## 3 Results
### Combined Effects of Fe\({}_{3}\)O\({}_{4}\) NPs and Chemotherapeutic Agents on Apoptosis  
To determine the effects of combined Fe\({}_{3}\)O\({}_{4}\) NPs and rapamycin/carboplatin, we chose to treat prostate cancer cells with 100 \(\upmu\)g/mL Fe\({}_{3}\)O\({}_{4}\) NPs and 10 nM rapamycin or 100 \(\upmu\)g/mL Fe\({}_{3}\)O\({}_{4}\) NPs and 10 \(\upmu\)M carboplatin. FCM analysis showed a strong induction of apoptosis by the combination of 100 \(\upmu\)g/mL Fe\({}_{3}\)O\({}_{4}\) NPs and 10 nM rapamycin in PC-3 cells (Figure 5A). The portions of Annexin V(+)/PI(-) and Annexin V(+)/PI(+) cells indicated the early and late stages of apoptosis. The quantitative data showed that the effects varied with cell type and chemotherapeutic agent (Figure 5B-E). Their combinations (ramamycincin and Fe\({}_{3}\)O\({}_{4}\) NPs in PC-3 cells, and carboplatin and Fe\({}_{3}\)O\({}_{4}\) NPs in DU145 cells) resulted in a significant increase in apoptotic cells compared to each chemotherapeutic drug alone (\(p<0.01\) and \(p<0.05\), respectively). In addition, to determine whether the effects of the combined treatment were synergistic, the CI was calculated based on the response additivity approach. When PC-3 cells were treated with Fe\({}_{3}\)O\({}_{4}\) NPs together with rapamycin or carboplatin, the CIs were 0.97 (Figure 5B) and 1.36 (Figure 5D), respectively; additionally, when DU145 cells were treated with Fe\({}_{3}\)O\({}_{4}\) NPs together with rapamycin or carboplatin, the CIs were 1.20 (Figure 5C) and 0.93 (Figure 5E), respectively. Synergistic effects were observed in PC-3 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs and rapamycin (Figure 5B) and in DU145 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs and carboplatin (Figure 5E). Figure 5: _Cont._Effects of Fe\({}_{3}\)O\({}_{4}\) NPs, Chemotherapeutic Agents, and Their Combinations on the Expression of MDR1, MRP1, and BCRP mRNA in Prostate Cancer Cells  
To determine whether the development of chemosensitivity in both cell lines was associated with decreased expression of ABC transporter genes, we examined the expression of _MDR1_, _ABCC1_, and _ABCG2_ mRNAs (Figure 6). _MDR1_ expression was not detected in both cell lines (data not shown). In both cells treated with MNPs and carboplatin/rapamycin, _ABCC1_ and _ABCG2_ levels were altered; however, these differences were not significant. Figure 5: Combined effect of Fe\({}_{3}\)O\({}_{4}\) NPs and chemotherapeutic agents on apoptosis. DU145 or PC-3 cells were treated with Fe\({}_{3}\)O\({}_{4}\) NPs, carboplatin, rapamycin or their combinations for 48 h. Cells were then harvested, stained with Annexin V and PI, and analyzed by FCM. (**A**) Representative case of FCM in PC3 cells. The portions surrounded by the line mean the early and late stages of apoptosis; (**B**) Comparison of apoptotic rates among PC-3 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs, rapamycin alone, or combination treatment; (**C**) Comparison of apoptotic rates among DU145 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs, rapamycin alone, or combination treatment; (**D**) Comparison of apoptotic rates among PC-3 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs, carboplatin alone, or combination treatment; (**E**) Comparison of apoptotic rates among DU145 cells treated with Fe\({}_{3}\)O\({}_{4}\) NPs, carboplatin alone, or combination treatment. * Significantly different from the group with chemotherapeutic agent alone at \(p<0.01\); ** significantly different from the group with chemotherapeutic agent alone at \(p<0.05\). Figure 6: _Cont._  
Effects of Fe\({}_{3}\)O\({}_{4}\) NPs, Chemotherapeutic Agents, and Their Combinations on NF-\(\kappa\)B Expression in Prostate Cancer Cells  
The effects of Fe\({}_{3}\)O\({}_{4}\) NPs, rapamycin, and carboplatin alone or in combination on NF-\(\kappa\)B expression in both cell lines were analyzed.