Protective and Antitumor Effects of Oxidal and Pyrucet in Hamsters with Experimental Graffi Tumor

The use of combination drug therapy instead of monotherapy is a new positive and promising approach in the treatment of cancer. Oxidal® and Pyrucet® are food supplements containing Methylene blue (USP), Caffeine (USP), Salicylic acid (USP) and Ethyl Acetoacetate and Ethyl Pyruvate respectively. The therapeutic significance of each of these compounds (their derivatives) is well established, but no data are available on their combined action in in vivo experimental tumor models. The aim of the present study was to investigate the in vivo protective effect of monoand combined experimental therapy with dietary supplements Oxidal® and Pyrucet® in the Graffi myeloid tumor model in hamsters. For this purpose, the two drugs were administered alone or in combination in two treatment regimens prophylactic (before) and therapeutic (simultaneously) with the transplantation of Graffi tumor cells. The protective effect was determined by the biometric parameters of tumor growth (transplantability, tumor size, mortality, mean survival time, survival Original Research Article Toshkova et al.; JPRI, 33(12): 1-19, 2021; Article no.JPRI.65906 2 rate) recorded during the experiment. The results showed a favorable effect of both drugs, administered alone or in combination, before and simultaneously with the transplantation of tumor cells on the appearance and development of Graffi tumor in hamsters. About 2-fold lower transplantability, prolongation of the latency period by 7 days, inhibition of tumor growth till 30 th day of experiments, reduced mortality and increased individual and overall survival time between 7 to 10 days were observed in the Graffi tumor-bearing hamsters with experimental therapy compared to control-Graffi tumor-bearing hamsters, without therapy. The obtained data revealed that Oxidal® and Pyrucet® could be a promising candidate for the treatment of tumor diseases.


INTRODUCTION
Globally, cancer is a major health problem associated with an annual increase in morbidity and mortality [1][2][3]. Despite advances in the early detection of tumors and in the use of chemotherapy, radiation and surgery to treat cancer, there are two main problems with therapy of neoplasms -(multiple) drug resistance and adverse side effects, some of which are lifethreatening or dose-limiting [4]. The search for alternative cancer treatments, as well as the repurposing of conventional drugs to new applications in oncology, is one of the most promising modern strategies to combat cancer. Attention is focused on long-established in clinical practice preparations with a well-known pharmacokinetic/pharmacodynamic profile and toxicity. Oxidal® and Pyrucet® are food supplements. Oxidal® includes Methylene blue (USP), Caffeine (USP) and Salicylic acid (USP), while Pyrucet® includes Ethyl Acetoacetate and Ethyl Pyruvate. The therapeutic potential of each of these compounds (their derivatives) is well established.
Methylene blue (MB) is apolyaromatic cationic dye of the phenothiazine group widely used as a diagnostic agent/histological dye with established clinical applications, including oncological diagnosis by tissue staining and for treatment of methemoglobinemia [5]. It has been extensively used as photosensitizing agent for photodynamic inactivation of RNA viruses, including human immunodeficiency virus (HIV), hepatitis B virus, and hepatitis C virus in plasma [6,7]. (MB) of cases with SARS-CoV-2 [8]. There is preventative role of Methylene blue] and inhibition of replication [9,10]. MB is also a promising photosensitizer for photodynamic therapy (PDT) against microbial cells, viruses, as well as cancer cells [11][12][13][14]. In in vivo trials in female BALB/c mice,the combined photodynamic/photothermal (PDT/PTT) therapies promoted complete tumor ablation and metastasis prevention while only PDT or PTT were unable to stop tumor development [15].
According to several separate studies, caffeine inhibited the growth of different cancer cell lines in vitro [16][17][18][19][20]. Okano et al. [16] found that caffeine inhibited the proliferation of hepato cellular carcinoma (HCC) cells. Liu et al. [17] showed significantly suppressed growth and viability of human gastric cancer cells and induction of apoptosis. Caffeine was cytotoxic to the human breast MCF-7 and MDA-MB-231 cancer cell lines. It induced oxidative stress and predominantly apoptosis in both cell lines [18]. The antitumor action of caffeine against human colorectal cancer (HT-29 and RKO) is linked in part to its anti-inflammatory properties [19]. Research of Wang et al. [20] indicated that treatment with acombination of caffeine and atorvastatin maybe an effective strategy forinhibiting the growth of human prostate cancer cells in vitro.
Salicylic acid (SA) is the major metabolite and active component of aspirin. Besides analgesic, antithrombotic and anti-inflammatory effects, use of aspirin is associated with a reduced risk of developing different cancers such as colon and rectal, pancreatic, breast, head and neck cancer, and etc. [21][22][23][24][25]. Vejselova and Kutlu [25] reported on the antitumor (apoptotic, cytotoxic, and growth-inhibiting effects) activity of the salicylic acid on A549 human lung adenocarcinoma cells. Acetylsalicylic acid and salicylic acid at 10 mM to 20 mM concentrations inhibit the viability and induce apoptosis of cervical cancer cells line (HeLa) [26]. The pathways by which aspirin and salicylic acid exert their anti-cancer effects are still not fully elucidated, although multiple mechanisms affecting enzyme activity (both COX-dependent and independent), transcription factors, cellular signalling and mitochondrial functions have been proposed [23,[27][28][29][30][31][32].
Ethyl pyruvate (EP) was recently identified as a  stable lipophilic ester of pyruvic acid, with  notoxicity and with significant pharmacological  benefits  and  antineoplastic activities. Ethylpyruvate (EP) suppressednon-smallcell lung cancer (NSCLC) cell growth, invasion and migration in vitro, and promotedapoptosis [33]. Blockade of HMGB1-mediated signaling pathway by EP effective lyinhibited diffuse large B-cell lymphoma (DLBCL) tumorigenesis in vitro and disease progression in mice in vivo [34]. Several reports demonstrated that EP treatment suppressed tumor progression and increased the overall surivial of animals in various in vivo tumor models, including hepatic, gastric, gall bladder cancer, lung and mesothelioma [35][36][37][38][39][40].
The combination therapy is one of new and interesting methods in cancer therapy researches. Combinations of multiple drugs in cancer treatments offer the potential for inhibiting multipletar gets and path ways simultaneous lytomore effective lykill cancer cells and prevent or delay the emergence of drug resistance [32,41,42].
The aim of the present study was to evaluate the antitumor influence of the dietary supplements Oxidal® and Pyrucet®, and their combination against Graffi tumor in hamsters. We report the results of two surveys in which prophylactic (before tumor implantation) and therapeutic (at time of tumor implantation) treatments were applied.

Experimental Animals
Hamsters 2-4 months aged, from both sexes of the race "Golden Syrian", weighing about 100 g were used in the experiments. They were bred in individual plastic cages with free access to food and water, in the vivarium of the Institute of Experimental Morphology, Pathology and Anthropology with Museum (IEMPAM), Bulgarian Academy of Sciences (BAS).

Experimental Design
The therapy in hamsters with experimental tumors was applied in two variants: prophylactic and therapeutic. In the prophylactic variant of therapy, the individual (Oxidal®, Pyrucet®) or combined (Oxidal® + Pyrucet®) treatment started on day 1 of the experiment and continued until day 20, after the transplantation of the tumor cells (a total of 27 days daily treatment of each hamster were performed). In the therapeutic variant, the administration of single (Oxidal®, Pyrucet®,) or combined (Oxidal® + Pyrucet®,) treatment began on the day of injection of hamsters with tumor cells and lasted 20 days (a total of 20 days daily treatment for hamsters were performed). For control, Graffi tumor bearing hamsters without therapy were used (Control group).
The experimental hamsters were randomly assigned to a total of 7 experimental groups as follows:

Biometric Parameters
The effect of the applied two variants of therapy with the studied dietary supplements Oxidal® and Pyrucet®, was evaluated by determining the biometric indicators of tumor growth for hamsters from all experimental groups. Transplantability (%)-number of hamsters that developed a tumor from all injected hamsters in the group. Tumor size (mm)-represents the average size of two mutually perpendicular diameters of the tumor (A-width and B-length), measured with a caliper. Mortality/Lethality (%) -number of dead hamsters compared to the total number of hamsters in the group. Average survival (days)-average number of days survival of all hamsters in the group. Individual survival rate was also measured in hamsters with experimental therapy.

Statistics
The statistical significance of the differences between the control and treatment groups was evaluated by using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test using the GraphPad Prism software package. Significance was defined at P<0.05.

RESULTS AND DISCUSSION
In this research the biometric tumor parameterstransplantability (%), tumor size (mm), lethality (%), mean and individual survival (days) in all experimental groups were analyzed.

Transplantability (%)
Transplantabilityi indicates in % the number of hamsters that developed tumors compared to the total number of injected hamsters in the group.On a daily basis following the 7 th day after injecting of Graffi tumor cells in the trial animals, via palpation of the skin at the injection site, the appearance of a tumor was reported. Transplantability data (%) in hamsters with therapy with Oxidal® (A), Pyrucet® (B) and Oxidal® + Pyrucet®(C) were presented in Fig.  1A, B&C.
From Fig. 1 (A, B &C) it could be seen that the transplantability percent in the control group (Gr. 7-TBH) without therapy was 20% on day 9 and 100% on day 12 after tumor cell transplantation.
In the hamsters from the experimental groups with applied single or combined therapy, the transplantability was reduced (the graphs were shifted to the right of those of the control (Fig.  1A, B &C).
For the hamsters from group 1 it was observed 22% transplantability on the 10 th , 44% on the 12 th , and 100% on the 16 th day. For group 2 the transplantability was 12.5% on the 9 th day, 25% on the 10 th day and 100% on the 19 th day (Fig.  1A).
Decreased transplantability was observed in hamsters from group 3 and group 4. The transplantability by groups was as follows: For group 3 -on the 9 th day -12.5%, on the 12 th day -25% and on the 16 th day -100% and for group 4 -on the 10 th day. 16.6%, on the 12 th day -50% and on the 17 th day -100% (Fig. 1B).
For hamsters from group 5 -on the 10 th day tumors appeared in 12.5%, on the 12 th day -in 25%, on the 13 th day -in 50%, on the 14 th dayin 62.5% and on 16 th day -in 100% of hamsters. For group 6 the data were as follows: on the 10 th day tumors appeared in 20%, on the 13 th day -in 60%, on the 17 th day -in 80% and on the 19 th day -in 100% of hamsters (Fig. 1C).
The chart showed that in control untreated Graffi tumor bearing hamsters (Gr. 7-TBH), the tumor appearance time (latent period) was within the interval of 9-12 days. In the other groups this interval was extended -in the range of 9-16 days for Gr. 1, Gr. 3 and Gr. 5, in the range of 9-19 days for Gr. 2 and Gr. 6, and in the range of 9-17 days for Gr. 4 (Fig. 1A, B &C). The positive effect in extended time for tumor appearance and the reduced percentage of transplantability in the drug-treated groups can be explained with the applied therapy.

Tumor Size (mm)
Tumor size in hamsters from experimental (Oxidal®, Pyrucet® and Oxidal® + Pyrucet® therapy) and control groups was measured regularly with a caliper for 30 days, after tumor injection. The data are presented in Fig. 2A, B& C.
In the hamsters from the control group (Gr.7-TBH), the tumor size increased progressively. The reported values were: 5.4 ± 2.5 mm on the 13 th day, 9.6 ± 3.91 mm on the 15 th day, 16 In the hamsters from the groups with applied single or combined therapy, the size of the tumors was smaller than that of the control (the graphs were shifted down from those of the control ( Fig. 2A, B& C).
For hamsters from group 1 (Oxi + Tu) ( Fig. 2A) a delay in tumor size was seen compared to the control group (Gr.7-TBH). The reported dimensions for hamsters of Gr.  (Fig. 2B).
In the hamsters with combination therapy from Gr.5 (Fig. 2C) a reduction in the size of the tumors was seen in comparison with the control (Gr.7-TBH). The reported dimensions for the Gr.

Mortality (%)
The mortality presents the percentage of dead animals in comparison with the total number of hamsters in the group, reported at certain intervals (30 Applied single or combination therapy with Oxidal® and Pyrucet® reduces the mortality rate in hamsters with Graffi tumor.

The Median Survival Time (Days)
The data in fig. 4 (Fig. 4).

Illustrative Images of Hamsters from the Trial Groups
In support of the metrics data in Fig. 2A, B&C, we also present photographic material (Fig. 6-9 and Fig. 10 Via palpation of the skin on the back of hamster, from Gr. 1 firmness with a size of 3-6 mm was felt, and after skin dissection it was observed a hyperemic zone with a tumor formation with the same size (Fig. 6A). In the case of a hamster from Gr. 2, a diffuse firmness with a size of 3-5 mm was palpated, and after skin dissection it was observed a hyperemic zone with a diameter approximately 1 cm and 2-3 single tumor formations with a size of 1-2 mm (Fig.6B).
In a hamster from Gr.3, no firmness was felt, and after dissection of the skin, no hyperemia was observed at the tumor injection site (Fig. 7A). When palpating the skin on the back of a hamster from Gr.4, firmness with a size 3-4 mm was established and after the skin dissection there was observed a clearly defined zone of hyperemia with centrally located formation of the same dimension (Fig.7B).
No firmness was felt when palpated the skin of hamster N1 from Gr.5 (Fig. 8A, left panel), but after dissection of the skin there was seen a hyperemic area with initial tumor formation. In hamster N2 from Gr.5, a seal with a size of 1-2 mm was palpated and after skin dissection an intensely colored hyperemic zone with a single tumor with a size of about 1-2 mm was observed (Fig. 8A, right panel).
Via palpation, it was established a firmness with a size of about 2-3 mm in hamster 1 from Gr.6 and after skin dissection a hyperemic zone and a single tumor nodule gray-whitish-colored with a size of about 2 mm was seen (Fig. 8B, left  panel). At the other hamster N2 from Gr.6 we found out a seal with an elongated shape, and after dissection of the skin, there was a pronounced elongated hyperemic zone with elongated in shape tumor, whitish-gray colored and 2-4 mm in size (Fig.8B, right panel).
In a hamster from the control group (Gr. 7), a tumor with a size of 10-12 mm was palpated on the back, and after skin dissection, a large hyperemic area with a well-formed solid tumor with a size of 10-12 mm was observed (Fig.9A).
Graffi-TBH without drug treatment on 30 th day after tumor cell transplantation was presented (Fig.9B). The macroscopic finding showed an oval in shape, subcutaneous solid tumor with a size of 2.5 /4.5 cm that covered half of the dorsal surface of the hamster. After skin dissection it was visible a grey to off-white in color tumor formation with uneven surface and hemorrhagic areas. A large tumor metastasis was seen in the right axillary region (Fig. 9B).
The results obtained from the current research (hamsters randomly selected from 7 experimental groups) indicate:  The tumors of hamsters that received experimental mono-or combined therapy with Oxidal® and Pyrucet® however were 2-3 times smaller in size than that of the control Gr. 7. This fact gives us reason to draw a conclusion with regards to the positive effect over the occurrence and development of Graffi tumor in hamsters with Oxidal® and Pyrucet® therapy.
In support of the data stated above for tumor size in Fig. 2A, B&C photo material from the relevant experimental groups were presented. .

. Hamsters of Gr. 3 (Pyr + tu) (12 days after injection of daily treatment with Pyrucet per os) (A) and Gr. 4 (tu + Pyr) (12 days after injection of tumor cells and 12-day daily treatment with
The photograph material in Fig. 10 ( therapy), 11 (Pyrucet® therapy) and 12 ( + Pyrucet® combination therapy) supported the results presented in Fig. 2A, B &C, in particular, in the groups with prophylactic or therap single or combination therapy with Pyrucet®, the tumor size was smaller compared to the untreated control groupbearing hamsters (comparative consideration of line above and middle line towards line below in Figs. 10, 11 & 12).

Hamsters of Gr. 3 (Pyr + tu) (12 days after injection of Graffi tumor cells and 19 days of daily treatment with Pyrucet per os) (A) and Gr. 4 (tu + Pyr) (12 days after injection of day daily treatment with Pyrucet® per os, started on the day of injection of the tumor cells) (B)
The photograph material in Fig. 10 (Oxidal® therapy) and 12 (Oxidal® combination therapy) supported the results presented in Fig. 2A, B &C, in particular, in the groups with prophylactic or therapeutic, single or combination therapy with Oxidal® and , the tumor size was smaller compared s evidence that these substances exhibit antitumor effects in in vitro human and animal model systems A different target mechanisms were observed for the different compounds -they act by influencing (activate/inhibit) cellular signaling pathways, regulatory proteins, transcription factors, mitochondrial functions, interfering with the cellular metabolism and with lysosomal function, etc. [16][17][18][19][20][28][29][30][31][32][33]40]. For example caffeine works in hepatocellular carcinoma (HCC) cel via activation of MEK/ERK/EGFR pathway (cell cycle arrest independent apoptosis) [16]; in human gastric cancer activating the caspase-9/caspase pathway [17]; in human breast MCF MB-231 cancer cell lines by interfering with the cellular metabolism and with lysosomal function [18]; in human colorectal cancer (HT RKO) lines is linked in part to its anti inflammatory properties [19]. In prostatecancer cells, the combination ofcaffeineandatorvastatindownregulatedphospho -Akt, phospho-Erk1/2, anti-apoptotic Bcl   [20]. To exert their chemopreventive effects aspirin and salicylic acid may target cell cycle regulatory proteins, both COX-dependent and independent enzyme activity, mitochondrial functions, etc. [28,29,30,31]. Aspirin's anti-cancer effect may occur through down regulation of c expression [28]. To exert their effects aspirin and salicylic acid may target cell cycle regulatory proteins, both dependent and independent enzyme activity, mitochondrial functions, etc. cancer effect may regulation of c-Myc gene .
Published evidence demonstrates that salicylic acid exerted antitumor effects (significantly inhibited cell proliferation accompanied by apoptosis induction) in liver cancer cells in part mediated by the nitric oxide (NO) pathway [29]. The cell structure damage and ultrastructural changes, concretely indicating apoptosis in A549 cells by using transmission electron (TEM) and confocal microscopy were also reported [25]. It was demonstrated that combination photodynamic cylic acid and methylene blue can improve the cell killing effectiveness of MB on the MB-23 [32]. Ethyl pyruvate (EP) mediated suppression lung cancer cell growth via inhibition HMGB1-RAGE signaling pathway NFκB/STAT3 pathway [33]. apoptosiss witch activity of EP may its anti-inflammatory action and suppress tumor development [39]. EP may suppress growth in the liver through its anti and apoptotic-inducing properties experiments, in trahepatic visible generated with in 2 weeks after injection of MC38 colorectal cancer cells. The Necrosis-toof EP may contribute to suppression of suppress tumor anti-inflammatory properties. In in vivo tumors were direct portal colorectal cancer cells. The obtained results showed that pretreatment with EP 30 min prior to infusion of tumor cells and continuing daily for 9 days inhibited tumor growth significantly in a dose-depend entmanner compared with untreated mice [40].
The combination therapy is one of new interesting methods in cancer therapy researches. It is believed that the multitarget approach in the treatment of tumors is important for the realization of successful antitumor therapy [32,41,42,44,45]. The combination therapy is one of new and interesting methods in cancer therapy It is believed that the multitarget approach in the treatment of tumors is important realization of successful antitumor therapy

CONCLUSION
In the present study the in vivo protective effect of dietary supplements Oxidal® and Pyrucet®, administered alone or in combination, in two treatment regimens in the Graffi hamsters were assessed. The results obtained by us demonstrate a protective antitumor effect of both food supplements, reported on the values of biometric parameters in all schemes and combinations of applied therapy. We observed inhibition of tumor transplantation and prolongation of the latency period, slow tu growth during the experiment, as well as reduced mortality. In addition, the mean and individual survival of hamsters with therapy was significantly increased compared to control untreated hamsters. The obtained results are Graffi tumor in results obtained ate a protective antitumor effect of both food supplements, reported on the values of biometric parameters in all schemes and combinations of applied therapy. We observed inhibition of tumor transplantation and prolongation of the latency period, slow tumor growth during the experiment, as well as reduced mortality. In addition, the mean and individual survival of hamsters with therapy was significantly increased compared to controluntreated hamsters. The obtained results are supported by rich illustrative material. positive effect has been attributed to the multi target mechanism of action of Pyrucet® on the tumor and to defense mechanisms of experimental animals after experimental therapy. Our data support the idea that dietary supplements Oxidal® and Pyrucet® may be promising candidates for the treatment of tumors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All experiments were carried out in accordance with the European Convention for the Protection ; Article no. JPRI.65906 Pyrucet®. Legend: Pyrucet® per os for dle row -Gr.2per os for 20 days, started hamsters with Graffi tumor, ve material.This positive effect has been attributed to the multi target mechanism of action of Oxidal® and on the tumor and to defense mechanisms of experimental animals after experimental therapy. Our data support the idea that dietary supplements Oxidal® and Pyrucet® may be promising candidates for the treatment of All experiments were carried out in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and approved by the National Veterinary Office in Bulgaria, regarding laboratory animals and animal welfare.