## 4 Discussion
A differential response between tumor and normal cells has been reported in some studies involving prostate cancer cells, supporting the design of new strategies in prostate cancer therapy [30]. In our previous report, Fe\({}_{3}\)O\({}_{4}\) NPs was found to enhance DTX-induced prostate cancer cell death, and ROS levels were found to increase with exposure, similar to the levels of 8-OH-dG, a marker of oxidative DNA damage with exposure in DU145 and PC-3 cells [12]. A slight difference of cell viability between DU145 and PC-3 was observed in this study although both cell lines showed to increase ROS production as same as the previous study [12]. In addition, Fe\({}_{3}\)O\({}_{4}\) NPs alone reduced the viability of LNCaP and PC-3 cells, but had little or no effect on the viability of DU145 and PrSC cells [12]. PrSC cells were prostate stromal cells, and used as normal control. These results suggestthat the cytotoxicity of Fe\({}_{3}\)O\({}_{4}\) NPs may be dependent on the cell type, which may be linked with the different redox state properties. Multiple pathways, including apoptosis, inflammation, and drug efflux pumps, are implicated in chemoresistance of prostate cancer [31]. The expression of NF-kB, a transcriptional factor for survival signaling pathways, increases in DU145 cells treated with DTX [16]. Fe\({}_{3}\)O\({}_{4}\) NPs were also found to enhance the inhibitory effects of DTX via suppression of NF-kB expression [12]. However, significant suppression of NF-kB expression was not observed in prostate cancer cell lines treated with a combination of Fe\({}_{3}\)O\({}_{4}\) NPs and rapamycin or carboplatin. In addition, ROS has been reported to up- or down-regulate the expression of p-glycoprotein in various cells [32; 33; 34]. Recently, AgNP treatment has also been reported to inhibit the efflux activity of drug-resistant cells [20]. The authors mentioned that it was necessary to clarify whether AgNPs exerted their inhibitory effects directly on the ABC transporter itself, through disruption of mitochondrial function and ATP production, or by transcriptional silencing of the _mdr1_-encoding genomic locus [20]. These findings suggest that NPs may have the potential to affect the expressions of ABC transporters in cells via various pathways. However, in this study, significant suppression of _ABCC1_ and _ABCG1_ expression was not observed in either cell line following combined treatment with Fe\({}_{3}\)O\({}_{4}\) NPs and rapamycin or carboplatin. Additional experiments are needed to clarify the mechanisms through which the effects of chemotherapies are enhanced by the combination treatment. In this study, we found that the characteristics of different cell lines may play a key role in determining the effects of Fe\({}_{3}\)O\({}_{4}\) NPs combined with rapamycin or carboplatin. The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway is a key signaling pathway in prostate cancer progression [35]. Rapamycin and its analogs (rapidogs) were the first identified inhibitors of the PI3K/Akt/mTOR pathway and were found to inhibit tumor growth in mouse xenograft models derived from PTEN\({}^{-/-}\) PC-3 and PTEN\({}^{+/-}\) DU145 cells [36]. However, rapamycin and rapalogs did not show clinical benefits as single agents because they did not inhibit mTOR complex 2 (mTORC2), which activates Akt in prostate cancer cells. In this study, Fe\({}_{3}\)O\({}_{4}\) NPs enhanced the inhibitory effects of rapamycin in both prostate cancer cell lines, and combination of Fe\({}_{3}\)O\({}_{4}\) NPs with other chemotherapeutic agents was more effective in PC-3 cells when a concentration of 100 \(\upmu\)g/mL Fe\({}_{3}\)O\({}_{4}\) NPs was used. In addition, combined treatment with Fe\({}_{3}\)O\({}_{4}\) NPs and rapamycin in PC-3 cells synergistically enhanced induction of apoptosis. Fe\({}_{3}\)O\({}_{4}\) NPs have been reported to induce AKT activation, demonstrating that this pathway is involved in cellular proliferation after exposure of Fe\({}_{3}\)O\({}_{4}\) NPs.