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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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FTI-induced apoptosis in AML cells is mediated by activation of caspase-3.FTI induced activation of caspase-3. Caspase-3 activity in BM AML cells from a representative patient, cultured for 24 h in medium alone (untreated) or treated with FTI or with FTI + caspase-3 inhibitor Z-DEVD-FMK (50 μM), were measured by flow cytometry using the fluorogenic caspase-3 specific substrate DEVD-AMC (A). Caspase-3 is activated after FTI exposure in AML cells. Caspase-3 production in BM AML cells from a representative case, cultured for 6 and 24h in control medium (−FTI) or treated with FTI (+ FTI), was measured by flow cytometry using the cell-permeable FITC-labeled peptide FAM-VAD-FMK in combination with PI to differentiate dead from live cells (B). After 6 and 24 h of FTI exposure, 33% and 55% of live AML cells (B, bottom right panels) are positive for FAM-VAD-FMK, respectively. (Panel C) FTI-mediated apoptosis of AML cells is partially reverted by caspase-3 inhibition. Flow cytometric detection of apoptosis from a representative AML patient who showed in vitro susceptibility to FTI (SCH 1 μM) (C). Abbreviations, see text.
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f3-tm-15-22: FTI-induced apoptosis in AML cells is mediated by activation of caspase-3.FTI induced activation of caspase-3. Caspase-3 activity in BM AML cells from a representative patient, cultured for 24 h in medium alone (untreated) or treated with FTI or with FTI + caspase-3 inhibitor Z-DEVD-FMK (50 μM), were measured by flow cytometry using the fluorogenic caspase-3 specific substrate DEVD-AMC (A). Caspase-3 is activated after FTI exposure in AML cells. Caspase-3 production in BM AML cells from a representative case, cultured for 6 and 24h in control medium (−FTI) or treated with FTI (+ FTI), was measured by flow cytometry using the cell-permeable FITC-labeled peptide FAM-VAD-FMK in combination with PI to differentiate dead from live cells (B). After 6 and 24 h of FTI exposure, 33% and 55% of live AML cells (B, bottom right panels) are positive for FAM-VAD-FMK, respectively. (Panel C) FTI-mediated apoptosis of AML cells is partially reverted by caspase-3 inhibition. Flow cytometric detection of apoptosis from a representative AML patient who showed in vitro susceptibility to FTI (SCH 1 μM) (C). Abbreviations, see text.

Mentions: FTIs could induce caspase-3 activation by flow cytometric measurement of MFI after cleavage of the specific fluorogenic substrate DEVD-AFC (mean percentage of positive cells after 6 and 24h culture, 25±4% and 38±4% vs 9±1% and 18±5%, FTI-treated and control AML cells, respectively; mean of 5 experiments). In addition, pre-incubation with caspase-3 inhibitor Z-DEVD-FMK allowed to partially abrogate FTI-mediated caspase activation (mean % ± SEM after 24 h exposure to FTI, 22±6; p=0.03) (Fig. 3A–B), and also to partially prevent FTI-mediated apoptosis (mean % of apoptosis ± SEM after FTI treatment, 36±4 vs 16.3±5, in absence and in presence of Z-DEVD-FMK respectively; p=0.01) (Fig. 3C).


In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells
FTI-induced apoptosis in AML cells is mediated by activation of caspase-3.FTI induced activation of caspase-3. Caspase-3 activity in BM AML cells from a representative patient, cultured for 24 h in medium alone (untreated) or treated with FTI or with FTI + caspase-3 inhibitor Z-DEVD-FMK (50 μM), were measured by flow cytometry using the fluorogenic caspase-3 specific substrate DEVD-AMC (A). Caspase-3 is activated after FTI exposure in AML cells. Caspase-3 production in BM AML cells from a representative case, cultured for 6 and 24h in control medium (−FTI) or treated with FTI (+ FTI), was measured by flow cytometry using the cell-permeable FITC-labeled peptide FAM-VAD-FMK in combination with PI to differentiate dead from live cells (B). After 6 and 24 h of FTI exposure, 33% and 55% of live AML cells (B, bottom right panels) are positive for FAM-VAD-FMK, respectively. (Panel C) FTI-mediated apoptosis of AML cells is partially reverted by caspase-3 inhibition. Flow cytometric detection of apoptosis from a representative AML patient who showed in vitro susceptibility to FTI (SCH 1 μM) (C). Abbreviations, see text.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120747&req=5

f3-tm-15-22: FTI-induced apoptosis in AML cells is mediated by activation of caspase-3.FTI induced activation of caspase-3. Caspase-3 activity in BM AML cells from a representative patient, cultured for 24 h in medium alone (untreated) or treated with FTI or with FTI + caspase-3 inhibitor Z-DEVD-FMK (50 μM), were measured by flow cytometry using the fluorogenic caspase-3 specific substrate DEVD-AMC (A). Caspase-3 is activated after FTI exposure in AML cells. Caspase-3 production in BM AML cells from a representative case, cultured for 6 and 24h in control medium (−FTI) or treated with FTI (+ FTI), was measured by flow cytometry using the cell-permeable FITC-labeled peptide FAM-VAD-FMK in combination with PI to differentiate dead from live cells (B). After 6 and 24 h of FTI exposure, 33% and 55% of live AML cells (B, bottom right panels) are positive for FAM-VAD-FMK, respectively. (Panel C) FTI-mediated apoptosis of AML cells is partially reverted by caspase-3 inhibition. Flow cytometric detection of apoptosis from a representative AML patient who showed in vitro susceptibility to FTI (SCH 1 μM) (C). Abbreviations, see text.
Mentions: FTIs could induce caspase-3 activation by flow cytometric measurement of MFI after cleavage of the specific fluorogenic substrate DEVD-AFC (mean percentage of positive cells after 6 and 24h culture, 25±4% and 38±4% vs 9±1% and 18±5%, FTI-treated and control AML cells, respectively; mean of 5 experiments). In addition, pre-incubation with caspase-3 inhibitor Z-DEVD-FMK allowed to partially abrogate FTI-mediated caspase activation (mean % ± SEM after 24 h exposure to FTI, 22±6; p=0.03) (Fig. 3A–B), and also to partially prevent FTI-mediated apoptosis (mean % of apoptosis ± SEM after FTI treatment, 36±4 vs 16.3±5, in absence and in presence of Z-DEVD-FMK respectively; p=0.01) (Fig. 3C).

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