|Year : 2021 | Volume
| Issue : 4 | Page : 202-209
A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells
Cing-Yu Chen1, Cheng-Hsun Wu2, King-Chuen Wu3, Lian-Ru Shiao1, Chin-Min Chuang4, Yuk-Man Leung1, Louis W C Chow5
1 Department of Physiology, China Medical University, Taichung, Taiwan
2 Department of Anatomy, China Medical University, Taichung, Taiwan
3 Department of Anesthesiology, Chang Gung Memorial Hospital; Chang Gung University of Science and Technology, Chiayi, Taiwan
4 Department of Emergency Medicine, China Medical University Hospital, Taichung, Taiwan
5 State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Taipa, Macau; UNIMED Medical Institute; Organisation for Oncology and Translational Research, Hong Kong, China
|Date of Submission||04-May-2021|
|Date of Decision||03-Jun-2021|
|Date of Acceptance||08-Jun-2021|
|Date of Web Publication||17-Aug-2021|
Prof. Yuk-Man Leung
Department of Physiology, China Medical University, Taichung
Source of Support: None, Conflict of Interest: None
Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 μM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 μM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 μM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.
Keywords: Apoptosis, breast cancer, BT-474 cells, Ca2+ overload, endoplasmic reticulum stress, gamma-linolenic acid, oxidative stress
|How to cite this article:|
Chen CY, Wu CH, Wu KC, Shiao LR, Chuang CM, Leung YM, Chow LW. A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells. Chin J Physiol 2021;64:202-9
|How to cite this URL:|
Chen CY, Wu CH, Wu KC, Shiao LR, Chuang CM, Leung YM, Chow LW. A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells. Chin J Physiol [serial online] 2021 [cited 2021 Oct 20];64:202-9. Available from: https://www.cjphysiology.org/text.asp?2021/64/4/202/324007
Cing-Yu Chen and Cheng-Hsuan Wu contributed equally as first authors.
Chin-Min Chuang, Yuk-Man Leung and Louis W.C. Chow contributed
equally as corresponding authors.
| Introduction|| |
Gamma-linolenic acid (GLA) could be obtained from oils of various vegetables, for example, evening primrose oil and hemp seed oil. It is also present in oats, barley, hemp seeds, and spirulina. GLA is a carboxylic acid having an 18-carbon chain and 3 cis double bonds. GLA has been demonstrated as an anticancer agent on its own right. GLA has been shown to cause inhibition of MDA-MB-435 breast cancer cell growth in vitro. GLA suppresses cell cycle progression in human colon cancer HT115 and breast cancer MCF7 cells via modulating phosphorylation and subsequent degradation of p27kip1 and p57kip2. The same group has later shown that GLA stimulates phosphorylation and translocation of peroxisome proliferator-activated receptors (PPARs) to the nucleus; removing PPARγ with antisense oligos abolished the suppressive effect of GLA on breast cancer cells. In examining expression of Her-2/neu (erbB-2) oncogene, an important regulator of development of many human cancer types, Menendez et al. demonstrated that GLA treatment markedly suppressed Her-2/neu protein levels in the Her-2/neu-overexpressing cell lines BT-474, SK-Br3, and MDA-MB-453 (breast cancer), SK-OV3 (ovarian cancer), and NCI-N87 gastrointestinal tumor cells. GLA caused mitochondria-dependent apoptotic death in 7,12-dimethylbenz anthracene-induced mammary gland carcinoma.
GLA has also been shown to enhance the actions of other anticancer drugs. In breast cancer patients taking tamoxifen alone or tamoxifen plus GLA, tumor biopsies results showed that the tamoxifen plus GLA group exhibited significantly more reduced estrogen receptor levels as compared to the tamoxifen alone group. The same group has later demonstrated that in nude mice implanted with MCF-7 B1M cell line (estrogen receptor-positive human breast cancer xenografts), dietary GLA enhances tamoxifen inhibitory effects on tumor growth and estrogen receptor expression.
In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. We found a basal level of GLA-induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, endoplasmic reticulum (ER) stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress, and we have delineated the underlying mechanism (opening of IP3 receptor-channel and ryanodine receptor-channel).
| Materials and Methods|| |
Materials and cell culture
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were purchased from Invitrogen Corporation (Carlsbad, CA, USA). GLA was from Cayman Chemical (Ann Arbor, MI, USA). Cyclopiazonic acid (CPA) and eicosapentaenoic acid (EPA) were from Sigma-Aldrich (St. Louis, MO, USA). Xestospongin C and ryanodine were purchased from Tocris (Bristol, UK). Fura-2 AM was purchased from Calbiochem-Millipore (Darmstadt, Germany). BT-474 cells and MDA-MB-231 cells were cultured at 37°C in 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and penicillin-streptomycin (100 units/ml, 100 μg/ml) (Invitrogen).
Assay of cell viability and apoptosis
Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were cultured in a 96-well plate at a density of 1.5 × 104/well and were then treated with various polyamines for 48 h. MTT (final concentration at 0.5 mg/ml) was subsequently added to each well and then further incubated for 4 h. The culture medium was then removed and 100 μl of dimethyl sulfoxide (DMSO) was added to each well for 15 min (with shaking) to dissolve the precipitates. The absorbance at 595 nm was measured using an ELISA reader and was used as an indicator of cell viability or metabolic activity. Thus, a decrease in MTT reduction implies cell death, decrease in cell proliferation, or loss of metabolic activity.
Apoptosis was examined by flow cytometry using an FITC annexin V apoptosis detection kit with propidium iodide (BioLegend, San Diego, CA, USA) according to manufacturer's instructions.
Microfluorimetric measurement of cytosolic Ca2+
Microfluorimetric measurement of cytosolic Ca2+ concentration was performed using fura-2 as the Ca2+-sensitive fluorescent dye as described previously. Briefly, cells were incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA, USA) for 1 h at 37°C and then washed in extracellular bath solution which contained (mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES (pH 7.4 adjusted with NaOH). When intracellular Ca2+ release was assayed, Ca2+-free solution was used. This Ca2+-free solution was the same as the extracellular bath solution mentioned above except that Ca2+ was omitted and 100 μM EGTA was supplemented. Cells were alternately excited with 340 nm and 380 nm (switching frequency at 1 Hz) using an optical filter changer (Lambda 10-2, Sutter Instruments, Novato, CA, USA). Emission was collected at 500 nm and images were captured using a CCD camera (CoolSnap HQ2, Photometrics, Tucson, AZ, USA) linked to an inverted Nikon TE2000-U microscope. Images were analyzed with an MAG Biosystems Software (Santa Fe, NM, USA). All imaging experiments were performed at room temperature (25°C). We measured and analyzed the 340/380 ratio changes at a region of interest of single cells within the microscopic views (regarded as one experiment) and then repeated the experiment a few more times to get the mean of all single cells examined.
Reactive oxygen species assay
After cells were treated with DMSO or 30 μM GLA for various time periods, they were incubated in serum-free DMEM containing 20 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA, Sigma, St. Louis, MO, USA) for 30 min at 37°C in the dark. After washing, cells were trypsinized for 3 min at 37°C and then washed again 3 more times in phosphate-buffered saline (PBS) by centrifugation. Cells were dispersed in PBS and put in polystyrene tubings for fluorescence-activated cell sorting. For each sample, 105 cells were analyzed using an FACSCanto flow cytometer system (BD Biosciences, San Jose, CA, USA). Data were analyzed by BD FACSDIVA™ software (BD Biosciences).
Western blotting was performed as described previously. Briefly, cells were washed in cold PBS, lysed for 30 min on ice with radioimmunoprecipitation assay buffer. Protein samples containing 30 μg protein were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 5% nonfat milk in PBS buffer to block nonspecific binding. The membranes were incubated with various primary antibodies: anti-actin (1:1000; GeneTex, Irvine, CA, USA), anti-CHOP, anti-eIF2-α, and anti-p-eIF2-α (all 1:1000; Cell Signaling, Danvers, MA, USA). Subsequently, the membranes were incubated with goat anti-rabbit or goat anti-mouse peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. The blots were visualized by enhanced chemiluminescence (ECL; Millipore, Darmstadt, Germany) using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).
Measurement of mitochondrial Ca2+
After cells were treated with DMSO or 30 μM GLA for various time periods, they were incubated in serum-free DMEM containing 5 μM Rhod 2 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37°C in the dark. Cells were then trypsinized for 3 min at 37°C and then washed again 3 more times in PBS by centrifugation. Cells were dispersed in PBS, put in polystyrene tubings for fluorescence-activated cell sorting, and then analyzed using a FACSCanto flow cytometer system (BD Biosciences, San Jose, CA, USA). Excitation and emission wavelengths were set at 488 and 576 nm, respectively. Data were analyzed by BD FACSDIVA™ software (BD Biosciences).
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was measured using a Mitochondrial Membrane Potential Assay Kit (#12664; Cell Signaling, Danvers, MA, USA). Briefly, cells were seeded at 5 × 104 cells per well and allowed to attach overnight. Cells were then treated with DMSO or 30 μM GLA for different time periods. After the incubation time, JC-1 (2 μM) was added to each well for 30 min. Fluorescence was then detected using a SpectraMax M2 multimode plate reader (Molecular Devices, San Jose, CA, USA) with excitation at 485 nm and emissions at 520 and 590 nm. Mitochondrial membrane potential was calculated using the ratio RFU of red emission (590 nm)/RFU of green emission (520 nm).
Data are presented as means ± standard error of mean. The unpaired or paired Student's t-test was used where appropriate to compare two groups. For comparison among multiple groups, one-way ANOVA with Tukey's HSD post hoc test was used to determine statistical significance. A value of P < 0.05 was considered to be significantly different.
| Results|| |
GLA treatment caused a concentration-dependent cell death [Figure 1]a. In subsequent experiments, we used a GLA concentration (30 μM) close to the basal level of plasma concentration in human studies., As shown in [Figure 1]b, incubation of BT-474 cells with 30 μM GLA resulted in a time-dependent cell death, so that at day 4, cell death was 43%. To investigate if GLA inflicted apoptotic cell death, we performed flow cytometry (annexin V/PI double staining) to confirm that GLA caused apoptosis at 72 h [Figure 1]c. GLA at 30 μM did not significantly decrease the viability of breast cancer MDA-MB-231 cells [Figure 2]. EPA (30 μM), which has been shown to have anticancer effects on prostate cancer cells and esophageal adenocarcinoma cells,, did not affect the viability of both BT-474 and MDA-MB-231 cells. These results suggest that the cytotoxic effect of GLA on BT-474 cells was not due to its fatty acid nature per se.
|Figure 1: Gamma-linolenic acid caused apoptotic cell death. (a) BT-474 cells were treated with different concentrations of gamma-linolenic acid for 4 days and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine cell viability. (b) BT-474 cells were treated with 30 μM gamma-linolenic acid for 1–4 days and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine cell viability. (c) BT-474 cells were treated with 30 μM gamma-linolenic acid for 3 days and apoptosis was assayed. Results are mean ± standard error of mean from four separate experiments. *Significantly (P < 0.05) different from control. **Significantly (P < 0.01) different from control.|
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|Figure 2: Effects of gamma-linolenic acid and eicosapentaenoic acid on viability of BT-474 cells and MDA-MB-231 cells. BT-474 cells and MDA-MB-231 cells were treated with DMSO, 30 μM gamma-linolenic acid, or 30 μM eicosapentaenoic acid for 4 days and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine cell viability. Results are mean ± standard error of mean from four separate experiments. *Significantly (P < 0.05) different from the DMSO control.|
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We then investigated whether GLA affected mitochondrial membrane potential. As shown in [Figure 3], GLA caused a decrease in mitochondrial membrane potential (depolarization) within 2 h; such drop in mitochondrial membrane potential continued up to 60 h (2.5 days). GLA also caused an increase in mitochondrial Ca2+ level, which began at 24 h and persisted until 60 h [Figure 4].
|Figure 3: Effect of gamma-linolenic acid on mitochondrial membrane potential. BT-474 cells were treated with 30 μM gamma-linolenic acid for different time periods and mitochondrial membrane potential was quantified by JC-1 fluorescence assay. Red emission/green emission ratio lower than control values indicates depolarization. Results are mean ± standard error of mean from three independent experiments. *Significantly (P < 0.05) different from control.|
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|Figure 4: Effect of gamma-linolenic acid on mitochondrial Ca2+. BT-474 cells were treated with 30 μM gamma-linolenic acid for different time periods and mitochondrial Ca2+ level was quantified by flow cytometry using Rhod 2 as fluorescence probe. Results are mean ± standard error of mean from three independent experiments. *Significantly (P < 0.05) different from control.|
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As GLA-induced elevation in mitochondrial Ca2+ level might have resulted from cytosolic Ca2+ overload, we next examined the effect of GLA on cytosolic Ca2+ homeostasis. The concentration–response effect of GLA on raising cytosolic Ca2+ level is shown in [Figure 5]a. We used 30 μM GLA in subsequent experiments and examined whether Ca2+ elevation resulted from intracellular Ca2+ release or Ca2+ influx. Results in [Figure 5]b show that the GLA-triggered Ca2+ signal was markedly stronger in the presence of extracellular Ca2+ than in the absence of extracellular Ca2+. These data suggest that GLA triggered both intracellular Ca2+ release and Ca2+ influx.
|Figure 5: Ca2+ signals stimulated by gamma-linolenic acid. (a) [Ca2+]i in BT-474 cells was examined in Ca2+-containing bath solution. The cells were stimulated with different concentrations of gamma-linolenic acid. (b) Gamma-linolenic acid caused both Ca2+ release and Ca2+ influx. BT-474 cells were bathed in Ca2+-containing solution or Ca2+-free solution and were exposed to 30 μM gamma-linolenic acid. Results are mean ± standard error of mean, each group having 23-39 cells from four separate experiments.|
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An intact intracellular Ca2+ pool is physiologically important for cell functions. We next examined the relation between GLA-releasable Ca2+ store and the Ca2+ store discharged by CPA, an inhibitor of sarcoplasmic/ER Ca2+ ATPase. [Figure 6]a and [Figure 6]b shows, respectively, CPA-induced Ca2+ store release without and with 30 μM GLA pretreatment, and it was obvious that after GLA pretreatment, the CPA-induced Ca2+ release was abolished. [Figure 6]c and [Figure 6]d shows, respectively, GLA-induced Ca2+ store release without and with 30 μM CPA pretreatment, and it could be shown that after CPA pretreatment, the GLA-induced Ca2+ release was much smaller. These observations suggest that the GLA- and CPA-dischargeable Ca2+ stores had a large extent of overlap.
|Figure 6: Gamma-linolenic acid depleted the cyclopiazonic acid-sensitive Ca2+ pool via Ca2+ release through RYR and IP3R. (a and b) BT-474 cells in Ca2+-free solution were pretreated with ethanol or 30 μM gamma-linolenic acid for 30 min before being stimulated by 30 μM cyclopiazonic acid. (c and d) Cells in Ca2+-free solution were pretreated with DMSO or 30 μM cyclopiazonic acid for 30 min before stimulated by 30 μM gamma-linolenic acid. (e) Cells in Ca2+-free solution were pretreated with DMSO, 100 μM ryanodine, or 3 μM XeC for 8 min before stimulated by 30 μM gamma-linolenic acid. Significant (P < 0.05) differences between the control DMSO group, the ryanodine group, and the XeC group began at 103 and 183 s, respectively. Results are mean ± standard error of mean, each group having 26–49 cells from 3–6 separate experiments.|
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We then examined how GLA caused Ca2+ release. Pretreatment with the 3 μM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 μM ryanodine (ryanodine receptor-channel blocker) suppressed GLA-triggered Ca2+ release [Figure 6]e, suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel.
Ca2+ store depletion would lead to ER stress. We next examined if GLA caused ER stress (increase in expression of p-eIF2α and CHOP, two typical ER stress markers). As shown in [Figure 7], both CPA (positive control) and GLA caused an increase in levels of p-eIF2α at 5 h but not 60 h; they both caused an increase in CHOP levels at 5 and 60 h. Whether GLA elicited reactive oxygen species (ROS) formation was investigated. Results in [Figure 8] show that GLA caused ROS formation at 5, 24, and 60 h.
|Figure 7: Effects of gamma-linolenic acid on ER stress marker expression. BT-474 cells were treated with 30 μM gamma-linolenic acid for 5 or 60 h and then ER stress markers were analyzed by Western blot. Results are mean ± standard error of mean from five separate experiments. *Significantly (P < 0.05) different from control.|
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|Figure 8: Gamma-linolenic acid triggered ROS formation. BT-474 cells were treated with 30 μM gamma-linolenic acid for different time periods and subject to ROS detection by flow cytometry. Results are mean ± standard error of mean from three separate experiments. *Significantly (P < 0.05) different from control.|
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| Discussion|| |
In one study, circulating concentrations of GLA in Caucasians and Asians ranged from 20 to 28 μM. In another clinical study, basal plasma GLA concentration was found to be around 33 μM. The concentration of GLA used in this work (30 μM) was within the range of basal plasma GLA concentrations in these clinical studies. Thus, we have provided evidence that basal concentrations of GLA may exert tonic suppression on breast cancer cell proliferation. In concordance with this notion, inflammatory breast cancer is correlated with decreased level of GLA in breast adipose tissue. In addition, in one clinical study, plasma level of dihomo-GLA (an elongated product of GLA) was found to be inversely correlated with risk of breast cancer and overall risk of cancer.
GLA significantly decreased the viability of BT-474 cells but not MDA-MB-231 cells. The cause of this difference is unknown. BT-474 cells are luminal-type breast cancer cells which express HER2 (HER2+) and both estrogen and progesterone receptors (HR+), while MDA-MB-231 cells are of basal type which do not express HER2 (HER2-) and lack estrogen and progesterone receptors (HR-). Whether the higher sensitivity of BT-474 than MDA-MB-231 cells to GLA toxicity is attributed to expression of both HER2 and hormonal receptors is unclear and warrants future investigation.
We have, for the first time, shown that GLA caused Ca2+ store depletion and results have suggested that GLA-triggered Ca2+ release was via opening of IP3 receptor-channel and ryanodine receptor-channel. Remarkably, GLA has been known to induce channel-type ionic currents in bilayer lipid membranes, hence this fatty acid may have other nonspecific actions on biological membranes to trigger Ca2+ fluxes.
Ca2+ store depletion led to ER stress. This is, to our best knowledge, the first report to show GLA could induce ER stress. It is noted α-linolenic acid (ALA) does not induce ER stress but instead protects several cell types from toxicity by inhibiting ER stress. For instance, ALA protects renal proximal tubular NRK-52E cells from lipotoxicity by attenuating ER stress. Stearic acid-inflicted ER stress and cell death were remarkably suppressed by ALA in primary rat hepatocytes.
We have shown that intracellular Ca2+ release and Ca2+ influx led to Ca2+ overload. Inevitably this led to consequent mitochondrial Ca2+ overload (via uptake of Ca2+ by mitochondria Ca2+ uniporter), ROS formation, and collapse of mitochondrial membrane potential. This mitochondria-dependent pathway of apoptosis, together with the abovementioned ER stress pathway, might contribute to the final apoptotic death of BT-474 cells (ER+ HER2+). GLA has been shown to downregulate HER2 expression in multiple cancer cell types including BT-474. In ER+ HER2− breast cancer MCF-7 cells, GLA also caused mitochondria-dependent apoptotic death.
GLA appears to target mitochondria in other cancer cell types. Dietary GLA could lead to reduction in Walker 256 carcinosarcoma size in rats, with apoptosis, necrosis, and changes in mitochondrial ultrastructure. The same group has also shown in in vitro experiments that GLA causes inhibition of mitochondrial complexes I + III and IV, ROS generation, drop in mitochondrial membrane potential, cytochrome c release, and DNA fragmentation in Walker 256 carcinoma cells. GLA also induces a decrease in mitochondrial membrane potential, ROS formation, Ca2+ overload, caspase activation, and eventually apoptosis in colon cancer LoVo and RKO cells. Interestingly, GLA reportedly has protective effects on noncancer cells. For instance, GLA suppresses transforming growth factor-β1-induced apoptosis in hepatocytes.
| Conclusion|| |
This is the first report to show that a basal level of GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel. GLA induced apoptotic death in BT-474 breast cancer cells by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress and oxidative stress.
Financial support and sponsorship
Y. M. L. would like to thank China Medical University, Taiwan, for providing fundings (CMU109-S-21). C. M. C and C. H. W thank China Medical University Hospital, Taiwan, for support (DMR-109-062; DMR-110-084). K.C.W would like to thank Chang Gung Memorial Hospital, Chiayi, for grant support (CMRPG6J0371; CMRPG6L0131).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]