Circulating microRNAs as novel biomarkers of ALK-positive nonsmall cell lung cancer and predictors of response to crizotinib therapy.

Circulating microRNAs are potential diagnostic and predictive biomarkers, but have not been investigated for patients with anaplastic lymphoma kinase (ALK)-positive lung cancer. In this exploratory study, we sought to identify potential plasma biomarkers for ALK-positive non-small cell lung cancer (NSCLC). A microRNA microarray was used to select ALK-related microRNAs in ALK-positive NSCLC (n = 3), ALK-negative NSCLC (n = 3), and healthy subjects (n = 3). Plasma levels of 21 microRNAs were differentially expressed for ALK-positive and ALK-negative NSCLC, including 14 down-regulated and 7 up-regulated microRNAs. We also identified 5s rRNA as the most stable endogenous control gene using geNorm and NormFinder algorithms. Candidate microRNAs in plasma from ALK-positive (n = 41) and ALK-negative NSCLC patients (n = 32) were quantified using real-time reverse transcriptase quantitative polymerase chain reaction. The expression levels of miR-28-5p, miR-362-5p, and miR-660-5p were all down-regulated in ALK-positive NSCLC, compared with ALK-negative NSCLC. The areas under the receiver operating characteristic curves of miR-28-5p, miR-362-5p, miR-660-5p, and 3-microRNAs panel were 0.873, 0.673, 0.760, and 0.876, respectively. The positive predictive values of miR-28-5p, miR-362-5p, and miR-660-5p were 96.43%, 80.77%, and 83.87%, respectively. Increased plasma levels of miR-660-5p after crizotinib treatment predicted good tumor response (p = 0.012). The pre-crizotinib levels of miR-362-5p were significantly associated with progression-free survival (p = 0.015). Thus, in this preliminary investigation, we identified a potential panel of 3 microRNAs for distinguishing between patients with ALK-positive and ALK-negative NSCLC. We also identified miR-660-5p and miR-362-5p as potential predictors for response to crizotinib treatment.

[The Properties of an Infrared Hybrid Single Beam Spectrum].

A hybrid single beam spectrum ø(α)=αø(b1)+(1-α)ø(b2)=αø0e(-Kb1)+(1-α)ø0e(-Kb2) is introduced as the combination of two single beam spectra ø(b1) and ø(b2) from the same sample but with different pathlengths (b(1) and b(2)), where α(0＜α＜1) is the hybrid coefficient. The intensity of hybrid spectrum ø(α) can be controlled easily to the desired point by simply choosing an appropriate α. The experimental results showed that hybrid spectrum ø(α) is very nearly identical to ø(b)=ø(0)e-K(b(2)-αb(2)+αb(b)) under appropriate conditions, namely ø(α)≈ø(b) , where ø(b) is the single beam spectrum of the real sample with the pathlength of b(2)-αb(2)+αb(1). Therefore, the desired single beam spectrum ø(b) can be obtained easily by choosing α and we no longer need to prepare IR sample with the thickness of b. Hybrid spectrum method shows valuable potential in application of eliminating background interference.

Gastrodin Ameliorates Oxidative Stress and Proinflammatory Response in Nonalcoholic Fatty Liver Disease through the AMPK/Nrf2 Pathway.

This study was designed to investigate the antioxidative, antiinflammatory and metabolism-regulating effects of gastrodin (GSTD) in the treatment of nonalcoholic fatty liver disease (NAFLD). Oleic acid (OA) was used to induce steatosis in HL-7702 cells; a high-fat or high-fat and high-cholesterol diet was used to induce NAFLD in mice and rats. Our results showed that GSTD significantly increased hepatic superoxide dismutase (SOD) but decreased reactive oxygen species (ROS)/malondialdehyde (MDA) and reduced the mRNA levels of proinflammatory cytokines both in vitro and in vivo. GSTD promoted the phosphorylation of nuclear factor erythroid-2-related factor-2 (Nrf2) at serine (Ser) 40, stimulated its nuclear translocation and increased hepatic expression of heme oxygenase-1 (HO-1). GSTD activated AMP-activated protein kinase (AMPK), suppressed hepatic steatosis, lowered serum triglyceride (TG)/glucose and decreased body weight gain in animals with NAFLD. The stimulating effects of GSTD on the Nrf2 pathway as well as its antioxidative/antiinflammatory activities were abolished by compound C in OA-treated HL-7702 cells. In summary, our results demonstrate that GSTD activates the AMPK/Nrf2 pathway, ameliorates oxidative stress/proinflammatory response and improves lipid metabolism in NAFLD. Our findings may support the future clinical application of GSTD for the treatment of NAFLD to reduce hepatic steatosis, oxidative stress and proinflammatory response.

Combination of icotinib, surgery, and internal-radiotherapy of a patient with lung cancer severely metastasized to the vertebrae bones with EGFR mutation: a case report.

A 48-year-old Chinese female was referred to us regarding EGFR-mutated advanced non-small cell lung cancer, and metastasis to left scapula and vertebrae bones which caused pathological fracture at T8 and T10 thoracic vertebrae. An aggressive combined therapy with icotinib, vertebrae operation, and radioactive particle implantation and immunotherapy was proposed to prevent paraplegia, relieve pain, and control the overall and local tumor lesions. No postoperative symptoms were seen after surgery, and the pain was significantly relieved. Icotinib merited a 31-month partial response with grade 1 diarrhea as its drug-related adverse event. High dose of icotinib was administered after pelvis lesion progression for 3 months with good tolerance. Combination therapy of icotinib, surgery, and internal radiation for metastases of the vertebrae bones from non-small cell lung cancer seems to be a very promising technique both for sufficient pain relief and for local control of the tumor, vertebrae operation can be an encouraging option for patients with EFGR positive mutation and good prognosis indicator.

Preparation and characterization of anti-GP73 monoclonal antibodies and development of double-antibody sandwich ELISA.

BACKGROUND: Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variables representing tumor growth and invasiveness. The aim of this study was to prepare a pair of monoclonal antibodys (mAbs) against GP73 and develop a newly designed double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis of HCC. MATERIALS AND METHODS: Produced by prokaryotic expression, the purified recombinant GP73 (rGP73), produced by prokaryotic expression, was used to immunize the Balb/c mice. Two hybridoma cell lines against GP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titers of anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealed that anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully established and validated by 119 HCC and 103 normal serum samples. RESULTS: showed that the detection limit of this method could reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those of in healthy controls (mean=70.92 ng/ml). CONCLUSIONS: Results of our study not only showed that sGP73 levels of HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratory homemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide a solid foundation for subsequent clinical applications.

Serum CEA level change and its significance before and after Gefitinib therapy on patients with advanced non-small cell lung cancer.

OBJECTIVE: The aim of this study was to explore change and significance of serum carcino-embryonic antigen (CEA) before and after gefitinib therapy in patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Forty patients with advanced NSCLCs in III~IV stages were selected as study objects given gefitinib therapy combined with routine local radiotherapy until tumor progression or intolerable toxicity. After treatment, all patients were divided into control and non-control groups according to the results of evaluation based on RECIST 1.1 (Response Evaluation Criteria in Solid Tumors in 2009). Peripheral fasting blood from all patients was collected in the early morning and serum CEA was assessed by electro-chemiluminescence immunoassay (ECLIA) before and after treatment. Before treatment, patients were divided into high CEA group (CEA level > 50 ng/mL) and low CEA group (CEA level ≤ 50 ng/mL). Adverse reactions were noted and progression-free survival (PFS) in both groups was recorded after long-term follow-up that ended in December, 2012. RESULTS: There was no difference between control and non-control groups in CEA level before treatment (P>0.05), whereas serum CEA decreased more markedly lower in the control group after treatment (P<0.01). All patients were divided into high CEA group (26) and low CEA group (14) according to serum CEA level. There was no statistically significant difference between two groups in adverse reactions (P>0.05) but the rate in former group was lower. Additionally, survival rates at 9 and 12 months in high CEA group were clearly higher than in the low CEA group (P<0.01). CONCLUSIONS: Serum CEA level can serve as a biochemical index to evaluate the prognosis with gefitinib treatment for NSCLC.

[Design, synthesis, and PPARgamma agonistic activity of novel indenone derivatives].

Agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are of interest as a treatment of type II diabetes, and indenone derivatives are a new class of non-TZD PPARgamma agonists. Based on existing indenone derivatives, a series of novel ones have been designed and synthesized. Meanwhile the structures have been comfirmed with 1H NMR and MS. Among them, 17b and 19 showed higher agonistic activities than rosiglitazone.

Toxoplasma gondii: enzyme-linked immunosorbent assay based on a recombinant multi-epitope peptide for distinguishing recent from past infection in human sera.

Enzyme-linked immunosorbent assays (ELISAs) based on a recombinant multi-epitope peptide (rMEP) were used in an attempt to differentiate pregnant women with Toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). The recombinant expression vector pET-32c-MEP encoding MEP constructed previously was expressed in Escherichia coli and the rMEP was purified as a bioactive fusion protein. The IgG-ELISA and IgM-ELISA based on the purified rMEP were developed, and used to detect IgG and IgM antibodies against Toxoplasma gondii in human sera. Immunoblot assays showed that the purified rMEP could be strongly recognized by IgM antibodies in the pooled sera from women with acute profiles, and by IgG antibodies in the pooled sera from women with chronic profiles. ELISA results also proved that the reactivities of IgG and IgM antibodies differed significantly in sera from women with acute and chronic profiles. Compared with two commercial ELISA tests for seradiagnosis of toxoplasmosis, the total concordance (including positive and negative sera) of this rMEP-based assay was 93.2% and 95.7% for the detection of IgG and IgM antibodies, respectively. Our study suggests that the rMEP protein could be used as the diagnostic antigen to differentiate recent from past infections in human toxoplasmosis.

Polymorphisms in interleukin-1B (IL-1B) and interleukin 1 receptor antagonist (IL-1RN) genes associate with gastric cancer risk in the Chinese population.

BACKGROUND: Gastric cancer is one of the most common malignancies afflicting the Chinese population. Polymorphisms in interleukin-1B (IL-1B) and interleukin-1 receptor antagonist (IL-1RN) genes have been associated with increased gastric cancer risk. AIMS: A case-control study enrolled 392 gastric cancer patients and 508 healthy were carried out to investigate the association between polymorphisms in IL-1B and IL-1RN and gastric cancer risk. METHODS: Polymerase chain reaction (PCR)-restriction fragment length polymorphism was used for detection of two potentially functional polymorphisms (IL-1B-31 and IL-1B-511) in the IL-1B gene promoter and PCR was used for detection of the variable tandem repeat in the second intron of IL-1RN. RESULTS: The data showed that the IL-1B-31CC genotype increased gastric cancer risk to an adjusted odd of 2.27 (95% CI, 1.49-3.46), IL-1B-31CT to 1.48 (95% CI, 1.01-2.16) and IL-1B-31CT/CC to 1.68 (95% CI, 1.17-2.40), while IL-1B-51TT genotype associated with increased gastric cancer risk to an adjusted odd of 2.53 (95% CI, 1.67-3.84), IL-1B-511TC to 1.45 (95% CI, 1.02-2.06), and IL-1B-511TC TT/TC to 1.72 (95% CI, 1.23, 2.39). Furthermore, IL-1RN heterogeneity genotype (IL-1RN2L) was associated with gastric cancer risk to an adjusted odd of 1.70 (95% CI, 1.05-2.74) compared to the wild-type homozygote (IL-1RNLL). In addition, H. pylori infection enhanced gastric cancer risk through these SNPs. CONCLUSIONS: The data from the current study demonstrated that the genotype CC or CT of IL-1B-31, TT or CT of IL-1B-511, and 2L of IL-1RN increased risk of gastric cancer in this Chinese population and the risk was further enhanced by H. pylori.

[Effect of recombinant adenovirus Ad-DT-A in targeted therapy for malignant cancer cell lines with loss of IGF2 imprinting].

OBJECTIVE: To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors. METHODS: The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors. RESULTS: The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%. CONCLUSIONS: The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

RNAi-mediated silencing of CD147 inhibits tumor cell proliferation, invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro.

BACKGROUND: CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer. METHODS: Short hairpin RNA (shRNA) expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively. RESULTS: Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin. CONCLUSION: CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.

[Erlotinib in the treatment of advanced non-small-cell lung cancer (NSCLC)].

OBJECTIVE: Erlotinib is a small-molecule inhibitor of EGFR tyrosine kinase, showing a significant improvement of survival in non-small-cell lung cancer (NSCLC) after the failure of front-line chemotherapy. The aim of this study was to evaluate the antitumor efficacy and toxicity of Erlotinib in the treatment of advanced NSCLC patients. METHODS: A total of 104 patients with advanced NSCLC admitted in our department during December 2006 to November 2008 were enrolled in this study. Eligible patients received oral Erlotinib 150 mg/d until disease progression or intolerable toxicity. Best clinical response was determined using RECIST criteria, the adverse events were evaluated according to the NCI criteria. RESULTS: The total effective rate was 27.9% (29/104) and the clinical benefit was 76.0% (79/104). The median progression-free survival was 5.1 months (95%CI 4.0 - 8.0). The median survival time was 13.1 months (95%CI 10.0 - 15.7). The 1-year survival rate was 61.5%. Significant survival benefit from erlobinib therapy was observed for patients with good personal status (HR 0.56, P = 0.006), adenocarcinoma (HR 0.43, P = 0.004) and skin rash (HR 0.46, P = 0.005). But patients with smoking (HR 2.75, P < 0.001) and liver metastasis (HR 2.91, P = 0.002) add the risk of death. The adverse events were mild (grade < or = 2), most common toxicities were skin rash in 73.1% (76/104) and diarrhea in 41.3% (43/104). Only 6.7% (7/104) patients got adverse events of grade > or = 3. CONCLUSION: Erlotinib is an effective and well-tolerated treatment option for advanced NSCLC and could offer an alternative for patients after the failure of first-line chemotherapy, unsuitable for or not wishing to receive chemotherapy.

[Effect of Rapamycin and Cyclosporin A on the expression of TLR5 and Foxp3 in allorejection].

AIM: To study the effect of Rapamycin (RAPA) and Cyclosporin A (CsA) on the expression of TLR5 and Foxp3 in allotransplantation model in vivo. METHODS: The murine model of skin allotransplantation was established, and divided into three groups, injected with CsA 10 mg/(kg.d), RAPA 1.5 mg/(kg.d)) and normal saline respectively for 14 consecutive days. The grafts survival was observed daily. The expression of TLR5 and Foxp3 mRNA was detected by RT-PCR. RESULTS: The survival time was obviously longer in RAPA and CsA treated groups than control group. The expression of TLR5 mRNA in allotransplantation treated with RAPA was higher than that in control group(P<0.05) and remained at a higher level than the other two groups until day 21. TLR5 showed the highest expression on day 10 among three groups, which was the top point of allorejection. Compared with CsA group, RAPA treatment in vivo caused the higher expression of Foxp3 mRNA(P<0.05), which remained at a high level after treatment stopped. However, in CsA group, Foxp3 mRNA expression increased on day 7 and 10, and then decreased significantly on day 14. TLR5 and Foxp3 expression was positively correlated among three groups. RAPA and CsA promoted the expression of TLR5 in T cells when treated with flagellin for 6 hours in vitro and RAPA has stronger effect. CONCLUSION: RAPA and CsA can promote the expression of TLR5 and Foxp3 in allotransplantation model in vivo and flagellin enhanced this effect in vitro.

Beta1- and beta2-adrenoceptors in basolateral nucleus of amygdala and their roles in consolidation of fear memory in rats.

It is known that beta-adrenoceptor (AR) in the basolateral nucleus of amygdala (BLA) plays an essential role in fear memory formation. However, the cellular and subcellular distributions of beta1- and beta2-ARs in the BLA and their roles in fear memory formation are poorly understood. Here, we report that both beta1- and beta2-ARs are predominantly expressed in BLA neurons but not in astrocytes. beta1-AR is distributed in the cell membrane and cytoplasm of neurons, whereas beta2-AR is localized not only in the cell membrane and cytoplasm but also in the nucleus. Intra-BLA infusion of the beta1-AR antagonist metoprolol and atenolol or the beta2-AR antagonist ICI118551 and butoxamine produces a severe deficit in 24-h auditory fear memory, leaving 1-h memory intact. Western-blot analysis reveals that the protein level of cytoplasmic beta1-AR significantly increases 2- and 4-h postconditioning, whereas that of cytoplasmic or nuclear beta2-AR is unchanged. The present results indicate that beta1- and beta2-ARs in the BLA have differential subcellular localizations and both are required for the consolidation of auditory fear memory.

[Study on extraction of coumarins in Cnidium monieri by supercritical CO2 and separation of compositions analysis].

OBJECTIVE: To analyze the chemical constituents of supercritical carbon dioxide extraction products from Cnidium monieri. METHOD: Four-factor and three-level orthogonal experimental design was used to optimize the SFE conditions as guided by the content of total coumarins in the extract. The chemical constituents were separated and identified by recrystalization. RESULT: Optimum extraction process was established: 25 MPa as extraction pressure, 50 degrees C as extraction temperature, 6.5 MPa as separation pressure and 60 degrees C as separation temperature. CONCLUSION: Changes in extraction pressure, temperature, time, pulverized degree and separation pressure affect the extracting results remarkably. The two kinds of chemical constituents were separated by recrystallization from C. monieri and identified by the methods of UV, IR, MS, NMR.