Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression, and induces apoptosis in HL-60 leukemic cells.
All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D(3) analogue, 20-epi-22-oxa-24a,26a,27a-tri-homo-1alpha,25(OH) 2D, (KH 1060), which belongs to the family of potent 20-epi-1,25(OH),D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9-cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.
Elstner
E
E
Division of Hematology-Oncology, UCLA School of Medicine, California 90048, USA.
Linker-Israeli
M
M
Umiel
T
T
Le
J
J
Grillier
I
I
Said
J
J
Shintaku
I P
IP
Krajewski
S
S
Reed
J C
JC
Binderup
L
L
Koeffler
H P
HP
D000954:Antigens, Surface / Q000032:analysis; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002117:Calcitriol / Q000008:administration & dosage / Q000031:analogs & derivatives; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002999:Clone Cells; D004357:Drug Synergism; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D010587:Phagocytosis / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2; D014212:Tretinoin / Q000008:administration & dosage; D051028:bcl-2-Associated X Protein
D000954:Antigens, Surface; C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C074279:KH 1060; D014212:Tretinoin; D002117:Calcitriol