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Evaluation of cytotoxic effects of fungal origin nanosilver particles on oral cancer cell lines: An in vitro study


1 Department of Conservative Dentistry & Endodontics, Al Badar Rural Dental College & Hospital, Kalaburgi, Karnataka, India
2 Department of Conservative dentistry & Endodontics, HKE's SN Institute of Dental Sciences and Research, Sedam Road, Kalaburgi, Karnataka, India
3 Consultant Endodontics, Director, RAK Dental Centre, Ras Al Khaima, UAE
4 Specialist Endodontics, Sharjah Specialty Dental Center, Sharjah, UAE
5 Department of Conservative Dentistry & Endodontics, SVS Institute of Dental Sciences, Mahbubnagar, Telangana, India
6 Department of Restorative, (Operative Dentistry), Jazan University, KSA, Saudi Arabia

Date of Submission05-Sep-2020
Date of Decision26-Dec-2020
Date of Acceptance03-Jan-2021
Date of Web Publication18-Aug-2021

Correspondence Address:
Kiran R Halkai,
Department of Conservative Dentistry and Endodontics, Al-Badar Rural Dental College and Hospital, Kalaburgi - 585 102
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1308_20

 > Abstract 


Background: Oral cancer is often associated with poor prognosis and it is found that conventional treatment options cause severe side effects, adjacent tissue disfigurement, and loss of function. Recently, silver nanoparticles (AgNPs) paved their path for cancer treatment.
Aim: This study aimed to investigate cytotoxic effects of fungal procured AgNPs on oral squamous cell carcinoma (SCC-9) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Methodology: The silver nanoparticles were biosynthesized using the fungi Fusarium semitectum. Cell lines were cultured in a 1:1 ratio of Dulbecco's Modified Eagle's medium and Ham's F12 medium and subcultured in a T-75 cm2 flask. Cell count was adjusted to 1 × 105 cells/ml; 50,000 cells/well were seeded into a 96 well plate and incubated at 37°C, for 24 h in 5% CO2 humidified conditions. AgNPs (1.75–50 μl/ml) were added to the plates and further incubated at 37°C for another 24 h. Medium containing cells without AgNPs were used as a control group. Later, 20 μl of MTT was added to each well and incubated for 6 h at above-mentioned conditions. About 0.1 mL of Dulbecco's Modified Eagle's Medium solution was added to each well to solubilize formazan. The absorbance was measured using a Tecan reader at 540 nm. The experiment was repeated thrice independently. The percentage (%) inhibition of growth and the AgNP's concentration that prevents the cell growth by 50% (IC50) were determined.
Results: Significant dose-dependent inhibition of the growth of SCC-9 cell lines was seen and IC50 was found at 12 μl/ml concentration of AgNPs.
Conclusion: Biosynthesized AgNPs of fungal origin exhibit effective anticancer properties against the SCC-9 cell line.

Keywords: Biosynthesis, cytotoxicity, fungal-derived nanosilver particles, oral cancer



How to cite this URL:
Halkai KR, Halkai R, Patil S, Alawadi J, Alawadhi WS, Marukala NR, Mohammad Albar NH, Indi S. Evaluation of cytotoxic effects of fungal origin nanosilver particles on oral cancer cell lines: An in vitro study. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324032




 > Introduction Top


Oral squamous cell carcinoma is the most common malignant tumor occurring in the head-and-neck region involving the structures such as the oral or nasal cavity, lips, lateral and ventral surfaces of the tongue, floor of the mouth, paranasal sinuses, and also pharynx or larynx.[1] Despite its occurrence in easily recognizable clinical sites, most of the patients present with advanced stages involving regional lymph node metastases with poor prognosis.[2] Irrespective of different treatment modalities of various combinations of procedures, such as diagnosis at an early stage, advanced surgical procedures, radiotherapy, and chemotherapy, the survival rate is poor and responsible for the increased death rate globally among all the tumors.[3] It involves the important anatomic structures associated with breathing, mastication, etc.; therefore, invasive treatment leads to adverse functional and cosmetic impairments.[4] Treatment of oral cancer with radiotherapy results in severe adverse effects such as osteoradionecrosis, xerostomia, increased rate of dental caries, and periodontitis. It also affects the adjacent healthy structures such as salivary glands, skin, and brain. Chemotherapy causes systemic manifestations such as the impairment of homeostasis, anemia, neutropenia, and/or thrombopenia, immune deficiency.[5]

Recently, nanotechnology has been advocated for the prevention, early diagnosis, and treatment of oral cancer for improving the prognosis rate. It has significantly revolutionized the cancer therapy.[6],[7] Nanoparticles are included in the diagnosis of cancer in the form of nanoshells, nanotubes, nanowires, nanodiamonds, magnetic nanoparticles, and nanosponges, etc.[8] Nanoparticle-assisted tumor biomarkers have been widely used for oral cancer diagnosis in recent days. NPs specifically target the tumor cells and biomarkers, therefore allowing more reliable early diagnosis of oral cancer with minimal damage to the adjacent healthy tissues. NPs also help in monitoring the prognosis of cancer therapy over time and destroys only the cancer cells.[9]

Nanoparticles have been used as therapeutic agents owing to their extremely small size, they have been incorporated in drug delivery systems. NPs provide great hope for improving the prognosis of oral cancer mainly by improving the properties of an existing therapeutic agent when used in combination for synergistic activity by increasing the stability, modifying the pharmacokinetics, decreasing the toxicity, etc., and when used solely they directly target the tumor cells.[10] NPs have been used as synergetic agents along with existing chemotherapeutic agents to overcome drug resistance and to increase the effectiveness of drugs targeting the cancer cells, while reducing their cytotoxic effects on healthy tissues.[11]

In recent days, biosynthesized silver nanoparticles have gained immense popularity due to their eco-friendly and being nonhazardous to human health. They can be effectively produced using fungi, bacteria, plant extracts, etc., without using any chemicals or tedious processing techniques, besides they are economical and exhibit excellent properties at much smaller doses and are biocompatible. Among them, fungal-derived AgNPs are widely used as antimicrobial and antitumor agents.[12]

The cytotoxicity of AgNPs has been studied using various cell models.[13],[14] These studies have shown that AgNPs interfere with the cellular functions of the target cells including the cancer cells and cause DNA damage, apoptosis, and cell death hence, they are indicated for cancer therapy.[15],[16]

In our previous study, the cytotoxic effect of fungal-derived biosynthesized silver nanoparticles was studied on normal human gingival fibroblast cells and the results showed that AgNPs are biocompatible toward normal cells and cytotoxicity was found to increase with increasing concentration dose of AgNPs and IC50 was found at 256 μg/ml.[17] The available literature on the effect of biosynthesized AgNPs against oral cancer cells is scarce; hence, the present study aims to evaluate the cytotoxic effect of fungal-derived AgNPs on oral cancer cells (SCC-9 cell line) and to determine whether they can be used as antitumor agents for oral cancer therapy.


 > Methodology Top


Silver nanoparticles were biosynthesized using the endophytic fungi Fusarium semitectum, as described in earlier studies.[12] In brief, for obtaining the fungal cultures, freshly obtained Ashwagandha (Withania somnifera) leaves were grown on Potato Dextrose Agar plates, and pure cultures obtained were then added to 100 ml of malt glucose yeast peptone broth and incubated for 72 h at 29°C until confluent growth. The fungal biomass was then filtered using a Whatman filter paper and cleansed with sterile distilled water. About 25 g of biomass was added to 100 ml of sterile distilled water which is further incubated for another 48 h. Then, 1 mM concentration of silver nitrate (AgNO3) solution was added to the fungal filtrate and kept under observation in dark at 29°C for 24 h. Characterization of AgNPs was done by visual observation, ultraviolet spectrum (T90+ UV–Vis, USA), transmission electron microscope (JEOL/JEM 2100, USA), and Fourier transform infrared spectroscopy (Thermo Nicolet Avatar 370, USA). The characterization studies showed the formation of a brown-colored solution and UV spectrum in a 420 mm range indicating the formation of AgNPs. The particles were found to be 1–10 nm in size and the FTIR spectrum showed the presence of fungal-derived biomolecules that act as stabilizers and capping agents for AgNPs. The pH of the solution was measured as 7.4 and stored in a refrigerator at 4°C until use. Before adding to the cell suspension, the temperature of the prepared NPs was elevated to 37°C in a warm water bath.

Cell lines and culture medium

American tissue cell cultures (ATCC) of human SCC cell lines (SCC-9 (ATCC CRL-1629)) and all the chemicals used in the study were obtained from HiMedia Laboratories Ltd., Mumbai, India. The stock cells were cultured in a solution of 1:1 ratio of Dulbecco's Modified Eagle's medium and Ham's F12 medium containing 1.2 g/L sodium bicarbonate, 0.5 mM sodium pyruvate, and 2.5 mM L-glutamine, supplemented with 400 mg/ml hydrocortisone and 100 mg/ml streptomycin in 5% CO2 atmosphere at 37°C.

After removing the medium, the cells were rinsed with TPVG solution containing 0.25% trypsin, 0.03% ethylenediaminetetraacetic acid, and 0.05% glucose in PBS. The solution was discarded and an additional 2 ml of the TPVG solution was added to the cells and kept at 37°C for the detachment of the cells followed by subculturing into a T-75 cm2 tissue culture flasks containing fresh culture medium. The viability of the cells was checked under an inverted microscope.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The cell count was adjusted to 1 × 105 cells/ml using the above-mentioned culture medium. The cell suspension (around 50,000 cells/well) was seeded into a 96-well microtiter plate, and incubation was carried out at 37°C, for 24 h. in 5% CO2 humidified conditions. After 24 h of incubation, the superficial layer was removed, and the monolayer was washed with the medium. About 100 μl of different concentrations of AgNPs (1.75–50 μl) were added to the microtiter plates containing cells and further incubated at 37°C for another 24 h. Culture medium containing an equal volume of untreated cells were used as control groups. Then, the medium in the wells was removed and 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml in PBS) was added to each well and incubated for 6 h at 37°C in a 5% CO2 atmosphere. The microplates show purple color due to the formation of solid crystals of formazan indicating the presence of viable cells. The supernatant was removed and 0.1 mL of Dulbecco's Modified Eagle's Medium solution was added to each well and kept for 15 min. The microplates were gently shaken to solubilize the formazan. The absorbance of each microplate well was measured using a microplate reader (BMR-100, Boeco, Hamburg, Germany) at 540 nm. The experiment was repeated thrice independently and the average of three readings was recorded as the final reading. The percentage (%) inhibition of growth was calculated using the equation mentioned below and the concentration doses of AgNPs that prevent the cell growth by 50% (IC50) values were produced from the dose–response curve.

Calculating percentage growth inhibition




 > Results Top


The results showed significant dose-dependent inhibition of the growth of SCC-9 cell lines. Minimum percentage inhibition of 7.34% was found at 1.75 concentration of AgNPs. Maximum inhibition of 88.46% was found at 50 μl of AgNPs. At a concentration of 12.5 μl/ml, the parentage inhibition was 52.37%. The IC50 was found around 12 μl concentration of AgNPs. Hence, AgNPs exhibit cytotoxicity on SCC-9 cells preventing their cell growth [Figure 1], [Table 1] and [Graph 1].
Figure 1: Microscopic observation of oral cancer (squamous cell carcinoma-9) cell lines treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (a) Normal cell line. (b) At 50 μl/ml concentration of AgNPs

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Table 1: Percentage (%) inhibition on human oral cancer (squamous cell carcinoma-9) cell lines by fungal-derived nanosilver particles

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 > Discussion Top


The prognosis of oral cancer is poor and unfortunately in the current treatment protocols, no approach can selectively bind to the cancer cells and prevent side effects and damage to the adjacent tissues.[18] Owing to improved biological and physical properties, AgNPs have emerged as a new perspective for the early detection and treatment of oral cancer. The incorporation of AgNPs for diagnosis and therapeutics has drastically reduced the mortality rate of oral cancer.[19]

The antitumor and antibacterial effects of AgNPs are due to their smaller size and spherical shapes, allowing them to exhibit potential direct contact to the target cell surfaces and initiate cytotoxic effects.[20] Literature shows many studies demonstrating the anticancer effects of biosynthesized AgNPs on different cell lines.[14],[15] However, the effect of fungal-derived AgNPs on oral cancer cells is rare; hence, the effect of biosynthesized AgNPs on SCC-9 cell lines was evaluated in the present study.

Cytotoxicity of AgNPs is extensively studied in in vitro models as it is easy to perform, regulate, interpret, and simulate in vivo conditions. MTT assay is the most commonly employed quantitative cytotoxic method, as it is simple to perform and yields reliable and reproducible results, hence used to determine the cytotoxic effects on SCC-9 cells in the present study.[21]

Han et al.[14] stated that biosynthesized AgNPs exhibited cytotoxic effects on human lung epithelial adenocarcinoma cell lines and IC50 was found at 20 g/ml while for the synthetic AgNPs, IC50 values were found to be 70 g/ml, signifying that biosynthesized AgNPs are effective at much smaller doses.

Dose-dependent antitumor effects were found in another study on human lung cancer (A549) and human breast cancer (MCF 7) cell lines at 100 μl/ml as IC50 value.[22] Similar results were showed in another study with lower concentrations (10–100 μg/mL), of mycosynthesized AgNps showed higher effectiveness in MCF-7 cells compared to the silver ions.[23] The results of our study are in accordance with the above-mentioned studies indicating the AgNPs produced using the fungi Fusarium semitectum showed effective antitumor effects on SCC-9 cell lines at much smaller concentrations ranging from 1.75 to 50 μl/ml. The IC50 for inhibiting the cancer cells was found at a much smaller dose of 12 μl/ml AgNPs. About >80% of cell growth inhibition was found at a concentration of 50 μl/ml, indicating that fungal-derived AgNPs can be used as antitumor agents in the treatment of oral carcinoma.

Mechanisms for the antitumor effects of AgNPs can be briefly summarized as follows. AgNPs enter the target cells by invagination through the cell membrane and localize inside the cell.[24] Besides, they also affect the cellular respiration by entering into the mitochondria and producing the reactive oxygen species, oxidative stress, induce apoptosis, and cause damage to DNA and ultimately killing the cancer cells.[15],[16]

Moreover, studies reported that AgNPs are identified as vascular permeability factors as they affect the vascular endothelial growth factor and inhibit angiogenesis within cancer cells.[25] Jun et al.[26] analyzed the morphology of cancer cells and suggested that biosynthesized AgNPs significantly induce cell death of cancer cells.

A study demonstrated the anticancer efficacy of biosynthesized AgNPs of both bacterial (B-AgNPs) and fungal-derived AgNPs (F-AgNPs) in human breast cancer MDA-MB-231 cells. Both AgNPs showed effective cytotoxicity against the cancer cells.[27] However, fungal-derived AgNPs exhibited stronger efficacy due to the fungal-derived enzymes acting as naturally occurring reducing and capping agents AgNPs. Targeting only the cancer cells is a crucial part of cancer treatment, biosynthesized AgNPs exhibited more cytotoxic effect in human lung carcinoma cells (A549) than noncancer human lung cells, signifying that AgNPs exhibit cell-specific cytotoxicity, due to the low pH in the cancer cells.[28] Therefore, fungal-derived biosynthesized AgNPs exhibit anticancer properties, indicating that they can be used as alternative agents for cancer therapy.[29] However, further research is needed for enhancing the target selectivity of fungal-derived AgNPs solely on cancer cells and to determine the biocompatibility and side effects of these particles using in vivo models and clinical trials.


 > Conclusion Top


Within the limits of present study, it is found that fungal-derived biosynthesized AgNPs exhibit effective antitumor efficacy against SCC-9 cell lines, indicating a new path for improving the prognosis of oral cancer therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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