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. 2017 Feb 7:12:1009-1018.
doi: 10.2147/IJN.S108577. eCollection 2017.

Nanoparticle-based hyperthermia distinctly impacts production of ROS, expression of Ki-67, TOP2A, and TPX2, and induction of apoptosis in pancreatic cancer

Robert Ludwig  1 , Francisco J Teran  2 , Ulf Teichgraeber  1 , Ingrid Hilger  1
Affiliations

Nanoparticle-based hyperthermia distinctly impacts production of ROS, expression of Ki-67, TOP2A, and TPX2, and induction of apoptosis in pancreatic cancer

Robert Ludwig et al. Int J Nanomedicine. .

Abstract

So far, the therapeutic outcome of hyperthermia has shown heterogeneous responses depending on how thermal stress is applied. We studied whether extrinsic heating (EH, hot air) and intrinsic heating (magnetic heating [MH] mediated by nanoparticles) induce distinct effects on pancreatic cancer cells (PANC-1 and BxPC-3 cells). The impact of MH (100 µg magnetic nanoparticles [MNP]/mL; H=23.9 kA/m; f=410 kHz) was always superior to that of EH. The thermal effects were confirmed by the following observations: 1) decreased number of vital cells, 2) altered expression of pro-caspases, and 3) production of reactive oxygen species, and 4) altered mRNA expression of Ki-67, TOP2A, and TPX2. The MH treatment of tumor xenografts significantly (P≤0.05) reduced tumor volumes. This means that different therapeutic outcomes of hyperthermia are related to the different responses cells exert to thermal stress. In particular, intratumoral MH is a valuable tool for the treatment of pancreatic cancers.

Keywords: heat dose; iron oxide nanoparticles; magnetic hyperthermia; nanomedicine; proliferation marker.

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Conflict of interest statement

Disclosure The authors report no conflict of interest in this work.

Figures

Figure 1
Figure 1
MH markedly reduces BxPC-3 cell viability compared to EH using comparable temperatures. Notes: Analysis of cell populations (vital, apoptotic, necrotic) via flow cytometry at 24 hours (upper panel) and 48 hours (lower panel) post-treatment with MH (left) or EH (right) at different temperatures. Controls: native cells (37°C) cells treated with MNP without heating (37°C MNP). Mean values and standard deviations (n≥3) given as percentage of investigated populations (10,000–20,000 cells). MNP concentration: 100 µg/mL. Mann–Whitney U-test: comparison of temperature effects (41°C vs 43°C and 43°C vs 47°C groups). *P≤0.05, **P≤0.01, or ***P≤0.001. Comparison of time effects: “a” indicates 43°C or 47°C (24 vs 48 hours) with P≤0.01. “b” Vital cells, 47°C, EH (24 vs 48 h) (no significant differences). Comparison of therapy modality effects (MH vs EH groups): numbers “1–11” indicate P≤0.01. Abbreviations: MH, magnetic heating; EH, extrinsic heating; MNP, magnetic nanoparticles; AMF, alternating magnetic field.
Figure 2
Figure 2
MH exhibits greater impact on apoptosis induction, ROS formation, and proliferation markers expression compared to EH. Notes: (A) Representative protein bands after immunoblotting of protein cell lysates (PANC-1 cells). β-actin: protein loading control. (B) Flow cytometry analysis of cells to detect ROS induction after staining with DCFH-DA (n≥3). (C) qRT-PCR analysis of proliferation markers. Data are depicted in relation to an expression level of 0. The expression level of 0 represents the relative mRNA expression of a gene based on B2M (reference mRNA expression in the cell) minus the mRNA expression of the respective gene in native, non-treated cells (37°C) (n≥6). MNP concentration: 100 µg/mL. (*P≤0.05 [Mann–Whitney U-test: treated vs native cells]; non-treated controls at 37°C). Results are expressed as mean values and standard errors. Abbreviations: MNP, magnetic nanoparticles; ROS, reactive oxygen species; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; EH, extrinsic heating; MH, magnetic heating; HSP, heat shock protein; AMF, alternating magnetic field; n.d., not determined; DCFH, 2′,7′-Dichlorofluorescin diacetate.
Figure 3
Figure 3
MH treatment at a temperature >43°C in PANC-1 xenografts resulted in a significant reduction of tumor volume compared to untreated tumors. Notes: (A) Relative tumor volumes of tumors of PANC-1 xenografts after treatment (CEM43T90: 137±118 min) compared to untreated tumors over a period of 28 days. (B) Efficiency of different treatment modalities observed by macroscopic tumor images. Arrow, area of intratumoral iron oxide deposition; arrowhead, distinctly shrunk tumor after treatment. (C) Tumor surface temperatures during the two consecutive treatments of representative PANC-1 xenografts. The amount of MF66 MNP applied: 0.2 mg Fe/100 mm3 of tumor volume. MNP/AMF: n=5; MNP: n=5; AMF: n=3; untreated: n=3. (*P≤0.05; Mann–Whitney U-test: MH treated vs untreated). Abbreviations: MNP, magnetic nanoparticles; AMF, alternating magnetic fields; d, day; min, minutes.
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