Disulfram/Copper Complex May Enhance Its Cytotoxic Effect on CD34+CD38- KG1-Alpha Cells By Down Regulating the Expression of HIF1-Alpha in the Co-Cultured MSCs
Authors: Xutao Guo , Zheng Haiqing , Pengcheng Shi , Yan Wang , Jiabao Liang , Feng Ru , Xu Bing 
Published: October 2019

Abstract
Backgroud We had reported that Disulfram/copper complex (DS/ Cu) had a potent and selective anti-leukemia property in vitro against leukemia stem-like cells (e.g., CD34+/CD38- KG1α and Kasumi-1 cells and primary CD34+ cells isolated from AML patients) as well as was highly effective in vivo in CD34+/CD38- leukemic cell-derived xenograft mouse models. Here, we report the in vitro activity of DS/Cu against CD34+/CD38- leukemia stem-like cells sorted from KG1α AML cell line by affecting the co-cultured AML bone marrow mesenchymal stromal cells. Methods KG1α cells were cultured and stained with hCD34-APC, hCD38- PE, and hCD123-PE-Cy7 to determine the percentage of cells expressing CD34, CD38, and CD123. To isolate CD38- cells, CD38 microbeads were used for the depletion of CD38+ cells. First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8) and monitor expression of HIF-1α expression by western blot. Next, the sorted CD34+CD38- KG1α cells were co-cultured with primary AML bone marrow MSCs isolated from a de novo AML patient in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 24 hr. The co-cultured cells were treated with DS/CU with/without YC1(HIF-1α inhibitor)、IDA(idarubicin) and then separated. CCK8 assay, fow cytometry were used to determine the cell proliferation and apoptosis, respectively. Finally, we used western bolting to explore the underlying mechanism of the cytotoxicity of DS/CU in the AML cells and the sensitizated effect in AML MSCs. Results First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8). As shown in Figure 1, DS/Cu (DS 1umol/L,Cu 0.5umol/l) administrated alone was unable to induce apoptosis in MSCs (P>0.05 vs untreated control; see below Figure 1). However, DS in combination with 0.5 μM Cu for 24 hrs resulted in signifcantly increased apoptosis in CD34+CD38- KG1α cells.( P<0.001 vs untreated control; see Figure 1) .The co-cultured cells were treated with DS/CU、IDA with/without YC1 and then separated. CCK8 assay, fow cytometry were used to determine the cell apoptosis, respectively. Treatment with DS/CU resulted in signifcantly increased apoptosis in the co-cultured CD34+/CD38- KG1α cells (Figure 2; P<0.05, DS/Cu vs either untreated control). Comparable phenomena were observed in co-cultured CD34+/CD38- KG1α cells treated with idarubicin. However, while treatment with YC1 and DS/CU had no signifcant effect on cell apoptosis (P>0.05 vs untreated control). Taken together, these results indicated that DS/CU was active against co-cultured CD34+/CD38- KG1α cells and the effect can be reversed by HIF1-α inhibitor(see Figure 2).We examined the cytotoxic effect of DS/Cu on CD34+/CD38- KG1α cells in different culture systems using the CCK-8. As shown in Figure 4, while treatment with DS/CU had significant inhibited proliferation on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P<0.05 vs un-co-cultured control). However, while treatment with DS/CU and CoCl2 (HIF-1α promoter) had no signifcant effect on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P>0.05 vs un-co-cultured control,see Figure 3).CD34+CD38- KG1α cells and MSCs separated from the co-cultured system were treated with DS/CU with/without YC1 and Cocl2, followed by Western blot analysis to monitor expression of HIF-1α. As shown at the figure 4, the expressions of HIF-1α and SDF-1α between the two cells were clearly down-regulated treated with DS/CU alone. And the expressions of HIF-1α and SDF-1α were up-regulated treated with DS/CU and CoCl2. However, when co-treated with YC1, the down-regulation of HIF-1α was reverse. DS/Cu down redulated the expressions of HIF1-α、SDF1-α、CXCR4 and VLA4 in the co-cultured CD34+CD38- KG1α cells and these effect could be reserved by HIF1-α inhibitor.Conclusion DS/Cu preferentially induces apoptosis of CD34+CD38- KG1α cells, but not MSCs. DS/Cu exhibited cytotoxicity in CD34+/CD38- KG1α cells, and MSCs enhances this effect. The mechanism may be related to the HIF1α/SDF1/CXCR4 and VLA4 pathway. DS/CU may enhance its inhibitory effect on CD34+CD38- KG1α cells by down regulating the expression of HIF1-α in MSCS.

Disclosures
No relevant conflicts of interest to declare.