Antineoplastic Efectiveness of Silver Nanoparticles Synthesized from Onopordum Acanthium L. extract (AgNPs-OAL) toward MDA-MB231 Breast Cancer Cells

The present research was done to investigate the anticancer properties of silver nanoparticles (AgNPs) fabricated using bioactive extract of Onopordum acanthium L. (AgNPs-OAL) against breast cancer cell MDA_MB231 in vitro. The determination studies of AgNPs-OAL were conrmed by X-ray diffraction (XRD), eld emission scanning electron microscopy (FESEM) analysis. Interestingly, FESEM image observed the spherical shape of AgNPs-OAL with the range of 1–100 nm. As AgNP-OAL exhibited signicant cytotoxicity properties on breast cancer MDA_MB231 cells with IC 50 values of 66.04 μg/mL, while lowing toxicity toward normal human embryonic kidney 293 (HEK293) cells with IC 50 values of 101.04 μg/mL was evaluated. Further, up-regulation of apoptotic Bax and CAD genes expressions were conrmed by quantitative real-time reverse transcription-PCR (qRT-PCR) technique results. Moreover, enhanced cell cycle population (sub-G1), annexin V/PI staining, acridine orange and ethidium bromide (AO/EB) staining, Hoescht 33258 dye, and generation of reactive oxygen species (ROS) observed in AgNP-OAL-treated MDA_MB231 cancer cells. The green-synthesized AgNP-OAL has promising anticancer eciency that can trigger apoptosis pathway in the MDA_MB231 breast cancer cells.


Introduction
According to the global cancer burden based on the GLOBOCAN 2020, it was estimated that 19.3 million new cancer cases and almost 10.0 million cancer deaths happened in 2020. Breast cancer is the most frequently diagnosed cancer, with almost 2.3 million new cases reported to be distinguished in 2020 [1]. A large number of drugs in chemotherapy suffer from different side effects such as enhancing multi-drug resistance (MDR), poor solubility, and undesirable damage to normal cells, which indicates as results of commonly therapeutic intervention strategies [2].
Fabrication of nanoparticles is mediated through a variety of chemical, physical, and biological strategies [8]. There is an increased interest in synthesis of nanoparticles by using green fabrication method using plants owning to their various advantages such as simple, cost effective, an environmentally eco-friendly, and do not require hazardous chemicals [9]. On the other hand, Onopordum acanthium L. is a owering plant belonging to the family Asteraceae, with rich phytochemical compounds. Onopordum acanthium L. displayed signi cant medical activities such as antiradical, antibacterial, antihypertensive, cardiotonic agent, antitumor, and anti-in ammatory activities [10].
In this context, we have explored the apoptotic activity of AgNPs using Onopordum acanthium L. (AgNPs-OAL) extract as reducing agent. Field emission scanning electron microscopy (FESEM) was utilized to identify the morphological feature of the nanoparticles. Crystalline nature of nanoparticles was explored using XRD, and the in vitro cytotoxicity was performed in MDA-MB231 (human breast cancer), and HEK293 (human embryonic kidney) cell lines upon treatment with prepared AgNPs-OAL. Moreover, cell cycle analysis, morphological properties of apoptotic/necrotic cells, ROS generation, apoptotic Bax and CAD gene expressions, and induction of apoptosis/necrosis were also evaluated by Annexin V/PI staining to explore the signi cant impacts of the AgNPs-OAL as anticancer agents.

Materials And Methods
Fabrication and determination of green AgNPs-OAL For preparation of plant extract, the powder form (50 grams) of dried plant Onopordum acanthium L. was added to 200 cc of solvent ethanol (50%), and centrifuged for 20 min at 14000 rpm. The resulting extract was ltered through a lter paper, and then stored in the refrigerator prior to use. The extract of Onopordum acanthium L. about 5 mL was added dropwise in 100 mL 1 mM silver nitrate (AgNO 3 , 99%) solution with constant stirring at room temperature. To collect the suspension, Onopordum acanthium L. extract-derived AgNPs (AgNPs-OAL) were centrifuged at 13000 rpm for 20 min. The determination studies were con rmed by X-ray diffraction (XRD), eld emission scanning electron microscopy (FESEM) analysis. The crystalline nature of AgNPs-OAL was characterized by XRD (Burker AXS D8 X-Ray diffractometer) instrument using Cu Kα radiation in 2θ range of 5°-80°. The particle morphology of prepared AgNPs-OAL was imaged and analyzed through FESEM (Philips XL30) instrument.
Cell culture MDA-MB231 (human breast cancer), HEK293 (human embryonic kidney) cell lines were procured from NCBI (National Cell Bank of Iran), Pasteur Institute of Iran. The cells were sustained in RPMI 1640 media pH = 7.2 with 10% FBS serum. The medium were added with 1% penicillin/streptomycin solution, followed by incubation at 5% CO2, 37°C.

Viability assay
The percentage of cell viability has been evaluated toward MDA-MB231 and HEK293 cell lines via MTT method followed by treatment with AgNPs-OAL according to previous standard protocol [11]. All the cells (1 × 10 4 cell /well) were grown in 96-well plates and exposed to various test doses (0, 0.625, 12.5, 25, 50, and 100 µg/mL) of AgNPs-OAL. After 24 hours incubation, MTT solution (5 mg/mL) was introduced into each well for 4 hours. The formazan crystals formed followed by adding MTT dye. To improve dissolving, DMSO solution was added to samples followed by recording the absorbance value at 570 nm using an ELISA microplate reader.

Analysis of apoptotic gene expressions
The apoptotic Bax and CAD gene expressions in AgNPs-OAL-treated cells were distinguished through SYBR Green real-time quantitative PCR at mRNA level. First, the cells were treated with AgNPs-OAL in IC 50 concentration overnight. RNA-isolation kit was used for the isolation of the total cellular RNA (Qiagen, Valencia, CA) based on manufacturer's protocols. Then, complementary DNA (cDNA) was fabricated in terms of the manufacturer's protocols using a PrimeScript™ RT Kit (Takara, Japan). The primers utilized for RT-PCR were: forward 5′ CGGCAACTTCAACTGGGG 3′ and reverse 5′ TCCAGCCCAACAGCCG 3′ for Bax, forward 5′ TGGCAGAGATCGGAGAGCAT 3′ and reverse 5′ TCCTTCCATCCCTTCAGAGACTT 3′ for CAD. Also, the sequence of the forward primer for Glyceraldehyde 3 phosphate Dehydrogenase (GAPDH) as a housekeeping gene was 5′ CCCACTCCTCCACCTTTGAC 3′ and that of reverse primer was 5′ CATACCAGGAAATGAGCTTGACAA 3′. The ratio formula (2 −ΔΔCt ) was used to assess the apoptotic gene expressions.
In vitro Apoptosis/necrosis assay The quanti cation of the mode of AgNPs-OAL-induced cell death in the MDA-MB231 cells were assessed using ow cytometry followed by staining with uorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) staining in terms of the kit's Protocol (Roch, Switzerland). For this, the cells (3 × 10 5 cells/well) were subjected to 66.04 µg/mL of AgNPs-OAL, allowed to grow for 24 h. Washing of the treated cells was done by PBS, re-suspended in binding buffer, and staining with propidium iodide and Annexin V followed by incubation in the dark. After incubated for 10 min, the percentage of the apoptotic and necrotic cells was ascertained through ow cytometer.
2',7'-dichloro dihydro uorescein diacetate (DCFH-DA) assay Intracellular ROS generation was distinguished using ow cytometry. The cells (MDA-MB231, 3 × 10 5 cell /well) were seeded in 6-well plates for 24 h followed by the addition of required concentration of AgNPs-OAL. The MDA-MB231 cells were washed with phosphate-buffered saline and then cells were exposed with DCFH-DA dye after a further treatment of 30 min. The stained cells were further investigated via ow cytometer for determination of ROS production.

Cell cycle analysis
To investigate the changes in cell cycle evaluation in AgNPs-OAL treated MDA-MB231 cells, ow cytometric analysis were determined. The MDA-MB231 cells were cultured in 6-well plates and exposed with 66.04 µg/mL of the AgNPs-OAL in the CO 2 incubator for 24h.
Further, the cells were harvested, and washed with PBS. After xation in cold ice 70% ethanol, the cells were then stained with propidium iodide, followed by treatment of RNase A (100 µg/ml). Finally, the DNA content of stained cells was measured through ow cytometer.
Morphology of apoptotic/necrotic cells Determination of apoptotic/necrotic cells was investigated by a dual acridine orange and ethidium bromide (AO/EB) and Hoechst nuclear staining. The MDA-MB231 cells were treated with 66.04 µg/mL of AgNPs-OAL. After washing with PBS, control and treated cells were xed by 4% formaldehyde and stained with 10 µL of AO/EB solution for 5 min incubation.
For the Hoechst 33258 staining, MDA-MB231 cells were subjected to six-well plating, overnight incubation with AgNPs-OAL, staining with Hoechst 33258 solutions for 5 min in the dark, followed by washing thrice with PBS. For observation, treated and control cells were visualized for their apoptotic to nuclei using uorescence microscopy image.

Result And Discussion
Characterization of AgNPs-OAL In recent decades, silver nanoparticle fabricated using biological methods show promising impact toward cancer cell lines. Plant mediated green strategy for AgNPs synthesis can be superior to fungal and bacterial-mediated strategies because of their accessibility, environment friendly procedures and safety [12][13][14]. In this investigation, Onopordum acanthium L. extract was used for the fabrication of AgNPs. As the Onopordum acanthium L. extract was mixed with silver nitrate solution, the variation in color was seen in the solution that converted into the dark brown overnight, indicating the fabrication of silver nanoparticles by phytoconstituents of Onopordum acanthium L. extract.
The re ection planes of the fabricated AgNPs-OAL were consistent with earlier investigations [15,16]. In order to nd out the shapes and morphology of the produced AgNPs-OAL, the eld emission scanning electron microscopy (FESEM) analysis was investigated. The AgNPs-OAL was spherically shaped with a size in the range of 1-100 nm (Fig. 2).

Cytotoxicity effect of AgNPs-OAL on MDA-MB-231cells
In the current investigation, cytotoxicity of AgNPs-OAL was examined toward human breast cancer (MDA-MB-231) human embryonic kidney (HEK293) cells at concentration of 6.25 to 100 µg/mL using MTT assay. Interestingly, AgNPs-OAL has exhibit signi cant dose-dependency on cell viability followed by increasing concentrations. The IC 50 values of AgNPs-OAL against MDA-MB231 and HEK293 cells were 66.04 and 101.04 µg/mL, respectively. The in vitro cell viability was exhibited as 85% in AgNPs-OAL at 12.5 µg/mL however; the percentage of viability was remarkably reduced as 45% at 100 µg/mL of nanoparticles (Fig. 3). Herein, we showed that MDA-MB-231 cells are more sensitive to AgNPs-OAL than normal HEK293 cells. Our results are in agreement with that of Gurunathan [19,20].

Measurement Of Reactive Oxygen Species Production
Reactive oxygen species (ROS) produced in normal cells and have roles in many physiological conditions [21]. Under excessiveness of ROS, a series of cellular events were occurring results in lipid peroxidation, DNA damaging, cell cycle arrest, and promote apoptosis pathway [22]. It has been reported that new strategies for targeting ROS contribute to the cancer therapy [23]. Various reports have elucidated the roles of different nanometals in triggering ROS content in different cancer cells [24,25]. Zhu et al. In our study, ROS production in treated MDA-MB231 cells was 71.7%, while the control cells were 4.08%.
In the current work, the ROS production in MDA-MB231 cells was illustrated in Fig. 4. Maximum production of ROS was closely exhibited for AgNPs-OAL, resulting in high cytotoxicity on MDA-MB231 cells as presented in the above results.

Agnps-oal Induce Apoptosis In Mda-mb231 Cells
Apoptosis represents a well de ned intracellular physiological cell death with often doesn't trigger in ammatory response [28]. Effector caspase 3 activation is required for e cient execution of apoptotic death [29]. Translocation and cleavage of caspase-activated DNAse (CAD) were activated by caspase 3, results in DNA fragmentation.
In the current study, we evaluated the roles of pro-apoptotic Bax and CAD regulation in relation to the apoptotic death of cancer cells [22]. The mRNA expression levels of genes in AgNPs-OAL -mediated apoptotic cells were displayed by qRT-PCR method, which is presented in Fig. 5. The expression of the CAD and pro-apoptotic Bax genes was found to enhance by a 4.04-fold and 1.38 fold in AgNPs-OAL treated MDA-MB231 cells, respectively.
The MDA-MB231 cells undergoing programmed cell death and necrosis were determined by the Annexin V/PI staining (Fig. 6). We found that the AgNPs-OAL enhanced the number of early and late apoptosis on MDA-MB231 by 47.8 % at 66.04 µg/mL. In the report conducted by Mohammadi-Ziveh et al., the apoptotic properties of Ag-S.Khuzistanica on the colorectal HT29 cancer cell line were investigated. They indicated that 375, 750, 1500, 3000 µg/mL of Ag-S.Khuzistanica can increase the apoptosis rate (~ 30%) along with the up-regulation of apoptotic index ratio. In our study, the apoptosis rate (47.8 %) was much higher after exposing to AgNPs-OAL as compared with the Mohammadi-Ziveh results (~ 30%) [30].
Considering the main pattern of nanoparticles in the apoptosis, the cell cycle results were conducted in the AgNPs-OAL -exposed MDA-MB231 cells. The cellular DNA of treated MDA-MB231 cells was stained with PI followed by testing the DNA content via ow cytometry assay. Herein, the proportion of AgNPs-OAL-exposed cells in the apoptotic cell population (sub-G1) phase was increased from 7.04-12.58% in MDA-MB231 cells (Fig. 7). A comparison of the Annexin V/PI staining and cell cycle results indicates that the exposure of MDA-MB231 cells with AgNPs-OAL cause to inhibition of sub-G1 phase and the upregulation of apoptosis by increasing early and late phase of apoptosis pathway.
Moreover, we employed a further examination to determine the possible stimulation role of AgNPs-OAL in apoptosis pathway. Given that the previous section demonstrated that the AgNPs-OAL has the capability of triggering an apoptosis pathway, the alterations in the nuclear morphology of apoptotic, necrotic cells was tested by AO/EB double staining and Hoescht 33258 after treated with AgNPs-OAL.
The Hoescht 33258 results display that AgNPs-OAL-treated cells has enhanced apoptosis via cell structure abnormality, bright color, and condensed chromatin (Fig. 8).
At concentration of 66.04 µg/mL, AO/EB double staining was investigated to discriminate the variations in apoptotic and normal cells. The Fig. 9 shows that treated cells display apoptosis by changing color of the nucleus into orange, while green nuclei represent untreated cells. Nanoparticles-exposed MDA-MB231 cells demonstrated an increase in the cell shrinkage and nuclear fragmentation as compared to untreated cells, exhibiting the promotion of apoptosis pathway by the fabricated AgNPs-OAL. Thus, the results exhibited that the AgNPs-OAL increased cell death, leading to the induction of apoptosis pathway.

Conclusion
In summary, plant-mediated AgNPs synthesis from Onopordum acanthium L. extract was generated by green fabrication route. Determinations of the obtained nanoparticles were conducted by FESEM and XRD analysis. The results of in vitro cytotoxicity assay, apoptotic genes expressions, cell cycle analysis, annexin V/PI staining, AO/EB staining, Hoescht 33258 staining, and generation of reactive oxygen species (ROS) exhibited that fabricated AgNPs could act as a promising strategy in treating breast cancer in future.

Con ict of interest
The authors declare no con ict of interest.
Author contributions 25. Khan BF, Hamidullah, Dwivedi S, Konwar R, Zubair S, and Owais M (2019) Potential of bacterial culture media in biofabrication of metal nanoparticles and the therapeutic potential of the assynthesized nanoparticles in conjunction with artemisinin against MDA-MB-231 breast cancer cells. J Cell Physiol 234 (5)    Percentage of (a) viable MDA-MB231 and (b) HEK293 cell lines exposed with AgNPs-OAL at different concentrations after overnight treatment. The data are presented as the mean ± SEM.

Figure 4
Effect of the produced AgNPs-OAL on ROS generation in untreated (a) and treated (b) MDA_MB231 cells utilizing DCFH-DA followed by ow cytometry.   Morphology changes of (a) Untreated and (b) treated MDA_MB231 cells with fabricated AgNPs-OAL following AO/EB dye under Fluorescence microscope.