is why for estimation of the significance in differences with the data of the control group the methods of nonparametric statistics were used: Mann Whitney U-test and Kolmogorov-Smirnov test. To analyze the correlation of the data of two samples, the Spearman correlation coefficient was used. Differences were considered to be statistically significant at p < 0.05.

2.6. Ethics

Data from PS were aggregated and de-linked from personal information related to patients, medical institutions, and pharmacies: these are anonymous data. Therefore, no ethical issues are posed by the use of these data for this study.

3. Results

3.1. Free Radical Activity of Blood Plasma

Free radical activity of blood plasma increased at all stages of carcinogenesis as compared with healthy people (Figure 1).

At the IIIrd and IVth stages a higher statistically significant increase in free radical activity was observed which greatly exceeds that at the Ist, IInd stages. The high level of Imax is likely due to the presence of distant metastases and this fact is reflected in the availability of reliable interconnection between these two parameters (r = 0.456). Thus, an increase is shown in free radical activity of blood plasma from the first stage of malignancies of epithelial tissues with further increase in subsequent stages which can be due to the detected activation of free radical oxidation at metastasis.

3.2. Oxidative Protein Modification

A significant activation of the total OMP was observed in malignant tumors of epithelial tissues, as evidenced by an increase in the content of carbonyl derivatives of proteins in the plasma, starting from the Ist stage of disease (by 71%, 146%, 259% and 82%, respectively) (Figure 2).

Special attention was paid to the activity of OMP at the initial and terminal stages of the disease, which demonstrate no great differences. But the analysis of the distribution of carbonyl derivatives of different wavelength showed a significant decrease of the OMP activity as compared to actually healthy people for the Ist stage of cancer (Figure 3).

A statistically significant reduction in absorption level is observed registered at 230 nm wavelength where aliphatic ADNPH of neutral nature are found. These compounds are the markers of protein fragmentation. On the contrary, the content of carbonyl derivatives at wavelengths of 270, 370 and 430 statistically significantly increases (Figure 3).

In the terminal (IV) stage of malignant neoplasms the level of phenylhydrazones, determined at 230, 370 and 530 nm, is significantly increased, that is, aliphatic ketone dinitrophenyl hydrazones are predominant which are the markers of protein aggregation characteristic of the later stages of oxidative stress (Figure 4).

With the development of the objective effect in patients after the 1st course of treatment a statistically significant reduction of free radical activity of blood plasma is observed, in average by 20% (Figure 5). In the subsequent progression of tumor a tendency is shown to increase in free radical activity of blood plasma after the 1st course of treatment.

With objective effect of polychemotherapy, the significant differences in the level of blood plasma OMP proteins with that before treatment were not found (Figure 6).

An increase in free radical activity of blood plasma in the absence of objective effect after polychemotherapy was accompanied by the activation of the total OMP―by 2.83 times in patients with recurrent colon cancer and by 2 times in patients with recurrent bladder cancer (Figure 6).

Analysis of phenylhydrazones distribution at different wavelengths, depending on the efficiency of cytostatic therapy, did not show significant changes in the case of disease progression (Figure 7, Figure 8).

Common to the cases of the objective effects of polychemotherapy has been a dramatic increase in the carbonyl derivatives recorded at 230 nm (Figure 9, Figure 10).

Figure 1. Free radical activity of the blood plasma of patients with malignant tumors of epithelial tissues. ―the group of actually healthy people the patients with different stages of carcinogenesis not previously subjected to anti-tumor treatment: ― Ist stage, ---―IInd stage, ―IIIrd stage, ―IVth stage. Note: *―the differences with indices of the control group are significant (р < 0.05).

Figure 2. Oxidative modification of blood plasma proteins of patients with malignant tumors of epithelial tissues. ―the group of actually healthy people, the patients with different stages of carcinogenesis not previously subjected to anti-tumor treatment: ---―Ist stage, ―IInd stage, ―IIIrd stage, ―IVth stage. Note: *―the differences with indicators of actually healthy people are significant (р < 0.05).

Figure 3. Spontaneous OMP blood plasma in patients with malignant tumors of epithelial tissues (Ist stage) as compared to actually healthy people. Note: *―the differences with indicators of actually healthy people are significant (р < 0.05).

Figure 4. Spontaneous OMP blood plasma in patients with malignant tumors of epithelial tissues (IVth stage) compared with actually healthy people. Note: *―the difference with indicators of actually healthy people is significant (р < 0.05).

Figure 5. Free radical activity of blood plasma in patients before treatment and after the 1st course of chemotherapy. ―patients with colon cancer before anti-tumor treatment and after the 1st course of polychemotherapy; ―patients with bladder cancer before anti-tumor treatment and after the 1st course of polychemotherapy; ―patients with colon cancer before anti-tumor treatment and with progression of the disease after the 1st course of polychemotherapy; ―patients with bladder cancer before treatment and with progression of the disease after the 1st course of polychemotherapy. Note: *―the differences with the values before chemotherapy are statistically significant (р < 0.05).

Figure 6. Oxidative modification of plasma proteins in patients before treatment and after the 1st course of polychemotherapy. ―patients with colon cancer before anti-tumor treatment and with objective effect after the 1st course of polychemotherapy; - --- -―patients with bladder cancer before anti-tumor treatment and with objective effect after the 1st course of polychemotherapy; ―patients with colon cancer before anti-tumor treatment and with progression of the disease after the 1st course of polychemotherapy; - ―patients with bladder cancer before anti-tumor treatment and with progression of the disease after the 1st course of polychemotherapy. Note: *―the differences with values before chemotherapy are statistically significant (р < 0.05).

Figure 7. Spontaneous OMP of blood plasma in patients with bowel cancer before treatment and after the 1st course of polychemotherapy in the absence of an objective treatment effect.

Figure 8. Spontaneous OMP of blood plasma in patients with bladder cancer before treatment and after the 1st course of polychemotherapy in the absence of an objective treatment effect.

Figure 9. Spontaneous OMP of blood plasma in patients with bowel cancer before treatment and after the 1st course of polychemotherapy in presence of the objective effect of treatment. Note: *―the differences with values before chemotherapy are statistically significant (р < 0.05).

Figure 10. Spontaneous OMP of blood plasma in patients with bladder cancer before treatment and after the 1st course of polychemotherapy in the presence of the objective effect of treatment. Note: *―the differences with values before chemotherapy are statistically significant (р < 0.05).

4. Discussion

Cancer is the fatal disease that is mostly determined at the later stages and accompanied with the appearance of metastasis. Moreover there are no objective markers to determine the efficacy of chemotherapy treatment. That is why the aim of investigation was to establish relationship between free radical activity in blood and tumor progression, from one side, and the correlation between investigating parameters and efficiency of chemotherapy, from another side. It is well known that the participation of ROS, in particular, of superoxide radical and hydrogen peroxide, involves in the regulation of cell proliferation [12] . Besides, in the processes of metastasis a significant role belongs to matrix metalloproteinases―the extra-cellular family of zinc-dependent endopeptidases capable of destroying all kinds of extra-cellular matrix proteins [13] . Regulation of the activity of these enzymes is also carried out by ROS [14] .

The proteins are one of the main substrates, affected by ROS. Carbonyl fragments of polypeptide chain, defined as 2,4-dinitrophenylhydrazones [15] , serve as the final oxidation products. The modified protein molecules are easier subjected to proteolysis with formation of peptides of average molecular weight which are one of the components of endogenous intoxication [16] . OMP generates new antigens and provokes an immune response [17] [18] . Products of such modification may cause secondary damage to other biomolecules [19] . The appearance of substantial amounts of modified proteins provokes the development of immune responses (macrophage activation and oxygen blast) not only to modified proteins but also to normal molecules. Inactivation of proteins occurs simultaneously with their modification [20] [21] .

In the early stages of oxidative stress the aldehyde phenylhydrazones are dominated (230 nm―aldehydes of neutral nature, 270 nm―aldehydes of the basic nature), and in the later stages―the ketone dinitrophenyl hydrazones of basic nature (370 nm) [22] . Under the effect of ROS the fragmentation of proteins can occur [23] .

The increase in the total level of OMP, accompanying the previously shown increase of free radical activity of blood plasma, at various stages of cancer is due to different fractions of carbonyl derivatives. In the initial stages of the disease this increase is due to aldehyde and ketone DNPH, indicating the realization of both the processes of fragmentation of proteins and their aggregation. In the terminal stage the increase in OMP is due to aliphatic ketone dinitrophenyl hydrazones which are the markers of protein aggregation characteristic of the later stages of oxidative stress.

With the development of the objective effect in patients after the 1st course of treatment a statistically significant reduction of free radical activity of blood plasma is observed. These results might be due to high level of reduced glutathione, low-molecular compound of nonprotein nature which has antiradical activity and to catalase activity in tumor cells, which is probably a factor of resistance of tumors to free radical stress and to entrance of these compounds into systemic circulation [24] . In the subsequent progression of tumor a tendency is shown to increase in free radical activity of blood plasma after the 1st course of chemotherapy treatment.

At 230 nm wavelength ADPH of neutral nature are determined characterizing the appearance of oxidized modified oligopeptides resulting from the destruction of cellular proteins. It can be assumed that these substances are formed as a result of destruction of malignant tumor cells in the presence of an objective effect of treatment.

5. Conclusion

A statistically significant increase in free radical activity and in degree of oxidative modification of blood plasma proteins is shown starting from the first stage of malignancy of epithelial tissues. In the initial stages of carcinogenesis the level of oxidative modification of blood plasma proteins increases both due to the aldehyde and ketone carbonyl derivatives which indicates the realization of both processes of protein fragmentation and of their aggregation. At the terminal stage of malignancies the increase in oxidative modification of proteins is due to aliphatic ketone dinitrophenyl hydrazones being the markers of protein aggregation characteristic of the late stages of oxidative stress. In the case of objective response to polychemotherapy, a statistically significant reduction of free radical activity of blood plasma was found after the first course of chemotherapy by cytostatics as well as a statistically significant increase in carbonyl derivatives registered at 230 nm which can serve as a marker of the efficiency of therapy.

Author Contributions

In the manuscript preparation, all authors contributed equally until submission.

Cite this paper
Erlykina, E. , Obukhova, L. and Kopytova, T. (2018) Free Radical Activity as Diagnostic and Prognostic Criteria in Solid Tumors and Their Therapy. Journal of Biosciences and Medicines, 6, 1-12. doi: 10.4236/jbm.2018.612001.
References

[1]   Semiglazov, V.V. and Topuzov, E.E. (2009) Breast Cancer. MEDpress-Inform, Moscow, 176. [In Russian]

[2]   Aschele, C., Lonardi, S. and Monfardini, S. (2002) Thymidylate Synthase Expression as a Predictor of Clinical Response to Fluoropyrimidine-Based Chemotherapy in Advanced Colorectal Cancer. Cancer Treatment Reviews, 28, 27-47.
https://doi.org/10.1053/ctrv.2002.0253

[3]   Libra, M., Navolanic, P.M., Talamini, R. and Toffoli, G. (2004) Thymidylate Synthetase mRNA Levels Are Increased in Liver Metastases of Colorectal Cancer Patients Resistant to Fluoropyrimidine-Based Chemotherapy. BMC Cancer, 1, 11-17.
https://doi.org/10.1186/1471-2407-4-11

[4]   Shain, А.А. (2004) Oncology. Iz-datel’skii centr Akademiya, Tyumen. [In Russian]

[5]   Miki, J., Furusato, B., Li, H., Gu, Y., Takahashi, H., Egawa, S., Sesterhenn, I.A., McLeod, D.G., Srivastava, S. and Rhim, J.S. (2007) Identification of Putative Stem Cell Markers, CD 133, and CXCR_4, in hTERT-Immortalised Primary Nonmalignant and Malignant Tumor_Derived Human Prostate Epithelial Cell Lines and in Prostate Cancer Specimens. Cancer Research, 67, 3153-3161.
https://doi.org/10.1158/0008-5472.CAN-06-4429

[6]   Naito, Y., Lee, M.-C., Kato, Y., Nagai, R. and Yoshikazu, Y. (2010) Oxidative Stress Markers. Anti-Aging Medicine, 7, 36-44.
https://doi.org/10.3793/jaam.7.36

[7]   Jones, L.A., Holmes, J.C. and Seligman, R.B. (1956) Spectrophotometric Studies of Some 2,4-Dinitrophenylhydrazones. Analytical Chemistry, 2, 191-198.
https://doi.org/10.1021/ac60110a013

[8]   Ho, E., Galougahi, K.K., Liu, C.-C., Bhindi, R. and Figtree, G.A. (2013) Biological Markers of Oxidative Stress: Applications to Cardiovascular Research and Practice. Redox Biology, 1, 483-491.
https://doi.org/10.1016/j.redox.2013.07.006

[9]   Kuz'mina, Е.I., Nelyubin, А.S. and Shchennikova, М.К. (1983) Application of Induced Chemoluminescence for Assessment of Free Radical Reactions in Biological Substrates. Mezhvuzovskii sbornik biokhimii i biofiziki mikroorganizmov. Gorky, 179-183. (In Russian)

[10]   Dubinina, Е.Е., Burmistrov, S.О., Khodov, D.А. and Porotov, I.G. (1995) Oxidative Modification of Human Blood Plasma Proteins, the Method of Its Determination. Voprosy meditsinskoi khimii, 1, 24-26. (In Russian)

[11]   Dubinina, Е.Е. (2006) Products of Oxygen Metabolism in Functional Activity of Cells (Life and Death, Creation and Destruction). Fiziologicheskie i kliniko-boikhimicheskie aspekty. SPb.: Meditsinskaya pressa, 440. (In Russian)

[12]   Sarsour, E.H., Kumar, M.G., Chaudhuri, L. and Goswami, P.C. (2009) Redox Control of the Cell Cycle in Health and Disease. Antioxidants & Redox Signaling, 12, 2985-3011.
https://doi.org/10.1089/ars.2009.2513

[13]   Chakraborti, S., Mandal, M., Das, S., Mandal, A. and Chakraborti, T. (2003) Regulation of Matrix Metalloproteinases: An Overview. Molecular and Cellular Biochemistry, 1-2, 269-285.
https://doi.org/10.1023/A:1026028303196

[14]   Browatzki, M., Larsen, D., Pfeiffer, C.A., Gehrke, S.G., Schmidt, J., Kranzhofer, A., Katus, H.A. and Kranzhofer, R. (2005) Angiotensin II Stimulates Matrix Metalloproteinase Secretion in Human Vascular Smooth Muscle Cells via Nuclear Factor-KappaB and Activator Protein 1 in a Redox-Sensitive Manner. R.J. Vasc. Res, 5, 415-423.

[15]   Shugalei, I.V. (2000) Chain Process of Peroxidation of Proteins—A Suitable Model for Investigation of Destructive Ability of Active Forms of Oxygen. Russkii zhurnal VICh/SPID i rodstvennye problem, 1, 77-78. (In Russian)

[16]   Pasechnik, I.N. (2004) Oxidative Stress as a Component of Formation of Critical States in Surgical Patients. (In Russian)

[17]   Squier, T.C. (2001) Oxidative Stress and Protein Aggregation during Biological Aging. Experimental Gerontology, 9, 1539-1550.
https://doi.org/10.1016/S0531-5565(01)00139-5

[18]   Lambeth, J.D. (2007) Nox Enzymes, ROS, and Chronic Disease: An Example of Antagonistic Pleiotropy. Free Radical Biology & Medicine, 3, 332-347.

[19]   Piroddi, M., Depunzio, I., Calabrese, V., Mancuso, C., Aisa, C.M., Binaglia, L., Minelli, A., Butterfield, A.D. and Galli, F. (2007) Oxidatively-Modified and Glycated Proteins as Candidate Pro-Inflammatory Toxins in Uremia and Dialysis Patients. Amino Acids, 4, 573-592.
https://doi.org/10.1007/s00726-006-0433-8

[20]   Winterbourn, C.C. (2000) Biomarkers of Myeloperoxidase-Derived Hypochlorous Acid Free Radic. Biology and Medicine, 5, 403-409.

[21]   Ryabov, G.А., Aziziv, G.А. and Pasechnik, I.N. (2002) Oxidative Stress and Endogenous Intoxication of Patients in Critical States. Vestnik Intensivnoi Terapii, 4, 4-7. (In Russian)

[22]   Tolochko, Z.S. and Spiridonov, V.К. (2010) Oxidative Modification of Proteins in Blood of Rats at Destruction of Capsaicin-Sensitive Nerves and Change in the Level of Nitrogen Oxide. Rossiskii fiziologicheskii zhurnal im. I.М. Sechenova, 1, 77-84. (In Russian)

[23]   Muravleva, L.Е., Molotov-Luchanskii, V.B. and Klyuev, D.А. (2010) Oxidative Modification of Proteins: Problems and Prospects of Investigation. Fundament. Issled, 1, 74-78. (In Russian)

[24]   Blokhin, Yu.D. (2004) Phenotype of Multiple Drug Stability of Tumor Cells Due to Destruction of the Program of Cell Death. Vestn. Ramn., 12, 16-20. (In Russian)

 
 
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