• Users Online: 326
  • Print this page
  • Email this page

Table of Contents
Year : 2022  |  Volume : 65  |  Issue : 1  |  Page : 30-36

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol], a natural phenolic compound found in the olive, alters Ca2+ signaling and viability in human HepG2 hepatoma cells

1 Department of Medicine, Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan
2 Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
3 Department of Pharmacy, Kaohsiung Veterans General Hospital Tainan Branch; Department of Pharmaceutical Science and Technology, Chung Hwa University of Medical Technology, Tainan, Taiwan
4 Departmentnt of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
5 Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung, Kaohsiung, Taiwan
6 Department of Pediatrics, Ping Tung Christian Hospital, Pingtung, Taiwan
7 Department of Nursing, Tzu Hui Institute of Technology, Pingtung, Taiwan
8 Department of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch; Department of Nursery, Chung Hwa University of Medical Technology, Tainan, Taiwan
9 Department of Nursery, Chung Hwa University of Medical Technology, Tainan; Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan
10 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Date of Submission24-Aug-2021
Date of Decision29-Nov-2021
Date of Acceptance23-Dec-2021
Date of Web Publication25-Feb-2022

Correspondence Address:
Dr. Chiang-Ting Chou
Department of Nursery, Chung Hwa University of Medical Technology, Tainan; Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi
Prof. Chung-Ren Jan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung
Dr. Lyh-Jyh Hao
Department of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch; Department of Nursery, Chung Hwa University of Medical and Technology, Tainan
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjp.cjp_74_21

Rights and Permissions

Hepatotoma is the leading type of primary liver cancer in adults and third cause of death in the world. Hydroxytyrosol is a natural phenol existing in olive (Olea europaea L.). Hydroxytyrosol is the chief ingredient of olive oil, which was early deemed to be the most robust antioxidant in olive oil. Hydroxytyrosol is known to inhibit various types of cancer by different methods. This study was aimed to delineate the action of hydroxytyrosol on viability and [Ca2+]i in HepG2 hepatoma cells. Fura-2 was used to detect [Ca2+]i, and WST-1 assays were applied to explore cell cytotoxicity. Hydroxytyrosol elicited [Ca2+]i raises. Eliminating external Ca2+ diminished the Ca2+ signal by 30%. Hydroxytyrosol-evoked Ca2+ influx was diminished by 20% by three inhibitors of store-operated Ca2+ channels and by a protein kinase C activator and an inhibitor. In the absence of Ca2+, thapsigargin eradicated hydroxytyrosol-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122, a PLC inhibitor, did not inhibit hydroxytyrosol-elicited [Ca2+]i raises. Hydroxytyrosol reduced cell viability. This cytotoxic action was not reversed by preincubation with BAPTA/AM, a cytosolic Ca2+ binder. In sum, in HepG2 hepatoma cells, hydroxytyrosol elicited [Ca2+]i raises by provoking PLC-unrelated discharge of Ca2+ from ER and Ca2+ influx through PKC-sensitive store-operated Ca2+ entry. In addition, hydroxytyrosol elicited Ca2+-dissociated cytotoxicity.

Keywords: Calcium, cell death, hepatoma cells, HepG2, hydroxytyrosol

How to cite this article:
Cheng HH, Liao WC, Lin RA, Chen IS, Wang JL, Chien JM, Kuo CC, Hao LJ, Chou CT, Jan CR. Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol], a natural phenolic compound found in the olive, alters Ca2+ signaling and viability in human HepG2 hepatoma cells. Chin J Physiol 2022;65:30-6

How to cite this URL:
Cheng HH, Liao WC, Lin RA, Chen IS, Wang JL, Chien JM, Kuo CC, Hao LJ, Chou CT, Jan CR. Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol], a natural phenolic compound found in the olive, alters Ca2+ signaling and viability in human HepG2 hepatoma cells. Chin J Physiol [serial online] 2022 [cited 2022 Aug 17];65:30-6. Available from: https://www.cjphysiology.org/text.asp?2022/65/1/30/338440

He-Hsiung Cheng, Wei-Chuan Liao, Rong-An Lin contributed equal to this work.

  Introduction Top

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol] is a natural phenolic compound existing in olive (Olea europaea L.). Olive oil possesses a main position in diets of regions in areas throughout the Mediterranean places.[1] Hydroxytyrosol is the chief ingredient of olive oil, olive mill waste water from olive oil manufactures, and exacted olive leaf, which was early thought to be the most robust in vitro antioxidant in olive oil.[2] Hydroxytyrosol is also synthesized in the human body during dopamine and tyramine metabolisms. In recent years, studies of hydroxytyrosol and similar phenols have increased because these chemicals may contribute to the avoidance of some abnormal situation including cardiovascular diseases and cancer.[3] The question remains as whether the dose ingested from food is high enough at the action site.

It was deemed that in cancer cells, hydroxytyrosol inhibited many oncogenic signaling pathways,[4],[5] prevented cardiovascular diseases and tumor,[5] improved insulin sensitivity in human,[6] prevented liver ischemia/reperfusion injury in mice,[7] and protected the cell signaling pathways in liver.[8] Based on these effect of harmful actions of hydroxytyrosol on liver function, we designed the present experiments to assess Ca2+ movement and death in human hepatoma cells. There are only two papers reported on PubMed showing that hydroxytyrosol increased [Ca2+]i. Hydroxytyrosol elevated [Ca2+]i in lymphomonocyte,[9] and in malignant mesothelioma cells.[10] Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca2+ channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca2+ channels.[10] The action of hydroxytyrosol on Ca2+ levels in HepG2 hepatoma cells remains unknown.

Ca2+ is a key second envoy in cell signaling. Fluctuations in [Ca2+]i may stimulate and modulate diverse cell signals such as proliferation, cytotoxicity, protein status, secretion, contraction, gene expression, apoptosis, neural plasticity, growth.[11] One of these is the inositol 1, 4, 5-trisphosphate/calcium (InsP3/Ca2+) signaling pathway that operates through primary mechanisms. Its primary role is to generate Ca2+. These Ca2+ rises generate the above processes through complex pathways, which are under intense investigations.

The main Ca2+ depots for the signal are extracellular solution and Ca2+ stored in organelles such as the endoplasmic reticulum (ER). Discharge of Ca2+ from ER may cause store-operated Ca2+ entry through cell membrane, which is a primary Ca2+ entry pathway in most nonexcitable cells.[12] ER is involved in releasing and modulating Ca2+ in many cellular responses.[13] One example is via opening of inositol 1, 4, 5-trisphosphate (IP3) receptors on ER membrane upon stimulation of phospholipase C (PLC).[14] Another Ca2+ influx route is via Ca2+ channels and receptors. Here, HepG2 human hepatoma cells were applied to delineate action of hydroxytyrosol on [Ca2+]i changes and cytotoxicity of liver cells. This cell line was chosen since it shows robust [Ca2+]i raises upon chemical triggering. Evidence shows that in this cell, [Ca2+]i raises were provoked by compounds such as solanine[15] and polyphyllin D.[16]

To this end, we used fura-2, a Ca2+-selective dye to evaluate [Ca2+]i. We delineated the [Ca2+]i raises, created the dose-signal relationships, and explored mechanisms of hydroxytyrosol-elicited discharge of internal Ca2+ and Ca2+ entry from external solution. Further, the action of hydroxytyrosol on viability and its connection to Ca2+ were surveyed.

  Materials and Methods Top


The chemicals 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/acetoxy methyl (BAPTA/AM) were acquired from Molecular Probes® (Eugene, OR, USA). The other chemicals were purchased from Sigma-Aldrich® (St. Louis, MO, USA).

Cell culture

Human hepatoma HepG2 cells were acquired from Bioresource Collection and Research Center (Taiwan). They were cultured in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin and were kept at 37°C in a humidified 5% CO2 atmosphere.

Solutions for [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 had similar chemicals as Ca2+-containing medium except that CaCl2 was substituted with 0.3 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM MgCl2. Hydroxytyrosol was dissolved in ethanol as a 2 M stock solution. The other chemicals were dissolved in water or common organic solvents. The concentration of organic solvents in the experimental solutions was below 0.1%, and did not affect [Ca2+]i or viability.

[Ca2+]i measurements

Cells grown confluent on 6-cm dishes were trypsinized. They were subsequently made into a suspension in culture medium at a concentration of 106/mL. Then, trypan blue exclusion was performed to check cell viability. The viability was routinely greater than 95% after the procedure. The suspended cells were subsequently incubated with fura-2/AM (2 μM) for 30 min at 25°C. Afterward, we washed cells with Ca2+-containing medium two times and cells were made into a suspension using Ca2+-containing medium at a concentration of 107/ml. Subsequently, we conducted fura-2 fluorescence measurements by adding cell suspension in a cuvette with continuous stirring. After rinsing with saline, the normal basal levels of Ca2+ were 50 ± 2 (mean ± standard deviation, n = 10). The cuvette had 0.5 million cells suspended in 1 mL of culture medium depending on the condition of the experiments. Fura-2 fluorescence was detected using a Shimadzu RF-5301PC spectrofluorophotometer right after 0.1 mL cell suspension was added to 0.9 mL Ca2+-containing or Ca2+-free medium, by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. Reagents were administered to the cuvette during the recording, by pausing the recording for 2 s to open and close the cuvette-containing chamber. We performed calibration of [Ca2+]i after the experiment was completed. Triton X-100 (0.1%) and CaCl2 (5 mM) were added to the cuvette to rupture cell membranes and obtain the maximal fura-2 fluorescence. Thereafter, the Ca2+ chelator EGTA (10 mM) was administered to chelate Ca2+ in the cuvette to obtain the minimal fura-2 fluorescence. Our experiments often completed within 20 min. Control experiments showed that cells bathed in a cuvette had a viability of 95% after 20 min. [Ca2+]i was calculated as previously described in literature.[17]

Mn2+ quenching assays

To determine whether Ca2+ influx occurred during stimulation with hydroxytyrosol, Mn2+ quenching of fura-2 fluorescence was conducted in Ca2+-containing medium containing MnCl2 (50 μM). To start an assay, MnCl2 was administered to cell suspension in the cuvette 30 s before starting the recording. Results were collected at excitation signal at 360 nm (Ca2+-insensitive) and emission signal at 510 nm at 1-s intervals as detailed in literature.[18]

Cell viability assays

Live cells were able to cleave tetrazolium salts by dehydrogenases. In contrast, dead cells were not. The tetrazolium dye 4-[3-[4-lodophenyl]-2-4(4- nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1; 10 μl pure solution) was added to cells after hydroxytyrosol treatment to detect cell viability, and cells were incubated for 30 min in a humidified atmosphere. Increases in the intensity of color of WST-1 were noticeably proportional to the number of live cells. Our assays were based on the manufacturer's instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). We seeded cells in 96-well plates at a density of 104/well in culture medium overnight in the presence of 0-100 μM hydroxytyrosol.

Furthermore, cells were preincubated with 5 μM BAPTA/AM for 1 hour before treatment with hydroxytyrosol in experiments using BAPTA/AM to chelate cytosolic Ca2+. The cells were washed once with Ca2+-containing medium and incubated with or without hydroxytyrosol for 24 hour. We detected the absorbance of samples (A450) by using an enzyme-linked immunosorbent assay (ELISA) reader. The optical density was normalized to the absorbance of unstimulated cells in each plate and presented as a percentage of the control response.


Results were presented as mean ± standard error of the mean (SEM) 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 significant difference procedure. P < 0.05 was considered significant.

  Results Top

[Figure 1] illustrates the chemical structure of hydroxytyrosol.
Figure 1: Chemical structure of hydroxytyrosol.

Click here to view

Hydroxytyrosol-induced Ca2+ responses

[Figure 2]a demonstrates resting [Ca2+]i was 50 ± 1 nM. At 50–200 μM, hydroxytyrosol elicited dose-associated raises in [Ca2+]i. At 200 μM, hydroxytyrosol evoked [Ca2+]i elevations of 30 ± 5 nM. This response saturated at 200 μM for the reason that 300 μM hydroxytyrosol failed to elicit a greater response (unshown). In the deficiency of Ca2+, hydroxytyrosol also evoked concentration-associated [Ca2+]i raises at 50-200 μM. At 200 μM, hydroxytyrosol evoked [Ca2+]i elevations of 20 ± 2 nM [Figure 1]b. Demonstrated in [Figure 1]c are dose-signal relationships. Removing extracelluar Ca2+ reduced the Ca2+ response by 30%.
Figure 2: Hydroxytyrosol elevated [Ca2+]i. (a) Hydroxytyrosol was administered to cells at 25 s. The level of hydroxytyrosol was depicted by arrows. The assays were conducted in Ca2+-containing medium. (b) Hydroxytyrosol increased [Ca2+]i in Ca2+-containing medium. Hydroxytyrosol was administered at 25 s Ca2+-free medium. (c) Dose-signal relationships of hydroxytyrosol-provoked [Ca2+]i raises in Ca2+-free or Ca2+-containing medium. Y axis represented the percentage of the area under the curve (25–250 s) of the [Ca2+]i raises elicited by 200 μM hydroxytyrosol in the presence of Ca2+. Results are mean ± standard error of the mean of three independent assays. *P < 0.05 in comparison to empty circles.

Click here to view

Mn2+ caused Ca2+ influx

The next set of assays was performed to demonstrate hydroxytyrosol-caused [Ca2+]i raises concerned Ca2+ entry. Mn2+ and Ca2+ penetrated cells via parallel pathways but Mn2+ smothers fluorescence at all excitation wavelengths.[18] Hence, decreases of fura-2 fluorescence excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+ suggests Ca2+ entry had occurred. [Figure 3] depicts 200 μM hydroxytyrosol provoked an instant reduction in the 360 nm excitation response that attained to a maximal level of 101 ± 2 units at 100 s. The data demonstrate Ca2+ influx participated in hydroxytyrosol-provoked [Ca2+]i raises.
Figure 3: Hydroxytyrosol provoked Mn2+ smothering of fluorescence. Assays were conducted in the presence of Ca2+. MnCl2 (50 μM) was administered to cells 1 min prior to assays. Of notice, the y axis is fluorescence intensity recorded at the Ca2+-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Trace a: no hydroxytyrosol. Trace b: hydroxytyrosol (200 μM) was administered as shown. Results are mean ± standard error of the mean of three separate experiments.

Click here to view

Modulation of hydroxytyrosol-caused Ca2+ responses

[Figure 2] confirms that hydroxytyrosol-evoked Ca2+ signal attained to saturation at 200 μM; consequently in this set of assays the signal provoked by 200 μM hydroxytyrosol was defined as control. Inhibitors of store-operated Ca entry; econzaole (0.5 μM), nifedipine (1 μM), or SKF96365 (5 μM); PKC modulators: phorbol 12-myristate 13 acetate (PMA; 1 nM; a PKC activator), GF109203X (2 μM; a PKC inhibitor); were administered 1 min prior to hydroxytyrosol (200 μM) in the presence of Ca2+. The five compounds hampered hydroxytyrosol-evoked [Ca2+]i raises by around 20% [Figure 4].
Figure 4: Inhibitory action of Ca2+ channel regulators on hydroxytyrosol-evoked [Ca2+]i raises. In modulator-pretreated groups, the chemical was administered 1 min prior to hydroxytyrosol (200 μM). The dose was 1 μM for nifedipine, 0.5 μM for econazole, 5 μM for SKF96365, 10 nM for PMA, and 2 μM for GF109203X. Results were presented as the percentage of control (1st column, 100%) that is the area under the curve (25-200 s) of 200 μM hydroxytyrosol-provoked [Ca2+]i raises in the presence of Ca2+ and were mean ± standard error of the mean of three independent analyses. *P < 0.05 in comparison to 1st column.

Click here to view

Intracellular depots of hydroxytyrosol-provoked Ca2+ signal

ER has been demonstrated to be the major Ca2+ repository in most cells.[12] Accordingly, the participation of ER in hydroxytyrosol-induced discharge of Ca2+ in HepG2 cells was undertaken. The assays were carried out in Ca2+-free medium. [Figure 5]a shows evidence for 1 μM thapsigargin, an ER Ca2+ pump inhibitor[19] provoked [Ca2+]i elevations of 30 ± 1 nM. Hydroxytyrosol (200 μM) administered at 500 s did not succeed to evoke a Ca2+ response. [Figure 5]b proves hydroxytyrosol (200 μM) provoked [Ca2+]i raises of 20 ± 2 nM. Thapsigargin administered at 500 s failed to elicit [Ca2+]i raises.
Figure 5: Thapsigargin participated in hydroxytyrosol-caused Ca2+ discharge. (a and b) Thapsigargin (1 μM) and hydroxytyrosol (200 μM) were administered at time shown. This set of assays was conducted in the absence Ca2+. Results were mean ± standard error of the mean of three independent analyses.

Click here to view

The part phospholipase C played in hydroxytyrosol-provoked Ca2+ discharge

PLC is a cytosolic protein that modulates liberation of Ca2+ from ER. For the reason that hydroxytyrosol discharged Ca2+ from ER, the part of PLC in this procedure was examined. U73122,[20] a suppressor of PLC, was utilized to survey if PLC was needed for hydroxytyrosol-provoked discharge of Ca2+ from ER. [Figure 6]a proves that ATP (10 μM) provoked [Ca2+]i elevations of 11 ± 2 nM. Stimulation of cells with ATP often triggers PLC activation which leads to [Ca2+]i elevations in most cells.[21] [Figure 6]b illustrated that pretreatment with 2 μM U73122 fell short to alter resting [Ca2+]i levels but entirely suppressed ATP-evoked [Ca2+]i elevations. These findings imply that U73122 successfully inhibited PLC. The results further depict pretreatment with U73122 and ATP failed to influence 200 μM hydroxytyrosol-evoked [Ca2+]i elevations. U73343 (2 μM), a chemical that was often utilized as an analog for U73122, did not inhibit ATP-evoked [Ca2+]i raises (unshown).
Figure 6: The part U73122 played in hydroxytyrosol-caused discharge of Ca2+. Assays were conducted in the absence of Ca2+. (a) ATP (10 μM) was administered at time indicated. (b) 1st column is 200 μM hydroxytyrosol-caused [Ca2+]i raises. 2nd column illustrates that 2 μM U73122 failed to alter the resting [Ca2+]i. 3rd column depicts 10 ATP-provoked [Ca2+]i raises. 4th column implicates that U73122 (treatment for 30 s) totally suppressed 10 μM ATP-caused [Ca2+]i raises. 5th depicts that U73122 and ATP pretreatment failed to alter 200 μM hydroxytyrosol-caused [Ca2+]i raises. Results were mean ± standard error of the mean of three independent assays. *P < 0.05 in comparison to first column.

Click here to view

Action of hydroxytyrosol on viability

For the reason that treatment with hydroxytyrosol evoked robust [Ca2+]i raises and abnormal [Ca2+]i raises might change viability of cells,[11] the next set of assays was conducted to observe action of hydroxytyrosol on cell viability. 0–100 μM hydroxytyrosol was added to cells overnight, and WST-1 analysis was analyzed. 40–100 μM hydroxytyrosol induced a dose-associated reduction of cell viability [Figure 6].

The following subject was whether hydroxytyrosol-elicited cytotoxicity and preceding [Ca2+]i raises were related. As such, experiments were designed to explore this question. BAPTA/AM, a Ca2+ chelator[22] was applied to cells to avoid [Ca2+]i raises throughout hydroxytyrosol pretreatment. After pretreatment with 5 μM BAPTA/AM, 200 μM hydroxytyrosol did not elicit [Ca2+]i raises (unshown). Obviously, BAPTA/AM successfully chelated cytosolic Ca2+. [Figure 7] further delineates that cell viability was not changed by 5 μM BAPTA/AM loading. This implicates 40–100 μM hydroxytyrosol-induced cytotoxicity failed to be reversed by BAPTA/AM preincubation.
Figure 7: Cytotoxicity of hydroxytyrosol. Cells pretreated with 0–100 μM hydroxytyrosol were kept for 24 h, and WST-1 analyses were conducted. Results were mean ± standard error of the mean of three separate assays. Findings are presented as percentage of control that is the raises in cell count in the absence of hydroxytyrosol. Control had 10,125 ± 456 cells/well prior to assays, and 13,455 ± 289 cells/well after preincubation overnight. *P < 0.05 in comparison to control. In all treatments, the cytosolic Ca2+ binder BAPTA/AM (5 μM) was administered to fura-2-loaded cells before incubation with hydroxytyrosol in the presence of Ca2+.

Click here to view

  Discussion Top

Our study suggests that hydroxytyrosol elicited [Ca2+]i raises. The Ca2+ response was compiled of Ca2+ influx and Ca2+ discharge since removing external Ca2+ diminished Ca2+ response by 30%. This proposal was confirmed by Mn2+ quenching data which meant that Ca2+ entry took place throughout hydroxytyrosol pretreatment.

The data illustrate that hydroxytyrosol-elicited [Ca2+]i elevations were suppressed by 20% by nifedipine, econazole or SKF96365. These three compounds were thought to exert inhibitory effect on store-operated Ca2+ entry.[23],[24],[25] As a result, hydroxytyrosol seems to induce Ca2+ influx via store-operated Ca2+ entry which was activated by discharge of Ca2+ depository.[26] Since these modulators did not fully abolished the Ca2+ influx, the remaining 10% of Ca2+ influx remains to be explored.

Protein kinases status and Ca2+ homeostasis are intimately related.[27] Our results prove that hydroxytyrosol-elicited [Ca2+]i raises were hampered by 20% by either increasing or decreasing PKC activity. This suggests hydroxytyrosol-provoked [Ca2+] rises required a stable PKC state.

Concerning the Ca2+ repository participated in hydroxytyrosol-induced Ca2+ discharge from stores, the thapsigargin-sensitive ER depot appeared to have a crucial part. Our findings further prove the Ca2+ discharge was through a PLC-dissociated route, since the discharge was unaltered when PLC was fully inhibited. The PLC-dissociated discharge might be due to other pathways. For instance, U73122 was demonstrated to block Ca2+ influx in an uncommon PLC-independent pathway that is involved in neutrophil activation.[28] The other pathways merit more studies.

Cytotoxicity might be influenced in a fashion depending on external or internal Ca2+.[29],[30] Our results illustrate that hydroxytyrosol elicited concentration-dependent cell death that is Ca2+-independent. Even though hydroxytyrosol-elicited Ca2+ response did not cause cell death, numerous downstream processes could be altered.[11] Our findings prove that hydroxytyrosol (40-100 μM) inhibited cell viability. This may help explain the harmful effects observed in vivo.

  Conclusion Top

Together, the findings suggest hydroxytyrosol provoked entry of Ca2+ through PKC-sensitive, store-operated Ca2+ entry and Ca2+ discharge from ER in a fashion unrelated to PLC. Hydroxytyrosol induced cell death that is Ca2+-independent. Since raises in [Ca2+]i can influence numerous cell events, prudence should be exercised in employing hydroxytyrosol for other investigation, and it is noteworthy hydroxytyrosol may be cytotoxic to liver cells at concentration around tens of μM ranges.


The authors acknowledge Kaohsiung Veterans General Hospital for providing infrastructure facilities.

Financial support and sponsorship

This research was supported by Chang Bing Show Chwan Memorial Hospital (BRD-109042) to HHC and Kaohsiung Veterans General Hospital (KSVGH110-157) to CRJ.

Conflicts of interest

There are no conflicts of interest.

  References Top

Rigacci S, Stefani M. Nutraceutical properties of olive oil polyphenols. An itinerary from cultured cells through animal models to humans. Int J Mol Sci 2016;17:E843.  Back to cited text no. 1
Romani A, Ieri F, Urciuoli S, Noce A, Marrone G, Nediani C, et al. Health effects of phenolic compounds found in extra-virgin olive oil, by-products, and leaf of Olea europaea L. Nutrients 2019;11:1776.  Back to cited text no. 2
Tuck KL, Hayball PJ. Major phenolic compounds in olive oil: Metabolism and health effects. J Nutr Biochem 2002;13:636-44.  Back to cited text no. 3
Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM, Binkoski AE, Hilpert KF, et al. Bioactive compounds in foods: Their role in the prevention of cardiovascular disease and cancer. Am J Med 2002;113 Suppl 9B:71S-88S.  Back to cited text no. 4
Vilaplana-Pérez C, Auñón D, García-Flores LA, Gil-Izquierdo A. Hydroxytyrosol and potential uses in cardiovascular diseases, cancer, and AIDS. Front Nutr 2014;1:18.  Back to cited text no. 5
de Bock M, Derraik JG, Brennan CM, Biggs JB, Morgan PE, Hodgkinson SC, et al. Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: A randomized, placebo-controlled, crossover trial. PLoS One 2013;8:e57622.  Back to cited text no. 6
Pan S, Liu L, Pan H, Ma Y, Wang D, Kang K, et al. Protective effects of hydroxytyrosol on liver ischemia/reperfusion injury in mice. Mol Nutr Food Res 2013;57:1218-27.  Back to cited text no. 7
Soto-Alarcon SA, Valenzuela R, Valenzuela A, Videla LA. Liver protective effects of extra virgin olive oil: Interaction between its chemical composition and the cell-signaling pathways involved in protection. Endocr Metab Immune Disord Drug Targets 2018;18:75-84.  Back to cited text no. 8
Palmerini CA, Carlini E, Saccardi C, Servili M, Montedoro G, Arienti G. Activity of olive oil phenols on lymphomonocyte cytosolic calcium. Mol Cell Biochem 2005;280:181-4.  Back to cited text no. 9
Marchetti C, Clericuzio M, Borghesi B, Cornara L, Ribulla S, Gosetti F, et al. Oleuropein-enriched olive leaf extract affects calcium dynamics and impairs viability of malignant mesothelioma cells. Evid Based Complement Alternat Med 2015;2015:908493.  Back to cited text no. 10
Berridge MJ. The inositol trisphosphate/calcium signaling pathway in health and disease. Physiol Rev 2016;96:1261-96.  Back to cited text no. 11
Bootman MD, Lipp P, Berridge MJ. The organisation and functions of local Ca2+ signals. J Cell Sci 2001;114:2213-22.  Back to cited text no. 12
Cho CH, Woo JS, Perez CF, Lee EH. A focus on extracellular Ca2+ entry into skeletal muscle. Exp Mol Med 2017;49:e378.  Back to cited text no. 13
Nomikos M, Kashir J, Lai FA. The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation. Biochem J 2017;474:3659-73.  Back to cited text no. 14
Gao SY, Wang QJ, Ji YB. Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells. World J Gastroenterol 2006;12:3359-67.  Back to cited text no. 15
Gao M, Cheung KL, Lau IP, Yu WS, Fung KP, Yu B, et al. Polyphyllin D induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization. Arch Toxicol 2012;86:741-52.  Back to cited text no. 16
Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440-50.  Back to cited text no. 17
Merritt JE, Jacob R, Hallam TJ. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem 1989;264:1522-7.  Back to cited text no. 18
Thastrup O, Cullen PJ, Drøbak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A 1990;87:2466-70.  Back to cited text no. 19
Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher SK. The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem 1991;266:23856-62.  Back to cited text no. 20
Kuroki M, Takeshige K, Minakami S. ATP-induced calcium mobilization in human neutrophils. Biochim Biophys Acta 1989;1012:103-6.  Back to cited text no. 21
Tsien RY. New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 1980;19:2396-404.  Back to cited text no. 22
Leung YM, Kwan CY. Current perspectives in the pharmacological studies of store-operated Ca2+ entry blockers. Jpn J Pharmacol 1999;81:253-8.  Back to cited text no. 23
Michel LY, Verkaart S, Latta F, Hoenderop JG, Bindels RJM. Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA. Cell Calcium 2017;65:52-62.  Back to cited text no. 24
Morita K, Sakakibara A, Kitayama S, Kumagai K, Tanne K, Dohi T. Pituitary adenylate cyclase-activating polypeptide induces a sustained increase in intracellular free Ca2+ concentration and catechol amine release by activating Ca2+ influx via receptor-stimulated Ca2+ entry, independent of store-operated Ca2+ channels, and voltage-dependent Ca2+ channels in bovine adrenal medullary chromaffin cells. J Pharmacol Exp Ther 2002;302:972-82.  Back to cited text no. 25
Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 1986;7:1-12.  Back to cited text no. 26
Korchak HM, Corkey BE, Yaney GC, Kilpatrick LE. Negative regulation of ligand-initiated Ca2+ uptake by PKC-beta II in differentiated HL60 cells. Am J Physiol Cell Physiol 2001;281:C514-23.  Back to cited text no. 27
Wang JP. U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation. Naunyn Schmiedebergs Arch Pharmacol 1996;353:599-605.  Back to cited text no. 28
Nagy G, Koncz A, Perl A. T cell activation-induced mitochondrial hyperpolarization is mediated by Ca2+- and redox-dependent production of nitric oxide. J Immunol 2003;171:5188-97.  Back to cited text no. 29
Das S, Bhattacharyya S, Ghosh S, Majumdar S. TNF-alpha induced altered signaling mechanism in human neutrophil. Mol Cell Biochem 1999;197:97-108.  Back to cited text no. 30


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
Materials and Me...
Article Figures

 Article Access Statistics
    PDF Downloaded195    
    Comments [Add]    

Recommend this journal