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Antiproliferative activity of PEP005, a novel ingenol angelate that modulates PKC functions, alone and in combination with cytotoxic agents in human colon cancer cells.

Benhadji KA, Serova M, Ghoul A, Cvitkovic E, Le Tourneau C, Ogbourne SM, Lokiec F, Calvo F, Hammel P, Faivre S, Raymond E - Br. J. Cancer (2008)

Bottom Line: The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent.In addition, PEP005 increased the phosphorylation of PKC delta and p38.In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U728, RayLab, Department of Medical Oncology, Beaujon University Hospital, APHP, Paris 7, 100 boulevard Général Leclerc, Clichy 92110, France.

ABSTRACT
PEP005 is a novel ingenol angelate that modulates protein kinases C (PKC) functions by activating PKC delta and inhibiting PKC alpha. This study assessed the antiproliferative effects of PEP005 alone and in combination with several other anticancer agents in a panel of 10 human cancer cell lines characterised for expression of several PKC isoforms. PEP005 displayed antiproliferative effects at clinically relevant concentrations with a unique cytotoxicity profile that differs from that of most other investigated cytotoxic agents, including staurosporine. In a subset of colon cancer cells, the IC(50) of PEP005 ranged from 0.01-140 microM. The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent. In Colo205 cells, apoptosis induction was observed at concentrations ranging from 0.03 to 3 microM. Exposure to PEP005 also induced accumulation of cells in the G1 phase of the cell cycle. In addition, PEP005 increased the phosphorylation of PKC delta and p38. In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects. Cell cycle blockage induced by PEP005 in late G1 lasted for up to 24 h and therefore a 24 h lag-time between PEP005 and subsequent exposure to cytotoxics was required to optimise PEP005 combinations with several anticancer agents. These data support further evaluation of PEP005 as an anticancer agent and may help to optimise clinical trials with PEP005-based combinations in patients with solid tumours.

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Cell cycle effects and apoptosis induction by PEP005 in colon cancer cells. Effects of increasing concentrations of PEP005 for 24 h (A) on cell cycle distribution in Colo205 cells detected by flow cytometry, SubG1, G0/G1, S, and G2/M phases of cell cycle. Effect of duration of exposure to PEP005 (B) in Colo205 cells were harvested after different incubation times with 0.3 μM PEP005. (C, D) Flow cytometry study of PEP005 induced apoptosis in Colo205 and HT29 cell lines. Apoptotic cells are distributed as: early apoptosis (Annexin V positive and PI negative) and late apoptosis (Annexin V and PI positive).
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fig3: Cell cycle effects and apoptosis induction by PEP005 in colon cancer cells. Effects of increasing concentrations of PEP005 for 24 h (A) on cell cycle distribution in Colo205 cells detected by flow cytometry, SubG1, G0/G1, S, and G2/M phases of cell cycle. Effect of duration of exposure to PEP005 (B) in Colo205 cells were harvested after different incubation times with 0.3 μM PEP005. (C, D) Flow cytometry study of PEP005 induced apoptosis in Colo205 and HT29 cell lines. Apoptotic cells are distributed as: early apoptosis (Annexin V positive and PI negative) and late apoptosis (Annexin V and PI positive).

Mentions: In Colo205 cells, which were the most PEP005 sensitive cells in our panel, we further analysed the cell cycle distribution using flow cytometry. Cells were incubated for 24 h with various concentrations of PEP005. Exposure to PEP005 concentrations ranging from 0.03 to 3.0 μM led to an inhibition of S-phase entry with an accumulation of cells in G1 and an increase of the sub-G1 fraction suggesting apoptosis (Figure 3A). Duration of exposure was shown to play a role in PEP005-induced effects on the cell cycle, the maximal inhibition being obtained for 24 h exposure (Figure 3B). In contrast to that observed in Colo205 cells, no change was observed in HT29 cells that appeared to be more resistant than Colo205 cells to PEP005. As the increase of the sub-G1 fraction suggested induction of apoptosis, we further confirmed apoptosis induction using double staining with annexin V-FITC and PI in cells exposed for 24 h to PEP005 at concentrations ranging 0.03–0.3 μM (Figure 3C). In Colo205 cells, we observed a significant increase of the early apoptosis fraction (annexin V positive, PI negative) as well as of the late apoptosis fraction (annexin V positive, PI positive). In contrast, no apoptosis was detectable in HT29 cells at concentrations ranging from 3 to 30 μM (Figure 3D).


Antiproliferative activity of PEP005, a novel ingenol angelate that modulates PKC functions, alone and in combination with cytotoxic agents in human colon cancer cells.

Benhadji KA, Serova M, Ghoul A, Cvitkovic E, Le Tourneau C, Ogbourne SM, Lokiec F, Calvo F, Hammel P, Faivre S, Raymond E - Br. J. Cancer (2008)

Cell cycle effects and apoptosis induction by PEP005 in colon cancer cells. Effects of increasing concentrations of PEP005 for 24 h (A) on cell cycle distribution in Colo205 cells detected by flow cytometry, SubG1, G0/G1, S, and G2/M phases of cell cycle. Effect of duration of exposure to PEP005 (B) in Colo205 cells were harvested after different incubation times with 0.3 μM PEP005. (C, D) Flow cytometry study of PEP005 induced apoptosis in Colo205 and HT29 cell lines. Apoptotic cells are distributed as: early apoptosis (Annexin V positive and PI negative) and late apoptosis (Annexin V and PI positive).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2600681&req=5

fig3: Cell cycle effects and apoptosis induction by PEP005 in colon cancer cells. Effects of increasing concentrations of PEP005 for 24 h (A) on cell cycle distribution in Colo205 cells detected by flow cytometry, SubG1, G0/G1, S, and G2/M phases of cell cycle. Effect of duration of exposure to PEP005 (B) in Colo205 cells were harvested after different incubation times with 0.3 μM PEP005. (C, D) Flow cytometry study of PEP005 induced apoptosis in Colo205 and HT29 cell lines. Apoptotic cells are distributed as: early apoptosis (Annexin V positive and PI negative) and late apoptosis (Annexin V and PI positive).
Mentions: In Colo205 cells, which were the most PEP005 sensitive cells in our panel, we further analysed the cell cycle distribution using flow cytometry. Cells were incubated for 24 h with various concentrations of PEP005. Exposure to PEP005 concentrations ranging from 0.03 to 3.0 μM led to an inhibition of S-phase entry with an accumulation of cells in G1 and an increase of the sub-G1 fraction suggesting apoptosis (Figure 3A). Duration of exposure was shown to play a role in PEP005-induced effects on the cell cycle, the maximal inhibition being obtained for 24 h exposure (Figure 3B). In contrast to that observed in Colo205 cells, no change was observed in HT29 cells that appeared to be more resistant than Colo205 cells to PEP005. As the increase of the sub-G1 fraction suggested induction of apoptosis, we further confirmed apoptosis induction using double staining with annexin V-FITC and PI in cells exposed for 24 h to PEP005 at concentrations ranging 0.03–0.3 μM (Figure 3C). In Colo205 cells, we observed a significant increase of the early apoptosis fraction (annexin V positive, PI negative) as well as of the late apoptosis fraction (annexin V positive, PI positive). In contrast, no apoptosis was detectable in HT29 cells at concentrations ranging from 3 to 30 μM (Figure 3D).

Bottom Line: The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent.In addition, PEP005 increased the phosphorylation of PKC delta and p38.In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U728, RayLab, Department of Medical Oncology, Beaujon University Hospital, APHP, Paris 7, 100 boulevard Général Leclerc, Clichy 92110, France.

ABSTRACT
PEP005 is a novel ingenol angelate that modulates protein kinases C (PKC) functions by activating PKC delta and inhibiting PKC alpha. This study assessed the antiproliferative effects of PEP005 alone and in combination with several other anticancer agents in a panel of 10 human cancer cell lines characterised for expression of several PKC isoforms. PEP005 displayed antiproliferative effects at clinically relevant concentrations with a unique cytotoxicity profile that differs from that of most other investigated cytotoxic agents, including staurosporine. In a subset of colon cancer cells, the IC(50) of PEP005 ranged from 0.01-140 microM. The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent. In Colo205 cells, apoptosis induction was observed at concentrations ranging from 0.03 to 3 microM. Exposure to PEP005 also induced accumulation of cells in the G1 phase of the cell cycle. In addition, PEP005 increased the phosphorylation of PKC delta and p38. In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects. Cell cycle blockage induced by PEP005 in late G1 lasted for up to 24 h and therefore a 24 h lag-time between PEP005 and subsequent exposure to cytotoxics was required to optimise PEP005 combinations with several anticancer agents. These data support further evaluation of PEP005 as an anticancer agent and may help to optimise clinical trials with PEP005-based combinations in patients with solid tumours.

Show MeSH
Related in: MedlinePlus