Affibody-displaying bio-nanocapsules effective in EGFR,typical biomarker,expressed in various cancer cells

The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (Z_EGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (Z_EGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of Z_EGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]₂-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]₂-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR.     

Dimeric HER2-specific affibody molecules inhibit proliferation of the SKBR-3 breast cancer cell line

HER2-specific affibody molecules in different formats have previously been shown to be useful tumor targeting agents for radionuclide-based imaging and therapy applications, but their biological effect on tumor cells is not well known. In this study, two dimeric ((Z_HER2:4)₂ and (Z_HER2:342)₂) and one monomeric (Z_HER2:342) HER2-specific affibody molecules are investigated with respect to biological activity. Both (Z_HER2:4)₂ and (Z_HER2:342)₂ were found to decrease the growth rate of SKBR-3 cells to the same extent as the antibody trastuzumab. When the substances were removed, the cells treated with the dimeric affibody molecules continued to be growth suppressed while the cells treated with trastuzumab immediately resumed normal proliferation. The effects of Z_HER2:342 were minor on both proliferation and cell signaling. The dimeric (Z_HER2:4)₂ and (Z_HER2:342)₂ both reduced growth of SKBR-3 cells and may prove therapeutically useful either by themselves or as carriers of radionuclides or other cytotoxic agents.     

The HER2-Binding Affibody Molecule (ZHER2∶342)2 Increases Radiosensitivity in SKBR-3 Cells

We have previously shown that the HER2-specific affibody molecule (Z_HER2∶342)₂ inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (Z_HER2∶342)₂ sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (Z_HER2∶342)₂ and 8 Gy (S_8Gy 0.006) was decreased by a factor four compared to the untreated (S_8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (Z_HER2∶342)₂. Treatment by (Z_HER2∶342)₂ strongly increased the levels of dimerized and phosphorylated HER2 even after 5 minutes of stimulation. The monomeric Z_HER2∶342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation.     

BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.     

Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells

The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2⁺) and MDA-MB-231 (Her2^?). IC₅₀ values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 μg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 μg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2⁺ cancer.     

负载Her-2多肽的DCIK细胞对乳腺癌细胞杀伤作用研究

本文观察利用树突状细胞(DC)呈递肿瘤抗原(Her-2多肽)的特性提高DCIK细胞对乳腺癌细胞的杀伤活性。提取外周血来源的有核细胞诱导分离出细胞因子诱导的杀伤细胞(CIK)和树突状细胞(DC),DC负载Her-2多肽后和CIK细胞共培养产生DCIK细胞,并鉴定其HLA基因型。分析三株肿瘤细胞(MDA-MB-231、SK-BR-3、MCF-7)HLA基因型和Her-2蛋白表达情况。用细胞毒试验(CCK-8法)测定DCIK细胞的对三株Her2表达不同的乳腺癌细胞株的杀伤活性。结果表明DCIK细胞对MDA-MB-31、SK-BR-3、MCF-7的杀伤率(效靶比10:1)分别为50.38%±3.25%、52.19%±3.25%、47.09%±2.41%。而负载Her-2多肽的DCIK细胞对MDA-MB-231、SK-BR-3、MCF-7的杀伤率分别为76.30%±1.74%(P<0.001)、55.70%±3.05%(P=0.0143)、47.67%±2.40%(P=0.6972)。实验证明负载Her-2多肽的DCIK细胞能显著提高对Her-2( )的乳腺癌细胞的杀伤作用,为乳腺癌患者进行过继免疫治疗提供了理论依据。     

Preparation and Identification of HER2 Radioactive Ligands and Imaging Study of Breast Cancer-Bearing Nude Mice

OBJECTIVE: A micro-molecule peptide TP1623 of ^99mTc-human epithelial growth factor receptor 2 (HER2) was prepared and the feasibility of using it as a HER2-positive molecular imaging agent for breast cancer was evaluated. METHODS: TP1623 was chemically synthesized and labeled with ^99mTc. The labeling ratio and stability were detected. HER2 expression levels of breast cancer cells (SKBR3 and MDA-MB-231) and cell binding activity were measured. Biodistribution of ^99mTC-TP1623 in normal mice was detected. SKBR3/MDA-MB-231-bearing nude mice models with high/low expressions of HER2 were established. Tumor tissues were stained with hematoxylin–eosin (HE) and measured by immunohistochemistry to confirm the formation of tumors and HER2 expression. SPECT imaging was conducted for HER2-overexpressing SKBR3-bearing nude mice. The T/NT ratio was calculated and compared with that of MDA-MB-231-bearing nude mice with low HER2 expression. The competitive inhibition image was used to discuss the specific binding of ^99mTc- TP1623 and the tumor. RESULTS: The labeling ratio of ^99mTc-TP1623, specific activity, and radiochemical purity (RCP) after 6 h at room temperature were (97.39 ± 0.23)%, (24.61 ± 0.06) TBq/mmol, and (93.25 ± 0.06)%, respectively. HER2 of SKBR3 and MDA-MB-231 cells showed high and low expression levels by immunohistochemistry, respectively. The in vitro receptor assays indicated that specific binding of TP1623 and HER2 was retained. Radioactivity in the brain was always at the lowest level, while the clearance rate of blood and the excretion rate of the kidneys were fast. HE staining showed that tumor cells were observed in SKBR3- and MDA-MB-231-bearing nude mice, with significant heteromorphism and increased mitotic count. The imaging of mice showed that targeted images could be made of ^99mTc-TP1623 in high HER2-expressing tumors, while no obvious development was shown in tumors in low HER2-expressing nude mice. No development was visible in tumors in competitive inhibition of imaging, which indicates the combination of ^99mTc-TP1623 and tumor was mediated by HER2. CONCLUSION: High labeling ratio and specific activity of ^99mTc-TP1623 is successfully prepared; it is a molecular imaging agent for HER2-positive tumors that has potential applicative value.     

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu-positive tumor cells

Targeting of non‐phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non‐pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti‐HER2/neu affibody on the surface. After co‐incubation with tumor cells for 3 h, the anti‐HER2/neu affibody‐presenting E. coli strain was selectively internalized into HER2/neu‐positive SKBR‐3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E‐mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor‐targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu‐positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier. Biotechnol. Bioeng. 2011; 108:1662–1672. © 2011 Wiley Periodicals, Inc.     

The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation

Background

Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation.

Results and Discussion

Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4’-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1–10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.

Conclusion

Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.     

HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z_HER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were?～70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm?～0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained?>?90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

Up-regulation of acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells

Expression of the HER2 oncogene is increased in approximately 30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase alpha (ACCalpha) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCalpha compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCalpha in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCalpha, and the HER2-mediated increase in ACCalpha and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCalpha. On the other hand, FASN and ACCalpha were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5'- and 3'-untranslated regions of both FASN and ACCalpha mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCalpha in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.     

Integrin α2β1 Mediates Tyrosine Phosphorylation of Vascular Endothelial Cadherin Induced by Invasive Breast Cancer Cells

The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α₂β₁ integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α₂β₁ integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β₁-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α₂β₁ integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Understanding Heterogeneity and Permeability of Brain Metastases in Murine Models of HER2-Positive Breast Cancer Through Magnetic Resonance Imaging: Implications for Detection and Therapy

OBJECTIVES: Brain metastases due to breast cancer are increasing, and the prognosis is poor. Lack of effective therapy is attributed to heterogeneity of breast cancers and their resulting metastases, as well as impermeability of the blood–brain barrier (BBB), which hinders delivery of therapeutics to the brain. This work investigates three experimental models of HER2 + breast cancer brain metastasis to better understand the inherent heterogeneity of the disease. We use magnetic resonance imaging (MRI) to quantify brain metastatic growth and explore its relationship with BBB permeability. DESIGN: Brain metastases due to breast cancer cells (SUM190-BR3, JIMT-1-BR3, or MDA-MB-231-BR-HER2) were imaged at 3 T using balanced steady-state free precession and contrast-enhanced T1-weighted spin echo sequences. The histology and immunohistochemistry corresponding to MRI were also analyzed. RESULTS: There were differences in metastatic tumor appearance by MRI, histology, and immunohistochemistry (Ki67, CD31, CD105) across the three models. The mean volume of an MDA-MB-231-BR-HER2 tumor was significantly larger compared to other models (F_2,12 = 5.845, P < .05); interestingly, this model also had a significantly higher proportion of Gd-impermeable tumors (F_2,12 = 22.18, P < .0001). Ki67 staining indicated that Gd-impermeable tumors had significantly more proliferative nuclei compared to Gd-permeable tumors (t[24] = 2.389, P < .05) in the MDA-MB-231-BR-HER2 model. CD31 and CD105 staining suggested no difference in new vasculature patterns between permeable and impermeable tumors in any model. CONCLUSION: Significant heterogeneity is present in these models of brain metastases from HER2 + breast cancer. Understanding this heterogeneity, especially as it relates to BBB permeability, is important for improvement in brain metastasis detection and treatment delivery.     

SARNP,a participant in mRNA splicing and export,negatively regulates E-cadherin expression via interaction with pinin

Triple-negative breast cancer (TNBC) is associated with a high mortality rate, which is related to the insufficient number of appropriate biomarkers and targets. Therefore, there is an urgent need to discover appropriate biomarkers and targets for TNBC. SARNP (Hcc-1 and CIP29) is highly expressed in several cancers. It binds to UAP56, an RNA helicase component of the TREX complex in messenger RNA (mRNA) splicing and export. However, the role of SARNP in mRNA splicing and export and in the progression of breast cancer, especially of TNBC, remains unknown. Therefore, we examined the role of SARNP in mRNA splicing and export and progression of TNBC. We confirmed that SARNP binds to UAP56 and Aly and that SARNP overexpression enhances mRNA splicing, whereas its knockdown suppressed mRNA export. The SARNP overexpression induced the proliferation of MCF7 cells, whereas its knockdown induced E-cadherin expression and downregulated vimentin and N-cadherin expressions in SK-BR-3 and MDA-MB-231 cells. SARNP downregulates E-cadherin expression by interaction with pinin. Mice injected with MDA-MB-231_shSARNP cells exhibited a significant reduction in tumor growth and lung metastasis compared with those injected with MDA-MB-231_shCon cells in vivo. These findings suggested that SARNP is involved in mRNA splicing and export. SARNP maintains mesenchymal phenotype by escaping from inhibitory interaction with pinin leading to the downregulation of E-cadherin expression.     

Caleosin-assembled oil bodies as a potential delivery nanocarrier

Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we have sought to use nanoscale artificial oil bodies (NOBs) as an alternative delivery carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu affibody with a high affinity towards the HER2/neu receptor. After overproduction in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in one step. Consequently, resulting NOBs had a zeta potential around −49 mV and ranged in size from 150 to 200 nm. Upon loading with a hydrophobic fluorescence dye, NOBs were found to be selectively internalized into HER2/neu-positive tumor cells. Further analyses showed that more than 90% cells were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. In addition, the in vitro assay revealed the release of the dye from NOBs in a slow and prolong manner. Overall, these results indicate the potential of Cal-based NOBs as a delivery vehicle.     

METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells

Previous research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as a promoter or a suppressor in the development of human breast cancer by MCF7, MDA-MB-231, and MDA-MB-468. To resolve these conflicting results we have investigated the role of this CAM in the progression of the three aforementioned cell lines plus one additional human breast cancer cell line, SK-BR-3. We transfected the SK-BR-3 cells with human METCAM/MUC18 cDNA to obtain G418-resistant clones, which expressed different levels of the protein and which were used to test the effect of human METCAM/MUC18 expression on in vitro motility, invasiveness, anchorage-independent colony formation in soft agar, disorganized growth in a 3D basement membrane culture assay, and in vivo tumorigenesis in athymic nude mice. Enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, and anchorage-independent colony formation of SK-BR-3 cells and favored disorganized growth of the cells in 3D basement membrane culture. Enforced expression also increased tumorigenicity and final tumor weights of SK-BR-3 clones/cells after subcutaneous injection of the cells under the left third nipple of female athymic nude mice. To understand the mechanisms, we also determined the expression of several downstream key effectors in the tumors. Tumor cells from METCAM/MUC18 expressing clones exhibited elevated expression of an anti-apoptotic and survival index (Bcl2), an aerobic glycolysis index (LDH-A), and pro-angiogenesis indexes (VEGF and VAGFR2). We concluded that human METCAM/MUC18 promotes the development of breast cancer cells by increasing an anti-apoptosis and survival pathway and augmenting aerobic glycolysis and angiogenesis.     

乳腺癌体外骨转移瘤3D模型的建立

目的:构建乳腺癌体外骨转移瘤3D模型。方法:新生CD-1小鼠的颅骨单独孵育为正常组,新生CD-1小鼠的颅骨与MDA-MB-231细胞低氧共孵育四天为模型组。通过扫描电镜(SEM)鉴定骨转移瘤模型,中性红染色法鉴定骨组织中的破骨细胞,硝酸银复染法观察骨的溶解,结晶紫染色法观察骨转移瘤模型中肿瘤细胞的生长。结果:正常组骨表面光滑完整;模型组骨组织表面黏附大量肿瘤细胞,破骨细胞活性增强,产生严重的溶骨,表面出现骨陷窝,骨纤维发生断裂。结论:成功建立了乳腺癌骨转移瘤体外3D模型,该模型能够模拟体内骨转移瘤微环境。     

Synthesis and Evaluation of Piperazine-Tethered Derivatives of Alepterolic Acid as Anticancer Agents

Alepterolic acid is a natural diterpenoid isolated from Aleuritopteris argentea with potential anti-cancer activity. In this study, alepterolic acid was modified to construct a series of arylformyl piperazinyl derivatives ( 3a – 3p ). The synthesized derivatives were fully characterized with HRMS, NMR, and IR. Four compounds with inhibition rate higher than 30 % at 10 μM ( 3f , 3n , 3g and 3k ) were further measured to obtain the IC₅₀ values against four cancer cell lines, including hepatoma cell lines HepG2, lung cancer cell lines A549, estrogen receptor-positive cell lines MCF7, and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 by MTT assay. It was found that these compounds were more effective to HepG2 and MDA-MB-231 cells, while less toxic to A549 and MCF7 cells, and compound 3n as the most toxic derivatve against MDA-MB-231 cell lines, with IC₅₀ value of 5.55±0.56 μM. Trypan blue staining and colony formation assay showed that compound 3n inhibited the growth of MDA-MB-231 cells and prevented colony formation. Hoechst staining, flow cytometry and western blot analysis revealed that compound 3n induced caspase-dependent apoptosis in MDA-MB-231 cells. Conclusively, compound 3n was demonstrated to be a potential anti-cancer lead compound for further investigation.