Systemic Evaluation of Mechanism of Cytotoxicity in Human Colon Cancer HCT-116 Cells of Silver Nanoparticles Synthesized Using Marine Algae Ulva lactuca Extract

In the current study, biogenic silver nanoparticles (U-AgNPs) were synthesized using marine green macro-algal Ulva lactuca extract, and evaluated mechanism behind its anticancer activity against the Human colon cancer (HCT-116). The biogenic U-AgNPs were characterized using various physiochemical techniques. The TEM micrographs confirmed the spherical morphology of synthesized U-AgNPs, with a mean size of 8–14 nm. EDX spectrum as well as ICP-OES confirmed that AgNPs was nearly 90% purity for silver. FTIR Spectra analysis of U-AgNPs confirmed U. lactuca extract bioactive molecules presence over U-AgNPs surface as a stabilizing agent, thereby improving biocompatibility. The cytotoxicity study revealed the dose dependent cell death in colon cancer cells with no loss of viability in normal human colon epithelial cells. Furthermore, the fluorescence micrographs of nucleus staining assay revealed the DNA fragmentation and nucleus condensation of cancer cells treated with U-AgNPs, indicating an apoptosis-mediated cell death. The western bolt and RT-PCR analysis of U-AgNPs treated cancer cells showed the rise in proapoptotic markers (P53, Bax, and P21) and decline in anti-apoptotic markers (Bcl-2), thus confirming the p53-dependent apoptosis mediated cell death in HCT-116. Overall, our study concluded that novel biogenic U-AgNPs nanoparticles, synthesized using marine green macro-algal U. lactuca extract showed efficient anticancer activity against HCT-116 cell line and hence could work as potential therapeutic agent for targeted anti-cancer therapy.


Introduction
Colorectal cancer is fourth among cancer-related deaths and the third commonly occurring malignancy amounting to up to 9.7% of all cancers as per the report of the International Agency for Research [1]. Reports have projected that colorectal cancer will spike up to 2.4 million by 2035. The conventional method of the treatment of colorectal cancer is chemotherapy mainly employing fluoropyrimidine 5-fluorouracil but suffers greatly by poor prognosis due to the side effects of chemotherapy and multi-drug resistance (MDR) as a consequence of low solubility and improper drug distribution [2,3]. Hence, there is a need for alternative intervention having a better therapeutic index with minimal side effects.
In the recent decade, nanoparticles have gained a lot of attention in the field of cancer therapy as drug delivery agents, therapeutics, as well as in cancer diagnosis by imaging [4][5][6][7][8]. Importantly, metal nanoparticles have like silver nanoparticles (AgNPs) have distinctive properties comparatively whose exploitation has led to the breakthrough in various domains such as biomedical diagnostics [9], food industry [10], therapeutic agent [11,12], drug delivery [13], and antimicrobial agent [14][15][16].
Silver nanoparticles possess an advantage over other metal and metal oxide nanoparticles due to their intrinsic biological properties such as antimicrobial property, antioxidant property, anti-tumorigenic property, anti-inflammatory property [4,17,18]. These distinctive properties of silver nanoparticles become instrumental in utilizing them in different sectors as air and water disinfectant, biomedical applications like drug delivery process, wound healing patches, and medical devices, and most industries like textiles, food industry, and animal husbandry [19,20]. Moreover, silver nanoparticles have certain constraints for their biomedical application such as their undesirable toxicity and surface oxidation in an oxygen-containing biological fluid [21,22]. Constrains of AgNPs of undesirable cytotoxicity and surface oxidation can be curbed by capping the silver nanoparticle surface with biomolecules [23][24][25].
In this study, we synthesized novel biogenic silver nanoparticles (U-AgNPs) using extract of a green macro-algal Ulva lactuca as green synthesis involved biomolecules as a reducing agent also acts as a capping agent helps in preventing undesirable cytotoxicity and surface oxidation. Further, the green synthesis provides an advantage over other methods by fabricating the nanoparticles with plant phytochemicals thus creating stable nanoparticle with uniform size [26,27]. Further, the U-AgNPs characterized using Surface Plasmon Resonance (SPR) using UV-Vis spectrophotometer, XRD analysis (X-ray diffraction), Transmission electron microscopy (TEM), Energy-dispersive X-ray (EDX), FTIR, ζ-potential, and dynamic light scattering (DLS) analysis. Further, U-AgNPs was analysed using ICP to determine the purity of silver. Finally, the anticancer potential of U-AgNPs was studied in human colon cancer cells (HCT-116) using various cytochemical analyses as well as the cytocompatibility in human epithelial (FHC cells) and hemocompatibility (in human RBC cells) were also evaluated.

Material
High-quality grade Silver nitrate (AgNO 3 ) was obtained from Hi-media Ltd (Mumbai, India). All chemicals and DMEM media used in the current study were of analytical grade and procured from Sigma Aldrich (MO, USA). Buffers and sample preparation were prepared in double distilled water. The glassware used in the study were thoroughly washed, dried and sterilized. Antibodies used for analysis were procured from Cell Signalling Technology (MA, USA).
All the cell lines used in the studied were procured from National Centre for Cell Science, Pune.

Sample Collection and Preparation
Ulva lactuca commonly known as Sea lettuce were collected and cleaned thoroughly under running water. The cleaned plant leaves were rigorously grounded with mortar pestle to form a smooth paste using distilled water, which was then centrifuged at 3000 rpm for 10 min. The pellet was removed and supernatant was mixed vigorously with ethyl acetate in a ratio of 1:1 (v/v). This mixture was allowed to stand still for phase separation. Ethyl acetate phase containing bioactive molecule was collected and dried using a rotary evaporator. The Final concentrated U. lactuca extract was stored at 4 °C until further used.

Synthesis and Characterization of Silver Nanoparticles (U-AgNPs)
AgNPs were synthesized using the concept of metal reduction by the plant extract at 25 °C. Here, 5 mM aqueous AgNO 3 solution (100 mL) was taken in a Flask where the aqueous U. lactuca Extract (1 mg/mL) is added dropwise with constant stirring until color of solution change to dark reddish/yellowish color indicating the formation of AgNPs. The preliminary confirmation of formation AgNPs was done using a UV-Vis spectrophotometer (Perkin Elmer, l-35). Transmission Electron microscopy (Joel-2800 F) was done to further confirm the size and morphology of synthesized AgNPs with EDX. The purity of Ag in silver nanoparticles was determined using PerkinElmer Optima 5300 DV inductively coupled plasma optical emission spectroscopy (ICP-OES) model where Standards silver solution is used to test silver purity in the test sample after suitable dilution. The size distribution and surface charge of the synthesized AgNPs was confirmed with dynamic particle size analyzer (Nano ZS, Malvern Instruments). An X-ray diffractometer (Rigaku-SmartLab) was performed to determine the crystallinity of the synthesized AgNPs. Further, FT-IR spectroscopy (Perkin Elmer, spectra-2) of biosynthesized AgNPs was carried out to study the chemical bond vibrations in AgNPs, to assure the presence of bioactive components or metabolites over its surface.

Anticancer Activity/Cytotoxicity
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay was carried out to evaluate the anticancer activity of synthesized U-AgNPs as per earlier reports [28,29]. This method utilizes the principle of metabolization of tetrazolium salt in viable cells and which gives and absorbance at 570 nm. In our study, we used colon cancer cell line HCT-116 and normal colon epithelial cells to study the cytotoxicity. The seeding of cells was done in a 96 well plate with the count of 5 × 10 4 cells/well, followed by the treatment of cells with varying U-AgNPs concentrations (0-1000 µM) using DMEM media. The treated cells were then kept for incubation at 37 °C for 24 h in a CO 2 incubator with 5% CO 2 and 95% humidity conditions. Afterwards, the cell were again treated with MTT (5 mg/mL) and kept for further 4 h. The crystals formed were dissolved in 200 µL of DMSO and the absorbance was measured at 570 nm with a reference filter as 655 nm.

Nuclear Staining Assay
In Brief, HCT-116 cells at a cell density of 2 × 10 5 viable cells per well were seeded in six-well plates and incubated in a CO 2 incubator for 24 h. After 24 h of incubation, culture media was replaced with a fresh medium containing U-AgNPs at the required concentration followed by incubation for 24 h with AgNPs. After the 24 h of treatment with U-AgNPs, cells were washed three times with cold PBS followed by cells staining using Hoechst 33,342 dye (10 µg/ mL) for 15 min. Afterwards, the stained cells were visualized using a fluorescence microscope.

RT-PCR Analysis
Total cell RNA was extracted from HCT-116 cells using Trizol Reagent (TRI reagent®, Sigma) as directed by manufacturer's procedure. The purified RNA was pellet stored in aliquots of 5 µL in RNA storage buffer at − 80 °C for further use. The cDNA first strand synthesis has been carried out using extracted RNA as template along with the oligo(dT)18 primer and reverse transcriptase (RevertAid™ H Minus First Strand cDNA Synthesis Kit). The cDNAs homologous of anti-apoptotic (Bcl-2) as well as pro-apoptotic markers (P53, Bax, and P21) and housekeeping gene (GAPDH) positive strand RNA were amplified for 30 cycles, using specific primers tabulated in Table 1. The conditions in automated thermal cycler are as follows annealing step at 60 °C for 40 s, extension step at 72 °C for 40 s, and denaturation step at 95 °C for 20 s. Negative control reaction was performed without reverse transcriptase for each sample. The electrophoretic was used separate PCR products are each sample for analyse by using DNA-500 or 1000 kit using a Microchip Electrophoresis System-MCE®-202 (Shimadzu, Japan).

Western Blot
Initially, HCT-116 cells at the cell density of 1 × 10 6 were cultured using 100 mm cell culture dishes using DMEM media in a CO 2 incubator for 24 h. Later, the old media was removed and the cells were replenished with fresh culture media containing U-AgNPs. The cells were again incubated for another 24 h and then harvested by trypsinization. After trypsinization, cells collected were washed twice with icecold 1X PBS and resuspended in lysis buffer at 4 °C for 45 min used for RIA (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5 mM deoxycholate, 0.1% SDS, 1% NP-40, 1 mM Na 3 VO 4 , 1 mM phenyl methane sulphonyl fluoride, 2 mM dl-dithiothreitol, 10 mM b-glycerophosphate, 50 mM NaF, 0.5% Triton X-100 and protease inhibitor cocktail). After the treatment with lysis buffer, the lysate was subjected to centrifugation at 14,000 rpm. The cell debris as pellet was removed and supernatant collected was used for Bradford method of protein estimation. A 10% SDS-PAGE was run for the determined protein samples and was transferred to the polyvinylidene difluoride membrane. Finally, the membrane was treated with antibodies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Bcl-2, Bax, p53, and p21 as per protocol where GAPDH is used as control.

UV-Visible Spectroscopy Analysis
The synthesis of biogenic U-AgNPs was carried by reduction of AgNO 3 (aqueous ionic form) into metal nanoparticles (solid). The change in color of the solution from green to dark yellow indicates the formation of U-AgNPs. However, to further confirm the synthesis of nanoparticle formation from plant extract, we performed UV-Vis absorption spectroscopy (Fig. 1). The spectra obtained from plant extract, silver nitrate solution, and AgNPs are shown in Fig. 1. While there was no peak observed for the extract and silver nitrate solution, a clear peak at 452 nm for AgNPs attributes to the surface plasmon resonance of nanoscale metallic silver.  : GGT CAC CAG GGC TGC TTT T  R: TTC CCG TTC TCA GCC TTG AC  p53  F: CCT CCT GGC CCC TGT CAT CTT  R: ACC TCC GTC ATG TGC TGT  Thus, the preliminary confirmation of U-AgNPS was done with SPR peak at 452 nm.

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
FTIR analysis of nanoparticles gives the idea about the local environment or change in the functional groups adsorbed on the surface of nanoparticles. The comparative FTIR Spectra of AgNO 3, U. lactuca, and U-AgNPs are shown in Fig. 2. The spectrum for aqueous extract of U. lactuca and AgNO 3 shows all the characteristic peaks of the compounds, whereas in the case of the synthesized U-AgNPs the peak at 1474/cm corresponds to the presence of nanoparticles as opposed to AgNO 3 . Also, as observed from Fig. 2, U-AgNPs exhibits a peak at 3045/cm which points to an existing ester (C-O) bond. The presence of a significant number of secondary metabolites in the aqueous extract of U. lactuca act as reducing agents. The carotenoids and tocopherol are the phytochemicals mainly considered responsible for the reduction of AgNO 3 to silver nanoparticles (U-AgNPs). The broadening of amide I band peak at 1984/cm indicates the capping of U-AgNPs during the synthesis process. This amide I band peak at 1984/cm is attributed to the carbonyl stretch in proteins. whereas the peak at 3387/cm is the characteristic of OH stretch in alcohol-based compounds. Hence, bioactive molecules of U. lactuca extract presence over the AgNPs surface as capping agent confirmed by FTIR analysis, thereby increasing the surface stability of AgNPs along with enhancing the biocompatibility as well as its anticancer activity [30,31].

X-ray Diffraction Analysis
The crystalline structure of synthesized U-AgNPs was confirmed with the X-ray Diffraction technique. The results (Fig. 3) show the comparative XRD patterns for plant extract powder, silver nitrate, and U-AgNPs. In Fig. 3  peak corresponding to 111 planes and was found to be 5.63 nm.

ζ-Potential
The net surface charge or ζ-potential of U-AgNPs was determined using a Zeta analyzer. From Fig. 4, it could be said that synthesized U-AgNPs are positively charged with a surface potential of + 12.5 ± 2.2 mv. This positive surface potential on U-AgNPs can be attributed to the effective coating of Ulva-lactuca and confirms the stability of U-AgNPs.

TEM, DLS Analysis and EDX Analysis
The morphological features of synthesized U-AgNPs were analysed using TEM and DLS particle size analyzer. As observed from Fig. 5A and B, the particles obtained are spherical with sizes ranging between 8 and 14 nm. Whereas, the hydrodynamic size of U-AgNPs using DLS analysis was found to be in the range of 10-20 nm (Fig. 5D). Furthermore, the electron diffraction pattern (SAED) of the AgNPs indicates the crystalline nature of nanoparticles, which confirms the XRD data (Fig. 3). Moreover, the purity of U-AgNPs were analysed using EDX and ICP-OES as described in the 'Materials and Method Section'. EDX spectrum obtained showed strong silver signal for U-AgNPs with 88% purity for silver with impurities like Fe (0.37%), and Phosphorous (4.0%), and Cl (7.3%) (Fig. 6). Further, the resulted obtained from ICP-OES also confirmed U-AgNPs is 87.2% pure which was in par with the data obtained by EDX with other impurities like Fe (0.42%), P (4.3%), Mg (0.08%), Ca (0.05%), Mn Ca (0.025%).

Cell-Viability Assay
The cell viability of HCT-116 cells was examined in presence of U-AgNPs, AgNO 3, and U. lactuca extract via MTT assay and colony-forming ability of cells. For the clonogenic assay, HCT-116 cells were treated separately with U-AgNPs, AgNO 3, and U. lactuca extract for 24 h. The results (Fig. 7 A) indicate a dose-dependent cell death in presence of U-AgNPs and AgNO 3 , respectively. Hence, we demonstrated that synthesized U-AgNPs exhibit higher antiproliferative potential against HCT-116 cells as compared to its precursor compounds viz. AgNO 3 and U. lactuca extract (Fig. 7 A). Moreover, the IC 50 value of HCT-116 cells treated with U-AgNPs, were derived from Fig. 7B and it was found to be 142 ± 0.45 µM. This concludes that U-AgNPs are mitigating the cell attachment to form the colony as compared to the AgNO 3 and U.lactuca extract.
MTT assay was carried out to further validate the findings of the clonogenic assay. As observed from Fig. 7B, a dose-dependent cell death in HCT-116 cells occurred with the cells treated with U-AgNPs. The cell death was found to be 85% with U-AgNPs as compared to the 23% with AgNO 3, for the same concentration (200 µM) after 24 h of treatment. Hence to further explore the underlying mechanisms attributing the cytotoxic behaviour of U-AgNPs against HCT-116 cells, we chose to work with lower U-AgNPs concentration of 50, 100, and 200 µM. However, with the anticancer effects of U-AgNPs, it becomes imperative to examine the cytocompatibility of U-AgNPs using a normal human epithelial cell line. Hence, the effect of U-AgNPs was studied in both normal human epithelial cell line (FHC cells) as well as in colon cancer HCT-116 cell lines using varying concentrations (0-1000 µM) of U-AgNPs for 24 h and cell viability was measured. The results indicate only a 20% loss of cell viability in normal human epithelial cell line (FHM) cells as compared to that colon cancer cell line HCT-116, which was found to be almost 95% at the same U-AgNPs concentration (Fig. 7C). There was a drastic decrease in cell viability of HCT-116 cells at the minimum starting concentration of U-AgNPs, which further reduced as the dose of AgNPs was increased. However, in the case of FHM cell lines, the cell viability was not affected as much and does not affect higher doses of AgNPs with an overall 20% decrease in viability (Fig. 7C). This difference in cell viability of FHM and HCT cell lines in presence of U-AgNPs confirms the selective toxicity of U-AgNPs against the cancer cell line while keeping the normal human epithelial cell line unaffected.

Nuclear Staining Assay
Apoptosis helps in maintaining the balance between healthy and unhealthy cells inside the human body, by successfully removing the defective cells from the body. Hence to understand the mechanism behind the antiproliferative activity of U-AgNPs against colon cancer cell lines, we performed a nuclear staining assay using Hoechst 33,342 dye. The Hoechst 33,342 dye binds to the DNA and emits blue fluorescence under UV illumination. This helps to visualize and analyze the nuclear morphology of the cell and determine any defect in cell morphology. The fluorescence micrographs of AgNPs treated cells stained with Hoechst 33,342 dye were obtained and the DNA of cells treated with AgNPs is fragmented and significant nucleus condensation was quite visible, whereas the morphology of untreated cell was found intact (Fig. 8). Further, we also found that the fragmentation and distorted morphology of the cell becomes much clearer and enhanced with the increased AgNPs in concentration (Fig. 8). As nuclear condensation and cell fragmentation are major indications of apoptosis-led cell death, we can assume that U-AgNPs treatment also leads to cancer cell death through apoptosis.

Apoptosis-Associated mRNA and Protein Expression
To confirm the apoptosis mediated cell death by U-AgNPs in the cancer cell, we examined the mRNA levels of apoptosis markers in HCT-116 cells after treatment with U-AgNPs where anti-apoptotic (Bcl-2) as well as proapoptotic markers (P53, Bax, and P21) were determined against GAPDH as control (Fig. 9). The RT-PCR analysis of changes in mRNA levels in cells treated with different concentrations of U-AgNPs (0-200 µM) was recorded. It was observed that anti-apoptotic makers Bcl2 levels were high in untreated cells or control sample, but the expression level of Bcl2 found to decreased with increasing concentration of U-AgNPs. On the other hand, P53, Bax, and P21 showed significantly upregulated expression after the treatment with U-AgNPs. The upregulation in the expression of these markers was shown to be dosedependent ( Fig. 9) confirming the selective anticancer activity of U-AgNPs via p53-dependent apoptosis against the colon cancer cell line [33].
Further, we performed western blot to determine expression level of anti-apoptotic and pro-apoptotic markers in HCT cells after treatment with U-AgNPs where the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a control (Fig. 10). As observed from Fig. 10, at lower U-AgNPs concentration (50 µM) no significant change in protein expression level in all the apoptotic markers was observed. However, with an increase in the concentration of U-AgNPs, while there was an enhancement in the phosphorylation level of the P38 protein, the ERK protein expression showed an insignificant change. Also, the expression level of pro-apoptotic markers P53, Bax, and P21 proteins saw a significant rise whereas anti-apoptotic marker Bcl-2 showed a decline in expression level.  The results obtained confirms that U-AgNPs promotes the increase in phosphorylation levels of P38 protein without significant effects on ERK, thereby promoting the upregulation of pro-apoptotic markers P53 and Bax with the downregulation of pro-apoptotic markers Bcl-2 leading to the increase in the ratio of Bax/Bcl-2 and activation of P21 resulting in further increase cell death. Moreover, Gopinath et al. have reported that Bax acts on voltage-dependent ion channels on mitochondria, thereby activating cytochrome-C release and promoting apoptosis [34]. Furthermore, Shahbazzadeh et al. have reported that cytotoxic of nano-silver on osteoblast G292 cancer cell line mediated by up-regulation of pro-apoptotic P53 which in-turn activates the transcription of P21 gene resulting in impeding the DNA replication and inhibiting cell proliferation by stopping the cell cycle at the G1, G2 or S phase [35]. Hence, the cytotoxic effect of AgNPs on HCT-116 cells is dose-dependent and induce via p53-dependent apoptosis. Furthermore, the mechanism of cytotoxicity findings provide necessary toxicological information for developing the better biological safety of nanomaterials for clinical application.

Conclusions
The current work demonstrated the synthesis of silver nanoparticles (U-AgNPs) by using a green macro-algal U. lactuca and established its selective anticancer activity via p53-dependent apoptosis against the colon cancer cell line (HCT-116) using various biochemical assays such as cell viability using MTT assay, Clonogenic cell survival assay, DAPI nucleus staining as well as western blotting where the expression level of various proapoptotic proteins and antiapoptotic proteins were determined. U-AgNPs showed dosedependent and specific cytotoxicity in HCT-116 confirmed by cell viability assay as no effect was seen in human colon epithelial cells. Further, nuclear staining assay confirmed the dose-dependent toxicity caused by B-AgNPs in HCT-116 cells via apoptosis. western blotting results demonstrated the increased expression of pro-apoptotic marker P53, Bax, and P21 against GAPDH agent, a housekeeping gene as control whereas Moreover, anti-apoptotic marker proteins expression (Bcl-2) showed a dose-dependent decrease with increase in the U-AgNPs concentration used for treatment. Thus, categorically prove that biogenically synthesised U-AgNPs mediate p53-dependent apoptosis in colon cancer cells HCT-116 via activation of proapoptotic proteins with and inhibition anti-apoptotic proteins via DNA damage. Hence, we categorically proved that biogenically silver nanoparticle (U-AgNPs) prepared using U. lactuca extract is not only an eco-friendly technique, perhaps also possesses anticancer activity potential making it a probable nanotherapeutic for colon cancer with understanding the underlying biological mechanism toxicity in cancer cells inducing apoptosis provides better clinical safety.