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Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget.

Qiu YQ, Yang CW, Lee YZ, Yang RB, Lee CH, Hsu HY, Chang CC, Lee SJ - Oncotarget (2015)

Bottom Line: Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds.Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b.Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Tylophorine compounds have been the focus of drug development for decades. Tylophorine derivatives exhibit anti-cancer activities but their cellular targets remain unknown. We used a biotinylated tylophorine derivative to probe for the interacting cellular target(s) of tylophorine. Tylophorine directly binds to caprin-1 and consequently enhances the recruitment of G3BP1, c-Myc mRNA, and cyclin D2 mRNA to form a ribonucleoprotein complex. Subsequently, this tylophorine targeted ribonucleoprotein complex is sequestered to the polysomal fractions and the protein expressions of the associated mRNA-transcripts are repressed. Caprin-1 depleted carcinoma cells become more resistant to tylophorine, associated with decreased formation of the ribonucleoprotein complex targeted by tylophorine. Consequently, tylophorine downregulates c-Myc and cyclins D1/D2, causing hypophosphorylation of Rb and suppression of both processing-body formation and the Warburg effect. Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds. Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b. Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

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The tylophorine compounds downregulate c-Myc protein levels and affect the expression of c-Myc target genes in carcinoma cells treated with tylophorine compounds in vitro or in vivoA. Tylophorine and a tylophorine-derived-dibenzoquinoline 33b (DBQ 33b) inhibited the protein expression of c-Myc in 13 carcinoma and 2 fibroblast cell lines. The results shown are representative of 3 independent experiments. B-D. The effects of tylophorine on the expression of c-Myc target genes. The results were obtained from an analysis of gene expression profiling with a cDNA-array (B) and verified by RT-qPCR (C). The genes regulated by the tylophorine compounds through c-Myc are listed by function and their upregulation or downregulation by c-Myc as determined in previous studies as indicated (D). Cells were treated with vehicle (0.1% DMSO), tylophorine (2 μM), or DBQ 33b (0.5 μM) for 24 h prior to lysis for western analysis with the indicated antibodies (A) or prior to mRNA extraction for gene expression profiling using the Illumina-HumanHT-12-v4 Expression-BeadChip (B) and RT-qPCR analysis (C). E & F. The protein (E) and mRNA (F) expression levels of c-Myc were downregulated in vivo in A549 xenograft tumors in mice by treatment with DBQ 33b [22]. (E) The protein expression of c-Myc detected by immunoblot analysis (upper panel) was quantitated and shown individually (lower panel). (F) The mRNA expression of c-Myc was analyzed by RT-qPCR; the products were shown in the upper panel, and the individual data were plotted in the lower panel. The relative expression levels of mRNA or protein were normalized with their respective internal loading control GAPDH. The sequences of the gene primer pairs used were listed in Supplemental Table 3. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.0001.
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Figure 6: The tylophorine compounds downregulate c-Myc protein levels and affect the expression of c-Myc target genes in carcinoma cells treated with tylophorine compounds in vitro or in vivoA. Tylophorine and a tylophorine-derived-dibenzoquinoline 33b (DBQ 33b) inhibited the protein expression of c-Myc in 13 carcinoma and 2 fibroblast cell lines. The results shown are representative of 3 independent experiments. B-D. The effects of tylophorine on the expression of c-Myc target genes. The results were obtained from an analysis of gene expression profiling with a cDNA-array (B) and verified by RT-qPCR (C). The genes regulated by the tylophorine compounds through c-Myc are listed by function and their upregulation or downregulation by c-Myc as determined in previous studies as indicated (D). Cells were treated with vehicle (0.1% DMSO), tylophorine (2 μM), or DBQ 33b (0.5 μM) for 24 h prior to lysis for western analysis with the indicated antibodies (A) or prior to mRNA extraction for gene expression profiling using the Illumina-HumanHT-12-v4 Expression-BeadChip (B) and RT-qPCR analysis (C). E & F. The protein (E) and mRNA (F) expression levels of c-Myc were downregulated in vivo in A549 xenograft tumors in mice by treatment with DBQ 33b [22]. (E) The protein expression of c-Myc detected by immunoblot analysis (upper panel) was quantitated and shown individually (lower panel). (F) The mRNA expression of c-Myc was analyzed by RT-qPCR; the products were shown in the upper panel, and the individual data were plotted in the lower panel. The relative expression levels of mRNA or protein were normalized with their respective internal loading control GAPDH. The sequences of the gene primer pairs used were listed in Supplemental Table 3. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.0001.

Mentions: Because tylophorine-related alkaloids potently inhibit the growth of a wide variety of drug-sensitive and drug-resistant cancer cell lines, we evaluated c-Myc protein expression levels in a panel of carcinoma cell lines (13 carcinoma cell lines from 9 types of tumors and 2 fibroblast cell lines) with tylophorine treatment. We observed the sustained blockade of c-Myc protein expression in each of the individual carcinoma cell lines upon treatment with tylophorine or its potent derivative DBQ 33b [23] but not in the fibroblast cell lines. c-Myc protein expression levels were reduced to 1-33% (Fig. 6A), whereas c-Myc mRNA levels were increased by 1.5-4.5-fold (Figs. 3A, 6B, & 6C). We also performed gene expression profiling and analysis of the c-Myc downstream pathways that were affected by tylophorine or DBQ 33b (Fig. 6B), as well as validation by RT-qPCR (Fig. 6C). Both compounds increased the expression of c-Myc mRNA by ~2-4-fold; the mRNA levels of genes that were upregulated by c-Myc were decreased (e.g., cyclin A2, cyclin B1, SERBP1, PRDX3, and POLR3G), and the mRNA levels of genes that were downregulated by c-Myc increased (e.g., GADD45A, and THBS1) (Fig. 6B and 6C). These results are consistent with the above observation that increased c-Myc mRNA levels and decreased c-Myc protein levels occurred in tylophorine-treated cells, which led to negative regulation of c-Myc effector pathways. The genes, whose expression was upregulated or downregulated by tylophorine and DBQ 33b, associated with the function of c-Myc in carcinogenesis are shown in Fig. 6D.


Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget.

Qiu YQ, Yang CW, Lee YZ, Yang RB, Lee CH, Hsu HY, Chang CC, Lee SJ - Oncotarget (2015)

The tylophorine compounds downregulate c-Myc protein levels and affect the expression of c-Myc target genes in carcinoma cells treated with tylophorine compounds in vitro or in vivoA. Tylophorine and a tylophorine-derived-dibenzoquinoline 33b (DBQ 33b) inhibited the protein expression of c-Myc in 13 carcinoma and 2 fibroblast cell lines. The results shown are representative of 3 independent experiments. B-D. The effects of tylophorine on the expression of c-Myc target genes. The results were obtained from an analysis of gene expression profiling with a cDNA-array (B) and verified by RT-qPCR (C). The genes regulated by the tylophorine compounds through c-Myc are listed by function and their upregulation or downregulation by c-Myc as determined in previous studies as indicated (D). Cells were treated with vehicle (0.1% DMSO), tylophorine (2 μM), or DBQ 33b (0.5 μM) for 24 h prior to lysis for western analysis with the indicated antibodies (A) or prior to mRNA extraction for gene expression profiling using the Illumina-HumanHT-12-v4 Expression-BeadChip (B) and RT-qPCR analysis (C). E & F. The protein (E) and mRNA (F) expression levels of c-Myc were downregulated in vivo in A549 xenograft tumors in mice by treatment with DBQ 33b [22]. (E) The protein expression of c-Myc detected by immunoblot analysis (upper panel) was quantitated and shown individually (lower panel). (F) The mRNA expression of c-Myc was analyzed by RT-qPCR; the products were shown in the upper panel, and the individual data were plotted in the lower panel. The relative expression levels of mRNA or protein were normalized with their respective internal loading control GAPDH. The sequences of the gene primer pairs used were listed in Supplemental Table 3. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.0001.
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Figure 6: The tylophorine compounds downregulate c-Myc protein levels and affect the expression of c-Myc target genes in carcinoma cells treated with tylophorine compounds in vitro or in vivoA. Tylophorine and a tylophorine-derived-dibenzoquinoline 33b (DBQ 33b) inhibited the protein expression of c-Myc in 13 carcinoma and 2 fibroblast cell lines. The results shown are representative of 3 independent experiments. B-D. The effects of tylophorine on the expression of c-Myc target genes. The results were obtained from an analysis of gene expression profiling with a cDNA-array (B) and verified by RT-qPCR (C). The genes regulated by the tylophorine compounds through c-Myc are listed by function and their upregulation or downregulation by c-Myc as determined in previous studies as indicated (D). Cells were treated with vehicle (0.1% DMSO), tylophorine (2 μM), or DBQ 33b (0.5 μM) for 24 h prior to lysis for western analysis with the indicated antibodies (A) or prior to mRNA extraction for gene expression profiling using the Illumina-HumanHT-12-v4 Expression-BeadChip (B) and RT-qPCR analysis (C). E & F. The protein (E) and mRNA (F) expression levels of c-Myc were downregulated in vivo in A549 xenograft tumors in mice by treatment with DBQ 33b [22]. (E) The protein expression of c-Myc detected by immunoblot analysis (upper panel) was quantitated and shown individually (lower panel). (F) The mRNA expression of c-Myc was analyzed by RT-qPCR; the products were shown in the upper panel, and the individual data were plotted in the lower panel. The relative expression levels of mRNA or protein were normalized with their respective internal loading control GAPDH. The sequences of the gene primer pairs used were listed in Supplemental Table 3. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.0001.
Mentions: Because tylophorine-related alkaloids potently inhibit the growth of a wide variety of drug-sensitive and drug-resistant cancer cell lines, we evaluated c-Myc protein expression levels in a panel of carcinoma cell lines (13 carcinoma cell lines from 9 types of tumors and 2 fibroblast cell lines) with tylophorine treatment. We observed the sustained blockade of c-Myc protein expression in each of the individual carcinoma cell lines upon treatment with tylophorine or its potent derivative DBQ 33b [23] but not in the fibroblast cell lines. c-Myc protein expression levels were reduced to 1-33% (Fig. 6A), whereas c-Myc mRNA levels were increased by 1.5-4.5-fold (Figs. 3A, 6B, & 6C). We also performed gene expression profiling and analysis of the c-Myc downstream pathways that were affected by tylophorine or DBQ 33b (Fig. 6B), as well as validation by RT-qPCR (Fig. 6C). Both compounds increased the expression of c-Myc mRNA by ~2-4-fold; the mRNA levels of genes that were upregulated by c-Myc were decreased (e.g., cyclin A2, cyclin B1, SERBP1, PRDX3, and POLR3G), and the mRNA levels of genes that were downregulated by c-Myc increased (e.g., GADD45A, and THBS1) (Fig. 6B and 6C). These results are consistent with the above observation that increased c-Myc mRNA levels and decreased c-Myc protein levels occurred in tylophorine-treated cells, which led to negative regulation of c-Myc effector pathways. The genes, whose expression was upregulated or downregulated by tylophorine and DBQ 33b, associated with the function of c-Myc in carcinogenesis are shown in Fig. 6D.

Bottom Line: Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds.Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b.Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Tylophorine compounds have been the focus of drug development for decades. Tylophorine derivatives exhibit anti-cancer activities but their cellular targets remain unknown. We used a biotinylated tylophorine derivative to probe for the interacting cellular target(s) of tylophorine. Tylophorine directly binds to caprin-1 and consequently enhances the recruitment of G3BP1, c-Myc mRNA, and cyclin D2 mRNA to form a ribonucleoprotein complex. Subsequently, this tylophorine targeted ribonucleoprotein complex is sequestered to the polysomal fractions and the protein expressions of the associated mRNA-transcripts are repressed. Caprin-1 depleted carcinoma cells become more resistant to tylophorine, associated with decreased formation of the ribonucleoprotein complex targeted by tylophorine. Consequently, tylophorine downregulates c-Myc and cyclins D1/D2, causing hypophosphorylation of Rb and suppression of both processing-body formation and the Warburg effect. Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds. Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b. Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

Show MeSH
Related in: MedlinePlus