MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

Introduction

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method

In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results

MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions

MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.     

Quercetin Suppresses Drug-Resistant Spheres via the p38 MAPK–Hsp27 Apoptotic Pathway in Oral Cancer Cells

Background

Treatment failure in oral squamous cell carcinoma (OSCC) leading to local recurrence(s) and metastases is mainly due to drug resistance. Cancer stem cells (CSCs) are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive.

Methods

A drug-resistant sphere (DRSP) model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo.

Results

Our data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis) and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27) via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu), suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC.

Conclusions

The p38 MAPK–Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis using Qu combined with Cis may be a treatment strategy to improve prognosis in patients with OSCC.     

Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance

Background

The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies.

Methodology/Principal Findings

In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo.

Conclusions/Significance

The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.     

EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Background

Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.

Methodology/Principal Findings

We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as “tumor spheres” and as “monolayers” in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.

Conclusions/Significance

EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.     

De-Regulated MicroRNAs in Pediatric Cancer Stem Cells Target Pathways Involved in Cell Proliferation,Cell Cycle and Development

Background

microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.

Methodology/Results

Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).

Conclusion

Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.     

Decreased expression of SATB2: a novel independent prognostic marker of worse outcome in laryngeal carcinoma patients

Background

To investigate the expression and role of special AT-rich sequence-binding protein-2 (SATB2) in laryngeal squamous cell carcinoma (LSCC) tissue and cell line (HEp2), and to evaluate the clinical and prognostic significance of SATB2 protein in patients with LSCC.

Methods

The expression of SATB2 was examined in LSCC tissue and HEp2 cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of SATB2 on the proliferation and tumor progression ability of HEp2 cells. Tumor formation assay in nude mice was used to analyze the effect of SATB2 on the tumorigenicity of HEp2 cells.

Results

The status of SATB2 protein in carcinoma tissues is much lower than that in paracarcinoma tissues. The overall survival of the patients with high SATB2 expression was significantly higher than the low SATB2 expression group. Lower or negative SATB2 expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of SATB2 in HEp2 cells inhibited cell proliferation and tumor progression ability, and down-regulation of SATB2 showed the opposite effects. Over-expression of SATB2 repressed the tumorigenicity of HEp2 cells by in vivo experiments. Moreover, multivariate analysis suggested that SATB2 expression might be an independent prognostic indicator for the survival of LSCC patients after curative surgery.

Conclusions

SATB2 might involve in the development and progression of LSCC as a tumor suppressor, and thereby may be a valuable prognostic marker for LSCC patients.     

Nonadhesive culture system as a model of rapid sphere formation with cancer stem cell properties

Background

Cancer stem cells (CSCs) play an important role in tumor initiation, progression, and metastasis and are responsible for high therapeutic failure rates. Identification and characterization of CSC are crucial for facilitating the monitoring, therapy, or prevention of cancer. Great efforts have been paid to develop a more effective methodology. Nevertheless, the ideal model for CSC research is still evolving. In this study, we created a nonadhesive culture system to enrich CSCs from human oral squamous cell carcinoma cell lines with sphere formation and to characterize their CSC properties further.

Methods

A nonadhesive culture system was designed to generate spheres from the SAS and OECM-1 cell lines. A subsequent investigation of their CSC properties, including stemness, self-renewal, and chemo- and radioresistance in vitro, as well as tumor initiation capacity in vivo, was also performed.

Results

Spheres were formed cost-effectively and time-efficiently within 5 to 7 days. Moreover, we proved that these spheres expressed putative stem cell markers and exhibited chemoradiotherapeutic resistance, in addition to tumor-initiating and self-renewal capabilities.

Conclusions

Using this nonadhesive culture system, we successfully established a rapid and cost-effective model that exhibits the characteristics of CSCs and can be used in cancer research.     

Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

Objective

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells.

Methods

High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays.

Results

Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting.

Conclusion

Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).     

LMNA Knock-Down Affects Differentiation and Progression of Human Neuroblastoma Cells

Background

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.

Methodology/Principal Findings

Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.

Conclusions/Significance

We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.     

Effect of CD44 Binding Peptide Conjugated to an Engineered Inert Matrix on Maintenance of Breast Cancer Stem Cells and Tumorsphere Formation

Introduction

As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs.

Methods

4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP).

Results

Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP.

Conclusion

CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.     

Noninvasive Identification of Viable Cell Populations in Docetaxel-Treated Breast Tumors Using Ferritin-Based Magnetic Resonance Imaging

Background

Cancer stem cells (CSCs) are highly tumorigenic and are responsible for tumor progression and chemoresistance. Noninvasive imaging methods for the visualization of CSC populations within tumors in vivo will have a considerable impact on the development of new CSC-targeting therapeutics.

Methodology/Principal Findings

In this study, human breast cancer stem cells (BCSCs) transduced with dual reporter genes (human ferritin heavy chain [FTH] and enhanced green fluorescence protein [EGFP]) were transplanted into NOD/SCID mice to allow noninvasive tracking of BCSC-derived populations. No changes in the properties of the BCSCs were observed due to ferritin overexpression. Magnetic resonance imaging (MRI) revealed significantly different signal intensities (R₂* values) between BCSCs and FTH-BCSCs in vitro and in vivo. In addition, distinct populations of pixels with high R₂* values were detected in docetaxel-treated FTH-BCSC tumors compared with control tumors, even before the tumor sizes changed. Histological analysis revealed that areas showing high R₂* values in docetaxel-treated FTH-BCSC tumors by MRI contained EGFP+/FTH+ viable cell populations with high percentages of CD44+/CD24− cells.

Conclusions/Significance

These findings suggest that ferritin-based MRI, which provides high spatial resolution and tissue contrast, can be used as a reliable method to identify viable cell populations derived from BCSCs after chemotherapy and may serve as a new tool to monitor the efficacy of CSC-targeting therapies in vivo.     

Lineage tracing of cardiac explant derived cells

Aims

Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs.

Methods

Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies.

Results

Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone.

Conclusions

This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.     

Efficient enrichment of hepatic cancer stem-like cells from a primary rat HCC model via a density gradient centrifugation-centered method

Background

Because few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs.

Methodology

After hepatic tumor cells (HTCs) were first isolated from diethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment (DTDA), they were separated into four fractions using percoll continuous gradient centrifugation (PCGC) and sequentially designated as fractions I–IV (FI–IV). Morphological characteristics, mRNA and protein levels of stem cell markers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and in vivo malignant capacities were determined for the cells of each fraction.

Findings

As the density of cells increased, 22.18%, 11.62%, 4.73% and 61.47% of primary cultured HTCs were segregated in FI–FIV, respectively. The cells from FIII (density between 1.041 and 1.062 g/ml) displayed a higher nuclear-cytoplasmic ratio and fewer organelles and expressed higher levels of stem cell markers (AFP, EpCAM and CD133) than cells from other fractions (P<0.01). Additionally, in vitro, the cells from FIII showed a greater capacity to self-renew, differentiate into mature HTCs, transit across membranes, close scratches, and carry resistance to chemotherapy than did cells from any other fraction; in vivo, injection of only 1×10⁴ cells from FIII could generate tumors not only in subcutaneous tissue but also in the livers of nude mice.

Conclusions

Through our novel method, HCSC-like cells were successfully enriched in FIII. This study will greatly contribute to two important areas of biological interest: CSC isolation and HCC therapy.     

Emulsified Phosphatidylserine,Simple and Effective Peptide Carrier for Induction of Potent Epitope-Specific T Cell Responses

Background

To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability.

Methodology/Principal Findings

Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c⁺CD11b⁺MHCII⁺ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo.

Conclusions/Significance

This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.     

Syndecan Binding Protein (SDCBP) Is Overexpressed in Estrogen Receptor Negative Breast Cancers,and Is a Potential Promoter for Tumor Proliferation

Background

Syndecan binding protein (SDCBP), an adapter protein containing PDZ domains, contributes to the tumorigenicity and metastasis of many malignant tumors, such as malignant melanoma. Our study aimed in revealing the expression profile of SDCBP in breast cancer (BCa) and its role in tumor cell proliferation, and then exploring its value in the targeted treatment of BCa.

Methodology/Principal Findings

We first evaluated the SDCBP expression by immunohistochemistry in normal breast and BCa tissues. Then we explored the expression profile of SDCBP in different BCa cell lines. By constructing SDCBP-silenced BCa cell clones, we further assessed the effects of SDCBP suppression on tumor cells in vitro by cell culture and in vivo by tumorigenicity. SDCBP expression was detected in 80.6% (n = 160) of BCa tissues, in contrast to its expression in 13% (n = 23) of normal breast tissues (P<0.001). Among the tumors, the level of its expression was positively correlated with histological grade and tumor staging while negatively correlated with the estrogen receptor (ER) expression. Higher expression of SDCBP was also noted in ER-negative BCa cell lines. It was also identified that SDCBP expression was down-regulated in a dose-dependent mode by 17-β estradiol in estrogen-responsive MCF-7. Furthermore, SDCBP silence inhibited ER-negative tumor cell growth in vivo and in vitro. Cell cycle studies showed that SDCBP silence increased G1 cell population and resulted in related cell-cycle-regulator changes: up-regulation of p21 and p27 while down-regulation of cyclin E.

Conclusion/Significance

Our results suggested that SDCBP played an important role in tumor growth of ER-negative BCas. In these tumors where the estrogen signaling pathway is not available, SDCBP probably contribute to tumor growth through an alternative signaling pathway by promoting tumor cells passing the G1/S checkpoint into the cell cycle. Suppression of SDCBP expression may have its potential to become a targeted therapy for ER-negative BCas.     

Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer

Background

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.

Methodology/Principal Findings

In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.

Conclusions/Significance

Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.     

RHPS4 G-Quadruplex Ligand Induces Anti-Proliferative Effects in Brain Tumor Cells

Background

Telomeric 3′ overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure.

Methods

We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels.

Results

Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo.

Conclusion

This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.