FormalPara Key Points

Treating tumor-bearing mice with ascorbate (AS) or acetylsalicylic acid (ASA), or ASA+AS, extended lifespan and ameliorated biochemical alterations.

ASA+AS exhibited superior antitumor efficacy than ASA or AS alone.

1 Introduction

Worldwide, cancer is one of the deadliest human diseases [1], with a higher mortality rate in developing countries than in developed countries [2]. Disturbingly, cancer causes the death of more than 8 million people per year [3]. Between 2020 and 2022, the global number of cancer-affected persons increased from 18 to 20 million. By 2035, it is estimated that cancer will affect 24 million people [1, 4] and will be the cause of death of 14.6 million people worldwide [5]; the number of affected people is expected to rise to 35 million by 2050 [6]. Cancer treatment involves oncological surgeries, radiation therapy, medical oncology, palliative care, and pain management [7]. Clinical chemotherapeutics are expensive and cause serious adverse effects, e.g. myelosuppression, anemia, and nephrotoxicity. As a result, there is a need to develop novel, effective, and well-tolerated alternatives [8].

Under cellular oxidative stress, when the oxidants rise at the cost of antioxidants, numerous antioxidants play key roles in controlling the concentrations of oxidants to avoid cellular damage. These antioxidants are either enzymatic, e.g. catalase, or non-enzymatic, e.g. ascorbate (AS) and glutathione (GSH) [9]. It has been established that oxidative stress and consequent inflammation are the main contributors to the development and progression of several diseases, including cancer [10]. The experimental evidence and observed efficacy of anti-inflammatory drugs demonstrate the link between cancer and inflammation [11]. Consequently, compounds with antioxidant and/or anti-inflammatory criteria are good prospective applicants for cancer treatment.

Acetylsalicylic acid (ASA) is a nonsteroidal anti-inflammatory drug (NSAID) applied to prevent blood clots and alleviate pain, fever, and inflammation. NSAIDs, such as aspirin, not only inhibit the production of prostaglandins but also have anti-inflammatory effects because they can neutralize a wide range of free radicals [12]. ASA acts mainly by disrupting the cyclooxygenase (COX) enzyme; however, recent studies have shown that aspirin also has other targets that can help fight cancer progression. ASA is beneficial in interfering with the energy metabolism of human breast and ovarian cancer cell lines. This is achieved by targeting key enzymes involved in the proliferation of cancer cells, both directly and through COX inhibition [13, 14]. Moreover, ASA has been found to inhibit cancer progression by interfering with proliferative pathways [15] and platelet-driven pro-carcinogenic activity [16], as demonstrated through in vitro enzymatic tests and cancer cell line investigations [17].

AS is a natural substance with antioxidant, anti-aging, and immunomodulatory properties [18]. In the 1970s, it was shown that 10% of terminally ill cancer patients who were administered 10 g of AS daily lived much longer than reasonable clinical expectation, while on the other hand, no cancer patients who were administered the conventional treatment without AS remained alive. Later, several investigations revealed that tumor cells tend to stop growing when exposed to high doses of AS. Moreover, research studies have suggested that taking AS supplements can impede tumor progression, metastasis, and cytokine production related to inflammation while enhancing the effects of chemotherapy [19]. AS is not harmful to healthy cells but it is found to be toxic to cancer cells. This property of AS makes it a promising option for potentially increasing the lifespan of cancer patients and improving their overall quality of life. Moreover, when used along with other anticancer agents, AS has been shown to enhance its effectiveness against cancer cells [18].

A recent study by El Sadda et al. [20] discovered that a combination of ASA and AS was a well-tolerated and effective treatment for hepatocellular carcinoma (HCC) rats, surpassing the effectiveness of doxorubicin treatment; however, testing this combination’s efficacy in treating other types of tumors has still not been investigated.

This study investigated the effectiveness of combining ASA and AS compared with treatment with ASA or AS alone in treating Ehrlich solid tumors in mice.

2 Materials and Methods

2.1 Chemicals

Chemicals and reagents of the highest available quality were used. AS (L-ascorbic acid) was purchased from Citreos Fine Chem (Surat, Gujarat, India), and ASA (75 mg tablets), under the tradename Aggrex (acetylsalicylic acid; Rameda, Sixth of October, Giza, Egypt), was obtained from a local pharmacy.

2.2 Experimental Animals

Adult female Swiss albino mice weighing 25–30 g were purchased from the Urology and Nephrology Centre, Mansoura University, Egypt. The mice were housed in a room with a controlled temperature (23 °C), humidity, and a 12-h light/dark cycle as per the guidelines outlined in the ‘Guide for the Care and Use of Laboratory Animals’ and approved by our Chemistry Department, Faculty of Science, Damietta University, Damietta, Egypt (Board no.: 200). The mice were kept in sterile plastic cages with a maximum of 10 animals per cage and had access to food and water ad libitum.

2.3 Tumor Cell Line

The Nile Center for Experimental Research in Mansoura, Egypt, gently provided the parent Ehrlich ascites carcinoma (EAC) cell line. To maintain the tumor cell line, 1×106 viable tumor cells in 1 mL saline were serially transplanted intraperitoneally into female Swiss albino mice every 7–10 days. For solid tumor induction, EAC cells were implanted subcutaneously into the right thigh of the hind limb, at a dose of 1×106 cells/mouse.

2.4 Animal Grouping

At random, the mice were grouped into eight groups (10 mice each). Four of these groups included healthy mice that were not injected with any solution (Healthy); healthy mice that were treated intraperitoneally with AS (4 g/kg) once daily for 10 consecutive days (AS); healthy mice that were treated by oral gavage with ASA (60 mg/kg) once daily for 10 consecutive days (ASA); and healthy mice that were treated intraperitoneally with AS (4 g/kg) and by oral gavage with ASA (60 mg/kg) once daily for 10 consecutive days (AS+ASA). Four additional groups studied the antitumor effects of AS, ASA, and AS with ASA on tumor-bearing mice. These groups included mice that were injected with EAC cells (subcutaneously with 1×106 tumor cells/mouse into the right thigh of the hind limb) [EAC]; mice bearing solid tumors that were treated intraperitoneally with AS (4 g/kg) once daily for 10 consecutive days, 10 days after solid tumor inoculation (EAC+AS); mice bearing solid tumors that were treated using oral gavage with ASA (60 mg/kg) once daily for 10 consecutive days, 10 days after solid tumor inoculation (EAC+ASA); and mice bearing solid tumors that were treated intraperitoneally with AS (4 g/kg) and by oral gavage with ASA (60 mg/kg) once daily for 10 consecutive days, 10 days after solid tumor inoculation (EAC+AS+ASA).

2.5 Samples

On day 21 of the experiment, six mice from each group were sacrificed, and a blood specimen and liver organ were obtained. The tumor was separated and weighed. One part of each blood sample, on ethylenediamine tetraacetic acid (EDTA), was used to measure the hemoglobin (Hb) level. The other portion of each sample was centrifuged and sera were used for assessing serum biochemical parameters. Livers were quickly dissected, rinsed with isotonic saline, and dried. Ten percent of liver tissue homogenates were then prepared in a cold phosphate buffer (weight/volume). After centrifugation, the supernatants were used to estimate hepatic lipid peroxidation and antioxidant parameters. The remaining mice (four in each group) were kept alive to measure the percentage increase in lifespan.

2.6 Measurements

2.6.1 Right Thigh Dimensions and Tumor Volume

Right thigh dimensions (length and width) and tumor volume (length and width of the femur solid tumor) were measured using a digital Vernier caliper. Tumor volume was calculated as follows: tumor volume (mm3) = 0.52 AB2, where A is the minor tumor axis and B is the major tumor axis [8].

2.6.2 Median Survival Time (MST) and Percentage Increase in Lifespan

Mice deaths were recorded daily from the day of tumor inoculation for 4 months, and MST and percentage increase in lifespan (%ILS) [21] were calculated:

$$\begin{gathered} {\text{MST in days }} = \, \left( {{\text{first mortality day }} + {\text{ last mortality day}}} \right) \, \times { 1}/{2} \hfill \\ \% {\text{ILS }} = \, \left( {\left[ {{\text{MST }}\left( {\text{treated group}} \right)/{\text{MST }}\left( {\text{control group}} \right)} \right] \, {-}{ 1}} \right) \, \times { 1}00. \hfill \\ \end{gathered}$$

2.6.3 Determination of Blood Hemoglobin Levels

Blood Hb levels were estimated in blood samples using the commercially available assay kit (Biodiagnostic, Dokki, Giza, Egypt). Briefly, via reacting with potassium ferricyanide and potassium cyanide, Hb is converted to cyanmethemoglobin, which can be measured at 540 nm.

2.6.4 Estimation of Oxidative Stress Parameters

The amount of malondialdehyde (MDA) was measured by thiobarbituric acid (TBA) assay, which is based on MDA reaction with TBA to give a red color absorbed at 535 nm [22]. Nitric oxide (NO) content was assayed per the method reported by Montgomery and Dymock [23]. Nitrite, in an acid medium, forms nitrous acid, which diazotizes sulfanilamide. Bright reddish-purple azo dye formed by diazotization product coupled with N-(1-naphthyl)ethylenediamine was measured at 540 nm. Estimation of GSH in tissue homogenate was carried out per the method of Beutler et al. [24] using Elman’s reagent, and catalase activity was determined using the method of Chance and Herbert [25]. The idea is for the production of chromic acetate via a reaction between dichromate and hydrogen peroxide in the presence of acetic acid under heating. The produced chromic acetate is measured calorimetrically at 620 nm. Determination of total antioxidant capacity (TAC) in liver tissue homogenate was performed according to the method of Koracevic et al. [26]. Via a Fenton reaction, Fe-EDTA interacts with hydrogen peroxide, forming hydroxyl radicals. These radicals break benzoate, leading to the liberation of thiobarbituric acid reactive species. Antioxidants in the tested sample suppress thiobarbituric acid reactive species. This reaction was measured spectrophotometrically and the inhibition of color development was defined as the antioxidant activity.

2.6.5 Serum Biochemical Parameters

Kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total bilirubin, creatinine, urea, total cholesterol, triglycerides, low-density lipoprotein-cholesterol (LDL-C), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) purchased from Biodiagnostic were utilized following the guidelines of the manufacturer. Albumin was measured by reacting with bromocresol green in an acidic medium to form a blue-green-colored complex read at 620 nm. AST and ALT activities were assessed by measuring the amount of oxaloacetate and pyruvate produced over a specific time after reacting with 2,4-dinitrophenyl hydrazine to form a brown color read at 546 nm. Bilirubin reacts with the diazonium salt of sulfanilic acid to produce azobilirubin read at 535 nm in an acid medium. Creatinine was estimated by reacting with alkaline-picrate, producing an orange complex read at 492 nm. To estimate urea level, ammonia released from urea under the action of urease was allowed to interact with a mixture of salicylate, sodium nitroprusside, and hypochlorite to produce a green color read at 630 nm.

Regarding the triglycerides test, glycerol produced under the lipase action on triglycerides was converted into glycerol-3-phosphate by glycerol kinase and then oxidized by glycerol phosphate oxidase, forming dihydroxyacetone phosphate and hydrogen peroxide. A reaction between hydrogen peroxide, 4-aminoantipyrene, and chlorophenol in the presence of peroxidase produced a red color read at 546 nm. In the total cholesterol test, cholesterol esters are converted by cholesterol esterase into free cholesterol, which is then oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide reacts with phenol and 4-aminoantipyrene to form a red dye read at 512 nm. To estimate LDL-C, serum was added to a precipitating reagent and centrifuged. Cholesterol in the supernatant was then assessed using the same enzymatic method for total cholesterol. Additionally, CK-MB activity was measured by following the rate of formation of nicotinamide adenine dinucleotide phosphate reduced form (NADPH) at 340 nm. Finally, LDH activity was measured by monitoring the rate of transformation of nicotinamide adenine dinucleotide reduced form/oxidized form (NADH/NAD+) at 340 nm.

2.7 Statistical Analysis

Statistically, results were expressed as mean ± standard deviation (SD) and the statistical analysis was performed using Instat software, version 3.10 (GraphPad, Inc., Sorrento Valley, San Diego, CA, USA). For comparing means of more than two groups, the statistical analysis was performed using one-way analysis of variance (ANOVA) for parameters with Gaussian distribution, followed by the Tukey–Kramer multiple comparisons test. For data with non-Gaussian distribution, the Kruskal–Wallis test was used, followed by Dunn’s test for multiple comparisons. For all analyses, p-values of < 0.05, < 0.01, and < 0.001 were assumed significant, very significant, and extremely significant, respectively.

3 Results

Tumor implanted in the right thigh of a mouse (Fig. 1) resulted in a 54.58% increase in the thigh’s dimensions, where its length and width were increased from 11.9 × 14.7 mm on day 11 from tumor inoculation to 16 × 16.9 mm on the day of sacrifice (Day 21). After treatment with ASA, the thigh’s dimensions were decreased by 2.9%; length and width decreased from 10.1 × 15.9 mm to (12 × 13 mm). Similarly, treatment with AS or the combination reduced the thigh’s dimensions by 20.53% and 21.8%, respectively (from 13.5 × 14.9 to 11.5 × 13.9 mm, and from 12.5 × 14.9 to 10.5 × 13.5 mm, respectively) (Table 1).

Fig. 1
figure 1

Parent cell line (ascitic Ehrlich) and solid Ehrlich tumor implanted in the right thigh of a mouse

Full size image
Table 1 Changes in the dimensions of the right thigh of tumor-bearing mice groups throughout the experimental period until sacrifice
Full size table

Effects on tumor weight and tumor volume are represented in Table 2. There were remarkable reductions in the mean weight and volume of the separated tumors from mice legs in all treated tumor-bearing mice groups. The mean tumor weight in the case of treatment with ASA was 1.055 g, with AS 0.945 g, and 0.823 g with the combination compared with 1.56 g in untreated tumor-bearing mice. Regarding tumor volume, treatment of the tumor-bearing mice with ASA, AS, and the combination diminished mean tumor volume from 1792 mm3 in untreated tumor-bearing mice to 1145, 1075, and 965.61 mm3, respectively. The improvements shown by the combination were significant (p < 0.05) and superior, and the improvements were better with AS than with ASA (Table 2).

Table 2 Mean weight and volume of solid tumors of the tumor-bearing mice groups on the day of sacrifice
Full size table

Median survival time (MST) and percentage increase in lifespan (%ILS) were calculated (Fig. 2) to evaluate the mortality rate by recording the mortality from the day of tumor inoculation daily for 4 months. Treatment with AS, ASA, and the combination prolonged the lifespan; these agents showed extended MST from 54 days in the EAC group to 84, 81.5, and 93.5 days in the treated EAC-bearing groups, respectively. They also showed a %ILS of 55.56, 50.93, and 73.15% in mice in the EAC-bearing groups treated with AS, ASA, and the combination, respectively, compared with the untreated tumor-bearing group. There was a significant increase in Hb content in the mice in the EAC-bearing group treated with the combination of AS and ASA (p < 0.01) compared with the untreated EAC group, which showed a significant (p < 0.001) decrease in Hb content compared with the healthy control group. However, the Hb content in mice in the EAC-bearing groups treated with either AS or ASA alone showed a non-significant increase (p > 0.05) compared with the untreated EAC group. Healthy groups administered AS, ASA, or the combination displayed similar Hb content to the healthy control group (Fig. 2).

Fig. 2
figure 2

Median survival time, percentage increase in lifespan, and mean blood hemoglobin levels of different mice groups. For MST and %ILS, n = 4 mice in each group, and for Hb level, n = 6 mice in each group. Results are expressed as mean ± SD. ap < 0.001 versus the healthy group; bp < 0.01 versus the EAC group. AS ascorbate, ASA acetylsalicylic acid, EAC Ehrlich ascites carcinoma, %ILS percentage increase in lifespan, Hb hemoglobin, SD standard deviation, MST mean survival time

Full size image

As illustrated in Table 3, liver function tests include serum ALT, AST, bilirubin, and albumin. There was a significant (p < 0.001) increase in both ALT and AST activities and an increase in bilirubin (p < 0.001) as well as a decrease (p < 0.05) in serum albumin in the EAC control group compared with the corresponding healthy control group. After treatment with AS, AST activity was decreased (p < 0.001) and albumin level was increased (p < 0.05); after ASA treatment, AST activity was reduced (p < 0.001); and after treatment with AS plus ASA, serum ALT and AST activities were decreased (p < 0.05 and p < 0.001, respectively) while albumin level was increased (p < 0.001) toward normal ranges. The effect of treatment with the combination was significantly (p < 0.01) improved than with ASA on albumin level. In healthy groups, which administered AS, ASA, or the combination, all liver function tests showed non-significant changes (p > 0.05) compared with the healthy group. In addition, kidney function tests included serum creatinine and blood urea. There were elevations (p < 0.001) in serum creatinine and blood urea values in the EAC group compared with the healthy control group. After treatment of tumor-bearing mice with AS, ASA, or the combination, there were decreases (p < 0.05 to p < 0.001) in creatinine and blood urea in all treated groups compared with the untreated EAC group. Compared with the EAC+ASA group, treatment of tumor-bearing mice with the combination showed a more significant (p < 0.001) improvement in both creatinine and urea levels, while AS alone showed a more significant (p < 0.05) improvement in urea levels. Regarding healthy groups administered AS, ASA, or the combination, creatinine and urea showed non-significant (p > 0.05) changes compared with the healthy group (Table 3).

Table 3 Mean levels of alanine aminotransferase, aspartate aminotransferase, albumin, bilirubin, creatinine, and urea in sera of different mice groups
Full size table

There were significant increases (p < 0.001) in cholesterol, LDL-C, and triglycerides in the EAC group, and significant decreases (p < 0.01 to p < 0.001) were noted in the EAC group after treatment with AS, ASA, or the combination of both. Compared with the treatment of tumor-bearing mice with AS or ASA, treatment with the combination showed improved (p < 0.001) outcomes in triglycerides. Administration of AS, ASA, or the combination to healthy mice resulted in non-significant differences (p > 0.05) in cholesterol, LDL-C, and triglyceride levels compared with the healthy control group. Regarding heart function tests, there was a significant increase (p < 0.001) in LDH and CK-MB activities in EAC controls compared with the healthy controls. After treatment, serum LDH activity was decreased (p < 0.001) in the treated tumor-bearing groups compared with EAC controls. Compared with the tumor-bearing mice treated with AS or ASA, treatment with the combination showed improved outcomes in LDH (p < 0.01 and p < 0.001, respectively), while healthy groups treated with AS, ASA, or the combination showed no significant differences (p > 0.05) in CK-MB and LDH activities compared with the healthy control group (Table 4).

Table 4 Mean levels of cholesterol, low-density lipoprotein-cholesterol, triglycerides, creatine kinase-MB, and lactate dehydrogenase in the sera of different mice groups
Full size table

As shown in Table 5, tumor progression was associated with a significant (p < 0.001) increase in MDA and NO levels, accompanied by remarkable decreases (p < 0.001) in antioxidant (TAC, GSH, and catalase) levels. Treatment of tumor-bearing mice with AS, ASA, or the combination caused significant decreases (p < 0.001) in MDA and NO levels, and increased the levels of all studied antioxidants (p < 0.05 to p < 0.001). Administration of the combination for tumor treatment was significantly successful (p < 0.001) in decreasing MDA and increasing (p < 0.001) the antioxidant levels in comparison with administration of AS or ASA alone for the same purpose. In the healthy mice groups, administration of AS increased catalase activity (p < 0.05), administration of AS or ASA decreased NO levels (p < 0.001), and administration of the combination reduced levels of MDA (p < 0.01) and NO (p < 0.001) and increased levels of TAC (p < 0.001), GSH (p < 0.001), and catalase (p < 0.05).

Table 5 Mean levels of malondialdehyde, nitric oxide, total antioxidant capacity, reduced glutathione, and catalase in liver tissue homogenate of different mice groups
Full size table

4 Discussion

First, we did not record any toxicity symptoms according to administration of ASA (at a dose of 60 mg/kg) or AS (at a dose of 4 g/kg), or their combination, to healthy mice, and they did not show any significant pathological changes in all the studied parameters. In contrast, they increased antioxidant levels, and decreased MDA and NO levels, suggesting that ASA- and AS-applied doses are well-tolerated for healthy mice. Our results are in unity with Shilpi et al. [18] and Bhattacharyya et al. [27], who reported that AS and ASA are well-tolerated for therapeutic use; however, long-term use or high doses of ASA may cause oxidative stress and result in gastrointestinal erosions and apoptotic lesions [27, 28].

By tumor implantation in the right thigh of a mouse, an increase of 54.58% was recorded in the thigh dimensions in the period from day 11 to day 21 (the day of sacrifice) due to uncontrolled tumor growth. After treatment with ASA, AS, or the combination, we detected a decrease in the thigh dimensions by 2.9%, 20.53%, and 21.8%, and reductions in mean volumes of the separated tumors from mice legs by 40%, 36%, and 46%, respectively, due to tumor shrinking. Undoubtedly, as presented, the regression in solid tumors caused by treatment with the combination was superior. These observations indicate that ASA, AS, or the combination can inhibit tumor growth; however, the combination is more powerful. In 2008, Chen and colleagues found that an intraperitoneal injection of AS (4 g/kg) reduced the size of aggressive glioblastoma, pancreatic, and ovarian tumors in mice by 41–53% [29].

One of the key factors in evaluating a successful compound for fighting cancer and tumors is its ability to prolong lifespan. An increase in lifespan of at least 25% is hypothesized as an effectual antitumor response [30]. Our current research shows that ASA, AS, and their combination may help prolong lifespan and fight cancer. The untreated tumor-bearing mice had an MST of 54 days. By treatment, the MST improved to 81.5, 84, and 93.5 days, with a %ILS of 50.93%, 55.56%, and 73.15%, for ASA, AS, and the combination, respectively. One cause behind the short lifespan may be tumor-associated oxidative stress that can cause surrounding healthy cell death, negatively affecting survival [20]. Furthermore, cancer cells thrive by depriving healthy cells of normal resources of living, which may make them die faster. They can also increase their production of enzymes that break down tissue. Therefore, if an antitumor compound could terminate the tumor growth, the lifespan could be increased. Our findings are supported by Shilpi and colleagues [18], who demonstrated that AS may extend survival and improve the quality of life of cancer patients. In addition, if administered with other anticancer agents, AS can synergize their anticancer action. In this context, AS improved the antitumor effects of aspirin. The delay in tumor cell division may be responsible for the prolonged lifespan of mice treated with ASA and/or AS [31].

Cancer chemotherapy can cause myelosuppression and anemia. Anemia is mainly caused by a drop in red blood cell (RBC) count or Hb synthesis due to variable factors, including deficient Fe, hemolysis, or myelopathic disorder [32]. Carcinogenesis generates toxic diffusible reactive oxygen species (ROS) that damage biomolecules and cells and contribute to malignant transformation. RBC membranes are vulnerable to oxidative stress due to their high polyunsaturated fatty acids and iron content, which catalyzes ROS production [33]. In the current study, anemia in tumor-bearing mice was ascertained by the decrease (p < 0.001) in Hb observed in the untreated EAC group. Treating mice with AS plus ASA resulted in a significant increase (p ˂ 0.01) in Hb content compared with untreated mice. Treating with either ASA or AS alone showed a non-significant increase. Our results are in line with Pandya et al. [34], Islam et al. [35], and Kandar et al. [36], who registered the return of Hb content to almost normal levels as a result of tumor treatment. Our findings suggest that combining AS and ASA can protect hematopoietic activity without myelotoxicity.

Several studies have shown that tumors cause functional disturbances in liver and kidney organs [37,38,39]. In this study, tumor inoculation increased (p < 0.001) ALT, AST, bilirubin, creatinine, and urea, and decreased (p < 0.05) serum albumin. These disturbances indicate hepato-nephrotoxicity due to tumor progression and increased free radical production, which results in oxidative stress in all tissues. These findings parallel those of Salem et al. [40] and Aboseada et al. [41], who demonstrated that EAC increased serum urea and creatinine levels in female mice. The increase in kidney function biomarkers may be linked to tumor-induced kidney damage. This can lead to a decrease in the glomerular filtration rate and renal tubular reabsorption, and consequently, this reduces the excretion of urea and creatinine, causing their levels to increase in the bloodstream [42]. After tumor treatment, all these elevated levels were decreased significantly, except bilirubin, while serum albumin levels were increased, suggesting hepato-renal protective effects. The combination of the anti-inflammatory properties of ASA and antioxidant properties of AS exhibited improved hepato-renal protective effects than AS or ASA, while the hepato-protective effects of AS or ASA were nearly similar. Compared with the EAC+ASA group, treatment of tumor-bearing mice with the combination treatment showed significant (p < 0.001) improvement in both creatinine and urea levels, while AS alone showed significant (p < 0.05) improvement in urea level. Consistent with this, Şahin et al. attributed the conservative impact of losartan against hepatic and renal destruction induced by acetaminophen to the antioxidant and anti-inflammatory characteristics [43].

Pathological changes in lipid profile (triglycerides, cholesterol, and LDL-C) were also recorded in EAC-bearing mice. Significant increases (p < 0.001) in cholesterol, LDL-C, and triglycerides in the EAC group and significant decreases (p < 0.001) in the EAC group after treatment were noted. Compared with treating tumor-bearing mice with AS or ASA, treatment with the combination showed improved (p < 0.001) outcomes. AS is involved in decreasing cholesterol since it plays a crucial role in breaking down cholesterol in the liver. Under its sufficiency, bile acids synthesis from cholesterol speeds up, consequently decreasing circulatory cholesterol [44].

When the heart experiences damage or stress due to low oxygen, it releases cardiac enzymes. LDH and CK are mainly released from damaged cardiac tissue, while AST primarily leaks from damaged hepatic tissues. However, AST also leaks from other damaged tissues, including cardiac tissue [45]. Saad et al. [5] reported CK and LDH elevations in their Ehrlich-induced oxidative tissue damage model. Our current work showed a significant increase (p < 0.001) in AST, LDH, and CK activities in EAC controls. These elevations may be explained by the rise in free radicals confirmed via the observed elevations in NO and MDA levels. They attack lipids in cellular membranes, increasing their permeability and leading to the outflow of these enzymes from the damaged membranes into the circulatory blood. This is linked to several studies on cardiotoxicity, including those by Basal et al. [46], Cui et al. [47], and Shosha et al. [48]. In addition, Kong et al. [49] found that the myocardium produces low levels of antioxidants, such as superoxide dismutase (SOD), catalase, and GSH, which makes it highly vulnerable to cardiac damage caused by free radicals. After different treatments, LDH and AST activities were decreased in the treated tumor-bearing groups. Compared with treating tumor-bearing mice with AS or ASA, treatment with the combination showed improved outcomes in LDH. Again, these improved outcomes may be attributed to gathering the anti-inflammatory properties of ASA and the antioxidant properties of AS together.

Previous studies have shown that free radicals play a role in the initiation and promotion of cancer. Patients with tumors have been found to have high MDA concentrations [50]. MDA can lead to modifications in the structures of cellular proteins, phospholipids, or nucleic acids through binding to their amine groups. In the nucleus, MDA can bind with the DNA nitrogenous bases, forming bridges between the strands and resulting in DNA replication stopping or initiating mutagenic effects [51]. Various diseases have been connected with high NO levels, e.g. diabetes [52], cardiomyopathy [48], etc. Oxygen binds excessive NO, producing free radicals (nitrite and peroxynitrite) [53]. Increased catalase activity offers protection versus cancer. It decreases cellular concentrations of reactive species, particularly hydrogen peroxide. This reverses ‘the malignant phenotype’. Overexpressed catalase in the breast cell line (MCF-7) impaired the ability of cancer cells to proliferate or migrate, and increased the sensitivity to chemotherapeutic agents such as cisplatin and doxorubicin [54]. GSH peroxidase deals with organic hydroperoxides and decomposes hydrogen peroxide in the presence of GSH [50]. GSH, a significant non-protein thiol in living organisms, has multiple roles as an antioxidant agent and can protect against cancer via decreasing oxidative stress. GSH aids in vitamin E regeneration that arrests lipid peroxidation in membranes. Furthermore, GSH can directly scavenge superoxide anion radicals [55].

Our results in the current study highlighted an association of EAC tumor progression with significant (p < 0.001) increases in MDA and NO levels, accompanied by remarkable decreases (p < 0.001) in antioxidant levels: TAC, GSH, and catalase. These findings confirm earlier comparable experimental findings by Aboseada et al. [41] regarding the oxidative stress environment in the tumor-host body, and consequently emphasize the inclusion of support oxidative stress and inflammation in cancer pathogenesis as important contributors. Compatible findings were also shown in HCC-bearing rats [56]. Cancer patients are prone to a platelet-related disorder called hyper-aggregability, which is a chief cause of thrombotic events in such patients [57]. Through impairment of the antioxidant defense system or by triggering platelet aggregation, cancer can increase lipid peroxidation. Platelet aggregation leads to the production of molecules such as thromboxane and prostaglandins, which are sources of free radicals. High NO and MDA in this study indicate inflammation with oxidative stress. An increase in NO may be attributed to its ability to inhibit platelet aggregation and adhesion. This finding is consistent with the findings reported by Saad et al. [30]. According to a previous study by David et al. [58], high levels of MDA can cause the release of many inflammatory cytokines, which then trigger the production of more cytokines and lead to increased oxidative stress [59]. Kalavacherla et al. [60] and Saad et al. [30] found a link between lipid peroxidation and inflammation and confirmed MDA as a sensitive marker for inflammation. They also demonstrated the involvement of oxidative stress in chronic inflammation.

If the body’s endogenous antioxidants become powerless to defend against ROS/reactive nitrogen species (RNS), exogenous antioxidants are necessary. The treatment of mice with tumors resulted in significant reductions (p < 0.001) in MDA and NO levels, alongside increases in the levels of all the studied antioxidants (p < 0.05 to p < 0.001). Administration of the combination of both ASA and AS was significantly more successful (p < 0.001) in reducing MDA levels and increasing antioxidant levels (p < 0.001) compared with only using AS or ASA for the same purpose. It has been suggested that NSAIDs, including ASA, not only inhibit prostaglandin synthesis but also possess anti-inflammatory properties due to their capacity to scavenge free radicals. These drugs are effective against a range of ROS, including O–2, OH–, and HOCl, as well as RNS such as NO and peroxynitrite (ONOO–) [12]. AS has been found to possess antioxidant properties that can help reduce the harmful damage caused by free radicals. When AS is ingested, it has electrons that it can give to reactive species, which then get neutralized to water and no longer pose a threat to the body [61]. The oxidized version of AS, dehydroascorbic acid, has a ring structure that helps balance out the positive charge and prevents it from harming healthy cells [62]. Elwood et al. reported that ASA inhibits cancer progression through interference with proliferative pathways, cancer-associated inflammation, and platelet-driven pro-carcinogenic activity, based on in vitro enzymatic assays and human cancer cell line studies [17]. AS has an antitumor effect due to several reasons. First, it can trigger a high rate of apoptotic pathways in tumor cells; second, it can cause pro-oxidant damage to the tumor cells that cannot be repaired; and lastly, AS can be oxidized in the plasma and turned into dehydroascorbic acid, which is highly toxic to tumor cells [18, 63, 64]. The oxidation/reduction capacity of AS is important in its biological activity. AS is critical for regulating many enzymes, including those involved in epigenetic and hypoxia-inducible factor (HIF) regulation. AS reduction to ions, e.g. iron, maintains various roles in catalysis. Reducing ferric to ferrous ions ensures Fe-dependent enzymatic processes are carried out, such as those that possess a role in DNA synthesis and/or epigenetics. One example is the post-translational modification of collagen, which makes AS necessary for connective tissue function, especially in blood vessel walls [62].

Another example is the post-translational regulation of HIF-1 levels by members from the hydroxylases family. Lacking AS or iron interferes with the activity of hydroxylases, resulting in more HIF-1 stabilization and activation [65]. HIF-1 regulates the transcription of many genes implicated in cancer biology, impacting cell immortality, angiogenesis, and resistance to therapy. High proliferation impairs cancer cells’ access to nutrients, leading to anaerobic cellular metabolism [66]. Vitamin C ensures the proper functioning of methylcytosine oxidase ten-eleven translocation (TET) proteins. AS affects TET proteins by enabling the reduction of ferric to ferrous ions and as a cofactor for correct enzyme folding. TET genes act as tumor suppressors, but their mutation in many cancers reduces expression. K-Ras oncogene inhibits TET expression, leading to decreased 5-hydroxymethylcytosine (5-hmC) levels and increased 5-methylcytosine (5-mC), resulting in reduced expression of pro-apoptotic genes [62]. The current study suggests that ASA and AS may work together to protect against cancer and prevent the creation of harmful free radicals. This protective effect could help keep healthy cells and organs safe from the damaging effects of reactive oxygen/nitrogen intermediates. Therefore, using the combination of antioxidants ASA and AS could be a potentially effective treatment to prevent cancer.

There are some limitations to our study. We were unable to use AS-deficient mice, and were unable to use animals other than mice with different solid tumors. Therefore, further research is necessary to validate the findings in bigger and more varied animal models.

5 Conclusions

In vivo, we treated tumor-bearing mice using ASA 60 mg/kg, AS 4 g/kg, or a combination of both for 10 consecutive days. This treatment improved the levels of ALT, AST, albumin, creatinine, urea, cholesterol, LDL-C, triglycerides, LDH, MDA, NO, TAC, GSH, and catalase, bringing them closer to normal ranges. The treatment decreased solid tumor weight and volume, reduced lipid peroxidation, enhanced antioxidant levels, protected liver, kidney, and heart tissues from tumor-induced damage, raised blood Hb levels, amended lipid profile, and extended lifespan. AS displayed improved antitumor impact than ASA, and the combination of both showed much improved outcomes. ASA and AS are low-cost, well-tolerated, readily available, and effective as anti-inflammatory, antioxidant, and antitumor agents. Therefore, combining ASA with AS might be helpful in cancer therapy after further investigations to validate our findings.