Acute myeloid leukemia(AML) is a heterogeneous disease characterized by the accu.mulation of immature myeloid progenitor cells in the bone marrow,compromising of normal hematopoi.esis and ultimately resulting in bone ...Acute myeloid leukemia(AML) is a heterogeneous disease characterized by the accu.mulation of immature myeloid progenitor cells in the bone marrow,compromising of normal hematopoi.esis and ultimately resulting in bone marrow failure.Chemotherapy is the mainstay treatment for all AML patients,however,drug resistance and clinical relapse limits its efficacy.The 5-year survival rate of AML patients is only 26.6%.Survival rates are even lower among patients ages 65 to 74 years(5.3%) and 75 years or older(1.6%).Therefore,exploring novel therapeutic agents is urgent for improving the outcome of patients with AML.Saponins are amphipathic glycosides found in traditional Chinese medicines.In the present study,we isolated a panel of saponins from Paris forrestii(Takht.) H.Li,a unique plant found in Tibet and Yunnan provinces,China.By examining their activities in suppressing acute myeloid leukemia cell proliferation,total saponins from Paris forrestii(TSPf) displayed more potent activity than individual ones.TSPf induced more than 40% AML cell apoptosis within 24 h and decreased the viability of all leukemia cell lines.TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation.TSPf downregulated pro-survival proteins Mcl-1,Bcl-xL and Bcl-2,but upreg.ulated the expression of tumor suppressor proteins p53,p27,Bax and Beclin 1.The AKT/mTOR signaling pathway is frequently over activated in various AML cells,and TSPf was found to suppress the activa.tion of both AKT and mTOR,but had no effects on their total protein expression.This was further con.firmed by the inactivation of 4 EBP-1 and p70 S6 K,two typical downstream signal molecules in the AKT/mTOR pathway.More specifically,TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6,a recently identified oncogene in AML.RNF6 activated AKT/mTOR,and consistently,knockdown of RNF6 led to inactivation of the AKT/mTOR pathway.Furthermore,TSPf suppressed the growth of AML xenografts in nude mice models.Oral administration of 100 mg · kg^(-1) body weight almost fully suppressed tumor growth within 14 d,without gross toxicity.This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway.Given its low toxicity,TSPf could be developed for the treatment of AML.展开更多
Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synt...Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.展开更多
基金supported by Natural Science Foundation of China(81770154)
文摘Acute myeloid leukemia(AML) is a heterogeneous disease characterized by the accu.mulation of immature myeloid progenitor cells in the bone marrow,compromising of normal hematopoi.esis and ultimately resulting in bone marrow failure.Chemotherapy is the mainstay treatment for all AML patients,however,drug resistance and clinical relapse limits its efficacy.The 5-year survival rate of AML patients is only 26.6%.Survival rates are even lower among patients ages 65 to 74 years(5.3%) and 75 years or older(1.6%).Therefore,exploring novel therapeutic agents is urgent for improving the outcome of patients with AML.Saponins are amphipathic glycosides found in traditional Chinese medicines.In the present study,we isolated a panel of saponins from Paris forrestii(Takht.) H.Li,a unique plant found in Tibet and Yunnan provinces,China.By examining their activities in suppressing acute myeloid leukemia cell proliferation,total saponins from Paris forrestii(TSPf) displayed more potent activity than individual ones.TSPf induced more than 40% AML cell apoptosis within 24 h and decreased the viability of all leukemia cell lines.TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation.TSPf downregulated pro-survival proteins Mcl-1,Bcl-xL and Bcl-2,but upreg.ulated the expression of tumor suppressor proteins p53,p27,Bax and Beclin 1.The AKT/mTOR signaling pathway is frequently over activated in various AML cells,and TSPf was found to suppress the activa.tion of both AKT and mTOR,but had no effects on their total protein expression.This was further con.firmed by the inactivation of 4 EBP-1 and p70 S6 K,two typical downstream signal molecules in the AKT/mTOR pathway.More specifically,TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6,a recently identified oncogene in AML.RNF6 activated AKT/mTOR,and consistently,knockdown of RNF6 led to inactivation of the AKT/mTOR pathway.Furthermore,TSPf suppressed the growth of AML xenografts in nude mice models.Oral administration of 100 mg · kg^(-1) body weight almost fully suppressed tumor growth within 14 d,without gross toxicity.This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway.Given its low toxicity,TSPf could be developed for the treatment of AML.
基金supported by the National Natural Science Foundation of China(Nos.81672744,81472252)Science and Technology Project of Guangdong Province(No.2016A020217007)Guangdong Esophageal Cancer Institute(No.M201606)
文摘Background: The mitogen-activated extracellular signal-regulated kinase 1/2(MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer(CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells,using a synthetic lethal short hairpin RNA(shRNA) screening approach.Methods: We infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183(RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF 183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA(siRNA) was used to knock down RNF183 transiently.Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay(ELISA) were used to evaluate the protein abundance. MTT assay,colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.Results: In the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8(IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.Conclusion: The RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.
