The role of zoledronic acid in the adjuvant treatment of breast cancer: current perspectives

Importance of the field: Breast cancer is one of the leading causes of cancer-related deaths in the world. Despite the significant improvements in the adjuvant treatment strategies of early-stage breast cancer, many patients experience relapse. Bisphosphonates are widely used in the treatment of bone metastasis of solid tumors and multiple myeloma, as well as in osteoporosis. The results of clinical studies of adjuvant treatment on early-stage hormone-receptor-positive breast-cancer patients under hormonal treatment – especially with the bisphosphonate zoledronic acid – caused excitement because they demonstrated an additive effect on decreasing disease relapses at bone or other sites. A number of clinical and in vitro and in vivo preclinical studies, which are either ongoing or have just ended, are investigating the mechanisms and antitumoral activity of bisphosphonates.

Areas covered in this review: The current literature on the preclinical and clinical studies into antitumoral effect and adjuvant treatment of bisphosphonates, especially zoledronic acid, are summarized. Data in the literature over the last two decades were also reviewed.

What the reader will gain: The reader will find a summary of preclinical and clinical studies of antitumoral effect and adjuvant treatment with bisphosphonates, in particular zoledronic acid, as well as ongoing trials about adjuvant treatment of breast cancer with zoledronic acid and ibandronate.

Take home message: Current evidence supports zoledronic acid as an effective treatment in adjuvant breast-cancer therapy for hormone-receptor-positive breast-cancer patients when added to hormonotherapy. Uncertainty about effects of zoledronic acid and other bisphosphonates will be clarified after completion of ongoing trials.     

白花丹素的体外肾毒性及酸性成纤维细胞生长因子对白花丹素所致肾损伤的保护作用

目的：观察白花丹素在体外对人胚肾细胞293的毒性,以及酸性成纤维细胞生长因子（aFGF）对白花丹素所致肾损伤的保护作用。方法：采用四噻唑蓝（MTT）法检测不同浓度白花丹素对人胚肾细胞293的毒性,以及aFGF对白花丹素IC50值的影响,并测定培养上清液中超氧化物歧化酶（SOD）、丙二酰二醛（MDA）和乳酸脱氢酶（LDH）的活性。结果：白花丹素在体外对人胚肾细胞293的毒性呈现剂量依赖性,24、48和72 h的IC50值分别为（150.421±3.014）、（88.426±1.965）和（84.811±1.035）μg/mL。在aFGF作用下,24、48和72 h的IC50值分别升至（342.624±2.887）（、176.835±1.097）和（133.278±1.124）μg/mL。aFGF保护组与白花丹素损伤组的SOD、MDA和LDH测定结果有显著性差异（P〈0.05）。结论：白花丹素可抑制人胚肾细胞293的增殖,对肾脏有一定的毒性。aFGF对白花丹素所致肾损伤有明显的保护作用。     

Bornyl caffeate induces apoptosis in human breast cancer cells in vitro via the ROS- and JNK-mediated pathways

Aim： To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms.
Methods： Human breast cancer cell line MCF-7 and other tumor cell lines （T47D, HepG2, HeLa, and PC12） were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential （MMP） was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species （ROS） were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis.

Results： Bornyl caffeate （10, 25, and 50 μmol/L） suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 （p38 inhibitor）, SP600125 （JNK inhibitor）, z-VAD （pan-caspase inhibitor） or the thiol antioxidant L-NAC.

Conclusion： Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways.     

美登木素对人乳头状甲状腺癌B-CPAP细胞增殖和凋亡的作用及机制

目的 探讨美登木素对人乳头状甲状腺癌B-CPAP细胞增殖、凋亡的影响及作用机制。方法 用不同浓度（0.19、0.76、3.05、12.21、48.83、195.31、781.25、3 125 nmol/L）的美登木素溶液处理B-CPAP细胞,SRB法检测B-CPAP细胞增殖情况。将B-CPAP细胞分为对照组及美登木素（0.1、1、10 nmol/L）组,光学显微镜观察B-CPAP细胞形态变化;流式细胞术检测B-CPAP细胞凋亡情况;Western blotting法测定B-CPAP细胞中B淋巴细胞瘤-2（Bcl-2）、半胱氨酸天冬氨酸蛋白酶-3前体（Procaspase-3）的蛋白表达量。结果 与对照组比较,美登木素能有效抑制B-CPAP细胞增殖,且呈浓度及时间相关性;随美登木素浓度增加B-CPAP细胞凋亡率逐渐升高,Bcl-2和Procaspase-3蛋白表达水平下降（P＜0.05）,呈明确的浓度相关性。结论 美登木素具有抑制B-CPAP细胞增殖及诱导凋亡的作用,其机制可能与调控Bcl-2和Procaspase-3蛋白有关。     

纳米雄黄对MCF-7乳腺癌干细胞的体外抑制作用

目的 研究纳米雄黄对乳腺癌干细胞的体外抑制作用.方法 以人乳腺癌MCF-7亲本细胞为对象,采用无血清培养法培养获得乳腺癌干细胞.以阿霉素(1 mg/L)为阳性对照,以相同质量浓度的水飞雄黄为参照,采用CCK-8法检测纳米雄黄对MCF-7亲本细胞及干细胞增殖的影响;借助悬浮球形成及分化实验、划痕实验、Transwell侵...     

Recombination adenovirus-mediated human lactoferrin cDNA inhibits the growth of human MCF-7 breast cancer cells

Objectives Human lactoferrin, an 80 kDa iron‐binding glycoprotein, has antitumour effects. We have explored the potential therapeutic role of re‐expressing human lactoferrin gene product in human breast cancer. Methods A recombinant adenovirus expressing the human lactoferrin cDNA (ad‐hLTF) was constructed and used to infect breast cancer cells. Key findings Seventy‐two hours after infection, ad‐hLTF had considerable cytotoxicity on MCF‐7 cells. A time‐course study showed that ad‐hLTF infection of MCF‐7 cells at 100 plaque‐forming units per cell increased the number of cells in G₀/G₁ phase and appeared markedly at Sub‐G₁ apoptotic peak. The presence of apoptotic cells was confirmed using Annexin V‐fluoresecein isothiocyanate apoptosis detection by flow cytometry. Ad‐hLTF also resulted in a decrease of Bcl‐2 protein and an increase in Bax protein. Conclusions Ad‐hLTF plays an important role in the induction of cell cycle arrest and apoptosis in MCF‐7 cells. The results demonstrated that ad‐hLTF could have potential benefits in the treatment of breast cancer.     

Allitridi induces apoptosis by affecting Bcl-2 expression and caspase-3 activity in human gastric cancer cells

INTRODUCTION Gastric cancer is the second leading cause of can-cer mortality in the world and is the leading cause ofcancer mortality in China[1]. Numerous epidemiologicalinvestigations have demonstrated that an inverse corre-lation between gastric cancer incidence and garlic con-sumption[2-8]. For example, Linqu County has gastriccancer rate that was 15 times higher than that of Cang-shan County in Shangdong Province[10], and the deathrate of gastric cancer in Linqu County was 7…     

化疗药体外干预诱导人乳腺癌细胞产生耐药性的研究

目的研究化疗药物高浓度冲击法和低浓度干预对乳腺癌细胞MDA-MB-231多药耐药性的影响。方法采用化疗药物紫杉醇、阿霉素高浓度冲击和紫杉醇低浓度短期干预MDA-MB-231细胞,SRB法检测细胞药敏性改变;HE染色检测细胞形态;免疫细胞化学检测P-gp蛋白表达变化;RT-PCR检测MDR1基因表达。结果阿霉素高浓度冲击后细胞未存活,紫杉醇高浓度冲击后细胞能存活并最终稳定生长(MDA-MB-231/a),SRB法检测IC50发现该细胞药敏性与野生型细胞(MDA-MB-231/w)无差异;紫杉醇低浓度短期干预后(MDA-MB-231/b)SRB法检测发现该细胞对多种化疗药的IC50比野生型略有提高;免疫细胞化学显示MDA-MB-231/aP-gp表达与野生型无差异,MDA-MB-231/bP-gp表达略有提高;RT-PCR检测发现MDA-MB-231/b中MDR1基因表达高于MDA-MB-231/w。结论化疗药物高浓度冲击法无法获得MDA-MB-231的耐药细胞,低浓度短期干预可以略微提高细胞的耐药性,提示低浓度持续诱导法可以获得MDA-MB-231的耐药细胞。     

来那替尼对人乳腺癌细胞增殖、凋亡的影响及其作用机制

目的:探讨来那替尼对人乳腺癌细胞增殖、凋亡的影响及其作用机制。方法:以0,0.5,1和2 μmol·L^-1四种浓度的来那替尼处理人乳腺癌细胞BT474,MTT法检测其对细胞增殖的影响;流式细胞术检测其对细胞生长周期及凋亡的影响;蛋白免疫印迹法检测其对细胞凋亡信号通路相关蛋白的表达水平。结果:MTT实验结果表明来那替尼可显著抑制人乳腺癌细胞的增殖,2 μmol·L^-1来那替尼处理96 h后,其细胞存活率为31%;流式细胞分析结果表明来那替尼可将BT474细胞阻滞在G₀/G₁期,2 μmol·L^-1来那替尼阻滞效果最为明显,处于G₀/G₁期的细胞百分比为76%;来那替尼也可促进细胞凋亡,2 μmol·L^-1剂量时凋亡率为19.3%。蛋白免疫印迹结果表明来那替尼可减弱细胞内AKT、mTOR的磷酸化蛋白表达水平。结论:来那替尼可能通过抑制细胞内PI3K-AKT-mTOR信号通路促进乳腺癌细胞凋亡,为乳腺癌的治疗提供理论依据。     

The combination of baicalin and baicalein enhances apoptosis via the ERK/p38 MAPK pathway in human breast cancer cells

Aim:

To examine whether the cell growth inhibitory effect of the combination of baicalin and baicalein is related to apoptosis. Moreover, to determine whether the expression of some apoptosis-related proteins is regulated by the ERK/p38 MAPK pathway.

Methods:

Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by acridine orange (AO) staining, DNA ladder assay and flow cytometric analysis. Apoptosis-related proteins were observed using Western blot analysis.

Results:

Compared with baicalin or baicalein alone, the combination treatment of baicalin (50 μmol/L) and baicalein (25 μmol/L) had an anti-proliferative effect in a time-dependent manner. Isobologram analysis demonstrated that the combination treatment had a synergistic effect. Moreover, apoptosis in MCF-7 cells was increased by 12% and 20% with the combination treatment at 24 h and 48 h, respectively. With the combination treatment in MCF-7 cells, cleaved caspase-3 and caspase-9 were observed, and the level of bcl-2 expression was decreased approximately 20% and 40% at 24 h and 48 h, respectively. The expression of bax and p53 were increased about 25% and 15% at 48 h, respectively. Moreover, the activation of caspase-3, -9 and the regulation of bcl-2, bax and p53 were related to ERK /p38 MAPK activation.

Conclusion:

In this study, apoptosis was enhanced by the combination treatment of baicalin and baicalein, which activated caspases-3 and caspase-9, downregulated the level of bcl-2 and upregulated the level of bax or p53 via the ERK/p38 MAPK pathway.     

Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell lung cancer cells in vitro

Aim： To assess the synergistic actions of lidamycin （LDM） and chloroquine （CQ）, a lysosomal enzyme inhibitor, in human non-small cell lung cancer （NSCLC） cells, and to elucidate the potential mechanisms. Methods： Human NSCLC cell lines A549 and H460 were treated with CQ and/or LDM. Cell proliferation was analyzed using MTI- assay and apoptosis was quantified using flow cytometry. Western blotting was used to detect the protein levels of caspase 3, PARP, Bcl-2, Bax, p53, LC3-1 and LC3-11. A H460 cell xenograft model in BALB/c nude mice was used to evaluate the anticancer efficacy of CQ and LDM in vivo. Results： Both LDM and CQ concentration-dependently suppressed the proliferation of A549 and H460 ceils in vitro （the ICso values of LDM were 1.70±0.75 and 0.043±0.026 nmol/L, respectively, while the IC50 values of CQ were 71.3±6.1 and 55.6±12.5 pmol/L, respectively）. CQ sensitized both NSCLC cell lines to LDM, and the majority of the coefficients of drug interaction （CDIs） for combination-doses were less than 1. The ratio of apoptosis of H460 cells induced by a combined treatment of CQ and LDM （77.0%±5.2%） was significantly higher than those caused by CQ （23.1%±4.2%） or by LDM （65.1%±4.1%） alone. Furthermore, the combined treatment markedly increased the cleaved PARP and cleaved caspase 3 in H460 cells, which were partly reversed by pretreatment with the caspase inhibitor zVAD.fmk, zVAD.fmk also partially reversed the inhibitory effect of the combination treatment on the proliferation of H460 cells. The combination therapy group had a notable increase in expression of Bax and a very slight decrease in expression of Bcl-2 and p53 protein. LDM alone scarcely affected the level of LC3-11 in H460 cells, but slightly reduced CQ-induced LC3-11 expression. 3-MA, an autophagy inhibitor also sensitized H460 cells to LDM. In nude mice bearing H460 cell xenograft, administration of LDM （25 pg/kg, iv） and CQ （60 mg/kg, ip） suppressed tumor growth by 57.14% and 73.02%, respectively. Conclusion： The synergistic anticancer effect of LDM and CQ in vitro results from activation of a caspase-dependent and p53- independent apoptosis pathway as well as inhibition of cytoprotective autophagy.     

RIP3 overexpression sensitizes human breast cancer cells to parthenolide in vitro via intracellular ROS accumulation

Aim： Receptor-interacting protein 3 （RIP3） is involved in tumor necrosis factor receptor signaling, and results in NF-KB-mediated prosurvival signaling and programmed cell death. The aim of this study was to determine whether overexpression of the RIP3 gene could sensitize human breast cancer cells to parthenolide in vitro. Methods： The expression of RIP3 mRNA in human breast cancer cell lines （MCF-7, MDA-MB-231, MDA-MB-435 and T47D） was detected using RT-PCR. Both MDA-MB-231 and MCF-7 cells were transfected with RIP3 expression or blank vectors via lentivirus. Cell viability was measured with MTT assay; intracellular ROS level and cell apoptosis were analyzed using flow cytometry. Results： RIP3 mRNA expression was not detected in the four human breast cancer cell lines tested. However, the transfection induced higher levels of RIP3 protein in MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of RIP3 decreased the IC50 values of parthenolide from 17.6 to 12.6 μmol/L in MCF-7 cells, and from 16.6 to 9.9 μmol/L in MDA-MB-231 cells. Moreover, overexpression of RIP3 significantly increased parthenolide-induced apoptosis and ROS accumulation in MCF-7 and MDA-MB-231 cells. Pretreatment with N-acetyl-cysteine abrogated the increased sensitivity of RIP3-transfected MCF-7 and MDA-MB-231 cells to parthenolide. Conclusion： Overexpression of RIP3 sensitizes MCF-7 and MDA-MB-231 breast cancer cells to parthenolide in vitro via intracellular ROS accumulation.     

红三叶异黄酮对人宫颈癌HeLa细胞的抑制作用

目的探讨红三叶异黄酮对人宫颈癌HeLa细胞的抑制作用及其作用机制。方法以MTT法检测红三叶异黄酮20、40、80μg/m L分别在24、48、72 h对人宫颈癌HeLa细胞增殖作用的影响,并在倒置显微镜下观察HeLa细胞形态学改变。流式细胞仪检测红三叶异黄酮20、40、80μg/m L对HeLa细胞凋亡的影响。实时荧光定量PCR仪检测红三叶异黄酮20、40、80μg/m L对HeLa细胞凋亡中调节基因Bax、Bcl-2表达水平的影响。结果红三叶异黄酮对人宫颈癌HeLa细胞的增殖抑制作用,表现为时间、浓度的相关性。红三叶异黄酮对HeLa细胞的形态学改变,表现为随药物浓度增加细胞固缩变小,并出现凋亡小体。流式细胞仪检测分析显示红三叶异黄酮能引起HeLa细胞凋亡,并随药物浓度的增加而明显,当红三叶异黄酮达到一定有效浓度时,诱导其细胞凋亡。通过调节Bax mRNA与Bcl-2 mRNA的表达,促进抑制HeLa细胞增殖,能使Bax表达上升、Bcl-2表达下降,且呈剂量相关性。结论红三叶异黄酮对人宫颈癌HeLa细胞的增殖具有抑制作用,并呈一定的剂量和时间相关性,提示通过调节Bax mRNA与Bcl-2 mRNA的表达,促进抑制HeLa细胞增殖。     

Environmentally relevant concentration of arsenic trioxide and humic acid promoted tumor progression of human cervical cancer cells: In vivo and in vitro studies

In a previous study, treatment at higher concentrations of arsenic trioxide or co‐exposure to arsenic trioxide and humic acid was found to be inhibited cell growth of cervical cancer cells (SiHa cells) by reactive oxygen species generation. However, treatment at lower concentrations slightly increased cell viability. Here, we investigate the enhancement of progression effects of environmentally relevant concentration of humic acid and arsenic trioxide in SiHa cell lines in vitro and in vivo by measuring cell proliferation, migration, invasion, and the carcinogenesis‐related protein (MMP‐2, MMP‐9, and VEGF‐A) expressions. SiHa cells treated with low concentrations of humic acid and arsenic trioxide alone or in co‐exposure significantly increased reactive oxygen species, glutathione levels, cell proliferation, scratch wound‐healing activities, migration abilities, and MMP‐2 expression as compared to the untreated control. In vivo the tumor volume of either single drug (humic acid or arsenic trioxide) or combined drug‐treated group was significantly larger than that of the control for an additional 45 days after tumor cell injection on the back of NOD/SCID mice. Levels of MMP‐2, MMP‐9, and VEGF‐A, also significantly increased compared to the control. Histopathologic effects of all tumor cells appeared round in cell shape with high mitosis, focal hyperkeratosis and epidermal hyperplasia in the skin, and some tumor growth in the muscle were observed. Our results may indicate that exposure to low concentrations of arsenic trioxide and humic acid is associated with the progression of cervical cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1121–1132, 2016.     

Synergistic anti-cancer activity of the combination of dihydroartemisinin and doxorubicin in breast cancer cells

BackgroundDihydroartemisinin (DHA) exhibits potent anti-malarial and anti-cancer activities. This study aimed to investigate the anti-proliferative effects of a combination of DHA and doxorubicin (DOX) on human breast cancer cells.MethodsMTT assay and the combination index (CI) were used to show the anti-proliferative effects and calculate the synergism potential, respectively. Flow cytometry assay was used to detect apoptosis and the intracellular accumulation of DOX. JC-1 staining was used to determine the mitochondrial membrane potential. Western blot analysis was used to detect the protein expression of some apoptosis-related molecules.ResultsAsynergistic anti-proliferative effect was found, and the enhanced anti-cancer activity was observed to be accompanied by the prompt onset of apoptosis in MCF-7 cells. The combinative treatment remarkably decreased the mitochondrial membrane potential and activated caspase cascades more than the mono-treatment. Pretreatment with DHAalso did not influence the accumulation of DOX in MCF-7 cells.ConclusionThis study presented a new opportunity to enhance the effectiveness of future treatment regimens of breast cancer using DOX.     

S-allylcysteine,a garlic derivative,suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro

Aim： To investigate the effects of S-allylcysteine （SAC）, a water-soluble garlic derivative, on human ovarian cancer cells in vitro. Methods： Human epithelial ovarian cancer cell line A2780 was tested. Cell proliferation was examined with CCK-8 and colony formation assays. Cell cycle was analyzed with flow cytometry. Cell apoptosis was studied using Hoeohst 33258 staining and Annexin V/PI staining with flow cytometry. The migration and invasion of A2780 cells were examined with transwell and wound healing assays. The expression of relevant proteins was detected with Western blot assays. Results： SAC （1-100 mmol/L） inhibited the proliferation of A2780 cells in dose- and time-dependent manners （the ICsovalue was approximately 25 mmoVL at 48 h, and less than 6.25 mmol/L at 96 h）. Furthermore, SAC dose-dependently inhibited the colony formation of A2780 cells. Treatment of A2780 cells with SAC resulted in G/S phase arrest and induced apoptosis, accompanied by decreased expression of pro-caspase-3, Parp-1 and Bcl-2, and increased expression of active caspase-3 and Bax. SAC treatment significantly reduced the migration of A2780 cells, and markedly decreased the protein expression of Wnt5a, p-AKT and c-Jun, which were the key proteins involved in proliferation and metastasis. Conclusion： SAC suppresses proliferation and induces apoptosis in A2780 ovarian cancer cells in vitro.