Limits...
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.

Show MeSH

Related in: MedlinePlus

ARC and sangivamycin inhibit PKC substrate phosphorylation, PKCδ kinase activity, RNA polymerase II phosphorylation and P-TEFb kinase activity. A) Representative blot of the effects of ARC, sangivamycin, toyocamycin, fludarabine and thioguanine on the endogenous PKC activity (upper panel) and TPA-stimulated PKC substrate phosphorylation (lower panel). Cells were incubated for 9 h with 100 μM drug. To stimulate PKC, 5 μM TPA was included during the last 2 h of incubation. Lysates were prepared and probed for PKC substrates containing phospho-serine. B) Activity of PKCδ to incorporate 32P from [γ-32P] ATP into PKC substrate peptide 2 in the presence of the indicated concentrations of drugs. C) Activity of the recombinant PKA catalytic subunit to phosphorylate the substrate Kemptide, in the presence of the indicated concentrations of drugs and the PKA inhibitor peptide. D) Upper panel, MCF7 cells were incubated with 100 μM ARC, sangivamycin, toyocamycin, fludarabine and thioguanine for 3 h before lysates were prepared and probed for phosphorylated RNA polymerase II. Lower panel, densitometric analysis of western blots. Data is presented as the total phosphorylated RNA polymerase as a propotion of actin band intensity. E) Purified P-TEFb was incubated with substrate (PDKtide) in the absence or presence of the indicated concentrations of drug. The effects of the drugs are presented as % of control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651907&req=5

Figure 4: ARC and sangivamycin inhibit PKC substrate phosphorylation, PKCδ kinase activity, RNA polymerase II phosphorylation and P-TEFb kinase activity. A) Representative blot of the effects of ARC, sangivamycin, toyocamycin, fludarabine and thioguanine on the endogenous PKC activity (upper panel) and TPA-stimulated PKC substrate phosphorylation (lower panel). Cells were incubated for 9 h with 100 μM drug. To stimulate PKC, 5 μM TPA was included during the last 2 h of incubation. Lysates were prepared and probed for PKC substrates containing phospho-serine. B) Activity of PKCδ to incorporate 32P from [γ-32P] ATP into PKC substrate peptide 2 in the presence of the indicated concentrations of drugs. C) Activity of the recombinant PKA catalytic subunit to phosphorylate the substrate Kemptide, in the presence of the indicated concentrations of drugs and the PKA inhibitor peptide. D) Upper panel, MCF7 cells were incubated with 100 μM ARC, sangivamycin, toyocamycin, fludarabine and thioguanine for 3 h before lysates were prepared and probed for phosphorylated RNA polymerase II. Lower panel, densitometric analysis of western blots. Data is presented as the total phosphorylated RNA polymerase as a propotion of actin band intensity. E) Purified P-TEFb was incubated with substrate (PDKtide) in the absence or presence of the indicated concentrations of drug. The effects of the drugs are presented as % of control.

Mentions: The parallel activities of ARC and sangivamycin in previous assays prompted further investigation into whether ARC treatment reproduces the classical activity of sangivamycin, inhibition of protein kinase C (PKC) [15]. Lysates were prepared from cells treated with 100 μM of each compound for 9 h in the presence or absence of the PKC activator, TPA (Fig. 4A). Western blots probed with an antibody which detects phosphorylated PKC substrates (anti-phospho-serine PKC substrate) illustrated that the levels of endogenous phosphorylated PKC substrate declined significantly after treatment with ARC and sangivamycin in both untreated and TPA exposed cells. Levels of phosphorylation were unaffected in cells treated with toyocamycin, fludarabine or thioguanine. We next investigated the ability of the adenosine analogs to inhibit the activity of recombinant PKCδ. ARC, sangivamycin and toyocamycin all inhibited phosphorylation of a PKC substrate peptide in the presence of [γ-32P] ATP (Fig. 4B). While sangivamycin is reported to be a potent inhibitor of PKC activity, it has little effect on protein kinase A. In a similar assay to that used above for PKC, none of the adenosine analogs had any effect on the ability of recombinant PKA catalytic subunit to phosphorylate its respective peptide substrate, Kemptide, whereas the control PKA inhibitor peptide completely prevented phosphorylation (Fig. 4C). Thus, both ARC and sangivamycin inhibit the kinase activity of PKC, but not of PKA.


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)

ARC and sangivamycin inhibit PKC substrate phosphorylation, PKCδ kinase activity, RNA polymerase II phosphorylation and P-TEFb kinase activity. A) Representative blot of the effects of ARC, sangivamycin, toyocamycin, fludarabine and thioguanine on the endogenous PKC activity (upper panel) and TPA-stimulated PKC substrate phosphorylation (lower panel). Cells were incubated for 9 h with 100 μM drug. To stimulate PKC, 5 μM TPA was included during the last 2 h of incubation. Lysates were prepared and probed for PKC substrates containing phospho-serine. B) Activity of PKCδ to incorporate 32P from [γ-32P] ATP into PKC substrate peptide 2 in the presence of the indicated concentrations of drugs. C) Activity of the recombinant PKA catalytic subunit to phosphorylate the substrate Kemptide, in the presence of the indicated concentrations of drugs and the PKA inhibitor peptide. D) Upper panel, MCF7 cells were incubated with 100 μM ARC, sangivamycin, toyocamycin, fludarabine and thioguanine for 3 h before lysates were prepared and probed for phosphorylated RNA polymerase II. Lower panel, densitometric analysis of western blots. Data is presented as the total phosphorylated RNA polymerase as a propotion of actin band intensity. E) Purified P-TEFb was incubated with substrate (PDKtide) in the absence or presence of the indicated concentrations of drug. The effects of the drugs are presented as % of control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651907&req=5

Figure 4: ARC and sangivamycin inhibit PKC substrate phosphorylation, PKCδ kinase activity, RNA polymerase II phosphorylation and P-TEFb kinase activity. A) Representative blot of the effects of ARC, sangivamycin, toyocamycin, fludarabine and thioguanine on the endogenous PKC activity (upper panel) and TPA-stimulated PKC substrate phosphorylation (lower panel). Cells were incubated for 9 h with 100 μM drug. To stimulate PKC, 5 μM TPA was included during the last 2 h of incubation. Lysates were prepared and probed for PKC substrates containing phospho-serine. B) Activity of PKCδ to incorporate 32P from [γ-32P] ATP into PKC substrate peptide 2 in the presence of the indicated concentrations of drugs. C) Activity of the recombinant PKA catalytic subunit to phosphorylate the substrate Kemptide, in the presence of the indicated concentrations of drugs and the PKA inhibitor peptide. D) Upper panel, MCF7 cells were incubated with 100 μM ARC, sangivamycin, toyocamycin, fludarabine and thioguanine for 3 h before lysates were prepared and probed for phosphorylated RNA polymerase II. Lower panel, densitometric analysis of western blots. Data is presented as the total phosphorylated RNA polymerase as a propotion of actin band intensity. E) Purified P-TEFb was incubated with substrate (PDKtide) in the absence or presence of the indicated concentrations of drug. The effects of the drugs are presented as % of control.
Mentions: The parallel activities of ARC and sangivamycin in previous assays prompted further investigation into whether ARC treatment reproduces the classical activity of sangivamycin, inhibition of protein kinase C (PKC) [15]. Lysates were prepared from cells treated with 100 μM of each compound for 9 h in the presence or absence of the PKC activator, TPA (Fig. 4A). Western blots probed with an antibody which detects phosphorylated PKC substrates (anti-phospho-serine PKC substrate) illustrated that the levels of endogenous phosphorylated PKC substrate declined significantly after treatment with ARC and sangivamycin in both untreated and TPA exposed cells. Levels of phosphorylation were unaffected in cells treated with toyocamycin, fludarabine or thioguanine. We next investigated the ability of the adenosine analogs to inhibit the activity of recombinant PKCδ. ARC, sangivamycin and toyocamycin all inhibited phosphorylation of a PKC substrate peptide in the presence of [γ-32P] ATP (Fig. 4B). While sangivamycin is reported to be a potent inhibitor of PKC activity, it has little effect on protein kinase A. In a similar assay to that used above for PKC, none of the adenosine analogs had any effect on the ability of recombinant PKA catalytic subunit to phosphorylate its respective peptide substrate, Kemptide, whereas the control PKA inhibitor peptide completely prevented phosphorylation (Fig. 4C). Thus, both ARC and sangivamycin inhibit the kinase activity of PKC, but not of PKA.

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