Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217–224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.     

Anti-laminin receptor antibody targeting of liposomes with encapsulated doxorubicin to human breast cancer cells in vitro

The tumor cell laminin receptor is a cell-surface protein that binds laminin with high affinity (Kd = 1.0 nM). The putative ligand-binding domain of the laminin receptor has been molecularly cloned and sequenced. In the present study, we used the predicted amino acid sequence of the laminin receptor to generate synthetic peptide antigens and produced immunoglobulin M (IgM) anti-laminin receptor monoclonal antibodies. The disulfide bond group of the IgM molecule was used to couple the antibodies to the surface of liposomes encapsulating doxorubicin. The anti-laminin receptor monoclonal antibodies coupled to the liposomes bound avidly to the surface of MDA-MB-435S (MDA-435) human breast carcinoma cells, which have high numbers of laminin receptors. These antibody-coupled liposomes exhibited a low degree of binding to Hs 578Bst (Hs 578) normal human breast epithelial cells, which express a low number of laminin receptors. Excess liposomes competed for the binding of unbound laminin to the tumor cell surface, and excess laminin competed for binding with the liposomes. Antibody-coupled liposomes encapsulating doxorubicin were specifically more efficient in inhibiting colony formation by MDA-435 cells in vitro than unbound doxorubicin or liposomes without anti-laminin receptor monoclonal antibodies. Unbound doxorubicin inhibited thymidine uptake by 10%-20% in both Hs 578 and MDA-435 cells, whereas the antibody-coupled liposomes encapsulating doxorubicin inhibited thymidine uptake by 90% in MDA-435 cells but only 15% in Hs 578 cells. Thus, use of anti-laminin receptor monoclonal antibodies coupled with liposomes encapsulating doxorubicin represents a new strategy for selective targeting of doxorubicin to carcinoma cells with exposed laminin receptors.     

Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump.

Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells. Confocal fluorescence microscopic observations demonstrated binding and subsequent internalization of rhodamine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells), with inhibition by free folic acid. Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted liposomes (FTLs) indicate that liposomal DOX is rapidly internalized, released in the cytoplasmic compartment, and, shortly thereafter, detected in the nucleus, the entire process lasting 1-2 h. FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis. Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOX. The in vitro cytotoxic activity obtained with DOX-loaded FTLs was 10-fold greater than that of the nontargeted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells. However, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX. It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression.     

Anisamide-targeted stealth liposomes: a potent carrier for targeting doxorubicin to human prostate cancer cells

Certain human malignancies including prostate cancer overexpress sigma receptor, a membrane bound protein that binds haloperidol and various other neuroleptics with high affinity. An anisamide derivatized ligand possesses high affinity for sigma receptors and we hypothesized that its incorporation into the liposomes encapsulating doxorubicin (DOX) can specifically target and deliver the drug to prostate cancer cells that overexpress sigma receptors. A polyethylene glycol phospholipid was derivatized with an anisamide ligand, which was then incorporated into the DOX-loaded liposome. The resulting anisamide-conjugated liposomal DOX showed significantly higher toxicity to DU-145 cells than non-targeted liposomal DOX, the IC50 being 1.8 microM and 14 microM respectively. The cytotoxicity of the targeted liposomal DOX, however, was significantly blocked by haloperidol, suggesting that the enhanced cytotoxicity was specifically mediated by the sigma receptors. Fluorescence imaging studies after intravenous (i.v.) administration showed that incorporation of anisamide into liposomes significantly improved their accumulation into the tumor. A weekly injection of the targeted liposomal DOX for 4 weeks at a dose of 7.5 mg/kg led to a significant growth inhibition of established DU-145 tumor in nude mice with minimal toxicity. Free DOX was effective, but associated with significant toxicities. The present study is the first to demonstrate the use of small molecular weight ligand for mediating efficient targeting of liposomal drugs to sigma receptor expressing prostate cancer cells both in vitro and in vivo.     

Metabolic fate of liposomal phosphatidylinositol in murine tumor cells: implications for the mechanism of tumor cell cytotoxicity

The mechanism of the previously reported cytotoxicity of liposomes containing plant phosphatidylinositol (PI) against numerous tumor cell lines was examined in detail by using liposomes containing synthetic PI specifically labeled either with radioactive myo-inositol, or in the sn-2 position with radioactive linoleic acid, oleic acid, or arachidonic acid. The uptake of liposomal PI by N4TG1 neuroblastoma cells increased with time and was dependent on the nature of the fatty acids. Uptake was highest with liposomal PI containing linoleic acid followed by arachidonic acid and then by oleic acid. The cellular fate of liposomal PI was determined by analysis of radioactive metabolites present in extracts of tumor cell lipids. Appearance of liposomal PI metabolic products in the tumor cells was correlated with thymidine uptake as a measure of viability. After 3 h incubation of cells with PI liposomes it was found that the release of both radioactive liposomal fatty acids (and probably also lyso-Pl) and radioactive diglycerides was correlated inversely with the cellular uptake of [methyl-3H]thymidine and uptake of [3H]myoinositol. An experiment in which liposomes were prepared both from animal Pl which contained predominantly saturated fatty acids in the sn-2 position and an increasing mole fraction of a synthetic Pl containing radioactive linoleic acid in the sn-2 position established that the amount of Pl containing linoleic acid in the sn-2 position could be correlated with a decrease in the amount of thymidine uptake by tumor cells. The above results clearly established that phospholipases A2 and C in the tumor cells were responsible for the formation of metabolites of liposomal Pl, and these metabolic products might have been responsible for cytotoxicity and cell death.     

Folate receptor targeted delivery of liposomal daunorubicin into tumor cells

BACKGROUND: The folate receptor (FR) is amplified in a wide variety of human tumors. Thus, targeting cytotoxic therapies to FR is a promising strategy for chemotherapy. MATERIALS AND METHODS: FR-targeted liposomal daunorubicin (f-L-DNR) was compared to non-targeted liposomal DNR (L-DNR) for cellular uptake and cytotoxicity in FR-expressing cells. Liposomal DNR retention was evaluated for liposomes loaded with either sodium citrate or ammonium sulfate as the trapping agent. The cellular uptake of liposomal DNR was determined by flow cytometry and fluorometry measurements while cytotoxicity was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Liposomal DNR retention was superior for liposomes prepared using ammonium sulfate. Cellular uptake of f-L-DNR in KB oral carcinoma cells, Chinese hamster ovary (CHO-FR-beta), and KG-1 human acute myelogenous leukemia cells were 9.4, 40, and 4,6-fold higher than non-targeted L-DNR, respectively. The cytotoxicity of f-L-DNR in KB and CHO-FR-beta cells was 18 times and 49 times higher than L-DNR, respectively. Both cellular uptake and cytotoxicity of f-L-DNR could be inhibited by 1 mM folic acid. CONCLUSION: FR-mediated delivery of liposomal DNR to FR-expressing cells increases DNR cellular uptake and cytotoxicity. Therefore, therapeutic evaluation in relevant animal models is warranted.     

乳腺癌细胞体外摄取2-脱氧葡萄糖荧光类似物

目的 观察2-脱氧葡萄糖(2-DG)荧光类似物2-N[7-硝基苯-2-乙二酸,34羟氨基]-2-脱氧葡萄糖(2-NBDG)被高表达葡萄糖转运蛋白1(GLUT-1)的乳腺癌细胞靶向摄取的情况.方法 应用逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测乳腺癌MDA-MB-231细胞GLUT-1 mRNA和蛋白的表达,采用Western blot法比较乳腺癌MDA-MB-231细胞和MCF-7细胞GLUT-1的蛋白表达量.应用2-NBDG孵育人乳腺癌MDA-MB-231细胞,采用荧光显微镜及流式细胞仪观察、分析对2-NBDG的摄取情况,比较MDA-MB-231和MCF-7细胞吸收2-NBDG量的差异.结果 RT-PCR和免疫组化检测结果显示,MDA-MB-231细胞高表达GLUT-1;Western blot检测结果进一步显示,MDA-MB-231细胞的GLUT-1表达(0.946 4±0.007)高于MCF-7(0.833±0.010).荧光成像及流式细胞仪分析结果显示,MDA-MB-231细胞能快速摄取2-NBDG,且加入50 mmol/L D-葡萄糖后,荧光强度降低了46.0%.2-NBDG孵育乳腺癌细胞20 min后,MDA-MB-231细胞荧光强度(25.10±0.57)明显高于MCF-7细胞(10.12±0.62).结论 2-NBDG能迅速被高表达GLUT-1的乳腺癌MDA-MB-231细胞靶向吸收.     

Increased intracellular drug accumulation and complete chemosensitization achieved in multidrug-resistant solid tumors by co-administering valspodar (PSC 833) with sterically stabilized liposomal doxorubicin

We have previously demonstrated that liposome encapsulation of doxorubicin (DOX) can alleviate adverse interactions with non-encapsulated DOX and the cyclosporine multidrug-resistant (MDR) modulator Valspodar. We have now investigated the behavior of different liposomal DOX formulations in MDA435LCC6/MDR-1 human breast cancer solid tumor xenograft models to identify liposome characteristics associated with enhanced therapeutic activity and the mechanism whereby increased chemosensitization is achieved. Toxicity studies incorporating conventional phosphatidylcholine (PC)/cholesterol (chol) and sterically stabilized (polyethylene glycol 2000 [PEG]-containing) formulations of DOX indicated that whereas PC/Chol DOX was approximately 3-fold more toxic in the presence of Valspodar, PEG containing distearoylglycerophosphocholine (DSPC)/Chol DOX was minimally affected. In mice bearing MDR tumors, co-administration of Valspodar and egg phosphocholine (EPC)/Chol DOX resulted in modest MDR modulation and efficacy, whereas the sterically stabilized formulation induced reductions in tumor growth equivalent to that achieved for drug-sensitive tumors treated with non-encapsulated DOX. Pharmacokinetic studies revealed a 2.5-fold increase in plasma DOX area under the curve (AUC) upon co-administration of Valspodar with EPC/Chol DOX whereas no such alterations were observed with the sterically stabilized liposomes. Compared to non-encapsulated DOX combined with Valspodar, improvements in efficacy and toxicity correlated with the extent to which liposomal DOX formulations were able to circumvent pharmacokinetic interactions. Confocal microscopy demonstrated that Valspodar increased cell-associated DOX which correlated with the level of anti-tumor efficacy.     

Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice

We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139). In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells. PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively. The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines. In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent. PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments. The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.     

Effect of natural flavonoids to reverse P-glycoprotein-related multidrug resistance in breast cancer cell cultures

The aim of the research was to evaluate the influence of two P-glycoprotein (P-gp) inhibitors silymarin and quercetin on anticancer drug doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) delivery into breast cancer cells (2D cultures) and cancer cell spheroids (3D cultures) at different pH. The cytotoxicity of the compounds was assessed using MTT assay. Spheroids were generated using magnetic 3D Bioprinting method. The uptake of DOX and PLD into monolayer-cultured cells and spheroids was assessed by fluorescence microscopy. Both tested flavonoids did not increase DOX and PLD levels into monolayer-cultured 4T1 cells and 4T1 cell spheroids. However, both silymarin and quercetin enhanced DOX and PLD uptake into JC cell cultures. Silymarin and quercetin may modulate DOX and PLD transport into monolayer-cultured cells and three-dimensional cancer cell cultures depending on P-gp activity.     

Ag-bearing liposomes engrafted with peptides that interact with CD11c/CD18 induce potent Ag-specific and antitumor immunity

Dendritic cells (DCs) play key role in eliciting antigen (Ag)-specific immune responses, and crucial to this is the uptake of Ag via surface receptors including the heterodimeric integrin CD11c/CD18. Here we report that CD11c/CD18-interacting peptides can be used as targeting moieties to deliver liposomal Ag to antigen presenting cells (APCs) and elicit Ag-specific and antitumor immunity. Two peptides of sequence related to human ICAM-4 and previously reported to bind CD11c/CD18, and a 12-mer cyclic peptide previously identified by phage display to bind CD11c/CD18, were produced synthetically, and tested for their ability to target liposomal Ag. The three peptides were designed to contain a shorter spacer to reduce steric hindrance, and a His-tag to enable engraftment onto liposomes incorporated with chelator lipid. Our results show that the three peptides, denoted as p17, p18 and p30, promote strong binding of liposomes to CD11c(+) and CD11b(+) cells in vitro and in vivo. Vaccination of mice with Ag-bearing liposomes engrafted with the peptides, particularly p18 and p30, induced Ag-specific T cell priming and antibody production. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA-derived plasma membrane vesicles (PMVs) engrafted with p18 and p30 peptide showed dramatic antitumor responses, inhibiting tumor growth/metastasis in both the lung and subcutaneous tumor models, with a high proportion of the mice apparently being "cured" of their tumors. The engraftment of p18 and p30 peptides onto liposomes and PMVs, thus provides an effective means to target Ags to DCs in vivo, for the development of effective cancer vaccines and immunotherapies.     

Doxorubicin in TAT peptide-modified multifunctional immunoliposomes demonstrates increased activity against both drug-sensitive and drug-resistant ovarian cancer models

Multidrug resistance (MDR) is a hallmark of cancer cells and a crucial factor in chemotherapy failure, cancer reappearance, and patient deterioration. We have previously described the physicochemical characteristics and the in vitro anticancer properties of a multifunctional doxorubicin-loaded liposomal formulation. Lipodox^®, a commercially available PEGylated liposomal doxorubicin, was made multifunctional by surface-decorating with a cell-penetrating peptide, TATp, conjugated to PEG₁₀₀₀-PE, to enhance liposomal cell uptake. A pH-sensitive polymer, PEG₂₀₀₀-Hz-PE, with a pH-sensitive hydrazone (Hz) bond to shield the peptide in the body and expose it only at the acidic tumor cell surface, was used as well. In addition, an anti-nucleosome monoclonal antibody 2C5 attached to a long-chain polymer to target nucleosomes overexpressed on the tumor cell surface was also present. Here, we report the in vitro cell uptake and cytotoxicity of the modified multifunctional immunoliposomes as well as the in vivo studies on tumor xenografts developed subcutaneously in nude mice with MDR and drug-sensitive human ovarian cancer cells (SKOV-3). Our results show the ability of multifunctional immunoliposomes to overcome MDR by enhancing cytotoxicity in drug-resistant cells, compared with non-modified liposomes. Furthermore, in comparison with the non-modified liposomes, upon intravenous injection of these multifunctional immunoliposomes into mice with tumor xenografts, a significant reduction in tumor growth and enhanced therapeutic efficacy of the drug in both drug-resistant and drug-sensitive mice was obtained. The use of “smart” multifunctional delivery systems may provide the basis for an effective strategy to develop, improve, and overcome MDR cancers in the future.     

Relative malignant potential of human breast carcinoma cell lines established from pleural effusions and a brain metastasis.

Three human breast cancer cell lines from pleural effusions (MDA-MB-435, MDA-MB-231, MDA-MB-468) and one from a brain metastasis (MDA-MB-361) were tested for growth potential in vitro (minimum serum requirements, growth in semisolid agarose), for tumorigenicity and metastasis in nude mice following injection into the mammary fatpad (m.f.p.) and intravenously (i.v.), and for formation of experimental brain metastasis following injection into the carotid artery (i.a.). Colony-forming efficiency of the breast cancer cells in dense agarose corresponded with metastatic potential of the m.f.p. tumors. The results consistently ranked the 4 cell lines as follows: MDA-MB-435 greater than MDA-MB-231 greater than MDA-MB-468 greater than MDA-MB-361, from the most to the least aggressive. The exception was for tumor growth in the brain after i.a. injection of cells, where MDA-MB-361 cells were ranked as second highest for tumor take, but with the slowest growth rate. These results indicate that in this series of breast carcinomas, the cells from a brain metastasis (MDA-MB-361) have a lower malignant potential than cells from visceral metastases.     

转铁蛋白修饰多柔比星脂质体靶向抑制乳腺癌细胞增殖的研究

目的：利用转铁蛋白（TF）修饰多柔比星脂质体,并探讨其对乳腺癌细胞增殖的影响。方法采用薄膜超声法制备脂质体,利用硫酸梯度法制备多柔比星脂质体,然后采用后插入法制备 TF修饰多柔比星脂质体。激光共聚焦显微镜检测乳腺癌细胞 MCF-7和 MDA-MB-231对 TF-多柔比星脂质体的摄取,四甲基偶氮唑盐比色法（MTT 法）检测 TF-多柔比星脂质体靶向作用 MCF-7和 MDA-MB-231细胞后的杀伤能力,软琼脂克隆集落实验检测 TF-多柔比星脂质体对 MCF-7和 MDA-MB-231细胞的生长抑制作用。结果激光共聚焦显微镜观察显示,MCF-7细胞和 MDA-MB-231细胞对 TF-多柔比星脂质体摄取率显著高于多柔比星脂质体;MTT 法检测结果则显示,TF-多柔比星脂质体对 MCF-7细胞[（20．8±3．2）μmol／L]和 MDA-MB-231细胞[（20．1±3．0）μmol／L]杀伤的 IC50显著低于多柔比星脂质体[（158．6±24．6）μmol／L;（160．1±25．1）μmol／L）]和游离多柔比星[（161．7±26．2）μmol／L;（166．9±27．0）μmol／L）],差异有统计学意义（F ＝116．03,P ＜0．001;F ＝75．29,P ＜0．001）。软琼脂克隆集落实验显示,TF-多柔比星脂质体对克隆集落的生长抑制显著优于多柔比星脂质体、游离多柔比星及对照组[MDA-MB-231细胞直径：（60．5±10．4）μm、（94．3±16．8）μm、（131．8±22．6）μm、（162．8±30．3）μm;MCF-7细胞直径：（31．8±5．5）μm、（62．1±11．1）μm、（108．6±18．6）μm、（157．4±29．3）μm],差异有统计学意义（F ＝87．17,P ＜0．0001;F ＝178．23,P ＜0．0001）。结论 TF-多柔比星脂质体对乳腺癌细胞体外增殖具有显著的抑制作用,能够高效、特异地杀死乳腺癌细胞,为体内治疗乳腺癌提供了一定的理论依据。     

Combining doxorubicin and liposomal anti-HER-2/NEU antisense oligodeoxynucleotides to treat HER-2/NEU-expressing MDA-MB-435 breast tumor model

This study assessed the in vivo therapeutic activity of an antisense molecule targeted against HER-2/neu expressing mRNA. Antisense activity was evaluated in female SCID/Rag2m mice bearing subcutaneous tumors derived from HER-2/neu-transfected MDA-MB-435 (MDA-MB-435(HER2)) cells, a transfected line derived from the human breast cancer MDA-MB-435 cell line. Animals were treated with free or liposome-encapsulated antisense. The area under the curve (AUC(0-24h)) of the liposomal formulated antisense was demonstrated to be more than 30-fold greater than that of free antisense following intravenous administration. Efficacy was determined by assessing changes in tumor growth rate as well as by an immunohistological end-point evaluating HER-2/neu expression. HER-2/neu protein expression was reduced in mice bearing HER-2/neu-transfected MDA-MB-435 tumors when treated with liposomal antisense. However, tumors in these mice grew at a faster rate than the control, a result that was interpreted to be a consequence of selection of a more rapidly proliferating HER-2/neu-negative subpopulation of cells. Effective control of the MDA-MB-435(HER2) tumors was achieved when antisense treatment was combined with doxorubicin. Tumors derived from animals treated with the combination of doxorubicin and the liposomal antisense against HER-2/neu exhibited no detectable levels of HER-2/neu expression. Antisense targeted against HER-2/neu mRNA was effective in reducing or eliminating HER-2/neu protein expression, and when combined wtih doxorubicin treatment was efficacious in the treatment of mice bearing HER-2/neu-overexpressing human xenograft tumors.     

Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models

We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.     

Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth

We have previously reported that infection with the non-pathogenic, tumor suppressive, wild-type adeno-associated virus type 2 (AAV2) inhibited proliferation of breast cancer-derived lines representing both weakly invasive (MCF-7 and MDA-MB-468), as well as aggressive (MDA-MB-231) cancer types. AAV2-induced death occurred via targeting pathways of apoptosis and necrosis. In contrast, normal human mammary epithelial cells were unaffected upon AAV2 infection. The current study characterizes AAV2 infection and subsequent death of the highly aggressive, triple-negative (ER⁻/PR⁻/HER2⁻) MDA-MB-435 cell line derived from metastatic human breast carcinoma. Monolayer MDA-MB-435 cultures infected with AAV2 underwent complete apoptotic cell death characterized by activation of caspases -7, -8, and -9 and PARP cleavage. Death was further correlated with active AAV2 genome replication and differential expression of viral non-structural proteins Rep78 and Rep52. Cell death coincided with increased entry into S and G₂ phases, upregulated expression of the proliferation markers Ki-67 and the monomeric form of c-Myc. Expression of the p16^INK4, p27^KIP1, p21^WAF1, and p53 tumor suppressors was downregulated, indicating marked S phase progression, but sharply contrasted with hypo-phosphorylated pRb. In parallel, MDA-MB-435 breast tumor xenografts which received intratumoral injections of AAV2 were growth retarded, displayed extensive areas of necrosis, and stained positively for c-Myc as well as cleaved caspase-8. Therefore, AAV2 induced death of MDA-MB-435 xenografts was modulated through activation of caspase-regulated death pathways in relation to signals for cell cycle controls. Our findings provide foundational studies for development of novel AAV2 based therapeutics for treating aggressive, triple-negative breast cancer types.     

Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.     

miR-6775-3p在乳腺癌细胞中的表达及其对乳腺癌细胞生物学行为的影响

背景与目的：微小RNA（microRNA,miRNA）与肿瘤的发生、发展过程密切相关。探讨miR-6775-3p在乳腺癌细胞系中的表达及其对乳腺癌细胞生物学行为的影响。方法：通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测4种乳腺癌细胞系MDA-MB-231、MDA-MB-453、MDA-MB-468和BT-549中miR-6775-3p的表达水平,选取miR-6775-3p表达水平最低的乳腺癌细胞系过表达miR-6775-3p后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法检测细胞的增殖情况,同时采用transwell迁移和侵袭实验分别检测细胞迁移和侵袭能力的变化。通过RTFQ-PCR和蛋白［质］印迹法（Western blot）检测miR-6775-3p过表达的乳腺癌细胞系中细胞周期蛋白依赖性蛋白激酶4（cyclin-dependent protein kinase 4,CDK4）和CDK6,以及侵袭转移标志物基质金属蛋白酶（matrix metalloproteinase,MMP）17和MMP24 mRNA以及蛋白的表达变化。结果：RTFQ-PCR结果显示,乳腺癌细胞系MDA-MB-453中miR-6775-3p的表达最低,在MDA-MB-453细胞中转染miR-6775-3p mimics后,miR-6775-3p的表达水平明显升高（P<0.001）。CCK-8实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的增殖能力明显降低（P<0.01）。Transwell迁移和侵袭实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的迁移（P<0.001）和侵袭能力（P<0.01）明显降低。RTFQ-PCR和Western blot实验结果显示,CDK4、CDK6及MMP17、MMP24的mRNA表达和蛋白水平均显著降低（P<0.01）。结论：miR-6775-3p可能抑制乳腺癌细胞的增殖、迁移和侵袭能力。