Four new steroidal glycosides from Lilium lancifolium Thunb. and their antitumor activity.

Four new steroidal glycosides (1-4), including two steroidal saponins named lililancifoloside B and C (1-2), one pregnane glycoside named lililancifoloside D (3), and one C22-steroidal lactone glycoside named lililancifoloside E (4), together with five known ones (5-9), were isolated from the bulbs of Lilium lancifolium Thunb. By using spectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS, the structures of 1-4 were elucidated. All isolates were tested for their cytotoxic potential against the MCF-7, MDA-MB-231, HepG2, and A549 cell lines. Compound 6 distinguished out among them, IC50 values of 3.31, 5.23, 1.78, and 1.49 µM against the four cell lines, respectively. Other compounds such as compound 3, 5, and 9 have also shown specific cytotoxic activity. Next, studies showed that compound 6 might cause HepG2 cells to undergo a cell cycle arrest during the G2/M phase and apoptosis.

Jaridon 6, a new diterpene from Rabdosia rubescens (Hemsl.) Hara, can display anti-gastric cancer resistance by inhibiting SIRT1 and inducing autophagy.

Tumor resistance is the main cause of treatment failure and is associated with many tumor factors. Jaridon 6, a new diterpene extracted from Rabdosia rubescens (Hemsl.) Hara, which has been previously extracted by our research team, has been tested having more obvious advantages in resistant tumor cells. However, its mechanism is unclear. In this study, we studied the effect and the specific mechanism of Jaridon 6 in resistant gastric cancer cells. Cytotoxicity test, colony test, western blotting, and nude test verified the anti-drug resistance ability of Jaridon 6 in the MGC803/PTX and MGC803/5-Fu cells. Jaridon 6 has shown obvious inhibitory effects in the sirtuin 1 (SIRT1) enzyme test. Transmission electron microscopy and immunofluorescence tests further proved the autophagic action of Jaridon 6. Jaridon 6 could inhibit the proliferation of the resistant gastric cancer cell in vivo and in vitro. Jaridon 6 inhibited SIRT1 enzyme and induced autophagy by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway. Thus, it may be considered for treating gastric cancer resistance by individual or combined administration, as an SIRT1 inhibitor and autophagy inducer.

[Up-Regulation of PI3K/Akt Signaling Pathway in H929 Cell Line by Doxycycline Not Beneficial to Its Anti-Myeloma Effect].

OBJECTIVE: To investigate the effect and possible mechanism of up-regulation of p-Akt by doxycycline (DOX) on myeloma cell line H929. METHODS: Multiple myeloma cell line H929 was treated with DOX at different concentrations for different times, and cell proliferation rate was measured by CCK-8 assay. The protein expression level of p-Akt, PTEN, p-PDK1, p-mTOR, p-GSK-3ß, and p-BAD was analyzed by Western blot. The mRNA levels of mTOR, BCL-2, and NF-κB was analyzed by RT-PCR. PI3K inhibitor Wortmannin was used to antagonize the up-regulation of p-Akt, and the cell proliferation and p-Akt protein expression level were analyzed by CCK-8 assay and Western blot respectively. RESULTS: DOX could inhibit the proliferation of H929 cells and up-regulate the expression of p-Akt at the same time. The protein levels of both p-PDK1 and PTEN in H929 cells did not alter significantly during DOX treatment. The expressions of p-BAD and p-GSK-3ß were up-regulated in H929 cells after treated with DOX, but the expression of p-mTOR was not altered. The mRNA levels of mTOR, BCL-2, and NF-κB in H929 were all down-regulated in H929 cells during DOX treatment. The effect up-regulating p-Akt level by DOX was suppressed when DOX combined with PI3K inhibitor Wortmannin and Wortmannin could enhance the inhibitory effect of DOX in H929 cells. CONCLUSION: DOX can activate PI3K/Akt signaling pathway in H929 cells, and antagonizing this effect of DOX may enhance its cytotoxicity to myeloma cells.

An effective classifier based on convolutional neural network and regularized extreme learning machine.

An effective classifier combining convolutional neural network and regularized extreme learning machine (called as CNN-RELM) is presented in this paper. Firstly, CNN-RELM trains the convolutional neural network (CNN) using the gradient descent method until the learning target accuracy reaches. Then the fully connected layer of CNN is replaced by regularized extreme learning machine (RELM) optimized by genetic algorithm and the rest layers of the CNN remain unchanged. The experiments on different face databases are given to evaluate the performance of CNN-RELM. The experimental results show that CNN-RELM is a feasible classifier and it outperforms CNN and RELM. Due to the uniting of CNN and RELM, CNN-RELM have the advantages of CNN and RELM and it is easier to learn and faster in testing.

Complement Receptor-Mediated Phagocytosis Induces Proinflammatory Cytokine Production in Murine Macrophages.

Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1ß, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.

Generation and characterization of a paclitaxel-resistant human gastric carcinoma cell line.

The main aim of this study was to establish a novel paclitaxel (PTX)-resistant human gastric carcinoma cell line and to investigate its biological significance. A cell line, MGC803/PTX, was established by gradually increasing PTX density on the basis of MGC803 over a period of 10 months. In addition, a pair of resistant cell lines (SW620 and SW620/PTX) were added to further explain the resistant mechanism of PTX. The drug resistance index and stability of MGC803/PTX cells were detected using the Cell Counting Kit-8 method. The morphological features were observed using inverted microscopy. Apoptosis was measured by flow cytometry (FCM) and Hoechst 33258 fluorescence staining. The distribution of the cell cycle was determined by FCM, and protein expressions of P-gp, Bcl-2, Bax, and PARP were detected by western blot analysis. When characterizing the resistance in vitro, we found that MGC803/PTX cells were 10.3-fold more resistant to PTX compared with MGC803 cells. In addition, MGC803/PTX cells showed cross-resistance to 5-fluorouracil and adriamycin. FCM and Hoechst 33258 fluorescence staining indicated that MGC803/PTX cells had a significantly lower percentage of apoptotic cells after treatment with PTX compared with MGC803 cells. Other differences between parental cells and resistant cells included morphology, proliferation rate, doubling time, cell cycle distribution, and colony-formation rate. Western blot analysis indicated that P-gp, Bcl-2, and PARP protein were more abundant in MGC803/PTX and SW620/PTX cells compared with MGC803 and SW620 cells, whereas Bax protein levels were lower in resistant cells. Furthermore, MGC803/PTX cells showed obvious resistance to PTX in vivo. To our knowledge, this is the first report on the establishment of a PTX-resistant MGC803 cell line, which is an important tool to explore the resistance of anticancer drugs and to overcome tumor drug resistance.

Folded short azapeptide for conformation switching-based fluorescence sensing.

Herein, we report a conformation switching-based fluorescence sensing scheme using dipeptide-based amidothioureas (azapeptides) that contain a folded ß-turn structure. Amidothiourea is equipped at its two termini with an electron acceptor and an electron donor or two fluorophores, such that it exhibits enhanced exciplex or excimer emission because of the turn structure in which the two termini are brought into close proximity; on the other hand, it exhibits a dramatic ratiometric fluorescence response upon anion binding to the thiourea moiety because of the resultant extended conformation of the anion binding complex.

Structure-Activity Relationship Studies of ß-Lactam-azide Analogues as Orally Active Antitumor Agents Targeting the Tubulin Colchicine Site.

We have synthesized a series of new ß-lactam-azide derivatives as orally active anti-tumor agents by targeting tubulin colchicine binding site and examined their structure activity relationship (SAR). Among them, compound 28 exhibited the most potent antiproliferative activity against MGC-803 cells with an IC50 value of 0.106 µM by induction of G2/M arrest and apoptosis and inhibition of the epithelial to mesenchymal transition. 28 acted as a novel inhibitor of tubulin polymerization by its binding to the colchicine site. SAR analysis revealed that a hydrogen atom at the C-3 position of the ß-lactam was required for the potent antiproliferative activity of ß-lactam-azide derivatives. Oral administration of compound 28 also effectively inhibited MGC-803 xenograft tumor growth in vivo in nude mice without causing significant loss of body weight. These results suggested that compound 28 is a promising orally active anticancer agent with potential for development of further clinical applications.

Profiling methyl jasmonate-responsive transcriptome for understanding induced systemic resistance in whitebark pine (Pinus albicaulis).

KEY MESSAGE: RNA-seq analysis on whitebark pine needles demonstrated that methyl jasmonate (MeJA)-triggered transcriptome re-programming substantially overlapped with defense responses against insects and fungal pathogens in Pinus species, increasing current knowledge regarding induced systemic resistance (ISR) to pathogens and pests in whitebark pine. Many whitebark pine populations are in steep decline due to high susceptibility to mountain pine beetle and the non-native white pine blister rust (WPBR). Resistance, including induced systemic resistance (ISR), is not well characterized in whitebark pine, narrowing the current options for increasing the success of restoration and breeding programs. Exogenous jasmonates are known to trigger ISR by activating the plant's immune system through regulation of gene expression to produce chemical defense compounds. This study reports profiles of whitebark pine needle transcriptomes, following methyl jasmonate (MeJA) treatment using RNA-seq. A MeJA-responsive transcriptome was de novo assembled and transcriptome profiling identified a set of differentially expressed genes (DEGs), revealing 1422 up- and 999 down-regulated transcripts with at least twofold change (FDR corrected p < 0.05) in needle tissues in response to MeJA application. GO analysis revealed that these DEGs have putative functions in plant defense signalling, transcription regulation, biosyntheses of secondary metabolites, and other biological processes. Lineage-specific expression of defense-related genes was characterized through comparison with MeJA signalling in model plants. In particular, MeJA-triggered transcriptome re-programming substantially overlapped with defense responses against WPBR and insects in related Pinus species, suggesting that MeJA may be used to improve whitebark pine resistance to pathogens/pests. Our study provides new insights into molecular mechanisms and metabolic pathways involved in whitebark pine ISR. DEGs identified in this study can be used as candidates to facilitate identification of genomic variation contributing to host resistance and aid in breeding selection of elite genotypes with better adaptive fitness to environmental stressors in this endangered tree species.

Gene expression profiling and pathway network analysis of anti-tumor activity by Jaridon 6 in esophageal cancer.

Jaridon 6, a novel ent-kaurene diterpenoid derived from Rabdosia rubescens (Hemsl.) Hara, possesses strong anti-tumor activity in esophageal cancer cells. In this study, we explored the underlying molecular events of the anti-tumor activity of Jaridon 6. Cell viability and apoptosis results obtained by flow cytometry confirmed the tumor inhibitory effect of Jaridon 6 in esophageal cancer cells. A cDNA microarray was performed and the observations were validated using quantitative reverse transcription polymerase chain reaction. The microarray data showed that 151 genes were differentially expressed between the untreated group and the Jaridon 6-treated group, among these were 57 upregulated genes, and 94 downregulated genes (P < 0.01, fold change threshold: 2). These included genes such as Wnt, peroxisome, and genes involved in chemokine signaling pathways. In addition, Western blot analysis demonstrated that Jaridon 6 regulated the expression of Wnt pathway proteins, including reduced levels of Dvl 2, survivin and cyclin D1, and increased levels of p-ß-catenin, and AXIN2 in EC109 and EC9706 esophageal cancer cells. In addition, recombinant murine Wnt3a could change the regulation of Jaridon 6 on Wnt pathway proteins. Immunohistochemical analysis indicated that the anti-tumor activity of Jaridon 6 was closely related to the Wnt signaling pathway in esophageal cancer cells.

Characterization of Five Novel Mitoviruses in the White Pine Blister Rust Fungus Cronartium ribicola.

The white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.) is an exotic invasive forest pathogen causing severe stem canker disease of native white pine trees (subgenus Strobus) in North America. The present study reports discovery of five novel mitoviruses in C. ribicola by deep RNA sequencing. The complete genome of each mitovirus was determined by rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (RT-PCR). A single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) was detected in each of the viral genomes using mitochondrial genetic codes. Phylogenetic analysis indicated that the C. ribicola mitoviruses (CrMV1 to CrMV5) are new putative species of the genus Mitovirus. qRT-PCR and RNA-Seq analyses revealed that viral RNAs were significantly increased in fungal mycelia in cankered pine stems compared to expression during two different stages of spore development, suggesting that viral genome replication and transcription benefit from active growth of the host fungus. CrMVs were widespread with relatively high levels of minor allele frequency (MAF) in western North America. As the first report of mitoviruses in the Class Pucciniomycetes, this work allows further investigation of the dynamics of a viral community in the WPBR pathosystem, including potential impacts that may affect pathogenicity and virulence of the host fungus.

Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (P<0.05). The apoptotic rate of the experimental group was markedly higher than the control group and blank group (P<0.01). The CCK-8 proliferation assay revealed that, compared with the control and blank groups, the proliferation of the EC109 cells in the experimental group was significantly inhibited (P<0.05). The wound healing assay revealed that the migration capacity of the cells in the experimental group was significantly decreased (P<0.05). The Transwell chamber assay demonstrated that the invasive ability of the EC109 cells in the experimental group was significantly decreased (P<0.01). These results revealed that ABCE1 is closely associated with cell proliferation, invasion and migration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

Changes of ribbon synapses number of cochlear hair cells in C57BL/6J mice with age(Δ).

To investigate the changes of ribbon synapses (RS) number in cochlear hair cells in C57BL/6J mice with age. Basilar membranes within the cochlea of C57BL/6J mice aged 2, 6, 10 and 12 months were harvested (5 mice in each age group). The presynaptic and postsynaptic membranes were subject to double immunohistochemical staining and observed with a laser confocal microscope. The number of RS in each segment of basilar membrane was counted by using 3D reconstruction technique. Compared with 2-month-old mice, reduction of RS number in basilar membrane inside cochlea mainly occurred to the basal turn among C57BL/6J mice aged 6 months. The number of RS in each turn among 10-month-old mice decreased considerably, and such decrease continued in the top turn and middle turn in mice aged 12 months. In contrast, the number of RS in the basal turn increased slightly. Reduction of RS probably takes place in the early stage of C57BL/6J mice presbycusis. Early prevention of presbycusis can be achieved through measures to mitigate the reduction of RS.

Radiosensitivity enhancement by arsenic trioxide in conjunction with hyperthermia in the EC-1 esophageal carcinoma cell line.

OBJECTIVE: To explore the effect on radiosensitivity of arsenic trioxide (As203) in conjunction with hyperthermia on the esophageal carcinoma EC-1 cell line. METHOD: Inhibition of EC-1 cell proliferation at different concentrations of As203 was assessed using the methyl thiazolyl blue colorimetric method (MTT method), with calculation of IC50 value and choice of 20% of the IC50 as the experimental drug concentration. Blank control, As203, hyperthermia, radiotherapy group, As203 + hyperthermia, As203 + radiotherapy, hyperthermia + radiotherapy and As203 + hyperthermia + radiotherapy groups were established, and the cell survival fraction (SF) was calculated from flat panel colony forming analysis, and fitted by the 'multitarget click mathematical model'. Flow cytometry (FCM) was used to detect changes in cell apoptosis and the cell cycle. RESULTS: As203 exerted inhibitory effects on proliferation of esophageal carcinoma EC-1 cells, with an IC50 of 18.7 µmol/L. After joint therapy of As203 + hyperthermia + radiotherapy, the results of FCM showed that cells could be arrested in the G2/M phase, and as the ratio of cells in G0/G1 and S phases decreased, cell death became more pronounced. CONCLUSION: As203 and hyperthermia exert radiosensitivity effects on esophageal carcinoma EC-1 cells, with synergy in combination. Mechanistically, As203 and hyperthermia mainly influence the cell cycle distribution of EC-1 esophageal carcinoma cells, decreasing the repair of sublethal damage and inducing apoptosis, thereby enhancing the killing effects of radioactive rays.

[Analysis of prognostic factors in 68 patients with cancer of unknown primary site].

OBJECTIVE: The aim of this study was to analyze the clinical characteristics and prognostic factors in patients with cancer of unknown primary site (CUP). METHODS: The clinical and follow-up data of 68 CUP patients (46 adenocarcinoma patients, 22 squamous cell carcinoma patients), were retrospectively analyzed. Univariate and multivariate analysis were conducted to determine the correlation of survival with clinical features, tumor markers, blood test, liver function and so on. RESULTS: The median survival time of the 68 CUP patients was 123 days. The results from univariate Cox regression analysis showed that the prognostic factors were related to a performance status, presence or absence of liver metastases, the number of metastatic sites, carcinoembryonic antigen (CEA), lactate dehydrogenase (LDH), hypoalbuminemia, hypohemoglobinemia and lymphocyte count. Multivariate Cox regression analysis of the clinical factors identified that a performance status (PS) ≥ 2, liver metastasis, elevated serum carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) levels, hypoalbuminemia (< 35 g/L) and lymphopenia (≤ 0.7 × 10(9)/L) were significant independent unfavorable predictive factors. Based on the number of the unfavorable predictive factors, we divided all the patients into three subgroups: subgroup involving 0-1 unfavorable factor, subgroup involving 2 - 3 unfavorable factors and subgroup involving 4 - 6 unfavorable factors. The median survival time was 390 days, 138 days and 77 days, respectively, in the 3 subgroups. Compared with the other two groups, the survival of the subgroup involving 0 - 1 unfavorable factor was significantly longer (P < 0.05), the survival between the subgroup involving 2 - 3 unfavorable factors and subgroup involving 4 - 6 unfavorable factors was not significantly different (P > 0.05). CONCLUSIONS: A performance status ≥ 2, liver metastasis, elevated serum carcinoembryonic antigen and lactate dehydrogenase levels, hypoalbuminemia and lymphopenia are independent unfavorable prognostic factors in patients with cancer of unknown primary site. The patients who had more than 2 unfavorable prognostic factors have a worse prognosis.

[Effects of Qingshi Cream on chronic dermatitis-eczema in mice].

OBJECTIVE: To explore the effects of Qingshi Cream, a compound traditional Chinese herbal medicine, on chronic dermatitis-eczema in mice induced by 2,4-dinitrochlorobenzene (DNCB). METHODS: Thirty BALB/C mice were randomly divided into vaseline group, 0.1% mometasone furoate cream group and Qingshi Cream group. Right ears of BALB/C mice were repeatedly challenged with 0.1% DNCB every three days for five times and previously sensitized with 7% DNCB to induce chronic dermatitis-eczema. Mice in different groups were applied Qingshi Cream, 0.1% mometasone furoate cream and vaseline respectively after each challenge. Weight difference of two ears, pathological change of right ear and dermal inflammatory cell number were used to assess the effects of the drugs. RESULTS: After the 5th challenge, weight differences of two ears in the 0.1% mometasone furoate cream group and the Qingshi Cream group were significantly decreased as compared with that in the vaseline group. Changes such as ear swelling thickening and cellular infiltration in dermis were observed, and these features seemed to be more significant in the vaseline group as compared with the 0.1% mometasone furoate cream group and the Qingshi Cream group. CONCLUSION: Qingshi Cream has an obvious effect in treatment of chronic dermatitis-eczema in mice.