Applicability of mitochondrial energy factors in accompanying therapy of lymphoproliferative diseases (experimental study)

Elena M. Frantsiyants, Irina V. Kaplieva, Valerija A. Bandovkina*, Lidia K. Trepitaki, Ekaterina I. Surikova, Irina V. Neskubina, Ju-lija A. Pogorelova, Natalia D. Cheryarina, Alla I. Shikhlyarova, Tat’jana I. Moiseenko, Maxim N. Duritskii, Sergey V. Tumanian, Yuriy V. Przhedetskiy, Viktoria V. Pozdnyakova. Applicability of mitochondrial energy factors in accompanying therapy of lymphoproliferative diseases (experimental study). Cardiome-try; Issue 20; November 2021; p. 29-33; DOI: 10.18137/cardiom-etry.2021.20.2933; Available from: issues/no20-november-2021/applicability-of-mitochondrial

Cancer is a process of “microevolution”, when the most adapted cell gains an advantage in survival among a heterogeneous cell population. Therefore, it is logical to assume that carcinomatous cells, which modulate their repertoire of defense mechanisms, are endowed with an advantage of constant proliferation and survival. Two features thereof imply the altered cell respiration and avoidance by the mechanisms of cell death to escape apoptosis, as a result of which cancer cells increase the mass of tissue and become resistant to clinical treatment regimens [1]. Mitochondria, which are the center of energy functions, are the key to the life and death of a cell [2]. Cell survival depends on various critical functions of the mitochondrial membrane, which shows significant morphological changes at the initial stages of apoptosis [3]. Cytochrome C is an evolutionarily highly conserved protein localized in the mitochondrial intermembrane space, and it is the last oxygen-receiving enzyme in the respiratory chains, and it is considered to be the final stage of the mitochondrial respiration. Internal apoptosis is closely linked with the permeability of the external mitochondrial membrane and the subsequent release of the cytochrome C protein into the cytosol, where it can participate in the activation of caspases through the formation of apoptosomes [4]. It has been revealed that there is a decrease in the level of cytochrome C in mitochondria under the influence of malignant growth against the background of comorbid pathologies [5]. Metabolic changes remain the key stage that assists in the transformation of a normal cell into a tumor phenotype. The supply of energy in cancer cells mainly occurs by anaerobic glycolysis. To limit the flow of excess energy, the transition of cell respiration from glycolysis to oxidative phosphorylation is a necessary step.

Therapeutic agents demonstrating anti-tumor specificity cause serious damage to normal cells, limiting in such a way their clinical effectiveness. The use of agents, which suppress or disrupt the bioenergetic profile of cancer cells, may be useful in the treatment of cancer [6]. Such methods include activation therapy [7], the use of xenon [8], and the accompanying cytochrome C therapy in the treatment of lung cancer in combination with chemotherapy drugs [9].

Despite the fact that there are a lot of studies devoted to the protective properties of antioxidants in chemotherapy, many unresolved issues remain high on the agenda. In this connection it should be mentioned that it is also important to identify their anti-carcinogenic activity and specify the optimal mode of their preventive use.

Recently medical drug Cytochrome C has been developed, which represents a high-molecular-weight iron-porphyrin compound, a conjugated protein, similar in its structure to hemoglobin (it consists of heme and a single peptide chain). Cytochrome C plays an important role in the biochemical redox processes in almost all aerobic organisms. These reactions occur with the participation of two mitochondrial enzymes: cytochrome oxidase and cytochrome reductase. Heme exhibits the properties either of an electron donor or its acceptor. It is highly reactive with oxygen radicals, such as peroxide or hydrogen peroxide, which is a strong oxidizer. Heme metabolites act as traps for the peroxide radical. Heme-containing cytochrome C is capable to neutralize free oxygen radicals in certain redox processes.

The aim herein is to study the effectiveness of the use of Cytochrome C at the early stages of the growth of Pliss lymphosarcoma in outbred albino rats.

Materials and methods

Our experimental studies were carried out in outbred albino male rats with an initial weight of 180-220 g delivered by the National Medical Research Centre of Oncology, Ministry of Health of the Russian Federation. The research in animals was conducted in accordance with the Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes and Order No. 267 “Approval of the Rules of Laboratory Practice” dated June, 19, 2003 issued by the Ministry of Health of the Russian Fed- 30 | Cardiometry | Issue 20. November 2021

eration. All rats were kept under the same conditions, 5 individuals in each standard plastic box, under the natural light conditions, at an ambient temperature of 22-26°C and free access to water and food. The observation of the animals was performed every day, including twice a week measurements of weight and volumes of subcutaneous tumor nodes.

Statistical processing of the obtained results was carried out using the Student’s parametric criterion with a PC with the STATISTICA 10.0 software and applying the nonparametric Wilcoxon-Mann-Whitney criterion test. All the results obtained were checked for their compliance with the law of normal distribution (the Shapiro-Wilk criterion). Some of the indicators were found to be in correspondence with the law, while the others were evaluated as inconsistent therewith. For the indicators with the normal distribution, the Student’s criterion was employed, and for those in disagreement with the normal distribution law, the Mann – Whitney criterion was applied. The data were presented as an arithmetic mean value ± standard error of the mean (M±m). The differences between the two samples were considered statistically significant at p<0.05.

Results Subcutaneous tumors were identifiable in all rats of the reference group (100%) who did not receive the medication treatment, as well as in most of the rats of the main test group (55%), who received Cytochrome C; at the same time, tumors were not identifiable in 45% of the animals in the main test group (see Table 1 herein). Against the background of the experimental medication treatment, all tumors in the rats of the main test group demonstrated their regression with time (see Tables 1-3 herein).

Moreover, in 73% of the cases, with full recovery of rats, i.e., the complete regression of the tumors was observed, and 3 rats died (27%). In dead animals, their tumors, despite their large size, became soft upon the use of Cytochrome C and were found fluctuated in palpation. When opening the tumors, their necrotic contents flowed out. In the reference group of the rats, the subcutaneous tumors grew throughout the experiment and had a dense-elastic consistency, and at necropsy the tumors had the standard appearance (see Table 1 herein).

The regression of the subcutaneous tumor nodes upon the actions and effects made by Cytochrome C has been evidenced not only by the smaller average tumor volumes in the rats of the main test group, but also by a decrease in the coefficient φ, which characterizes an average specific rate of tumor growth (see Table 2 herein). φ is an integral indicator that objectively reflects the growth of tumors during the entire observation period. It is determined by the formula as follows: φ (t1, t2) = (ln F (t2) / F (t1)) / (t2-t1), where F (t1) is the volume of the tumor at the beginning of the interval; F (t2) is the volume of the tumor at the end of the interval; and t2 – t1 is the time interval.

The effectiveness of the experimental medication therapy was evaluated by the percentage of the tumor growth inhibition (PTGI) and the coefficient of therapy effectiveness (γ).

By this means PTGI = (K-Op) / K × 100, where K is the average volume of tumors in rats of the reference group, Op is the average volume of tumors in rats of the test group.

Table 1

Effect made by Cytochrome C on production of experimental subcutaneous tumors and dynamics of their growth in rats inoculated with Pliss lymphosarcoma

groups indicators	Reference group (rats with a growing tumor)	Main test group with cytochrome C medication (rats with a regressing tumor)
number of rats with tumor, pc.	10	11
number of rats without tumor, pc.	–	9
number of rats/dead rats (in main test group), pc.	10 (100%)	8 / 3
time of the production of tumors (survivors/ dead), day of the experiment	7.2±0.8	11.3±2.5 / 6.8±1.2
Final volume of tumors (survivors/dead), in main test group, cm3	92.5±9.4	23.6±4.7 / 53.8±6.1

Table 2

Change in the rate of tumor growth (φ) in rats with Pliss lymphosarcoma, arb.u. (М±m)

Day of the experiment Group of aimals	10	13	16	20	23	27	29
Reference	1.03±0.34	1.48±0.50	0.88±0.05	0.68±0.03	0.57±0.02	–	–
Cytochrome C	0	0.77*±0.05	0.52*±0.09	0.27*±0.17	0.17 *±0.17	0.04±0.22	0.04±0.17

Note: *statistically significant difference compared with the reference

Table 3

Dynamics of PTGI and γ in rats with Pliss lymphosarcoma

Day of the experiment Coefficients	8	10	13	16	20	23
PTGI, %	– 267.65	0	11.40	3.13	55.11	67.79
γ, arb.u.	7.50	15.63	6.77	19.27	35.99	33.14

γ = C (control, reference) / K (experiment), where K is the coefficient of change in the volume of the tumor; K = Vt / Vo, where Vt is the volume of the tumor after the therapy in the studied time; Vo is the initial volume of the tumor.

On day 8 of the experiment, the size of subcutaneous tumor nodes in the rats treated with Cytochrome C was significantly larger than the respective reference values (see Table 3 herein).

On day 10, the sizes of tumors in both groups were evaluated as follows: in the rats treated with Cytochrome C and in those without medication the sizes were found to be identical and practically did not differ from each other within 16 days of the experiment. From day 20, the tumors in the rats of the main test group began to intensively regress (see Table 3 herein). When evaluating the antitumor effectiveness of the Cytochrome C medication, it was revealed that the effectiveness of the treatment was at a minimum on day 8 and 13 of the experiment and at a maximum at the end of the observation period on day 20 and 23 (see Table 3 herein).

Apparently, Cytochrome C has produced its anti-tumor effect in different ways: in 45% of the rats, the drug immediately suppressed the development of subcutaneous tumors, and in 55% of the cases, the medication effect has been demonstrated with a delay: its anti-tumor effect is cumulative. Before a certain point in time, the subcutaneously transplanted tumors continued to grow, however when the concentration of Cytochrome C reached a certain “therapeutic” value, necrosis of the tumor cells occurred. In most of the animals, the body was able to cope with the necrotic masses and eliminate them without negative consequences: the rats demonstrated their recovery. In 3 rats, the organism was not capable to cope with the consequences of necrosis, despite the fact that the tumor tissue was lysed, it remained in the body. Finally, intoxication with the products of the tumor decay led to the death of the animals. According to the results of the study, a 32 | Cardiometry | Issue 20. November 2021

patent for the invention was duly obtained by our research team [10].

Conclusions

Thus, Cytochrome C, used according to the therapeutic and preventive schedule, has shown a pronounced anti-tumor effect. In 45% of the cases, the medical drug has prevented the development of Pliss lymphosarcoma, in 55% of the cases tumors have developed, but subsequently, from day 20, they have demonstrated regression, when we have recorded 73% of the animals with complete recovery and 27% with well-marked lysis of the tumor tissue. Perhaps, the effectiveness of the drug can be increased if an inert medium is used to protect the protein from proteolytic cleavage, when it enters blood, against the background of detoxification drugs. The obtained data support the reasonability of the recommendation for using cytochrome C in the complex treatment of cancer as a promising factor of the accompanying therapy.

Statement on ethical issues

Research involving people and/or animals is in full compliance with current national and international ethical standards.

Conflict of interest

None declared.

Author contributions

The authors read the ICMJE criteria for authorship and approved the final manuscript.