Exploration of tumor growth regression of quinoa and chia oil nanocapsules via the control of PIK3CA and MYC expression, anti-inflammation and cell proliferation inhibition, and their hepatorenal safety in rat breast cancer model

Background The second most common cancer in the world is breast cancer. Chemotherapy is used to treat breast cancer, but instances of multidrug resistance, targets that are not selective, and physicochemical issues raise doubts about its efficacy. So, the exploration of chemopreventive agents from efficient natural sources has been required. The chia and quinoa seeds have health‑promoting activities that include cardio‑protective, antidiabetic, and antican‑ cer effects. Given the paramount importance of their oils and their potential bioactivities, this work aimed to assess the repressive effect of their oil nanocapsules against mammary tumors in rats. Rat models of chemically induced mammary tumors were gavaged with chia and quinoa nanocapsules for one month. The repressive effect of nano‑ capsules was studied by quantifying TNF‑α, assessing the gene expression of proto‑oncogenes (PIK3CA and MYC) using qRT‑PCR, and analyzing the cell cycle in mammary tissue. Results The studies clarified that the inhibition of tumors in response to quinoa and chia nanocapsules was associ‑ ated with a reduction in TNF‑α levels, proliferation capability, and motivation for apoptosis. Furthermore, quinoa and chia nanocapsule management repressed the activation of the MYC and PIK3CA genes. As well as nanocapsules modulated the liver enzymes and kidney function alterations induced in mammary tumor animals. Meanwhile, both oils’ nanocapsules do not have an impact on the liver and kidneys of healthy rats. Conclusions The findings indicate that quinoa and chia nanocapsules are safe and can reduce tumor growth, sug‑ gesting a potential natural therapeutic target for breast cancer treatment.

Page 2 of 15 El makawy et al.Bulletin of the National Research Centre (2024) 48:7

Background
In Egypt, there are over 22,000 new cases diagnosed with cancer annually, accounting for 33% of all female cancer cases.Given the growing population, shifting population pyramid, and adoption of a more Westernized lifestyle, this is predicted to increase exponentially over the coming years.Breast cancer (BC) is one of the most important cancers in Egyptian women (Abdelaziz et al. 2021).
According to the last global cancer statistics, it was the second-leading cause of cancer-related deaths in 2018.In 2018, more than two million women were affected by this disease, with a mortality rate of 6.6%, resulting in more than 500 thousand deaths of women (Bray et al. 2020).Chemotherapy resistance is defined as a medication's low ability to generate a positive response during treatment and is one of the primary causes of chemotherapy failure (Vasan et al. 2019).Chemotherapy resistance affects the choice of chemotherapy in recurrence cases and presents a challenge to the treatment of neoplasms.Multidrug resistance is a characteristic of cells that can endure chemotherapy treatment and decrease drug absorption, which encourages the efflux of anticancer medications from tumor cells.Membrane transporter protein activity is dependent on MDR, which can explain why some cancer cells are resistant to chemotherapy (Nedeljkovic and Damjanovic 2019).
In terms of genetic and biochemical processes, cancer has several characteristics that may cause the deregulation of signaling pathways like p53 and nuclear factor-B (NF-B), accelerating cancer development (Aunan et al. 2017).The immune system's primary building block is inflammation, where chronic inflammation is thought to be a sign of cancer development and has the potential to cause a variety of complex changes at the molecular, cellular, and hampered healing processes.Also, the ROS/ RNS random production causes mutations, decreases DNA repair efficiency, and significantly increases cytokine/chemokine release, as well as pathophysiologic protein synthesis and signaling pathways (Kubatka et al. 2021).
The pleiotropic proinflammatory cytokine that is encoded by tumor necrosis factor (TNF) is important for controlling immune cells.The cytokine was implicated in the perception of a broad range of biological processes, such as cell division, proliferation, death, lipid metabolism, and coagulation.TNF was discovered to be an endotoxin-induced serum factor that contributes to several malignancies, including necrosis.TNF-α has been associated with breast cancer developmental stages and metastasis (Rana et al. 2021).
A highly complex process is involved when tumor tissue transforms regularly over the course of years or even decades of life.Strong stresses that have the potential to damage DNA, persistent inflammation, extensive interaction between relevant molecular pathways, and cellular cross talk between adjacent tissues all play a role in this process.Based on modifiable risk factors, which are essential in cancer prevention, the majority of cancer cases are naturally sporadic (Kapinova et al. 2018).
The discovery of chemopreventive agents from efficient natural sources has received a lot of scientific attention in recent years.Through various cellular and molecular methods, a wide range of phytoconstituents have been investigated for their potential to inhibit the development of carcinogenesis both in vitro and in vivo (Kaur et al. 2018).
Due to their superior nutritional value and wide range of bioactive compounds, chia (Salvia hispanica L.) and quinoa (Chenopodium quinoa Willd.)seeds have regained popularity in developed nations (Pellegrini et al. 2018a, b).Natural antioxidants like tocopherols, phytosterols, carotenoids, and phenolic compounds are abundant in chia and quinoa seeds (Pereira et al. 2019;Marineli et al. 2014).Omega-3 and omega-6 fatty acids can be found naturally in chia (Salvia hispanica L.) seeds.A sizeable amount of dietary fiber, proteins with high biological value, tocopherols and polyphenols, vitamins, carotenoids, and minerals are also present in them.Because of their health-promoting properties, which include effects on diabetes prevention, cancer prevention, and cardioprotection, chia seeds have seen a sharp increase in popularity in recent years (Camara et al. 2020).Also, Shaer and Al-Abbas (2022) concluded that chia nanoparticles are a hopeful adjuvant therapy in breast cancer cells.Quinoa (Chenopodium quinoa Willd) grains are renowned for their superior nutritional value and potential health advantages.They contain high levels of dietary fiber, unsaturated fatty acids, and minerals, and their protein content ranges from 12 to 20% (Carvalho et al. 2020).In addition, quinoa is rich in polyphenols, phytosterols, flavonoids, and vitamins E, B, and C, which have positive health effects (Carvalho et al. 2019).Quinoa has been identified in numerous studies as a protective agent against a wide range of illnesses, including cancer, allergies, and inflammatory diseases.Additionally, they may lower the risk of cardiovascular diseases (Heck et al. 2019).A few subsequent in vitro investigations have indicated the antitumor properties of quinoa seeds.Quinoa seeds exhibit a promising cytotoxic effect against liver cancer cell lines (Mohamed et al. 2019), and human cervical carcinoma cell lines (Pasko et al. 2019).In addition, Mollaei et al. (2021) proposed quinoa as a latent complementary agent for lung cancer therapy that could induce apoptosis in lung cancer cells (A549) through the increased ratio of BAX/BCL2.
The tumorigenic perspective of an oncogene is well defined by how well it collaborates with other driver alterations to alter a cell's structure in complicated ways (Bell et al. 2019).One of the most frequently altered genes in cancer is phosphatidylinositol-3 kinases (PIK3), which are heterodimeric lipid kinases that encode the catalytic subunit p110 of PI3K.Activated PI3K is essential to tumor development by regulating a variety of cellular processes, including metabolism, genomic stability, cell motility, and proliferation (Venetis et al. 2020).The MYC oncogene family consists of three members, C-MYC, MYCN, and MYCL, which encode c-Myc, N-Myc, and L-Myc, respectively.The Myc oncoproteins are members of the family of super-transcription factors, which may control at least 15% of all genome transcription.A variety of biological functions, such as cell survival, differentiation, and proliferation, as well as immune monitoring, are controlled by the principal downstream effectors of Myc (Chen et al. 2018).MYC deregulation is linked to poor outcomes and contributes to the onset and spread of breast cancer.The loss of tumor suppressors and the activation of oncogenic pathways are associated with MYC deregulation in breast cancer, which is caused by a variety of mechanisms including gene amplification, transcriptional regulators, and protein and mRNA stabilization (Kalkat et al. 2017;Ahmadi et al. 2021).
The phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene encodes for the catalytic subunit of PI3K (p110 protein).Numerous forms of human cancer, including colon, breast, brain, liver, stomach, and lung cancers, have been linked to gene insertions, deletions, and somatic missense mutations in this gene.It was suggested that somatic PIK3CA mutations could increase the kinase activity of the protein change the cellular structure.As the most frequently mutated oncogene in breast cancer, PIK3CA mutations have been linked to breast cancer.According to current theories, 20-30% of all cases of human breast cancer have PIK3CA mutations (Alqahtani et al. 2019).
Treatment decisions for breast cancer must take into account the patient's preference for alternative treatments such as CAM in addition to traditional care options like surgery, radiation, chemotherapy, and endocrine therapy.As a means of boosting immune system function, managing cancer symptoms, preventing cancer from returning, and reducing emotional distress.Up to 80% of breast cancer patients reported including complementary and alternative therapies in their treatment plans.In addition, CAM therapy use was 36% higher among breast cancer survivors than in general patients.So, the study aimed to investigate the potential role of quinoa and chia nanocapsules in repressing inflammation, proto-oncogene expression variation, and cell cycle proliferation in rats' breast cancer.In addition, to determine the safety of quinoa and chia nanocapsules by assessing liver and kidney functions.

Oil extraction
Oil was extracted from 500 g of seeds powdered by soaking them in petroleum ether 40-60.The rotary evaporator was used to evaporate the solvent under a vacuum at 35 °C.The extraction was carried out until it was exhausted.To create saponifiable fractions for GC/MS analysis, the dried solvent-free extract was employed (El Makawy et al. 2022).

Unsaponifiable compound isolation
Oil was saponified by heating 5 g of oil at 95 °C for 1 h while being mixed with 50 ml of 1 N ethanolic KOH. 100 cc of distilled water was added when the mixture had cooled, and it was thoroughly mixed.The resultant solution was extracted twice using 100 ml of diethyl ether in a decanter funnel.Every extraction stage involved collecting the top organic layer, washing it two times with 75 ml of distilled water, once with 100 ml of 0.5 N ethanolic KOH, and then neutralizing it with 100 ml of distilled water.After that, the organic layers were dried with Na2SO4, and the solution was filtered and evaporated using a vacuum oven at 45 °C (Tavakoli et al. 2019).

Nanocapsule preparation and characterization
Sodium alginate solution was used as the aqueous phase in the manufacturing process of nanocapsules, according to El Makawy et al. (2022).Transmission electron microscopy (TEM) was applied to assess the nanocapsule's dimensions and morphology.

Animals
The experiment of the study was done on 96 healthy female Wistar rats from the National Research Centre animal house, weighing between 130 and 150 g.They were exposed to a 12 h day/night cycle at an ambient temperature of 22 3 °C and a humidity of 50 ± 10% with free access to food and water.Animals were kept for acclimatization for about 14 days.In all animal operations, the handling and use of experimental animals were done in accordance with the standards set by the National Institutes of Health (NIH) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).All experiments were performed in line with the ethical guidelines approved by the Medical Research Ethics Committee of the National Research Centre, Egypt, for Experimental Animals (No.19164).

Experimental design
Firstly, thirty-six rats were distributed to six groups, six rats each, and treated as the follows: distilled water and considered a negative control; 100 mg/kg b.w.corn oil as a vehicle; 100 and 200 mg/kg b.w.Quinoa oil nanocapsules; 100 and 200 mg/kg b.w.chia oil nanocapsules.Secondly, sixteen female rats were injected subcutaneously into the mammary region with a single dose of 80 mg/ kg b.w.DMBA dissolved in 0.5 ml corn oil.Then, after four months of treatment, rat breast cancer models were divided into six groups of 10 animals each: DMBA group animals remained without treatment; 5-Flu group animals were managed with a dose of 20 mg/Kg b.w./day 5-fluorouracil; chia oil nanocapsules two groups were treated with chia oil nanocapsules 100 and 200 mg/kg b.w./day; the other two groups were administered with quinoa oil nanocapsules at a dose of 100 and 200 mg/kg b.w./day.For all treatments, the animals were administered orally for one month.

Anesthesia and tissue collection
Once the course of treatment had ended, the rats were anaesthetized through IP injections of xylazine (40 mg/ kg b.w.) and ketamine (4 mg/kg b.w.), according to Bhatia et al. (2022).According to Jekl et al. (2005), blood was withdrawn from the inferior vena cava in heparinized tubes and centrifuged for 10 min at 5000 rpm.Then, plasma was collected and retained in aliquots at -80 °C.Besides, rats were killed by cervical dislocation, and afterward, PBS was pumped into them to collect the required tissues.

Determination of the serum biochemical parameters
Activities of enzyme markers in the liver and kidney, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, uric acid, urea, and albumin in serum, were determined.All the analyses were performed in triplicate for every sample using available commercial kits from Bio Diagnostic (Egypt).

Quantification of TNF-α in mammary gland tissues as measured by ELISA
The level of TNF-α was determined by an enzyme-linked immunosorbent assay (ELISA) test using a rat tumor necrosis factor α (TNF-α) kit (SL 0722Ra, Sunlong Biotech Co., Ltd.) as described by the kit manufacturer.

Assessment of PIK3CA and MYC gene expression by quantitative real-time PCR
Samples of inguinal mammary glands were homogenized in an Easy Red Total RNA Extraction Kit (Intronbio, Korea), then RNA was extracted according to the manufacturer's instructions.The yield and quality of RNA were analyzed using a NanoDrop ™ 1000 spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis.RNA (1000 ng) was treated with the RNase-free DNase kit (Promega) to remove any genomic DNA contamination, then cDNA was synthesized using the First Strand cDNA Synthesis Kit (Thermo Scientific).Two oncogenes (PIK3Ca and MYC) were used in the study, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control (Table 1).qRT-PCR was performed in a 15 µl reaction containing cDNA, TOPreal ™ qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics), forward and reverse primers (10 pmol/μl) (Macrogen), and nuclease-free water.The resulting data were normalized to GAPDH and analyzed using the 2 − ΔΔCT method (Livak and Schmittgen 2001).
Histopathological tissue preparation and cell cycle analysis: Inguinal mammary gland samples were fixed for 72 h in 10% neutral-buffered formalin, then dehydrated using alcohols, cleaned with xylene, and embedded in paraffin wax.Using a microtome, 5-m-thick sections were sliced and stained with Feulgen.The image analysis of stained DNA was done using the computerized Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd., Cambridge, UK).Cell cycle analysis was performed on a real-time image from the microscope that we visualized on the video monitor.The normal control samples were analyzed first to establish the reference values.The optical density of the selected nuclei in each microscopic field is measured and automatically converted by the system into a cell phase in the life cycle.Up until the appropriate number of nuclei (100-150) was measured, numerous fields were chosen.Percentages of cells in the first growth phase (G1), proliferating cells (S), and second growth phase (G2) were calculated and determined automatically by the system.

Statistical analysis
The statistical analysis was carried out using SPSS software (SPSS, IBM, Chicago, IL, USA).The results were displayed using means and the standard error of means (SEM).An analysis of variance (ANOVA) and a Duncan post hoc descriptive test were used in the statistical analysis, with P ≤ 0.05 being recorded as statistically significant.

Nanocapsules characterization
The nanocapsule examination by TEM showed that quinoa nanocapsules were spherical, and the mean particle sizes ranged between 5 and30 nm.The chia nanocapsules were polygonal, with sizes ranging between 10 and 35 nm.

The impact of chia and quinoa oil nanocapsules on serum TNF-alpha levels
The data in Fig. 1 represent the values of serum TNF-α in all experimental groups.The results of the serum TNF-α analysis revealed that there were no significant differences between the corn oil and the negative control.Although quinoa and chia nanocapsules induced a significant increase in the levels of TNF-α (P ≤ 0.05) in healthy animals as compared to the control.In breast tumor animals, the TNF-α content showed a highly significant increase, while treatment with 5-Flu, quinoa, and chia nanocapsules at two doses significantly (P ≤ 0.05) attenuated the elevation of TNF-α levels in tumor tissue.

Cell cycle analysis
Figure 2 clarifies the analysis of the cell cycle of rat breast tumor cells before and after management with quinoa, chia nanocapsules, and 5-Flu.Quinoa and chia nanocapsules-treated animals with two doses of 100 and 200 mg/kg showed a typical DNA pattern that was  characterized by sub-G1, G1, S, and G2/M cell cycle phases.The population of cell nuclei containing a sub-G1 complement of DNA representing the apoptotic cells was high in all treated groups.In chia nanocapsules, the low-dose treatment was 67% and the highdose was 71%: in quinoa nanocapsule-treated animals, was 50% for the low dose and 29% for the high-dose, and in 5-Flu-treated animals, was 87%.Quinoa nanocapsules at low dose decreased the percentage of cell population in the G1 phase from 60% in tumor tissue to 45%, and chia capsules to 31% and 28%, as well as 5-Flu, which decreased the cell population to 12%.On the other hand, the percentage of cells in the S phase was very low in the quinoa and chia-treated groups compared to tumor group.In addition, all treatments triggered an associated decrease in the proportion of cells in the cell cycle G2/M phase.These findings proposed that quinoa low dose and chia two doses, as well as 5-Flu, induced G1-phase cell cycle arrest.

Proto-oncogenes expression
Figure 3 illustrates the effect of chia and quinoa nanocapsules on MYC gene expression in the mammary gland tissue of healthy and tumor-model rats.No obvious difference was detected in the MYC gene expression between the control and tumor groups.Compared to the tumor group, quinoa, chia, and 5-Flu significantly reduced MYC expression (P ≤ 0.05).There was no difference in MYC expression between the breast cells of animals treated with the high dosage of quinoa and those treated with the 5-Flu.
Figure 4 shows the influence of chia and quinoa nanocapsules on PIK3ca expression in the mammary gland tissue of healthy and tumor-model rats.As depicted in Fig. 4, DMBA significantly increased Pik3ca expression as compared with the control group.Interestingly, treatment with 5-Flu significantly increased Pik3ca expression as compared with the DMBA group.On the contrary, treatment with chia and quinoa reduced the expression of PIK3ca more than in DMBA and 5-Flutreated groups.Both quinoa and chia nanocapsules (100 mg/kg) significantly decreased the PIK3ca expression more than in 200 mg/kg (P ≤ 0.05).

Effect of chia and quinoa oil nanocapsules on liver functions
Data analyses of the effect of quinoa and chia oils nanocapsules on the liver function of the DMBA-breast cancer rat model are illustrated in Table 4.The administration of chia and quinoa oil nanocapsules to healthy animals did not display a significant alteration in the ALT and AST serum levels, while significantly increasing the serum ALP level (P ≤ 0.05) as compared to the vehicle.Meanwhile, in the breast tumor model, chia nanocapsules exhibited a dose-dependent reduction in the elevation of ALT, AST, and ALP.However, the quinoa nanocapsules significantly diminished the level of ALT and nonsignificantly decreased the levels of AST and ALP as compared to the DMBA-treated group.Moreover, 5-Flu management in breast cancer models significantly (P ≤ 0.05) diminished the levels of serum ALT, AST, and ALP as compared to breast tumor animals.

The effect of quinoa and chia oil nanocapsules on kidney function
As shown in Table 5, chia and quinoa nanocapsules did not affect the serum creatinine levels in healthy rats.Low doses of chia and quinoa oil nanocapsules did not induce Fig. 3 The effect of chia and quinoa oil nanocapsules and 5-flurouracil on Myc expression in mammary gland tissues of DMBA-treated rats (n = 6) for all tested groups Fig. 4 The effect of chia and quinoa oil nanocapsules and 5-flurouracil on Pik3 expression in mammary gland tissues of DMBA-treated rats (n = 6) for all tested groups  significant change in the serum levels of uric acid and urea, while the high doses significantly elevated the uric acid, urea, and albumin levels as compared to the control.In breast tumor tissues, levels of serum creatinine, uric acid, urea, and albumin were significantly higher (P ≤ 0.05) than in normal tissue.Meanwhile, chia and quinoa oil nanocapsule management reduced the rise in creatinine, urea, and albumin levels in a breast cancer rat model but did not diminish the uric acid level.

Discussion
The global effort to prevent and treat breast cancer is being made by scientists.Rapid technological progress produces synthetic medicines, but due to their harmful side effects, phytomedicine has gained great attention in recent years (Laskar et al. 2020).Natural ingredient medications for the treatment of various forms of cancer depend on modifying multiple pathways, including cellular proliferation, differentiation, apoptosis, angiogenesis, and metastasis.These phytochemicals have shown anticarcinogenic capabilities by preventing the onset, development, and progression of cancer (Muhammad et al. 2022).The current study investigates the role of chia and quinoa oil encapsulation in breast cancer repression.The findings showed that rats exposed to either of the two oil nanocapsules had a lower risk of developing breast cancer.Numerous studies have supported the anticancer properties of chia and quinoa.Cardenas et al. (2018) reported that chia seeds are a promising source of protein fractions with anticancer activity because they have a high protein content and a good amino acid profile.Chia seed extract has been shown to be effective in treating cancer (Mutar and Alsadooni 2019).Liu et al. (2020) found that quinoa seed is rich in phenolic components that can be responsible for their superior antioxidant, anti-inflammatory, and antitumor activities.
Our results revealed that squalene is the major component of the unsaponifiable fraction of quinoa oil.Squalene is a metabolite involved in the biosynthesis of cholesterol.It is established that it has in vitro anticancer activities, enhances the action of the immune system against tumors, and protects breast cells against the accumulation of mutagenic lesions in their DNA (Palaniyandi et al. 2018;Sanchez-Quesada et al. 2022).
The presence of inflammatory cells and inflammatory mediators, such as cytokines, in the cancer microenvironment is one aspect of cancer-related inflammation that contributes to the disease's progression.Of all the cytokines, tumor necrosis factor-alpha (TNF-α) is a significant multifunctional inflammatory cytokine that regulates immune responses, survival, and apoptosis through a variety of pleiotropic effects.Numerous cell types, including fibroblasts, Kupffer cells, keratinocytes, infiltrating inflammatory cells, and tumor cells have been reported to be capable of releasing TNF-α.By binding to its receptors, that can increase the production of growth factors, proteases, and cytokines, such as TNF-α receptor 1 (TNFR1) and TNF-α receptor 2 (TNFR2) (Cai et al. 2017).In breast cancer, high proinflammatory cytokine (TNF-α) expression levels are associated with increased cell proliferation, a higher malignancy grade, and a higher incidence of metastasis (Mercogliano et al. 2020).Quinoa and chia seed nanocapsule treatments reduced the serum TNF-α content of breast cancer animals.These results were supported by Ferreira et al. (2018) and Ahmed et al. (2021), who confirmed that chia seeds reduced plasma IL-6 and TNF-α levels as well as ROS levels.Abdel-Wahhab et al. (2021) found that quinoa supplementation inhibits the overproduction of inflammatory mediators, as evidenced by a significant decrease in serum TNFα, IL-1β, IL-6 levels in cyclophosphamideintoxicated mice.The abundance of antioxidants, such as linolenic acid, may be to blame (Kulczynski et al. 2019).Also, the phytosterol squalene present in quinoa may be responsible for its observed anti-inflammatory activity.It was suggested that squalene has the potential to prevent the over-activation of neutrophils, monocytes, and macrophages via targeting pro-and anti-inflammatory pathways (Ibrahim and Naina Mohamed 2021).
The present study showed a significant increase in Pik3ca expression in tumor tissue as compared to the control.The phosphatidylinositol-3-kinase (PI3K) pathway plays a main role in the development and progression of breast cancer.The Pik3ca gene is one of the most frequently mutated genes in breast cancer, which leads to PI3K activation (Dong et al. 2021).Recently, PI3K inhibitors can be used as a strategy for cancer treatment alone or combined with other strategies of cancer therapy (Alowiri et al. 2019).Herein, treatment with chia and quinoa nanocapsules showed a significant decrease in Pik3ca expression, suggesting their capability to inhibit the PI3K pathway.This was in concurrence with (Wang et al. 2017).They suggested that KIF20A knockdown brought cell cycle arrest in the G0/G1 phase and stimulated apoptosis by deactivating the PI3K/Akt pathway via c-Myc and triggering apoptosis.
Normal biological functions like cell proliferation and apoptosis depend on MYC control and transcriptional activity.MYC plays an important regulatory role in the occurrence of breast cancer, and its amplification can be used as a predictor of diagnosis (Liu et al. 2021).More than half of human malignancies have been proven to be caused by the deregulation of the MYC oncogene (García-Gutierrez et al. 2019).In healthy cells, expression of the endogenous MYC gene is elevated in response to diverse mitogenic and developmental signals.The MYC protein functions as a transcription factor that integrates these signals into wide changes in gene expression and prompt cell growth and proliferation.Many of the genetic alterations that occur in tumors decouple MYC expression from its normal regulatory constraints, thereby resulting in an elevation of MYC proteins that are less sensitive to normal and extracellular signals (Schaub et al. 2018).
Data from our study showed that there was no obvious change detected in MYC gene expression levels between the control and breast tumor groups.The exact mechanism by which the oncoprotein contributes to the development, maintenance, and progression of cancer is still unknown, despite evidence that MYC dysregulation is causally involved in these processes.It might accomplish this through a variety of pathways, including those that promote angiogenesis, reduce apoptosis, regulate stem cell production, and boost cell proliferation (Beaulieu et al. 2020).
It was suggested that up-or down-regulation of MYC mRNA levels controls the entering and exiting of the cell cycle.MYC stimulates cell cycle mainly through the repression of cell cycle inhibitors.MYC stimulates the cell cycle progression through the regulation of many genes related to cell cycle control (Ahmadi et al. 2021).Our research results revealed that chia and quinoa nanocapsules, as well as 5-Flu treatment, lowered MYC mRNA expression and arrested the cell cycle in G1 phase.The decrease in MYC levels and accumulation of p53 are typical responses to reducing cell damage.MYC promotes apoptosis by indirectly increasing p53 levels, which in turn induces p53 to block MYC production (Ahmadi et al. 2021).Taken together, the results of the current study and of our earlier research (El Makawy et al. 2022, 2023) were in agreement with the above-mentioned mechanism.
One of the physiological alterations in cancer cells is the absence of apoptotic mechanisms, a characteristic associated with enhanced cell proliferation (Hamza et al. 2022).The result of cell cycle analysis clarified that the tumor tissue showed an increase in cell proliferation, while quinoa and chia nanocapsule management displayed a decrease in cell proliferation, clarifying an increase of apoptosis.Cell death is a fundamental biological process necessary for cellular development, and its degeneration is linked to the pathogenesis, etiology, and treatment of many degenerative diseases such as cancer (Zhang et al. 2016).This indicated that the quinoa and chia nanocapsules have the ability to treat breast cancer in rats' models.In agreement with our results, Shaer and Al-Abbas (2022) concluded that chia seeds loaded with PLGA-PEG nanoparticles are a promising adjuvant therapy in breast cancer cells via exerting apoptosis.According to Albadri [Albadri (2020) and Mollaei et al. (2021), quinoa may have lethal effects on the breast cancer cell lines MCF-7 and MDA-MB231 through an apoptotic mechanism.In DMBA-treated animals receiving Salvadora persica fruit, Hamza et al. (2022) found that an increase in cell death and a decrease in cell proliferation would result in enough cell turnover to reduce the number of potentially cancerous cells and prevent tumor growth.In addition, Hashem et al. (2022) concluded that the majority of natural products target intrinsic apoptotic signaling pathways, which result in excessive intracellular signals that set off mitochondrial-initiated processes that kill cancer cells.Luca et al. (2020) reported that the essential oils of different plants have been demonstrated to stop the cell cycle by modulating the expression of cell cycle regulatory proteins.In the present study, the cell cycle analysis of mammary gland tissues indicated that quinoa and chia nanocapsules arrested the cell cycle at the G0/G1 phase.A similar observation regarding cell cycle arrest at different phases was established by Pani et al. (2022).They indicated that individual drugs, curcumin, ellagic acid, quercetin, and resveratrol effectively arrested the cell cycle at the S phase, while the combination of drugs arrested the cycle at the G2/M phase.
Regarding the impact of quinoa and chia nanocapsules on liver and kidney functions, the findings showed that chia oil nanocapsules did not significantly change the ALT and AST blood levels in healthy animals but did lower them in tumor-model rat liver and kidney tissues.According to Mohammed and Basuny (2020), rats fed chia seed oils for eight weeks did not experience any appreciable alterations in the activity of their liver enzymes.In contrast, ALT and AST levels in healthy rats were reduced by quinoa nanocapsules.This result was in line with that of Ng and Wang (2021), who noted that because a high level of these markers is associated with liver damage, a drop in ALT and AST levels caused by quinoa eating is likely to be advantageous to liver health.Quinoa's capacity to protect the liver has also been proven in various studies using animals.Quinoa attenuated the liver function in rats managed with a high-cholesterol diet, CCl4, nicotine, a high-fructose diet, and infected fish (Alghamdi 2018; Halaby et al. 2017;Al-Qabba et al. 2020;Saxena et al. 2017;Ali 2019;Mohamed et al. 2019;Ahmed et al. 2020).The results of the current study also revealed that chia and quinoa nanocapsules did not affect creatinine levels, but their high doses increased the levels of urea, uric acid, and albumin in healthy rats and moderated the hepatotoxicity brought on by tumor models.According to Demir and Bilgic (2019), a chiarich diet does not affect renal function parameters.

Conclusions
We can infer that quinoa and chia nanocapsules are riskfree and can inhibit tumor growth by lowering TNF-α levels, decreasing the ability of cells to proliferate, and leading to apoptosis.Furthermore, the management of nanocapsules repressed the activation of the MYC and Pik3ca genes.MYC elevation signifies one of the possible mechanisms through which breast tumors progress resistance to PI3K pathway-specific targeted therapies.As products of oncogenes, MYC and PIK3CA are wellestablished oncoproteins that contribute to breast oncogenesis.Meanwhile, results confirmed that both oils' nanocapsules do not impact healthy rats' livers and kidneys.So we can advise that quinoa and chia nanocapsules are safe and promising adjuvant therapies for breast cancer treatment.However, additional assessments are required to validate our findings and ensure their safety.There was a research limitation due to the paucity of prior research investigating the assessment of these oils in vivo potential anticancer properties.

Fig. 1
Fig.1The levels of serum TNF-α in healthy and tumor rats.Data presented as mean ± SE (n = 6) for all tested groups.Different letters show significant difference among groups in a column (P ≤ 0.05)

Table 1
Sequence of primers used in the study

Table 2
GC/MS analysis of the unsaponifiable fraction of quinoa oil R t : Retention time (min); M + molecular weight; BP: base peak

Table 3
GC/MS analysis of the unsaponifiable fraction of chia oil R t : Retention time (min); M + molecular weight; BP: base peak

Table 4
Effect of chia and quinoa oil nanocapsules on liver functions

Table 5
Effect of chia and quinoa oil nanocapsules on kidney functions