Skip to main content
Beni-Suef University Journal of Basic and Applied Sciences
Download PDF

Chitosan nanoparticles, camel milk exosomes and/or Sorafenib induce apoptosis, inhibit tumor cells migration and angiogenesis and ameliorate the associated liver damage in Ehrlich ascites carcinoma-bearing mice

Beni-Suef University Journal of Basic and Applied Sciences volume 13, Article number: 74 (2024) Cite this article

Abstract

Background

It is crucial to improve cancer patients' quality of life by developing medications that can treat cancer with minimum adverse effects. This study aimed to evaluate the therapeutic effect of chitosan nanoparticles (CNPs) and camel milk exosomes (CMEs) alone or in combination with Sorafenib (SOR) on Ehrlich ascites carcinoma (EAC)-bearing mice and to assess whether EAC-associated liver injury would be ameliorated due to this combination. Liver function and oxidant/antioxidant status were determined spectrophotometrically, while the levels of inflammatory cytokines were estimated by enzyme-linked immunosorbent assay. Gene expression was detected using real-time polymerase chain reaction.

Results

The tumor burden in EAC-bearing mice was reduced after treatment with CNPs ± CMEs ± SOR as indicated by (1) reduced ascetic fluid volume and tumor-cell viability; (2) induction of apoptosis [high p53, BCL2 associated X (Bax), caspase 3, low B-cell leukemia/lymphoma 2 protein (Bcl2)]; (3) increased intracellular reactive oxygen species; (4) decreased migration [high matrix metalloproteinase 9 (MMP9) and low TIMP metallopeptidase inhibitor 1 (TIMP1)]; (5) declined angiogenesis [low vascular endothelial growth factor (VEGF). These treatments also reduced liver injury induced by EAC as noticed by (1) restored liver function indices [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and albumin]; (2) restored redox balance [low malondialdehyde (MDA) levels and high superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities]; (3) increased antioxidant gene expression [high nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1)]; (4) declined inflammation [low interleukin-1β (IL1β) and tumor necrosis factor alpha (TNFα) levels), and (5) enhanced structure of liver. SOR + CNPs-treated mice showed the most improvement, followed by SOR + CMEs-treated animals.

Conclusions

Based on these findings, we determined that CNPs and CMEs enhanced SOR's anticancer efficacy and had an ameliorative role against EAC-induced liver injuries.

Graphic abstract

1 Background

Cancer has been a major health concern, and it is predicted to overtake heart disease and accidents as the main cause of mortality in the next several decades. The breast adenocarcinoma used to create the Ehrlich ascites carcinoma (EAC) is an example of an in vivo experimental model which was commonly used to assess tumor etiology and create products with anticancer activity [1, 2]. To facilitate tumor development, EAC causes a local inflammatory response with cumulative vascular permeability, leading to severe edema, progressive ascitic fluid production, and cellular migration [3]. EAC cells rapidly divided, filling the peritoneal cavity with ascitic fluid and without treatment, the animal might die 17–18 days following EAC injection [4]. Not only do EAC cells have a limited lifespan, but they lack a particular antigen for tumor transplantation [5]. Even though EAC cells are often found in the peritoneal cavity, there have been reports of these cells causing damage to the liver [6,7,8].

Sorafenib (SOR), an oral multi-kinase inhibitor, is among the chemotherapies that showed potent anticancer effects against hepatocellular carcinoma (HCC) and EAC, and this effect was mediated through a wide variety of molecular pathways [7, 9]. Despite SOR being considered a standard anticancer drug for HCC, its usage is limited, due to side effects including tiredness, loss of appetite, hypertension, and hand-foot syndrome in addition to the potential for SOR resistance to develop [10]. This means that despite SOR’s efficacy in extending survival times, it also carries significant risks which should be weighed against its potential benefits. The use of SOR in combination with natural supplements containing antioxidant and anti-inflammatory characteristics makes these adverse effects far more manageable [7, 9, 11, 12]. These supplements may also improve the SOR's effectiveness by supplying extra nutrients that assist in SOR pharmacokinetic and pharmacodynamic effects [13].

Chitosan is a biodegradable polymer of carbohydrates generated from chitin that possesses powerful anti-tumor, immune-stimulating, and antibacterial properties [14]. Chitosan is a natural substance that has fewer adverse effects than synthesized medications, but its restricted biological uses are a result of its high molecular weight and water insolubility [15]. To address these drawbacks, chitosan, and other natural ingredients were administered as nanoparticles [16]. Chitosan nanoparticles (CNPs) effectively killed human liver cancer HepG2 [17,18,19], and BEL7402 cells [20]. According to certain research, CNPs had an anticancer effect via increasing antioxidant enzyme activity and reducing oxidative stress [19, 21, 22], induction of necrosis [19, 20, 23], apoptosis [18], and anti-angiogenesis effect [23]. The effects of CNPs on EAC have been studied, and the evidence suggests that they have anticancer effects [24,25,26]. CNPs encapsulated with polyphenon-E have anticancer activity against Ehrlich solid tumors in mice [27]. Additionally, a carboxymethyl chitosan-based copolymer exhibited antitumor activity against EAC [28].

Exosomes, small, extracellular vesicles (ranging in size from 50 to 120 nm) that are released from cells, play a crucial role in intercellular communication due to their high concentration of proteins, lipids, and a wide range of RNAs (mRNAs, miRNAs, and long noncoding RNAs) [29, 30]. Camel milk exosomes (CMEs) effectively alleviated cyclophosphamide-induced immunosuppression and diabetic nephropathy in rats by activating endogenous antioxidant enzymes and inhibiting ROS [31, 32]. Moreover, they inhibited the growth of some Gram-negative bacteria and the yeast Candida albicans [33]. CMEs have also been shown to have an anti-tumor impact through their ability to reduce inflammation, oxidative stress, metastasis, and increase apoptosis in a large variety of cancer cells including breast cancer (MCF7), liver cancer (HepaRG, HepG2), colon cancer (CaCo2), pancreas cancer (PANC1), and leukemia (Hl60) cells [34,35,36,37,38]. Additionally, combining CMEs with hesperidin and tamoxifen significantly improved their anti-tumor efficacy against MCF7 xenografts in mice [35].

The therapeutic effect of the CNPs or SOR against EAC was previously documented, however, further to our knowledge no research evaluated the effect of CMEs alone or in combination with CNPs or SOR on EAC. The purpose of this study was to determine if CNPs and CMEs alone or in combination with SOR had any therapeutic effects on mice suffering EAC burden and the associated liver injury.

2 Methods

Before conducting this experiment, we obtained ethical approval from the research ethics committee at Kafrelsheikh University with a license number of KFS-IACUC/154/2023. All procedures were carried out in compliance with the standards set out by ARRIVE. https://arriveguidelines.org/arrive-guidelines.

2.1 Preparation and characterizations of CNPs

Chitosan, prepared from carb shells in the form of powder (moisture more than 10%, Marine Hydrocolloids Company, Meron, India), was treated with sodium TPP to get CNPs as previously detailed [39]. The CNPs' dimensions and morphology were analyzed with a 100 kV JEOL transmission electron microscopy (TEM, JEM-2100). The dried CNPs were sonicated in ethanol for a short time to break up the aggregates, and then 200 µl of CNPs were put on a carbon-coated copper grid covered with nitrocellulose for direct TEM examination.

2.2 CMEs isolation and characterization

Mixed milk samples were collected from three healthy Falahi camels (Camelus dromedaries, 10–12 years old, and 3 breeding cycles) at mid-lactation period from a national farm in Marsa Mattrouh, Egypt. Exosomes were isolated by ultracentrifugation at 100,000g using Optima L-90okay ultracentrifuge (Beckman Coulter, California, USA) as previously detailed [33]. In brief, milk samples underwent four successive cool (4 °C) centrifugation rounds; the first at 5000g/15 min; the second at 13,000g/30 min; the third and fourth at 100,000g/90 min/each. The first two rounds were performed to eliminate debris, fat globules, and casein, while the last two rounds were to precipitate CMEs. After collecting CME pellets, we resuspended them in PBS at a concentration of 6 mg/mL, centrifuged them at 5000 rpm for 30 min twice with the 100 kDa filters, and then stored the resulting solution at 4 °C. Similar to CNPs, the size, and morphology of CMEs were determined using TEM. Further confirmation was performed using flow cytometrical detection (Attune, Applied Biosystem, Foster City, California, USA) of exosomes specific CD63 and CD81 (1:200, Santa Cruz, Heidelberg, Germany) and a standard Nanobead calibration kit containing beads (Technologies Drive Fisher, Florida. USA).

2.3 Experimental design

Female Swiss albino mice (n = 56, 19–24 g, and 9–11 weeks old) were kept at standardized laboratory conditions with a basal diet and water ad libitum. Animals were randomly allocated into 8 groups (7 mice/ group). Control (Cnt) group, mice were orally administered PBS (300 µl/ mouse). EAC group, mice were injected intraperitoneally (IP) with 1 million EAC cells (200 µL/mouse) and left for 2 weeks. The source of these cells was from Cancer Biology Unit, Cairo, Egypt. In brief, ascitic fluid containing EAC cells was aspirated from EAC mice (purchased from Cancer Biology Unit) on Day(D)14 following injection with EAC cells [1]. CNPs group, EAC mice were orally treated with 300 µl CNPs (2.5 mg/Kg body weight) for 7 days starting from D4 to D10 of EAC injection [20]. CMEs group, EAC mice were orally given 300 µl CMEs (50 mg/kg) twice at D4 and D10 [40]. SOR group, EAC mice were orally treated with 300 µl SOR (30 mg/kg) for 7 days starting from D4 to D10 of EAC injection [7]. Co-treated groups, mice were co-treated with SOR and CNPs (SOR + CNPs group), SOR and CMEs (SOR and CMEs group), or CNPS and CMEs (CNPS + CMEs group) as previously described.

At the end of the experiment (D14), animals were weighed, the average body weight change (g) was calculated, and blood samples were collected from the medial canthus of the eye using a clean capillary tube. Serum was obtained by centrifugation (3000g/15 min) and was kept in a − 20 °C freezer until further analysis. Livers were taken soon after exsanguination, with some specimens being fixed in 10% formalin (for histopathology) and the rest being homogenized (for ELISA and spectrophotometry) or frozen at − 80 °C (for gene expression).

2.4 Tumor burden indicators

The amount of ascites fluid containing EAC cells aspirated from the peritoneal cavity of each mouse before dissection at (D14) was recorded in a graded centrifuge tube. The total, viable, and non-viable EAC cells were counted using a Neubauer hemocytometer after the cells were suspended in sterile isotonic saline [1].

2.5 Intracellular reactive oxygen species

The levels of intracellular ROS were fluorometrically estimated in examined tumor cells as previously detailed [41] and as detailed by manufacturer’s guidelines (Sigma-Aldrich, Saint Louisan, MA, USA, Cat# MAK144). This assay monitors the conversion of non-fluorescent probe 2,7-dichlorofluorescein diacetate (DCFDA) into the fluorescent DCF. In this assay, H2O2 was added to EAC cells to induce oxidative stress. Briefly, EAC cells were exposed to 25 µM H2O2 after 2 h after being treated with each therapy CNPs ± CMEs ± SOR for 24 h. After a brief PBS wash, DCFDA (5 μM) was added, and the cells were left to rest in the dark at 37 °C for half an hour. We quantified intracellular ROS levels as a percentage of the control by measuring the fluorescence intensity of DCFDA in a fluorescent microplate reader (485 nm excitation and 535 nm emission).

2.6 Serum and tissue biochemical assays

Using commercially available kits (Biomed Diagnostics, Cairo, Egypt), we determined the concentrations of serum albumin, aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) to assess liver damage. For the determination of oxidative and antioxidant status, homogenates of the liver were made using the methods previously described [42]. The levels of lipid peroxide malondialdehyde (MDA) and the activities of endogenous antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were quantified calorimetrically in liver homogenates using commercially available kits (Biodiagnostics, Cairo, Egypt) and as previously detailed [43, 44]. Since epinephrine cannot be auto-oxidized into adrenochrome at a pH of 10.2, its quantity may be measured with the help of SOD. An indication of CAT activity is the rate of H2O2 degradation at 240 nm. GPx activity was evaluated in liver homogenates, as previously reported [45].

2.7 Determination of IL1β and TNFα concentrations by ELISA

Liver tissues were first digested by RIPA lysis buffer and their protein contents were measured by a BCA kit with a standard of bovine serum albumin (Thermo Scientific, Waltham, Massachusetts, USA). The concentrations of IL1β and TNFα were measured using rat IL1β and TNFα ELISA Kits purchased from MyBioSource (San Diego, USA) with Cat # of MBS825017 and MBS2507393, respectively, as described by the manufacturer. All measurements were recorded by microplate reader at optical density (OD) = 450 nm. Coefficient of variation of intra-assay and inter-assay precision for IL1β and TNFα were 5–6% and 5.5–6.5%, respectively.

2.8 Detection of relative gene expression by qPCR

Expression of p53, Bax, caspase 3, Bcl2, MMP9, TIMP1, and VEGF genes were determined in EAC cells, but Nrf2, and HO-1 genes were detected in liver tissue using qPCR. We first extracted total RNA using Trizol reagent (Invitrogen, Waltham, Massachusetts, USA, Cat# 15596026) and used RevertAid H Minus Reverse (Thermo Scientific, #EP04 51) to get cDNA. The qPCR assay and data analysis were carried out using a Piko qPCR thermal cycler (Thermo Scientific, USA) and its integrated software. We prepared a PCR mixture (20 μl) including 10 μl Syber Green (Thermo Scientific), 2 μl cDNA, 1 μl from each primer (Table 1), and 6 μl RNase water. We maintained the specified temperature range and time as instructed by the manufacturer. Normalization of gene expression was performed using GAPDH as an endogenous reference and calculated by the 2−ΔΔCt method.

Table 1 Primers used in qPCR
Full size table

2.9 Histopathology

Liver tissues were fixed in 10% formalin for 24 h. Then to dry the liver tissue, an increasing series of ethanol was used. The tissues were then cleaned in xylene, embedded in paraffin wax, and sectioned using a microtome at a thickness of 5 µm. Tissue slides were then stained with eosin and hematoxylin, and the results were analyzed and photographed using microscope with automatic camera (Olympus, Japan).

2.10 Statistical analysis

The data were presented as the mean (±) the standard error mean (SEM) and analyzed using a one-way analysis of variance (ANOVA), followed by the Tukey as a post hoc test to detect significance based on multiple comparisons. We used GraphPad Prism 8 to perform all statistical analyses. Statistically significant values were determined to be those with P < 0.05.

3 Results

3.1 CNPs and CMEs characterization

Using TEM, CNPs looked like uniformly sized spheres with diameters ranging from 160 to 300 nm (Fig. 1A), while CMEs took the form of nanovesicles with average dimensions of 40 to 110 nm (Fig. 1B). Flow cytometry revealed higher expression of the exosomal marker proteins CD63 (84.25%) and CD81 (80.40%) (Fig. 1C).

Fig. 1
figure 1

CNPs and CMEs characterization. As shown by TEM, the produced CNPs range in size from 160 to 300 nm (A), while the size range of isolated CMEs was 40 to 110 nm (B). The expression of specific exosomal CD63 and CD81 proteins was detected by flow cytometry analysis (C)

Full size image

3.2 Effects of treatments with CNPs ± CMEs ± SOR on EAC burden

3.2.1 Effect of treatments on tumor indices

To assess the impact of different treatments on tumor burden, we measured the body weight, the volume of ascitic fluid, and the number of viable and non-viable EAC cells in the ascitic fluid aspirated from mice. Table 2 summarizes the results of this analysis. Accumulation of ascetic fluid indicates a rise in EAC cells and tumor burden, as well as an increase in body weight. Indeed, the untreated EAC group exhibited significantly (P < 0.05) elevated body weight than all other groups. However, compared to the EAC group, the groups that received treatment had significantly (P < 0.05) reduced body weight. The lowest weight was observed in the SOR + CNPs group, followed by the SOR + CMEs, SOR, and then CNPs + CMEs groups.

Table 2 Effect of different therapies CNPs and/or CMEs ± SOR on body weight change and tumor indices
Full size table

Expectedly, the amount of ascitic fluid produced by the tumors was significantly (P < 0.05) larger in the EAC group compared to the other treated groups. The treatment groups with the lowest ascitic fluid volume were those given SOR + CNPs, followed by SOR + CMEs, then SOR, CNPs + CMEs, CNPs, and lastly the CMEs group. Total and live tumor cell counts were significantly (P < 0.05) lower in the treatment groups compared to the EAC group. The SOR + CNPs group had the lowest live EAC count, followed by the SOR + CMEs group, the SOR group, the CNPs + CMEs group, the CNPs group, and finally the CMEs group. However, SOR + CNPs, SOR + CMEs, and SOR had significantly (P < 0.05) more dead cells compared to the other treated groups.

3.2.2 Effect of treatments on apoptosis and intracellular ROS of EAC cells

Real-time PCR was utilized to check the effect of treatments on the expression of genes related to apoptosis (p53, Bax, and caspase3) and cell survival (Bcl2) and the results revealed that the treatments significantly (P < 0.05) increased the expression of apoptotic genes and significantly (P < 0.05) decreased the expression of the anti-apoptotic gene in all treated groups. The most effective treatment was SOR + CNPs, followed by SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs, compared to the EAC group (Fig. 2).

Fig. 2
figure 2

The effect of CNPs and/or CMEs ± SOR on the expression of apoptotic (Bax, p53, and caspase 3) and anti-apoptotic (Bcl2) genes and intracellular ROS levels in EAC cells. Values are presented as fold change mean ± SEM (n = 3/group). Values with different letters [a (highest value) − f (lowest value)] are significantly different at P < 0.05. Each group was measured against every other group. EAC: Ehrlich ascetic carcinoma; CNPs, chitosan nanoparticles; CMEs: camel milk exosomes; SOR: sorafenib

Full size image

The higher levels of intracellular ROS could be considered as a major mechanism by which the majority of anticancer agents induce apoptosis in cancer cells. To check this hypothesis, the intracellular ROS levels were quantified in EAC cells of all groups, and the results were presented in Fig. 2. EAC cells treated alone with H2O2 (as an inducer of intracellular ROS) exhibited significantly (P ≤ 0.05) greater intracellular ROS levels than the control EAC cells. Cells treated with CMEs, CNPs, and SOR alone or in combination showed a substantial rise (P < 0.05) in intracellular ROS levels, with the following order of levels: SOR + CNPs, SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs, in comparison to control cells. However, ROS produced by these treatments remained significantly lower than those induced by H2O2.

3.2.3 Effect of treatments on EAC cells migration and angiogenesis

Different treatments inhibited EAC cells migration and angiogenesis as revealed by significantly (P < 0.05) downregulated expression of the migration-related MMP9 gene and angiogenesis-related VEGF gene and significantly (P < 0.05) upregulated expression of the anti-migration TIMP1 gene in treated groups, with best effect for SOR + CNPs, followed by SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs, compared to the untreated EAC group (Fig. 3).

Fig. 3
figure 3

Quantitative PCR analysis of the effect of CNPS, CMEs, and SOR on the expression of migration-related (MMP9 and TIMP1) and angiogenesis-related (VEGF) genes in EAC cells. Values are presented as fold change mean ± SEM (n = 3/group). Values with different letters [a (highest value) − f (lowest value)] are significantly different at P < 0.05. Each group was measured against every other group. EAC: Ehrlich ascetic carcinoma; CNPs, chitosan nanoparticles; CMEs: camel milk exosomes; SOR: sorafenib

Full size image

3.3 Effects of treatments with CNPs ± CMEs ± SOR on EAC-associated liver injury

3.3.1 Effect of treatments on liver function

Serum levels of AST, ALT, and ALP were all significantly (P < 0.05) greater in untreated EAC mice, but albumin was significantly (P < 0.05) lower than in treated groups. Among the many treatments tested, SOR + CNPs resulted in the greatest enhancement in these serum biochemical markers, followed by SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs. However, these indicators remained significantly (P < 0.05) higher in the treated groups than the control group, except the SOR + CNPs and SOR + CMEs group which showed insignificant increases (Table 3).

Table 3 Effect of CNPs and/or CMEs ± SOR on liver function
Full size table

3.3.2 Effect of treatments on redox balance and inflammation

The liver of the untreated EAC bearing mice had significantly (P < 0.05) higher levels of lipid peroxidation marker MDA and significantly (P < 0.05) lower activities of antioxidant enzymes (SOD, CAT, and GPx) associated with significantly (P ≤ 0.05) downregulated expression of Nrf2 and HO-1 compared to all other groups (Fig. 4). All treatments recovered these markers to levels comparable to the control groups with best improvement (lowest MDA and highest antioxidant-related parameters) observed in SOR + CNPs, followed by SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs.

Fig. 4
figure 4

Effect of CNPs and/or CMEs ± SOR on oxidative (MDA) and antioxidant (SOD, CAT. GPx, NrF2, HO-1) parameters and inflammatory cytokines (IL1β and TNFα) in the liver of EAC bearing mice. Values are presented as mean ± SEM (n = 3–5/group). Values with different letters [a (highest value) – g (lowest value)] are significantly different at P < 0.05. Each group was measured against every other group. EAC: Ehrlich ascetic carcinoma; CNPs, chitosan nanoparticles; CMEs: camel milk exosomes; SOR: sorafenib

Full size image

Treatment effects on inflammatory cytokines were evaluated by ELISA, which revealed significantly (P < 0.05) elevated levels of IL1β and TNFα in the EAC group compared to the other groups. All treatments brought IL1β and TNFα levels back to levels near the normal, with SOR + CNPs showing the greatest improvement followed by SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs (Fig. 4).

3.3.3 Effect of treatments on liver structure

The effects of different treatments on the liver histopathology of EAC cells are illustrated in Fig. 5. The control group’s liver had normal tissue structure, with hepatocytes of polygonal shape forming cords and separated by blood-filled spaces that extended from the intact central vein. However, untreated EAC mice had a significant region of hepatocellular necrosis and aggregates of pleomorphic, hyperchromatic, and darkly basophilic EAC cells in the perivascular area surrounding the dilated and congested central vein. When compared to the EAC group, all six treated groups exhibited remarkable structural improvement in liver histology. The CNPs group exhibited perivascular small focal area of EAC aggregation, slight hepatocellular necrosis, and moderate congestion in the central vein. The liver of the CMEs group showed a perivascular large focal area of EAC cell aggregation, moderate hepatocellular necrosis, and mild congested portal vein. The liver of the SOR group showed intact hepatocytes separated by dilated blood sinusoids radiating from the central vein and marked reduction in EAC cell aggregation. The liver of the SOR + CNPs group showed marked regression of EAC cells with mild vacuolar degeneration of hepatocytes and congested central vein. There was a moderate localized region of pleomorphic cells with mild degenerative alterations in the liver of the SOR + CMEs and CNPs + CMEs group.

Fig. 5
figure 5

Representative photomicrographs of liver sections stained with H&E (scale bars = 50 µm). The control group (Cnt) shows hepatocytes arranged in a cord-like pattern (H), separated by blood sinusoids (black arrows) radiating from an intact central vein (CV). The EAC group shows a perivascular aggregation of EAC cells (arrowheads) surrounded by an area of hepatic necrosis (N) and congested portal vein (black arrow). The CNPs group shows a perivascular small focal area of EAC aggregation (arrowhead), hepatocellular necrosis (N), and congestion in the central vein (CV). The CMEs group shows a perivascular large focal area of EAC cell aggregation (arrowhead), hepatocellular necrosis (N), and mild congested portal vein (black arrows). The SOR group shows intact hepatocytes (H) separated by dilated blood sinusoids (black arrow) radiating from the central vein (CV) and a marked reduction in EAC cell aggregation (arrowhead). The SOR + CNPs group shows marked regression of EAC cells (arrowhead), slight hepatocyte degeneration (D), and congested central vein (CV). The SOR + CMEs and CNPs + CMEs groups show a moderate EAC cell aggregate (arrowheads) with mild to moderate degenerative alterations in the liver

Full size image

4 Discussion

SOR is a powerful anticancer drug, but its long-term use is constrained by its side effects. Because of their significant antioxidant, anti-inflammatory, and apoptotic potentials, CNPs and CMEs exert anticancer effects against many different types of cancer cells [18, 21, 34,35,36,37]. Aiming for a synergistic impact while reducing the dose, toxicity, and drug resistance of chemotherapeutic medicines, the strategy of drug combination has been extensively employed in the treatment of cancer [49]. Combining SOR with antioxidant-rich natural supplements has been shown to boost its therapeutic value and mitigate its negative effects [7, 9, 11, 12]. The results of the current investigation corroborated these emerging tendencies by demonstrating the efficacy of either CNPs or CMEs as adjuvants with SOR in cancer treatment. This is the first report of its kind, to our knowledge, indicating combining SOR with CNPs or CMEs may improve anticancer efficacy while decreasing adverse effects in EAC-bearing mice.

Treatment with CNPs ± CMEs ± SOR substantially reduced tumor burden as evidenced by lower body weight, ascites fluid volume, and viable and total EAC cell count, in the treated EAC mice compared to the untreated EAC mice. Our findings also showed that the number of dead EAC cells was enhanced after these co-treatments. Similarly, previous studies also reported potent anticancer potentials for SOR alone or in combination with amygdalin (Vit B12) [7] and for CNPs against EAC cells [24,25,26]. SOR, as a multi-kinase inhibitor, can directly inhibit cancer cell proliferation through targeting RAF/MEK/ERK pathway and elevation of intracellular ROS [50]. CNPs' positive charge may be responsible for their direct anticancer potential by allowing them to cling to the negatively charged membrane of tumor cells and cause membrane destruction and organelle disruption [26]. CMEs can also directly kill cancer cells via induction of apoptosis [34,35,36,37]. In the present study, we found a significant elevation in the intracellular ROS levels following treatments with SOR, CNPs, and CMEs alone or in combination with the following order from highest to lowest levels: SOR + CNPs, SOR + CMEs, SOR, CNPs + CMEs, CNPs, and CMEs. Together, treatment with CNPs ± CMEs ± SOR may exert their anticancer action by directly inhibiting tumor cell growth via many signaling pathways that work synergistically during co-treatments. However, indirect effects through induction of immunity or inflammation could also be not ignored as revealed by another study [51].

Cancer cells restrict apoptosis association factors in their microenvironments to protect themselves from death [52]. Therefore, the majority of cancer drugs work by stopping cancer cell division or killing them off via induction of apoptosis [2, 53]. In the present study, we found that co-treatment with SOR and CNPs or CMEs induced the upregulation of the apoptotic genes (p53, Bax, caspase 3) and the downregulation of the anti-apoptotic Bcl2 gene. Consistent with our findings, individual treatment with SOR or CNPs combat cancer cells via triggering apoptosis [11, 19, 39]. Furthermore, CMEs induced apoptosis in HepG2, MCF7, HepaRG, PANC1, and HL60 cells either alone or in conjunction with anticancer medicines (doxorubicin or tamoxifen), demonstrating powerful antiproliferative potential [33, 36,37,38, 54, 55].

Inducing oxidative stress inside cancer cells and tumor microenvironments is another mechanism of action for the vast majority of chemotherapy drugs [52, 56, 57]. We also observed increased intracellular ROS production in EAC cells exposed to SOR, CNPs, and CMEs each alone or in combination. According to these findings, treatments may inhibit EAC cells by inducing the oxidative stress pathway. Similarly, prior research has shown that SOR, CNPs, and CMEs can each be used alone to induce apoptosis in cancer cells by ROS over release [11, 19, 20, 33]. Damage to macromolecules (proteins and lipids) and DNA from an excess of ROS causes oxidative damage to the cell's components, most notably the membrane, mitochondria, and nucleus, and ultimately results in cell death [58]. Since phospholipids are the membrane's primary structural component, it is believed that ROS-dependent lipid degradation generated by therapies is the main cause of loss of membrane integrity preceding apoptosis. Similarly, TEM analysis of cancer cells treated with CNPs showed the existence of holes in cell membranes [20].

Cancer cells need an abundance of oxygen and glucose to survive. Cancer cells, sensing a lack of oxygen and/or glucose in the tumor microenvironment, induce VEGF and begin the process of angiogenesis, which paves the way for metastasis [59]. Moreover, the migration-related MMP9 gene is responsible for activating TGFβ, which in turn increases the expression of the angiogenesis-related VEGF gene [60]. We also found a significantly increased expression of MMP9 and VEGF and a significantly decreased expression of the anti-migratory TIMP1 gene in the untreated EAC cells. In support, Attia, et al. [7] and El Bakary, et al. [6] also found that MMP9 and VEGF expressions were significantly increased in EAC mice. Moreover, we found that this elevated expression was declined after treatment with SOR alone or in combination with CNPs or CMEs. This is evidence that the treatments are able to reduce angiogenesis in the tumor microenvironment and migration of EAC cells. Consistent with our results, CMEs alone also inhibited VEGF and MMP9 and upregulated TIMP1 in HepaRG cells [36]. Similar molecular changes were also observed in the livers of EAC-bearing mice following treatment with SOR alone or in combination with amygdalin [7].

Previous research suggested that EAC cells may go from the peritoneal cavity to other organs including the liver, causing hepatic damage and over-release of AST, ALT, and ALP enzymes into the bloodstream [7, 8]. These results are corroborated by our observation that liver function enzymes were significantly higher in the EAC group than in the control group. Treatments with SOR or CNPs resulted in a decrease in these liver enzymes in the blood of EAC-bearing mice [7, 19]. We also observed a substantial decrease in the blood levels of these enzymes after administering CNPs ± CMEs ± SOR compared to EAC bearing mice. Albumin is another vital hepatic marker for monitoring the development of liver injury. Its level was much lower in EAC than in control mice. In support, decreased level of albumin was also observed in hepatic dysfunction cases of EAC-bearing mice [7, 8]. Again, the administration of SOR alone or with CNPs or CMEs elevated this level in EAC mice.

In the current work, oxidative stress was prevented in the livers of EAC-bearing mice by various treatments as evidenced by decreased the lipid peroxidation marker (MDA) and elevated the activities of antioxidant enzymes (SOD, CAT, GPx) and the upregulated expression of antioxidant-related Nrf2 and HO-1. In agreement with our findings, administration of SOR alone or with amygdalin restored redox balance (low MDA and high SOD, GSH, and Nrf2) in the liver of EAC mice [7]. A similar antioxidant effect was observed in the liver of the HCC rat model after treatment with CNPs [19] and in MCF7 xenograft following treatment of rats with CMEs [54]. Comparable antioxidant effects were shown in tumor-bearing mice and immunocompromised rats when CMEs were used alone or in conjunction with anticancer treatments [32, 55]. Moreover, human and cow milk exosomes exert antioxidant potential that protects against oxidative stress in intestinal crypt epithelial cells [61, 62]. So the puzzle now is how these medications create ROS in EAC cells while having anti-oxidant properties in the liver. We attribute this disparity in outcomes to the nature of target cells. Before addressing this issue, it is important to note that the greater ROS levels in the liver of untreated EAC mice may be attributable to migratory EAC cells. As a result, when these cells perished, the ROS emission was reduced, and the antioxidant status rose. Indeed, histological investigation indicated fewer migratory EAC cells in the liver of treated EAC mice compared to untreated EAC mice, indicating restored redox equilibrium owing to a decrease in ROS source (EAC cells). However, the situation is different when these treatments are applied on separate cancer cells (as in cancer cell lines and EAC cells) in such cases the treatments induced ROS overproduction to kill these cells. While it's true that cancer cells may stay alive by reducing their generation of ROS, organ cancers are complicated by the presence of normal (healthy) cells and other elements of the tumor microenvironment that produce their own ROS. This elevated ROS repressed antioxidant enzyme activities in the untreated EAC group. On the other hand, CNPs and CMEs greatly increased the activity of these enzymes, scavenging the accumulated free radicals and protecting the hepatic tissue from oxidative damage. This assertion warrants attention, since chemotherapeutic drugs that induce oxidative stress seem to be linked to chemo-resistance in clinical applications [63]. Because of this, CNPs and CMEs may be used in the future as adjuvants to chemotherapeutic medicines to mitigate the oxidative stress caused by these drugs.

Inflammation may be triggered by cancer cells in two ways: by producing cytokines directly [64] or by physically damaging normal tissue [65]. Because inflammation plays such a crucial role in tumorigenesis and metastasis, various anticancer medicines were developed to target inflammation-related pathways. CNPs ± CMEs ± SOR were also shown to have an anti-inflammatory impact, as evidenced by lower IL1β and TNFα levels in the liver of in the treated EAC mice compared to the untreated group. A similar anti-inflammatory effect was reported for CNPs and CMEs on the EAC solid tumor in mice and MCF7 xenografts tumor in rats [26, 54].

Histopathological examination of slides prepared from the liver of the EAC group revealed hepatocellular necrosis and aggregates of pleomorphic, hyperchromatic, and darkly basophilic EAC cells in the perivascular area surrounding the dilated and congested central vein. Similar histological alterations were observed by other studies in the liver of the untreated EAC mice [6,7,8], confirming the liver injury by EAC cells. The above-mentioned hepatic structural changes were ameliorated after treatments with SOR, CNPS, and CMEs alone or in combination. In agreement, co-administration of SOR and amygdalin or individual treatment with CNPs improved the hepatic damage induced by EAC in mice [7] and by HCC in rats [19].

Animals treated with SOR in combination with CNPs or CMEs, with better effect SOR + CNPs, showed the best anticancer effect (as revealed by lower tumor burden indices, EAC migration and angiogenesis, and higher apoptosis and ROS) with the least hepatic injury (as indicated by lower functional and structural damage and ROS and higher antioxidant and anti-inflammatory properties). Regarding the individual therapy, the SOR group fared best, followed by the CNPs group, and lastly the CMEs group. Hence, to maximize therapeutic potentials, either CNPs or CMEs should be used as adjuvants for SOR in cancer therapy. However, before recommending that we should address the following limitations: (1) the study used only one type of cancer (EAC), which may limit the applicability of the findings to other cancers, (2) the study did not measure the long-term effects of CNPs and CMEs on tumor growth, survival, and quality of life of the mice due to short life span of EAC cells, (3) the study did not explore the actual mechanisms of action of CNPs and CMEs on EAC cells and how they interact with SOR at the protein level. Therefore, additional in-depth molecular analysis at the protein level is necessary for in vitro and in vivo studies including several types of cancer cells.

5 Conclusions

Treatment of EAC-bearing mice with CNPs ± CMEs ± SOR reduced tumor burden (decreased body weight, ascetic fluid, live EAC cells and increased dead EAC cells, apoptosis, and ROS), and relieved liver injury caused by EAC (restored redox balance and damaged liver tissues and improved anti-inflammatory effects). The combined groups showed the best therapeutic effects with maximum potential for SOR + CNPs followed by SOR + CMEs. Thus, CNPs and CMEs may boost SOR's EAC anticancer impact and might be utilized as a SOR adjuvant in cancer treatment. However, further experiments are required on SOR + CNPs and SOR + CMEs co-therapy to develop a unique cancer-targeting method.

Availability of data and materials

The data supporting the present findings are contained within the manuscript.

Abbreviations

ALP:

Alkaline phosphatase

ALT:

Alanine transaminase

AST:

Aspartate transaminase

Bax:

BCL2 associated X

Bcl2:

B-cell leukemia/lymphoma 2 protein

CAT:

Catalase

CMEs:

Camel milk exosomes

CNPs:

Chitosan nanoparticles

EAC:

Ehrlich ascites carcinoma

GPx:

Glutathione peroxidase

HO-1:

Heme oxygenase-1

IL1β:

Interleukin-1β

MDA:

Malondialdehyde

MMP9:

Matrix metalloproteinase 9

Nrf2:

Nuclear factor erythroid 2-related factor 2

qPCR:

Real-time PCR

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

SOR:

Sorafenib

TIMP1:

TIMP metallopeptidase inhibitor 1

TNFα:

Tumor necrosis factor alpha

VEGF:

Vascular endothelial growth factor

References

  1. El-Magd MA, Khamis A, Nasr Eldeen SK, Ibrahim WM, Salama AF (2017) Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma. Biomed Pharmacother 92:870–878

    Article  CAS  PubMed  Google Scholar 

  2. Elsayed SA, Harrypersad S, Sahyon HA, El-Magd MA, Walsby CJ (2020) Ruthenium(II)/(III) DMSO-Based Complexes of 2-Aminophenyl Benzimidazole with In Vitro and In Vivo Anticancer Activity. Molecules 25:4284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mutar TF, Tousson E, Hafez E, Abo Gazia M, Salem SB (2020) Ameliorative effects of vitamin B17 on the kidney against Ehrlich ascites carcinoma induced renal toxicity in mice. Environ Toxicol 35:528–537

    Article  CAS  PubMed  Google Scholar 

  4. Magdy A, Sadaka E, Hanafy N, El-Magd MA, Allahloubi N, El Kemary M (2020) Green tea ameliorates the side effects of the silver nanoparticles treatment of Ehrlich ascites tumor in mice. Mol Cell Toxicol 16:271–282

    Article  CAS  Google Scholar 

  5. Zeferino RC, Mota NSRS, Grinevicius VMAS, Costa AJG, Silva FRMB, Pich CT, Pedrosa RC (2018) P-24 - Benznidazole induce oxidative stress, DNA damage and apoptosis in Balb-c mice-bearing Ehrlich ascites tumor. Free Radical Biol Med 120:S52

    Article  Google Scholar 

  6. El Bakary NM, Alsharkawy AZ, Shouaib ZA, Barakat EMS (2020) Role of bee venom and melittin on restraining angiogenesis and metastasis in γ-irradiated solid ehrlich carcinoma-bearing mice. Integr Cancer Ther 19:1534735420944476

    Article  PubMed  PubMed Central  Google Scholar 

  7. Attia AA, Salama AF, Eldiasty JG, Mosallam SAE-R, El-Naggar SA, El-Magd MA, Nasser HM, Elmetwalli A (2022) Amygdalin potentiates the anti-cancer effect of Sorafenib on Ehrlich ascites carcinoma and ameliorates the associated liver damage. Sci Rep 12:6494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tousson E, Hafez E, Abo Gazia MM, Salem SB, Mutar TF (2020) Hepatic ameliorative role of vitamin B17 against Ehrlich ascites carcinoma-induced liver toxicity. Environ Sci Pollut Res Int 27:9236–9246

    Article  CAS  PubMed  Google Scholar 

  9. Elmetwalli A, Hashish SM, Hassan MG, El-Magd MA, El-Naggar SA, Tolba AM, Salama AF (2023) Modulation of the oxidative damage, inflammation, and apoptosis-related genes by dicinnamoyl-L-tartaric acid in liver cancer. Naunyn Schmiedeberg’s Arch Pharmacol 396:3087–3099

    Article  CAS  Google Scholar 

  10. Zou X, Fan W, Xue M, Li J (2021) Evaluation of the benefits of TACE combined with sorafenib for hepatocellular carcinoma based on untreatable TACE (unTACEable) progression. Cancer Manag Res 13:4013–4029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mansour GH, El-Magd MA, Mahfouz DH, Abdelhamid IA, Mohamed MF, Ibrahim NS, Abdel HA, Wahab A, Elzayat EM (2021) Bee venom and its active component Melittin synergistically potentiate the anticancer effect of Sorafenib against HepG2 cells. Bioorgan Chem. https://doi.org/10.1016/j.bioorg.2021.105329

    Article  Google Scholar 

  12. Mahfouz DH, EL-Magd MA, Mansour GH, Abdel Wahab AH, Abdelhamid IA, Elzayat E (2021) Therapeutic potential of snake venom, l-amino oxidase and sorafenib in hepatocellular carcinoma. Mol Cell Toxicol. https://doi.org/10.1007/s13273-021-00151-8

    Article  Google Scholar 

  13. Yao J, Man S, Dong H, Yang L, Ma L, Gao W (2018) Combinatorial treatment of Rhizoma Paridis saponins and sorafenib overcomes the intolerance of sorafenib. J Steroid Biochem Mol Biol 183:159–166

    Article  CAS  PubMed  Google Scholar 

  14. Tsai GJ, Su WH (1999) Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot 62:239–243

    Article  CAS  PubMed  Google Scholar 

  15. Torchilin VP (2006) Micellar nanocarriers: pharmaceutical perspectives. Pharm Res 24:1

    Article  PubMed  Google Scholar 

  16. Nel A, Xia T, Madler L, Li N (2006) Toxic potential of materials at the nanolevel. Science (New York, N.Y.) 311:622–627

    Article  CAS  PubMed  Google Scholar 

  17. Subhapradha N, Shanmugam A (2017) Fabrication of β-chitosan nanoparticles and its anticancer potential against human hepatoma cells. Int J Biol Macromol 94:194–201

    Article  CAS  PubMed  Google Scholar 

  18. Loutfy S, Alam El-Din H, Elberry M, Allam N, Hasanin M, Abdellah A (2016) Synthesis, characterization and cytotoxic evaluation of chitosan nanoparticles: in vitro liver cancer model. Adv Nat Sci Nanosci Nanotechnol 7:035008

    Article  Google Scholar 

  19. Elkeiy M, Khamis A, El-Gamal M, Abo Gazia M, Zalat Z, El-Magd M (2018) Chitosan nanoparticles from Artemia salina inhibit progression of hepatocellular carcinoma in vitro and in vivo. Environ Sci Pollut Res Int 27:19016–19028

    Article  PubMed  Google Scholar 

  20. Qi L, Xu Z, Chen M (2007) In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles. Eur J Cancer (Oxford, England : 1990) 43:184–193

    Article  CAS  Google Scholar 

  21. Subhapradha N, Shanmugam V, Shanmugam A (2017) Chitosan nanoparticles from marine squid protect liver cells against N-diethylnitrosoamine-induced hepatocellular carcinoma. Carbohyd Polym 171:18–26

    Article  CAS  Google Scholar 

  22. El-Denshary E, Aljawish A, El-Nekeety A, Hassan N, Saleh R, Rihn B, Abdel-Wahhab M (2015) Possible synergistic effect and antioxidant properties of chitosan nanoparticles and quercetin against carbon tetrachloride-induce hepatotoxicity in rats. Soft Nanosci Lett 5:36–51

    Article  Google Scholar 

  23. Xu Y, Wen Z, Xu Z (2009) Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism. Anticancer Res 29:5103–5109

    CAS  PubMed  Google Scholar 

  24. Salahuddin N, Elbarbary AA, Alkabes HA (2017) Quinazolinone derivatives loaded polypyrrole/chitosan core-shell nanoparticles with different morphologies: antibacterial and anticancer activities. NANO 12:1750002

    Article  CAS  Google Scholar 

  25. Gebreel DT, Elatrash AM, Atta FA, Tousson E, Allam S (2021) Roles of chitosan nanoparticles on ehrlich ascites carcinoma induced changes in some blood parameters. Asian Oncol Res J 3:213–219

    Google Scholar 

  26. Ahmed N, Said U, Meky N, Amohamed M (2018) Role of chitosan nanoparticles as anti-angiogenic in mice bearing Ehrlich carcinoma. Oncol Res Rev 15: 233–241

    Google Scholar 

  27. Othman AI, El-Sherbiny IM, ElMissiry MA, Ali DA, AbdElhakim E (2018) Polyphenon-E encapsulated into chitosan nanoparticles inhibited proliferation and growth of Ehrlich solid tumor in mice. Egypt J Basic Appl Sci 5:110–120

    Google Scholar 

  28. El-Far M, Elshal M, Refaat M, El-Sherbiny IM (2011) Antitumor activity and antioxidant role of a novel water-soluble carboxymethyl chitosan-based copolymer. Drug Dev Ind Pharm 37:1481–1490

    Article  CAS  PubMed  Google Scholar 

  29. Yin K, Wang S, Zhao RC (2019) Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm. Biomark Res 7:8

    Article  PubMed  PubMed Central  Google Scholar 

  30. Alzahrani FA, El-Magd MA, Abdelfattah-Hassan A, Saleh AA, Saadeldin IM, El-Shetry ES, Badawy AA, Alkarim S (2018) Potential effect of exosomes derived from cancer stem cells and MSCs on progression of DEN-induced HCC in rats. Stem Cells Int. https://doi.org/10.1155/2018/8058979

    Article  PubMed  PubMed Central  Google Scholar 

  31. Shaban AM, Raslan M, Qahl SH, Elsayed K, Abdelhameed MS, Oyouni AAA, Al-Amer OM, Hammouda O, El-Magd MA (2022) Ameliorative effects of camel milk and its exosomes on diabetic nephropathy in rats. Membranes 12:1060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ibrahim HM, Mohammed-Geba K, Tawfic AA, El-Magd MA (2019) Camel milk exosomes modulate cyclophosphamide-induced oxidative stress and immuno-toxicity in rats. Food Funct 10:7523–7532

    Article  CAS  PubMed  Google Scholar 

  33. Shaban AM, Raslan M, Sharawi ZW, Abdelhameed MS, Hammouda O, El-Masry HM, Elsayed KNM, El-Magd MA (2023) Antibacterial, antifungal, and anticancer effects of camel milk exosomes: an in vitro study. Vet Sci 10(2):24

    Google Scholar 

  34. Badawy AA, El-Magd MA, AlSadrah SA (2018) Therapeutic effect of camel milk and its exosomes on MCF7 cells in vitro and in vivo. Integr Cancer Ther 17:1235–1246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Badawy AA, Othman RQA, El-Magd MA (2021) Effect of combined therapy with camel milk-derived exosomes, tamoxifen, and hesperidin on breast cancer. Mol Cell Toxicol 26:107–115

    Google Scholar 

  36. El-Kattawy AM, Algezawy O, Alfaifi MY, Noseer EA, Hawsawi YM, Alzahrani OR, Algarni A, Kahilo KA, El-Magd MA (2021) Therapeutic potential of camel milk exosomes against HepaRG cells with potent apoptotic, anti-inflammatory, and anti-angiogenesis effects for colostrum exosomes. Biomed Pharmacother 143:112220

    Article  CAS  PubMed  Google Scholar 

  37. Othman R, Badawy A, Alruwaili M, El-Magd M (2021) Camel milk exosomes potentiate the anticancer effect of doxorubicin on multidrug-resistant human leukemia hl60 cells in vitro and in vivo. Pak J Med Health Sci 15:3313–3320

    Article  Google Scholar 

  38. Ali M, Elsayed GR, Salama MF, El-Magd MA (2022) Camel milk exosomes had a selective anticancer effect on PANC1 cells and a proliferative effect on H6c7 cells. Kafrelsheikh Vet Med J 20:1–5

    Article  Google Scholar 

  39. Alzahani FE-M (2022) Mohammed Abu co-treatment of caffeic acid phenethyl ester with chitosan nanoparticles inhibits DNA methylation in HepG2 Cells. Pak J Zool. https://doi.org/10.17582/journal.pjz/20220407050442

    Article  Google Scholar 

  40. Munagala R, Aqil F, Jeyabalan J, Gupta RC (2016) Bovine milk-derived exosomes for drug delivery. Cancer Lett 371:48–61

    Article  CAS  PubMed  Google Scholar 

  41. Zedan AMG, Sakran MI, Bahattab O, Hawsawi YM, Al-Amer O, Oyouni AAA, Nasr Eldeen SK, El-Magd MA (2021) Oriental hornet (Vespa orientalis) larval extracts induce antiproliferative, antioxidant, anti-inflammatory, and anti-migratory effects on MCF7 cells. Molecules 26:3303. https://doi.org/10.3390/molecules26113303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mohamed AE, El-Magd MA, El-Said KS, El-Sharnouby M, Tousson EM, Salama AF (2021) Potential therapeutic effect of thymoquinone and/or bee pollen on fluvastatin-induced hepatitis in rats. Sci Rep 11:15688

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. El-Bayomi KM, Saleh AA, Awad A, El-Tarabany MS, El-Qaliouby HS, Afifi M, El-Komy S, Essawi WM, Almadaly EA, El-Magd MA (2018) Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes. Reprod Fertil Dev 30:487–497

    Article  CAS  PubMed  Google Scholar 

  44. Saleh AA, El-Magd MA (2018) Beneficial effects of dietary silver nanoparticles and silver nitrate on broiler nutrition. Environ Sci Pollut Res 25:27031–27038

    Article  CAS  Google Scholar 

  45. Hafeman DG, Sunde RA, Hoekstra WG (1974) Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J Nutr 104:580–587

    Article  CAS  PubMed  Google Scholar 

  46. Shalini S, Nikolic A, Wilson CH, Puccini J, Sladojevic N, Finnie J, Dorstyn L, Kumar S (2016) Caspase-2 deficiency accelerates chemically induced liver cancer in mice. Cell Death Differ 23:1727–1736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. McCracken JM, Jiang L, Deshpande KT, O’Neil MF, Pritchard MT (2016) Differential effects of hyaluronan synthase 3 deficiency after acute vs chronic liver injury in mice. Fibrogenesis Tissue Repair 9:4

    Article  PubMed  PubMed Central  Google Scholar 

  48. Sallam AA, El-Magd MA, Ahmed MM, Ghamry HI, Alshahrani MY, Hegazy RA, Magdy A, El-Fotoh MFA (2022) Quercetin alleviated multi-walled carbon nanotubes-induced neurotoxicity in mice through inhibition of oxidation, inflammation, and pyroptosis. Biomed Pharmacother 151:113160

    Article  CAS  PubMed  Google Scholar 

  49. Khamis AA, Ali EM, Abd El-Moneim MA, Abd-Alhaseeb MM, El-Magd MA, Salim EI (2018) Hesperidin, piperine and bee venom synergistically potentiate the anticancer effect of tamoxifen against breast cancer cells. Biomed Pharmacother 105:1335–1343

    Article  CAS  PubMed  Google Scholar 

  50. Ma Y, Xu R, Liu X, Zhang Y, Song L, Cai S, Zhou S, Xie Y, Li A, Cao W et al (2021) LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells. Int J Med Sci 18:1456–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Samudrala PK, Augustine BB, Kasala ER, Bodduluru LN, Barua C, Lahkar M (2015) Evaluation of antitumor activity and antioxidant status of Alternanthera brasiliana against Ehrlich ascites carcinoma in Swiss albino mice. Pharmacogn Res 7:66–73

    Article  Google Scholar 

  52. Abdelwahab MA, El-Barbary AA, El-Said KS, Betiha M, Elkholy HM, Chiellini E, El-Magd MA (2019) Functionalization of poly(3-hydroxybutyrate) with different thiol compounds inhibits MDM2–p53 interactions in MCF7 cells. J Appl Polym Sci 136:46924

    Article  Google Scholar 

  53. Khamis AAA, Ali EMM, El-Moneim MAA, Abd-Alhaseeb MM, El-Magd MA, Salim EI (2018) Hesperidin, piperine and bee venom synergistically potentiate the anticancer effect of tamoxifen against breast cancer cells. Biomed Pharmacother 105:1335–1343

    Article  CAS  PubMed  Google Scholar 

  54. Badawy AA, El-Magd MA, AlSadrah SA (2018) Therapeutic effect of camel milk and its exosomes on MCF7 cells in vitro and in vivo. Integr Cancer Ther 7:1235–1246

    Article  Google Scholar 

  55. Badawy AA, Othman RQA, El-Magd MA (2021) Effect of combined therapy with camel milk-derived exosomes, tamoxifen, and hesperidin on breast cancer. Mol Cell Toxicol. https://doi.org/10.1007/s13273-021-00163-4

    Article  Google Scholar 

  56. Awad MG, Ali RA, Abd El-Monem DD, El-Magd MA (2020) Graviola leaves extract enhances the anticancer effect of cisplatin on various cancer cell lines. Mol Cell Toxicol 16:385–399. https://doi.org/10.1007/s13273-020-00092-8

    Article  CAS  Google Scholar 

  57. Badawy A, Hassanean H, Ibrahim AK, Habib ES, El- Magd MA, Ahmed SA (2019) Isolates from Thymelaea hirsuta inhibit progression of hepatocellular carcinoma in vitro and in vivo. Nat Prod Res 35:1799–1807

    Article  PubMed  Google Scholar 

  58. Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 279:34682–34690

    Article  CAS  PubMed  Google Scholar 

  59. Yang SF, Yang WE, Kuo WH, Chang HR, Chu SC, Hsieh YS (2008) Antimetastatic potentials of flavones on oral cancer cell via an inhibition of matrix-degrading proteases. Arch Oral Biol 53:287–294

    Article  CAS  PubMed  Google Scholar 

  60. Westergren-Thorsson G, Bagher M, Andersson-Sjöland A, Thiman L, Löfdahl CG, Hallgren O, Bjermer L, Larsson-Callerfelt AK (2018) VEGF synthesis is induced by prostacyclin and TGF-β in distal lung fibroblasts from COPD patients and control subjects: Implications for pulmonary vascular remodelling. Respirology (Carlton, Vic) 23:68–75

    Article  PubMed  Google Scholar 

  61. Wang L, Shi Z, Wang X, Mu S, Xu X, Shen L, Li P (2020) Protective effects of bovine milk exosomes against oxidative stress in IEC-6 cells. Eur J Nutr 60(1):317–327

    Google Scholar 

  62. Feng X, Chen X, Zheng X, Zhu H, Qi Q, Liu S, Zhang H, Che J (2021) Latest trend of milk derived exosomes: cargos, functions, and applications. Front Nutr 8:747294–747294

    Article  PubMed  PubMed Central  Google Scholar 

  63. Kim E-K, Jang M, Song M-J, Kim D, Kim Y, Jang HH (2019) Redox-mediated mechanism of chemoresistance in cancer cells. Antioxidants 8:471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Bonavita E, Galdiero MR, Jaillon S, Mantovani A (2015) Phagocytes as corrupted policemen in cancer-related inflammation. Adv Cancer Res 128:141–171

    Article  CAS  PubMed  Google Scholar 

  65. Chauhan VP, Martin JD, Liu H, Lacorre DA, Jain SR, Kozin SV, Stylianopoulos T, Mousa AS, Han X, Adstamongkonkul P et al (2013) Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat Commun 4:2516

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

Authors did not receive any external fund.

Author information

Authors and Affiliations

  1. Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt

    Amr A. Tawfic, Hany M. Ibrahim & Khaled Mohammed-Geba

  2. Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelshiekh University, Kafr El-Shiekh, Egypt

    Mohammed A. El-Magd

Authors
  1. Amr A. Tawfic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hany M. Ibrahim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Khaled Mohammed-Geba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mohammed A. El-Magd
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AT, HI, KG Conceptualization: HI & ME, methodology: AT & ME, Formal analysis and investigation: AT, HI, KG, ME, writing—original draft preparation: AT, Writing—review and editing: HI, KG, ME, supervision, validation and final editing: HI, KG, ME, all authors commented on previous versions of the manuscript, read and approved the final manuscript.

Corresponding author

Correspondence to Mohammed A. El-Magd.

Ethics declarations

Ethics approval and consent to participate

The Animal Ethical Committee at the Faculty of Veterinary Medicine, Kafrelsheik University gave its approval to the research, which was carried out in accordance with the principles outlined in the Declaration of Helsinki. An ethical license (KFS-IACUC/112/2021) was issued by the committee.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tawfic, A.A., Ibrahim, H.M., Mohammed-Geba, K. et al. Chitosan nanoparticles, camel milk exosomes and/or Sorafenib induce apoptosis, inhibit tumor cells migration and angiogenesis and ameliorate the associated liver damage in Ehrlich ascites carcinoma-bearing mice. Beni-Suef Univ J Basic Appl Sci 13, 74 (2024). https://doi.org/10.1186/s43088-024-00535-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s43088-024-00535-4

Keywords