Cytotoxicity of green - synthesized silver nanoparticles by Adansonia digitata fruit extract against HTC116 and SW480 human colon cancer cell lines

: Nanoparticles synthesized from plants are being explored for cancer treatment therapies all over the world. This study reported the eco - friendly and low - cost method for the green synthesis of silver nanoparticles ( AgNPs ) from Adansonia digitata fruit as a reducing and capping agent. The anti - cancer potential of synthesized parti cles was explored against HTC116 and SW480 colon cancer cell lines. Prepared AgNPs were characterized by ultraviolet - visible spectroscopy, zeta potential, tra nsmission electronic microscopy, scanning electronic microscopy, Fourier transform infrared, and energy dispersive spectrum. The cytotoxicity was determined with the 3 -( 4,5 - dimethylthiazol - 2 - yl )- 2,5 - diphenyltetrazolium bromide assay and expression levels of four genes ( CTNNB1 , APC , LRP5 , and LRP6 ) were checked by reverse transcription polymerase chain reaction. The sharp peak of surface plasmon resonance at 400 nm con ﬁ rms the formation of AgNPs. Dynamic light scattering showed average sizes of 16.34 nm with a polydispersity index of 0.193. A. digitata AgNPs were spherical with slight aggregated. AgNPs were more cytotoxic than A. digitata extract and decrease the expression of CTNNB1 and LRP6 genes while LRP5 gene expression was increased in both cell lines. APC gene expression was decreased in SW480 but increased in HTC116 with treatment. Overall, this study suggested that AgNPs synthesized by A. digi tata fruit extract can be an attractive candidate for antic ancer applications.


Introduction
Silver nanoparticles (AgNPs) possess many attractive properties due to which many scientific communities are showing interest in their therapeutic applications [1]. AgNPs are being used in many commercial items, such as soap catheters and bandages to curb infection against pathogenic agents [2][3][4][5]. They show promising results in nanomedicine as drug delivery vectors, theranostics agents, and anti-cancer agents [1,[6][7][8]. AgNPs can be synthesized by many chemical and physical methods, which may have some shortcomings as some of these methods need high energy or may produce toxic by-products [9,10]. Particularly, chemically produced nanoparticles give out many harmful chemicals, which are not encouraging for biomedical uses. These issues or disadvantages were solved by preparing AgNPs by green methods by using biological assets, such as plants [11,12], microbes [13], algae [14], and yeasts [15]. However, plants are mostly used due to their low cost and the presence of rich bio-reducing agents [11,12]. The oxidation process of various biomolecules present in plants is mainly responsible for the reduction of silver ions to AgNPs [11]. The synthesized AgNP stability mostly depends on the nature of biomolecules present in the extract. AgNPs' size and shape depend on the concentration of plant extract as increases in plant extract lead to the formation of a large number of nanoparticles [16]. All plant parts, such as flowers, bark, leaves, fruits, and seed, have the capacity to synthesize nanoparticles in an eco-friendly manner [17]. Some of the most recent studies showing cytotoxicity of green-synthesized nanoparticles using various parts of plants against various cancer cell lines are listed in Table 1.
Due to promising cytotoxicity activity, AgNPs are attaining interest worldwide to achieve an effective cancer treatment. The unique optical properties, size, and conductivity of these particles play an essential role in cytotoxic capacity and drug delivery for treating cancer in addition to cancer diagnosis [3,8]. Nanoparticles synthesized by medicinal plants are being explored for cancer treatment therapies because these particles have shown a controlled effect on many cancer cell lines [11,31].
International Agency for Research on Cancer placed colorectal cancer (CRC) as the third most commonly diagnosed malignancy in men and the second place in women [32]. The advanced stage of CRC is treated by only one approved drug, that is 5-fluorouracil, and its treatment is still a major challenge for researchers [33]. It has been found that AgNPs can induce DNA damage and chromosomal aberrations in many cancer cell lines [34].
In the present study, we explored the anticancer potential of green-synthesized AgNPs from Adansonia digitata fruit extract on HTC116 and SW480 colon cancer cell lines. A. digitata is full of nutrients and is used to treat many diseases, such as diarrhea, malaria, and microbial infections [35,36]. Its excellent antioxidant content reveals its high antimicrobial, antiviral antioxidant, and anti-inflammatory properties. Its fruit is rich in flavonoids, phytosterols, amino acids, fatty acids, vitamins, and minerals [35,36]. A. digitata fruit pulp is very rich in vitamin C and antioxidants. A. digitata fruit is approved as a nutritional product by statutory bodies for use in certain nutritional products [37]. The European Commission authorized it, and the United States has recognized it as a food supplement [38,39]. Demand for A. digitata fruit has increased in the cosmetic industry due to its fatty acid content. Although green synthesis of AgNPs from A. digitata has been reported in the literature, the anticancer potential of the green-synthesized A. digitata AgNPs is not reported. This novel study describes the green synthesis of AgNPs by a simple and eco-friendly process by using A. digitata fruit extract and unveiled its anticancer activities against different colon cancer cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay followed by gene-expression profiling methods of four genes (CTNNB1, APC, LRP5, and LRP6).

Fruit extract
Fresh A. digitata cultivated in the Kordofan region of Sudan was included in this study. Twenty grams of fruit pulp were soaked in 200 mL of distilled water and boiled for 30 min. The extract was filtered after cooling and centrifuged at 10,000 rpm for 10 min. The collected supernatant was stored at 4°C.

Biosynthesis of AgNPs
Silver nitrate (Fisher) solution was mixed with the abovementioned fruit extract with a final concentration of 5 mM. The mixture was heated at 60°C for 10 min. The reduction of silver ions to AgNPs was observed by a color change from light yellow to brown and was confirmed by measuring absorbance from 300 to 600 nm with the help of a ultraviolet (UV)-visible spectrophotometer (Perkin Elmer, Lambda 25).

Characterization of green-synthesized
A. digitata AgNPs The first step to examine the green synthesis of A. digitata AgNPs was by monitoring the color change followed by recording absorbance from 300 to 600 nm to get the absorbance peak. Then, the average size of A. digitata AgNPs was recorded by analyzing zeta potential (ZEN3600, Malvern) by electrophoretic light scattering (ELS) in a disposable folded capillary cell (DTS1060). The A. digitata AgNPs were loaded on a carbon-coated copper grid and analyzed on transmission electron microscopy (TEM) JEM-1400 plus JEOL and field emission scanning electron microscopy (SEM) JSM-7610F (Joel). Samples of A. digitata fruit extract and AgNPs were placed directly in potassium bromide cells and analyzed for bio-reducing functional groups recorded by infrared spectroscopy (Nicolet 6700; Thermo Scientific) the range from 4,000 to 400 cm −1 .
Energy-dispersive X-ray spectroscopy was used to validate the presence of specific elements present in the sample.

Cell culture
Human colon cancer cell lines HTC116 and SW480 provided by Central Research Laboratory King Saud University were cultured in Eagle's minimum essential medium (Stem Cell Technologies) under humidified incubator having with 5% CO 2 . Trypsin (Sigma) was used to harvest the cells followed by washing in phosphate buffered saline (PBS) (Sigma) and then used for further experiments.

Cytotoxic activity of green-synthesized A. digitata AgNPs
Cancer cells were seeded in a 96-well plate at a density of 2 × 10 5 cells/well in 100 µL of optimized medium grown to a density of 2 × 10 4 cells/well for 24 h and then exposed to different test concentrations of A. digitata extract and green-synthesized A. digitata AgNPs separately for 48 h. Finally, 100 µL of MTT (Sigma Aldrich, UK) 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. One hundred microliters of dimethyl sulfoxide (Ajax Finechem Pty Ltd, Australia) were added to dissolve formazan crystals. The 96-wellplate was also shaken for 15 min in the dark to help dissolve the formazan crystals. The optical density (OD) of each treatment was measured at absorbance 490 nm using a 96-well plate reader (Molecular Devices; SPECTRA max-PLUS384). Each experiment was performed in four replicates. Values of optical densities were normalized according to the control (untreated cells).

Gene expression analysis
Gene expression analysis was done after treating cells with IC 50 of green-synthesized A. digitata AgNPs and A. digitata fruit extract separately. Cells were incubated for 24 h and then were harvested for RNA extraction.

RNA isolation and RT-PCR
Reverse transcription polymerase chain reaction (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 carried out. The GAPDH gene was used as an internal control. The oligonucleotide sequences are listed in Table 2. 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).
Relative mRNA expression levels were then normalized by using the mRNA level of the reference gene (GAPDH) as an endogenous control in each sample. mRNA data were analyzed using the comparative Ct method.
3 Results and discussion

Characterizations of green-synthesized A. digitata AgNPs
The formation of A. digitata AgNPs was detected by a color change from light yellow to brown, which happened due to the reduction of AgNO 3 to form AgNPs. We synthesize spherical AgNPs in 10 min by mixing silver nitrate solution at 60°C with fruit extract, which was     prepared by incubation fruit pulp in boiling water and left overnight, thus accompanying the green chemistry policy. High temperature decreases the diameter of AgNP due to the increasing intensity of the surface plasmon resonance (SPR) band as a result of the bathochromic shift [40]. The sharp peak of SPR at 400 nm in the UV region confirms the formation of AgNPs (Figure 1). This technique is considered one of the basic and quick methods to explore AgNPs [41]. The peak of absorbance is due to the vibration of electrons in synthesized AgNPs [42]. Normally, AgNPs show absorption peaks in the range of 380-470 nm [43]. Green synthesis of A. digitata-mediated AgNPs was confirmed by dynamic light scattering (DLS), which showed average sizes of 16.34 nm, as shown in Figure 2. The polydispersity index was 0.193, indicating small size distribution and homogeneous population of prepared AgNPs [41]. Polydispersity index less than 0.3 specifies monodispersed and can be used for pharmaceutical purposes [44]. Zeta potential was −0.39 as shown in Figure 3. Nanoparticles' interface in deionized water is negatively charged. Generally, the surface charge appears after nanoparticle preparation, due to the ionizing groups present in the medium [45]. The charge gained by dispersed nanoparticles plays a major role in the stability preventing aggregation [46]. The surface morphology, size, and 3D structure of green-synthesized A. digitata AgNPs were observed by TEM and SEM (Figure 4). TEM results clearly show that AgNPs were spherical and with a size range from 9 to 20 nm, which agrees with the results shown by DLS. The most recent studies listed in Table 1 show almost the same size and shape of AgNPs. TEM is an ultrathin image displaying a 2D structure while SEM demonstrates the 3D structure of a material [47]. Although the spherical structure of A. digitata AgNPs was confirmed by SEM micrograph, the recorded size was in the range of 32.8-37.8 nm, indicating the cluster or aggregation of A. digitata AgNPs [41]. The energy-dispersive spectrum (EDX) of the A. digitata AgNPs exhibited strong signals for the sodium, potassium, and chlorine regions. The presence of other elements sulfur and magnesium was also observed in the spectrum ( Figure 5).

Fourier transform infrared spectroscopy (FT-IR)
Fruit extract of A. digitata is very rich in flavonoid, tannin, saponin, alkaloid, and phenols [35,36]. These secondary metabolites help in the reduction process and also act as a capping agent due to which nanoparticles do not aggregate once formed [48]. FT-IR measurements were carried out on A. digitata extract and its reduced form to recognize the changes in the bonds followed by reduction of the metal precursors and capping of A. digitata AgNPs using an FT-IR spectrophotometer. As displayed in Figure 6a  Most of the peaks that appeared in the A. digitata extract disappeared after the synthesis of A. digitata AgNPs. Based on the FT-IR analysis, it is confirmed that the broad peaks of phenols and proteins act as reducing stabilizing and capping agents and for AgNPs. Our results are in agreement with previous studies showing that the

Cancer-cell cytotoxicity
We use the MTT assay to examine the impact of A. digitata AgNPs and A. digitata fruit extract on the cell viability of cancer cell lines. Both extracts decrease the cell viability in a dose-dependent manner in the range from 3.12 to 100 μg·mL −1 against human colon cancer cell lines HTC116 and SW480. IC 50 values of test samples show higher cytotoxicity of A. digitata AgNPs against SW480 followed by HTC116. Remarkable differences in cell cytotoxicity were shown between A. digitata AgNPs and A. digitata fruit extract (Figure 7a and b). At all fixed concentrations, the cytotoxicity effect of the A. digitata AgNPs was higher than the A. digitata extract as shown in Figure 7a and b. The overall results showed that greensynthesized nanoparticles were more active than pure fruit extract on colon cell lines' death and showed strong cytotoxicity. Many studies have highlighted the effective role of AgNPs against any kind of cancer in vitro and in vivo [49]. Generally, the zeta potential, which depends on the surface charge, is important for the stability of the nanoparticle suspension and is also a major factor in the initial adsorption of nanoparticle on the cell membrane, and the zeta potential and size thus affect the nanoparticle toxicity. The size, shape, and charge of synthesized AgNPs in the current study were acceptable to reveal anticancer properties as small size particles are more active in showing the anticancer activity [49]. The size of an AgNP is the main factor to determine its toxicity toward biological systems as particles that are small in size can pass through cells or subcellular organelles of a cell line [50]. Gliga et al. [51] reported the size-dependent cytotoxicity against human cancer cell lines showing small AgNPs as more cytotoxic as compared to larger particles. AgNPs are proven safe for healthy cells while toxic against cancerous cells.
Several experiments in vitro and in vivo have shown that AgNPs can up-or downregulate the expression of many key genes and regulate important signaling pathways to control the cell proliferation and viability of cancer cells [52][53][54].
Abnormal stimulation of the WNT/β-catenin signaling pathway is observed in CRCs with activating mutations in the catenin beta 1 (CTNNB1) gene encoding β-catenin, inactivating adenomatous polyposis coli (APC) gene mutations [55]. Also, the low-density lipoprotein receptor-related protein 6 (LRP6), an indispensable co-receptor for WNT, is overexpressed in colorectal adenocarcinomas [56]. Currently, many clinical trials are being done to see the responses of therapies inhibiting Wnt/β-catenin signaling pathways in patients with CRC. However, chemoresistance is one of the major challenges in these trials. In this study, the expression level of these four genes CTNNB1, APC, LRP5, and LRP6 was measured after treating the cells with A. digitata AgNPs and A. digitata fruit extract to explore its possible role in anticancer mechanisms. Figure 8 presents the results as a chart showing gene expression fold change in the control (without treated) cancerous colorectal cell line and treated cell line. Normally SW480 expresses only mutant APC and whereas HCT116 expresses wild-type APC but mutant CTNNB1 [57]. Mutation in APC and CTNNB1 occurs in more than 80% of colon tumors and is one of the initial events that contribute to colon cancer origin [58]. APC acts as a negative regulator of the canonical WNT signaling pathway through proteasomal degradation of β-catenin, which is expressed by the CTNNB1 gene [59].
A. digitata AgNPs and its aqueous extract show low expression of the APC gene in SW480 as compared to untreated cells, but it was slightly more expressed in HCT116 cells on treatment. Expression of the CTNNB1 gene was decreased in both types of cell lines on treatment. These observations clearly show the inhibitory effect of A. digitata AgNPs and its aqueous extract against chosen cell lines. Alteration in low-density lipoprotein-related receptors 5 and 6 (LRP5/6) genes are linked to the development of cancer in humans [60]. The expression of LRP5 can regulate the expression of serotonin by inhibiting the expression of tryptophan hydroxylase 1 (Tph1), which is the rate-limiting biosynthetic enzyme for serotonin [61]. Serotonin has been found to promote CRC by the modulation of DNA repair mechanisms and immune response [62]. LRP5 was highly expressed in both cell lines after treatment with A. digitata AgNPs and A. digitata extract. Anti-cancer potential of both extracts in our results can be revealed by an elevated level of low-density lipoprotein-related receptors 5 since LRP5 can downregulate tyrosine hydroxylase and decrease serotonin levels [63]. We find a remarkable decrease in expression of the LRP6 gene in both cell lines with treatment. Reducing LRP6 expression has been reported to inhibit the cell proliferation and delay tumor growth [64]. Also, LRP6 is a co-receptor for WNT and is overexpressed in colorectal adenocarcinomas in association with increased WNT/β-catenin signaling [65].

Conclusions
Studies showing molecular mechanisms behind the anticancer potential of green-synthesized AgNPs are limited. AgNPs were successfully synthesized from A. digitata fruit extract, which acts as reducing, capping, and stabilizing agents in the process. The synthesis procedure was simple, low cost, and eco-friendly. SPR at 400 nm confirms the formation of AgNPs. Analytical characterization, such as TEM, SEM, and ELS, supports the overall morphology of synthesized AgNPs. A. digitata AgNPs and A. digitata fruit extract showed potent anti-cancer potential.