International Journal of

Molecular Sciences

Article
Evaluation of the Cytotoxic Effect of Pd2Spm against Prostate
Cancer through Vibrational Microspectroscopies
Raquel C. Laginha 1 , Clara B. Martins 1 , Ana L. C. Brandão 1 , Joana Marques 1 , M. Paula M. Marques 1,2 ,
Luís A. E. Batista de Carvalho 1, * , Inês P. Santos 1,† and Ana L. M. Batista de Carvalho 1,†

1 Molecular Physical-Chemistry R&D Unit, Department of Chemistry, University of Coimbra,

3004-535 Coimbra, Portugal
2 Department of Life Sciences, Faculty of Science and Technology, University of Coimbra,
3000-456 Coimbra, Portugal
* Correspondence: labc@ci.uc.pt; Tel.: +351-239854462
† These authors contributed equally to this work.

Abstract: Regarding the development of new antineoplastic agents, with a view to assess the selective
antitumoral potential which aims at causing irreversible damage to cancer cells while preserving
the integrity of their healthy counterparts, it is essential to evaluate the cytotoxic effects in both
healthy and malignant human cell lines. In this study, a complex with two Pd(II) centers linked by
the biogenic polyamine spermine (Pd2 Spm) was tested on healthy (PNT-2) and cancer (LNCaP and
PC-3) prostate human cell lines, using cisplatin as a reference. To understand the mechanisms of
action of both cisplatin and Pd2 Spm at a molecular level, Fourier Transform Infrared (FTIR) and
Raman microspectroscopies were used. Principal component analysis was applied to the vibrational
data, revealing the major metabolic changes caused by each drug, which were found to rely on DNA,
lipids, and proteins, acting as biomarkers of drug impact. The main changes were observed between
the B-DNA native conformation and either Z-DNA or A-DNA, with a higher effect on lipids having
Citation: Laginha, R.C.; Martins, C.B.;
been detected in the presence of cisplatin as compared to Pd2 Spm. In turn, the Pd-agent showed a
Brandão, A.L.C.; Marques, J.;
Marques, M.P.M.;
more significant impact on proteins.
Batista de Carvalho, L.A.E.;
Santos, I.P.; Keywords: prostate cancer; cisplatin; palladium(II); Raman microspectroscopy; FTIR microspectroscopy
Batista de Carvalho, A.L.M.
Evaluation of the Cytotoxic Effect of
Pd2 Spm against Prostate Cancer
through Vibrational 1. Introduction
Microspectroscopies. Int. J. Mol. Sci. It is estimated that prostate cancer was responsible for more than 375,000 deaths in
2023, 24, 1888. https://doi.org/
2020. This is the second most common type of cancer, after lung cancer, the fifth with the
10.3390/ijms24031888
highest mortality rate in individuals with prostate, and the first in prevalence in the last five
Academic Editor: Francesco D’Amico years [1]. Prostate cancer can be classified under several category types: (1) adenocarcinoma,
which may be divided into acinar or ductal adenocarcinomas; (2) transitional cell and
Received: 22 December 2022
(3) squamous cell carcinomas; (4) small cell prostate cancer; (5) sarcoma; and (6) lymphoma
Revised: 13 January 2023
tumors, with acinar adenocarcinoma being the most common [2]. Prostate cancer screening
Accepted: 16 January 2023
of asymptomatic individuals with prostate is carried out through blood analysis to assess
Published: 18 January 2023
prostate specific antigen (PSA) levels followed or not by digital rectal examination. Patients
with positive findings undergo transrectal imaging techniques (ultrasound or magnetic
resonance) and biopsy. The histopathological analysis of the biopsy enables the conclusive
Copyright: © 2023 by the authors. diagnosis of the type of tumor [3].
Licensee MDPI, Basel, Switzerland. The treatment of choice for prostate cancer patients depends on various factors, such
This article is an open access article as the type of tumor and its staging. When detected at an early stage, the patient can either
distributed under the terms and be kept under active surveillance/watchful waiting or proceed to radiotherapy, which may
conditions of the Creative Commons be combined with adjuvant or neoadjuvant androgen suppression hormone therapy. This
Attribution (CC BY) license (https:// consists of reducing the levels of testosterone and dihydrotestosterone (DHT) as a way
creativecommons.org/licenses/by/ of inhibiting tumor growth. When there is no tumor growth reduction after hormonal
4.0/).

Int. J. Mol. Sci. 2023, 24, 1888. https://doi.org/10.3390/ijms24031888 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 1888 2 of 14

castration (castration-resistant prostate cancer, CRPC), chemotherapy might be necessary.

If treatment fails, patients may undergo prostatectomy [3,4].
There are several antineoplastic agents currently approved for clinical use against
CRPC, such as docetaxel, which is frequently used in combination with prednisone or even
in a cocktail with other drugs like darolutamide [5,6]. Prednisone may also be administered
in combination with the well-known compound cabazitaxel to treat metastatic castration
resistant prostate cancer (mCRPC) when patients have already undergone chemotherapy
that included docetaxel and became resistant [7,8]. These types of chemotherapeutic drugs,
docetaxel and cabazitaxel, belong to the category of microtubule targeting compounds,
specifically taxane anticancer agents, with their mechanism of action relying on the mi-
crotubules’ stabilization or increased polymerization [9]. Additionally, there are studies
with promising results for inorganic agents, namely with platinum(II) and palladium(II)
metal centers, against metastatic and non-metastatic CRPC [4,10]. Platinum agents have
a different mode of action when compared to antimitotic targeting agents. They form
adducts with DNA by direct binding between the metal ion and the nitrogen from the
purine and pyrimidine bases, leading to disruption of the replication mechanisms and
inducing cell death by apoptosis. Cisplatin (cis-Pt(NH3 )2 Cl2 ) was first synthesized in 1845
by Michele Peyrone, its cell growth-inhibiting effect having been discovered serendipi-
tously by Barnett Rosenberg in 1960. In the 1970s, it was tested and approved for clinical
use to fight ovarian and metastatic testicular cancers. Due to the positive outcomes of
cisplatin as a chemotherapeutic drug, the development of inorganic agents has been more
significant over the years [11,12]. Nevertheless, drug toxicity, adverse side effects, and
cellular resistance mechanisms are three of the faced challenges when using conventional
metal-based anticancer drugs [3,4]. To suppress these limitations, in the last decades,
several Pt-based compounds, such as Picoplatin (Pt(II), BTP-114 (cisplatin pro-drug) and
Satraplatin (Pt(IV)—oral platinum analogue) have undergone clinical trials for prostate
cancer treatment [13], although none of these have yet been approved for clinical use due
to low efficacy or severe side-effects.
In order to overcome these deleterious side effects and improve efficacy, polynuclear
Pt(II) and Pd(II) chelates with biogenic polyamines have been developed over the past
years [14]. Linear alkyl polyamines, such as putrescine, spermidine, and spermine, are
polycations essential for cell growth but are also recognized for their association to neoplas-
tic processes. They promote higher flexibility to the metal complexes (relative to cisplatin
or carboplatin) and higher rates of interstrand cross-links [15–17]. Besides the structural
similarity of these transition metals, Pd(II) complexes are more reactive than Pt(II), which
correlates to faster reactions and higher lability. This is corroborated by the ligand field
theory, the higher energy splitting in 5d orbitals in Pt compared to 4d orbitals in Pd leads
to less nucleus–electron interaction in d orbitals, enabling the donation of two electrons to
the ligands, giving rise to more stable bonds, usually less labile [14,17–19].
For an in-depth knowledge of the complexes and determination of structural–activity
relationships (SAR’s), vibrational spectroscopy techniques may be used. Fourier transform
infrared (FTIR) and Raman are non-destructive, non-invasive, cost-effective, reproducible,
and highly accurate tools, very suitable for understanding the impact of the chemotherapeu-
tic agents on cells, at a molecular level. These two vibrational methods allow to understand
a drug’s metabolic impact under different conditions (e.g., drug-treated, or untreated cells).
Therefore, both FTIR and Raman have been extensively applied in biomedical research,
particularly to analyze the biochemical composition of cell lines or tissues and to classify
malignant and healthy tissue in the early-cancer diagnosis or surgical guidance, allowing a
better understanding of the disease onset at the biological and chemical levels [20–28].
Within the various available healthy and carcinogenic prostate cell lines, three can
be highlighted: (1) PNT-2 cell line constituted by normal prostatic-epithelial cells, thus
considered a healthy cell line; (2) LNCaP cell line, which corresponds to prostate adeno-
carcinoma cells sensitive to androgen levels; and (3) PC-3 cell line, are CRPC epithelial
cells of adenocarcinoma unresponsive to decreasing androgen levels [4,29]. Some studies
 considered a healthy cell line; (2) LNCaP cell line, which corresponds to prosta
adenocarcinoma cells sensitive to androgen levels; and (3) PC-3 cell line, are CRP
epithelial cells of adenocarcinoma unresponsive to decreasing androgen levels [4,29
Some studies have reported higher values of HIF1α protein in PC-3 than in LNCaP, sin
Int. J. Mol. Sci. 2023, 24, 1888 3 of 14
this is likely a CRPC contributively of metastatic and chemo-resistance, a protein which
responsible to regulate the transcription of DNA [30].
haveVibrational
reported higher spectroscopy has been
values of HIF1α proteinapplied
in PC-3 to prostate
than in LNCaP,cancer
sinceto distinguish
this is likely a norm
from
CRPC contributively of metastatic and chemo-resistance, a protein which is responsiblefingerprin
cancer tissues [31–35], allowing the identification of biochemical to
especially
regulate therelated to DNA
transcription and lipids,
of DNA [30]. leading to an accurate evaluation of the sample. F
prostate cells lines,
Vibrational the highhas
spectroscopy wavenumbers
been applied have been cancer
to prostate used toto fully characterize
distinguish normal the PC
from cancer tissues [31–35], allowing the identification of biochemical
cell line [36] and to study the use of lipid altering drugs against PC-3 and PNT-2 fingerprints espe- cell lin
cially related to DNA and lipids, leading to an accurate evaluation
[37]. FTIR microspectroscopy was used to analyze and classify several prostate of the sample. For cancer ce
prostate cells lines, the high wavenumbers have been used to fully characterize the PC-3
lines [38], and a few microFTIR studies are to be found in the literature reporting the u
cell line [36] and to study the use of lipid altering drugs against PC-3 and PNT-2 cell
oflines
anticancer
[37]. FTIRagents such as paclitaxel,
microspectroscopy was useddoxorubicin,
to analyze andorclassify
methrotrexate against
several prostate the PC-3 ce
cancer
line [39,40].
cell lines [38], and a few microFTIR studies are to be found in the literature reporting the
use ofThis work aims
anticancer agentsatsuch
evaluating cell doxorubicin,
as paclitaxel, viability in orPNT-2, LNCaP,against
methrotrexate and PC-3
the PC-3prostate ce
cell line [39,40].
lines in the presence of cisplatin and Pd2Spm, a complex with two Pd(II) centers linked b
This work
the biogenic aims at evaluating
polyamine spermine cell viability
(Figure 1),inwhich
PNT-2, LNCaP,
has shown and PC-3 prostate
increased cell activi
cytotoxic
lines in the presence of cisplatin and Pd Spm, a complex with two Pd(II) centers linked by
against several human cancer cells 2(e.g., triple-negative breast cancer) [21] (at their ha
the biogenic polyamine spermine (Figure 1), which has shown increased cytotoxic activity
maximal inhibitory
against several humanconcentration (ICtriple-negative
cancer cells (e.g., 50) values). FTIR and Raman microspectroscopi
breast cancer) [21] (at their half
were applied to identify possible biomarkers
maximal inhibitory concentration (IC50 ) values). FTIR of cytotoxicity, aiming at the developme
and Raman microspectroscopies
of
were new andto identify
applied improved chemotherapeutic
possible drugs. aiming
biomarkers of cytotoxicity, The at metabolic profileof of eac
the development
administered
new and improved drugchemotherapeutic
against each prostatedrugs. cancer cell lineprofile
The metabolic (LNCaP andadministered
of each PC-3) was assesse
drug
as against
well as theeach prostate
drugs´ cancer
impact oncell line (LNCaP
a healthy and PC-3)
prostate was(PNT-2).
cell line assessed, as well as the
drugs´ impact on a healthy prostate cell line (PNT-2).

Figure 1. Structural representation of the Pt(II) and Pd(II) complexes presently studied.
Figure 1. Structural representation of the Pt(II) and Pd(II) complexes presently studied.
2. Results and Discussion
2.2.1.
Results and
Cytotoxic Discussion
Evaluation
The cytotoxic
2.1. Cytotoxic effect of cisplatin and Pd2 Spm was evaluated through the MTT assay,
Evaluation
against two prostate cancer cell lines (PC-3 and LNCaP) and one healthy prostate cell line
The cytotoxic effect of cisplatin and Pd2Spm was evaluated through the MTT assa
(PNT-2) (Figure 2). A clear separation between dose–response curves for prostate cancer
against two prostate
and non-cancer cancer
cells was cell lines
observed for Pd(PC-3 and LNCaP) and one healthy prostate cell lin
2 Spm at 24, 48, and 72 h incubation times. For
(PNT-2)
cisplatin,(Figure 2). A clear
a slight overlap separation
between between
all the three dose–response
cell lines was detected at 24 curves
and 48for prostate
h. Table 1 canc
and non-cancer
comprises the IC50cells was
values observed
obtained for Pd2and
for cisplatin Spm Pdat
2 24,
Spm 48,
for and
24, 48, 72
and h incubation
72 h incubation times. F
periods. For cisplatin, the values obtained were lower for all time points when
cisplatin, a slight overlap between all the three cell lines was detected at 24 and 48 h. Tab compared
1tocomprises
Pd2 Spm. The healthy
the IC50 cells (PNT-2)
values were more
obtained for sensitive
cisplatinto and
both Pt(II)/Pd(II)
Pd2Spm forcompounds
24, 48, and 72
when compared to the corresponding cancer cells (PC-3 and LNCaP). This is indicative of a
incubation periods. For cisplatin, the values obtained were lower for all time points whe
non-discriminatory cytotoxic effect induced by these metal-based drugs. Values previously
compared
reported for tocisplatin
Pd2Spm.forThe healthy
PC-3, LNCaP, cells
and(PNT-2) werePC-3–1.0
PNT-2 were: more sensitive
µM [41]to (72both Pt(II)/Pd(I
h, SRB
compounds
method) 10.6 µM [42] (48 h, MTT method), LNCaP—3.7 µM [41] (48 h, SRB method), and This
when compared to the corresponding cancer cells (PC-3 and LNCaP).
indicative
PNT-2–8.5 of µMa[43]
non-discriminatory
(96 h, MTT method), cytotoxic effectsome
which reflects induced by these
variability, oftenmetal-based
due to the drug
type of method as well as the experimental design. The higher effect of cisplatin in the
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 14

Values previously reported for cisplatin for PC-3, LNCaP, and PNT-2 were: PC-3–1.0 µM
Int. J. Mol. Sci. 2023, 24, 1888 4 of 14
[41] (72 h, SRB method) 10.6 µM [42] (48 h, MTT method), LNCaP—3.7 µM [41] (48 h, SRB
method), and PNT-2–8.5 µM [43] (96 h, MTT method), which reflects some variability,
often due to the type of method as well as the experimental design. The higher effect of
hormone-insensitive PC-3 cells, whenPC-3
cisplatin in the hormone-insensitive compared to the compared
cells, when hormone-sensitive LNCaP, was also
to the hormone-sensitive
presently
LNCaP, wasidentified (Table 1).
also presently identified (Table 1).

Figure2.2. Dose–response
Figure Dose–response curves
curves of
of cisplatin
cisplatin and
and Pd
Pd22Spm
Spm against
against prostate
prostate cancer
cancer (PC-3,
(PC-3, red
red line;
line;
LNCaP,blue
LNCaP, blue line)
line) andand prostate
prostate non-cancer
non-cancer (PNT-2,
(PNT-2, black black line)
line) cell cellatlines,
lines, 24, 48,atand
24,7248, and 72 h
h incubation
incubation times. Data are expressed as mean ± SEM, n = 4.
times. Data are expressed as mean ± SEM, n = 4.

Table 1. Half maximal inhibitory concentration (IC50, µM) of cisplatin and Pd2Spm against prostate
Table Half maximal
cancer1.(PC-3 inhibitory
and LNCaP) concentration
and prostate (IC50(PNT-2)
non-cancer , µM) ofcell
cisplatin and
lines, at 24,Pd 2 Spm
48, and against prostate
72 h incubation
cancer
times. (PC-3 and LNCaP) and prostate non-cancer (PNT-2) cell lines, at 24, 48, and 72 h incubation
times.
Drug Time PC-3 LNCaP PNT-2
Drug
Cisplatin 24 h Time 25.6 PC-3 13.6 LNCaP PNT-2
13.2
Cisplatin 48 h 24 h 3.0 25.6 7.3 13.6 5.213.2
72 h 48 h 0.7 3.0 2.7 7.3 2.45.2
72 h 0.7 2.7 2.4
Pd2Spm 24 h 31.3 76.0 6.6
Pd2 Spm 48 h 24 h 27.9 31.3 27.0 76.0 9.06.6
48 h 27.9 27.0 9.0
72 h 31.2 28.5 5.4
72 h 31.2 28.5 5.4

2.2. Raman and FTIR Microspectroscopy Characterization

2.2. Raman and FTIR Microspectroscopy Characterization
When probing the biochemical signatures of cell lines by Raman and FTIR
When probing the
microspectroscopies, biochemical
a large number ofsignatures
data pointsofarecell linesrequired.
usually by Raman and
This FTIR mi-
is important
crospectroscopies,
in order to average a large number of
the resulting data points
spectral are usually
signatures required.
from each This iscell
individual important
and to
in order to average the resulting spectral signatures from each individual
obtain a representative spectral signature of the sample (Figure 3), thus minimizing cell andthe
to
obtain a representative spectral signature of the sample (Figure 3), thus minimizing
effect of the cell cycle profile (predominantly G1 under normal conditions) as previously
the effect of the cell cycle profile (predominantly G1 under normal conditions) as pre-
reported [39]. In this study, both FTIR and Raman data were acquired through a randomly
viously reported [39]. In this study, both FTIR and Raman data were acquired through
distributed cell population consisting of cells exhibiting a high spectral heterogeneity due
a randomly distributed cell population consisting of cells exhibiting a high spectral
to different stages of the cell cycle.
heterogeneity due to different stages of the cell cycle.
Figure 3 depicts the average Raman and FTIR spectra obtained for the three prostate
cell lines (PNT-2, LNCaP, and PC-3) under the following conditions: (1) untreated cells
(control), and (2) treated cells (either with cisplatin or Pd2Spm). In Table S1
(Supplementary Material), the assignments for all the signals observed in the FTIR and
Raman spectra for control cells of PNT-2, LNCaP, and PC-3 cell lines are identified
 optimal method for cellular fixation in order to preserve the sample in a similar
physiologic state condition, avoiding contaminations in the fixation process. Formalin
leads to the formation of methylene bridges between aldehyde groups from formaldehyde
and the primary and secondary amines of cellular proteins, which allows to maintain a
similar constitution to that of in vivo cells. A slightly less intense signal from the infrared
Int. J. Mol. Sci. 2023, 24, 1888 5 of 14
spectra is expected from this fixation, caused by the conformational change of proteins
and lipidic breakdown.

Figure 3.
Figure 3. Mean
Mean Raman
Raman(500–1800
(500–1800cmcm−−11) and infrared (1000–1800 cm−−11))spectra
(1000–1800 cm spectra for
for untreated/control
untreated/control
(blue line), cisplatin-treated (green line), and Pd
(blue line), cisplatin-treated (green line), and Pd2 2 Spm-treated (red line) cells.

Figure 3 depicts
Principal componentsthe average
analysis Raman
(PCA) and of FTIR
the FTIRspectra
andobtained for the three prostate
Raman spectroscopic results
cell
waslines (PNT-2, in
performed LNCaP,
orderand to PC-3)
unveilunder the following
the chemical conditions:
differences (1) untreated
between control cells
and
(control), and cells
drug-treated (2) treated
for eachcells (either
cell with cisplatin
line under study (Figure or Pd24).Spm). In Table
Overall, S1 (Supplementary
a good discrimination
Material),
was attained the by
assignments
FTIR between for all the signals
controls, observed in and
Pd2Spm-treated, the FTIR and Ramancells
cisplatin-treated spectra
for
for
eachcontrol cells
cell line of PNT-2,
(Figure 4A,C,E), LNCaP, and PC-3not
this separation cell linessoare
being clearidentified (including
for the Raman signals
data (Figure
only detected
4B,D,F). by infrared,
A slight and specific
overlap between DNA and
the scores of theprotein
controlconformational
and drug-treated rearrangements
classes was
promoted by drug exposure) [21,22]. The spectra for untreated cells
expected, evidencing that the drugs did not affect the cells substantially towards complete are in accordance with
published data based on FTIR microspectroscopy [38,40].
destruction [44]. From Figure 4A, it is clear that the main discrimination between control,
The cells were
Pd2Spm-treated, andfixed with a 4% formaldehyde
cisplatin-treated LNCaP cells in solution (formalin),
the FTIR spectra considered
was along the
principal
optimal
componentsmethod for cellular
2 (PC2) and 3fixation
(PC3) in order to preserve
(explaining 39.8% and the sample
11.3% in ofa total
similar physio-
variance,
logic state condition,
respectively). From these avoiding
scores contaminations
and loadings, it isinpossible the fixation process.
to observe thatFormalin leads
discrimination
to the formation
between of methylene
the drug-treated bridges
vs. control between
LNCaP cellsaldehyde
in FTIR isgroupsmainly from formaldehyde
attributed to: drug-
and the LNCaP
treated primarycellsandshowing
secondary more amines of cellular
contribution, than proteins, which allows
control LNCaP to maintain
cells, from amide IIIa
similar
β-sheets at 1228 cm ; amide II ((δ(CN-H)/ν(CN)), lipids (ν(CC), ν(CN), δ(CHinfrared
constitution to
−1 that of in vivo cells. A slightly less intense signal from the 2)), and
spectra
proteinsis(δ(CH
expected from this
2), ν(=C=C=) fixation,
conjugated caused
) at 1547 cm−1by the conformational
; carbohydrates (δ(CH2)) change
at 1157 ofcm
proteins
−1 and

and lipidic breakdown.

1315 cm ; and guanine (ν(CC)ring) at 1315 cm . Drug-induced conformational changes
−1 −1

werePrincipal components
also detected: the bands analysis
attributed(PCA) of the IFTIR
to amide and Raman
anti-parallel spectroscopic
β-sheets (1691 cm−1results
) were
was performed
stronger in order to unveil
in the drug-treated the chemical
cells, reflecting a cleardifferences betweenThe
effect on proteins. control
LNCaP and drug-
control
treated cells for each cell line under study (Figure 4). Overall,
group showed a higher intensity than drug-treated cells in the bands assigned to proteins a good discrimination
was attained
(ν(CC), by FTIR
ν(CN)), between controls,
phospholipids (νs(PO2−),Pdν(CC),
2 Spm-treated,
ν(CO)), and and cisplatin-treated cells for
carbohydrates (δ(OCH),
each
ν(CC), ν(CO)glycogen), detected at 1050 cm and 1074 cm , which may be indicative data
cell line (Figure 4A,C,E), this separation
−1 not being−1 so clear for the Raman of a
(Figure 4B,D,F). with
drug interaction A slight overlapmembrane
the cellular between the scores of the
(phospholipid control and drug-treated
moieties).
classes was expected, evidencing that the drugs did not affect the cells substantially
towards complete destruction [44]. From Figure 4A, it is clear that the main discrimi-
nation between control, Pd2 Spm-treated, and cisplatin-treated LNCaP cells in the FTIR
spectra was along principal components 2 (PC2) and 3 (PC3) (explaining 39.8% and
11.3% of total variance, respectively). From these scores and loadings, it is possible to
observe that discrimination between the drug-treated vs. control LNCaP cells in FTIR is
mainly attributed to: drug-treated LNCaP cells showing more contribution, than control
LNCaP cells, from amide III β-sheets at 1228 cm−1 ; amide II ((δ(CN-H)/ν(CN)), lipids
(ν(CC), ν(CN), δ(CH2 )), and proteins (δ(CH2 ), ν(=C=C=)conjugated ) at 1547 cm−1 ; carbo-
hydrates (δ(CH2 )) at 1157 cm−1 and 1315 cm−1 ; and guanine (ν(CC)ring ) at 1315 cm−1 .
Drug-induced conformational changes were also detected: the bands attributed to amide
I anti-parallel β-sheets (1691 cm−1 ) were stronger in the drug-treated cells, reflecting
a clear effect on proteins. The LNCaP control group showed a higher intensity than
Int. J. Mol. Sci. 2023, 24, 1888 6 of 14

drug-treated cells in the bands assigned to proteins (ν(CC), ν(CN)), phospholipids

(νs (PO2 − ), ν(CC), ν(CO)), and carbohydrates (δ(OCH), ν(CC), ν(CO)glycogen ), detected
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 14
at 1050 cm−1 and 1074 cm−1 , which may be indicative of a drug interaction with the
cellular membrane (phospholipid moieties).

Figure 4.4.PCA
Figure PCAscores and and
scores loading plots ofplots
loading FTIR ((A,C,E);
of FTIR1000–1800
((A,C,E);cm cm−1 ) and 500–
−1) and Raman ((B,D,F);
1000–1800 Raman
1800 cm −1) data for cisplatin
− 1 and Pd 2Spm-treated prostate cancer cell lines LNCaP (A,B) and PC-3
((B,D,F); 500–1800 cm ) data for cisplatin and Pd2 Spm-treated prostate cancer cell lines LNCaP
(C,D), and non-cancer cell line PNT-2 (E,F) vs. their respective controls. (For clarity the loadings are
(A,B) and PC-3 (C,D), and non-cancer cell line PNT-2 (E,F) vs. their respective controls. (For
offset, the dashed horizontal lines indicating zero loading).
clarity the loadings are offset, the dashed horizontal lines indicating zero loading).
Regarding the Raman spectra of the LNCaP cells, the classes were not as distinctly
separated as in FTIR (Figure 4B): PC1 and PC2 providing a slight separation and
explaining 31.4% and 24.8% of the total variance, respectively. As for FTIR, the LNCaP
control cells showed a wider dispersion in both principal components relative to the
Int. J. Mol. Sci. 2023, 24, 1888 7 of 14

Regarding the Raman spectra of the LNCaP cells, the classes were not as distinctly
separated as in FTIR (Figure 4B): PC1 and PC2 providing a slight separation and explaining
31.4% and 24.8% of the total variance, respectively. As for FTIR, the LNCaP control cells
showed a wider dispersion in both principal components relative to the drug-treated cells.
Relative to the control, drug-treated LNCaP cells evidenced: a higher contribution from
Z-DNA (ν(OPO)backbone ) at 749 cm−1 ; proteins (ν(CN)) at 1128 cm−1 ; guanine (ν(CC)ring )
at 1328 cm−1 ; cytosine, guanine, and thymine at 1347 cm−1 ; adenine, guanine, thymine,
glycoproteins (δ(CH3 )), and lipids/acyl chains (δ(CH2 )) at 1379 cm−1 ; and amide I antipar-
allel β-sheets at 1680 cm−1 . The drug-treated LNCaP cells have also suffered a shift of the
phenylalanine band from 1001 cm−1 to 1008 cm−1 . The DNA (OPO) backbone elongation
is the most sensitive peak to recognize cell death since this indicates a collapse of the
phosphodiester bonds in the double helix [21].
LNCaP control cells showed a higher contribution, compared to drug-treated cells,
from B-DNA/deoxyribose (ν(CO)) and protein/lipids/carbohydrates (ν(CC), ν(CN), and
ν(CC), ν(CO)) at 1064 cm−1 ; RNA/adenine and cytosine (ν(CC)ring ) at 1297 cm−1 ; lipids
(δ(CH2 )) at 1440 cm−1 ; and amide I (random coil) at 1655 cm−1 .
Regarding the PC-3 cell line, a separation between Pd2 Spm-treated cells vs. the
remaining groups (control and cisplatin-treated being overlapped) was clear in the
FTIR spectra (Figure 4C), along a combination of PC1 and PC2 (representing 45.1%
and 26.5% of total data variance, respectively). This separation is mainly due to a
stronger contribution, in Pd2 Spm-treated PC-3 cells (compared to PC-3 controls and
PC-3 cisplatin-treated cells), from phenylalanine (δ(CH), ν(O–CH3 )), phospholipids
(ν(CC), δ(CH)), and carbohydrates (ν(CC), ν(CO),ν(C–OH)) at 1033 cm−1 ; porphyrins
(ν(C=C)) at 1527 cm−1 ; amide II (δ(CN-H)/ν(CN)) at 1551 cm−1 ; and amide I (random
coil) at 1659 cm−1 .
From the Raman spectra, separation between Pd2 Spm-treated PC-3 cells vs. PC-3
control and PC-3 cisplatin-treated cells was obtained along PC5 (5.0% of total variance,
Figure 4D). As in FTIR, PC-3 controls and cisplatin-treated PC-3 cells were slightly
overlapped. The Pd2 Spm-treated PC-3 cells presented a higher contribution from cyto-
sine and adenine (ν(CC)ring ) at 779 cm−1 ; guanine at 1333 cm−1 ; adenine and guanine
(ν(CC)ring ) at 1571 cm−1 ; phenylalanine (νs (CC)ring ) at 1003 cm−1 ; lipids (δ(CH2 )) at
1433 cm−1 and (ν(C=C)) at 1650 cm−1 ; and amide I (α-helix) at 1651 cm−1 .
Finally, regarding the non-cancer prostate cell line, PNT-2, it is visible that the
discrimination between the three groups (control, Pd2 -treated and cisplatin-treated
cells) in the FTIR spectra occurs along both PC2 and PC3, covering 7.1% and 4.9% of
total data variance, respectively (Figure 4E). The drug-treated PNT-2 cells show a higher
contribution from lipids (δ(CH2 )) at 1453 cm−1 ; porphyrins (ν(C=C)) at 1526 cm−1 ;
and random coil and β-sheets of amide I at 1650 cm−1 . When compared to the drug-
treated samples, the PNT-2 control cells evidence a higher contribution from proteins
and membrane lipids (δ(CH2 ), ρ(CH2 )) at 1402 cm−1 ; lipids (δ(CH2 ) at 1455 cm−1 );
porphyrins (ν(C=C) at 1522 cm−1 ) and nucleic acids (νs (PO2 - )B-DNA at 1086 cm−1 ),
adenine and cytosine (ν(CC)ring at 1577 cm−1 ).
From PNT-2 Raman data (Figure 4F), it is possible to separate the control from the drug-
treated cells (Pd2 Spm and cisplatin-treated cells are overlapping) along PC4 (which covers
10.3% of the total variance). The drug-exposed PNT-2 cells show a higher contribution
from thymine (ν(CC)ring ) at 1602 cm−1 , and proteins (ν(CS), τ(CC)tyrosine at 644 cm−1 ,
δ(C=CH)phenylalanine at 1604 cm−1 , and amide I (random coil) at 1648 cm−1 ).
From Figure 4A,E, it is visible that the FTIR scores from Pd2 Spm-treated LNCaP
and PNT-2 cells display an intermediate position relative to the cisplatin-treated and
control samples, as opposed to the PC-3 cells. This may suggest that different metabolic
pathways might be involved in the cytotoxic activity of both Pt-drugs drugs. The same
intermediate position of Pd2 Spm-treated cells is visible in the Raman scores of LNCaP
(Figure 4B), although this effect is not evident for the other cell lines when probed by
Raman spectroscopy.
Int. J. Mol. Sci. 2023, 24, 1888 8 of 14

For a deeper interpretation of the effect of cisplatin and Pd2 Spm on prostate healthy
vs. cancer cell lines (PNT-2 vs. PC-3 and LNCaP), the principal component analysis of
the Raman and FTIR data is shown in Figure 5. For the Pd2 Spm-treated PC-3 vs. PNT-2
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 14
(Figure 5A,B), the FTIR spectra were distinctly separated along PC3 (explaining 4.2% of
the total variance). Based on the Raman data, these groups were separated along PC1
(which covered 48.0% of the data variance). Therefore, it was clear that the Pd2 Spm-treated
covered 48.0% of the data variance). Therefore, it was clear that the Pd2Spm-treated PC-3
PC-3 cells presented a higher contribution from proteins (ν(O-CH3 ) and δ(CH)phenylalanine
cells presented 1; a higher contribution from proteins (ν(O-CH 3) and δ(CH)phenylalanine at 1029
at 1029 cm−ν(CN),
cm-1; ν(CC), ν(CC),δ(CH ν(CN), δ(CH ) at 1152 cm−1 ) in FTIR; a blue-shift of ν(CC), ν(CO),
2) at 1152 cm2−1) in FTIR; a blue-shift of ν(CC), ν(CO), ν(C–OH)
ν(C–OH) from phenylalanine, phospholipids, and carbohydrates to Raman; −1 in Raman;
1008 cmlipids
from phenylalanine, phospholipids, and carbohydrates to 1008 cm−1 in
lipids − 1 − 1
(δ(CH2(δ(CH 2 ), ν(=C=C=)
), ν(=C=C=) 1152 cm−1), at
conjugated) at conjugated 1152
and δ(CH cm , andcm
2) at 1323 δ(CH ) at 1323
−1, in2FTIR, and cm
at 1442, in
cmFTIR,
−1 and
at 14422))cm − 1 (δ(CH − 1
(δ(CH in Raman; 2 )) inI Raman;
amide amide
(antiparallel I (antiparallel
β-sheets at 1672 cm β-sheets at 1672
in FTIR and cm coil
random inatFTIR and
−1

1659 cm-1coil
random in Raman);
at 1659ν(C=O)cm−1 in of amino
Raman); acids’ side chain
ν(C=O) at 1691acids’
of amino cm−1 inside
FTIR; at 1691 cm−1 in
and guanine
chain
(ν(CC)and
FTIR; ring) at 1323 cm(ν(CC)
guanine −1 in FTIR. −1 in FTIR.
ring ) at 1323 cm

PCA scores and loading plots((A,C,E,G);

of FTIR 1000–1800
((A,C,E,G); − 1 ) and Raman
Figure
Figure 5.5.PCA scores and loading plots of FTIR cm−11000–1800
) and Ramancm ((B,D,F,H);
((B,D,F,H); −1) data forcm
500–1800 cm500–1800 − 1
cisplatin- andfor
) data Pd2cisplatin-
Spm-treatedand
prostate cancer cell lines
Pd2 Spm-treated PC-3 (A–D)
prostate cancerandcell lines
LNCaP (E,F,G,H) vs. the non-cancer cell line PNT-2. (For clarity, the loadings are offset,
PC-3 (A–D) and LNCaP (E,F,G,H) vs. the non-cancer cell line PNT-2. (For clarity, the loadings the dashed
horizontal lines indicating zero loading).
are offset, the dashed horizontal lines indicating zero loading).
Int. J. Mol. Sci. 2023, 24, 1888 9 of 14

Regarding the Pd2 Spm-treated PNT-2 vs. PC-3 cells, the former presented a higher
contribution, in the FTIR spectra, from B-DNA ν(CO)deoxyribose at 1058 cm−1 and
νs (PO2 − ) at 1085 cm−1 , amide II (δ(CN-H), ν(CN)) at 1542 cm−1 , amide I (parallel
β-sheet) at 1639 cm−1 , and amide I (random coil) at 1649 cm−1 . From the Raman
spectra, PNT-2 presented higher intensity from B-DNA/nucleic bases and tryptophan
(ν(CC)ring ) at 678 cm−1 , B-DNA/deoxythimine and tryptophan (ν(CC)ring ) and Z-DNA
(ν(OPO)backbone ) at 753 cm−1 , nucleic acids (ν(CC)ring from cytosine, thymine, and
uracil) at 784 cm−1 , ν(CC), ν(CO), ν(C-OH) from phenylalanine, phospholipids, and
carbohydrates at 1000 cm−1 , proteins and lipids (ν(C=C), ν(C=N)) at 1583 cm−1 .
When comparing cisplatin-treated PC-3 cells vs. cisplatin-treated PNT-2 cells
(Figure 5C,D), separation was obtained along PC2 in FTIR (32.4% of total variance), and
along PC1 and PC3 in Raman (corresponding to 29.5% and 18.8%, respectively). The
major biomarkers for separation were: PNT-2 showed a higher intensity in the bands
assigned to phenylalanine (δ(CH)) at 1024 cm−1 in FTIR and at 1001 cm−1 and 1603 cm−1
in Raman; B-DNA/deoxythimine at 677 cm−1 and 752 cm−1 ; tryptophan (ν(CC)ring )
and porphyrin (ν(C=C)) at 1556 cm−1 in Raman. PC-3 cell line presented a higher
contribution from A-DNA (δ(CH2 ) and ν(C=O)) at 1414 cm−1 and 1708 cm−1 in FTIR,
and in Raman, showed the band assigned to phospholipids and phenylalanine blue-
shifted to 1008 cm−1 , and a higher contribution from RNA/ribose (ν(CO)), proteins
(ν(CN)), and lipids (ν(CC)acyl (trans conformation) ) at 1132 cm−1 , amide III/α-helix and
lipids (ω(CH2 ), t(CH2 )) at 1277 cm−1 , RNA/adenine and cytosine at 1300 cm−1 , and
amide I random coil at 1659 cm−1 .
It is noteworthy that even though the FTIR loadings of the principal components that
separate the PNT-2 vs. PC-3 cells treated with either Pd2 Spm or cisplatin (Figure 5A,C,
respectively) display a similar information, with the bands ascribed to aromatic lipids
and proteins (at ca. 1466 cm−1 ) being more clearly observed in the cisplatin-treated
PC-3 cells than in the Pd2 Spm-treated ones. Moreover, it is noticeable that the Pd2 Spm-
treated healthy prostate cell line PNT-2 displays a strong contribution from B-DNA
deoxyribose (ca. 1059 cm−1 ) and B-DNA phosphates (ca. 1087 cm−1 ) (Figure 5A),
which is not verified for the prostate cancer cell line PC-3. When these cells were
treated with cisplatin, PNT-2 showed a lower contribution from B-DNA deoxyribose
and B-DNA phosphates than PC-3 (Figure 5C). Using Raman spectroscopy, the first
principal component (PC1) in the Pd2 Spm-treated cells was relevant for PC-3 vs. PNT-2
separation (Figure 5B) and its loading is comparable to the PC1 loading of the cisplatin-
treated cells (Figure 5D). However, for the latter, the third principal component was
more determinant for discrimination of these two cell lines than PC1 alone, giving some
insight into the effect of cisplatin—namely PNT-2 cells displaying a stronger intensity of
B-DNA/deoxythimine (ca. 753 cm−1 ), RNA/ribose (ca. 1131 cm−1 ) than PC-3 cells.
Regarding the LNCaP vs. PNT-2 Pd2 Spm-treated cells, a clear separation was obtained
with both FTIR and Raman, along PC1 (75.3% and 43.4% of the total variance, respectively)
(Figure 5E,F). The main contributions for this separation were: a higher intensity of the
bands assigned to B-DNA/deoxyribose (at 1059 cm−1 and 1086 cm−1 in FTIR), amide I
(β-sheets) at 1638 cm−1 in FTIR and at 1675 cm−1 in Raman, amide II at 1545 cm−1 in FTIR
and aromatic lipids (at 1465 cm−1 in FTIR), for Pd2 Spm-treated LNCaP cells as compared
to Pd2 Spm-treated PNT-2.
Similarly to cisplatin-treated LNCaP vs. PNT-2 cells (Figure 5G,H), a good separa-
tion was obtained along PC1 in FTIR (covering 78.4% of total variance) and along PC2
in Raman (corresponding to 26.6% of total variance). Relative to cisplatin, the PNT-2
cells showed a higher intensity from B-DNA (νs (PO2 - )) at 1087 cm−1 in FTIR and at
1095 cm−1 in Raman; phenylalanine (δ(CH)) at 1031 cm−1 in FTIR and at 1171 cm−1 in
Raman; phospholipids (δ(CH)) and carbohydrates (ν(CC), ν(CO), ν(COH)) at 1031 cm−1
in FTIR and at 1001 cm−1 in Raman; lipids (δ(CH2 )) at 1439 cm−1 ; and amide I (ran-
dom coil) at 1657 cm−1 in Raman. In contrast, the Raman signal from cisplatin-treated
LNCaP cells showed a stronger contribution, relative to PNT-2, from RNA/ribose
Int. J. Mol. Sci. 2023, 24, 1888 10 of 14

(ν(CO)), lipids (ν(CC)acyl-trans ), and proteins (ν(CN)) at 1133 cm−1 , tyrosine proteins
(ν(C=C), ν(C=N)) and lipids (ν(C=C), ν(C=N)) at 1588 cm−1 .
It is noteworthy that for LNCaP vs. PNT-2, the principal components that separate
both cell lines treated with either Pd2 Spm (PC1 in Figure 5E) or cisplatin (PC1 in Figure 5G)
are very similar in FTIR. The main difference is the band assigned to amide II at 1545 cm−1 ,
which plays a more important role in cisplatin-treated than in Pd2 Spm-treated LNCaP cells.

3. Materials and Methods

3.1. Chemicals and Solutions
Cisplatin (cis-dichlorodiammine platinum(II), >99.9%), (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl-sulphoxide (DMSO), peni-
cillin/streptomycin 100× solution, phosphate buffered saline (PBS), potassium tetra-
chloropalladate(II) (K2 PdCl4 , >99.9%), RPMI-1640 medium, N,N´-bis(3-aminopropyl)-
1,4-diaminobutane (spermine, free base, Spm), Trypan blue (0.4% w/v), trypsin-EDTA
(1×), as well as solvents, inorganic salts and acids, were purchased from Sigma-Aldrich
Chemical S.A. (Sintra, Portugal). Fetal bovine serum (FBS) was obtained from Gibco-
Life Technologies (Porto, Portugal). All reagents were of analytical grade. Pd2 Spm was
synthesized according to published procedures [45] optimized by the authors [46].
For drug administration, initial stock solutions of cisplatin (320 µM) were prepared
in PBS and Pd2 Spm (320 µM) was prepared in PBS with a 10% (v/v) DMSO. All solutions
were sterile filtered and stored at −20 ◦ C.

3.2. Cell Culture

The human prostate cancer (PC-3 and LNCaP) and non-tumorigenic (PNT-2) cell lines
were obtained from the ECCAC Culture Collections and supplied by Sigma-Aldrich Chem-
ical S.A. (Sintra, Portugal). They were cultured as monolayers, at 37 ◦ C, in a humidified
atmosphere of 5% CO2 . The cultures were maintained in RPMI-1640 culture medium,
supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin/streptomycin.
The cells were subcultured at 80% confluence, using 0.05% trypsin-EDTA (1×) in PBS.

3.3. Cytotoxic Evaluation

For all cell lines (PC-3, LNCaP and PNT-2), cell cultures were established in 96-well
plates (100 µL/well) at a density of 3 × 104 cells/cm2 , and were allowed to attach for 24 h.
Triplicates were treated for different incubation periods (three independent experiments)
with a range of concentrations between 2 and 32 µM of the tested compounds (cisplatin
and Pd2 Spm). According to the population doubling time for all cell lines (ca. 24 h), 24, 48,
and 72 h timepoints after drug administration were chosen.
In order to evaluate cell viability, the MTT assay was used. Briefly, at each time-
point, the growth media was removed, cells were washed with PBS and MTT solution
(2.5 mg/mL) was added to each well (100 µL) [47]. After 3 h of incubation at 37 ◦ C, the
formazan crystals were solubilized in DMSO (100 µL), and the absorbance was measured
at 570 nm.

3.4. Sample Preparation for Vibrational Microspectroscopies

Upon harvesting by trypsinization, all prostate cell lines (PC-3, LNCaP, and PNT-2)
were centrifuged and the pellet was resuspended in culture medium and seeded, at
a concentration of 3 × 104 cells/cm2 , on optical substrates suitable for either FTIR or
Raman data collection, respectively, CaF2 disks (Crystran UV-grade, 1 mm × 13 mm) or
MgF2 disks (Crystran Raman-grade, 1 mm × 13 mm), which were previously washed
with 70% ethanol. After incubation for 24 h (allowing the cells to adhere), each of the
tested drugs (cisplatin or Pd2 Spm) were added according to the respective 50% cell
growth inhibition values (IC50 ) for each cell line, and the cells were allowed to culture
for a further 48 h. The growth medium was then removed, the cells were washed twice
with 0.9% NaCl (w/v), fixed in 4% formalin (diluted in 0.9% NaCl from the commercial
Int. J. Mol. Sci. 2023, 24, 1888 11 of 14

neutral buffered formaldehyde solution) for 10 min, and washed several times with
deionized water (to remove any residual salt) [48]. The disks were allowed to air-dry
prior to spectroscopic analysis.
All samples were prepared in duplicate, in two independent experiments.

3.5. FTIR Microspectroscopy

FTIR acquisitions were performed using a Bruker Hyperion 2000 microscope with a
liquid nitrogen-cooled mercury-cadmium-telluride (MCT) detector, in transmission mode
using a 15× Cassegrain both condenser and objective, coupled to a Bruker Optics Vertex 70
spectrometer, both purged by CO2 -free dry air. Each spectrum was the sum of 128 scans, at
4 cm−1 resolution, and the 3-term Blackman–Harris apodization function was applied.

3.6. Raman Microspectroscopy

Raman microspectroscopy acquisition was performed using a WITec confocal Raman
microscopy system Alpha 300 R coupled to an ultra-high-throughput spectrometer (UHTS)
300 VIS-NIR, using a 532 nm diode-pumped solid-state laser. The laser power on the
sample was kept at 28 mW and the measurements were achieved using a 100×/0.8 Zeiss
Epiplan objective with 10 accumulations of 10 s per spectrum for PC-3 cell line, and 15
accumulations of 10 s per spectrum for LNCaP and PNT-2 cell lines.

3.7. Data Processing and Statistical Analysis

Cells viability data results are expressed as mean ± standard error of the mean (SEM)
and compared with non-treated controls. Statistical analysis was carried out through
one-way ANOVA followed by Tukey’s multiple comparison test. A p-value < 0.05 was
considered statistically significant.
The microFTIR spectral data, processed by the OPUS 9.1 software, was atmospherically
compensated and corrected for changes in background intensity. The quality of the spectra
was assessed based on the intensity of the amide I band. The spectra were corrected for
Mie scattering using an Extended Multiplicative Signal Correction (EMSC) function with
20 iterations [49]. The analyzed spectra were truncated from 1000 to 1800 cm−1 and vector
normalized. These operations were performed in Matlab2020b (MathWorks).
The microRaman spectral data were obtained using the Project FIVE software (WITec).
In order to reduce the spectral noise, principal component noise removal was performed, re-
taining the first 20 principal components. The spectral data were cropped to the fingerprint
region, 500–1800 cm−1 , and further corrected for cosmic ray removal, background sub-
traction (polynomial order fitting of 2nd order), Savitzky–Golay smooth filtered (window
width of 5 points), and the area was normalized. The data pre-processing was performed
using the Quasar Spectroscopy 1.6.0 software [50,51]. In order to analyze the spectral infor-
mation between cell line classes and to identify possible biomarkers, principal component
analysis was done. The order of the principal components (PC) denoted their importance
in relation to the data set variance, PC1 corresponding to the highest variance present in
the data.

4. Conclusions
The development of new and improved chemotherapeutic drugs is aimed at achieving
both efficacy and selectivity, i.e., leading to irreversible damage to cancer cells while
maintaining the integrity of healthy cells. In addition, acquired resistance to treatment
should be minimized, as well as deleterious side effects, by exploring novel pathways
of cytotoxicity. The impact of Pd2 Spm towards human prostate cancer cell lines (LNCaP
and PC-3) and human prostate non-cancer cells (PNT-2) was studied (and compared to
cisplatin´s effect), through the evaluation of the cellular viability (by biological assays)
coupled to spectroscopic measurements of the cells’ biochemical profile (by FTIR and
Raman microspectroscopies).
Int. J. Mol. Sci. 2023, 24, 1888 12 of 14

A non-discriminatory cytotoxic effect induced by both Pt- and Pd-agents was ob-
served in all tested cell lines. Still, considering the least possible damage to non-cancer
cells (PNT-2) prompted by drug exposure, cisplatin appeared to be the best choice when
compared to Pd2 Spm after 48 h of drug administration (IC50 = 5.2 µM vs. 9.0 µM).
Concerning prostate cancer, an IC50 of 3.0 µM was obtained for the PC-3 cell line, which
is lower than the 5.2 µM value found for the PNT-2 non-cancerous cells.
Regarding the spectroscopic data, drug-treated cells evidenced key changes such as:
native B-DNA to Z- or A-DNA conformations; shifts to high wavenumbers of bands from
phenylalanine (νs (CC)ring ) and phospholipids (δ(CH)). Additionally, the ν(C=O) signal
from ester groups suggested a clear perturbation of the cellular membrane, particularly in
the nonpolar part of the phospholipids, which may affect the membrane integrity.
The different cytotoxic activity induced by cisplatin vs. Pd2 Spm is suggested by
the intermediate position that Pd2 Spm-treated LNCaP and PNT-2 cells present relative
to the corresponding cisplatin-treated and control samples. A higher amount of lipids
was detected in the presence of cisplatin as compared to the Pd-agent, suggesting a drug
interaction with the cellular membrane. Additionally, Pd2 Spm showed a more signifi-
cant impact on proteins and carbohydrates. Regarding the Pd2 Spm-treated non-cancer
PNT-2 cells, a strong contribution from B-DNA deoxyribose and B-DNA phosphates was
found. However, when PNT-2 and PC-3 cell lines were treated with cisplatin, the former
evidenced a lower contribution from B-DNA deoxyribose and B-DNA phosphates, while
the PC-3 cells show a conformational change from native B-DNA to A-DNA (indicative
of DNA disruption).
MicroFTIR and microRaman are two powerful analytical techniques which enabled
us to conclude that the two drugs currently tested behave differently against the human
prostate cell lines under study. These findings on the metabolic impact of anticancer Pt-
and Pd-complexes are expected to contribute to the development of new and optimized
metal-based agents for specific cancer types as well as subtypes.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms24031888/s1.
Author Contributions: Conceptualization, A.L.M.B.d.C., J.M. and I.P.S.; methodology, R.C.L., C.B.M.,
A.L.C.B., A.L.M.B.d.C. and I.P.S.; software, R.C.L., A.L.M.B.d.C. and I.P.S.; validation, R.C.L., I.P.S.
and A.L.M.B.d.C.; formal analysis, R.C.L., I.P.S. and A.L.M.B.d.C.; investigation, R.C.L., C.B.M.,
A.L.C.B., J.M., M.P.M.M., L.A.E.B.d.C., I.P.S. and A.L.M.B.d.C.; resources, L.A.E.B.d.C.; data curation,
I.P.S.; writing—original draft preparation, R.C.L., I.P.S. and A.L.M.B.d.C.; writing—review and
editing, R.C.L., C.B.M., A.L.C.B., J.M., M.P.M.M., L.A.E.B.d.C., I.P.S. and A.L.M.B.d.C.; visualization,
R.C.L., C.B.M., A.L.C.B., J.M., M.P.M.M., L.A.E.B.d.C., I.P.S. and A.L.M.B.d.C.; project administration,
L.A.E.B.d.C., I.P.S. and A.L.M.B.d.C.; supervision, A.L.M.B.d.C., I.P.S., J.M. and L.A.E.B.d.C.; funding
acquisition, L.A.E.B.d.C. All authors have read and agreed to the published version of the manuscript.
Funding: This work was developed within the Molecular Physical-Chemistry R&D Unit (UIDB/00070/2020
and UIDP/00070/2020) financed by the Portuguese Foundation for Science and Technology (FCT).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Ana Rita Cunha is acknowledged for her support in sample collection.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2023, 24, 1888 13 of 14

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