Sensitization of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ROS production

Abstract

Side effect of radiation therapy (RT) remains the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Taken together, these results identify CONPs as a potentially novel RT-sensitizer as well as protectant for improving pancreatic cancer treatment.

From the clinical editor: Pancreatic tumors remain some of the most notoriously treatment-unresponsive malignancies. Cerium oxide nanoparticles may be capable of sensitizing such cells to radiotherapy, as demonstrated in this study.

Figure 1

Acidic pH is relatively resistant to H₂O₂ production compared to neutral pH. Water (pH 7.4) and PBS at indicated pHs were irradiated at indicated doses and H₂O₂ production was examined by Amplex Red Assay. *P<0.05 compared to water exposed to that RT dose. B, TEM analysis of CONPs. Left panel shows TEM image of the CONPs of size between 5–8 nm (inset, high magnification images). Right panel shows selected area of electron diffraction pattern of the CONPs where A (111), B (200), C (220), D (311), E (222) and F (400) are the different crystal planes of fluorite crystal structure.

Figure 2

At neutral pH CONPs generally decrease RT-induced H₂O₂ production. (A & B) CONP suspensions of serial concentrations up to 200 μM in water or PBS at neutral pH (Figure S2) were irradiated at indicated doses. H₂O₂ production was determined at indicated time points post-RT (see response to all the concentrations and time-course response in Figure S1A & S1B). (C&D) Water was irradiated at indicated doses. After 1 hour CONPs were added up to 200 μM. H₂O₂ production was then determined at indicated time points post-RT (see response to all the concentrations in Figure S1C & S1D). (E&F) Water was irradiated at indicated doses. After 24 hours CONPs were added up to 200 μM. H₂O₂ production was then determined at indicated time points post-RT (see response to all the concentrations in Figure 21E & S1F).*P<0.05 compared to 0 μM CONP at that time point.

Figure 3

Under acidic conditions CONPs enhances H₂O₂ production and lose H₂O₂ scavenging activity. (A–D) CONPs of serial concentrations (up to 200 μM) were included in acidic PBS solutions at indicated pHs for 24 hours followed by RT at indicated doses. H₂O₂ production was determined at indicated time points post-RT (see response to all the concentrations and time-course response in Figure S4A–D). *P<0.01 compared to 0 μM at the same time point. (E&F) CONPs with predominant Ce³⁺ and Ce⁴⁺ on the surface were included in water at indicated pHs for indicated periods of time before concentration of superoxide radical (E) or H₂O₂ (F) was determined using a SOD assay kit and Amplex Red assay kit, respectively as described in Methods.

Figure 4

CONP treatment prior to, but not post, RT increase RT-induced ROS levels in acidic pancreatic cancer cells and decrease RT-induced ROS levels in neutral pancreatic normal cells. (A&B) The cancer (L3.6pl) and normal hTERT-HPNE cells were treated with (CONP) or without (Ctrl) CONPs for 24 hours followed by 5 Gy RT. ROS levels were then determined and compared between the cells at indicated times. Relative fold changes were normalized to the control groups. (C&D) The cells were treated with 5 Gy RT for 24 hours prior to CONP treatment. ROS levels were then determined and compared between the cells at indicated times. Relative fold changes are normalized to the control groups. *P < 0.001. The acidic cancer cellular environment relative to the neutral normal cellular environment was confirmed (see Figure S5)

Figure 5

CONP pretreatment selectively sensitizes pancreatic cancer cells to RT-induced cell death in culture. (A) Indicated cells were pre-treated with 10 μM CONPs for 24 hours followed by RT at 5 Gy. Cell viability was determined 96 hours post-RT. Cell death was normalized to untreated group. (B & C) L3.6pl cells were treated similarly as in A. Immediately after the treatment, cells were detached, replated and grown for 7 days before colonies were counted.

Figure 6

CONPs enhance tumor cell apoptosis in vivo. (A–D) Histologic evaluations using hematoxylin and eosin (H&E) staining. (E–H) TUNEL staining of apoptosis cells in situ. Tumor cell implantation and treatment of mice are described in Table 1. Tumor tissues along with adjacent normal pancreatic tissues were collected at the time when mice were sacrificed. Formalin-fixed and paraffin-embedded, immediately adjacent tissue sections were used for the staining. Immunopositive cells for TUNEL staining were observed over 10 individual slides for each condition and quantified using the NIH ImageJ software to determine the number of TUNEL positive cells per field of view.