The indole-3-carbinol cyclic tetrameric derivative CTet inhibits cell proliferation via overexpression of p21/CDKN1A in both estrogen receptor-positive and triple-negative breast cancer cell lines

Introduction

Indole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3''-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in γ-cyclodextrin (γ-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study.

Methods

Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored.

Results

CTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 μM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters.

Conclusions

Our data support CTet formulated with γ-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.     

3-bromopyruvate enhanced daunorubicin-induced cytotoxicity involved in monocarboxylate transporter 1 in breast cancer cells

Increasing evidence demonstrates that the hexokinase inhibitor 3-bromopyruvate (3-BrPA) induces the cell apoptotic death by inhibiting ATP generation in human cancer cells. Interestingly, some tumor cell lines are less sensitive to 3-BrPA-induced apoptosis than others. Moreover, the molecular mechanism of 3-BrPA-trigged apoptosis is unclear. In the present study, we examined the effects of 3-BrPA on the viability of the breast cancer cell lines MDA-MB-231 and MCF-7. We further investigated the potential roles of monocarboxylate transporter 1 (MCT1) in drug accumulation and efflux of breast cancer cells. Finally, we explored whether 3-BrPA enhanced daunorubicin (DNR)-induced cytotoxicity through regulation of MCT1 in breast cancer cells. MTT and colony formation assays were used to measure cell viability. Western blot analysis, flow cytometric analysis and fluorescent microscopy were used to determine the molecular mechanism of actions of MCT1 in different breast cancer cell lines. Whole-body bioluminescence imaging was used to investigate the effect of 3-BrPA in vivo. We found that 3-BrPA significantly inhibited cell growth and induced apoptosis in MCF-7 cell line, but not in MDA-MB-231 cells. Moreover, we observed that 3-BrPA efficiently enhanced DNR-induced cytotoxicity in MCF-7 cells by inhibiting the activity of ATP-dependent efflux pumps. We also found that MCT1 overexpression increased the efficacy of 3-BrPA in MDA-MB-231 cells. 3-BrPA markedly suppressed subcutaneous tumor growth in combination with DNR in nude mice implanted with MCF-7 cells. Lastly, our whole-body bioluminescence imaging data indicated that 3-BrPA promoted DNR accumulation in tumors. These findings collectively suggest that 3-BrPA enhanced DNR antitumor activity in breast cancer cells involved MCT-1, suggesting that inhibition of glycolysis could be an effective therapeutic approach for breast cancer treatment.     

CIP2A is a target of bortezomib in human triple negative breast cancer cells

Introduction

Triple negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets, such as hormone receptors or human epidermal growth factor receptor type 2 (HER2); therefore, prognosis is poor. Bortezomib, a proteasome inhibitor, may exert efficacy in TNBC through its multiple cellular effects. Here, we tested the efficacy of bortezomib and examined the drug mechanism in breast cancer cells.

Methods

Five breast cancer cell lines: TNBC HCC-1937, MDA-MB-231, and MDA-MB-468; HER2-overexpressing MDA-MB-453; and estrogen receptor positive MCF-7 were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western Blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interfering RNA (siRNA). In vivo efficacy of bortezomib was tested in nude mice with breast cancer xenografts. Immunohistochemical study was performed on tumor tissues from patients with TNBC.

Results

Bortezomib induced significant apoptosis, which was independent of its proteasome inhibition, in the three TNBC cell lines, but not in MDA-MB-453 or MCF-7 cells. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. We showed that bortezomib inhibited CIP2A in association with p-Akt downregulation in a dose- and time-dependent manner in all sensitive TNBC cells, whereas no alterations in CIP2A expression and p-Akt were noted in bortezomib-resistant cells. Overexpression of CIP2A upregulated p-Akt and protected MDA-MB-231 and MDA-MB-468 cells from bortezomib-induced apoptosis, whereas silencing CIP2A by siRNA overcame the resistance to bortezomib-induced apoptosis in MCF-7 cells. In addition, bortezomib downregulated CIP2A mRNA but did not affect the degradation of CIP2A protein. Furthermore, bortezomib exerted in vivo antitumor activity in HCC-1937 xenografted tumors, but not in MCF-7 tumors. Bortezomib downregulated CIP2A expression in the HCC-1937 tumors but not in the MCF-7 tumors. Importantly, CIP2A expression is readily detectable in tumor samples from TNBC patients.

Conclusions

CIP2A is a major determinant mediating bortezomib-induced apoptosis in TNBC cells. CIP2A may thus be a potential therapeutic target in TNBC.     

Repression of breast cancer cell growth by proteasome inhibitors in vitro: impact of mitogen-activated protein kinase phosphatase 1

Mitogen activated protein kinase phosphatase-1 (MKP-1) has emerged as an important protein mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies, especially proteasome inhibitors. In this in vitro study, we utilized the breast cancer epithelial cell lines MCF-7 and MDA-MB-231, in comparison to MCF-10A control cells, to examine the impact of MKP-1 on breast cancer cell growth and repression by proteasome inhibitors. We confirm that proteasome inhibitors MG-132 and bortezomib induce MKP-1 protein upregulation and we show that one of the ways in which bortezomib increases MKP-1 in breast cancer cells, in addition to inhibition of ubiquitin-proteasome system, is via upregulation of MKP-1 mRNA expression in p38 MAPK-mediated manner. Notably, these effects are specific to cancer cells, as bortezomib activated p38 MAPK and induced MKP-1 in MCF-7 and MDA-MB-231 breast cancer cells, but not in control cells (MCF-10A). We took a dual approach toward targeting MKP-1 to show that bortezomib-induced effects are enhanced. Firstly, treatment with the non-specific MKP-1 inhibitor triptolide reduces breast cancer cell growth and augments proteasome inhibitor-induced effects. Secondly, specific knock-down of MKP-1 with siRNA significantly repressed cell viability by reduced cyclin D1 expression, and enhanced repression of cancer cell growth by proteasome inhibitors. Taken together, these results indicate that removing the unwanted (MKP-1-inducing) effects of bortezomib significantly improves the efficacy of proteasome inhibition in breast cancer cells. Thus, future development of drugs targeting MKP-1 offer promise of combination therapies with reduced toxicity and enhanced cell death in breast cancer.     

鱼藤素通过FZD7调控Wnt通路抑制三阴性乳腺癌细胞侵袭、迁移、EMT的实验研究

目的:探讨鱼藤素对三阴性乳腺癌（TNBC）细胞增殖、侵袭、迁移和上皮-间质转化（EMT）的抑制作用及可能的机制。方法:体外培养人TNBC细胞系（MDA-MB-231和BT-20）,加入鱼藤素（5、10、20 μmol/L）诱导后,采用CCK-8法检测MDA-MB-231和BT-20细胞的存活率;克隆形成实验检测细胞克隆形成能力;流式细胞术分析细胞凋亡情况;Transwell实验检测细胞迁移和侵袭能力;Western Blot检测细胞中卷曲同源物7（FZD7）、Ki-67、E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）、β-连结蛋白（β-catenin）、c-myc蛋白表达。利用FZD7过表达载体质粒（pcDNA-FZD7）转染建立FZD7过表达细胞,进一步研究FZD7过表达对鱼藤素抗TNBC作用的影响;裸鼠成瘤实验检测鱼藤素对MDA-MB-231细胞体内生长的影响。结果:与对照组（0 μmol/L）相比,鱼藤素（5、10、20 μmol/L）呈剂量依赖性的显著降低MDA-MB-231和BT-20细胞存活率,并显著增加MDA-MB-231和BT-20细胞的凋亡率（P＜0.05）。与对照组相比,鱼藤素可显著降低MDA-MB-231和BT-20细胞增殖活力、克隆形成能力、迁移与侵袭能力和细胞内Vimentin、FZD7、β-catenin、c-myc蛋白表达,升高细胞凋亡率和细胞内E-cadherin蛋白表达（P＜0.05）;过表达FZD7可逆转鱼藤素对TNBC细胞恶性表型的影响。体内实验中,与对照组相比,鱼藤素组裸鼠的肿瘤体积和肿瘤质量、肿瘤中FZD7蛋白表达和FZD7、Ki-67阳性表达显著降低（P＜0.05）,与鱼藤素组和鱼藤素+pcDNA-NC组相比,鱼藤素+pcDNA-FZD7组肿瘤体积和肿瘤质量、肿瘤中FZD7蛋白表达和FZD7、Ki-67阳性表达显著升高（P＜0.05）。结论:鱼藤素可抑制TNBC细胞增殖、迁移、侵袭和EMT,并诱导细胞凋亡,其作用机制可能与下调FZD7的表达,抑制Wnt/β-catenin信号通路的活化有关。     

Silymarin inhibits proliferation of human breast cancer cells via regulation of the MAPK signaling pathway and induction of apoptosis

Silymarin is a purified mixture of four isomeric flavonoids extracted from the seeds and fruit of the milk thistle plant, Silybum marianus (L.). Silymarin exhibits a wide variety of biological effects and is commonly used in traditional medicine. Therefore, the anticancer effects of silymarin on human breast cancer cells were investigated to determine its pharmacological mechanisms in vitro and in vivo. The viability and proliferation of MDA-MB- 231 and MCF-7 breast cancer cells were investigated using MTT and wound healing assays. Silymarin decreased the viability and proliferation of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner. The number of apoptotic bodies, as shown by DAPI staining, was increased in a concentration-dependent manner, indicating that silymarin induces apoptosis. Additionally, changes in the expression levels of apoptosis-related proteins were demonstrated in human breast cancer cells using western blotting. Silymarin increased the levels of Bax, cleaved poly-ADP ribose polymerase, cleaved caspase-9 and phosphorylated (p-)JNK, and decreased the levels of Bcl-2, p-P38 and p-ERK1/2. Furthermore, the inhibitory effects of silymarin on MCF-7 tumor growth were investigated. In mice treated with silymarin for 3 weeks (25 and 50 mg/kg), MCF-7 tumor growth was inhibited without organ toxicity. In MCF-7 tumors, silymarin induced apoptosis and decreased p-ERK1/2 levels, as assessed using a TUNEL assay and immunohistochemistry. These results indicated that silymarin inhibited breast cancer cell proliferation both in vitro and in vivo by modulating the MAPK signaling pathway. Therefore, silymarin may potentially be used as a chemo-preventive or therapeutic agent.     

Contrasting effect of recombinant human erythropoietin on breast cancer cell response to cisplatin induced cytotoxicity

Background

Human recombinant erythropoietin (rHuEpo) that is used for the treatment of the chemotherapy-induced anaemia in cancer patients was shown to cause detrimental effects on the course of disease due to increased adverse events inflicting patient’s survival, potentially related to rHuEpo-induced cancer progression. In this study, we elucidate the effect of rHuEpo administration on breast cancer cell proliferation and gene expression after cisplatin (cDDP) induced cytotoxicity.

Materials and methods

Two breast carcinoma models, MCF-7 and MDA-MB-231 cell lines, were used differing in oestrogen (ER) and progesterone (PR) receptors and p53 status. Cells were cultured with or without rHuEpo for 24 h or 9 weeks and their growth characteristics after cDDP treatment were assessed together with expression of genes involved in the p53-signaling pathway.

Results

Short-term exposure of breast cancer cells to rHuEpo lowers their proliferation and reduces cDDP cytotoxic potency. In contrast, long-term exposure of MCF-7 cells to rHuEpo increases proliferation and predisposes MCF-7 cells to cDDP cytotoxicity, but has no effect on MDA-MB-231 cells. MDA-MB-231 cells show altered level of ERK phosphorylation, indicating involvement of MAPK signalling pathway. Gene expression analysis of p53-dependent genes and bcl-2 gene family members confirmed differences between long and short-term rHuEpo effects, indicating the most prominent changes in BCL2 and BAD expression.

Conclusions

Proliferation and survival characteristics of MCF-7 cells are reversely modulated by the length of the rHuEpo exposure. On the other hand, MDA-MB-231 cells are almost irresponsive to long-term rHuEpo, supposedly due to the mutated p53 and ER(+)/PR(−) status. The p53 and ER/PR status may predict tumour response on rHuEpo and cDDP treatment.     

The role of the polyamine catabolic enzymes SSAT and SMO in the synergistic effects of standard chemotherapeutic agents with a polyamine analogue in human breast cancer cell lines

Introduction

Polyamine analogues have demonstrated significant activity against human breast cancer cell lines as single agents as well as in combination with other cytotoxic drugs. This study evaluates the ability of a polyamine analogue N ¹,N ¹¹-bis(ethyl)norspermine (BENSpm) to synergize with six standard chemotherapeutic agents, 5-fluorouracil (FU), fluorodeoxyuridine, cis-diaminechloroplatinum(II) (C-DDP), paclitaxel, docetaxel, and vinorelbine.

Materials and methods

Four human breast cancer cell lines (MDA-MB-231, MCF-7, Hs578t, and T47D) and one immortalized, non-tumorigenic mammary epithelial cell line (MCF-10A) were used for in vitro combination studies with BENSpm and cytotoxic drugs. Xenograft mice models generated with MDA-MB-231 cells were used for in vivo studies with BENSpm and paclitaxel.

Results and conclusion

BENSpm exhibited synergistic inhibitory effect on cell proliferation in combination with 5-FU or paclitaxel in human breast cancer cell lines (MDA-MB-231 and MCF-7) and was either antagonistic or less effective in the non-tumorigenic MCF-10A cell line. Synergism was highest with 120 h concomitant treatment or pre-treatment with BENSpm for 24 h followed by concomitant treatment for 96 additional hours. Since the cytotoxic effects of many polyamine analogues and cytotoxic agents are believed to act, in part, through induction of the polyamine catabolic enzymes SSAT and SMO, the role of these enzymes on synergistic response was evaluated in MDA-MB-231 and MCF-7 treated with BENSpm and 5-FU or paclitaxel. Combination treatments of BENSpm with 5-FU or paclitaxel resulted in induction of SSAT mRNA and activity in both cell lines compared to either drug alone, while SMO mRNA and activity were increased only in MDA-MB-231 cells. Induction was greater with BENSpm/paclitaxel combination than BENSpm/5-FU. Further, RNAi studies demonstrated that both SSAT and SMO play a significant role in the response of MDA-MB-231 cells to treatment with BENSpm and 5-FU or paclitaxel. In MCF-7 cells, only SSAT appears to be involved in the response to these treatments. In an effort to translate combination studies from in vitro to in vivo, and to form a basis for clinical setting, the in vivo therapeutic efficacy of BENSpm alone and in combination with paclitaxel on tumor regression was evaluated in xenograft mice models generated with MDA-MB-231 cells. Intraperitoneal exposure to BENSpm or taxol singly and in combination for 4 weeks resulted in significant inhibition in tumor growth. These findings help elucidate the mechanisms involved in synergistic drug response and support combinations of polyamine analogues with chemotherapeutic agents which could potentially be used in the treatment of breast cancer.     

The Novel C24D Synthetic Polypeptide Inhibits Binding of Placenta Immunosuppressive Ferritin to Human T Cells and Elicits Anti–Breast Cancer Immunity In Vitro and In Vivo

Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain–like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti–breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2⁺) or T47D (HLA-A2⁻) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti–MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2⁺) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.Abbreviations: CTLs, cytotoxic T lymphocytes; E:T, effector/target; FITC, fluorescein isothiocyanate; IFN-γ, interferon-γ; IL, interleukin; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PLF, placental type isoferritin; PLIF, placenta immunosuppressive ferritin; TNBC, triple-negative breast cancer; TNF-α, tumor necrosis factor–α     

Enhancement of ionizing radiation response by histamine in vitro and in vivo in human breast cancer

The radioprotective potential of histamine on healthy tissue has been previously demonstrated. The aims of this work were to investigate the combinatorial effect of histamine or its receptor ligands and gamma radiation in vitro on the radiobiological response of 2 breast cancer cell lines (MDA-MB-231 and MCF-7), to explore the potential molecular mechanisms of the radiosensitizing action and to evaluate the histamine-induced radiosensitization in vivo in a triple negative breast cancer model. Results indicate that histamine significantly increased the radiosensitivity of MDA-MB-231 and MCF-7 cells. This effect was mimicked by the H₁R agonist 2-(3-(trifluoromethyl)phenyl)histamine and the H₄R agonists (Clobenpropit and VUF8430) in MDA-MB-231 and MCF-7 cells, respectively. Histamine and its agonists enhanced radiation-induced oxidative DNA damage, DNA double-strand breaks, apoptosis and senescence. These effects were associated with increased production of reactive oxygen species, which correlated with the inhibition of catalase, glutathione peroxidase and superoxide dismutase activities in MDA-MB-231 cells. Histamine was able also to potentiate in vivo the anti-tumoral effect of radiation, increasing the exponential tumor doubling time. We conclude that histamine increased radiation response of breast cancer cells, suggesting that it could be used as a potential adjuvant to enhance the efficacy of radiotherapy.     

Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells

Breast cancer has a propensity to metastasize to bone, thus causing pathological fractures. Bisphosphonates are established drugs in the treatment of bone metastasis that inhibit osteoclast activity and interrupt the vicious cycle of osteoclast–tumor cell interactions. We evaluated the direct effects of zoledronic acid on estrogen receptor (ER)-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells. While zoledronic acid (100 μM) inhibited MDA-MB-231 cell proliferation after 72 h, and induced apoptosis via activation of caspase-3 and -7, it had only minor effects on MCF-7 cells. In addition, zoledronic acid induced apoptosis by up-regulating TNF-related apoptosis-inducing ligand (TRAIL) in MDA-MB-231 cells (p < 0.01), but had no effect on the expression of its decoy receptor osteoprotegerin (OPG). In MCF-7 cells, both cytokines were suppressed by zoledronic acid. In conclusion, zoledronic acid enhanced the TRAIL-to-OPG ratio in TRAIL-sensitive MDA-MB-231 cells, indicating that the TRAIL/OPG cytokine system is a bisphosphonate-responsive target in breast cancer.     

The expression of growth hormone-releasing hormone (GHRH) and its receptor splice variants in human breast cancer lines; the evaluation of signaling mechanisms in the stimulation of cell proliferation

Antagonists of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers including breast cancer, xenografted into nude mice or cultured in vitro. Splice variants (SVs) of receptors for GHRH have been found in several human cancers and cancer cell lines. The antiproliferative actions of GHRH antagonists could be mediated in part through these SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and SVs of its receptors in human breast cancer cell lines MCF-7, MCF-7MIII, MDA-MB-231, MDA-MB-435, MDA-MB-468, and T47D. mRNA for GHRH was present in all lines tested. mRNA for SV₁ isoform of GHRH receptors was found in MCF-7MIII, MDA-MB-468, and T47D; and for SV₂ isoform in MCF-7MIII and T47D cell lines. In proliferation studies in vitro, the growth of T47D cells was stimulated by GHRH and dose-dependently inhibited by GHRH antagonist JV-1-38. H89 (protein kinase A inhibitor), bisindolylmaleimide I (protein kinase C [PKC] inhibitor) and verapamil (voltage-dependent calcium channel blocker) inhibited the GHRH-stimulated proliferation of T47D cells. The GHRH antagonist JV-1-38 suppressed the T47D cell growth in vitro stimulated by PKC activator (phorbol-12-myristate-13-acetate). The stimulation of T47D cells by GHRH was followed by an increase in cAMP production and GHRH antagonist JV-1-38 competitively inhibited this effect. Our results suggest that SVs of GHRH receptors could mediate the responses to GHRH and GHRH antagonists in breast cancer through Ca²⁺-, cAMP- and PKC-dependent mechanisms. The presence of SV₁ of GHRH receptors in human cancers provides a rationale for antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.     

BTG2 inhibits the proliferation, invasion, and apoptosis of MDA-MB-231 triple-negative breast cancer cells

The purposes of this study were to investigate the effects of B cell translocation gene 2 (BTG2) on the proliferation, apoptosis, and invasion of triple-negative breast cancer and to provide an experimental basis for the future treatment of human triple-negative breast cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the MDA-MB-231 human triple-negative breast cancer cell line using lipofection. Then, relevant changes in the biological characteristics of the BTG2-expressing cell line were analyzed using MTT (tetrazolium blue), flow cytometry, and Transwell invasion chamber assays. Additionally, the effects of BTG2 expression on cyclin D1, caspase 3, and matrix metalloproteinases 1/2 (MMP-1/-2) expression were analyzed. Cell proliferation was significantly lower in the pcDNA3.1-BTG2-transfected group compared to the empty vector and blank control groups (p?<?0.05). There was no significant difference between the empty vector and blank control groups. FCM results demonstrated that there were significantly more cells in the G1 phase of the cell cycle and fewer S phase cells in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p?<?0.05). Additionally, the proportion of cells that migrated across the membrane was significantly lower in the pcDNA3.1-BTG2 group than in the empty vector and blank control groups (p?<?0.05). Cyclin D1 and MMP-1/-2 expression were significantly lower in MDA-MB-231 cells transfected with pcDNA3.1-BTG2 as compared to the empty vector and blank control groups (p?<?0.05). Caspase 3 expression was significantly higher in MDA-MB-231 cells from the pcDNA3.1-BTG2 group compared to the empty vector and blank control groups (p?<?0.05). In conclusion, BTG2 may inhibit MDA-MB-231 proliferation and promote apoptosis. Additionally, BTG2 may also inhibit the invasion of MDA-MB-231 human triple-negative breast cancer cells.     

环状RNA hsa_circ_0058514在三阴性乳腺癌中的表达及作用研究

背景与目的：环状RNA（circular RNA，circRNA）是一类具有重要调节潜能的非编码RNA，参与多种肿瘤的发生、发展，但对于三阴性乳腺癌（triple-negative breast cancer，TNBC）尚未见报道。该研究旨在探讨环状RNA hsa_circ_0058514在TNBC发生、发展中的作用。方法：采用RNA测序（RNA sequencing，RNA-seq）对4对TNBC组织和癌旁组织进行分析，采用实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR） 对20对TNBC组织和癌旁组织以及正常人乳腺上皮细胞MCF-10A和TNBC细胞MDA-MB-231和BT-549中hsa_circ_0058514的表达进行验证。将干扰质粒pLL3.7-sh-circ转染TNBC细胞MDA-MB-231和BT-549后，采用RTFQ-PCR检测细胞中hsa_circ_0058514的表达；采用细胞计数试剂盒（cell counting kit-8，CCK-8）和EdU实验检测细胞增殖；采用划痕和Transwell小室实验分别检测细胞迁移和侵袭；采用流式细胞术检测细胞凋亡和细胞周期；采用蛋白质印迹法（Western blot）检测细胞周期蛋白E1（cyclin E1，CCNE1）和细胞周期蛋白依赖性激酶2（cyclin-dependent kinase 2，CDK2）蛋白的表达。结果：环状RNA hsa_circ_0058514在TNBC组织和细胞中显著高表达（P<0.001，P<0.01）；转染pLL3.7-sh-circ后，TNBC细胞中hsa_circ_0058514的表达量显著低于对照组（P<0.001）。下调hsa_circ_0058514后，TNBC细胞增殖、迁移及侵袭能力下降，并促进细胞凋亡，导致细胞周期阻滞。Western blot结果显示，转染pLL3.7-sh-circ后，CCNE1和CDK2蛋白的表达下调（P<0.05）。结论：环状RNA hsa_circ_0058514在TNBC组织和细胞中均高表达，其在TNBC发生、发展中可能起到癌基因的作用，并有望成为TNBC治疗的新靶点。     

PPAPDC1A在体内外对乳腺癌细胞增殖、侵袭和转移能力的影响

目的:通过构建稳定过表达和干扰PPAPDC1A的乳腺癌细胞株,探讨PPAPDC1A对乳腺癌细胞增殖、侵袭和转移能力的影响。方法:利用CCK-8和Transwell实验检测PPAPDC1A稳定过表达和干扰后对乳腺癌细胞体外增殖和侵袭能力的影响。采用裸鼠皮下成瘤实验检测PPAPDC1A对乳腺癌细胞体内增殖和裸鼠致瘤性的作用。利用免疫组织化学染色法检测各组肿瘤组织中Ki-67的表达。通过裸鼠尾静脉注射实验检测PPAPDC1A对乳腺癌MCF-7细胞和MDA-MB-231细胞体内转移能力的影响。结果:成功建立稳定过表达PPAPDC1A的乳腺癌MCF-7细胞株和稳定干扰PPAPDC1A的乳腺癌MDA-MB-231细胞株;CCK-8和Transwell实验结果显示,与MCF-7和MCF-7-Vector细胞株相比,MCF-7-PPAPDC1A细胞株的生长速度显著增快,穿膜细胞数量多（P＜0.05）;与此相反,MDA-MB-231-shPPAPDC1A组细胞的生长速度和穿膜细胞数明显少于MDA-MB-231-shNC和MDA-MB-231 细胞株（P＜0.05）。动物实验结果显示,与MCF-7-Vector组相比,MCF-7-PPAPDC1A组的肿瘤生长速度较快,肿瘤的体积较大,Ki-67的阳性率高,肺转移灶的数目增多（P＜0.05）;与此相反,与MDA-MB-231-shNC组相比MDA-MB-231-shPPAPDC1A组的肿瘤生长速度较慢,肿瘤的体积较小,Ki-67的阳性率低,肺转移灶的数目减少（P＜0.05）。结论:PPAPDC1A对乳腺癌细胞的增殖、侵袭和转移有促进作用。     

BU-32: a novel proteasome inhibitor for breast cancer

Introduction

Proteasome inhibition provides an attractive approach to cancer therapy and may have application in the treatment of breast cancer. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade^®, also called PS-341) in metastatic breast cancer patients have shown limited activity when used as a single agent. This underscores the need to find new and more efficacious proteasome inhibitors. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499-S) using in vitro and in vivo breast cancer models.

Methods

We have recently synthesized a novel proteasome inhibitor (BU-32) and tested its growth inhibitory effects in different breast cancer cells including MCF-7, MDA-MB-231, and SKBR3 by in vitro cytotoxicity and proteasomal inhibition assays. The apoptotic potential of BU32 was tested using flow cytometry and analyzing cell cycle regulatory proteins. In vivo tumor xenograft studies for solid tumor as well as tumor metastasis were conducted using MDA-MB-231-GFP cells.

Results

We report for the first time that BU-32 exhibits strong cytotoxicity in a panel of cell lines: MDA-MB-231 (IC₅₀ = 5.8 nM), SKBR3 (IC₅₀ = 5.7 nM) and MCF-7 cells (IC₅₀ = 5.8 nM). It downregulates a wide array of angiogenic marker genes and upregulates apoptotic markers, including Bid and Bax. Incubation of MDA-MB-231 cells with BU-32 results in the accumulation of cell cycle inhibitor proteins p21 and p27 and stabilization of the tumor suppressor protein p53. Studies in in vivo solid tumor and metastasis models show significant effect with a 0.06 mg/kg dose of BU-32 and marked reduction in tumor burden in the skeleton.

Conclusions

We have shown that BU-32 is effective in cultured breast cancer cells and in breast cancer xenografts. The results suggest its potential benefit in breast cancer treatment.     

Effects of metformin on breast cancer cell proliferation,the AMPK pathway and the cell cycle

Aim

The aim of this study was to compare the effects and mechanisms of action of metformin on estrogen receptor (ER)-positive and ER-negative breast cancer cell lines.

Methods

The anti-proliferative effects of metformin, and of the direct activator of adenosine monophosphate-activated protein kinase (AMPK), A-769662, on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) breast cancer cell lines were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a yellow tetrazole) assays. Fluorescence-activated cell sorting was also used to examine the effect of metformin on the cell cycle. Finally, phosphorylation of the metformin target AMPK, and of its potential downstream targets including acetyl-CoA carboxylase (ACC), p53, p70-S6K and Raptor, was examined using immunoblotting.

Results

Metformin and A-769662 caused significant, concentration-dependent suppression of cell proliferation with G1 cell cycle arrest in both MCF-7 and MDA-MB-231 cells. The proliferation suppression effect was more profound in MCF-7 cells. A concentration-dependent phosphorylation of AMPK was detected following metformin treatment, as was phosphorylation of ACC in both cell lines, but not p53, p70-S6k or Raptor.

Conclusion

Metformin acts as a growth inhibitor in both ER-positive and ER-negative breast cancer cells in vitro, and arrests cells in G1 phase, particularly in the ER-positive MCF-7 cells. The effect is likely to be mediated by AMPK activation, in part by inhibition of fatty acid synthesis via ACC phosphorylation.     

The anti-progestin RU-486 inhibits viability of MCF-7 breast cancer cells by suppressing WNT1

The Wnt signaling pathway is activated in over 50% of women with breast cancer and contributes to tumor progression. Here, we investigated the effects of RU-486 on Wnt signaling in breast cancer cell lines. RU-486 reduced viability of the progesterone receptor-positive MCF-7 and T-47D cells, but had no effect on the triple-negative MDA-MB-231 cells. Furthermore, RU-486 suppressed WNT1 expression of MCF-7 cells by 99%. The addition of recombinant WNT1 partially reversed the RU-486-dependent inhibition of viability in MCF-7, but not in T-47D cells. In conclusion, we identified WNT1 as a novel mediator of the anti-tumor effects of RU-486 in MCF-7 cells.