Figure 1.
Effects of zoledronic acid (ZOL) on SACC-83 in vitro and in vivo.
A After ZOL treatment, the total number of live SACC-83 cells was markedly decreased both dose and time dependently. B Cell viability assessed by Cell Counting Kit-8 assay after treatment with ZOL. C Representative phase-contrast images of SACC-83 cells treated with 50 µM ZOL at the indicated time points. Scale bar = 100 µm. D Colonies were stained with crystal violet, followed by scoring of colony numbers. E The growth curves of tumors in different group after the first treatment. ZOL inhibited the growth of SACC-83 xenograft tumor (n = 5 tumors). Error bars show the mean ± standard deviation. *Differences with p<0.05 are considered statistically significant.
Figure 2.
Cell cycle arrest induced by zoledronic acid (ZOL).
A After 24B Effect of ZOL on cell cycle regulatory genes. SACC-83 cells were treated with ZOL (0–100 µM) for 24 h. Expression of cyclin D1, p21CIP1, p27KIP1 and p53 was measured by western blotting.
Figure 3.
Zoledronic acid induces apoptosis after 48 h of treatment.
A Annexin-V/propidium iodide double staining was performed to determine the apoptosis rate. Data are representative a result from three independent experiments. B, C 4,6-Diamidino-2-phenylindole dihydrochloride stained (Scale bar, 100 µm) and terminal transferase deoxyuridine triphosphate nick end labeled (Scale bar, 50 µm) cells were observed under a fluorescence microscope. D Expression of apoptosis related proteins was detected by western blotting.
Figure 4.
Zoledronic acid (ZOL) increased reactive oxygen species (ROS) production in SACC-83 cells.
A ROS levels were measured by flow cytometry after incubation with dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. B N-acetylcysteine (NAC) eliminated ZOL-induced ROS generation. Cells were protected by NAC (2 mM) with 12 h pretreatment when co-incubated with ZOL for another 48 h. ROS levels were determined by DCFH-DA staining. C, D Cells were pretreated with NAC for 12 h, then co-treated with or without 50 µM ZOL for another 48 h. The impact of ZOL and NAC on cell viability and apoptosis were determined by Cell Counting Kit-8 assay and 4,6-diamidino-2-phenylindole dihydrochloride staining, respectively. E Cells were pretreated with NAC for 12 h, then co-treated with or without ZOL for 14 d. The number of colonies formed was measured. All experiments were performed independently in triplicate per experimental point; representative results are shown. *Differences with p<0.05 are considered statistically significant.
Figure 5.
Inhibition of zoledronic acid (ZOL)-induced autophagy partially reversed SACC-83 apoptosis and inhibition of colony formation.
A SACC-83 cells were treated with various doses of ZOL for 48 h and then LC-3B and Atg7 protein were determined by western blotting. B Cells were pretreated with 5 mM 3-methyladenine (3-MA) for 12 h before exposure to 50 µM ZOL for 48 h, then immunofluorescent staining for monodansyl cadaverine (MDC) and LC-3B was observed under a confocal microscope. Scale bar = 50 µm. C Impact of 5 µM 3-MA on the viability, apoptosis and colony formation of ZOL treated cells was measured by Cell Counting Kit-8 (CCK-8) assay, 4,6-diamidino-2-phenylindole dihydrochloride staining and a colony formation assay, respectively. D Cells were transfected with Atg7 or Beclin-1 small interfering RNA (siRNA) for 24 h before exposure to 50 mM ZOL for 48 h. Cell viability was measured by CCK-8 assay. E Effect of Beclin-1 siRNA on apoptotic cell death and colony formation. All data are representative of at least three independent experiments. *Differences with p<0.05 are considered statistically significant.
Figure 6.
Zoledronic acid (ZOL) enhances cisplatin/paclitaxel-induced cytotoxicity in SACC-83 cells.
Cells were co-treated with ZOL and cisplatin/paclitaxel for 48 h. A Cell viability was examined by Cell Counting Kit-8 assay. B Isobolographic analysis of the cytotoxic effects of ZOL and cisplatin/paclitaxel on SACC-83 cells.