Cisplatin radiosensitizes radioresistant human mesenchymal stem cells

Abstract

Cisplatin-based chemo-radiotherapy is widely used to treat cancers with often severe therapy-associated late toxicities. While mesenchymal stem cells (MSCs) were shown to aid regeneration of cisplatin- or radiation-induced tissue lesions, the effect of the combined treatment on the stem cells remains unknown. Here we demonstrate that cisplatin treatment radiosensitized human bone marrow-derived MSCs in a dose-dependent manner and increased levels of radiation-induced apoptosis. However, the defining stem cell properties of MSCs remained largely intact after cisplatin-based chemo-radiation, and stem cell motility, adhesion, surface marker expression and the characteristic differentiation potential were not significantly influenced. The increased cisplatin-mediated radiosensitivity was associated with a cell cycle shift of MSCs towards the radiosensitive G2/M phase and increased residual DNA double-strand breaks. These data demonstrate for the first time a dose-dependent radiosensitization effect of MSCs by cisplatin. Clinically, the observed increase in radiation sensitivity and subsequent loss of regenerative MSCs may contribute to the often severe late toxicities observed after cisplatin-based chemo-radiotherapy in cancer patients.

Keywords: DNA double-strand breaks; cisplatin; mesenchymal stem cells; radiosensitization; radiotherapy.

Figure 1. Cisplatin pre-treatment radiosensitizes MSCs

Clonogenic survival assays for MSCs and HS68 fibroblasts pre-treated with 200ng/mL or 1000ng/mL cisplatin 48 prior to irradiation. ^*P<0.05, ^**P<0.01 (paired Student's t-test). Table represents sensitizer enhancement ratios for each cell line.

Figure 2. MSC adhesion and motility is unaffected by cisplatin-based chemo-radiation

(A) Adhesion kinetics of MSCs and HS68 fibroblasts after radiation. (B) Average velocity of MSCs and HS68 fibroblasts after treatment with cisplatin, IR or a combined treatment. ^*P<0.05.

Figure 3. Cisplatin-based chemo-radiation does not alter morphology and surface marker expression of MSCs

(A) Microscopic images of unstained MSCs and HS68 fibroblasts showing no visible changes in morphology after cisplatin-based chemo-radiation. 10x objective, scale bar 100μm. (B) FACS histograms of defining MSC surface markers at 48 hours after cisplatin-based chemo-radiation.

Figure 4. Cisplatin-based chemo-radiation does not affect the differentiation potential of MSCs

(A) Alcian blue staining for chondrogenic MSC differentiation after treatment with cisplatin and radiation. 2x objective, scale bar 1000μm. (B) BODIPY staining for adipogenic differentiation in MSCs. Relative staining intensities were measured to quantify adipogenic and chondrogenic differentiation levels. 2x objective, scale bar 2000μm. ^*P<0.05, ^**P<0.01.

Figure 5. Cisplatin-based chemo-radiation increases apoptosis and G2 phase arrest

(A) Cell cycle distribution of MSCs and HS68 fibroblasts at 48 hours after cisplatin-based chemo-radiation. (B) Apoptosis levels of MSCs and HS68 fibroblasts after cisplatin-based chemo-radiation. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 6. Cisplatin pre-treatment causes prolonged DNA damage signaling and increased radiation-induced DNA double-strand breaks in MSCs

(A) γH2AX foci numbers in MSCs and HS68 fibroblasts after cisplatin-based chemo-radiation. ^*P<0.05, ^**P<0.01. (B) Western blot analyses of various proteins regulating cell cycle checkpoints and double-strand DNA break repair at 2 and 24 hours after irradiation with 6 Gy.