hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

<div><p>Background</p><p>The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.</p> <p>Principal Findings</p><p>The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process. </p> <p>Conclusions/Significance</p><p>Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.</p> </div

hERG channel expression suppressed by BmKKx2 in erythroid differentiation of K562 cells.

<p>(A) γ-globin expression in K562 cells during the differentiation with or without BmKKx2 shown by quantitative real-time PCR. (B) hERG channel expression in K562 cells during the differentiation process with or without BmKKx2 shown by quantitative real-time PCR. **<i>p</i><0.01 (Student’s <i>t</i>-test). Symbols and associated error bars represent means ± SD for three independent experiments (C) hERG and γ-globin expression in K562 cells with or without BmKKx2 tested by the western blotting analysis. Both HERG1 and HERG1B isoforms were detected [24]. </p

Scorpion toxin BmKKx2 primary structure and its pharmacological effect on the hERG channel.

<p>(A) the sequence alignments between scorpion toxins BmKKx2 and BeKm-1 [11,19]. (B) The pharmacological effect of BmKKx2 on hERG channels. The hERG channels were transfected in HEK 293 cells, and their current traces were shown in the absence (control) or presence 10 nM BmKKx2. (C) Dose-dependence curve of BmKKx2 on hERG channels expressed in HEK 293 cells. Symbols and associated error bars represent means ± SD for several cells (n=5). (D) The current trace of hERG potassium channels in absence of BmKKx2 in wild type K562 cells; (E) The pharmacological effect of BmKKx2 on hERG channels in wild type K562 cells. Current traces were shown in the absence (control) or presence 1 μM BmKKx2 .</p

Induction of apoptosis by BmKKx2 during the erythroid differentiation of K562 cells.

<p>(A-B) K562 cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. K562 cells were treated with BmKKx2, and BSA was used as the control for 48 h (A). K562 cells were treated with Ara-C (1 μM) and Ara-C (1 μM)+ BmKKx2 for 48 h (B). Flow cytometry data show representative results from one of three independent experiments. (C) Apoptotic cells were stained with annexin V-FITC and PI analyzed by flow cytometry. Values were mean ± SD from three experiments. *<i>p</i><0.05 (Student’s <i>t</i>-test).</p

No effect of BmKKx2 on the erythroid differentiation in the hERG channel-deficient K562 cells.

<p>(A) hERG channel expression in hERG-silenced and control cells shown by quantitative real-time PCR. (B) hERG channel expression shown by western blotting analysis. HSC70 was used as the endogenous control. (C) BmKKx2 enhancing the differentiation in the lentiviral vector-infected control cells. (D) BmKKx2 with no effect on the differentiation of the hERG-silenced cells. K562 cells in (C) and (D) were treated with Ara-C for 48 h. The right panel of (C) and (D) showed the mean values of GPA fluorescence from three independent samples. **<i>p</i><0.01 (Student’s <i>t</i>-test).</p

BmKKx2 binding causing the Ca²⁺ concentration decrease during the erythroid differentiation of K562 cells.

<p>(A) Intracellular Ca²⁺ was stained by Fluo-8, and the Ca²⁺ concentration was measured by flow cytometric analysis. The mean values were mean ± SD from three independent experiments. **<i>p</i><0.01 (Student’s <i>t</i>-test). (B) Flow cytometric analysis of Fluo-8 fluorescence intensity in K562 cells with or without BmKKx2 after Ara-C induced for 24 h.</p

Characterization of MicroRNA Expression Profiles and the Discovery of Novel MicroRNAs Involved in Cancer during Human Embryonic Development

<div><p>MicroRNAs (miRNAs), approximately 22-nucleotide non-coding RNA molecules, regulate a variety of pivotal physiological or pathological processes, including embryonic development and tumorigenesis. To obtain comprehensive expression profiles of miRNAs in human embryos, we characterized miRNA expression in weeks 4-6 of human embryonic development using miRNA microarrays and identified 50 human-embryo-specific miRNAs (HES-miRNAs). Furthermore, we selected three non-conserved or primate-specific miRNAs, hsa-miR-638, -720, and -1280, and examined their expression levels in various normal and tumor tissues. The results show that expression of most miRNAs is extremely low during early human embryonic development. In addition, the expression of some non-conserved or primate-specific miRNAs is significantly different between tumor and the corresponding normal tissue samples, suggesting that the miRNAs are closely related to the pathological processes of various tumors. This study presents the first comprehensive overview of miRNA expression during human embryonic development and offers immediate evidence of the relationship between human early embryonic development and tumorigenesis.</p> </div

Statistical analyses of three HES-miRNAs (hsa-miR-638, -720, and -1280) expression using <i>in situ</i> hybridization

<p>High-density multiple organ tumor and normal tissue microarrays containing 500 tissue-dots with 18 tumor types and normal corresponding control tissues. Digoxigenin-labeled locked nucleic acid (LNA) probes (LNA-638, LNA-720, and LNA-1280) were used to specifically detect the miRNA expression on the tissue chip. (A) Aberrant expression of hsa-miR-638 on tissue microarray. Significant up-regulation of miR-638 can be observed in hepatocellular liver cancer and cervix uteri squamous cell carcinoma. miR-638 down-regulation is found in stomach adenocarcinoma versus the corresponding normal tissues (p=0.0092, p=0.0003, and p=0.0095, respectively) (B) Aberrant expression of hsa-miR-720 on tissue microarray. Hsa-miR-720 is significantly upregulated in cervix uteri squamous cell carcinoma, lung squamous cell adenocarcinoma, ovary adenocarcinoma, and urothelial carcinoma versus the corresponding normal tissues (p=0.0086, p=0.0386, p=0.0404, and p=0.035, respectively). Hsa-miR-720 is significantly downregulated in intestinal mucosa malignant tissues versus normal skin tissues (p=0.0017). (C) Aberrant expression of hsa-miR-1280 on tissue microarray. Hsa-miR-1280 is downregulated in squamous cell carcinoma, intestinal mucosa malignant tissue, and pancreatic adenocarcinoma versus the corresponding normal tissues (p<0.0001, p=0.0009, and p=0.0270, respectively). Hsa-miR-1280 is upregulated significantly in cervix uteri squamous cell carcinoma tissues versus normal cervix uteri tissues (p=0.01076). MiRNA expression levels (y-axis) with a score=0, 1, 2, or 3, indicate negative, weak, medium, or strong staining intensity, respectively. n indicates the numbers of the specimens studied. NT indicates normal tissue samples; CT indicates carcinoma tissue samples. Two-tailed student <i>t</i> tests were performed to compare miRNA expression in normal and cancerous tissues.</p

hsa-miR-638 expression in stomach adenocarcinoma and corresponding normal tissues.

<p>Panels a through t show stomach adenocarcinoma samples and panels u through z show normal stomach tissue. The blue staining signal indicates expression of hsa-miR-638. The red staining shows the cell nucleus (scale 200μm as Figure 5a). j2 shows higher magnification (5x) of the indicated area in figure 5j, and z2 shows higher magnification (5x) of the indicated area in figure 5z.</p

Hierarchical clustering analyses of the expression of 50 HES-miRNAs

<p>(A) Fifty HES-miRNAs were divided into 3 clusters: clusters a, b, and c. The arrow shows the miRNAs that were selected to further validate the microarray data using microRNA qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069230#pone-0069230-g003" target="_blank">Figure 3</a>). The asterisk shows the non-conserved or primate-specific HES-miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069230#pone.0069230.s004" target="_blank">Figure S4</a>). The hollow triangle indicates miRNAs harborring the same seed region sequence among cluster c. (B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069230#pone-0069230-g002" target="_blank">Figure 2B</a> shows the mature sequence of six miRNAs (miR-17, -106a, -106b, -20a, -20b, -93) with the identical seed region tagged with light red shadow. (C) Pie chart shows functional pattern analysis (Gene Ontology) of the conserved targets (1245 transcripts), predicted by TargetScan (<a href="http://www.targetscan.org/vert_60/" target="_blank"><u>http://www.targetscan.org/vert_60/</u></a>), of the six miRNAs.</p