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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 2  |  Page : 60-67

Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells


1 Department of Medicine, Chang Bing Show Chwan Memorial Hospital, Changhua County, Taiwan
2 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung; Department of Pharmacy, Tajen University, Pingtung, Taiwan
3 Department of Surgery, Kaohsiung Veterans General Hospital; Department of Physical Therapy, Shu-Zen Junior College of Medicine and Management, Kaohsiung, Taiwan
4 Department of Nursing, Tzu Hui Institute of Technology, Pingtung, Taiwan
5 Department of Endocrinology and Metabolism, Kaohsiung Veterans General Hospital Tainan Branch; Chung Hwa University of Medical Technology, Tainan, Taiwan
6 Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus; Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital Chiayi Branch, Puzi City, Chiayi County, Taiwan
7 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Date of Submission07-Feb-2020
Date of Acceptance18-Mar-2020
Date of Web Publication27-Apr-2020

Correspondence Address:
Dr. Chiang-Ting Chou
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus, Puzi City, 61363, Chiayi County
Taiwan
Dr. Chung-Ren Jan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362
Taiwan
Dr. Lyh-Jyh Hao
Department of Endocrinology and Metabolism, Kaohsiung Veterans General Hospital Tainan Branch, Tainan 71051
Taiwan
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Source of Support: This work was supported by RD.105001 from Chang Bing Show Chwan Memorial Hospital, Changhua County 50544, Taiwan., Conflict of Interest: None


DOI: 10.4103/CJP.CJP_14_20

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  Abstract 

Tectorigenin, a traditional Chinese medicine, is isolated from the flower of plants such as Pueraria thomsonii Benth. It is an O-methylated isoflavone, a type of flavonoid. Previous studies have shown that tectorigenin evoked various physiological responses in different models, but the effect of tectorigenin on cytosolic-free Ca2+ levels ([Ca2+]i) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca2+ signal transduction and viability in Madin–Darby Canine Kidney (MDCK) renal tubular cells. [Ca2+]i in suspended cells were measured by applying the fluorescent Ca2+-sensitive probe fura-2. Viability was explored by using water-soluble tetrazolium-1 as a fluorescent dye. Tectorigenin at concentrations of 5–50 μM induced [Ca2+]i rises. Ca2+ removal reduced the signal by approximately 20%. Tectorigenin (50 μM) induced Mn2+ influx suggesting of Ca2+ entry. Tectorigenin-induced Ca2+ entry was inhibited by 10% by three inhibitors of store-operated Ca2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca2+]i rises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca2+]i rises. Tectorigenin at concentrations between 10 and 60 μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl did not reverse tectorigenin's cytotoxicity. Our data suggest that, in MDCK cells, tectorigenin evoked [Ca2+]i rises and induced cell death that was not associated with [Ca2+]i rises. Therefore, tectorigenin may be a Ca2+-independent cytotoxic agent for kidney cells.

Keywords: Ca2+ signal transduction, Madin-Darby Canine Kidney, tectorigenin, viability


How to cite this article:
Cheng HH, Liang WZ, Liao WC, Kuo CC, Hao LJ, Chou CT, Jan CR. Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells. Chin J Physiol 2020;63:60-7

How to cite this URL:
Cheng HH, Liang WZ, Liao WC, Kuo CC, Hao LJ, Chou CT, Jan CR. Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells. Chin J Physiol [serial online] 2020 [cited 2020 May 29];63:60-7. Available from: http://www.cjphysiology.org/text.asp?2020/63/2/60/283346

He-Hsiung Cheng, Wei-Zhe Liang and Wei-Chuan Liao contributed equally to this work.



  Introduction Top


Tectorigenin, an isoflavoloid compound, is isolated from the dried flower of plants such as Pueraria thomsonii Benth.[1],[2],[3] It is an O-methylated isoflavone, a type of flavonoid. The cellular action of tectorigenin includes modulation of adipogenic differentiation and adipocytokines secretion via peroxisome proliferator-activated receptor gamma and IκB kinase/nuclear factor-κB signaling pathways,[4] suppression of the inflammation of lipopolysaccharide (LPS)-evoked acute lung injury in mice,[5] repression of palmitate-caused endothelial insulin resistance via targeting reactive oxygen species-associated inflammation and insulin receptor substrate 1 pathway,[6] enhancement of miR-338 expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis,[7] and blockade of interferon-gamma/LPS-induced inflammatory responses in murine macrophage RAW 264.7 cells.[8]

Regarding cancer research, tectorigenin has been shown to inhibit the inflammation-evoked epithelial-mesenchymal transition in a co-culture model of human lung carcinoma,[9] sensitize paclitaxel-resistant human ovarian cancer cells,[10] cause anti-proliferative on hepatic stellate cells,[11] inhibit epidermal growth factor-induced activation of phospholipase C (PLC) proliferation in human epidermoid carcinoma cells,[12] induce apoptosis in human promyelocytic leukemia HL-60 cells,[13] and suppress osteosarcoma cell migration.[14] Inin vivo studies, tectorigenin inhibited tumor growth in systemic malignancies.[15] Besides cytotoxic effects in various models, the protective effect of tectorigenin on oxidative stress induced by hydrogen peroxide was shown.[16] Tectorigenin also showed therapeutic effects on liver fibrosis in rats.[17] However, the effect of tectorigenin on physiological responses in renal tubular cells is still unclear.

In terms of tectorigenin-induced Ca2+ signaling, tectorigenin was shown to evoke [Ca2+]i rises in HepG2 human hepatocellular carcinoma cells[18] and isolated rat hepatocytes.[19] However, these two studies did not explore the pathways underlying the Ca2+ signal. Ca2+ is a special cation in that it plays a key second messenger role in almost every cell type found. Many cell responses are triggered by a transient change in cytosolic-free Ca2+ levels ([Ca2+]i), such as contraction, fertilization, hormone and fluid secretion, muscle contraction, apoptosis, protein activation, and neural plasticity.[20] In resting cells, [Ca2+]i is usually maintained at 50–100 nM.[21] [Ca2+]i may rise via Ca2+ entry from extracellular solution and/or Ca2+ release from intracellular stores such as the endoplasmic reticulum, mitochondria, and lysosome.[21],[22] Several pathways are involved in Ca2+ influx such as receptors and Ca2+ channels including the store-operated Ca2+ entry channels.[21],[22] Activation of PLC can trigger the production of inositol 1, 4, 5-trisphosphate (IP3) that releases Ca2+ via the IP3 receptors on the endoplasmic reticulum membrane.[21],[22] Given the importance of Ca2+ signaling, it is crucial to unveil the pathways of the Ca2+ signal evoked by an agonist.

The aim of this study was to examine whether tectorigenin changed Ca2+ signal transduction and viability in Madin–Darby Canine Kidney (MDCK) renal tubular cells. The MDCK cells are commonly applied for kidney studies. Literature shows that [Ca2+]i rises can be induced in MDCK cells stimulated by various compounds such as angiotensin II,[23] diindolylmethane,[24] and thymol.[25] The Ca2+-selective fluorescent dye fura-2 was applied as a probe to detect [Ca2+]i. The effect of tectorigenin on viability was measured by using water-soluble tetrazolium-1 (WST-1), a fluorescent indicator of mitochondrial activity.


  Materials and Methods Top


Chemicals

Tectorigenin [Figure 1]a and other reagents were purchased from Sigma-Aldrich® (St. Louis, MO, USA) unless otherwise indicated. The reagents for cell culture were purchased from Gibco® (Gaithersburg, MD, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/AM) and 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM) were purchased from Molecular Probes® (Eugene, OR, USA).
Figure 1: Effect of tectorigenin (a) on [Ca2+]i in fura-2-loaded cells. (b) Tectorigenin was added at 25 s. The concentration of tectorigenin was indicated. The experiments were performed in Ca2+-containing medium. Y axis is the [Ca2+]i induced by tectorigenin in Ca2+-containing medium. (c) Effect of tectorigenin on [Ca2+]i in the absence of extracellular Ca2+. Tectorigenin was added at 25 s in Ca2+-free medium. Y axis is the [Ca2+]i rise induced by tectorigenin in Ca2+-free medium. (d) Concentration-response plots of tectorigenin-induced [Ca2+]i rises in the presence or absence of extracellular Ca2+. Y axis is the percentage of the net (baseline subtracted) area under the curve (25–250 s) of the [Ca2+]i rises induced by 50 µM tectorigenin in Ca2+-containing medium. Data are mean ± stand error of the mean of three independent experiments. *P < 0.05 compared to open circles.

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Cell culture

MDCK cells obtained from Bioresource Collection and Research Center (Taiwan) were cultured in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified air containing 5% CO2.

Solutions used in [Ca2+]i measurements

Ca2+-containing medium (pH 7.4) had 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 mM glucose. Ca2+-free medium contained similar chemicals as Ca2+-containing medium except that CaCl2 was replaced with 0.3 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM MgCl2. Tectorigenin was dissolved in absolute alcohol as a 0.1 M stock solution. The other chemicals were dissolved in water, ethanol, or dimethyl sulfoxide. The concentration of organic solvents in the experimental solutions did not exceed 0.1%, and did not affect viability or basal [Ca2+]i.

[Ca2+]i measurements

We measured [Ca2+]i abiding by protocols described previously.[23],[24],[25] Confluent cells grown on 6-cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 106 cell/ml. Cell viability was determined by trypan blue exclusion. The viability was >95% after the treatment. The cells were subsequently loaded with 2 μM fura-2/AM for 30 min at 25°C in the same medium. After loading, the cells were washed with Ca2+-containing medium twice and were made into a suspension in Ca2+-containing medium at a concentration of 107 cell/ml. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer immediately after 0.1 ml cell suspension was added to 0.9 ml Ca2+-containing or Ca2+-free medium, by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. During the recording, reagents were added to the cuvette by pausing the recording for 2 s to open and close the cuvette-containing chamber. For calibration of [Ca2+]i, after completion of the experiments, the detergent Triton X-100 (0.1%) and CaCl2 (5 mM) were added to the cuvette to obtain the maximal fura-2 fluorescence. Then, the Ca2+ chelator EGTA (10 mM) was added to chelate Ca2+ in the cuvette to obtain the minimal fura-2 fluorescence. Control experiments showed that cells bathed in a cuvette had a viability of 95% after 20 min of fluorescence measurements. [Ca2+]i was calculated as previously described.[26]

Mn2+ quenching of fura-2 fluorescence was performed in Ca2+-containing medium containing 50 μM MnCl2. MnCl2 was added to cell suspension in the cuvette 30 s before the fluorescence recoding was started. Data were recorded at an excitation signal at 360 nm (Ca2+-insensitive) and emission signal at 510 nm at 1-s intervals as described previously.[27]

Cell viability analyses

The measurement of cell viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases. Increases in the amount of developed color correlated proportionally with the number of live cells. Assays were performed according to the manufacturer's instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). The cells were seeded in 96-well plates at a density of 104 cell/well in culture medium for 24 h in the presence of tectorigenin. The cell viability detecting reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1; 10 μl pure solution) was added to samples after tectorigenin treatment, and the cells were incubated for 30 min in a humidified atmosphere. The cells were incubated with/without tectorigenin for 24 h. In the experiments using BAPTA/AM to chelate cytosolic Ca2+ to inhibit [Ca2+]i rises, the cells were treated with 5 μM BAPTA/AM for 1 h prior to incubation with tectorigenin. The cells were washed once with Ca2+-containing medium and incubated with/without tectorigenin for 24 h. The absorbance of samples (A450) was determined using a multiwall plate reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value. The absorbance of samples (A450) was determined using an enzyme-linked immunosorbent assay (ELISA) reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value.

Statistical analysis

Data were reported as mean ± standard error of the mean of three independent experiments. Data were analyzed by one-way analysis of variances using the Statistical Analysis System (SAS®, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey's honestly significantly difference procedure. P <0.05 was considered statistically significant.


  Results Top


Effect of tectorigenin on [Ca2+]i in Madin-Darby Canine Kidney cells

[Figure 1]b shows that the basal [Ca2+]i level was 52 ± 2 nM. At 5–50 μM, tectorigenin induced concentration-dependent rises in [Ca2+]i. At a concentration of 50 μM, tectorigenin induced [Ca2+]i rises of 95 ± 5 nM. This signal was saturated at 50 μM because 100 μM tectorigenin did not induce a larger response (not shown). In Ca2+-free medium, tectorigenin also induced concentration-dependent rises in [Ca2+]i at 5–50 μM. At 50 μM, tectorigenin induced rises in [Ca2+]i of 71 ± 2 nM [Figure 1]c. [Figure 1]d shows the concentration–response relationship. The half-maximal effective concentration (EC50) value was 10 ± 2 μM in Ca2+-containing medium or 20 ± 3 μM in Ca2+-free medium, respectively, by fitting to a Hill equation. Ca2+ removal reduced the Ca2+ signal by approximately 20%. [Figure 1] shows that tectorigenin-induced Ca2+ response saturated at 50 μM; thus, 50 μM tectorigenin was used in the following experiments as control.

Tectorigenin-induced Mn2+ influx in Madin–Darby Canine Kidney cells

Further experiments were conducted to determine the role of Ca2+ influx in tectorigenin-induced [Ca2+]i rises. Evidence shows that Mn2+ and Ca2+ share similar movement pathways, however, it differs from Ca2+ by quenching fura-2 fluorescence at all excitation wavelengths.[27] The fura-2 fluorescence at the excitation wavelength of 360 nm is insensitive to Ca2+, therefore, quenching of fura-2 fluorescence excited at 360 nm by Mn2+ suggests that Ca2+ influx occurs. [Figure 2] shows that 50 μM tectorigenin evoked an instant decrease in the 360 nm excitation signal that reached a maximum value of 101 ± 2 arbitrary units at 250 s.
Figure 2: Effect of tectorigenin on Ca2+ influx by measuring Mn2+ quenching of fura-2 fluorescence. Experiments were performed in Ca2+-containing medium. MnCl2 (50 µM) was added to cells 1 min before fluorescence measurements. The y axis is fluorescence intensity (in arbitrary units) measured at the Ca2+-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Trace a: Control, without tectorigenin. Trace b: Tectorigenin (50 µM) was added as indicated. Data are mean ± standard error of the mean of three independent experiments

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Regulation of tectorigenin-induced [Ca2+]i rises in Madin–Darby Canine Kidney cells

In Ca2+-containing medium, 1 min before addition of tectorigenin, phorbol 12-myristate 13 acetate (PMA; 1 nM; a protein kinase C (PKC) activator), GF109203X (2 μM; a PKC inhibitor), econazole (0.5 μM), nifedipine (1 μM), or SKF96365 (5 μM) was administered. All the chemicals except PMA and GF109203X inhibited tectorigenin-induced [Ca2+]i rises by approximately 10% [Figure 3].
Figure 3: Effect of Ca2+ channel modulators on tectorigenin-induced [Ca2+]i rises. In blocker or modulator-treated groups, the reagent was added 1 min before tectorigenin (50 µM). The concentration was 1 µM for nifedipine, 0.5 µM for econazole, 5 µM for SKF96365, 10 nM for phorbol 12-myristate 13 acetate, and 2 µM for GF109203X. Data are expressed as the percentage of control (1st column) that is the area under the curve (25–200 s) of 50 µM tectorigenin-induced [Ca2+]i rises in Ca2+-containing medium, and are mean ± standard error of the mean of three independent experiments. *P < 0.05 compared to the 1st column

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Sources of tectorigenin-induced Ca2+ release in Madin–Darby Canine Kidney cells

Effort was made to examine the organelles responsible for tectorigenin-induced [Ca2+]i rises. Literature shows that the endoplasmic reticulum is a key Ca2+ store[21],[22] in most cells. In the following experiments, the role of the endoplasmic reticulum in tectorigenin-evoked Ca2+ release in MDCK cells was explored. The experiments were conducted in Ca2+-free medium to prevent the interference of Ca2+ influx. [Figure 4]a shows that 1 μM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor,[28] induced [Ca2+]i rises of 40 ± 1 nM. Tectorigenin (50 μM) added at 500 s induced a small Ca2+ signal of 10 ± 2 nM. [Figure 4]b shows that tectorigenin (50 μM) induced [Ca2+]i rises of 55 ± 2 nM. Thapsigargin added at 500 s failed to induce [Ca2+]i rises. It appears that the endoplasmic reticulum played a pivotal role in tectorigenin-evoked Ca2+ release from organelles.
Figure 4: Effect of thapsigargin on tectorigenin-induced Ca2+ release. (a and b) Thapsigargin (TG; 1 μM) and tectorigenin (50 μM) were added at time points indicated. Experiments were performed in Ca2+-free medium. Data are mean ± standard error of the mean of three independent experiments

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A role of phospholipase C in tectorigenin-induced [Ca2+]i rises in Madin–Darby Canine Kidney cells

PLC can activate the release of Ca2+ from the endoplasmic reticulum via IP3.[21],[22] Therefore, the role of PLC in tectorigenin-induced Ca2+ release was explored. The PLC inhibitor U73122[29] was applied to examine if the activation of PLC was needed for tectorigenin-induced Ca2+ release. [Figure 5]a shows that ATP (10 μM) induced [Ca2+]i rises of 41 ± 2 nM. ATP is a PLC-dependent agonist of [Ca2+]i rises in most cell types.[30] [Figure 5]b shows that incubation with 2 μM U73122 did not change basal [Ca2+]i, but abolished ATP-induced [Ca2+]i rises. This suggests that U73122 effectively suppressed PLC activity. The data also show that incubation with 2 μM U73122 partly inhibited the combination of ATP and tectorigenin-induced [Ca2+]i rises by half. Furthermore, ATP and tectorigenin did not induce [Ca2+]i rises synergistically. The U73122 analog U73343 which does not have inhibition on PLC was used as control for U73122. Our findings suggest that U73343 (2 μM) did not change ATP-induced [Ca2+]i responses (not shown). It suggests that tectorigenin-induced Ca2+ release from the endoplasmic reticulum required PLC activation.
Figure 5: Effect of U73122 on tectorigenin-induced Ca2+ release. Experiments were performed in Ca2+-free medium. (a) Adenosine triphosphate (10 μM) was added at 25 s. (b) The first column is 50 μM tectorigenin-induced [Ca2+]i rises. The second column shows that 2 μM U73122 did not alter basal [Ca2+]i. The third column shows ATP-induced [Ca2+]i rises. The fourth column shows that U73122 pretreatment for 60 s abolished ATP-induced [Ca2+]i rises (*P < 0.05 compared to 3rd column). The fifth column shows that U73122 (incubation for 60 s) and adenosine triphosphate (incubation for 30 s) pretreatment inhibited 50 μM tectorigenin-induced [Ca2+]i rises. The sixth column shows that combination of adenosine triphosphate (10 μM) tectorigenin (50 μM) did not induce [Ca2+]i rises synergistically. Data are mean ± standard error of the mean of three independent experiments

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Cytotoxic effect of tectorigenin on Madin–Darby Canine Kidney cells

Considering uncontrolled [Ca2+]i rises may change cell viability,[22] experiments were conducted to explore the action of tectorigenin on the viability of MDCK cells. Cells were treated with 0–60 μM tectorigenin for 24 h, and the tetrazolium assay was performed. In the presence of 10–60 μM tectorigenin, cell viability decreased concentration dependently [Figure 6]. The IC50(half maximal inhibitory concentration) value of tectorigenin was approximately 45.5 μM in MDCK cells.
Figure 6: Cytotoxic effect of tectorigenin. (a) Following 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl (BAPTA/AM) treatment, cells were incubated with fura-2-acetoxy methyl as described in “Materials and methods.” Then, [Ca2+]i measurements were conducted in Ca2+-containing medium. Tectorigenin was added as indicated. (b) Cells were treated with 0–60 µM tectorigenin for 24 h, and cell viability assay was performed. Data are mean ± standard error of the mean of three independent experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control response that is the increase in cell numbers in tectorigenin-free groups. Control had 10,545 ± 285 cells/well before experiments, and had 13852 ± 845 cells/well after incubation for 24 h. *P < 0.05 compared to control. In each group, the Ca2+ chelator BAPTA/AM (5 µM) was added to cells followed by treatment with tectorigenin in medium. Cell viability assay was subsequently performed.

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No effect of 1,2-bis(2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl on preventing tectorigenin-evoked cell death

Question arose whether the tectorigenin-evoked cell death was induced by preceding [Ca2+]i responses. The intracellular Ca2+ chelator BAPTA/AM[31] was widely appliedin vitro experiments to suppress [Ca2+]i rises. After treatment with 5 μM BAPTA/AM, tectorigenin (50 μM) did not evoke [Ca2+]i rises in BAPTA/AM-treated cells in both Ca2+-containing and Ca2+-free solutions [Figure 6]a. This suggests that BAPTA loading for 25 h still effectively chelated cytosolic Ca2+. [Figure 6]b also shows that 5 μM BAPTA/AM loading did not alter the control value of cell viability. In the presence of 10–60 μM tectorigenin, BAPTA/AM loading did not reverse tectorigenin-induced cell death.


  Discussion Top


With regard to the effect of tectorigenin on [Ca2+]i, the literature is meager. Evidence shows that tectorigenin affected Ca2+ homeostasis in HepG2 cells[18] and isolated rat hepatocytes.[19] However, the underlying mechanisms were not clear. This study represents the first attempt to explore the effect of tectorigenin on Ca2+ signaling and viability in MDCK cells. Our study shows that tectorigenin increased [Ca2+]i in MDCK cells. The Ca2+ signal was composed of Ca2+ entry and Ca2+ release because the signal was reduced by 20% by removing extracellular Ca2+. The Mn2+ quenching data also suggest that Ca2+ influx occurred during tectorigenin incubation.

Our findings show that tectorigenin-evoked [Ca2+]i rises were inhibited by 10% by econazole, nifedipine, and SKF96365. These chemicals are often used to suppress store-operated Ca2+ entry.[32],[33],[34] Our data suggest that tectorigenin-caused Ca2+ entry partly went through store-operated Ca2+ entry. Because 20% of tectorigenin-induced [Ca2+]i rises was via Ca2+ influx, this influx appears to involve store-operated Ca2+ entry and other unknown pathways. Studies have shown that in MDCK cells, three are some plasmamemmal cation channels that participate in Ca2+ signaling, including transient receptor potential channels.[35] As selective blockers are still lacking for these channels, the pathways underlying tectorigenin-induced Ca2+ influx in MDCK cells deserve further investigation.

The interactive relationship between protein kinases and Ca2+ homeostasis has been established. For instance, atrial G protein-activated K+ channels activity was regulated by different Ca2+-dependent PKC isoforms in a receptor-specific manner.[36] Fluvastatin was reported to inhibit Rab5-mediated slow delayed rectifier K+ currents (IKs) internalization caused by chronic Ca2+-dependent PKC activation.[37] In contrast, Ca2+-independent PKC isoform was thought to be activatedin vitro andin vivo by ingenol 3,20 dibenzoate.[38] Our data show that tectorigenin-evoked [Ca2+]i rises were not inhibited by enhancing or inhibiting PKC activity. This suggests that tectorigenin induced a PKC-independent [Ca2+]i signal.

The sesquiterpene lactone thapsigargin is found in the plant Thapsia garganica, and is one of the major constituents of the roots and fruits of this Mediterranean species. Thapsigargin has a strong inhibitory effect on the Ca2+ pump on the endoplasmic reticulum and is widely applied to study Ca2+ homeostasis.[39] Our data suggest that the Ca2+ stores involved in tectorigenin-induced Ca2+ release appear to be the thapsigargin-sensitive endoplasmic reticulum store. However, thapsigargin did not completely inhibit tectorigenin-induced Ca2+ release, therefore, tectorigenin may also release Ca2+ from other stores such as lysosome and mitochondria.[22]

The data further show that the Ca2+ release was via a PLC-dependent pathway because the release was suppressed by 50% when PLC activity was inhibited. A key mechanism that underlies the release of Ca2+ from the endoplasmic reticulum is through Ca2+ release via the IP3 receptors on the endoplasmic reticulum membrane.[21],[22] IP3 and diacylglycerol (DAG) are produced when phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane is hydrolyzed by PLC. IP3 and DAG are crucial second messengers modulating different cellular responses. Therefore, it suggests that PLC activated by tectorigenin, an isoflavoloid compound, can trigger the production of IP3 that releases Ca2+ via the IP3 receptors on the endoplasmic reticulum membrane. As only half of tectorigenin-induced Ca2+ release was via PLC-associated pathways, other mechanisms might be involved such as the PLA2 pathways. For example, literature shows that agonist-mediated activation of PLA2 initiates Ca2+ mobilization in intestinal longitudinal smooth muscle in an IP3-independent manner.[40]

Cell viability can be affected in a Ca2+-associated[41] or Ca2+-dissociated fashion.[42] Our data implicate that tectorigenin evoked cell death in a concentration-dependent, Ca2+-independent manner. As Ca2+ movement changes numerous cellular responses, tectorigenin may alter the physiology of MDCK cells in a Ca2+-dependent manner that awaits further investigation. Several studies were performed to explore the plasma level of tectorigenin in animal models. The plasma level of tectorigenin may reach ~10 μM.[43],[44] This level may be expected to go much higher in patients with liver or kidney disorders or taking higher doses.[43],[44] Our results suggest that tectorigenin at a concentration of 10 μM started to induce cell death and the IC50 was approximately 45.5 μM in MDCK cells. Thus, for some groups of kidney patients, our data may be clinically relevant.

Together, the results show that tectorigenin induced Ca2+ influx via PKC-insensitive store-operated Ca2+ entry and also Ca2+ release from the endoplasmic reticulum in a PLC-dependent manner. Tectorigenin also caused Ca2+-independent cell death. The effect of tectorigenin on [Ca2+]i and viability in MDCK cells should contribute to the pharmacology of tectorigenin. Our data may help otherin vitro researches to interpret their results because an alteration of [Ca2+]i may change many cellular responses.

Acknowledgment

This work was supported by RD-105001 from Chang Bing Show Chwan Memorial Hospital, Changhua County 50544, Taiwan.

Financial support and sponsorship

This work was supported by RD-105001 from Chang Bing Show Chwan Memorial Hospital, Changhua County 50544, Taiwan.

Conflicts of interest

There are no conflicts of interest.



 
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