Research Article Evaluation of the Response of HOS and Saos-2 Osteosarcoma Cell Lines When Exposed to Different Sizes and Concentrations of Silver Nanoparticles
Konstantinos Michalakis ,1,2,3 Athina Bakopoulou ,1 Eleni Papachristou,1
Dimitra Vasilaki ,1 Alexandros Tsouknidas ,4 Nikolaos Michailidis ,5
and Elaine Johnstone6
1School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece 2Tufts University, Boston, MA, USA 3University of Oxford, Oxford, UK 4Laboratory for Biomaterials and Computational Mechanics, Department of Mechanical Engineering, University of Western Macedonia, Kozani, Greece 5Department of Mechanical Engineering, School of Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece 6Department of Oncology, University of Oxford, Oxford, UK
Correspondence should be addressed to Konstantinos Michalakis; firstname.lastname@example.org
Received 12 September 2021; Revised 20 November 2021; Accepted 22 November 2021; Published 13 December 2021
Academic Editor: Aziz ur Rehman Aziz
Copyright © 2021 Konstantinos Michalakis et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Osteosarcoma is considered to be a highly malignant tumor affecting primarily long bones. It metastasizes widely, primarily to the lungs, resulting in poor survival rates of between 19 and 30%. Standard treatment consists of surgical removal of the affected site, with neoadjuvant and adjuvant chemotherapy commonly used, with the usual side effects and complications. There is a need for new treatments in this area, and silver nanoparticles (AgNPs) are one potential avenue for exploration. AgNPs have been found to possess antitumor and cytotoxic activity in vitro, by demonstrating decreased viability of cancer cells through cell cycle arrest and subsequent apoptosis. Integral to these pathways is tumor protein p53, a tumor suppressor which plays a critical role in maintaining genome stability by regulating cell division, after DNA damage. The purpose of this study was to determine if p53 mediates any difference in the response of the osteosarcoma cells in vitro when different sizes and concentrations of AgNPs are administered. Two cell lines were studied: p53-expressing HOS cells and p53-deficient Saos-2 cells. The results of this study suggest that the presence of protein p53 significantly affects the efficacy of AgNPs on osteosarcoma cells.
Osteosarcoma is considered a relatively uncommon malig- nant disease. Nevertheless, it is the most common cancer arising from bone . It usually affects adolescents and young adults. In recent years, much advancement has been made in treating osteosarcoma, which combines surgery, chemotherapy, and sometimes radiotherapy. Currently, the 5-year survival rate for patients diagnosed with osteosar-
coma is 60-70% [2–5]. The chemotherapy agents employed include cisplatin, doxorubicin, ifosfamide, and methotrexate. Other cytotoxic agents such as etoposide and different com- binations have also been suggested in the literature . Nev- ertheless, the use of these drugs has several side effects and complications including neutropenia, mouth ulcers, fatigue, severe diarrhea, nausea, and vomiting. The side effects can be very serious and commonly require hospitalization. Car- diomyopathies and irreversible lung fibrosis have also been
Hindawi BioMed Research International Volume 2021, Article ID 5013065, 16 pages https://doi.org/10.1155/2021/5013065
described, illustrating that severe side effects present a major drawback for the use of chemotherapeutic agents . This along with therapeutic limitations, due to the systemic cyto- toxic effects, has motivated scientists to start exploring dif- ferent directions in an attempt to find innovative therapies for several types of cancer, including osteosarcoma [8–15]. Some novel therapeutic agents have been tested for that pur- pose, including tumor microenvironment inhibitors, which target signal-transduction pathways and immunomodula- tory agents. Methods for overcoming resistance mechanisms as well as new delivery mechanisms have also been tested . One of these avenues of interest is silver nanoparticles. Although the exact action by which AgNPs act on cells is not fully understood, it is speculated that a Trojan horse mecha- nism is involved . Upon entering the cell, the AgNPs release silver ions in the cytoplasm which then induce the formation of ROS, thus causing an imbalance of the cell’s redox homeostasis [18, 19]. It is not known yet whether the observed oxidative damage is due to the action of AgNPs per se, accumulation of silver ions in the cytoplasm, or a combination of both [20, 21] (Figure 1). A recent in vitro study testing the antibacterial effect of AgNPs with different sizes has shown that smallest-sized AgNPs are more effica- cious on two different types of Gram-negative bacteria . According to Gliga et al., smaller AgNPs are more active due to the increased Ag ion release from the increased total surface area  (Figure 2).
Tumor protein p53, whose gene TP53 is located on the short arm of chromosome 17, plays a critical role in regulating cell division, after DNA damage occurs. It is crucial in deter- mining if the DNA damage can be repaired or if the cell will undergo apoptosis [24, 25]. When DNA damage in the form of a double-strand break occurs, there is recruitment of ATM serine protein kinases and/or ATR kinases, which are then activated. These kinases phosphorylate p53, leading the protein to evade degradation by ubiquitin. As a result, the levels of p53 increase markedly; the protein is stabilized and activates transcription of p21(Cip1/Waf1) . The latter acts by binding and inhibiting the activity of several complexes, including cyclin E-CDK2, cyclin E-CDK1, and cyclin E- CDK4/6, and prevents cell cycle progression at phase G1 [27, 28]. This arrest gives time to the cell to repair the damage of the DNA. Furthermore, p53 is responsible for the production of DNA repair enzymes and proapoptotic proteins .
In this way, p53 acts as a tumor suppressor, and its inac- tivation seems to play a key role in the development of human cancer. For the pivotal role in maintaining genome integrity, p53 has been named “guardian of the genome” . If DNA is damaged and p53 is present and functional, the cell cycle arrests in phase G1. On the contrary, in the absence of functional p53, cells continue to grow and divide. The p53 protein is unique in the sense that it exists in very small quantities in normal cells, due to its instability and rapid degradation. Mouse models have shown that the absence of p53 is associated with the development of several types of tumors . Furthermore, p53 is mutated in more than half of all human cancers, and in more than 80% of tumors, there is a p53 signaling pathway disruption of some kind [32–34].
Several human osteosarcoma cell lines have been isolated so far, including the HOS, U-2OS, MG-63, G-292, and Saos- 2. An analysis of these cell lines with p53 genomic probes has revealed some key differences. p53 was found to be pres- ent in G-292, MG-63, HOS, and U-2OS cell lines, with a rearrangement in the first intron of the gene described in G-292 and MG-63. A point mutation within the p53 coding sequence has been described in HOS cells which results in overproduction of mutant p53 [35, 36].