Journal Pre-proof

Tetra-cationic platinum(II) porphyrins like a candidate

photosensitizers to bind, selective and drug delivery for metastatic
melanoma

Gabriela Klein Couto, Bruna Silveira Pacheco, Victoria

Mascarenhas Borba, João Carlos Rodrigues Junior, Thaís Larré
Oliveira, Natália Vieira Segatto, Fabiana Kommling Seixas,
Thiago V. Acunha, Bernardo Iglesias, Tiago Collares

PII: S1011-1344(19)31190-X
DOI: https://doi.org/10.1016/j.jphotobiol.2019.111725
Reference: JPB 111725

To appear in: Journal of Photochemistry & Photobiology, B: Biology

Received date: 7 September 2019

Revised date: 1 November 2019
Accepted date: 25 November 2019

Please cite this article as: G.K. Couto, B.S. Pacheco, V.M. Borba, et al., Tetra-cationic
platinum(II) porphyrins like a candidate photosensitizers to bind, selective and drug
delivery for metastatic melanoma, Journal of Photochemistry & Photobiology, B:
Biology(2019), https://doi.org/10.1016/j.jphotobiol.2019.111725

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© 2019 Published by Elsevier.

 Journal Pre-proof

Tetra-cationic platinum(II) porphyrins like a candidate photosensitizers to

bind, selective and drug delivery for metastatic melanoma
Gabriela Klein Couto,a Bruna Silveira Pachecoa, Victoria Mascarenhas Borbaa, João
Carlos Rodrigues Juniora, Thaís Larré Oliveiraa, Natália Vieira Segattoa, Fabiana
Kommling Seixasa, Thiago V. Acunhab, Bernardo Iglesias b* and Tiago Collaresa*

a
Molecular and Cellular Oncology Research Group, Cancer Biotechnology Laboratory,
Technological Development Center, Federal University of Pelotas, Pelotas, Brazil.
b
Laboratory of Bioinorganic and Porphyrinoid Materials, Chemistry Department, Federal
University of Santa Maria, Santa Maria, Brazil.

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Authors email:

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Couto, GK (gaby_kc@yahoo.com.br)
Pacheco, BS (pacheco.sbruna@gmail.com)
Borba, VM (victoriamborba2@hotmail.com)
Junior, JCR (jcrodriguesjr@hotmail.com)
Oliveira, TL (thais.larreoliveira@gmail.com)
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Segatto, NV (naty_segatto@hotmail.com)
Seixas, FK (seixas.fk@gmail.com)
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Acunha, TV (thiagovacunha@hotmail.com)
Iglesias, B (bernardopgq@gmail.com)
Collares, T (collares.t@gmail.com)
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* Corresponding authors: Dr. Tiago Collares (collares.t@gmail.com) and Bernardo A.

Iglesias (bernardopgq@gmail.com).

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ABSTRACT

Photodynamic therapy (PDT) is an expanding treatment modality due to its minimally

invasive localized activity and few adverse effects. This therapy requires photosensitive
compounds, which have high sensitivity to light exposure. Thus, in this work, the in
vitro antitumor activity of meso-tetra(3- and 4-pyridyl)porphyrins (3-TPyP and 4-
TPyP) in metastatic melanoma cell (WM1366 line) and non-tumoral Ovarian lineage
Chinese Hamister (CHO) was evaluated using photodynamic process. Cell viability
tests, molecular docking, annexin V, confocal microscopy and qRT-PCR were
performed. Our results show that both porphyrins inhibited the viability of metastatic
melanoma cells when exposed to light and did not alter viability in the dark. In addition,
they did not demonstrate cytotoxicity in non-tumor cells. Molecular coupling
demonstrated platinum porphyrin affinity for the N-terminal region of APO B-100,

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LDL receptor, and therefore of the cells under study. Genes such as Caspase 3 and 9,
P21, Bax / BCL2, MnSod and GSH showed increased expression. For meta isomer 3-
PtTPyP treatment, caspase-9 and caspase-3 expression levels showed a 4.89 and 3.23-
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fold increase, respectively, while for the para isomer 4-PtTPyP, this change was 3.77
and 12.16-fold, respectively. We also observed an upregulated expression of p21, a
protein well-known by its action in cell cycle arrest in a p53-dependent manner.
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Conclusion: 3-PtTPyP and 4-PtTPyP demonstrated antitumor effect on WM1366 cells,
inducing apoptosis and significant alteration of cell cytoskeleton actin. Our work shows
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that platinum(II) porphyrins may be promising photosensitizers for the treatment of

metastatic melanoma by PDT.
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KEYWORDS: Platinum(II) porphyrins; Photosensitizer; Molecular docking; LDL

receptor; APO B-100, Cancer.
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1. Introduction
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Melanoma is considered the most aggressive type of skin cancer, due to its late
diagnosis in most cases [1]. It can be cured when diagnosed in the early stages,
however, it is very likely to develop metastases if this diagnosis is late. The treatment of
this pathology is chosen depending on the tumor staging. The advanced stages are very
difficult to be treated with the currently available therapies, including chemo and
immunotherapy, highlighting the need for new and selective treatments [2–4].
According to World Cancer Research Fund, this neoplasy is the 19 th most commonly
occurring cancer. In 2018, nearly 300,000 new cases were diagnosed [5]. According to
American Cancer Society, 96,480 new cases are estimated to be diagnosed in 2019 and
7,230 deaths from melanoma are expected. And in Brazil, for biennium 2018/2019, an
estimated 6,260 new cases of skin cancer of the melanoma type [6].

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In this context, alternative therapies have been studied to optimize the treatment
of aggressive pathologies such as cancer [7]. Photodynamic therapy (PDT) has been
shown to be a promising alternative in this regard. It has its action based on three main
points, being: the use of sensitizers, light and oxygen molecule, to induce cellular
damage. It is characterized by being a minimally invasive and a tumor-selective method,
besides presenting a decrease of the adverse effects to the patient [8,9].

Currently, new molecules have been found that have ideal properties to be used as
photosensitizers in PDT. In this sense, some specific characteristics need to be observed
in these molecules so that they can be used as photosensitizers. Among these
characteristics we highlight potentiated photostability, good solubility in physiological

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medium, high generation of reactive oxygen species (ROS), selectivity and high
phototoxicity [10].

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A widely studied example is the porphyrins due to their structure in the form of a
ring with 18 conjugated π electrons, which is why their porphyrin derivatives absorb
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light, proving their photophysical properties as well as their ability to accumulate in the
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tumor selectively [11]. In order to potentiate the action of these structures, associations
with some inorganic compounds, such as platinum(II) complexes, have been described
[12]. This association is justified by the fact that, in isolation, such metals demonstrate
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potent anti-tumor action properties, and thus, the union between porphyrins and metal-
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transition coordination compounds may offer the possibility of synergistic effects as

well as decrease observed adverse effects for each drug separately [12]. Thus, the aim of
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this study was to analyze the anti-tumor action of isomeric tetra-

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cationic(pyridyl)porphyrins containing peripheral attached platinum(II) complexes at

the meta and para positions on metastatic melanoma cell line using photoactivated
compounds.

2. Materials and methods

2.1. Tetra-cationic porphyrin photosensitizers 3-PtTPyP and 4-PtTPyP

The meso-tetra(3- and 4-pyridyl)porphyrins (3-TPyP and 4-TPyP) were

purchased from Frontier Scientific® (Logan, Utah, USA). Peripheral platinated(II)

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porphyrin hexafluorophosphate compound (3-PtTPyP and 4-PtTPyP; Figure 1) were

synthesized, fully characterized and reported in some publications in the literature [12–
15] (see supplementary information section – Figs. S1-S7 and Table S1). All Pt(II)-
porphyrins tested in this study are soluble in DMSO and stable in this solution (see
supplementary information section – Figs. S8-S9). Stock solutions were prepared in
anhydrous dimethyl sulfoxide at concentration of 1 mg/mL (Sigma-Aldrich®).

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Fig. 1. Structural representation of free-base platinum(II) peripheral porphyrins 3-PtTPyP and 4-PtTPyP
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used in this study. The hexafluorophosphate counter-ions are omitted for more clarity.
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2.2. Cell culture and reagents

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The WM1366 cell line kindly provided by Universidade de Ribeirão Preto, SP-
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Brazil and a non-tumor cell line derived from the ovary of the Chinese hamster (CHO),
were obtained from the Rio de Janeiro Cell Bank (PABCAM, Federal University of Rio
de Janeiro, RJ, Brazil). They were grown in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), respectively, obtained
from Vitrocell Embriolife (Campinas, Brazil) and Gibco (Grand Island, NY, USA).
Cells were grown under controlled atmosphere at 37°C, 95% humidity and 5% CO 2 .

2.3. Experimental groups and photodynamic assay

The cells were divided into two distinct groups: light and dark conditions. Each
group was treated with five different concentrations (0.563, 1.125, 5.625, 11.25, 28.125
and 56.25 nM) of the two platinum(II) porphyrins, 3-PtTPyP and 4-PtTPyP. After

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adherence of the cells to the wells, they were divided into separate groups and treated
with the different concentrations previously presented of the proposed compounds. As
these compounds are activated by the incidence of light, these were divided into two
groups (light and dark), where the light group was activated by phototherapy for 30
min. In the irradiation light conditions, the porphyrins were exposed to white-light (400
to 800 nm range, consisting of a 100 W LED lamp system) with a fluence rate of 50
mW/cm2 , for 30 min (total light dose of 45 J/cm2 ), according to a method in the
literature [16]. After exposure to light for 30 min the plates were put back into the
incubator and the following tests were performed 24 h after exposure to light (Figure 2).

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3-PtTPyP and 4-PtTPyP
Photosensitizers

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Fig. 2. Scheme of treatment protocol of WM1366 cells with 3-PtTPyP and 4-PtTPyP photosensitizers in
photodynamic therapy.

2.4. Cell proliferation assay

WM1366 and CHO cells were seeded in 96-well culture plates at a density of 2.0
x 104 /well (200 μL/well) with different concentrations of the molecules. The negative
control consisted in 200 μL/well of medium, and the control of the vehicle in 200
μL/well of medium and DMSO (with concentration less than 0.5%). Cell proliferation
was evaluated 24 hours after the activation of the compounds by the light irradiation.
After the incubation period, the MTT salt (tetrazolium salt [3- (4,5-dimethylthiazol-2-

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yl) -2,5-diphenyltetrazolium bromide]) was added to each well (5 mg MTT/ml). Then,

absorbance was measured using a spectrophotometer (Thermo Plate TP-Reader) at a
wavelength of at 495nm. Percent of growth inhibition was determined by the formula:

% inhibition = (Abs492 treated cells / Abs492 control cells) × 100

2.5. Molecular Docking

Given the importance of the binding between our molecules (3-PtTPyP and 4-
PtTPyP) in the mechanism of selectivity of tumor cells, molecular docking analyzes
were performed. For the creation of the target receptor we used the sequence

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APOB_HUMAN (P04114) that was obtained from the UniProt website (Cathy H. Wu et
al., 2006) and its domains were analyzed by the SMART platform (Ivica Letunic et al.,

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2004) to obtain the N-terminal domain amino acid sequence (LPD_N), which was
predicted at the 46-598 position with its E-value of 6.97e-140. This represents a
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conserved region that has been found in several lipid carrier proteins, including
vitellogenin, triglyceride microsomal transfer protein and Apolipoprotein B-100 (Abdul
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Hafeez Khan et al., 2010). The receptor was modeled by homology using CPHmodels-
3.0 (Morten Nielsen et al., 2010) with its Z-score of 59.41. The model used was 1LSH
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protein refined lipovitelin molecular structure with a resolution of 1.9 Å, with its E-
Value of 5e-05 and 99% convergence with BLAST-protein search. The preparation of
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the molecules began with the 2D creation of the molecules, prepared by ChemDraw
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2018 software, the molecules were prepared as ligands for anchoring using the
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Molecular Operating Environment (MOE; Chemical computing Group, Montreal,

receptor
Canada) software. The protonation state was adjusted to pH 7. Our was prepared
by the Schröndinger Maestro Protein Preparation Wizard (Protein Preparation Wizard,
Schröndinger, LLC, New York, NY, 2015). Where hydrogens were added, completing
the missing loops and finally minimizing their energy and optimizing the protein.
Molecular anchoring was performed using the GOLD software (G. Jones, P. Willett, R.
C. Glen, A. R. Leach, R. Taylor, J. Mol. Biol. 1997, 267, 727).

Anchoring results were obtained through fitness values, in which the higher the
value, the better the interaction of anchoring with the complexes is defined. For this
work we used the ChemPLP algorithm. To validate the results obtained by docking, we
used a low-density lipoprotein (LDL) analog, which binds at the Apo B-100 site causing

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it to trigger the membrane LDL-R binding cycle. The analog chosen was amino acid
0582
sequence DLKKLVKEVLKESQLPTVMDFRKFSRNYQ 0610 of name B0582 [17].
This amino acid sequence was taken from the N-terminal region of APO B-100 itself
and was modeled by CPHmodels-3.0 (Morten Nielsen et al., 2010). After modeling, the
GOLD molecular anchor program was used to simulate the binding of B0582 to our
target protein. For this simulation was used the standard mode of the software, we used
the target protein without solvent and with added hydrogen atoms.

2.6. Confocal microscopy analysis

Changes in cell membrane were identified using DAPI (4,6-diamidino-2-

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phenylindole) staining, which forms a fluorescent complex with double-stranded DNA,
and Texas red that stains the cell's actin. Cells seeded in a 96-well plate were treated

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with 4.501 and 3.012 nM of 3-PtTPyP and 4-PtTPyP porphyrins, respectively. After
24 h of treatment the white-light source was applied, and cells were incubated for 24 h.
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After treatment, the cells were washed three times in phosphate buffered saline (PBS),
fixed and stained according to the manufacturer's protocol. Cell morphology was
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examined by SP8 confocal microscopy (Leica Microsystems ©). DAPI dye emission:
~460nm. Texas red dye emission: ~615nm. Cell morphology was examined by confocal
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microscopy at a magnification of 400 x.

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2.7. RNA extraction and qRT-PCR

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Total mRNA was extracted of cells using TRIzol (Invitrogen™, Carlsbad, USA)
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followed by DNase treatment with DNA-free® kit (Ambion™, USA) and mRNA
quantification by Nanovue Plus Spectrophotometer™ (GE®). The cDNA synthesis was
performed using High Capacity cDNA Reverse Transcription kit (Applied
Biosystems™, UK) according to the manufacturer’s protocol. The amplification was
made with UltraSYBR Mix (COWIN Bioscience Co., Pequim, China) using the
Stratagene Mx3005P and the sequence of primers used are indicated in Table 1. Gene
expression were normalized using glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) as a reference gene and the conditions for the reactions included 95°C for 15
s, 60°C for 60 s and 72°C for 30 s. The 2ΔΔCT (Delta–Delta Comparative Threshold)
method was used to normalize the fold change in gene expressions. Control used to

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calculate ΔΔCT was the group that received only DMEM, without porphyrin and
without light.

Table 1. Primer sequences for qRT-PCR used in this study.

Primer Sequence 5’ → 3’
MnSod For GGAAGCCATCAAACGTGACT
MnSod Rev CTGATTTGGACAAGCAGCAA
P21 For TGTCCGTCAGAACCCATGC
P21 Rev AAAGTCGAAGTTCCATCGCTC
Casp 9 For GTCTCAATGCCACAGTCCAG
Casp 9 Rev TGTACATGCAGCAAACCTC

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Casp 3 For CAGTGGAGGCCGACTTCTTG
Casp 3 Rev TGGCACAAAGCGATCGGAT
GSHR For CCCGATGTATCACGCAGTTA
GSHR Rev pr
TTCACTGCAACAGCAAAACC
ATGCGTCCACCAAGAAGC
Bax For
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Bax Rev ACGGCGGCAATCATCCTC
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BCL-2 For GGTGGGGTCATGTGTGTGG

BCL-2 Rev CCGTTCAGGTACTCAGTCATCC
iNOS For ACAAGCCTACCCCTCCAGAT
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iNOS Rev TCCCGTCAGTTGGTAGGT

GAPDH For GGATTTGGTCGTATTGGG
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GAPDH Rev TCGCTCCTGGAAGATGG

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2.8. Annexin V by flow cytometry

The ability of the different treatments to induce apoptosis against metastatic

melanoma cells was assessed by flow cytometry using the Muse ™ Annexin V & Dead
Cell Assay Kit (EMD Millipore Corporation). For this analysis, WM1366 cells were
plated in 12-well plates at a density of 1.0 x 105 cells per well. After 24 h of adhesion,
the cells were incubated with the IC 50 concentrations of the porphyrin molecules.
Twenty-four hours after treatment, the cells were treated with light for 30 min (the dark
group stayed away from the light). After light exposure, the cells were washed with
PBS, trypsinized and centrifuged at 1200 rpm for 10 min. After centrifugation, 1.0 × 105
cells were stained according to the manufacturer's instructions and analyzed using the
Muse Cell Analyzer (EMD Millipore Corporation).

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2.9. Cell cycle analysis

In order to assess possible mechanisms involved in the decreased cell viability,
such as cell cycle arrest, we performed flow cytometry analysis with the objective of
identifying cell populations at different stages of the cell cycle after different treatments.
For this analysis, WM1366 cells were plated in 12-well plates at a density of 2.0 x 105
cells per well. After 24 h, the cells were incubated with the IC 50 concentrations of each
of the molecules. After 24 h, the cells underwent photodynamic therapy for 30 min.
After light irradiation process, cells were detached, fixed with 70% ethanol and stained
according to the manufacturer's protocol. Measurement of DNA content was analyzed

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by propidium iodide staining using the Guava Cell Cycle reagent kit (Merck Millipore
Corporation) and analyzed in the Muse Cell Analyzer (EMD Millipore Corporation).

2.10 Statistical analysis pr

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Data are presented as mean and standard deviation (SD). Comparative analyzes
were performed using one-way analysis of variance (ANOVA). Tukey's post hoc
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method was employed for multiple comparisons. All statistical analyzes were performed
with GraphPad Prism and p <0.05 was considered statistically significant. For the the
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cell cycle analysis, the bidirectional analysis of variance (ANOVA) was used.
Bonferroni's post hoc method was used for multiple comparisons.
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3. Results
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3.1. Platinum(II) porphyrins as good candidates for photosensitizers

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With the cell proliferation assay, it was observed that platinum (II) porphyrins
were activated in the presence of light (Fig. 3A and B) at most concentrations tested.
Cellular inhibition was more expressive for 3-PtTPyP and 4-PtTPyP molecules at the
two highest concentrations (5.625 and 56.25 nM). In addition, we calculated the IC50 of
the molecules for subsequent testing (Table 2). Also, we tested as porphyrins in the
CHO cell at the same IC50 concentrations calculated for the 3-PtTPyP and 4-PtTPyP
porphyrins. In addition to the IC50 concentration, we added a concentration above and
below this value, and these compounds were not toxic to normal cells (Figures 3E-H).
Graphs 3C-D and 3G-H show the test performed with porphyrins in the dark and we can
see that all concentrations were well below 50% inhibition. In addition, due to the low

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A B C D
3 - P t T P y P - W M 1 3 6 6 L ig h t 4 - P t T P y P - W M 1 3 6 6 L ig h t 3 -P tT P y P - W M 1 3 6 6 D a rk 4 -P tT P y P - W M 1 3 6 6 D a rk
g e e ,f 100
100 100 100

% c e ll g r o w t h in h ib it io n
% c e ll g r o w t h in h ib it io n

% c e ll g r o w t h in h ib it io n

% c e ll g r o w t h in h ib it io n
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C o n c e n t r a t io n ( n M ) C o n c e n t r a t io n ( n M ) C o n c e n t r a t io n ( n M ) C o n c e n t r a t io n ( n M )

E 3 - P t T P y P - C H O L ig h t F 4 - P t T P y P - C H O L ig h t G 3 -P tT P y P - C H O D a rk H 4 -P tT P y P - C H O D a rk

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solubility and aggregate formation, all assays without platinum (II) peripheral
complexes could not be performed.

Fig. 3. Effect of light action on photosensitizing molecule on cell proliferation. Porphyrin -treated and untreated cells
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were irradiated for 30 minutes. Dark group went totally in the dark for the same time. The grap h shows the comparison
within the light and dark group of 3-PtTPyP and 4-PtTPyP porphyrins. Data are expressed as mean ± SD of three
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independent times performed in triplicate. Fig. 3A-D: WM1366 cell line was treated with 3-PtTPyP (Fig. A and B) and
4-PtTPyP (Fig.C and D) platinum (II) porphyrins at 5 different concentrations. Control group received no treatment
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with porphyrins. Fig. 3E-F: Effect of light on the photosensitizing molecule on cell proliferation of non -tumor cells. The
CHO cell line was treated with 3-PtTPyP (Fig. E and G) and 4-PtTPyP (Fig. F and H) platinum (II) porphyrins at a
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concentration above the IC 50 and one below the predetermined IC 50 of each of the molecules. The concentrations
used were: 5.62, 4.50 and 2.81 nM for 3-PtTPyP and 5.62, 3.01 and 2.81 for 4-PtTPyP. For comparison different letters
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in the chart denote significant difference between the groups. p <0.05 was considered significant.
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Table 2. IC50 values of molecules 3-PtTPyP and 4-PtTPyP after 24h of light exposition against
WM1366 line.

Porphyrin IC50 (nM)

3-PtTPyP 4.501±0.58

4-PtTPyP 3.012±0.27

3.2. Peripheral platinum(II) porphyrins Bind to LDL Receptor

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After running the molecular docking tests, we recognized as better the position
of the analog B0582 that obtained the highest fitness which was 93.2881 with the
ChemPLP algorithm. Based on these values, we chose the positions with the closest
proximity of this score to the 3-PtTPyP and 4-PtTPyP molecules. It was also taken into
consideration the pose in which it had greater interaction with lysines, because
according to Guevara et al. this analog has high affinity for lysines. We anchored the 3-
PtTPyP and 4-PtTPyP molecules in the region of the binding site that B0582 was
anchored to, having amino acid residues at a distance of 20 Å considered. Anchoring
results can be seen in table 3 and figures 4A-F. For this study of docking molecular we
use the algorithms in the default mode directly in the GOLD software, we use the

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fitness function like parameters for screening poses. This function indicates the ability

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of the ligand compound interact directly with the target protein, the higher this value
better they are because they indicate the affinity of bond of these poses and the protein.
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This score was calculated by the forces of the bonds between the ligand and the protein
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for example, van der Waals forces, electrostatics and hydrophobics interactions.

We used as valid the poses of the compounds in which they obtained a high
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fitness score and obtained bonds with amino acids present in the active site of the
protein.
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Table 3. Anchor Results of APO B-100 E LDL Receptor Molecules .

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MOLECULE - AMINO ACIDS FITNESS COMMON AMINO

PROTEIN ACIDS WITH B0582
B0582- Apo B-100 ASP168, PRO172, 93.2881 -
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ARG174, LEU182,
LYS184, LYS409,
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PRO412, LYS462,
LYS488, LYS492.

3-PtTPyP - Apo B-100 ARG174, ILE177, 90.7311 ARG174, LEU182,

LEU180, ALA181, LYS184 e LYS492
LEU182, LYS184,
GLU490, LYS492 e
ARG522.

4-PtTPyP-Apo B-100 ASP168, ARG169, 68.5542 ASP168, PRO172,

LYS171, PRO172, LYS409 e PRO412
LYS409, PRO412.

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Fig 4. Molecular docking results. Fig. A and D represent the 3D molecular structure of platinum porphyrins 3-PtTPyP
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and 4-PtTPyP and their amino acid linkages of the B0582 LDL analog. Fig B and E demonstrate the binding of
platinum porphyrins to the N-terminal region of APO B-100. Fig C and F demonstrate the similarity of B0582
anchoring of 3-PtTPyP and 4-PtTPyP molecules to the N-terminal region of APO B-100.
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3.3. Platinum(II) porphyrins alter cellular actin organization

To analyze the morphological alterations of the treated WM1366 cells with a

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cytotoxic concentration of the 3-PtTPyP and 4-PtTPyP porphyrins, we observed that in

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the control group (without treatment) the stress fibers appeared thin and diffuse (Figure
5A). After being treated, the cells appeared to be rounded, a general thickening of the
membrane actin fibers (Figures 5B and C) and significant ruffling occurred at the edge
of their plasma membrane was also observed. Additional morphological changes as
microspikes were observed (Figure 5C) on the cell surface of some cells.
Such changes mentioned above were not identified in dark group cells (Figures 5D, E
and F).

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Control 3-PtTPyP Light 4-PtTPyP Light

A Light B C

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D
Control Dark
E
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3-PtTPyP Dark
F
4-PtTPyP Dark
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Fig. 5. (A-F): Morphological analysis after treatment with 3-PtTPyP and 4-PtTPyP
photosensitizers. WM1366 cells were stained Texas Red and DAPI-cytoplasm and nucleus,
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respectively. In the image we can observe a uniform cytoplasm in the control group (Figs. 5A and
D) and in the treatments (3-PtTPyP and 4-PtTPyP) we observe a reorganization of actin filaments
(arrows fig. 5B). A general thickening of actin fibers in the membrane, ruffling occurred at the
border of its plasma membrane (arrows fig. 5B) and microspikes (arrows fig. 5C) was observed on
the cell surface of some cells. These changes were not identified in dark group cells (Figs. 5D, E
and F), respectively.

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3.4. Pt(II)-porphyrins induces apoptosis

We use annexin V to detect apoptotic cells because of its ability to bind to

phosphatidylserine, an apoptosis marker when it is on the outer leaflet of the plasma
membrane. The annexin V double staining assay and dead cells allow to differentiate
between early apoptosis, late apoptosis, dead / debris and living cell populations. Early
or late apoptosis rates (Fig. 6A-D and 7A-D) assessed by flow cytometry showed that 3-
PtTPyP and 4-PtTPyP porphyrins at concentrations of 4.50 nM and 3,012 nM induced
a percentage of the total. of apoptotic cells of 37.18% and 13.5%, respectively (total
apoptosis = early and late apoptosis), indicating that the photodynamic process using Pt

f
oo
(II) -pyrphyrins as photosensitizers is efficient. In addition, we observed that the dark,
light control and light control groups did not significantly induce apoptosis in both
molecules (Figures 6 pr
and 7) (P <0.05).
e-
Pr
al
u rn
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A C o n tro l B 3 -P tT P y P D a rk

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C o n tr o l L ig h t 3 -P tT P y P L ig h t
C D

pr
e-
Pr
al
u rn
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E
3 -P tT P y P L IG H T / D A R K

4 0
c e lls

* *

3 0
T o ta l a p o p to tic

2 0

1 0
%

0
l

k
o

r
tr

ig

a
ig

D
n

L
o

P
P
C

y
y
tr

P
P
n

tT
tT
o

-P
C

-P

3
3

T re a tm e n t

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Fig. 6. Induction of apoptosis by porphyrin 3-PtTPyP. WM1366 cells were evaluated for apoptosis by
annexin V staining under light and dark group conditions at the IC 50 concentration of the compound.
The graph (Fig. E) shows the total percentage of apoptotic cells. Figures A -D show % late and recent
apoptosis in each of the groups. Porphyrin 3-PtTPyP with white light dosage had a significant increase in
apoptosis when compared to all other groups (**) denotes p <0.007.

f
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pr
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C o n tro l B 4 -P tT P y P D a rk
A

f
C o n tr o l L ig h t 4 -P tT P y P L ig h t
C D

oo
pr
e-
Pr
al

E
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4 -P tT P y P L IG H T / D A R K

1 5

*
c e lls
u T o ta l a p o p to tic

1 0
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5
%

0
l

k
o

r
tr

ig

a
ig

D
n

L
o

P
P
C

y
y
tr

P
P
n

tT
tT
o

-P
C

-P

4
4

T re a tm e n t

Fig. 7. Porphyrin 4-PtTPyP apoptosis induction. WM1366 cells were evaluated for apoptosis by annexin
V staining under light and dark group conditions at the IC 50 concentration of the compound. The graph
(fig. E) shows the total percentage of apoptotic cells. Figures A -D show % of late and recent apoptosis in

17
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each of the groups. Porphyrin 4-PtTPyP with white light dosage had a significant increase in apoptosis
when compared to all other groups (*) denotes p <0.03.

3.5. Tetra-cationic platinum(II) porphyrins alter expression level of genes related to

apoptosis and oxidative stress

In order to further investigate the platinum(II) porphyrins photodynamic therapy

mechanisms of apoptosis induction in the melanoma cell line, evidenced by the annexin
V assay, the relative mRNA expression of the p21, BAX, Bcl-2, caspase 9, caspase 3,
MnSOD, iNOS and Glutathione reductase (GSHR) genes were assessed by qRT-PCR.
As shown in Figures 8-10, both porphyrins (3-PtTPyP and 4-PtTPyP) significantly

f
altered the expression levels of all genes analyzed in metastatic melanoma cell

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(WM1366) after photodynamic conditions (exposure to light) (p <0.001). Interestingly,
we also obtained a discrete increase in GSHR expression in the dark group of 3-PtTPyP
porphyrin (p <0.001), Fig. 8-G. As expected, the increased relative expression of
caspase 3, caspase 9 and P21 genes as well as the upregulated Bax/Bcl-2 ratio
pr
e-
corroborate with the apoptotic results obtained in the annexin V test. Further, elevated
Pr

iNOS levels in porphyrin 4-PtTPyP (Fig. 10-B) indicate that oxidative stress may be
related to a possible cause of apoptosis and cell death.
al

A B C
C a s p a s e 3 - 3 -P tT P y P C a s p a s e 9 - 3 -P tT P y P P 2 1 - 3 -P tT P y P

20 10 15
R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n
R e la tiv e g e n e e x p r e s s io n
rn

8
15
10 **
6
10 *
4
5
5
****
u

0 0 0
l
l

t
t
l

t
t

t
t

rk
rk
rk

o
o
o

h
h

h
h

h
h
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tr
tr
tr

ig

a
ig

ig
g

ig
ig
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D
D
D

n
n
n

L
L

L
L
L

o
o
o

l
l
o

C
C
C

o
o
tr

tr
tr
n

n
o

o
c

T r e a tm e n t T r e a tm e n t T r e a tm e n t

D E F G
B A X - 3 -P tT P y P B C l2 - 3 - P t T P y P M n S O D - 3 -P tT P y P G S H R - 3 -P tT P y P
20 5 60
R e la tiv e g e n e e x p r e s s io n

10
R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n

**
15
4 ***
40
3
10 5

** 2 ***
20
5
1

0 0 0 0
l

rk

rk
o

t
h

rk

l
o

t
h

rk
o

o
tr

h
ig

a
ig

tr

ig

a
ig

tr

ig

a
ig

tr

ig

a
ig
D
n

D
n
L

D
n
L

D
n
L

L
o

L
o

o
l

o
l
C

l
C

l
o

o
tr

tr

tr

tr
n

n
o

o
C

T r e a tm e n t T r e a tm e n t T r e a tm e n t T r e a tm e n t

Fig. 8. Platinum(II) porphyrin 3-PtTPyP increased expression of Caspase 3, Caspase 9, P21, BAX, BCL2,
MnSOD and GSHR genes in metastatic melanoma line WM1366 24 h after being submitted to white-light

18
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irradiation. The gene expression profile was determined by qRT-PCR and the data were normalized using the
GAPDH levels. 3-PtTPyP had a significant increase in the expression of all evaluated genes when compared to
the other groups. (****) denotes p <0.0001, (***) denotes p <0.0005, (**) denotes p <0.004, (*) p <0.04. when
compared to the light and dark group of the same molecule. Three independent experiments were performed in
triplicate.

A C a s p a s e 3 - 4 -P tT P y P B C a s p a s e 9 - 4 -P tT P y P C P 2 1 - 4 -P tT P y P

20 10 15

R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n
****
G e n e e x p r e s s io n

15 ****
10

10 5 ***
5
5

0 0 0

l
l

t
t

t
t

rk

rk
rk

o
o

h
h

h
h

tr

tr
tr

ig

ig

a
ig

ig

ig
ig

D
D

n
n

L
L

L
L

o
o

l
l

C
C

o
o

tr

tr
tr

n
n

o
o

C
C

C o n c e n t r a t io n (  M ) T r e a tm e n t T r e a tm e n t

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D E F G
B A X - 4 -P tT P y P B C l2 - 4 - P t T P y P M n S O D - 4 -P tT P y P G S H R - 4 -P tT P y P

20 10 60 10
R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n

R e la tiv e g e n e e x p r e s s io n
15

10
**

****
pr 40

20
****

5
**
e-
5

0 0 0 0
l

l
t

t
t

t
rk

rk

rk

rk
o

o
h

h
h

h
tr

tr

tr

tr
ig

ig

ig

ig

a
ig

ig

ig

ig
D

D
n

n
L

L
L

L
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o
l

l
C

C
o

o
tr

tr

tr

tr
n

n
Pr
o

o
C

C
T r e a tm e n t T r e a tm e n t T r e a tm e n t T r e a tm e n t

Fig. 9 - Platinum(II) porphyrin 4-PtTPyP increased expression of Caspase 3, Caspase 9, P21, BAX, BCL2,
al

MnSOD and GSHR genes in metastatic melanoma line WM1366 24 h after being submitted to white -light
irradiation. The gene expression profile was determined by qRT-PCR and the data were normalized using the
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GAPDH levels. 4-PtTPyP had a significant increase in the expression of all evaluated genes when compared to
the other groups. (****) denotes p <0.0001, (***) denotes p <0.0005, (**) denotes p <0.004, (*) p <0.04. when
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compared to the light and dark group of the same molecule. Three independent experiments were performed in
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triplicate.

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iN O S - 3 -P tT P y P iN O S - 4 -P tT P y P
A B
e x p r e s s io n 5 5

e x p r e s s io n
4 4

3 3
g e n e

g e n e
* * *
2 2
R e la tiv e

R e la tiv e
1 1

0 0
l

t
k

k
o

h
r

r
tr

tr
ig

a
ig

ig

a
ig
D
n

D
n
L

L
o

o
l

l
C

o
tr

tr
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n
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C

C
T re a tm e n t T re a tm e n t

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C N itr it e a n d n itr a te le v e ls

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0 .6
(  m o l)

0 .4

pr
N O x

0 .2
e-
0 .0
Pr t

t
k

k
h

h
r

r
a

ig

a
ig

ig
D

D
L

L
l

P
P

P
o

y
tr

y
tr

P
P

P
n

tT

tT
tT

tT
o

-P

-P
C

-P

-P
C

4
3

4
al

T re a tm e n t
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Fig. 10. The platinum(II) porphyrins 3-PtTPyP and 4-PtTPyP did not alter the expression of the iNOS
gene (Figs. 10A and B) in the metastatic melanoma line WM1366 and did not induce production of nitric
u

oxide in the cell medium (Fig. 10C) 24 hours after submitted to white-light irradiation. The concentration
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tested was that of the IC50 of each of the molecules. Data are expressed as mean ± SD of three
independent times, performed in triplicate. There was no significant difference in the light and dark
groups.

3.6. Peripheral platinum(II) porphyrins don’t arrest the cell cycle

Cells at the different stages of the cell cycle (G0/ 1, S and G2/M) were analyzed
by flow cytometry and are shown in Figure 11. The results showed that although an
increased expression of the P21 gene (involved in the cell cycle) was observed in the
qRT-PCR, we did not have a significant stop in the G0/G1, S and G2/M cycle between
the groups evaluated.

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Fig. 11. The platinum(II) porphyrins 3-PtTPyP and 4-PtTPyP did not alter the cell cycle. The groups had
no significant difference in cell cycle arrest.
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Pr

4. Discussion

Results obtained in WM1366 after treatment with 3-PtTPyP and 4-PtTPyP

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showed that both porphyrins were not toxic to non-tumor cells (CHO cell line), as they
showed low levels of inhibition in the cytotoxicity test. These findings suggest a
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possible selectivity of porphyrin compounds towards tumor cells, which could be

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explained by the tendency of binding of the photosensitizers, preferably, with low

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density lipoproteins (LDL) [18,19]. Firestone et al. demonstrated that neoplastic cells
have higher uptake of LDL and with this a greater expression of these receptors [20].
Our molecular docking data corroborate this suggestion as our 3-PtTPyP and 4-
PtTPyP porphyrins demonstrated a binding profile with the target. This protein was
chosen because an important part of the circulating LDL binds to Apo B-100 to bind to
the LDL receptor present on the cell membrane. The peptide sequence called B0582
(analogous to LDL) showed the binding of the N-terminal region of APO B-100 +
B0582 and allowed us to compare the interaction of porphyrins with this target [17].
Our results demonstrate that platinum porphyrins 3-PtTPyP and 4-PtTPyP bound very
similarly to B0582 in the target protein, especially 3-PtTPyP, which had a fitness score
of 90.73 (very close to the B0582 score that was 93.28) and amino acid binding:

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ARG174, ILE177, LEU180, ALA181, LEU182, LYS184, GLU490, LYS492 and

ARG522. Of which four of these amino acids were the same in the binding of B0582 to
N-terminal region (ARG174, LEU182, LYS184 and LYS492).
Porphyrin 4-PtTPyP had a score of 68.55 fitness and amino acid binding
ASP168, ARG169, LYS171, PRO172, LYS409, PRO412. In this platinum porphyrin
we also find four amino acids in common with B0582 (ASP168, PRO172, LYS409 and
PRO412). These results corroborate our in vitro findings, described above, where we
observed a selectivity for the tumor lineage. As with 3-PtTPyP we can suggest that 4-
PtTPyP platinum porphyrin is a photosensitizer-like candidate due to the stability of
hydrophobic bonds with amino acids. These molecular anchor studies have also been

f
described in the same vein to predict specific receptor affinities in order to improve drug

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delivery or induce possible selectivity. Bazcaran and co-workers conducted anchor
studies with the ALK gene for non-small-cell lung cancer to try to improve the action of
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drugs for this cancer. [21]. Similarly, Xu et al. Evaluated by molecular anchorage the
e-
receptor RXR as a pharmacological route for acute promyelocytic leukemia [22]. Still a
review published by our group demonstrated the importance of using molecular docking
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to predict interactions between molecules and their receptors in the development of new
drug candidates [4].
al

It is still important to highlight the fact that the molecules did not present
cytotoxicity in all dark groups confirming the action of the molecules 3-PtTPyP and 4-
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PtTPyP only when exposed to light. In addition, other evidence leads us to believe that
one of the main acting species may be singlet oxygen (1 O2 ) produced by the light action
u

in the presence of Pt(II)-porphyrins studied here [12,23], as reported in the literature on

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photoinactivation processes of microorganisms by the same platinum(II) porphyrins

[16,24].

Morphological changes were observed in WM1366 cells treated with 3-PtTPyP

and 4-PtTPyP porphyrins. The reorganization of actin fibers can be observed after
treatment with photodynamic therapy, such as microspikes on the surface of the
membrane and formation of filopodia. It can be said that these modifications in actin
from the cytoskeleton of the cell are important for the defense mechanism and cell death
[25]. Garg and collaborators in a study with retinal pigmented epithelium cells obtained
findings corroborating ours [26]. As well as Zhao and co-workers who observed such
modifications in the actin in bovine pulmonary artery endothelial cells (BPAEC), which

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underwent stress caused by hydrogen peroxide (H2 O2 ), stress similar to that caused by
anti-cancer drugs such as cisplatin [27–29]. Other studies in lung cells (A549 and H460)
have also confirmed our findings related to actin cytoskeleton rearrangement [30]. In
our study these changes were observed only in the light-exposed groups treated with
porphyrins 3-PtTPyP and 4-PtTPyP, probably by ROS generation. In the light/dark
and 3-PtTPyP and 4-PtTPyP dark control groups we did not observe these
modifications. With these results we can state that the molecules are activated only in
white-light conditions and that only light exposure has no action on the cells.

We accessed the effect of porphyrins 3-PtTPyP and 4-PtTPyP in the apoptosis

induction of metastatic melanoma cells in vitro. Treatments with both Pt(II)-porphyrins

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after light exposure increased the number of total apoptotic cells evidenced by their
phosphatidylserine externalization, when compared to the control group and to the

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groups in the dark conditions. This evidence of phosphatidylserine externalization taken
together with the finds towards cytoskeleton actin reorganization demonstrated by
e-
Texas red staining strongly indicate that porphyrins 3-PtTPyP and 4-PtTPyP induce
apoptosis in metastatic melanoma cells in vitro after light activation. In addition, the
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fact that porphyrins 3-PtTPyP and 4-PtTPyP treatments did not have an effect in the
cell cycle of WM1366 cells could further indicate that the mechanism of growth
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inhibition of these compounds is linked to apoptosis induction rather than cell cycle
arrest. Deregulations in the apoptotic process can generate cellular disorders that are
rn

related to several pathologies, such as cancer [31,32]. Thus, the ability to modulate this
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cellular death mechanism presents great potential in the development of new

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oncological therapies. Cells undergoing death by apoptosis experience some cellular

alterations known as apoptotic hallmarks, that include actin reorganization,
phosphatidylserine externalization, chromatin condensation and membrane
permeabilization [33].

Our results corroborate with finds in the literature, where the apoptotic activity of
different porphyrins as photosensitizers in photodynamic therapy has been demonstrated
in several human cell lines, including lung carcinoma [34], tongue squamous carcinoma
[35], breast adenocarcinoma [36] and gastric cancer [37]. The mechanisms involved in
porphyrin`s induction of apoptosis are usually related to increased ROS production and
caspase activation [35,36]. Hematoporphyrin monomethyl ether, for instance, caused
apoptosis by increased production of intracellular reactive oxygen species as well as

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caspase 3 activity in human tongue squamous cell carcinoma (Tca8113) after

photodynamic therapy [35].

Gene expression profile observed through qPCR in WM1366 cells exposed to

platinum(II) porphyrin derivatives followed by white-light activation also indicate
induction of programmed cell death by apoptosis, corroborating with results obtained by
confocal microscopy and annexin V analysis. Apoptosis intrinsic pathway is related
with mitochondrial damage and oxidative stress, both induced by photosensitizer
molecules [38]. Initiator and executor caspases involved in this pathway [39] were
upregulated in response to porphyrin treatments when compared with negative control
group that received only medium. For meta isomer 3-PtTPyP treatment, caspase-9 and

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caspase-3 expression levels showed a 4.89 and 3.23-fold increase, respectively, while
for the para isomer 4-PtTPyP, this change was 3.77 and 12.16-fold, respectively. We

pr
also observed an upregulated expression of p21, a protein well-known by its action in
cell cycle arrest in a p53-dependent manner. Despite no cell cycle arrest has been
e-
induced by tetra-platinated(II) porphyrins, several authors have reported a direct
involvement of p21 in apoptosis induction in a p53-independent mechanism [40].
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Mitochondrial permeability and cytochrome c liberation, involved in apoptosis

intrinsic pathway, are regulated by proteins from Bcl-2 family [39]. We observed an
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increased expression of both anti-apoptotic and pro-apoptotic regulators, BCL-2 and

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BAX, respectively, in WM1366 cells treated with porphyrins. However, when we

analyzed the BAX/BCL-2 ratio, an up-regulation was observed, indicating a pro-
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apoptotic response induced by photodynamic therapy, also in accordance with other

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analyses performed here. Considering that the Bcl-2 family comprises 25 proteins, and
among them, BAX and BCL-2 represent its major members, the ratio BAX/BCL-2 has
been used as one of the hallmarks in the apoptotic process as well as a predictive and
prognostic marker for cancer treatments [35].

Upregulation of MnSOD expression profile is in accordance with low levels of

nitrite and nitrate, molecules that act inactivating this enzyme. High levels of MnSOD
are reported as a marker of oxidative stress; direct or indirectly, through ROS
detoxification, this enzyme prevents formation of nitrites and nitrates [41]. Unchanged
levels of iNOS may be related with cytoskeletal reorganization during apoptosis [42]
and also could explain the stable rates observed for NOx. Similarly, oxidative stress is
also marked by an increased rate of oxidized glutathione [43], which may explain high
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levels of glutathione reductase observed in our study as a defense mechanism against

cell redox imbalance. Although most of our results are concordant, it is important to
highlight that besides transcriptional regulation, several targets that we have evaluated
in this study are also subject to post-transcriptional and translational regulations.

5. Conclusion
In this article we investigate possible mechanisms of action involved in the anti-
tumor activity of tetra-cationic porphyrins 3-PtTPyP and 4-PtTPyP, submitted to
white-light irradiation conditions. Our results suggest that both platinum(II) porphyrins

f
induced apoptosis via activation of caspases 3 and 9. This cell death by apoptosis was

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confirmed by the Annexin V assay. In addition, the reorganization of actin observed in
the groups treated with porphyrins corroborates with death by apoptosis. Moreover, the
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in silico study indicated that both platinum(II) porphyrins are promisors as drug-
e-
delivery strategy, since they presented affinity to N-terminal region of ApoB-100.
Additional assays will be performed on different cell lines in order to further extend the
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application spectrum of platinum porphyrins. Also, modifications in the chemical

structure of these porphyrins, as different metallic centers and new platinum derivatives
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will be studied.
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6. Funding and Acknowledgments

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This study was financed in part by the CAPES/PROEX - Finance Code 001,
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CNPq and FAPERGS. Bernardo A. Iglesias also to thanks the CNPq Universal Grants
409150/2018-5 and PQ Grants 304711/2018-7.

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[12] T.T. Tasso, T.M. Tsubone, M.S. Baptista, L.M. Mattiazzi, T. V. Acunha, B.A.
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[13] J.A. Naue, S.H. Toma, J.A. Bonacin, K. Araki, H.E. Toma, Probing the binding
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Highlights

 Platinum(II) porphyrins are good candidates for photosensitizer.

 Platinum(II) porphyrins alter cellular actin organization.
 Platinum(II) porphyrins increased the number of total apoptotic cells.
 Caspases 3 and 9 are involved in the mechanism of death of platinum
porphyrins.
 Platinum(II) porphyrins have affinity for LDL receptor

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