease the serum VEGF level and to inhibit tumor growth and metastasis specifically.

Study on the mechanism of acupoint sticking therapy with Chuan fuling for treatment of asthma model rats / 中国针灸

<p><b>OBJECTIVE</b>To investigate the mechanism of the acupoint sticking therapy with Chuanfuling for preventing and treating asthma.</p><p><b>METHODS</b>Thirty male SD rats were randomly divided into a control group (normal saline, p.i. +no acupoint sticking+ normal saline, spray inhalation), model group (normal saline with ovalbumin, p.i. +no acupoint sticking+ normal saline with ovalbumin, spray inhalation), and acupoint sticking group (normal saline with ovalbumin, p.i. +acupoint sticking with Chuan fuling+normal saline with ovalbumin, spray inhalation), 10 rats in each group. The incubation period of nodding breath, symptom of asthmatic attack, expression level of interleukin-4 mRNA (IL-4 mRNA) and interferon-gamma mRNA (IF-gamma mRNA), as well as pathological changes on the middle leaf of right lung, were observed in each group.</p><p><b>RESULTS</b>(1) Comparing with the control group, the model group was showed that the expression level of IL-4 mRNA in the peripheral blood cells (PBMC) was increased, while hyperemia, edema and eosinocyte (EOS) invasion of lung tissue was more serious (P < 0.01). (2) Comparing with the model group, the acupoint sticking group was showed that the expression level of IL-4 mRNA in PBMC was decreased, the incubation period of nodding breath was prolonged for induced asthma on the fifth and seventh time with lower frequency, while in the lung tissue EOS invasion was reduced (P < 0.05), but there were no significant changes on the hyperemia and edema (P > 0.05).</p><p><b>CONCLUSION</b>Acupoint sticking for treating asthma of model rats with Chuanfuling can inhibit the expression level of IL-4 mRNA in PBMC, and the release of the inflammatory mediator and cytokine from the EOS to the air passage, in order to reduce the injury of epithelial layer and high reaction on the air passage.</p>