**2.3 Photodynamic therapy**

Cells were cultivated in 6-well plates at a density of 1 × 10<sup>6</sup> cells/well, at 37°C in a 5% CO2 atmosphere, and incubated for 18 hours for cell adhesion. After plating, cells were incubated with AlPcS4 for 1 hour at 37°C in an atmosphere containing 5% CO2. Then, they were washed with PBS to remove the photosensitizer not absorbed by the cells. Irradiation was performed by using a LED dispositive (Biopdi/IRRAD-LED) λ = 660 nm. Each well was exposed to 25 mW, an energy density of 5 J/cm2. Immediately after treatment, all groups were incubated with 25 μM Click-iT™ Metabolic Glycoprotein Labeling (according to the manufacturer's instructions, Thermo Fisher Scientific™- **Table 1**) for 24 and 48 hours to evaluate the changes in the protein glycosylation process by the Golgi complex (**Figure 1**). At the end of the incubation periods, the cells were scraped, added into 5 ml tubes, and centrifuged at 5259 g at 4°C for 5 minutes for cell sedimentation. After this, cells were resuspended and washed in PBS 2 times, fixed with 4% paraformaldehyde in PBS for 15 minutes, washed two more times with PBS, and permeabilized with 0.25% Triton x-100 in PBS for 15 minutes, washed with 3% BSA in PBS twice, and then incubated with an FTIC-conjugated antibody for one hour while diluted 1: 1000 in a Click-iT reaction buffer for one hour. At the end of the incubation period, it was washed


#### **Table 1.**

*The markers used to evaluate glycoproteins.*

#### **Figure 1.**

*Scheme of the experimental procedure. C – Control; PS – Photosensitizer; L – Led only; T – Treatment = PS + Led. Created with BioRender.com.*

with PBS, resuspended in the Click-iT reaction buffer, and read by BD AccuriC6 Plus flow cytometer. All experiments were performed in duplicate (n = 6).

### **2.4 Statistical analysis**

The data presented are in the form of mean and standard deviation, compared by the two-way ANOVA test and confirmed by the Tukey test. Statistical significance was admitted with P < 0.05 with \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001 being considered significant. Experiments were performed in three independent replications with n = 8. GraphPad Prism 6® software was used to per-form statistical and graphical analyses.

### **3. Results**

The evaluation of the glycoprotein synthesis by Flow Cytometry demonstrates that the modified sialic acid glycoproteins (Click-iT® ManNAz - **Figure 2**) in the treatment group present a higher fluorescence intensity in 24 hours, concerning the O-GLcNAz-modified glycoproteins and the O- glycans linked. The HEp-2 strain presents the synthesis of sialic acid-modified glycoproteins and O-GlcNAzmodified effectively in the first 24 hours; after 48 hours, a decrease in the synthesis of these glycoproteins is observed (**Figures 3** and **4**). They were probably modified due to the action of glycosidases and glycosyltransferases, changing their structures. O-linked glycans are less fluorescent in the first 24 hours; however, in the group treated within 48 hours, an increase in the synthesis of these glycoproteins is observed, which can be considered a possible target for photodynamic treatment.

#### **Figure 2.**

*Analysis of cells treated with Click-iT®-ManNAz, the graph shows the fluorescence intensity of the cells of the control, LED, and treatment groups in the periods of 24 and 48 hours. The treatment group showed a high fluorescence intensity within 24 hours, with a severe fluorescence reduction occurring in all groups within 48 hours.*

*Can PDT Alter the Glycosylation of the Tumor Cell Membrane? DOI: http://dx.doi.org/10.5772/intechopen.94172*

#### **Figure 3.**

*Analysis of cells treated with Click-iT®-GlcNAz, the graph shows the fluorescence intensity of the cells of the control, led, and treatment groups in the periods of 24 and 48 hours. The fluorescence intensity in all groups in the 24 hours is the same for all. Within 48 hours it is possible to observe an increase in fluorescence intensity in the treatment group compared the control and LED groups.*

#### **Figure 4.**

*Analysis of cells treated with Click-iT®-GalNAz, the graph shows the fluorescence intensity of the cells of the control, LED, and treatment groups in the periods of 24 and 48 hours. The treatment group, compared to the other groups, has a high fluorescence intensity.*

#### **4. Discussion**

The use of the technique called metabolic oligosaccharide engineering (metabolic oligosaccharide engineering) allows for the labeling of glycans with probes for visualization in cells by enriching specific types of glycoconjugates for proteomic analysis. This methodology promotes the metabolic labeling of glycans with a specific reactive functional group, the azide. Azide-labeled carbohydrates are endocytosed

by cells and integrated with glycan biosynthesis in various glycoconjugates. The cells are incubated for periods of 24 to 72 hours to allow the synthesis of surface glycoproteins to be monitored [11, 12].

According to prior research [13, 14], glycosylation markers can assist in cancer detection and monitoring since the malignant transformation of cancer cells associated with changes in cell glycosylation are associated with tumor progression and, finally, metastasis. The schemes shown above demonstrate the biosynthesis of glycoproteins in a normal cell (**Figure 5**). The results obtained demonstrate indirectly that the photodynamic treatment altered the glycosylation of proteins in the lattice, consequently compromising the glycosylation in the Golgi and the insertion of glycoproteins in the plasma membrane (**Figure 6**). The statement concerning the reticule is based on previous data obtained by our group, which demonstrated changes in the reticular tubular network and the presence of surface glycoproteins N-acetyl glucosamine terminals [15, 16].

The glycoproteins, when sent via vesicle traffic to the Golgi complex, change with the removal of mannose residues, the addition of N-acetyl glucosamine, galactose, and sialic acid. The addition of carbohydrates is associated with the function that the glycoprotein will play on the cell surface.

The glycosylation markers can be used for cancer detection and monitoring, since changes in cell glycosylation are associated with the transformation of cancer cells into glycosylation, tumor progression, and, finally, metastasis [13, 14]. The schemes shown above demonstrate the biosynthesis of glycoproteins in a normal cell (**Figures 7** and **8**). The results obtained indirectly demonstrate that the photodynamic treatment altered the glycosylation of proteins in the lattice, consequently compromising the glycosylation in the Golgi and the insertion of glycoproteins in the plasma membrane. The reticule statement is based on previous data obtained by our group [15], which demonstrated changes in the reticular tubular network.

The photodynamic treatment action on surface glycans has a significant impact on cell signaling and the regulation of cell-tumor cell adhesion and cell-matrix

#### **Figure 5.**

*Membrane glycoprotein biosynthesis scheme. The monosaccharide complexed with azide (Click-iT™ Metabolic Glycoprotein Labeling Reagent), crosses the plasma membrane, becoming available in the cytoplasm. Golgi, responsible for the glycosylation process, captures the monosaccharide, which will be used in the processing of membrane protein, coming from the rough endoplasmic reticulum. After the incorporation of the labeled monosaccharide into the protein, the vesicle is released and fused to the plasma membrane, exposing the glycoprotein, allowing its detection by microscopy or cytometry. Created with BioRender.com.*

*Can PDT Alter the Glycosylation of the Tumor Cell Membrane? DOI: http://dx.doi.org/10.5772/intechopen.94172*

#### **Figure 6.**

*Membrane glycoprotein biosynthesis scheme after PDT. The monosaccharide complexed with azide (Click-iT™ metabolic glycoprotein labeling reagent), crosses the plasma membrane, becoming available in the cytoplasm. Golgi, responsible for the glycosylation process, captures the monosaccharide, which will be used in the processing of membrane protein, coming from the rough endoplasmic reticulum, but after PDT changes the sequence of monosaccharides, modifies the glycan modifying the glycoprotein. After the incorporation of the labeled monosaccharide into the protein, the vesicle is released and fused to the plasma membrane, exposing the glycoprotein, allowing its detection by microscopy or cytometry. Created with BioRender.com.*

#### **Figure 7.**

*Glycosylation scheme in the rough endoplasmic reticulum. Created with BioRender.com.*

interaction, compromising the interaction between cancer cells and the tumor microenvironment.

An exciting result refers to the AlPcS4 group, with reduced glycosylation of proteins with mannose and galactose terminal monosaccharides, when compared to the control group. This data suggests that phthalocyanine endocytosed by the tumor cell requires more lysosomes to be degraded, a demand supplied by Golgi, which releases transport vesicles with acid hydrolases to the lysosomes via the mannose −6-phosphate receptor. For the substitution of mannose by galactose, mannosidase action must occur; however, as there is a need for more hydrolases and more transporters, the final carbohydrate does not change.

In the 48 hours, the AlPcS4 and LED groups show similar behavior to the control group, indicating that, after the period of interaction with the photosensitizer and

### *Photodynamic Therapy - From Basic Science to Clinical Research*

**Figure 8.** *Glycosylation scheme in the Golgi apparatus. Created with BioRender.com.*

the action of light, the cell restores its synthesis process. Reinforcing the information that light and separate photosensitizers cannot cause damage to cells.
