唑来膦酸体外抗人鼻咽癌细胞作用研究
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摘要
背景与目的
鼻咽癌(Nasopharyngeal carcinoma, NPC)为中国华南地区常见的恶性肿瘤,放疗或联合化放疗是主要治疗手段。早期患者单纯放疗5年生存率高达80%以上,晚期患者5年生存仅25%～50%,局部复发及远处转移仍是治疗失败的主要原因,骨转移是最常见转移部位。如何有效控制远处转移、提高局部控制率是目前研究的热点。
唑来膦酸属于第3代双膦酸盐类药物,在目前临床应用的双膦酸盐中具有最强的抗骨重吸收能力。在乳腺癌、前列腺癌、骨髓瘤等肿瘤细胞系中,体内外试验已证明唑来膦酸能通过抑制肿瘤细胞的增殖、分化,诱导细胞凋亡,降低肿瘤细胞的迁移、侵袭能力,抑制肿瘤新生血管形成,刺激??T淋巴细胞增殖等多种途径直接或间接发挥其抗肿瘤的作用。唑来膦酸与多种化疗药物联合比单用化疗药物可取得更好的抗肿瘤作用。
已有临床研究对唑来膦酸的抗肿瘤作用仍存在争论。奥地利乳腺癌和结直肠癌研究组-12 (The Austrian Breast & Colorectal Cancer Study Group Trial 12, ABCSG-12)的结果表明,唑来膦酸提高早期乳腺癌的无病生存率和无复发生存率。而英国的AZURE研究显示唑来膦酸并没有给乳腺癌患者生存获益,未能提高患者的无病生存率和总生存率。唑来膦酸对鼻咽肿瘤的临床治疗作用,以及对鼻咽癌细胞系的抗癌作用尚未有研究报道。
本课题研究重点主要是通过体外实验,观察唑来膦酸对人低分化鼻咽癌HNE-1细胞系增殖、凋亡诱导,侵袭、迁移和管道形成能力的影响,并探讨相关的作用机制,为进步探索唑来膦酸治疗鼻咽癌提供理论依据。
方法
第一部分唑来膦酸对鼻咽癌HNE-1细胞增殖和凋亡诱导的影响:
1 MTT试验检测不同浓度唑来膦酸对HNE-1细胞的增殖抑制作用;
2流式细胞术检测不同浓度唑来膦酸对HNE-1细胞的凋亡诱导作用;
3流式细胞术检测不同浓度唑来膦酸对HNE-1细胞周期分布的影响作用;
4原位末端标记法(TUNEL)检测不同浓度唑来膦酸对HNE-1细胞凋亡诱导作用;
5实时定量聚合酶链反应(RT-Q-PCR)检测不同浓度唑来膦酸对HNE-1细胞Bcl-2、Bax、Bad和Caspase9 mRNA水平的影响;
6 Western免疫印迹法(Western Blot)检测不同浓度唑来膦酸对HNE-1细胞Bcl-2、Bax、Bad和Caspase9蛋白的影响。
第二部分唑来膦酸对鼻咽癌HNE-1细胞迁移、侵袭和血管拟态形成的影响:
1 Transwell迁移实验检测不同浓度唑来膦酸对HNE-1细胞迁移能力的影响;
2 Transwell侵袭实验检测不同浓度唑来膦酸对HNE-1细胞侵袭能力的影响;
3不同浓度唑来膦酸对HNE-1细胞血管拟态形成的影响;
4明胶酶谱实验检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9活性的影响;
5酶联免疫吸附实验(ELISA)检测不同浓度唑来膦酸对HNE-1细胞释放VEGF的影响;
6逆转录聚合酶链反应(RT-PCR)检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9和VEGF mRNA水平的影响;
7 Western Blot法检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9和VEGF蛋白的影响。
结果
第一部分
1与唑来膦酸对照组(0?mol/L)比较,不同浓度唑来膦酸处理组(2.5?mol/L、5?mol/L、10?mol/L、20?mol/L、40?mol/L)作用24hr,对HNE-1细胞增殖无抑制作用(P>0.05);作用48hr和72hr均能明显抑制HNE-1细胞增殖,其中48hr增殖抑制率分别为(3.43±2.46)%、(5.29±4.56)%、(27.25±14.97)%、(32.94±7.54)%和(38.44±11.54)%, 72hr增殖抑制率分别为(8.67±3.22)%、(11.79±0.69)%、(32.58±2.28)%、(39.95±1.95)%和(44.79±2.21)%,与对照组比较差异均有统计学意义(P<0.05);但抑制增殖能力与药物浓度和作用时间未呈时效和量效关系(P>0.05)。
2 FCM法检测不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞,24hr未能明显诱导细胞凋亡(P>0.05);48hr早期凋亡率分别为4.0%、14.4%、14.7%和14.1%,与对照组比较差异有统计学意义(P<0.05);72hr早期凋亡率分别为4.5%、11.8%、11.4%和27.8%,与对照组比较差异有统计学意义(P<0.05)。
3 FCM法检测检测不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)作用HNE-1细胞48hr,20?mol/L、40?mol/L组HNE-1细胞的S期细胞较对照组比例升高,差异有统计学意义(P<0.01);而10?mol/L组则不明显。
4 TUNEL法染色显示,凋亡细胞核呈棕褐色颗粒,细胞多呈圆形、少数形状不规则。不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)作用HNE-1细胞48hr,凋亡指数分别为(3.33±4.25)%、(4.45±2.56)%、(5.55±2.87)%和(5.12±3.14)%,与对照组比较差异无统计学意义(P>0.05);作用72hr凋亡指数分别为(5.00±5.84)%、(11.67±3.33)%、(16.67±5.29)%和(26.11±4.92)%,与对照组比较差异有统计学意义(P<0.05)。
5 RT-Q-PCR结果显示,与对照组比较,不同浓度(1??mol/L、2??mol/L、4??mol/L)唑来膦酸处理HNE-1细胞48hr,Bad基因的表达量分别为1.77±0.10、3.04±0.24和3.52±0.06;Bcl-2基因的表达量分别为1.03±0.07、0.97±0.11和0.33±0.19;Bax基因的表达量分别为1.61±0.34、3.36±0.36和4.90±1.89;Caspase9基因的表达量分别2.59±0.12、2.63±0.14和3.39±0.52;与对照组比较差异有统计学意义(P<0.05)。
6 Western Blot结果显示,经不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)处理HNE-1细胞48hr,Bax,Bad和Caspase?9的蛋白表达均有不同程度的升高,Bcl-2的蛋白表达则下降。Gel-Pro Analyzer 4.0软件分析结果显示,Bad蛋白表达的IOD值分别为606.27±34.46、414.40±3.90、1218.20±11.41和1346.00±18.70;Bax蛋白表达的IOD值分别为270.60±17.25、249.00±15.06、390.87±22.24和573.17±19.04;Caspase?9蛋白表达的IOD值分别为731.43±1.80、1595.53±21.12、1552.10±64.81和1622.80±58.60;Bcl-2蛋白表达的IOD值分别为1175.90±10.86、1246.53±41.35、343.43±9.06和158.27±6.56;与对照组比较差异均有统计学意义(P<0.05)。
第二部分
1不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,其迁移细胞数分别为121.80±7.09、61.60±10.29、27.80±7.89和19.80±3.35,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05),其穿膜细胞分别降低(49.80±0.56)%、(77.38±0.25)%和(82.82±1.24)%。
2不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,其侵袭细胞数分别为75.80±2.59、54.80±5.36、44.60±6.43和38.60±8.23,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异统计学上也有意义(P<0.05),其穿膜细胞分别降低(28.87±1.29)%、(42.93±2.36)%和(53.69±5.13)%。
3不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞48hr,HNE-1细胞小管状结构形成的数量分别为118.0±9.85、96.00±3.61、59.66±14.36和42.33±2.23,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05)。
4明胶酶谱试验结果显示,40?mol/L唑来膦酸作用HNE-1细胞24hr,抑制MMP2和MMP9的酶活性,但10?mol/L、20?mol/L组则抑制不明显。
5不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr, HNE-1细胞上清中VEGF浓度分别为(5263.86±89.17)pg/ml、(4626.16±30.26)pg/ml、(4154.65±39.65)pg/ml和(1907.76±171.38)pg/ml,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05)。
6 RT-PCR结果显示,不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,VEGF、MMP2、MMP9三者mRNA的表达均有不同程度的降低。Gel-Pro Analyzer 4.0软件分析结果显示,VEGF mRNA表达的IOD值分别为5.17±0.17、4.72±0.30、2.43±0.21和1.13±0.02;MMP2 mRNA表达的IOD值分别为4.82±0.13、4.02±0.06、3.59±0.03和2.18±0.02;MMP9 mRNA表达的IOD值分别为5.27±0.17、4.99±0.01、4.20±0.01和2.52±0.04;与对照组比较,差异均有统计学意义(P<0.05)。
7 Western Blot结果显示,不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,VEGF、MMP2和MMP9蛋白表达均有不同程度的降低。Gel-Pro Analyzer 4.0软件分析结果显示,VEGF蛋白表达的IOD值分别为2155.60±85.13、1954.33±119.14、1490.76±82.24和1116.30±75.17;MMP2蛋白表达的IOD值分别为511.58±26.27、161.33±12.60、164.87±14.12和56.41±6.03;MMP9蛋白表达的IOD值分别为353.99±22.81、184.43±7.23、93.09±6.65和43.31±5.73;与对照组比较,差异均有统计学意义(P<0.05)。
结论
1唑来膦酸体外能抑制人鼻咽癌HNE-1细胞增殖、诱导HNE-1细胞凋亡、干扰HNE-1细胞周期分布;
2唑来膦酸通过下调HNE-1细胞Bcl-2 mRNA和蛋白的表达,上调Bax、Bad和Caspase9 mRNA和蛋白表达,诱导其凋亡。
3唑来膦酸体外能抑制HNE-1细胞的迁移、侵袭和血管拟态形成;
4唑来膦酸通过抑制HNE-1细胞VEGF的分泌,抑制MMP2和MMP9酶活性发挥其抗迁移、侵袭和血管拟态形成作用。
Background and Objective
Nasopharyngeal 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.
Methods
Human 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.
Results
Part One
1 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 Two
1 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).
Conclusions
1 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.
鼻咽癌(Nasopharyngeal carcinoma, NPC)为中国华南地区常见的恶性肿瘤,放疗或联合化放疗是主要治疗手段。早期患者单纯放疗5年生存率高达80%以上,晚期患者5年生存仅25%～50%,局部复发及远处转移仍是治疗失败的主要原因,骨转移是最常见转移部位。如何有效控制远处转移、提高局部控制率是目前研究的热点。
唑来膦酸属于第3代双膦酸盐类药物,在目前临床应用的双膦酸盐中具有最强的抗骨重吸收能力。在乳腺癌、前列腺癌、骨髓瘤等肿瘤细胞系中,体内外试验已证明唑来膦酸能通过抑制肿瘤细胞的增殖、分化,诱导细胞凋亡,降低肿瘤细胞的迁移、侵袭能力,抑制肿瘤新生血管形成,刺激??T淋巴细胞增殖等多种途径直接或间接发挥其抗肿瘤的作用。唑来膦酸与多种化疗药物联合比单用化疗药物可取得更好的抗肿瘤作用。
已有临床研究对唑来膦酸的抗肿瘤作用仍存在争论。奥地利乳腺癌和结直肠癌研究组-12 (The Austrian Breast & Colorectal Cancer Study Group Trial 12, ABCSG-12)的结果表明,唑来膦酸提高早期乳腺癌的无病生存率和无复发生存率。而英国的AZURE研究显示唑来膦酸并没有给乳腺癌患者生存获益,未能提高患者的无病生存率和总生存率。唑来膦酸对鼻咽肿瘤的临床治疗作用,以及对鼻咽癌细胞系的抗癌作用尚未有研究报道。
本课题研究重点主要是通过体外实验,观察唑来膦酸对人低分化鼻咽癌HNE-1细胞系增殖、凋亡诱导,侵袭、迁移和管道形成能力的影响,并探讨相关的作用机制,为进步探索唑来膦酸治疗鼻咽癌提供理论依据。
方法
第一部分唑来膦酸对鼻咽癌HNE-1细胞增殖和凋亡诱导的影响:
1 MTT试验检测不同浓度唑来膦酸对HNE-1细胞的增殖抑制作用;
2流式细胞术检测不同浓度唑来膦酸对HNE-1细胞的凋亡诱导作用;
3流式细胞术检测不同浓度唑来膦酸对HNE-1细胞周期分布的影响作用;
4原位末端标记法(TUNEL)检测不同浓度唑来膦酸对HNE-1细胞凋亡诱导作用;
5实时定量聚合酶链反应(RT-Q-PCR)检测不同浓度唑来膦酸对HNE-1细胞Bcl-2、Bax、Bad和Caspase9 mRNA水平的影响;
6 Western免疫印迹法(Western Blot)检测不同浓度唑来膦酸对HNE-1细胞Bcl-2、Bax、Bad和Caspase9蛋白的影响。
第二部分唑来膦酸对鼻咽癌HNE-1细胞迁移、侵袭和血管拟态形成的影响:
1 Transwell迁移实验检测不同浓度唑来膦酸对HNE-1细胞迁移能力的影响;
2 Transwell侵袭实验检测不同浓度唑来膦酸对HNE-1细胞侵袭能力的影响;
3不同浓度唑来膦酸对HNE-1细胞血管拟态形成的影响;
4明胶酶谱实验检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9活性的影响;
5酶联免疫吸附实验(ELISA)检测不同浓度唑来膦酸对HNE-1细胞释放VEGF的影响;
6逆转录聚合酶链反应(RT-PCR)检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9和VEGF mRNA水平的影响;
7 Western Blot法检测不同浓度唑来膦酸对HNE-1细胞MMP2、MMP9和VEGF蛋白的影响。
结果
第一部分
1与唑来膦酸对照组(0?mol/L)比较,不同浓度唑来膦酸处理组(2.5?mol/L、5?mol/L、10?mol/L、20?mol/L、40?mol/L)作用24hr,对HNE-1细胞增殖无抑制作用(P>0.05);作用48hr和72hr均能明显抑制HNE-1细胞增殖,其中48hr增殖抑制率分别为(3.43±2.46)%、(5.29±4.56)%、(27.25±14.97)%、(32.94±7.54)%和(38.44±11.54)%, 72hr增殖抑制率分别为(8.67±3.22)%、(11.79±0.69)%、(32.58±2.28)%、(39.95±1.95)%和(44.79±2.21)%,与对照组比较差异均有统计学意义(P<0.05);但抑制增殖能力与药物浓度和作用时间未呈时效和量效关系(P>0.05)。
2 FCM法检测不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞,24hr未能明显诱导细胞凋亡(P>0.05);48hr早期凋亡率分别为4.0%、14.4%、14.7%和14.1%,与对照组比较差异有统计学意义(P<0.05);72hr早期凋亡率分别为4.5%、11.8%、11.4%和27.8%,与对照组比较差异有统计学意义(P<0.05)。
3 FCM法检测检测不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)作用HNE-1细胞48hr,20?mol/L、40?mol/L组HNE-1细胞的S期细胞较对照组比例升高,差异有统计学意义(P<0.01);而10?mol/L组则不明显。
4 TUNEL法染色显示,凋亡细胞核呈棕褐色颗粒,细胞多呈圆形、少数形状不规则。不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)作用HNE-1细胞48hr,凋亡指数分别为(3.33±4.25)%、(4.45±2.56)%、(5.55±2.87)%和(5.12±3.14)%,与对照组比较差异无统计学意义(P>0.05);作用72hr凋亡指数分别为(5.00±5.84)%、(11.67±3.33)%、(16.67±5.29)%和(26.11±4.92)%,与对照组比较差异有统计学意义(P<0.05)。
5 RT-Q-PCR结果显示,与对照组比较,不同浓度(1??mol/L、2??mol/L、4??mol/L)唑来膦酸处理HNE-1细胞48hr,Bad基因的表达量分别为1.77±0.10、3.04±0.24和3.52±0.06;Bcl-2基因的表达量分别为1.03±0.07、0.97±0.11和0.33±0.19;Bax基因的表达量分别为1.61±0.34、3.36±0.36和4.90±1.89;Caspase9基因的表达量分别2.59±0.12、2.63±0.14和3.39±0.52;与对照组比较差异有统计学意义(P<0.05)。
6 Western Blot结果显示,经不同浓度唑来膦酸(0?mol/L、10?mol/L、20?mol/L、40?mol/L)处理HNE-1细胞48hr,Bax,Bad和Caspase?9的蛋白表达均有不同程度的升高,Bcl-2的蛋白表达则下降。Gel-Pro Analyzer 4.0软件分析结果显示,Bad蛋白表达的IOD值分别为606.27±34.46、414.40±3.90、1218.20±11.41和1346.00±18.70;Bax蛋白表达的IOD值分别为270.60±17.25、249.00±15.06、390.87±22.24和573.17±19.04;Caspase?9蛋白表达的IOD值分别为731.43±1.80、1595.53±21.12、1552.10±64.81和1622.80±58.60;Bcl-2蛋白表达的IOD值分别为1175.90±10.86、1246.53±41.35、343.43±9.06和158.27±6.56;与对照组比较差异均有统计学意义(P<0.05)。
第二部分
1不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,其迁移细胞数分别为121.80±7.09、61.60±10.29、27.80±7.89和19.80±3.35,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05),其穿膜细胞分别降低(49.80±0.56)%、(77.38±0.25)%和(82.82±1.24)%。
2不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,其侵袭细胞数分别为75.80±2.59、54.80±5.36、44.60±6.43和38.60±8.23,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异统计学上也有意义(P<0.05),其穿膜细胞分别降低(28.87±1.29)%、(42.93±2.36)%和(53.69±5.13)%。
3不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞48hr,HNE-1细胞小管状结构形成的数量分别为118.0±9.85、96.00±3.61、59.66±14.36和42.33±2.23,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05)。
4明胶酶谱试验结果显示,40?mol/L唑来膦酸作用HNE-1细胞24hr,抑制MMP2和MMP9的酶活性,但10?mol/L、20?mol/L组则抑制不明显。
5不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr, HNE-1细胞上清中VEGF浓度分别为(5263.86±89.17)pg/ml、(4626.16±30.26)pg/ml、(4154.65±39.65)pg/ml和(1907.76±171.38)pg/ml,与对照组比较,差异有统计学意义(P<0.01);且不同浓度组间差异也有统计学意义(P<0.05)。
6 RT-PCR结果显示,不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,VEGF、MMP2、MMP9三者mRNA的表达均有不同程度的降低。Gel-Pro Analyzer 4.0软件分析结果显示,VEGF mRNA表达的IOD值分别为5.17±0.17、4.72±0.30、2.43±0.21和1.13±0.02;MMP2 mRNA表达的IOD值分别为4.82±0.13、4.02±0.06、3.59±0.03和2.18±0.02;MMP9 mRNA表达的IOD值分别为5.27±0.17、4.99±0.01、4.20±0.01和2.52±0.04;与对照组比较,差异均有统计学意义(P<0.05)。
7 Western Blot结果显示,不同浓度(0?mol/L、10?mol/L、20?mol/L、40?mol/L)唑来膦酸处理HNE-1细胞24hr,VEGF、MMP2和MMP9蛋白表达均有不同程度的降低。Gel-Pro Analyzer 4.0软件分析结果显示,VEGF蛋白表达的IOD值分别为2155.60±85.13、1954.33±119.14、1490.76±82.24和1116.30±75.17;MMP2蛋白表达的IOD值分别为511.58±26.27、161.33±12.60、164.87±14.12和56.41±6.03;MMP9蛋白表达的IOD值分别为353.99±22.81、184.43±7.23、93.09±6.65和43.31±5.73;与对照组比较,差异均有统计学意义(P<0.05)。
结论
1唑来膦酸体外能抑制人鼻咽癌HNE-1细胞增殖、诱导HNE-1细胞凋亡、干扰HNE-1细胞周期分布;
2唑来膦酸通过下调HNE-1细胞Bcl-2 mRNA和蛋白的表达,上调Bax、Bad和Caspase9 mRNA和蛋白表达,诱导其凋亡。
3唑来膦酸体外能抑制HNE-1细胞的迁移、侵袭和血管拟态形成;
4唑来膦酸通过抑制HNE-1细胞VEGF的分泌,抑制MMP2和MMP9酶活性发挥其抗迁移、侵袭和血管拟态形成作用。
Background and Objective
Nasopharyngeal 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.
Methods
Human 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.
Results
Part One
1 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 Two
1 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).
Conclusions
1 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.
引文
[1]万德森主编.临床肿瘤学.北京:科学出版社. 2005,1090.
[2] Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2006, 15(10):1765-1777. PMID:17035381
[3] Arango BA, Castrellon AB, Perez CA, et al. Nasopharyngeal carcinoma: alternative treatment options after disease progression. Expert Rev Anticancer Ther. 2010, 10(3): 377-386. PMID: 20214519
[4] Caponigro F, Longo F, Ionna F, et al. Treatment approaches to nasopharyngeal carcinoma: a review. Anticancer Drugs. 2010, 21(5): 471-477. PMID: 20124988
[5] Razak AR, Siu LL, Liu FF, et al. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer. 2010, 46(11): 1967-1978. PMID: 20451372
[6] Bensouda Y, Kaikani W, Ahbeddou N, et al. Treatment for metastatic nasopharyngeal carcinoma. Eur Ann Otorhinolaryngol Head Neck Dis. [Epub ahead of print]. PMID: 21177151. (doi:10.1016/j.anorl.2010.10.003)
[7] Grewal VS, Fayans EP. Bisphosphonate-associated osteonecrosis: a clinician's reference to patient management. Todays FDA. 2008, 20(8):38-41, 43-46. PMID: 18767452
[8] Markose G, Mackenzie FR, Currie WJ, et al. Bisphosphonate osteonecrosis: a protocol for surgical management. Br J Oral Maxillofac Surg. 2009, 47(4):294-297. PMID: 19231046
[9] Russell RG. Bisphosphonates: mode of action and pharmacology. Pediatrics.2007,119( Suppl 2):S150-162. PMID: 17332236
[10] Rogers MJ, Gordon S, Benford HL, et al. Cellular and molecular mechanisms of action of bisphosphonates. Cancer. 2000, 88(12 Suppl):2961-2978. PMID: 10898340
[11] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007, 14(20):2126-2135. PMID: 17691952
[12] Green JR. Bisphosphonates: Preclinical Review. Oncologist. 2004, 9(Suppl 4):3-13. PMID: 15459425
[13] Montague R, Hart CA, George NJ, et al. Differential inhibition of invasion and proliferation by bisphosphonates: anti-metastatic potential of Zoledronic acid in prostate cancer. Eur Urol. 2004, 46(3):389-340. PMID: 15306113
[14] Boissier S, Ferreras M, Peyruchaud O, et al. Bisphosphonates Inhibit Breast and Prostate Carcinoma Cell Invasion, an Early Event in the Formation of Bone Metastases. Cancer Res. 2000, 60(11):2949-2954. PMID: 10850442
[15] Yamada J, Tsuno NH, Kitayama J, et al. Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation. J Surg Res. 2009, 151(1):115-120. PMID: 18619615
[16] Hasmim M, Bieler G, Rüegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost. 2007, 5(1):166-173. PMID: 17059425
[17] Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology. Cancer Res. 2007, 67(1):5-8. PMID: 17210676
[18] Sato K, Kimura S, Segawa H, et al. Cytotoxic effects of gammadelta T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy. Int J Cancer. 2005, 116(1):94-99. PMID: 15756684
[19] Ural AU, Avcu F, Candir M, et al.In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52. PMID: 16925824
[20] Chang JW, Hsieh JJ, Shen YC, et al. Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells. Cancer Lett. 2009, 278(1):17-26. PMID: 19233551
[21] M?nkk?nen H, Auriola S, Lehenkari P, et al. A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates. Br J Pharmacol. 2006, 147(4):437-445. PMID: 16402039
[22] Goffinet M, Thoulouzan M, Pradines A, et al. Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells. BMC Cancer. 2006, 6:60. PMID: 16539721
[23] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects.Br J Cancer. 2003, 88(10):1631-1640. PMID: 12771933
[24] R?ikk?nen J, M?nkk?nen H, Auriola S, et al. Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells. Biochem Pharmacol. 2010, 79(5):777-783. PMID: 19819230
[25] Li YY, Chang JW, Liu YC, Wang CH,et al. Zoledronic acid induces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression. Anticancer Drugs. 2011, 22(1):89-98. PMID: 20926944
[26] Moriceau G, Ory B, Mitrofan L, et al. Zoledronic acid potentiates mTOR inhibition and abolishes the resistance of osteosarcoma cells to RAD001 (Everolimus): pivotal role of the prenylation process. Cancer Res. 2010, 70(24):10329-10339. PMID: 20971812
[27] Zarogoulidis K, Boutsikou E, Zarogoulidis P, et al. The impact of zoledronic acid therapy in survival of lung cancer patients with bone metastasis. Int J Cancer. 2009, 125(7): 1705-1709. PMID: 19521984
[28] Morgan GJ, Davies FE, Gregory WM, et al. First-line treatment with zoledronic acid as compared with clodronic acid in multiple myeloma (MRC Myeloma IX): a randomised controlled trial. Lancet. 2010, 376(9757):1989-1999. PMID: 21131037
[29] Moschetta M, Di Pietro G, Ria R, et al. Bortezomib and zoledronic acid on angiogenic and vasculogenic activities of bone marrow macrophages in patients with multiple myeloma. Eur J Cancer. 2010, 46(2):420-429. PMID: 19914061
[30] Saad F, Gleason DM, Murray R, et al. A randomized, placebo-controlled trial of zoledronicacid in patients with hormone-refractory metastatic prostate carcinoma. J Natl Cancer Inst. 2002, 94(19):1458-1468. PMID: 12359855
[31] Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med. 2009, 360(7):679-691. PMID: 19213681
[32] Eidtmann H, de Boer R, Bundred N, et al. Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study. Ann Oncol. 2010, 21(11):2188-2194. PMID: 20444845
[33] Coleman RE,Thorpe HC,Cameron D,et al.Adjuvant Treatment with Zoledronic Acid in Stage II/III Breast Cancer. The AZURE Trial (BIG 01/04).SABCS 2010; Abstract S4-5.
[34] Pandya KJ, Gajra A, Warsi GM, et al.Multicenter, randomized, phase 2 study of zoledronic acid in combination with docetaxel and carboplatin in patients with unresectable stage IIIB or stage IV non-small cell lung cancer. Lung Cancer. 2010, 67(3):330-338. PMID: 19493585
[1] Jagdev SP, Coleman RE, Shipman CM, et al. The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer. 2001, 84(8):1126-1134. PMID: 11308265
[2] Zwolak P, Manivel JC, Jasinski P, et al. Cytotoxic effect of zoledronic acid-loaded bone cement on giant cell tumor, multiple myeloma, and renal cell carcinoma cell lines. J Bone Joint Surg Am. 2010, 92(1):162-168.PMID: 20048108
[3] Clyburn RD, Reid P, Evans CA, et al. Increased anti-tumour effects of doxorubicin and zoledronic acid in prostate cancer cells in vitro: supporting the benefits of combination therapy.Cancer Chemother Pharmacol. 2010, 65(5):969-978.PMID: 19730863
[4] Matsumoto S, Kimura S, Segawa H, et al. Efficacy of the third-generation bisphosphonate, zoledronic acid alone and combined with anti-cancer agents against small cell lung cancer cell lines.Lung Cancer. 2005, 47(1):31-39. PMID: 15603852
[5] Karabulut B, Karaca B, Varol U, et al. Enhancing cytotoxic and apoptotic effect in OVCAR-3 and MDAH-2774 cells with all-trans retinoic acid and zoledronic acid: a paradigm of synergistic molecular targeting treatment for ovarian cancer. J Exp Clin Cancer Res. 2010, 29(1):102. PMID: 20673323
[6] M?rten A, Lilienfeld-Toal M, Büchler MW, et al. Zoledronic acid has direct antiproliferative and antimetastatic effect on pancreatic carcinoma cells and acts as an antigen for delta2 gamma/delta T cells. J Immunother. 2007, 30(4):370-377. PMID: 17457212
[7] Chuah C, Barnes DJ, Kwok M, et al. Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells. Leukemia. 2005, 19(11): 1896-1904. PMID: 16167056
[8] Koto K, Murata H, Kimura S, et al.Zoledronic acid inhibits proliferation of human fibrosarcoma cells with induction of apoptosis, and shows combined effects with otheranticancer agents.Oncol Rep. 2010, 24(1):233-239. PMID: 20514467
[9] Algur E, Macklis RM, H?feli UO.Synergistic cytotoxic effects of zoledronic acid and radiation in human prostate cancer and myeloma cell lines.Int J Radiat Oncol Biol Phys. 2005, 61(2):535-542. PMID: 15667977
[10] Ural AU, Avcu F, Candir M, et al. In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52. PMID: 16925824
[11] Neville-Webbe HL, Coleman RE, Holen I. Combined effects of the bisphosphonate, zoledronic acid and the aromatase inhibitor letrozole on breast cancer cells in vitro: evidence of synergistic interaction.Br J Cancer. 2010, 102(6):1010-1017. PMID: 20160726
[12] Rachner TD, Singh SK, Schoppet M, et al. Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells. Cancer Lett. 2010, 287(1):109-116. PMID: 19577359
[13] Li YY, Chang JW, Chou WC, et al. Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival For non-small-cell lung cancers. Lung Cancer. 2008, 59(2): 180-191. PMID: 17900752
[14] Ory B, Blanchard F, Battaglia S, et al. Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status. Mol Pharmacol. 2007, 71(1):333-343. PMID: 17050806
[15] Almubarak H, Jones A, Chaisuparat R, et al. Zoledronic acid directly suppresses cell proliferation and induces apoptosis in highly tumorigenic prostate and breast cancers. J Carcinog. 2011, 10:2.PMID: 21297922
[16]张卫东,韩轶鹏,宗志红,等.唑来膦酸对肺癌95D细胞周期阻滞和诱导凋亡的作用机制.中华肿瘤杂志. 2009, 31(6):418-422.
[17] Iguchi T, Miyakawa Y, Saito K, et al. Zoledronate-induced S phase arrest and apoptosis accompanied by DNA damage and activation of the ATM/Chk1/cdc25 pathway in human osteosarcoma cells. Int J Oncol. 2007, 31(2): 285-291. PMID: 17611684
[18] Romani AA, Desenzani S, Morganti MM, et al. Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells.Biochem Pharmacol. 2009, 78(2):133-141. PMID: 19464430
[19] Silva KL, Vasconcellos DV, Castro ED, et al. Bisphosphonates induce apoptosis in CLL cells independently of MDR phenotype.Cancer Chemother Pharmacol. 2008, 62(1):165-171. PMID: 18335218
[20] Rachner TD, Singh SK, Schoppet M, et al. Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells.Cancer Lett. 2010, 287(1):109-116. PMID: 19577359
[21] Kawata E, Ashihara E, Nakagawa Y,et al. A combination of a DNA-chimera siRNA against PLK-1 and zoledronic acid suppresses the growth of malignant mesothelioma cells in vitro.Cancer Lett. 2010,294(2):245-253. PMID: 20206440.
[22] Corso A, Ferretti E, Lunghi M, et al.Zoledronic acid down-regulates adhesion molecules of bone marrow stromal cells in multiple myeloma: a possible mechanism for its antitumor effect. Cancer. 2005,104(1):118-125.PMID: 15895374.
[23] Marra M, Santini D, Meo G,et al. Cyr61 downmodulation potentiates the anticancer effects of zoledronic acid in androgen-independent prostate cancer cells. Int J Cancer. 2009,125(9):2004-2013.PMID: 19530242.
[24] Li YY, Chang JW, Liu YC,et al.Zoledronic acid induces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression. Anticancer Drugs. 2011,22(1):89-98.PMID: 20926944.
[25] Constantinou C, Papas KA, Constantinou AI. Caspase-independent pathways of programmed cell death: the unraveling of new targets of cancer therapy? Curr Cancer Drug Targets. 2009, 9(6): 717-728.PMID: 19754356
[26] Mitrofan LM, Castells FB, Pelkonen J, et al. Lysosomal-mitochondrial axis in zoledronic acid-induced apoptosis in human follicular lymphoma cells. J Biol Chem. 2010, 285(3): 1967-1979. PMID: 19875454
[27] Ullen A, Schwarz S, Lennartsson L, et al. Zoledronic acid induces caspase-dependent apoptosis in renal cancer cell lines. Scand J Urol Nephrol. 2009, 43(2): 98-103. PMID: 19101859
[28] Caraglia M, Marra M, Leonetti C, et al. R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Aktinactivation and reduced Bcl-2 and bad phosphorylation. J Cell Physiol. 2007, 211(2): 533-543. PMID: 17192846
[29] Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells. Cell Biol Int. 2009,33(2):239-246.PMID: 19103299
[30] Sewing L, Steinberg F, Schmidt H, et al. The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis. Apoptosis. 2008, 13(6): 782-789.PMID: 18437576
[1] Kingsley LA, Fournier PG, Chirgwin JM, et al. Molecular biology of bone metastasis. Mol Cancer Ther. 2007, 6(10):2609-2617. PMID: 17938257
[2] Arango BA, Castrellon AB, Perez CA, et al. Nasopharyngeal carcinoma: alternative treatment options after disease progression. Expert Rev Anticancer Ther. 2010, 10(3): 377-386. PMID: 20214519
[3] Caponigro F, Longo F, Ionna F, et al. Treatment approaches to nasopharyngeal carcinoma: a review. Anticancer Drugs. 2010, 21(5): 471-477. PMID: 20124988
[4] Razak AR, Siu LL, Liu FF, et al. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer. 2010, 46(11): 1967-1978. PMID: 20451372
[5] Chiang AC, MassaguéJ. Molecular basis of metastasis. N Engl J Med. 2008, 359(26): 2814-2823. PMID: 19109576
[6] Harel R, Angelov L. Spine metastases: current treatments and future directions. Eur J Cancer. 2010, 46(15): 2696-2707. PMID: 20627705
[7] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007, 14(20):2126-2135. PMID: 17691952
[8] Aft R, Naughton M, Trinkaus K, et al. Effect of zoledronic acid on disseminated tumour cells in women with locally advanced breast cancer: an open label, randomised, phase 2 trial. Lancet Oncol. 2010, 11(5): 421-428. PMID: 20362507
[9] Rack B, Jückstock J, Genss EM, et al. Effect of zoledronate on persisting isolated tumour cells in patients with early breast cancer. Anticancer Res. 2010, 30(5): 1807-1813. PMID: 20592383
[10] Haddad RI, Shin DM. Recent advances in head and neck cancer. N Engl J Med. 2008, 359(11):1143-1154.PMID: 18784104
[11] Vermorken JB, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med. 2008, 359(11): 1116-1127. PMID: 18784101
[12] Facchini G, Caraglia M, Morabito A, et al. Metronomic administration of zoledronic acidand taxotere combination in castration resistant prostate cancer patients: Phase I ZANTE trial. Cancer Biol Ther. 2010, 10(6):543-548. PMID: 20657175
[13]董梅,冯奉仪,张阳,等.唑来膦酸治疗实体瘤骨转移或多发性骨髓瘤中重度骨痛的Ⅲ期临床研究.中华肿瘤杂志. 2008, 30(3):215-220.
[14] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects. Br J Cancer. 2003, 88(10): 1631-1640. PMID: 12771933
[15] Montague R, Hart CA, George NJ, et al. Differential inhibition of invasion and proliferation by bisphosphonates: anti-metastatic potential of Zoledronic acid in prostate cancer. Eur Urol. 2004, 46(3):389-401. PMID: 15306113
[16] Boissier S, Ferreras M, Peyruchaud O, et al. Bisphosphonates Inhibit Breast and Prostate Carcinoma Cell Invasion, an Early Event in the Formation of Bone Metastases Cancer Res. 2000, 60(11): 2949-2454. PMID: 10850442
[17] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010, 141(1):52-67. PMID: 20371345
[18] Gialeli C, Theocharis AD, Karamanos NK. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011, 278(1):16-27. PMID: 21087457
[19] Radisky ES, Radisky DC.Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer.J Mammary Gland Biol Neoplasia. 2010, 15(2):201-212.PMID: 20440544
[20]李明明,张卫东,宗志红,等.唑来膦酸对肺癌95D细胞增殖和侵袭抑制.中国肺癌杂志, 2009, 12(3):198-202.
[21]成伟,周维国.喉癌组织中HIF-1α和COX-2及VEGF与微血管密度的关系及临床意义.临床耳鼻咽喉头颈外科杂志. 2010, 24(18): 820-822.
[22] Shibata MA, Ambati J, Shibata E, et al. The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model. BMC Med. 2010, 8:69. PMID: 21047425
[23] Soltau J, Zirrgiebel U, Esser N, et al. Antitumoral and antiangiogenic efficacy of bisphosphonates in vitro and in a murine RENCA model. Anticancer Res. 2008, 28(2A):933-941. PMID: 18507039.
[24] Santini D,Vincenzi B, Galluzzo S,et al. Repeated Intermittent Low-Dose Therapy with Zoledronic Acid Induces an Early, Sustained, and Long-Lasting Decrease of Peripheral Vascular Endothelial Growth Factor Levels in Cancer Patients. Clin Cancer Res. 2007, 13(15 pt 1): 4482-4486. PMID: 17671133
[25] Folkman J: Clinical applications of research on angiogenesis.Seminars in Medicine of the Beth Israel Hospital, Boston.New Engl J Med.1995, 333(26):1757-1763.PMID:7491141
[26] Maniotis AJ, Folberg R, Hess A, et al.Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol. 1999,155(3):739-752. PMID:10487832
[27]王鸿彪,林英城,曾德,等.人参皂甙Rg3体外抗鼻咽癌HNE-1细胞管道形成的作用.中华肿瘤杂志,2010,32(10):739-742
[28] Wang JY, Sun T, Zhao XL, et al.Functional significance of VEGF-a in human ovarian carcinoma: role in vasculogenic mimicry.Cancer Biol Ther. 2008,7(5):758-766. PMID:18376140
[29] Basu GD, Liang WS, Stephan DA, et al.A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells.Breast Cancer Res. 2006,8(6):R69.PMID:17156488
[30] Mei J, Gao Y, Zhang L, et al.VEGF-siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vitro.Exp Oncol. 2008,30(1):29-34.PMID:18438338
[1] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007,14(20):2126-2135.
[2] Clézardin P, Ebetino FH, Fournier PG. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res.2005, 65(12):4971-4974.
[3] M?nkk?nen H, Auriola S, Lehenkari P, et al. A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates.Br J Pharmacol.2006, 147(4):437-445.
[4] Caraglia M, Marra M, Leonetti C, et al. R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Akt inactivation and reduced Bcl-2 and bad phosphorylation.J Cell Physiol. 2007,211(2):533-543.
[5] Goffinet M, Thoulouzan M, Pradines A,et al. Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells.BMC Cancer. 2006,6:60.
[6] Kubista B, Trieb K, Sevelda F, et al. Anticancer Effects of Zoledronic Acid against Human Osteosarcoma Cells.J Orthop Res.2006,24(6):1145-1152.
[7] Ory B, Blanchard F, Battaglia S, et al. Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status. Mol Pharmacol. 2007, 71(1):333-343.
[8] Green JR. Bisphosphonates: Preclinical Review. Oncologist.2004, 9 Suppl 4:3-13.
[9] Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells. Cell Biol Int.2009, 33(2):239-246.
[10] Fabbri F, Brigliadori G, Carloni S,et al. Zoledronic acid increases docetaxel cytotoxicity through pMEK and Mcl-1 inhibition in a hormone-sensitive prostate carcinoma cell line. J Transl Med. 2008,6:43
[11] Brubaker KD, Brown LG, Vessella RL, et al. Administration of zoledronic acid enhances the effects of docetaxel on growth of prostate cancer in the bone environment. BMC Cancer. 2006,6:15
[12] Ottewell PD, Deux B, M?nkk?nen H, et al.Differential Effect of Doxorubicin and Zoledronic Acid on Intraosseous versus Extraosseous Breast Tumor Growth In vivo.Clin Cancer Res. 2008,14(14):4658-4666.
[13] Lu S, Zhang J, Zhou Z, et al. Synergistic inhibitory activity of zoledronate and paclitaxel on bone metastasis in nude mice.Oncol Rep.2008,20(3):581-587.
[14] Ural AU, Avcu F, Candir M, et al.In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52.
[15] Chang JW, Hsieh JJ, Shen YC, et al. Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells. Cancer Lett.2009, 278(1):17-26.
[16] Winter MC, Holen I, Coleman RE. Exploring the anti-tumour activity of bisphosphonates in early breast cancer. Cancer Treat Rev. 2008, 34(5):453-475.
[17] Boissier S, Ferreras M, Peyruchaud o, et al. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases.Cancer Res. 2000,60(11):2949-2954.
[18] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects.Br J Cancer.2003, 88(10):1631-1640.
[19] Leto G, Badalamenti G, Arcara C, et al. Effects of zoledronic acid on proteinase plasma levels in patients with bone metastases.Anticancer Res. 2006, 26(1A):23-26.
[20] Kingsley LA, Fournier PG, Chirgwin JM, et al.Molecular biology of bone metastasis.Mol Cancer Ther. 2007,6(10):2609-2617.
[21] Viereck V, Emons G, Lauck V, et al. Bisphosphonates Pamidronate and Zoledronic Acid Stimulate Osteoprotegerin Production by Primary Human osteoblasts.Biochem Biophys Res Commun.2002,291(3):680-686.
[22] Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology.Cancer Res, 2007.67(1):5-8.
[23] Sato K, Kimura S, Segawa H, et al. Cytotoxic effects of gammadelta T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy.Int J Cancer. 2005, 116(1):94-99.
[24] Bezzi M,Hasmim M,Bieler G, et al. Zoledronate Sensitizes Endothelial Cells to Tumor Necrosis Factor-induced Programmed Cell Death.J Biol Chem. 2003, 31,278(44): 43603- 43614.
[25] Yamada J, Tsuno NH, Kitayama J, et al. Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation.J Surg Res.2009, 151(1):115-120.
[26] Hasmim M, Bieler G, Rüegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost.2007, 5(1):166-173.
[27] Ferretti G, Fabi A, Carlini P, et al. Zoledronic-acid-induced circulating level modifications of angiogenic factors, metalloproteinases and proinflammatory cytokines in metastatic breast cancer patients. Oncology. 2005, 69(1):35-43.
[28] Powles T, Paterson A, McCloskey E, et al. Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer [ISRCTN83688026].Breast Cancer Res.2006,8(2):R13.
[29] Ha TC, Li H. Meta-analysis of clodronate and breast cancer survival.Br J Cancer. 2007, 96(12):1796-1801.
[30] Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer.N Engl J Med.2009,360(7):679-691.
[2] Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2006, 15(10):1765-1777. PMID:17035381
[3] Arango BA, Castrellon AB, Perez CA, et al. Nasopharyngeal carcinoma: alternative treatment options after disease progression. Expert Rev Anticancer Ther. 2010, 10(3): 377-386. PMID: 20214519
[4] Caponigro F, Longo F, Ionna F, et al. Treatment approaches to nasopharyngeal carcinoma: a review. Anticancer Drugs. 2010, 21(5): 471-477. PMID: 20124988
[5] Razak AR, Siu LL, Liu FF, et al. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer. 2010, 46(11): 1967-1978. PMID: 20451372
[6] Bensouda Y, Kaikani W, Ahbeddou N, et al. Treatment for metastatic nasopharyngeal carcinoma. Eur Ann Otorhinolaryngol Head Neck Dis. [Epub ahead of print]. PMID: 21177151. (doi:10.1016/j.anorl.2010.10.003)
[7] Grewal VS, Fayans EP. Bisphosphonate-associated osteonecrosis: a clinician's reference to patient management. Todays FDA. 2008, 20(8):38-41, 43-46. PMID: 18767452
[8] Markose G, Mackenzie FR, Currie WJ, et al. Bisphosphonate osteonecrosis: a protocol for surgical management. Br J Oral Maxillofac Surg. 2009, 47(4):294-297. PMID: 19231046
[9] Russell RG. Bisphosphonates: mode of action and pharmacology. Pediatrics.2007,119( Suppl 2):S150-162. PMID: 17332236
[10] Rogers MJ, Gordon S, Benford HL, et al. Cellular and molecular mechanisms of action of bisphosphonates. Cancer. 2000, 88(12 Suppl):2961-2978. PMID: 10898340
[11] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007, 14(20):2126-2135. PMID: 17691952
[12] Green JR. Bisphosphonates: Preclinical Review. Oncologist. 2004, 9(Suppl 4):3-13. PMID: 15459425
[13] Montague R, Hart CA, George NJ, et al. Differential inhibition of invasion and proliferation by bisphosphonates: anti-metastatic potential of Zoledronic acid in prostate cancer. Eur Urol. 2004, 46(3):389-340. PMID: 15306113
[14] Boissier S, Ferreras M, Peyruchaud O, et al. Bisphosphonates Inhibit Breast and Prostate Carcinoma Cell Invasion, an Early Event in the Formation of Bone Metastases. Cancer Res. 2000, 60(11):2949-2954. PMID: 10850442
[15] Yamada J, Tsuno NH, Kitayama J, et al. Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation. J Surg Res. 2009, 151(1):115-120. PMID: 18619615
[16] Hasmim M, Bieler G, Rüegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost. 2007, 5(1):166-173. PMID: 17059425
[17] Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology. Cancer Res. 2007, 67(1):5-8. PMID: 17210676
[18] Sato K, Kimura S, Segawa H, et al. Cytotoxic effects of gammadelta T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy. Int J Cancer. 2005, 116(1):94-99. PMID: 15756684
[19] Ural AU, Avcu F, Candir M, et al.In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52. PMID: 16925824
[20] Chang JW, Hsieh JJ, Shen YC, et al. Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells. Cancer Lett. 2009, 278(1):17-26. PMID: 19233551
[21] M?nkk?nen H, Auriola S, Lehenkari P, et al. A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates. Br J Pharmacol. 2006, 147(4):437-445. PMID: 16402039
[22] Goffinet M, Thoulouzan M, Pradines A, et al. Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells. BMC Cancer. 2006, 6:60. PMID: 16539721
[23] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects.Br J Cancer. 2003, 88(10):1631-1640. PMID: 12771933
[24] R?ikk?nen J, M?nkk?nen H, Auriola S, et al. Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells. Biochem Pharmacol. 2010, 79(5):777-783. PMID: 19819230
[25] Li YY, Chang JW, Liu YC, Wang CH,et al. Zoledronic acid induces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression. Anticancer Drugs. 2011, 22(1):89-98. PMID: 20926944
[26] Moriceau G, Ory B, Mitrofan L, et al. Zoledronic acid potentiates mTOR inhibition and abolishes the resistance of osteosarcoma cells to RAD001 (Everolimus): pivotal role of the prenylation process. Cancer Res. 2010, 70(24):10329-10339. PMID: 20971812
[27] Zarogoulidis K, Boutsikou E, Zarogoulidis P, et al. The impact of zoledronic acid therapy in survival of lung cancer patients with bone metastasis. Int J Cancer. 2009, 125(7): 1705-1709. PMID: 19521984
[28] Morgan GJ, Davies FE, Gregory WM, et al. First-line treatment with zoledronic acid as compared with clodronic acid in multiple myeloma (MRC Myeloma IX): a randomised controlled trial. Lancet. 2010, 376(9757):1989-1999. PMID: 21131037
[29] Moschetta M, Di Pietro G, Ria R, et al. Bortezomib and zoledronic acid on angiogenic and vasculogenic activities of bone marrow macrophages in patients with multiple myeloma. Eur J Cancer. 2010, 46(2):420-429. PMID: 19914061
[30] Saad F, Gleason DM, Murray R, et al. A randomized, placebo-controlled trial of zoledronicacid in patients with hormone-refractory metastatic prostate carcinoma. J Natl Cancer Inst. 2002, 94(19):1458-1468. PMID: 12359855
[31] Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med. 2009, 360(7):679-691. PMID: 19213681
[32] Eidtmann H, de Boer R, Bundred N, et al. Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study. Ann Oncol. 2010, 21(11):2188-2194. PMID: 20444845
[33] Coleman RE,Thorpe HC,Cameron D,et al.Adjuvant Treatment with Zoledronic Acid in Stage II/III Breast Cancer. The AZURE Trial (BIG 01/04).SABCS 2010; Abstract S4-5.
[34] Pandya KJ, Gajra A, Warsi GM, et al.Multicenter, randomized, phase 2 study of zoledronic acid in combination with docetaxel and carboplatin in patients with unresectable stage IIIB or stage IV non-small cell lung cancer. Lung Cancer. 2010, 67(3):330-338. PMID: 19493585
[1] Jagdev SP, Coleman RE, Shipman CM, et al. The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer. 2001, 84(8):1126-1134. PMID: 11308265
[2] Zwolak P, Manivel JC, Jasinski P, et al. Cytotoxic effect of zoledronic acid-loaded bone cement on giant cell tumor, multiple myeloma, and renal cell carcinoma cell lines. J Bone Joint Surg Am. 2010, 92(1):162-168.PMID: 20048108
[3] Clyburn RD, Reid P, Evans CA, et al. Increased anti-tumour effects of doxorubicin and zoledronic acid in prostate cancer cells in vitro: supporting the benefits of combination therapy.Cancer Chemother Pharmacol. 2010, 65(5):969-978.PMID: 19730863
[4] Matsumoto S, Kimura S, Segawa H, et al. Efficacy of the third-generation bisphosphonate, zoledronic acid alone and combined with anti-cancer agents against small cell lung cancer cell lines.Lung Cancer. 2005, 47(1):31-39. PMID: 15603852
[5] Karabulut B, Karaca B, Varol U, et al. Enhancing cytotoxic and apoptotic effect in OVCAR-3 and MDAH-2774 cells with all-trans retinoic acid and zoledronic acid: a paradigm of synergistic molecular targeting treatment for ovarian cancer. J Exp Clin Cancer Res. 2010, 29(1):102. PMID: 20673323
[6] M?rten A, Lilienfeld-Toal M, Büchler MW, et al. Zoledronic acid has direct antiproliferative and antimetastatic effect on pancreatic carcinoma cells and acts as an antigen for delta2 gamma/delta T cells. J Immunother. 2007, 30(4):370-377. PMID: 17457212
[7] Chuah C, Barnes DJ, Kwok M, et al. Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells. Leukemia. 2005, 19(11): 1896-1904. PMID: 16167056
[8] Koto K, Murata H, Kimura S, et al.Zoledronic acid inhibits proliferation of human fibrosarcoma cells with induction of apoptosis, and shows combined effects with otheranticancer agents.Oncol Rep. 2010, 24(1):233-239. PMID: 20514467
[9] Algur E, Macklis RM, H?feli UO.Synergistic cytotoxic effects of zoledronic acid and radiation in human prostate cancer and myeloma cell lines.Int J Radiat Oncol Biol Phys. 2005, 61(2):535-542. PMID: 15667977
[10] Ural AU, Avcu F, Candir M, et al. In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52. PMID: 16925824
[11] Neville-Webbe HL, Coleman RE, Holen I. Combined effects of the bisphosphonate, zoledronic acid and the aromatase inhibitor letrozole on breast cancer cells in vitro: evidence of synergistic interaction.Br J Cancer. 2010, 102(6):1010-1017. PMID: 20160726
[12] Rachner TD, Singh SK, Schoppet M, et al. Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells. Cancer Lett. 2010, 287(1):109-116. PMID: 19577359
[13] Li YY, Chang JW, Chou WC, et al. Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival For non-small-cell lung cancers. Lung Cancer. 2008, 59(2): 180-191. PMID: 17900752
[14] Ory B, Blanchard F, Battaglia S, et al. Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status. Mol Pharmacol. 2007, 71(1):333-343. PMID: 17050806
[15] Almubarak H, Jones A, Chaisuparat R, et al. Zoledronic acid directly suppresses cell proliferation and induces apoptosis in highly tumorigenic prostate and breast cancers. J Carcinog. 2011, 10:2.PMID: 21297922
[16]张卫东,韩轶鹏,宗志红,等.唑来膦酸对肺癌95D细胞周期阻滞和诱导凋亡的作用机制.中华肿瘤杂志. 2009, 31(6):418-422.
[17] Iguchi T, Miyakawa Y, Saito K, et al. Zoledronate-induced S phase arrest and apoptosis accompanied by DNA damage and activation of the ATM/Chk1/cdc25 pathway in human osteosarcoma cells. Int J Oncol. 2007, 31(2): 285-291. PMID: 17611684
[18] Romani AA, Desenzani S, Morganti MM, et al. Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells.Biochem Pharmacol. 2009, 78(2):133-141. PMID: 19464430
[19] Silva KL, Vasconcellos DV, Castro ED, et al. Bisphosphonates induce apoptosis in CLL cells independently of MDR phenotype.Cancer Chemother Pharmacol. 2008, 62(1):165-171. PMID: 18335218
[20] Rachner TD, Singh SK, Schoppet M, et al. Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells.Cancer Lett. 2010, 287(1):109-116. PMID: 19577359
[21] Kawata E, Ashihara E, Nakagawa Y,et al. A combination of a DNA-chimera siRNA against PLK-1 and zoledronic acid suppresses the growth of malignant mesothelioma cells in vitro.Cancer Lett. 2010,294(2):245-253. PMID: 20206440.
[22] Corso A, Ferretti E, Lunghi M, et al.Zoledronic acid down-regulates adhesion molecules of bone marrow stromal cells in multiple myeloma: a possible mechanism for its antitumor effect. Cancer. 2005,104(1):118-125.PMID: 15895374.
[23] Marra M, Santini D, Meo G,et al. Cyr61 downmodulation potentiates the anticancer effects of zoledronic acid in androgen-independent prostate cancer cells. Int J Cancer. 2009,125(9):2004-2013.PMID: 19530242.
[24] Li YY, Chang JW, Liu YC,et al.Zoledronic acid induces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression. Anticancer Drugs. 2011,22(1):89-98.PMID: 20926944.
[25] Constantinou C, Papas KA, Constantinou AI. Caspase-independent pathways of programmed cell death: the unraveling of new targets of cancer therapy? Curr Cancer Drug Targets. 2009, 9(6): 717-728.PMID: 19754356
[26] Mitrofan LM, Castells FB, Pelkonen J, et al. Lysosomal-mitochondrial axis in zoledronic acid-induced apoptosis in human follicular lymphoma cells. J Biol Chem. 2010, 285(3): 1967-1979. PMID: 19875454
[27] Ullen A, Schwarz S, Lennartsson L, et al. Zoledronic acid induces caspase-dependent apoptosis in renal cancer cell lines. Scand J Urol Nephrol. 2009, 43(2): 98-103. PMID: 19101859
[28] Caraglia M, Marra M, Leonetti C, et al. R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Aktinactivation and reduced Bcl-2 and bad phosphorylation. J Cell Physiol. 2007, 211(2): 533-543. PMID: 17192846
[29] Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells. Cell Biol Int. 2009,33(2):239-246.PMID: 19103299
[30] Sewing L, Steinberg F, Schmidt H, et al. The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis. Apoptosis. 2008, 13(6): 782-789.PMID: 18437576
[1] Kingsley LA, Fournier PG, Chirgwin JM, et al. Molecular biology of bone metastasis. Mol Cancer Ther. 2007, 6(10):2609-2617. PMID: 17938257
[2] Arango BA, Castrellon AB, Perez CA, et al. Nasopharyngeal carcinoma: alternative treatment options after disease progression. Expert Rev Anticancer Ther. 2010, 10(3): 377-386. PMID: 20214519
[3] Caponigro F, Longo F, Ionna F, et al. Treatment approaches to nasopharyngeal carcinoma: a review. Anticancer Drugs. 2010, 21(5): 471-477. PMID: 20124988
[4] Razak AR, Siu LL, Liu FF, et al. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer. 2010, 46(11): 1967-1978. PMID: 20451372
[5] Chiang AC, MassaguéJ. Molecular basis of metastasis. N Engl J Med. 2008, 359(26): 2814-2823. PMID: 19109576
[6] Harel R, Angelov L. Spine metastases: current treatments and future directions. Eur J Cancer. 2010, 46(15): 2696-2707. PMID: 20627705
[7] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007, 14(20):2126-2135. PMID: 17691952
[8] Aft R, Naughton M, Trinkaus K, et al. Effect of zoledronic acid on disseminated tumour cells in women with locally advanced breast cancer: an open label, randomised, phase 2 trial. Lancet Oncol. 2010, 11(5): 421-428. PMID: 20362507
[9] Rack B, Jückstock J, Genss EM, et al. Effect of zoledronate on persisting isolated tumour cells in patients with early breast cancer. Anticancer Res. 2010, 30(5): 1807-1813. PMID: 20592383
[10] Haddad RI, Shin DM. Recent advances in head and neck cancer. N Engl J Med. 2008, 359(11):1143-1154.PMID: 18784104
[11] Vermorken JB, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med. 2008, 359(11): 1116-1127. PMID: 18784101
[12] Facchini G, Caraglia M, Morabito A, et al. Metronomic administration of zoledronic acidand taxotere combination in castration resistant prostate cancer patients: Phase I ZANTE trial. Cancer Biol Ther. 2010, 10(6):543-548. PMID: 20657175
[13]董梅,冯奉仪,张阳,等.唑来膦酸治疗实体瘤骨转移或多发性骨髓瘤中重度骨痛的Ⅲ期临床研究.中华肿瘤杂志. 2008, 30(3):215-220.
[14] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects. Br J Cancer. 2003, 88(10): 1631-1640. PMID: 12771933
[15] Montague R, Hart CA, George NJ, et al. Differential inhibition of invasion and proliferation by bisphosphonates: anti-metastatic potential of Zoledronic acid in prostate cancer. Eur Urol. 2004, 46(3):389-401. PMID: 15306113
[16] Boissier S, Ferreras M, Peyruchaud O, et al. Bisphosphonates Inhibit Breast and Prostate Carcinoma Cell Invasion, an Early Event in the Formation of Bone Metastases Cancer Res. 2000, 60(11): 2949-2454. PMID: 10850442
[17] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010, 141(1):52-67. PMID: 20371345
[18] Gialeli C, Theocharis AD, Karamanos NK. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011, 278(1):16-27. PMID: 21087457
[19] Radisky ES, Radisky DC.Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer.J Mammary Gland Biol Neoplasia. 2010, 15(2):201-212.PMID: 20440544
[20]李明明,张卫东,宗志红,等.唑来膦酸对肺癌95D细胞增殖和侵袭抑制.中国肺癌杂志, 2009, 12(3):198-202.
[21]成伟,周维国.喉癌组织中HIF-1α和COX-2及VEGF与微血管密度的关系及临床意义.临床耳鼻咽喉头颈外科杂志. 2010, 24(18): 820-822.
[22] Shibata MA, Ambati J, Shibata E, et al. The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model. BMC Med. 2010, 8:69. PMID: 21047425
[23] Soltau J, Zirrgiebel U, Esser N, et al. Antitumoral and antiangiogenic efficacy of bisphosphonates in vitro and in a murine RENCA model. Anticancer Res. 2008, 28(2A):933-941. PMID: 18507039.
[24] Santini D,Vincenzi B, Galluzzo S,et al. Repeated Intermittent Low-Dose Therapy with Zoledronic Acid Induces an Early, Sustained, and Long-Lasting Decrease of Peripheral Vascular Endothelial Growth Factor Levels in Cancer Patients. Clin Cancer Res. 2007, 13(15 pt 1): 4482-4486. PMID: 17671133
[25] Folkman J: Clinical applications of research on angiogenesis.Seminars in Medicine of the Beth Israel Hospital, Boston.New Engl J Med.1995, 333(26):1757-1763.PMID:7491141
[26] Maniotis AJ, Folberg R, Hess A, et al.Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol. 1999,155(3):739-752. PMID:10487832
[27]王鸿彪,林英城,曾德,等.人参皂甙Rg3体外抗鼻咽癌HNE-1细胞管道形成的作用.中华肿瘤杂志,2010,32(10):739-742
[28] Wang JY, Sun T, Zhao XL, et al.Functional significance of VEGF-a in human ovarian carcinoma: role in vasculogenic mimicry.Cancer Biol Ther. 2008,7(5):758-766. PMID:18376140
[29] Basu GD, Liang WS, Stephan DA, et al.A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells.Breast Cancer Res. 2006,8(6):R69.PMID:17156488
[30] Mei J, Gao Y, Zhang L, et al.VEGF-siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vitro.Exp Oncol. 2008,30(1):29-34.PMID:18438338
[1] Yuasa T, Kimura S, Ashihara E, et al. Zoledronic acid - a multiplicity of anti-cancer action. Curr Med Chem. 2007,14(20):2126-2135.
[2] Clézardin P, Ebetino FH, Fournier PG. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res.2005, 65(12):4971-4974.
[3] M?nkk?nen H, Auriola S, Lehenkari P, et al. A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates.Br J Pharmacol.2006, 147(4):437-445.
[4] Caraglia M, Marra M, Leonetti C, et al. R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Akt inactivation and reduced Bcl-2 and bad phosphorylation.J Cell Physiol. 2007,211(2):533-543.
[5] Goffinet M, Thoulouzan M, Pradines A,et al. Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells.BMC Cancer. 2006,6:60.
[6] Kubista B, Trieb K, Sevelda F, et al. Anticancer Effects of Zoledronic Acid against Human Osteosarcoma Cells.J Orthop Res.2006,24(6):1145-1152.
[7] Ory B, Blanchard F, Battaglia S, et al. Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status. Mol Pharmacol. 2007, 71(1):333-343.
[8] Green JR. Bisphosphonates: Preclinical Review. Oncologist.2004, 9 Suppl 4:3-13.
[9] Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells. Cell Biol Int.2009, 33(2):239-246.
[10] Fabbri F, Brigliadori G, Carloni S,et al. Zoledronic acid increases docetaxel cytotoxicity through pMEK and Mcl-1 inhibition in a hormone-sensitive prostate carcinoma cell line. J Transl Med. 2008,6:43
[11] Brubaker KD, Brown LG, Vessella RL, et al. Administration of zoledronic acid enhances the effects of docetaxel on growth of prostate cancer in the bone environment. BMC Cancer. 2006,6:15
[12] Ottewell PD, Deux B, M?nkk?nen H, et al.Differential Effect of Doxorubicin and Zoledronic Acid on Intraosseous versus Extraosseous Breast Tumor Growth In vivo.Clin Cancer Res. 2008,14(14):4658-4666.
[13] Lu S, Zhang J, Zhou Z, et al. Synergistic inhibitory activity of zoledronate and paclitaxel on bone metastasis in nude mice.Oncol Rep.2008,20(3):581-587.
[14] Ural AU, Avcu F, Candir M, et al.In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells. Breast Cancer Res. 2006, 8(4):R52.
[15] Chang JW, Hsieh JJ, Shen YC, et al. Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells. Cancer Lett.2009, 278(1):17-26.
[16] Winter MC, Holen I, Coleman RE. Exploring the anti-tumour activity of bisphosphonates in early breast cancer. Cancer Treat Rev. 2008, 34(5):453-475.
[17] Boissier S, Ferreras M, Peyruchaud o, et al. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases.Cancer Res. 2000,60(11):2949-2954.
[18] Denoyelle C, Hong L, Vannier JP, et al. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects.Br J Cancer.2003, 88(10):1631-1640.
[19] Leto G, Badalamenti G, Arcara C, et al. Effects of zoledronic acid on proteinase plasma levels in patients with bone metastases.Anticancer Res. 2006, 26(1A):23-26.
[20] Kingsley LA, Fournier PG, Chirgwin JM, et al.Molecular biology of bone metastasis.Mol Cancer Ther. 2007,6(10):2609-2617.
[21] Viereck V, Emons G, Lauck V, et al. Bisphosphonates Pamidronate and Zoledronic Acid Stimulate Osteoprotegerin Production by Primary Human osteoblasts.Biochem Biophys Res Commun.2002,291(3):680-686.
[22] Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology.Cancer Res, 2007.67(1):5-8.
[23] Sato K, Kimura S, Segawa H, et al. Cytotoxic effects of gammadelta T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy.Int J Cancer. 2005, 116(1):94-99.
[24] Bezzi M,Hasmim M,Bieler G, et al. Zoledronate Sensitizes Endothelial Cells to Tumor Necrosis Factor-induced Programmed Cell Death.J Biol Chem. 2003, 31,278(44): 43603- 43614.
[25] Yamada J, Tsuno NH, Kitayama J, et al. Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation.J Surg Res.2009, 151(1):115-120.
[26] Hasmim M, Bieler G, Rüegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost.2007, 5(1):166-173.
[27] Ferretti G, Fabi A, Carlini P, et al. Zoledronic-acid-induced circulating level modifications of angiogenic factors, metalloproteinases and proinflammatory cytokines in metastatic breast cancer patients. Oncology. 2005, 69(1):35-43.
[28] Powles T, Paterson A, McCloskey E, et al. Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer [ISRCTN83688026].Breast Cancer Res.2006,8(2):R13.
[29] Ha TC, Li H. Meta-analysis of clodronate and breast cancer survival.Br J Cancer. 2007, 96(12):1796-1801.
[30] Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer.N Engl J Med.2009,360(7):679-691.