Analysis of Biomolecular Changes in HeLa Cervical Cancer Cell Line Induced by Interaction with [Pd(dach)Cl₂]

Abstract

Transition metal complexes have been used in medicine for several decades, but their intracellular effects are not yet fully elucidated. Therefore, in this study, we investigate biomolecular changes induced by a palladium(II) complex in cervical carcinoma (HeLa) cells as a model to study the subtle changes caused by transition metal ions ingested by the cells. The impact of dichloro(1,2-diaminocyclohexane)palladium(II), [Pd(dach)Cl₂], was studied by synchrotron radiation-based Fourier transform infrared (SR FTIR) spectroscopy, a powerful tool for studying alterations in cellular components’ biochemical composition and biomolecular secondary structure on a single-cell level. A spectral analysis, complemented by statistics, revealed that the Pd(II) complex considerably affected all major types of macromolecules in HeLa cells and induced structural changes in proteins through an increased formation of cross-β-sheets and causes structural rearrangement in deoxyribonucleic acid (DNA) through potential chromosome fragmentation. Although a certain level of lipid peroxidation was detectable by SR FTIR spectroscopy and confirmed by an analysis of cellular lipids by matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry, the oxidative stress is not a significant mechanism by which Pd(II) expresses the effect on the HeLa cells.

1. Introduction

Several transition metal complexes have been used as pharmaceuticals over the decades and many more are currently in development and clinical trial testing. They are used as anti-inflammatory, anti-cancer, and antiviral therapeutics [1,2,3]. Metal complexes’ immense potential in treating various cancer forms has earned widespread global attention. Cisplatin (cis-dichlorodiammineplatinum(II), cis-[PtCl₂(NH₃)₂]) has been the most used metallodrug for treating many different types of cancer in the past several decades [4,5,6,7]. However, cisplatin and other platinum-based therapeutics have many disadvantages, such as renal toxicity, neurotoxicity, ototoxicity, myelosuppression, nausea, vomiting, hair loss, gradual resistance forming, and poor solubility in liquid mediums [8,9]. Palladium(II) complexes have a similar metal centre to platinum(II) complexes [10], but they can interact with biomolecular targets in diverse ways depending on the ligand structure and there are several molecular targets for Pd complexes other than nucleic acids, including the mitochondria and ribosome proteins (review in [11]). However, it has been assumed that Pd(II) complexes induce cancer cell cycle arrest in a different stage compared to Pt complexes. Substituted polyamines have been shown to coordinate efficiently with Pd(II) and to act as modulators of the hydrophilic/lipophilic properties of the resulting chelates, thus improving the administration and bioavailability of transition metal complexes [12]. On the other hand, transition metals can be highly toxic against healthy cells, which might result in organ damage, nephrotoxicity, ototoxicity, and other side effects [13], but it is postulated that replacement of Pt with Pd in a metallodrug can help overcome them [11].

Although they have been used for decades as therapeutic agents, the effect of transition metal complexes on cells, which includes the entire cellular regulatory systems, has yet to be fully elucidated. Synchrotron radiation-based Fourier transform infrared (SR FTIR) spectroscopy, a label-free method for elucidating biomolecular changes, has a high potential and sensitivity, with a highly brilliant light source that provides higher spectral and spatial resolution at a cellular level [14]. SR FTIR has been used for the study of nanocomposite systems [15] and has been applied for profiling of biomolecules inside various cells and tissues [16]. SR FTIR spectroscopy can elucidate the drug’s effect on individual cellular biomolecules without their separation from the cellular milieu. In this work, we applied SR FTIR spectroscopy to study the effect of different concentrations of dichloro(1,2-diaminocyclohexane)palladium(II) ([Pd(dach)Cl₂], or Pd(II) complex, Figure 1) on lipids, proteins, or nucleic acids in HeLa cervical carcinoma cells, and the obtained results demonstrate clearly the influence of the Pd(II) complex on the protein and nucleic acid structure in the HeLa cell line, resulting in a higher overall content of proteins with β-sheets and changing the structure of DNA.

2. Results

2.1. Cytotoxicity of Pd(II) Complex

The effects of Pd(II) complex treatment on the HeLa cancer cells are presented in Figure 2. The survival rate of cells decreased to 78% compared to the control after Pd(II) treatment at a concentration of 85.76 µmol/L, which demonstrates rather low cytotoxicity of the Pd(II) complex on this tumour cell line, which gives us the opportunity to analyse intracellular effects in this concentration range.

2.2. SR FTIR Spectroscopy

A spectral analysis, including the second derivative (17 smoothing points, third polynomial order, and vector normalisation), was implemented for major fingerprint areas of the SR FTIR spectrum of whole cells: 3050–2800 cm⁻¹ lipid area, 1800–1480 cm⁻¹ proteins and esters, and 1480–980 cm⁻¹ nucleic acids and carbohydrate region. The structural characterisation of the synthesised Pd(II) complex is given in Figures S1–S3. As the Pd(II) complex induces changes in all three areas, they will be presented individually. To statistically evaluate the results and compare the influence of various treatments, we applied a principal component analysis (PCA).

2.2.1. Changes in the Lipid Region

FTIR lipid spectral regions of untreated cancer cells and cells treated with three different concentrations of the Pd(II) complex were recorded and are presented in Figure 3a. The bands at ~2956–2960 cm⁻¹, ~2929–2935 cm⁻¹, and 2871–2873 cm⁻¹ are assigned to ν_asCH₃, ν_asCH₂, and ν_sCH₃, respectively. Differences can be seen in symmetric (ν_s) and asymmetric (ν_as) stretching vibrations of CH₃ groups and asymmetric stretching vibrations of CH₂ groups. Although the ν_sCH₂ vibration in cells is detectable when the SR FTIR spectra of lyophilised cells are analysed [17], this band is missing in the spectra of cells acquired with formalin-fixed cells [18]. In the ν_asCH₃ region, the Pd concentration of 68.6 µM (Pd 2) exhibits the same peak position as the control cells (~2958 cm⁻¹), while the treatment intensity is slightly lower than the controls. The lowest Pd complex concentration of 34.3 µM (Pd 1) shifts the ν_asCH₃ signal peak towards a higher wavenumber (~2960 cm⁻¹). It shows a higher intensity, while the highest concentration of 102.9 µM (Pd 3) shifts the absorption maximum towards a lower wavenumber (~2956 cm⁻¹) and demonstrates lower intensity than the control. The same absorption maximum shift trend is also present in the ν_asCH₂ region, with the Pd 1 maximum being at ~2935 cm⁻¹, Pd 2 and control at ~2933 cm⁻¹, and Pd 3 at ~2935 cm⁻¹. The band position at ~2873 cm⁻¹ (ν_sCH3) was the same for all samples except for the highest Pd complex concentration, which shifted the maximum towards a lower wavenumber, ~2871 cm⁻¹. As the shifts in the ν_asCH₃ and ν_asCH₂ position might indicate the interaction with the Pd(II) complex, the changes in their intensity ratio might imply lipid oxidation, similar to our previous results obtained in ovarian cancer cells treated with a Ru complex [19], but to a somewhat lesser extent.

The PCA score plot (Figure 3b) indicates the PC1 and PC2 differentiation for the samples treated with the highest and lowest Pd complex concentrations. On the other hand, the cellular lipids treated with the Pd 1 and Pd 2 concentrations differ from the control over the PC2. The statistical analysis confirmed concentration-dependent differences detected in HeLa cells treated with various concentrations of the Pd(II) complex, and the maximum contribution to the observed differences between all samples was found in the region from ~2935 to ~2825 cm⁻¹ (PC1) and at ~3000 cm⁻¹ (PC2) (PC loadings are given in Figure 3c). The latter corresponds to the =C-H stretching vibrations [20], indicating changes in the saturation level upon the Pd(II) treatment.

Several band area ratios were calculated to examine potential Pd(II) complex-induced lipid damage, and changes in the level of unsaturated lipids. Changes in the content of lipid-related functional groups can be associated with oxidative stress-induced lipid degradation [21]. Therefore, we determined the ratio of the asymmetric vibration of CH₂ and CH₃. The ratio of ν_as(CH₂)/ν_as(CH₃) significantly increased after cells were treated with the highest concentration of the Pd complex, implying that the Pd(II) induced lipid peroxidation in HeLa cells (Figure 3d).

To confirm the observed changes in the lipid region, we performed a positive-ion MALDI TOF MS analysis of the lipid extract of the cells treated with a Pd complex at a concentration of 20 µg/mL. Signals of the lipids and phospholipids arise from the proton or sodium adducts and represent singly positively charged ions, as described previously [22]. Several m/z regions can be assigned to a specific phospholipid group. They are indicated in Figure S4 in the lipids’ positive MALDI TOF mass spectra. Only several signals are labelled by their position, corresponding to the differences between the samples. The signal at m/z 1049.3 might arise from phosphatidylinositol phosphate (PIP), but its identity could not be confirmed. The signal at m/z 494.3 [23] most likely corresponds to the proton adduct of lyso-phosphatidylcholine (LPC) 16:1 (the numbers represent the total number of carbons in the fatty acid chains and the number of double bonds), whereas that at m/z 568.3 is the sodium adduct of diacylglycerol (DAG) 36:0 [24]. Significant changes in other m/z regions are not detectable. Finally, at the low-mass region of the spectra of Pd(II)-treated cells, the disappearance of specific lipid-related signals can be observed simultaneously with the appearance of the PIP signal at m/z 1049 in Pd(II)-treated cells.

2.2.2. Changes in the Protein Region

The spectral region associated with the protein groups can be found inside the 1700–1480 cm⁻¹ region. Here, there are two primary bands: the so-called Amide I (at ~1650 cm⁻¹) that arises from C=O stretching vibration and the less intense Amide II band, which occurs from bending N-H vibration (usually around 1540 cm⁻¹) [25].

The Pd(II) treatment caused significant changes in the protein spectral region, in the intensity and position of the Amide I and Amide II bands (Figure 4a). Changes in the peak area imply a change in secondary structures. At the same time, the wavenumber shift implies a change in intermolecular interactions, i.e., hydrogen bonds [26,27]. Treated cells exhibit a peak shift to a lower wavenumber in the Amide I region, with rising complex concentration. The absorption maximum of the control sample is at ~1653 cm⁻¹, Pd 1 and Pd 2 are at ~1651 cm⁻¹, and Pd 3 is at 1649 cm⁻¹. The most prominent peak area is observed for sample Pd 1, followed by the control sample and samples Pd 2 and Pd 3. The Amide II band exhibits fewer changes than the Amide I band. The lowest Pd(II) complex concentration peak treatment of cells shifts the Amide I band towards a higher wavenumber (~1541 cm⁻¹) compared to the three other samples, which had an absorption maximum of ~1537 cm⁻¹.

Since the most bands that can be attributed to secondary structure elements are allocated in the Amide I region, we inspected this band in more detail. The second derivative of the Amide I region (Figure 5a, region: 1700–1600 cm⁻¹) reveals specific changes in protein secondary structures upon the action of the Pd(II) complex. Shifts in the absorption maxima corresponding to cumulative changes in the protein secondary structure are confirmed by analysing the second derivative of the Amide I region and the corresponding PC score plots (Figure 5a and Figure 5b, respectively). The most prominent structural change was determined at the signal at ~1653 cm⁻¹ related to the α-helix [28], as also demonstrated by the PC-loading maximum (Figure 5c). This result confirms the above-indicated findings obtained by analysing averaged SR FTIR spectra (Figure 4a). Additionally, changes in the intensity and the position of signals corresponding to parallel and intermolecular β-sheets (~1630 cm⁻¹ and ~1605 cm⁻¹, respectively) [25,28] are observed. Structural changes in the proteins’ secondary structure are confirmed by PCA, which also showed a maximum in the PC2 at about ~1545 cm⁻¹ (Figure 4c and Figure 5c). These changes also showed a Pd(II) concentration dependence.

Finally, the PC1 × PC2 score plot (Figure 4b and Figure 5b) showed significant differences between the PC1 and PC2 components, and the most expressed differences were observed between the control and Pd 3-treated HeLa cells. PC1 and PC2 components of Pd 1- and Pd 2-treated cells overlap and are distributed between the PC scores of the control and Pd 3-treated cells.

2.2.3. Changes in the Nucleic Acid and Carbohydrate Region

As Pd(II) complexes are known to interact with DNA and RNA through the electrostatic interactions with a phosphate backbone [10], we expected to find significant changes in the nucleic acid spectral region. Symmetric and asymmetric phosphate regions were the most affected. Still, changes can also be seen in the higher-wavenumber carbohydrate region (for better visual clarity, these two regions are separately presented in Figure 6).

After Pd(II) treatment, a slight increase in intensity was detected in the band at ~1396 cm⁻¹ corresponding to the COO¯ symmetric stretching vibration [17], as well as a wavenumber shift from ~1398 cm⁻¹, implying structural changes (Figure 6a). This carboxylic group band arises from polysaccharides [27] with a small contribution of vibrations of the terminal groups of protein, i.e., amino acids [29]. The band at ~1452 cm⁻¹ assigned to the CH₂ and CH₃ groups’ deformation vibration showed a concentration-dependent decrease in intensity. It is shifted toward lower wavenumbers, indicating structural changes in carbohydrates, lipids, and proteins [25]. The statistical analysis of the signals corresponding to carbohydrate vibrations was performed. A clear Pd(II)-concentration-dependent differentiation was found over the PC1 (Figure 7a). The maximum total variations in this region were identified at ~1458 cm⁻¹ (PC1 and PC2), which corresponds to the shifts in the CH₂ or CH₃ deformation vibrations, as discussed above.

The phosphate region (Figure 6b) and changes in peak intensity can be detected in both asymmetric (~1246 cm⁻¹) and symmetric (~1084 cm⁻¹) phosphate bond stretching vibrations, which mostly correspond to cellular nucleic acids [30]. The position of the asymmetric phosphate bond region changes towards lower wavenumbers (~1246 to 1236 cm⁻¹) after treating cells with Pd 2 and Pd 3 concentrations (Figure 6b). In contrast, the lowest concentration does not affect this band. HeLa treatment with Pd 2 concentration results in a decrease in the intensity of this band, whereas Pd 3 results in a significant intensity increase.

The band at ~1084 cm⁻¹ assigned to symmetric phosphate stretching increases in intensity after the treatment with Pd 1 and Pd 2 concentrations, but the treatment of cells with the highest Pd(II) concentration results in an increase in the signal intensity and the shift towards a lower wavenumber (~1059 cm⁻¹). This band also might correspond to C-O stretching as an indicator of cholesterol [31], implying a potential impact of the Pd(II) on cholesterol metabolism in treated cells.

The PCA of the phosphate region was performed for asymmetric and symmetric areas separately to emphasise the changes induced by various concentrations of the Pd(II) complex. The PC1 × PC2 plot (Figure 7c) demonstrated a differentiation of the Pd 2- and Pd 3-treated cells over PC1, whereas the control and Pd 1 treatment are scattered and cover almost the same area. The loading plot (Figure 7d) shows group variations in the signals, giving maxima at ~1175 cm⁻¹ (PC2) and at ~1160 cm⁻¹ (PC1 and PC2) and a minimum at ~1250 cm⁻¹ (PC2), which arise from the phosphate asymmetric vibrations. Symmetric phosphate bands showed the segregation of samples treated with Pd 2 and Pd 3 from the control and Pd 1-treated cells over PC2 (Figure 7e). The highest total variability in this region was detected at ~1000 cm⁻¹ (PC2, Figure 7f). Other changes are detectable, but without a clear statistical significance in the signal intensities.

3. Discussion

In this work, we have identified significant changes in all major classes of biomolecules induced by the treatment of HeLa cells with the Pd(II) complex. Although considered an analogue to Pt complexes in terms of their potential anti-cancer activity, the investigated Pd(II) complex demonstrated a mild cytotoxic effect, which is highly advantageous for studying its intracellular effects. Mild cytotoxicity expressed on the HeLa cells and many viable cells proves that this complex is suitable as a model system for studying biomolecular structural changes.

For SR FTIR spectroscopy, we used the formalin fixation/ethanol dehydration protocol. Although numerous fixation protocols were tested, none of them appeared to be ideal for all biomolecular vibrational regions, and each affects the signals to a certain extent, and the changes are visible either through the change in the signal intensity or the position [18]. However, since the same protocol was used to prepare the control and treated cells for SR FTIR spectroscopy, their spectra can be compared and the observed differences assigned to the Pd(II) treatment.

The literature data indicate that Pd interacts mainly with nucleic acids [32,33], but the precise mechanism of action is not fully elucidated. Furthermore, the binding kinetics and stability of the complexes formed between Pd ions and S-, O-, or N-donor molecules [34,35,36] most likely differ from their interaction with the same donors inside the cells because of the complexity of the cellular environment and the interaction among the individual cellular components.

According to the acquired SR FTIR spectral information, the investigated Pd(II) complex impacts all main spectral regions by inducing a change in the concentration of individual functional groups or alterations caused by the interaction with new species or a drug [25,37]. Changes in the protein region can be related to the Pd(II) concentration used to treat the cells, which confirms that observed changes are specific to the treatment of HeLa with this agent. The signal intensity of the left half of the Amide I band (1750–1650 cm⁻¹, antiparallel β-sheets) decreased with the treatment, whereas the signal increase is detected in the region of parallel β-sheets (Figure 4a and Figure 5a). These changes imply alterations in the organisation of the native beta sheets to hyper-arranged cross-β-sheets after Pd(II) treatment [25,28]. Structural changes result in a loss of protein function, which might accelerate the B-DNA structural changes (see below) and increase the number of the DNA-double-strand breaks. The exact mechanism of these changes remains to be further clarified.

Along with the changes in the proteins upon the interaction of cells with the Pd(II) complex, changes in the nucleic acid structure are also detected by SR FTIR spectroscopy. Significant effects were found in the phosphate region, characteristic of changes in the DNA structure, particularly in the thymine- and deoxyribose-associated bands. Changes in the position of the asymmetric stretching phosphate vibration (~1246 cm⁻¹) imply a local disorder of B-DNA structure [38]. In contrast, concentration-dependent changes in the signal at ~1083 cm⁻¹ might indicate chromatin fragmentation, i.e., a higher number of double-strand breaks as observed in an irradiated COLO 679 melanoma cell line [30].

Pd(II) complexes interact with DNA primarily through intercalation and interaction with the DNA groove [32,33]. At the same time, the metal core itself can form stable bonds with the phosphate backbone and nitrogen atoms in nuclear bases [39,40]. Despite this potential for direct interaction, Pd(II) complexes are generally less stable than Pt(II) complexes and are known to undergo hydrolysis in cells faster [41,42]. As a result, the changes seen in the nucleic acid region are likely not only a result of direct Pd(II)–DNA interactions and interactions mediated through the proteins.

An additional mechanism through which Pd(II) complexes might act is stimulating reactive oxygen species (ROS) production and boosting intracellular oxidative processes. An increase in the ROS and the products of oxidation might lead the cell into apoptosis [43,44,45,46]. Previous research shows that Pd(II) complexes exhibit this mode of action independently or in combination with other therapeutics or radiation [47,48,49]. One of the main signs of oxidative damage in cells is lipid peroxidation, and its indicator is the ν_as(CH₂)/ν_as(CH₃) ratio [21,50,51]. The ratio of ν_as(CH₂)/ν_as(CH₃) in HeLa cells significantly increased after Pd 3 treatment (Figure 3d), suggesting that lipid degradation occurred after Pd(II) treatment of the highest concentration, possibly through ROS-induced damage. In addition, the disappearance of specific lipid-related signals in the low-mass region and the appearance of the PIP signal at m/z 1049.3 in Pd(II)-treated cells (Figure S4) most likely arise from the increased lipid turnover and activation/deactivation of corresponding signalling pathways, which further confirm the observed changes by SR FTIR spectroscopy.

4. Materials and Methods

4.1. Reagents

Human cervical carcinoma cells (HeLa) were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). All chemicals used in this work were purchased from Sigma-Aldrich, GmbH (Taufkirchen, Germany).

4.2. Synthesis and Characterisation of [Pd(dach)Cl₂]

The complex, [Pd(dach)Cl₂] (Figure 1 and Figures S1–S3, Table S1), was a generous gift from Prof. Biljana Petrović (Faculty of Natural Sciences, University of Kragujevac, Kragujevac, Serbia). It was synthesised as described in the literature for Pt complexes [52] and characterised by UV-Vis, NMR spectroscopy, and MALDI TOF mass spectrometry (Axima Performance, Shimadzu, Shimadzu Europe, Duisburg, Germany).

4.3. Determination of Cell Viability

HeLa cells were cultured in a DMEM medium at 37 °C and 5% CO₂. Cytotoxicity of the Pd(II) complex was determined 48 h after treating the cells with increasing concentrations of the Pd(II) complex (in the range from 0.34 to 85.76 μmol/L) using a sulforhodamine B (SRB) assay according to a procedure described elsewhere [53]. The absorbance was measured at 550 nm with a reference wavelength of 690 nm in a microplate reader (Wallac VICTOR2 1420 Multilabel counter, PerkinElmer, Turku, Finland). The results were presented as a percentage of cell viability determined according to the following equation:

$$\mathrm{Cell}\mathrm{viability}\left(\%\right)={\displaystyle \frac{\mathrm{Absorbance}\mathrm{of}\mathrm{sample}}{\mathrm{Absorbance}\mathrm{of}\mathrm{control}}}\times 100$$

Two biological replicates were tested in quadruplicate, and a Student’s t-test was used to analyse the significance of the differences between samples. The differences were significant when a p-value was less than 0.05. The results were presented as the mean ± standard deviation.

4.4. Lipid Extraction and Analysis

Lipids were extracted from the cells prepared in the same way as described for cytotoxicity determination. After the cells were incubated with the Pd(II) complex for 48 h, total lipid extraction from HeLa cells was obtained according to a modified protocol of Bligh and Dyer [54].

The effect of the Pd(II) complex on cell lipids was analysed using matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Mass spectrometry measurements were performed using Axima Performance MALDI-TOF/TOF from Shimadzu (Shimadzu Corp., Duisburg, Gemany). The instrument had a variable repetition rate of N₂—a 50 Hz laser. The spectra were acquired in the reflectron mode in the m/z range from 300 to 1500. Each spectrum represented the average of a minimum of 100 individual laser shots.

4.5. Preparation of Cells for SR FTIR Spectroscopy

For the SR FTIR spectroscopy preparation, cells, at the density of 10,000 (control) or 20,000 (treated) cells per sample, were grown on a round CaF₂ carrier, 10 mm ø and 0.5 mm thick, polished window and treated with 34.3, 68.6, and 102.9 µmol/L of [Pd(dach)Cl₂] for 24 h. These concentrations proved to be optimal for structural change characterisation, as they were high enough to affect cell metabolism and low enough to keep most cells alive and available for a further analysis. The cell medium was removed, and cells were washed three times with a sterile physiological solution (0.9% NaCl). Cells were fixed in a 4% formaline solution in PBS for 15 min, after which cells were dehydrated by 75%, 95%, and absolute ethanol for 5 min.

4.6. SR FTIR Spectroscopic Measurements

After the treatment, changes in cell biomolecules were analysed by SR FTIR spectroscopy (Synchrotron ALBA, MIRAS beamline, Barcelona, Spain) [55]. Synchrotron light as an IR source was coupled to a Vertex 70v spectrometer, 3000 Hyperion microscope, and mercury cadmium telluride detector. The aperture of the FTIR microscope was set to a single-cell size (15 × 15 µm²), and 60 cells were analysed from each group in 3 replicates. Each single-cell spectrum was acquired by co-adding 256 spectra at a 4 cm⁻¹ resolution. Spectra for treated and control cells were collected in the 4000–900 cm⁻¹ mid-infrared range. The OPUS 8.2 (Bruker, Germany) software package was used for data acquisition.

The spectral analysis, including rubber band baseline correction and vector normalisation for every single cell, was implemented for three different areas: 3050–2800 cm⁻¹ lipid area, 1800–1480 cm⁻¹ proteins and esters, and 1480–900 cm⁻¹ nucleic acids and carbohydrate region. The second derivative (17 smoothing points, third polynomial order, and vector normalisation) was determined for the Amide I protein region (1700–1600 cm⁻¹). The principal component analysis (PCA) for each data set was performed. All spectral processing and statistical analyses were executed using Orange software (Bioinformatics Laboratory of the University of Ljubljana [56], Version 3.34.0), with the Quasar data analysis package, Version 1.7.0 [57].

5. Conclusions

Using a holistic approach, we have provided a glimpse into the complexity of the intracellular interaction of the transition metal Pd(II) complex and demonstrated that Pd(II) affects the structure of proteins by increasing the formation of intermolecular β-sheets, which might, in turn, affect the function of the most probable protein target for Pd(II). As additional support to this conclusion, the changes in the spectral region of NAs imply the fragmentation of chromosomes, i.e., a higher number of double-strand breaks. Altogether, we show that the interaction mechanism of Pd(II) with cells goes through the specific interaction with biomolecules and not by increasing the level of oxidative stress. Our findings contribute to further bioinorganic chemistry development and possibly enable the identification of the target molecules for future metallodrug therapy.