Indole phytoalexin derivatives induce mitochondrialmediated apoptosis in human colorectal carcinoma cells

AIM To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.METHODS Changes in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and Brd U assay,allowing us to choose the most potent substance.Cell cycle alterations were analyzed using flow cytometric analysis.The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry,as well as acridine orange/propidium iodide(AO/PI)staining.The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis,such as poly(ADP-ribose)polymerase(PARP),caspase-3 and-9,cytochrome c,Bcl-2 family proteins,and also the integrity of the mitochondrial membrane.To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry.RESULTS Among the ten tested compounds,compound K-453{(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol}exhibited the most potent activity with IC50=32.22±1.14μmol/L in human colorectal HCT116 cells and was thus selected for further studies.Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G1 DNA content,which is considered as a marker of apoptotic cell death.The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining.The apoptosis was associated with the loss of mitochondrial membrane potential,PARP cleavage,caspase-3 and caspase-9 activation,release of cytochrome c,as well as changes in the levels of Bcl-2family members.Moreover,flow cytometry showed that compound K-453 stimulates phosphorylation of p38MAPK but decreases phosphorylation of Akt and Erk1/2.Activation of p38 MAPK was also confirmed using Western blot analysis.This analysis also revealed downregulation of NF-κB1(p50)and Rel A(p65)proteins and the loss of their anti-apoptotic activity.CONCLUSION In our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.

Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus

Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expression profile of P.massoniana infested by Bursaphelenchus xylophilus.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistancelike protein,and the gene encoding a putative nematode resistance protein.Increased infestation pressure(number of nematodes inoculated)caused a continuous decline in the gene expression of stem samples.An infestation of P.massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.A downregulation of auxin responsive family protein was observed in infested samples,which resulted in a suppression of plant growth.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and growth regulation was observed in P.massoniana,together with a decrease in the levels of phytoalexinlike secondary substances,all of which resulted in withering and ultimately death of P.massoniana.

Oryzalexin S biosynthesis:a cross-stitched disappearing pathway

Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.