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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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Antiproliferative effects of PEP005 in cells, according to PKC expression. (A) Cytotoxicity of PEP005 after 1, 24, and 48 h exposures in Colo205 cells. (B) Modulation of PKC δ and p38 phosphorylation by PEP005 in Colo205 cells. Colo205 cells were treated with PEP005 (0.3 μM) for the indicated time. Protein extracts were then analysed for PKC δ and p38 phosphorylation. (C) Correlation between PKC levels and sensitivity to PEP005. Expression levels of PKC α, β, δ, and ɛ were correlated with IC50 of PEP005 in 10 cancer cell lines (•, colon cancer cell lines; ○, other cell lines). All values from the densitometric analysis of western blot are standardised with respect to Colo205. The r2 value is the correlation coefficient calculated by linear regression analysis.
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fig2: Antiproliferative effects of PEP005 in cells, according to PKC expression. (A) Cytotoxicity of PEP005 after 1, 24, and 48 h exposures in Colo205 cells. (B) Modulation of PKC δ and p38 phosphorylation by PEP005 in Colo205 cells. Colo205 cells were treated with PEP005 (0.3 μM) for the indicated time. Protein extracts were then analysed for PKC δ and p38 phosphorylation. (C) Correlation between PKC levels and sensitivity to PEP005. Expression levels of PKC α, β, δ, and ɛ were correlated with IC50 of PEP005 in 10 cancer cell lines (•, colon cancer cell lines; ○, other cell lines). All values from the densitometric analysis of western blot are standardised with respect to Colo205. The r2 value is the correlation coefficient calculated by linear regression analysis.

Mentions: We first evaluated the expression of PKC isoforms α, β, δ, and ɛ in a panel of 10 human cancer cell lines representing common human cancers (breast, lung, colon, and ovarian cancer). The mRNA and protein expression levels of PKCs were assessed by RT–PCR (data not shown) and western blot analysis. Protein kinase C isoforms α and δ, which are targets of PEP005, were expressed at various levels in the panel of cell lines. On the basis of these results, we selected four colon cancer cell lines for further experiments. Those cells were considered as representative as they displayed different levels of PKCα and PKCδ expression. The antiproliferative effects were assessed by MTT assay for durations of exposure varying between 1 and 48 h (Table 1). In our panel of colon cancer cells, exposure to PEP005 for 48 h led to higher antiproliferative effects than shorter exposure times (Table 1 and Figure 2A). Colo205, HCC2998, HCT116, and HT29 cells displayed IC50 values of 0.01, 30.0, 120.0, and 140.0 μM after 48 h PEP005 exposure, respectively. Previous data showed no relevant change in the expression of PKC isoforms under treatment with PEP005 in cancer cells but that PEP005 may activate PKC and inhibit PKC phosphorylation (Serova et al, 2008). As expected from our previous results, we observed that exposure of Colo205 cells to 3 μM PEP005 for 20 min increased the phosphorylation of PKCδ and activated p38 (Figure 2B). Semiquantitative western blot analysis of expression of PKC isoforms α, β, δ, and ɛ was performed showing that most cancer cells in our panel expressed various levels of PKC. Attempts were made to determine whether baseline expressions of those PKC isoforms correlated with sensitivity to PEP005. In this study, we further found no statistically significant correlation between sensitivity to PEP005, and PKC isoforms expression was detectable neither in our panel of colon cancer cell lines nor in the extended panel of 10 human cancer cell lines (Figure 2C). The cytotoxicity profile of PEP005 was compared to that of various chemotherapeutic agents including oxaliplatin, cisplatin, doxorubicin, SN38, 5FU, gemcitabine, vinorelbine, docetaxel, and staurosporine in our panel of colon cancer cell lines. The IC50 values are reported in Table 1. In our study, PEP005 displayed a cytotoxicity profile that differs from that of most cytotoxic agents. Interestingly, Colo205 cells were more sensitive to PEP005 than to 5FU and oxaliplatin.


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)

Antiproliferative effects of PEP005 in cells, according to PKC expression. (A) Cytotoxicity of PEP005 after 1, 24, and 48 h exposures in Colo205 cells. (B) Modulation of PKC δ and p38 phosphorylation by PEP005 in Colo205 cells. Colo205 cells were treated with PEP005 (0.3 μM) for the indicated time. Protein extracts were then analysed for PKC δ and p38 phosphorylation. (C) Correlation between PKC levels and sensitivity to PEP005. Expression levels of PKC α, β, δ, and ɛ were correlated with IC50 of PEP005 in 10 cancer cell lines (•, colon cancer cell lines; ○, other cell lines). All values from the densitometric analysis of western blot are standardised with respect to Colo205. The r2 value is the correlation coefficient calculated by linear regression analysis.
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Related In: Results  -  Collection

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fig2: Antiproliferative effects of PEP005 in cells, according to PKC expression. (A) Cytotoxicity of PEP005 after 1, 24, and 48 h exposures in Colo205 cells. (B) Modulation of PKC δ and p38 phosphorylation by PEP005 in Colo205 cells. Colo205 cells were treated with PEP005 (0.3 μM) for the indicated time. Protein extracts were then analysed for PKC δ and p38 phosphorylation. (C) Correlation between PKC levels and sensitivity to PEP005. Expression levels of PKC α, β, δ, and ɛ were correlated with IC50 of PEP005 in 10 cancer cell lines (•, colon cancer cell lines; ○, other cell lines). All values from the densitometric analysis of western blot are standardised with respect to Colo205. The r2 value is the correlation coefficient calculated by linear regression analysis.
Mentions: We first evaluated the expression of PKC isoforms α, β, δ, and ɛ in a panel of 10 human cancer cell lines representing common human cancers (breast, lung, colon, and ovarian cancer). The mRNA and protein expression levels of PKCs were assessed by RT–PCR (data not shown) and western blot analysis. Protein kinase C isoforms α and δ, which are targets of PEP005, were expressed at various levels in the panel of cell lines. On the basis of these results, we selected four colon cancer cell lines for further experiments. Those cells were considered as representative as they displayed different levels of PKCα and PKCδ expression. The antiproliferative effects were assessed by MTT assay for durations of exposure varying between 1 and 48 h (Table 1). In our panel of colon cancer cells, exposure to PEP005 for 48 h led to higher antiproliferative effects than shorter exposure times (Table 1 and Figure 2A). Colo205, HCC2998, HCT116, and HT29 cells displayed IC50 values of 0.01, 30.0, 120.0, and 140.0 μM after 48 h PEP005 exposure, respectively. Previous data showed no relevant change in the expression of PKC isoforms under treatment with PEP005 in cancer cells but that PEP005 may activate PKC and inhibit PKC phosphorylation (Serova et al, 2008). As expected from our previous results, we observed that exposure of Colo205 cells to 3 μM PEP005 for 20 min increased the phosphorylation of PKCδ and activated p38 (Figure 2B). Semiquantitative western blot analysis of expression of PKC isoforms α, β, δ, and ɛ was performed showing that most cancer cells in our panel expressed various levels of PKC. Attempts were made to determine whether baseline expressions of those PKC isoforms correlated with sensitivity to PEP005. In this study, we further found no statistically significant correlation between sensitivity to PEP005, and PKC isoforms expression was detectable neither in our panel of colon cancer cell lines nor in the extended panel of 10 human cancer cell lines (Figure 2C). The cytotoxicity profile of PEP005 was compared to that of various chemotherapeutic agents including oxaliplatin, cisplatin, doxorubicin, SN38, 5FU, gemcitabine, vinorelbine, docetaxel, and staurosporine in our panel of colon cancer cell lines. The IC50 values are reported in Table 1. In our study, PEP005 displayed a cytotoxicity profile that differs from that of most cytotoxic agents. Interestingly, Colo205 cells were more sensitive to PEP005 than to 5FU and oxaliplatin.

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