PAR2 deficiency tunes inflammatory microenvironment to magnify STING signalling for mitigating cancer metastasis via anionic CRISPR/Cas9 nanoparticles.

Metastasis is one of the most significant causes for deterioration of breast cancer, contributing to the clinical failure of anti-tumour drugs. Excessive inflammatory responses intensively promote the occurrence and development of tumour, while protease-activated receptor 2 (PAR2) as a cell membrane receptor actively participates in both tumour cell functions and inflammatory responses. However, rare investigations linked PAR2-mediated inflammatory environment to tumour progression. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is an emerging and powerful gene editing technique and can be applied for probing the new role of PAR2 in breast cancer metastasis, but it still needs the development of an efficient and safe delivery system. This work constructed anionic bovine serum albumin (BSA) nanoparticles to encapsulate CRISPR/Cas9 plasmid encoding PAR2 sgRNA and Cas9 (tBSA/Cas9-PAR2) for triggering PAR2 deficiency. tBSA/Cas9-PAR2 remarkably promoted CRISPR/Cas9 to enter and transfect both inflammatory and cancer cells, initiating precise PAR2 gene editing in vitro and in vivo. PAR2 deficiency by tBSA/Cas9-PAR2 effectively suppressed NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signalling in inflammatory microenvironment to magnify stimulator of interferon genes (STING) signalling, reactive oxygen species (ROS) accumulation and epithelial-mesenchymal transition (EMT) reversal, consequently preventing breast cancer metastasis. Therefore, this study not only demonstrated the involvement and underlying mechanism of PAR2 in tumour progression via modulating inflammatory microenvironment, but also suggested PAR2 deficiency by tBSA/Cas9-PAR2 as an attractive therapeutic strategy candidate for breast cancer metastasis.

The Yin-Yang roles of protease-activated receptors in inflammatory signalling and diseases.

Inflammatory diseases have become increasingly prevalent throughout the world. Coronavirus disease 2019 (COVID-19), which has recently become pandemic, also exhibits hyperinflammation and cytokine release syndrome. To address inflammation-related diseases, numerous molecular targets have been explored in preclinical studies and clinical trials. Among them, the protease-activated receptors (PARs) that belong to G protein-coupled receptors are one of the most popular classes of drug targets, participating in inflammatory signalling and diseases. PARs activation can trigger downstream intracellular signalling to modulate a variety of inflammatory responses in multiple systems, including nervous, respiratory, digestive, circulatory, urinary and immune systems. Importantly, there are the Yin-Yang effects, comprising anti- and pro-inflammatory roles, of PARs activation in different types of inflammations, and these are context-dependent. Alternatively, it was recently revealed that PARs not only modulate inflammatory-related tumour progression, but also participate in inflammatory cytokine release related to COVID-19 via direct interaction with severe acute respiratory syndrome coronavirus 2 protein, suggesting that PARs also participate in other diseases via inflammatory responses. In this review, we renew and discuss the findings of PARs as molecular targets for treating inflammatory diseases, highlighting the novel roles of PARs and facilitating a better understanding of their designated values in the specific inflammatory environment.

PAR2 blockade reverses osimertinib resistance in non-small-cell lung cancer cells via attenuating ERK-mediated EMT and PD-L1 expression.

Osimertinib, as the third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), is a first-line molecularly targeted drug for non-small cell lung cancer (NSCLC). However, the emergence of therapeutic resistance to osimertinib markedly impairs its efficiency and efficacy, leading to the failure of clinical applications. Novel molecular targets and drugs are urgently needed for reversing osimertinib resistance in NSCLC. Protease-activated receptor 2 (PAR2) that belongs to a subfamily of G protein-coupled receptors can stimulate the transactivation of EGFR to regulate multiple cellular signalling, actively participating in tumour progression. This study firstly discovered that PAR2 expression was notably enhanced when NSCLC cells became resistant to osimertinib. A PAR2 inhibitor facilitated osimertinib to attenuate EGFR transactivation, ERK phosphorylation, EMT and PD-L1 expression which were associated to osimertinib resistance. The combination of the PAR2 inhibitor and osimertinib also notably blocked cell viability, migration, 3D sphere formation and in vivo tumour growth whereas osimertinib itself lost such inhibitory effects in osimertinib-resistant NSCLC cells. Importantly, this reversal effect of PAR2 blockade was uncovered to depend on ERK-mediated EMT and PD-L1, since inhibition of ß-arrestin or ERK, which could be modulated by PAR2, sensitized osimertinib to prevent EMT, PD-L1 expression and consequently overcame osimertinib resistance. Thus, this study demonstrated that PAR2 antagonism could limit ERK-mediated EMT and immune checkpoints, consequently attenuating EGFR transactivation and reactivate osimertinib. It suggested that PAR2 may be a novel drug target for osimertinib resistance, and PAR2 inhibition may be a promising strategy candidate for reversing EGFR-TKI resistance in NSCLC.

Targeting PAR2 Overcomes Gefitinib Resistance in Non-Small-Cell Lung Cancer Cells Through Inhibition of EGFR Transactivation.

Drug resistance can notably restrict clinical applications of gefitinib that is a commonly used EGFR-tyrosine kinase inhibitors (EGFR-TKIs) for non-small cell lung cancer (NSCLC). The attempts in exploring novel drug targets and reversal strategies are still needed, since gefitinib resistance has not been fully addressed. Protease-activated receptor 2 (PAR2), a G protein-coupled receptor, possesses a transactivation with EGFR to initiate a variety of intracellular signal transductions, but there is a lack of investigations on the role of PAR2 in gefitinib resistance. This study established that protease-activated receptor 2 (PAR2), actively participated in NSCLC resistant to gefitinib. PAR2 expression was significantly up-regulated when NSCLC cells or tumor tissues became gefitinib resistance. PAR2 inhibition notably enhanced gefitinib to modulate EGFR transactivation, cell viability, migration and apoptosis in gefitinib-sensitive and-resistant NSCLC cells, suggesting its reversal effects in gefitinib resistance. Meanwhile, the combination of a PAR2 inhibitor (P2pal-18S) and gefitinib largely blocked ERK phosphorylation and epithelial-mesenchymal transition (EMT) compared to gefitinib alone. Importantly, we probed its underlying mechanism and uncovered that PAR2 blockade sensitized gefitinib and reversed its resistance mainly via ß-arrestin-EGFR-ERK signaling axis. These effects of PAR2 inhibition were further confirmed by the in vivo study which showed that P2pal-18S reactivated gefitinib to inhibit tumor growth via restricting ERK activation. Taken together, this study could not only reveal a new mechanism of receptor-mediated transactivation to modulate drug resistance, but also provide a novel drug target and direction for overcoming gefitinib resistance in NSCLC.

PAMs inhibits monoamine oxidase a activity and reduces glioma tumor growth, a potential adjuvant treatment for glioma.

BACKGROUND: Monoamine oxidase (MAO) A catalyzes oxidative deamination of monoamine neurotransmitters and dietary amines and regulates brain development and functions. Recently, we showed that MAO A mediates the progression and migration of glioma and MAO A inhibitors reduce glioma cell growth. Glioblastoma (GBM) is a common and most malignant brain tumor which is difficult to treat. Temozolomide (TMZ) is the current standard chemotherapy for glioma, but tumors usually become resistant and recur. So far, no effective therapy for TMZ-resistant glioma is available. Natural plant antimicrobial solution (PAMs) is a Chinese herbal medicine which has been used for decades without toxicity and has multiple medical functions including anti- inflammatory effects. Here, we report the effects of PAMs on glioblastoma growth. METHODS: The growth of TMZ -sensitive (U251S),-resistant (U251R) human glioma cells, and mouse glioma cell line GL-26 were assessed by MTS colorimetric assay, colony formation, and cell migration assays. Male C57BL/6 mice were implanted subcutaneously or intracranial with luciferase-positive mouse glioma GL-26 cells and treated with vehicle; MAO A inhibitor clorgyline (10 mg/kg); TMZ (1 mg/kg); PAMs (48 mg/kg) alone or in combination with TMZ (1 mg/kg) for 14 days. At the end of the treatment, mice were sacrificed, MAO A catalytic activity in tumors was measured, and tumor sizes were determined by imaging and weight. RESULTS: These results show that PAMs inhibits MAO A catalytic activity in all three glioma cell lines studied U251S, U251R, and GL-26. PAMs reduced glioma growth and has greater effects in combination with low dose of TMZ than PAMS or TMZ alone in all three cell lines as shown by MTS, colony formation, and cell migration assays. Using the subcutaneous or intracranial GL-26 glioma mouse model, PAMs reduced the tumor growth and MAO A activity, similar to the MAO A inhibitor clorgyline. Combining PAMs with non-toxic dose TMZ increased survival to a greater extent than those of PAMs or TMZ alone. CONCLUSIONS: This is the first study which suggests that PAMs alone or co-administration with low doses of TMZ may be a potential adjuvant to reduce the toxicity of TMZ and to abrogate drug resistance for the effective treatment of glioma.

G Protein-coupled Receptors in Cancer Stem Cells.

G protein-coupled receptors (GPCRs) are highly expressed on a variety of tumour tissues while several GPCR exogenous ligands become marketed pharmaceuticals. In recent decades, cancer stem cells (CSCs) become widely investigated drug targets for cancer therapy but the underlying mechanism is still not fully elucidated. There are vigorous participations of GPCRs in CSCs-related signalling and functions, such as biomarkers for CSCs, activation of Wnt, Hedgehog (HH) and other signalling to facilitate CSCs progressions. This relationship can not only uncover a novel molecular mechanism for GPCR-mediated cancer cell functions but also assist our understanding of maintaining and modulating CSCs. Moreover, GPCR antagonists and monoclonal antibodies could be applied to impair CSCs functions and consequently attenuate tumour growth, some of which have been undergoing clinical studies and are anticipated to turn into marketed anticancer drugs. Therefore, this review summarizes and provides sufficient evidences on the regulation of GPCR signalling in the maintenance, differentiation and pluripotency of CSCs, suggesting that targeting GPCRs on the surface of CSCs could be potential therapeutic strategies for cancer therapy.

Down-regulation expression of TGFB2-AS1 inhibits the proliferation, migration, invasion and induces apoptosis in HepG2 cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality and without effective prognosis. Previous study has been confirmed that the abnormal expression of long non-coding RNAs (lncRNAs) TGFB2-AS1 was involved in tumorigenesis. However, the biological functions of TGFB2-AS1 in hepatocellular carcinoma (HCC) remain largely unclear. OBJECTIVE: We comprehensively assess the clinical significance of TGFB2-AS1 and investigate the biological functions of TGFB2-AS1 on HCC HepG2 cells. METHODS: We firstly confirmed the expression of TGFB2-AS1 between tumor and normal tissues using public available transcriptome data. We analyzed the clinical significance of TGFB2-AS1 using the TCGA HCC datasets. The biological functions of TGFB2-AS1 on HCC HepG2 cells were explored by multiple in vitro assays. RESULTS: We found that TGFB2-AS1 was remarkably increased in HCC tissues (P = 0.00148) and exhibited a potential predictive marker for HCC, with an area under curve (AUC) of 0.708 (P = 0.0034) using the fifty pairs of matched HCC tissues of TCGA. Besides, higher expression of TGFB2-AS1 in HCC tissues was identified as being positively associated with advanced tumor (P = 0.012) and disease stage (P = 0.009) in 355 HCC cases using independent sample nonparametric test. Downregulation of TGFB2-AS1 expression significantly restrained proliferation (P < 0.01) and impaired colony formation (P < 0.05). Furthermore, TGFB2-AS1 depletion remarkably promoted the apoptosis of HepG2 cells (P < 0.05) and inhibited migration and invasion (P < 0.01). CONCLUSION: Taken together, these findings suggested that TGFB2-AS1 might serve as a potential therapeutic target for HCC.

Transcriptome profiling analysis of differentially expressed mRNAs and lncRNAs in HepG2 cells treated with peptide 9R-P201.

OBJECTIVE: To characterize the transcriptome profile of hepatocellular carcinoma (HCC) HepG2 cells treated with peptide 9R-P201 for further functional verification and HCC drug development. RESULTS: 1557 mRNAs (1125 upregulated and 432 downregulated) and 881 lncRNAs (640 upregulated and 241 downregulated) with significant differential expression were identified using RNA-seq. The qRT-PCR results showed that the differential expression of several mRNAs and lncRNAs coincided with the RNA-seq results. Differentially expressed mRNAs and lncRNAs presented a significant difference in genomic characteristics but no preference under 9R-P201 treatment compared with control. The GO and KEGG functional enrichment analyses showed that differentially expressed mRNAs and lncRNAs remarkably enriched in cancer-related biological processes and signaling pathways. Finally, we screened out 33 TFs, 273 lncRNAs and 94 target genes with high degree interaction which were remarkably associated with the tumorigenesis and progression of cancers using betweenness centrality analysis. CONCLUSION: These findings offer novel insights into the mechanism of 9R-P201 in HepG2 cells and provide new opportunities for the future 9R-P201-based drug development and the treatment of hepatocellular carcinoma.

A novel peptide, 9R-P201, strongly inhibits the viability, proliferation and migration of liver cancer HepG2 cells and induces apoptosis by down-regulation of FoxM1 expression.

Overexpression of FoxM1 was closely related to the proliferation, metastasis, chemo-resistance and poor prognosis of various cancers. FoxM1 was regarded as the Achilles' heel of cancer and a potential target for anti-cancer drug discovery. We previously obtained several high affinity peptides from the phage random library against the DNA binding domain of FoxM1c (FoxM1c-DBD) protein. Here in this paper, we found that 9R-P201, one of the novel peptides, showed stronger inhibition to HepG2 cancer cells than those of DU145, HUVEC and L-02 cells with an IC50 of 43.6µg/ml (13.1µM). The peptide was highly effective to liver cancer cells with an IC50 for L-02 cells of 2855.9µg/ml. We confirmed that 9R-P201 aggregated in the cell nucleus and the expression of FoxM1 was significantly down-regulated at both transcriptional and translational levels in HepG2 cells, leading to the suppression of cell proliferation, migration, angiogenesis, and induction of apoptosis. Whole genomic RNA sequencing analysis revealed that 357 genes were significantly and differentially expressed, most of them were enriched in cancer-associated biological processes. Finally, treatment of HepG2 xenografts with 9R-P201 resulted in growth inhibition and down-regulation of foxM1 expression in tumors. Collectively, our findings suggested that 9R-P201 could strongly inhibit the viability, proliferation and migration of liver cancer HepG2 cells and induce apoptosis by down-regulation of FoxM1 and regulation of related gene expression in signal transduction passways. Thus, 9R-P201 holds great potential as a lead anti-cancer drug targeting FoxM1.

[Virtual screening and molecular simulations of antisense peptides targeting MT1-MMP].

Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) plays the pivotal role in tumor development and metastasis, so it is a promising drug target in malignancy. To acquire MT1-MMP specific binding peptides, we first analyzed MMPs sequences to find the divergent and specific sequence of MT1-MMP by bioinformatics approach, then set the specific sequence as the sense peptide target and designed antisense peptide library. Finally, by means of molecular docking, molecular dynamics simulation and in vitro cell assays, we screened the antisense peptide library against MT1-MMP and further studied the obtained specific peptides. Here, we identified the divergent and specific sequence of AYIREGHE (Named MT1-loop) located in MT1-MMP loop by multiple sequence alignment and established the antisense peptides library with capacity of 1 536 sequences. After two rounds of virtual screening, we obtained five antisense peptides with Rerankscores in the top for further screening. They all interacted with MT1-MMP, and docked well at the active site composed of MT1-loop sequence. Analysis of the affinities of these five antisense peptides to other MMPs (MMP1-3, MMP7-13, MMP14 HPX, MMP16) revealed that the peptide FVTFPYIR was more specific to MT1-MMP. Molecular dynamics simulation showed that the peptide FVTFPYIR might affect the stability of MT1-MMP and thus have effects on its activities. Meanwhile, the peptide FVTFPYIR could specifically inhibit the growth of MG63 and MDA-MB-231 tumor cells both of which expressed MT1-MMP. The work provides a new insight and way for the development of antitumor lead peptides targeting MT1-MMP.

Systemic Screening of Strains of the Lion's Mane Medicinal Mushroom Hericium erinaceus (Higher Basidiomycetes) and Its Protective Effects on Aß-Triggered Neurotoxicity in PC12 Cells.

Hericium erinaceus possesses multiple medicinal values. To date, however, there have been few studies of the systemic screening of H. erinaceus strains, and the neuroprotective effects of H. erinaceus prepared from homogenized, fresh fruiting bodies are not fully understood. In this study, 4 random primers were selected and used in random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) to screen and evaluate the genetic diversity of 19 commercial strains of H. erinaceus from different localities in China. A total of 66 bands were obtained, and the percentage of polymorphic loci reached 80.30%. Five dendrograms were constructed based on RAPD by Jaccard cluster and within-group linkage analysis. Primer S20 as well as all 4 primers had great potential as specific primers for RAPD-PCR molecular identification and differentiation of H. erinaceus strains. Based on the results of submerged culture and fruiting body cultivation, strains HT-N, HT-J1, HT-C, and HT-M were identified as superior among the 19 H. erinaceus strains. Further study showed that the oral preparation of homogenized, fresh fruiting bodies of H. erinaceus could attenuate the Aß25-35-triggered damage in PC12 cells by significantly increasing cell viability and by decreasing the release of lactate dehydrogenase. In conclusion, RAPD-PCR combined with liquid and solid cultures can be used well in the screening and identification of H. erinaceus strains, and products prepared from homogenized, fresh fruiting bodies of H. erinaceus had neuroprotective effects on PC12 cells.

Identification and analysis of the regulatory network of Myc and microRNAs from high-throughput experimental data.

As a transcription factor, c-Myc exerts significant influence in cancer development by regulating transcription of a large number of target genes including microRNAs. However, details of regulatory networks composed of Myc, microRNAs, and microRNA target genes are still unclear. Here, at system level, we built a comprehensive Myc-regulated miRNAs (Myc-miRNAs) regulatory network through the integration of experimentally validated high-throughput data and computational predictions. Using miRNA genomic information with ChIP-PET, we identified 30 Myc-miRNAs and found most of these Myc-miRNAs target genes were significantly enriched in cell cycle, apoptosis, cell proliferation GO terms and Myc-regulated signaling pathways, using gene sets enrichment analysis. We found most Myc-miRNAs involved in Myc-related cancer pathways expressed abnormally in Myc-associated tumors through the integration of diverse types of experimental data. Based upon Myc target genes identified by ChIP-chip assays, we identified that 1031 Myc-miRNAs feed-forward loops (FFLs) were significantly different from those obtained by chance; also, 11 high-quality FFLs were extracted from experimentally validated interactions. Finally, we built the miRNA-protein interaction network of experimentally validated Myc-miRNAs and discussed the more complex network composed of several FFLs networks. As shown in this study, we performed comprehensive analysis of the Myc-miRNAs regulatory network and provided potential Myc-miRNAs target genes which were involved in Myc pathway and cancer-related biological processes.

Epitope mapping of metuximab on CD147 using phage display and molecular docking.

Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23-30), I37, D45, E84, V88, EPMGTANIQLH (92-102), VPP (131-133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

[Selection and interaction of Ni2+ metal-binding peptides].

Ni2+ binding peptides were selected from phage random dodecapeptide library by metal affinity chromatography. After four rounds of biopanning, phage amplification and DNA sequencing, a group of peptide sequences were obtained. GenBank blast found no homogenous sequences, Clustal W analysis showed no motifs but they were really riched in histidines and contained di- or more histidines(his). Affinity assays of selected metal-binding phages for various metal-charged NTA resins and the experiments of E. coli suppression and detoxification gave positive results for Ni2+ binding peptides: strong affinities for Ni2+ were found for Ni2+ binding peptide displayed phages, as well as for other metals (Cu2+, Co2+, Zn2+, Cr2+, Cd2+); affinities of the binding peptides for Cu2+, Ni2+, Co2+ and Zn2+ were much higher than that of Cd2+ and Cr2+; in addition, Ni2+ binding peptides displayed phages had effects on E. coli as to enhance the tolerance and detoxification of E. coli for heavy metals when exposed to Ni2+ and Cd2+. The interactions of meal binding peptides for heavy metals were also disclosed by microscopic observation. The research offered great values for the study of the interaction between metals and peptides, as well as in other areas such as heavy metal bioremediation.

Selection of novel nickel-binding peptides from flagella displayed secondary peptide library.

Nickel (Ni) performs its biological or toxic functions in nickel-protein coordination form. Novel Ni-binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni-chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni-binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni-binding peptides may provide lead molecules for Ni-chelation and applications thereof.

A conformation-constrained peptide library based on insect defensin A.

Here, we reported a conformation-constrained peptide library, that was constructed based on the scaffold of a 29 amino acids peptide derived from insect defensin A. The peptide scaffold was designed utilizing the InsightII molecular modeling software and then displayed on M13 filamentous bacteriophage by fusion with coat protein III. The library was constructed by randomization of seven positions located within the two loops of the peptide scaffold generating approximately 8.3 x 10(8) transformants. Sequences from 14 randomly selected phage clones indicated that the distribution of nucleotides and amino acids paralleled with the expected frequency. Screening against the target proteins: tumor necrosis factor alpha, TNF receptor 1, TNF receptor 2 and monoclonal antibody against BMP-2 showed significant enrichment in all cases. The results presented here show that the reconstructed insect defensin A domain will be a promising non-antibody protein scaffold for the presentation of a phage-displayed constrained peptide library.