Zoledronic acid augments the radiosensitivity of cancer cells through perturbing S- and M-phase cyclins and p21CIP1 expression

Abstract

Radiotherapy and adjuvant chemotherapy have become the standard treatments for multiple types of cancer. Although cancer cells are usually sensitive to radiotherapy, metastasis and local failure still occur mainly due to developed resistance to radiotherapy. Thus, it is critical to improve therapeutics for cancer treatment. The present study demonstrated that third-generation bisphosphonate zoledronic acid (ZOL), even at a low concentration, augments the radiosensitivity of cancer cells exposed to ionizing radiation (IR) by inducing S-phase arrest and subsequently promoting apoptosis. This function of ZOL was associated with elevated levels of cyclin A and cyclin B in the S and M phases, as well as decreased p21^CIP1 expression. In addition, ZOL also inhibited malignant the invasiveness of cancer cells. Notably, these effects could be enhanced concurrently with IR. The present data indicated that combined treatment with ZOL plus IR may be a novel technique to augment the radiosensitivity of cancer cells.

Keywords: radioresistance; radiosensitizing effects; zoledronic acid.

Figure 1.

ZOL affects the cell proliferation of (A) CNE-2 and (B) HNE-1 cells in a dose-dependent manner. PBS (vehicle control) or ZOL (2–32 µM) was added to the cell culture medium for 48 or 72 h. MTT assay was used to evaluate the cell viability. The experiments were repeated three times. *P<0.05, **P<0.01 compared with the vehicle control group. ZOL, zoledronic acid.

Figure 2.

ZOL inhibits clonogenic survival synergistically with IR in (A) CNE-2 and (B) HNE-1 cells. Cells were pretreated with vehicle control or 2 µM of ZOL for 6 h prior to exposure to IR at the indicated doses. The medium was changed the next day and the cells were subsequently incubated in normoxic conditions for 12–14 days prior to staining. Cell colonies were stained and counted, and the relative surviving fraction was calculated. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 compared with the control group. ZOL, zoledronic acid; IR, ionizing radiation.

Figure 3.

Effects of ZOL and IR on cell cycle distribution. (A) CNE-2 and (B) HNE-1 cells were pretreated with 2 µM of ZOL for 6 h, and then irradiated at 4 Gy. The medium was changed the next day. Cell cycle distribution was measured by flow cytometry at 24 h post-irradiation. **P<0.01 compared with the control group. ZOL, zoledronic acid; IR, ionizing radiation.

Figure 4.

ZOL increases the protein levels of cyclin A and cyclin B, and decreases p21^CIP1 expression. CNE-2 and HNE-1 cells were treated with 2 µM of ZOL for 6 h, and then irradiated at 4-Gy single fractions. Western blot analysis was performed following IR for 24 h using antibodies against the indicated proteins. ZOL, zoledronic acid; IR, ionizing radiation.

Figure 5.

ZOL decreases the mobility and invasiveness of cancer cells. These effects were more significant when combined with IR. (A) CNE-2 and (B) HNE-1 cells were treated with 2 µM of ZOL 24 h after plating and then irradiated at 4 Gy 6 h later. Cells were subjected to invasion assays. Representative fields (magnification, ×100) of invaded cells are shown. (C) Quantitative analysis from three independent experiments. Data are presented as the mean ± standard deviation. ***P<0.001 compared with the control. ZOL, zoledronic acid; IR, ionizing radiation.