Modulation of Ara-C Cytotoxicity by Coadministration with Antisignalling Drugs in HL60 and Ara-C-Resistant HL60/Ara-C Cells
Ara-C (cytosine arabinoside) is one of the most effective drugs in the treatment of acute myeloid leukemia (AML). Ara-C is phosphorylated to Ara-CTP (cytosine arabinoside triphosphate) which inhibits DNA polymerase and induces DNA strand breaks after incorporation into DNA. There is increasing evidence that ara-C also affects some cellular signal transduction pathways; it induces transcription/expression of c-fos, c-jun, NF kappaB, protein kinase C δ, mitogen-activated protein kinase and cyclin E and downregulates cyclin-dependent-kinase-2 and retinoblastoma protein phosphorylation. These events may contribute to ara-C cytotoxicity. The antisignalling drugs all-trans retinoic acid (ATRA), staurosporine, quercetin and bryostatin-1 have recently been shown to enhance ara-C cytotoxicity.
We chose 7 different antisignalling agents [ATRA and the kinase inhibitors quercetin, genistein, CGP 52411, tyrphostin A48, nordihydroguaiaretic acid (NDGA) and staurosporine] and compared their ara-C sensitising potencies with those of hydroxyurea (HU), arabinosyl-2-fluoroadenine (F-Ara-A) and 2-chlorodeoxyadenosine (2-CdA), agents currently in use as “second line treatment” in resistant and relapsed AML. Cytotoxicity was assessed by the tetrazolium (MTT) assay in HL60 cells and a newly derived ara-C-resistant subline (HL60/ara-C) in at least three separate experiments. Supraadditive cytotoxicity was found for combinations containing ATRA, CGP 52411, tyrphostin A48 and HU in both cell lines, for 2-CdA, staurosporine and NDGA in HL60, and for F-Ara-A in HL60/ara-C cells. Quercetin and genistein did not sensitise cells against ara-C.
To elucidate the mechanism of sensitisation in HL60 cells we studied the influence of the modulators of ara-C cytotoxicity on cellular markers of apoptosis. After 4h-coincubation we measured cell size (volume), DNA loss by flow cytometry (sub-G1 peak) and DNA fragmentation by conventional gel electrophoresis. Tyrphostin A48, NDGA, ATRA and HU increased ara-C-induced apoptosis, whereas staurosporine did not affect it. CGP 52411 decreased the effect of ara-C on apoptotic indicators after 4h-, but no longer after 12h-coincubation.
The results suggest that antisignalling drugs such as ATRA, CGP 52411, tyrphostin A48, staurosporine and NDGA may be efficacious alternatives to the already clinically applied ara-C modulators. Among the clinically used ara-C modulators, HU sensitised more potently against ara-C than F-Ara-A and 2-CdA.
KeywordsGemcitabine Thymidine Etoposide Cytosine Catechol
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