The concenTraTion of ceruloplasmin in blood of Tumor-bearing raTs afTer adminisTraTion of a dirhenium(iii) compound and cisplaTin

Contemporary investigations of mechanisms of resistance to platinides showed the key role of copper metabolism in cancer patients and proposed possible methods to attenuate the resistance by modulation of copper transporting mechanisms. In this vein, investigation of ceruloplasmin (Cp) levels – the main coppercontaining protein in blood, in experiments with tumor-containing animals upon cytostatics administration is topical and has great importance. the concentration of Cp was measured in the serum of tumor-bearing rats with ordinary (t8) and resistant to cisplatin (t8*) Guerin’s carcinoma upon administration of cisplatin and quadruple bonding dirhenium(III) compound dichlorotetra-μ-isobutyratodirhenium(ІІІ) (I) in different medicamental forms. It was shown that development of tumor in t8 group led to increasing of concentration of Cp in 3.7 times and in T8* group – more than in 8 times in comparison to control, confirming the essential role of Cp in the formation of resistance phenomenon. administration of cisplatin together with I led to effective inhibition of tumor in groups with t8 and t8*, indicating decreased resistance in the group t8*. Greater reduction of Cp levels was observed in the groups with t8* upon administration of the rhenium-plati num antitumor system, than in groups with t8, that underlines the importance of further investigations of the dirhenium(III) compounds in the resistance to cytostatics cancer models. Some mechanisms concerning the regulation of copper homeostasis and properties of nano-composites are discussed.


Contemporary investigations of mechanisms of resistance to platinides showed the key role of copper metabolism in cancer patients and proposed possible methods to attenuate the resistance by modulation of copper transporting mechanisms. In this vein, investigation of ceruloplasmin (Cp) levels -the main coppercontaining protein in blood, in experiments with tumor-containing animals upon cytostatics administration is topical and has great importance. the concentration of Cp was measured in the serum of tumor-bearing rats with ordinary (t8) and resistant to cisplatin (t8*) Guerin's carcinoma upon administration of cisplatin and quadruple bonding dirhenium(III) compound dichlorotetra-μ-isobutyratodirhenium(ІІІ) (I) in different medicamental forms. It was shown that development of tumor in t8 group led to increasing of concentration of Cp in
times and in T8* group -more than in 8 times in comparison to control, confirming the essential role of Cp in the formation of resistance phenomenon. administration of cisplatin together with I led to effective inhibition of tumor in groups with t8 and t8*, indicating decreased resistance in the group t8*. Greater reduction of Cp levels was observed in the groups with t8* upon administration of the rhenium-plati num antitumor system, than in groups with t8, that underlines the importance of further investigations of the dirhenium(III) compounds in the resistance to cytostatics cancer models. Some mechanisms concerning the regulation of copper homeostasis and properties of nano-composites are discussed. k e y w o r d s: ceruloplasmin, cluster rhenium(III) compound, Guerin's carcinoma, resistance to cisplatin. C eruloplasmin (Cp) is a copper-containing protein that includes 40-70% of the whole plasma copper. It possesses the enzymatic activity as ferroxidase and takes part in oxidizing of bivalent iron. Cp belongs to proteins of acute phase and serves as a marker of inflammation [1].
Changing of Cp concentrations in blood was shown under different diseases. The reducing of Cp level was noticed under Wilson's, Menson's diseases, copper deficiencies and aceruloplasmia [2]. Also, the Cp level is changing under cancer development: concentration of Cp in blood was increased under oncological diseases of different etiologies and increased Cp level was also found in the tissues of onco-patients [3].
Contemporary knowledge about Cp functions in cancerogenesis includes its role as a prognostic marker of some types of cancer [4]; as an adipokine which is overexpressed in adipose tissue of obese subjects [5]; as a substance that promotes tumor progressions through influence on the glycolytic process in cancer cells [6].
It is well-known that resistance to platinides, especially to cisplatin (cPt), is a negative phenomenon in the oncological practice and includes some complicated mechanisms, among which blockade of the transport of platinides is considered to be the most ultimate. The expression of the human copper transporter 1 (hCtr1), which transports also platinides, was found to be significantly reduced in cisplatin-resistant cancer cells. Moreover, one of the recently found tools to overcome cPt resistance was to introduce cPt together with copper -chelating agents [7][8][9].
In our previous works, the anticancer and antioxidant properties of cluster rhenium(III) compounds which were administered to tumor-bearing animals separately, with cPt, (Re -Pt system), in liposomal forms and in the forms of solid lipid nanoparticles in the model of ordinary (T8) and resistant to cPt (T8*) Guerin's carcinoma was shown [10][11][12]17]. But the concentration of Cp in these works was not investigated. Thus, the aim of the work was to investigate the influence of the introduction of cPt and a cluster rhenium(III) compound in the form of nanoliposomes and solid lipid nanoparticles on the concentration of Cp in the blood of tumor-bearing rats under development of T8 and T8*.
All animal experiments were performed in accordance with the rules of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 1986). Rats without tumor were control group (control, No 1). Tumor growth was modeled by transplantation of 20% suspension of T8 or T8* to healthy rats (groups T8, No 2 and T8*, No 5 respectively).
A single intraperitoneal injection of cPt in solution (sl) or nanoliposomal (nl) forms at a dose 1.75 µmol/kg was performed on the 9 th day after tumors transplantation, groups: T8+cPt sl, No 3; T8*+cPt sl, No 6; T8+cPt nl, No 4; T8*+cPt nl, No 7. The Re-Pt antitumor system was introduced with separately introductions of the components when cPt was introduced in the same mode and quantities as in groups 3, 6 and 4, 7 and on the 3 rd day after tumor transplantation i was administrated ten times in liposomes in the dose of 7 µmol/kg, groups: Experimental animals were decapitated under chloroform anesthesia on the 21 st day. Blood was collected in test tubes. The serum was obtained by natu ral plasma coagulation. The content of the Cp was investigated in the serum using the Ravin method and the standard reagent set (Private Joint-Stock Company Reagent, Ukraine). The degree of reduction of oxidative stress in plasma of experimental animals was evaluated as the ratio of the concentration of MDA in plasma of experimental animals to the concentration of MDA in plasma of rats with Guerin's carcinoma without any administration (ordinary or, respectively, resistant) [11,17]. The results were statistically processed using the Student's t-test. Differences in values were considered statistically significant at P ≤ 0.05.

results and discussion
Development of ordinary Guerin's carcinoma T8 led to increasing of concentration of Cp in 3.7 times in comparison to control (groups No 2 and No 1), Table 1.
At the same time, Cp concentration in serum of T8* -bearing rats increased in 8.5 times in comparison to control. Thus, development of a cPt-resistant tumor T8* led to increasing of Cp concentration in 2.3 times more intensively in comparison to ordinary tumor T8. These findings may support the upper cited works about essential role of Cp in the formation of resistance phenomenon in cancer cells [7][8][9].
Introduction of cPt solutions to tumor-bearing rats showed different directions of changing of Cp levels in T8 and T8* groups of animals: if introduction of cPt to T8 group led to increasing of Cp level on 22.9% in comparison to T8 group, the same introduction to T8* group led to decreasing of Cp on 27.0% in comparison to T8* group of animals. To our knowledge, such an essential difference in elevation or dropping of Cp levels as a reply to cisplatin solution introductions is first demonstrated.
Introductions of liposomes loaded with cPt led to higher efficacy of tumor inhibition in T8* group t a b l e 1. the concentration of Cp in serum, tumors inhibition and reducing of plasma mDa concentration in comparison to t8 and t8* groups under cPt introduction *Р < 0.001 relative to control, # Р < 0.05 relative to group T8, ## Р < 0.001 relative to group T8* In fact, liposomal forms of drugs enter the cytoplasm, bypassing active import. Reducing of tumors always was accompanied by the decreasing of oxidative stress, especially under application of the liposomal drugs [12,17], that is also confirmed by data presented in Table 1. In both kinds of tumors, introductions of liposomal forms of cisplatin led to more essential decreasing of the intensity of peroxide oxidation of lipids (POL).
Introduction of the Re-Pt system in liposomal forms (groups 8, 9, 10) led to very effective inhibition of T8 ( Table 2) that was also shown earlier [11,17].
Effective inhibition of tumor in groups with T8 (No 8, No 9, No 10) and T8* (No 11, No 12, No 13 that is an example of resistance overcoming) coincided with relatively low concentrations of Cp (68.5-204.0% to control) and (70.0-135.0% to control) respectively. Also, the decreasing of POL intensity in blood of experimental animals is observed.
Comparing data of Tables 1 and 2 we may conclude that introducing of the rhenium compound i in any form together with cisplatin led to the lowering of Cp together with effective inhibition of the tumors as ordinary, as resistant to cisplatin.
Especially interesting is to compare groups No 3 and No 8; No 6 and No 11. In all these ext a b l e 2. the concentration of Cp in serum, inhibition of tumors and reducing of plasma mDa concentration in comparison to t8 and t8* after re-Pt system introduction *Р < 0.001 relative to control, # Р < 0.05 relative to group T8, ## Р < 0.001 relative to group T8* Thus, according to the aim of the work, we have shown that development of a cPt-resistant tumor T8* led to increasing of Cp concentration in 2.3 times more intensively in comparison to ordinary tumor T8; introduction of cPt solutions to tumorbearing rats showed different directions of changing of Cp levels in T8 and T8* groups of animals; introductions of liposomes loaded with cPt led to higher levels of Cp in both T8 and T8* groups in comparison to groups with free cPt introductions; introducing of the rhenium compound i in any form together with cisplatin led to the lowering of Cp in comparison to groups with cPt introductions.
If also to compare the average Cp level between groups of tumor-bearing rats shown in Table 3, it is evident that in the groups No 8, No 9 and No 10 there were level reduction in average by 36.0% in comparison to T8 (Table 1,

group No 2).
Greater reduction of Cp levels was observed in the groups No 11,No 12 and No 13 with T8*, where the Re-Pt system was introduced. There was a reduction of Cp levels in average on 77.0% in comparison to T8* (Table 1, group No 5). The comparison with appropriate groups where i was introduced together with cisplatin, also showed the larger reduction of the Cp concentration in groups with T8* than in groups with T8. It means that i influence on the copper homeostasis of both types of tumors, but in somehow different manner that underlines the importance of following investigations of the dirhenium(III) com-pounds in the cancer models with resistance to cytostatics.
Recent findings revealed that hCtr1 regulates intracellular copper homeostasis, which, in turn, controls hCtr1 expression via a homeostatic feedback loop [7,9]. The copper-lowering agents increased the expression of hCtr1, subsequently resensitizing tumor cells to platinum therapy. Does the rhenium compound i play somehow the role of the copperlowering agent in these experiments? We have no any supplying facts for this now. But if so, the hypothesis that resistance to platinum therapy can be reversed through the use of a copper-lowering agent as a rhenium compound like i is very promising. But we know, that mechanism of platinum compounds transport are multifactorial and any stage of the mechanisms underlying cisplatin transport (both import and export) and retention in the tumor cells are important determinants and may be affected by i.