1. Introduction
Cancer is one of the most prominent health problems of our age and, although it used to be ranked as seventh and eighth among terminal illnesses in the early part of the century, today it is ranked as second, following cardiac diseases in many countries of the world including Turkey. Treatment methods of cancer usually include chemotherapy, radiotherapy, surgery, and immunotherapy and one or several of these methods is/are used in the treatment according to individual characteristics of patients diagnosed with cancer and the severity of illness. Chemotherapy is a treatment method that has selective lethal effects, especially against multiplying cells, and is performed with natural or synthetic chemical and biological agents, and hormones. The aim of chemotherapy is to extend patients’ life expectancy and provide a better quality of life. However, there are chemotherapy-related difficulties and toxic effects depending on the method used [
1]. Antioxidant substances have been commonly used in recent studies in order to enhance the immune system and reduce cytotoxic effects [
2]. Hazardous chemicals’ wastes containing cisplatin (CP) are released into the environment in various departments of hospital laboratories and engineering departments. In addition, the chemical waste of anticancer agents of industrial and healthcare institutions can pose a vital risk to nontarget species in the aquatic environment due to cytotoxic, genotoxic, mutagenic, and teratogenic effects. When environmental problems are considered, nonhazardous chemicals in nano-technological products gain importance for the continuation of human and animal health. The common types of nonhazardous and natural chemicals, such as propolis, clay, sugar, etc., are used in the nano-formulations. However, it is reported that nanotechnology-applied products might be more effective than conventional products [
3]. With the recent advances in nanotechnological studies, nanomaterials are widely preferred for several industrial and pharmaceutical applications [
4,
5].
Nanotechnological effect is provided through using materials (such as chitosan) that inhibit enzyme activation and DNA synthesis [
6]. The CP is one of the most commonly used chemotherapeutic drugs and a platinum-based chemotherapy substance making the bacteria-DNA synthesis impossible and, thus, leading to the death of cells that cannot repair the DNA. It causes a number of cytotoxic side effects while producing these effects. The side effects of its clinical use were primarily reported as nephrotoxicity and hepatotoxicity [
2,
7]. The increase in liver enzymes in serum and bilirubin is an indicator of liver dysfunction [
8]. CP hepatotoxicity indicates that the cytochrome P450-2E1 enzyme worsens toxicity even further with the increase in expression level [
9]. Histopathological changes are sinus dilatation and infiltration of inflammatory cells around the portal area, as well as necrosis and degeneration of hepatocytes [
10,
11]. These conditions, caused by oxidative stress, may lead to even worse situations. As a matter of fact, reactive oxygen species (ROS) causing the development of oxidative stress may induce apoptosis by means of the intrinsic and extrinsic pathways [
12]. In the extrinsic pathway of apoptosis, ROS are formed by the Fas ligand as an upward stream for the activation of Fas by phosphorylation. This condition is necessary not only for the induction of apoptosis, but also for the post-mortality domain related with caspase 8 and Fas [
13]. While the ROS in the intrinsic pathway facilitate the release of cytochrome C by activating Bcl-2 and Bcl-xL, which are known pore-stabilizing proteins, they inhibit the proteins that are pore-destabilizing (Bcl-2 associated protein X, death receptor signal Bcl-2) [
14]. Indeed, high levels of ROS and CP may cause apoptosis and necrosis in cells with cancer [
15,
16,
17]. ROS may also induce necrosis via autophagia [
18].
The term nanotechnology was used for the first time to define the ultra-fine production technology in 1974 [
19,
20]. Nanotechnology provides progress in molecular, atomic, and macromolecular areas [
20]. The purpose of nanotechnology is to increase the effectiveness of materials by using the change in their size and to enable nanosize to result in a better effect in the fields of biology and medicine [
21]. As a matter of fact, when active ingredients are present in the nano structure, they increase the stability of substances due to their protective effects on oxidant agents and other components or enzymes [
20,
22,
23]. In recent years, polymeric nanoparticles with unique properties have been preferred for use in different industrial applications. The use of biodegradable and biocompatible polymer matrix-based nanoparticles released into soil, water, and air without pollution plays an important role in reducing the environmental burden. Plastic waste is a major environmental concern, so biodegradable polymer matrix-based nanoparticles with a small size (1 to 100 nanometers) and a high surface area are an environmentally friendly alternative to conventional plastic. However, biodegradable polymeric nanoparticles have played a significant role in recent research, as they undergo complete degradation and have a less negative impact on the environment [
24,
25]. The presence of biopolymers has been important for the environment and nanotechnology to ensure the uniform distribution of nanoparticles, prevent their aggregation, and increase stability. For this purpose, chitosan, a green material, was preferred as a biopolymer matrix to prevent the formation of secondary pollutants [
26]. Chitosan is a biocompatible polymer for interaction of the material and the body and it is also used for the interaction between material and body due to the presence of free amino groups, which favor the interaction with cells. It can be chemically modified to prepare the nanocarrier, which is controlled and adsorbed in the body [
27,
28].
Propolis, which is a natural beekeeping product, due to the beneficial effects of its compounds, has been used for a lot of pharmacological and biological activities [
29]. When administered orally, propolis is nontoxic and rarely causes allergic reactions. The allergic reactions of propolis are more common after topical administration. Propolis is considered an occupational contact allergen for 0.76–4.3% of beekeepers [
30,
31]. Nanoscale encapsulation of propolis with chitosan was conducted in the present study. There are studies in the literature indicating that the nanoscale drugs have little allergic effects [
32,
33] or side effects [
34]. Moreover, supporting studies also take part in the literature suggesting that chitosan encapsulation ameliorates hypersensitivity [
33,
34,
35,
36]. Biomaterial-based nano-propolis (NP) can be synthesized by different techniques such as hydrothermal, sonochemical, microwave, and solvothermal [
37,
38,
39,
40]. There are several studies on biomaterial-based NP in different applications, such as biomedical and purification of wastewater, and relatively few studies on the preparation of propolis-based NP.
In this study, propolis-based nanoparticles, which are a natural material with a homogeneous distribution in the polymer matrix, were synthesized by the green sonochemical method. The low cost and nontoxic, natural materials attract the attention of nanotechnology studies. For this reason, the propolis is a green candidate for these new nanomaterials. Having many important effects, propolis dissolves in water slightly. On the other hand, NP may prove more effective by increasing the dissolvability of propolis. Studies on NP usually remain limited to studies on bacteria. As a matter of fact, in studies on
E. coli, it has been reported that even a very small amount of NP inhibits the development of
E. coli [
41,
42]. It was reported that antimicrobial activity of NP was much more effective than propolis [
21]. In addition, antitumor studies have revealed that the antiproliferative effects of NP are much better than propolis [
3]. The hypoglycemic effect of NP has been investigated, as well. It was concluded that NP, used in the treatment of diabetic rats induced by streptozotocin, is effective in the renewal of damaged β-cells and reduction in blood glucose (GLU) level [
43]. In the light of all these studies, the basis of our hypothesis was based on the assumption that the nanoparticle structure of propolis, which is known to be effective on oxidative damage, could be more effective. Studies are needed to confirm the effectiveness of different doses of NP in different types of cancer in order to reduce the effects of the damage caused by CP, which is one of the most common drugs used in the treatment of cancer that significantly affects human health.
This study was designed for the purpose of determining the effects of NP in rats induced by CP on performance (FI, BW and BWC) and some biochemical parameters and, also antioxidant status and Bcl-2 and Bax protein expression levels in liver and kidney tissues.
3. Discussion
CP is one of the most important chemotherapeutic agents. However, side effects of CP restrict its usage and effectiveness. Hepatotoxicity, nephrotoxicity, decrease in sperm, hair loss, nausea and vomiting are some important side effects of it. The studies have usually suggested that CP has a toxic effect with a mechanism caused by oxidative damage. It has been reported that the CP causes the production of free oxygen radicals and nuclear Factor kappa B activation and increases the adenosine A1 receptor synthesis [
44]. In addition, it is reported that CP inhibits the DNA transcription by making cruciate ligaments in the chain. The effect of CP on nephrotoxicity can be explained with the fact that inosine, which is an adenosine metabolite, creates an oxidative damage leading to the production of free radicals while being metabolized by xanthine oxidase [
44,
45].
In this study, the effect on FI of CP was investigated and it was determined that the FI was statistically reduced by CP (
Table 1). Similar to the present study; previous studies on CP reported that FI was significantly reduced by CP in rats [
46]. It was reported that CP has direct toxic effects on the stomach [
46]. Similarly, the study conducted by Malik et al. [
46] found that FI was significantly reduced by CP 6 mg/kg BW in rats. They associated this effect of CP with the fact that it causes gas accumulation in the stomach and emptying of the stomach was delayed due to this accumulation of gas. It was seen that propolis and NP applications positively affected the decrease in FI caused by CP toxicity, and FI in the CP + Propolis and CP + NP-30 groups was similar to the control group between days 1 and 21. When examining BW and BWC, it was determined that the CP group had significantly lower values than the control group between days 1 and 21. When examining the values of the CP + Propolis group between days 1 and 21, it was determined that there was a significant increase compared to the CP group. It was observed that although there was an improvement in FI and an increase in BW in the CP + NP groups compared to the CP group, there was a more significant improvement in the CP + Propolis group. This was associated with the fact that propolis increased FI and especially resin, wax, honey and vanillin in its structure [
47,
48] caused the flavour increase. In addition, the presence of significant differences between propolis doses of nano groups (10 and 30 mg/kg BW) and the propolis group (100 mg/kg BW) was associated with the fact that flavour-increasing substances were partially fewer than the nano groups. Improvements in FI reflect on BW values, as well. Additionally, improvements in FI and BW were associated with flavones in the structure of propolis [
49].
When examining biochemical parameters in the present study, it was determined that GLU, AST, ALP, albumin and BUN values were statistically different in the CP group. However, the CP + Propolis and CP + NP application significantly changed this difference in a positive way (
Table 2). In a study conducted on rats with liver damage induced by CP, Cetin et al. [
50] investigated the effects of grape seed and essential thyme oil on some serum blood parameters. They determined that CP caused a statistically significant increase in serum ALT and AST levels. The most important characteristics of the curative substances is that they have antioxidant compounds (especially phenolic compounds) as in propolis. In a previous study conducted with propolis, it was reported that ALP and AST levels of male rats in which oxidative stress was induced with alcohol caused a significant decrease in the group in which propolis was applied [
51]. The effect of CP is observed not only to increase in liver enzymes, but also on renal toxicity. When examining creatinine and BUN values in the present study, it was found that creatinine and BUN values of the CP group were significantly higher than other groups; whereas, BUN values of the CP + Propolis and CP + NP groups were significantly lower than the CP group. In the study conducted by Katanic et al. [
52] using spirea (Filipendula Ulmaria) extract to eliminate the side effects of CP, it was reported that CP significantly increased ALT, AST, and ALP values, which are among liver function tests, and creatinine and BUN values, which are among renal function tests, compared to the control group; on the other hand, the aforementioned extract (derived from the root area) decreased these parameters compared to the group to which CP was administered alone, but did not decrease the total protein, which is similar to the present study. The fact that propolis and NP had therapeutic effects against the negative effects of CP on blood parameters was associated with effective antioxidant compounds in their structure [
53,
54].
Antioxidant and anticancer effects of propolis have become the subject of recent studies [
55,
56]. In studies conducted with propolis and other antioxidant substances, it has been reported that propolis reduces the formation of free radicals and lipid peroxidation by means of flavonoids, which are available in structure of it [
55,
57]. Flavonoids display an antioxidant property by chelating with trace elements or radicals [
58]. It is reported that flavonoids protect unsaturated fatty acids against oxidants in cell membranes, just like ascorbates [
59]. Caffeic acid phenethyl ester (CAPE), one of the main ingredients of propolis, blocks the production of ROS [
60]. The results of previous studies reported that CP caused damage in the liver tissue by increasing the MDA level and decreasing antioxidant enzymes; however, antioxidant contributions minimized that damage [
50,
61]. In a study using grape seed extract to determine whether or not especially effective phenolic compounds could reduce liver damage [
50], it was found that superoxide dismutase and GSPx activities in the liver tissue were significantly higher in the CP + grape seed extract group compared to the CP alone. In the present study, similar results were obtained in the liver tissue (
Table 3). As a matter of fact, when examining the MDA level in the liver and kidney tissues, it was determined that the CP group was significantly higher than the other groups; whereas, GSH, GSH-Px and CAT levels decreased. It was found that the MDA level of the liver and kidney tissues of the CP + Propolis group significantly decreased compared to the CP group; whereas, GSH, GSH-Px and CAT levels increased significantly in the liver and kidneys (
Table 3). It is reported that various flavonoids and phenolic substances in propolis act like vitamin C and prevent lipids and other compounds from oxidating or they have the ability of scavenging free radicals by interrupting the oxidative damage process [
51,
57]. In addition, flavonoids inhibit the activity of enzyme systems including lipid peroxidation, thrombocyte aggregation, capillary permeability, fragility, cyclooxygenase and lipoxygenase [
59]. In parallel with the results of previous studies [
51,
57], the results of this study showed that propolis reduced oxidative damage caused by CP toxicity in tissues. In the study, propolis was used in the level of 100 mg/kg BW and the results were found to be effective on the antioxidant system. On the other hand, the effectiveness of NP, which was used at lower doses (10 and 30 mg/kg BW) than propolis (100 mg/kg BW) was compared with propolis. In the obtained results, it was primarily determined that CP + NP groups, similarly with the propolis group, reduced MDA levels in all tissues compared to the CP group and both CP + NP groups significantly increased GSH, GSH-Px and CAT levels in all tissues. It was found that liver and kidney MDA levels in the CP + NP groups were similar to the control group and GSH, GSH-Px and kidney CAT levels in the liver and kidney tissues also had similar results to the control group. These results showed that both doses of the NP application were effective on the recovery of oxidative damage caused by CP in tissues and it was found that especially the NP dose of 30 mg/kg BW was relatively more effective. In previous studies, the effect of nano-selenium on testicle tissues, in which oxidative damage was induced with CP, was examined and it was determined that nano-selenium increased antioxidant enzyme activities in testicle tissue [
62]. In a study, which was injected as intraperitoneal at a dose 5 mg/kg of Paclitaxel diluted in 1 mL saline once a week for four weeks, it was reported that it caused increases in 8—OHdG and DNA damage according to the control group in rats, but administration of propolis, at a dose of 50 mg/kg dissolved in 1 mL distilled water orally once daily for four weeks, alleviated the toxic effect of Paclitaxel by diminishing oxidation state and DNA damage, preserving cell energy [
63]. The content of the ration used in the study is given in
Table 4. When examining the content of propolis in this study, it was observed that the content of flavonoids was high (
Table 5 and
Table 6). Due to that content, it was observed to have a strong antioxidant property and show that effect by preventing lipid peroxidation caused by CP toxicity.
Molecules such as calcium, ceramide and the Bcl-2 family, as well as proteins such as p53, caspase and cytochrome-c, and also mitochondrials, usually have an important role in the regulation of apoptosis. Whether or not a cell tends toward apoptosis depends on the heterodymus or homodymus form of the Bcl-2 family genes. The Bcl-2 family consists of proapoptotic and antiapoptotic groups. If proapoptotic proteins are greater in a cell, the cell tends toward apoptosis. If antiapoptotic proteins are greater, the cell tends toward apoptosis less [
64,
65]. While Bax is a proapoptotic member, Bcl-2 is an antiapoptotic member. In this study, these two important parameters were examined to determine apoptosis. It is known that CP induces apoptosis in CP-sensitive cells by activating Bax, leads to the release of cytochrome C into cytosol, and activates caspase. In addition, Bcl-2 protein regulated in CP-resistant cells has been revealed to be an important factor in CP resistance [
66]. In the present study [
66], it was found out that CP apparently induced apoptosis because of Bax protein increases in the liver and kidney tissues, whereas Bcl-2 protein decreased (
Figure 1,
Figure 2,
Figure 4 and
Figure 5), which is compatible with the literature. In the CP-related apoptosis; propolis and NP applications increased the release of Bcl-2 protein and decreased the release of Bax protein. In a study, in which galangin was used to prevent the renal tubular damage induced by CP in rats [
67], it was determined that CP increased the expression of Bax, which is a proapoptotic protein, and decreased the expression of Bcl-2, which is an antiapoptotic protein. On the other hand, the use of galangin reversed that situation. According to the results of the present study, it was determined that apoptosis caused by CP in the liver and kidney tissues was reversed with the propolis and NP applications (
Figure 1,
Figure 2,
Figure 4 and
Figure 5). In addition, when examining the Bax/Bcl-2 rate, it was determined that there were significant differences between CP + NP-30 and propolis groups in terms of the liver and kidney tissues and the CP + NP-30 group gave effective results by significantly reducing the Bax level in tissues (
Figure 3 and
Figure 6). According to the results acquired, the fact that especially the second dose (30 mg/kg BW) of NP was much more effective than free propolis (100 mg/kg BW) at the cellular level was associated with the significant increase in propolis activity by nanotechnology, which thus made it possible to obtain better results despite using lower doses of propolis [
54].
Oršolić et al. [
68] administered propolis (50 mg/kg, ip) 7 and 3 days before inoculation of the Ehrlich ascites tumor (EAT) cells (2 × 10
6, ip) in mice, and then applied the cisplatin (5 or 10 mg/kg, ip) 3 days after the inoculation of EAT cells at 37 °C and 43 °C. After the experimental period, they reported that the combination of propolis + cisplatin 5 mg/kg at 37 °C resulted in tumor growth inhibition and increased the survival of mice; propolis also reduced the toxic and genotoxic effect of cisplatin on normal cells without affecting the cytotoxicity of cisplatin on EAT cells. In another study, which was designed to investigate the in vitro anticancer effect of propolis ethanolic extract (PEE) and its protective role against methotrexate (MTX) toxicity in the Ehrlich acid carcinoma (EAC) experimental model, Salem et al. [
69] reported that the PEE prompted cytotoxic effects in cancer cell lines and antitumor effects against the EAC mice model by reducing the tumor volume and count of viable tumor cells, with a significant increase in the life period of mice. Production of NP and its utilization in cancer cases are a new approach together with the progress of nanotechnology.