Synthesis and photodynamic activity of unsymmetrical A3B tetraarylporphyrins functionalized with l-glutamate and their Zn(II) and Cu(II) metal complex derivatives

Article abstract of DOI:10.1016/j.biopha.2016.05.010

Four novel unsymmetrical A₃B porphyrins 1, 2, 3 and 4 were synthesized following Lindsey procedure. Porphyrins 3 and 4 include one and three l-glutamate groups, respectively, and all porphyrins were metallated with Zn(II) (1a-4a) or Cu(II) (1b-4b). Porphyrins and metalloporphyrins presented values of singlet oxygen quantum yields (ΦD) ranging from 0.21 to 0.67. The tetraaryl derivatives in this study showed phototoxicity in SiHa cells with IC₅₀ values ranging from <0.01 to 6.56 ± 0.11 μM, the metalloporphyrin 4a showed the lowest IC₅₀ value. Comparing the phototoxic activity between all porphyrins, functionalization of porphyrins with glutamate increased 100 times phototoxic activity (1 (IC₅₀ 4.81 ± 0.34 μM) vs. 3 (IC₅₀ 0.04 ± 0.02 μM) and 2 (IC₅₀ 5.19 ± 0.42 μM) vs. 4 (IC₅₀ 0.05 ± 0.01 μM)). This increased activity could be attributed to reduced hydrophobicity and increased Φδ, given by functionalization with l-glutamate. Metalloporphyrins 3a (IC₅₀ 0.04 ± 0.01 μM) and 4a (IC₅₀ <0.01 μM) presented the best values of phototoxic activity. Therefore, functionalization and zinc metalation increased the phototoxic activity. SiHa cells treated with porphyrins 3, 4, 3a and 4a at a final concentration of 10 μM, showed increased activity of caspase-3 enzyme compared to the negative control; indicating the induction of apoptosis. Differential gene expression pattern in SiHa cells was determined; treatments with metalloporphyrins 4a and 4b were performed, respectively, comparing the expression with untreated control. Treatments in both cases showed similar gene expression pattern in upregulated genes, since they share about 25 biological pathways and a large number of genes. According to the new photophysical properties related to the structural improvement and phototoxic activity, these molecules may have the potential application as photosensitizers in the photodynamic therapy.

Full text of DOI:10.1016/j.biopha.2016.05.010

Biomedicine & Pharmacotherapy 82 (2016) 327–336
Available online at
ScienceDirect
www.sciencedirect.com
Synthesis and photodynamic activity of unsymmetrical A3B
tetraarylporphyrins functionalized with
and Cu(II) metal complex derivatives
L-glutamate and their Zn(II)
Eder U. Arredondo-Espinoza, Susana T. López-Cortina, Mónica A. Ramírez-Cabrera,
Isaías Balderas-Rentería*
Universidad Autónoma de Nuevo León, Laboratorio de Ingeniería Genética y Genómica, Facultad de Ciencias Químicas, Av. Universidad s/n, Cd. Universitaria,
6
6451 San Nicolás de los Garza, N.L., Mexico
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 27 December 2015
Received in revised form 7 May 2016
Accepted 9 May 2016
3
Four novel unsymmetrical A B porphyrins 1, 2, 3 and 4 were synthesized following Lindsey procedure.
Porphyrins 3 and 4 include one and three l-glutamate groups, respectively, and all porphyrins were
metallated with Zn(II) (1a–4a) or Cu(II) (1b–4b). Porphyrins and metalloporphyrins presented values of
singlet oxygen quantum yields (
FD) ranging from 0.21 to 0.67. The tetraaryl derivatives in this study
Keywords:
showed phototoxicity in SiHa cells with IC50 values ranging from <0.01 to 6.56 ꢀ 0.11
m
M, the
L
-glutamate
metalloporphyrin 4a showed the lowest IC50 value. Comparing the phototoxic activity between all
porphyrins, functionalization of porphyrins with glutamate increased 100 times phototoxic activity (1
Metalloporphyrins
Photodynamic therapy
Photosensitizer
(
IC50 4.81 ꢀ0.34
This increased activity could be attributed to reduced hydrophobicity and increased FD, given by
M) and 4a (IC50 <0.01 M)
m
M) vs. 3 (IC50 0.04 ꢀ 0.02
m
M) and 2 (IC50 5.19 ꢀ 0.42
m
M) vs. 4 (IC50 0.05 ꢀ 0.01
mM)).
Unsymmetrical porphyrins
functionalization with l-glutamate. Metalloporphyrins 3a (IC50 0.04 ꢀ 0.01
m
m
presented the best values of phototoxic activity. Therefore, functionalization and zinc metalation
increased the phototoxic activity. SiHa cells treated with porphyrins 3, 4, 3a and 4a at a final
concentration of 10 mM, showed increased activity of caspase-3 enzyme compared to the negative
control; indicating the induction of apoptosis. Differential gene expression pattern in SiHa cells was
determined; treatments with metalloporphyrins 4a and 4b were performed, respectively, comparing the
expression with untreated control. Treatments in both cases showed similar gene expression pattern in
upregulated genes, since they share about 25 biological pathways and a large number of genes. According
to the new photophysical properties related to the structural improvement and phototoxic activity, these
molecules may have the potential application as photosensitizers in the photodynamic therapy.
ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
repeatedly, induces an inflammatory response leading to the
development of an important immune system reaction and can
Photodynamic therapy (PDT) is a minimally invasive procedure
also be combined with other cancer treatments [2].
and a promising new approach to fight cancer. PDT destroys cancer
Porphyrins are PS’s agents used in PDT. Porphyrins (aromatic
macrocycles formed by four pyrrole rings joined together by
interpyrrolic methine bridges) [3,4] are interesting molecules with
key roles in many biological processes [5], and they have also been
used for various applications, such as catalysis [6], chemical
sensors [7] and photodynamic therapy [8].
An ideal photosensitizer should satisfy important require-
ments, such as high purity and stability, obtained by means of a
simple synthesis under laboratory conditions, photoactivity at
wavelengths between 630 and 800 nm, solubility in biological
fluids with selective uptake in tumour cells, optimal absorption,
distribution, metabolism and excretion (ADME) properties,
cells by photochemical generation of reactive oxygen species (ROS)
1
mainly singlet oxygen ( O
2
), these species are generated by the
interaction of molecular oxygen with a photosensitizing agent (PS)
excited by irradiation of visible light [1]. The advantages of PDT are
as follows: the PS’s agents show selectivity to cancer cells;
phototoxic effect is localized and occurs only in the place that is
irradiated, thereby minimizing side effects; therapy can be applied
*
Corresponding author.
E-mail address: isaias.balderasrn@uanl.edu.mx (I. Balderas-Rentería).
http://dx.doi.org/10.1016/j.biopha.2016.05.010
753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
0

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E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
3
Scheme 1. Synthesis of A B porphyrins 1–4 by Lindsey method.
minimal toxicity in the absence of light, without yield of toxic
metabolites and also no mutagenic or carcinogenic effects [9].
Porphyrins are PS’s agents used in PDT, and it has been proved
2.2. General procedure for the synthesis of unsymmetrical A
porphyrins
3
B
that the nature and location of substituents in the meso and
b
According to Geier and Lindsey [16] method, porphyrin
synthesis was performed via condensation of the corresponding
aromatic aldehydes and pyrrole under mixed-acid catalysis.
positions of the porphyrins have an effect on their spectral,
photophysical and biological characteristics. Some porphyrins
possess affinity for tumour cells and they absorb light in the visible
range, however, high hydrophobicity is a therapeutic limitation.
This problem can be solved by adding hydrophilic substituents,
such as sugars, hydroxyl group, sulphates, phosphonates, peptides,
etc. [10–14]. Another option for improving the properties of the
photosensitizers is the formation of complexes with metals
bonded inside the tetrapyrrole ring, which causes the modification
of the photophysical properties of the photosensitizers [15].
3 2
BF (OEt) (0.17 mg, 0.01.2 mM) and trifluoro acetic acid (TFA)
ꢁ
(51 mg, 0.45 mM) were added to a solution of aldehyde A
(3.75 mM), aldehyde B (1.25 mM) and freshly distilled pyrrole
(335 mg, 5 mM) in 300 ml of CH
temperature for 2 h under inert atmosphere (N
2
Cl
2
; the mixture was kept at room
). Then 2,3-
2
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (57 mg, 0.251 mM)
was added, and the mixture was kept at room temperature for 2 h.
The solvent was evaporated and the crude material was purified on
a chromatography column using silica gel as stationary phase and
mixtures of hexanes, dichloromethane and isopropanol as eluent
to give porphyrins 1–4 as purple solids (Scheme 1, Table 1).
3
In this study, we synthesized four unsymmetrical A B tetraaryl
porphyrins; two of them functionalized with dimethyl 2-amino-
pentanedioate (l-glutamate) and two obtained without any
modification of the former aldehydes (p-methoxybenzaldehyde
and methyl p-formylbenzoate). The corresponding zinc and copper
metalloporphyrins were also obtained. We evaluated the photo-
dynamic activity of 12 tetraaryl derivatives, four porphyrins (1, 2, 3,
2.2.1. 5,10,15-Tris(p-methoxyphenyl)-20-(p-
methoxycarbonylphenyl)-21H,23H-porphyrin, (1)
The general procedure was followed using p-methoxybenzal-
dehyde as aldehyde A (510 mg, 3.75 mM) and methyl p-formyl-
benzoate as aldehyde B (205 mg, 1.25 mM), yielding 228 mg (24%
yield) of 1 after column chromatography purification (hexane:
4
) and eight metalloporphyrins (1a–4a and 1b–4b). The quantum
yield of singlet oxygen ( ), dark cytotoxicity, phototoxicity of all
F
D
derivatives and induction of apoptosis of derivatives 3, 3a, 4 and 4a
was evaluated. A study of differential gene expression with DNA
microarrays was also performed, and differential gene expression
was compared after treatment with derivatives 4a and 4b.
dichloromethane 3:7 as eluent). UV–vis (CH
2
Cl
2
) lmax: 423, 517,
1
3
544, 593 and 649 nm. H NMR (CDCl ) d
ꢂ2.77 (s, 2H), 4.08 (s, 9H),
4.11 (s, 3H), 7.28 (d, J = 7.5 Hz, 6H), 8.12 (d, J = 7.5 Hz, 6H), 8.3 (d,
J = 7.5 Hz, 2H), 8.43 (d, J = 7.5 Hz, 2H), 8.78 (d, J = 4 Hz, 2H), 8.88 (s,
4H), 8.89 (d, J = 4 Hz, 2 H).
2. Materials and methods
2.1. Materials
Table 1
Phenyl substituents in A
method.
UV–vis measurements were performed with a Perkin Elmer
3
B porphyrins 1–4,synthesized by Lindsey
Lambda-12 Spectrometer. NMR spectra were recorded on a
1
5
00 MHz Varian-NMR System, H NMR spectra at 500 MHz with
Porphyrin
R
1
R
2
CDCl as solvent and tetramethylsilane as internal standard.
3
1
2
3
CH
3
CH
3
CH
3
O-
OC(O)-
OC(O)-
CH
CH
3
OC(O)-
O-
Coupling constants (J) are given in Hz. Mass spectrometric
measurements were performed on a JEOL JMS700 instrument.
CH Cl was dried by distillation over lithium aluminium hydride.
2 2
3
Solvents were used after distillation at normal pressure. All
reactants were of reagent grade from Sigma-Aldrich and Merck as
it corresponds. Chromatography glass column packed with silica
gel 60 (0.04–0.063 mm) was used. We use the cell line of cervical
cancer (SiHa cell) obtained from the American Type Culture
Collection (ATCC) (Rockville, MD, USA), with a catalog number
ATCC: HTB-35, for dark cytotoxicity, phototoxic activity, apoptosis
and DNA microchip assays.
4
CH
3
OC(O)-

E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
329
2.2.2. 5,10,15-Tris(p-methoxycarbonylphenyl)-20-(p-
chromatography column using silica gel as stationary phase and
methoxyphenyl)-21H,23H-porphyrin, (2)
AcOEt as eluent to give 846 mg (92% yield) of p-formyl benzoic acid,
1
The general procedure was followed using methyl p-formyl
benzoate as aldehyde A (616 mg, 3.75 mM) and p-methoxyben-
zaldehyde as aldehyde B (170 mg, 1.25 mM), producing 202 mg
5, as a beige solid. H NMR (CDCl
J = 8 Hz, 2H), 10.13 (s, 1H).
3
) d 7.29 (d, J = 8 Hz, 2H), 8.26 (d,
(
(
4
(
20% yield) of 2 after column chromatography purification
hexane:dichloromethane 3:7 as eluent). UV–vis (CH Cl
ꢂ2.8 (s, 2H), 4.07
s, 3H), 4.10 (s, 9H), 7.28 (d, J = 8 Hz, 2H), 8.11 (d, J = 8 Hz, 2H), 8.29 (d,
J = 8 Hz, 6H), 8.43 (d, J = 8 Hz, 6H), 8.79 (d, J = 4.5 Hz, 2H), 8.81 (s,
2.3.2. Synthesis of dimethyl 2-aminopentanedioate, (6)
2
2
)
l
max
:
3
l-glutamic acid (1 g, 7 mM) was dissolved in 30 ml of CH OH,
1
25, 516, 552, 591 and 647 nm. H NMR (CDCl
3
)
d
and SOCl (1.19 g, 10 mM) was added dropwise. The reaction
mixture was then allowed to stir at room temperature for 18 h, the
solvent was evaporated and the crude product washed with ethyl
4H), 8.91 (d, J = 4.5 Hz, 2H).
ether. The product was dissolved in AcOEt, then Et
3
N (506 mg,
5
mM) was added and the mixture was stirred for 18 h at room
2.2.3. 5,10,15-Tris(p-methoxyphenyl)-20-(p-(dimethyl-2-
temperature, after white precipitate was formed, the reaction
aminopentanedioate)-carbonylphenyl)-21H,23H-porphyrin, (3)
The general procedure was followed using p-methoxybenzal-
dehyde as aldehyde A (510 mg, 3.75 mM) and dimethyl 2-(4-
mixture was filtered and the solvent evaporated to give 437 mg of
1
compound 6 (70% yield). H NMR (CDCl
3
)
d
2.32 (m, 2H), 2.43 (t,
2H), 3.7 (s, 6H), 4.21 (s, 2H) and 4.23 (t, 1H).
formylbenzamido)pentanedioate, 7, as aldehyde
.25 mM), producing 294 mg (26% yield) of 3 after column
chromatography purification (dichloromethane:isopropanol 98:2
B (385 mg,
1
2.3.3. Synthesis of dimethyl 2-(4-formylbenzamide)pentanedioate, (7)
The synthesis was performed following the typical conditions
for amide formation [21]. p-Formyl benzoic acid 5 (1 g, 6.6 mM)
and dimethyl 2-aminopentanedioate 6 (1.76 mg, 10 mM) were
placed in a flask under inert nitrogen atmosphere, then N,N -
dicyclohexylcarbodiimide (1.36 g, 6.6 mM) and 4-dimethylamino-
1
as eluent). UV–vis (CH
NMR (CDCl
ꢂ2.76 (s, 2H), 1.21 (q, 2H), 1.25 (t, 2H), 4.11 (s, 9H),
.12 (s, 6H), 7.27 (d, J = 8.5 Hz, 6H), 8.14 (d, J = 8.5 Hz, 6H), 8.35 (d,
J = 8.5 Hz, 2H), 8.45 (d, J = 8.5 Hz, 2H), 8.81 (d, J = 4 Hz, 2H), 8.90 (s,
2 2
Cl ) lmax: 421, 518, 555, 593 and 649 nm. H
3
) d
0
4
4H), 8.92 (d, J = 4, 2H).
2 2
pyridine (147 mg, 1.2 mM) were dissolved in 40 ml of dry CH Cl ,
transferred via cannula into the reaction flask and then allowed to
stir for 20 h at room temperature. The crude reaction was filtered
and washed with AcOEt and the filtrate was evaporated. After
purification on a chromatography column using silica gel as
2
.2.4. 5,10,15-Tris(p-(dimethyl-2-aminopentanedioate)-
carbonylphenyl)-20-(p-methoxyphenyl)-21H,23H-porphyrin, (4)
The general procedure was followed using dimethyl 2-(4-
formylbenzamido)pentanedioate, 7, as aldehyde
.75 mM) and p-methoxybenzaldehyde as aldehyde B (170 mg,
.25 mM), producing 390 mg (25% yield) of 4 after column
chromatography purification (dichloromethane:isopropanol 98:2
A
(1.15 g,
stationary phase and AcOEt as eluent, 673 mg of 7 were obtained
1
3
1
(33% yield) as a beige solid. H NMR (CDCl
3
)
d
2.18 (t, 2H), 2.39 (m,
2H), 3.69 (s, 3H), 3.76 (s, 3H), 4.64 (t, 1H), 6.24 (s, 2H), 7.99 (d,
J = 8 Hz, 2H), 8.26 (d, J = 8 Hz, 2H), 10.11 (s, 1H)
1
as eluent). UV–vis (CH
NMR (CDCl
ꢂ2.77 (s, 2H), 1.25 (q, 6H), 1.28 (t, 6H), 4.10 (s, 3H),
.12 (s, 18H), 7.27 (d, J = 8 Hz, 2H), 8.15 (d, J = 8 Hz, 2H), 8.32 (d,
J = 8 Hz, 6H), 8.42 (d, J = 8 Hz, 6H), 8.81 (d, J = 4.5 Hz, 2H), 8.82 (s,
2 2
Cl ) lmax: 424, 517, 557, 592 and 648 nm. H
3
)
d
2.4. General procedure for the synthesis of metalloporphyrins
4
Metalloporphyrins 1a–4a and 1b–4b were obtained by metal-
4H), 8.91 (d, J = 4.5 Hz, 2H).
lation of porphyrins with Zn(II) and Cu(II) as follows: 0.1 mM of the
corresponding porphyrin were dissolved in 10 ml of CH
2 2
Cl . A
2.3. General procedure for the synthesis of dimethyl 2-(4-
solution of 0.2 mM of the metal acetate in 10 ml of CH OH was
3
formylbenzamido) pentanedioate
prepared. The solutions were mixed and then stirred for 2 h at
room temperature. Finally, the solvent was evaporated and the
metalloporphyrins purified on a chromatography column using
The synthetic route that includes at first the coupling reaction
between p-formylbenzoic acid, 5, and dimethyl 2-aminopentano-
dioate, 6, following the typical conditions for amide formation
2 2
silica gel as stationary phase and CH Cl as eluent (Scheme 3) [18].
(Scheme 2) [17], obtaining dimethyl 2-(p-formylbenzamide)
2.4.1. 5,10,15-Tris(p-methoxyphenyl)-20-(p-
pentanedioate, 7.
methoxycarbonylphenyl)-porphyrinate zinc(II) complex, (1a)
The general procedure was followed using porphyrin
1
2
.3.1. Synthesis of p-formyl benzoic acid, (5)
Methyl p-formyl benzoate (1 g, 6 mM) was dissolved in 40 ml of
(76.23 mg, 0.1 mM) and zinc acetate (36.69 mg, 0.2 mM), producing
58 mg (71% yield) of 1a. UV–vis (CH Cl max: 422, 550 and
3
592 nm H NMR (CDCl ) d 4.09 (s, 9H), 4.11 (s, 3H), 7.28 (d, J = 8.7 Hz,
2
2
) l
1
CH
3
OH and KOH (617 mg, 11 mM) was dissolved in 10 ml of H
2
O,
the solutions were mixed together and allowed to stir at room
temperature for 18 h. The pH of the solution was adjusted to 2 by
6H), 8.12 (d, J = 8.7 Hz, 6H), 8.29 (d, J = 8.1, 2H), 8.42 (d, J = 8.1 Hz,
2H), 8.87 (d, J = 4.8 Hz, 2H), 8.98 (s, 4H), 8.99 (d, J = 4.8 Hz, 2H).
3
the addition of HCl (20% in CH OH) and then filtered. The solvent
was evaporated and the crude material was purified on a
Scheme 2. Synthesis of dimethyl 2-(4 formylbenzamide) pentanedioate, 7.

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E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
Scheme 3. Synthesis of metalloporphyrins 1a–4a and 1b–4 b.
2.4.2. 5,10,15-Tris(p-methoxyphenyl)-20-(p-
3
(CDCl ) d 1.25 (q, 6H), 1.28 (t, 6H), 4.10 (s, 3H), 4.12 (s, 18H), 7.27 (d,
methoxycarbonylphenyl)-porphyrinate copper(II) complex, (1b)
J = 8 Hz, 2H), 8.15 (d, J = 8 Hz, 2H), 8.32 (d, J = 8 Hz, 6H), 8.42 (d,
J = 8 Hz, 6H), 8.91 (d, J = 4.5 Hz, 2H), 8.92 (s, 4H), 8.98 (d, J = 4.5 Hz,
2H).
The general procedure was followed using porphyrin
1
(
76.23 mg, 0.1 mM) and copper(II) acetate (36 mg, 0.2 mM),
producing 45 mg (55% yield) of 1b. UV–vis (CH Cl max: 418,
2
2
) l
5
40 and 578 nm.
2.4.8. 5,10,15-Tris(p-(dimethyl-2-aminopentanedioate)-
carbonylphenyl)-20-(p-methoxyphenyl)-porphyrinate copper(II)
complex, (4b)
2.4.3. 5,10,15-Tris(p-methoxycarbonylphenyl)-20-(p-
methoxyphenyl)-porphyrinate zinc(II) complex, (2a)
The general procedure was followed using porphyrin
81.83 mg, 0.1 mM) and zinc acetate (37 mg, 0.2 mM), producing
The general procedure using porphyrin 4 (125 mg, 0.1 mM) and
copper(II) acetate (36 mg, 0.2 mM), producing 72 mg (56% yield) of
4b. UV–vis (CH Cl ) lmax: 419, 540 and 575 nm was followed.
2 2
2
(
7
2 mg (82% yield) of 2a. UV–vis (CH
2
Cl
2
) lmax: 420, 551 and
1
592 nm. H NMR (CDCl
3
)
d
4.09 (s, 2H), 4.10 (s, 9H), 7.28 (d,
2.5. Photodynamic activity
J = 8.4 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.1 Hz, 6H), 8.43 (d,
J = 8.1 Hz, 6H), 8.88 (d, J = 4.8 Hz, 2H), 8.90 (s, 4H), 9.02 (d, J = 4.8 Hz,
2.5.1. Quantum yields of singlet oxygen
2
H).
Quantum yields of singlet oxygen with the method of
illumination in the steady state were determined, comparing
the scavenger consumption rates of a reference photosensitizer
and the compounds under study [19].
2.4.4. 5,10,15-Tris(p-methoxycarbonylphenyl)-20-(p-
methoxyphenyl)-porphyrinate copper(II) complex, (2b)
The general procedure was followed using porphyrin
81.83 mg, 0.1 mM) and copper(II) acetate (36.32 mg, 0.2 mM),
producing 75 mg (85% yield) of 2b. UV–vis (CH Cl ) lmax: 417, 539
2 2
2
5,10,15,20-tetra(4-hydroxyphenyl)-porphyrin (p-TPPH) was
used as reference photosensitizer with a quantum yield of 0.56
using methanol as solvent, 1,3-diphenylisobenzofuran (DPBF) was
(
and 577 nm.
used as scavenger [20]. The singlet oxygen quantum yields (
were calculated by the equation: (sample) = (ksample/kref
(ref) is the singlet oxygen quantum yield
for the reference (p-TPPH) in methanol ( (ref) = 0.56), ksample and
ref are the DPBF photobleaching rates in the presence of the
D
F )
) (Aref/
F
D
ꢁ
2.4.5. 5,10,15-Tris(p-methoxyphenyl)-20-(p-(dimethyl-2-
A
sample
)
ꢁ F
D D
(ref), where F
aminopentanedioate)-carbonylphenyl)-porphyrinate zinc(II)
complex, (3a)
F
D
k
The general procedure was followed using porphyrin 3 (91 mg,
samples and reference, respectively; Aref and Asample are the
absorbance at Q band of the samples and reference, respectively.
0
.1 mM) and zinc acetate (37 mg, 0.2 mM), producing 64 mg (67%
1
yield) of 3a. UV–vis (CH
2
Cl
2
)
l
max: 424, 555 and 598 nm. H NMR
D
Determination of F was performed as follows: 2.5 ml of
(
CDCl 1.21 (q, 2H), 1.25 (t, 2H), 4.12 (s, 9H), 4.13 (s, 6H), 7.27 (d,
3
)
d
acidified methanol was placed with an appropriate amount of the
test compound to an optical density of 1.00 ꢀ 0.02 in the Soret
J = 8.5 Hz, 6H), 8.14 (d, J = 8.5 Hz, 6H), 8.35 (d, J = 8.5 Hz, 2H), 8.45 (d,
J = 8.5 Hz, 2H), 8.91 (d, J = 4 Hz, 2H), 9.01 (s, 4H), 9.02 (d, J = 4 Hz, 2H).
band. Aliquots of 1,3-diphenylisobenzofuran in acetonitrile (25 ml)
were added to an optical density of 1.00 ꢀ 0.02 at 410 nm. The
solution was irradiated with visible light (LED Lamp ATLED Apollo
BR40 35W true white). Finally, the optical density at 410 nm was
determined at different times (25, 50, 75 and 100 s) in a GENESYS
20 spectrophotometer model (THERMO SPECTRONIC).
2
.4.6. 5,10,15-Tris(p-methoxyphenyl)-20-(p-(dimethyl-2-
aminopentanedioate)-carbonylphenyl)-porphyrinate copper(II)
complex, (3b)
The general procedure was followed using porphyrin 3 (91 mg,
0
.1 mM) and copper(II) acetate (36 mg, 0.2 mM), producing 63 mg
(
66% yield) of 3b. UV–vis (CH Cl max: 415, 546 and 572 nm.
2
2
)
l
2.5.2. Cytotoxic studies
The phototoxic effects of the porphyrin derivatives were
determined on human adenocarcinoma SiHa cells maintained in
MEM supplemented with 10% fetal bovine serum at 37 C in a
2
.4.7. 5,10,15-Tris(p-(dimethyl-2-aminopentanedioate)-
ꢃ
carbonylphenyl)-20-(p-methoxyphenyl)-porphyrinate zinc(II)
complex, (4a)
2
humidified 5% CO atmosphere. The viability study was assessed
3
The general procedure was followed using porphyrin 4 (125 mg,
.1 mM) and zinc acetate (37 mg, 0.2 mM), producing 57 mg (44%
using the WST-1 assay. Briefly, 5 ꢄ10 cells were seeded on to 96-
well plates and incubated for 24 h to grow. After this incubation
period, the cells were treated with different concentrations of the
0
1
2 2
yield) of 4a. UV–vis (CH Cl ) lmax: 422, 550 and 591 nm. H NMR

E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
331
compound and incubated for 24 h. Later, the medium was replaced
by PBS and the cells were irradiated under visible light (LED Lamp
ATLED Apollo BR40 35W true white, 0.3 m away from the culture
plate) for 2 h. At the end of this time, the cells were incubated for
route that includes at first the coupling reaction between p-
formylbenzoic acid, 5, and dimethyl 2-aminopentanodioate, 6,
following the typical conditions for amide formation (Scheme 2),
obtaining dimethyl 2-(p-formylbenzamide)pentanedioate, 7, (33%
yield). Dimethyl 2-aminopentanodioate 6 was previously synthe-
sized by the esterification of l-glutamic acid, using thionyl chloride
to obtain the corresponding dichloride followed by the esterifica-
tion with methyl alcohol; the spectral data obtained for this
product are in agreement with those described earlier [23].
Once dimethyl 2-(p-formylbenzamide)pentanedioate, 7, was
obtained, we finished the synthetic route obtaining functionalized
24 h; WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetra-
zolio]-1,3-benzene disulfonate) was then added to each well
ꢃ
(
10
m
l) for 2 h at 37 C. Optical densities were measured at 450 nm
using a Universal Microplate Reader ELx800 (Biotek Instrument,
Vermont, USA). [11]. As a positive control Triton X-100 at 1% in PBS
was used, the negative control was culture medium without any
treatment and as blank, reaction MEM medium and WST-1 was
used, we also used a control of the solvent (isopropanol/
dichloromethane, 2:1), the final concentration of solvent in each
reaction well was 0.5% (v/v) or less. The dark cytotoxicity was
determined as phototoxicity except that irradiation was omitted.
Different concentrations of the compounds to be evaluated were
3
unsymmetrical A B porphyrins, 3 and 4 (26% and 25% yield,
respectively) by Lindsey procedure having p-methoxy benzalde-
hyde as the other aldehyde in the reaction mixture with pyrrole,
with the corresponding stoichiometric ratio depending on which
group was considered as A or B meso group (Scheme 1 and Table 1).
This procedure is different from other reports in which the
functionalized porphyrins are synthesized by first obtaining the
free base porphyrin and then performing diverse types of
functionalization, sometimes having disadvantages related to
the reactivity of the other meso substituents or with the porphyrin
macrocycle itself [24–26]. The route reported here could have the
advantage of identifying the functionalization group since the
beginning and then continue to the formation of the porphyrin
macrocycle which is easily identified by the characteristic UV–vis
absorption pattern, a high intensity Soret band around 420 nm and
four low intensity Q bands Qy(1,0), Qy (0,0), Qx(1,0) and Qx(0,0)
between 480 and 700 nm (Table 2) [3].
prepared from a stock of 200
methane 2:1, and then was complete to 1 ml with medium MEM.
The tested concentrations were 10, 5, 1, 0.1 and 0.01 M.
mM in 30 ml isopropanol/dichloro-
m
2.5.3. Apoptosis
Activity of caspase-3 was performed as apoptosis assay
TM
following the procedure of commercial kit (The ApoTarget
Protease Assay Caspase-3, Invitrogen). The compound under study
(
3, 4, 3a and 4a) and podophyllotoxin as positive control at a final
concentration of its IC50 were exposed on the SiHa cells
podophyllotoxin IC50 0.02 M on Hela cells), untreated cells
were used as negative control [21].
(
m
1
The H NMR data of new porphyrins show all the expected
2
.5.4. DNA microarrays
SiHa cells were treated for 24 h with the metalloporphyrin 4a
signals. The free base porphyrins showed that highly shielded peak
at around ꢂ2 and ꢂ3 ppm is the N ꢂꢂ H at the center of porphyrin
ligand in comparison with metalloporphyrins as this peak
disappeared after complexation of porphyrin with metal because
and 4b, respectively, at a concentration of 10 mM. Untreated cells
were used for the comparison of gene expression after treatment.
The cells were incubated for 4 h, followed by the conventional RNA
extraction, then the complementary DNA (cDNA) labeled with
fluorophores was synthesized. The cDNA was hybridized in the
microarray, after hybridization the microarray was scanned.
The RNA was extracted with Trizol reagent (Invitrogen). RNA
samples were run on an agarose gel 1% to identify RNA integrity.
RNA concentrations were determined with a NanoDrop (Thermo-
scientific) spectrophotometer. The Chipshot kit (Promega) was
1
the two H atom are replaced by metal ion. The H NMR data of
cupper metalloporphyrins were not obtained due to paramagnetic
metal nature [18]. However, the structure of all the synthesized
metalloporphyrins was also assured because the former free base
porphyrin in each case was characterized and there was a
significant difference of the UV–vis pattern as described later.
The absorption spectra of metalloporphyrins is characterized by a
strong near-UV Soret band, around 420 nm, and two weaker bands,
the Q(1-0) and Q(0-0), around 539 and 598 nm, these are in
consequence of the square symmetry introduced by the metal
(Table 2) [3,5].
used, following the Promega Protocol (Z3604) ꢂ20
mg of total RNA
was used to obtain aminoallyl-cDNA and subsequent purification.
To obtain quantitative values of each signal of fluorescence in
the microarray slide, the image analysis was performed using the
software GenArise. The upregulated and downregulated genes
with a z-score greater or lesser than ꢀ2 standard deviations as
applicable were considered significant for the bioinformatics
analysis. By means of DAVID (The Database for Annotation,
Visualization program and Integrated Discovery) virtual platform,
biological pathways were identified, in which upregulated and
downregulated genes were presented [22].
It is important to notice the moderate reduction of hydropho-
bicity of porphyrins 3 and 4 compared with 1 and 2, as it was
observed at first by the Rfs on TLC (CH Cl -iPrOH, 98-2),1, Rf = 0.75,
2 2
Table 2
Photophysical properties of porphyrins and metalloporphyrins.
b
Porphyrin
UV–visible parameter (
l
max/nm)a
F
D
ꢀ SD
Soret Band
Q Bands
3
. Results and discussion
1
2
3
4
1
2
3a
4a
1
2
3
423
425
421
424
422
420
424
422
418
417
415
419
517, 544, 593, 649
516, 552, 591, 647
518, 555, 593, 649
517, 557, 592, 648
550, 592
551, 592
555, 598
550, 591
540, 578
0.47 ꢀ 0.05
0.42 ꢀ 0.02
0.50 ꢀ 0.04
0.46 ꢀ 0.03
0.66 ꢀ 0.02
0.67 ꢀ 0.03
0.61 ꢀ 0.03
0.56 ꢀ 0.03
0.21 ꢀ 0.04
0.21 ꢀ 0.03
0.25 ꢀ 0.04
0.22 ꢀ 0.01
3.1. Synthesis and characterization
a
a
In order to obtain unsymmetrical A
3
B porphyrins, 1 and 2, we
followed the procedure described by Lindsey and co-workers
Scheme 1 and Table 1), via acid-catalyzed condensation of pyrrole
(
b
b
b
and the corresponding aldehydes followed by oxidation with DDQ.
The purification of each reaction mixture by chromatography
column allowed us to obtain two new unsymmetrical porphyrins,1
and 2 (24% and 20% yield, respectively).
539, 577
546, 572
540, 575
4b
a
In dichloromethane.
Our interest on obtaining A
3
B unsymmetrical porphyrins
b
Using (p-THPP,
D
F = 0.56 in methanol) as the reference.
functionalized by amino acids lead us to propose the synthetic

3
32
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2
, Rf = 0.64, 3, Rf = 0.35 and 4, Rf = 0.22. We also calculate the Log P
for 48 h in the dark. The results are shown in dose–response curves
(Fig. 1a). Functionalized porphyrins and corresponding metallated
with zinc 3, 4, 3a and 4a had the higher values of dark cytotoxicity,
1
using Chem BioDraw , 1, Log P = 9.74, 2, Log P = 9.91, 3, Log P = 8.28
and 4, Log P = 7.35. These two parameters suggest that porphyrins 3
and 4 are less lipophilic than 1 and 2. The decrease in the Log P
values modifies the balance between hydrophilic and hydrophobic
character of the compounds, and the presence of l-glutamate in the
structure as substituent of the meso groups of porphyrins 3 and 4,
improves the high hydrophobicity of porphyrin core [10–14], and it
seems to be important especially for the possible application as
photosensitizers and the permeation across biological membranes
that could be attributed to
determined) [19].
a higher cellular uptake (not
Phototoxicity was also determined in SiHa cells, after cells were
exposed to tetraaryl derivatives for 24 h, the cells were irradiated
with visible light for 2 h (Fig. 1b). IC50 values were determined by
extrapolating the results presented in dose-response curves
(Table 3). IC50 values were calculated for all compounds studied.
Comparing the phototoxic activity of porphyrins, it can be
observed that functionalization of porphyrins with glutamate
[27,28].
Porphyrins bearing amino acid moieties have been reported to
have interesting characteristics for PDT, because of their ability to
stablish specific interactions with cell membranes and to improve
the solubility in aqueous solutions [29,30]. The synthesis of a
cationic porphyrin modified glutamic acid protected either by an
N-Boc or an N-Fmoc group was reported, emphasizing the great
interest of these porphyrins as inhibitors of human telomerases,
DNA intercalators, components of artificial receptors and photo-
sensitizers in photodynamic therapy [31]. Based on these and other
similar publications in which it is clear that the relevance of the
presence of amino acid moieties as part of the meso substituents of
porphyrins, the synthesis and characterization of porphyrins 3 and
increased 100 times phototoxic activity (1 (IC50 4.81 ꢀ0.34
3 (IC50 0.04 ꢀ 0.02 M) and 2 (IC50 5.19 ꢀ 0.42 M) vs. 4 (IC50
0.05 ꢀ 0.01 M)). This increased activity can be attributed to
moderate reduced hydrophobicity, increased cellular uptake and
increased , which is given by functionalization with glutamate.
It has been reported that the addition of hydrophilic substituents
increases the recognition of the membrane, cellular uptake and can
address the photosensitizers to tumour cells [10,15,19,36]. Metal-
lation with zinc increased phototoxic activity compared with the
corresponding porphyrins. Metallation with copper decreased
phototoxic activity compared with the corresponding porphyrins.
mM) vs.
m
m
m
F
D
4
functionalized with l-glutamate represent an important contri-
bution.
In addition to these four new porphyrins whose photophysical
Metalloporphyrins 3a (IC50 0.04 ꢀ 0.01
mM) and 4a (IC50 < 0.01
M) presented the best values of phototoxic activity. Therefore,
m
functionalization and zinc metallation increased phototoxic
activity.
Phototoxicity results presented in this paper are similar to
porphyrins synthesized and evaluated by other authors. Wang
et al., functionalized meso-tetra(4-aminophenyl)porphyrin with
the amino acid valine, the functionalized porphyrin showed
and biological characteristics could be compared between each
other in order to establish the effect of the glutamate function-
alization group, we consider to complete the study by synthesizing
the corresponding Zn(II) and Cu(II) metalloporphyrins (Scheme 3).
Zinc porphyrins have been proved as good photosensitizers [32],
while some copper porphyrins presented average photodynamic
activity [33]. Metalloporphyrins 1a–4a and 1b–4b were obtained
following the synthetic procedure that results in the coordination
of the corresponding metal with nitrogen atoms located inside the
porphyrin core, due to the reaction of the free base porphyrin with
the metal salt (acetate); after purification, the metalloporphyrins
were obtained in 44–85% yields.
highlighted phototoxicity (from a concentration of 1 mM) after
irradiation with visible light on MCF-7 cells [30]. In a study
presented by Banfi et al., synthesized seven tetraaryl porphyrins
with substituents meso hydroxyphenyl and methoxyphenyl and
phototoxic activity was evaluated in HTC 116 cells of colorectal
carcinoma, IC50 values ranging between 0.004 and 13
m
M were
obtained; phototoxicity values against Photofrin , which pre-
sented an IC50 of 0.073 g/ml are compared [37]. According to
these results and those presented by Banerjee et al., who reported
1
m
3.2. Quantum yield of singlet oxygen
1
an IC50 value of 4.3 mM for Photofrin on HeLa cells [38], we can
Porphyrins and metalloporphyrins presented values of
F
D
conclude that functionalized porphyrins 3 and 4 and the zinc
metalloporphyrins 3a and 4a could be more phototoxic than
Photofrin .
ranging from 0.21 to 0.67 metalloporphyrins copper 1b, 2b, 3b and
b had the lowest of values, this is because the paramagnetic
1
4
F
D
metal deactivate the excited states and shorten life time of the
triplet state, while zinc metalloporphyrins 1a, 2a, 3a and 4a
Other authors also reported the activity of other photo-
sensitizers, which are considered for most of the phototoxic
showed the highest values of
F
D
, increasing the intersystem
photosensitizers, the p-THPP with an IC50 of 0.0045
mM to HTC 116
crossing and lifetime of triplet state, attributed to diamagnetic
metal (Table 2) [15].
cells [37] and temoporfin with an IC50 of 0.0076 mM on the same
cells [39].
Zinc metalloporphyrins have the highest values of singlet
oxygen; these values are similar to other porphyrins and metal-
loporphyrins presented by other authors, Cheng-Liang et al.
The fact that the zinc metalloporphyrin 4a presented an
IC50 < 0.01 M in SiHa cells makes it a promising PS agent. Králová
et al., synthesized glycol functionalized porphyrins and assessed
their activity in HL60 leukaemia cells and 4T1 breast cancer cells
m
reported
D
F values between 0.45 and 0.68 for symmetrical and
unsymmetrical porphyrins with meso substituents p-hydroxy-
phenyl and p-aminophenyl and Nifiatis et al. reported halogen
with IC50 values ranging from 0.033 to 0.093 and 0.093–0.143 mM,
respectively [10]. In another study presented by Laville et al.,
glycosylated porphyrins were synthesized and evaluated in
tetraaryl porphyrins in the meso-phenyl group
D
F values between
0
.63 and 0.96 [19,32]. As reported by others, in the present work,
retinoblastoma cells with IC50 values between 0.06 and 0.65 mM
compounds with higher values of FD are considered better
photosensitizers. The increase in the quantum yield of singlet
oxygen can be attributed to the increased hydrophilicity [34,35].
[40]. These results are comparable to those obtained in this work.
3.4. Induction of apoptosis
3.3. Cytotoxic effects
The activity of caspase-3 in SiHa cells after treated with
porphyrins (3, 4, 3a, 4a) to a final concentration of 10
mM and using
The dark cytotoxicity of porphyrins and metalloporphyrins in
podophyllotoxin as positive control, was determined. Fifty
mg of
SiHa cells was determined by WST-1 assay. The cells were exposed
to all tetraaryl derivatives at different concentrations below 10
lysate protein were used to determine activity of such enzyme.
Caspase-3 enzyme hydrolyses the substrate tetrapeptide DEVD-
mM

E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
333
Fig.1. Dark citotoxicity (a) and Phototoxicity (b) dose-response curve obtained in SiHa cells following exposure to different tetraayl porphyrin derivatives. Data are expressed
as mean value ꢀ standard error of the mean value of three independent experiments, each performed in quintuplicate. The colorimetric WST-1 test was used for cell viability
estimation.
pNA, releasing p-nitroaniline, which is indicative of the activity of
Table 3
the enzyme. The absorbance values at 405 nm are shown in Table 4.
Activity of caspase-3 enzyme indicates an apoptotic process.
SiHa cells treated with porphyrins 3, 4, 3a and 4a at a final
IC50 phototoxic values of porphyrins and metalloporphyrins in SiHa cells, obtained
from three different experiments with five replicates.
Porphyrin
IC50
(m
M) ꢀ SD
concentration of 10 mM, showed increased activity of caspase-3
enzyme compared to the negative control, but lower compared to
treatment with podophyllotoxin; the activity of caspase-3 which
Dark cytotoxicity
Phototoxicity
1
2
3
4
7.22 ꢀ 0.05
7.49 ꢀ 0.18
3.11 ꢀ 0.11
2.94 ꢀ 0.08
7.32 ꢀ 0.08
7.25 ꢀ 0.17
3.07 ꢀ 0.27
2.53 ꢀ 0.65
7.55 ꢀ 0.13
6.92 ꢀ 0.16
7.07 ꢀ 0.04
4.82 ꢀ 0.36
4.81 ꢀ 0.34
5.19 ꢀ 0.42
0.04 ꢀ 0.02
0.05 ꢀ 0.01
2.39 ꢀ 0.30
2.93 ꢀ 0.21
0.04 ꢀ 0.001
<0.01
6.56 ꢀ 0.11
2.25 ꢀ 0.04
0.60 ꢀ 0.03
2.78 ꢀ 0.04
Table 4
Absorbance values at 405 nm indicate the activity of caspase-3 enzyme.
1a
2a
3a
4a
1b
2b
3b
4b
Treatments
Abs ꢀ SD (405 nm)
3
4
3a
4a
0.298 ꢀ 0.027
0.373 ꢀ 0.011
0.328 ꢀ 0.033
0.384 ꢀ 0.021
0.489 ꢀ 0.034
0.117 ꢀ 0.006
Podophyllotoxin
Control

3
34
E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
Table 5
indicates the presence of apoptosis was observed. Apoptosis
requires activation of endonucleases, DNA degradation and
activation of caspases. Programmed cell death is often related to
loss of mitochondrial function, which is reflected by a decrease in
mitochondrial membrane potential and release of cytochrome C. It
is known that the accumulation of ROS can be a cause of altered
mitochondrial function. Some reports have documented the
presence of apoptosis generated by the treatment of various
photosensitizers [37,41,42], so the results obtained in the present
work are consistent with those studies.
Biological pathways shown by DAVID where the upregulated genes are involved.
Pathways
No. Upregulated genesa
Porphyrin 4a
Porphyrin 4b
Systemic lupus erythematosus
MAPK signaling pathway
Calcium signaling pathway
Prion diseases
Vascular smooth muscle contraction
Apoptosis
Long-term depression
Hedgehog signaling pathway
Ribosome
16
25
17
6
20
22
15
5
12
10
8
10
11
7
Inoue et al., found that treatment with 5-aminolevulinic acid
decreased mitochondrial membrane potential and produce cell
death caused primarily by apoptosis [43], Tochigi et al., mentioned
7
8
9
5
10
–
Fructose and mannose metabolism
Proteasome
6
–
2 2
that low concentrations of H O activated preferably caspase-
Insulin signaling pathway
Adipocytokine signaling pathway
Heparan sulfate biosynthesis
Long-term potentiation
Melanogenesis
Arachidonic acid metabolism
RIG-I-like receptor signaling pathway
Phosphatidylinositol signaling system
Endocytosis
Chronic myeloid leukemia
Dilated cardiomyopathy
T cell receptor signaling pathway
Neurotrophin signaling pathway
Type II diabetes mellitus
Pathways in cancer
12
7
12
–
dependent apoptotic pathway while high concentrations of H
2 2
O
induce both, apoptotic cell death and caspase independent
necrotic process [44]. In addition, nitric oxide is involved in the
photodynamic therapy, as it can either stimulate or inhibit
apoptosis, acting on the NF-jB/Snail/RKIP survival/anti-apoptotic
loop where low dose of PDT induces low levels of nitric oxide,
which causes stimulation of the anti-apoptotic pathway after
mentioned, whereas at higher doses of PDT stimulates high levels
of nitric oxide, which inhibits the pathway and therefore
stimulates apoptosis [45].
Kessel mentioned that overexpression of anti-apoptotic Bcl-2
protein leads to stabilization of the pro-apoptotic Bax protein. After
photodamage, Bax excess is available to initiate interaction with
the mitochondrial membrane, resulting in the release of cyto-
chrome C; a trigger apoptosis [47], therefore the crucial factors in
determining the type of cell death after PDT are: the type of cell,
the subcellular localization of the PS, and the dose of light to
activate the PS [47,48].
4
3
7
9
10
9
6
–
7
–
7
–
14
7
16
8
8
8
9
9
10
5
–
–
22
4
21
–
Ether lipid metabolism
Base excision repair
GnRH signaling pathway
Non-small cell lung cancer
Inositol phosphate metabolism
Melanoma
4
–
8
–
5
6
5
–
6
–
Gap junction
7
7
Maturity onset diabetes of the young
Dorso-ventral axis formation
VEGF signaling pathway
Starch and sucrose metabolism
Bladder cancer
Regulation of actin cytoskeleton
Axon guidance
Fc gamma R-mediated phagocytosis
Nucleotide excision repair
Alzheimer’s disease
Glutathione metabolism
Natural killer cell mediated cytotoxicity
Tight junction
Parkinson’s disease
B cell receptor signaling pathway
Glioma
3
5
3
3
3.5. Analysis of differential gene expression (DNA microarray)
6
6
4
–
4
–
Analysing differential gene expression in SiHa cells was
14
9
7
–
determined; two treatments with metalloporphyrins 4a and 4b
were performed respectively, comparing the expression with
untreated control. For treatment with metalloporphyrin 4b, 113
genes were downregulated and 1882 genes were found as
upregulated, whereas treatment with the metalloporphyrin 4a,
12
–
4
–
15
6
4
–
11
10
9
45 genes were downregulated and 1841 genes were found as
–
upregulated. A bioinformatic analysis of the upregulated and
downregulated genes using the DAVID virtual tool was performed,
associating these genes to metabolic and biological pathways;
allowing observing changes in gene expression from treatment. For
the treatment with metalloporphyrin 4a, the tool yielded 42
biological pathways involved in the case of the upregulated genes
and no interaction in biological pathways was found in down-
regulated genes and treatment with metalloporphyrin 4b, the tool
yielded 35 biological pathways involved in the case of the
upregulated genes and three biological pathways for down-
regulated genes (Table 5).
Treatments in both cases showed similar gene expression
pattern in upregulated genes since they share about 25 biological
pathways and a large number of genes. This evidence may suggests
that the mechanism by which the treatments have their cytotoxic
activity is similar, therefore the difference in the phototoxic
activity of the two treatments may be due to other factors, such as
the quantum yield of singlet oxygen due to the chemical nature of
the metal, the log P, cellular uptake, among others, and not the
mechanism by which they perform their cytotoxic activity.
However, results of microarrays have a qualitative character and
must be verified by a quantitative technique, such as real-time PCR.
Regarding the upregulated genes, B-cell lymphoma 2 (Bcl-2
gene) was found and some studies have reported overexpression of
–
–
6
–
5
a
Upregulated genes with a z-score greater or lesser than ꢀ 2 SD.
this gene following treatment with PDT [49,50], the Bcl-2 gene
controls permeability of the outer membrane after photodynamic
injury, also induce the release of cytochrome C and, thus, induces
apoptosis [46,9]. Other upregulated gene involved in apoptosis is
tumour necrosis factor receptor, the signaling pathway of this
receptor and FAS-associated APO2/TRIAL induces apoptosis with
an independent mechanism of p-53 [51].
Other upregulated genes (DNA fragmentation factor 40 kDa and
4
5 kDa) are nucleases that mediate DNA fragmentation and
chromatin condensation during apoptosis, some genes have
important roles in apoptosis (caspase 6, tumour necrosis factor
receptor, inhibitor of kappa light polypeptide promoter gene in B
cells). Other upregulated found genes (Heat shock 70, growth
arrest and Jun D) have cell function in response to stress;
particularly heat shock 70 protein prevents cell death by inhibiting
the aggregation of cellular proteins [50].
Upregulated genes involved in the MAPK signaling pathway
was found, these enzymes are critical components of cellular

E.U. Arredondo-Espinoza et al. / Biomedicine & Pharmacotherapy 82 (2016) 327–336
335
signaling network that ultimately regulates gene expression in
References
response to extracellular stimuli. The three families of MAPK are
well known: the extracellular signal-regulated kinases (ERKs),
protein kinases c-Jun N-terminal kinases activated by stress (JNK/
SAPKs), and p38 MAPK. Each of these enzymes is a target for
phosphorylation cascades wherein the sequential activation of
three kinases is a common signaling pathway. The JNK and p38
proteins are key signs of stress and inflammatory mediators.
Activation of the MAPK pathway appears to play an important role
in the induction of apoptosis [52].
Other upregulated genes involved in the activation of apoptosis
genes are the calcium signaling pathway, since it has been shown
to increase intracellular calcium released from the endoplasmic
reticulum and mitochondria after treatment with PDT, that it is
involved in initiation and development of apoptosis [52].
Overexpression of the proto-oncogene JunD gene was also
found, this gene protein complex interacts with transcription
factor activator protein 1 (AP-1), whose function is related to the
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