Shikonin Derivatives from Onsoma visianii Decrease Expression of Phosphorylated STAT3 in Leukemia Cells and Exert Antitumor Activity

Abstract

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from Onosma visianii Clem (Boraginaceae), in BCL1, mouse CLL cells and JVM-13, human B-PLL cells was explored in this study. The cytotoxicity of shikonin derivatives was measured by an MTT test. Cell death, proliferation, cell cycle, and expression of molecules that control these processes were analyzed by flow cytometry. Expression of STAT3-regulated genes was analyzed by real-time q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The antitumor effects of shikonin derivatives in vivo were analyzed, using flow cytometry, by detection of leukemia cells in the peripheral blood and spleens of mice intravenously injected with BCL1 cells. The two most potent derivatives, isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS), induced cell cycle disturbances and apoptosis, inhibited proliferation, and decreased expression of phospho-STAT3 and downstream-regulated molecules in BCL1 and JVM-13 cells. IBS and MBS decreased the percentage of leukemia cells in vivo. The link between the decrease in phosphorylated STAT3 by MBS and IBS and BCL1 cell death was confirmed by detection of enhanced cell death after addition of AG490, an inhibitor of Jak2 kinase. It seems that IBS and MBS, by decreasing STAT3 phosphorylation, trigger apoptosis, inhibit cell proliferation, and attenuate leukemia cell stemness.

Keywords: BCL1; JVM-13; Onosma visianii; STAT3; isobutyrylshikonin; α-methylbutyrylshikonin.

Figure 1

Dose- and time-dependent cytotoxicity of isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS) on the BCL1 cell line. Graphs of (a) BCL1 and (b) JVM-13 cells’ survival after 24 and 48 h growth in the presence of IBS and MBS determined by MTT assay. All data are presented as mean values from three independent experiments performed in triplicate.

Figure 2

IBS and MBS induce apoptotic death of leukemia cells. Apoptosis of untreated and IBS- and MBS-treated BCL1 and JVM-13 cells for 24 h (a) evaluated by flow cytometry using Annexin V (FITC) and PI double staining. (b) Representative dot plots illustrate populations of viable (AnnV− PI-), early apoptotic (Ann V+ PI−), late apoptotic (AnnV+ PI+), and necrotic (AnnV− PI+) BCL1 cells treated with IBS or MBS. Percentage of Mcl-1 and Noxa positive (c) JVM-13 and (e) BCL1 cells evaluated by flow cytometry. mRNA expression of Bax and Bcl-2 in (d) JVM-13 and Bax and (f) caspase-3 expression in BCL1 cells treated by IBS and MBS quantified by qRT-PCR. (g) Immunofluorescence staining for Bax (green) and cleaved caspase-3 (green) together with DNA staining with DAPI (blue) in BCL1 cells incubated with MBS (2 µg/mL) for 24 h, as well as in untreated cells (magnification × 200). The data are presented as means + SDs of a three independent experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences between treated and untreated cells.

Figure 3

IBS and MBS attenuate the expression of Ki67 in BCL1 and JVM-13 cells. (a) Percentage of Ki-67 positive BCL1 and JVM-13 cells exposed to IBS and MBS (concentrations = 1 and 2 µg/mL) for 24 h determined by flow cytometry presented as the mean + SD from three independent experiments. Data were analyzed with Student’s t-test: * p < 0.05; ** p < 0.01, # p < 0.05 (indicates differences between two doses of MBS). (b) Representative histograms of Ki67 expression (mean fluorescence intensity) in BCL1 and JVM-13 cells.

Figure 4

IBS and MBS induce cell cycle disturbances in JVM-13 and BCL1 cells. Distribution of (a) BCL1 and (b) JVM-13 cells in different phases of the cell cycle after treatment with IBS and MBS (concentrations = 1 µg/mL and 2 µg/mL) was determined by flow cytometry after staining with Vybrant^® DyeCycle™ Ruby stain. Results are expressed as the percentage of cells in different phases of the cell cycle. (c) mRNA expression of cyclin D3 quantified by RT-PCR in BCL1 cells after treatment with IBS and MBS (2 µg/mL) for 24 h. (d) Percentage of JVM-13 cells expressing cyclin D3 after treatment with two different concentrations of IBS and MBS (1 µg/mL and 2 µg/mL) for 24 h determined by flow cytometry. (e) Percentage of p16-, p21-, and p27-positive BCL1 cells analyzed by flow cytometry after 24 h treatment with IBS and MBS. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 indicates a significant difference from untreated cells.

Figure 5

Treatment with IBS and MBS attenuates the expression of pSTAT3- and STAT3-regulated genes in BCL1 and JVM-13 cells. (a) Percentages of pSTAT3 positive BCL1 and JVM-13 cells determined by flow cytometry 24 h after treatment with IBS and MBS and representative histograms of pSTAT3 expression in JVM-13 cells. (b) mRNA expression of c-Myc, Nanog, and Oct4 in BCL1 cells and c-Myc, Nanog, Sox2 and Oct4 in JVM-13 cells treated with IBS and MBS quantified by RT-PCR. (c) BCL1 cells’ survival after 24 h growth in the presence of IBS and MBS, with or without the addition of Jak2 inhibitor AG490 (tyrphostin), determined by MTT assay. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Treatment with IBS and MBS inhibits BCL1 leukemia growth in vivo. Mononuclear cells from (a) peripheral blood and (b) spleens of untreated and IBS- and MBS-treated mice 20 days after intravenous BCL1 cell injection (1 × 10⁶). The percentage of CD5+CD19+ BCL1 cells is presented as the mean ± SD. * p < 0.05, ** p < 0.01. (c) AST, ALT, urea, and creatinin levels were determined in the serum 90 days after the BCL1 injection in untreated and IBS- and MBS-treated mice, presented as the mean ± SD (eight animals per group).