Apoptotic Genes’ Expression in an Ovarian Cancer Cell Line (A2780) Exposed to Green Synthesized Cerium Oxide Nanoemulsion

Introduction: Cancer can be defined as an illness of reformed gene expression. There are numerous agents affecting gene expression and altering cellular activities. Nanotechnology has offered the possibility of modulating tumor suppressor genes’ expression, improving kinetics of gene-targeted therapeutics, and simplifying drug delivery to tumors and across bio-complexes. Methods: Gene expressions of caspase3, 8 and 9, Bax and Bcl2 were assessed after RNA extraction and cDNA synthesis by real-time polymerase chain reaction (PCR) in an ovarian cancerous cell line (A2780). Real-time PCR was employed to determine the change fold of apoptotic genes in the cells exposed to the biosynthesized CeO 2 -NPs ( cerium oxide nanoparticles) at the dose of 0, 7, 9, and 11 μg/mL after 24 hours of incubation. Results: Our findings displayed a significant increase in the antioxidant genes’ expression in the A2870 cells. The results exhibited that the biosynthesized CeO 2 -NPs could enhance the Bax/ Bcl2 ratio in a dose-dependent way. Also, the expression of caspase3, 8, and 9 up-regulated significantly under the dose of 11 μg/mL. Conclusion: Considering the effects of the bio-fabricated CeO 2 -NPs on the expression of apoptotic genes in ovarian cancerous cell lines, these nanoparticles (NPs) may be employed in pharmacology to develop new anti-cancer medications.

surface area, and good bio-compatibility.
There are many methods for nanoparticle synthesis, among which green methods applying plants are more important because of their low toxicity, high safety, and being eco-friendly. Extracts from several plants such Ribes khorasanicum, 37 Handelia trichophylla, 38 Tribulus terrestris, 39 and other herbal species have been used for the synthesis of NPs. Brassica napus from the Brassicaceae family is an important plant in the food and agriculture industries. The oil of canola (B. napus), as an important major oilseed crop, is used in many food products.
Nanoemulsions are sub-micron-sized emulsions used to deliver drugs to cancer cells and have been extensively considered in recent years. Ovarian cancer is the seventh frequently identified cancer among women. 40 Rare studies have been performed on the effects of cerium nanoemulsions on cancer cells. This investigation aimed to estimate the gene expression of a number of apoptotic genes in the A2780 cell line exposed to various concentrations of cerium oxide nanoemulsions.

Experimental Materials
PCR Master Mix, SYBR Green PCR Master Mix, and cDNA synthesis Kit were obtained by Qiagen (Germany).

Extraction and Preparation of Cerium Oxide Nanoemulsions
With the purpose of providing the extract, 10 g of B. napus pollen grains powder was added to distilled water and mixed. The combination was heated for 60 minutes at 40°C and then filtered. The Ce(NO3) 3 •6H 2 O solution in 90 mL distilled water and 10 mL pollen extract was stirred for 2 hours at 40°C. Subsequently, the precipitate was separated and heated at 500°C for 2 hours to obtain the nanoceria. Since cerium oxide NPs are insoluble in water, its use is somehow limited in various fields. In order to increase their bioavailability, nanoemulsions were formed from cerium oxide NPs. To prepare the nanoemulsions, 5 mg of the NPs was suspended in polysorbate (tween) 20 (100 µL) and tween 80 (100 µL) and then added to 500 μL of ethylene glycol and completely mixed. Finally, it was reached to 50 mL by adding distilled water. 15 Cell Culture A2780, the human ovarian cancer cell line, was obtained from National Pasteur Institute (Iran) and maintained in DMEM medium. Cells were cultured, and ∼1×10 4 cells/ well were planted into 96-well dishes. 41 A2780 cells were treated with the bio-fabricated CeO 2 NPs to check their cytotoxicity.

Expression of Apoptotic Genes
The gene expressions of caspase 3, 8, and 9, Bcl2, and Bax were determined in the ovarian cancer cells (A2780) treated with the fabricated NPs. The cells were seeded at 5×10 3 cells/mL concentration in RMPI 1640 containing fetal bovine serum (FBS) and Pen-Strep. After that, the cells were treated with 0, 7, 9, and 11 µg/mL concentrations of the NPs for 48 hours. Then the treated cells were washed by Phosphate Buffered Saline (PBS). The gene expressions of caspase3, 8, and 9, Bcl2, and Bax were evaluated by quantitative reverse transcription real time polymerase chain reaction (RT-qPCR) and the primers displayed in Table 1.

RNA Extraction
After 48 hours of treatment with CeO 2 NPs, total RNA was obtained from A2780 cells. For this, one mL of the RNXplus solution was added to cells. Later, chloroform was added, and centrifuge was performed at 4°C to extract RNA, which was quantified by a plate-reader (Epoch, Biotek, Winooski, VT, UK).
cDNA Synthesis cDNA was produced from the total extracted RNA by Fermentase Kit (Germany). The mixture was kept for one cycle at 37 °C/15 min, one cycle at 85 °C/5 seconds, and one cycle at 4°C/5 min. Furthermore, RT-enzyme-free samples were applied to detect pollution of samples.

Real-Time Polymerase Chain Reaction
The expression of apoptotic genes was evaluated. Thermal program was set as a primary step (95°C/2 min) subsequently 30 cycles (95°C/15 s; 56.4°C/20 s, and 72°C/30 s). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was employed as the reference gene. Deionized water was used as the negative control.

Results
In this research, a human ovarian cancer cell line (A2780) was used to test the effects of biosynthesized CeO 2 NPs on the expression of apoptosis-related genes (bax, bcl2, and a number of caspases). The gene expression of the caspases, Bcl2, and Bax upregulated in the cancerous cells exposed to the various concentrations (7, 9, and 11 μg/mL) of the prepared CeO 2 -NPs. The fold-change of Caspase-3 treated with 11 μg/mL of CeO 2 -NPs was meaningful (P < 0.001) compared to non-treated cells (Figure 1). Also, an increase in the expressions of caspase 8 and caspase 9 was observed upon treatment with the synthesized cerium oxide nanoemulsions (Figures 2 and 3).
Our results also demonstrated that the biosynthesized CeO 2 NPs could raise the Bax/Bcl2 ratio in a dosagedependent manner (Figures 4 and 5).

Discussion
This study aimed to produce cerium oxide nanoemulsions and characterize their biological features, with a special focus on the applications of cerium oxide nanoemulsions as anti-tumor cytotoxic agents. Natural materials and their derivatives are safer than artificial materials and drugs to be used to generate biologically applicable therapeutics. 42 Medicinal plants have efficient biological complexes in their construction, which work as reducing factors during the synthesis process of nanomaterials. 43,44 Brassica napus L. is one of the important natural herbs with cytotoxicity against different types of tumor cells. In this study, the green-synthesized CeO 2 -NPs produced from B. napus L. revealed high anticancer activity. Regarding the enormous biomedical and anti-cancer activities of NPs, CeO 2 -NPs have been suggested as anticancer agents in many investigations. 45 The anti-proliferative effect has been noted as one of the anticancer mechanisms attributed to cell cycle and apoptosis induction in cancer cells. 46 We here showed that the CeO 2 -NPs significantly modulated the expressions of apoptotic genes in the cancerous A2780 cell line, as shown by RT-PCR. Among the key agents contributing to apoptosis regulation is Bcl-2, as well as other processes such as cytochrome C and caspase-9 activation.
The ratio of bax/bcl2 expression governs the progression of apoptosis and therefore can influence cancer development. 47,48 Caspase 3 and caspase 9 are two important players in the progression of intrinsic and extrinsic apoptotic routes, respectively. 49 Our results here were in accordance with prior studies indicating the bax/ bcl2 ratio as an indicator for cancer progression. 50,51 In the present research, the prepared CeO 2 -NPs up-regulated caspase 3 and 9 expressions, inhibiting the proliferation of A2780 cells. This suggested that the apoptotic path stimulated by bax/bcl2 and caspase 3 could participate

Conclusion
In this investigation, the expression of apoptosis-related genes was investigated in the A2780 ovarian cancerous cell line exposed to biosynthesized CeO 2 -NPs. The produced NPs enhanced the expression of caspase 3, 8, and 9 and upregulated the Bax/Bcl2 ratio in the assessed cancerous cells. Therefore, these NPs can have therapeutic potential for treating disorders such as cancer.
Author contribution MA designed and performed experiments, MS analyzed data and AE wrote the paper and supervisor of research.

Ethical Approval
Not applicable.

Competing Interests
There is no conflict of interest to declare.