唑来膦酸在乳腺癌辅助治疗中的作用及其机制

双膦酸盐是治疗恶性肿瘤骨转移的有效药物之一,其作用机制是抑制破骨细胞介导的骨质重吸收.最新研究发现,新一代的双膦酸盐唑来膦酸可以诱导肿瘤细胞凋亡和抑制肿瘤血管新生,从而发挥直接或间接的抗肿瘤作用.有研究发现,对乳腺癌采用常用化疗药物序贯唑来膦酸治疗,可发挥协同抗肿瘤效应.唑来膦酸-弗隆辅助协同试验(Zometa-Femara Adjuvant Synergy Trial, ZO-FAST)和奥地利乳腺癌和结直肠癌研究小组-12(Austrian Breast and Colorectal Cancer Study Group-12, ABCSG-12)的最新研究结果显示,唑来膦酸在辅助治疗中与内分泌药物联用可显著提高患者的无病生存率和总生存率,而与辅助化疗联合使用的效果还有待唑来膦酸联合新辅助化疗降低复发(Neo-Adjuvant Zoledronic Acid to Reduce Recurrence,AZURE)临床试验予以证实.这些结果均表明,唑来膦酸用于乳腺癌的辅助治疗可以降低肿瘤的复发和转移.对唑来膦酸的最佳剂量和疗程尚有待进一步的研究予以确认.     

唑来膦酸在早期乳腺癌中的抗肿瘤研究进展

早期乳腺癌在接受辅助治疗（化疗和内分泌治疗）时均会对患者骨密度造成不良影响,加速骨丢失。第三代双磷酸盐—唑来膦酸其作用机制是抑制破骨细胞介导的骨质重吸收,主要用于恶性肿瘤骨转移引起的高钙血症。唑来膦酸-弗隆辅助协同试验（ZO-FAST）显示唑来膦酸在早期乳腺癌辅助内分泌治疗同时使用不仅可有效防止骨质丢失,还具有明显降低肿瘤复发的作用。奥地利乳腺癌和结直肠癌研究小组-12（ABCSG-12）试验结果同样表明唑来膦酸在联合内分泌治疗时可显著降低患者的疾病进展风险和死亡风险。除此之外,临床前实验和临床试验也证实唑来膦酸联合化疗也具有协同的抗肿瘤作用。唑来膦酸联合新辅助化疗降低复发（AZURE试验）,对于绝经5年以上和年龄>60岁的人群,辅助化疗加唑来膦酸显著减低疾病进展和死亡风险。在ABCSG-12试验中同样发现对于年龄>40岁的患者,唑来膦酸可明显降低复发风险,而年龄≤40岁患者却未在唑来膦酸的治疗中获益。这些结果表明唑来膦酸在雌激素低水平（自然的或治疗后结果）的早期乳腺癌患者中易于发挥抗肿瘤作用。目前对于唑来膦酸的最佳剂量和持续时间尚有待于进一步的研究确认,相信随着相关临床试验结果的公布可以提供更充足的证据来支持唑来膦酸在早期乳腺癌的使用。      

唑来膦酸在乳腺癌辅助治疗中的临床应用及机制探讨

第三代双膦酸盐类药物唑来膦酸是治疗乳腺癌骨转移公认的标准药物之一,但其在乳腺癌辅助治疗中的应用地位尚存在争议。目前临床研究结果表明,唑来膦酸辅助治疗不仅可以提高骨密度．而且可以降低乳腺癌的复发和转移。在低雌激素水平环境下,唑来膦酸可使早期乳腺癌患者得到生存获益．而且早期应用的益处优于延迟应用。唑来膦酸通过抑制肿瘤转移过程中的多条途径而发挥抗肿瘤作用。但唑来膦酸的最佳剂量、治疗周期及持续时间尚有待进一步的研究予以确认。     

唑来膦酸联合靶向及免疫调节药物的抗肿瘤研究进展

唑来膦酸是第三代含氮双磷酸盐,在临床中广泛用于治疗骨疾病。既往研究发现唑来膦酸在体内外对多种肿瘤细胞均有抗肿瘤作用,同时可以协同增加化疗、内分泌治疗、放疗等抗肿瘤疗效。最近研究发现,唑来膦酸亦可以协同增加靶向及免疫调节药物的抗肿瘤疗效,其抗肿瘤作用机制与抑制甲羟戊酸代谢途径、影响肿瘤信号通路、调节免疫反应、抗血管生成等有关。本文综述了唑来膦酸联合靶向及免疫调节药物在抗肿瘤治疗方面的研究进展。     

唑来膦酸抗肿瘤作用的研究进展

唑来膦酸作为第三代双膦酸盐药物已广泛用于恶性实体瘤骨转移相关事件的防治。进一步研究发现,唑来膦酸可通过抑制甲羟戊酸途径中焦磷酸合酶的活性,使异戊烯焦磷酸大量蓄积,同时异戊烯焦磷酸与细胞内一磷酸腺苷结合形成三磷酸腺苷分解类似物蓄积于细胞中,从而改变细胞周期蛋白和凋亡蛋白水平,抑制肿瘤细胞生长并促进其凋亡,具有一定的直接和/ 或间接抗肿瘤作用。有关肺癌、乳腺癌、前列腺癌及肝癌等前期临床试验提示唑来膦酸与细胞毒药物、内分泌药物及靶向药物联合具有一定程度的协同抗肿瘤作用,并且在抗肿瘤药物之后序贯应用效果更好。一些相关的临床试验正在进行中。      

唑来膦酸抗肿瘤血管生成作用研究进展

唑来膦酸是第三代双膦酸盐药物,主要用于治疗恶性高钙血症及骨转移癌。目前的研究发现,除抑制骨吸收外唑来膦酸还具有直接和间接的抗肿瘤活性。其中抑制肿瘤血管形成是其间接抗肿瘤作用机制之一。其抗肿瘤血管生成作用可能机制如下：（1）抑制肿瘤细胞及肿瘤间质细胞分泌VEGF,调节VEGF-VEGFR自分泌环抑制血管生成;（2）抑制血管内皮细胞迁移、黏附作用;（3）诱导循环内皮细胞祖细胞凋亡,抑制肿瘤细胞及肿瘤浸润巨噬细胞分泌MMPs;（4）抑制肿瘤细胞血管生成拟态。但唑来膦酸抗肿瘤活性的用药剂量不同于目前治疗骨转移的剂量,其抗肿瘤血管生成拟态的作用机制尚未明确,需要进一步研究。     

唑来膦酸治疗乳腺癌及其骨转移的研究进展

唑来膦酸是第三代含氮双膦酸盐类药物的典型代表,在已上市的双膦酸盐类药物中其综合疗效最好,不仅可以通过抑制甲羟戊酸途径直接作用于破骨细胞,也可直接或间接对肿瘤细胞产生抑制作用.乳腺癌属于易发生骨转移的一类恶性肿瘤,大量研究表明唑来膦酸对乳腺癌骨转移具有一定的疗效,可缓解骨痛,目前其已被纳入乳腺癌骨转移的临床常规治疗中,同时唑来膦酸在辅助性内分泌治疗乳腺癌时亦发挥重要的作用.本文就唑来膦酸对乳腺癌骨转移治疗方面的相关研究进展做一综述.     

唑来膦酸在乳腺癌中抗肿瘤作用的研究进展

唑来膦酸(ZOL)是第三代双膦酸盐类药物的典型代表,在已上市的双膦酸盐类药物中应用最广泛、综合疗效最好,现已作为乳腺癌骨转移的常规治疗药物.多项临床前研究及临床研究已证实,唑来膦酸对肿瘤细胞有直接或间接的抑制作用,联合其他辅助治疗(化疗和内分泌治疗)可发挥协同作用,对改善乳腺癌患者术后生存、抑制复发及转移具有重要作用,本文就上述研究进展作一综述.     

唑来膦酸对乳腺癌细胞系MCF-7的凋亡作用及其机制

目的:研究唑来膦酸对乳腺癌MCF-7细胞的凋亡作用和途径.方法:培养乳腺癌MCF-7细胞株,用不同浓度梯度的唑来膦酸进行处理,MTT、Annexin V-PI双染法进行生长抑制和凋亡情况的检测和分析,Western blot检测相关蛋白表达情况.结果:唑来膦酸作用于MCF-7细胞后,MTT、Annexin V-PI双染法检测发现药物对细胞呈明显的生长抑制作用,且有明显的剂量依赖性.当作用时间为48h时抑制作用最为明显.Western blot检测发现,使用唑来膦酸后,Bcl-2、Bcl-XL和Survivin 3个凋亡抑制蛋白表达水平相较于未处理组(NM)表达降低,促凋亡蛋白Bax表达明显升高.同时,Caspase-3和Caspase-9相较于未处理组,表达水平也有明显的升高,并都呈现剂量相关性.结论:唑来膦酸通过线粒体凋亡途径促进乳腺癌MCF-7细胞凋亡,且具有剂量依赖性.     

VEGF基因沉默联合唑来膦酸抑制胃癌细胞增殖和迁移的机制

目的:探讨靶向抑制VEGF基因联合唑来膦酸对胃癌细胞增殖、迁移及STAT3信号通路的影响。方法:采用LipofectamineTM2000将si-VEGF转染至胃癌BGC-823细胞中,以RT-PCR和Western blot 检测其转染效果。转染后,加入不同浓度的唑来膦酸共同培养48 h,MTT法检测si-VEGF联合唑来膦酸对BGC-823细胞增殖的影响,并检测唑来膦酸的IC50;将细胞随机分为si-NC组（转染阴性对照）、si-NC＋唑来膦酸组（转染阴性对照后,给予唑来膦酸处理）、si-VEGF组（转染si-VEGF）和si-VEGF+唑来膦酸组（转染si-VEGF后,给予唑来膦酸处理）,Transwell小室实验检测细胞的侵袭和迁移能力,Western blot检测细胞中STAT3、p-STAT3和MMP-2蛋白的表达情况。结果:转染si-VEGF后成功下调BGC-823细胞中VEGF mRNA和蛋白的表达,si-VEGF联合唑来膦酸对BGC-823 细胞增殖的抑制作用明显高于唑来膦酸单独作用,且唑来膦酸的IC50为62.94 μmol/L。与si-NC组相比,si-NC+唑来膦酸组、si-VEGF组和si-VEGF+唑来膦酸组的侵袭细胞数及迁移细胞数均明显减少,且p-STAT3和MMP-2蛋白表达明显降低,而STAT3表达差异不明显。其中,si-VEGF+唑来膦酸组的作用效果明显高于si-NC+唑来膦酸组或si-VEGF组。结论:靶向抑制VEGF基因联合唑来膦酸能够协调抑制胃癌细胞增殖和迁移,其作用机制可能与抑制STAT3信号通路有关。     

Significance of combination therapy of zoledronic acid and gemcitabine on pancreatic cancer

In the present study, we examined the cytotoxic effects of combination therapy with zoledronic acid (ZOL) and gemcitabine (GEM) on pancreatic cancer cells in vitro and in vivo. Four human pancreatic cancer cell lines were treated with ZOL, GEM or a combination of both, and the effects of the respective drug regimens on cell proliferation, invasion and matrix metalloproteinase (MMP) expression were examined. A pancreatic cancer cell line was also intrasplenically or orthotopically implanted into athymic mice and the effects of these drugs on tumor metastasis and growth in vivo were evaluated by histological and immunohistochemical analyses. Combination treatment with low doses of ZOL and GEM efficiently inhibited the proliferation (P < 0.001) and invasion (P < 0.001) of pancreatic cancer cells in vitro. Western blotting assay revealed that MMP-2 and MMP-9 expression levels were decreased after ZOL treatment. In vivo, combined treatment significantly inhibited tumor growth (P < 0.05) and the development of liver metastasis (P < 0.05). These data revealed that ZOL and GEM, when used in combination, have significant antitumor, anti-metastatic and anti-angiogenic effects on pancreatic cancer cells. The present study is the first to report the significance of the combination treatment of ZOL and GEM in pancreatic cancer using an in vivo model. These data are promising for the future application of this drug regimen in patients with pancreatic cancer.     

Role of zoledronic acid in oncolytic virotherapy: Promotion of antitumor effect and prevention of bone destruction

Osteosarcoma is an aggressive malignant bone tumor that causes bone destruction. Although tumor‐specific replicating oncolytic adenovirus OBP‐301 induces an antitumor effect in an osteosarcoma tumor, it cannot prevent bone destruction. Zoledronic acid (ZOL) is a clinically available agent that inhibits bone destruction. In this study, we investigated the potential of combination therapy with OBP‐301 and ZOL against osteosarcomas with bone destruction. The antitumor activity of OBP‐301 and ZOL in monotherapy or combination therapy was assessed using three human osteosarcoma cell lines (143B, MNNG/HOS, SaOS‐2). The cytotoxic effect of OBP‐301 and/or ZOL was measured by assay of cell apoptosis. The effect of OBP‐301 and ZOL on osteoclast activation was investigated. The potential of combination therapy against tumor growth and bone destruction was analyzed using an orthotopic 143B osteosarcoma xenograft tumor model. OBP‐301 and ZOL decreased the viability of human osteosarcoma cells. Combination therapy with OBP‐301 and ZOL displayed a synergistic antitumor effect, in which OBP‐301 promoted apoptosis through suppression of anti‐apoptotic myeloid cell leukemia 1 (MCL1). Combination therapy significantly inhibited tumor‐mediated osteoclast activation, tumor growth and bone destruction compared to monotherapy. These results suggest that combination therapy of OBP‐301 and ZOL suppresses osteosarcoma progression via suppression of MCL1 and osteoclast activation.     

Cytotoxic effects of gammadelta T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy

Nitrogen containing-bisphosphonates (N-BPs), widely used to treat bone diseases, have direct antitumor effects via the inactivation of Ras proteins. In addition to the direct antitumor activities, N-BPs expand gammadeltaT cells, which exhibit major histocompatibility complex-unrestricted lytic activity. BPs accumulate intermediate metabolites which may be tumor antigens in target cells. The purpose of our study was to clarify the cytotoxicity of gammadelta T cells expanded ex vivo by the most potent N-BP, zoledronate (ZOL). Especially, we focused on the importance of pretreatment against target cells also with ZOL; 1 microM ZOL plus IL-2 increased the absolute number of gammadeltaT cells 298-768 fold for 14 days incubation. The small cell lung cancer and fibrosarcoma cell lines pretreated with 5 microM ZOL showed a marked increase in sensitivity to lysis by gammadeltaT cells. While, untreated cell lines were much less sensitive to lysis by gdT cells. Video microscopy clearly demonstrated that gammadeltaT cells killed target cells pre-treated with ZOL within 3 hr. Pretreatment with 80 microg/kg ZOL also significantly enhanced the antitumor activity of gammadeltaT cells in mice xenografted with SBC-5 cells. These findings show that ZOL significantly stimulated the proliferation of gammadeltaT cells and that gammadeltaT cells required pre-treatment with ZOL for cytotoxic activity against target cells.     

Zoledronic acid, a third-generation bisphosphonate, inhibits cellular growth and induces apoptosis in oral carcinoma cell lines

Bisphosphonates (BPs) inhibit bone resorption by preventing osteoclast maturation and apoptosis induction. Recently, BPs have also been shown to have antitumor effects against various types of carcinomas in vitro and in vivo. In this study, we investigated the antitumor effect of zoledronic acid (ZOL), a third generation bisphosphonate, on proliferation, cell cycle and apoptosis of oral cancer cells. Direct antitumor effects of ZOL against four oral carcinoma cell lines (squamous cell carcinoma, HSC3, HSC4, SCCKN; salivary adenocarcinoma, HSY) were measured by WST assay. Apoptosis-related molecules were analyzed by Western blot analysis and cell cycle was analyzed by flow cytometry. ZOL had a dose-dependent antitumor effect in the four oral cancer cell lines. ZOL activated caspase-3, -8 and -9 and induced cellular apoptosis. Western blot analysis showed that ZOL increased cleaved anti-human poly(ADP-ribose) polymerase expression and decreased Bcl-2 and Bid expression. Treatment with ZOL increased the number of cells in apoptosis, sub G1 phase and S phase, and reduced the number of cells in the G0/G1 and G2/M phase in a concentration-dependent manner. ZOL inhibits cell proliferation and induces apoptosis of oral cancer cells in vitro. These findings suggest that ZOL might be beneficial in the treatment of oral carcinoma patients.     

Zoledronic acid inhibits visceral metastases in the 4T1/luc mouse breast cancer model.

PURPOSE: It is established that bisphosphonates (BPs), specific inhibitors of osteoclasts, have beneficial effects on bone metastases of breast cancer. In addition, recent studies have reported that BPs have anticancer effects and suppress visceral metastases, too. However, the results of clinical studies are still conflicting. In the present study, we examined the effects of the BP zoledronic acid (ZOL), one of the most potent BPs currently available, on visceral metastases of breast cancer using an animal model in which mouse breast cancer cells 4T1/luc implanted at the orthotopic mammary fat pad spontaneously metastasize to multiple organs including bone, lung, and liver in female BALB/c mice. Experimental Design and RESULTS: The 4T1/luc-bearing mice received single or four i.v. injections of ZOL (0.5 or 5 microg/mouse) during the whole experimental period. Bone metastases were reduced by the ZOL treatment. More importantly, ZOL significantly suppressed lung and liver metastases. Furthermore, ZOL prolonged overall survival of the tumor-bearing mice. Of interest, apoptosis in 4T1/luc cells colonized in bone was increased by ZOL; however, those in lung were not changed. In vitro studies demonstrated that ZOL inhibited cell migration and invasion and promoted apoptosis of 4T1/luc cells. CONCLUSIONS: These results are consistent with the notion that ZOL affects breast cancer metastasis to visceral organs as well as bone. These effects of ZOL may be attributable to inhibition of migration and invasion of breast cancer cells. Clinical relevance of our experimental results needs to be determined in breast cancer patients with visceral metastases.     

Anti-tumor and anti-osteolysis effects of the metronomic use of zoledronic acid in primary and metastatic breast cancer mouse models

This study aims to determine the effect of metronomic (0.0125 mg/kg twice a week for 4 weeks) zoledronic acid (ZOL) on cancer propagation and osteolysis against both metastatic and primary breast cancer in mice model. From our results, metronomic ZOL resulted in a significant reduction of tumor burden and did not promote lung or liver metastasis. The metronomic ZOL appeared to be more effective than the conventional regimen (0.1 mg/kg once in 4 weeks) in reducing breast cancer tumor burden, and regulating its movement to lung and liver. This dosing schedule of ZOL showed great potential against metastatic breast cancer.     

Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival for non-small-cell lung cancers

BACKGROUND: Although zoledronic acid (ZOL), a third-generation nitrogen-containing bisphosphonate, has been identified as an attractive therapeutic agent against breast cancer, prostate cancer, multiple myeloma as well as small-cell lung cancer (SCLC), as best as we are aware, the anti-tumor effect of ZOL upon non-small-cell lung cancer (NSCLC) remains to be effectively investigated. This study examined the effects of ZOL upon the line-1 tumor cell, using a murine lung adenocarcinoma cell line similar to the behavior of human lung adenocarcinoma. METHODS: We investigated the anti-tumor effects of ZOL (3-100 microM) on line-1 tumor cells in vitro, including cellular proliferation, by means of an MTT assay, cell-cycle analysis by flow cytometry and by assessing the level of apoptosis by annexin V/propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) staining. Further, we evaluated the growth and survival of line-1 tumor cells following ZOL treatment (1 microg/kg/week) using an animal model. We also examined the in vivo cell-cycle pattern using lacZ-expressing line-1 cells (line-1/lacZ). RESULTS: ZOL significantly slowed the line-1 tumor growth in a dose-dependent manner in vitro. The treated line-1 tumor cells typically arrested at the S/G2/M-phase of the cell-cycle following ZOL exposure, but no apoptotic cells could be detected by either annexin V/PI or DAPI staining. When the ZOL was washed out, the drug-inhibited cells continued to proliferate again and the cell-cycle prolongation elicited earlier by the drug, then disappeared. Within 72-96 h following drug removal, the cell-cycle of the treated cells revealed a similar distribution to that of the untreated controls. In vivo studies demonstrated that ZOL significantly slowed the line-1 tumor growth. Indeed, mice lived significantly longer when they had been ZOL-treated than was the case for untreated mice (p<0.05). Using line-1/lacZ cells, the in vivo cell-cycle distribution of line-1 tumor cells subsequent to ZOL exposure revealed S/G2/M-phase arrest that was identical to the in vitro culture. CONCLUSIONS: ZOL maintains the potential to reduce tumor burden and prolong survival for murine pulmonary adenocarcinoma. The flow cytometrical analysis of cell-cycle demonstrated that ZOL induces no apoptosis but is able to arrest line-1 tumor cells at the S/G2/M-phase. Although the clinical relevance of these results warrants verification for human lung cancer patients, ZOL combined with chemotherapy and/or radiotherapy appears to be a new therapeutic strategy for the effective treatment of NSCLC.