Introduction: Zinc oxide nanoparticles (ZnONPs) are one of the most interesting metal oxide nanoparticles due to their easy functionalization, biocompatibility, and anticancer impact. The current study was designated to evaluate the in vitro and in vivo anticancer potency of biologically synthesized ZnONPs. Methods: Fenugreek seeds’ extract was used to prepare ZnONPs, and then characterized by scanning electron microscope (SEM), energy dispersive X-ray (EDX), X-ray diffraction (XRD), UV-V spectroscopy and transmission electron microscope (TEM). The in vitro antitumor activity of biogenic ZnONPs against different cancer cells was evaluated by MTT assay. In addition, their anticancer activities alone or in combination with Doxorubicin were investigated against EAC model using intraperitoneal injection day after day. Results: Biologically synthesized ZnONPs showed a cytotoxic potency against different cancer cell lines combined with lower toxicity against normal cells. Further, the in vivo study revealed that the treatment by ZnONPs alone or combined with doxorubicin hampered the proliferation of EAC in mice by lowering the ascetic volume and the number of viable tumor cells. Moreover, ZnONPs alone or combined with doxorubicin induced the cell cycle arrest at G0/G1 phase and apoptosis by up-regulating the expression of caspase-3 and Bax and down-regulating the expression of Bcl-2 proteins. Conclusion: Our study indicated that the biogenic ZnONPs could be instructive to future cancer treatment research.

Cell cycle Ehrlich ascites carcinoma Fenugreek seeds extract Zinc oxide nanoparticles

The chemotherapeutic drugs are considered as the primary option for different malignant tumors. These anticancer agents are designated to simply kill the cancer cells in a non-specific manner, leading to its distribution and accumulation in healthy tissues and resulting in undesirable and severe side effects1. Therefore, novel treatment approaches have been employed to minimize the severity of side effects such as nanotechnology. Nanotechnology has introduced nanoparticle systems for cancer treatment, which have the ability to selectively eradicate the cancer cells and at the same time save the normal cells from the drug toxicity2.

Zinc oxide nanoparticles (ZnONPs) are one of the most important inorganic metal oxide nanoparticles, which are employed in different biomedical applications such as cancer therapy due to its impressive properties, nontoxicity and biocompatibility3. Despite there being many approaches used in the manufacture of ZnO nanostructure, they are related to certain constraints such as toxic chemicals, high operating costs and energy needs4. Therefore, replacing these approaches with simple, cost-effective and environmentally sustainable systems is becoming increasingly important. The green synthesis of ZnONPs using natural plant extracts attracted the attention of the researchers because plant-mediated synthesis is environmentally friendly, inexpensive, readily available, yields the highest quantity of NPs and has a wide range of metabolites to help effectively produce and cap NPs5.

Fenugreek seed (Trigonella foenum graecum) is an annual plant belonging to the family of Leguminosae. Such seeds have therapeutic properties for anorexia, antidiabetic, hepatoprotective influence, anticancerial, antibacterial and gastric stimulant. Fenugreek seeds contain various components such as diosgenin, saponin, coumarin, a variety of alkaloids such astrigonellin, gentianin and carpaine, polyphenol compounds such as rhaponticin and isovitexin, and flavonoids6. Such biomolecules contain different functional groups capable of forming nanoparticles. The resulting nanoparticles are then safe against further reactions and aggregations, which increases its steadiness. Previous studies suggested that fenugreek seeds extract is rich in galactomannan —polysaccharides that have reducing functional groups which may have an important role in nanoparticles preparation, in addition to, containing phenolic compounds that may act as capping and stabilizing agents for the prepared nanoparticles7. The objective of this study is to introduce a new and simple green synthesis method for preparing the nanostructure of ZnO using fenugreek extract as a reducing agent and an effective stabilizer, rather than using harmful, reducing and costly stabilizing agents such as surfactants and polymers, and investigating their potential anticancer activity in vitro against various lines of cancer cells and in vivo using Ehrlich ascites carcinoma model.

Fenugreek seeds (Giza 1, Egyptian cultivar) were purchased from the local market of Tanta, Egypt. Ethanol, sodium hydroxide and zinc nitrate. 6H₂O were purchased from El-Nasr pharmaceutical chemicals company, Egypt. 3(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),Trichloroacetic acid (TCA), Thiobarbituric acid (TBA), 5,5'- dithiobis 2- nitrobenzoic acid (DTNB), 1-chloro-2,4 dinitrobenzene (CDNB) and reduced glutathione (GSH) were purchased from Thermo Fisher, Germany. Adricin (doxorubicin, 2 mg/mL) was obtained from EIMC United Pharmaceutics, Cairo, Egypt. All other chemicals were obtained in analytical grades.

The fenugreek seeds were used for the preparation of 5% aqueous extract according to the method of Ramamurthy with some modifications7. The mixture was boiled for 30 minutes on a magnetic stirrer and filtered to eliminate the unwanted residue after cooling using the Whatman filter paper, and the clear supernatant extract was used for the synthesis of 3D hierarchical flower-like nanostructure of ZnO.

Zinc oxide nanoparticles were prepared with some modifications in the methods8, 9. In brief, 20 mL of fenugreek aqueous extract was added dropwise to 80 mL Zn (NO₃)₂. 6H₂O (0.025 M) under continuous vigorous stirring at room temperature for 1 hour. The pH of the mixture was subsequently converted to 12 using NaOH (2 M) with continuous stirring for another 1 hour. This yielded a yellowish-white solution. A white ZnO powder was separated by centrifugation at 10000 rpm for 20 minutes, washed with doubly distilled H₂O, then with absolute ethanol three times to eliminate the impurities, dried in oven at 60 ^oC overnight, and collected for characterization.

The crystalline size and phase identity of synthesized ZnONPs were characterized by a GNR-X-ray diffractometer (APD 2000 PRO) with Cu-Kα radiation (Voltage = 40 kV, Current = 30 mA, λ = 1.5406, scan rate of 2 min^-1 and scan range of 2 θ from 10 — 90^o)10. Using the PerkinElmer spectrophotometer, the KBr technique was employed to check the Fourier transform infrared (FTIR) spectra in which the dried sample was grounded to powder with a mesh sieve and mixed 1/1 with vacuum dried KBr powder to make a compressed pellet with subsequent recording of infrared spectrum11. The surface morphology of samples was studied by transmission electron microscope (TEM), JEOL-TEM “100-SX” and scanning electron microscope (SEM), JeoL-JSM- 6510 TV12. The UV–Vis absorption spectrum of the sample was measured using the Shimadzu 2100S UV–Vis spectrophotometer (Japan) with a 1.0-cm-path length cell. Sample suspension was prepared by dispersing 1mg of ZnONPs in 1ml absolute ethanol by sonication, then diluted 20 times and absolute ethanol was used as a blank13, 14. The composition of elements such as Zn and O was verified by a dispersive energy X-ray spectra (EDX)15.

Four different types of cancer cells (EAC, MCF-7, HCT-116 and HepG2) and normal cells (WISH)were used to study the cell anticancer activity and cytotoxicity against ZnONPs. The cells were purchased from the cell culture department, VACSERA (Cairo, Egypt) and cultured according to the standard protocol. The HCT-116, EAC and WISH cells were grown in RPMI-1640, while MCF-7 and HepG-2 cells were grown in DMEM medium (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/mL) (Cambrex Bio Science Verviers, Belgium). All cells were maintained at 37 °C in a 5% CO₂ humidified incubator.

The cytotoxicity of the green synthesized ZnONPs was assessed by 3-(4, 5-di Methyl Thiazol-2-yl) 2,5-diphenyl Tetra-zolium bromide (MTT) assay. In brief, the cells were seeded into 96-well plates at density 1 x 10⁴ cells/well (1 x 10⁵ cells/mL) and incubated at 37 °C in a CO₂ incubator to develop a complete monolayer sheet. After 24 hours, the cells were subjected to treatment with different concentrations of ZnONPs and doxorubicin (5 – 100 µg/mL), and incubated for an additional 48 hours. The wells containing medium without drug were served as control, and triplicates were maintained for all concentrations. After the incubation, 200 µL of 0.5 mg/mL 3-(4, 5-di Methyl Thiazol-2-yl) 2,5-diphenyl Tetra-zolium bromide (MTT) (Sigma-Aldrich, USA) was added to each well and incubated in 5% CO₂ incubator for 4 hours. The formazan crystals of MTT reduction were solubilized in dimethylsulfoxide (DMSO) and then the absorbance was measured at 570 nm using microplate ELISA reader16.

The animal experiments were performed according to the guidelines for the care and use of laboratory animals approved by Research Ethical Committee (Faculty of Science, Tanta University, Egypt), which is also in agreement with the guidelines of the National Institutes of Health (NIH). Number of ethical committee is (Rec-Sci-Tu-0317). Ehrlich Ascites Carcinoma (EAC) bearing donor mouse was provided by the National Cancer Institute (Cairo University, Egypt). The tumor line was propagated in female mice in the ascetic form by sequential passages.

Sixty adult female Swiss albino mice weighing 18 – 30 g, purchased from the breeding unit of Egyptian Organization for Biological Products and Vaccines (Abbassia, Cairo), were used throughout this work and provided with standard diet and tap water ad libitum. They were divided into six groups (n = 10). The G1, control group were injected in day "0", then day other day with 0.1 ml of isotonic saline; G2, ZnONPs group were injected day other day with ZnONPs in a dose of 20 mg/Kg body weight17; G3, EAC group were injected once with 0.1 ml of 1 x 10⁶ EAC cells/mouse in day "0", then with saline day other day18; G4, EAC mice injected day other day with doxorubicin in a dose of 2 mg/Kg body weight19; G5, EAC mice injected day other day with ZnONPs in a dose of 20 mg/ Kg body weight; G6, EAC mice injected day other day with a combination of doxorubicin (2 mg/Kg body weight) and ZnONPs (20 mg/Kg body weight). All treatments were performed using intraperitoneal injection for 2 weeks. Treatment with doxorubicin and ZnONPs were begun after 24 hours of tumor induction.

On the 14^th day of the experiment, the animals were subjected to light anesthesia with ether, and the blood samples were collected and left to clot at room temperature, then centrifuged at 3000 rpm for 10 mins. The supernatant was withdrawn and stored at 20 ^oC for further serum parameters. After blood withdrawal, the animals were sacrificed, and the ascetic fluid was withdrawn from the peritoneal cavity and the viability of cells was checked by 0.4% trypan blue. The livers and kidneys were excised, washed in isotonic saline, then divided into two parts. The first part was fixed in 10% neutral formalin and kept for histopathological examination. The second part was homogenized and kept at -20 ⁰C for further biochemical parameters.

Diagnostic kits for measuring serum ALT, AST, albumin, urea, and creatinine were purchased from a biodiagnostic company in Giza, Egypt. ALT, AST, albumin, and urea were estimated according to20, while creatinine was determined according to21.

The extent of lipid peroxidation in tissue homogenate was estimated by measuring the production of malondialdehyde (MDA) as an indicator of oxidative stress. MDA was determined by incubating the samples with 0.67% thiobarbituric acid and measuring the pink-colored complex product TBARS at 532 nm22. The results were expressed as n moles of MDA/g tissue using the extinction coefficient (Ɛ) equal to 156000 M^-1 cm^-1. Reduced glutathione was determined according to the method of Beutler23, which is based on the reduction of 5, 5^´-dithiobis-2-nitrobenzoic acid (DTNB) by reduced glutathione to give a yellow-colored TNB chromophore, and its absorbance can be measured at 412 nm.

In addition, the antioxidant enzymes including catalase was assessed by monitoring the decomposition of H₂O₂ at 240 nm as described by Beers24. Concisely, 3 mL buffered H₂O₂ were pipetted in 3.0 cm quartz cuvette and 10 µL of sample was added, then mixed by inversion. The absorbance was measured at zero time and after one minute at 240 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the decomposition of 1 µmol H₂O₂/min under assay condition by using an extinction coefficient of 0.0394 mm^-1 cm^-1.

Glutathione peroxidase (GPx) activity was measured based on the method of Rotruck25, which depends on the reaction between the remaining glutathione after the action of Gpx and Elmann^,s reagent (DTNB) to give yellow colored complex which absorbed at 412 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the consumption of 1 µmol GSH per min.

Glutathione S-transferase (GST) activity was estimated by measuring the absorbance of dinitrophenyl thioether adduct formed due to conjugation between GSH and 1-chloro-2, 4 dinitrobenzene (CDNB) at 340 nm26. One unit of enzyme activity was defined as the amount of enzyme producing 1nmol of product per min using an extinction coefficient of 9.6 mM^-1 cm^-1.

The analysis of DNA content by flow cytometry was conducted according to the method of27 to support the cytotoxicity of ZnONPs towards EAC cells. In brief, EAC cells were washed twice with PBS and centrifuged. The supernatant was discarded and the cells were fixed in 500 µL of 70% ice-cold ethanol for at least 2 hours at -20 ^ºC. The mixture was centrifuged at 1000 rpm for 5 mins to remove the ethanol. The fixed cells were washed with PBS and mixed with 500 µL of PI (propidium iodide) staining solution. Before analysis, the stained cells were incubated in darkness for 30 – 60 mins at room temperature. Then the samples were analyzed using Accuri C6 Flow Cytometer (Becton Dickinson, Sunnyvale, CA, USA).

Immunoblotting was conducted following the method reported by28. Briefly, the total cell proteins were isolated by RIPA lysis buffer and the concentration total protein concentration was measured by Bradford assay29. Equal amounts of protein (20 µg) were separated on 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk, then incubated with anti-Bcl₂ (Abcam, AB59348), anti-Bax (Abcam, AB3191) and anti-Caspase3 (Abcam, AB184787) (1:1000) overnight at 4 ^oC. After washing, the membranes were incubated with an HRP secondary antibody (Abcam, AB97051) for 1 hour, then the chemiluminescent ECL substrate (PerkinElmer, USA, NEL103E001EA) was applied to the plot to detect the protein bands. Finally, these bands were analyzed by using image J software, and β-actin (Abcam, AB8226) was used as internal control30.

Small pieces of liver and kidney tissues which isolated from different experimental groups were fixed in 10% formalin and then dehydrated using different concentrations of alcohol. The dehydrated tissues were embedded into paraffin, sectioned for 4 mm thick and mounted on the glass slides. The tissue sections were stained by hematoxylin and eosin and examined by light microscope31.

All data were expressed as mean ± SD of three replicates from each experimental group. For comparison between groups, the data were analyzed by one-way of variance (ANOVA), followed by Tukey’s test in GraphPad Prism Software 6 (San Diego, CA). Probability values of less than 0.05 (P < 0.05) were considered to be statistically significant.

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Zinc oxide nanoparticles were prepared by using fenugreek seeds aqueous extract that formed a cloudy solution when added to a colorless solution of zinc nitrate. 6H2O, then turned into a yellowish white precipitate after adding sodium hydroxide. The SEM image of the prepared ZnONPs demonstrated a 3D hierarchical flower-like nanostructure with an average diameter of 1.7 μm (Figure 1Ⅰ). The elemental composition of the prepared sample was analyzed by Energy-dispersive X-ray. The spectrum in Figure 1Ⅱ revealed that the 3D flower-like nanostructure of ZnO contains three elements, namely zinc, oxygen and carbon, the atomic percentages of these elements being 20.59%, 45.01% and 34.40% respectively. Further, the XRD pattern of the green synthesized ZnONPs revealed various peaks at 2θ values of 31.92°, 34.42°, 36.31°, 47.52°, 56.68°, 62.93° and 67.98° which corresponded to (100), (002), (101), (102), (110), (103) and (112) crystal planes respectively (Figure 1 Ⅲ). The average crystalline size of ZnONPs, derived from the FWHM of a more intense peak corresponding to the (101) plane (located at 36.31°) was estimated to be 15.41 nm using Scherrer’s formula. FTIR (Figure 1ⅣA) was recorded to identify the most important functional groups on the fenugreek extract to examine their role in the manufacture and capping of ZnO 3D flower-like structure. The spectrum showed a high absorption peak of 3446 cm^-1and a strong band at a frequency of 1657 cm^–1. In addition to that, another small band appeared at 1446 cm^–1. For ZnO, a broad band appeared at 420 cm^-1 beside the main bands existing in the extract with a slight shift (Figure 1ⅣB). UV-Vis spectroscopy is an effective method in the affirmation of nanostructures' optical properties. UV-Vis spectroscopy initially confirmed the formation of ZnONPs within a range of 300 – 600 nm. The absorption spectrum of the 3D flower-like nanostructure of ZnO showed a distinctive band at 375 nm (Figure 1Ⅴ). TEM of the green synthesized ZnONPs was conducted to further investigate the topography structure of the prepared nanoparticles. TEM image (Figure 1Ⅵ) revealed a flower-like shape which is in good agreement with the findings of SEM. Moreover, these images show ZnO nanospheres with variable diameters, which were assembled into nanorods. Such nanorods were self-assembled to the 3D hierarchical flower-like structure. In addition to that, the polydispersity index and zeta potential of the prepared ZnONPs was studied using dynamic light scattering measurements as shown in Fig. S1 and S2 (supplementary data) respectively.

The cytotoxicity of ZnONPs was investigated by MTT assay using diverse cancer cell lines and doxorubicin as a chemotherapeutic guide drug. The data represented in Figure 2 showed an inverse relation between ZnONPs concentration and the viability of cancer cells, and the inhibitory concentration was found to be 27.09, 12.35, 20.7 and 12.05 µg/mL for EAC, MCF-7, HCT-116 and HepG-2 cells respectively. In contrast, the results revealed that there is a non-significant effect on normal cells (WISH) upon ZnONPs exposure up to 30 µg/mL. On the other hand, doxorubicin showed a cytotoxic effect on both cancer and normal cells.

Table 1

Groups	Body weight at start (g)	Body weight at sacrifice (g)	% of body weight change
Control	20.6 ± 6.0	21.56 ± 4.4	4.6
ZnONPs	21 ± 2.5	22.2 ± 0.5	5.7
EAC	21.68 ± 2.0	28.6 ± 3.1a	24.36
EAC + DOX	22.36 ± 1.6	22.9 ± 0.7b	2.5
EAC + ZnONPs	22.56 ± 2.2	24.15 ± 0.8b	7.04
EAC + DOX + ZnONPs	20.9 ± 1.7	21.3 ± 1.0b	2.03

Mice were treated 24 hours after inoculation of EAC (1 x 10⁶ cells) with ZnONPs (20 mg/kg, i.p, day other day) and doxorubicin (2 mg/kg, i.p, day other day) for 2 weeks. Results are expressed as mean ± SD. ^a P significant difference compared to control group, ^b P significant difference compared to EAC group.