LncRNA H19 Participates in Leukemia Inhibitory Factor Mediated Stemness Promotion in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy and the third leading cause of cancer-related death worldwide. Cancer stem cells (CSCs) were considered to play important roles in the genesis and development of many tumors. In recent years, it has been observed that leukemia inhibitory factor (LIF) might be involved in the regulation of stemness in cancer cells. In this study, we observed that LIF could increase the spheroid formation and stemness marker expression (inculding Nanog and SOX2) in CRC cell lines, such as HCT116 and Caco2 cells. Meanwhile, we also observed that LIF could upregulate LncRNA H19 expression via PI3K/AKT pathway. Knockdown of the expression of LncRNA H19 could decrease the spheroid formation and SOX2 expression in LIF-treated HCT116 and Caco2 cells, and thereby LncRNA H19 knockdown could compensate for the stemness enhancement effects induced by LIF. Our results indicated that LncRNA H19 might participate in the stemness promotion of LIF in CRC cells.

MCP-1/CCR2 axis is involved in the regulation of γδT cells in lupus nephritis.

γδT cells are important innate immune cells that are involved in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis (LN) is a serious complication of SLE, characterized by the accumulation of immune cells (including γδT cells) in the target organs to participate in the disease process. Therefore, clarifying how γδT cells chemotactically migrate to target organs may be a key to developing therapeutic methods against LN. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in LN patients and healthy controls. Real-time polymerase chain reaction (RT-PCR) and flow cytometry were used to measure the expression of chemokine receptors on the surface of γδT cells. The chemotactic migration ability of γδT cells was detected by Transwell assay. Signalling pathway activation of γδT cells was detected by Automated Capillary Electrophoresis Immunoassay and flow cytometry. The serum levels of chemokines, including monocyte chemoattractant protein-1 (MCP-1) in LN patients, were significantly increased. CCR2, the receptor of MCP-1, was also highly expressed on the surface of peripheral γδT cells in LN patients. In addition, the exogenous addition of MCP-1 can enhance chemotactic migration of γδT cells in LN patients. MCP-1 could activate STAT3 signalling in LN patients' peripheral γδT cells. γδT cells might participate in the pathogenesis of LN through MCP-1/CCR2 axis. This finding provides new opportunities for developing treatment methods against LN by targeting MCP-1/CCR2 axis.

Leukemia Inhibitory Factor Impairs the Function of Peripheral γδT Cells in Patients with Colorectal Cancer.

Immunotherapeutic strategies are recognized as promising treatment methods for colorectal cancer (CRC). αßT cell-mediated cytotoxicity is tolerated by cancer cells with low MHC class I expression; therefore, γδT cell-based cancer immunotherapy has generated increasing interest as a potential treatment option. To enhance the potency of γδT cell-based immunotherapy, the key factors involved in the regulation of γδT cells in CRC need to be identified along with devising ways to overcome potential hurdles. In this study, we observed that leukemia inhibitory factor (LIF), the serum level of which was highly increased in those with solid tumors, could specifically attenuate the cytotoxic function of peripheral γδT cells in patients with CRC. We observed that in patients with CRC, the expression levels of perforin and granzyme were significantly decreased in the recombinant human LIF (rhLIF)-treated peripheral γδT cells, whereas that of the LIF receptor (LIFR) was higher. The regulation of the cytotoxic function of the γδT cells by rhLIF was effected mainly through the STAT3 signaling pathway, which caused an increase in the expression levels of interleukin (IL)-17, COX-2, and prostaglandin E2 (PGE2). Our results revealed that rhLIF could impair the function of γδT cells in CRC patients by reducing the cytotoxic function and increasing the expression of tumor-promoting molecules, such as IL-17, COX-2, and PGE2.

CX3CR1 might be a promising predictor of systemic lupus erythematosus patients with pulmonary fibrosis.

The inflammatory process in systemic lupus erythematosus (SLE) affects many organs including the lungs. Chemokines are suggested to play important roles in the pathogenesis of SLE with pulmonary fibrosis (PF). In the present study, our objective is to evaluate the correlation between chemokines and PF in SLE patients. Transcriptome sequencing analysis was used to find the different expressed genes between SLE patients with PF and without PF. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in SLE patients and healthy controls. Expression of CX3CR1 was measured by real-time polymerase chain reaction (PCR) and flow cytometer. Sixteen differentially chemokine genes were found to be associated to SLE with PF. Meanwhile, the upregulation of C-X3-C motif chemokine receptor 1 (CX3CR1) and its ligand, CX3C chemokine ligand 1 (CX3CL1) were observed in SLE patients with PF than that of SLE patients without PF and healthy control. Phenotypic analysis also showed that the surface expression of CX3CR1 increased in PBMCs from SLE patients with PF. Our observations indicated that CX3CL1/CX3CR1 axis is associated with PF in SLE. CX3CR1 might be a promising predictor of SLE with PF and the interactions between CX3CL1 and CX3CR1 might provide potential candidate target for the treatment of SLE with PF.

Enrichment and separation of high-polar compounds from Saussurea obvallata using solid-phase extraction combining with offline two-dimensional liquid chromatography.

The high-polar compounds from natural products are often used as medicines due to their good bioactivities. However, owing to the complexity and diversity of their structure, the separation of high-polar compounds is still a challenging work. For this, an efficient method for enrichment and separation of the high-polar compounds from Saussurea obvallata was developed. First, the target compounds were enriched from the total extract using a solid-phase extraction method. An offline two-dimensional liquid chromatography method was used for the separation of pure compounds from the enriched sample. After optimization of chromatographic conditions, high separation selectivity of target compounds was obtained on a polar-modified C18 column and a HILIC XAmide column. Hence, a two-dimensional reversed-phase × hydrophilic interaction liquid chromatography system was constructed and enlarged from the analytical level to the preparative level. In the first dimension, four fractions were obtained on the XCharge C18 column with a recovery rate of 71.2%. In the second-dimension preparation on the XAmide column, eight high-polar compounds with more than 96% purity were isolated. All compounds were isolated from Saussurea obvallata for the first time. The results demonstrated that this developed strategy is effective for preparative-scale isolation of high-polar compounds from natural products.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

PTEN-Induced Putative Kinase 1 (PINK1)/Parkin-Mediated Mitophagy Protects PC12 Cells Against Cisplatin-Induced Neurotoxicity.

BACKGROUND The pathogenesis of chemotherapy-induced neuropathy, a dose-dependent adverse effect of cisplatin, involves mitochondrial dysfunction. PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy removes damaged mitochondria under various pathological conditions. The objective of this study was to determine mitophagy status and its effects on mitochondrial function and neuronal cell damage after cisplatin treatment using an in vitro model of cisplatin-induced neurotoxicity. MATERIAL AND METHODS PC12 cells were transfected with Parkin or Parkin siRNA using lentiviral particles and Lipofectamine 3000™, respectively, and then were exposed to 10 µM cisplatin. The expression of autophagic proteins was measured by Western blot analysis. Mitophagy in PC12 cells was detected by confocal microscopy analysis of mitochondria-lysosomes colocalization and autophagic flux. The effects of PINK1/Parkin-mediated mitophagy on cisplatin-induced neurotoxicity were assessed via mitochondrial function, neuritic length, nuclear diameter, and apoptosis. RESULTS Cisplatin activated PINK1/Parkin-mediated mitophagy in PC12 cells. Autophagic flux analysis revealed that cisplatin inhibits the late stage of the autophagic process. The knockdown of Parkin suppressed cisplatin-induced mitophagy, aggravating cisplatin-induced depolarization of mitochondria, cellular ATP deficits, reactive oxygen species outburst, neuritic shortening, nuclear diameter reduction, and apoptosis, while Parkin overexpression enhanced mitophagy and reversed these effects. CONCLUSIONS PINK1/Parkin-regulated mitophagy can protect against cisplatin-related neurotoxicity, suggesting therapeutic enhancement of mitophagy as a potential intervention for cisplatin-induced peripheral neuropathies. The interference of cisplatin with autophagosome-lysosome fusion may be partly responsible for cisplatin-induced neurotoxicity.

Emodin is a Potential Drug Targeting CD44-positive Hepatocellular Cancer.

BACKGROUND: Liver cancer is one of the most prevalent forms of cancer of the digestive system in our country. The most common subtype of this disease is hepatocellular carcinoma (HCC). Currently, treatment options for HCC patients include surgical resection, liver transplantation, radiofrequency ablation, chemoembolization, and biologic-targeted therapy. However, the efficacy of these treatments is suboptimal, as they are prone to drug resistance, metastasis, spread, and recurrence. These attributes are closely related to cancer stem cells (CSCs). Therefore, the utilization of drugs targeting CSCs may effectively inhibit the development and recurrence of HCC. METHODS: HepG2 and Huh7 cells were used to analyze the antitumor activity of emodin by quantifying cell growth and metastasis, as well as to study its effect on stemness. RESULTS: Emodin effectively suppressed the growth and movement of HCC cells. Emodin also significantly inhibited the proliferation of CD44-positive hepatoma cells. CONCLUSION: Emodin shows promise as a potential therapeutic agent for HCC by targeting CD44-- positive hepatoma cells.

Polyphyllin VII is a Potential Drug Targeting CD44 Positive Colon Cancer Cells.

BACKGROUND: Current therapies for colon cancer are hindered by treatment failure and recurrence, mainly due to colon cancer stem cells (CSCs). Thus, treatment using drugs targeting CSCs should be effective in eliminating colon cancer cells and impeding cancer recurrence. OBJECTIVE: This study aimed to test if PPVII can be a potent drug candidate for the treatment of colon cancer by targeting CD44 positive colon cancer cells. METHODS: In this study, we first demonstrated that CD44 is highly expressed in colon cancer tissues by TCGA/GTEX database analysis and immunohistochemical staining. RESULTS: In this study, we first demonstrated that CD44 is highly expressed in colon cancer tissues by TCGA/GTEX database analysis. CD44 had high accuracy as a diagnostic and predictive index for colorectal cancer through receiver operating characteristic curve (ROC) analysis. At the same time, survival curve analysis also showed that the high expression of CD44 was associated with poor prognosis in patients with colon cancer. CD44's higher expression in colon cancer tissues was further confirmed by immunohistochemical staining; the positive rate of CD44 expression was 87.95%. Then, one of the constituents that derives from the root of Paris polyphylla, Polyphyllin VII (PPVII), has been confirmed to inhibit the migration of colon cancer cells. Our results also demonstrated that PPVII could inhibit the sphere-forming ability of colon cancer cells. Further experiment results showed that PPVII could downregulate the expression of CD44 in colon cancer cells. In addition, PPVII was proved to have inhibitory effects against CD44 positive colon cancer cells. CONCLUSION: Therefore, PPVII might be a potent candidate reagent for the treatment of colon cancer by targeting CD44 positive colon cancer cells.

Bioactivity-guided isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata (DC.) Edgew. Using affinity solid phase extraction assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea obvallata (DC.) Edgew. is a traditional Tibetan medicine used for the treatment of inflammation-related diseases, but the scientific validation was very limited. AIM OF THE STUDY: This study aimed to rapid screen and targeted isolate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata extract. MATERIALS AND METHODS: An efficient ligand-fishing method based on affinity solid phase extraction (A-SPE) combining with HPLC was developed. The identified COX-2 inhibitors were separated using preparative liquid chromatography. In vitro COX-2 inhibition assays were employed to confirm the inhibitory activities of the isolated compounds. In addition, the effect of the isolated compounds on the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-induced RAW 264.7 were evaluated. RESULTS: A total of four phenylpropanoids, isolariciresinol, syringaresinol, pinoresinol and balanophonin were targeted isolated as COX-2 inhibitors with IC50 values of 36.4 ± 2.6 µM, 23.1 ± 1.8 µM, 3.6 ± 0.3 µM and 12.1 ± 0.9 µM, respectively. The isolated compounds significantly inhibited LPS-induced NO production in a dose-dependent manner. And, the results of the inhibitory effect on the release of PGE2 and the expression of COX-2 in LPS-induced macrophages were consistent with A-SPE analysis. CONCLUSION: The present work demonstrated that the developed A-SPE-HPLC method could successfully targeted isolated COX-2 inhibitors from S. obvallata extract. And, the isolation results indicated that the therapeutic effect of S. obvallata on inflammation-related diseases was partly based on the COX-2 active ingredients.

Non-structure protein ORF1ab (NSP8) in SARS-CoV-2 contains potential γδT cell epitopes.

Upon activation by the pathogen through T-cell receptors (TCRs), γδT cells suppress the pathogenic replication and thus play important roles against viral infections. Targeting SARS-CoV-2 via γδT cells provides alternative therapeutic strategies. However, little is known about the recognition of SARS-CoV-2 antigens by γδT cells. We discovered a specific Vγ9/δ2 CDR3 by analyzing γδT cells derived from the patients infected by SARS-CoV-2. Using a cell model exogenously expressing γδ-TCR established, we further screened the structural motifs within the CDR3 responsible for binding to γδ-TCR. Importantly, these sequences were mapped to NSP8, a non-structural protein in SARS-CoV-2. Our results suggest that NSP8 mediates the recognition by γδT cells and thus could serve as a potential target for vaccines.

miRNAs in anti-cancer drug resistance of non-small cell lung cancer: Recent advances and future potential.

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors worldwide. Clinical success is suboptimal owing to late diagnosis, limited treatment options, high recurrence rates, and the development of drug resistance. MicroRNAs (miRNAs), a range of small endogenous non-coding RNAs that are 22 nucleotides in length, have emerged as one of the most important players in cancer initiation and progression in recent decades. Current evidence has revealed the pivotal roles of miRNAs in regulating cell proliferation, migration, invasion, and metastasis in NSCLC. Recently, several studies have demonstrated that miRNAs are strongly associated with resistance to anti-cancer drugs, ranging from traditional chemotherapeutic and immunotherapy drugs to anti-vascular drugs, and even during radiotherapy. In this review, we briefly introduce the mechanism of miRNA dysregulation and resistance to anti-tumor therapy in NSCLC, and summarize the role of miRNAs in the malignant process of NSCLC. We then discuss studies of resistance-related miRNAs in chemotherapy, radiotherapy, targeted therapy, immunotherapy, and anti-vascular therapy in NSCLC. Finally, we will explore the application prospects of miRNA, an emerging small molecule, for future anti-tumor therapy. This review is the first to summarize the latest research progress on miRNAs in anti-cancer drug resistance based on drug classification, and to discuss their potential clinical applications.

Comprehensive screening and separation of cyclooxygenase-2 inhibitors from Pterocephalus hookeri by affinity solid-phase extraction coupled with preparative high-performance liquid chromatography.

Pterocephalus hookeri, a classical Tibetan herb, is mainly used to treat rheumatoid arthritis (RA) and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. A novel strategy for screening and target separating COX-2 inhibitors from the extracts of P. hookeri based on affinity solid-phase extraction (ASPE) column combined with preparative high-performance liquid chromatography (pre-HPLC) was successfully developed. The potential COX-2 inhibitors of P. hookeri were screened and recognized by the ASPE-HPLC system, which strategy is to analyze the compounds isolated by the ASPE column. Then, the active compounds were targeted separated by pre-HPLC according to real-time chromatograms. The control drugs celecoxib and glipizide were analyzed to verify the specificity and accuracy of the developed method. As a result, two pure compounds with COX-2 binding affinities were successfully separated from P. hookeri. They were characterized as swertisin and scopoletin using 1H- and 13C NMR spectroscopy, and the in vitro COX-2 inhibitory activities were verified. Compounds with COX-2 inhibitory activities could be screened and targeted separated from crude extracts by this strategy, which indicated that the proposed method was feasible, robust and effective for rapid separation of COX-2 inhibitors from natural products.

MicroRNAs: Emerging oncogenic and tumor-suppressive regulators, biomarkers and therapeutic targets in lung cancer.

Lung cancer is one of the most common solid tumors worldwide and the leading cause of cancer-related deaths, causing a devastating impact on human health. The clinical prognosis of lung cancer is usually restricted by delayed diagnosis and resistance to anticancer therapies. MicroRNAs, a range of small endogenous noncoding RNAs 22 nucleotides in length, have emerged as one of the most important players in cancer initiation and progression in recent decades. Current evidence reveals pivotal roles of microRNAs in regulating cell proliferation, migration, invasion and metastasis in lung cancer. An increasing number of preclinical and clinical studies have also explored the potential of microRNAs as promising biomarkers and new therapeutic targets for lung cancer. The current review summarizes the most recent progress on the functional mechanisms of microRNAs involved in lung cancer development and progression and further discusses the clinical application of miRNAs as putative therapeutic targets for molecular diagnosis and prognostic prediction in lung cancer.

Detection of circulating genetically abnormal cells using 4-color fluorescence in situ hybridization for the early detection of lung cancer.

PURPOSE: Available biomarkers lack sensitivity for an early lung cancer. Circulating genetically abnormal cells (CACs) occur early in tumorigenesis. To determine the diagnostic value of CACs in blood detected by 4-color fluorescence in situ hybridization (FISH) for lung cancer. METHODS: This was a prospective study of patients with pulmonary nodules ≤ 30 mm detected between 10/2019 and 01/2020 at four tertiary hospitals in China. All patients underwent a pathological examination of lung nodules found by imaging and were grouped as malignant and benign. CACs were detected by 4-color FISH. Patients were divided into the training and validation cohorts. Receiver operating characteristics analysis was used to analyze the diagnosis value of CACs. RESULTS: A total of 205 participants were enrolled. Using a cut-off value of ≥ 3, blood CACs achieved areas under the curve (AUCs) of 0.887, 0.823, and 0.823 for lung cancer in the training and validation cohorts, and all patients, respectively. CACs had high diagnostic values across all tumor sizes and imaging lesion types. CACs were decreased after surgery (median, 4 vs. 1, P < 0.001) in the validation set. The CAC status between blood and tissues was highly consistent (kappa = 0.909, P < 0.001). The AUC of CAC (0.823) was higher than that of CEA (0.478), SCC (0.516), NSE (0.506), ProGRP (0.519), and CYFRA21-1 (0.535) (all P < 0.001). CONCLUSION: CACs might have a high value for the early diagnosis of lung cancer. These findings might need to be validated in future studies. Evidence suggested homology in genetic aberrations between the CACs and the tumor cells.

Data on the involvement of Meox1 in balloon-injury-induced neointima formation of rats.

In the previous report, Meox1 was found to promote SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade (Wu et al., 2017) [1]. Here, we presented new original data on the involvement of Mesoderm/mesenchyme homeobox gene l (Meox1) in balloon-injury-induced neointima formation of rat. In rat carotid artery balloon injury model to induce vascular remodeling, Meox1 was induced in vascular smooth muscle cell (SMCs) of rat carotid arteries. Most proliferating cell nuclear antigen (PCNA)-positive cells also expressed Meox1. These data suggested that Meox1 may be involved in SMCs proliferation during injury-induced neointima formation. Furthermore, knocked down its expression in injured arteries by adenoviral delivery of Meox1 short hairpin RNA (shRNA) (shMeox1), neointima formation was significantly inhibited. Elastin staining also confirmed the reduction of neointima in Meox1 shRNA-transduced arteries. Moreover, knockdown of Meox1 decreased the collagen production/deposition that was significantly increased in neointima induced by balloon injury.

Mesoderm/mesenchyme homeobox gene l promotes vascular smooth muscle cell phenotypic modulation and vascular remodeling.

AIMS: To investigate the role of mesoderm/mesenchyme homeobox gene l (Meox1) in vascular smooth muscle cells (SMCs) phenotypic modulation during vascular remodeling. METHODS AND RESULTS: By using immunostaining, Western blot, and histological analyses, we found that Meox1 was up-regulated in PDGF-BB-treated SMCs in vitro and balloon injury-induced arterial SMCs in vivo. Meox1 knockdown by shRNA restored the expression of contractile SMCs phenotype markers including smooth muscle α-actin (α-SMA) and calponin. In contrast, overexpression of Moex1 inhibited α-SMA and calponin expressions while inducing the expressions of synthetic SMCs phenotype markers such as matrix gla protein, osteopontin, and proliferating cell nuclear antigen. Mechanistically, Meox1 mediated the SMCs phenotypic modulation through FAK-ERK1/2 signaling, which appears to induce autophagy in SMCs. In vivo, knockdown of Meox1 attenuated injury-induced neointima formation and promoted SMCs contractile proteins expressions. Meox1 knockdown also reduced the number of proliferating SMCs, suggesting that Meox1 was important for SMCs proliferation in vivo. Moreover, knockdown of Meox1 attenuated ERK1/2 signaling and autophagy markers expressions, suggesting that Meox1 may promote SMCs phenotypic modulation via ERK1/2 signaling-autophagy in vivo. CONCLUSION: Our data indicated that Meox1 promotes SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade. Thus, targeting Meox1 may be an attractive approach for treating proliferating vascular diseases.