Anticancer drug screening of natural products:In vitro cytotoxicity assays,techniques,and challenges

Natural products include several diverse compounds that have been found to be effective against cancer.Discovering anticancer compounds in nature is a multistep and complex process that requires pre-clinical and clinical studies.Only a few of the available natural products are used to treat cancer since most of them have very high complexity and low bioavailability.Therefore,the process of anticancer drug discovery requires a straightforward and effective method to assess anticancer activity using in vitro assays.This review summarizes various cell-based assays and techniques used to measure cell viability,migration,and apoptosis,focusing in particular on the principles,mechanisms,advantages,and disadvantages of each assay to provide a preliminary platform for cancer drug discovery.

Anti-melanoma action of small molecular peptides derived from Brucea javanica(L.)Merr.globulin in vitro

Objective:The morbidity of malignant melanoma keeps increasing annually.It has high risks of metastasis,drug resistance,and poor prognosis in clinics.Moreover,the available medicines used commonly,such as dacarbazine,temozolomide,the v-Raf murine sarcoma viral oncogene homolog B1(BRAF)inhibitor vemurafenib,and the programmed cell death protein 1 inhibitor pembrolizumab,have some limitations at some extent.Therefore,a more effective therapeutic strategy is still urgently necessary.Methods:In this study,Brucea javanica(L.)Merr.globulins were hydrolyzed with pepsin,then ultra-filtrated to collect small molecular peptides(≤3 kDa).The peptides were then analyzed by antiproliferative assay,cell-cycle distribution,apoptosis assay,and in vitro wound-scratch assay.Finally,western blotting was conducted to elucidate the underlying anti-melanoma mechanism.Results:The small molecular peptide from B.javanica significantly inhibited malignant melanoma cell proliferation with the IC_(50) of 2.72 mg/mL for 72 h.Further analysis indicated that B.javanica peptides arrested cell cycle at the S and G2/M phases and induced apoptosis by upregulating p21,p53,Bax,caspase-3,and cleaved PARP while downregulating Bcl-2 expression.The inhibitory migration effects were also confirmed by wound-healing assay.Conclusion:The small molecular biopeptides from B.javanica may be a promising bioactive agent candidate for melanoma treatment.

Network pharmacology and preliminary cell screening studies on the anti-liver cancer activity of Nauclea Officinalis

Objective:To explore the mechanism of Nauclea Officinalis of anti-liver cancer effect based on network pharmacology,and to preliminarily verify anti-liver cancer activity of Nauclea Officinalis through cell screening.Methods:Network pharmacology was used to screen for common targets of Nauclea Officinalis and liver cancer,protein-protein interaction(PPI)network was constructed,and enrichment analysis and mechanism prediction were conductd.Molecular docking of main active ingredients of Nauclea Officinalis with core targets was made.Preliminary verification was performed by in vitro cell experiments such as CCK8,cell apoptosis,and PCR.Results:After the screening,14 active ingredients of Nauclea Officinalis were obtained,with 587 related targets.After mapping with liver cancer targets,there were 288 common targets,mainly including TP53,SRC,STAT3,and other core targets.Among them,compounds such as strictosamide,pumiloside and vincosamide may be potential active ingredients of Nauclea Officinalis of anti-liver cancer effect.They may participate in protein phosphorylation and negative regulation of the apoptosis process by mediating cancer pathways,PI3K/Akt and EGFR tyrosine kinase inhibitors resistance signaling pathways to play an anti-liver cancer role;molecular docking results showd that active ingredients of Nauclea Officinalis had a stable binding with liver cancer core targets;in vitro cell experiments showd that main ingredient strictosamide of Nauclea Officinalis had cytotoxicity against liver cancer cells,inhibited liver cancer cell proliferation(P<0.001),down-regulated gene expression of liver cancer HepG2 cells SRC,STAT3,MAPK3(P<0.05),and induced liver cancer cell apoptosis(P<0.001).Conclusion:This study preliminarily explores the potential mechanism of active ingredients of Nauclea Officinalis against liver cancer and its preliminary pharmacological effects,providing a theoretical basis for the study of Nauclea Officinalis of anti-liver cancer mechanism.

MEDICINAL PLANTS OF THE GENUS LEONURUS AND SEVENTEEN OF THEIR ISOLATED CONSTITUENTS SCREENED FOR EFFECTS ON PPARa,β/δ, and γ IN AN IN VITRO LUCIFERASE REPORTER GENE ASSAY

Leonurus japonicus Houtt.is used in TCM to treat the metabolic syndrome.However,up to now,no active constituents could be identified.Here we describe the isolation of 17 dominant constituents of L.japonicus and the related European herb Leonurus cardiaca L.-namely7R-chloro-6-desoxy-harpagide,ajugol,campneoside II,chicoric acid,ferulic acid,harpagide,isoacteoside,

Comparison of Different MARs(Matrix Attachment Regions) Effect on Transgene Expression

Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.

Current technologies to identify protein kinase substrates in high throughput

Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.