Figure 1.
20(OH)D3 and 1,20(OH)2D3 have less calcemic effect in comparison to 1,25(OH)2D3.
In vivo testing of calcemic effects of 20(OH)D3 and 1,20(OH)2D3 in comparison to 1,25(OH)2D3.
Figure 2.
Novel secosteroids and steroidal 5.7-diene compounds have anti-proliferative effects on leukemia cells.
The anti-proliferative effects of novel secosteroidal and steroidal 5,7-diene compounds on Mel mouse erythroleukemia (A), human K562 myeloid (B), HL-60 human promyelocytic (C), U937 promonocytic (D) leukemias. After treatment for 7 days with different compounds (10−7 M), cells were stained with trypan blue and the number of viable cells counted. Statistically significant effects are marked as follow *p<0.05; **p<0.01; ***p<0.001.
Figure 3.
Novel secosteroids and steroidal 5.7-diene induce erythroid differentiation in leukemia cells.
Induction of erythroid differentiation determined was from the the concentration of hemoglobin in an equal number of cells. Cells were treated for 7 days with different compounds (10−7 M), stained with benzidine solution and the amount of hemoglobin as hemin in Mel (A) or K562 (B) leukemia cells was determined by measuring the absorbance at 600 nm after lysis of the cells. ***p<0.001. C.The number of benzidine positive K562 cells after 7 days of treatment with different compounds (10−7 M). Cells were stained with benzidine solution and the number of benzidine positive (blue) cells was determined by counting 200 cells per microscopic field. The results are present as % of benzidine positive cells in comparison to total cell number. *p<0.05; **p<0.01; ***p<0.001.
Figure 4.
Novel secosteroids and steroidal 5.7-diene induce monocytic differentiation in HL-60 (A, C) and U937 cells (B, D).
A and B. Cells were treated for 7 days with different compounds (10−7 M), stained with NBT and the number of NBT positive (blue) cells was determined counting 200 cells per field, and expressed as a % of the total cells. Induction of differentiation measured by the absorbance at 715 nm after lysis of the equal number of NBT stained cells is in C and D. *p<0.05; **p<0.01; ***p<0.001.
Figure 5.
Novel secosteroidal and steroidal 5.7-diene induce the expression of differentiation genes in HL-60 cells.
Real time PCR analysis of the expression of CD11b and transferrin genes in HL-60 cells. Cells were treated for 3 days with graded concentrations of 1,25(OH)2D3 (A), 20(OH)D3 (B), 20,23(OH)2D3 (C) and 1,20(OH)2D3 (D), mRNA was isolated and the gene expression analyzed. *p<0.05; **p<0.01; ***p<0.001 determined with one way ANOVA.
Figure 6.
Novel secosteroids and steroidal 5.7-diene induce the expression of markers of differentiation in HL-60 cells.
Flow cytometry analysis of CD11b protein expression in HL-60 cells. Cells were treated for 5 days with 10−6 M 1,25(OH)2D3 (A), 20(OH)D3 (B), 20,23(OH)2D3 (C), 1,20(OH)2D3 (D), pD (E) and 20(OH)pD (F), mRNA was isolated and the % of cells expressing CD11b protein determined. *p<0.05; **p<0.01; ***p<0.001.
Figure 7.
Novel secosteroids and steroidal 5.7-diene arrest HL-60 cells in G1/G0 phase of the cell cycle.
Cell cycle analysis of the HL-60 human promyelocytic human leukemia shows arrest in G1/G0 phase (A). Statistically significant inhibition of S (B) and G2/M (C) phases is also evident for 1,25(OH)2D3, 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 but absent for pD and 20(OH)pD. Representative flow cytometry analysis for 20,23(OH)2D3 is in D. *p<0.05; **p<0.01; ***p<0.001.