hERG1 K+ Channels Regulated Shedding of Leukemia Cell-Derived Microvesicles
Authors: Zheng Fang , Li Huiyu , Li Juanjuan , Du Wen , Wang Ningfang , Chen Xiangjun , Lu Cong , Huang Shiang 
Published: October 2019

Abstract
Abstract 4641
Microvesicles (MVs) are submicrometric membrane fragments and shed from not only activated platelets and tumor infiltrating cells but also from malignant cancer cells. They can “hijack” membrane components and engulf cytoplasmic contents from their cellular origin. Leukemia cell-derived MVs have been shown to positively correlate with leukemia proliferation, chemotaxis, adhesion, survival, angiogenesis, and implication for disease progression. However, only scarce information is available in the molecular basis by which MVs facilitate leukemogenesis. Our previous work and other recent studies suggest that human ether à-go-go-related gene1 (hERG1) K+ channels are constitutively expressed in leukemia cells, and regulate cell proliferation, invasiveness, neoangiogenesis, as well as aggravation clinical prognosis. Given the attributes of MVs and hERG1 K+ channels in leukemia progression, we therefore aimed to investigate a possible regulatory effect of hERG1 K+ channels upon MVs as a mean to uncover molecular events involved in promotion of leukemogenesis by MVs.
HL-60 and K562 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Cells in mid-logarithmic growth were harvested and then recultivated in serum-free RPMI 1640 for 24h. MVs were isolated from culture supernatants using a two-step differential centrifugation process at 4°C. An initial centrifugation at 2,000 rpm for 30 min was used to pellet out the treated cells. The cell-free supernatants were then centrifuged at 20,000 rpm for 2 h to isolate the MVs pellet. The quantity of shedded MVs was measured by BCA protein assay, and characterization of MVs was assessed by flow cytometry. Chemotaxis and adhesion assay were performed to evaluate the effects of hERG1 K+ channels upon MVs in leukemia.
The protein content of MVs isolated from supernatants of K562 cells was significantly higher than that from supernatants of HL-60 cells (p<0.05). The MV fractions contained 871.5μ g ± 256.7 protein/106 cells in supernatants derived from K562 cells and 625.0μ g ± 270.9 protein/106 cells in supernatants of HL-60 cells. We next determined the molecular profile of MVs isolated from different cell supernatants. The results showed that in addition to the myeloid lineage antigen (CD11b), MVs contained hERG1 K+ channels, which are highly expressed in leukemia cells and appear of exceptional importance in favoring leukemogenesis. Interestingly, inhibition of hERG1 K+ channels led to a rapid reduction of MV fractions in supernatants, suggesting that the activity of hERG1 K+ channels are related with the shedding of MVs. Considering MVs are important constituents of the leukemia microenvironment, we confirmed that MVs created a positive feedback loops to facilitate leukemogenesis. Exposure to MVs with different concentrations, the amount of hERG1 protein expressed on leukemia cell surface was in turn moderately enhanced. With MVs simulation, we also observed that the migration of leukemia cells was significantly increased, and noticed that MVs significantly enhanced their adhesion to HUVECs. Of interest, by blocking hERG1 K+ channels with E-4031 impaired the ability of MVs to enhance chemotaxis of K562 and HL-60 cells, decreased by 28.7%±3.3 and 23.5%±3.0, separately. In addition, following E-4031 and MVs treatment in leukemia cells, adhesion of K562 cells to HUVECs was strikingly inhibited by 33.1%±3.1 compared with MVs simulation. Similar results were observed with HL-60 cells.
The present study demonstrates that leukemia cell-derived MVs can “hijack” their parental cell membrane hERG1 K+ channels. Importantly, we have uncovered that hERG1 K+ channels regulated the shedding of MVs and modulated the biological effects of MVs upon leukemia cells. We believe that this new correlation among MVs, hERG1 K+ channels and leukemia can be exploited to both better understand leukemia progression and also suggest novel therapies for this disease, such as control MVs’ biological effects by anti-MVs drugs.

Disclosures:
No relevant conflicts of interest to declare.