Influence of ozone therapy on sensitivity of cervical cancer cells to chemical and radiation therapy

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The article presents the results of oxygenation influence on sensitivity to chemical therapy of cervical cancer in patients with IIb stage. The criterion for the effectiveness of oxygenation were determined by immunohistological analysis of nitrotyrosine – a marker of oxidative stress. 

On the background of ozone therapy in a large number of patients was recorded the complete disappearance of the tumor, as well as a significant decrease in the number of nitrotyrosine-positive cells. 

Therefore , it is testified that ozone therapy improve s the efficiency of chemical therapy of cervical cancer by reducing thecellular hypoxia of tumor.

Cervical cancer (CC) today remains one of the most common malignant neoplasms (MNO). In the structure of the incidence of reproductive system organs in women, cervical cancer ranks second after endometrial cancer. 

Modern advances in molecular biology, genetics, biochemistry, immunology and virology make it possible to significantly expand the understanding of the molecular genetic nature of cancer, to better understand the pathogenetic mechanisms of tumor growth, and contribute to the improvement of traditional methods of fighting cancer (surgery, radiation therapy, and chemotherapy).

At all stages of the development of the neoplastic process, tumor cells are in conditions of hypoxia [1]. At the same time, due to the activation of intracellular mechanisms, they acquire resistance to the damaging effect of ionizing radiation and anticancer drugs [2]. 

Therefore, an active search is under way for drugs capable of increasing the sensitivity of tumor cells to therapeutic effects. 

In this regard, electron -withdrawing compounds (metronidazole) and inhibitors of post-radiation DNA repair ( 8-chloro-caffeine, 8-bromo-caffeine ), hyperbaric oxygenation, artificial hyperglycemia, local hyperthermia and chemotherapy were found to be effective [3].

The aim of the investigation was to study the effect of oxygenation on the sensitivity to chemoradiation therapy for cervical cancer and to assess changes in the expression of nitrotyrosine in cells in cervical cancer, as well as against the background of transrectal insufflation of the ozone-oxygen mixture.

This disease is one of the most common malignant neoplasms in women [4]. 

Transrectal insufflation of an ozone-oxygen mixture was used as a method of oxygenation. Earlier it was shown that with this method of administration ozone is low-toxic [5], disintegrates in the cells of the intestinal epithelium [6] and increases the antioxidant protection in them [7]. 

During hypoxia, free radicals are formed in cells. The final product of the interaction of radicals NO and О  – nitrotyrosine – is a marker indicating the level of oxidative stress [8]. 

In cancer patients, it has been shown that the level of nitrotyrosine correlates with the prognosis of survival in bladder cancer [9] and is not a prognostic marker for ovarian cancer [10].

Materials and methods

The study was conducted in 53 patients aged 24 to 63 years with stage IIb (T2bN0M0) disease, in which the tumor invaded the cervix with invasion of the parametrium, but without spreading to nearby lymph nodes or distant organs. All patients received chemoradiation therapy. 

The main group consisted of 31 patients who, before each irradiation session, underwent transrectal insufflation of an ozone-oxygen mixture using the Medozon-BM apparatus (Nizhny Novgorod) with an ozone supply at the output of 10 μ / ml per 1.5 L (total dose – 15 mg).

The control group consisted of 22 patients who underwent complex treatment according to the same plan, but did not undergo ozone therapy. 

Three weeks after chemoradiation therapy, all 53 patients underwent radical hysterectomy with bilateral iliac lymphadenectomy by the Wertheim-Meigs method. Isolated tumors were fixed in 10% formalin and embedded in paraffin according to the standard technique.

Morphological changes were assessed on paraffin sections 3-5 μm thick, stained with hematoxylin and eosin. LSAB kit (DAKO) was used for immunohistochemical analysis of nitrotyrosine localization (Chemicon). 

Immunohistochemical reactions were visualized with a solution containing aminoethylcorbazole. The sections were embedded in glycerol-gelatin.

Results and its discussion

In 44.8% of patients under the influence of ozone therapy, tumor structures were not determined; in the control group, such patients accounted for 27.2%. 

The differences are statistically significant (p <0.01). 

Changes in the stage of the tumor process were not recorded in 3% of patients after ozone therapy and 4.5% in the control group (p> 0.05). 

The remaining patients showed tumor regression, which was more pronounced against the background of ozone therapy.

The numerous effects of hypoxia on the tumor include: selection of genotypes that support survival in conditions of lack of oxygen (for example, with the p53 mutation [11], changes in the expression of genes that suppress apoptosis [2], autophagy [12], enhancement of anabolic processes [13], an increase in mediated the tyrosine kinase cascade of reactions [14], angiogenesis [15] and the formation of free radicals [16]

The data obtained indicate an increase in the sensitivity of tumor cells to chemoradiation therapy under conditions of oxygenation under the influence of ozone therapy, which probably occurred as a result of the suppression of all the above negative the consequences of hypoxia.

Immunohistochemical determination of the localization of nitrotyrosine in patients without ozone therapy showed that in tumors of different patients, the number of nitrotyrosine + -cells is very different and ranges from 30 to 80%. 

At the same time, the number of nitrotyrosine + -cells does not correlate with the severity of the tumor process, which is consistent with the literature data [9, 10]. In patients who underwent ozone therapy, the number of nitrotyrosine + cells in the collected material is significantly lower and ranged from 2 to 23%. 

It is important to note that in the control group, nitrotyrosine was predominantly determined in the nuclei of cells, and in the material obtained from patients after ozone therapy in a larger number of cells, it was localized in the cytoplasm. 

Reducing the amount of nitrotyrosine+ -cells in tumors in patients with oxygenation is probably associated with the predominant death of these cells during chemoradiation therapy. 

Thus, it was experimentally shown that when doxirubicin was administered against the background of thermo-radiotherapy, the peak in the number of nitrotyrosine + cells in the tumor was determined after 4 hours and their number significantly decreased by 8 hours with an increase in the number of cells dying by apoptosis [17].

Thus, it was  shown that ozone therapy increases the effectiveness of chemoradiation therapy for cervical cancer by reducing hypoxia of tumor cells.

LITERATURE

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3. Maksimov S.Ya., Guseinov LD, Baranov S.B. Chemoradiation therapy for cervical cancer and cancer of the uterine body // Practical Oncology. – 2008. – T. 9, No. 1. – S. 39-46.

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9. Soini Y., Haapasaari K., Vaarala M. et al. 8-hydroxydeguanosine and nitrotyrosine are prognostic factors in urinary bladder carcinoma // Int J Clin Exp Pathol. – 2011. – Vol. 4, No. 3. – P. 267-275.

10. Karihtala P., Soini Y., Vaskivuo L. et al. DNA adduct 8-hydroxydeoxyguanosine, a novel putative marker of prognostic significance in ovarian carcinoma – Int J Gynecol Cancer. – 2009. – Vol. 19, No. 6. – P. 1047-51.

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12. Rouschop KM et al. The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5 // J. Clin. Invest. – 2010. – Vol. 120. – P. 127-141.

13. Cairns RA, Harris IS, Mak TW Regulation of cancer cell metabolism // Nature Rev. Cancer. – 2011. – Vol. 11. – P. 85-95.

14. Wang Y. Ohh M. Oxygen-mediated endocytosis in cancer // J. Cell. Mol. Med. – 2010. – Vol. 14. – P. 496-503.

15. Semenza GL Hypoxia, clonal selection, and the role of HIF-1 in tumor progression // Crit. Rev. Biochem. Mol. Biol. – 2000. – Vol. 35. – P. 71-103.

16. Guzy RD et al. Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing // Cell. Metab. – 2005. – Vol. 1. – P. 401-408.

17. Solazzo S, Muneeb A, Schor-Bardach R et al. Liposomal Doxorubicin Increases Radiofrequency Ablation-induced Tumor Destruction by Increasing Cellular Oxidative and Nitrative Stress and Accelerating Apoptotic Pathways // Radiology. – 2010. – Vol. 255. – P. 62-74.

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