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The Pivotal Role of Intracellular Calcium in Oxaliplatin-Induced Inhibition of Neurite Outgrowth but Not Cell Death in Differentiated PC12 Cells Miki Takeshita,† Yoshiko Banno,*,‡ Mitsuhiro Nakamura,† Mayuko Otsuka,† Hitomi Teramachi,† Teruo Tsuchiya,† and Yoshinori Itoh§ †
Department of Pharmacy Practice and Science, Gifu Pharmaceutical University, Gifu 501-1196, Japan Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu 501-1193, Japan § Department of Pharmacy, Gifu University Hospital, 1-1 Yanagido, Gifu 501-1194, Japan ‡
ABSTRACT: The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGFmediated increase in the intracellular calcium concentration [Ca2+]i. A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca2+]i. The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca2+]i and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin.
’ INTRODUCTION Oxaliplatin is a third-generation platinum-based anticancer agent that displays reduced renal toxicity and gastrointestinal side effects1 and is effective for treating a variety of cancers, including colorectal,24 ovarian,5 cervical,6 gastric,7 esophageal,8 nonsmall cell lung,9 pancreatic,10 germ cell,11 and gall bladder cancer.12 In particular, oxaliplatin is highly effective for treating colorectal cancer when used in combination with fluoropyrimidine analogues such as 5-fluorouracil and capecitabine; oxaliplatin has emerged as a key drug in treating advanced colorectal cancer. However, during the course of oxaliplatin chemotherapy, the patient frequently develops peripheral neuropathy, which decreases patient quality of life and leads to dose reductions or the discontinuation of chemotherapy.1316 Oxaliplatin-induced peripheral neuropathy includes acute cold-induced dysesthesia and chronic cumulative dose-related paralysis.15,16 Although the precise mechanisms underlying the neurotoxicity of oxaliplatin remain to be clarified, in vitro studies have reported a role for sodium channels in the oxaliplatin-induced enhancement of neuronal excitability attributed to the modulation of voltage-gated sodium channels1719 and the reduction of the amplitude of voltage-gated sodium currents via oxalate-mediated intracellular calcium chelation20 or without effects on intracellular calcium.21 r 2011 American Chemical Society
Altered cellular metabolism and axoplasmic transport after the accumulation of oxaliplatin in sensory neurons is a possible mechanism underlying oxaliplatin-induced chronic neuropathy.22 Several agents have been examined for preventative effects against oxaliplatin-induced peripheral neuropathy in both human and experimental animals, and some of them such as N-acetylcysteine and reduced glutathione exert significant protective effects on peripheral nerves against chronic oxaliplatin therapy.2331 Conversely, Gamelin et al.32 reported that both acute and chronic peripheral neuropathy are markedly reduced by the intravenous infusion of a mixture of calcium gluconate and magnesium sulfate (Ca/Mg) before and after oxaliplatin injection. Oxaliplatin gets accumulated in the cells, where it is biotransformed into oxalate and dichloro-(1,2-diaminocyclohexane)platinum [Pt(DACH)Cl2].33 They speculated that the oxaliplatin metabolite oxalate is responsible for the acute neuropathy induced by oxaliplatin, as oxalate chelates calcium and magnesium ions in sensory neurons. However, Hochster et al.34 claimed that oxaliplatin chemotherapy patients who received Ca/Mg exhibited a significantly lower response rate than did the placebo group patients. In contrast, Received: April 19, 2011 Published: October 07, 2011 1845
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Chemical Research in Toxicology Gamelin et al.35 reported no differences in the response rate (50% in Ca/Mg vs 53% in placebo) or in the progression-free (12 vs 12 months) and overall survival (25.1 vs 25.5 months). They also reported a significantly lower frequency and grade of oxaliplatin neurotoxicity (5 vs 24% for grade 3 symptoms), suggesting that Ca/Mg is effective in reducing oxaliplatin-induced neurotoxicity, although the underlying mechanism is not clear. Neurite extension and retraction are important processes in the establishment of neuronal networks during development. A number of neuronal models have been used for mechanistic studies and as screens for detecting potential neurotoxicants. In particular, rat pheochromocytoma PC12 cells have been widely used in both neurobiological and neurotoxicological studies as a model of neuronal differentiation. An important feature of PC12 cells is that they respond to nerve growth factor (NGF) with a drastic change in phenotype and acquire a number of properties characteristic of sympathetic neurons.36 Differentiation of PC12 cells is most often assessed by semiquantitative or quantitative morphological methods including determining the extent of neurite growth or neurite length. In this study, to elucidate the characteristics of oxaliplatin neurotoxicity and the molecular mechanisms underlying the protective effects of Ca/Mg, we investigated the effects of Ca/Mg on the oxaliplatin-induced inhibition of NGFinduced neurite outgrowth and extension in PC12 cells. We also determined whether Ca/Mg treatment affects the cell death induced by oxaliplatin in PC12 cells as well as in the human colon cancer cell line HCT-116.
’ EXPERIMENTAL PROCEDURES Materials. U0126 and bisindolylmaleimide I (BIM) were purchased from Watman Calbiochem (Nottingham, United Kingdom). BAPTA/AM was obtained from Sigma Chemical Co. (St. Louis, MO). Oxaliplatin (Elplat injection 50 mg) was obtained from Yakult Co., Ltd. (Tokyo, Japan). 3-(4,5-Dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque (Kyoto, Japan). NGF was purchased from Alomone Laboratories (Jerusalem, Israel). Quest Fluo-8AM was obtained from ABD Bioquest (Sunnyvale, CA). Rabbit polyclonal antibody against Tyr204-phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was obtained from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibodies against ERK1/2 and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody against growth-associated protein-43 (GAP-43) was purchased from Chemicon International, Inc. (Temecula, CA). Antirabbit IgG and antimouse IgG antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare (Tokyo). Calcium gluconate and magnesium sulfate were obtained from Sigma Chemical Co. Ca/Mg solution was prepared as a mixture of calcium gluconate and magnesium sulfate. All other reagents were obtained from standard commercial sources. Cell Culture and Differentiation Assay. The PC12 cell line was a gift from Dr. Y. Sugimoto (Shirakawa Institute of Animal Genetics, Fukushima, Japan). We cultured the PC12 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 5% horse serum, 100 U/mL penicillin G, and 100 U/mL streptomycin, as described previously.37 The cells were grown to the subconfluent stage at 37 °C in a humidified atmosphere containing 5% CO2. The PC12 cells were plated (0.75 105 cells/dish) onto 35 mm tissue culture plates coated with collagen type IV (BD BioCoat, Bradford, MA) and grown in DMEM. Two days after plating, the medium was replaced with DMEM containing 1.0% FBS, and cells were cultured for 6 h. A concentrated stock of NGF was then added to the 1.0% FBS culture medium to give a final concentration of 50 ng/mL.
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After 48 h of incubation, various inhibitors were added, and the cells were incubated for an additional 24 h. Cells exposed to vehicle alone (1.0% FBS culture medium) were used as controls. Morphometric analysis was performed on digitized images of live cells taken under phase-contrast illumination with an Olympus IMT2 inverted microscope linked to a digital CCD camera (Tokyo). Images of three fields per well were taken with a mean of 300 cells per field. The neurite length was measured by manually tracing the length of the distance between the cell periphery and the tip of the longest neurite for each cell in a field under the microscope. Data from the three fields in each well were pooled. Experiments were repeated at least three times using cultures prepared on different days.
Determination of the Intracellular Calcium Concentration ([Ca2+]i). PC12 cells (5 105 cells/mL) were cultured for 24 h in a 12well plate. Afterward, the growth medium was removed, and the cells were loaded in the washing buffer solution composed of 134 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4) and supplemented with 1 μM Quest Fluo-8AM ester at 37 °C for 1 h. After incubation, cells were washed three times in the same dye-free buffer. The dishes were transferred to fluorescence microplate readers (SpectraMaxM3/M4, Molecular Devices) and maintained at 37 °C. Then, NGF (50 ng/mL) was added to the buffer, and fluorescence of Fluo-8 was monitored at 10 s intervals using filters set to 490 nm excitation/525 nm emission. To assess the inhibition of the NGFevoked increase in [Ca2+]i, PC12 cells were pretreated with oxaliplatin (0.5 μM), oxalate (0.5 μM), or BAPTA/AM (5 μM) for 30 min after loading with Quest Fluo-8AM, and the responses of cells to NGF (50 ng/mL) alone were measured and compared to those in drugtreated cells in separate experiments. Ionomycin (1 μM) was added at the end of each experiment to assess the fluorescence of the calciumsaturated probe in each well. [Ca2+]i data are expressed as the fluorescence of the calcium levels elevated by NGF relative to the fluorescence of the saturated calcium levels in each well. To visualize the increase in [Ca2+]i, cells were loaded with 1 μM Quest Fluo-8AM in serum-free DMEM for 1 h. The cells pretreated with or without oxaliplatin (0.5 μM), oxalate (0.5 μM), and/or Ca/Mg (5.4 mM calcium gluconate/1.5 mM magnesium sulfate) were washed twice with the washing buffer and then stimulated with NGF (50 ng/ mL). A fluorescence analysis was performed with a fluorescence microscope (BIOREVO, Keyence) linked to a digital CCD camera using fluorescent channel (480 nm excitation/520 nm emission). The number of fluorescent cells and total cells were determined for 2 min at 10 s intervals after NGF stimulation in three fields per well by using automatic cell counting software (BZ-H1C). The number of cells that exhibited a fluorescent intensity of 150 or more was counted, and the number of fluorescent cells was expressed as a percentage of the total cells. In control cells without NGF stimulation, the fluorescent intensity was 50 or less. Western Blot Analysis. After being grown to subconfluence in growth medium in 60 mm dishes, the PC12 cells were incubated for 2 h in serum-free DMEM and pretreated with individual inhibitors for 30 min before the addition of NGF (final concentration, 50 ng/mL). At the indicated time, the cells were harvested in ice-cold lysis buffer (1% Nonidet P-40, 0.5% sodium cholate, 1% sodium dodecyl sulfate, 1 mM ethyleneglycol-N,N,N0 ,N0 -tetraacetic acid, 1 mM O,O0 -bis(2aminoethyl)-ethyleneglycol-N,N,N0 ,N0 -tetraacetic acid, 150 mM NaCl, 20 mM HEPES, 3 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 20 μg/mL leupeptin, 20 mM β-glycerophosphate, 1 mM sodium fluoride, and 1 mM sodium O-vanadate; pH 7.4) and then sonicated. Protein concentrations were determined using the Bradford protein assay reagent (BioRad, Hercules, CA) with bovine serum albumin used as a standard. The total cell lysates (1050 μg of protein) were subjected to electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA). 1846
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Figure 1. Representative photographs (A) and data on neurite length (B and C) and [Ca2+]i (D) showing the inhibitory effects of oxaliplatin (L-OHP), oxalate, and BAPTA/AM in PC12 cells. The differentiated PC12 cells were incubated with or without 0.5 μM L-OHP or oxalate (A) or 0.11 μM L-OHP (B) or oxalate (C) for 24 h, and then, phase-contrast images were photographed. The neurite length was measured from each photograph using Axio Vision software. Data represent the means ( SEMs of 56 experiments. **P < 0.01 as compared with NGF (+) alone. (D) The undifferentiated PC12 cells were pretreated with or without L-OHP (0.5 μM), oxalate (0.5 μM), or BAPTA/AM (5 μM) for 30 min, incubated with Fluo-8AM (1 μM) for 1 h, and stimulated with 50 ng/mL NGF. The fluorescence of free Fluo-8 was determined at the indicated times. The increases in the free calcium concentration are expressed as arbitrary units of fluorescence (dF) (left side panel) and relative to the fluorescence values of the calcium-saturated probe as assessed using ionomycin (right side panel). Each point represents the mean ( SEM of 56 experiments. The membranes were then blocked using 5% bovine serum albumin. The phosphorylation of ERK1/2 and GAP-43 was measured by immunoblotting with antibodies specific to the phosphorylated and total proteins. After repeated washes, the bound antibodies were detected using the ECL Western blotting detection system (GE Healthcare UK Ltd., Buckinghamshire, United Kingdom). The protein band density was determined with a densitometer (Atto Densitograph, version 2.5; Atto, Ltd., Tokyo). Cell Viability Measurement. To measure cell viability, NGFdifferentiated PC12 cells or HCT-116 cells (American Type Culture Collection, Manassas, VA) were plated at a density of 0.5 104 cells per well in 96-well plates. The cells were incubated with oxaliplatin in the absence or presence of Ca/Mg (3.6 mM calcium gluconate/1 mM magnesium sulfate) for 48 h. Cell viability was assessed using standard MTT assays, according to the manufacturer's instructions.
Apoptosis Assay. Apoptosis was assessed by Annexin-V staining using a commercial apoptosis assay kit (MEBCYTO-apoptosis kit, Medical & Biological Lab. CO., Ltd., Nagoya, Japan). The cells were cultured on plastic plates at 1.0 104 cells/plate. At 48 h after seeding, the cells were incubated with oxaliplatin in the absence or presence of Ca/Mg for 48 h, after which the cells were washed twice with phosphate-buffered saline and incubated in 100 μL of binding buffer containing fluorescent isothiocyanate (FITC)-labeled Annexin-V (10 μL) for 30 min in the dark. The cells were then washed twice with binding buffer and visualized at an excitation wavelength of 490 nm and an emission wavelength of 615 nm using a fluorescent microscope. Statistical Analyses. Data are expressed as the mean ( SEM. The significance of differences was determined by stepdown one-way ANOVA, followed by the StudentNewmanKeuls test for comparisons with the 1847
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control using the ySTAT (2006) software (Medical Book Publication Co. Ltd., Tokyo, Japan). A p value of
