Matrine induces mitochondrial apoptosis in cisplatin-resistant non-small cell lung cancer cells via suppression of ß-catenin/survivin signaling.

Matrine is an alkaloid isolated from Sophora flavescens and shows anticancer activities. The present study was carried out to determine the cytotoxic effects of matrine on cisplatin-resistant non-small cell lung cancer (NSCLC) cells and the associated molecular mechanisms. Parental and cisplatin-resistant A549 and H460 NSCLC cells were treated with 1 or 2 g/l of matrine for 48 h, and cell viability and apoptosis were assessed. ß-catenin-mediated transcriptional activity, mitochondrial membrane potential (ΔΨm) changes, activation of caspases, and survivin expression were examined. The effect of overexpression of survivin on the anticancer activity of matrine was investigated. Compared to the parental cells, cisplatin-resistant NSCLC cells showed increased ß-catenin transcriptional activity. Matrine treatment resulted in a significant reduction in ß-catenin activation and survivin expression in the cisplatin-resistant cells. Matrine caused apoptotic death in the cisplatin-resistant NSCLC cells, coupled with loss of ΔΨm and activation of caspase-9 and -3. Matrine-induced apoptosis of the cisplatin-resistant NSCLC cells was significantly reversed by overexpression of survivin. In conclusion, matrine exposure induces mitochondrial apoptosis in cisplatin-resistant NSCLC cells, which is largely mediated through inactivation of ß-catenin/survivin signaling. Further investigation of the therapeutic benefit of matrine in overcoming cisplatin resistance in NSCLC is warranted.

Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell-cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina-tion with chemotherapeutic agents for NSLC.

[Construction and characterization of EGFP reporter gene labeled Sindbis virus].

OBJECTIVE: To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV). METHODS: The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV. RESULTS: We successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability. CONCLUSION: The labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.

[Preparation and identification of a recombinant hepatitis C virus (HCV) based on sindbis virus vector].

OBJECTIVE: To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector. METHODS: The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA). RESULTS: The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2. CONCLUSION: The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.

Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.

[FAIMS of trace volatile organic compounds].

FAIMS is a fast and high sensitive technique for detecting trace volatile organic compounds. Spectra of acetone, benzene and toluene were obtained on a homemade high-field asymmetric waveform ion mobility spectrometer and they can be easily separated in the spectra. Three xylene isomeric compounds were also successfully separated in FAIMS. Effect of carrier gas flow rate on the ion intensity was analyzed, and the optimal flow rate of carrier gas was 220 L x h(-1) which can be used for the optimization of FAIMS instrument. The detection limit for acetone is 100 ng x L(-1) that is an order of magnitude lower than the foreign traditional IMS.

[IL-1ß can promote proliferation and migration of Hepa1-6 cells and impair IFNα-induced cell growth inhibition in vitro].

AIM: To analyze the effect of recombinant IL-1ß on proliferation, migration, and the effect on IFNα induced cell growth inhibtion. METHODS: The vector pLIVE-mIL-1ß was transfected into Hepa1-6 cells mediated by transIT-LT1. Gene expression level of IL-1ß was analyzed by RT-PCR and Sandwich ELISA. Cell migration was assessed using wound healing assay. RESULTS: IL-1ß significantly stimulated proliferation and migration of Hepa1-6 cells. However, expression of IL-1ß significantly down-regulated growth inhibition inducecd by IFNα. CONCLUSION: The recombinant vector could stably express IL-1ß and promote in vitro proliferation, migration, and impair IFNα-induced cell growth inhibition.

[The expression of O6-methylguanine DNA methyltransferase and p53 in non-small cell lung cancer and the association with the prognosis].

OBJECTIVE: To investigate the expression and role of O(6)-methylguanine DNA methyltransferase (MGMT) and p53 in non-small cell lung cancer (NSCLC) and the association with prognosis. METHODS: Immunohistochemical method was used to investigate the expression of MGMT and p53 in NSCLC specimens from 110 cases and in 20 cases of benign lung diseases as the control. The association of their expression with the prognosis of the 110 patients was evaluated. RESULTS: The positive expression of MGMT in NSCLC and benign lung diseases was 41.8% (46/110) and 80% (16/20) (χ(2) = 9.89, P < 0.05), respectively. The positive expression of p53 in NSCLC and benign lung diseases were 56.4% (62/110) and 0% (0/20) (χ(2) = 21.551, P < 0.05), respectively. There was a significant association between expression of MGMT with smoking, and lymph node metastasis (χ(2) = 12.107, P < 0.05; χ(2) = 6.512P < 0.05). There was also a significant association between expression of p53 with smoking and lymph node metastasis (χ(2) = 6.330, P < 0.05; χ(2) = 7.909, P < 0.05). A negative correlation was observed between the expression of MGMT and that of p53 protein in NSCLC (R(S) = -0.592, P < 0.05). The 5-year survival rate and median survival time of 110 cases was 10.9% (12/110), and (30.4 ± 0.6) months. In 46 cases with positive expression of MGMT the 5-year survival rate was 0% (0/110) and median survival was (25.9 ± 0.4) months, which were lower than those in the 64 patients with negative expression of MGMT [18.8% (12/64), (32.4 ± 0.7) months], Log rank test χ(2) = 23.569, P < 0.05. In the 62 patients with positive expression of p53, the 5-year survival rate and median survival were 4.8% (3/62) and (30.4 ± 1.2) months, which were lower than those in the 48 cases with negative expression of p53 [18.8% (9/48), (30.5 ± 1.1) months], Log rank test χ(2) = 5.521, P < 0.05. CONCLUSION: The loss of expression of MGMT may lead to activation of the wild-type p53. They may participate in lung carcinomatosis, and may predict prognosis in patients with NSCLC.

Isolation and full-length sequence analysis of Armigeres subalbatus totivirus, the first totivirus isolate from mosquitoes representing a proposed novel genus (Artivirus) of the family Totiviridae.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a -1 frameshift. Elements favouring -1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

[Establishment of AFP promoter operated murine IL-1beta recombinant vector and its expression in H22 cells].

AIM: To establish a hepatoma specific murine IL-1beta (mIL-1beta) expression vector operated by AFP promoter and analysis its expression in H22 cell. METHODS: The chimeric operating sequence composed of the minimal AFP promoter and CMV enhancer(ECMV) was prepared through SOE-PCR. The sequence was inserted to replace the conventional enhancer and promoter in pIRES2-EGFP to establish the novel hepatoma specific vector p(afp)IRES2-EGFP. Full length of murine IL-1beta was amplified through RT-PCR by pfu DNA polymerase followed by cloning to establish the recombinant pIRES2-EGFP-mIL-1beta expression vector verified through PCR, restriction enzyme assay, DNA sequencing and cell transfection. p(afp)IRES2-EGFP-mIL-1beta was tranfected into H22 hepatoma cells and YAC-1 lymphoma cells in a transient transfection system mediated by jetPEI. Expression of the vector was observed under fluorescent microscope 48 h after transfection. Expression level of mIL-1beta was detected by RT-PCR. RESULTS: A 537 bp chimeric AFP promoter and ECMV was yield and inserted to establish a novel hepatoma specific vector p(afp)IRES2-EGFP proved by restriction enzyme assay, DNA sequencing and transfection. Full length murine IL-1beta was then amplified and cloned to establish the recombinant expression vector p(afp)IRES2-EGFP-mIL-1beta verified through repeated clony PCR, restriction enzyme assay by EcoR I and Xho I, DNA sequencing and transfection. Purified p(afp)IRES2-EGFP-mIL-1beta was transiently transfected into H22 cells and YAC-1 cells by jetPEI, and bright green fluorescence was only seen on the surface of H22 cells, indicating that p(afp)IRES2-EGFP-mIL-1beta can specifically express target gene within the murine hepatoma cells. Simutaneously, the expression level of mIL-1beta was markedly elevated in H22/mIL-1beta in RT-PCR assay. CONCLUSION: We successfully prepared a hepatoma specific expression vector named p(afp)IRES2-EGFP-mIL-1beta that could expression high level of murine IL-1beta in a transient transfection system.

[The first report of Kadipiro virus isolation in China].

5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.

[A new member of Brevidensovirus, 0507JS11 virus isolated from Culex mosquitoes collected in Xinjiang].

OBJECTIVE: To probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy. METHODS: 0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis. RESULTS: 0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus. CONCLUSION: 0507JS11 virus is a new member in Brevidensovirus.

[Isolation and primary identification of viruses in mosquitoes in the south of Xinjiang].

OBJECTIVE: To isolate viruses from mosquitoes in the south of Xinjiang and identify these viruses primarily. METHODS: A total of 13 491 mosquitoes were collected in the south of Xinjiang from Jul to Aug, 2005. These mosquitoes were divided into 130 groups and grinded respectively. The supernates were inoculated in C6/36 and Vero cells. Viruses isolated were detected, the genomic nucleic types by electrophoresis of viral genomes and the morphologies observed under electronmicroscope. RESULTS: All 42 viruses were isolated, which caused CPEs on C6/36 but not on Vero cells. 27 viruses showed similar genomic profiles with 12 dsRNA segments. 1 virus displayed genomic profile with 10 dsRNA segments. 5 viruses took on similar genomic profiles with about 4 kbp DNA band. 9 viruses did not get any taxonomy information. Electromicroscopic pictures of these viruses revealed that above four types of viruses had distinguished morphologies indicating different virus species. CONCLUSION: There should be several virus species in the mosquitoes in the south of Xinjiang. dsRNA virus with 12 genomic segments should play analysis a predominant role in the south of Xinjiang.

[Isolation and identification of Banna virus from mosquito for the first time in Inner Mongolia].

OBJECTIVE: To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region. METHODS: A strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar). RESULTS: The new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%. CONCLUSION: The virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.

[Isolation and identification of Japanese encephalitis virus in Tanghe County, Henan Province].

OBJECTIVE: To isolate Japanese encephalitis virus (JEV) from mosquitoes collected in Tanghe County, Henan province and analyze the genotype of the newly isolated JEV strains and the characteristics of amino acid in the E gene. METHODS: Viruses were isolated from mosquitoes collected in 2004 and identified by biological, serological and molecular biologic methods. PrM and E segments of the newly isolated JEV were amplified by RT-PCR, the PCR products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by Clustal X (1.8) program, MEGA 3.1 and GENEDOS (3.2). RESULTS: Totally 3722 mosquitoes were collected including Culex, Armigeres, Aedes, Anopheline. Three new JEV strains isolated from Culex belonged to genotype 1. The homologue of nucleotide and amino acid of E gene between new JEV strains and live attenuated vaccine strain SA14-14-2 was 86.9-87.7% and 95.2%-97.0%, respectively. Totally there were 12 common sites of amino acid differences in E gene between them. CONCLUSION: Newly isolated viruses in Henan province belonged to JEV genotype 1. It suggests that the vaccine strain SA14-14-2 currently used for preventing JE is able to protect people from JEV infection, although there are some amino acid differences between them.

Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China.

Ten virus isolates belonging to species Getah virus (GETV) have been obtained during surveys for arboviruses in China since 1964. Seven of these isolates (YN0540, YN0542, SH05-6, SH05-15, SH05-16, SH05-17 and GS10-2) were obtained during the current study. The full-length sequences of three Chinese isolates (M1, isolated in 1964; HB0234, isolated in 2002; YN0540, isolated in 2005) were determined. The full-length sequences of these isolates were respectively 11 696, 11 686 and 11 690 nt, and showed more than 97 % intraspecies identity. Deletions were found in the capsid protein of strain M1 and non-structural protein nsP3 of strain HB0234. The E2 gene and 3' UTR of all ten isolates were also characterized. The E2 gene of the Chinese GETV isolates showed nucleotide sequence identities of 98-100 % when compared with other GETV isolates. In the 3' UTR of the Chinese isolates, an insertion of 10 consecutive adenine residues (nt 189-198) appeared in strain M1, and 9 or 3 consecutive adenines were found towards the 3' end of the third RES in strains SH05-6 and SH05-15, respectively. The 3' UTRs of the Chinese isolates showed a deletion between positions 45 and 54 and nucleotide transitions at positions 43, 64 and 148. Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates. The isolation of GETV from various provinces in China and also in Russia and Mongolia (including regions of the northern tundra) are an indication of changes in the world distribution of this re-emerging virus.

[New type of cytoplasmic polyhedrosis virus isolated from mosquitoes in China].

0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.

[0507JS60 virus isolated in Xinjiang was identified as Liaoning virus].

0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.

[Detection of Banna virus-specific nucleic acid with TaqMan RT-PCR assay].

BACKGROUND: To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus. METHODS: Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated. RESULTS: All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation. CONCLUSION: The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.

[Isolation and identification of arboviruses in Hebei Province].

BACKGROUND: To study the arboviruses carried by mosquitoes collected in Hebei Province. METHODS: Samples were collected from mosquito active sites and stored in liquid nitrogen till use. Pools of 20 to 30 mosquitoes were ground after sterilization, centrifugal supernant was inoculated onto C6/36 cell, cytopathic effect was observed for three sequential passages. Positive isolates were identified by IFA and RT-PCR. RESULTS: Totally 1310 mosquitoes were collected from two villages of She county, Hebei province. They were divided into 46 pools and ground respectively. Thirteen positive isolates were obtained. Two isolates reacted with alphaviral antibodies and were amplified by alphaviral primers, nucleotide sequence showed the highest homology (98%) to Getah virus (AY702913.1), so the two isolates were identified as Getah virus. CONCLUSION: Getah virus was isolated from mosquitoes in Hebei Province. This is the first report of isolating Getah virus from inland of China.