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ANTITUMOR ACTIVITY OF B-GLUCAN ISOLATED FROM DATE FRUITS ON CANCER CELLS IN VIVO
H.M. Al-Khuzaayx, Y.H. Al-Juraisy2, A.F. Hasan3*, E. Tousson4
1 Department of Science, College of Basic Education, Mustansiriyah University, Baghdad, Iraq;
2 Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq;
3 Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq;
4 Department of Zoology, Faculty of Science, Tanta University, Egypt.
* Corresponding author: [email protected]. iq
Abstract. Discovering alternate methods to treat cancer has been the focus of several investigations. The preventive benefits of p-glucan against liver damage, toxicity, and alteration in antinuclear antibody (ANA), alpha-fetoprotein (AFP), and anti-double strand DNA antibody (anti-dsDNA) caused by Ehrlich ascites carcinoma (EAC) are investigated in this study. A total of 40 mice weighing between 20-25 g, were divided randomly into four groups: the control group, the p-glucan group (200 mg/kg bw/day for two weeks), the EAC group, and the EAC+P-glucan group. The most recent research demonstrated that EAC damaged the liver and increased serum levels of AFP, anti-dsDNA, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP). However, compared to the control, serum total proteins and albumin levels considerably decreased. EAC therapy with p-glucan enhanced liver structure and function. As a result, it is possible that suggests that p-glucan can help prevent and treat liver toxicity.
Keywords: p-glucan, cancer, mice, anticancer, liver function.
List of Abbreviations
AFP - Alpha-fetoprotein Anti-dsDNA - Anti-double strand DNA antibody
EAC - Ehrlich ascites carcinoma ALT - Alanine transaminase AST - Aspartate transaminase ALP - Alkaline phosphatase BG - P-glucan
NCI - National Cancer Institute EVC - Egypt Vaccine Establishment's
Introduction
Cancer is a set of illnesses that kill millions of people worldwide over an extended period of time (Al-Khuzaay et al., 2019). Cancer's extremely aggressive nature, dismal prognosis, and short survival rate make it a serious global public health problem even today. Breast cancer and liver cancer are two of the most deadly cancers worldwide, with 2.26 million and 2.21 million new cases, respectively, in 2020. Breast cancer is a serious condition that results in 46,000 deaths and 1.41 million new diagnoses annually (Oeffinger et al., 2015). Breast cancer is the second largest cause of death for women
and has a high rate of morbidity and mortality (Pagani et al., 2010). Ehrlich ascites carcinoma (EAC) is initially hyperdiploid, highly transplantable, never regressing, rapidly proliferating, having a shorter life span, and being fully malignant; this condition is sometimes referred to as an undifferentiated carcinoma (Tousson et al., 2022). Chemotherapy is the most often used type of cancer therapy; it causes apoptosis, which kills malignant development cells; yet, it has a significant negative effect on patients' quality of life and is believed to be a direct cause of death (Tousson et al., 2014). Date palm (Phoenix dactylifera L.), is a major crop in the Middle East, including Iraq (Al-Juraisy et al., 2016). p-glucan is a type of p-D-glucose polysaccharides that is found in the cell walls of bacteria, fungi, plants, algae, and grains in nature. They have radically diverse physicochem-ical characteristics depending on the source (Chaichian et al., 2020; Murphy et al., 2020). BG is a common natural ingredient found in foods, medicines, and cosmetics (Gulmen et al., 2011). BG are some of the polysaccharides present in the structural components of the cell walls of different microorganisms, such as fungus, yeast, and bacteria (Murphy et al., 2020).
P-glucans are used to treat a number of diseases, including as cancer, HIV/AIDS, diabetes, hypercholesterolemia, and P-glucans modulate the immune system (Choromanska et al., 2018). P-glucans possess anticancer qualities and the ability to stimulate the immune system; nevertheless, the precise method via which P-glucan eliminates cancer cells is unclear and complex (Hasan et al., 2023).
Materials and Methods
Preparation of plant
P-glucan was first separated by hot water extraction from the dried fruits of the phoenix dac-tylifera, and it was then purified using gel filtration chromatography and ion exchange.
Induction of cancer cells in mice
Mice with EAC were donated by Cairo University's Egyptian National Cancer Institute (NCI). Mice harboring cancer have had EAC cells suspended in sterile isotonic saline. In order to generate EAC, each mouse received an intraperitoneal injection of 2.5 million viable EAC cells.
Animals and experimental design
40 female Swiss albino mice about 20-25 g were removed from the Egypt Vaccine Establishment's (EVC) animal house colony. The animals were kept in a reasonably sticky environment with a 12-hour light/dark cycle, commercial food, and water for around 14 days. The ambient temperature range was 22 to 25 °C. Guidelines for this work were determined by the Moral Committee of the Tanta University Faculty of Science, which was supported by the Institutional Animal Care and Use Committee (IACUC - SCI - TU - 00179).
Four mouse groups (Gp1-Gp4) were created and dispersed evenly.
GP 1: control group. GP2: P-glucan group. The mice were given 200 mg/kg per day of P-glucan orally for 14 Days. GP3: EAC group; mice received roughly 2,500,000 Ehrlich cells intraperitoneally (I.P.) per mouse (Mutar et al., 2020). GP4: (EAC+P-glucan) group; mice were given 2,500,000 EAC cells per mouse via hypodermic injection to cause tumors (EAC); then
on the second day, P-glucan was given orally for 14 days.
Tissue and blood sampling
The mice in the infected group had their peritoneal cavity fluid cells of the EAC removed after 14 days of tumor inoculation. The tumor cells were within these fluid cells. Furthermore, all of the mice in each group received an intra-peritoneal injection of sodium pentobarbital (less than 100 mg/kg) to induce anesthesia. After a cardiovascular puncture, blood samples were obtained and spun at 3000 rpm for 20 minutes. For biochemical analysis, the separated serum was kept in storage at -20 °C. Samples of liver were removed for histological analysis, cleaned in saline, and then preserved in 10% neutral buffer formalin.
Measurement of serum liver function
Serum AST and ALT activities were evaluated with the approved method by Reitman and Frankel (1957). El-Aarag and his colleagues (2021) and Doumas and his colleagues (1971) specified tests to measure the amount of albumin and the activity of alkaline phosphatase in sera, respectively. Saggu et al. (2014) detected the levels of serum total proteins, although.
Measurement of tumor marker and anti-dsDNA antibodies and antinuclear antibodies (ANA) levels
According to Abd Eldaim et al. (2021), an automated quantitative enzyme linked fluorescence assay (ELFA) was used to determine the concentrations of serum alpha-fetoprotein (AFP) using mini-VIDAS® AFP (Biomerieux, Marcy - L'Etoile, France). Helix Diagnostics provided enzyme immunoassay (EIA) kits for anti-ssDNA and anti-dsDNA antibody detection (West Sacramento, CA). According to El-Masry and his colleagues (2020), the ANA Screen ELISA test system is an ELISA for the identification of IgG class antibodies to ANA in serum (Kumar et al., 2009).
Histological studies
Each mouse's liver was sectioned using paraffin wax, a tiny portion was stored in 10% neu-
tral buffer formalin fixative, and some of it was stained with eosin and hematoxylin for histo-logical examination (Tousson et al., 2016).
Ethics
The study design was approved by the Institutional Ethical Committee for Animal Care and Use (code: IACUC - S CI-TU-0241).
Statistical analysis
The Statistical Package for the Social Sciences (SPSS software version 16) was used to analyze the findings. The data were displayed as mean ± standard error of the mean (SEM) and subjected to one-way analysis of variance (ANOVA) and Dunnett test statistical analysis. Comparisons using the Dunnett test were used to determine how significant the differences between the groups were. To compare the significant difference between groups, an unpaired T-test was used. P < 0.05 was established as the threshold for statistical significance.
Results
Impact of P-glucan on cytological examinations
Following the injection of Ehrlich cells into several groups, cytological analyses of the tumor cells from the mice's ascites fluid are displayed in Fig. 1A. The ascites fluid cells in the EAC group showed that the amount of peritoneal fluid increased, the number of mitotic cells increased, and there were many tumor cells with enlarged nuclei. Additionally, the architectural organization of the cells was disorganized, there was a significant degree of cellular anaplasia, there was pleomorphism and anisocytosis, there was nuclear vascularity, and there was hyperchromasia (1B). However, compared to the EAC group, the treated EAC mice with P-glucan (EAC+P-glu-can) showed reduced rates of death, mitotic cells, and cellular changes (Fig. 1C & 1D).
Impact of fP-glucan on serum liver functions test
Table 1 showed that, in comparison to the control and P-glucan groups, mice injected
with Ehrlich cells (EAC) had considerably lower levels of albumin and total proteins and significantly higher levels of ALT, AST, and ALP. Comparing EAC+P-glucan to untreated EAC, there was a substantial (P < < 0.05) drop in ALT, AST, and ALP, as well as an increase in albumin and total proteins (Table 1).
Changes of AFP and ANA and anti-dsDNA levels
The EAC group had significantly greater serum levels of AFP compared to the control and P-glucan groups. Compared to the EAC group, the EAC+P-glucan groups treated with P-glucan showed a significant reduction in AFP (Table 2). ANA serum levels were substantially lower in the EAC+P-glucan group (treated EAC with P-glucan) than in the EAC group, but significantly higher in the EAC group when compared to the control and P-glucan groups (Table 2). Serum levels of anti-dsDNA were considerably greater in the EAC group than in the control and P-glucan groups. However, the EAC treated with P-glucan (EAC+P-glucan) groups demonstrated a significant decrease in anti-dsDNA compared to the EAC group.
Impact of P-glucan on histological liver
The histological changes in the liver sections of the four groups were displayed in Figure 2A-2D. Both the control and P-glucan-treated animals' liver sections included hepatocytes with the characteristic polygonal shape, noticeable round nuclei, eosinophilic cytoplasm, and sparsely spaced hepatic sinusoids situated in between the hepatic cords. Additionally, a precise pattern of Kupffer cells was constructed (Fig. 2A & 2B). However, the liver slices from the EAC revealed a large number of inflammatory cells, significant hepatocyte cytoplasmic vacuolization, prominent widespread necrosis, and hepatic cord degeneration (Fig. 2C). Following P-glucan therapy of EAC, moderate inflammatory cells and cellular infiltrations were seen (Fig. 2D).
A
C
Fig. 1. Impact of p-glucan on cytological examinations of cancer cells. A: EAC mice. B: Ehrlich ascites cells smear from untreated group reveals many tumor cells, mitosis, nuclear expansion, and considerable degree of cellular anaplasia, using Giemsa stain. C: B-glucan treatment in EAC mice models. D: Giemsa staining of Ehrlich cells from ascites fluid in mice treated with the p-glucan group reveals a significant number of apop-totic cells, a decrease in mitotic cells, and minimal tumor cells
Table 1
Determination of the liver enzymes (AST, ALT, ALP, albumin, and total protein)
in the experimental groups
Mean ± Standard error
Groups ALT AST ALP Albumin TP
(U / L) (U / L) (U / L) (g / dL) (g / dL)
Control 37.1# ± 1.49 132.7# ± 7.12 133.6# ± 9.18 3.80# ± 0.21 7.20# ± 0.30
ß-glucan 34.6# ± 1.40 121.0# ± 5.30 133.8# ± 8.88 5.08# ± 0.30 7.22# ± 0.33
EAC 90.0* ± 3.42 206.5* ± 8.09 230.9* ± 9.50 4.06*± 0.30 6.15* ± 0.50
EAC + ß-glucan 40.5# ± 1.88 160.7#* ± 9.02 161.3#* ± 9.79 4.70# ± 0.19 6.71#* ± 0.20
Note: * p < 0.05 shows a noteworthy variation from the control group. # p < 0.05 - the EAC group is much different from this.
Table 2
Determination of AFP and ANA and anti-dsDNA levels
Groups Mean ± Standard error
AFP ANA (iu /ml) Anti-dsDNA (u / Ml)
Control 0.12# ± 0.003 0.11# ± 0.002 9.80# ± 0.68
ß-glucan 0.13# ± 0.005 0.12# ± 0.007 8.30# ± 0.66
EAC 3.12* ± 0.009 0.31* ± 0.008 34.6* ± 0.051
EAC + ß-glucan 0.50# ± 0.004 0.17# ± 0.004 19.7#* ± 0.99
Note: * p < 0.05 shows a noteworthy variation from the control group. # p < 0.05 - the EAC group is much different from this.
Fig. 2. Impact of P-glucan on histological structure. Photomicrographs of liver histological sections from the various experimental groups A and B show that the hepatocytes (Hp) and central vein (Cv) have their normal structures in the control (Gl) and P-glucan (G2) liver sections, respectively. C: in the EAC group (G3), there were clearly visible vacuolated hepatocytes, inflammatory cells (arrows), and diffuse necrosis of the hepatic tissue. D: hepatic tissue with mild widespread necrosis (arrowheads) in EAC+P-glucan (G4)
Discussion
EAC is one of the experimental breast tumors developed from spontaneous mouse ade-nocarcinoma, and it is regarded as a weakly differentiated malignant tumor that is transplantable. Segura et al. (2000) reported that EAC has been utilized to research the anti-tumor effects of several chemical compounds, both natural and artificial. In the current study, EAC induced elevations in tumor marker AFP, ANA, anti-dsDNA reflecting its inflammatory effect in female mice.
However, treatments with P-glucan depleted the elevations in AFP, ANA and anti-dsDNA levels. This confirmed the findings of Bruce et al. (2008). According to our findings, EAC caused decreases in albumin and total proteins as well as increases in ALT, AST, and ALP. These outcomes align with the findings of Hal-dar et al. (2010). As seen in the EAC group, the rise of liver enzymes is a sign of declining hepatic functioning brought on by the spread of
cancer. It was given careful consideration in reports on the ameliorative effects of medications and natural products in contemporary investigations, especially when it came to dietary therapy for EAC. The hepatic ameliorative benefits of P-glucan against EAC were clearly demonstrated, as evidenced by increases of albumin and total proteins and decreases of blood AST, ALT, and ALP levels. The hepatoprotective effect of P-glucan is supported by the decrease in AST and ALT activity.
Recent research indicates that EAC raised anti-dsDNA and ANA levels; P-glucan treatment of EAC restored these levels to normal. These results corroborated the findings of El-Masry et al. (2020) and Ahmed et al. (2019), who discovered that in mice, EST increased ANA and anti-dsDNA. Liver damage can be caused by viruses, alcohol, auto-immune diseases, chemicals, and other reasons. Furthermore, it has been documented that EAC-bear-ing mice cause a severe hepatic condition that
can result in liver fibrosis (Abd-Eldaim et al., 2021; Tousson et al., 2020). However, they could have an impact on how gut hormones are regulated. ß-glucans' potential to regulate inflammation via immunostimulatory pathways might be connected to their anticancer properties (Baldassano et al., 2017; Wang et al., 2020; Chan et al., 2009).
Most anticancer drugs function by subjecting tumor cells to oxidative stress, which is thought to be the primary cause of most macro-molecular alterations in the cell. Reactive oxygen species may harm proteins, membrane li-pids, DNA, and other macromolecules (Matos et al., 2019). The immuno-modulating property of ß-glucan can activate macrophages, phagocytose the pathogen, and generate proinflam-matory cytokines, all of which can boost the immune system. They significantly affect the intestinal flora, which is beneficial to the organism's balance (Vetvicka et al., 2021).
The current study found that EAC led to hepatic dysfunction as seen by increased serum ALT, AST, ALP, and AFP activities and decreased blood levels of albumin and total protein. These results corroborated previous research by Aldubayan et al. (2019) and Tousson et al. (2020) that discovered liver damage from EAC. Considering that hepatotoxicity was identified by histological examination of the liver tissues of EAC-bearing mice and that the results were corroborated of Tousson et al. (2020), when discovered that EAC damaged the liver and killed the hepatocytes that were responsible for releasing their enzymes into the
plasma, it's probable that the hepatic tissue injuries are the origin of these elevations in liver enzyme activity. Consistent with the results of Aldubayan et al. (2019) and EI-Masry et al. (2020), he asserted that Ehrlich tumors raise AFP levels in mice's serum because they could trigger an inflammatory response; our findings showed that EAC did indeed raise AFP levels in mice's serum. Conversely, P-glucan therapies for EAC exacerbated these alterations in the liver's physiological state. Elevated levels of serum AST, ALT, and ALP might potentially reduce the amount of enzyme leakage into the bloodstream and provide significant evidence for their protective effects on hepatocytes against membrane fragility and stabilize hepatic cellular membrane damage.
Conclusions
EAC simultaneously reduced total protein and albumin levels and increased serum ALT, AST, ALP, AFP, ANA, and anti-dsDNA activities, changing the makeup and function of the liver. On the other hand, EAC therapy with P-glucan reduced the changes in AFP, ANA, anti-dsDNA, and liver structure, reducing the effects of EAC on liver function and structure and demonstrating the significant hepatoprotective potential of P-glucan.
Acknowledgments
The authors would like to thank Mustansiri-yah University (www.uomustansiriyah.edu.iq) Baghdad-Iraq for its support in the present work.
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