Voltage-Gated Proton Channel in Human Glioblastoma Multiforme

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Letter

Voltage-gated proton channels in human glioblastoma multiforme cells Luisa Ribeiro-Silva, Fernanda Oliveira Queiroz, Annielle Mendes Brito da Silva, Aparecida Emiko Hirata, and Manoel Arcisio-Miranda ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00083 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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ACS Chemical Neuroscience

Voltage-gated proton channels in human glioblastoma multiforme cells

Luisa Ribeiro-Silva,† Fernanda Oliveira Queiroz, † Annielle Mendes Brito da Silva, † Aparecida Emiko Hirata,‡ Manoel Arcisio-Miranda†*

†

Laboratório de Neurobiologia Estrutural e Funcional (LaNEF), Departamento de Biofísica, Escola

Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil ‡

Departamento de Fisiologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São

Paulo, SP, Brasil.

Keywords: proton channel, intracellular pH, glioma

*

Correspondence to:

Manoel Arcisio-Miranda, Ph.D. Laboratório de Neurobiologia Estrutural e Funcional, Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu, 862 – 7° andar, 04023-060, São Paulo, SP, Brasil, Phone: +55-11-5576-4848 x2336, E-mail: [email protected]

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ABSTRACT Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, consequently their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ∆pH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival and migration indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM.


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Voltage-sensitive proton currents were first identified in snail neurons 1 and subsequently recognized in a number of different cell types, including dinoflagelates, neutrophils, skeletal muscle, spermatozoid, osteoclast and microglia 2–7. In 2006, a gene encoding an ion channel responsible for those voltage-sensitive proton currents was identified in human and mouse. 8,9. Structure-function studies revealed that, in contrast with other voltage-sensitive ion channels, the proton channel (HV1) lacks the classical pore domain 8. The HV1 channel consists of four transmembrane segments (S1 – S4) that correspond to the voltage-sensor domains of other voltage-gated ion channels, an acidic and prolinerich N-terminal, and a coiled C-terminal. The S4 segment has positive charges responsible to sense the voltage changes across the membrane. Also, HV1 functional unit is a dimer, formed by the association of the C-terminal of two monomers in an α-helical coiled-coil configuration. Although mechanisms of cooperativity between the monomers are described, each monomer can act independently and has its own pathway for the transport of protons 10,11. Biophysical studies show that the HV1 channels have particular properties: (i) they are remarkably selective for protons; (ii) besides being voltage dependent its open probability is also ∆pH-dependent, rising with extracellular pH increase and intracellular pH decrease; and (iii) its current can be inhibited by divalent cations such as Cd2+ and Zn2+ 12–14. The most prominent function of the HV1 channel is regulating the intracellular pH during the 'respiratory burst' of phagocytes 15. However, other diverse functions like airway acid secretion, histamine secretion, sperm motility, and brain damage in ischemic stroke have emerged 5,16–18. More recent works have also demonstrated the involvement of the HV1 channels in the progression and survival of different tumors, such as breast cancer, colorectal cancer, glioma and B-cell lymphoma 19–22. In these types of cancer, the expression of HV1 channels has been implicated in more malignant tumors and associated with a worse prognosis. Furthermore, it has been shown that a truncated isoform of the HV1 channel is more expressed in malignant B cells 22. This shorter isoform confers some advantages to cancer cell migration and proliferation.



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Glioblastoma multiforme (GBM) is the most common and lethal human brain tumor, and is characterized by extensive cell migration and invasion leading to a poor prognosis 23. It is known that hypoxia can increase tumor aggressiveness and cause radiation and chemotherapy resistance, and this effect can be mediated by ion channels 24. The hypoxic ambient makes tumor cells have a high glycolytic activity and produce acidic metabolites, consequently cells need to extrude more protons in order to avoid intracellular acidification. We hypothesized that the HV1 channels may be one of the pathways involved in intracellular pH regulation in GBM. Herein we report the functional expression, contribution to pH regulation and importance to cell survival and migration of the HV1 channels in T98G cells, a human GBM cell line with astrocytic origin. To detect whether HV1 channels are functionally expressed in GBM cells, we performed wholecell patch clamp in T98G cells and mouse primary astrocytes. Under ∆pH of ~2 units and at +40 mV applied-voltage, the probability to find the HV1 channels in its open and conductive state is high 25. Thus, a single pulse of 2s from -80 mV to +40 mV was used and an outward proton current was elicited in T98G cells, but not in astrocytes (Figure 1). As T98G has an astrocytic origin 26, the functional expression of proton channels is one possible adaptation to a transformed and more malignant tissue. Moreover, Wu et al. have also demonstrated that mouse astrocytes do not express HV1 channels and do not show outward proton currents 18.


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Figure 1. Functional expression of HV1 in Glioblastoma Multiforme. (A) Outward proton current induced by a single 2s voltage pulse from -80 to +40 mV in a whole-cell voltage clamp configuration are observed in T98G cells, but not in astrocytes. (B) Average current densities at steady-state in T98G cells (n = 14) and astrocytes (n = 4). Data are expressed as mean ± S.E.M. * p < 0.05. unpaired Student’s t test.

Two main properties characterize the HV1 channels, being blocked by divalent cations and opening dependent of the pH gradient. To further verify that the proton currents observed in T98G are conducted by HV1 channels, these properties were explored in electrophysiological recordings applying pulses of 2s from -80 mV to +80 mV (Figure 2A). Addition of 100 µmol L-1 ZnCl2 inhibited the proton current by 80% (Figure 2B), in consonance with previous reports 14,18. Furthermore, altering the pH of the pipette solution from 5.5 to 7.4, and maintaining the bath solution pH at 7.4, shifted the currentvoltage curve to more positive voltages (Figure 2C). Additionally, for ∆pH of 1.9 the reversal potential



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was -53.3 ± 5.7 mV (data not shown), its difference from the predicted for a proton selective membrane from the Nernst potential can be accounted for by proton depletion 25.

Figure 2. Biophysical and pharmacological characterization of outward proton currents in T98G. (A) Currents evoked by 20 mV steps from -80 mV to +80 mV in T98G cells with pHpipette = 5.5 (control), after addition of 100 µmol L-1 ZnCl2 in the bath solution and with pHpipette = 7.4. All registers were done in the whole-cell voltage clamp configuration and pHbath = 7.4. (B) Mean I-V relationship of outward, steady state proton currents decreased after addition of ZnCl2 (n = 4) in relation to control (n = 4). (C) Changing the pHpipette from 5.5 (n = 15) to 7.4 (n = 8) shifted the mean I-V relationship to the right. Data are expressed as mean ± S.E.M.

Tumors are known to have a more acidic extracellular pH (~ 6.8) than normal tissue 27, because either their cells produce and extrude more protons, and/or the extracellular acid is not as efficiently cleared from their microenvironment. There are several pathways from which cells can export protons, such as the Na+/H+ exchanger or the proton pumps. It has been shown that the HV1 channel can also serve as one of these pathways in tumor cells 19,21. To determine if HV1 channels also contribute to intracellular pH (pHi) regulation in T98G, cells were loaded with the pH-sensitive fluorescent probe, 6 ACS Paragon Plus Environment

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BCECF-AM and pHi was measured in the presence or not of 100 µmol L-1 ZnCl2. Blocking of the HV1 channel by Zn2+ significantly reduced the pHi from 7.25 ± 0.01 (n = 235 cells) to 6.95 ± 0.03 (n = 242 cells) in a sodium-free solution (Figure 3), however in presence of sodium Zn2+ didn’t affect pHi. We hypothesized that this may be due to an upregulation of Na+/H+-exchanger 28, which activity might be sufficient to maintain cell alkalinity in the presence of sodium and Zn2+. Thus, suggesting that HV1 channels extrude protons in order to prevent intracellular acidification in T98G cells under certain conditions.

Figure 3. HV1 regulates intracellular pH in GBM. T98G cells were loaded with the pH-sensitive dye BCECF-AM and the fluorescence ratio (F490/F440) measured. (A) Representative ratio images of T98G cells in the absence (top) and presence of 100 µmol L-1 ZnCl2 (bottom) in the bath solution containing (left) or not sodium(right). (B) Mean cytosolic pH values of T98G cells with (clear column) or without blocking of HV1 channels by ZnCl2. Data are expressed as mean ± S.E.M. * p < 0.05. unpaired Student’s t test.

The lower acidic extracellular pH in tumor tissue gives an advantage in proliferation and growth to cancerous cells compared to normal cells 29,30. In sight of this, we analyzed if the HV1 channels contributes to survival of human GBM. First, astrocytes and T98G cells were incubated for 24 hours with various concentrations of ZnCl2 and their viability was tested with the MTT assay (Figure 4A). 7 ACS Paragon Plus Environment


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Zn2+ exhibited a striking selectivity to tumor cells, significantly reducing cell viability in T98G in a concentration dependent manner, and not affecting astrocytes. Cell size (FSC-H) and granularity (SSCH) were analyzed by flow cytometry. Cells treated with high concentrations of ZnCl2 presented an increase in cell granularity (Figure 4C), possibly due to increased autophagosome formation. In addition, staining with Propidium Iodide (PI), a cell death marker, showed a decrease in the percentage of live cells with increasing concentrations of ZnCl2 (Figure 4B). Taken together, these data may suggest that intracellular acidification caused by blocking of HV1 channels promotes cell death in T98G cells.

Figure 4. Effect of ZnCl2 in the survival and viability of T98G cells. (A) Percentage of viable cells of T98G and astrocytes after 24 hours exposure to various ZnCl2 concentrations, as assayed by the MTT test. Data are expressed as mean ± S.E.M. (B) Increasing concentrations of ZnCl2 showed a higher percentage of cells stained by Propidium Iodide. (C) Cell morphology analysis by flow cytometry exhibited an increase of granularity with high concentrations of ZnCl2. Data are representative of three independent assays. 8 ACS Paragon Plus Environment

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The main feature of malignancy in a tumor is the ability to migrate and invade other tissues. Since it has already been shown that the HV1 channel regulates migration of colorectal and breast cancer cells 20,21, we next assessed if these channels also participate in this process in T98G cells, using a modified version of the Boyden chamber method. As figure 5 shows, after 16 hours, 100 µmol L-1 ZnCl2 reduced significantly the number of migrated cells (Figure 5), which may be due to decreased cell viability by blocking of HV1 channels, further studies are needed to rule out this possibility. This data may suggest that HV1 channels contribute to the malignant characteristic of GBM cells.



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Figure 5. Effect of ZnCl2 in cell migration of T98G. (A) Representative images of migrated cells using a modified version of the Boyden chamber assay and stained with Hoechst 33342, in the presence or not of 100 µmol L-1 ZnCl2 for 16 hours. (B) ZnCl2 reduced significantly the percentage of migrated cells. Data are expressed as mean ± S.E.M. and are representative of three independent assays. * p < 0.05. unpaired Student’s t test.

We have provided evidence that HV1 is functionally expressed in glioblastoma multiforme cells, by recording voltage-sensitive proton current and showing Zn2+ blockage and current ∆pH-dependent shift. Furthermore, the expression and activity of the HV1 channel may contribute to pHi regulation, cell survival, and migration of human T98G cells. Thus, as suggested in other types of cancer, the HV1 channels may contribute to the pathology of glioblastoma multiforme tumors, being correlated with metastatic tendency and poor prognosis. Therefore, the HV1 channel may be a new therapeutic target for glioblastoma multiforme tumors.

EXPERIMENTAL SECTION Mouse primary astrocyte culture We obtained primary mouse astrocytes from 0-1 day old C57BL/6 mice pups obtained at Centro de Desenvolvimento de Modelos Experimentais para Biologia e Medicina (CEDEME;Unifesp). All animal experiments were carried out in compliance with the NIH-Guidelines for the care and use of laboratory animals, and approved by the Animal Ethics Committee of UNIFESP. Briefly, the pup brains were removed from the skull, and the cerebellum, olfactory bulbs, and meninges removed. The remaining cerebral cortex was minced and washed three times with Hank's balanced salt solution (HBSS) with 10% fetal bovine serum (FBS), three times with HBSS without FBS and once with versene solution (in mmol L-1: 2.7 KCl, 1.8 KH2PO4, 137 NaCl, 10 Na2HPO4, 0.68 EDTA). The tissue was digested in 0.25% trypsin-EDTA at 37ºC for 25 min. Afterwards, the washes 10 ACS Paragon Plus Environment

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were repeated once again. To achieve maximum dissociation, the tissue was passed through firepolished pasteur pipettes with different tip diameters. The cells were centrifuged at 1500 rpm for 4 min. The supernatant was discarded, while the pellet was resuspended in HBSS with FBS and plated in 75cm2 tissue-culture flasks pre-treated with 50 µg mL-1 poli-L-lysine. The culture was incubated at 37ºC and 5% CO2 in DMEM/F12 medium (50%:50%; v:v) supplemented with 10% FBS and 100 U mL-1 penicillin, 100 mg mL-1 mg/ml streptomycin. Fourteen days after plating, the flask was agitated at 200 rpm on an orbital shaker overnight to dislodge contaminating oligodendrocytes and microglia. Astrocytes were trypsinized and plated onto glass coverslips.

T98G cell culture The human glioblastoma multiforme cell line T98G (kindly provided by Dr. Fernando A. Oliveira) was cultured at 37 ºC in 5% CO2 with DMEM medium (Sigma Aldrich, USA), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg mL-1 streptomycin and 2 mmol L-1 Lglutamine.

Electrophysiological recordings Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Currents recordings were made with an Axoclamp 200B patch-clamp amplifier. Data were filtered at 5 kHz and sampled at 20 kHz. Patch electrodes of 5- 10 MΩ were fabricated from glass capillaries (Perfecta, Brasil). The electrodes were filled with the following solution (in mmol L-1): 70 NMDG, 1 EGTA, 150 MES, 70 glucose (270 mOsm), pH 5.5. For pipette solution with pH 7.4, MES was replaced for HEPES. The extracellular solution contained (in mmol L-1): 1 CaCl2, 100 HEPES, 1 MgCl2, 75 NMDG, 70 glucose (288 mOsm), pH 7.4. The solutions were adjusted using MES for pH 5.5 and using CsOH for pH 7.4. The whole-cell series resistance was compensated by 70%.



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Intracellular pH measurements Intracellular pH measurements were made with the pH-sensitive probe BCECF-AM (Molecular Probes, USA). Cells were incubated with 2 µmol L-1 BCECF-AM in DMEM medium at 37 ºC for 30 min. After loading, the cells were washed three times with HEPES buffer and remained in the same solution. The HEPES buffer sodium-free contained (in mmol L-1): 1 CaCl2, 100 HEPES, 1 MgCl2, 75 NMDG, 70 glucose, pH 7.4. The HEPES buffer with sodium contained (in mmol L-1): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 20 HEPES, pH 7.4. The dye was excited at 490 and 440 nm wavelengths and emission was recorded at 535 nm wavelength using a TIRF microscope (Leica Microsystems, Germany). The exposure time was 100 ms and pictures were taken every 10 sec. Calibration of fluorescence versus pH was performed by equilibration of external and internal pH with 10 µmol L-1 nigericin in a high K+ buffer with a range of pH from 5.5 to 8.5.

Cell viability assay Cell viability was assayed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 31. 1x104 cells per well were plated in 96-well plates and cultured overnight. Following, the medium were replaced by a range of ZnCl2 concentrations, and the cells were cultured for 24h. After the incubation period, 0.1 mg mL-1 of MTT was added to each well and the plates were further incubated at 37 ºC for 2h. The supernatants were carefully removed and 100 µL of dimethyl sulfoxide (DMSO, Synth Lab, Brasil) was added to each well. The optical density was measured at 570 nm with a microplate reader (Bio Tek Instruments, USA).

Flow Cytometry Cell death assay was performed using Propidium Iodide staining. For this purpose, T98G cells were seeded on 24-well plates at a density of 1x105 cells/well. 24h after plating the medium was replaced, including a range of ZnCl2 concentrations. Following 24h of incubation the cells were 12 ACS Paragon Plus Environment

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trypsinized with trypsin-EDTA at 37°C for 2 min. Then, 10% FBS medium was added to terminate the trypsinization process, and the cells were gently pipetted up and down to avoid the formation of cell clumps. The cells were transferred into a 1.5 mL tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, then the cells were washed with 500 µL binding buffer (in mmol L-1: 10 Hepes, 140 NaCl and 2.5 CaCl2, pH 7.4) and centrifuged again. Cells were resuspended in 50µL of binding buffer and stained for 5 minutes with 0.5 µL propidium iodide solution (1 mg mL-1 diluted in water, Sigma-Aldrich, USA). Finally, 300 µL of binding buffer were added and cells were immediately analyzed using FACS-Calibur flow cytometer (Becton Dickinson, USA). A total of 10,000 cells were acquired per sample and the data were analyzed with FlowJo V10 software (Tree Star, USA).

Migration assay In vitro migration assay was performed using transwell inserts in 24 well plates. A total of 2x104 cells in 100 µL DMEM were seeded on the upper chamber of a 33.6 mm2-transwell with 8.0 µm-pore polycarbonate membrane inserts (Greiner Bio-One, Austria). Cells were allowed to adhere for 4 h before treatment with 100 µmol L-1 ZnCl2. DMEM (600 µL) with 5% FBS was added to the outer chamber as a chemoattractant. The plates were incubated for 16 h at 37 ºC in 5% CO2. Cells in the upper chamber were gently removed with a cotton swab, followed by staining with Hoechst 33342 (Molecular Probes, USA) for 10 min. Each experiment was conducted three independent times.

Statistical analysis Results are presented as media ± standard error of mean. Parametric statistics tests were used ANOVA of two way with Bonferroni post test or t Student test. In all tests P < 0.05 was considered for statistical difference to be considered.



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ACKNOWLEDGMENT This study was supported by research grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Processo Fapesp n° 2010/52077-9 to M.A.-M. and Processo Fapesp n° 2011/52099-5 to A.E.H.). L.R.-S. is a Fapesp fellowship recipient (Processo Fapesp n° 2013/26005-9). F.O.Q. and A.M.B.S. are Capes fellowship recipients.

AUTHOR CONTRIBUTIONS L.R.-S., F.O.Q., and A.M.B.S. performed and analyzed the experiments. M.A.-M. and A.E.H. supervised the project. L.R.-S, A.M.B.S., and M.A-M. wrote the manuscript. All authors reviewed the manuscript.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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Voltage-Gated Proton Channel in Human Glioblastoma Multiforme

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