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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 105-115

Inflection point in glioma growth and angiogenesis driven by potassium channels


1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Molecular Diagnostics Labs, Scintilla Bio-MARC, Bengaluru, Karnataka, India
2 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Bengaluru, Karnataka, India

Date of Web Publication27-Jun-2019

Correspondence Address:
Dr. Divya Khaitan
Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Bengaluru, Karnataka
India
Dr. Nagendra Ningaraj
Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Molecular Diagnostics Labs, Scintilla Bio-MARC, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/glioma.glioma_12_19

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  Abstract 

Background and Aim: The overexpression and alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) that encodes large-conductance calcium-activated voltage-sensitive potassium (BKCa) channels are implicated in the development of human cancers. Dysfunctional angiogenesis in hypoxic tumors is a challenge to intravenous anticancer drug treatments. Hypoxic factors also lead to abnormal vascular functions posing hurdle for anticancer drug delivery to tumors. The aim of this study was to explore the role of BKCachannels in tumor angiogenesis, specifically with regard to release of vascular endothelial growth factor (VEGF). Materials and Methods: We subjected the glioma cells under hypoxia and normoxia and studied the expression and activity of BKCachannels in in vitro and in vivo tumor models. Then, we studied the proangiogenic factor, VEGF, in tumors and monitored the neoangiogenic process. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University, Atlanta, GA, USA (approved No. A0706007_01) on July 20, 2007. Results: We presented in vivo and cell based in vitro experimental evidence on the direct and indirect interactions of BKCachannels with VEGF signaling. There was evidence that under hypoxia, glioma cells overexpressed KCNMA1 and increased VEGF secretion. By inhibiting KCNMA1, we showed that VEGF secretion was significantly reduced, thus potentially controlling angiogenesis, which has implications for vascular permeability and anticancer drug delivery. Moreover, there were differences in alternate splicing of KCNMA1 between normal and malignant cells under hypoxia and normoxia. Conclusion: We conclude that BKCachannels regulate hypoxia-induced angiogenesis. Therefore, serious effort is needed to better understand the molecular mechanisms of BKCachannelopathies triggering angiogenesis and progression of glioma. The modulators of BKCachannels could be viable in new anticancer therapeutics.

Keywords: Angiogenesis, anthracyclines, glioma, HIF-1, hypoxia, Maxi-K/calcium-activated voltage-sensitive potassium channels, vascular endothelial growth factor, vascular endothelial growth factor receptor


How to cite this article:
Ningaraj N, Khaitan D. Inflection point in glioma growth and angiogenesis driven by potassium channels. Glioma 2019;2:105-15

How to cite this URL:
Ningaraj N, Khaitan D. Inflection point in glioma growth and angiogenesis driven by potassium channels. Glioma [serial online] 2019 [cited 2022 Nov 28];2:105-15. Available from: http://www.jglioma.com/text.asp?2019/2/2/105/261671


  Introduction Top


Cancer is a chronic disease characterized by uncontrolled cell growth.[1] Cancer cells typically go through four stages – initiation, proliferation, invasion, and metastasis. There are over 100 different types of cancer, and each is classified by the type of cells that are initially affected.[2] Cancer cells divide uncontrollably to form lumps or masses of tissue called tumors that can grow and interfere with several bodily functions. Cell signaling involving vascular endothelial growth factor (VEGF) and its VEGF receptor (VEGFR) plays a major role in cancer progression by promoting new blood vessels formation called neoangiogenesis.[3] Disruption of the genes encoding either VEGF or any of the three receptors of the VEGF family results in embryonic lethality because of failure of blood vessel development.[4] VEGFR2 is the main signal transducing VEGFR for angiogenesis and mitogenesis of endothelial cells, which is directly related to cell cancer. VEGFs are combined with VEGFRs to activate the VEGF signaling cascade leading to angiogenesis. As shown in [Table 1], specific isoforms of VEGFs are combined with specific VEGFRs to regulate several critical cell functions and also impact on human health and diseases.[5],[6],[7],[8]
Table 1: Major types of vascular endothelial growth factor receptors with their biological functions

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VEGF can be detected in both plasma and serum samples of patients, with much higher level in serum. Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors.[9] Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.[10],[11] The autocrine VEGF signaling is crucial for tumor initiation and transformation into highly aggressive cancers.[3] The blocking of autocrine VEGF secretion provides a promising strategy to develop new therapeutic approaches.[12] VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis, such as cancer, psoriasis, and rheumatoid arthritis. Direct role of VEGF in tumor growth has been shown using dominant negative VEGFRs to block in vivo proliferation, as well as blocking antibodies to VEGF or to VEGFR2.[13] Interference with VEGF function by targeting the VEGF signaling pathway is a major interest in drug development for blocking angiogenesis in primary and metastatic brain tumors [Figure 1].
Figure 1: Adapted from vascular endothelial growth factor signaling pathway showing a key role for calcium-activated potassium channel subunit alpha-1/calcium-activated voltage-sensitive potassium channels expression in tumor cells increases vascular endothelial growth factor secretion thus mediating enhanced endothelial cell proliferation and migration. Vascular endothelial growth factor also caused an increase in calcium-activated voltage-sensitive potassium channel expression in human brain microvasculature endothelial cell line cells [Figure 5]B and glioma cells strengthening the argument that vascular endothelial growth factor-calcium-activated voltage-sensitive potassium channel play a tandem role in neoangiogenesis in brain tumors. This alleged vascular endothelial growth factor-calcium-activated voltage-sensitive potassium channel interplay appears to be exacerbated glioma in a hypoxic microenvironment. V: Vascular, N: Normoxia, A: Vascular endothelial growth factor binding to vascular endothelial growth factor A receptors, M: Migrating endothelial cells

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The calcium-activated voltage-sensitive potassium (BKCa) channels interact with a variety of proteins both at the plasma membrane and with intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. However, the role of BKCa channels in tumor microenvironment including hypoxia is yet to be explored. Hypoxia promotes vessel growth by upregulating multiple proangiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology.[14] In general, uncontrolled cancer growth and subsequent neoangiogenesis lead to hypoxic tumor microenvironment.[15] VEGF expression increases dramatically in hypoxic conditions due to a number of activated oncogenes that are overexpressed in hypoxia. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis.[16] Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors.[17] The second-generation multitargeted tyrosine kinase inhibitor targets VEGFR, platelet-derived growth factor receptor, and c-kit as key proteins responsible for tumor growth and survival.[18] Pazopanib exhibits good potency against all of the human VEGFRs and closely relate to tyrosine receptor kinases in vitro and demonstrates antitumor activity in several human tumor xenografts. Therefore, VEGFRs are attractive therapeutic targets.[19]

Recent work has shown the central role of K+ channels affect multiple conditions of the tumor microenvironment including hypoxia and adenosine.[20] It has long been known that the interaction of tumor cells with their host microenvironment, including endothelial cells and the extracellular matrix, plays an important role in tumor growth and invasion.[21],[22] Hypoxia induces the transcriptional activation of signaling pathways and regulates tumor growth through differential alternative splicing.[23] Nonetheless, very little is known about the effect of hypoxia on the alternative splicing of calcium-activated potassium channel subunit alpha-1 (KCNMA1) either in tumor cells or in endothelial cells. Understanding the role of hypoxia in KCNMA1 splicing is extremely crucial to study blood–brain tumor barrier (BTB) function and improve drug delivery. Our previous studies[24],[25],[26],[27],[28],[29],[30] have revealed that human brain microvascular endothelial cells adjacent to glioma cells overexpress BKCa channels, as opposed to human brain microvascular endothelial cells in normal brain. Our aim is to seek whether the tumor cells (with or without physical contact) overexpress KCNMA1 or its splice variants to increase secretion of VEGF to induce angiogenesis.


  Materials and Methods Top


Cell culture

U-87 MG (U-87), Hs683, and LN-18 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and maintained in minimum essential medium Eagle with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Coculture in monolayer

The established human brain microvasculature endothelial cell line (HCMEC/D3) of normal brain endothelial phenotype, kindly provided by Dr. Weksler (Weill Medical College, Cornell University, Ithaca, NY, USA). As described by us earlier,[31] HCMEC/D3 cells labeled with PKH2 were cocultured with parental and transfected U-87 and Hs683 cells in rat collagen-1-coated tissue culture dishes for 24–72 h. One set of cocultured cells was placed in an incubator (Thermo Fisher Scientific, Bangalore, India) with 95% N2 and 5% CO2 to induce hypoxia while the other set maintained in an incubator with 5% CO2 to achieve normoxia. The cell lines grown individually were used as a reference in the proposed experiments. Hypoxia was monitored using 2-(2-nitro-1H-imidazol-1-yl)-N-(2, 2, 3, 3,3-pentafluoropropyl) acetamide (EF5; Thermo Fisher Scientific, Waltham, MA, USA) by flow cytometry as described in the literature.[32],[33]

Inhibition of KCNMA1 using tetracycline-repressor system regulated small hairpin RNA

To minimize the leakiness, we engineered a vector containing three Tet-repressor binding sites and used H1 promoter instead of the widely used cytomegalovirus promoter. Red arrow indicates the area of shKCNMA1 intervention [Figure 2]A. Briefly, the KCNMA1 siRNA sequence (Ambion, siRNA ID: 112,882) was used to construct long complementary DNA oligonucleotides (containing Hin dIII and Bam HI sites on their respective 5' and 3' ends). The oligonucleotides obtained from Thermo Fisher Scientific were amplified and then TOPO-cloned into the polymerase chain reaction (PCR) 4-TOPO vector and constructs confirmed by sequencing. The fragments were then subcloned into the Tet-On pcDNA4/H1-3TetR vector to create the small hairpin RNA (shRNA) plasmid pcDNA4/H1-3TetR/shKCNMA1. U-87 MG/TR cells (stably expressing the Tet repressor) were transfected with the shRNA plasmid and stably selected using Zeocin (Sigma, St. Louis, MO, USA). Doxycycline (Dox) was added for 48 h to induce shRNA and downregulation of KCNMA1- translated BKCa channels [Figure 2]B and [Figure 3].
Figure 2: Cloning and expression of calcium-activated potassium channel subunit alpha-1 (KCNMA1) small hairpin RNA (shRNA). (A) We engineered a vector containing three Tet-repressor binding sites and used H1 promoter instead of the widely used cytomegalovirus promoter. (B) Addition of doxycycline for 48 h resulted in induction of shRNA and downregulation of KCNMA1-translated calcium-activated voltage-sensitive potassium (BKCa) channels (last two lanes). TetR: Tetracycline

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Figure 3: The small hairpin RNA (shRNA) blocked calcium-activated potassium channel subunit alpha-1 (KCNMA1) expression resulting in inhibition of invasion (A and B) Cell numbers counted in transfected U-87 MG cells (U-87/TR) and compared with untransfected cells. Data are expressed as the mean ± standard deviation, and analyzed by analysis of variance. *P < 0.05, vs. U-87/TR group. All experiments were performed in triplicate. Dox: Doxycycline

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KCNMA1 cloning

To overexpress KCNMA1 and α-subunit of BKCa channel protein, two oligonucleotides: 5'-GAA GCT TAT GGC TGT TGA TGG GTG TTT-3' and 5'-GTC TAG AGG GGA AAT GAG TGG CAG ATA-3', which are adjacent to the start and stop codon of the human BKCa channel α-subunit sequence (NM 002247.2), were used to amplify a 3.7 kb fragment.

To generate a stable cell line, 2 × 105 U-87 cells were transfected with 2 μg of pcDNA6/KCNMA1 using lipofectamine transfection reagent (Invitrogen). Stable clones were selected for 2 weeks in a cell culture medium (Becton, Dickinson and Company, Durham, NC, USA) containing 5 μg/mL blasticidin.

Quantitative PCR

The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizing gene to correct for differences in the amount of RNAs in each sample. Gene-specific primers and probes for KCNMA1 and GAPDH were obtained from ABI TaqMan Gene Expression Assay (HMBS, Hs006090297_m1; TLR9, Hs900152973_m1) (Applied Biosystems). The quantitative PCR and subsequent data analysis were performed using the M × 4000, a multiplex Quantitative PCR System (Stratagene, Amsterdam, The Netherlands). The quantitative PCR reactions were performed and fluorescence measured during the 60°C-step for each cycle. Data were calculated by comparative Ct method.[34] The KCNAM1 expression level was normalized to the expression level of GAPDH.

Membrane potential assay

The functional activity of BKCa channels in parental and transfected U-87 cells was measured using the fluorometric imaging plate reader membrane potential assay kit and a specific BKCa channel activator (50 μM), NS-1619/NS-004 (1-[5-chloro-2-hydroxyphenyl]-5-trifluoromethyl-1,3-dihydro-2-benzimidazol-2-one) provided by NeuroSearch A/S (Copenhagen, Denmark), selective agonists of BKCa channels, on the FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) as described by us previously.[30],[31]

Detection of KCNMA

1 in coculture under normoxia and hypoxia


Since there is not much known based on our literature search on the expression of BKCa channels in cocultured brain endothelial cells with the U-87 MG cells, we studied the expression of BKCa channel α-subunit in hypoxic and normoxic conditions using flow cytometry and fluorescence microscopy. The parental and transfected U-87 MG cells were cocultured with HCMEC/D3 cells labeled with live cell staining dye PKH2 (green fluorescence). The coculture was subjected to normoxic and hypoxic conditions for 48 h and the cells were stained with BKCa channel α-subunit antibody. Goat anti-rabbit IgG labeled with phycoerythrin were purchased from Abcam (ab72465, ab97070) and Life-Technologies (P2771MP). Phycoerythrin-labeled secondary antibody was used for detection of BKCa channel α-subunit. The staining protocol was similar for immunofluorescence and flow cytometry. However, the cells were trypsinized and fixed as single cells for flow cytometry analysis, while the cells grew on coverslips for microscopy. Endothelial cells showing green (PKH2) and red (for BKCa channel) fluorescence were gated, and flow cytometric histograms plotted for red fluorescence and mean fluorescence intensity (represents the BKCa channel expression) were calculated. The cells were acquired on the guava flow cytometer (Tree Star Inc., Ashland, OR, USA) and analyzed using FlowJo Software (Tree Star Inc.). Phycoerythrin-labeled secondary antibody was used for detection of BKCa channel α-subunit. The cells were acquired on the Guava flow cytometer and analyzed using FlowJo software (Tree Star Inc.).

Detection of glioma cell growth and invasion

Growth and invasion assays were measured in parental and transfected U-87 cells using BD BioCoat™ growth factor reduced insert plates (Matrigel™ Invasion Chamber 12-well plates) obtained from Becton, Dickinson Company.[35]

Detection of VEGF secretion and HCMEC/D3 cell proliferation

To prepare conditioned media (CM), untransfected KCNMA1 and KCNMA1 vE22 overexpressing U-87 MG cells were used, with HCMEC/D3 as the control. Cells (1.5 × 106 cells/dish) were seeded on culture dishes. After incubation for 24 h, the cell layer was washed with phosphate-buffered saline (twice) to remove serum components. The dishes were then incubated with endothelial growth media (Lonza, Cohasset, MN, USA) under hypoxic conditions (to facilitate VEGF secretion). A separate set of dishes were treated with Iberiotoxin (10 nM) obtained from Sigma. After incubation for 48 h, the culture medium was collected and centrifuged at 1.12 × g for 5 min and filtered through a 0.2-μm filter to remove cells and cell debris, and the filtrate served as CM. As described by us,[31] we determined the role of VEGF in modulating endothelial cell proliferation via altered BKCa channel expression, brain microvascular endothelial cells (HCMEC/D3, gift from Dr. Weksler, Cornell University, Cornell, NY, USA) were grown in the presence of VEGF (secreted), added VEGF (Sigma) and iberiotoxin.

VEGF levels in CM were measured using human VEGF enzyme-linked immunosorbent assay kit (Invitrogen). The shKCNMA1 with or without Dox (D9891, Sigma) at 1 mg/mL was added to HCMEC/D3 cells, grown in the media containing VEGF. The cell proliferation and invasion were measured to delineate the role of VEGF in KCNMA1. Cell proliferation was determined by growth curve and MTT assay, and BKCa channel expression by flow cytometry as described by us.[35] In noncontact experiments, HCMEC/D3 cells were grown in CM derived from parental and transfected U-87 cells. For contact experiments, labeled-HCMEC/D3 cells were grown with parental and transfected U-87 MG cell line and labeled-HCMEC/D3 cells counted on hemocytometer using Nikon inverted fluorescence microscope (Nikon Inc., Melville, NY, USA).

Detection of glioma growth and invasion

We studied the effect of BKCa channels on the invasiveness of glioma cells by pharmacological inhibition using iberiotoxin and by shRNA-mediated knockdown of its α-subunit. Invasion was assessed using the Matrigel invasion assay as described by us.[31] BD BioCoat™ growth factor reduced insert plates (Matrigel™ Invasion Chamber; Becton, Dickinson and Company) were prepared by rehydrating the BD Matrigel™ matrix coating in the inserts. Dulbecco's modified Eagle medium containing chemoattractant (1% fetal bovine serum) was added to the lower wells of the plate, and cell suspension in serum-free medium was added in each insert well. For induction of shRNA, Dox (1 mg/mL) was added to the cell culture medium in both upper and lower chambers along with cells and chemoattractant solution. Cells on the lower surface of the insert were stained with crystal violet and each transwell membrane mounted on a microscopic slide for visualization and analysis. The number of tumor cells that migrated from the upper to the lower side of the filter was counted. The data were expressed as the percent invasion through the membrane relative to the migration through the control membrane. Invasion percent = mean number of treated cells invading through the Matrigel insert membrane/mean number of cells migrating through control insert membrane × 100.

Detection of tumor cell-induced VEGF secretion in endothelial cells

To test this hypothesis, HCMEC/D3 cells were incubated with CM obtained from U-87 MG cells overexpressing KCNMA1 and KCNMA1 vE22. Internal controls were HCMEC/D3 cells grown in the absence of CM and cells treated with VEGF (Sigma). Following 48 h of treatment with CM, cells were trypsinized, counted, and fixed with 4% paraformaldehyde and stained with BKCa channel α-subunit antibody for flow cytometry assay and microscopy following immunostaining.

Vascular endothelial growth factor measurement

To determine whether modulation of KCNMA1 affects VEGF secretion, parental and transfected cells were seeded into six-well cell clusters at 3 × 106 cells per well. After 48 h of incubation, the cell culture medium was collected to measure secreted VEGF using mouse monoclonal antibodies against vimentin (1:100; Thermo Fisher Scientific, Fremont, CA, USA), and cells were subsequently detached and counted. We measured VEGF levels in culture media obtained from wild-type and KCNMA1 shRNA transfected U-87 MG cells to establish a role for BKCa channels in altering VEGF secretion. VEGF levels were measured using the human VEGF ELISA kit (Invitrogen) and expressed as picograms per million cells (pg/106).

Detection of BKCa channel expression, activity, KCNMA1 splicing in hypoxia

The effect of hypoxia on BKCa channel expression was studied by immunofluorescence staining of glioma cells growing under hypoxia for 48 h (95% N2 and 5% CO2) and normoxia (5% CO2). Immunoblotting of BKCa channel protein was performed on paraformaldehyde-fixed cells, which were permeabilized with 0.05% Triton-×100 in phosphate-buffered saline for 8 min on ice. Membranes were washed with Tween-tris-buffered saline four times for 15 min each and incubated with anti-mouse peroxidase-conjugated secondary antibody (1:1000 dilution; Santa Cruz, CA, USA) for BKCa channel detection and anti-rabbit peroxidase-conjugated secondary antibody (1:5000 dilution; Amersham, CA, USA) for 1 h at 37°C. Cells were then washed and incubated with fluorescein isothiocyanate-labeled secondary antibody for 60 min at 4°C. Images were acquired using a Nikon inverted microscope, Photometrics coolsnap HQ2 charge couple device camera, and Metamorph software. RNA was extracted using TRIzol reagent (Invitrogen). Primer pairs spanning the entire gene (27 exons) were used to identify differential splicing events under normoxia and hypoxia by quantitative PCR. We also measured the invasive potential of cells exposed to hypoxia and normoxia.

Detection of KCNMA1 modulation on tumor formation

We injected 2 × 106 parental and transfected U-87 cells subcutaneously into both the flanks of 4–6-week-old female athymic nude mice (weighing 18–20 g, n = 5/group) obtained from Charles River (Woburn, MA, USA). Mice were randomly assigned into control and treatment groups. Tumors were measured once a week for 12 weeks, and volume calculated using the formula (V = 0.523 [l × b × h]), where l, b, and h are the length, width, and height of the tumor, respectively. The mice were euthanized by carbon dioxide (air displacement rate 10%–30%/min) inhalant when tumor volume reached 500 mm3. The rodent study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University, Atlanta, GA, USA (approved No. A0706007_01) on July 20, 2007. The animal experiments were performed in accordance with Memorial Health University Medical Center, Mercer Institutional Animal Care and Use Committee approval, and in compliance with Association for Assessment and Accreditation of Laboratory Animal Care policies and guidelines.

Statistical analysis

One-way analysis of variance and multitest correction for P values were applied to all the in vitro(in triplicate) and >in vivo(n = 5/group) experimental data. P values determined in cells (low-grade vs. high-grade glioma as well as shKCNMA1 transfected vs. non-transfected U87 cells) acted as the candidate variable in the analysis of variance model. The error bars shown in figures represent the standard deviation (SD). The SPSS software 20 (IBM, Armonk, NY, USA) was used for the analyses. All the in vitro(in triplicate repeated twice) and >in vivo(n = 5/group) experimental data were subjected to one-way analysis of variance, nonparametric Z-test, and multitest correction for P values in tissue type (low-grade vs. high-grade glioma as well as shKCNMA1-transfected vs. nontransfected U-87 cells) as the candidate variable in the one-way analysis of variance model. The error bars shown in the figures represent the SD. P < 0.05 was considered statistically significant.


  Results Top


Effect of BKCa channels/KCNMA1 on VEGF secretion and HCMEC/D3 cell proliferation

We observed a significant increase (P < 0.05) in human brain microvascular endothelial cells (HCMEC/D3) proliferation in the presence of CM obtained from U-87 MG cells overexpressing KCNMA1 vE22 [Figure 4]A, compared to HCMEC/D3 cells grown in the absence of VEGF. In contrast, CM from KCNMA1-silenced U-87 MG cells did not affect HCMEC/D3 cell proliferation. BKCa channel expression increased nearly two-fold in endothelial cells grown in CM from U-87 MG cells overexpressing KCNMA1 vE22 [Figure 4]B. These results suggested that tumor cells secrete certain growth factors including VEGF, which alter BKCa channels expression on endothelial cells that might stimulate their proliferation. VEGF also caused an increase in BKCa channel expression in HCMEC/D3 cells.
Figure 4: Conditioned media: Human brain microvasculature endothelial cell line (HCMEC/D3) cell proliferation, calcium-activated voltage-sensitive potassium (BKCa) channel expression. (A) A significant increase in endothelial cell proliferation in the presence of conditioned media obtained from U-87 MG cells overexpressing calcium-activated potassium channel subunit alpha-1 (KCNMA1) vE22, compared to HCMEC/D3 cells grown in the absence of vascular endothelial growth factors (*P < 0.05). In contrast, conditioned media from KCNMA1-silenced U-87 MG cells did not affect HCMEC/D3 line cell proliferation. Data are expressed as the mean ± standard deviation and analyzed by one-way analysis of variance. All experiments were performed in triplicate with appropriate controls. (B) BKCachannel expression increased nearly 2-fold in endothelial cells grown in conditioned media from U-87 MG cells overexpressing KCNMA1 vE22. Numbers in the brackets represent the total number of BKCachannels-expressing cells

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Effect of hypoxia on BKCa channel expression and activity

As seen in [Figure 5]A, high-grade glioma cell lines LN-18 and U-87 MG expressed higher levels of BKCa channel α-subunit when exposed to hypoxic conditions. The increased expression of BKCa channel α-subunit under hypoxia corroborated with increased activity of BKCa channels [Figure 5]B. We also measured the invasive potential of cells exposed to hypoxia and demonstrated that tumor cells were more invasive under hypoxia compared to normoxia. Furthermore, the migration of U-87 MG and LN-18 cells in hypoxia was decreased by 90% when BKCa channels were inhibited by Iberiotoxin [Figure 5]C. These results suggest that hypoxia increases BKCa channel-mediated migration of glioma cells.
Figure 5: Calcium-activated potassium channel subunit alpha-1 (KCNMA1) in tumor microenvironment. (A) High-grade glioma cell lines LN-18 and U-87 MG expressed higher levels of calcium-activated voltage-sensitive potassium (BKCa) channel α-subunit when exposed to hypoxic conditions. (B) The increased expression of BKCachannel α-subunit under hypoxia corroborated with increased activity of BKCachannels. (C) The invasive/migration potential of cells exposed to hypoxia was greater under hypoxia compared to normoxia. Data are expressed as the mean ± standard deviation and analyzed by analysis of variance. All experiments were performed in triplicate

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Effect of KCNMA1/BKCa channel suppression on glioma invasion and growth

The shRNA-induced inhibition of KCNMA1 expression significantly inhibited the invasion of transfected U-87 MG cells compared to untransfected cells (P < 0.01) [Figure 3]A. In addition, shRNA-mediated silencing of KCNMA1 showed a 70% reduction in growth of U-87 MG cells [Figure 3]B as compared to untransfected U-87 MG cells. We found that the invasive potential of glioma cells exposed to hypoxia was greater under hypoxia compared to normoxia.

Effect of KCNMA1/BKCa channel expression and vascular endothelial growth factor on endothelial cell proliferation

We measured VEGF levels in culture media obtained from wild-type and KCNMA1 shRNA transfected U-87 MG cells to establish a role for BKCa channels in altering VEGF secretion. Addition of VEGF increased endothelial cell proliferation, which was inhibited by Iberiotoxin [Figure 6]A, suggesting a role for BKCa channels in VEGF-mediated endothelial cell proliferation. VEGF also caused an increase in BKCa channel expression in HCMEC/D3 cells [Figure 6]B. A significant decrease in VEGF secretion was observed in U-87 cells where KCNMA1 was silenced, and in Hs683 cells, which is known to express lower level of (KCNMA1) as compared with U-87 MG cells (P < 0.01) [Figure 6]C. These results suggest that VEGF secretion may be influenced by KCNMA1 expression levels.
Figure 6: Effect of calcium-activated potassium channel subunit alpha-1 (KCNMA1)/calcium-activated voltage-sensitive potassium (BKCa) expression on glioma cells on vascular endothelial growth factors (VEGF) secretion and endothelial cell proliferation. (A) Addition of VEGF increased endothelial cell proliferation, which was inhibited by iberiotoxin. (B) VEGF addition increased BKCachannel expression in human brain microvasculature endothelial cell line cells. (C) A significant decrease in VEGF secretion was observed in U-87 cells where KCNMA1 was silenced, and in Hs683 cells, which is known to express low level of KCNMA1 as compared with U-87 MG cells (*P < 0.01). Data are expressed as the mean ± standard deviation, and analyzed by analysis of variance. All experiments were performed in triplicate. shRNA: Small hairpin RNA

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Hypoxia and calcium-activated KCNMA1 splicing

The effect of hypoxia on KCNMA1 splicing was studied by exposing glioma and endothelial cells to hypoxia and normoxia as described above. RNA was extracted using TRIzol reagent (Invitrogen). Primer pairs spanning the entire gene (27 exons) were used to identify differential splicing events under normoxia and hypoxia by quantitative PCR. [Figure 7] shows the differential expression of exon (PCR product intensities and additional bands, shown by yellow arrowheads) under normoxia and hypoxia, which suggests that alternative splicing of KCNMA1 might be stimulated by hypoxia.
Figure 7: Calcium-activated potassium channel subunit alpha-1 splicing in hypoxia and normoxia – alternative splicing of calcium-activated potassium channel subunit alpha-1 seen in glioma cells (Hs683, U-87, LN-18, and endothelial cells (human brain microvasculature endothelial cell line) potentially stimulated by hypoxia and normoxia. A differential expression of exons (quantitative polymerase chain reaction product intensities and additional bands, shown by yellow arrowheads) under normoxia and hypoxia are shown. Number of samples (1–11) run on the gel is indicated

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In vivo effect of KCNMA1 inhibition on tumorigenicity

As compared to the untransfected group [Figure 8]A, shKCNMA1-transfected group showed reduced tumor growth [Figure 8]B upper panel]. Interestingly, the removal of Dox on week 6 reactivated the KCNMA1 promoting tumor growth [Figure 8]B bottom panel]. In wild-type group, tumor growth formation and growth were swift and reached (500 mm3) by 4 weeks, prompting euthanasia, compared to negligible tumor growth in the KCNMA1-silenced group, which remained insignificant until the 6th week when Dox was withdrawn. However, from the 6th week onward tumor growth was dramatic and reached 350 mm3 by the 11th week, prompting euthanasia.
Figure 8: Study of calcium-activated potassium channel subunit alpha-1 (KCNMA1) expression modulation and tumorigenicity. (A) Representative images of nude mice with parental and transfected U-87 MG xenografts. Black arrows indicate tumor. (B) Tumor volume during 12 weeks of the study. Mice with wild-type U87 cells developed tumors within 3 weeks. However, mice with shKCNMA1-transfected U87 cells developed tumors only when doxycycline was withdrawn in the 6th week. Blue arrow indicates the time point when the doxycycline was withdrawn

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  Discussion Top


BKCa channels are overexpressed on brain tumor capillary endothelial cells and serve as the convergence point for various BTB-permeability-related signaling pathways.[24],[25],[26],[27],[28],[29],[30],[31] These critical observations prompted us to determine whether these channels play an important role in BTB permeability regulation in human brain tumor xenograft where permeability increase was caused by NS-1619 that was attenuated by iberiotoxin (IC50= 68 nM), a selective inhibitor of BKCa channels.[28] VEGF has been shown to increase BBB permeability by affecting the tight junctions between endothelial cells, i.e., via the paracellular route.[28] Nevertheless, it is not clear whether VEGF has any direct effect on BKCa channel [Figure 1] activation that has been shown to trigger endothelial transcytotic vesicles leading to increased vesicular transport.

BKCa channels and hypoxia

Hypoxia upregulates (Ca2+)i, activates BKCa channels[36] and induces VEGF in melanoma,[37] all of which are necessary for the tumor growth, invasion, metastasis, and angiogenesis. Studies have shown that VEGF acts as an autocrine growth and survival factor for VEGFR-expressing tumor cells.[3] It is known that VEGF effect is attributable to a paracrine mechanism by tumor cells[38] and is responsible for initiating signal transduction pathways within the endothelial cells. Autophosphorylation of key sites within the cytoplasmic domain of VEGFR induces several cellular signaling pathways leading to angiogenesis.[39] More specifically, IP3 mobilizes (Ca2+)i, which activates BKCa channels and increases proliferation of endothelial cells. In addition, PI3K pathway-mediated release of nitric oxide (NO) from endothelial cells has been shown to be an essential mediator of VEGF-A (VEGF165)-induced angiogenesis. We have shown that NO activates BKCa channels on endothelial cells[28] leading to their proliferation and increased BTB permeability in glioma models. The effect of BKCa channels on tumor cells are shown to be directly affecting number of different cellular processes such as progression, perturbation of membrane potential, sensitivity to (Ca2+)i balance, cell proliferation, invasion and metastasis, and survival.[40] In addition a slight increase in (Ca2+)i activates BKCa channels directly.[41] The investigation of the role of BKCa channels in association with VEGF in tumor cells in promoting angiogenesis is long overdue. The regulatory network between BKCa channels and key processes such as angiogenesis that shape the tumor microenvironment could provide the scientific basis for the implementation of novel anticancer strategies. We propose that VEGF via endothelial NO synthase, (Ca+2)i, and NO activates BKCa channels and increases proliferation of endothelial cells.

It has long been known that the interaction of tumor cells with their host microenvironment, including endothelial cells and the extracellular matrix, plays an important role in tumor growth and invasion.[42] Hypoxia induces the transcriptional activation of signaling pathways and regulates tumor growth.[43] Tumor-endothelial cell interaction in hypoxia has been shown to cause differential alternative splicing.[44] Nonetheless, very little is known about the effect of hypoxia on the alternative splicing of KCNMA1 either in tumor cells or in endothelial cells. Understanding the role of hypoxia in KCNMA1 splicing is extremely crucial as glioma cells interact with brain microvascular endothelial cells to migrate to normal brain and form microsatellites. These processes have implications on the BTB function and drug delivery. Our previous studies[28],[29],[30],[31] have revealed that human brain microvascular endothelial cells adjacent to glioma cells overexpress BKCa channels, as opposed to human brain microvascular endothelial cells in normal brain. Even so, whether the tumor cell (with or without physical contact) secretes certain factors that induce endothelial cells to overexpress KCNMA1 or its splice variants remains to be thoroughly investigated. Several studies have shown that endothelial cells proliferate following stimulation by VEGF and/or basic fibroblast growth factor to form new blood vessels.[45],[46] Also, interleukin-8 or C-X-C motif chemokine ligand 8 assists in accelerating the proliferation and migration of the cancerous cells. It also inhibits apoptosis of the cells which leads to the continual survival of the cells even in the most extremes of condition, where the cells are not able to derive an optimum amount of oxygen and nutrition from the blood plasma. This is precisely the reason for which a number of anticancer drugs are not able to control invading cancer cells. A review discussed how pediatric brain tumors regulate angiogenesis to obtain a vascular supply, what types of inhibitors are available, how different classes of inhibitors work, the types of resistance possible, how rapidly these inhibitors may work, and what surrogate markers of activity are available to follow responses.[47]

Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer.[48] BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway.[49] Our work shows an association between the BKCa channel isoform expression and VEGF secretion by glioma cells, which is exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery. We unraveled the expression pattern of KCNMA1 and VEGF in tumor microenvironment [Figure 5] and splicing patterns of KCNMA1 [Figure 7] under normoxia and hypoxia alone and in coculture with brain microvascular endothelial cells (HCMEC/D3).

Other researchers have found that the modulation of BKCa channel activity or expression by the hypoxic environment may participate in the acquisition of the aggressive phenotype observed in glioblastoma multiforme cells residing in a hypoxic environment.[50] Channelopathy due to altered ion channel expression/function exacerbates angiogenesis signaling.[21] We showed the association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia [Figure 6]. Thus, altered tumor vasculature might affect vascular permeability for anticancer drug delivery. There is a need to study the mechanism how KCNMA1 and its splice variant KCNMA1 Δ E2 expression under normoxia and hypoxia affect neovascularization. For example, we used U-87 cells alone and in coculture with brain endothelial cells to study the role of KCNMA1 alternative splicing in glioma biology. Therefore, discovery and validation of KCNMA1 alternate splice variants in brain tumors may serve as new tools for the diagnosis and classification of brain tumor patients with high risk of aggressive phenotype like glioblastoma multiforme. Such splice variations in a number of genes were shown to increase malignancy and could precede clinical cancer diagnosis.[51] We constructed a potential VEGF signaling pathway adapted from KEGG-VEGF signaling pathway by activation and suppression of KCNMA1 in U-87 cells. Then, we reported that FLT1, HMCN1 were upregulated by KCNMA1 activation and AKT2, KDR, MAPK11, PXN, SPHK1, NFAT5, and NFATC1 were downregulated when KCNMA1 was suppressed.[40]

The pathway suggests that KCNMA1/BKCa channels appear to play a role in VEGF secretion and neovascularization in brain tumors. Hence, it may be expected that use of BKCa channels modulators may be considered for developing novel therapeutic strategies against brain tumors.

The presence of hypoxia appears to be a common feature of brain tumors and primarily responsible for their poor response to treatment.[21] Hypoxia is also shown to cause the leakage of tumor blood vessels by inducing VEGF expression, which promotes new blood vessel formation (angiogenesis) and increases vascular permeability during tumor growth.[14] Hypoxia transforms tumor cells to be more malignant, aggressive, genetically unstable, and less susceptible to apoptosis, rendering resistance to a wide range of anticancer therapies. Our data showed that brain tumors cells exposed to hypoxia may also induce distinct molecular changes like the overexpression of BKCa channels/KCNMA1 splice variants and increased VEGF secretion that promotes neoangiogenesis. Cancer is more likely be caused by channelopathy along with other oncogenic factors.[52] The brain tumor microenvironment alters BKCa channels expression on endothelial cells that might stimulate their proliferation by increased VEGF secretion by tumor cells.


  Conclusion Top


Perhaps, the discovery and validation of brain-specific metastasis-associated KCNMA1 alternate splice variants will serve as new tools for the diagnosis and prognosis of aggressive glioma phenotype. Our results clearly show that KCNMA1 exacerbate glioma growth and angiogenesis in hypoxia. Taken together, these experiments show a critical role of KCNMA1 expression on VEGF secretion by glioma cells and endothelial cell proliferation in normoxia and hypoxia. These studies will contribute to a fundamental understanding of the role of KCNMA1 in glioma microenvironment and angiogenesis.

Financial support and sponsorship

The authors would like to thank the Scintilla Group, Bengaluru, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; and National Institutes of Health, Bethesda, MD, USA for providing the opportunity and Innovative research grant support from Georgia Cancer Coalition, Atlanta, GA, USA.

Institutional review board statement

The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University, Atlanta, GA, USA (approved No. A0706007_01) on July 20, 2007.

Conflicts of interest

There are no conflicts of interest.



 
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