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Table of Contents
Year : 2019  |  Volume : 2  |  Issue : 1  |  Page : 46-54

Evidence of calcium-activated potassium channel subunit alpha-1 as a key promoter of glioma growth and tumorigenicity

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Bengaluru, Karnataka, India
2 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research; Department of Molecular Oncology, Scintilla Bio-MARC Pvt. Ltd., Bengaluru, Karnataka, India

Date of Web Publication1-Apr-2019

Correspondence Address:
Dr. Nagendra Ningaraj
Scintilla Academy for Applied Sciences' Education and Research, Bengaluru - 560 060, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/glioma.glioma_44_18

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Background and Aim: Mechanisms of glioma progression are poorly understood. Upregulation of calcium-activated potassium channel subunit alpha-1 (KCNMA1), which encodes the α-subunit of maxi-calcium-activated potassium (BKCa) channels, is shown to be a novel mechanism for the malignant phenotype of brain tumor cells. The aim of this study was to establish the functional role of KCNMA1 in glioma biology. Materials and Methods: U-87-MG (U-87) cells were transfected to increase BKCa channel expression and activity. Glioma cell proliferation, invasiveness, and transendothelial migration were then measured. BKCa channels were blocked with iberiotoxin or short hairpin RNA (shRNA), which significantly inhibited K+ currents and growth of U-87 cells. It was tested whether KCNMA1 overexpression enhanced tumorogenecity in glioma xenograft mouse models by injecting wild-type and KCNMA1- overexpressing U87-MG cells. In parallel experiment, it was studied whether shRNA KCNMA1-expressing U-87 cells show attenuated glioma growth in mice. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA on July 20, 2007. Results: The effect of KCNMA1 overexpression in glioma growth as well as on associated cell biology functions such as proliferation, invasion, and migration was presented in this study. Messenger RNA and protein analyses revealed that KCNMA1 was amplified in 90% of high-grade gliomas and in high-grade glioma cell line U-87. In contrast, KCNMA1 amplification was not found in normal brain tissues. These data indicate that KCNMA1 plays critical role in glioma biology by interacting with several cellular processes. The data demonstrate that KCNMA1 amplification drives glioma cell proliferation and growth, which can be attenuated by its downregulation. Conclusion: KCNMA1 is a regulator of glioma cell proliferation and growth and thus qualifies as a promising diagnostic and therapeutic target in the treatment of glioma.

Keywords: Calcium-activated potassium channel subunit alpha-1, glioma, maxi-calcium-activated potassium channels, potassium channels, tumorigenicity

How to cite this article:
Khaitan D, Ningaraj N. Evidence of calcium-activated potassium channel subunit alpha-1 as a key promoter of glioma growth and tumorigenicity. Glioma 2019;2:46-54

How to cite this URL:
Khaitan D, Ningaraj N. Evidence of calcium-activated potassium channel subunit alpha-1 as a key promoter of glioma growth and tumorigenicity. Glioma [serial online] 2019 [cited 2022 Aug 13];2:46-54. Available from: http://www.jglioma.com/text.asp?2019/2/1/46/255151

  Introduction Top

Brain tumors are the most common type of solid tumor in both children and adults. It is estimated that 78,980 new cases of primary malignant and nonmalignant brain tumor and other central nervous system tumors will be diagnosed in the United States in 2019. This includes an estimated 23,830 primary malignant brain tumors and 55,150 nonmalignant brain tumors.[1] The most common form of malignant glioma is glioblastoma multiforme (GBM). The treatment of brain tumors is problematic, in terms of both cure rates and long-term quality of life.[2] Gliomas are insidious due to their highly invasive and destructive manifestation, and thus, GBM patients' median survival is <15 months.[3] The low-grade gliomas often manifest as astrocytomas or anaplastic astrocytomas and sometimes progress dramatically to a highly malignant GBMs. There is a critical need to develop reliable biomarkers for the early detection and successful treatment to attenuate gliomas' progression into GBM. Gene expression profiling studies have identified many GBM specific genes, involved in epigenetic inactivation, that drive glioma transformation.[4] GBM has distinct gene expression pattern among different histological types and grades of gliomas.[5] Increasing evidence shows that potassium channels are integral part of glioma cell growth and survival.[6]

The importance of voltage-gated potassium channels in tumor biology has aroused great interest in recognition of ion channels as potential targets for tumor therapy.[5],[6],[7],[8] Several potassium channels have been implicated in tumor progression and cell proliferation. Specifically, large-conductance, voltage-sensitive, Ca2+-activated potassium (BKCa) channels are overexpressed in human glioma cells.[7] Calcium-activated potassium channel subunit alpha-1 (KCNMA1) encodes the α-subunit of the BKCa channels. They play a key role in cellular functions and have recently emerged as regulators of tumorigenesis. These channels respond to changes in intracellular calcium ([Ca2+]i) and membrane potential, and their expression correlates with increased malignancy in gliomas.[8],[9],[10] Studies have shown that pharmacological inhibition of BKCa channel by iberiotoxin abolished the activation of K+ ion channel currents and attenuated migration of glioma cells.[11],[12],[13] Elucidation of the molecular mechanisms of KCNMA1 regulation is critical for the understanding of a variety of physiological and pathological conditions. The aim of this study was to study the effect of KCNMA1 modulation in glioma cells, thus offering a promising biomarker for early diagnosis and prognostication of high-grade gliomas.

  Materials and Methods Top

Cell culture

U-87 MG (U-87) 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). The rodent study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA on July 20, 2007.

Clinical samples

Human normal brain (n = 1) and glioma tissues (n = 5) were obtained from Memorial Health University Medical Center and Comprehensive Human Tissue Network, University of Alabama, USA. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

Calcium-activated potassium channel subunit alpha-1 cloning for overexpression

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. The primers contained Hin dIII and Xba I restriction sites for cloning. The cDNA was synthesized using Superscript III reverse transcriptase and 1 μg of total RNA from A172 glioma cells, representing grade IV glioma (GBM). Polymerase chain reaction (PCR) was carried out using Amplitaq Gold (ABI-Applied Biosystems, Carlsbad, CA, USA) polymerase, 1 μL of cDNA, annealing temperature of 58°C, and 35 cycles. The PCR product was run on a 1% agarose-TAE gel and the band corresponding to 3.7 kb excised, gel purified, and cloned into pCR4-TOPO vector (Invitrogen). The entire cloned fragment was sequenced using multiple primers to ensure whether each sequence has a good overlap with the adjacent sequence. This sequence was then compared to the KCNMA1 sequence NM 002247.2 (NCBI, Bethesda, MD, USA). Subsequently, the TOPO clones were digested with Hin dIII and Xba I to release the 3.7 kb fragment, which was gel purified and ligated into the Hin dIII-Xba I site of pcDNA6-V5/HIS expression vector (Invitrogen). The resulting plasmid pcDNA6/KCNMA1-expressing KCNMA1 from a T7 promoter containing blasticidin resistance marker was used for selection of stable transfectants.

Generation of stable cell line expressing calcium-activated potassium channel subunit alpha-1

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 medium containing 5 μg/mL blasticidine.

Cloning of calcium-activated potassium channel subunit alpha-1 in short hairpin RNA expression vector: pcDNA4/TO/HisA (Invitrogen) was modified for the expression of KCNMA1 short hairpin RNA (shRNA) by cloning the H1 promoter from PRNATin-H1.2/Neo vector (GenScript, Piscataway, NJ, USA) using Bgl II and Hin dIII as reported,[11] replacing the cytomegalovirus (CMV) promoter. This yielded the vector pcDNA4/H1.2. The KCNMA1 shRNA sequence was derived from Silencer® small-interfering RNA (siRNA) (Ambion, siRNA ID: 112882) and constructed as long complementary oligos (KCNMA1 si FW-cgt act tca atg aca ata ttt caa gag aat att gtc att gaa gta cgt ctt ttt t and KCNMA1 si RV-aaa aaa gac gta ctt caa tga caa tat tct ctt gaa ata ttg tca ttg aag tac g, containing Hin dIII and Bam HI sites on their respective 5' and 3' ends). The oligos were mixed at 100 μM, heated and amplified through one round of PCR (Amplitaq Gold, Applied Biosystems, Carlsbad, CA, USA), and then TOPO-cloned into the pCR4-TOPO vector (Invitrogen), sequenced and then subcloned via Hin dIII and Bam HI into pcDNA4/H1.2 to create pcDNA4/H1.2/shKCNMA1.

Selection of stably-expressing calcium-activated potassium channel subunit alpha-1 short hairpin RNA cell line

2.5 × 105 U-87 cells were seeded into a single well of a 6-well plate and transfected with 5 μg pcDNA4/H1.2/shKCNMA1 using Fugene 6 (Promega, Madison, WI, USA). After 24 h, cells were selected with 400 μg/mL Zeocin (Sigma, St. Louis, MO, USA) for the following 10 days. The remaining stable cells were then expanded.

Quantitative polymerase chain reaction assay

Total RNA was extracted from cells grown to 75% confluency using TRIzol® total RNA isolation reagent (Sigma). Total RNA was also isolated from the frozen human tissues disrupted using a Dounce homogenizer and TRIzol. The cDNA for reverse transcription polymerase chain reaction (PCR) was generated by the SuperScript™First-Strand Synthesis System according to the manufacturer's instructions (Invitrogen). PCR was carried out in a total volume of 20 μL, containing 0.2 mM dNTPs, 1 mM MgCl2, and 1 unit of AmpliTaq Gold DNA Polymerase. Thirty-two amplification cycles were performed (Applied Biosystems Thermal Controller) using a denaturing temperature of 95°C for 20 s, an annealing temperature of 58°C for 30 s, and a primer extension at 72°C for 20 s. Each amplification experiment also included two negative PCR controls, a no-RNA control from reverse transcription procedures and a no-cDNA RNase-free water control. Following amplification, 20 μL of the samples were separated via electrophoresis on a 2% agarose gel.

The house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizing gene to correct for differences in the amount of RNA 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 (qPCR) and subsequent data analysis were performed using the Mx4000 Multiplex Quantitative PCR System (Stratagene, Amsterdam, The Netherlands). The qPCR reactions were performed in 20 μL with 5 μL cDNA, 10 μL Absolute qPCR ROX Mix (ABgene, Courtaboeuf, France), and 1 μL sense and antisense primers with probes. The reaction conditions were 50°C for 2 min, 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Fluorescence was measured during the 60°C step for each cycle. Data were calculated by comparative Ct method.[12] The expression level of KCNAM1 was normalized to the expression level of GAPDH.

Western blot assay

Parental and transfected U-87 cells were seeded at a density of 5 × 105 cells/6-well plate. After 48 h seeding, cells were trypsinized, washed with phosphate-buffered saline (PBS), and resuspended in cell lysis buffer (Invitrogen) for 30 min at 4°C. The cells were centrifuged at 12,000 × g for 15 min, supernatant mixed with an equal volume of 2× sodium dodecyl sulfate (SDS) lamelli buffer, boiled for 10 min, and stored for future use. Equal amount of protein from each sample was loaded and resolved on a 7.5 cm × 7.5 cm SDS-polyacrylamide gel at a constant potential gradient of 10 V/cm for 2–4 h to ensure that the bromophenol blue dye-front completely passes through the gel. A dual vertical gel unit (Novex™ from Fisher Scientific, Pittsburgh, PA, USA) was used in this study. Electrophoresis buffer Tris-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Tris 12.1 g, HEPES 23.8 g, SDS 1 g; dissolved in 1 L; pH 8.0) was used. Following separation of proteins, they were blotted to polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA) in transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol [v/v]) overnight at 14 V. The blots were blocked with 1% bovine serum albumin in Tween-Tris-buffered saline (TTBS) (15 mM Tris-HCl pH 7.5, NaCl 0.9%, and 0.1% Tween-20) for 1 h at room temperature (25–28°C). Membranes were incubated with a mouse monoclonal antibody (in 5% w/v nonfat dairy milk) anti-α-subunit of BKCa channel antibody (University of California, Davis, CA, USA) and anti-GAPDH antibody (anti-rabbit at dilution 1:1000) (Cell Signaling, Danvers, MA, USA) for 1 h in TTBS and 1% bovine serum albumin. Membranes were washed with TTBS four times for 15 min each and incubated with antimouse peroxidase-conjugated secondary antibody (1:1000 dilution; Santa Cruz, CA, USA) for BKCa channel detection and antirabbit peroxidase-conjugated secondary antibody (1:5000 dilution; Amersham, CA, USA) for 1 h at 37°C. Blots were washed and developed using ECL chemiluminescence detection reagent (Pierce, Rockford, IL, USA).

Membrane potential assay

The functional activity of BKCa channels in parental and transfected U-87 cells was measured using the FLIPR Membrane Potential Assay Kit on the FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) as described by us previously.[13],[14] Briefly, the cells were seeded in sterile, clear flat bottom, 96-well plates (Corning Inc., MA, USA) at a density of 4 × 104 cells/well and cultured to achieve a monolayer within 48 h. The monolayer of cells was incubated with the membrane potential assay reagent for 30 min before loading the test compounds. Membrane potential studies was performed with 100 μM of BKCa channel activator 1-(5-chloro-2-hydroxyphenyl)-5-trifluoromethyl-1,3-dihydro-2-benzimidazol-2-one (NS-1619) (NeuroSearch NA, Ballerup, Denmark) with or without BKCa channel inhibitor iberiotoxin (10 nM) (Sigma). The FlexStation was set up using the following parameters: excitation: 530 nm, emission: 565 nm, and emission cutoff: 550 nm wavelengths. Observations and recordings were made for 300 s after adding NS-1619 and iberiotoxin.

Cell proliferation detection

Parental and transfected U-87 cells were seeded at a density of 1 × 105 cells/6-well plate, which was taken as the initial value (N0) for all growth kinetics measurements. Cells were harvested at 24-h intervals up to 72 h by trypsinization. Cell were counted using Nexcelom Bioscience Cellometer Counter (Lawrence, MA, USA). The cells were fixed in 80% ethanol for cell cycle analysis. A minimum of six readings spread over three independent experiments were taken for determining the proliferation rate (pR).

pR = Nt/N0, where N0 is number of cells at 0 h; and Nt is number of cells at time “t.”

The mean doubling time of untreated cells was calculated from the exponential region of growth curves, i.e., between 24 h and 72 h.

Cell cycle detection

Flow cytometric measurements were performed on cells fixed with 80% ethanol overnight at 4°C. Fixed cells were washed twice with PBS, treated with 200 μg/mL RNase-A for 30 min at 37°C, and stained with 50 μg/mL propidium iodide (Sigma) in PBS. Cell cycle distribution was studied with the help of Guava flow cytometer (Austin, TX, USA) using the FlowJo software (Guava flow cytometer, Austin, TX, USA) for acquisition and analysis.

Matrigel invasion detection

BD BioCoat™ growth factor reduced insert plates (Matrigel™ Invasion Chamber 12-well plates) were prepared by rehydrating the BD Matrigel™ matrix (Becton Dickinson, Franklin Lake, NJ, USA) coating in inserts with 0.5 mL of culture medium for 2 h at 37°C. The rehydration solution was carefully removed from the inserts, 0.75 mL Dulbecco's Modified Eagle's Medium containing chemoattractant (1% fetal bovine serum) was added to the lower wells of the plate, and 0.5 mL of cell suspension (2.5 × 104 cells, in serum-free medium-containing 0.1% bovine serum albumin) was added to each insert well. For invasion inhibition assays, 10 nM of iberiotoxin was added to the cell culture medium in both upper and lower chambers along with cells and chemoattractant solution. For induction of shRNA, doxycycline (1 mg/mL) was added to cell culture medium in both the upper and lower chambers along with cells and chemoattractant solution. Uncoated insert plates, included as migration controls, were used without rehydration. Invasion assay plates were incubated for 20–22 h at 37°C. Following incubation, the noninvading cells were carefully removed by scrubbing the upper surface of the insert. The cells on the lower surface of the insert were stained with crystal violet (Sigma) and each transwell membrane mounted on a microscopic slide for visualization and analysis. The number of tumor cells that have migrated from the upper to the lower side of the filter was counted using a phase-contrast microscope (Olympus, Central Valley, PA, USA) in the central area of the filter by a technician blinded to this study. Data were expressed as the percent invasion through the membrane relative to the migration through the control membrane.

% Invasion = mean number of transfected cells invading through the Matrigel insert membrane/mean number of nontransfected cells migrating through the Matrigel insert membrane.

Effect of maxi-calcium-activated potassium channel 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 nude mice (n = 5/group), weighing 20–22 g, obtained from Charles River Laboratories (Charles River, MA, USA). Tumors were measured once a week for 12 weeks, and the volume calculated using the formula V = 0.523 (l × b × h), where l refers to length, b refers to breadth, and h refers to height. When tumor volume reached 500 mm3, mice were euthanized. With approval from the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA, on July 20, 2007, and in compliance with the Association for the Accreditation of Laboratory Animal Care policies and guidelines, the animal experiments were performed.

Ki-67 immunostaining and hematoxylin and eosin staining

Tumor tissues (U-87 xenografts) from euthanized mouse were collected, mounted in optimal cutting temperature (OCT® or Cryomatrix® from BD Biosciences, San Diego, CA, USA) frozen in dry ice, and sectioned (10-μm thick) using a cryostat (Leica Biosystems, Buffalo Grove, IL, USA). For immunohistochemistry, sections were fixed in cold 100% acetone for 3 min, air-dried, and incubated with various primary and secondary antibodies (1:1000 dilution) obtained from ProSci Inc., Poway, CA, USA, to block nonspecific proteins at room temperature for 4 h. Subsequently, slides were washed three times in PBS and incubated with secondary antibody at room temperature for 1 h. Finally, alkaline phosphatase or hydrogen peroxide polymer-3-amino-9-ethylcarbazole chromogen substrate kits (Sigma) were used as per the manufacturer's instructions (Lab Vision Corporation, Fremont, CA, USA). Slides were counterstained with hematoxylin and sections were mounted using Vectashield (Vector Laboratories, Burlington, CA, USA) mounting medium and imaged using Olympus microscope (Olympus, Central Valley, PA, USA). The proliferation index was determined by calculating the number of Ki-67-positive cells per total number of cells in ten fields at ×40 and expressed as a percentage.

Statistical analysis

The SPSS software (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 versus high-grade glioma as well as shKCNMA1-transfected versus -nontransfected U-87 cells) as the candidate variable in the one-way analysis of variance model. The error bars shown in figures represent the standard deviation (SD). A P < 0.05 was considered statistically significant.

  Results Top

Overexpression of calcium-activated potassium channel subunit alpha-1 in human glioma cell lines and tissues

To validate the biological significance of KCNMA1 in glioma biology, KCNMA1 expression levels were modulated in a well-studied high-grade glioma cell line U-87 cell line. Western blot showed relatively higher protein level of BKCa channel (α-subunit) in human glioma biopsies compared to human normal brain. We measured the levels of KCNMA1 in various glioma cell lines, representing low- and high-grade gliomas by qPCR. The U-87 and 118 and A-172 cell lines express significantly (P < 0.01) high levels of KCNMA1 and higher membrane potential activities compared to Hs683, which is a low-grade glioma cell line [Figure 1]. It is well established that human tumor cell lines differ in genotypic and phenotypic characteristics from the tumor tissue. Therefore, to validate results with cell lines, we used human normal brain and glioma tissues to determine the strength of KCNMA1 and BKCa channel expressions. Both KCNMA1 and BKCa channel protein expressions were higher (P < 0.001) in brain tumor tissues as compared to normal brain, and differential expression of the protein was observed among different grades of glioma tissues [Figure 1]B. Three out of five human glioma samples analyzed showed overexpression of BKCa channel protein [Figure 1]B-c, d, and f compared with normal human brain [Figure 1]B-b.
Figure 1: Qualitative and quantitative measurement of BKCa channels. (A) BKCa channel (α-subunit) protein expression was measured in human glioma biopsies (a: normal brain, b–f: glioma samples) by Western blot assay; (B) KCNMA1 mRNA was measured in HBMVEC (bar 1 from left) and varying grades of glioma cell lines (Hs683: low-grade glioma cell line-bar 2 from left), LN29 (bar 3 from left), SW1080 (bar 4 from left), U-87 MG (bar 5 from left), U-118 (bar 6 from left) by qPCR-Significantly (P < 0.01) high levels of KCNMA1 mRNA were found in high grade glioma cells compared to Hs683 and HBMVEC, X-axis is the relative quantification of KCNMA1 mRNA expression in glioma cells relative to reference HBMVEC cells; (C) BKCa channel activity was measured in LN29 (a), SW1080 (b), U-87 MG (c), U-118 (d), and A172 (e) using membrane potential assay. Significantly (P < 0.01) higher BKCa channel activities (measured by membrane potential assay using FlexStation) were recorded in high-grade glioma cells compared to HBMVEC. The error bars shown in B represent the standard deviation. BKCa: Maxi-calcium-activated potassium, RUF: Relative florescence unit, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, HBMVEC: Human brain microvascular endothelial vascular cells, NS-1619: 1-(5-chloro-2-hydroxyphenyl)-5-trifluoromethyl-1,3-dihydro-2-benzimidazol-2-one, KCNMA1: Calcium-activated potassium channel subunit alpha-1, qPCR: Quantitative polymerase chain reaction

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Characterization of calcium-activated potassium channel subunit alpha-1-transfected U-87-MG cells

Previous studies detected expression of KCNMA1 in U-87 cells,[15],[16],[17] and we confirmed whether KCNMA1 is translated to BKCa channel protein in U-87 cells. To assess potential role of KCNMA1 glioma biology, U-87 cells were stably transfected with shKCNMA1 and pCDNA6 KCNMA1 or U-87/shKCNMA1 [Figure 2]A. The effect of loss or gain of KCNMA1 in cell function was studied. U-87/shKCNMA1 cells showed 75% decrease in mRNA levels, while a 3-fold increase in mRNA level was observed in U-87 cells stably overexpressing KCNMA1 [Figure 2]B. Western blot results correlated well with the mRNA levels [Figure 2]C. To confirm whether the overexpressed BKCa channel protein exhibits increased functional activity and downregulated BKCa channel protein shows decreased channel activity, we performed membrane potential assays. A two-fold increase in channel activity was observed when the activator (100 μM NS-1619) was added to cells overexpressing the wild-type KCNMA1 compared to control vector-transfected U-87 cells [P < 0.05; [Figure 2]D. In contrast, 90% reduction in the BKCa channel activity was observed in U-87shKCNMA1-transfected cells [Figure 2]D.
Figure 2: Schematic representation of KCNMA1 cloning approach. (A) PCR cloning strategy for KCNMA1; (B) KCNMA1 mRNA expression by qPCR; (C) BKCa channel expression by Western blot assay; (D) BKCa channel activity by membrane potential assay in transfected and nontransfected U-87 cells. A two-fold increase in channel activity was observed when the activator (100 μM NS-1619) was added to cells overexpressing the wild-type KCNMA1 compared to control vector transfected U-87 cells (P < 0.05). The error bars shown in B and D represent the standard deviation. BKCa: Maxi-calcium-activated potassium, KCNMA1: Calcium-activated potassium channel subunit alpha-1, qPCR: Quantitative polymerase chain reaction

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In vitro effects of calcium-activated potassium channel subunit alpha-1 transfection on glioma cell proliferation

Since BKCa channels are known to enhance invasion and proliferation of glioma cells, we examined the effect of KCNMA1 knockdown and overexpression on U-87 glioma cell proliferation and invasion. We also studied the role of BKCa channels on the invasiveness of U-87 cells by shRNA-mediated knockdown of KCNMA1 and overexpression of KCNMA1. Invasion was assessed using the Matrigel invasion assay. The KCNMA1-overexpressing cells exhibited a 2.5-fold increase in invasion while silencing of KCNMA1 (U-87/shKCNMA1) reduced the invasion by nearly 3-fold, demonstrating the specificity of KCNMA1 function in glioma invasion [Figure 3]A. To study the effect of KCNMA1 on glioma cell proliferation, we compared the growth rates of U-87 vector only cells versus U-87 KCNMA1 overexpression and U-87shKCNMA1 cells. A marked increase in the proliferation rate (P < 0.001) of KCNMA1-transfected cells compared to U-87 cells alone was observed, suggesting that KCNMA1 has a profound effect on glioma cell proliferation. In addition, stable knockdown of KCNMA1 resulted in a 70% reduction (P < 0.001) in growth of U-87 cells as compared to nontransfected U-87 cells [Figure 3]B. To determine whether the cessation of cell growth in the KCNMA1 knockdown was due to arrest of cells in a particular phase of the cell cycle, the DNA content was analyzed by flow cytometry. U-87 cells overexpressing KCNMA1 showed higher proportion of cells in S phase (53%) and significantly reduced G2 phase (19%). In contrast, U-87 shRNA-transfected cells showed a higher proportion of cells in G1 phase (69%) at 24 h [Figure 3]C. Interestingly, the cell cycle for U-87 cells overexpressing KCNMA1 was similar to U-87-nontransfected cells at further time points. However, a marked increase in S phase (31%) at 48 h and G2 phase (33%) at 72 h in U-87 shRNA-transfected cells was observed as compared to U-87-nontransfected cells [Figure 3]C.
Figure 3: In vitro effects of KCNMA1 modification on glioma cell functions. (A–C) Effect of BKCa channels on glioma migration (A), proliferation (B), and cell cycle (C). The arrows indicate the glioma cells in the G2 phase of the cell cycle. The error bars shown in A and B represent the standard deviation

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Effect of maxi-calcium-activated potassium channel expression modulation on tumorigenicity

Tumor size was monitored and measured over a period of 12 weeks in female nude mice (n = 5) injected subcutaneously with U-87 cells stably expressing the KCNAM1 and U-87 vector only cells. The tumor growth rate and size of the tumors in each mouse population were quite dramatic. U-87 cells overexpressing KCNMA1 formed significantly (P < 0.001) larger tumors over 28 days as compared to nontransfected control glioma cells [Figure 4]A. The tumor volume of U-87-KCNMA1 was nearly 4.5 times greater than the control group injected with nontransfected control glioma cells [Figure 4]A. We found statistically significant increase in mean tumor volume comparing transfected to control mice tumor size (1336 ± 258.36 mm3 versus 300 ± 67 mm3; nonparametric Z-test = 2.205, P = 0.0270). In a complimentary experiment, the effect of KCNMA1 downregulation in U-87 cells and U-87shKCNMA1 cells was studied by injecting these cells subcutaneously into the flanks of 4–6-week-old female nude mice (n = 5/group). Although all the control animals formed tumors (100% tumor take rate), mice injected with U-87shKCNMA1 cells had a much lower proportion of tumor take where 50% of the animals developed tumors. Even up to 3 months after tumor cell injection, the mice group with U-87shKCNMA1 did not develop tumors. The growth rate and size of the tumors in each population were quite distinct. U-87 control cells formed significantly larger tumors after 3 weeks (mean tumor volume 96 ± 17 mm3, P < 0.001) of injection, whereas the tumors that did develop from U-87shKCNMA1 cells were nearly three times smaller. Interestingly the mice that did develop tumor from U-87shKCNMA1 cells showed a reduction in mean tumor volume after 3 weeks of tumor injection and tumor-free survival after 5 weeks until 90 days [Figure 4]A. This tumorigenicity experiment clearly demonstrates the tumorigenic role of KCNMA1 in U-87 cells. To the best of our knowledge, we are the first laboratory to report the tumorigenic role of KCNMA1 in animal models. To study whether overexpression of KCNMA1 promotes cell proliferation and its downregulation attenuates it, the tumor sections from U-87 xenografts from nude mice were immunostained with anti-Ki-67 antibody and slides read by a pathologist, who was blinded to the study. The number of Ki-67 positive cells was significantly higher in sections obtained from tumors developed by KCNMA1-transfected cells compared with nontransfected cells (36.7% ± 4.2% vs. 24.9% ± 3.2%, P < 0.01). In contrast, the tumors that developed from shKCNMA1-transfected U-87 cells in mice showed remarkable decrease in Ki-67-positive cells (6.6% ± 2.5%) [Figure 4]B. The hematoxylin and eosin staining of U-87 xenografts is shown in [Figure 4]B. More importantly, tumor sections from U-87shKCNMA1 × enografts showed necrotic regions [Figure 4]B. We measured KCNMA1 mRNA levels in parental and transfected U-87 × enografts to confirm the role of KCNMA1 in cell proliferation (Ki-67-positive cells) and tumorigenicity. The observed changes in tumorigenicity and Ki-67 labeling were further verified with the qPCR results [Figure 4]C.
Figure 4: In vivo tumorgenicity. (A) Study of BKCa channel modulation and tumorigenicity; subcutaneous tumors in mice of control (a), U-87 MG KCNMA1 (b), and U-87 M shKCNMA1 groups (c). Arrows indicate the location of subcutaneous tumors. (B) Representative images of Ki-67 immunostaining (i) and hematoxylin and eosin staining – arrows indicate the Ki67-positive cells (designate growth fraction of glioma cells) tumor sections of subcutaneous tumors in mice as shown in A and (ii) of U-87 xenografts in nude mice. (C) KCNMA1 mRNA levels in parental and transfected U-87 MG xenografts in nude mice. The error bars shown in A and C represent the standard deviation. BKCa: Maxi-calcium-activated potassium, KCNMA1: Calcium-activated potassium channel subunit alpha-1

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  Discussion Top

One of the intriguing features of gliomas is their ability to diffuse and aggressively invade the normal brain. This makes complete surgical tumor resection extremely difficult. Several studies have shown the critical role of BKCa channels in glioma cell function.[4],[5],[6],[16],[17],[18],[19],[20] Specifically, studies have emphasized the importance of BKCa channels in cell size and volume regulation in glioma cell migration and invasion.[21],[22],[23],[24],[25],[26],[27],[28] Earlier, we showed that iberiotoxin inhibits BKCa channel activity in brain tumor cells.[13],[14],[20],[21] Others have also reported the functional role of KCNMA1 in brain tumor,[22],[23],[24],[25],[26] breast cancer,[27],[28],[29],[30],[31] and osteoblasts.[32] More recently, we reported that by knocking down the KCNMA1 gene with siRNA, pharmacological function of BKCa channel is decreased.[33] In the present study, we designed a strategy that allows synthesis of shRNA in a RNA polymerase III (Pol III) promoter H1 as described[11],[34],[35] to efficiently inhibit KCNMA1 and established a glioma cell line with a downregulated KCNMA1 to analyze its role in cell size, proliferation, cell cycle, invasion, transendothelial migration, and tumorigenesis. To understand the mechanisms involved in above cellular functions, we employed a shRNA-mediated silencing of KCNMA1 in U-87 cells using the RNA Pol III promoter H1 in transfected cells. RNA pol III promoter has the advantage of directing the synthesis of small, noncoding transcripts whose 3' ends are defined by termination within a stretch of 3–4 thymidines.[36],[37],[38] Thus, we avoided the KCNMA1 knockdown-mediated toxicity in U-87 cells. To study the rationale that glioma cells develop migratory potential, we overexpressed KCNMA1 in U-87 cells. Our data clearly suggest that glioma cells overexpressing KCNMA1 have a significant increase in transendothelial migration and invasion while KCNMA1 downregulation or with iberiotoxin[14] attenuates the transendothelial migration, suggesting the role of KCNMA1 in glioma cell migration [Figure 3]A. This observation is also consistent with our previous studies[7],[13],[14] as well as other reports.[31],[32],[33] The signaling pathway(s) through which BKCa channels would affect glioma cell migration and invasion has not been examined thus far. However, findings presented through our expression array experiment on transfected high-grade glioma cells suggest that BKCa channels may serve this role in glioma cells via matrix metalloproteinase-3, hypoxia-inducible protein 2, and calcium binding protein 1 genes among other genes.[33] Pharmacological inhibition of BKCa channels with either 100 nM iberiotoxin or shRNA channels greatly reduce the ability of glioma cells to transverse a transwell barrier, a condition that mimics the spatial constraints encountered by cells as they invade the narrow spaces of brain.

To elucidate whether this difference in results could be due to nonspecific effects of the drugs used to inhibit BK channels, we extended our pharmacological studies using shRNA; short strings of oligonucleotides targeted to bind and induce degradation of specific mRNAs, thus preventing protein translation and expression of the protein of interest. We inserted the shRNA sequences into a plasmid vector, which, upon transfection, results in constant transcription of the shRNA constructs in transfected cells and overcomes the need for subsequent transfections to replenish shRNA supply. Using this approach, we were able to demonstrate a 90% knockdown in BKCa channel expression. This is an improvement in transfection efficiency (100%) compared to a reported low level of transfection efficiency, which did not allow the investigators to use their transfection system for performing biological assays.[35],[36] A biological inhibition study using shRNA and CMV promoter has demonstrated 70% knockdown of BKCa channel expression in glioma cells. However, the relatively low transfection efficiency disallowed use of the system for any biological assays such as transwell migration, cell proliferation, and tumorgenicity. In fact, it has been shown that unmodified CMV promoter is not suitable for shRNA expression as reported by Bruening et al.,[36] as the loss of target gene expression may not lead to protein downregulation. It was also reported that cellular stress induced by arsenite, osmotic shock, heat shock, and hydrogen peroxide-induced stress-activated protein kinases (SARKs/JNKs) causes upregulation of the CMV promoter, leading to erroneous results.[36] In addition, the chronic silencing or knockout of KCNMA1 gene that encodes the pore forming α-subunit BKCa channels is lethal to cancer cell lines because it regulates cell proliferation, calcium, and volume regulation in the cell.[37],[38]

Using a membrane potential assay, we examined the functional activity of BKCa channels. The BKCa channel activity increased in U-87 cells transfected to overexpress KCNMA1 while the activity was significantly reduced when KCNMA1 was downregulated with shRNA. These studies unequivocally identified currents targeted by NS-1619 and iberiotoxin as currents emanating from BKCa channels. Therefore, the role of BKCa channels in invasion and migration was verified by pharmacological (with specific modulators) as well as biological agent (shRNA). Above all, the transfected U-87 cells showed significant differences in tumorigenicity. It appears that our laboratory is the first one to report the tumorigenic potential of stably-transfected high-grade glioma cell line (U-87).

Ion channels are implicated in the development of several cancers. We and others have demonstrated that BKCa channel is overexpressed in gliomas and plays a regulatory role in glioma invasion and migration.[39] Identifying the most optimal and novel biomarker for low-grade glioma transformation to high-grade glioma is ideal yet challenging because of the multifactorial nature of the disease. The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Here, we present persuasive data that KCNMA1, which encodes for the pore forming α-subunit of BKCa channel, is overexpressed in high-grade gliomas. Perhaps, the high-grade gliomas are likely to exploit tumor microenvironmental factors in brain by overexpressing KCNMA1 and gain functional advantages over normal cells. There is now evidence that KCa-Ca2+ channel complexes found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration, and capacity to develop metastases.[24],[25] The generation of KCNMA1 knockdown technique in cancer cell lines allowed us to study the role of KCNMA1 in glioma biology. We suppressed KCNMA1 expression to study migration, proliferation, and tumorigenicity using KCNMA1 knockdown in a well-studied high-grade glioma cell line U-87. In a complementary experiment, we overexpressed KCNMA1 in U-87 MG cells to perform the above-mentioned studies.

We recently reported the biological significance of up- and down-regulation of KCNMA1 on associated genes; using the Affymetrix Human Genome U133 Plus 2.0 Array. The analyses of U-87 cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion. Some of the highly downregulated genes by suppression but upregulated genes by overexpression of KCNMA1 were shown.[36] Recent studies show involvement of epigenetic changes in glioma[4] and potassium channels in the progression of cancer to a more malignant phenotype.[36] These recent findings support our rationale that KCNMA1 plays critical roles in the cellular functions of GBM.

Furthermore, we designed siRNAs for KCNMA1 and screened for the most efficient siRNA and successfully generated shRNA and stable clones in U-87 cells. The modulation of KCNMA1 expression either by overexpressing or downregulating the transcription of the gene, which was reflected in the modified protein expression levels by western blot and functional activity as the KCNMA1-transcribed BKCa channels responded differently to NS-1619. We stably overexpressed KCNMA1 in U-87 cells and found that KCNMA1 overexpression results in significant increase in cell proliferation, invasion, and functional activity in vitro. More importantly, KCNMA1 overexpression resulted in significant increase in tumorigenicity while the shRNA-mediated downregulation of KCNMA1 resulted in significant attenuation of tumorigenicity. The shKCNMA12-transfected U-87 cells exhibited reduced cell proliferation and invasion, perturbed cell cycle, and altered functional BKCa channel activity, demonstrating the critical role of KCNMA1 in glioma cell biology.

The tumorigenicity data agreed with in vitro data wherein anti-KCNMA1 shRNA in U87 cells in subcutaneously mouse model showed attenuated tumor intake as well as significantly reduced tumor growth. This study clearly demonstrated that KCNMA1 expression is essential for glioma proliferation and growth. Similar studies are planned in intracranial human glioma model developed using KCNMA1 shRNA-transfected U-87 cells. More importantly, we found increased KCNMA1 levels in high-grade human glioma samples, which supported the in vitro and in vivo experiments. We expect to screen number of human glioma samples of various grades to confirm whether KCNMA1 will ultimately serve as a transformation biomarker of high-grade gliomas. In the future, we seek to correlate KCNMA1 and BKCa channel expression levels with clinical variables associated with glioma grade and patient prognosis. Perhaps, by targeting BKCa channels, we might increase anticancer drug delivery[39] and control glioma growth. Moreover, the data available from TCGA and The Protein  Atlas More Details refers to testing of KCNMA1 and BKCa channels as a pathological marker in cancer.[40]

Financial support and sponsorship

This work was supported by the Memorial Health University Medical Center faculty funding and Georgia Cancer Coalition award in part to NN.

Institutional review board statement

The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA on July 20, 2007.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

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