The antimicrobial peptide chensinin-1b alleviates the inflammatory response by targeting the TLR4/NF-κB signaling pathway and inhibits Pseudomonas aeruginosa infection and LPS-mediated sepsis.

Excessive inflammatory responses are a major contributor to the high mortality associated with sepsis, a prevalent global complication. Therefore, the potential therapeutic strategy for sepsis involves targeting macrophages and reducing proinflammatory cytokine release. Chensinin-1b, an analog of the natural antimicrobial peptide derived from Rana chensinensis skin secretion, exhibits broad-spectrum antibacterial activity and adopts a random coil conformation in both PBS and membrane solution. By efficiently neutralizing LPS, chensinin-1b holds promise in alleviating LPS-induced inflammatory responses. In this study, we established a mouse septic shock model by exposing mice to multiple-drug-resistant Pseudomonas aeruginosa, as well as an endotoxin-mediated sepsis model induced by LPS. Administering chensinin-1b significantly prolonged the survival of the experimental mice, concurrently mitigating inflammatory responses and reducing organ damage. Additionally, we investigated the anti-inflammatory mechanism of chensinin-1b using a constructed LPS-induced mouse macrophage RAW264.7 inflammatory model. Our findings demonstrated that chensinin-1b effectively mitigated the excessive activation of the TLR4/NF-κB signaling pathway by directly neutralizing extracellular LPS, thus ameliorating the inflammatory response. Moreover, upon blocking the TLR4 signaling pathway, chensinin-1b further reduced the release of proinflammatory cytokines induced by LPS, indicating alternative modes of regulation. Notably, chensinin-1b rapidly entered RAW264.7 cells within 30 min via endocytosis, diffusing into the cytoplasm while retaining its anti-inflammatory properties intracellularly. Although further investigations are warranted to comprehensively elucidate the intracellular anti-inflammatory mechanism of chensinin-1b, our findings substantiate its possession of anti-inflammatory properties both intracellularly and extracellularly. Thus, chensinin-1b emerges as a promising candidate for mitigating excessive inflammatory responses associated with sepsis.

Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Tyrosine kinase inhibitors, such as imatinib, have been used as first-line therapy for the treatment of GISTs. Although these drugs have achieved considerable efficacy in some patients, reports of resistance and recurrence have emerged. Extracellular signal-regulated kinase 1/2 (ERK1/2) protein, as a member of the mitogen-activated protein kinase (MAPK) family, is a core molecule of this signaling pathway. Nowadays, research reports on the important clinical and prognostic value of phosphorylated-ERK (P-ERK) and phosphorylated-MAPK/ERK kinase (P-MEK) proteins closely related to raf kinase inhibitor protein (RKIP) have gradually emerged in digestive tract tumors such as gastric cancer, colon cancer, and pancreatic cancer. However, literature on the expression of these downstream proteins combined with RKIP in GIST is scarce. This study will focus on this aspect and search for answers to the problem. AIM: To detect the expression of RKIP, P-ERK, and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease. Try to establish a new prognosis evaluation model using RKIP and P-ERK in combination with analysis and its prognosis evaluation efficacy. METHODS: The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information. These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020. Immunohistochemical method was used to detect the expression of RKIP, P-ERK, and P-MEK proteins in GIST tissue samples from these patients. Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data. A Nomogram was used to represent the new prognostic evaluation model. The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors, based on two risk classification systems [the new risk grade model vs the modified National Institutes of Health (NIH) 2008 risk classification system]. Receiver operating characteristic (ROC) curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems. RESULTS: In GIST tissues, RKIP protein showed positive expression in the cytoplasm and cell membrane, appearing as brownish-yellow or brown granules. The expression of RKIP was related to GIST tumor size, NIH grade, and mucosal invasion. P-ERK protein exhibited heterogeneous distribution in GIST cells, mainly in the cytoplasm, with occasional presence in the nucleus, and appeared as brownish-yellow granules, and the expression of P-ERK protein was associated with GIST tumor size, mitotic count, mucosal invasion, and NIH grade. Meanwhile, RKIP protein expression was negatively correlated with P-ERK expression. The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis. However, RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance. Furthermore, we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model. ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system. CONCLUSION: Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size, NIH 2008 staging, and tumor invasion, and P-ERK expression was also related to mitotic count. The expression of the two proteins had a certain negative correlation. The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients. The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.

Potent antitumoral effects of targeted promoter-driven oncolytic adenovirus armed with Dm-dNK for breast cancer in vitro and in vivo.

Currently, no curative treatments are available for late-stage metastatic or recurrent breast cancer, because the cancer tolerates both chemotherapy and endocrine therapy. In this study, we investigated the feasibility of a dual-regulated oncolytic adenoviral vector with a novel suicide gene to treat breast cancer. Following targeted gene virotherapy of conditionally replicating adenoviruses (CRAds), the novel suicide gene of multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-DNK) was inserted into the double-regulated oncolytic adenovirus SG500 to ensure more safety and enhanced antitumor activity against breast cancer both in vitro and in vivo. Selective replication, cell-killing efficacy, and cytotoxicity, combined with chemotherapeutics were investigated in several breast cell lines (MDA-MB-231 and MCF-7), normal cells (WI-38 and MRC-5), and human (MDA-MB-231) tumor models in vivo. The double-regulated SG500-dNK had high cell-killing activity in breast cancer. Replication was similar to wild-type in breast cells and was attenuated in normal cells. SG500-dNK combined with the chemotherapeutics (E)-5-(2-bromovinyl)-2'-deoxyuridine (Bvdu) and 2',2'-difluoro-deoxycytidine (dFdC) resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in vitro or in breast xenografts in vivo. These data suggest that the novel oncolytic variant SG500-dNK is a promising candidate for targeting breast tumors specifically when combined with chemotherapeutics.

Conditionally replicating adenovirus SG500-expressed mutant Dm-dNK gene for breast cancer therapy.

The purpose of this analysis was to investigate the enzyme activity and specificity of using adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants in combination with gemcitabine. Compared with herpes simplex type 1 thymidine kinase (HSV-TK) and other known dNKs, this Dm-dNK enzyme has a broader substrate specificity and a higher catalytic rate. We created the Dm-dNK mutants (dNKmu) by site-directed mutagenesis at the sites of 244E, 245S, 251S and 252R, with the last 10 amino acids in the amino acid sequence randomly mutated. We evaluated the enzyme activity and substrate specificity. The engineered enzymes showed a relative increase in phosphorylation in the nucleoside analogs of BVDU ((E)-5­(2-Bromovinyl)-2'-deoxyuridine) or gemcitabine (DFDC, 2',2'-difluoro-deoxycytidine) compared with the wild-type enzyme. The dNKmu enzymes were expressed in the breast cancer cell lines MDA-MB-231 (ER-) and MCF7 (ER+). In studying the sensitivity of the cell lines to DFDC, conditionally replicative adenovirus (CRAd) SG500-dNKmu showed higher expression and enzymatic activity than the replication-defective adenovirus SG500 in cancer cells, but with less cytotoxicity to cancer cells than that of SG500. Our data suggest that the triple phosphorylated DFDC catalyzed by dNKmu inhibited the replication of adenovirus with a simultaneous positive therapeutic effect to cancer cells. Therefore, concomitant use of the SG500­dNKmu and DFDC could be a novel targeted strategy in suicide gene therapy with safe control of excessive virus replication.

Survivin regulates the expression of VEGF-C in lymphatic metastasis of breast cancer.

BACKGROUND: As a known regulator of apoptosis, survivin has positive relationship with lymphatic metastasis in breast cancer. This study aims to detect the difference in expression between survivin and vascular endothelial growth factor-C (VEGF-C) in treated breast cancer cells and tissues, and to analyze the correlation among survivin, VEGF-C and lymphatic metastasis. METHODS: Plasmid with survivin and VEGF-C shRNA and lentivirus with survivin gene were constructed and transfected into breast cancer cell ZR-75-30. Then the expressions of the two genes were examined using western blot analysis and real-time PCR. The change of invasiveness of breast cancer cells was assessed using matrigel invasion assay. Using immunohistochemistry, the expression of survivin and VEGF-C were analyzed in 108 clinical breast cancer cases with breast cancer tissue and lymph node. RESULTS: Survivin regulated the expression of VEGF-C at both protein and mRNA levels in breast cancer cells. Immunohistochemical analysis showed that the level of VEGF-C expression was significantly related with that of survivin in breast cancer tissues (p<0.05). VEGF-C was found to participate in the process of breast cancer cells invasion mediated by survivin. The co-expression of the two and the single expression of any one took significant difference in positive lymph node (p<0.05). CONCLUSIONS: Survivin takes an important part in regulating the expression of VEGF-C. VEGF-C could influence the invasive ability mediated by survivin. The co-expression of survivin and VEGF-C is more statistically significant to assess lymphatic metastasis in breast cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9193530897100952.

Antitumor effects of oncolytic adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase in colorectal cancer.

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) was applied as a cancer gene therapeutic approach. To improve the antitumor effect of Dm-dNK, a novel suicide gene system based on an oncolytic adenovirus vector was developed to produce therapeutic effects towards colorectal cancer cells. We constructed an oncolytic adenoviral vector (ZD55), which was designed by deletion of the E1B-55 kDa gene for selective replication in tumor cells, containing suicide gene (Dm-dNK) driven by a cytomegalovirus promoter. We analysed the expression and activity of Dm-dNK in colorectal cancer cells (HCT-116 and SW620) via reverse transcription (RT)-PCR and enzyme assay. We assessed selective cytotoxic effects of Dm-dNK with the presence of the analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), difluorodeoxycytidine (dFdC) or 1-ß-D-arabinofuranosylthymine (ara-T) by MTT and FACS; the variation of oncolytic adenovirus was detected by titer assay and western blot analysis. Our data showed that ZD55-Dm-dNK mediated high expression of Dm-dNK in HCT-116 and SW620 cancer cell lines and low levels of expression in WI-38 and MRC-5 normal cells, strong cytotoxicity was observed only in tumor cells after ZD55-Dm-dNK infection combined with nucleoside analogs (NA). When ZD55-Dm-dNK was combined with BVDU or dFdC, it produced a synergistic inhibitive effect of adenovirus replication while maintaining specific cancer cell killing activity. The results suggest that the novel oncolytic virus ZD55-Dm-dNK, in combination with NA, has potential as an efficient selective antitumoral agent and may produce synergistic effects in safe control of adenovirus, which is a new promising therapeutic for colorectal cancer.

[Effects of interleukin-1beta on nitric oxide production and ATP synthesis in rat surplus hepatocytes after partial hepatectomy].

OBJECTIVE: To detect the nitric oxide (NO) production and energy metabolism of the interleukin (IL)-1beta-treated residual hepatocytes from rats after partial hepatectomy. METHODS: Forty rats were equally divided into partial hepatectomies (PH) group and control group. In the control group the rats were otherwise matched and underwent sham surgeries. The residual hepatocytes were separated by the collagenase perfusion method. The hepatocytes were cultured with cytokines such as IL-1beta. The production of NO in the two groups were measured with Griess reagent method, the production of inducible nitric oxide synthase (iNOS) protein detected with Western blot, the content of the nucleotide in the hepatocytes detected with high-performance liquid chromatography, and the content of the ketone body in the hepatocytes of the two groups determined with the enzymatic method. Afterwards the ketone body ratio (acetoacetate/beta-hydroxy butyrate, KBR) was calculated. RESULTS: The production of NO in the PH group was twice as much as that in the Sham group. IL-1beta decreased the content of ATP and the KBR in the hepatocytes of both groups, and the decrease magni tude in the PH group was significantly larger than that in the Sham group. After the injection of L-arginine, the production of NO in the hepatocytes in the PH group increased, and the level of ATP and KBR decreased. N(G)-methyl-L-arginine (L-NMMA), the inhibitor of NO synthase, inhibited the production of NO and reversed the decrease of ATP and KBR. CONCLUSION: After partial hepatectomy, increased NO production in the hepatocytes after the treatment of interleukin-1beta may disturb the function of mitochondria by inhibiting the synthesis of ATP.