双膦酸盐类药物CP对胃癌细胞生长的影响及机制研究
|
摘要
目的：胃癌是全球最常见的恶性肿瘤之一，流行病学调查显示，目前胃癌的发病率虽呈下降的趋势，但其预后情况仍不容乐观，全球范围内的五年生存率仍低于24%。对于局部进展期和晚期胃癌来讲，化疗仍是治疗的主要手段。多年来胃癌的化疗似乎进入“瓶颈”阶段，目前氟尿嘧啶类和铂类药物仍然是胃癌化疗的基础，联合蒽环类还是紫杉类尚有争议，因此为降低胃癌死亡率或延长胃癌患者生存期，探索低毒、有效的拮抗胃癌的新药是当前研究的重点与热点。
双膦酸盐类药物一般用于治疗各种原因所致的骨吸收性疾病。目前临床前研究表明，双膦酸盐类药物具有直接及间接抗肿瘤作用；临床研究则证实伴有骨转移的肿瘤病人应用双膦酸盐，可使病人从中明显获益。在对多发性骨髓瘤、前列腺癌、乳腺癌、胰腺癌等肿瘤的研究中发现双膦酸盐类药物确实具有直接抗肿瘤效应，其主要是通过抑制肿瘤增殖和诱导细胞凋亡发挥抗肿瘤作用的。以往的研究表明，由于结构上的不同，不同的双膦酸盐类药物的抗肿瘤效应亦不相同。在体外，其抑制肿瘤细胞增殖的作用强度是由其侧链基团所决定的，其R2侧链越为复杂，则其抗肿瘤效应越强。但有关双膦酸盐类药物抗肿瘤的细胞内信号途径目前不完全清楚，且其对胃癌的抗肿瘤研究少见报道。
细胞外信号调节激酶1/2（extracellular signa1regulated kinase1/2，ERK1/2）是广泛存在于真核细胞内的一类丝氨酸/苏氨酸蛋白激酶，属丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）家族成员。ERK1/2信号通路被激活后，可以对细胞内的底物蛋白或转录因子发挥作用，引起底物蛋白活化和转录增强，从而调控细胞的增殖、分化、凋亡等。以往的研究表明，ERK1/2信号通路可以通过线粒体及死亡受体细胞凋亡途径发挥对凋亡的调控作用。ERK1/2信号通路参与多种药物诱导的细胞凋亡，其亦有可能参与双膦酸盐类药物诱导的细胞凋亡，有关ERK1/2信号通路是否参与双膦酸盐类药物诱导的胃癌细胞凋亡，目前尚未见报道。
本实验，我们对新合成的双膦酸盐类药物CP进行研究，其化学名称为[2-(6-氨基-嘌呤-9-基)-1-羟基-膦酰乙基]膦酸，在化学结构上，除了含氮之外，其R2侧链增加了腺嘌呤，结构相对复杂（见Fig.1.1）。在体外，应用CP对胃癌SGC-7901细胞进行干预；并构建裸鼠胃癌移植瘤，应用CP进行体内研究，分析ERK1/2信号通路蛋白的表达变化与细胞凋亡之间的关系，以探讨ERK1/2信号通路与双膦酸盐类药物CP抗胃癌作用间的关系，揭示其细胞内信号转导机制，为双膦酸盐类药物在胃癌化学治疗中提供新的理论和实验依据。
方法：
1双膦酸盐类药物CP对胃癌细胞生长影响的研究。
培养人胃癌细胞株（SGC-7901、BGC-823、MKN-45和MKN-28)和人结肠癌细胞株(Lovo、HT-29)以不同浓度的CP（20，40，80，160μmol/L）作用于细胞，采用MTT法观察CP对细胞生长的影响，并计算CP的半数抑制浓度（IC50）。对胃癌SGC-7901细胞进行药物CP干预实验，MTT法观察不同作用时间点（0、1、3、6、12和24h），不同药物浓度（10、20、40和80μmol/L）对细胞生长的影响。流式细胞术（flow cytometry，FCM）检测CP对细胞周期及凋亡的影响，酶联免疫法（ELISA法）检测细胞凋亡的DNA片断，Western blot检测CP对SGC-7901细胞Bcl-2、Bax、Bad、caspase-3、caspase-9、PARP蛋白表达的影响，进一步凋亡酶活性分析CP作用后caspase-3、caspase-9活性的变化，观察CP在体外抑制胃癌细胞增殖及诱导凋亡中的作用。
2双膦酸盐类药物CP对胃癌细胞ERK1/2信号通路影响的研究。
应用40μmol/L的CP对胃癌SGC-7901细胞进行干预，Western blot检测CP作用不同时间（0、0.5、1、3、6、12和24h），细胞中ERK1/2、p-ERK1/2、p38、p-p38、JNK、p-JNK、MEK、p-MEK、Raf-1、p-Raf-1蛋白的表达水平。RT-PCR法检测ERK1/2mRNA水平。应用ERK1/2通路抑制剂PD98059及ERK1/2干扰RNA对细胞进行干预，将实验细胞分为8组：CP（40μmol/L）组；PD98059（20μmol/L）组；CP（40μmol/L）＋PD98059（20μmol/L）组；ERK1/2siRNA（25nmol/L）组；control siRNA（25nmol/L）组；CP（40μmol/L）＋control siRNA（25nmol/L）组；CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组；对照组。MTT法检测各组细胞增殖活力；FCM检测各组sub-G1期细胞百分比；凋亡酶活性分析，测定各组caspase-9活性；western blot分析各组PARP、cleaved PARP、ERK1/2、p-ERK1/2的蛋白表达水平。最终探讨ERK1/2通路与CP抑制胃癌细胞增殖及诱导凋亡之间的关系。
3双膦酸盐类药物CP对人胃癌裸鼠移植瘤生长的影响及机制研究。
将人胃癌SGC-7901细胞接种于12只BABL/C/nu/nu裸鼠（4周龄，雌性，15-17g）左侧腋窝皮下（细胞数2×106/部位），构建胃癌裸鼠移植瘤模型，将裸鼠随机分为2组，对照组及CP组，每组6只。接种后第5天开始，每日腹腔注射0.16%碳酸氢钠溶液及CP（200μg/kg/day，浓度为1mmol/L）至第30天，每周秤量裸鼠体重2次，每3天测量移植瘤的最长径及最短径，计算肿瘤体积并同时绘制肿瘤生长曲线，体积按公式V=ab2/2计算（a，b分别为肿瘤的最长径、最短径）。第30天处死裸鼠，取瘤体及裸鼠脏器，秤瘤重。免疫组织化学检测PCNA及Ki67在移植瘤中的表达，TUNEL法检测裸鼠移植瘤中细胞凋亡情况，Western blot ECL法检测移植瘤ERK1/2、pERK1/2、JNK、pJNK、p38、p-p38蛋白表达变化，HE染色后，光镜下观察移植瘤及裸鼠肝、肾脏器的改变。
结果：
1双膦酸盐类药物CP对胃癌细胞生长影响的研究。
1.1MTT法测定胃癌细胞生长抑制。用MTT法观察CP对胃癌及结肠癌细胞生长的抑制率，计算CP的半数抑制浓度为40μmol/L。用10~80μmol/LCP作用于胃癌SGC-7901、MKN-28、BGC-823细胞株1、3、6、12和24h后，可抑制胃癌细胞的生长，并呈时间和浓度依赖性，CP作用后对胃癌正常粘膜细胞GES-1无抑制作用。
1.2流式细胞术测定细胞凋亡和细胞周期。40μmol/L CP作用0、6、12、24h后能诱导胃癌SGC-7901细胞发生凋亡，在流式DNA组方图上表现为亚G1峰的出现，其出现于G0/G1峰前，此峰代表细胞凋亡。并呈时间依赖性改变细胞周期的分布，使G1期细胞、S期细胞比例降低，sub-G1期、G2/M期细胞比例增高。Annexin V/PI双染法流式细胞术分析显示：随时间延长，Annexin V阳性/PI阴性细胞逐渐增多，具有时效关系。
1.3ELISA法检测细胞凋亡的DNA片断。40μmol/L CP作用胃癌SGC-7901细胞0、6、12、24h后，酶联免疫法（ELISA法）检测细胞的DNA片段。随着CP作用时间的延长，可见DNA片段逐渐增多，反映凋亡细胞逐渐增多。
1.4CP对SGC-7901细胞Bcl-2、Bax、Bad、caspase-3、-9、PARP蛋白表达的影响。40μmol/L CP作用SGC-7901细胞0、6、12和24h后，cleavedcaspase-3、cleaved caspase-9、cleaved PARP、Bax、Bad表达水平逐渐升高，而Bcl-2蛋白水平逐渐下降，差异有显著性（P<0.05）；Bax/Bcl-2比率逐渐升高。
1.5凋亡酶活性分析。胃癌SGC-7901细胞在40μmol/L CP作用0、6、12、24h后，caspase-3、caspase-9的活性呈时间依赖性的增高，（P<0.05）
2双膦酸盐类药物CP对胃癌细胞ERK1/2信号通路影响的研究。
2.1CP上调胃癌细胞ERK1/2信号通路蛋白的表达。40μmol/L的CP作用胃癌SGC-7901细胞不同时间点（0、0.5、1、3、6、12、24h），可见p-ERK1/2蛋白表达逐渐增高，其上游相关蛋白p-raf-1，p-MEK1/2蛋白表达亦逐渐增高，而MAPK其他两条p38及JNK蛋白表达无变化。40μmol/L的CP作用胃癌SGC-7901细胞不同时间点（0、6、12、24h），可见ERK1/2mRNA水平逐渐增高。
2.2PD98059、ERK1/2siRNA减弱了CP抑制胃癌细胞增殖的作用。CP、PD98059及ERK1/2siRNA单独或两者联合作用于胃癌SGC-7901细胞24h后，经MTT分析，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的细胞活力较单独应用CP（40μmol/L）组增强，可见PD98059及ERK1/2siRNA削弱了CP抑制胃癌细胞增殖作用。
2.3PD98059、ERK1/2siRNA抑制了CP诱导胃癌细胞凋亡的作用
流式细胞术分析CP处理后的亚二倍体峰期（sub-G1期）胃癌细胞的百分比发现，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的sub-G1细胞占比较单独应用CP（40μmol/L）组明显降低，说明PD98059和ERK1/2siRNA均抑制了CP诱导胃癌细胞凋亡的作用。
2.4CP、PD98059、ERK1/2siRNA对caspase-9活性及PARP表达的影响。凋亡酶caspase-9活性分析显示，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的caspase-9活性较CP（40μmol/L）组显著降低。同时western blot分析PARP蛋白表达，PD98059和ERK1/2siRNA均降低了CP诱导的cleaved PARP蛋白表达水平。
2.5CP、PD98059、ERK1/2siRNA对ERK1/2蛋白表达的影响。CP（40μmol/L）、PD98059（20μmol/L）及ERK1/2siRNA（25nmol/L）单独或两者联合作用于胃癌SGC-7901细胞24h后，可见PD98059及ERK1/2siRNA部分的抑制了CP对ERK1/2的激活。
3双膦酸盐类药物CP对人胃癌裸鼠移植瘤生长的影响及机制研究。
在裸鼠的皮下，应用胃癌SGC-7901细胞，成功构建出人胃癌裸鼠移植瘤模型，成瘤率达100％。和对照组相比，CP组移植瘤的生长速度明显减慢，有统计学差异(P<0.01)，CP处理组裸鼠移植瘤第9、15、21、27、30天的平均体积分别为51±10.17mm3、254.17±38.78mm3、358.33±43.89mm3、462.5±52.22mm3及595±55.76mm3，对照组的相应平均体积分别为119.83±8.35mm3、299.66±30.81mm3、646.33±47.50mm3、1101.57±60.47mm3及1298.33±66.16mm3，CP处理组的裸鼠移植瘤瘤体的平均瘤重为0.67±0.09g，显著低于对照组的1.10±0.16g(P<0.05)。免疫组织化学检测，CP组PCNA和Ki-67在移植瘤组织中的表达，显著低于对照组(P<0.05)。TUNEL检测显示，与对照组相比，CP组移植瘤的凋亡率增加，两者有显著性差异（P<0.05）。CP移植瘤组pERK1/2的表达水平较对照组升高，JNK、pJNK、p38、p-p38蛋白的表达无明显改变。光镜下观察两组裸鼠肝、肾也无药物性损害改变。
结论：
1双膦酸盐类药物CP呈时间、剂量依赖性的抑制胃癌SGC-7901细胞增殖并诱导凋亡。
2双膦酸盐类药物CP可能通过阻滞细胞周期于G2/M期、抑制抗凋亡蛋白Bcl-2的表达并上调caspase-3、caspase-9蛋白的表达诱导细胞凋亡，从而抑制胃癌SGC-7901细胞增殖。
3双膦酸盐类药物CP处理可激活ERK1/2信号通路，ERK1/2的磷酸化蛋白表达增高。
4ERK1/2抑制剂PD98059及ERK1/2siRNA下调CP抑制胃癌细胞生长及诱导凋亡作用。
5双膦酸盐类药物CP可在体内、体外上调ERK1/2信号通路相关蛋白的表达，ERK1/2信号通路可能参与CP诱导的胃癌细胞凋亡。
6双膦酸盐类药物CP在体内抑制人胃癌裸鼠皮下移植瘤的生长，且无明显的药物不良反应，为双膦酸盐类药物用于胃癌的化学治疗提供了理论依据。
Objectives: Gastric cancer is one of the most common malignant tumorin the world.In recent years the incidence of gastric cancer is a downwardtrend, but its prognosis no obvious change.The five-year survival rate is stillless than24%in the worldwide. Chemotherapy is a major treatment means oflocal advanced and advanced gastric cancer. Over the years, gastric cancerchemotherapy seems to enter the stage of "bottleneck". Fluorouracil andplatinum drugs still is the foundation of gastric cancer chemotherapy, if jointanthracycline compounds or taxanes is controversial. Therefore to reduce themortality and prolong survival in patients with gastric cancer, to explore lowtoxicity and effective new drugs antagonist gastric cancer is the hot spots ofthe current research.
Bisphosphonates drugs commonly used in the treatment of benign andmalignant bone resorption disorder. Preclinical studies have shown that atpresent, bisphosphonates have direct and indirect antitumor effect. Clinicalstudies have confirmed tumor patients with bone metastasis appliedbisphosphonates can make the patient obtain significantly benefit from it. Thedirect anti-tumor effect of bisphosphonates has been confirmed in multiplemyeloma, prostate cancer, breast cancer, and pancreatic cancer, its antitumormechanism mainly through inhibiting tumor growth and induce apoptosis.Due to structural differences, different bisphosphonates anti-tumor effect isalso different, the results showed that the bisphosphonates restrain cancer cellin vitro proliferation effect intensity is determined by its side chain groups.Bisphosphonates with complex side chain groups have the stronger inhibitioneffect than simple one. The intracellular signaling pathways ofbisphosphonates is not entirely clear. Up to now, few works focused on theproliferation and apoptosis induced by bisphosphonate CP in gastric cancer cells has not yet been reported.
The extracellular-signa1regulated kinase (ERK1/2) is widely exists ineukaryotic cells of serine/threonine protein kinase, is a family members ofmitogen activated protein kinase (MAPK). From the perspective of the signaltransduction, ERK1/2may activate substrate proteins in the cytoplasm andnucleus, cause the expression of specific proteins or activation, regulation ofcell proliferation, differentiation, apoptosis, etc. ERK1/2signaling pathwaysplay the role of promoting apoptosis or inhibiting apoptosis by endogenousand exogenous cell apoptosis pathway. ERK1/2signaling pathway may beinvolved in the apoptosis induced by bisphosphonate CP. The relevance ofERK1/2signal pathways and cell apoptosis induced by bisphosphonate CP ingastric cancer has not been reported.
To study the new synthesis bisphosphonate drug CP, which chemicalname [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonicacid (CP), on human gastric cancer SGC-7901cells. Thus the objectives of thepresent study were:
1To study the effect of CP, a new bisphosphonate derivative, onproliferation and apoptosis of human gastric cancer SGC-7901cells, and toexplore its signal pathway as well.
2To investigate the role of ERK1/2signal transduction pathways in theprocess of proliferation and apoptosis induced by CP.
3To observe the growth inhibitory effect and side effect of CP on humangastric cancer tumor xenografts, and to elucidate the possible mechanisminvolving in these effects.
Methods：
1The effects of CP on gastric cancer cells growth. Human gastric cancercell lines (SGC-7901, BGC-823, MKN-45and MKN-28) and colon cancercell lines (Lo Vo, HT-29) were cultured in RPMI-1640medium containing10%fetal bovine serum. CP dissolved in0.16%sodium bicarbonate, wasadded to the medium, with a final concentration of20,40,80or160μmol/L.MTT assay was used to determine the influence of CP on proliferation of cells and calculate the half inhibitory concentration (IC50). MTT assay was used todetermine the influence of CP on proliferation of SGC-7901cells at0,1,3,612,24hours with a final concentration of10,20,40, or80μmol/L. The cellcycle distribution and apoptosis were detected by flow cytometry, furthermore,the DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). The expressions of Bcl-2, Bax, Bad,caspase-3,-9, PARP protein were detected by Western blot. Meanwhile, theapoptotic enzyme activity analysis of caspase-3,-9were done after the CPtreatment24hour in gastric cancer SGC-7901cell. To observe the function ofinhibiting gastric cancer cell proliferation and induce apoptosis by CP in vitro.
2The effects of CP on ERK1/2pathways in gastric cancer cell. Humangastric cancer SGC-7901cell were treated with40μmol/L CP. The expressionsof ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, MEK, p-MEK, Raf-1,p-Raf-1protein were detected by Western blot at0,0.5,1,3,612, and24hours. The expression of ERK1/2mRNA were detected by RT-PCR after40μmol/L CP treatment at0,6,12,24hours. Transfection experiment Cell isdivided into8group: CP (40μmol/L) group, PD98059(20μmol/L) group, CP(40μmol/L)+PD98059(20μmol/L) group, ERK siRNA (25nmol/L) group,control siRNA (25nmol/L) group, CP (40μmol/L)+control siRNA (25nmol/L)group, CP (40μmol/L)+ERK siRNA (25nmol/L) group and control group. Theproliferation activity of each group determined by MTT. FCM was used todetect sub-G1phase cell percentage in each group. Meanwhile, the apoptoticenzyme activity analysis of caspase-9were done in each group. Theexpressions of PARP, cleaved-PARP, ERK1/2, p-ERK1/2protein weredetected by Western blot.
3The effect of CP on gastric cancer nude mouse xenografts growth andits mechanism. Each of12intact female athymic nude mice (BABL/c, nu/nu,4week old,15-17g) was inoculated with SGC-7901cells (2×106/site)subcutaneously on the left axillary fossa. The animals were divided into twogroups (control and CP group). The CP group received CP (200μg/kg/day),while the control group was given vehicle (0.16%sodium bicarbonate) on day 5after inoculation and repeated daily for30day. The shortest and longestdiameter of the tumor were measured with slide gaud at3-day intervals, andtumor volume (mm3) was calculated using the following standard formula:(the longest diameter)×(the shortest diameter)2/2. On day30, mice weresacrificed and all tumor tissue samples were collected and weighed. The liverand kidneys were also dissected, fixed in10%formalin, stained withhematoxylin-eosin for histological examination. The expressions of PCNAand Ki67in tumor tissue were detected by immunohistochemistry.Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)was used to examine cell apoptosis in xenograft tissue. The expression ofERK1/2, pERK1/2, JNK, pJNK, p38, p-p38was assessed by Western blot.Pathological routine HE staining was used to observe the change in nudemouse transplantation tumor and liver and kidney.
Results：
1The effects of CP on gastric cancer cells growth.
1.1Cell growth inhibition were determined by MTT. With a determined byMTT method to observe the CP for different cell growth inhibition rate, theIC50of CP at48h was about40μmol/L. CP inhibited the growth of humangastric cancer cell lines (SGC-7901, BGC-823, MKN-45) in a dose-andtime-dependent manner after incubated with10-80μmol/L CP for24hours.The normal mucosa cells of stomach (GES-1) have no inhibition by CP.
1.2Cell cycle distribution and apoptosis were detected by flow cytometry. CPinduced apoptosis of gastric cancer SGC-7901cells. After incubation with CPfor24h, distinct apoptosis was observed on a DNA histogram as subdiploid orpre-G1peak, pre-G1peak was especially remarkable at24hours. Cell-cycleanalysis by flow cytometry showed that CP increased the proportion of cellsin the G2/M phase and decreased the proportion in the S phase of the cellcycle. Annexin V/PI double staining FCM analysis showed that annexin Vpositive and PI negative cells, which on behalf of apoptosis, graduallyincrease with the extension of time.
1.3The DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). After40μmol/L CP treatment for0,6,12,24hours, the DNA fragments of cell apoptosis was determined by ELISA. Theresults showed that DNA fragments increase gradually as the extension of CPincubation time, which reflect the apoptosis cells increase gradually。
1.4The effects of CP on protein expressions of Bcl-2, Bax, Bad, caspase-3,-9,PARP. After40μmol/L CP treatment for0,6,12,24hours,40μmol/L, theexpressions of Bax, Bad, cleaved caspase-3, cleaved caspase-9, PARP proteinwere increased gradually detected by Western blot. In contrast, the expressionlevel of Bcl-2protein decreased gradually.There are significant difference (P <0.05). The ratio of Bax to Bcl-2increased.
1.5The activity analysis of caspase-3,-9. The apoptotic enzyme activityanalysis of caspase-3,-9were done after the40μmol/LCP treatment24hourin gastric cancer SGC-7901cell. The activity of caspase-3, caspase-9wasincreased with time dependence (P <0.05).
2The effects of CP on ERK1/2pathways in gastric cancer cell.
2.1CP induced ERK1/2signal pathways activated in gastric cancer cell. After40μmol/L CP treatment for0,0.5,1,3,6,12,24hours, the expressions ofp-ERK1/2, p-raf-1, p-MEK1/2protein were increased gradually detected byWestern blot. The expressions of p-38and JNK protein which stand for theother MAPK signal pathways protein. In addition,40μmol/L CP significantlyup-regulated the expression of ERK1/2mRNA.
2.2PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP. After CP (40μmol/L), PD98059(20μmol/L) andERK siRNA (25nmol/L) alone or both combined incubation gastric cancerSGC-7901cells for24h, the cell viability was enhanced in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group than CP (40μmol/L) alone. So we can draw a conclusionthat PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP.
2.3PD98059and ERK1/2siRNA inhibited cell apoptosis induced by CP.Compared with CP alone group, the proportion of sub-G1phase cells analyzed by flow cytometry were significantly decreased in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group.
2.4CP, PD98059and ERK1/2siRNA influence the activity of caspase-9andexpression of PARP protein. The apoptotic enzyme activity analysis showedthat the caspase-9activity was decreased in CP+PD98059group andCP+ERK1/2siRNA group than CP alone group. Meanwhile, the expression ofPARP protein was decreased in CP+PD98059group and CP+ERK1/2siRNAgroup than CP alone group.
2.5CP, PD98059and ERK1/2siRNA affect ERK1/2protein expression. AfterCP (40μmol/L), PD98059(20μmol/L) and ERK siRNA (25nmol/L) alone orboth combined treatment gastric cancer SGC-7901cells for24h, theERK1/2signal pathways activated induced by CP were inhibited partially byPD98059and ERK1/2siRNA.
3The effect of CP on gastric cancer nude mouse xenografts growth and itsmechanism. The model of esophageal cancer xenograft in nude mice wassuccessfully established, the tumorigenic rate in nude mice injected withSGC-7901cells was100%. Intraperitoneal injection of CP significantlysuppressed tumor growth of the xenografts (P<0.01). On day9,15,21,27and30, the mean volume of the xenograft in the CP group was51±10.17mm3,254.17±38.78mm3,358.33±43.89mm3,462.5±52.22mm3and595±55.76mm3, respectively, significantly smaller than that (119.83±8.35mm3,299.66±30.81mm3,646.33±47.50mm3,1101.57±60.47mm3and1298.33±66.16mm3, correspondingly) in the control group. The tumor weight of theCP group was0.67±0.09g, significantly lower than that of the control group(1.10±0.16g)(P<0.05). Immunohistochemical detection showed that thePCNA and Ki-67expression of xenografts tissue in CP group is significantlylower than control group (P<0.05). Compared with the control group,apoptosis cells of CP group increased in xenografts tumor, both havesignificant difference (P<0.05). Compared with the control group, theexpression level of pERK1/2is higher in CP group, the expression level of JNK, pJNK, p38p-p38has no obvious change. Then observe two groups ofnude mice liver, kidney and no abnormal change.
Conclusions:
1CP was demonstrated to lead to a dose-and time-dependent inhibitionproliferation and inducing apoptosis in gastric cancer cells.
2CP may change the cell cycle distribution, inhibit expression of antiapoptotic proteins Bcl-2and raise caspase-3,-9protein expression which caninduce apoptosis and inhibit growth in gastric cancer cell.
3CP may activate ERK1/2signal pathway, increased phosphorylationprotein expression of ERK1/2.
4ERK1/2inhibitor PD98059and ERK1/2siRNA abrogated partially theability of growth and apoptosis induce by CP in gastric cancer cell
5Bisphosphonate CP can increase the expression of proteins involved inERK1/2signaling pathways in vitro and in vivo. ERK1/2signaling pathwaymay be involved in CP induced gastric cancer cell apoptosis.
6This experiment confirmed that the bisphosphonate CP can inhibitgrowth of gastric cancer nude mice subcutaneous transplantation tumor invivo, and no obvious side effects, and provides a theoretical basis forbisphosphonate CP in the chemical treatment of gastric cancer.
双膦酸盐类药物一般用于治疗各种原因所致的骨吸收性疾病。目前临床前研究表明，双膦酸盐类药物具有直接及间接抗肿瘤作用；临床研究则证实伴有骨转移的肿瘤病人应用双膦酸盐，可使病人从中明显获益。在对多发性骨髓瘤、前列腺癌、乳腺癌、胰腺癌等肿瘤的研究中发现双膦酸盐类药物确实具有直接抗肿瘤效应，其主要是通过抑制肿瘤增殖和诱导细胞凋亡发挥抗肿瘤作用的。以往的研究表明，由于结构上的不同，不同的双膦酸盐类药物的抗肿瘤效应亦不相同。在体外，其抑制肿瘤细胞增殖的作用强度是由其侧链基团所决定的，其R2侧链越为复杂，则其抗肿瘤效应越强。但有关双膦酸盐类药物抗肿瘤的细胞内信号途径目前不完全清楚，且其对胃癌的抗肿瘤研究少见报道。
细胞外信号调节激酶1/2（extracellular signa1regulated kinase1/2，ERK1/2）是广泛存在于真核细胞内的一类丝氨酸/苏氨酸蛋白激酶，属丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）家族成员。ERK1/2信号通路被激活后，可以对细胞内的底物蛋白或转录因子发挥作用，引起底物蛋白活化和转录增强，从而调控细胞的增殖、分化、凋亡等。以往的研究表明，ERK1/2信号通路可以通过线粒体及死亡受体细胞凋亡途径发挥对凋亡的调控作用。ERK1/2信号通路参与多种药物诱导的细胞凋亡，其亦有可能参与双膦酸盐类药物诱导的细胞凋亡，有关ERK1/2信号通路是否参与双膦酸盐类药物诱导的胃癌细胞凋亡，目前尚未见报道。
本实验，我们对新合成的双膦酸盐类药物CP进行研究，其化学名称为[2-(6-氨基-嘌呤-9-基)-1-羟基-膦酰乙基]膦酸，在化学结构上，除了含氮之外，其R2侧链增加了腺嘌呤，结构相对复杂（见Fig.1.1）。在体外，应用CP对胃癌SGC-7901细胞进行干预；并构建裸鼠胃癌移植瘤，应用CP进行体内研究，分析ERK1/2信号通路蛋白的表达变化与细胞凋亡之间的关系，以探讨ERK1/2信号通路与双膦酸盐类药物CP抗胃癌作用间的关系，揭示其细胞内信号转导机制，为双膦酸盐类药物在胃癌化学治疗中提供新的理论和实验依据。
方法：
1双膦酸盐类药物CP对胃癌细胞生长影响的研究。
培养人胃癌细胞株（SGC-7901、BGC-823、MKN-45和MKN-28)和人结肠癌细胞株(Lovo、HT-29)以不同浓度的CP（20，40，80，160μmol/L）作用于细胞，采用MTT法观察CP对细胞生长的影响，并计算CP的半数抑制浓度（IC50）。对胃癌SGC-7901细胞进行药物CP干预实验，MTT法观察不同作用时间点（0、1、3、6、12和24h），不同药物浓度（10、20、40和80μmol/L）对细胞生长的影响。流式细胞术（flow cytometry，FCM）检测CP对细胞周期及凋亡的影响，酶联免疫法（ELISA法）检测细胞凋亡的DNA片断，Western blot检测CP对SGC-7901细胞Bcl-2、Bax、Bad、caspase-3、caspase-9、PARP蛋白表达的影响，进一步凋亡酶活性分析CP作用后caspase-3、caspase-9活性的变化，观察CP在体外抑制胃癌细胞增殖及诱导凋亡中的作用。
2双膦酸盐类药物CP对胃癌细胞ERK1/2信号通路影响的研究。
应用40μmol/L的CP对胃癌SGC-7901细胞进行干预，Western blot检测CP作用不同时间（0、0.5、1、3、6、12和24h），细胞中ERK1/2、p-ERK1/2、p38、p-p38、JNK、p-JNK、MEK、p-MEK、Raf-1、p-Raf-1蛋白的表达水平。RT-PCR法检测ERK1/2mRNA水平。应用ERK1/2通路抑制剂PD98059及ERK1/2干扰RNA对细胞进行干预，将实验细胞分为8组：CP（40μmol/L）组；PD98059（20μmol/L）组；CP（40μmol/L）＋PD98059（20μmol/L）组；ERK1/2siRNA（25nmol/L）组；control siRNA（25nmol/L）组；CP（40μmol/L）＋control siRNA（25nmol/L）组；CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组；对照组。MTT法检测各组细胞增殖活力；FCM检测各组sub-G1期细胞百分比；凋亡酶活性分析，测定各组caspase-9活性；western blot分析各组PARP、cleaved PARP、ERK1/2、p-ERK1/2的蛋白表达水平。最终探讨ERK1/2通路与CP抑制胃癌细胞增殖及诱导凋亡之间的关系。
3双膦酸盐类药物CP对人胃癌裸鼠移植瘤生长的影响及机制研究。
将人胃癌SGC-7901细胞接种于12只BABL/C/nu/nu裸鼠（4周龄，雌性，15-17g）左侧腋窝皮下（细胞数2×106/部位），构建胃癌裸鼠移植瘤模型，将裸鼠随机分为2组，对照组及CP组，每组6只。接种后第5天开始，每日腹腔注射0.16%碳酸氢钠溶液及CP（200μg/kg/day，浓度为1mmol/L）至第30天，每周秤量裸鼠体重2次，每3天测量移植瘤的最长径及最短径，计算肿瘤体积并同时绘制肿瘤生长曲线，体积按公式V=ab2/2计算（a，b分别为肿瘤的最长径、最短径）。第30天处死裸鼠，取瘤体及裸鼠脏器，秤瘤重。免疫组织化学检测PCNA及Ki67在移植瘤中的表达，TUNEL法检测裸鼠移植瘤中细胞凋亡情况，Western blot ECL法检测移植瘤ERK1/2、pERK1/2、JNK、pJNK、p38、p-p38蛋白表达变化，HE染色后，光镜下观察移植瘤及裸鼠肝、肾脏器的改变。
结果：
1双膦酸盐类药物CP对胃癌细胞生长影响的研究。
1.1MTT法测定胃癌细胞生长抑制。用MTT法观察CP对胃癌及结肠癌细胞生长的抑制率，计算CP的半数抑制浓度为40μmol/L。用10~80μmol/LCP作用于胃癌SGC-7901、MKN-28、BGC-823细胞株1、3、6、12和24h后，可抑制胃癌细胞的生长，并呈时间和浓度依赖性，CP作用后对胃癌正常粘膜细胞GES-1无抑制作用。
1.2流式细胞术测定细胞凋亡和细胞周期。40μmol/L CP作用0、6、12、24h后能诱导胃癌SGC-7901细胞发生凋亡，在流式DNA组方图上表现为亚G1峰的出现，其出现于G0/G1峰前，此峰代表细胞凋亡。并呈时间依赖性改变细胞周期的分布，使G1期细胞、S期细胞比例降低，sub-G1期、G2/M期细胞比例增高。Annexin V/PI双染法流式细胞术分析显示：随时间延长，Annexin V阳性/PI阴性细胞逐渐增多，具有时效关系。
1.3ELISA法检测细胞凋亡的DNA片断。40μmol/L CP作用胃癌SGC-7901细胞0、6、12、24h后，酶联免疫法（ELISA法）检测细胞的DNA片段。随着CP作用时间的延长，可见DNA片段逐渐增多，反映凋亡细胞逐渐增多。
1.4CP对SGC-7901细胞Bcl-2、Bax、Bad、caspase-3、-9、PARP蛋白表达的影响。40μmol/L CP作用SGC-7901细胞0、6、12和24h后，cleavedcaspase-3、cleaved caspase-9、cleaved PARP、Bax、Bad表达水平逐渐升高，而Bcl-2蛋白水平逐渐下降，差异有显著性（P<0.05）；Bax/Bcl-2比率逐渐升高。
1.5凋亡酶活性分析。胃癌SGC-7901细胞在40μmol/L CP作用0、6、12、24h后，caspase-3、caspase-9的活性呈时间依赖性的增高，（P<0.05）
2双膦酸盐类药物CP对胃癌细胞ERK1/2信号通路影响的研究。
2.1CP上调胃癌细胞ERK1/2信号通路蛋白的表达。40μmol/L的CP作用胃癌SGC-7901细胞不同时间点（0、0.5、1、3、6、12、24h），可见p-ERK1/2蛋白表达逐渐增高，其上游相关蛋白p-raf-1，p-MEK1/2蛋白表达亦逐渐增高，而MAPK其他两条p38及JNK蛋白表达无变化。40μmol/L的CP作用胃癌SGC-7901细胞不同时间点（0、6、12、24h），可见ERK1/2mRNA水平逐渐增高。
2.2PD98059、ERK1/2siRNA减弱了CP抑制胃癌细胞增殖的作用。CP、PD98059及ERK1/2siRNA单独或两者联合作用于胃癌SGC-7901细胞24h后，经MTT分析，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的细胞活力较单独应用CP（40μmol/L）组增强，可见PD98059及ERK1/2siRNA削弱了CP抑制胃癌细胞增殖作用。
2.3PD98059、ERK1/2siRNA抑制了CP诱导胃癌细胞凋亡的作用
流式细胞术分析CP处理后的亚二倍体峰期（sub-G1期）胃癌细胞的百分比发现，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的sub-G1细胞占比较单独应用CP（40μmol/L）组明显降低，说明PD98059和ERK1/2siRNA均抑制了CP诱导胃癌细胞凋亡的作用。
2.4CP、PD98059、ERK1/2siRNA对caspase-9活性及PARP表达的影响。凋亡酶caspase-9活性分析显示，CP（40μmol/L）＋PD98059（20μmol/L）组和CP（40μmol/L）＋ERK1/2siRNA（25nmol/L）组的caspase-9活性较CP（40μmol/L）组显著降低。同时western blot分析PARP蛋白表达，PD98059和ERK1/2siRNA均降低了CP诱导的cleaved PARP蛋白表达水平。
2.5CP、PD98059、ERK1/2siRNA对ERK1/2蛋白表达的影响。CP（40μmol/L）、PD98059（20μmol/L）及ERK1/2siRNA（25nmol/L）单独或两者联合作用于胃癌SGC-7901细胞24h后，可见PD98059及ERK1/2siRNA部分的抑制了CP对ERK1/2的激活。
3双膦酸盐类药物CP对人胃癌裸鼠移植瘤生长的影响及机制研究。
在裸鼠的皮下，应用胃癌SGC-7901细胞，成功构建出人胃癌裸鼠移植瘤模型，成瘤率达100％。和对照组相比，CP组移植瘤的生长速度明显减慢，有统计学差异(P<0.01)，CP处理组裸鼠移植瘤第9、15、21、27、30天的平均体积分别为51±10.17mm3、254.17±38.78mm3、358.33±43.89mm3、462.5±52.22mm3及595±55.76mm3，对照组的相应平均体积分别为119.83±8.35mm3、299.66±30.81mm3、646.33±47.50mm3、1101.57±60.47mm3及1298.33±66.16mm3，CP处理组的裸鼠移植瘤瘤体的平均瘤重为0.67±0.09g，显著低于对照组的1.10±0.16g(P<0.05)。免疫组织化学检测，CP组PCNA和Ki-67在移植瘤组织中的表达，显著低于对照组(P<0.05)。TUNEL检测显示，与对照组相比，CP组移植瘤的凋亡率增加，两者有显著性差异（P<0.05）。CP移植瘤组pERK1/2的表达水平较对照组升高，JNK、pJNK、p38、p-p38蛋白的表达无明显改变。光镜下观察两组裸鼠肝、肾也无药物性损害改变。
结论：
1双膦酸盐类药物CP呈时间、剂量依赖性的抑制胃癌SGC-7901细胞增殖并诱导凋亡。
2双膦酸盐类药物CP可能通过阻滞细胞周期于G2/M期、抑制抗凋亡蛋白Bcl-2的表达并上调caspase-3、caspase-9蛋白的表达诱导细胞凋亡，从而抑制胃癌SGC-7901细胞增殖。
3双膦酸盐类药物CP处理可激活ERK1/2信号通路，ERK1/2的磷酸化蛋白表达增高。
4ERK1/2抑制剂PD98059及ERK1/2siRNA下调CP抑制胃癌细胞生长及诱导凋亡作用。
5双膦酸盐类药物CP可在体内、体外上调ERK1/2信号通路相关蛋白的表达，ERK1/2信号通路可能参与CP诱导的胃癌细胞凋亡。
6双膦酸盐类药物CP在体内抑制人胃癌裸鼠皮下移植瘤的生长，且无明显的药物不良反应，为双膦酸盐类药物用于胃癌的化学治疗提供了理论依据。
Objectives: Gastric cancer is one of the most common malignant tumorin the world.In recent years the incidence of gastric cancer is a downwardtrend, but its prognosis no obvious change.The five-year survival rate is stillless than24%in the worldwide. Chemotherapy is a major treatment means oflocal advanced and advanced gastric cancer. Over the years, gastric cancerchemotherapy seems to enter the stage of "bottleneck". Fluorouracil andplatinum drugs still is the foundation of gastric cancer chemotherapy, if jointanthracycline compounds or taxanes is controversial. Therefore to reduce themortality and prolong survival in patients with gastric cancer, to explore lowtoxicity and effective new drugs antagonist gastric cancer is the hot spots ofthe current research.
Bisphosphonates drugs commonly used in the treatment of benign andmalignant bone resorption disorder. Preclinical studies have shown that atpresent, bisphosphonates have direct and indirect antitumor effect. Clinicalstudies have confirmed tumor patients with bone metastasis appliedbisphosphonates can make the patient obtain significantly benefit from it. Thedirect anti-tumor effect of bisphosphonates has been confirmed in multiplemyeloma, prostate cancer, breast cancer, and pancreatic cancer, its antitumormechanism mainly through inhibiting tumor growth and induce apoptosis.Due to structural differences, different bisphosphonates anti-tumor effect isalso different, the results showed that the bisphosphonates restrain cancer cellin vitro proliferation effect intensity is determined by its side chain groups.Bisphosphonates with complex side chain groups have the stronger inhibitioneffect than simple one. The intracellular signaling pathways ofbisphosphonates is not entirely clear. Up to now, few works focused on theproliferation and apoptosis induced by bisphosphonate CP in gastric cancer cells has not yet been reported.
The extracellular-signa1regulated kinase (ERK1/2) is widely exists ineukaryotic cells of serine/threonine protein kinase, is a family members ofmitogen activated protein kinase (MAPK). From the perspective of the signaltransduction, ERK1/2may activate substrate proteins in the cytoplasm andnucleus, cause the expression of specific proteins or activation, regulation ofcell proliferation, differentiation, apoptosis, etc. ERK1/2signaling pathwaysplay the role of promoting apoptosis or inhibiting apoptosis by endogenousand exogenous cell apoptosis pathway. ERK1/2signaling pathway may beinvolved in the apoptosis induced by bisphosphonate CP. The relevance ofERK1/2signal pathways and cell apoptosis induced by bisphosphonate CP ingastric cancer has not been reported.
To study the new synthesis bisphosphonate drug CP, which chemicalname [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonicacid (CP), on human gastric cancer SGC-7901cells. Thus the objectives of thepresent study were:
1To study the effect of CP, a new bisphosphonate derivative, onproliferation and apoptosis of human gastric cancer SGC-7901cells, and toexplore its signal pathway as well.
2To investigate the role of ERK1/2signal transduction pathways in theprocess of proliferation and apoptosis induced by CP.
3To observe the growth inhibitory effect and side effect of CP on humangastric cancer tumor xenografts, and to elucidate the possible mechanisminvolving in these effects.
Methods：
1The effects of CP on gastric cancer cells growth. Human gastric cancercell lines (SGC-7901, BGC-823, MKN-45and MKN-28) and colon cancercell lines (Lo Vo, HT-29) were cultured in RPMI-1640medium containing10%fetal bovine serum. CP dissolved in0.16%sodium bicarbonate, wasadded to the medium, with a final concentration of20,40,80or160μmol/L.MTT assay was used to determine the influence of CP on proliferation of cells and calculate the half inhibitory concentration (IC50). MTT assay was used todetermine the influence of CP on proliferation of SGC-7901cells at0,1,3,612,24hours with a final concentration of10,20,40, or80μmol/L. The cellcycle distribution and apoptosis were detected by flow cytometry, furthermore,the DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). The expressions of Bcl-2, Bax, Bad,caspase-3,-9, PARP protein were detected by Western blot. Meanwhile, theapoptotic enzyme activity analysis of caspase-3,-9were done after the CPtreatment24hour in gastric cancer SGC-7901cell. To observe the function ofinhibiting gastric cancer cell proliferation and induce apoptosis by CP in vitro.
2The effects of CP on ERK1/2pathways in gastric cancer cell. Humangastric cancer SGC-7901cell were treated with40μmol/L CP. The expressionsof ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, MEK, p-MEK, Raf-1,p-Raf-1protein were detected by Western blot at0,0.5,1,3,612, and24hours. The expression of ERK1/2mRNA were detected by RT-PCR after40μmol/L CP treatment at0,6,12,24hours. Transfection experiment Cell isdivided into8group: CP (40μmol/L) group, PD98059(20μmol/L) group, CP(40μmol/L)+PD98059(20μmol/L) group, ERK siRNA (25nmol/L) group,control siRNA (25nmol/L) group, CP (40μmol/L)+control siRNA (25nmol/L)group, CP (40μmol/L)+ERK siRNA (25nmol/L) group and control group. Theproliferation activity of each group determined by MTT. FCM was used todetect sub-G1phase cell percentage in each group. Meanwhile, the apoptoticenzyme activity analysis of caspase-9were done in each group. Theexpressions of PARP, cleaved-PARP, ERK1/2, p-ERK1/2protein weredetected by Western blot.
3The effect of CP on gastric cancer nude mouse xenografts growth andits mechanism. Each of12intact female athymic nude mice (BABL/c, nu/nu,4week old,15-17g) was inoculated with SGC-7901cells (2×106/site)subcutaneously on the left axillary fossa. The animals were divided into twogroups (control and CP group). The CP group received CP (200μg/kg/day),while the control group was given vehicle (0.16%sodium bicarbonate) on day 5after inoculation and repeated daily for30day. The shortest and longestdiameter of the tumor were measured with slide gaud at3-day intervals, andtumor volume (mm3) was calculated using the following standard formula:(the longest diameter)×(the shortest diameter)2/2. On day30, mice weresacrificed and all tumor tissue samples were collected and weighed. The liverand kidneys were also dissected, fixed in10%formalin, stained withhematoxylin-eosin for histological examination. The expressions of PCNAand Ki67in tumor tissue were detected by immunohistochemistry.Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)was used to examine cell apoptosis in xenograft tissue. The expression ofERK1/2, pERK1/2, JNK, pJNK, p38, p-p38was assessed by Western blot.Pathological routine HE staining was used to observe the change in nudemouse transplantation tumor and liver and kidney.
Results：
1The effects of CP on gastric cancer cells growth.
1.1Cell growth inhibition were determined by MTT. With a determined byMTT method to observe the CP for different cell growth inhibition rate, theIC50of CP at48h was about40μmol/L. CP inhibited the growth of humangastric cancer cell lines (SGC-7901, BGC-823, MKN-45) in a dose-andtime-dependent manner after incubated with10-80μmol/L CP for24hours.The normal mucosa cells of stomach (GES-1) have no inhibition by CP.
1.2Cell cycle distribution and apoptosis were detected by flow cytometry. CPinduced apoptosis of gastric cancer SGC-7901cells. After incubation with CPfor24h, distinct apoptosis was observed on a DNA histogram as subdiploid orpre-G1peak, pre-G1peak was especially remarkable at24hours. Cell-cycleanalysis by flow cytometry showed that CP increased the proportion of cellsin the G2/M phase and decreased the proportion in the S phase of the cellcycle. Annexin V/PI double staining FCM analysis showed that annexin Vpositive and PI negative cells, which on behalf of apoptosis, graduallyincrease with the extension of time.
1.3The DNA fragments of cell apoptosis was determined by enzyme-linkedimmunosorbent assay (ELISA). After40μmol/L CP treatment for0,6,12,24hours, the DNA fragments of cell apoptosis was determined by ELISA. Theresults showed that DNA fragments increase gradually as the extension of CPincubation time, which reflect the apoptosis cells increase gradually。
1.4The effects of CP on protein expressions of Bcl-2, Bax, Bad, caspase-3,-9,PARP. After40μmol/L CP treatment for0,6,12,24hours,40μmol/L, theexpressions of Bax, Bad, cleaved caspase-3, cleaved caspase-9, PARP proteinwere increased gradually detected by Western blot. In contrast, the expressionlevel of Bcl-2protein decreased gradually.There are significant difference (P <0.05). The ratio of Bax to Bcl-2increased.
1.5The activity analysis of caspase-3,-9. The apoptotic enzyme activityanalysis of caspase-3,-9were done after the40μmol/LCP treatment24hourin gastric cancer SGC-7901cell. The activity of caspase-3, caspase-9wasincreased with time dependence (P <0.05).
2The effects of CP on ERK1/2pathways in gastric cancer cell.
2.1CP induced ERK1/2signal pathways activated in gastric cancer cell. After40μmol/L CP treatment for0,0.5,1,3,6,12,24hours, the expressions ofp-ERK1/2, p-raf-1, p-MEK1/2protein were increased gradually detected byWestern blot. The expressions of p-38and JNK protein which stand for theother MAPK signal pathways protein. In addition,40μmol/L CP significantlyup-regulated the expression of ERK1/2mRNA.
2.2PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP. After CP (40μmol/L), PD98059(20μmol/L) andERK siRNA (25nmol/L) alone or both combined incubation gastric cancerSGC-7901cells for24h, the cell viability was enhanced in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group than CP (40μmol/L) alone. So we can draw a conclusionthat PD98059and ERK1/2siRNA decreased the role of inhibition cellproliferation produced by CP.
2.3PD98059and ERK1/2siRNA inhibited cell apoptosis induced by CP.Compared with CP alone group, the proportion of sub-G1phase cells analyzed by flow cytometry were significantly decreased in CP(40μmol/L)+PD98059(20μmol/L) group and CP (40μmol/L)+ERK1/2siRNA(25nmol/L) group.
2.4CP, PD98059and ERK1/2siRNA influence the activity of caspase-9andexpression of PARP protein. The apoptotic enzyme activity analysis showedthat the caspase-9activity was decreased in CP+PD98059group andCP+ERK1/2siRNA group than CP alone group. Meanwhile, the expression ofPARP protein was decreased in CP+PD98059group and CP+ERK1/2siRNAgroup than CP alone group.
2.5CP, PD98059and ERK1/2siRNA affect ERK1/2protein expression. AfterCP (40μmol/L), PD98059(20μmol/L) and ERK siRNA (25nmol/L) alone orboth combined treatment gastric cancer SGC-7901cells for24h, theERK1/2signal pathways activated induced by CP were inhibited partially byPD98059and ERK1/2siRNA.
3The effect of CP on gastric cancer nude mouse xenografts growth and itsmechanism. The model of esophageal cancer xenograft in nude mice wassuccessfully established, the tumorigenic rate in nude mice injected withSGC-7901cells was100%. Intraperitoneal injection of CP significantlysuppressed tumor growth of the xenografts (P<0.01). On day9,15,21,27and30, the mean volume of the xenograft in the CP group was51±10.17mm3,254.17±38.78mm3,358.33±43.89mm3,462.5±52.22mm3and595±55.76mm3, respectively, significantly smaller than that (119.83±8.35mm3,299.66±30.81mm3,646.33±47.50mm3,1101.57±60.47mm3and1298.33±66.16mm3, correspondingly) in the control group. The tumor weight of theCP group was0.67±0.09g, significantly lower than that of the control group(1.10±0.16g)(P<0.05). Immunohistochemical detection showed that thePCNA and Ki-67expression of xenografts tissue in CP group is significantlylower than control group (P<0.05). Compared with the control group,apoptosis cells of CP group increased in xenografts tumor, both havesignificant difference (P<0.05). Compared with the control group, theexpression level of pERK1/2is higher in CP group, the expression level of JNK, pJNK, p38p-p38has no obvious change. Then observe two groups ofnude mice liver, kidney and no abnormal change.
Conclusions:
1CP was demonstrated to lead to a dose-and time-dependent inhibitionproliferation and inducing apoptosis in gastric cancer cells.
2CP may change the cell cycle distribution, inhibit expression of antiapoptotic proteins Bcl-2and raise caspase-3,-9protein expression which caninduce apoptosis and inhibit growth in gastric cancer cell.
3CP may activate ERK1/2signal pathway, increased phosphorylationprotein expression of ERK1/2.
4ERK1/2inhibitor PD98059and ERK1/2siRNA abrogated partially theability of growth and apoptosis induce by CP in gastric cancer cell
5Bisphosphonate CP can increase the expression of proteins involved inERK1/2signaling pathways in vitro and in vivo. ERK1/2signaling pathwaymay be involved in CP induced gastric cancer cell apoptosis.
6This experiment confirmed that the bisphosphonate CP can inhibitgrowth of gastric cancer nude mice subcutaneous transplantation tumor invivo, and no obvious side effects, and provides a theoretical basis forbisphosphonate CP in the chemical treatment of gastric cancer.
引文
1Berenson JR. Treatment of hypercalcemia of malignancy withBisphosphonates. Semin Oncol,2002,29:12~18
2Reszka AA, Rodan GA. Bisphosphonate mechanism of action. CurrRheumatol Rep,2003,5:65~74
3Yoneda T, Michigami T, Yi B, et al. Use of bisphosphonates for thetreatment of bone metastasis in experimental animail models. Cancer TreatRev,1999,25:293~299
4Nielsen OS, Munro AJ, Tannock IF. Bone metastases: Pathophysiologyand management policy. J Clin Oncol,1991,9(3):509
5Dominique H, Benjamin O, Francois G, et al. Bisphosphonates: newtherapeutic agents for the treatment of bone tumors. Trends in MolecularMed,2004,10(7):337~343
6Kohno N, Aogi K, Minami H, et al. Zoledronic acid significantly reducesskeletal complications compared with placebo in Japanese women withbone metastases from breast cancer: a randomized, placebo-controlled trial.J Clin Oncol,2005,23:3314~3321
7Luckman SP, Hughes DE, Coxon FP, et al. Nitrogen-containingbisphosphonates inhibit the mevalonate pathway and preventpost-translational prenylation of GTP-binding proteins, including Ras. JBone Miner Res,1998,13:581~589
8Van Beek E, Pieterman E, Cohen L, et al. Nitrogen-containingbisphosphonates inhibit isopentyl pyrophosphate isomerase/farnesylpyrophosphate synthase activity with relative potencies corresponding totheir antiresorptive potencies in vitro and in vivo. Biochem Biophys ResCommun,1999,255:491~494
9Shipman CM, Rogers MJ, Apperley JF, et al. Bisphosphonates induceapoptosis in human myeloma cell lines: a novel anti-tumour activity. Br JHaematol,1997,98:665~672
10Tassone P, Tagliaferri P, Viscomi C, et al. Zoledronic acid inducesantiproliferative and apoptotic effects in human pancreatic cancer cells invitro. Br J Cancer,2003,88:1971~1978
11Lee MV, Fong EM, Singer FR, et al. Bisphosphonate treatment inhibits thegrowth of prostrate cancer cells. Cancer Res,2001,61:2602~2608
12Senaratne SG, Pirianov G, Mansi JL, et al. Bisphosphonates induceapoptosis in human breast cancer cell lines. Br J Cancer,2000,82:1459~1468
13Pazianas M, Abrahamsen B, Eiken PA, et al. Reduced colon cancerincidence and mortality in postmenopausal women treated with an oralbisphosphonate. Danish National Register Based Cohort Study.Osteoporos Int,2012,23:2693~2701
14Bundred N. Antiresorptive therapies in oncology and their effects oncancer progression. Cancer Treat Rev,2012,38:776~786
15Rodrigues P, Hering FO, Meller A. Adjuvant effect of IV clodronate on thedelay of bone metastasis in high-risk prostate cancer patients: aprospective study. Cancer Res Treat,2011,43:231~235
16Daubine F, Lebot R, Marquez M, et al. Treatment of bone metastasis inprostate cancer: efficacy of a novel polybisphosphonate. Anticancer Res,2011,31:4141~4145
17Murray AW. Recycling the cell cycle: Cyclins revisited. Cell,2004,116:221~234
18Pietenpol JA, Stewart ZA. Cell cycle checkpoint signaling: Cell cyclearrest versus apoptosis. Toxicology,2002,181:475~481
19李文艺.细胞凋亡研究进展.畜牧与饲料科学.2010,31（11~12）：17~19
20Adams JM, Cory S. The Bcl-2protein family: arbiters of cell survival.Science,1998,281:1322~1326
21Mohanty IR, Arya DS, Gupta SK. Withania somnifera providescardioprotection and attenuates ischemia-reperfusion induced apoptosis.Clin Nutr,2008,27:635~642
22Tamm I, Schriever F, Dorken B. Apoptosis: implications of basic researchfor clinical oncology. Lancet Oncol,2001,2:33~42
23Scatena R, Bottoni P, Botta G, et a1. The role of mitochondria inpharmacotoxicology: a reevaluation of an old, newly emerging topic. Am JPhysiol Cell Physiol,2007,293(1):C12~21
24Hacker G, Paschen SA. Therapeutic targets in the mitochondrial apoptoticPathway. Expert Opin Ther Targets,2007,11:515~526
25Cryns V, Yuan J. Protease to die for. Genes Dev,1998,12(11):1551~1570
26Nicholson DW, Ali A, Thornberry NA, et a1. Identification and inhibitionof the ICE/CED-3protease necessary for mammalian apoptosis. Nature,1995,376:37~43
27Schreiber V, Danlzcr R, Ame JC, et al. Poly(ADP-ribose): novel functionsfor an old molecule. Nat Rev Mol Cell Biol,2006,7:517~528
28Jagtap C, Zabo S. Poly (ADP-ribose) polymerase and the therapeuticeffects of its inhibitors. Nat Rev Drug Discov,2005,4:421~440
29Virag L. Structure and function of poly(ADP-ribosc) polymerase-1: role inoxidative stress-related pathologies. Curr Vasc Pharmacol,2005,3:209~214
1Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature2001;410:37~40
2Hagan S, Garcia R, Dhillon A, et al. Raf kinase inhibitor protein regulationof raf and MAPK signaling. Methods Enzymol,2005,407:248~259
3Gong N, Li G, Xiao J, et al. Impact of MAPK cascade pathway and P53pathway upon liver transplant. J Huazhong Univ Sci Technolog Med Sci,2005,25(5):555~557
4Prashant KM, Narayana K, Neha S, et al. Interplay between MEK-ERKsignaling, cyclin D1, and cyclin-dependent kinase5regulates cell cyclereentry and apoptosis of neurons. Molecular Biology of the Cell,2012,23:3722~3730
5Chambon JP, Soule J, Pomies P, et al. Tail regression in Ciona intestinalis(Prochordate) involves a Caspase-dependent apoptosis event associatedwith ERK activation. Development,2002,129:3105~3114
6Stefanelli C, Tantini B, Fattori M, et al. Caspase activation inetoposide-treated fibroblasts is correlated to ERK phosphorylation andboth events are blocked by polyamine depletion. FEBS Lett,2002,527:223~228
7Tang D, Wu D, Hirao A, et al. ERK activation mediates cell cycle arrestand apoptosis after DNA damage independently of p53. J Biol Chem,2002,277:12710~12717
8Kim GS, Hong JS, Kim SW, et al. Leptin induces apoptosis viaERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells.J Biol Chem,2003,278:21920~21929
9Yang R, Piperdi S, Gorlick R. Activation of the RAF mitogen-activatedprotein extracellular signal-regulated kinase kinase extracellularsignal-regulated kinase pathway mediates apoptosis induced bychelerythrine in osteosarcoma. Clin Cancer Res,2008,14:6396~6404
10Park H, Im JY, Kim J, et al. Effects of apicidin，a histone deacetylaseinhibitor，on the regulation of apoptosis in H-ras-transformed breastepithelial cells．Int J Mol Med,2008,21(3):325~333
11Aprigliano I, Dudas J, Ramadori G, et al. Atorvastatin induces apoptosisby a caspase-9-dependent pathway: an in vitro study on activated rathepatic stellate cells. Liver Int,2008,28(4):546~557
12Kim YK, Kim HJ, Kwon CH, et al. Role of ERK activation incisplatin-induced apoptosis in OK renal epithelial cells. J Appl Toxicol,2005,25(5):374~382
13Hoda MR, Popken G. Mitogenic and anti-apoptotic actions ofadipocyte-derived hormone leptin in prostate cancer cells． BJU Int,2008,102(3):383~388
14Wang YF, Jiang CC, Kiejda KA, et al. Apoptosis induction in humanmelanoma cells by inhibition of MEK is caspase-independent andmediated by the Bcl-2family members PUMA, Bim, and Mcl-1. ClinCancer Res,2007,13(16):4934~4942
1Yamagishi S, Riichiro A, Yosuke I, et al. Minodronate, a newly developednitrogen-containing bisphosphonate, suppresses melanoma growth andimproves survival in nude mice by blocking vascular endothelial growthfactor signaling. American J of Pathology,2004,165:1865~1874
2Croucher PI, De Hendrik R, Perry MJ, et a1. Zoledronie acid treatment of5T2MM-bearing mice inhibits the development of myeloma bone disease：evidence for decreased osteolysis, tumor burden and angiogenesis, andincreased survival. J Bone Miner Res,2003,18(3):482~492
3Wong FW. Immunohistochemical antibody Ki67. Gynecol Obster,1994,37(2):123
4Wojciech S, Alicja Z. Proliferating cell nuclear antigen (PCNA): a keyfactor in DNA replication and cell cycle regulation.Annals of Botany,2011,107:1127~1140
5戴玲,刘淑俊,宛凤玲等，帕米磷酸二钠及埃本磷酸二钠对前列腺癌细胞系DU145-LNCaP体外生长的抑制作用.实用癌症杂志,2004,9(19):474~477
6Coxon JP, Oades GM, Kirby RS, et al. Zoledronic acid induces apoptosisand inhibits adhesion to mineralized matrix in prostate cancer cells viainhibition of protein prenylation. BJU Int,2004,94(1):164~170
7Cheng Y Y, Huang L, Lee K M, et al. Bisphosphonates induce apoptosis ofstromal tumor cells in giant cell tumor of bone. Calcif Tissue Int,2004,75(1):71~77
8Jagdev SP, Coleman RE, Shipman CM, et al. The bisphosphonate,zoledronic acid, induces apoptosis of breast cancer cells: evidence forsynergy with paclitaxel. Br J Cancer,2001,84:1126
9Ito M, Amizuka N, Nakajima T, et al. Ultrastructural and cytochemicalstudies on cell death of osteoclasts induced by bisphosphonate treat-ment.Bone1999;25:447~452
10Selander KS, Monkkonen J, Karhukorpi EK, et al. Characteristics ofclodronate-induced apoptosis in osteoclasts and macrophages. MolPharmacol,1996;50:1127~1138
11Swanson KM, Hohl RJ. Anti-cancer therapy: targeting the mevalonatepathway. Curr Cancer Drug Targets,2006,6(1):15~37
12张秀群.双膦酸盐在多发性骨髓瘤治疗中的免疫调节作用及抗肿瘤作用研究进展国外医学输血及血液分册,2002(25),3:225~257
13Hosfield DJ, ZhangY, Dougan DR, et al. Structural basis forbisphosphonate-mediated inhibition of isoprenoid biosynthesis. J BiolChem,2004,279(10):8526~8529
14林秀峰,俞惠新,谭成等.双膦酸盐对肺癌细胞增殖的影响.中国肺癌杂志,2005,8(6):510~513
15郑天虎，郭庆杰，王福梅等．细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡。现代肿瘤医学，2006，14(11):1341
1Berenson JR. Treatment of hypercalcemia of malignancy withBisphosphonates. Semin Oncol,2002,29:12~18
2Reszka AA, Rodan GA. Bisphosphonate mechanism of action. CurrRheumatol Rep,2003,5:65~74
3Yoneda T, Michigami T, Yi B, et al. Use of bisphosphonates for thetreatment of bone metastasis in experimental animal models. Cancer TreatRev,1999,25:293~299
4Nielsen OS, Munro AJ, Tannock IF. Bone metastases: Pathophysiologyand management policy. J Clin Oncol,1991,9(3):509
5Fleisch H, Russell RG, Francis MD. Diphosphonates inhibithydroxyapatite dissolution in vitro and bone resorption in tissue cultureand in vivo. Science,1969,165(3899):1262~1264
6Bukowski JF, Dascher CC, Das H. Alternative bisphosphonate targets andmechanisms of action. Biochem Biophys Res Commun,2005,328:746~750
7王娟，牛银波，孔祥鹤等．双膦酸盐对抗骨质疏松的研究进展.现代生物医学进展，2010，10(16):3131~3140
8Dominique H, Benjamin O, Francois G, et al. Bisphosphonates: newtherapeutic agents for the treatment of bone tumors. Trends in MolecularMed,2004,10(7):337~343
9Daubine F, LeGall C, Gasser J, et al. Antitumor effects of clinical dosingregimens of bisphosphonates in experimental breast cancer bonemetastasis. J Natl Cancer Inst,2007,99(4):322~330
10Nakajima H, Magae J, Tsuruga M, et al. Induction ofmitochondria-dependent apoptosis through the inhibition of mevalonatepathway in human breast cancer cells by YM529, a new third generationbisphosphonate. Cancer Letts,2007,253:89~96
11Sandrine B, Mercedes F, Olivier P, et al. Bisphosphonates inhibit breastand prostate carcinoma cell invasion, an early event in the formation ofbone metastases. Cancer Res,2000,60:2949~2954
12Almubarak H, Jones A, Chaisuparat R, et al. Zoledronic acid directlysuppresses cell proliferation and induces apoptosis in highly tumorigenicprostate and breast cancers. J Carcinog,2011,10:2
13R ikk nen J, M nkk nen H, Auriola S, et al. Mevalonate pathwayintermediates downregulate zoledronic acid-induced isopentenylpyrophosphate and ATP analog formation in human breast cancer cells.Biochem Pharmacol,2010,79(5):777~783
14Santini D, Martini F, Fratto ME, et al. In vivo effects of zoledronic acid onperipheral gamma delta T lymphocytes in early breast cancer patients.Cancer Immunol Immunother,2009,58(1):31~38
15Coscia M, Quaglino E, Iezzi M, et al. Zoledronic acid repolarizestumour-associated macrophages and inhibits mammary carcinogenesis bytargeting the mevalonate pathway. J Cell Mol Med,2010,14:2803~2815
16Benzaid I, Monkkonen H, Stresing V, et al. High phospho-antigen levels inbisphosphonate-treated human breast tumors promoteV{gamma}9V{delta}2T-cell chemotaxis and cytotoxicity in vivo. CancerRes,2011,71:4562~72
17Ma YG, Liu WC, Dong S, et al. Activation of BK(Ca) channels inzoledronic acid-induced apoptosis of MDA-MB-231breast cancer cells.Plos One,2012,7(5): e37451
18Misso G, Porru M, Stoppacciaro A, et al. Evaluation of the in vitro and invivo anti-angiogenic effects of denosumab and zoledronic acid. CancerBiol Ther,2012,13(14):1491~1500
19Green J, Clezardin P. The molecular basis of bisphosphonate activity: apreclinical perspective. Semin Oncol,2010,37(Suppl.1):S3~11
20Roelofs AJ, Hulley PA, Meijer A, et a1. Selective inhibition of Rabprenylation by a phosphonocarboxylate analogue of risedronate inducesapoptosis, but not S phase arrest, in human myeloma cells. Int J Cancer,2006,119(6):1254~1261
21Baulch BC, Molloy TJ, Yeh SL, et a1. Inhibitors of the mevalonatepathway as potential therapeutic agents in multiple myeloma. Leuk Res,2007,31(3):341~352
22Guenther A, Gordon S, Tiemann M, et a1. The bisphosphonate zoledronicacid has anti myeloma activity in vivo by inhibition of proteinprenylation．Int J Cancer,2010,126(1):239~246
23Croucher PI, De Hendrik R, Perry MJ, et a1. Zoledronie acid treatment of5T2MM-bearing mice inhibits the development of myeloma bone disease：evidence for decreased osteolysis，tumor burden and angiogenesis，andincreased survival．J Bone Miner Res,2003,18(3):482~492
24Corso A, Ferretti E, Lunghi M, et a1. Zoledronic acid down-regulatesadhesion molecules of bone marrow stromal cells in multiple myeloma：apossible mechanism for its antitumor effect. Cancer,2005,104(1):118~125
25Kunzmann V, Bauer E, Feurle J, et a1. Stimulation of gamma delta T cellsby aminobisphosphonates and induction of antiplasma cell activity inmultiple myeloma. Blood,2000,96(2):384~392
26Roelofs AJ, Jauhiainen M, Monkkonen H, et a1. Peripheral bloodmonocytes are responsible for gamma delta T cell activation induced byzoledronic acid through accumulation of IPP/DMAPP. Br J Haematol,2009,144(2):245~250
27Benzaid I, Monkkonen H, Stresing V, et a1. High phospho antigen levelsin bisphosphonate-treated human breast tumors promoteVgamma9Vdeha2T cell chemotaxis and cytotoxicity in vivo. Cancer Res,2011,71(13):4562~4572
28Abe Y, Muto M, Nieda M, et a1. Clinical and immunological evaluationof zoledronate-activated Vgamma9deha T-cell based immunotherapy forpatients with multiple myeloma. Exp Hematol,2009,37(8):956~968
29Coscia M, Quaglino E, Iezzi M, et a1. Zoledronic acid repolarizesTumour-associated macrophages and inhibits mammary carcinogenesis bytargeting the mevalonate pathway．J Cell Mol Med,2010,14(12)：2803~2815
30Eva C, Lisha GB, Janna EQ, et al. Zoledronic acid exhibits inhibitoryeffects on osteoblastic and osteolytic metastases of prostate cancer.Clinical Cancer Research,2003,9:295~306
31Asahi H, Mizokami A, Miwa S, et al. Bisphosphonate induces apoptosisand inhibits pro-osteoclastic gene expression in prostate cancer cells. Int JUrol,2006,13(5):593~600
32Coxon JP, Oades GM, Kirby RS, et al. Zoledronic acid induces apoptosisand inhibits adhesion to mineralized matrix in prostate cancer cells viainhibit ion of protein prenylation. BJU Int,2004,94(1):164~170
33Clezardin P, Fournier P, Boissier S, et al. In vitro and in vivo anti tumoreffects of bisphosphonates. Curr Med Chem,2003,10(2):173~180
34Green JR. Anti tumor effects of bisphosphonates. Cancer,2003,97(Suppl.3): S840~S847
35Dumon JC, Journe F, Kheddoumi N, et al. Cytostatic and apoptotic effectsof bisphosphonates on prostate cancer cells. Eur Urol,2004,45:521~528.
36Naoe M, Ogawa Y, Takeshita K, et al. Zoledronate stimulates gamma deltaT cells in prostate cancer patients. Oncol Res,2010,18(10):493~501
37Kubista B, Trieb K, Sevelda F, et al. Anticancer effects of zoledronic acidagainst human osteosareoma cells. J Orthop Res,2006,24:1145~1152
38Kubo T, Shimose S, MatsuoT, et al. Inhibitory effects of a newBisphosphonate, minodronateon, proliferation and invasion of a variety ofmalignant bone tumor cells. J Orthop Res,2006,24:1138~1144
39Tsang WP, Ho FY, Fung KP, et al. P53-RI75H mutant gains new functionin regulation of doxorubie in induced apoptosis. Int J Cancer,2005,114:331~336
40Cheng YY, Huang L, Lee KM, et al. Alendronate regulates cell invasionand MMP-2secretion in human osteosarcoma cell lines Pediatr BloodCancer,2004,42(5):410~415
41Clezardin P. Bisphosphonates’ antitumor activity: an unravelled side of amultifaceted drug class. Bone,2011,48:71~79
42Di Salvatore M, Orlandi A, Bagala C, et al. Anti-tumor andanti-angiogenetic effects of zoledronic acid on human non-small-cell lungcancer cell line. Cell Prolif,2011,44(2):139~146
43林秀峰,俞惠新,谭成等.双膦酸盐对肺癌细胞增殖的影响.中国肺癌杂志,2005,8(6):510~513
44Yamagishi S, Riichiro A, Yosuke I, et al. Minodronate, a newly developednitrogen-containing bisphosphonate, suppresses melanoma growth andimproves survival in nude mice by blocking vascular endothelial growthfactor signaling. Am J Pathol,2004,165:1865~1874
45Edwin MP, William LD, James G. The emerging role of bisphosphonatesin prostate cancer. Am J Ther,2004,11:60~73
46韩露艳,费素娟,陈复兴等。唑来膦酸对人末梢血γδT细胞杀伤胃癌细胞株SGC-7901作用的影响。世界华人消化杂志,2009,17(2):181~185
47韦金英，王烨，哈雪梅等。双膦酸盐类药物抑癌机制的初步研究。中国细胞生物学学报，2010，32(4):606-612
48Caraglia M, Santini D, Marra M, et al. Emerging anti cancer molecularmechanisms of aminobisphosphonates. Endocr Relat Cancer,2006,13(1):7~26
49Budman DR, Calabro A. Zoledronic acid (Zometa) enhances the cytotoxiceffect of gemcitabine and fluvastatin: in vitro isobologram studies withconventional and non conventional cytotoxic agents. Oncology,2006,70(2):147~153
50Eville-Webbe HL, Rostami-Hodjegan A, Evans CA, et al. Sequence andschedule dependent enhancement of zoledronic acid induced apoptosis bydoxorubicin in breast and prostate cancer cell. Int J Cancer,2005,113:364~371
51Neville-Webbe HL, E vans CA, Coleman RE, et al. Mechanisms of thesynergistic interaction between the bisphosphonate zoledronic acid and thechemotherapy agent paclitaxel in breast cancer cells in vitro. Tumour Biol,2006,27(2):92~103
52Michailidou M, Brown HK, Lefley DV, et al. Microvascular endothelialcell responses in vitro and in vivo: modulation by zoledronic acid andpaclitaxel? J Vasc Res,2010,47:481~493
53Ullen A, Lennartsson L, Harmenberg U, et al. Additive/synergisticantitumoral effects on prostate cancer cells in vitro following treatmentwith a combination of docetaxel and zoledronic acid. Acta Oncol,2005,44(6):644~650
54Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acidcombination triggers apoptosis synergistically through down regulatinganti apoptotic Bcl-2protein level in hormone-refractory prostate cancercells.Cell Biol Int,2009,33(2):239~246
55Clyburn RD, Reid P, Evans CA, et al. Increased anti-tumour effects ofdoxorubicin and zoledronic acid in prostate cancer cells in vitro:supporting the benefits of combination therapy. Cancer ChemotherPharmacol,2010,65:969~978
56Ottewell PD, M nkk nen H, Jones M, et al. Antitumor effects ofdoxorubicin followed by zoledronic acid in a mouse model of breastcancer. J Natl Cancer Inst,2008,100(16):1167~1178
57Neville-Webbe HL, Coleman RE, Holen I. Combined effects of thebisphosphonate, zoledronic acid and the aromatase inhibitor letrozole onbreast cancer cells in vitro: evidence of synergistic interaction. Br J Cancer,2010,102(6):1010~1017
58Koto K, Murata H, Kimura S, et al. Zoledronic acid inhibits proliferationof human fibrosarcoma cells with induction of apoptosis, and showscombined effects with other anticancer agents. Oncol Rep，2010，24(1):233~239
59Ozturk OH, Bozcuk H, Burgucu D, et al. Cisplatin cytotoxicity isenhanced with zoledronic acid in A549lung cancer cell line: preliminaryresults of an in vitro study. Cell Biol Int,2007,31(9):1069~1071
60Charehbili A, VandeVen S, Gerrit-Jan L, et al. NEOZOTAC:Efficacyresults from a phaseIII randomized trial with neoadjuvant chemotherapy(TAC) with or without zoledronic acid for patients with HER2-negativelarge resectable or stage II or III breast cancer(BC)-a Dutch Breast CancerTrialists' Group (BOOG) study. J Clin Oncol,2013,31(suppl):1028
61Winter MC, Wilson C, Syddall SP, et al. Neoadjuvant chemotherapy withor without zoledronic acid in early breast cancer-a randomized biomarkerpilot study. Clin Cancer Res,2013,19(10):2755~2765
62Coleman RE, Winter MC, Cameron D, et al. The effects of addingzoledronic acid to neoadjuvant chemotherapy on tumour response:exploratory evidence for direct anti-tumour activity in breast cancer. Br JCancer,2010,102(7):1099~1105
63Escudier B, Eisen T, Stadler WM, et al. Target Study Group. N.Engl.J.Med,2007,356:125~134
64Zekri J, Mansour M, Karim SY. The anti-tumour effects of zoledronic acid.J Bon Onco DOI:10.1016/j.jbo.2013.12.001
2Reszka AA, Rodan GA. Bisphosphonate mechanism of action. CurrRheumatol Rep,2003,5:65~74
3Yoneda T, Michigami T, Yi B, et al. Use of bisphosphonates for thetreatment of bone metastasis in experimental animail models. Cancer TreatRev,1999,25:293~299
4Nielsen OS, Munro AJ, Tannock IF. Bone metastases: Pathophysiologyand management policy. J Clin Oncol,1991,9(3):509
5Dominique H, Benjamin O, Francois G, et al. Bisphosphonates: newtherapeutic agents for the treatment of bone tumors. Trends in MolecularMed,2004,10(7):337~343
6Kohno N, Aogi K, Minami H, et al. Zoledronic acid significantly reducesskeletal complications compared with placebo in Japanese women withbone metastases from breast cancer: a randomized, placebo-controlled trial.J Clin Oncol,2005,23:3314~3321
7Luckman SP, Hughes DE, Coxon FP, et al. Nitrogen-containingbisphosphonates inhibit the mevalonate pathway and preventpost-translational prenylation of GTP-binding proteins, including Ras. JBone Miner Res,1998,13:581~589
8Van Beek E, Pieterman E, Cohen L, et al. Nitrogen-containingbisphosphonates inhibit isopentyl pyrophosphate isomerase/farnesylpyrophosphate synthase activity with relative potencies corresponding totheir antiresorptive potencies in vitro and in vivo. Biochem Biophys ResCommun,1999,255:491~494
9Shipman CM, Rogers MJ, Apperley JF, et al. Bisphosphonates induceapoptosis in human myeloma cell lines: a novel anti-tumour activity. Br JHaematol,1997,98:665~672
10Tassone P, Tagliaferri P, Viscomi C, et al. Zoledronic acid inducesantiproliferative and apoptotic effects in human pancreatic cancer cells invitro. Br J Cancer,2003,88:1971~1978
11Lee MV, Fong EM, Singer FR, et al. Bisphosphonate treatment inhibits thegrowth of prostrate cancer cells. Cancer Res,2001,61:2602~2608
12Senaratne SG, Pirianov G, Mansi JL, et al. Bisphosphonates induceapoptosis in human breast cancer cell lines. Br J Cancer,2000,82:1459~1468
13Pazianas M, Abrahamsen B, Eiken PA, et al. Reduced colon cancerincidence and mortality in postmenopausal women treated with an oralbisphosphonate. Danish National Register Based Cohort Study.Osteoporos Int,2012,23:2693~2701
14Bundred N. Antiresorptive therapies in oncology and their effects oncancer progression. Cancer Treat Rev,2012,38:776~786
15Rodrigues P, Hering FO, Meller A. Adjuvant effect of IV clodronate on thedelay of bone metastasis in high-risk prostate cancer patients: aprospective study. Cancer Res Treat,2011,43:231~235
16Daubine F, Lebot R, Marquez M, et al. Treatment of bone metastasis inprostate cancer: efficacy of a novel polybisphosphonate. Anticancer Res,2011,31:4141~4145
17Murray AW. Recycling the cell cycle: Cyclins revisited. Cell,2004,116:221~234
18Pietenpol JA, Stewart ZA. Cell cycle checkpoint signaling: Cell cyclearrest versus apoptosis. Toxicology,2002,181:475~481
19李文艺.细胞凋亡研究进展.畜牧与饲料科学.2010,31（11~12）：17~19
20Adams JM, Cory S. The Bcl-2protein family: arbiters of cell survival.Science,1998,281:1322~1326
21Mohanty IR, Arya DS, Gupta SK. Withania somnifera providescardioprotection and attenuates ischemia-reperfusion induced apoptosis.Clin Nutr,2008,27:635~642
22Tamm I, Schriever F, Dorken B. Apoptosis: implications of basic researchfor clinical oncology. Lancet Oncol,2001,2:33~42
23Scatena R, Bottoni P, Botta G, et a1. The role of mitochondria inpharmacotoxicology: a reevaluation of an old, newly emerging topic. Am JPhysiol Cell Physiol,2007,293(1):C12~21
24Hacker G, Paschen SA. Therapeutic targets in the mitochondrial apoptoticPathway. Expert Opin Ther Targets,2007,11:515~526
25Cryns V, Yuan J. Protease to die for. Genes Dev,1998,12(11):1551~1570
26Nicholson DW, Ali A, Thornberry NA, et a1. Identification and inhibitionof the ICE/CED-3protease necessary for mammalian apoptosis. Nature,1995,376:37~43
27Schreiber V, Danlzcr R, Ame JC, et al. Poly(ADP-ribose): novel functionsfor an old molecule. Nat Rev Mol Cell Biol,2006,7:517~528
28Jagtap C, Zabo S. Poly (ADP-ribose) polymerase and the therapeuticeffects of its inhibitors. Nat Rev Drug Discov,2005,4:421~440
29Virag L. Structure and function of poly(ADP-ribosc) polymerase-1: role inoxidative stress-related pathologies. Curr Vasc Pharmacol,2005,3:209~214
1Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature2001;410:37~40
2Hagan S, Garcia R, Dhillon A, et al. Raf kinase inhibitor protein regulationof raf and MAPK signaling. Methods Enzymol,2005,407:248~259
3Gong N, Li G, Xiao J, et al. Impact of MAPK cascade pathway and P53pathway upon liver transplant. J Huazhong Univ Sci Technolog Med Sci,2005,25(5):555~557
4Prashant KM, Narayana K, Neha S, et al. Interplay between MEK-ERKsignaling, cyclin D1, and cyclin-dependent kinase5regulates cell cyclereentry and apoptosis of neurons. Molecular Biology of the Cell,2012,23:3722~3730
5Chambon JP, Soule J, Pomies P, et al. Tail regression in Ciona intestinalis(Prochordate) involves a Caspase-dependent apoptosis event associatedwith ERK activation. Development,2002,129:3105~3114
6Stefanelli C, Tantini B, Fattori M, et al. Caspase activation inetoposide-treated fibroblasts is correlated to ERK phosphorylation andboth events are blocked by polyamine depletion. FEBS Lett,2002,527:223~228
7Tang D, Wu D, Hirao A, et al. ERK activation mediates cell cycle arrestand apoptosis after DNA damage independently of p53. J Biol Chem,2002,277:12710~12717
8Kim GS, Hong JS, Kim SW, et al. Leptin induces apoptosis viaERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells.J Biol Chem,2003,278:21920~21929
9Yang R, Piperdi S, Gorlick R. Activation of the RAF mitogen-activatedprotein extracellular signal-regulated kinase kinase extracellularsignal-regulated kinase pathway mediates apoptosis induced bychelerythrine in osteosarcoma. Clin Cancer Res,2008,14:6396~6404
10Park H, Im JY, Kim J, et al. Effects of apicidin，a histone deacetylaseinhibitor，on the regulation of apoptosis in H-ras-transformed breastepithelial cells．Int J Mol Med,2008,21(3):325~333
11Aprigliano I, Dudas J, Ramadori G, et al. Atorvastatin induces apoptosisby a caspase-9-dependent pathway: an in vitro study on activated rathepatic stellate cells. Liver Int,2008,28(4):546~557
12Kim YK, Kim HJ, Kwon CH, et al. Role of ERK activation incisplatin-induced apoptosis in OK renal epithelial cells. J Appl Toxicol,2005,25(5):374~382
13Hoda MR, Popken G. Mitogenic and anti-apoptotic actions ofadipocyte-derived hormone leptin in prostate cancer cells． BJU Int,2008,102(3):383~388
14Wang YF, Jiang CC, Kiejda KA, et al. Apoptosis induction in humanmelanoma cells by inhibition of MEK is caspase-independent andmediated by the Bcl-2family members PUMA, Bim, and Mcl-1. ClinCancer Res,2007,13(16):4934~4942
1Yamagishi S, Riichiro A, Yosuke I, et al. Minodronate, a newly developednitrogen-containing bisphosphonate, suppresses melanoma growth andimproves survival in nude mice by blocking vascular endothelial growthfactor signaling. American J of Pathology,2004,165:1865~1874
2Croucher PI, De Hendrik R, Perry MJ, et a1. Zoledronie acid treatment of5T2MM-bearing mice inhibits the development of myeloma bone disease：evidence for decreased osteolysis, tumor burden and angiogenesis, andincreased survival. J Bone Miner Res,2003,18(3):482~492
3Wong FW. Immunohistochemical antibody Ki67. Gynecol Obster,1994,37(2):123
4Wojciech S, Alicja Z. Proliferating cell nuclear antigen (PCNA): a keyfactor in DNA replication and cell cycle regulation.Annals of Botany,2011,107:1127~1140
5戴玲,刘淑俊,宛凤玲等，帕米磷酸二钠及埃本磷酸二钠对前列腺癌细胞系DU145-LNCaP体外生长的抑制作用.实用癌症杂志,2004,9(19):474~477
6Coxon JP, Oades GM, Kirby RS, et al. Zoledronic acid induces apoptosisand inhibits adhesion to mineralized matrix in prostate cancer cells viainhibition of protein prenylation. BJU Int,2004,94(1):164~170
7Cheng Y Y, Huang L, Lee K M, et al. Bisphosphonates induce apoptosis ofstromal tumor cells in giant cell tumor of bone. Calcif Tissue Int,2004,75(1):71~77
8Jagdev SP, Coleman RE, Shipman CM, et al. The bisphosphonate,zoledronic acid, induces apoptosis of breast cancer cells: evidence forsynergy with paclitaxel. Br J Cancer,2001,84:1126
9Ito M, Amizuka N, Nakajima T, et al. Ultrastructural and cytochemicalstudies on cell death of osteoclasts induced by bisphosphonate treat-ment.Bone1999;25:447~452
10Selander KS, Monkkonen J, Karhukorpi EK, et al. Characteristics ofclodronate-induced apoptosis in osteoclasts and macrophages. MolPharmacol,1996;50:1127~1138
11Swanson KM, Hohl RJ. Anti-cancer therapy: targeting the mevalonatepathway. Curr Cancer Drug Targets,2006,6(1):15~37
12张秀群.双膦酸盐在多发性骨髓瘤治疗中的免疫调节作用及抗肿瘤作用研究进展国外医学输血及血液分册,2002(25),3:225~257
13Hosfield DJ, ZhangY, Dougan DR, et al. Structural basis forbisphosphonate-mediated inhibition of isoprenoid biosynthesis. J BiolChem,2004,279(10):8526~8529
14林秀峰,俞惠新,谭成等.双膦酸盐对肺癌细胞增殖的影响.中国肺癌杂志,2005,8(6):510~513
15郑天虎，郭庆杰，王福梅等．细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡。现代肿瘤医学，2006，14(11):1341
1Berenson JR. Treatment of hypercalcemia of malignancy withBisphosphonates. Semin Oncol,2002,29:12~18
2Reszka AA, Rodan GA. Bisphosphonate mechanism of action. CurrRheumatol Rep,2003,5:65~74
3Yoneda T, Michigami T, Yi B, et al. Use of bisphosphonates for thetreatment of bone metastasis in experimental animal models. Cancer TreatRev,1999,25:293~299
4Nielsen OS, Munro AJ, Tannock IF. Bone metastases: Pathophysiologyand management policy. J Clin Oncol,1991,9(3):509
5Fleisch H, Russell RG, Francis MD. Diphosphonates inhibithydroxyapatite dissolution in vitro and bone resorption in tissue cultureand in vivo. Science,1969,165(3899):1262~1264
6Bukowski JF, Dascher CC, Das H. Alternative bisphosphonate targets andmechanisms of action. Biochem Biophys Res Commun,2005,328:746~750
7王娟，牛银波，孔祥鹤等．双膦酸盐对抗骨质疏松的研究进展.现代生物医学进展，2010，10(16):3131~3140
8Dominique H, Benjamin O, Francois G, et al. Bisphosphonates: newtherapeutic agents for the treatment of bone tumors. Trends in MolecularMed,2004,10(7):337~343
9Daubine F, LeGall C, Gasser J, et al. Antitumor effects of clinical dosingregimens of bisphosphonates in experimental breast cancer bonemetastasis. J Natl Cancer Inst,2007,99(4):322~330
10Nakajima H, Magae J, Tsuruga M, et al. Induction ofmitochondria-dependent apoptosis through the inhibition of mevalonatepathway in human breast cancer cells by YM529, a new third generationbisphosphonate. Cancer Letts,2007,253:89~96
11Sandrine B, Mercedes F, Olivier P, et al. Bisphosphonates inhibit breastand prostate carcinoma cell invasion, an early event in the formation ofbone metastases. Cancer Res,2000,60:2949~2954
12Almubarak H, Jones A, Chaisuparat R, et al. Zoledronic acid directlysuppresses cell proliferation and induces apoptosis in highly tumorigenicprostate and breast cancers. J Carcinog,2011,10:2
13R ikk nen J, M nkk nen H, Auriola S, et al. Mevalonate pathwayintermediates downregulate zoledronic acid-induced isopentenylpyrophosphate and ATP analog formation in human breast cancer cells.Biochem Pharmacol,2010,79(5):777~783
14Santini D, Martini F, Fratto ME, et al. In vivo effects of zoledronic acid onperipheral gamma delta T lymphocytes in early breast cancer patients.Cancer Immunol Immunother,2009,58(1):31~38
15Coscia M, Quaglino E, Iezzi M, et al. Zoledronic acid repolarizestumour-associated macrophages and inhibits mammary carcinogenesis bytargeting the mevalonate pathway. J Cell Mol Med,2010,14:2803~2815
16Benzaid I, Monkkonen H, Stresing V, et al. High phospho-antigen levels inbisphosphonate-treated human breast tumors promoteV{gamma}9V{delta}2T-cell chemotaxis and cytotoxicity in vivo. CancerRes,2011,71:4562~72
17Ma YG, Liu WC, Dong S, et al. Activation of BK(Ca) channels inzoledronic acid-induced apoptosis of MDA-MB-231breast cancer cells.Plos One,2012,7(5): e37451
18Misso G, Porru M, Stoppacciaro A, et al. Evaluation of the in vitro and invivo anti-angiogenic effects of denosumab and zoledronic acid. CancerBiol Ther,2012,13(14):1491~1500
19Green J, Clezardin P. The molecular basis of bisphosphonate activity: apreclinical perspective. Semin Oncol,2010,37(Suppl.1):S3~11
20Roelofs AJ, Hulley PA, Meijer A, et a1. Selective inhibition of Rabprenylation by a phosphonocarboxylate analogue of risedronate inducesapoptosis, but not S phase arrest, in human myeloma cells. Int J Cancer,2006,119(6):1254~1261
21Baulch BC, Molloy TJ, Yeh SL, et a1. Inhibitors of the mevalonatepathway as potential therapeutic agents in multiple myeloma. Leuk Res,2007,31(3):341~352
22Guenther A, Gordon S, Tiemann M, et a1. The bisphosphonate zoledronicacid has anti myeloma activity in vivo by inhibition of proteinprenylation．Int J Cancer,2010,126(1):239~246
23Croucher PI, De Hendrik R, Perry MJ, et a1. Zoledronie acid treatment of5T2MM-bearing mice inhibits the development of myeloma bone disease：evidence for decreased osteolysis，tumor burden and angiogenesis，andincreased survival．J Bone Miner Res,2003,18(3):482~492
24Corso A, Ferretti E, Lunghi M, et a1. Zoledronic acid down-regulatesadhesion molecules of bone marrow stromal cells in multiple myeloma：apossible mechanism for its antitumor effect. Cancer,2005,104(1):118~125
25Kunzmann V, Bauer E, Feurle J, et a1. Stimulation of gamma delta T cellsby aminobisphosphonates and induction of antiplasma cell activity inmultiple myeloma. Blood,2000,96(2):384~392
26Roelofs AJ, Jauhiainen M, Monkkonen H, et a1. Peripheral bloodmonocytes are responsible for gamma delta T cell activation induced byzoledronic acid through accumulation of IPP/DMAPP. Br J Haematol,2009,144(2):245~250
27Benzaid I, Monkkonen H, Stresing V, et a1. High phospho antigen levelsin bisphosphonate-treated human breast tumors promoteVgamma9Vdeha2T cell chemotaxis and cytotoxicity in vivo. Cancer Res,2011,71(13):4562~4572
28Abe Y, Muto M, Nieda M, et a1. Clinical and immunological evaluationof zoledronate-activated Vgamma9deha T-cell based immunotherapy forpatients with multiple myeloma. Exp Hematol,2009,37(8):956~968
29Coscia M, Quaglino E, Iezzi M, et a1. Zoledronic acid repolarizesTumour-associated macrophages and inhibits mammary carcinogenesis bytargeting the mevalonate pathway．J Cell Mol Med,2010,14(12)：2803~2815
30Eva C, Lisha GB, Janna EQ, et al. Zoledronic acid exhibits inhibitoryeffects on osteoblastic and osteolytic metastases of prostate cancer.Clinical Cancer Research,2003,9:295~306
31Asahi H, Mizokami A, Miwa S, et al. Bisphosphonate induces apoptosisand inhibits pro-osteoclastic gene expression in prostate cancer cells. Int JUrol,2006,13(5):593~600
32Coxon JP, Oades GM, Kirby RS, et al. Zoledronic acid induces apoptosisand inhibits adhesion to mineralized matrix in prostate cancer cells viainhibit ion of protein prenylation. BJU Int,2004,94(1):164~170
33Clezardin P, Fournier P, Boissier S, et al. In vitro and in vivo anti tumoreffects of bisphosphonates. Curr Med Chem,2003,10(2):173~180
34Green JR. Anti tumor effects of bisphosphonates. Cancer,2003,97(Suppl.3): S840~S847
35Dumon JC, Journe F, Kheddoumi N, et al. Cytostatic and apoptotic effectsof bisphosphonates on prostate cancer cells. Eur Urol,2004,45:521~528.
36Naoe M, Ogawa Y, Takeshita K, et al. Zoledronate stimulates gamma deltaT cells in prostate cancer patients. Oncol Res,2010,18(10):493~501
37Kubista B, Trieb K, Sevelda F, et al. Anticancer effects of zoledronic acidagainst human osteosareoma cells. J Orthop Res,2006,24:1145~1152
38Kubo T, Shimose S, MatsuoT, et al. Inhibitory effects of a newBisphosphonate, minodronateon, proliferation and invasion of a variety ofmalignant bone tumor cells. J Orthop Res,2006,24:1138~1144
39Tsang WP, Ho FY, Fung KP, et al. P53-RI75H mutant gains new functionin regulation of doxorubie in induced apoptosis. Int J Cancer,2005,114:331~336
40Cheng YY, Huang L, Lee KM, et al. Alendronate regulates cell invasionand MMP-2secretion in human osteosarcoma cell lines Pediatr BloodCancer,2004,42(5):410~415
41Clezardin P. Bisphosphonates’ antitumor activity: an unravelled side of amultifaceted drug class. Bone,2011,48:71~79
42Di Salvatore M, Orlandi A, Bagala C, et al. Anti-tumor andanti-angiogenetic effects of zoledronic acid on human non-small-cell lungcancer cell line. Cell Prolif,2011,44(2):139~146
43林秀峰,俞惠新,谭成等.双膦酸盐对肺癌细胞增殖的影响.中国肺癌杂志,2005,8(6):510~513
44Yamagishi S, Riichiro A, Yosuke I, et al. Minodronate, a newly developednitrogen-containing bisphosphonate, suppresses melanoma growth andimproves survival in nude mice by blocking vascular endothelial growthfactor signaling. Am J Pathol,2004,165:1865~1874
45Edwin MP, William LD, James G. The emerging role of bisphosphonatesin prostate cancer. Am J Ther,2004,11:60~73
46韩露艳,费素娟,陈复兴等。唑来膦酸对人末梢血γδT细胞杀伤胃癌细胞株SGC-7901作用的影响。世界华人消化杂志,2009,17(2):181~185
47韦金英，王烨，哈雪梅等。双膦酸盐类药物抑癌机制的初步研究。中国细胞生物学学报，2010，32(4):606-612
48Caraglia M, Santini D, Marra M, et al. Emerging anti cancer molecularmechanisms of aminobisphosphonates. Endocr Relat Cancer,2006,13(1):7~26
49Budman DR, Calabro A. Zoledronic acid (Zometa) enhances the cytotoxiceffect of gemcitabine and fluvastatin: in vitro isobologram studies withconventional and non conventional cytotoxic agents. Oncology,2006,70(2):147~153
50Eville-Webbe HL, Rostami-Hodjegan A, Evans CA, et al. Sequence andschedule dependent enhancement of zoledronic acid induced apoptosis bydoxorubicin in breast and prostate cancer cell. Int J Cancer,2005,113:364~371
51Neville-Webbe HL, E vans CA, Coleman RE, et al. Mechanisms of thesynergistic interaction between the bisphosphonate zoledronic acid and thechemotherapy agent paclitaxel in breast cancer cells in vitro. Tumour Biol,2006,27(2):92~103
52Michailidou M, Brown HK, Lefley DV, et al. Microvascular endothelialcell responses in vitro and in vivo: modulation by zoledronic acid andpaclitaxel? J Vasc Res,2010,47:481~493
53Ullen A, Lennartsson L, Harmenberg U, et al. Additive/synergisticantitumoral effects on prostate cancer cells in vitro following treatmentwith a combination of docetaxel and zoledronic acid. Acta Oncol,2005,44(6):644~650
54Karabulut B, Erten C, Gul MK, et al. Docetaxel/zoledronic acidcombination triggers apoptosis synergistically through down regulatinganti apoptotic Bcl-2protein level in hormone-refractory prostate cancercells.Cell Biol Int,2009,33(2):239~246
55Clyburn RD, Reid P, Evans CA, et al. Increased anti-tumour effects ofdoxorubicin and zoledronic acid in prostate cancer cells in vitro:supporting the benefits of combination therapy. Cancer ChemotherPharmacol,2010,65:969~978
56Ottewell PD, M nkk nen H, Jones M, et al. Antitumor effects ofdoxorubicin followed by zoledronic acid in a mouse model of breastcancer. J Natl Cancer Inst,2008,100(16):1167~1178
57Neville-Webbe HL, Coleman RE, Holen I. Combined effects of thebisphosphonate, zoledronic acid and the aromatase inhibitor letrozole onbreast cancer cells in vitro: evidence of synergistic interaction. Br J Cancer,2010,102(6):1010~1017
58Koto K, Murata H, Kimura S, et al. Zoledronic acid inhibits proliferationof human fibrosarcoma cells with induction of apoptosis, and showscombined effects with other anticancer agents. Oncol Rep，2010，24(1):233~239
59Ozturk OH, Bozcuk H, Burgucu D, et al. Cisplatin cytotoxicity isenhanced with zoledronic acid in A549lung cancer cell line: preliminaryresults of an in vitro study. Cell Biol Int,2007,31(9):1069~1071
60Charehbili A, VandeVen S, Gerrit-Jan L, et al. NEOZOTAC:Efficacyresults from a phaseIII randomized trial with neoadjuvant chemotherapy(TAC) with or without zoledronic acid for patients with HER2-negativelarge resectable or stage II or III breast cancer(BC)-a Dutch Breast CancerTrialists' Group (BOOG) study. J Clin Oncol,2013,31(suppl):1028
61Winter MC, Wilson C, Syddall SP, et al. Neoadjuvant chemotherapy withor without zoledronic acid in early breast cancer-a randomized biomarkerpilot study. Clin Cancer Res,2013,19(10):2755~2765
62Coleman RE, Winter MC, Cameron D, et al. The effects of addingzoledronic acid to neoadjuvant chemotherapy on tumour response:exploratory evidence for direct anti-tumour activity in breast cancer. Br JCancer,2010,102(7):1099~1105
63Escudier B, Eisen T, Stadler WM, et al. Target Study Group. N.Engl.J.Med,2007,356:125~134
64Zekri J, Mansour M, Karim SY. The anti-tumour effects of zoledronic acid.J Bon Onco DOI:10.1016/j.jbo.2013.12.001