A Novel Magnetic Nanoparticle Drug Carrier for Enhanced Cancer Chemotherapy

Background

Magnetic nanoparticles (NPs) loaded with antitumor drugs in combination with an external magnetic field (EMF)-guided delivery can improve the efficacy of treatment and may decrease serious side effects. The purpose of this study was 1) to investigate application of PEG modified GMNPs (PGMNPs) as a drug carrier of the chemotherapy compound doxorubicin (DOX) in vitro; 2) to evaluate the therapeutic efficiency of DOX-conjugated PGMNPs (DOX-PGMNPs) using an EMF-guided delivery in vivo.

Methods

First, DOX-PGMNPs were synthesized and the cytotoxicity of DOX-PGMNPs was assessed in vitro. Second, upon intravenous administration of DOX-PMGPNs to H22 hepatoma cell tumor-bearing mice, the DOX biodistribution in different organs (tissues) was measured. The antitumor activity was evaluated using different treatment strategies such as DOX-PMGPNs or DOX-PMGPNs with an EMF-guided delivery (DOX-PGMNPs-M).

Results

The relative tumor volumes in DOX-PGMNPs-M, DOX-PGMNPs, and DOX groups were 5.46±1.48, 9.22±1.51, and 14.8±1.64, respectively (each p<0.05), following treatment for 33 days. The life span of tumor-bearing mice treated with DOX-PGMNPs-M, DOX-PGMNPs, and DOX were 74.8±9.95, 66.1±13.5, and 31.3±3.31 days, respectively (each p<0.05).

Conclusion

This simple and adaptive nanoparticle design may accommodate chemotherapy for drug delivery optimization and in vivo drug-target definition in system biology profiling, increasing the margin of safety in treatment of cancers in the near future.     

Selective Androgen Receptor Modulators (SARMs) Negatively Regulate Triple-Negative Breast Cancer Growth and Epithelial:Mesenchymal Stem Cell Signaling

Introduction

The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75–95% of estrogen receptor (ER)-positive and 40–70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.

Materials and Methods

Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action.

Results

Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.

Conclusion

1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.     

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background

A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.

Methods

In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.

Results

In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.

Conclusions

Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.     

NAHA, a novel hydroxamic acid-derivative, inhibits growth and angiogenesis of breast cancer in vitro and in vivo

Background

We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo.

Methodology/Principal Findings

Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors.

Conclusion

NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Tumor Vasculature-Targeted Recombinant Mutated Human TNF-α Enhanced the Antitumor Activity of Doxorubicin by Increasing Tumor Vessel Permeability in Mouse Xenograft Models

Objective

Increasing evidence suggests that, when used in combination, tumor necrosis factor-α (TNF-α) synergizes with traditional chemotherapeutic drugs to exert a heightened antitumor effect. The present study investigated the antitumor efficacy of recombinant mutated human TNF-α specifically targeted to the tumor vasculature (RGD-rmhTNF-α) combined with the chemotherapeutic agent doxorubicin in 2 murine allografted tumor models.

Methods

Mice bearing hepatoma or sarcoma allografted tumors were treated with various doses of RGD-rmhTNF-α alone or in combination with doxorubicin (2 mg/kg). We then evaluated tumor growth and tumor vessel permeability as well as intratumoral levels of RGD-rmhTNF-α and doxorubicin.

Results

RGD-rmhTNF-α treatment enhanced the permeability of the tumor vessels and increased intratumoral doxorubicin levels. In addition, intratumoral RGD-rmhTNF-α levels were significantly higher than that of rmhTNF-α. In both of the tested tumor models, administering RGD-rmhTNF-α in combination with doxorubicin resulted in an enhanced antitumor response compared to either treatment alone. Double-agent combination treatment of doxorubicin with 50,000 IU/kg RGD-rmhTNF-α induced stronger antitumor effects on H22 allografted tumor-bearing mice than the single doxorubicin agent alone. Moreover, doxorubicin with 10,000 IU/kg RGD-rmhTNF-α synergized to inhibit tumor growth in S180 allografted tumor-bearing mice.

Conclusions

These results suggest that targeted delivery of low doses of RGD-rmhTNF-α into the tumor vasculature increases the antitumor efficacy of chemotherapeutic drugs.     

Targeted Delivery of Doxorubicin-Loaded Poly (ε-caprolactone)-b-Poly (N-vinylpyrrolidone) Micelles Enhances Antitumor Effect in Lymphoma

Background

The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL₆₃-b-PNVP₉₀ was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma.

Methods

In this study, we have used micelles of amphiphilic PCL₆₃-b-PNVP₉₀ block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma.

Results

DOX loaded PCL₆₃-b-PNVP₉₀ block copolymer micelles (DOX-PCL₆₃-b-PNVP₉₀) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Dalton''s lymphoma, DL). DOX-PCL₆₃-b-PNVP₉₀ demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL₆₃-b-PNVP₉₀ demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments.

Conclusion

Unlike free DOX, DOX-PCL₆₃-b-PNVP₉₀ does not show cytotoxicity against normal cells. DOX-PCL₆₃-b-PNVP₉₀ prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.     

Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl

Background

The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice.

Methodology and Results

The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers.

Conclusion and Significance

These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.     

Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging

Purpose

Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging.

Experimental Design

Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis.

Results

Affibody-DyLight conjugates showed high affinity to HER2 (K_D = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue.

Conclusions

The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.     

Hypoxia Regulates CD44 and Its Variant Isoforms through HIF-1α in Triple Negative Breast Cancer

Background

The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization.

Methods and Findings

The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of ¹²⁵I-radiolabeled CD44 antibody.

Conclusions

Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Mitotic Arrest and Apoptosis in Breast Cancer Cells Induced by Origanum majorana Extract: Upregulation of TNF-α and Downregulation of Survivin and Mutant p53

Background

In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231.

Results

We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic–resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation.

Conclusion

Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use.     

Assessment of Tracer 99mTc(V)-DMSA Uptake as a Measure of Tumor Cell Proliferation In Vitro

Purpose

To examine whether ^99mTc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation.

Methods

Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with ^99mTc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-³H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.

Results

In all cell lines rates of ^99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-³H]thymidine incorporation and expression of PCNA and Ki-67.

Conclusion

Rates of ^99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.     

Antitumor activity of Noscapine in combination with Doxorubicin in triple negative breast cancer

Background

The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC).

Methods

TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues.

Results

Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC₅₀ values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups.

Conclusions

Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.     

A Novel Compound NSC745885 Exerts an Anti-Tumor Effect on Tongue Cancer SAS Cells In Vitro and In Vivo

Objective

Oral squamous cell carcinoma (OSCC) is a prevalent cancer, especially in developing countries. Anthracyclines and their anthraquinone derivatives, such as doxorubicin, exhibit a cell growth inhibitory effect and have been used as anti-cancer drugs for many years. However, the cardiotoxicity of anthracycline antibiotics is a major concern in their clinical application. NSC745885 is a novel compound synthesized from 1,2-diaminoanthraquinone, which subsequently reacts with thionyl chloride and triethylamine. The present study aimed to investigate the anti-oral cancer potential and the safety of NSC745885.

Methods

We investigated the anti-cancer potential of NSC745885 in oral squamous carcinoma cell lines and in an in vivo oral cancer xenograft mouse model. The expression of apoptotic related genes were evaluated by real-time RT-PCR and western bloting, and the in vivo assessment of apoptotic marker were measured by immunohistochemical staining. The anti-tumor efficiency and safety between doxorubicin and NSC745885 were also compared.

Results

Our results demonstrated that NSC745885 exhibits anti-oral cancer activity through the induction of apoptosis in cancer cells and in tumor-bearing mice, and this treatment did not induce marked toxicity in experimental mice. This compound also exhibits a comparable anti-tumor efficiency and a higher safety in experimental mice when compared to doxorubicin.

Conclusions

The data of this study provide evidence for NSC745885 as a potential novel therapeutic drug for the treatment of human OSCC.