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

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.


FTI-induced inhibition of CML cell viability is mediated by NO pathway.FTIs induce iNOS mRNA signal amplification in KG1a and AML cells cultured for 48h. Ethidium bromide RT-PCR products (iNOS and GAPDH) are shown (A). FTIs cause increased expression of iNOS protein in KG1a cell line and 4 AML patients. Actin is used as control (B). γ-MM-arg partially preventes FTI-mediated apoptosis. Flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from a CML patient treated with FTI (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120747&req=5

f5-tm-15-22: FTI-induced inhibition of CML cell viability is mediated by NO pathway.FTIs induce iNOS mRNA signal amplification in KG1a and AML cells cultured for 48h. Ethidium bromide RT-PCR products (iNOS and GAPDH) are shown (A). FTIs cause increased expression of iNOS protein in KG1a cell line and 4 AML patients. Actin is used as control (B). γ-MM-arg partially preventes FTI-mediated apoptosis. Flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from a CML patient treated with FTI (C).

Mentions: Unstimulated primary total AML BM cells expressed iNOS mRNA by PCR detection. Because iNOS expression in total BM AML cells might be related to non-leukemic cells, immature CD34+ KG1a cells were tested for the expression of iNOS mRNA after 48 h culture. A stronger amplification of iNOS was detected, and also after FTIs stimulation (Fig. 5A). Similarly, immunoblot of cell lysates showed lower levels of iNOS at baseline, and a 10-fold increased after FTIs stimulation in both primary total BM AML cells and KG1a cells (Fig. 5B). Using the cell-permeable fluorescent indicator DAF-2 DA, NO levels increased 40% after FTI exposure from basal levels detected in AML BM cells cultured in control medium without FTI (p<0.001), and γ-MM-arg partially blocked FTI-mediated NO production in CML cells (Fig 5C). Inhibition of NO synthesis by pretreatment of AML cells with γ-MM-arg (500 μM), a competitive inhibitor of iNOS, did not affect the inhibitory effect on cell growth and apoptosis of FTIs (data not shown). However, when FTIs were used at IC10, γ-MM-arg partially prevented FTI effects (mean % ± SEM cell growth and apoptosis after FTI exposure, 31±2 and 21±4 vs 46±1 and 31±3 in absence and in presence of γ-MM-arg, respectively; p=0.01 and p=0.03) (Fig. 5C).


In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells
FTI-induced inhibition of CML cell viability is mediated by NO pathway.FTIs induce iNOS mRNA signal amplification in KG1a and AML cells cultured for 48h. Ethidium bromide RT-PCR products (iNOS and GAPDH) are shown (A). FTIs cause increased expression of iNOS protein in KG1a cell line and 4 AML patients. Actin is used as control (B). γ-MM-arg partially preventes FTI-mediated apoptosis. Flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from a CML patient treated with FTI (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-tm-15-22: FTI-induced inhibition of CML cell viability is mediated by NO pathway.FTIs induce iNOS mRNA signal amplification in KG1a and AML cells cultured for 48h. Ethidium bromide RT-PCR products (iNOS and GAPDH) are shown (A). FTIs cause increased expression of iNOS protein in KG1a cell line and 4 AML patients. Actin is used as control (B). γ-MM-arg partially preventes FTI-mediated apoptosis. Flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from a CML patient treated with FTI (C).
Mentions: Unstimulated primary total AML BM cells expressed iNOS mRNA by PCR detection. Because iNOS expression in total BM AML cells might be related to non-leukemic cells, immature CD34+ KG1a cells were tested for the expression of iNOS mRNA after 48 h culture. A stronger amplification of iNOS was detected, and also after FTIs stimulation (Fig. 5A). Similarly, immunoblot of cell lysates showed lower levels of iNOS at baseline, and a 10-fold increased after FTIs stimulation in both primary total BM AML cells and KG1a cells (Fig. 5B). Using the cell-permeable fluorescent indicator DAF-2 DA, NO levels increased 40% after FTI exposure from basal levels detected in AML BM cells cultured in control medium without FTI (p<0.001), and γ-MM-arg partially blocked FTI-mediated NO production in CML cells (Fig 5C). Inhibition of NO synthesis by pretreatment of AML cells with γ-MM-arg (500 μM), a competitive inhibitor of iNOS, did not affect the inhibitory effect on cell growth and apoptosis of FTIs (data not shown). However, when FTIs were used at IC10, γ-MM-arg partially prevented FTI effects (mean % ± SEM cell growth and apoptosis after FTI exposure, 31±2 and 21±4 vs 46±1 and 31±3 in absence and in presence of γ-MM-arg, respectively; p=0.01 and p=0.03) (Fig. 5C).

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.