Anti - colon cancer activities of green - synthesized Moringa oleifera – AgNPs against human colon cancer cells

: The anticancer activity of silver nanoparticles ( AgNPs ) is well known to be synthesized using green synthesized methods, although its mechanism of action is not understood fully. Moringa oleifera leaves were used as reducing and stabilizing agents to synthesize AgNPs. Green - synthesized AgNPs were characterized using ultra violet - visible spectroscopy, dynamic light scattering, transmission electronic microscopy, scanning electronic microscopy, Fourier transform infrared, and energy - dis persive X - ray spectroscopy analyses. The synthesized nanoparticles were then characterized by their anticancer properties by performing a 3 -( 4,5 - dimethylthiazol - 2 - yl )-2,5 - diphenyltetrazolium bromide assay. The real - time polymerase chain reaction was used to check the expres sion levels of the four genes ( β - catenin, adenomatous polyposis coli ( APC ) , and lipoprotein receptor - related pro teins 5 and 6 ( LRP5/6 )) . The synthesized nanoparticles were 25 nm on average and spherical in shape and aggre gated form. Noteworthy cytotoxicity is how green - synthe sized M. oleifera – AgNPs were observed in comparison with the M. oleifera leaf extract against a cancerous cell line. The M. oleifera – AgNPs decreased the expression of CTNNB1 and LRP6 genes, while the LRP5 gene expression increased in both cell lines. With treatment, the APC gene expression decreased in SW480 but increased in HTC116. Our results imply that AgNPs synthesized by M. oleifera extract could be an ideal strategy to combat colon cancer.


Introduction
Silver ions have been used in medicine historically, although silver nanoparticles (AgNPs) offer more possibilities for use in many types of medical treatments for humans [1]. AgNPs are the most widely used particles among metallic nanoparticles, accounting for almost 25% [2]. AgNPs are being incorporated into many surgical instruments daily, such as in face masks and even in bone-cementing materials, for their excellent antibacterial, antifungal, and antiviral properties [3,4]. Indeed, AgNPs are replacing silver sulfadiazine for wound-healing treatments [4,5]. Chemical methods produce AgNPs, but the reagents used in these processes usually are expensive and toxic to humans [6].
In recent tests of biological sources such as plants, seaweeds, insects, and microorganisms, silver ions were successfully reduced to AgNPs by reducing the capacities of the metabolites present in them [7][8][9][10][11]. AgNP synthesis via plant extract is better than other biological extracts in terms of availability, low toxicity, and the presence of rich phytochemicals such as alkaloids, flavonoids, flavones, terpenoids, terpenes, polysaccharides, phenolics, saponins, and tannins [12]. In addition, plant extracts are reducing and stabilizing agents during the AgNP synthesis process. The AgNPs synthesized through biologic approaches maintain a homogenous chemical composition and show promising results against many cancer cell lines [13]. Therefore, green-synthesis methods based on green chemistry offer an alternative to the chemical and physical synthesis of AgNPs used for anticancer treatments [7].
Although many studies have reported on the antitumor properties of AgNPs, their mechanism of action is not fully understood. The cytotoxicity of greensynthesized AgNPs depends on their size and shape: those synthesized from plant sources are usually spherical and cytotoxic against many human cancer cell lines [14]. Due to their small size, AgNPs can directly contact cell surfaces to initiate DNA damage and change the gene expression, which may result in cell death [14,15].
Colorectal cancer (CRC) was the second deadliest and third most common malignant tumor worldwide in 2020 [16]. The occurrence of this disease is much higher in Saudi Arabia as it is at the topmost in men and the third in females [17]. In this study, we investigated the cytotoxicity of AgNPs synthesized via aqueous extracts from Moringa oleifera leaves against two human colon cancer cell lines, SW480 and HTC116.

M. oleifera leaf extract
Fresh M. oleifera leaves that were cultivated in the Riyadh region of Saudi Arabia were included in this study. Taxonomic identification of M. oleifera was confirmed by Dr. Mona S. Alwhibi in the Botany and Microbiology Department, College of Science, King Saud University. Fifteen grams of fresh leaves were boiled in 150 mL of distilled water (1:10 weight/volume ratio) for 20 min. After cooling, the extract was filtered. The collected filtrate was used for the green synthesis of AgNPs.

Preparation of green-synthesized M. oleifera-AgNPs
Freshly prepared leaf extract was mixed with a silver nitrate solution, with a final concentration of 5 mM. The mixture was incubated at room temperature until a reduction of silver ions to AgNPs was observed based on color change. The preparation of AgNPs was confirmed by ultraviolet (UV)-visible (vis) spectrophotometer by measuring the absorbance of the mixture, from 300 to 600 nm.

Characterization of green-synthesized M. oleifera-AgNPs
At first, the green synthesis of M. oleifera-AgNPs was achieved by monitoring the color change, from green to brown, followed by recording the absorbance peak, from 300 to 600 nm. Then, the average size of the M. oleifera-AgNPs was recorded by analyzing the zeta potential and using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). By bioreducing the functional groups of M. oleifera leaves, the extracts were recorded using infrared spectroscopy. Energy-dispersive X-ray spectroscopy validated the presence of specific elements in the sample.

Cell culture
The human colon cancer cell lines, HTC116 and SW480, were cultured in Eagle minimum essential medium under a humidified incubator with 5% CO 2 . Trypsin was used to harvest the cells, followed by washing in PBS, and then was used for further experiments.

Cytotoxic activity of green-synthesized M. oleifera-AgNPs
Cancer cells were seeded in a 96-well plate at a density of 2 × 10 5 cells·well −1 in 100 µL optimized medium, grown to a density of 2 × 10 4 cells·well −1 for 24 h, and then exposed to various test concentrations of digitate extract and green-synthesized digitate-AgNPs separately for 48 h. Finally, 100 µL of MTT was added at 37°C at a final concentration of 5 mg‧mL −1 . The 96-well plate was kept in the dark for 2 h before the medium-containing MTT was removed. About 100 µL of DMSO was added to dissolve the formasane crystals. The 96-well plate was also shaken in the dark for 15 min, to help dissolve the formasane crystals. The optical density of each treatment was measured at a 490 nm absorbance, using a 96-well plate reader (Molecular Devices, SPECTRA max, PLUS384). Each experiment was performed in four replicates. The values of the optical densities were normalized according to the control (untreated cells).

Gene-expression analysis
Gene-expression analysis was performed after treating the cells with IC 50 of green-synthesized M. oleifera-AgNPs and M. oleifera extract separately. The cells were incubated for 24 h and then harvested for RNA extraction.

RNA isolation and real-time polymerase chain reaction (RT-PCR)
An RT-PCR was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The cDNA was stored at −20°C until the RT-PCR experiment was conducted. The GAPDH gene was used as an internal control. The oligonucleotide sequences are listed in Table 1. The RT-PCR was done on a LightCycler ViiA 7 Instrument (ViiA 7, Thermo Fisher Scientific). The data were obtained using LightCycler ViiA 7 software 1.0 (ViiA 7, Thermo Fisher Scientific). The relative mRNA expression levels were then normalized by using the mRNA level of the reference gene (GAPDH) as an endogenous control in each sample. The mRNA data were analyzed using the comparative Ct method.
3 Results and discussion

Characterizations of green-synthesized M. oleifera-AgNPs
A color change from green to brown first indicated the synthesis of the green-synthesized M. oleifera-AgNPs that formed by reducing the silver ion, with the help of the reducing agents present in the M. oleifera leaf extract. The absorption peak was found at 380 nm of the UV-vis spectrum, as shown in Figure 1. The AgNPs  are the most interesting of all the metal NPs, due to silver's strong plasmonic interaction with light with sharply localized surface plasmon resonance (LSPR) bands [18]. The absorption peak position of AgNPs is in the range of 380-470 nm subjected to the size and shape of the particle [19].
The LSPR peak wavelength of AgNPs can be in the range from most visible to the near-infrared region, which can fluctuate by changing the shape, size, and dielectric environment of the NPs [20]. We use the aqueous extract of M. oleifera leaves as these leaves are very rich in protein which aids in bio-reducing of metal ions to nanoparticles. A leaf extract of M. oleifera is also very rich in phenolic compounds, flavonoids, and antioxidant-like vitamins C and A [21][22][23]. These compounds act as good reducing and capping agents to stabilize NPs and control their aggregates, once they are formed [24]. Figure 2 shows that the average size of green-synthesized M. oleifera-AgNPs, which is shown by dynamic light scattering (DLS), was 197 nm, with a polydispersity index (PdI) of 0.334. The average uniformity of an NP in a solution is estimated by the PdI value, in that a larger value indicates a larger size distribution of NPs in a solution. The aggregation of NPs is also indicated by PdI value since a sample is monodispersing if the PdI value is less than 0.1 [25,26]. Thus, the values shown by DLS and PdI in our results indicate little aggregation of NPs.
The TEM and SEM were used to gauge the surface morphology, size, and three-dimensional structure of greensynthesized M. oleifera-AgNPs, as shown in Figure 3. TEM shows an ultrathin image that indicates a twodimensional structure, and SEM shows the three-dimensional structure of a material [27]. NPs were spherical with an average size of 25 nm, as shown by TEM images. Similar shapes were displayed using SEM images, but their sizes were in the range of 88.8-92 nm, which clearly shows aggregated, green-synthesized M. oleifera-AgNPs in the sample solution and agrees with DLS results. The energy-dispersive spectrum (EDX) of the M. oleifera-AgNPs exhibited strong signals for potassium, silver, and the chlorine regions ( Figure 4a shows the peaks visible on spectra, and Figure 4b shows the quantitative results).

Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy was used to measure both the M. oleifera leaf extract and its reduced form, M. oleifera-AgNP solution, and to identify the changes in the bonds that may have occurred due to reduced silver ions and capped AgNPs. The amount of absorption peaks for both solutions is shown in Figure 5a

Colon-cancer cell cytotoxicity
Both M. oleifera leaf extract and M. oleifera-AgNPs were tested for cytotoxicity against the HTC116 and SW480 human cancer cell lines by using an MTT assay, as shown  in Figure 6a and b. Both solutions decreased cell viability in a dose-dependent manner with an increased concentration from 3.12 to 100 μg‧mL −1 . M. oleifera-AgNP was found to be more cytotoxic than M. oleifera leaf extract at all concentrations. The IC 50 recorded for M. oleifera-AgNP against HTC116 was 70 μg‧mL −1 while SW480 recorded 100 μg‧mL −1 . Our results generally agree with many studies that highlight the efficacy of AgNPs against different types of cancer cell lines [7,29]. In the current study, the morphology of green-synthesized AgNPs was acceptable to reveal anticancer properties [29].
Green-synthesized AgNPs are used in many biomedical applications. AgNPs possess excellent antitumor potential by regulating the expression of many of the key genes that relate to numerous signaling pathways linked to oxidative stress, cell proliferation, DNA damage, and the cell-cycle arrest of cancer cells [30][31][32]. The Wnt/β-catenin signaling pathway is altered in almost 90% of CRC patients, which makes it a critical therapeutic target. Therapies that inhibit Wnt/β-catenin signaling pathways are being performed in many clinical trials to monitor the response of patients, but chemoresistance is a major hurdle [33].
We examined the expression level of some key regulators of the Wnt/β-catenin signaling pathway after treating the colon-cancer cell lines (HTC116 and SW480) with M. oleifera leaf extract and green-synthesized M. oleifera-AgNPs. These pathways play a major role in many biological processes, such as embryogenesis and tissue homeostasis; however, excessive activation of this pathway has been reported in most human malignancies, including CRC [34,35]. Almost 90% of CRC patients have mutations in some downstream components, such as adenomatous polyposis coli (APC) and β-catenin of the Wnt signaling pathway [36,37]. Also, mutations in the lipoprotein receptor-related proteins 5 and 6 (LRP5/6) coreceptors are detected in CRC patients frequently [38]. The CTNNB1 and APC genes and LRP5 and LRP6 were included in the study. Figure 7 shows the gene-expression fold change in the control (without treatment) versus treated cell line.
The APC gene's expression decreased in cell line SW 480 for treatment with both leaf and green-synthesized AgNP extracts but was least effective in cell line HTC116. The mutant APC gene is expressed in SW480 while HCT116 expresses only wild-type APC [39]. Green-synthesized AgNPs decreased the expression of the CTNNB1 gene remarkably, in both types of cell lines, as compared to leaf extract. This finding clearly indicates the inhibitory effect of green-synthesized AgNPs. The LRP5 expression level was slightly increased in both cell lines after treatment with green-synthesized AgNPs. The LRP5 gene expression can inhibit the tryptophan hydroxylase 1 expression level, which is the rate-limiting biosynthetic enzyme for serotonin [40]. Hence, LRP5 can regulate the low expression of serotonin by downregulating tyrosine hydroxylase. Serotonin is known to promote CRC by modulating DNA repair and the immune response [41,42]. Our results confirm that increasing the expression level of the LRP5 gene of green-synthesized AgNPs has anticancer potential. However, the expression level of the LRP6 gene was remarkably decreased in treated cell lines. The low expression level of the LRP6 gene has been linked to constrained cancer cell proliferation and delayed tumor growth in humans [43]. As a coreceptor for Wnt, the high expression of LRP6 is associated with increased Wnt/β-catenin signaling in colorectal adenocarcinomas [44].

Conclusion
M. oleifera. leaf extract acts as a reducing and stabilizing agent for the green synthesis of AgNPs. At first, greensynthesized AgNPs were ascertained through the color change from green to brown with UV spectra absorption peak at 380 nm. M. oleifera-AgNPs were spherical in shape with an average size of 25 nm, which is acceptable to show potent anticancer activity against human cancer cell lines HTC116 and SW480. Further study is needed to explore its therapeutic effect by targeting Wnt/β-catenin signaling pathways to overcome chemoresistance in patients.  Rasha Ramadan, Ramesa Shafi Bhat: writingoriginal draft and data analysis.

Conflict of interest:
Authors state no conflict of interest.