FT-IR Spectroscopy Study in Early Diagnosis of Skin Cancer

Results and Discussion

FT-IR spectra (4000-400 cm⁻¹) of normal healthy tissue (N), BCC, MM and NEV biopsies are shown in Figure 1. The spectra of BCC, MM, and NEV show variations in intensities and frequency shifts in wavenumbers of the absorption bands, in comparison to the healthy tissues.

The absorption bands of the stretching vibrations of vOH and vNH groups of glycosaminoglycans, proteins, and collagen of the skin, as well as the stretching vibration bands of the water molecules in the cells appear in the spectral region between 4000 and 3000 cm⁻¹ as previously reported (12-18). In Figure 1, the broad band at approximately 3484 cm⁻¹ in the spectra of N and NEV is assigned to the stretching vibration of vOH groups of water molecules, and to the vOH of polysaccharides of the hyaluronic acid (HA). This band is decreased in the cases of BCC and MM due to the skin dehydration and to the HA chain degradation. The intense band at 3290 cm⁻¹ is assigned to the stretching vibration of vNH groups of proteins and it shows that the protein formulation is in the form of Amide A, as previously shown (18, 19). Deconvolution analysis in this region showed that this vNH band consisted of 5 absorption bands (spectra not included here), which correspond to stretching vibrations of vNH groups of proteins and vOH of glycosaminoglycans, which interact with each other and form various strengths of hydrogen bonds.

The band at approximately 3062 cm⁻¹ indicates that most of the proteins have the configuration of amide B. In the case of amide B, the β-sheet protein structure predominates (19). This means that the effect of the NH group of the peptide bond –NHCO- is stronger than C=O, unlike in amide A, where the effect of C=O in the peptide bond is stronger. The coexistence of both A and B protein conformations illustrates the prevalence of different hydrogen bonds that hold the protein strands together (14, 19). As it is known, the hydrogen bond is important in stabilizing the protein helix and any change implies that the physiological environment has changed. We have found that these changes are very important and constitute a basic criterion in order to characterize the disease and its progression (10-18).

The bands in the spectral region between 3000 cm⁻¹ to 2870 cm⁻¹ (Figure 1) are assigned to the symmetric and antisymmetric stretching vibrations of methyl (v_asCH₃, v_sCH₃) and to methylene (v_asCH₂, v_sCH₂) groups of lipids, proteins, and glycosides (9-12, 15). A considerable increase of the intensity of these bands is observed in the spectra of MM and NEV, and less in BCC spectra, indicating that the environment of the membrane has changed and the surrounding medium has become more lipophilic (15). Deconvolution of BCC, MM, and NEV spectra showed the production of a new band at 2892 cm⁻¹ in this region, which is assigned to the presence of a branched alkyl chain. The formation of branched alkyl chain indicated that, through the metabolic pathways of the disease, free alkyl radicals were produced, which interacted with each other to give final stable dimer products. Moreover, increase of the intensities of the symmetric absorptions of vsCH₂ groups confirmed the higher order configuration of the lipid membranes, indicating the production of amyloids, due to cancer formation (11, 12, 16).

The spectral region 1800-700 cm⁻¹ contains information about the secondary structure of proteins (Figure 1). The new band at 1744 cm⁻¹ in N and BCC spectra appears as a shoulder, but in MM and NEV has much higher intensity and is assigned to aldehyde group (-CHO), which is formed as a result of lipid peroxidation (16, 17). The high intensity band near 1650 cm⁻¹ is assigned to vC=O vibrations of the amide I of the peptide bond (-NHCO-) of proteins (16-19). For MM and NEV samples, this band, upon deconvolution, was split into two bands at 1650 cm⁻¹ and 1633 cm⁻¹, due to the presence of both α-helix and random coil protein conformations. The next intense band at about 1550 cm⁻¹ is assigned to amide II of the proteins β-turns and it suggests that the collagen has α-helix configuration. This band also was shifted to lower frequencies, from 1544 cm⁻¹ for healthy cells to 1536 cm-1 for BCC, 1540 cm⁻¹ for MM, and 1549 cm⁻¹ for NEV, indicating a damage of the native structure of proteins. Noticeable changes were also observed in the spectral region of 1250-1000 cm⁻¹, including the absorptions of the -PO₂- and -C-O-C- group vibrations, where an oxygen atom is linked to two carbon atoms of the sugar moiety of glycosaminoglycans together with the exocyclic C-O-C inter-molecule groups (19). The bands near 1150-1165 cm⁻¹ are attributed to -C-O-C bonds of the sugar ring of HA and to sugar ring of DNA (20, 21). The band at 1056 cm⁻¹ is assigned to exocyclic -C-O-C- (oxygen bridge). The absorption bands of the C-C and C-OH appear at 1160 cm⁻¹. As it is shown in Figure 1, this band increased in intensity in the spectra of BCC, MM, and NEV, indicating that there was substantial reduction in the amount of hydrogen-bonded C-OH groups of cellular proteins, as well as an accumulation of carbohydrates as a result of glycosylation during the cancer progression. This band was also observed in the cancer regions of breast and colon, and metastatic bone cancers (10, 12, 22).

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Figure 1.

FT-IR spectra of skin tissues: normal healthy tissue (N), basal cell carcinoma (BCC), melanoma (MM) and nevus (NEV), in the region 4000-400 cm⁻¹.

Figure 2 shows the FT-IR spectra of normal healthy tissue (N), basal call carcinoma (BCC), melanoma (MM) and nevus (NEV) specimens. Region 900-800 cm⁻¹ reveals information on the configuration of the sugar-phospahte groups of DNA backbone. The absorption bands at 841 cm⁻¹ and 815 cm⁻¹ are assigned to sugar-phosphate vibrations of backbone strands of DNA and correspond to B-DNA and Z-DNA, respectively (23, 24). In the case of MM, only the cancerous Z-DNA conformation was observed, while in BCC both B-DNA and Z-DNA forms were found. In the normal tissue the band of B-DNA was masked from other absorptions. The high intensity band at 871 cm⁻¹ is attributed to tyrosine amino acid (25, 26).

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Figure 2.

FT-IR spectra of skin tissues: normal healthy tissue (N), basal cell carcinoma (BCC), melanoma (MM) and nevus (NEV), in the region 900-800 cm⁻¹.

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Figure 3.

FT-IR spectra of normal skin tissue (N) subtracted from the spectra of melanoma (MM) tissue (MM-N).

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Figure 4.

Reactions of hyaluronic acid (HA) with hydroxyl free radicals (HO*). The site of HA* radical formation is random and one monomer is shown here, which is denoted by the dashed lines. The final byproducts 5 and 6 are stable and are produced from the free radical 2.

From the above spectroscopic results it is shown that the disease affects the structure of the skin at a molecular level. In order to understand the behavior of the disease, we subtracted the spectra of the normal tissue (N) from the spectra of melanoma (MM), as it is shown in Figure 3. In the subtracted spectra of MM-N, the vOH and vNH bands are negative, suggesting the dehydration and collagen degradation of MM tissue, respectively. On the contrary, the absorption bands in the region of 1200-900 cm⁻¹, which correspond to the -C-O-C, C-OH groups, showed increased intensity. These findings lead to the suggestion that during melanoma progression the glycosylation of the tissue is increased. In order to explain the above spectral changes, we have to take in consideration that during melanoma development free hydroxyl radicals are produced, which play a crucial role in glycosaminoglycans fragmentation. The hydroxyl free radicals react with HA by subtracting hydrogen atoms and lead towards the formation of hyaluronic acid free radical (HA*) (Figure 4).

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Figure 5.

FT-IR spectra of basal cell carcinoma (BCC) subtracted from the spectra of melanoma (MM) tissue (MM-BCC).

In Figure 4, the formed HA* free radicals (Figure 4, product 1) are not stable and are decomposed to give a series of more stable products (Figure 4, products 5 and 6) or interact with molecular oxygen to give peroxides (Figure 4, product 4). These reactions could explain the observed reduction in the intensity of the band at 1054 cm⁻¹ (Figure 5, negative absorption at MM-N spectra). The produced HA* can also interact with other radicals of the cell macromolecules to produce branched or cross-linked polymers as it was detected by the band at 2892 cm⁻¹, assigned to vCH₂. The formed products are similar to those that were also produced upon irradiation of polysaccharides or by using cupric ions (Cu²⁺) and ascorbic acid (27-29). The glycosylation has also been shown in other cancers, such as breast and metastatic bone cancers (12, 22, 30). Furthermore, HA and its CD44 cellular receptors, and the receptor for HA-mediated motility/intracellular HA-binding protein have also been linked to cancer progression and metastasis (31-34). The intensity of the bands of amide I at 1634 cm⁻¹ and amide II at 1542 cm⁻¹ was reduced in MM, suggesting that the proteins had a radon coil configuration and increased active sites. These sites may react with free radicals and finally lead to fragmentation of collagen chain.

The spectrum of BCC and the spectra of subtraction of BCC from MM spectrum (MM-BCC) are shown in Figure 5, in the region of 4000-2820 cm⁻¹. The 3293 cm⁻¹ band of BCC assigned to vNH₂ became negative in MM-BCC, whereas the high-intensity band at 1744 cm⁻¹, which is not shown in BCC spectra or is shown as shoulder, indicated that a higher amount of aldehydes is produced in MM, due to peroxidation of lipids. This aldehydic band at 1744 cm⁻¹ is observed in all cancer samples and is formed by lipid-hydroxyl free radical initiation reactions following the peroxidation of lipid radicals (18, 35). The bands of amide I at 1634 cm⁻¹ and amide II at 1542 cm⁻¹, after subtraction, showed negative absorptions for MM, suggesting that in BCC more collagen is produced, in agreement with literature (36, 37). Another important difference was observed in the region 1200-900 cm⁻¹, where the bands that characterize the presence of glycosaminoglycans are found. Especially, the band at approximately 1048 cm⁻¹ in MM was reduced, while in BCC was strong. This band is due to carbon-oxygen-carbon bridges (C-O-C) and is assigned to C-O vibrations of HA. The absorption at 1048 cm⁻¹ could be used as a “marker band” of MM, and due to the presence of many HA-chain breaks it might discriminate melanoma from non-melanoma cancers. In the DNA region (Figure 2), only the Z-DNA form is detected in MM, while in BCC both B-DNA and Z-DNA are present in the spectra of the skin.

The increase of absorption band intensities of methyl and methylene groups of alkyl chains might be attributed to the fact that the environment becomes more lipophilic, as a result of lipid peroxidation, aldehyde and amyloid protein formation (10-12, 19). This lipophilic environment changes the permeability of cell membranes, leading to inactivation of the skin's defense systems. It has been previously shown (2) that, in skin cancers, the concentrations of the lipophilic antioxidants a-tocopherol, β-carotene, and ubiquinol-10, is much higher than in the normal skin. This fact may suggest that the lipophilic environment dissolves these antioxidants and thus, the skin is not any more protected.