Invest Clin 63(3): 243 - 261, 2022 https://doi.org/10.54817/IC.v63n3a04

Corresponding author: Gustavo Benaim . Laboratorio de Señalización Celular y Bioquímica de Parásitos, Instituto

de Estudios Avanzados (IDEA) and Instituto de Biología Experimental, Universidad Central de Venezuela (UCV),

Caracas, Venezuela. Tel: +584143060359; E-mail: gbenaim@gmail.com

Effect of tetrahydroquinoline derivatives

on the intracellular Ca2+ homeostasis

in breast cancer cells (MCF-7) and its

relationship with apoptosis.

Semer Maksoud1, Adriana Mayora2, Laura Colman3, Felipe Sojo4,5, Adriana A. Pimentel6,

Vladimir V. Kouznetsov7, Diego R. Merchán-Arenas7, Ángel H. Romero8,

Francisco Arvelo4,5, Juan Bautista De Sanctis9 and Gustavo Benaim10,11

1Department of Neurology and Experimental Therapeutics and Molecular Imaging

Laboratory, Massachusetts General Hospital, MA 02129, USA.

2Instituto de Medicina Experimental. Facultad de Medicina. Universidad Central de

Venezuela.

3Instituto Pasteur, Montevideo, Uruguay.

4Centro de Biociencias, Fundación Instituto de Estudios Avanzados (IDEA). Caracas,

Venezuela.

5Laboratorio de Cultivo de Tejidos y Biología de Tumores, Instituto de Biología

Experimental, Universidad Central de Venezuela (UCV), Caracas, Venezuela.

6Facultad de Farmacia, Universidad Central de Venezuela. Caracas, Venezuela.

7Laboratorio de Química Orgánica y Biomolecular (CMN), Universidad Industrial de

Santander. Parque Tecnológico Guatiguara, Piedecuesta. Colombia.

8Cátedra de Química General, Facultad de Farmacia, Universidad Central de Venezuela.

Caracas, Venezuela.

9Institute of Molecular and Translational Medicine. Palacky Unversity.

Olomouc. Czech Republic.

10Laboratorio de Señalización Celular y Bioquímica de Parásitos. Instituto de Estudios

Avanzados (IDEA). Caracas, Venezuela.

11Instituto de Biología Experimental, Universidad Central de Venezuela (UCV). Caracas.

Venezuela.

Key words: Ca2+; apoptosis; tetrahydroquinolines; mitocondria; SERCA; NF-κB.

Abstract. Tetrahydroquinoline derivatives are interesting structures exhib-

iting a wide range of biological activities, including antitumor effects. In this

investigation, the effect of the synthesized tetrahydroquinolines JS-56 and JS-92

on apoptosis, intracellular Ca2+ concentration ([Ca2+]i), and the sarco(endo)plas-

mic reticulum Ca2+-ATPase (SERCA) activity was determined on MCF-7 breast

cancer cells. Colorimetric assays were used to assess MCF-7 cells viability and

SERCA activity. Fura-2 and rhodamine 123 were used to measure the intracellu-

lar Ca2+ concentration and the mitochondrial electrochemical potential, respec-

244 Maksoud et al.

Investigación Clínica 63(3): 2022

Efecto de derivados de tetrahidroquinolinas sobre la

homeostasis del Ca2+ intracelular en células de cáncer de mama

(MCF-7) y su relación con la apoptosis.

Invest Clin 2022; 63 (3): 243 – 261

Palabras clave: Ca2+, apoptosis, tetrahidroquinolinas, Mitocondria, SERCA, NF-κB.

Resumen. Los derivados de tetrahidroquinolina son estructuras interesan-

tes que exhiben una amplia gama de actividades biológicas, incluyendo efectos

antitumorales. Se determinó el efecto de las tetrahidroquinolinas sintetizadas

JS-56 y JS-92 sobre la apoptosis, concentración intracelular de Ca2+ ([Ca2+]i) y

la actividad Ca2+-ATPasa del retículo sarco(endo)plásmico (SERCA) en células de

cáncer de mama MCF-7. Se usaron ensayos colorimétricos para evaluar la viabili-

dad de las células MCF-7 y la actividad SERCA. Se emplearon Fura-2 y rodamina

123 para medir la concentración de Ca2+ intracelular y el potencial electroquí-

mico mitocondrial, respectivamente. El ensayo TUNEL se utilizó para analizar la

fragmentación del ADN, mientras que la actividad de caspasas y la expresión géni-

ca dependiente de NF-κB se evaluaron mediante luminiscencia. Modelos in silico

permitieron el análisis del acoplamiento molecular. Estos compuestos aumentan

la concentración de Ca2+ intracelular; la principal contribución es la entrada de

Ca2+ desde el medio extracelular. Tanto JS-56 como JS-92 inhiben la actividad de

SERCA y disipan el potencial electroquímico mitocondrial a través de procesos

dependientes e independientes de la captación de Ca2+ por este orgánulo. Ade-

más, JS-56 y JS-92 generan citotoxicidad en células MCF-7. El efecto de JS-92 es

mayor que JS-56. Ambos compuestos activan las caspasas 7 y 9, provocan la frag-

mentación del ADN y potencian el efecto del 12-miristato-13-acetato de forbol en

la expresión génica dependiente de NF-κB. El análisis de acoplamiento molecular

sugiere que ambos compuestos tienen una alta interacción con SERCA, similar

a la tapsigargina. Ambos derivados de tetrahidroquinolina indujeron la muerte

celular a través de una combinación de eventos apoptóticos, aumento de [Ca2+]i

e inhibición de la actividad SERCA por interacción directa.

Received: 06-04-2022 Accepted: 10-06-2022

tively. TUNEL assay was used to analyze DNA fragmentation, while caspase activi-

ty and NF-κB-dependent gene expression were assessed by luminescence. In silico

models were used for molecular docking analysis. These compounds increase

intracellular Ca2+ concentration; the main contribution is the Ca2+ entry from

the extracellular milieu. Both JS-56 and JS-92 inhibit the activity of SERCA and

dissipate the mitochondrial electrochemical potential through processes depen-

dent and independent of the Ca2+ uptake by this organelle. Furthermore, JS-56

and JS-92 generate cytotoxicity in MCF-7 cells. The effect of JS-92 is higher than

JS-56. Both compounds activate caspases 7 and 9, cause DNA fragmentation,

and potentiate the effect of phorbol 12-myristate-13-acetate on NF-κB-dependent

gene expression. Molecular docking analysis suggests that both compounds have

a high interaction for SERCA, similar to thapsigargin. Both tetrahydroquinoline

derivatives induced cell death through a combination of apoptotic events, in-

crease [Ca2+]i, and inhibit SERCA activity by direct interaction.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 245

Vol. 63(3): 243 - 261, 2022

INTRODUCTION

Breast cancer is the most frequently

diagnosed cancer and the leading cause of

cancer-related death in women worldwide,

with an estimated 2.3 million new cases per

year and approximately more than 685,000

deaths by 2020 1. Among treatments against

this disease is the use of tamoxifen (Nolva-

dex®) for cancer cells expressing estrogen

receptors or trastuzumab (Herceptin®) for

HER-2+ mammary tumors 2. Of particular in-

terest, numerous investigations evaluate the

potential of natural or synthetic compounds

that stimulate apoptosis in breast cancer

cells by disturbing intracellular Ca2+ homeo-

stasis 3-9.

Ca2+ possesses an essential regulatory

role in differentiation, secretion, contrac-

tion, transcription, phosphorylation, and

apoptosis processes. A piece of large cell ma-

chinery composed of different proteins and

organelles contributes to the regulation of

intracellular Ca2+ concentration ([Ca2+]i) 10.

Among them is the sarco(endo)plasmic re-

ticulum Ca2+-ATPase (SERCA), which allows

active transport and a considerable accu-

mulation of this cation at the endoplasmic

reticulum (ER). Active transport maintains

[Ca2+] in the ER. This concentration is ap-

proximately three orders of magnitude high-

er (millimolar range) than cytoplasm [Ca2+]

(~100 nanomolar), and similar to the ex-

tracellular milieu 11. Apoptosis is directly

related to Ca2+ transfer from ER to mito-

chondria. Ca2+ depletion in ER can generate

a phenomenon called “ER stress” in which

the reduction of protein folding capacity,

and consequent accumulation of these mis-

folded proteins, initiates different processes

promoting cell death 12,13. The electropho-

retic uniporter (MCU) induces Calcium ac-

cumulation in the mitochondria. This [Ca2+]

increase generates an important change in

electrochemical potential (Δψm) 14 which

may enable membrane permeabilization

with consequent release of cytochrome c,

caspase-9 activation and cell death 15.

In this investigation, the effect of tetra-

hydroquinolines JS-56 and JS-92 (Fig. 1) was

evaluated on Ca2+ homeostasis and apopto-

sis induction in breast cancer cells MCF-7.

The tested compounds were prepared from

inexpensive and commercially available isat-

in, 2-aminobenzonitrile (or 4-bromoaniline)

and trans-isoeugenol, a major constituent of

clove oil using a previously published two-

step procedure 16,17. These tetrahydroquino-

lines have been shown to exert antitumor

activity against various cancer cell lines, in-

cluding MCF-7, SKBR3, PC3 and Hela cells

16,17; the aim of the study is to ascertain the

mechanism of apoptosis induction by the

compounds. Both compounds induced an in-

Fig. 1. Chemical structure of the tetrahydroquinolines JS-56 and JS-92.

246 Maksoud et al.

Investigación Clínica 63(3): 2022

crease in [Ca2+]i caused in part through SER-

CA inhibition, combined with the dissipation

of mitochondrial Δψm. Molecular docking

analysis showed that these compounds have

similar ligand-protein interactions with SER-

CA when compared to thapsigargin (Tg).

Furthermore, these tetrahydroquinolines

generated apoptosis via caspase activation

and DNA fragmentation. Both compounds

enhanced the effect of phorbol 12-myristate-

13-acetate (PMA) on the NF-κB-dependent

gene expression.

MATERIALS AND METHODS

Chemicals

The preparation of 8’-ciano-4’-(4-hydroxy-

3-methoxyphenyl)-3’-methyl-3’,4’-dihydro-1’H-

spiro[indoline-3,2’-quinolin]-2-one (JS-56) and

6’-bromo-4’-(4-hydroxy-3-methoxyphenyl)-3’-

methyl-3’,4’-dihydro-1’H-spiro[indoline-3,2’-

quinolin]-2-one (JS-92) was performed by fol-

lowing our previous reports which include the

reaction of isatin and 2-aminobenzonitrile or

4-bromoaniline to give the respective ketimine-

isatin derivatives and the subsequent acid-

catalyzed cycloaddition reaction with trans-

isoeugenol 16,17. Both compounds were purified

by silica gel column chromatography and ob-

tained as stable solids. Their structures were

confirmed by various spectroscopic techniques

such as EI-MS, 1H NMR, 13C NMR, and IR, test-

ing each sample as a pure chemical substance

in the following biological experiments.

Cell culture

The human breast cancer cell line MCF-

7 was grown in Dulbecco’s Modified Eagle

Medium (DMEM), supplemented with peni-

cillin (100 U/mL), streptomycin (10,000

μg/mL), 1% GlutaMAX™ and 10% fetal bo-

vine serum (FBS). These cells were kept in

a humidified incubator at 37°C and in an at-

mosphere of 5% CO2.

Cytotoxicity assays

To evaluate the cytotoxicity in MCF-

7 cells, the colorimetric reduction assay of

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-

razolium bromide (MTT) was performed 18.

Ten thousand cells were seeded in 96-well

plates for 48 h allowing confluence. Cells

were exposed to JS-56 and JS-92 for 24 h,

at concentrations ranging from 1 to 50 μM.

In all cases, both tetrahydroquinolines were

dissolved in dimethyl sulfoxide (DMSO). The

final concentration of this solvent was equal

to or less than 0.5%.

All appropriate controls were developed

with 0.5% DMSO. After treatment, cells were

incubated with 0.4 mg/mL of MTT for 2 h at

37°C. Then, the supernatant was removed, and

the formazan crystals were dissolved by adding

50 μL of DMSO. Absorbance was measured in a

multi-detection microplate reader (Sinergy HT,

Bio-Tek) at 570 nm. The IC50 value was defined

as the tetrahydroquinoline concentration caus-

ing a 50% reduction in absorbance compared

to the control of untreated cells, using the

Graphpad Prism 5.0 software.

Measurement of intracellular Ca2+

concentration

Measurements of [Ca2+]i in MCF-7 cells

were made using the fluorescent indicator

Fura-2, essentially as previously described 19.

Briefly, a suspension of 1x106 cells/mL was

prepared in a medium containing 138 mM

NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2,

5 mM glucose, 10 mM HEPES and 0.1% al-

bumin (extracellular medium), loading cells

with 200 μM sulfinpyrazone, 0.025% plu-

ronic acid, 0.1% albumin, and 2 μM Fura 2

acetoxymethyl ester (Fura 2-AM) for 30 min

at 25°C, in darkness and constant agitation.

After loading, cells were incubated for 15

minutes in the extracellular medium and

washed again using the same solution. When

indicated, these experiments were also

performed using the same wash buffer but

without CaCl2 (by addition of 2 mM EGTA).

Fura-2 fluorescence on loaded cells suspen-

sion was monitored through a Perkin Elmer

LS 55 spectrofluorimeter with a chopper

device. The excitation wavelengths were set

at 340 and 380 nm, while the emission was

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 247

Vol. 63(3): 243 - 261, 2022

measured at 510 nm. All experiments were

carried out at 30°C, with constant agitation

in a stirred cuvette.

Intracellular Ca2+ was calculated ac-

cording to Grynkiewicz et al. 20, using the

following equation: [Ca2+]I = Kd x (R-Rmin/

Rmax-R) x Fmin(380)/Fmax(380) where Kd is the dis-

sociation constant of Fura-2 (224 nM). R is

the ratio of emission fluorescence intensities

to excitation lengths of 340/380 nm. Rmin

and Rmax are ratios at 0 Ca2+ (6 μM digitonin

plus 4 mM EGTA) and saturating Ca2+ (2 mM

CaCl2), respectively. Fmin(380)/Fmax(380) are fluo-

rescent values of cells exposed to digitonin

plus 4 mM EGTA and 2 mM CaCl2, represent-

ing minimal and maximal fluorescent values.

Purification of Ca2+-ATPase from the

sarcoplasmic reticulum

The sarcoplasmic reticulum was ob-

tained from the white skeletal muscle of

the hind legs of rabbits, according to the

method described by Eletr and Inesi 21. The

vesicles derived from the sarcoplasmic retic-

ulum are highly enriched in Ca2+-ATPase (ap-

proximately 90%). To avoid Ca2+ retention

in the lumen of the vesicles, the ionophore

A-23187 (1 μM) was added.

Evaluation of SERCA activity

The purified enzyme (2 μg/mL) was in-

cubated for 45 min at 37°C and continuous

agitation in a final volume of 250 μL of a buf-

fer containing 100 mM KCl, 20 mM MOPS-

Tris (pH 7.0), 1 mM EGTA, 10 mM MgCl2,

4 mM ATP, 1 μM A23817 and 1 mM CaCl2,

obtaining 10 μM of Ca2+ in the medium and

reaching an optimal activity of the enzyme.

Inorganic phosphate production was quanti-

fied using the Fiske and Subbarow 22 colo-

rimetric assay, modified by applying ferrous

sulfate as a reducing agent 23.

Determination of the mitochondrial

electrochemical potential (∆ψm)

Mitochondrial membrane potential

changes were measured using the fluores-

cent marker rhodamine 123, as previously

described 24, with the modifications intro-

duced by Pimentel et al. 6. Rhodamine 123

is a cationic fluorescent dye that allows the

evaluation of the electrochemical potential of

this organelle when distributed according to

the Δψm. Rhodamine has a maximum excita-

tion peak of 488 nm and a maximum emis-

sion peak of 530 nm. For these experiments

3 x 105 cells/mL were resuspended in a me-

dium containing 138 mM NaCl, 5 mM KCl,

1 mM MgCl2, 1.5 mM CaCl2, 5 mM glucose,

10 mM HEPES-KOH, pH 7.4 and then loaded

with rhodamine 123 (20 μg/mL) for 45 min

at 30°C. All measurements were made on a

HITACHI-L-7000 spectrofluorimeter at 30˚C

with continuous stirring.

Determination of caspases 7 and 9 activity

We used Caspase-Glo 3/7 and Caspase-

Glo 9 kits (Promega, Madison, WI) and de-

veloped the respective tests following the

manufacturer’s instructions. Since MCF-7

cells do not possess caspase 3 25, the results

shown correspond to the effect of the dif-

ferent compounds on caspase 7 activity. In

a 96-well plate, 10,000 cells/well were seed-

ed and incubated for 48 h at 37°C in a 5%

CO2 atmosphere. Then, cells were exposed

to different treatments for 24 h. Then, the

cells were lysed, and the luminescence was

measured with a multidetector microplate

reader (Synergy HT, Bio-Tek). Staurosporine

(Stau, 1 μM) was applied as an apoptosis ac-

tivator (positive control), and 0.5% DMSO

was used as a negative control.

Determination of DNA fragmentation

DNA fragmentation was analyzed through

the “dead-end fluorimetric TUNEL (TdT-medi-

ated dUTP Nick-End labeling) system” (Prome-

ga) kit. Cells were double-labeled with Fluores-

cein-12-dUTP (a specific marker of fragmented

DNA) and propidium iodide (DNA marker). Ac-

cording to manufacturing instructions, chang-

es in fluorescence intensity were detected by

flow cytometry (Epics XL Beckman Coulter,

FL). Cells incubated with staurosporine (1 μM)

were used as a positive control.

248 Maksoud et al.

Investigación Clínica 63(3): 2022

Analysis of NF-κB-dependent gene expression

In this assay, HeLa tumor cells trans-

fected with a luciferase reporter gene whose

expression is under control of a modified

IL-6 promoter, which only contains a critical

binding site for NF-κB, were used. The lucif-

erase activity was determined in a luminom-

eter. Five thousand cells/well were seeded in

96-well plates and incubated for 48 h. Sub-

sequently, the cells were exposed to all treat-

ments and corresponding controls for 24 h.

Then, the cells were lysed, the luciferase sub-

strate was added, and the product was quan-

tified by luminescence. Incubation with cul-

ture medium represents the negative control

while phorbol 12-myristate 13-acetate (100

nM) was used as a positive control.

Molecular docking procedure

A 3D crystal structure from SERCA in

complex with BHQ (2,5-ditert-butylbenzene-1,4-

diol) and Tg was downloaded from Protein

Data Bank under PDB code 2AGV. The pro-

tein corresponds to sarco(endo)plasmic re-

ticulum Ca2+-ATPase from Oryctolagus cu-

niculus species, and it was crystallized with a

resolution of 2.4 Å 26. The protein presented

six co-crystallized chemical ligands: two mol-

ecules of BHQ, two molecules of Tg, and two

sodium atoms. Four active sites (two for BHQ

and the other two for Tg) were generated on

the protein: two on α-chain and the other two

on β-chain. The 3D structure was imported

into the Swiss-PdbViewer v4.0.1 software 27.

Hydrogen atoms were added to the protein

structure, and minimization calculations

were carried out on protein structure via AM-

BER force field, using the conjugate gradient

method with an RMA gradient of 0.01 kcal/

Åmol on Swiss-PdbViewer v4.0.1 software.

The prepared protein was exported to an Ar-

gusLab v.4.0.1 program package and saved

as an Agl document. Four active sites were

constructed on the SERCA protein derived

from BHQ and Tg inhibitors. It is well docu-

mented that both compounds, BHQ and Tg,

act as SERCA inhibitors. Specifically, BHQ is

an L-type Ca2+ channel inhibitor, blocks Ca2+

transport by inducing superoxide anion pro-

duction 28. On the contrary, Tg inhibits all

SERCA isozymes at similar concentrations29.

Both inhibitors are bound to the transmem-

brane domain of the enzyme close to the

membrane/cytosolic interface.

Once the protein was prepared, the two

tested tetrahydroquinolines JS56 and JS92

were built using ArgusLab v4.0 30, optimiz-

ing their respective geometries employing

B3LYP/6-31G(d,p) 31. Then, molecular dock-

ing of the two compounds and crystallized

inhibitors (BHQ and TG) over the four active

SERCA sites was performed employing the

ArgusLab (v4.0.1) package program under

Windows 7.0 environment, applying AMBER

force field. The docking was achieved through

their respective grid map dimensions and a

grid point spacing of 0.375 Å. A flexible li-

gand model was used in the docking and

subsequent optimization scheme. Different

orientations of the ligands were scanned and

ranked from their energy scores. Reproduc-

ibility of the calculated affinity and the mini-

mum energy pose was evaluated through 10

replicates for each quinoline and crystallized

inhibitor (BHQ or TS) 32. The ChemScore

scoring function was selected for the evalu-

ation of ligand binding modes. Settings for

genetic algorithm runs were kept at their de-

fault values; that is, the population size was

100, the selection pressure 1.1, and the num-

ber of operations was 100,000. Affinity energy

is reported as the mean of the 10 replicates.

The lowest energy poses (strongest-docking)

for each ligand in each target protein are

summarized in Table 1.

RESULTS

Effect of tetrahydroquinolines JS-56 and

JS-92 on cytotoxicity in MCF-7 cells

First, the possible cytotoxicity generated

by JS-56 and JS-92 on MCF-7 breast cancer

cells was evaluated through an MTT assay af-

ter being treated for 24 h with different con-

centrations of these tetrahydroquinolines. It

can be observed (Fig. 2) that both compounds

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 249

Vol. 63(3): 243 - 261, 2022

Table 1

Binding energies (kcal/mol), H-interactions, and hydrophobic interactions from molecular

docking on the four studied active sites.

Binding

site

Molecule Binding energy

(kcal/mol)

H-interactions (Å) Intermolecular interactions

TG1 JS-56 -13.1366 Two hydrogen bonding

(2.8596 and 2.1804 Å)

between the phenolic

hydrogen of the ligand

and Ile-829 residue.

a) H-π interaction with

Phe-834 and Phe-256.

b) Hydrophobic interactions

with Met-838, Tyr-837, Phe-834,

Ile-829, Leu-828, Pro-827,

Leu-828, Ile-765, Gln-259,

Leu-260 and Val-263.

JS-92 -12.5189 Hydrogen bonding

(2.8988 Å) between the

phenolic hydrogen of

the ligand and Phe-834

residue

a) H-π interaction with

Phe-236 and Phe-834.

b) Hydrophobic interactions

with Met838, Ile829, Pro827,

Leu828, and 765Ile.

Tg -14.1626 No hydrogen bonding a) No π-π interactions.

b) Hydrophobic interactions

with Met-838, Phe-834, Ile-829,

Leu-828, Pro-827, Phe-776,

Val-773, Val-772, Val-769, Asn-768,

Ile-765, Ile-761, Pro-308, Ile-267,

Val-263, Leu-260, Gln-259,

Phe-256, Leu-253,

Leu-249 residues.

Fig. 2. Effect of tetrahydroquinolines JS-56 and JS-92 on MCF-7 cell viability. MCF-7 cells were treated with

different concentrations of these compounds. Each point represents the mean ± SD of three indepen-

dent experiments. IC50 values obtained for JS-56 and JS-92 were 9.74 μM and 5.03 μM, respectively.

250 Maksoud et al.

Investigación Clínica 63(3): 2022

induced cytotoxicity in MCF-7 cells in a dose-

dependent manner, with an IC50 of 9.74 μM and

5.03 μM for JS-56 and JS-92, respectively. At a

concentration of 50 μM, JS-56 decreased cell

viability by approximately 88%. At the same

time, JS-92 exerted a more substantial effect

than JS-56 on this cell line, being able to re-

duce almost 100% of MCF-7 cell viability at the

same concentration.

Effect of tetrahydroquinolines JS-56 and

JS-92 on intracellular Ca2+ mobilization

To study the effect of these tetrahydro-

quinolines on the [Ca2+]i, cells were loaded

with the Ca2+ fluorescent indicator Fura-2. The

addition of JS-56 induced a rapid increase in

[Ca2+]i, reaching a peak followed by a continu-

ous decrease (Fig. 3A), as a consequence of

[Ca2+]i regulation carried out by the cellular

homeostatic machinery until it reached a new

steady-state Ca2+ level, which is greater than

the original basal level. This response is charac-

teristic of compounds capable of inducing the

release of Ca2+ from the ER, with the conse-

quent opening of plasma membrane Ca2+ chan-

nels (SOCE) activated by emptying this intra-

cellular reservoir 19,33. The effect observed upon

the addition of thapsigargin (Tg) (Fig. 3B), a

potent SERCA inhibitor, was similar to that ob-

served with JS-56. However, the maximum Ca2+

peak achieved with Tg was higher than JS-56.

Additionally, when comparing Figures 3A and

3B, the addition of Tg after JS-56 (Fig. 3A) still

induced a slight increase in [Ca2+]i. However,

the tetrahydroquinoline did not exert any dis-

cernible effect after Tg addition (Fig. 3B).

The same experiments were performed

in the absence of extracellular Ca2+ to deter-

mine the origin of the cation increase. Under

these experimental conditions, the maximum

Ca2+ peaks obtained with JS-56 (Fig. 3C) or

Tg (Fig. 3D) were considerably lower than

Fig. 3. Effect of tetrahydroquinoline JS-56 and thapsigargin (Tg) on [Ca2+]i in MCF-7 cells. The arrows in-

dicate the moment in which JS-56 (20 μM) and Tg (2 μM) were added, in the presence (Fig. A and

B) or absence (EGTA, Fig. C and D) of 2 mM CaCl2. The curves are representative of at least three

independent experiments.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 251

Vol. 63(3): 243 - 261, 2022

those reached in the presence of extracellu-

lar Ca2+ (Figs. 3A and 3B). Furthermore, the

Ca2+ levels obtained, after the maximum peak

and the subsequent decrease, were equal to

the basal levels (Figs. 3C and 3D). Moreover,

when Tg was added after JS-56, a minimal rise

in [Ca2+]i was detected, indicating again that,

different from Tg, the tetrahydroquinoline

was not able to release all the Ca2+ from the

ER (Fig. 3D). Likewise, the effect of the tet-

rahydroquinoline JS-92 was evaluated under

the same experimental conditions, obtaining

essentially the same overall results, as can be

observed in Fig. 4.

Effect of tetrahydroquinolines JS-56

and JS-92 on SERCA activity

To identify a possible mechanism of ac-

tion of these compounds, which could explain

their effect on the increase in [Ca2+]i, the

ability to inhibit SERCA activity was exam-

ined, considering that the results obtained

were very similar to those found by the use

of Tg. Fig. 5 shows the effect of increasing

concentrations of JS-56 and JS-92 on ATPase

activity. Both tetrahydroquinolines inhibited

SERCA activity in a dose-dependent man-

ner. The maximum inhibitory effect of JS-

56 and JS-92 on SERCA activity was 56 and

51%, respectively. Albeit none of the tetrahy-

droquinolines could completely inhibit the

SERCA activity, these results demonstrate

that part of the effect of these compounds

on [Ca2+]i elevation was undoubtedly due to

SERCA activity inhibition.

Effect of tetrahydroquinolines JS-56

and JS-92 on the mitochondrial

electrochemical potential

Mitochondria use their electrochemical

membrane potential (Δψm) to accumulate

Ca2+ through the electrophoretic Ca2+ uni-

porter (MCU). The effects of the tetrahydro-

quinolines and Tg on the mitochondrial Δψ

were studied by using the fluorescent mark-

er rhodamine 123. This fluorescent indica-

Fig. 4. Effect of tetrahydroquinoline JS-92 and thapsigargin (Tg) on [Ca2+]i in MCF-7 cells. The arrows indi-

cate the addition of JS-92 (20 μM) and Tg (2 μM), in the presence (Fig. A and B) or absence (EGTA,

Fig. C and D) of 2 mM CaCl2. The curves are representative of at least three independent experiments.

252 Maksoud et al.

Investigación Clínica 63(3): 2022

tor accumulates in the mitochondrial matrix

according to the electrochemical gradient.

Thus, an increase in fluorescence can be ob-

served due to the mitochondrial H+ gradient

dissipation, concomitantly with the release

of rhodamine 123 from the mitochondria to

the cytoplasm and then to the extracellular

milieu. Results from Fig. 6 demonstrate that

both JS-56 and JS-92 (20 μM) and Tg (2 μM)

produced a rapid and pronounced elevation

in fluorescence due to rhodamine 123 release

from mitochondria. However, the increment

in rhodamine 123 fluorescence caused by Tg

was more significant than those produced by

the tetrahydroquinolines (Figs. 6B and 6D).

It can also be noticed that JS-92 (Fig. 6C)

generated an elevation of rhodamine 123

fluorescence higher than JS-56 (Fig. 6A).

The effect of Tg after the addition

of the tetrahydroquinolines was less pro-

nounced (Figs. 6A and 6C). It is worthwhile

to mention that this fluorescence elevation

with both Tg and tetrahydroquinolines was

expected since it was due to the partial dis-

sipation of mitochondrial Δψ, generated by

the entry of Ca2+ released from the ER6. On

the other hand, after Tg application, the

addition of JS-56 and JS-92 led to an addi-

tional boost in fluorescence (Figs. 6B and

6D), indicating a superior Δψ dissipation,

which cannot be merely explained by the en-

trance of Ca2+ released from the ER. Thus,

these results suggest that both tetrahydro-

quinolines can directly affect the mitochon-

dria by Ca2+-independent mechanisms. As

expected, the positive control, carbonyl cy-

anide-4-(trifluoromethoxy)phenylhydrazone

(FCCP), generated a complete collapse of

the electrochemical gradient.

Effect of tetrahydroquinolines JS-56 and

JS-92 on the activity of caspases 7 and 9

We then evaluated the possible effect

of these tetrahydroquinolines on the induc-

tion of apoptosis in MCF-7 cells. Accordingly,

the activation of caspase 9, a known caspase

that initiates apoptosis, was assessed. The

1/2 IC50, IC50, and 2 IC50 obtained in the

MTT assay were used for both compounds.

As shown in Fig. 7 (upper panel), both JS-

56 and JS-92 significantly enhanced the ac-

tivity of caspase-9 compared to the control

in a dose-dependent manner. Sphingosine

(Sph), which promotes an increase in [Ca2+]

i in MCF-7 cells 33, and 2,5-di-t-butyl-1,4-

benzohydroquinone (BHQ), a known SERCA

reversible-inhibitor 34, also elevated the ac-

tivity of this initiating caspase. The positive

control Stau, which activates apoptosis in

MCF-7 cells 35, effectively activated caspase

9 at a level similar to the IC50 of the tetrahy-

droquinolines.

Fig. 5. Effect of tetrahydroquinolines JS-56 and JS-92 on SERCA activity. Each point represents the mean ±

SD of three independent experiments.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 253

Vol. 63(3): 243 - 261, 2022

Likewise, treatment of MCF-7 cells with

both tetrahydroquinolines raised the activity

of caspase 7 (Fig. 7, lower panel). Similar re-

sults were detected with the positive control

Stau and the SERCA inhibitor BHQ. The ac-

tivation-induced by JS-92 was slightly higher

compared to the rest of the compounds. This

effector caspase will be responsible for cleav-

ing specific cell targets to promote apopto-

sis in these cells.

Effect of tetrahydroquinolines JS-56 and

JS-92 on DNA fragmentation

An apoptotic cell is typically character-

ized by: changes in cell morphology, phos-

phatidylserine externalization, cytoplasm

contraction, shrinkage of the nucleus, and

DNA fragmentation 15. As a complement to

the results obtained in caspases 7 and 9 ac-

tivity assays, DNA fragmentation was ana-

lyzed through a TUNEL assay using Stau as a

positive control. In Fig. 8, cells were divided

into four populations displaying the percent-

age of cells in necrosis (propidium iodide,

IP+), apoptosis (fluorescein isothiocyanate,

FITC+), and alive (negative for both mark-

ers); apoptotic cells can either be IP- or IP+.

Apoptotic cells exhibit DNA fragmentation,

which allows FITC incorporation. However,

a subset of these apoptotic cells is also IP+,

denoting that these also have a loss of plas-

ma membrane integrity, corresponding to a

Fig. 6. Effect of tetrahydroquinolines JS-56 and JS-92 on the mitochondrial electrochemical potential in

MCF-7 cells. The fluorescent marker rhodamine 123 was used for these experiments, expressing re-

sults in arbitrary units (AU) of fluorescence. The arrows indicate the moment of the addition of the

different effectors: JS-56 or JS-92 (20 μM), thapsigargin (Tg, 2 μM), and FCCP (10 μM). The curves

are representative of at least three independent experiments.

254 Maksoud et al.

Investigación Clínica 63(3): 2022

later stage of apoptosis in which cells can

no longer control the passage of elements

across the membrane, with eventual cell ly-

sis in the final stage. The quantitative data is

represented in Table 2.

JS-56 treatment (10 μM) for 24 h in-

duced apoptosis, with 65.9% of FITC+ IP-

cells in this stage and only 4.3% of FITC+ IP+

cells. Treatment with Stau (1 μM) and JS-92

(5 μM) produced a significant percentage of

FITC+ IP- (31.5 and 46.5% for Stau and JS-

92, respectively) and FITC+ IP+ cells (60.6

and 35.5% for Stau and JS-92, respectively).

Hence, JS-92 has a more potent effect than

JS-56, which measurements of caspase ac-

tivity have also evidenced. These results are

statistically significant compared to the neg-

ative control (DMSO-treated MCF-7 cells).

Effect of tetrahydroquinolines JS-56

and JS-92 on NF-κB-dependent gene

expression

The nuclear transcription factor NF-κB

regulates numerous genes involved in multi-

ple cellular processes such as apoptosis, cell

proliferation, and differentiation. To evalu-

ate NF-κB-dependent gene expression, HeLa

tumor cells transfected with a luciferase re-

porter gene under the control of IL-6 pro-

moter, which contains critical binding sites

for NF-κB, were used.

Tetrahydroquinolines JS-56 and JS-92,

when added alone, were not able to increase

the NF-κB-dependent gene expression, with

values similar to those observed in the nega-

tive control (Fig. 9). The luminescence mea-

sured in the negative control corresponds

to the basal NF-κB-dependent gene expres-

sion on MCF-7 cells. The NF-κB-dependent

gene expression is elevated when cells were

treated with 100 nM of PMA; it has been

Table 2

Percentage of living, apoptotic, and necrotic cells after treatment with JS-56 and JS-92.

Treatment group

Percentage

Living cells Apoptosis

(IP-)

Apoptosis

(IP+)

Necrosis

Control DMSO 93.5 ± 4.6 0.9 ± 0.6 0.1 ± 0.1 4.1 ± 1.2

Staurosporine 3.9 ± 1.3 31.5 ± 7.5 60.6 ± 6.8 1.2 ± 0.5

JS-56 25.5 ± 9.3 65.9 ± 4.7 4.3 ± 2.8 1.9 ± 1.6

JS-92 14.6 ± 6.3 46.5 ± 5.5 35.5 ± 4.8 3.4 ± 1.8

Fig. 7. Effect of tetrahydroquinolines JS-56 and JS-

92 on the activity of caspases 9 (initiator)

and 7 (effector). Stau (staurosporine), BHQ

(2,5-di-t-butyl-1,4-benzohydroquinone) and

Sph (sphingosine). The bars represent the

mean ± SD derived from three independent

experiments. * and ** p < 0.05 compared

to the control of untreated cells or treated

with JS-56, respectively.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 255

Vol. 63(3): 243 - 261, 2022

reported that PKC can activate IκBs kinase

complexes (IKKs), which eventually leads

to NF-κB activation 36. Interestingly, the re-

sults demonstrate that the NF-κB-dependent

gene expression increased significantly when

cells were treated with PMA plus JS-56 or

JS-92, indicating an apparent effect of both

compounds on the PKC-stimulated NF-κB-

dependent gene expression.

Molecular docking

The experimental results on SERCA

activity obtained for the tetrahydroquino-

lines JS-56 and JS-92 were confirmed using

molecular docking for SERCA protein (PDB

code 2AGV). Interestingly, both Tg and BHQ

SERCA inhibitors showed practically the

same disposition as crystallized inhibitors

at the respective protein binding sites, re-

flecting the computational package’s poten-

tial for this molecular docking. In general,

the tetrahydroquinolines showed a higher

preference for Tg binding sites than for the

BHQ active site. This effect generates a li-

gand-protein complex with binding energies

between -12 and -13 kcal/mol at the active

sites of Tg; this is in contrast to weak dock-

ing energies found at the BHQ active site for

both tetrahydroquinolines. Particularly, ex-

perimental results have demonstrated that

Tg exerted a more potent inhibitory activity

(IC50 of 0.2 nM) than BHQ (IC50 of 20 nM)

26. The preference of these large tetrahydro-

quinolines for Tg binding sites may be as-

sociated with the larger box size than BHQ

active sites, which may facilitate the stabi-

lization of the inhibitor-protein complex for

these bulky and inflexible molecules. It is

Fig. 8. Effect of tetrahydroquinolines JS-56 and JS-92 on DNA fragmentation in MCF-7 cells. The MCF-7 cells

were incubated with Fluorescein-12-dUTP (fragmented DNA marker) and Propidium Iodide (IP, core

marker). Cells were treated with staurosporine (1 μM), JS-56 (10 μM), and JS-92 (5 μM). The bars

represent the mean ± SD derived from three independent experiments. * p < 0.0001 compared to the

control of cells treated with DMSO.

Fig. 9. Effect of tetrahydroquinolines JS-56 and JS-

92 on NF-κB-dependent gene expression.

HeLa tumor cells were transfected with a

luciferase reporter gene under the control

of the IL-6 promoter, which contains criti-

cal binding sites for NF-κB. Control, untrea-

ted MCF-7 cells; PMA, phorbol 12-myristate

13-acetate. * and ** p<0.05 compared to

the control of untreated cells or treated

with PMA, respectively.

256 Maksoud et al.

Investigación Clínica 63(3): 2022

essential to mention that our analysis was

focused on data obtained for the Tg active

site from chain-α, which exhibited the lower

binding energy complex.

DISCUSSION

Tetrahydroquinolines derivatives are an

essential group of natural or synthetic com-

pounds showing a wide range of biological

activities. Numerous compounds capable of

inducing a Ca2+ signal followed by a cellular

response have been reported. Ca2+ is one of

the essential ionic constituents in the body.

It has a central regulatory role in differentia-

tion, secretion, contraction, transcription,

phosphorylation, and apoptosis processes

10. Ca2+ is the component required by milk

to calcify newborn bones and teeth and

modulates the proliferation, differentiation,

and apoptosis of mammary gland epithelial

cells 37. The modulation of Ca2+ homeosta-

sis and its signaling could represent a viable

therapeutic approach in the treatment and/

or prevention of breast cancer. Therefore,

regulators of Ca2+ homeostasis or its signal-

ing in mammary gland tumors are possible

targets for drugs. In this investigation, we

used the tetrahydroquinolines JS-56 and JS-

92, modified derivatives from clove and cin-

namon plants, to study the effect of these

drugs on Ca2+ homeostasis and its possible

relationship with apoptosis in breast cancer

cells MCF-7.

In the interest of evaluating the cyto-

toxicity of tetrahydroquinolines JS-56 and

JS-92 on MCF-7 breast cancer cells, the MTT

assay was developed, allowing the determi-

nation of mitochondrial viability on treated

cells. Both compounds are cytotoxic to MCF-

7 cells in a dose-dependent manner, with an

IC50 of 9.74 μM and 5.031 μM for JS-56 and

JS-92, respectively. This difference may be

related to the bromine atom that JS-92, in-

stead of the cyanide atom present in JS-56.

IC50 values for these compounds in MCF-7

cells are significantly lower than those re-

ported previously in human dermis fibro-

blast, 92.87 μM and 42.48 μM for JS-56 and

JS-92, respectively 17.

Through fluorimetric techniques, we

were able to perform measurements of intra-

cellular Ca2+ in MCF-7 cells, aiming to find

possible mechanisms of action for these com-

pounds. The results revealed that JS-56 and

JS-92 promote an increase in [Ca2+]i. Like-

wise, the Tg effect on [Ca2+]i is diminished

when added after the impact generated by

the tetrahydroquinolines, compared to when

it is initially added, allowing us to conclude

that these compounds promote the release

of Ca2+ from a compartment sensitive to Tg,

namely the ER. Similar experiments were

done but using a solution without CaCl2. Un-

der these experimental conditions, the maxi-

mum Ca2+ peaks detected with JS-56, JS-92

or Tg are considerably reduced compared to

those achieved in the presence of extracellu-

lar Ca2+, and instead, returning to the basal

level. These results show an influx of Ca2+

from the outside. This effect may be caused

by a store-operated Ca2+ Entry (SOCE) or a

capacitive Ca2+ entry induced by emptying

Ca2+ from the ER 19,33.

The lack of an additive effect of Tg with

tetrahydroquinolines on the [Ca2+]i suggests

a possible similar mechanism of action. The

possible inhibitory potential of these com-

pounds on SERCA activity was examined ac-

cordingly. Both tetrahydroquinolines inhibit

the enzyme activity with similar maximum

inhibitory effects of 56 and 51% for JS-56

and JS-92, respectively. This effect also in-

dicates that different from Tg, these com-

pounds could not completely inhibit the

ATPase activity. However, part of the overall

effect on the onset of apoptosis is the partial

inhibition of SERCA activity. As it has been

shown that Tg can induce ER stress via SER-

CA inhibition, JS-56 and JS-92 are likely po-

tential candidates for generating ER stress

through this identical mechanism. In this

context, it is worth mentioning that SERCA

targeting could represent an effective thera-

peutic strategy in treating pathogens and

cancer cells 38.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 257

Vol. 63(3): 243 - 261, 2022

The effect of both compounds on the

mitochondrial membrane Δψm was deter-

mined since the accumulation of mitochon-

drial Ca2+ is a necessary event in the initia-

tion of apoptosis via the intrinsic pathway.

The primary source of the cation derives

from its transfer from the ER 39. The results

revealed that both tetrahydroquinolines

generate an increase in rhodamine 123 fluo-

rescence. Although the increment in rhoda-

mine fluorescence caused by Tg was higher

than the compounds, its effect after the ad-

dition of the tetrahydroquinolines was less

pronounced, suggesting that they share part

of the mechanism of action on mitochondria.

The fluorescence increase after treating the

cells with both Tg and tetrahydroquinolines

was expected due to the partial dissipation

of mitochondrial Δψm. This change is gen-

erated by the entry of Ca2+ released from

the ER 6. When both tetrahydroquinolines

were added after Tg, an additional increase

in fluorescence was triggered, indicating a

superior dissipation of the Δψ. These unex-

pected results suggested that JS-56 and JS-

92 induced a cell response independent of

[Ca2+]i. The effects are also independent of

the Ca2+ electrophoretic uniporter. These

effects have been reported previously with

concentrations higher than 1 μM 40. Accord-

ingly, both tetrahydroquinolines, similar to

the SERCA inhibitor, could interact with the

mitochondria, generating a widespread ef-

fect.

Given that the Δψm loss of the mito-

chondrial membrane is a hallmark event of

apoptosis, the presumed activation of char-

acteristic processes associated with pro-

grammed cell death after applying the tet-

rahydroquinolines was investigated. In this

study, it was demonstrated that tetrahydro-

quinolines JS-56 and JS-92 are capable of

activating caspases 7 and 9 and promoting

DNA fragmentation. Activation of caspases

and the percentage of cells with DNA frag-

mentation was higher for JS-92, which coin-

cides with its superior cytotoxic effect. DNA

fragmentation in conjunction with caspase

activation and the results demonstrating

that these tetrahydroquinolines cause the

dissipation of mitochondria Δψm to support

the conclusion that these compounds acti-

vate apoptosis in MCF-7 cells. Additionally,

after the inhibition of SERCA and the conse-

quent reduction of the [Ca2+] at the ER, mis-

folded proteins could accumulate. The un-

folded protein response could be triggered,

leading the cells to death 12,13. By suppress-

ing this enzyme’s activity and promoting the

cation’s entry into the mitochondria, the

release of pro-apoptotic factors from this or-

ganelle would be facilitated 15. Thus, part of

the potential anticancer effect of these com-

pounds would be mediated through SERCA

inhibition. Based on the accumulated evi-

dence, we suggest that disturbances of the

Ca2+ signal induced by these tetrahydroquin-

olines would trigger apoptosis by promoting

ER stress and the release of proapoptotic

factors from the mitochondria after the en-

try of Ca2+ into this organelle.

According to our results, JS-56 and JS-

92 failed to elevate the NF-κB-dependent

gene expression. However, NF-κB-dependent

gene expression was augmented when cells

were treated with PMA, an activator of PKC

which upregulates NF-κB 35. Interestingly, the

NF-κB-dependent gene expression is further

elevated when cells were exposed to the com-

bination of PMA and tetrahydroquinolines,

possibly because these compounds enhance

the affinity of PKC for PMA or modulate the

PMA activity making it more potent and/or

stable. Since NF-κB is a regulator of the ex-

pression of multiple genes, additional experi-

ments are needed to determine its putative

participation in pro-apoptotic events. We do

not rule out non-specific binding of other

transcription factors to this NF-κB binding

site on the IL-6 promoter. Future research

will confirm the participation of this tran-

scription factor by determination of cytoplas-

mic/nuclear levels of p65 as well as confirma-

tion of these results in breast cancer cells.

Based on the results that both tetrahy-

droquinolines may interact with the Tg bind-

258 Maksoud et al.

Investigación Clínica 63(3): 2022

ing site of SERCA, we used a docking mod-

el to test the possibility of interaction. It

should be noted that both tetrahydroquino-

lines exhibited docking energies higher than

Tg, indicating that the Tg-Ca2+-ATPase com-

plex is more stable than the quinoline-Ca2+-

ATPase complex (Table 1). These theoretical

findings are in good agreement with experi-

mental data, where Tg exhibited the higher

experimental inhibitory activity (IC50 of 0.2

nM) compared to the tested tetrahydroquin-

olines (IC50 of 60 and 100 µM for JS-56 and

JS-92, respectively). The high stabilization

of the Tg-Ca2+-ATPase complex can be asso-

ciated with diverse intermolecular interac-

tions with residues located in the Tg1 active

site, including (i) a large number of hydro-

phobic interactions with at least 20 residues

in chain A and 13 residues in chain B; (ii)

from one to two strong H-interactions with

Ile-829 and Phe-834 for JS-56 and JS-92, re-

spectively; and (iii) H-π interactions between

phenolic, quinolinic, or indolic rings with

specific phenyl rings in Phe-834, Phe-256

or Tyr-837 (Table 1 and Fig. 10). However,

it is essential to mention that these diverse

interactions found for these tetrahydroquin-

olines are located in turn to a specific re-

Fig. 10. Molecular docking of the tetrahydroquinolines JS-56 and JS-92 on the TG1 active site. (A) TG1 (gray

ribbons) and TG2 (blue ribbons) active sites into SERCA protein; (B) Location of JS-56 (cyan color)

and JS-92 (orange color) into the TG1 active site pocket of SERCA protein; (C) Superimposition

between JS-56 (cyan) and JS-92 (orange) with TG1 substrate (yellow color); (D) Disposition and in-

teractions of JS-56 (cyan) (D) and JS-92 (orange) (E) into TG1 active site. Tentative H-π interactions

between phenolic, indolic, or quinolinic rings of JS-56 and JS-92 with Phe-236 (gray color), Phe-834

(blue color), and Phe-256 (green color) residues.

Apoptotic effect of tetrahydroquinoline derivatives in breast cancer cells 259

Vol. 63(3): 243 - 261, 2022

gion of the active site. At the same time, Tg,

bearing a highly flexible octanoic acid resi-

due at C2, makes hydrophobic contacts with

non-polar patches on the outer surface of

SERCA’s trans-membrane domain and pos-

sibly with surrounding lipids, facilitating the

stabilization of the ligand-protein complex.

The molecular docking analysis shows that

hydrophobic ligands containing long and

flexible side chains are needed to design ef-

fective inhibitors of the Ca2+-ATPase enzyme

due to the high hydrophobic and extended

nature of the Ca2+-ATPase active site. Future

studies will continue to evaluate these types

of compounds and their effect on Ca2+ and

apoptosis, developing new analogs with dif-

ferent types of chemical modifications on a

more diverse panel of breast cancer cells and

other types of malignancies.

Both tetrahydroquinolines derivatives,

JS-56 and JS-92, induced cell death in MCF-

7 cells through a combination of events.

These events include the elevation of the

[Ca2+]i, inhibiting SERCA via direct interac-

tion, partially damaging the mitochondria,

which induced activation of apoptotic path-

ways and consequently MCF-7 cell death.

Therefore, these results show the potential

that these types of compounds would have

in anti-neoplastic treatment in humans. It

is hoped that more powerful analogs can be

developed in the future.

Funding

This work was supported by [Fondo Na-

cional de Ciencia, Tecnología e Investigación,

Venezuela (FONACIT)] (Grants 2017000274

and 2018000010), and the [Consejo de De-

sarrollo Científico y Humanístico-Univer-

sidad Central de Venezuela (CDCH-UCV)]

(Grant PG-03-8728-2013/2) to G.B.

Conflict of interest

The authors declare no conflicts of in-

terest.

Authors contributions

SM: Investigation, Methodology, Data

curation, Writing – original draft, Writing –

review & editing. AM: Methodology, Writing

– review & editing. LC: Methodology, Writing

– review & editing. FS: In silico analysis, Writ-

ing – review & editing. AP: NF-κB gene expres-

sion analysis. VK: In silico analysis, isolation

and purification of natural compounds. DMA:

Isolation and purification of natural com-

pounds. AR: Methodology, Writing – review &

editing. FA: In silico analysis, Writing – review

& editing. JS: Flow cytometry analysis, Writ-

ing – review & editing. GB: Conceptualiza-

tion, Formal analysis, Supervision, Funding

acquisition, Writing – review & editing.

Author’s ORCID numbers

• Semer Maksoud:

0000-0001-9773-9415

• Adriana Mayora:

0000-0002-6817-2066

• Laura Colman:

0000-0002-5188-880X

• Felipe Sojo:

0000-0002-6559-4845

• Adriana A. Pimentel:

0000-0002-8862-9318

• Vladimir V. Kouznetsov:

0000-0003-1417-8355

• Diego R. Merchán-Arenas:

0000-0001-9243-5914

• Ángel H. Romero:

0000-0001-8747-5153V

• Francisco Arvelo:

0000-0003-1590-358x

• Juan Bautista De Sanctis:

0000-0002-5480-4608

• Gustavo Benaim:

0000-0002-9359-5546

260 Maksoud et al.

Investigación Clínica 63(3): 2022

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