Molecular Mechanism Of Trichomicin From Fungi Inhibiting The Growth Of Colorectal Cancer

Trichomicin is a novel small-molecule compound isolated from Trichoderma hazianum and was granted a patent license(No.ZL 200510082974 X).Orally bioavailable Trichomincin(60 mg/kg)regresses human colorectal cancer(HT-29)xenografts,and the inhibition rate was 62.70%according to the tumor volume,57.44%according to the tumor weight respectively.In addition,compared with the control group and the positive drug group,the levels of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNFa)in t tissue of the Trichomicin treated group were significantly reduced.However,it is still unclear whether Trichomicin exerts anti-tumor effects through their cytotoxicity or by targeting the cytokine network in the tumor microenvironment(TME).To further illuminate the antitumor molecular mechanism of Trichomicin in colorectal cancer(CRC),it was explored the effect of Trichomincin to human CRC cells(HCT-116,HT-29),colon cancer-associated fibroblasts(CAFs),and human acute mononuclear leukemia cells(THP-1,can be used as research pattern of the macrophages cells in vitro)in vitro.According to the result of pharmacokinetics study in vivo,the maximum plasma concentration(Cmax)of Trichomicin is 4.8 μM.So we first detected Trichomicin cytotoxicity to HT-29,HCT-116 and THP-1 cells in 0-10 μmol/L concentration range by MTT assay.The results showed that there was no obvious cytotoxicity to the above cells even treated with 10 μmol/L of Trichomicin.This shows that the anti-tumor effect of Trichomicin in vivo is not due to its own cytotoxicity.It was confirmed that IL-6 and TNFa in the tumors treated with Trichomicin is reduced in our previous research.IL-6 and TNFa in the tumor microenvironment mainly secreted by tumor associated macrophage(TAM),we further analyze the effect of Trichomicin on the transcription and secretion of IL-6 and TNFa in macrophages under different stimulation conditions by Real-time qPCR and Enzyme Linked Immunosorbent Assay.The results showed that different concentrations of Trichomicin(1 μmol/L,5 μmol/L,10 μmol/L)inhibited the transcription and secretion of IL-6 and TNFa in macrophages in a dose-dependent manner.5μmol/L of Trichomicin can exert an inhibitory effect,and when treated with 10μmol/L of Trichomicin,the level of transcripts and secretion of IL-6 and TNFα in macrophages stimulated by Lipopolysaccharide(LPS)or CRC cells culture supernatants reduced by 90%or more.Since signal transducer and activator of transcription 3(Stat3)and nuclear factor κB(NF-κB)are driven by IL-6 and TNFa in CRC,it was further detected whether Trichomicin can target Stat3 and NF-κB signal pathways in CRC and stromal cells via western blotting.The results showed that Trichomicin inhibited IL-6-induced Stat3 signaling pathways in different CRC cells in a dose-dependent manner,and phosphorylation level of Stat3 induced by IL-6 in HT-29 and HCT-116 cells was lower than background level under 10 μmol/L of Trichomicin.The inhibition to Stat3 phosphorylation by Trichomicin is stronger than the Stat3 inhibitor BP-1-102 in same concentration.Similarly,Trichomicin inhibited NF-KB-related signaling pathways induced by TNFa in a dose-dependent manner,whereas Stat3 inhibitor BP-1-102 had no effect on NF-KB-related signaling pathways.Trichomicin also had similar inhibitory effects on Stat3 and NF-κB signaling pathways in cancer stromal cells(TAM and CAFs)stimulated by different CRC cells culture supernatants.This shows that Trichomicin inhibit tumor growth via targeting multiple pathways in tumor and stromal cells.To further confirm whether Trichomicin can directly target IKK/NF-κB and Stat3 signaling pathway proteins,we performed cellular knockdowns of NF-κB p65 by siRNA in HCT-116 cells.We found that Trichomicin still inhibited Stat3 phosphorylation after NF-κB p65 knockdowns in HCT-116 cells.This result confirmed that Trichomicin can target Stat3 and NF-κB p65 respectively.Finally,we explored the effect of Trichomicin on the expression of PD-L1 in CRC and stromal cells.We found that Trichomicin significantly inhibited PD-L1 expression indifferent cells.It needs to be further verified whether Trichomicin can directly target PD-L1,or only caused by the inhibition of Stat3.Taken together,this study demonstrates that the new structural compound,Trichomicin,can inhibit the growth of colorectal cancer via reducing the secretion of pro-inflammatory cytokines IL-6 and TNFa in tumor-associated macrophages,and inhibiting JAK/Stat3 and IKK/NF-κB signaling pathways in CRC and stromal cells.It has the potential to become as oral therapeutic agent for colorectal cancer.