Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Dihydroartemisinin inhibits restenosis after balloon angioplasty via circHSPA4/miR-19a-5p axis.

Dihydroartemisinin (DHA) inhibits restenosis following balloon angioplasty. However, data on the mechanisms of DHA activity in restenosis remains scant. Here, we investigated the role of circRNAs in mediating the inhibitory activity of DHA in neointimal formation. We used total RNA sequencing data to profile the expression of mRNA, circRNA and small RNA in sham, vascular balloon injury (VBI) and DHA-treated groups. CCK8 and EdU assays were employed to analyze cell proliferation, while qRT-PCR and Western blot were used to analyze the RNA or protein expression. In addition, we used RNA immunoprecipitation and luciferase reporter assay to assess the binding of circHSPA4 with miR-19a-5p. RNA sequencing demonstrated that circHSPA4 was upregulated in VBI. Treatment with DHA effectively suppressed the upregulation of the circHSPA4. In addition, analysis of platelet-derived growth family factor bb (PDGFbb)-induced HA-VSMCs showed upregulation of circHSPA4, which was associated with cell proliferation and differentiation. CircHSPA4 was shown to induce dedifferentiation and proliferation of smooth muscle cells. PDGFBB-induced overexpression of CircHSPA4 in HA-VSMCs led to suppression of miR-19a-5p, a phenomenon that was reversed by DHA, in concentration-dependent fashion. In addition, miR-19a-5p reduced the dedifferentiation and proliferation of the smooth muscle cells. Our data demonstrated that CircHSPA4 regulates proliferation and differentiation of smooth muscle cells. DHA and miR-19a-5p modulates CircHSPA4 and can be used as coated drugs on balloon catheter to improve the success rate of vascular remodeling.

Phase separation at the nanoscale quantified by dcFCCS.

Liquid-liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

Effects of arbuscular mycorrhizal fungi on accumulation and translocation of selenium in winter wheat.

BACKGROUND: Selenium (Se) is an essential micronutrient for humans and animals, but not for plants. Generally, cereals including wheat and rice are the main source of dietary Se for humans. Although arbuscular mycorrhizal fungi (AMF) are ubiquitous soil microbes and commonly develop symbionts with winter wheat (Triticum aestivum L.), the influence of AMF on accumulation and translocation of Se during developmental cycle of winter wheat is still unclear. RESULTS: Based on a pot trial, the present results indicated that the effects of AMF on grain Se concentration in winter wheat depend on the Se species spiked in the soil and that Rhizophagus intraradices (Ri) significantly enhanced grain Se concentration under selenite treatment. Moreover, inoculation of AMF significantly increased grain Se content under selenite and selenate treatments. The enhanced grain Se content of mycorrhizal wheat could be attributed to (i) apparently increased root growth of mycorrhizal wheat at jointing could absorb more Se for translocating to aerial tissues and consequently result in significantly higher stalk Se content and (ii) enhancing Se translocation from vegetative tissues to grains. The present study showed that AMF significantly (P < 0.05) increased pre-anthesis Se uptake under selenate treatment and post-anthesis Se uptake under selenite treatment. CONCLUSION: The present study indicated the feasibility of inoculation of AMF for increasing grain Se concentration under selenite treatment and enhancing the efficiency of biofortification of Se under selenate treatments. © 2022 Society of Chemical Industry.

Dynamic distribution and accumulation of PAHs in winter wheat during whole plant growth: Field investigation.

A field investigation was conducted to study the dynamic distribution and accumulation of polycyclic aromatic hydrocarbons (PAHs) in winter wheat in the surrounds of a coal-fired power plant. During March to June 2019, various tissues of winter wheat and the corresponding rhizosphere soil were collected for determination of PAHs. A clear spatial downward trend was found in concentration of Σ15PAHs in rhizosphere soil and wheat grain (194-237 µg kg-1 DM) with the increasing distance from the coal-fired power plant. Moreover, Σ15PAHs concentration in rhizosphere soil (1081 µg kg-1 DM), root (464 µg kg-1 DM) and stem (365 µg kg-1 DM) of winter wheat at regreening stage and leaf (323 µg kg-1 DM) at anthesis stage were significantly (p < 0.001) higher than that (895, 432, 287 and 265 µg kg-1 DM) at maturity stage, respectively. From regreening to maturity stage, root concentration factors (RCF) of 3- and 4-ring PAHs exhibited an increasing trend but the 5-ring PAHs showed an apparently downward trend. However, stem concentration factors (SCF) of 3- and 4-ring PAHs showed a decrease trend while the 5- and 6-ring showed first down and then stable trend. There were positive linear relationship between logKow and logSCF at anthesis (r = 0.681, p < 0.05) and maturity stage (r = 0.751, p < 0.05). Based on linear regression analysis, PAHs in grain mainly came from the transfer of vegetative tissues, and the contribution of PAHs from stem and leaf to grain was higher than that from root. In addition, the present study also found that the physicochemical properties of PAHs play a crucial role in transfer of PAHs from root to vegetative tissues and then to grain. The present research provided more comprehensive information on the fate of PAHs in winter wheat and the safety of the agricultural products.

Up-regulation of MSH6 is associated with temozolomide resistance in human glioblastoma.

The impact of DNA mismatch repair (MMR) on resistance to temozolomide (TMZ) therapy in patients with glioblastoma (GBM) is recently reported but the mechanisms are not understood. We aim to analyze the correlation between MMR function and the acquired TMZ resistance in GBM using both relevant clinical samples and TMZ resistant cells. First we found increased expression of MSH6, one of key components of MMR, in recurrent GBM patients' samples who underwent TMZ chemotherapy, comparing with those matched samples collected at the time of diagnosis. Using the cellular models of acquired resistance to TMZ, we further confirmed the up-regulation of MSH6 in TMZ resistant cells. Moreover, a TCGA dataset contains a large cohort of GBM clinical samples with or without TMZ treatment reinforced the increased expression of MSH6 and other MMR genes after long-term TMZ chemotherapy, which may resulted in MMR dysfunction and acquired TMZ resistance. Our results suggest that increased expression of MSH6, or other MMR, may be a new mechanism contributing to the acquired resistance during TMZ therapy; and may serve as an indicator to the resistance in GBM.

STAT3 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin.

The JAK-STAT3 signaling pathway is one of the critical pathways regulating cell proliferation, differentiation, and apoptosis. Myocardin is regarded as a key mediator for the change of smooth muscle phenotypes. However, the relationship between STAT3 and myocardin in the vascular smooth muscle cell (VSMC) phenotypic switch has not been investigated. The goal of this study was to investigate the molecular mechanism by which STAT3 affects the myocardin-regulated VSMC phenotypic switch. Data presented in this study demonstrated that STAT3 was rapidly up-regulated after stimulation with VEGF. Inhibition of the STAT3 activation process impaired VSMC proliferation and enhanced the expression of VSMC contractile genes by increasing serum-response factor binding to the CArG-containing regions of VSMC-specific contractile genes. In contrast, the interaction between serum-response factor and its co-activator myocardin was reduced by overexpression of STAT3. In addition, treated VEGF inhibited the transcription activity of myocardin, and overexpression of STAT3 inhibited myocardin-induced up-regulation of VSMC contractile phenotype-specific genes. Although myocardin and STAT3 are negatively correlated, interestingly, both of them can enhance the expression of VEGF, suggesting a feedback loop to regulate the VSMC phenotypic switch. Taken together, these results indicate that the JAK-STAT3 signaling pathway plays a key role in controlling the phenotypic switch of VSMCs through the interactions between STAT3 and myocardin by various coordinated gene regulation pathways and feedback loops.

An effective cytokine adjuvant vaccine induces autologous T-cell response against colon cancer in an animal model.

BACKGROUND: Despite recent advances in early detection and improvements in chemotherapy for colon cancer, the patients still face poor prognosis of postoperative recurrence and metastasis, the median survival for patients with metastatic colorectal cancer is approximately 22-24 months. Some immunotherapeutic approaches had been attempted in colon cancer patients to significantly increase overall survival. A vaccine based approach has shown a novel direction for colon cancer prevention and therapy. METHODS: In this study, the experiments were designed including prevention and therapeutic stages in order to attain effect against tumor recurrence in clinical settings. The anti-tumor efficacy of a novel cytokine adjuvant vaccine that contained cytokines GM-CSF and IL-2 and inactivated colon CT26.WT whole cell antigen was evaluated in BALB/c mouse tumor models by measuring tumor growth post vaccination and the survival time of tumor-bearing mice, analyzing the expression and distribution of CD4, CD8, CD11c, CD80, CD86 and CD83 positive cells in control and treated mice by flow cytometry and immunochemistry. The tumor-specific cytotoxic T cells (CTL) were analyzed by tumor proliferation and the lactic dehydrogenates (LDH) release assays. IFN-γ, IL-2 and GM-CSF secretion in serum was assayed by ELISA. RESULTS: Our results suggested that cytokine adjuvant vaccine significantly inhibited tumor growth and extended the survival period at least 160d. It was found that the levels of CD8 + T and the tumor-specific cytotoxicity were significantly higher in prevention and treatment group vaccinated by cytokine adjuvant vaccine. CD8 + T cells play a key role in anti-tumor response. CONCLUSIONS: The novel GM-CSF and IL-2 based adjuvant vaccine effectively activated autologous T-cell response and represented a promising immunotherapeutic approach for patients with colon cancer.

Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation.

Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered by EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor.

MRTF-A and STAT3 promote MDA-MB-231 cell migration via hypermethylating BRSM1.

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism of metastasis is still not fully understood. We now report that both MRTF-A and STAT3 play important roles in migration of MDA-MB-231 breast cancer cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers urokinase-type plasminogen activator (uPA) and osteopontin (OPN) and inhibiting the expression of breast cancer metastasis suppressor 1 (BRMS1). Luciferase reporter assays demonstrated that MRTF-A and STAT3 do not affect transcription of the BRMS1 promoter. Instead, we identified a newly molecular mechanism by which MRTF-A and STAT3 synergistically controlled MDA-MB-231 cell migration by recruiting DNMT1 to hypermethylate the promoter of BRMS1 and thus affect the expression of BRMS1. Interestingly, physical interaction between MRTF-A and STAT3 synergistically promotes the transactivity of DNMT1 by binding to the GAS element within the DNMT1 promoter. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.

Human chorionic gonadotropin decreases human breast cancer cell proliferation and promotes differentiation.

Human chorionic gonadotropin (hCG) is a glycoprotein produced by placental trophoblasts. Previous studies indicated that hCG could be responsible for the pregnancy-induced protection against breast cancer in women. It is reported that hCG decreases proliferation and invasion of breast cancer MCF-7 cells. Our research also demonstrates that hCG can reduce the proliferation of MCF-7 cells by downregulating the expression of proliferation markers, proliferating cell nuclear antigen (PCNA), and proliferation-related Ki-67 antigen (Ki-67). Interestingly, we find here that hCG elevates the state of cellular differentiation, as characterized by the upregulation of differentiation markers, ß-casein, cytokeratin-18 (CK-18), and E-cadherin. Inhibition of hCG secretion or luteinizing hormone/hCG receptors (LH/hCGRs) synthesis can weaken the effect of hCG on the induction of cell differentiation. Furthermore, hCG can suppress the expression of estrogen receptor alpha. hCG activated receptor-mediated cyclic adenosine monophosphate/protein kinase A signaling pathway. These findings indicated that a protective effect of hCG against breast cancer may be associated with its growth inhibitory and differentiation induction function in breast cancer cells.

Mass spectrometry in measurement of thyroid biomarkers.

In 2022, the number of patients with thyroid disease in China exceeded 200 million (10 million with hyperthyroidism, 90 million with hypothyroidism, and 100 million with other thyroid disease such as goiter, thyroid nodules, and thyroid cancer). Well-established markers include FT3, FT4, TT3, TT4, and TSH tested by a number of immunoassay methods. This approach is based on the primary binding of antigen with antibody and a subsequent secondary chemical reaction that provides an indirect measure. The use of traceable standards for quantitation remains an important factor to ensure inter-assay reliability and precision. Recently, mass spectrometry (MS) has received considerable attention as an analytic tool due to high resolution and quantitative accuracy. In addition, MS allows for sensitive determination of low-abundance markers making it ideal for development of traceable standards. Furthermore, this technology will allow for the development of highly accurate thyroid biomarker assays to facilitate diagnosis, enable early treatment and improve outcomes. Herein, we provide a systematic review and summary of MS in enhancing the analysis of thyroid biomarkers.

Studies on the Increasing Saltiness and Antioxidant Effects of Peanut Protein Maillard Reaction Products.

The salt taste-enhancing and antioxidant effect of the Maillard reaction on peanut protein hydrolysates (PPH) was explored. The multi-spectroscopic and sensory analysis results showed that the Maillard reaction products (MRPs) of hexose (glucose and galactose) had slower reaction rates than those of pentose (xylose and arabinose), but stronger umami and increasing saltiness effects. The Maillard reaction can improve the flavor of PPH, and the galactose-Maillard reaction product (Ga-MRP) has the best umami and salinity-enhancing effects. The measured molecular weight of Ga-MRP were all below 3000 Da, among which the molecular weights between 500-3000 Da accounted for 46.7%. The products produced during the Maillard reaction process resulted in a decrease in brightness and an increase in red value of Ga-MRP. The amino acid analysis results revealed that compared with PPH, the content of salty and umami amino acids in Ga-MRPs decreased, but their proportion in total free amino acids increased, and the content of bitter amino acids decreased. In addition, the Maillard reaction enhances the reducing ability, DPPH radical scavenging ability, and Fe2+ chelating ability of PPH. Therefore, the Maillard reaction product of peanut protein can be expected to be used as a substitute for salt seasoning, with excellent antioxidant properties.

Effects of epidermal growth factor receptor and phosphatase and tensin homologue gene expression on the inhibition of U87MG glioblastoma cell proliferation induced by protein kinase inhibitors.

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

Research and design of an intelligent fish tank system.

In order to improve the hardware configuration and interaction mode of the fish tank system and realize the diversification of client functions, the purpose of real-time remote monitoring and management is achieved. A set of IoT intelligent fish tank system composed of sensor unit, signal processing unit and wireless transmission unit was designed. The system improves the algorithm of the data collected by the sensor, and proposes an improved first-order lag average filtering algorithm. The system uses composite collection information, intelligent processing, chart data analysis and other methods to transmit the processed data to the cloud server through the WIFI communication module. An APP is designed on the remote monitoring and control end, and a visual data interface of the smart fish tank is made, and the user can modify the environmental parameters conducive to the biological survival inside the fish tank through the APP, it brings great convenience to the family fish tank, and the test shows that the system network is stable and fast in response, and the overall purpose of the intelligent fish tank system is achieved.

Effects of extrusion on physical properties of glutinous rice and its application in the improvement of quality characteristics of glutinous rice products.

This study investigated the effects of extrusion on the physical properties of glutinous rice and addressed the challenges associated with its hardened texture and reduced taste in glutinous rice products by adding extruded glutinous rice to assess their anti-retrogradation effect compared with different improvers. Glutinous rice flour with different gelatinization degrees was obtained by changing the initial moisture content of glutinous rice grains before extrusion, and their physicochemical properties and the effect of adding them to rice products were analyzed. Results showed that with the increase in moisture content, the viscosity, water absorption index of extruded glutinous rice flour, and product viscosity increased, while the gelatinization degree, water solubility index, and product elasticity decreased, and the hardness of the rice products showed a trend of first decreasing and then increasing. Twenty percent moisture content of glutinous rice products showed the best properties mentioned above. The effects of adding different improvers on retrogradation degree, quality characteristics, microstructure, and moisture migration of glutinous rice products were analyzed by texture profile analysis, sensory evaluation, scanning electron microscopy, and low-field nuclear magnetic resonance. It was found that soybean polysaccharides, xanthan gum, and extruded glutinous rice flour had better anti-retrogradation effects, while colloid and soybean polysaccharides provided a tighter and more three-dimensional internal structure to the rice products. Our study showed that extruded glutinous rice flour had good anti-retrogradation properties and little effect on flavor and taste, but it would increase the roughness and viscosity of the products, which had advantages and disadvantages compared with other improvers.

Increased expression of calcium-sensing receptors in atherosclerosis confers hypersensitivity to acute myocardial infarction in rats.

Acute myocardial infarction (AMI) is a leading cause of death worldwide. Most cases of AMI result from coronary atherosclerosis (AS). The pathogenic mechanisms underlying AS lesions and AMI are incompletely understood. Calcium-sensing receptors (CaSR) belong to a family of G-protein-coupled receptors. We previously discovered that CaSR was expressed in the heart tissue of adult rats. CaSR may contribute to AMI in AS. We initially established a rat model of AS by injection of vitamin D(3) and feeding with a high-fat diet. Isoproterenol (ISO) was used to induce AMI. The MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), cardiac troponin T (cTnT), tetrazolium chloride staining, and cardiac function parameters were selected as indicators of myocardial damage or necrosis. Cardiac apoptosis was analyzed by transferase dUTP nick-end labeling (TUNEL) assay. Expression of CaSR, Bcl-2, Bax, caspase-3, p-ERK1/2, p-JNK, and p-p38 were determined by Western blot analysis. Compared with the control group, levels of cTnT, CK-MB, and LDH; number of TUNEL-positive cells; and expression of CaSR, Bax, caspase-3, p-ERK1/2, p-JNK and p-p38, were significantly increased, whereas cardiac function and expression of Bcl-2 were decreased markedly in isoproterenol (ISO)-treated group (C/ISO) and AS groups. These changes were significant in the AS/ISO group than in the C/ISO group or AS group. The upregulation of CaSR during AS formation renders hypersensitivity to AMI. Activation of the pro-apoptotic mitochondria pathway and JNK-p38 MAPK pathway triggered by increased expression of CaSR may be one of molecular mechanisms underlying AMI in AS.

Involvement of calcium-sensing receptor in oxLDL-induced MMP-2 production in vascular smooth muscle cells via PI3K/Akt pathway.

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.

Alisol A attenuates malignant phenotypes of colorectal cancer cells by inactivating PI3K/Akt signaling.

Despite the advancement in the diagnosis and therapeutic strategies for colorectal cancer, the outcomes of patients with colorectal cancer remain unsatisfactory. Alisol A is a natural constituent of Alismatis rhizoma (zexie) and has demonstrated anti-cancer properties; however, the function of Alisol A in colorectal cancer is still unknown. In the present study, the effect of Alisol A on colorectal cancer progression was investigated. MTT and colony formation assays showed that treatment with Alisol A repressed colorectal cancer cell proliferation in a dose-dependent manner. Similarly, western blot analysis demonstrated that Alisol A upregulated E-cadherin protein expression levels, but downregulated N-cadherin and Vimentin protein expression levels in colorectal cancer cells. In addition, the number of cells in G0/G1 phase was enhanced, while that of S phase was reduced in Alisol A-treated colorectal cancer cells. Apoptosis and pyroptosis of colorectal cancer cells were stimulated following treatment with Alisol A. Alisol A suppressed the migration ability of colorectal cancer cells in a dose-dependent manner. Moreover, Alisol A increased the chemotherapeutic sensitivity of colorectal cancer cells to cisplatin. Mechanically, western blot analysis confirmed that Alisol A repressed the phosphorylation levels of PI3K, Akt and mTOR in colorectal cancer cells. The Akt activator, SC79 reversed the effect of Alisol A on colorectal cancer cell proliferation and apoptosis. In conclusion, Alisol A induced an inhibitory effect on colorectal cancer progression by inactivating PI3K/Akt signaling.