Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

Abstract

Background: Radiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells.

Materials and methods: Uptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer.

Results: These hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors.

Conclusion: These results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.

Keywords: FePt nanoparticles; human copper transporter 1; mitochondrial targeting; radiation resistance; reactive oxygen species.

Figure 1

γ-GCS overexpressed SR3A-13 and SR3A-14 cells exhibited the radioresistant phenomena and enhanced uptake of FePt NPs. (A) Western blotting analysis of γ-GCS and hCtr1 protein levels in SR3A, SR3A-13 and SR3A-14 cells. β-actin was used as a loading control. (B) Representative colony formation photographs of SR3A, SR3A-13 and SR3A-14 cells irradiated with or without 8 Gy radiation. Noted that number of cells per dish initially plated varied with the dose so that the number of colonies surviving was in the range that could be counted conveniently. (C) Cell survival curves for SR3A series cells exposed to radiation. The surviving fractions of SR3A-13 and SR3A-14 cells were significantly higher than that of the control SR3A cells. (D) Total iron and platinum content determined by ICP-OES (***P < 0.001). Error bars represent ± S.D. (E) Representative TEM images of SR3A, SR3A-13 and SR3A-14 cells treated with FePt NPs. Note that FePt NPs were mainly found in vesicles located in the cytoplasm. Shown in the bottom are high power view of images in red squares shown above.

Figure 2

Mitochondrial morphology and function were altered in FePt NPs-treated SR3A-13 and SR3A-14 cells. (A) TEM observations of mitochondria in cells as indicated treated with 1 mg/mL FePt NPs for 24 hours. Black arrow, normal mitochondrial; red arrowheads, abnormal mitochondria with increasing membrane density and loss of ridges. (B) Measurements of mitochondrial respiratory function, OCR, by Seahorse XF24 analyzer in SR3A, SR3A-13 and SR3A-14 cells treated with FePt NPs. The OCR values in SR3A-13 and SR3A-14 cells were significantly lower than that in SR3A cells, in a time-dependent manner. (C) Similar findings as in (B), but in concentration-dependent manner.

Figure 3

Enhancements of ROS production and radiosensitivity in SR3A-13 and SR3A-14 cells treated with 1 mg/mL FePt NPs for 24 hours and then irradiated with X-rays. (A) Flow cytometry analysis of ROS. Note that the combination treatment groups had significantly enhanced ROS levels than X-rays alone or control group especially in SR3A-13 and SR3A-14 cells. The result of MFI was presented as mean ± SD of three dependent experiments. ***p < 0.01. (B) Mitochondrial OCR by Seahorse XF24 analyzer showing mitochondrial functions were nearly totally abolished in SR3A-13 and SR3A-14 cells treated with FePt NPs and X-rays. (C) Representative photographs of colony formations of SR3A-13 and SR3A-14 cells treated with FePt NPs without (top row) or with (bottom) radiation (6 Gy). Noted that cell numbers seeded were different in 0 Gy and 6 Gy groups (500 vs 15,000 cells). (D) Cell survival curves of SR3A, SR3A-13 and SR3A-14 cells exposed to radiation with or without FePt NPs.

Figure 4

Elevated hCtr1 expression confers enhanced uptake/transport activity of FePt NPs and induces mitochondria dysfunction. (A) Western blotting analysis of γ-GCSh and hCtr1 protein levels in SR3A-hCtr1-WT cells. β-actin was used as a loading control. (B) The uptake/transport of 1 mg/mL FePt NPs for 24 hrs was significantly increased in SR3A-hCtr1-WT cells as compared with SR3A cells by ICP-OES measurement (***P < 0.001). Error bars represent ± S.D. (C) Representative TEM images of SR3A-hCtr1-WT cells treated with FePt NPs (left). Shown in the right is high-power view of image in red square demonstrating abnormal mitochondria in SR3A-hCtr1-WT cells with increasing membrane density and losing ridges after FePt NPs treatment (red arrowheads). (D) The OCR levels were significantly decreased in SR3A-hCtr1-WT cells treated with FePt NPs, in a time (left)- and concentration (right)-dependent manner.

Figure 5

hCtr1 expression significantly enhances FePt NPs-induced radiosensitivity. (A) SR3A-hCtr1-WT cells were treated with 1 mg/mL FePt NPs for 24 hours then irradiated with or without X-rays. Clonogenic assay shows significant decrease of surviving colony numbers in the combination treatment (left). Surviving fractions in these treatments shown (right). (B) ROS was considerably increased after combined treatment of FePt NPs and X-rays in SR3A-hCtr1-WT cells. (C) OCR measured by Seahorse XF24 analyzer was markedly attenuated in SR3A-hCtr1-WT cells treated with FePt NPs and X-rays.

Figure 6

Enhancement of radiation therapy efficacy by utilizing FePt NPs in SR3A-hCtr1-WT-bearing mice under various treatments. (A) Schematic drawing of experimental design for assessing the efficacy of FePt NPs and irradiation (6 Gy) in vivo. (B) Representative H&E and immunostainings of hCtr1 and iron in tumor tissues of SR3A-hCtr1-WT-bearing mice, square was the region magnified 400X in each tumor sections. (C) Tumor growth inhibition of SR3A-hCtr1-WT-subcutaneous xenograft. Growth reduction was seen in the FePt NPs- and X-rays irradiation-treated groups (*p < 0.05; **p < 0.01), but greater reduction was seen in the group of combined treatment with FePt NPs and X-rays (***P<0.01). Error bars represent ± S.D. (D) No significant changes of body weights of the mice among all the treatment groups.

Scheme 1

The schematic illustration of the GSH-overexpressed small-cell lung cancer cells exhibit elevated expression of hCtr1 and are resistant to X-rays irradiation. Radiation resistance can be overcome by enhanced uptake/transport of FePt NPs due to the overexpressed hCtr1 through the mechanisms of ROS outburst and mitochondria dysfunction.