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ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC.

Stockwin LH, Yu SX, Stotler H, Hollingshead MG, Newton DL - BMC Cancer (2009)

Bottom Line: In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.Structure-based homology searches were used to identify those compounds with similarity to ARC.Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Therapeutics Program, SAIC-Frederick Inc, NCI- Frederick, Frederick, MD 21702, USA. Stockwin@ncifcrf.gov

ABSTRACT

Background: The nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.

Methods: Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays.

Results: Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance.

Conclusion: Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

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The cytotoxic effects of ARC towards cells are similar to that of sangivamycin and toyocamycin. A) Time course analysis of ARC on 14C-leucine viability of HL60 cells (upper panel) and MCF7 cells (lower panel). Data from at least 2 replicate experiments each with triplicate points were pooled and the SEM are shown when they are greater than the symbol. B) Time course analysis of ARC on a panel of 12 cell lines representative of 6 different tumor types. C) Comparison of the cytotoxic effects of ARC over 6 days with those of fludarabine (Flud), toyocamycin (TOY), sangivamycin (SAN) and thioguanine (TG) towards HL60 (upper panel) and MCF7 (lower panel) cells. The IC50 defined as the concentration of drug required to inhibit 14C-leucine viability by 50% relative to control treated cells determined from at least two pooled experiments is plotted versus time. D) Comparison of activity of ARC with sangivamycin and toyocamycin over a panel of cell lines reveals a high correlation coefficient between ARC and sangivamcin. In all panels, cells were incubated with varying concentrations (0.1 – 1000 μM) of the indicated drug.
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Figure 2: The cytotoxic effects of ARC towards cells are similar to that of sangivamycin and toyocamycin. A) Time course analysis of ARC on 14C-leucine viability of HL60 cells (upper panel) and MCF7 cells (lower panel). Data from at least 2 replicate experiments each with triplicate points were pooled and the SEM are shown when they are greater than the symbol. B) Time course analysis of ARC on a panel of 12 cell lines representative of 6 different tumor types. C) Comparison of the cytotoxic effects of ARC over 6 days with those of fludarabine (Flud), toyocamycin (TOY), sangivamycin (SAN) and thioguanine (TG) towards HL60 (upper panel) and MCF7 (lower panel) cells. The IC50 defined as the concentration of drug required to inhibit 14C-leucine viability by 50% relative to control treated cells determined from at least two pooled experiments is plotted versus time. D) Comparison of activity of ARC with sangivamycin and toyocamycin over a panel of cell lines reveals a high correlation coefficient between ARC and sangivamcin. In all panels, cells were incubated with varying concentrations (0.1 – 1000 μM) of the indicated drug.

Mentions: ARC was evaluated in the context of 14C-leucine viability assays using two cell lines, HL60 and MCF7 (Fig. 2). In both lines, ARC was shown to have activity in the nanomolar range (Fig. 2A). The maximal effect occurred within the first 24 h of incubation for HL60 cells, while for MCF7 cells, the maximal effect did not occur until 3 days of incubation. Expanding this study to a panel of diverse tumor lines (Fig. 2B) confirmed nanomolar activity in all cell lines with a 20fold variance at day 1 (IC50 range 20 to 400 nM) which declined to 11fold (4.5 to 50 nM) by day 6 showing that relative cell line susceptibility gradually disappears with prolonged incubation. Following this, HL60 and MCF7 cells were treated with all 5 compounds and IC50 values determined over the same extended time course (1–6 days) (Fig. 2C). Results demonstrated that in both lines, the time course of cytotoxicity was similar between ARC, sangivamycin and toyocamycin, and markedly different from fludarabine and thioguanine. Having highlighted general similarities in IC50 for ARC, sangivamycin and toyocamycin in HL60 and MCF7 cells, the study was then extended to a panel of cell lines (Fig. 2D). Calculation of correlation coefficients showed high similarity in activity between ARC and sangivamycin (correlation coefficient, 0.87), but not between ARC and toyocamycin (correlation coefficient, 0.25). These experiments suggest for the 5 compounds, ARC and sangivamycin are the closest relatives in terms of growth inhibitory effects.


ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC.

Stockwin LH, Yu SX, Stotler H, Hollingshead MG, Newton DL - BMC Cancer (2009)

The cytotoxic effects of ARC towards cells are similar to that of sangivamycin and toyocamycin. A) Time course analysis of ARC on 14C-leucine viability of HL60 cells (upper panel) and MCF7 cells (lower panel). Data from at least 2 replicate experiments each with triplicate points were pooled and the SEM are shown when they are greater than the symbol. B) Time course analysis of ARC on a panel of 12 cell lines representative of 6 different tumor types. C) Comparison of the cytotoxic effects of ARC over 6 days with those of fludarabine (Flud), toyocamycin (TOY), sangivamycin (SAN) and thioguanine (TG) towards HL60 (upper panel) and MCF7 (lower panel) cells. The IC50 defined as the concentration of drug required to inhibit 14C-leucine viability by 50% relative to control treated cells determined from at least two pooled experiments is plotted versus time. D) Comparison of activity of ARC with sangivamycin and toyocamycin over a panel of cell lines reveals a high correlation coefficient between ARC and sangivamcin. In all panels, cells were incubated with varying concentrations (0.1 – 1000 μM) of the indicated drug.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: The cytotoxic effects of ARC towards cells are similar to that of sangivamycin and toyocamycin. A) Time course analysis of ARC on 14C-leucine viability of HL60 cells (upper panel) and MCF7 cells (lower panel). Data from at least 2 replicate experiments each with triplicate points were pooled and the SEM are shown when they are greater than the symbol. B) Time course analysis of ARC on a panel of 12 cell lines representative of 6 different tumor types. C) Comparison of the cytotoxic effects of ARC over 6 days with those of fludarabine (Flud), toyocamycin (TOY), sangivamycin (SAN) and thioguanine (TG) towards HL60 (upper panel) and MCF7 (lower panel) cells. The IC50 defined as the concentration of drug required to inhibit 14C-leucine viability by 50% relative to control treated cells determined from at least two pooled experiments is plotted versus time. D) Comparison of activity of ARC with sangivamycin and toyocamycin over a panel of cell lines reveals a high correlation coefficient between ARC and sangivamcin. In all panels, cells were incubated with varying concentrations (0.1 – 1000 μM) of the indicated drug.
Mentions: ARC was evaluated in the context of 14C-leucine viability assays using two cell lines, HL60 and MCF7 (Fig. 2). In both lines, ARC was shown to have activity in the nanomolar range (Fig. 2A). The maximal effect occurred within the first 24 h of incubation for HL60 cells, while for MCF7 cells, the maximal effect did not occur until 3 days of incubation. Expanding this study to a panel of diverse tumor lines (Fig. 2B) confirmed nanomolar activity in all cell lines with a 20fold variance at day 1 (IC50 range 20 to 400 nM) which declined to 11fold (4.5 to 50 nM) by day 6 showing that relative cell line susceptibility gradually disappears with prolonged incubation. Following this, HL60 and MCF7 cells were treated with all 5 compounds and IC50 values determined over the same extended time course (1–6 days) (Fig. 2C). Results demonstrated that in both lines, the time course of cytotoxicity was similar between ARC, sangivamycin and toyocamycin, and markedly different from fludarabine and thioguanine. Having highlighted general similarities in IC50 for ARC, sangivamycin and toyocamycin in HL60 and MCF7 cells, the study was then extended to a panel of cell lines (Fig. 2D). Calculation of correlation coefficients showed high similarity in activity between ARC and sangivamycin (correlation coefficient, 0.87), but not between ARC and toyocamycin (correlation coefficient, 0.25). These experiments suggest for the 5 compounds, ARC and sangivamycin are the closest relatives in terms of growth inhibitory effects.

Bottom Line: In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.Structure-based homology searches were used to identify those compounds with similarity to ARC.Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Therapeutics Program, SAIC-Frederick Inc, NCI- Frederick, Frederick, MD 21702, USA. Stockwin@ncifcrf.gov

ABSTRACT

Background: The nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines.

Methods: Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays.

Results: Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance.

Conclusion: Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

Show MeSH
Related in: MedlinePlus