Abstract
Background/Aim: Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated the anticancer potential of Fe3O4 and Fe3O4core Aushell nanoparticles as hyperthermic agents in terms of cytotoxicity and studied the expression of cellular markers of proliferation (changes in mRNA levels via real-time polymerase chain reaction). Materials and Methods: The human breast cancer cell line SK-BR-1 was incubated with either Fe3O4 or Fe3O4core Aushell nanoparticles stabilized with tryptophan, prior to hyperthermia treatment. The normal HEK293 cell line was used as a control. Toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to estimate possible toxic effects of the tested nanoparticles. After RNA extraction and cDNA synthesis, mRNA expression of three indicators of proliferation, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), was investigated. Results: At each concentration tested, Fe3O4core Aushell nanoparticles showed greater toxicity compared to Fe3O4, while SK-BR-3 cells were more susceptible to their cytotoxic effects compared to the HEK293 cell line. The expression of Ki-67, TOP2A and TPX2 was reduced in SK-BR-3 cells by both Fe3O4 or Fe3O4core Aushell nanoparticles compared to untreated cells, while the only observed change in HEK293 cells was the up-regulation of TOP2A. Conclusion: Both Fe3O4core Aushell and Fe3O4 NPs exhibit increased cytotoxicity to the cancer cell line tested (SK-BR-3) compared to HEK293 cells. The down-regulation in SK-BR-3 cells of the three proliferative markers studied, Ki-67, TOP2A and TPX2, after incubation with NPs suggests that cells that survived thermal destruction were not actively proliferating.
Hyperthermia represents a local adjuvant anticancer strategy which relies on the increase of temperature beyond the physiological level, typically to 40-43°C (1). Throughout the years, several means of hyperthermia have been used to achieve maximum effect on cancer cells while at the same time efforts to spare normal tissue are constantly being made. Currently, clinical trials and basic hyperthermia research focus on understanding the pleiotropic effects of hyperthermia, and technical research targeted at effective means of heat delivery (2, 3). Hyperthermic applications have been tested for several cancers including prostate cancer (4), liver cancer (5) while considerable research has been made in locally advanced breast cancer (BC) (6) and recurrent metastatic BC (7).
Among the different categories of tested hyperthermic agents, nanoparticles (NPs) have shown promising results. Their small size and their ability to penetrate cancer tissue along with their unique physicochemical characteristics have made them valuable candidates for hyperthermia treatments. One of the most studied types of NP, iron oxide in the form of magnetite (Fe3O4), have already been tested in several hyperthermia applications (8).
Current literature supports that the basic mechanism of action of magnetite-based hyperthermia is mediated via the production of reactive oxygen species (ROS) (9), which ultimately leads to cellular death if the repair mechanisms do not suffice to overcome the thermal damage. However, current data suggest that treatment of malignant cells with hyperthermia can result in an aggressive phenotype; the treated cells tend to grow faster and become resistant to the applied treatments (10). Thus, to counter these effects, hyperthermia is typically not applied as a monotherapy but as part of a therapeutic scheme that involves radiation, chemotherapy, or other treatments (11). In parallel, the search for a hyperthermic agent that will limit the proliferative potential of cancer cells is also an important goal. Due to the promising results of Fe3O4 NPs in several applications (12) the investigation of their impact on cellular proliferation markers remains a major clinical question.
In our previous work, we have shown that Fe3O4 and Fe3O4core Aushell NPs stabilized with tryptophan mostly affect cancer cells, and normal cells are damaged to a lesser extent (13). Moreover, we showed that the underlying mechanism of action was the induction of the apoptotic pathway. A distinct characteristic of the NPs tested in our study is that the magnetite cores are surrounded by Au shells, since magnetite (Fe3O4core) alone can be highly toxic (14). In this study, our aim was not only to expand our results in a human cancer cell line but also to investigate the effects of Fe3O4 and Fe3O4core Aushell NPs on the surviving cells. For this, we selected three genes, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), that are widely known as proliferation markers in cancer and investigated the potential changes upon exposure to Fe3O4core Aushell NPs and hyperthermia.
Materials and Methods
NP preparation. The NPs used (Fe3O4 and Fe3O4core Aushell) were obtained through chemical reduction as described in our previous study (13).
Cell culture. ATCC (Manassas, VA, USA) supplied SK-BR-3 cell line (breast cancer) and HEK293 (embryonic kidney cells) as a control. Both cell lines were cultured with DMEM High Glucose (BioSera, Shanghai, PR China) with 10% FBS (PAN Biotech, Aidenbach, Germany) and 100 U/ml penicillin and 100 g/ml streptomycin.
Hyperthermia. The hyperthermia session used the same conditions as in our previous published work (13). Both types of NP tested contain magnetite (Fe3O4), which allows the tested NPs to act as hyperthermic agents upon exposure to appropriate electromagnetic frequencies (13). The hyperthermia session was performed using a water loaded circular waveguide applicator with a diameter of 7 cm (WR-340 waveguide; Microwave Techniques LLC, Gorham, ME, USA). The device was operated as a 433 MHz microwave heater. The microwave device had an emission power of 100 W RMS. However, the transmitted power in our case was at the level of 15-20 W for 4 min. Both cell lines (SK-BR-3 and HEK293) upon exposure to the electromagnetic frequencies reached a temperature of 43°C which was maintained for 20 min. The detailed process is described in a previous study (14).
Cytotoxicity assay. To perform the cytotoxicity study, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter96, AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA) was performed as described in (15). The SK-BR-3 and HEK293 cells were seeded at approximately 5,000 cells/well in a 96-well plate and were incubated with either Fe3O4coreAushell or Fe3O4 NPs for 24 h. A range of concentrations was tested (minimum: 100 μg/ml, maximum concentration: 500 μg/ml). Each condition was tested in triplicate. The optical densities were read at 490 nm. Absorbances were normalized with respect to the untreated control culture to calculate changes in cell viability. All experiments were performed in duplicate.
RNA extraction, cDNA synthesis and real-time polymerase chain reaction (PCR). Prior to RNA extraction from a 6-well plate, cells were incubated with 400 μg/ml of only one type of NP for 24 h was performed. For the RNA extraction, the NucleoZOL method was chosen (Macherey-Nagel, Düren, Germany) (13).
For the cDNA synthesis, PrimeScript First-Strand cDNA kit (Takara Bio Europe SAS, Saint-Germain-en-Laye France) was used following the procedure described in (13). For the real-time PCR, KAPA SYBR FAST qPCR mix (KAPA BIOSYSTEMS, Cape Town, South Africa) was used.
The sequences of Ki-67, TOP2A, TPX2 and GAPDH primers are provided in Table I; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the gene of reference. Every reaction was performed in duplicates aiming to ensure sufficient reproducibility, while gene expression was normalized to the expression of GAPDH. Gene expression was calculated using the 2−ΔΔCt formula (13, 15).
Primer sequences used for polymerase chain reaction for marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2).
Statistical analysis. One-way analysis of variance was chosen for statistical analysis using GraphPad v.8 (GraphPad Software, San Diego, CA, USA). p-Values below 0.05 were considered statistically significant.
Results
Toxicity of Fe3O4coreAushell and Fe3O4 NPs. The toxicity of Fe3O4coreAushell and Fe3O4 NPs was investigated with and without hyperthermia treatment in HEK293 and SK-BR-3 at five concentrations (100, 200, 300, 400 and 500 μg/ml) using the MTS assay. As shown in our results, both NP types tested exerted dose-dependent toxicity (Figure 1) in both cell lines tested. Regarding HEK293 cells, the maximum reduction in viability was observed with the highest concentration (500 μg/ml) after hyperthermia with Fe3O4 NPs (60% viability). Hyperthermia after incubation with Fe3O4coreAushell resulted in smaller reduction of viability (77% viability) at the maximum concentration (500 μg/ml). Toxicity was higher with Fe3O4core NPs (viability of 63% at a concentration of 500 μg/ml) (Figure 1A). The cancer cell line SK-BR-3 showed a sharper decrease in viability. After hyperthermia, the viability was reduced to 42% and 28% using 500 μg/ml Fe3O4coreAushell and Fe3O4core NPs, respectively. Without hyperthermia treatment, similarly to HEK293, viability was increased compared to hyperthermia-treated SK-BR-3 cells. The highest toxicity was observed for Fe3O4core NPs (63% at 500 μg/ml) while Fe3O4coreAushell remained less toxic at all concentrations tested (Figure 1B).
Graphical illustrations of the viability of HEK293 and SK-BR-3 cells after incubation with Fe3O4coreAushell and Fe3O4 nanoparticles with (A) and without (B) hyperthermic treatment. Viability is shown relative to that of cells that were not incubated with nanoparticles. Statistically significant differences from the control were estimated using one-way analysis of variance at: *p<0.05, **p<0.01 and ***p<0.001.
Effect of Fe3O4coreAushell and Fe3O4 NPs on Ki-67, TOP2A and TPX2 expression. To analyze the levels of mRNA of Ki-67, TOP2A and TPX2, we performed real-time PCR for both HEK293 and SK-BR-3 cells incubated with Fe3O4coreAushell and Fe3O4 NPs (400 μg/ml) after hyperthermia. Hyperthermia of HEK293 cells incubated with Fe3O4coreAushell NPs did not cause any statistically significant change in the expression of Ki-67, TOP2A and TPX2 (Figure 2A) compared to untreated cells. On the contrary, in SK-BR-3 cells (Figure 2B) resulted in statistically significant down-regulation of Ki-67, TOP2A and TPX2 compared to untreated cells. Regarding the effects of Fe3O4 NPs in HEK293 cells, no statistically significant changes were observed for Ki-67 and TPX2 but a statistically significant increase in TOP2A expression was observed. Regarding SK-BR-3, the observed effects were like those of Fe3O4coreAushell NPs: Ki-67, TOP2A and TPX2 showed a statistically significant down-regulation.
Graphical illustrations of the fold-change mRNA expression of proliferation markers, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), in HEK293 (A) and SK-BR-3 (B) cells after incubation with 400 μg/ml Fe3O4coreAushell and Fe3O4 nanoparticles after hyperthermia treatment compared to untreated cells. Significantly different from the control by one-way analysis of variance at: *p<0.05, **p<0.01 and ***p<0.001.
Discussion
As shown by our results, the tested Fe3O4 and Fe3O4coreAushell NPs had a toxic effect on SK-BR-3 cells following hyperthermia effect. The observed toxicity was dose-dependent, with the maximum observed toxicity at 500 μg/ml for both Fe3O4 and Fe3O4coreAushell NPs. This finding supports the notion that these NPs can act as hyperthermic agents against SK-BR-3 cells. However, due to the increased toxicity of non-hyperthermic Fe3O4, not only in SK-BR-3 but also in HEK293 cells, Fe3O4coreAushell NPs can be considered more promising hyperthermic agents. Fe3O4coreAushell NPs being more promising may possibly be attributed to the intrinsic toxicity of Fe as has already been demonstrated in literature (16, 17). Furthermore, Fe3O4coreAushell NPs have been shown to have a selective effect on cancer cell lines as suggested by the viability results; at the highest concentration tested, 500 μg/ml, viability in HEK293 cells did not drop below 73%. Both NP types tested have been stabilized with tryptophan and our previous study (13) have shown that tryptophan is advantageous for increased toxicity against cancer cell lines. The mechanism behind this may be the different metabolism of tryptophan by malignant cells (18).
We further examined the effects of the tested NPs on the expression of Ki-67, TOP2A and TPX2, which have been recognized as important markers of cancer cell proliferation (19-21). Of the different concentrations tested in the toxicity assay, the most promising was 400 μg/ml due to its minimal effect on HEK293 cell line (viability 80%) and its profound toxicity towards SK-BR-3 (viability 51%).
Ki-67 is strongly linked with the proliferation and growth of cancer cells and currently is widely used as a marker in pathology. Its expression is increased in malignant tissues and signifies a poor prognosis (22). As our results show, both Fe3O4coreAushell and Fe3O4 NPs resulted in down-regulation of Ki-67 expression in SK-BR-3 cells. This finding is in accordance with a previous study (23) that showed that magnetic hyperthermia resulted in the decrease of Ki-67 expression. It is reasonable to believe that since Ki-67 reflects the percentage of actively proliferating cells (24), an effective treatment should result in the decrease of Ki-67 expression. Importantly, it has been shown that the lack of a decrease of Ki-67 after neoadjuvant chemotherapy in BC results in unfavorable outcomes (25). As our viability results indicate, at 400 μg/ml, approximately half the SK-BR-3 cell population was destroyed, and the cells that survived thermal destruction were not proliferating (as suggested by the reduced Ki-67 expression).
The next proliferation marker that we tested, TOP2A, was also down-regulated in SK-BR-3 with both Fe3O4coreAushell and Fe3O4 NPs. Interestingly, a change, namely up-regulation, was also observed for normal HEK293 cells. Although we did not further investigate the involved mechanism, this might indicate that the hyperthermic exposure mobilized the cell’s repair mechanisms. As shown by the MTS results, the viability of HEK293 remained higher after hyperthermia compared to the SK-BR-3 cell line and the increase of TOP2A mRNA (Fe3O4 NPs only) may reflect the treated cells response to induced cellular damage. TOP2A is known to be crucial for chromatin remodeling and crucially interacts with bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) to preserve genomic architecture (26). Similarly to Ki-67, we deem that its down-regulation in SK-BR-3 cells may be attributed to the extensive damage that was induced after hyperthermia.
The next gene studied, TPX2, is a recognized biomarker in several malignancies including BC (20, 27). Its increased expression has been associated with increased metastatic potential (28) and with poor patient outcomes (29). Our results showed both Fe3O4coreAushell and Fe3O4 NPs resulted in the down-regulation of TPX2 after hyperthermia treatment. Interestingly, TPX2 has been proposed as a potential target in BC and its inhibition resulted in the apoptotic death of treated cells via the PI3K/AKT/P21 pathway (30). In our previous study, both Fe3O4coreAushell and Fe3O4 NPs induced apoptosis in the investigated cell lines (13), while down-regulation of TPX2 has also been observed in magnetic hyperthermia (24). Moreover, our finding of down-regulation of TPX2 is in accordance with the reduced expression of Ki-67 and TOP2A, collectively indicating that the SK-BR-3 cells that survived hyperthermia and NP treatment were not in a state of active proliferation and thus the effects of the NPs are not limited to thermally destroying cancer cells.
Conclusion
Our results show that both Fe3O4coreAushell and Fe3O4 NPs exhibit a hyperthermic effect on SK-BR-3 cells, with Fe3O4coreAushell NPs inducing a greater reduction in viability compared to Fe3O4 NPs. This can be attributed to the more biocompatible nature of Au as opposed to Fe, which is known for its toxicity, and due to the use of tryptophan as a stabilizer. Moreover, the three genes used as proliferation markers were down-regulated after cells were incubated with either Fe3O4coreAushell or Fe3O4 NPs, a finding suggesting that after hyperthermia, cells were not actively proliferating. Further studies should be conducted to examine these effects in vivo, along with the possible effects of hyperthermia with Fe3O4coreAushell on other tumor characteristics and, most importantly, their effect on tumor metastatic potential.
Footnotes
Authors’ Contributions
Conceptualization: S.G., H.K. and M.G.; data curation: V.M.B. and MG; methodology: D.I.L and S.G.; supervision: V.K., G.A. and M.G.; original draft: I.M., N.V. and N.L.; writing, review, and editing: K.S. and M.G. All Authors have read and agreed to the published version of the article.
Funding
This research was funded by the Hellenic Society of Medical Oncology (HESMO), grant number 8953.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received March 8, 2024.
- Revision received May 3, 2024.
- Accepted May 8, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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