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In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells

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ABSTRACT

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

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FasR/FasL pathway is not involved in FTI-induced apoptosis in AML cells.Flow cytometric analysis of FasR and FasL expression in CD34+ AML cells from a representative case, untreated and treated with FTI (A). FTI-mediated inhibition of AML cell viability does not correlate with FasR and FasL expression on CD34+ AML cells. Cumulative mean ± SEM CD34+ Fas-R+ and C34+ Fas-L+ are reported (B). Caspase–8 is not activated by FTIs. AML cells untreated and treated with FTI after 48 h of culture. (C). AML cells were grown for 48 h with FTIs at IC50 and with FTIs + Fas-receptor triggering inhibitor Fas:Fc or caspase-8 inhibitor IETD-FMK (all used at 50 μM). AML cell viability was measured by colorimetric MTT assay. Columns represent cumulative mean ± SEM of cell viability of 5 experiments performed with all FTIs (D).
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f4-tm-15-22: FasR/FasL pathway is not involved in FTI-induced apoptosis in AML cells.Flow cytometric analysis of FasR and FasL expression in CD34+ AML cells from a representative case, untreated and treated with FTI (A). FTI-mediated inhibition of AML cell viability does not correlate with FasR and FasL expression on CD34+ AML cells. Cumulative mean ± SEM CD34+ Fas-R+ and C34+ Fas-L+ are reported (B). Caspase–8 is not activated by FTIs. AML cells untreated and treated with FTI after 48 h of culture. (C). AML cells were grown for 48 h with FTIs at IC50 and with FTIs + Fas-receptor triggering inhibitor Fas:Fc or caspase-8 inhibitor IETD-FMK (all used at 50 μM). AML cell viability was measured by colorimetric MTT assay. Columns represent cumulative mean ± SEM of cell viability of 5 experiments performed with all FTIs (D).

Mentions: Fas-R and Fas-L expression on AML cells exposed to FTIs was analysed by flow cytometry, but no variations were observed (21±4% and 23±8% in the absence and presence of FTIs, respectively) (Fig. 4A). Moreover, FTI–sensitive and FTI-resistant AML cells displayed similar expression of Fas-R (mean % ± SEM, 39±4 vs 49±6, respectively; p=0.5) and Fas-L (mean % ± SEM, 49±4 vs 52±3, respectively; p=0.7) (Fig. 4B). No modifications of caspase-8 activity were detected in AML cells after 48 h FTIs exposure by flow cytometry and intracellular caspase staining (Fig. 4C). To further exclude the involvement of Fas-R/Fas-L system in FTI-mediated apoptosis, prior to exposure to FTIs, AML cells were preincubated for 2 h with selective Fas-R/Fas-L inhibitors. As expected, no variations were observed in FTI-mediated inhibition of cell growth and apoptosis after pretreatment with Fas-receptor antagonist and caspase-8 inhibitor (mean % ± SEM of cell growth after FTI exposure, 41.6±3 and 43.3±3 without and with Fas:Fc vs 38±2 and 39±1 without and with IETD-FMK vs 42.3±3 and 46.6±3 without and with ZYVAD-FMK, respectively) (Fig. 4D).


In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells
FasR/FasL pathway is not involved in FTI-induced apoptosis in AML cells.Flow cytometric analysis of FasR and FasL expression in CD34+ AML cells from a representative case, untreated and treated with FTI (A). FTI-mediated inhibition of AML cell viability does not correlate with FasR and FasL expression on CD34+ AML cells. Cumulative mean ± SEM CD34+ Fas-R+ and C34+ Fas-L+ are reported (B). Caspase–8 is not activated by FTIs. AML cells untreated and treated with FTI after 48 h of culture. (C). AML cells were grown for 48 h with FTIs at IC50 and with FTIs + Fas-receptor triggering inhibitor Fas:Fc or caspase-8 inhibitor IETD-FMK (all used at 50 μM). AML cell viability was measured by colorimetric MTT assay. Columns represent cumulative mean ± SEM of cell viability of 5 experiments performed with all FTIs (D).
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f4-tm-15-22: FasR/FasL pathway is not involved in FTI-induced apoptosis in AML cells.Flow cytometric analysis of FasR and FasL expression in CD34+ AML cells from a representative case, untreated and treated with FTI (A). FTI-mediated inhibition of AML cell viability does not correlate with FasR and FasL expression on CD34+ AML cells. Cumulative mean ± SEM CD34+ Fas-R+ and C34+ Fas-L+ are reported (B). Caspase–8 is not activated by FTIs. AML cells untreated and treated with FTI after 48 h of culture. (C). AML cells were grown for 48 h with FTIs at IC50 and with FTIs + Fas-receptor triggering inhibitor Fas:Fc or caspase-8 inhibitor IETD-FMK (all used at 50 μM). AML cell viability was measured by colorimetric MTT assay. Columns represent cumulative mean ± SEM of cell viability of 5 experiments performed with all FTIs (D).
Mentions: Fas-R and Fas-L expression on AML cells exposed to FTIs was analysed by flow cytometry, but no variations were observed (21±4% and 23±8% in the absence and presence of FTIs, respectively) (Fig. 4A). Moreover, FTI–sensitive and FTI-resistant AML cells displayed similar expression of Fas-R (mean % ± SEM, 39±4 vs 49±6, respectively; p=0.5) and Fas-L (mean % ± SEM, 49±4 vs 52±3, respectively; p=0.7) (Fig. 4B). No modifications of caspase-8 activity were detected in AML cells after 48 h FTIs exposure by flow cytometry and intracellular caspase staining (Fig. 4C). To further exclude the involvement of Fas-R/Fas-L system in FTI-mediated apoptosis, prior to exposure to FTIs, AML cells were preincubated for 2 h with selective Fas-R/Fas-L inhibitors. As expected, no variations were observed in FTI-mediated inhibition of cell growth and apoptosis after pretreatment with Fas-receptor antagonist and caspase-8 inhibitor (mean % ± SEM of cell growth after FTI exposure, 41.6±3 and 43.3±3 without and with Fas:Fc vs 38±2 and 39±1 without and with IETD-FMK vs 42.3±3 and 46.6±3 without and with ZYVAD-FMK, respectively) (Fig. 4D).

View Article: PubMed Central - PubMed

ABSTRACT

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.

No MeSH data available.


Related in: MedlinePlus