Aishah E. Albalawi, Norah A. Althobaiti, Reem Hasaballah Alhasani, Sultan F. Alnomasy

1Faculty of Science, Department of Biology, University of Tabuk, Tabuk 47913, Saudi Arabia

2Biology Department, College of Science and Humanities, Al Quwaiiyah, Shaqra University, Al Quwaiiyah 19257, Saudi Arabia

3Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia

4Department of Medical Laboratories Sciences, College of Applied Medical Sciences, Al-Quwayiyah, Shaqra University, Riyadh, Saudi Arabia

ABSTRACT

KEYWORDS: Cancer; Ehrlich solid tumors; Treatment; Pistacia atlantica; Herbal medicines; Natural product; in vivo; Mice

1. Introduction

Among the many diseases that threaten human health and life, cancer is one of the most important reasons for death in the world[1]. The disease is characterized by the disruption of the normal cell cycle, which causes unplanned cell growth and subsequently the absence of differentiation, characterized as malignant developments[2]. Chemotherapy has long been one of the most important cancer therapies, which can be used alone or in combination with other treatments, such as surgical processes and radiotherapies[3]. At present, a wide range of chemical drugs are used to prevent and treat various types of cancers; however,the studies demonstrated that the current synthetic medications are accompanied by some adverse side effects such as damage to the normal tissues and cells, inhibition of bone marrow function,nausea, vomiting, alopecia, etc[4,5]. Therefore, the search for a new anticancer drug, especially from high-performance and lowtoxicity sources, has always been of interest to researchers. In recent years, herbs and their derivatives and products have attracted attention due to their high effectiveness, low toxicity, availability,and cheapness, as alternative drugs in the treatment of cancers[6,7].Wild pistachio [Pistacia atlantica (P. atlantica) Desf.], from the Anacardiaceae family, broadly grows in central Asia, particularly in Saudi Arabia, Syria, etc[8]. The plant has various therapeutic potential as traditional medicine and is used to treat or improve some diseases and conditions such as digestive problems,vomiting, diuretic, demulcent, dyspepsia, skin diseases, wound healing, disorders of liver and kidney, gastrointestinal disorders,toothache diseases, etc[9]. In addition, it also has several other pharmacological properties such as antinociceptive, antidiabetic,anti-inflammatory, antiproliferative, hepatoprotective, antioxidant,immunomodulatory, antimicrobial, etc[9,10]. Several studies also have reported the in vitro and in vivo anticancer activity of different parts of this plant against colonic adenocarcinoma (COLO205),human gastric carcinoma (AGS), human colorectal adenocarcinoma cell (HT29 cells), cervix adenocarcinoma (HeLa cells), etc[10-12].The Ehrlich tumor as a murine mammary adenocarcinoma is a solid form of the tumor which is broadly used in the assessment of efficacy and innovation of new anti-tumor agents[13,14]. The current survey was designed to assess the anti-tumor effects of P. atlantica methanolic extract (PAME) compared with cyclophosphamide(CP) against the Ehrlich solid tumors (EST) in mice.

2. Materials and methods

2.1. Reagents and drugs

All chemicals, Folin-Ciocalteau’s reagent, as well as Mayer and Dragendorff’s were obtained from Sigma-Aldrich (St. Louis, MO).MyBio-Source Mice Carcinoembryonic Antigen Elisa Kit was obtained from MyBio-Source, San Diego, USA. Liver and kidney enzymes kits were prepared from Roche, Germany. The mini-VIDAS?AFP kit was prepared from bioMérieux, Marcyl’Etoile,France. The oxidative stress kits, the antioxidant enzymes kits, and TNF-α ELISA kit were purchased from Abcam, USA. RNeasy tissue kit and the complementary DNA (cDNA) synthesis kit were prepared from Qiagen Co, Germany. All the chemicals used were of analytical grade.

2.2. Plant material

Fruits of P. atlantica were purchased from a marketplace in Riyadh city, Saudi Arabia and after being determined by a botanist,a sample of voucher was deposited at the herbarium of Shaqra University, Saudi Arabia for further experiments.

2.3. Preparation of methanolic extract

By percolation method, 200 g of the dried fruits were extracted with methanol for 72 h at 21 ℃. Next, the obtained extracts were passed over a filter paper and were evaporated by a rotary evaporator in a vacuum at 55 ℃ and preserved at ?20 ℃ until examination[15].

2.4. Phytochemical analysis

The primary phytochemical analysis of the PAME was done to test alkaloids, tannins, glycosides, saponins, and flavonoids,including Mayer and Dragendorff’s reagents to identify the alkaloids, Mg and HCl for identification of flavonoids, 1% gelatin along with 10% NaCl solutions to determine the tannin, the combination of FeCl2and H2SO4to examine the glycosides, and saponin determined by the capacity to produce suds[16].

2.4.1. Total phenol content

Total phenol content was studied using the Folin-Ciocalteau’s reagent colorimetric method using gallic acid as a standard[17].In brief, 0.2 mL of PAME was added to Folin-Ciocalteu solution and then 2 mL of sodium carbonate was added to the combination. Followed by the incubation (30 min) in the darkness,the absorbance of the mixture was recorded at 760 nm using a spectrophotometer (Jenway 6715, Cole-Parmer, Eaton Socon, UK);the results were described as mg gallic acid (GAE)/g dry weight.

2.4.2. Total flavonoid content

The content of total flavonoid was assessed based on the technique explained by Phuyal et al.[18] using aluminum chloride(AlCl32%) colorimetric and quercetin as a standard. Briefly,0.2 mL of extract or standard solution was added to 0.2 mL of aluminum chloride and 0.1 mL of 33% aqueous acetic acid and stirred well. In the end, the combination was reached a volume of 5 mL with ethanol. Lastly, the absorbance was measured at 430 nm and the total flavonoid was gained through a standard curve as mg quercetin equivalent per gram dry weight (mg QE/g DW).

2.4.3. Tannin condensed contents

The method described by Broadhurst and Jones[19] was used to determine the tannin condensed contents of PAME. After mixing the extract and control with vanillin-HCl (5 mL), the absorbance was measured at 510 nm. The content was presented as mg catechin (CE)/g DW.

2.5. Anti-tumor effects of PAME

2.5.1. Animals

Fifty-six female Swiss albino mice weighing 20-25 g and aged 6–8 weeks old were used to establish the animal model of EST.Mice were held in a colony room under a 12:12 h light/ dark cycle at (21 ± 2) ℃ with ad libitum access to food and water.

2.5.2. The Ehrlich ascites tumor cell line and induction of EST in mice

The cell line of the Ehrlich ascites tumor was prepared from the American Type Tissue Culture Collection (Manassas, USA) and was adjusted into 2.5×106cells/mL in sterile saline solution using a Neubauer hemocytometer.

2.5.3. Study design

Swiss albino mice were divided into seven groups (8 mice per group). At first, EST was established in 32 mice by intramuscular injection of 200 μL of cells (2.5×106cells/mL) in the right thigh of mice[20]. On the 5th day after the EST inoculation, the mice were randomly divided into groups including non-EST mice receiving normal saline (C1), EST mice receiving normal saline(C2), EST mice treated with cyclophosphamide (CP) (5 mg/kg)intraperitoneally once a day for 14 d (C3), EST mice treated with PAME 50 mg/kg orally once a day for 2 weeks (Ex1), and EST mice treated with PAME 100 mg/kg orally once a day for 2 weeks(Ex2). The selection of doses of PAME was based on the pilot experiments and previous study[21]. Furthermore, two groups of healthy mice were used to evaluate the sub-acute toxicity of PAME including mice treated with PAME 50 mg/kg orally once a day for 2 weeks (C4), mice treated with PAME 100 mg/kg orally once a day for 2 weeks (C5)[13].

2.5.4. Blood sampling

At first, the mice were euthanized with ketamine (100 mg/kg)along with xylazine (10 mg/kg). Then, their abdomen cavities were opened. Blood samples were obtained from the heart of mice.After centrifuging the blood samples at 6 000 rpm for 10 min, the obtained sera were separated and kept at ?80 ℃ until examination.

2.5.5. Body weight changes

Mice were weighed on day 5 and day 19. The percentage of the obtained weight was assessed using the equation[21]: % weight gain= (Mice weight on 19th day/Mice weight on 5th day?1)×100.

2.5.6. Tumor sampling

After sterile aseptically removing of tumors in mice of C2, C3,Ex1, and Ex2 groups, they were weighed, and their dimensions were recorded. For further analysis, tumors were equally divided into two parts: the first part was kept at ?80 ℃ for molecular tests,and the second part was kept at ? 20 ℃ for other tests.

2.5.7. Tumor growth inhibition

The antitumor activity of PAME was measured by calculating the tumor volume and tumor growth inhibition rate. Tumor volume was calculated by the vernier caliper after the 7th day of the treatment by the equation: tumor volume (mm3) = 4π(A/2)2×(B/2). So that, A and B are the small and big tumor axes. Tumor growth inhibition rate was determined based on the below formula:

Tumor growth inhibition rate (%)= (The mean tumor weight of control group?the mean tumor weight of treated group)/The mean tumor weight of control group C2×100[13].

2.5.8. Evaluation of the tumor markers

The level of alpha-fetoprotein (AFP) was determined using a mini-VIDAS?AFP kit based on the manufacturer’s protocols. In addition, the serum level of carcinoembryonic antigen (CEA) was also evaluated by MyBio-Source Mice Carcinoembryonic Antigen Elisa Kit.

2.5.9. Evaluation of serum levels of liver enzymes

Serum levels of some liver enzymes including alanine aminotransferase(ALT) and aspartate aminotransferase (AST) in mice of all groups were measured using the commercial diagnostic kits[22].

2.5.10. Evaluation of serum levels of kidney enzymes

The level of serum creatinine (Cr) and blood urea nitrogen (BUN)were determined in mice of all groups using the commercial diagnostic kits[22].

2.6. Cellular mechanisms

2.6.1. Evaluation of the oxidative stress markers

To evaluate the lipid peroxidation, tumor homogenates were studied by biodiagnostic analysis kits based on the malondialdehyde(MDA) production using the thiobarbituric acid (TBA) methodology explained by of El-Aarag et al.[23]; in addition, NO production(Abcam, USA) was measured in the hepatic suspension technique defined by Green et al.[24]. Briefly, in this method, the thiobarbituric acid reacts with MDA in the liver homogenate to produce a thiobarbituric acid reactive product. Then, the absorbance was calculated at 534 nm.

2.6.2. Evaluation of the antioxidant enzymes

The levels of some enzymes involved in antioxidant activities such as glutathione peroxidase (GPx), catalase enzyme (CAT), and superoxide dismutase enzyme (SOD) activities were determined using the commercial kits and based on the method described by Weydert[25], El-Aarag[26], and Sun et al.[27], respectively.

2.6.3. Measurement of tumor necrosis factor-alpha (TNF-α)level

TNF-α levels in tumor homogenates were examined by the mice TNF-ααELISA kit based on the manufacturer’s protocol.

2.6.4. Evaluation of the apoptosis-regulatory gene expression

The expression levels of some apoptosis-regulatory genes such as caspase-3, Bcl-2, and Bax were evaluated by the quantitative realtime PCR. In brief, the total RNA of tumor tissue was extracted by an RNeasy tissue kit based on the guidelines of the manufacturer.The cDNA was synthesized using the random primers according to the manufacturer’s instructions. The synthesized cDNA was then used for real-time PCR by SYBR green. The thermal program of reaction was 96 ℃ for 7 min, 40 cycles of 95 ℃ for 10 s, and 56 ℃for 30 s, respectively. Lastly, the 2?ΔΔCtwas calculated using the iQTM5 optical system software (Bio-Rad, Hercules, CA). The housekeeping gene was β-actin. Table 1 displays oligonucleotide primers which were used for real-time PCR[28].

Table 1. The sequence of primers used for real-time PCR.

2.7. Statistical analysis

The obtained data were presented as mean ± standard deviation.SPSS statistical software version, 22.0 (SPSS Inc., Chicago, IL,USA) was used for analysis. One-way ANOVA with Turkey’s post hoc test was used to assess differences between experimental groups.

2.8. Ethical statement

This study was approved by the Ethical Committee of Scientific Research, Al-Quwayiyah, Shaqra University, Saudi Arabia (No.20201123).

3. Results

3.1. Phytochemical analysis

The results exhibited the existence of a high volume of tannins,terpenoids, glycosides, and flavonoids as well as the lack of the alkaloids and saponins in the tested herb.

3.2. Secondary metabolites contents

The results showed that the total flavonoid, phenolic, and tannin content was 3.68 (mg QE/g DW), 74.6 (mg GEA/g DW), and 2.34(mg CE/g DW), respectively.

3.3. Body weight evaluation

Figure 1 demonstrates that EST mice treated with CP and PAME at 50 and 100 mg/kg showed a significant decline in body weight(P<0.05) when compared with that of untreated EST mice in the C2 control group.

Figure 1. Mean of % of weight gains. C1: non-Ehrlich solid tumors (EST)and non-treated mice; C2: EST mice receiving normal saline; C3: EST mice treated with cyclophosphamide; C4: healthy mice receiving the PAME at a dose of 50 mg/kg; C5: healthy mice receiving the PAME at a dose of 100 mg/kg; Ex1: EST mice treated with PAME 50 mg/kg; Ex2: EST mice treated with PAME 100 mg/kg. Data are expressed as mean±SD. *:P<0.001, significant difference compared with the C2 group.

Figure 2. Serum level of alpha-fetoprotein (AFP) (A) and carcinoembryonic antigen (CEA) (B). Data are expressed as mean±SD. #P<0.001 significant difference compared with the C1 group; *P<0.001 significant difference compared with the C2 group.

3.4. Tumor growth inhibition

Compared with the C2 group [(2.86±0.12) g], treatment wtih PAME significantly decreased the tumor volume in a dosedependent manner in EST mice (P<0.05) [50 mg/kg: (1.93±0.07)g, 100 mg/kg: (1.12±0.05) g]. The tumor inhibition rate was 32.5% and 60.8% after treatment with PAME at 50 and 100 mg/kg,respectively.

3.5. Evaluation of tumor markers

The results revealed that in the C2 group, the serum level of CEA and AFP was significantly elevated compared with the C1 group(P<0.001). As shown in Figure 2, the level of CEA and AFP was significantly declined in mice bearing EST treated with CP and PAME at 50 and 100 mg/kg when compared with the mice of the C2 group (P<0.001).

3.6. Evaluation of serum level of liver enzymes

As shown in Figure 3, the serum level of AST and ALT was significantly increased in the control group of C2 (P<0.001);whereas CP and PAME at 50 and 100 mg/kg significantly reduced the level of ALT and AST (P<0.001) compared with the C2 group. The findings also exhibited that after the oral administration of healthy mice with PAME at 50 (C4) and 100 (C5) mg/kg for 14 d, the levels of AST and ALT showed no significant difference compared with the control group C1.

3.7. Evaluation of serum level of kidney enzymes

Figure 4 demonstrates that the serum level of BUN and Cr was significantly increased in the control group of C2; whereas CP and PAME at 50 and 100 mg/kg significantly reduced the level of BUN and Cr (P<0.001) compared with the mice of the C2 group. The findings also exhibited that after the oral administration of healthy mice with PAME at 50 (C4) and 100 (C5) mg/kg for 14 d, the level of BUN and Cr showed no significant difference in comparison with the control group C1.

3.8. Evaluation of the oxidative and antioxidant markers

As shown in Figure 5, the level of tumor MDA and NO was significantly elevated in the C2 group; while CP and PAME at 50 and 100 mg/kg/day significantly reduced MDA and NO (P<0.05).The level of GPx, CAT, and SOD was significantly declined in the C2 group. However, PAME at 50 and 100 mg/kg/day significantly increased the level of GPx, CAT, and SOD (P<0.05).

3.9. Measurement of the TNF-α level

The results exhibited that the level of TNF-α in mice of the C2 control group was significantly increased; whereas treatment with CP and PAME at 50 and 100 mg/kg/day significantly decreased the level of TNF-α in mice (P<0.05) (Figure 6).

Figure 3. Serum level of (A) aspartate aminotransferase (AST) and (B) alanine aminotransferase (ALT). Data are expressed as mean±SD. #P<0.001 significant difference compared with the C1 group; *P<0.001 significant difference compared with the C2 group.

Figure 4. Serum level of (A) blood urea nitrogen (BUN) and (B) creatinine (Cr). Data are expressed as mean±SD. #P<0.001 significant difference compared with the C1 group; *P<0.001 significant difference compared with the C2 group.

Figure 5. Tumor level of the malondialdehyde (MDA) and nitric oxide (NO), glutathione peroxidase (GPx), catalase enzyme (CAT), and superoxide dismutase enzyme activity (SOD). Data are expressed as mean±SD. #P<0.001 significant difference compared with the C1 group; *P<0.05 significant difference compared with the C2 group.

Figure 6. TNF-α level. Data are expressed as mean±SD. #P<0.001 significant difference compared with the C1 group; *P<0.05 significant difference compared with the C2 group.

3.10. Evaluation of the apoptosis-regulatory gene expression

The expression of the caspase-3 gene was significantly upregulated in tumor tissues (P<0.001), with 3.21-, 2.58- and 3.10-fold after treatment of CP and PAME at 50 and 100 mg/kg,respectively. The expression of the Bax gene was also significantly(P<0.001) up-regulated in tumor tissues (P<0.001), by 3.42-, 2.79-and 3.60- fold after treatment of CP and PAME at 50 and 100 mg/kg, respectively. The expression level of Bcl-2 was downregulated after treatment of PAME.

4. Discussion

In many parts of the world, cancer is the 2nd principal reason for death after cardiovascular disease. Current problems in the use of chemotherapy and radiation therapy and the numerous side effects,as well as the resistance of cancer cells to common therapies, have led researchers to tend to new drugs with greater effectiveness and less toxicity[1-3]. Nature is an amazing source of suitable new drug compounds with great chemical diversity[29,30]. Therefore,in the present study, we aimed to evaluate the anti-tumor effects of PAME against the Ehrlich ascites carcinoma as a spontaneous breast carcinoma model in mice.

Our findings revealed that treatment of mice bearing EST with PAME at 50 and 100 mg/kg significantly decreased the tumor weight and volume. Moreover, tumor inhibition rate was 32.5% and 60.8%, respectively. Considering the anticancer activity of P.atlantica, Shafiei et al. have demonstrated cytotoxic effects of P.atlantica gum resin against human bile duct cancer (CC50value of 15.3 μg/mL), pancreatic carcinoma (CC50value of 11.5 μg/mL),gastric adenocarcinoma (CC50value of 8.1 μg/mL), and COLO205 cells (CC50value of 5.2 μg/mL)[12]. In the other study, Hashemi et al. have reported the cytotoxic effects of P. atlantica ethanolic extract against AGS (IC50value of 382 μg/mL) and HeLa cells (IC50value of 332 μg/mL)[31].

Today, serum levels of AFP and CEA are considered as the main tumor markers that become raised during some cancers development[32]. The elevation of these markers might indicate the liver and renal damage induced by the invasion of cancer cells. Our results demonstrated that the serum levels of CEA and AFP were significantly increased in the control group of C2, while PAME at 50 and 100 mg/kg significantly reduced the level of CEA and AFP(P<0.001).

Studies demonstrated that the cancer cells interrupted the metabolism of the normal liver cell which elevated the activity of serum enzymes, the destruction of hepatocytes by the invasion of cancer cells results in the release of AST and ALT into the plasma and subsequently the elevation of these liver enzymes[33].In line with previous studies[34,35], we found that the serum level of AST and ALT was significantly increased in mice bearing EST, indicating that EST results in severe hepatocellular damage.However, treatment of PAME at 50 and 100 mg/kg significantly reduced the level of ALT and AST (P<0.001).

Previous studies have demonstrated that EST impairs renal function and results in increasing blood BUN and Cr[36]. Similarly,we found that the serum level of BUN and Cr was significantly increased in mice bearing EST; whereas PAME at 50 and 100 mg/kg significantly reduced the level of BUN and Cr (P<0.001).

It has been proven that oxidative stress is mainly involved in the development of cancers and cardiovascular diseases[37]. Nowadays,studies demonstrated that antioxidants derived from natural products are potentially able to protect human beings from damages due to oxidative stress[38]. Our findings revealed that the levels of tumor MDA and NO were significantly elevated in the C2 group,which are significantly (P<0.05) reduced by PAME at 50 and 100 mg/kg. The levels of GPx, CAT, and SOD were significantly declined in the C2 group, which were significantly (P<0.05)increased by PAME at 50 and 100 mg/kg. In line with the current results, several studies reported the antioxidant effects of various parts of P. atlantica by DPPH-based radical scavenging, nitric oxide scavenging, β-carotene bleaching tests, etc[10,39]. Previous investigations have shown that the potent antioxidant effect of the plant is due to the presence of some phenolic (e.g. galloylquinic acid, quinic acid, gallic acid) and terpenes compounds whose antioxidant effects have already been proven[10,40].

Cancer cells can lead to serious damage as well as hypoxia in normal tissues, not only by physically damaging the cells but also by stimulating the production of some pro-inflammatory cytokines[41]. The results of the present study are similar to the previous study[42]. The level of pro-inflammatory cytokine TNF-α in EST mice was significantly increased; whereas treatment of PAME at 50 and 100 mg/kg significantly (P<0.05) decreased the level of TNF-α in mice. The main mechanism is its ability to scavenge free radicals as well as its anti-inflammatory effect[43].

Programmed-cell death which also called “apoptosis” is a form of cell death that occurs in response to various stressors such as physiological, pathological, or cytotoxic stimuli in the body.Apoptosis plays a role in different stages of a living organism’s biological evolution and can cause various diseases if it is out of control[44]. One of the main processes in tumorigenesis and cancers is the inhibition of apoptosis which is vital for cancer cells to keep their uncontrollable multiplying. Consequently, induction of apoptosis is considered a main approach to treating cancer[45].

In this study, the expression level of some apoptotic genes(caspase-3, Bcl-2, and Bax) in tumor tissues was evaluated by quantitative real-time PCR. Our findings exhibited the expression of caspase-3 and Bax gene was significantly increased in tumor tissues (P<0.001) after treatment of PAME at 50 and 100 mg/kg. In contrast, the expression level of Bcl-2 was downregulated.Similarly, in the study conducted by Shafiei et al., P. atlantica gum resin extract showed their anticancer effects against KMBC,PANC-1, CRL-1739, and COLO205 cancer cells through the upregulation of caspase-9, caspase-3, and Bax, as well as the downregulation of Bcl-2 genes[12]. Amiri et al. have also reported that P. atlantica ethanolic extract increased the expression of Bax and caspase-3 genes and down-regulated the Bcl-2 expression in human prostate cancer pc3 cells after 48 h exposure[11].

As a limitation of the study, we didn’t separate the main and effective compounds of the plant that may have anti-cancer effects or explore other possible anti-cancer mechanisms, neither, which will be studied in our further experiment.

In conclusion, our study revealed that P. atlantica methanolic extract could reduce tumor volume, body weight, tumor markers(AFP and CEA), serum level of liver and kidney enzymes, MDA,NO, and TNF-α level; whereas PAME at 50 and 100 mg/kg/day significantly increased the level of antioxidant enzymes of GPx, CAT, and SOD in EST mice. Therefore, the extract might be considered as an alternative anticancer agent against tumors,however, additional studies especially in the clinical setting are required to confirm this finding.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

The authors would like to thank the Deanship of Scientific Research at Shaqra University for supporting this work.

Authors’ contributions

AEA and NAA contributed in study design, conceptualization,methodology, and investigation. RHA and SFA performed the experiments and analyzed data. AEA, SFA and NAA supervised the study and wrote the original draft of the manuscript. All authors have read and approved the final manuscript.