HERG1 K+ Channels Regulate Leukemia Angiogenesis through VEGF Signaling In Leukemic Cells.
Authors: Huang Shiang , Li Huiyu , Guo Dongmei , Jie Shenghua , Liang Kaiwei , Zheng Fang 
Published: October 2019

Abstract
Abstract 1036

Background:
Angiogenesis has been shown as an important process in hematological malignancies. It consists in endothelial proliferation, migration, and tube formation following pro-angiogenic factors realizing, specially the vascular endothelial growth factor (VEGF). The human ether-à-go-go-related gene encodes the major subunit of the K+ channels which has three isotypes (hERG1,hERG2,hERG3) in human. Our previous studies demonstrated that HERG1 K+ channels were aberrantly expressed in leukemia cells and leukemia stem cells (LSCs) which appears to be important in favoring leukemia progression. However, the functions of HERG1 K+ channels on the leukemia-induced angiogenesis has not been investigated. The purpose of this study is to investigate the role of HERG1K+ channels on leukemia angiogenesis and the possible mechanism.

Methods:
First, prepare conditional media: K562, HL-60 cells were cultured in RPMI 1640 medium containing 10% FBS and various concentration (0, 10, 20, 40μM) of E-4031, a HERG1 K+ channel special inhibitor. Second, HUVECs were seeded in conditional media and HUVECs proliferation was measured by the CCK-8 cell proliferation assay. HUVECs cell migration assay was performed by Boyden chamber. The angiogenesis of HUVECs cultured in conditional media were measured by endothelial tube formation in Matrigel in vitro. The role of HERG1K+ channels on VEGF secreted by leukemic cells was determined by ELISA; The role of HERG1K+ channels on VEGF mRNA expression of leukemic cells was determined by RT-PCR.

Results:
The proliferation assay showed that the OD value of HUVEC poliferation was 0.91±0.13 in the control group when HUVECs was cultured with E-4031-free conditioned media from K562 cells. While in the experimental group, the OD value were 0.71±0.18, 0.67±0.12, 0.60±0.08, respectively for different concentrations of E-4031 (10, 20 and 40μM). We obtained similar results using human HL-60 cells. The HUVEC proliferation was significantly inhibited when the conditioned media were treated with different concentrations of E-4031 (P < 0.05). The migration results also showed that when cultured in conditioned media generated from untreated K562 cells, the number of HUVECs that migrated through the filters were 22 cells. In the media from 20μM E4031-pretreated K562 cells, the migrated HUVEC numbers were 8.The leukemia induced significant migration of HUVECs, which was significantly higher than that cells treated with E-4031(P<0.001). We observed that HUVECs cultured with medium from leukemia formed a tube-like structure or tube network. Culturing HUVECs with medium from K-562 plus 20μM of E-4031 led to 70% decrease in tube network. These indicated that HERG1 K+ channels could influence the cross-talk between the leukemic cells and endothelial cells. Blockage of HERG K+ channels decreased the ability of leukemic cells to induce HUVEC proliferation, migration and tube formation.
Our ELISA results showed that VEGF levels in conditional media was about 311±12.35pg/ml; Blockage of HERG K+ channels led to decrease secretion of VEGF(126±10.23 pg/ml) by K-562 cells(P < 0.05). RT-PCR showed that K-562 cells treated with E-4031 had a relatively lower mRNA expression of VEGF than that cells untreated with E-4031. We obtained similar results using human HL-60 cells.

Conclusion:
HERG1K+ channels have a promoting effect on leukemia angiogenesis, the mechanism of which may be that HERG1 K+ channels increase the level of VEGF expressions at both protein and mRNA level. And these may be critical steps of HERG1 K+ channels on leukemia angiogenesis. These findings suggested that HERG1 K+ channels may be a potential target of anti-angiogenesis.

Disclosures:
No relevant conflicts of interest to declare.