Study Of Anti-tumor Eeffects Of Zoledronic Acid On Human Nasopharyngeal Carcinoma Cell Line In Vitro

Background and ObjectiveNasopharyngeal carcinoma (NPC) is a common malignancy in southern China. Radiotherapy or combined chemoradiotherapy is the main treatment. 5-year survival rate was higher than 80% for patients in early stage treated with radiotherapy alone, while in patients with advanced disease was only 25% to 50%. Local recurrence and distant metastasis remain the main reasons for treatment failure. Bone metastasis is the most common sites of metastases. How to effectively control distant metastasis and improve the local control rate is currently a hot-spot for research.Zoledronic acid, the third generation of bisphosphonates, harbored the most potent effect of anti-reabsorption in the bisphosphonate family. In vitro or in vivo experiments in breast cancer, prostate cancer and myeloma cell lines suggested that zoledronic acid had direct or indirect anti-tumor effects through inhibiting tumor proliferation, inducing cell apoptosis, impairing migration and invasion, modulating immune response mediating by ??T cells and anti-angiogenesis effect. A synergic anti-tumor effect was also found when zoledronic acid combined with a number of conventional chemotherapeutics.Clinical studies about the anti-tumor effects of zoledronic acid had inconsistent results. Austrian Breast and Colorectal Cancer Study Group -12 (ABCSG-12) study showed that zoledronic acid improved disease-free survival and recurrence-free survival in early breast cancer. While the AZURE study turned out that zoledronic acid did not demonstrate survival benefit in breast cancer patients. Zoledronic acid for treatment of NPC, even whether it could act as an anti-tumor agent in nasopharyngeal carcinoma cell line had not been reported yet.In this study, we explored the anti-neoplastic effects of zoledronic acid on human nasopharyngeal carcinoma HNE-1 cell in vitro, focused mainly on cell proliferation, apoptosis, invasion, migration and vascular mimicry, and explored relevant mechanisms. This study aimed at providing a theoretical basis for the application of zoledronic acid in nasopharyngeal carcinoma. MethodsHuman nasopharyngeal carcinoma cell HNE-1 was exposed to various concentrations (0?mol/L -40??mol/L) of zoledronic acid.In part one, we investigated the cytotoxic effect by means of an MTT assay, apoptosis and cell cycle detected by flow cytometry. Morphological change was detected by TUNEL assay. We further explored mRNA expression prolife of Bcl-2, Bax, Bad and Caspase9 with realtime quantitative polymerase chain reaction (RT-Q-PCR) and the protein expression levels by Western blot.In part two, we examined the invasion and migration of HNE-1 cells by Transwell assay, vascular mimicry by tube-like structure formation assay, secretion of vascular endothelial growth factor (VEGF) detected using an ELISA assay, gelatinase activity determined by gelatine zymography. We further analyzed matrix metalloproteinase 2 and 9 (MMP2, MMP9) and VEGF mRNA expressions with reverse transcription polymerase chain reaction (RT-PCR) and the protein expression levels by Western blot.ResultsPart One1 Compared to the control group, different concentrations of ZOL ranged from 2.5?mol/L to 40?mol/L did not show an effect on the proliferation of HNE-1 cells at the time point of 24hr. The anti-proliferative effect was found over a period of incubation of 48hr as well as 72hr. The inhibitive rate of various concentrations of ZOL at 48hr was (3.43±2.46)%, (5.29±4.56)%, (27.25±14.97)%, (32.94±7.54)% and (38.44±11.54)%, respectively. While at 72hr, the inhibitive rate was respectively (8.67±3.22)%, (11.79±0.69)%, (32.58±2.28)%, (39.95±1.95)% and (44.79±2.21)%. But the inhibitive rates were not shown in a dose-dependent or time-dependent manner (P>0.05).2 Different concentrations of ZOL (0?mol/L, 10?mol/L, 20?mol/L, 40?mol/L) were not able to induce apoptosis in HNE-1 cells incubated for 24hr (P>0.05). The rate of early apoptosis was 4.0%, 14.4%, 14.7% and 14.1% over an incubation time of 48hr (P<0.05), and 4.5%, 11.8%, 11.4% and 27.8% over an incubation time of 72hr. (P<0.05).3 After incubated with ZOL for 48hr, HNE-1 cells treated with the concentrations of 20μmol/L and 40μmol/L showed a cell cycle arrest in S phases. But ZOL did not modulate cell cycle in the concentrations of 10μmol/L.4 By the TUNEL method, positive staining was showed as brown granular substance located in the nucleus. Apoptotic cells were mostly round with a small number of cells in irregular shapes. The apoptotic index for different concentrations of ZOL (0μmol/Lμμ10μmol/Lμμ20μmol/Lμμ40μmol/L) was (3.33±4.25) %, (4.45±2.56) %, (5.55±2.87) % and (5.12±3.14) % over an incubation time of 48hr (P>0.05). The apoptotic index over an incubation time of 72hr was respectively (5.00±5.84) %, (11.67±3.33) %, (16.67±5.29) % and (26.11±4.92) % (P<0.05).5 Compared to the control group, the mRNA expression levels in the treated groups were all with a significant change. The value for Bad was 1.77±0.10, 3.04±0.24 and 3.52±0.06 (P<0.05), for Bcl-2 was 1.03±0.07, 0.97±0.11 and 0.33±0.19 (P<0.05), for Caspase9 was 2.59±0.12, 2.63±0.14 and 3.39±0.52 (P<0.05).6 Compared to the control group, the protein expressions of Bax, Bad and Caspase9 in the treated groups were upregulated while Bcl-2 was downregulated. Analyzed by the Gel-Pro Analyzer 4.0 software, the IOD value for Bad was 606.27±34.46, 414.40±3.90, 1218.20±11.41 and 1346.00±18.70 (P<0.05), for Bax was 270.60±17.25, 249.00±15.06, 390.87±22.24 and 573.17±19.04 (P<0.05), for Caspase9 was 731.43±1.80, 1595.53±21.12, 1552.10±64.81 and 1622.80±58.60 (P<0.05), for Bcl-2 was 1175.90±10.86, 1246.53±41.35, 343.43±9.06 and 158.27±6.56 (P<0.05).Part Two1 Treatment of HNE-1 cells for 24hr with different concentrations of ZOL, from 10μmol/L to 40μmol/L, dramatically inhibited cell migration in a dose-dependent manner. Compared with the untreated group, the migrating cell number of treated groups was respectively decreased by (49.80±0.56) %, (77.38±0.25) % and (82.82±1.24) % (P<0.01). 2 Treatment of HNE-1 cells for 24hr with different concentrations of ZOL, from 10μmol/L to 40μmol/L, dramatically inhibited cell invasion in a dose-dependent manner. Compared with the untreated group, the invading cell number of treated groups was respectively decreased by (28.87±1.29) %, (42.93±2.36) % and (53.69±5.13) % (P<0.01).3 After treatment for 48hr with different concentrations of ZOL (0μmol/L, 10μmol/L, 20μmol/L, 40μmol/L), the number of tubules formed by HNE-1 cells was 118.0±9.85, 96.00±3.61, 59.66±14.36 and 42.33±2.23. The inhibitive effect on vascular mimicry was showed in a dose-dependent manner (P<0.05).4 Gelatine zymography showed gelatinase activity of MMP2 and MMP9 was inhibited significantly but only in cells treated under the concentration of 40μmol/L.5 After 24hr exposed to different concentrations (0μmol/L, 10μmol/L, 20μmol/L, 40μmol/L) of ZOL, the secretion of VEGF in the supernatant were (5263.86±89.17) pg/ml, (4626.16±30.26)pg/ml, (4154.65±39.65)pg/ml and (1907.76±171.38) pg/ml (P<0.01). The secretion was significantly inhibited in a dose-dependent manner (P<0.05)6 Compared to the control group, the mRNA expression of VEGF, MMP2 and MMP9 in the treated groups were downregulated to some extent. Analyzed by the Gel-Pro Analyzer 4.0 software, the IOD value for VEGF was 5.17±0.17, 4.72±0.30μμ2.43±0.21and 1.13±0.02 (P<0.05), for MMP2 was 4.82±0.13μμ4.02±0.06, 3.59±0.03 and 2.18±0.02 (P<0.05), for MMP9 was 5.27±0.17, 4.99±0.01, 4.20±0.01 and 2.52±0.04 (P<0.05).7 Compared to the control group, the protein expressions of VEGF, MMP2 and MMP9 in the treated groups were downregulated. Analyzed by the Gel-Pro Analyzer 4.0 software, the IOD value for VEGF was 2155.60±85.13, 1954.33±119.14, 1490.76±82.24 and 1116.30±75.17 (P<0.05), for MMP2 was 511.58±26.27, 161.33±12.60, 164.87±14.12 and 56.41±6.03 (P<0.05), for MMP9 was 353.99±22.81, 184.43±7.23, 93.09±6.65 and 43.31±5.73 (P<0.05).Conclusions1 Zoledronic acid inhibited proliferation, induced apoptosis and modulated cell cycle in HNE-1 cell line in vitro.2 Zoledronic acid induced apoptosis through upregulating genes of Bax, Bad and Caspase9 and downregulating Bcl-2 gene both in the mRNA and protein levels.3 Zoledronic acid inhibited migration, invasion and vascular mimicry of HNE-1 cell line in vitro.4 Zoledronic acid inhibited migration, invasion and vascular mimicry by suppressing the secretion of VEGF, the activity of MMP2 and MMP9 and the expression of VEGF, MMP2 and MMP9.