The Pan-Cancer Analysis Uncovers the Prognostic and Immunotherapeutic Significance of CD19 as an Immune Marker in Tumor.

Background: The specific cytotoxic effects of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy have led to impressive outcomes in individuals previously treated for B-cell malignancies. However, the specific biological role of CD19(+) target cells, which exert antitumor immunity against some solid tumors, remains to be elucidated. Methods: We collected information regarding the level of CD19 mRNA and protein expression from various databases including The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) for both tumor and normal samples. To evaluate the patient's prognosis according to CD19 expression, a Kaplan-Meier (KM) analysis and univariate Cox regression were performed. Furthermore, using the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using the Expression Data (ESTIMATE) algorithm, we estimated the ratio of immune cells infiltrating malignant tumor tissues. Afterward, the GSCALite repository was employed to evaluate the vulnerability of tumors expressing CD19 to drugs used in chemotherapy. To validate the results in clinical samples of certain cancer types, immunohistochemistry was then performed. Results: Most tumor types exhibited CD19 expression differently, apart from colon adenocarcinoma (COAD). The early diagnostic value of CD19 has been demonstrated in 9 different tumor types, and the overexpression of CD19 has the potential to extend the survival duration of patients. Multiple tumors showed a positive correlation between CD19 expression and tumor mutation burden (TMB), microsatellite instability (MSI), and ESTIMATE score. Furthermore, a direct association was discovered between the expression of CD19 and the infiltration of immune cells, particularly in cases of breast invasive carcinoma (BRCA). Moreover, CD19 is highly sensitive to a variety of chemotherapy drugs. Conclusion: The study reveals the potential of CD19 as both a predictive biomarker and a target for different cancer immunotherapies.

Network pharmacology and experimental validation to study the potential mechanism of Tongguanteng injection in regulating apoptosis in osteosarcoma.

OBJECTIVE: The main objectives of this study were to identify the active components of Tongguanteng injection (TGT) and investigate the preclinical efficacy and mechanism of TGT on osteosarcoma using a combination of network pharmacology and experimental validation. METHODS: To identify the active constituents and targets of TGT against osteosarcoma using network pharmacology, we constructed a network consisting of an 'active ingredient-disease-target-pathway' and a protein-protein interaction (PPI) network. The target organ network was utilized to investigate the distribution of core targets in tissues. Afterwards, the core targets underwent Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The binding energy between receptors and ligands was compared using molecular docking. In addition, SwissADME was employed to forecast the pharmacokinetic characteristics of the substances. Finally, real-time polymerase chain reaction (RT-PCR), cell proliferation assay, morphological analysis, apoptosis assay, mitochondrial membrane potential (MMP) detection, and Western blotting were utilized to confirm the potential mechanisms of TGT treatment in osteosarcoma cell lines 143B and SAOS2. RESULTS: A total of 54 chemical constituents of TGT and 71 targets associated with osteosarcoma were acquired. Through the molecular docking technology, Tenacigenin B, Marsdekoiside, Taraxasterol, Tenacissoside G, Tenacissoside L, and Tenacissoside J were identified as the primary active components of TGT among the various compounds. Analysis of target organs suggests that TGT may play an anti-osteosarcoma role through immune regulation. The GO and KEGG enrichment analysis revealed that TGT could trigger osteosarcoma cell apoptosis by inhibiting the HIF-1 signalling pathway and modulating PD-1 expression and the PD-1 checkpoint pathway in cancer. SwissADME database predicted that Tenacigenin B and Taraxasterol had the best drug-likeness. In vitro studies also demonstrated that TGT suppressed the activity and induced alterations in the morphology of osteosarcoma cells. It decreased MMP levels, triggered apoptosis by increasing Bax expression and Caspase-3 activity, and decreased Bcl-2 expression, thereby exerting an anti-osteosarcoma effect. In the meantime, RT-PCR tests demonstrated that TGT could control immune response against tumors and hinder the proliferation and spread of cancerous cells by impacting the levels of critical factors, including JUN, HSP90AA1, HDAC1, and CDK1. CONCLUSION: The study accurately anticipated the active components, targets, and pathways of TGT in the management of osteosarcoma. The molecular mechanism of TGT-induced apoptosis in osteosarcoma cells was demonstrated by in vitro experiments. These results provide theoretical and technical support for TGT as a clinical adjuvant drug for osteosarcoma.

[Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification].

The present study aimed to explore the main active components and underlying mechanisms of Marsdenia tenacissima in the treatment of ovarian cancer(OC) through network pharmacology, molecular docking, and in vitro cell experiments. The active components of M. tenacissima were obtained from the literature search, and their potential targets were obtained from SwissTargetPrediction. The OC-related targets were retrieved from Therapeutic Target Database(TTD), Online Mendelian Inheritance in Man(OMIM), GeneCards, and PharmGKB. The common targets of the drug and the disease were screened out by Venn diagram. Cytoscape was used to construct an "active component-target-disease" network, and the core components were screened out according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened out according to the node degree. GO and KEGG enrichment analyses of potential therapeutic targets were carried out with DAVID database. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock. Finally, the anti-OC activity of M. tenacissima extract was verified based on SKOV3 cells in vitro. The PI3K/AKT signaling pathway was selected for in vitro experimental verification according to the results of GO function and KEGG pathway analyses. Network pharmacology results showed that 39 active components, such as kaempferol, 11α-O-benzoyl-12ß-O-acetyltenacigenin B, and drevogenin Q, were screened out, involving 25 core targets such as AKT1, VEGFA, and EGFR, and the PI3K-AKT signaling pathway was the main pathway of target protein enrichment. The results of molecular docking also showed that the top ten core components showed good binding affinity to the top ten core targets. The results of in vitro experiments showed that M. tenacissima extract could significantly inhibit the proliferation of OC cells, induce apoptosis of OC cells through the mitochondrial pathway, and down-regulate the expression of proteins related to the PI3K/AKT signaling pathway. This study shows that M. tenacissima has the characteristics of multi-component, multi-target, and multi-pathway synergistic effect in the treatment of OC, which provides a theoretical basis for in-depth research on the material basis, mechanism, and clinical application.

Tongguanteng injection reverses paclitaxel resistance via upregulation of TAB1 expression in ovarian cancer in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongguanteng injection (TGT), the water extract from the stem of the Traditional Chinese hebal medicine of Marsdenia tenacissima (Roxb.) Wight et Arn. has been used as anticancer remedy for decades. TGT was not only used in the treatment of many malignant cancers extensively, but also an adjuvant anticancer drug with chemotherapeutics clinically. AIM OF THE STUDY: To evaluate the effects of TGT on reversing paclitaxel (PTX) resistance and investigate the potential mechanism related to TAB1 in ovarian cancer (OC) in vitro and in vivo. MATERIALS AND METHODS: The synergistic effect and reversal ratio were determined by CCK8 assay and median-effect principle after the combination of TGT and PTX in OC A2780 and its PTX-resistant (A2780/T) cells. The biological functions in cell apoptosis, migration and invasion of A2780/T cells treated by PTX 4 µM with TGT 20, 40, 80 mg⋅mL-1 for 24 h were evaluated by colony formation, flow cytometry, wound healing and transwell assays. Proteomics technique and bioinformatic analysis were used to indentify the change of TAB1 expression in A2780/T cells induced by TGT. The association between TAB1 expression and human OC was analyzed by gene expression databases. In A2780/T cells, western blotting and colony formation assays were used to investigate the relationship between TAB1 expression and PTX resistance after TAB1 overexpression by TAB1 plasmids. The mechanism of TGT and PTX regulating TAB1 and its related proteins were explored by western blotting and flow cytometry assays after TAB1 knock-down using siTAB1. Moreover, TUNEL staining, immunohistochemistry (IHC) and histopathology were used to observe the antitumor effects, TAB1 and p-p38 expression and the tissues impairments in nude mice xenograft model established by A2780/T cells after the co-treatment with TGT and PTX by in vivo. RESULTS: TGT combined with PTX showed the synergistic effect (CI<1), which could reverse the IC50 values of PTX in OC A2780 and A2780/T cells about 23.50 and 6.44 times, respectively. Besides, TGT combined with PTX could significantly inhibit the migration, invasion and promote apoptosis of A2780/T cells. We identified that TGT could induce TAB1 expression in A2780/T cells by proteomics analysis. TAB1 downregulation was significantly associated with tumorigenesis and poor prognosis in OC patients and PTX resistance in A2780/T cells. Furthermore, TGT could activate TAB1/TAK1/p38 MAPK signaling pathway targeting TAB1 and regulate the expression of Bax, Bcl-2 proteins to improve the sensitivity of A2780/T cells to PTX. TGT combined with PTX also showed a greater inhibition in tumor growth than PTX monotherapy in vivo. These promising results show the efficacy of TGT in reversing PTX resistance and provide a potential strategy that targeting TAB1/TAK1/p38 MAPK signaling pathway may improve the chemotherapy sensitivity in OC. CONCLUSIONS: Our results revealed that Tongguanteng injection could reverse paclitaxel resistance and the potential mechanism might be associated with the activation of TAB1/TAK1/p38 MAPK signaling pathway in OC in vitro and in vivo. TAB1 might be a pivotal target for reversing PTX resistance. This study will provide a theoretical basis for the combination of Tongguanteng injection and paclitaxel in clinic.

IiWRKY34 positively regulates yield, lignan biosynthesis and stress tolerance in Isatis indigotica.

Yield potential, pharmaceutical compounds production and stress tolerance capacity are 3 classes of traits that determine the quality of medicinal plants. The autotetraploid Isatis indigotica has greater yield, higher bioactive lignan accumulation and enhanced stress tolerance compared with its diploid progenitor. Here we show that the transcription factor IiWRKY34, with higher expression levels in tetraploid than in diploid I. indigotica, has large pleiotropic effects on an array of traits, including biomass growth rates, lignan biosynthesis, as well as salt and drought stress tolerance. Integrated analysis of transcriptome and metabolome profiling demonstrated that IiWRKY34 expression had far-reaching consequences on both primary and secondary metabolism, reprograming carbon flux towards phenylpropanoids, such as lignans and flavonoids. Transcript-metabolite correlation analysis was applied to construct the regulatory network of IiWRKY34 for lignan biosynthesis. One candidate target Ii4CL3, a key rate-limiting enzyme of lignan biosynthesis as indicated in our previous study, has been demonstrated to indeed be activated by IiWRKY34. Collectively, the results indicate that the differentially expressed IiWRKY34 has contributed significantly to the polyploidy vigor of I. indigotica, and manipulation of this gene will facilitate comprehensive improvements of I. indigotica herb.

SmMYC2b Enhances Tanshinone Accumulation in Salvia miltiorrhiza by Activating Pathway Genes and Promoting Lateral Root Development.

Salvia miltiorrhiza Bunge (Lamiaceae) is an economically important medicinal plant as well as an emerging model plant. Our previous studies indicate that SmMYC2b is a positive transcription factor that can affect the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza. Moreover, MYC2s are well known to induce the development of lateral roots. As tanshinones are mainly distributed in the periderm, the promotion of lateral root development probably leads to increased accumulation of tanshinones. In this paper, we firstly discovered that SmMYC2b played a dual regulatory role in effectively enhancing the tanshinone accumulation by activating tanshinone biosynthetic pathway and promoting lateral root development. The expression levels of the previously studied pathway genes SmCPS1, SmKSL1, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 dramatically increased. In addition, SmMYC2b was proved to exhibit a similar function as other homologs in promoting lateral root development, which increased the tanshinone produced tissue and further enhanced the biosynthesis of tanshinones. RNA-seq assays revealed that SmMYC2b-regulated genes comprised 30.6% (1,901 of 6,210) of JA-responsive genes, confirming that SmMYC2b played a crucial role in transcriptional regulation of JA-regulated genes. Overall, we concluded that SmMYC2b could enhance tanshinone accumulation by activating the tanshinone biosynthetic pathway and promoting lateral root development. Our study provides an effective approach to enhance the production of desired tanshinones and enriches our knowledge of the related regulatory network.

Author Correction: SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-Wide Identification and Characterization of Salvia miltiorrhiza Laccases Reveal Potential Targets for Salvianolic Acid B Biosynthesis.

Laccases are widely distributed in plant kingdom catalyzing the polymerization of lignin monolignols. Rosmarinic acid (RA) has a lignin monolignol-like structure and is converted into salvianolic acid B (SAB), which is a representatively effective hydrophilic compound of a well-known medicinal plant Salvia miltiorrhiza and also the final compound of phenolic acids metabolism pathway in the plant. But the roles of laccases in the biosynthesis of SAB are poorly understood. This work systematically characterizes S. miltiorrhiza laccase (SmLAC) gene family and identifies the SAB-specific candidates. Totally, 29 laccase candidates (SmLAC1-SmLAC29) are found to contain three signature Cu-oxidase domains. They present relatively low sequence identity and diverse intron-exon patterns. The phylogenetic clustering of laccases from S. miltiorrhiza and other ten plants indicates that the 29 SmLACs can be divided into seven groups, revealing potential distinct functions. Existence of diverse cis regulatory elements in the SmLACs promoters suggests putative interactions with transcription factors. Seven SmLACs are found to be potential targets of miR397. Putative glycosylation sites and phosphorylation sites are identified in SmLAC amino acid sequences. Moreover, the expression profile of SmLACs in different organs and tissues deciphers that 5 SmLACs (SmLAC7/8/20/27/28) are expressed preferentially in roots, adding the evidence that they may be involved in the phenylpropanoid metabolic pathway. Besides, silencing of SmLAC7, SmLAC20 and SmLAC28, and overexpression of SmLAC7 and SmLAC20 in the hairy roots of S. miltiorrhiza result in diversification of SAB, signifying that SmLAC7 and SmLAC20 take roles in SAB biosynthesis. The results of this study lay a foundation for further elucidation of laccase functions in S. miltiorrhiza, and add to the knowledge for SAB biosynthesis in S. miltiorrhiza.

Molecular cloning, bioinformatics analysis, and transcriptional profiling of JAZ1 and JAZ2 from Salvia miltiorrhiza.

Production of major effective metabolites, tanshinones and lithospermic acid B (LAB), was dramatically enhanced by exogenous jasmonate (JA) treatment in Salvia miltiorrhiza. However, the molecular mechanism of such metabolic activation in S. miltiorrhiza has not been elucidated yet. Here, we focused on jasmonate ZIM-domain (JAZ) proteins that act as repressors of JA signaling. Open reading frames of two novel genes, SmJAZ1 and SmJAZ2, from S. miltiorrhiza were amplified according to the annotation of S. miltiorrhiza transcriptome. Compared to plant JAZs, SmJAZ1 and SmJAZ2 were clustered into different groups by phylogenetic analysis. Organ expression pattern was studied by real-time quantitative PCR (RT-qPCR), showing higher transcription level of both genes in stems than roots and leaves. The two SmJAZs responded to methyl jasmonate at early stage and the transcriptional level significantly increased at 4 H. Our experimental results indicate that SmJAZ1 and SmJAZ2 are JA responsive and presented similar expression trend in JA response. The whole research will certainly facilitate further characterization of JAs effect on effective metabolites and help to ultimately achieve high yield of target compounds (tanshinones and LAB).

SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge, which contains tanshinones and phenolic acids as major classes of bioactive components, is one of the most widely used herbs in traditional Chinese medicine. Production of tanshinones and phenolic acids is enhanced by methyl jasmonate (MeJA). Transcription factor MYC2 is the switch of jasmontes signaling in plants. Here, we focused on two novel JA-inducible genes in S. miltiorrhiza, designated as SmMYC2a and SmMYC2b, which were localized in the nucleus. SmMYC2a and SmMYC2b were also discovered to interact with SmJAZ1 and SmJAZ2, implying that the two MYC2s might function as direct targets of JAZ proteins. Ectopic RNA interference (RNAi)-mediated knockdown experiments suggested that SmMYC2a/b affected multiple genes in tanshinone and phenolic acid biosynthetic pathway. Besides, the accumulation of tanshinones and phenolic acids was impaired by the loss of function in SmMYC2a/b. Meanwhile, SmMYC2a could bind with an E-box motif within SmHCT6 and SmCYP98A14 promoters, while SmMYC2b bound with an E-box motif within SmCYP98A14 promoter, through which the regulation of phenolic acid biosynthetic pathway might achieve. Together, these results suggest that SmMYC2a and SmMYC2b are JAZ-interacting transcription factors that positively regulate the biosynthesis of tanshinones and Sal B with similar but irreplaceable effects.