Differential effects of Ca(2+) on bisphosphonate-induced growth inhibition in breast cancer and mesothelioma cells

Bisphosphonates are widely clinically used inhibitors of bone resorption. Pre-clinical studies indicate that bisphosphonates also inhibit the growth of various cancer cells in vitro, but their in vivo anti-cancer activity varies greatly, depending on the tumor type. We compared the various cellular effects of bisphosphonates in breast cancer and mesothelioma cells, with differences in growth inhibition responses to bisphosphonate-treatment in vivo. We show that the growth inhibitory effects of nitrogen-containing bisphosphonates are significantly affected by excess Ca(2+) in a cell- and bisphosphonate-specific fashion. Furthermore, excess pyrophosphate-resembling bisphosphonates prevent nitrogen-containing-bisphosphonate-induced accumulation of unprenylated Rap1A, p38 phosphorylation and growth inhibition in human MDA-MB-231 breast cancer and mouse AB-12 mesothelioma cells. For some, but not all tested, pyrophosphate-resembling bisphosphonate: nitrogen-containing bisphosphonate combinations these results may be partially explained by the ability of the excess pyrophosphate-resembling bisphosphonates to chelate Ca(2+). In mice, subcutaneous AB-12 and MDA-MB-231 tumors exhibit positive staining for Ca(2+) minerals, as revealed with Von Kossa stainings. We further show that the AB-12 tumors accumulate significantly more of the bone scanning bisphosphonate, Tc99m-medronate, as compared with MDA-MB-231 tumors. In conclusion, our results suggest that Ca(2+) regulates the growth inhibitory effects of bisphosphonates in a target cell and drug-specific fashion. These findings may be of physiological relevance since many tumor types are calcified. They further suggest that bisphosphonates can accumulate in tumors that are growing at the visceral sites and that differences in tumor accumulation of bisphosphonates may regulate their in vivo sensitivity to these drugs.     

Pamidronate inhibits antiapoptotic bcl-2 expression through inhibition of the mevalonate pathway in prostate cancer PC-3 cells

Bisphosphonates are expected to be efficacious to prevent the growth of metastatic cancer in bone tissue. Bone metastases often occur in patients with various cancers, such as breast, lung and prostate cancer. Bcl-2 is a potent antiapoptotic protein and its expression is known to be closely related to its function. In this study, to investigate the effect of bisphosphonates on cancer cells, we focused on bcl-2 expression in bisphosphonate-treated prostate cancer cells. First, we observed that bcl-2 mRNA expression in PC-3 was significantly inhibited to 12% of the control level by treatment with 100 μM pamidronate for 12 h. Inhibition was seen in cells treated with nitrogen-containing bisphosphonates, which have the ability to inhibit isoprenoid biosynthesis via the mevalonate pathway, but not in non-nitrogen-containing etidronate. Simultaneous treatment with geranylgeraniol, an intermediate of the mevalonate pathway, significantly blocked inhibition by pamidronate, and treatment with geranylgeranyl transferase inhibitor GGTI-286 also suppressed bcl-2 mRNA expression. Furthermore, pamidronate inhibited the translocation of Rap1 protein to the membrane fraction, suggesting that a change in posttranslational modification of Rap1 occurred in treated cells. Finally, knockdown of Rap1 by siRNA resulted in the inhibition of bcl-2 expression. These results strongly indicate that bcl-2 reduction in bisphosphonate-treated PC-3 cells is dependent on inhibition of the mevalonate pathway. The inhibitory effect of bisphosphonates on bcl-2 expression shown in prostate cancer cell line should be tested in animal experiments and clinical studies.     

Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells

Increasing evidence is accumulating that zoledronic acid (ZOL), a nitrogen-containing bisphosphonate (N-BP), is able to affect tumor cells by inhibiting the enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway (MVP). The consequent accumulation of unprenylated proteins is believed to largely account for the cytotoxic effects of ZOL. FPPS inhibition leads also to the accumulation of isopentenyl pyrophosphate (IPP) and the apoptotic ATP analog, ApppI, but the role of this mechanism in the cytotoxic action of bisphosphonates is less clear. Since treatment with MVP intermediates has been shown to overcome N-BP-induced apoptosis via rescuing protein prenylation, our aim here was to determine their mechanism of action on ZOL-induced IPP/ApppI accumulation.Interestingly, the results revealed that ZOL-induced IPP/ApppI accumulation in MCF-7 cells were decreased by farnesol, and almost completely blocked by geranylgeraniol and geranylpyrophosphate. The functionality of the regulatory enzymes of IPP and ApppI, IPP isomerase and aminoacyl-tRNA-synthase, respectively, or protein levels of FPPS were not affected by the treatments. However, the protein levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and unprenylated Rap1A were observed to be strongly downregulated by geranylgeraniol and geranylpyrophosphate.This study represents a novel insight into the mechanism of action of MVP intermediates on the regulation of MVP after FPPS inhibition. The data implies that in addition to the previously reported effects on rescuing protein prenylation, MVP intermediates can preserve cell activity by inhibiting the accumulation of IPP/ApppI via HMGR downregulation. This supports the hypothesis that IPP/ApppI formation is a significant mechanism in the anticancer action of ZOL.     

Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis

Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [14C]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran (but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [14C]zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [14C]zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.     

25-Methoxyhispidol A, a novel triterpenoid of Poncirus trifoliata, inhibits cell growth via the modulation of EGFR/c-Src signaling pathway in MDA-MB-231 human breast cancer cells

The fruit of Poncirus trifoliata (Rutaceae) has been used a medicinal food and traditional medicine. Recently we reported the isolation of 25-methoxyhispidol A (25-MHA) as a novel triterpenoid from the immature fruit of P. trifoliata with the potential growth inhibition of cancer cells. However, the molecular mechanisms on the anti-proliferative activity in cancer cells remain to be elucidated. In the present study, we investigated the anti-proliferative activity and mechanisms of actions mediated by 25-MHA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. 25-MHA exhibited the growth inhibitory activity against MDA-MB-231 cells with the cell cycle arrest in the G0/G1 phase. The cell cycle arrest in the G0/G1 by 25-MHA was well correlated with the downregulation of cyclin D1, cyclin dependent kinase (CDK4), CDK2, cyclin A, phosphorylated retinoblastoma protein (pRb), and induction of cdk inhibitor p21^WAF1/Cip1 protein. 25-MHA also suppressed the activation of c-Src/epidermal growth factor receptor (EGFR)/Akt signaling, and consequently led to the inactivation of mTOR and its downstream signal molecules including 4E-binding protein (4E-BP) and p70 S6 kinase. These findings suggest that 25-MHA-mediated inhibitory activity of human breast cancer cell growth might be related with the cell cycle arrest and modulation of signal transduction pathways.     

Anti-tumor potential of 15,16-dihydrotanshinone I against breast adenocarcinoma through inducing G1 arrest and apoptosis

Chemotherapeutic drugs are usually designed to induce cancer cell death via cell cycle arrest and/or apoptosis pathways. In this study, we used the chemical drug 15,16-dihydrotanshinone I (DHTS) to inhibit breast cancer cell proliferation and tumor growth, and investigate the underlying molecular mechanisms. Human breast cancer cell lines MCF-7 and MDA-MB-231 were both used in this study, and DHTS was found to significantly decrease cell proliferation by a dose-dependent manner in both cells. Flow cytometry indicated that DHTS induced G1 phase arrest in synchronous MCF-7 and MDA-MB-231 cells. When analyzing the expression of cell cycle-related proteins, we found that DHTS reduced cyclin D1, cyclin D3, cyclin E, and CDK4 expression, and increased CDK inhibitor p27 expression in a dose-dependent manner. In addition, DHTS inhibited the kinase activities of CDK2 and CDK4 by an immunocomplex kinase assay. In addition, DHTS also induced apoptosis in both cells through mainly mitochondrial apoptosis pathways. We found that DHTS decreased the anti-apoptotic protein Bcl-xL level and increased the loss of mitochondria membrane potential and the amount of cytochrome c released. Moreover, DHTS activated caspase-9, caspase-3, and caspase-7 and caused cell apoptosis. The fact that DHTS-induced apoptosis could be blocked by pretreating cells with pan-caspase inhibitor confirmed that it is mediated through activation of the caspase-3-dependent pathway. In a nude mice xenograft experiment, DHTS significantly inhibited the tumor growth of MDA-MB-231 cells. Taken together, these results suggest that DHTS can inhibit human breast cancer cell proliferation and tumor growth, and might have potential chemotherapeutic applications.     

活化Notch3信号通路抑制MDA-MB-231细胞的增殖

目的 活化Notch3信号可以通过上调p27的表达抑制MDA-MB-231细胞的增殖.方法 用脂质体转染法将质粒转入体外培养的MDA-MB-231细胞,用Western印迹法和RT-PCR检测Notch3和p27基因的表达用CCK-8检测细胞增殖率;用平板克隆检测细胞克隆形成率;用流式细胞仪检测细胞周期.结果 Western印迹法和RT-PCR检测结果显示,在MDA-MB-231细胞中表达Notch3后,p27的表达上调.过表达Notch3后的MDA-MB-231细胞增殖减慢,第3天起差异均有统计学意义(P＜0.01);克隆形成率降低,差异有统计学意义(P＜0.05);细胞周期阻滞在G0/G1期,转目的基因组G0/G1期细胞所占比例(72.47±1.75)％与对照组(60.16±3.29)％相比差异有统计学意义(P＜0.01).结论 活化的Notch3信号通路可以上调p27表达,使细胞周期阻滞于G0/G1期,抑制MDA-MB-231乳腺癌细胞的增殖.这一发现或许能为三阴性乳腺癌的分子靶向治疗提供新的思路.     

Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53

The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death.     

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.     

Farnesyl pyrophosphate synthase: real-time kinetics and inhibition by nitrogen-containing bisphosphonates in a scintillation assay

A mix-and-read FlashPlate (PerkinElmer, Waltham, MA) assay for the enzyme farnesyl pyrophosphate (FPP) synthase (FPPS) was developed to rapidly measure both steps in the synthesis of FPP from dimethylallyl pyrophosphate (DMAPP). The assay used either DMAPP or geranyl pyrophosphate (GPP) and [(3)H]isopentenyl pyrophosphate ([(3)H]IPP) as substrates, and measured the FPPS-catalyzed conversion of these into [(3)H]FPP or [(3)H]GPP by capturing the products onto a phospholipid-coated scintillating microtiter plate and monitoring the product formation in a charge coupled device imager. The Michaelis-Menten parameters-k(cat) GPP (38/min), K(m) IPP (0.6 microM), and K(m) GPP (0.7 microM)-were consistent with previous studies using difficult phase separation techniques. The 50% inhibitory concentrations of various nitrogen-containing bisphosphonates (N-BPs) were determined and were also consistent with prior literature. Without precedent, weaker inhibition (5 microM) of the non-N-BPs was also detected. In preincubation studies, the potency of the N-BPs, and specifically zoledronate, increased slowly over time by 100-fold. This potency shift was reversed significantly by the inclusion of GPP with zoledronate. Zoledronate was uncompetitive with respect to IPP. Thus, these studies were consistent with prior structural and thermodynamic studies, and suggest a rapid formation of a lower-affinity complex between zoledronate and the GPP binding site, followed by the formation of a very tight complex of zoledronate and enzyme, which excludes further binding of GPP. Furthermore, one of the substrates from the first step in the catalytic cycle, DMAPP, was identified as a 1 microM inhibitor of the second step of the catalysis, suggesting that the FPP two-step synthesis is regulated by DMAPP.     

野马追总黄酮的体内外抗乳腺癌活性及机制研究

目的 探究野马追总黄酮(TFE)的体内外抗乳腺癌活性及相关的作用机制。方法 MTT法检测TFE对乳腺癌细胞(MCF-7、T47D、MDA-MB-231、MDA-MB-468)和人正常乳腺上皮细胞MCF-10A增殖的影响;流式细胞术检测TFE对乳腺癌细胞周期和凋亡的影响;吖啶橙(AO)染色法检测TFE对细胞自噬的影响;Western blot检测细胞周期、凋亡、自噬相关蛋白的变化,以及PI3K/Akt信号通路相关蛋白的变化;建立MDA-MB-231细胞的裸鼠移植瘤模型,观察TFE的体内抗肿瘤活性。结果 TFE能明显抑制乳腺癌细胞增殖,并呈剂量依赖性,而对人正常乳腺上皮细胞无显著的抑制作用;TFE能增加G₂/M期细胞比例,上调p-cdc-2蛋白表达,下调cdc-2和Cyclin B1蛋白表达;TFE能显著增加乳腺癌细胞凋亡比例,上调促凋亡蛋白Bad和Bax表达,下调抗凋亡蛋白Bcl-2表达;TFV能够诱导乳腺癌细胞产生自噬,自噬相关蛋白LC3-II表达上升,p62表达降低;TFE能够抑制显著抑制PI3K和Akt的磷酸化水平;TFE能够在体内抑制裸鼠肿瘤的体积。结论 TFE通过抑制PI3K/Akt信号通路,阻滞细胞周期、诱导凋亡和自噬,进而在体内外抑制乳腺癌细胞的生长。     

Bisphosphonate-induced ATP analog formation and its effect on inhibition of cancer cell growth

Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 micromol/l zoledronic acid or 500 micromol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1-100 micromol/l zoledronic acid or 10-2000 micromol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.     

The anti-proliferative inhibition of ellipticine in human breast mda-mb-231 cancer cells is through cell cycle arrest and apoptosis induction

Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here we report the mechanism of apoptosis induction and cell cycle arrest by ellipticine in human breast MDA-MB-231 cancer cells. Ellipticine treatment arrested MDA-MB-231 cells at the G2/M phase after 6 h of treatment. This effect was strongly associated with a concomitant decrease in the level of cyclin B1, Cdc25 and Cdc2, and increase in phospho-Cdc2 (Tyr15). In addition, ellipticine also induced apoptosis in MDA-MB-231 cells, as determined by using both DNA fragmentation and Annexin-V staining assay. Ellipticine increased the expression of Bax, but decreased the level of Bcl-2, Bcl-XL and X-linked inhibitor of apoptosis protein (XIAP), and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c, and activation of caspase-9 and -3). In addition, pre-treatment of cells with caspase-9 inhibitor inhibited ellipticine-induced cell proliferation and apoptosis, indicating that caspase-9 activation was involved in MDA-MB-231 cell apoptosis induced by ellipticine. Taken together, our study suggests that the inhibition of cell cycle progression signaling and initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of ellipticine in MDA-MB-231 cells.     

Effects of phosphorylation of immunomodulatory agent FTY720 (fingolimod) on antiproliferative activity against breast and colon cancer cells

FTY720 (fingolimod), a novel immunosuppressant, was found to become biologically activated by phosphorylation into FTY720-1-phosphate (FTY720-P), which is a high-affinity agonist for sphingosine-1-phosphate (sphingosine-1-P)-receptors. FTY720 has also been reported to have a strong antitumor activity. The association between the phosphorylation of FTY720 and the growth inhibition of FTY720 against cancer cells are still not completely understood. In this study, we investigated the effects of FTY720, sphingosine, and their related compounds on the proliferation of human breast cancer cell lines (MCF-7, MDA-MB-231 and Sk-Br-3) and human colon cancer cell lines (HCT-116 and SW620). Non-phosphorylated FTY720, sphingosine and an FTY720 derivative, ISP-I-55, showed significant growth inhibition against these cells, with IC50 values of 5-20 microM at 48 h post-drug treatment. We confirmed that FTY720 induces the activation of a major mitogen-activated protein kinase, JNK, without the activation of p38 and down-regulation of phospho-ERK in MCF-7 breast cancer cells. In contrast, the phosphorylated derivatives, FTY720-P and sphingosine-1-P, as well as a phosphinane FTY720 derivative, cFTY720-P, did not inhibit the growth of the cells in the concentration range of 5-50 microM, whereas FTY720-P and sphingosine-1-P slightly induced the growth of MCF-7 cells. Combining FTY720 with dimethylsphingosine, a sphingosine kinase inhibitor, augmented the inhibitory effect of FTY720. These results indicate that the antiproliferative activity of FTY720 does not result from its phosphorylation, either endogenous or exogenous.     

New insights into the molecular mechanisms of action of bisphosphonates

Bisphosphonates are currently the most important and effective class of anti-resorptive drugs available, but the exact molecular mechanisms by which they inhibit osteoclast-mediated bone resorption have only recently been identified. Due to the targeting of bisphosphonates to bone mineral and the ability of osteoclasts to release bone-bound bisphosphonate, a direct effect on mature osteoclasts appears to be the most important route of action. As a result of recent discoveries concerning their molecular mechanism of action, bisphosphonates can be grouped into two classes. The simple bisphosphonates that closely resemble PPi (such as clodronate, etidronate and tiludronate) can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, resulting in induction of osteoclast apoptosis. By contrast, the more potent, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate and zoledronate) appear to act as analogues of isoprenoid diphosphate lipids, thereby inhibiting FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for the post-translational farnesylation and geranylgeranylation of small GTPase signalling proteins. Loss of bone-resorptive activity and osteoclast apoptosis is due primarily to loss of geranylgeranylated small GTPases. Identification of FPP synthase as the target of nitrogen-containing bisphosphonates has also helped explain the molecular basis for the adverse effects of these agents in the GI tract and on the immune system.     

Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231

The effects of berberine on the behavior of breast tumors have not yet been established. To determine whether this compound is useful in the treatment of breast cancer, we analyzed the impact of berberine on the human breast cancer cell lines MCF-7 and MDA-MB-231 cells. Berberine was added to proliferating MCF-7 and MDA-MB-231 cells in culture. Following treatment, changes in cell growth characteristics such as proliferation, cell cycle duration, and the degree of apoptosis were assayed. Following berberine treatment, a time-dependent reduction in proliferation was observed in both cell lines at differing concentrations: 20 microM for MCF-7 and 10 microM for MDA-MB-231 cells. Annexin V staining showed an increase in apoptosis in both cell lines of 31 % in MCF-7 and 12 % in MDA-MB-231 cells compared to their respective controls. In addition, 12 % of the MCF-7 cells were arrested at G0/G1, compared to 62 % of control cells. These results demonstrate that treatment with berberine inhibits growth in both MDA-MB-231 and MCF-7 cells. In addition, they show that this partly occurs through the induction of apoptosis in MDA-MB-231 cells, and through both cell cycle arrest and induction of apoptosis in MCF-7 cells. Thus, berberine may be a novel therapeutic drug for breast cancer.     

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G(2)/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G(2) exit and initiation of mitosis, namely the p34(CDC2)/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21(WAF), proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (DeltaPsi) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.     

海参粘多糖对肿瘤细胞介导的凝血过程的影响

目的探究海参粘多糖(hGAG)对MDA-MB-231乳腺癌细胞介导的凝血过程的影响及其作用机制。方法采用MDA-MB-231乳腺癌细胞为模型,观察经不同浓度的hGAG处理后的肿瘤细胞对血浆复钙时间的影响;采用S-2222发光底物检测凝血因子Xa(FXa)的生成;以Fluo-4/AM为荧光探针,在荧光酶标仪下检测细胞Ca2+的变化;流式细胞仪检测细胞表面组织因子(TF),采用real-time PCR和Westernblot的方法分别对TF的mRNA和蛋白水平进行测定;并观察hGAG对AKT、MAPK、NF-κB等信号通路的影响。结果hGAG 0.01～1μmol.L-1可以呈剂量依赖性的方式延长肿瘤细胞诱导的血浆复钙时间,并降低FXa的生成,对Ca2+无明显影响,而能够下调TF的mRNA和蛋白表达,抑制p38的磷酸化而对其它信号通路无明显影响。结论 hGAG通过下调TF的表达及p38的磷酸化而抑制MDA-MB-231细胞诱导的凝血过程。