Calreticulin (CALR) promotes ionophore-induced microneme secretion in Toxoplasma gondii.

The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER) that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly downregulated (p < 0.05). The cellular biological functions of the downregulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.

To Assess the Role of microRNA-451 in the Progression and Metastasis of Colorectal Cancer.

The study aimed to determine the expression of miR451 in colorectal cancer (CRC) subjects with CRC cells, and the role of miR451 in colorectal cancer cells. In October 2020, ATC purchased CRC and normal mucosal cell lines of CRC and implanted them in DMEM with 10% fetal serum. The suitability of the HT29 cell line is verified using the STR profile. In an incubator with 5% CO2, enlarged cells were placed at 37 °C. TCGA data was used to select the top 120 patients with a high voice and the lowest 120 patients with a low voice. Cells were collected and coated with Annexin V and PE according to the manufacturer's instructions after 24.0 h. After that, the cells were separated. Cells were also tested using flow cytometry. HCT-120 cells were transplanted into a concentration of 5×105/ml cells in 6-source plates. HCT120 cells in the experimental group were combined with miR451 mimics, miR451 inhibitors, or miR451 miR + SMAD4B for 12 h at 37 °C, and cells were collected 24 h later at 37 °C. The sample was injected with 5 ml of Annexin VFITC and PE. Compared with normal colorectal mucosal cells, CRC cell lines decreased miR451 expression levels (fetal human cells (FHC) and HCoEpiC). Then, the HCT120 cells were transfected with miR451 inhibitors, and 72 h after transfection, say of miR451 was normal. There was a significant decrease in cell function in the miR451mimic groups, but an increase when the miR451 was blocked. The proliferation of cancer cells was prevented and chemotherapy was effective when miR451 was overexpressed. The SMAD4 gene provides instructions for making a protein involved in transmitting chemical signals from the cell surface to the nucleus. The SMAD4B expression was tested by RT-qPCR and Western blotting after 72.0 h of transmission. The mRNA and protein expression of SMAD4B decreased significantly when miR451 was significantly higher than when inhibited, as revealed in the results of this study. Seventy-two hours after transplantation, mRNA levels and SMAD4B proteins were measured in HCT120 cells. In addition, the researchers in this study investigated whether miR451 was associated with SMAD4B-directed control of CRC growth and migration. It was found that SMAD4B is highly expressed in both CRC and para-cancer tissues while using the TCGA database to detect SMAD4B expression. Patients with CRC with SMAD4B have a severe prognosis. MiR451 is sensitive to depressive disorders by targeting SMAD4B, according to these studies. We found that miR451 inhibited cell growth and migration, made CRC cells more readily available in chemotherapy, and did so by targeting SMAD4B. The findings suggest that miR451 and its genetic predisposition, SMAD4B, may help predict the prognosis and course of cancer patients. Treatments that target the miR451/SMAD4B axis may be helpful to people with CRC.

Treatment response, survival, and safety profile of camrelizumab plus apatinib regimen as third-line treatment in metastatic gastric cancer patients.

BACKGROUND: Camrelizumab, as a PD-1 inhibitor on the market recently, presents favorable therapeutic efficacy in several advanced cancers, while its application in metastatic gastric cancer (mGC) lacks data. This study aimed to assess treatment response, survival profile, and adverse events of camrelizumab plus apatinib regimen as third-line treatment in mGC patients. METHODS: Nineteen mGC patients who received camrelizumab plus apatinib as third-line treatment were analyzed in this observational study. Subsequently, treatment response and adverse events were documented, then progression-free survival (PFS) and overall survival (OS) were calculated. RESULTS: No (0.0%) patient achieved complete response; 5 (26.3%) patients achieved partial response; 8 (42.1%) patients had stable disease; 6 (31.6%) patients had progressive disease, resulting in objective response rate and disease control rate of 26.3% and 68.4%, respectively. Meanwhile, the median PFS and OS were 7.0 (95%CI: 2.9-11.0) months and 10.0 (95%CI: 7.4-12.6) months, accordingly. Besides, multiple metastases linked with worse PFS (P = 0.029) and OS (P = 0.021); Eastern Cooperative Oncology Group performance status (ECOG PS) score 1 (vs. 0) related to shorter OS (P = 0.030). Worth noting, the common adverse events were fatigue (42.1%), anemia (42.1%), neutropenia (42.1%), leukopenia (36.8%), pruritus (31.6%), proteinuria (31.6%), nausea and vomiting (31.6%), reactive capillary hemangioma (31.6%) and thrombocytopenia (31.6%). Meanwhile, grade 3-4 adverse events only included: thrombocytopenia (5.3%), hypertension (5.3%), and proteinuria (5.3%). CONCLUSION: Camrelizumab plus apatinib as third-line treatment achieves satisfactory therapeutic efficacy and survival profile with generally manageable adverse events in mGC patients.

Activation of WNT/CTNNB1/TCF7L2 in Epstein-Barr virus-positive gastric cancer regulates epithelial mesenchymal transition.

Gastric cancer (GC) is the fifth most frequently diagnosed malignant tumour and the third leading cause of cancer-related death. EBV infection is one of the major causes of GC, accounting for approximately 10% of GC cases. Studies revealed that EBV infection could induce malignancy through multilevel mechanisms, including epigenetics state alterations, miRNA regulations, etc. However, it is poorly understood if EBV positive GC has any characteristic oncogenic singling activation. By comparing the transcriptomic of EB positive and negative cell lines, TCGA GC samples, and GC single cells, we identified that canonical WNT signalling is commonly activated in EBV positive GC. Next, we proved that the WNT activation is crucial for the invasiveness of the GC via regulating epithelial-mesenchymal transition (EMT) gene expressions. Then we confirmed that the WNT signalling activation in EBV + GC is through the WNT/CTNNB1 (ß-catenin)/TCF7L2 axis with the expression of TCF7L2 indicating worse outcome and more metastasis in EBV+ GCs. Finally, we found that KLF5 is potentially involved as a cooperative transcription factor in the early phase of the EMT process through single-cell pseudotime analysis.

GPX8 is transcriptionally regulated by FOXC1 and promotes the growth of gastric cancer cells through activating the Wnt signaling pathway.

BACKGROUND: Glutathione Peroxidase 8 (GPX8) as a member of the glutathione peroxidase (GPx) family plays an important role in anti-oxidation. Besides, dysregulation of GPX8 has been found in gastric cancer, but its detailed molecular mechanism in gastric cancer has not been reported. METHODS: Our study detected the expression of GPX8 in gastric cancer tissues and cell lines using immunohistochemistry (IHC), western blot and qRT-PCR, and determined the effect of GPX8 on gastric cancer cells using CCK-8, colony formation, transwell migration and invasion assays. Besides, the effect of GPX8 on the Wnt signaling pathway was determined by western blot. Furthermore, the transcription factor of GPX8 was identified by bioinformatics methods, dual luciferase reporter and chromatin immunoprecipitation (CHIP) assays. In addition, the effect of GPX8 on tumor formation was measured by IHC and western blot. RESULTS: The over-expression of GPX8 was observed in gastric cancer tissues and cells, which facilitated the proliferation, migration and invasion of gastric cancer cells as well as the tumor growth. GPX8 knockdown effectively inhibited the growth of gastric cancer cells and tumors. Moreover, GPX8 could activate the Wnt signaling pathway to promote the cellular proliferation, migration and invasion through. Furthermore, FOXC1 was identified as a transcription factor of GPX8 and mediated GPX8 expression to affect cell development processes. CONCLUSIONS: These findings contribute to understanding the molecular mechanism of GPX8 in gastric cancer. Additionally, GPX8 can be a potential biomarker for gastric cancer therapy.

A novel panel of stool-based DNA biomarkers for early screening of colorectal neoplasms in a Chinese population.

PURPOSE: The mortality of colorectal cancer ranked fifth in China according to cancer statistics in 2015. Cancer screening had been repeatedly proved to play a vital role in decreasing the incidence and mortality of colorectal cancer, but the existing screening methods could not meet the requirements. So it is of urgent need to develop a non-invasive, convenient and accurate screening method. METHODS: In this study, stool samples were collected from 102 colorectal cancer, 20 colorectal adenoma, 6 hyperplastic polyps patients and 105 normal controls, and stool DNA was extracted for detection of methylation (BMP3, NDRG4, SDC2 and SFRP2) and KRAS mutations. Meanwhile, hemoglobin in stool samples was detected by immunoassays. Then, the logistic regression model used for classification was built with these biomarkers, and a ROC curve was drawn to evaluate the performance of each biomarker and the panel of them. Meanwhile, conventional serum biomarkers were detected for the comparison of positive rate in colorectal cancer between serum biomarkers and stool DNA biomarkers. RESULTS: As a result, a classification model built with methylation of SDC2 and SFRP2, KRAS mutations and hemoglobin showed a sensitivity of 91.4% for colorectal cancer and 60% for adenoma with the specificity of 86.1%. Compared with it, most of the conventional serum biomarkers showed a sensitivity of less than 20% for colorectal cancer which was significantly lower than stool DNA biomarkers. CONCLUSIONS: A novel panel comprised of stool DNA biomarkers was of much higher sensitivity and specificity in early screening of colorectal neoplasms than conventional serum biomarkers.

The ubiquitin ligase CHIP modulates cellular behaviors of gastric cancer cells by regulating TRAF2.

BACKGROUND: CHIP is an E3 ubiquitin ligase that plays contrast roles in diverse human malignancies, depending on its targets. To date, the mechanisms underlying the function of CHIP in gastric cancer remains unclear. Here, we aim to further clarify the effects of CHIP on the development and progression of gastric cancer and explore its potential target. METHODS: Stably transfected CHIP-shRNA and TRAF2-shRNA AGS gastric cancer cell lines were established using Lipofectamine 2000. Cell growth was measured by an xCelligence real-time monitoring system and colony formation assay. Cell proliferation was detected using CCK-8, Ki-67, or CFSE assays. Apoptosis was detected by TUNEL assay or Annexin V/PI-staining followed by flow cytometric analysis. Cell cycle distribution was detected by PI-staining followed by flow cytometric analysis. Cell migration and invasion abilities were measured by a real-time xCelligence system, Transwell insert, and scratch assays. The expression of cell cycle-related proteins, apoptosis-related proteins, AKT, ERK, NF-κB signaling subunits, MMP2, MMP9, and Integrin ß-1 were detected by Western blotting analysis. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Gastric cancer tissue microarray was analyzed to investigate the expression of both CHIP and TRAF2, and their clinical significance. RESULTS: The CHIP-silencing in the AGS cells was oncogenic evidenced by the appearance of capable of anchorage-independent growth. The CHIP-silencing significantly enhanced the AGS cell proliferation capability likely due to the induced phosphorylation of ERK. The CHIP-silencing significantly inhibited apoptosis due to increased expression of Bcl-2. The CHIP-silencing promoted the AGS cell migration and invasion abilities, likely by regulating the expression of Integrin ß-1. TRAF2 expression was markedly decreased in the CHIP-overexpressing cells at protein level, but not at mRNA level. The TRAF2-silencing markedly inhibited the proliferation ability of the AGS cells, the defected cell proliferation and enhanced apoptosis were involved in. The TRAF2-silencing also attenuated the cell migration and invasion capacities of the AGS cells. Furthermore, the expression of CHIP was downregulated while the expression of TRAF2 was upregulated in gastric cancer tissues. TRAF2 expression is independent prognostic factors of gastric cancer. The expression of CHIP and TRAF2 was negatively correlated in the gastric cancer tissue. Lower CHIP or higher TRAF2 was significantly linked to shorter overall survival in gastric cancer patients. CONCLUSIONS: TRAF2 influenced diverse aspects of cellular behavior of gastric cancer cells, including cell growth, migration, and invasion, which was in contrast to the functions of CHIP. TRAF2 could be considered as an independent prognostic factor in gastric cancer patients. It is possible that TRAF2 was a substrate of CHIP and CHIP regulated the TRAF2/NF-κB axis, which modulated diverse cellular behaviors in the AGS gastric cancer cells.

RelB plays an oncogenic role and conveys chemo-resistance to DLD-1 colon cancer cells.

BACKGROUND: Nuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemo-sensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. However, the significance of RelB in colorectal cancer (CRC) remains unclear. Here, we systematically explored the functions of the alternative NF-κB subunit RelB in colon cancer cells and its underlying mechanism. METHODS: Stably transfected RelB-shRNA DLD-1 cells were established using Lipofectamine 2000. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Cell growth was monitored by an x-Celligence system. Cell proliferation was analyzed by a CCK-8 and a Brdu proliferation assay. Response to 5-FU was assessed by an x-Celligence system. Cell apoptosis and cell cycle was detected using flow cytometry analyses. Cell migration and invasion abilities were detected by an x-Celligence system, Transwell inserts, and wound-healing assays. RelB expression and its clinical significance were analyzed using the CRC tissue microarray. The expression of NF-κB signaling subunits, AKT/mTOR signaling molecules, cell cycle related proteins, MMP2, MMP9, and Integrin ß-1 were measured by Western blotting analyses. RESULTS: The RelB-silencing inhibited cell growth of DLD-1 cells. The RelB-silencing exerted the anti-proliferative by downregulation of AKT/mTOR signaling. The RelB-silencing caused G0-G1 cell cycle arrested likely due to decreasing the expression of Cyclin D1 and CDK4, concomitant with increased expression of p27Kip1. The RelB-silencing enhanced cytotoxic effect of 5-FU and induced cell accumulation in S-phase. The RelB-silencing impaired the migration and invasion potential of DLD-1 cells, which was related to downregulation of MMP2, MMP9, and Integrin ß-1. Importantly, the RelB expression was correlated with depth of tumor invasion, lymph node metastasis, metastasis stage, and pTNM stage. High-RelB expression was significantly correlated with poor overall survival in CRC patients. CONCLUSION: Our studies here provided evidence that RelB plays an oncogenic role and conveys chemo-resistance to 5-FU. RelB can be considered as an independent indicator of prognosis in CRC.

Ethyl 4-[(E)-(2-hy-droxy-benzyl-idene)amino]-piperidine-1-carboxyl-ate.

In the title compound, C(15)H(20)N(2)O(3), the piperidine ring adopts a chair conformation, although the amide N atom is almost planar (bond angle sum = 359.7°). The mol-ecule adopts an E conformation about the C=N bond, which allows for the formation of an intra-molecular O-H⋯N hydrogen bond. In the crystal, mol-ecules are linked by C-H⋯O inter-ations, resulting in C(6) chains propagating in [010].