An ascidian Polycarpa aurata-derived pan-inhibitor against coronaviruses targeting Mpro.

Coronaviruses (CoVs) are responsible for a wide range of illnesses in both animals and human. The main protease (Mpro) of CoVs is an attractive drug target, owing its critical and highly conserved role in viral replication. Here, we developed and refined an enzymatic technique to identify putative Mpro inhibitors from 189 marine chemicals and 46 terrestrial natural products. The IC50 values of Polycarpine (1a), a marine natural substance we studied and synthesized, are 30.0 ± 2.5 nM for SARS-CoV-2 Mpro and 0.12 ± 0.05 µM for PEDV Mpro. Our research further demonstrated that pretreatment with Polycarpine (1a) inhibited the betacoronavirus SARS-CoV-2 and alphacoronavirus PEDV multiplication in Vero-E6 cells. As a result, Polycarpine (1a), a pan-inhibitor of Mpro, will function as an effective and promising antiviral option to combat CoVs infection and as a foundation for further therapeutic research.

Development and validation of prognostic nomogram based on log odds of positive lymph nodes for patients with gastric signet ring cell carcinoma.

OBJECTIVE: Our aims were to establish novel nomogram models, which directly targeted patients with signet ring cell carcinoma (SRC), for individualized prediction of overall survival (OS) rate and cancer-specific survival (CSS). METHODS: We selected 1,365 SRC patients diagnosed from 2010 to 2015 from Surveillance, Epidemiology and End Results (SEER) database, and then randomly partitioned them into a training cohort and a validation cohort. Independent predicted indicators, which were identified by using univariate testing and multivariate analyses, were used to construct our prognostic nomogram models. Three methods, Harrell concordance index (C-index), receiver operating characteristics (ROC) curve and calibration curve, were used to assess the ability of discrimination and predictive accuracy. Integrated discrimination improvement (IDI), net reclassification improvement (NRI) and decision curve analysis (DCA) were used to assess clinical utility of our nomogram models. RESULTS: Six independent predicted indicators, age, race, log odds of positive lymph nodes (LODDS), T stage, M stage and tumor size, were associated with OS rate. Nevertheless, only five independent predicted indicators were associated with CSS except race. The developed nomograms based on those independent predicted factors showed reliable discrimination. C-index of our nomogram for OS and CSS was 0.760 and 0.763, which were higher than American Joint Committee on Cancer (AJCC) 8th edition tumor-node-metastasis (TNM) staging system (0.734 and 0.741, respectively). C-index of validation cohort for OS was 0.757 and for CSS was 0.773. The calibration curves also performed good consistency. IDI, NRI and DCA showed the nomograms for both OS and CSS had a comparable clinical utility than the TNM staging system. CONCLUSIONS: The novel nomogram models based on LODDS provided satisfying predictive ability of SRC both in OS and CSS than AJCC 8th edition TNM staging system alone.

Gene therapy of gastric cancer using LIGHT-secreting human umbilical cord blood-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have the ability to migrate into tumors and therefore are potential vehicles for the therapy of malignant diseases. In this study, we investigated the use of umbilical cord blood mesenchymal stem cells (UCB-MSCs) as carriers for a constant source of transgenic LIGHT (TNFSF14) to target tumor cells in vivo. METHODS: Lentiviral vectors carrying LIGHT genes were constructed, producing viral particles with a titer of 2 × 10(8) TU/L. Fourteen days after UCB-MSCs transfected by LIGHT gene packaged lentivirus had been injected into mouse gastric cancer models, the expression levels of LIGHT mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Then the tumors' approximate volumes were measured. RESULTS: The treatment with MSC-LIGHT demonstrated a strong suppressive effect on tumor growth compared to treatment with MSC and NaCl (p < 0.001). Examination of pathological sections of the tumor tissues showed that the areas of tumor necrocis in the MSC-LIGHT group were larger than those in the MSC group. Moreover, we found that MSCs with LIGHT were able to significantly induce apoptosis of tumor cells. The expression levels of LIGHT mRNA and protein were significantly higher in the UCB-MSCs with the LIGHT gene than the levels in UCB-MSCs (p < 0.001). CONCLUSION: These results suggest that UCB-MSCs carrying the LIGHT gene have the potential to be used as effective delivery vehicles in the treatment of gastric cancers.

TNF-α expression in the UCB-MSCs as stable source inhibits gastric cancers growth in nude mice.

Mesenchymal stem cells (MSCs) are potentially vehicles for therapy of malignant diseases. In our study, we investigated whether UCB-MSCs are capable to carry TNF-α to target tumor cells in vivo. The human gastric cancer cells SGC-7901 were subcutaneously injected into the abdomen near groins of 15 nude mice to establish experiment tumor models. MSC-TNF-α demonstrated a strong suppressive effect on the tumor growth compared with MSC and NaCl. Thus, MSC-TNF-α can obviously inhibit Gastric cancers growth in nude mice, indicating that UCB-MSCs may have the potential to become a prevention approach against gastric cancer.

Prognostic Value of Tumor Deposit Counts in Patients with Stage III Colorectal Cancer: A Population-Based Study.

OBJECTIVE: To investigate the prognostic value of tumor deposits (TDs) counts in stage III colorectal cancer (CRC) patients and develop a prognostic nomogram. METHODS: Data on stage III CRC patients from 2010 to 2015 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. The Kaplan-Meier analysis was used to assess differences in survival outcomes among patients. The Cox regression analysis was performed to establish the independent prognostic factors for cancer-specific survival and to establish a nomogram. The nomograms' performance was evaluated by calibration plots and concordance index (C-index). Decision curve analysis (DCA) was used to assess the clinical utility of the prediction model. RESULTS: A total of 23,345 CRC patients were included in this study, and 3,578 (15.3%) had TDs. Cox multivariate regression analyses revealed that age, race, histological tumor grade, the administered chemotherapy, pathological type, T-stage, CEA, N-stage, peripheral nerve invasion, and TDs were independent prognostic factors. Patients with many TDs (=0/1-4, HR: 1.325,/≥5 HR: 2.223) had poorer cancer-specific survival. The prognostic value of the number of TDs was comparable to that of lymph node metastasis. The C-indices of the nomogram were superior to TNM staging in training (0.730 vs 0.646) and validation (0.714 vs 0.636) groups. DCA revealed that the nomogram had a higher clinical net benefit compared to TNM staging. CONCLUSIONS: TDs count is an adverse prognostic factor for stage III CRC patients. Furthermore, the TDs-based nomogram can accurately predict the prognostic outcomes for stage III CRC.

[Effect of synergistic polarization macrophage modulated by N-terminal domain of a2 vacuolar ATPase and macrophage colony stimulating factor on proliferation of gastric cancer cells].

OBJECTIVE: To investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase (a2NTD) and macrophage colony-stimulating factor (M-CSF) on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells. METHODS: Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups: the control group (RPMI 1640), the experimental group I (M-CSF 100 µg/L), the experimental group II (a2NTD 500 µg/L) and the experimental group III (a2NTD 500 µg/L plus M-CSF 100 µg/L). After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901. RESULTS: The expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups (+). The mean absorbance (A) was 0.092 ± 0.005 in control group, 0.095 ± 0.006 in group I, 0.094 ± 0.005 in group II, 0.094 ± 0.005 in group III, and no significant differences were observed among 4 groups (all P>0.05). Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group (-) with A 0.025 ± 0.004; it was normal in groupI and group II (+) with A 0.191 ± 0.012 in group I and 0.197 ± 0.136 in group II (P=0.212), and it was up-regulated significantly in group III (+++) with A 0.285 ± 0.011. There were significant differences between either two groups except group I and group II (all P<0.01). Secretion of IL-10 in group I and group II [(85.65 ± 13.64) ng/L and (87.77 ± 14.25) ng/L] was significantly higher compared with control group [(71.67 ± 7.56) ng/L, P<0.01]. Secretion of IL-12 in group I and group II [(9.91 ± 1.50) ng/L and (10.15 ± 1.80) ng/L] was significantly lower compared with control group [(16.87 ± 1.10) ng/L, P<0.01]. Secretion of IL-10 in group III [(116.98 ± 14.27) ng/L] was the highest, and secretion of IL-12 [(5.31 ± 0.88) ng/L] was the lowest (all P<0.01). There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group III showed the greatest impact compared with other 3 groups (P<0.01). CONCLUSIONS: a2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.

High expression of NEDD9 predicts adverse outcomes of colorectal cancer patients.

NEDD9, a member of Crk-associated substrate (CAS) family, is highly expressed in multiple cancer types and involved cancer cell adhesion, migration, invasion. The prognostic value of NEDD9 has not been evaluated before. The aim of this study was to evaluate the association between NEDD9 expression and survival in colorectal cancer (CRC) patients. NEDD9 expression was analyzed by immunohistochemistry in 92 patients with CRC. Patients were followed-up annually by telephone or at outpatient clinic. The results revealed that high expression of NEDD9 in 68/92 CRC samples, compared with 12/92 normal tissues (P<0.01). Correlation analysis showed high level of expression of NEDD9 was significantly correlated with advanced TNM stage (P=0.014), pT grade (P=0.009), pN (P=0.013) and pM status (P=0.047). Patients with a higher NEDD9 expression had a significantly shorter overall survival (OS) (P<0.01). The multivariate analysis revealed that NEDD9 expression could serve as an independent predictive factor of OS. Our finding demonstrated the potential value of NEDD9 expression level as a prognostic molecular marker and a target for new therapies for CRC patients.

MicroRNA-107 promotes proliferation of gastric cancer cells by targeting cyclin dependent kinase 8.

BACKGROUND: The biological processes and molecular mechanisms underlying miR-107 remain unclear in gastric cancer(GC). In this study, we aimed to investigate the expression, biological functions and mechanisms of miR-107 in GC. METHODS: Quantitative real-time RT-PCR was used to test miR-107 expression. MTT and colony formation assays were conducted to explore the potential function of miR-107 in human GC cell line SGC7901. The target gene was determined by bioinformatic algorithms, dual luciferase reporter assay, RT-PCR and Western blot. RESULTS: Expression of miR-107 was significantly elevated in GC cell line than that in gastric epithelial cell line(p = 0.012). We found that miR-107 inhibitor transfection significantly decreased the proliferation of GC cell line, and clone formation rate of miR-107 inhibitor transfected group was significantly lower than that of control group. Luciferase assays using a reporter carrying a putative miR-107 target site in the 3'untranslated region (3'-UTR) of cyclin dependent kinase 8 (CDK8) revealed that miR-107 directly targets CDK8. The expression level of CDK8 mRNA and protein in miR-107 inhibitor transfected GC cell line was significantly decreased compared with control group. CONCLUSION: Our findings indicate that miR-107 is upregulated in GC and affects the proliferation of GC cells, partially through the regulation of CDK8. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_164.

[NF-κb inhibitor PDTC enhances tumor necrosis factor α-induced apoptosis of gastric cancer cell SGC-7901].

OBJECTIVE: To investigate the effect of PDTC (inhibitor of NF-κb) on apoptosis of human gastric cancer cell line SGC-7901 induced by tumor necrosis factor α (TNF-α) and explore the related mechanisms. METHODS: After the treatment with different concentrations of PDTC, TNF-α or PDTC combined with TNF-α on gastric cancer cell line SGC-7901, the growth inhibition of SGC-7901 was measured by MTT assay. Hoechst was used to assess SGC-7901 cell apoptosis. The protein expressions of survivin and caspase-3 were detected by Western blot assay. RESULTS: The growth inhibition rate of SGC-7901 induced by PDTC (15, 30, 60, 100 µmol/L) was (12.14±0.91)%, (20.00±1.11)%, (37.63±1.01)% and (41.46±1.07)%. Different concentrations of PDTC all inhibited the growth of SGC-7901 significantly (all P<0.01), The growth inhibition rate of SGC-7901 induced by 25 mg/L TNF-α was (2.38±0.67)%, which could not significantly inhibit the growth of SGC-7901 [control (1.50±0.81)%], while TNF-α of 50, 100, 150 mg/L could inhibit the growth of SGC-7901 significantly [(4.53±0.85)%, (4.43±0.70)% and (4.74±1.07)%, all P<0.05]. PDTC (15 µmol/L) combined with TNF-α (25, 50, 100, 150 mg/L) significantly increased the cell growth inhibition rate compared with TNF-α alone or PDTC 15 µmol/L alone (all P<0.01). Hoechst assay showed that 100 mg/L TNF-α, 15 µmol/L PDTC and combination of above two all induced cell apoptosis (P<0.01), and the combination group had significantly higher percentage of cell apoptosis (P<0.01). Survivin protein was significantly down-regulated in combination group as compared with single TNF-α (100 mg/L) group, but was not significant down-regulated as compared with single PDTC (15 µmol/L) group. Caspase-3 protein expression was significantly increased in combination group as compared with other two groups. CONCLUSION: PDTC can enhance the cell apoptosis induced by TNF-α, which may be associated with the blocking of TNF-α-activated NF-κB signaling pathway by PDTC, the down-regulation of survivin expression, and up-regulation of caspase-3 expression.

[Study of inhibiting and killing effects of transgenic LIGHT human umbilical cord blood mesenchymal stem cells on stomach cancer].

OBJECTIVE: To study the inhibition and killing effect of transgenic LIGHT umbilical cord blood mesenchymal stem cells (UCBMSCs) on stomach carcinoma. METHODS: The LIGHT gene was recombined to construct the transfer plasmid pGC-FU-LIGHT by infusion technique. The 293T cells were co-transfected with the transfer plasmid pGC-FU-LIGHT, the construction plasmid Helper 1.0 and the envelope plasmid Helper 2.0 with the help of lipofectamine 2000 to produce lentiviral particles. Transgenic UCBMSCs(MSC-LIGHT) and empty carrier UCBMSCs (MSC) were obtained. Human gastric cancer cell SGC-7901 was injected into nude mice subcutaneously groin. The model of transplanted human gastric cancer cell SGC-7901 in nude mice was established. Tumorigenesis nude mice were separated into three groups randomly with 5 in each group: MSC-LIGHT group, MSC group, and NS group. Three groups of nude mice were injected around the tumor with MSC-LIGHT, MSC and NS every other day for 3 times. Four weeks later, the transplanted gastric cancer volume was measured. The expressions of LIGHT in the three groups were determined by RT-PCR and ELISA method. The necrosis area in the tumors was calculated under pathological examination. RESULTS: The average volume of transplanted tumor was(0.45±0.25) cm(3) in MSG-LIGHT group, (0.64±0.36) cm(3) in MSG group, and(1.21±0.79) cm(3) in NS group, and the difference was statistically significant(P<0.05). The LIGHT mRNA was 2.96±0.27, 1.23±0.47, and 0.73±0.10 respectively. The LIGHT protein was(167.89±2.31), (73.22±5.74), and (49.66±5.25) ng/L. The differences were all statistically significant among the three groups(both P<0.01). Pathological examination showed that the necrosis area was largest in MSC-LIGHT group. CONCLUSION: Transgenic UCBMSCs secret LIGHT in a paracrine manner, which has inhibition and killing effects on stomach carcinoma.