Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs.
magnetic nanoparticles; active targeting; antifouling; breast cancer; magnetic resonance imaging
To obtain positive contrast based on T1 weighting from magnetic iron oxide nanoparticle (IONP) using ultrashort echo time (UTE) imaging and investigate quantitative relationship between positive contrast and the core size and concentration of IONPs.
Materials and Methods
Solutions of IONPs with different core sizes and concentrations were prepared. T1 and T2 relaxation times of IONPs were measured using the inversion recovery turbo spin echo (TSE) and multi-echo spin echo sequences at 3 Tesla. T1-weighted UTE gradient echo and T2-weighted TSE sequences were used to image IONP samples. U87MG glioblastoma cells bound with arginine-glycine-aspartic acid (RGD) peptide and IONP conjugates were scanned using UTE, T1 and T2-weighted sequences.
Positive contrast was obtained by UTE imaging from IONPs with different core sizes and concentrations. The relative-contrast-to-water ratio of UTE images was three to four times higher than those of T2-weighted TSE images. The signal intensity increases as the function of the core size and concentration. Positive contrast was also evident in cell samples bound with RGD-IONPs.
UTE imaging allows for imaging of IONPs and IONP bound tumor cells with positive contrast and provides contrast enhancement and potential quantification of IONPs in molecular imaging applications.
magnetic nanoparticle; magnetic resonance imaging; iron oxide; ultrashort TE; molecular Imaging
A novel handheld probe based on a microelectromechanical systems (MEMS) scanning mirror for three-dimensional (3D) fluorescence molecular tomography (FMT) is described. The miniaturized probe consists of a MEMS mirror for delivering an excitation light beam to multiple preselected points at the tissue surface and an optical fiber array for collecting the fluorescent emission light from the tissue. Several phantom experiments based on indocyanine green, an FDA approved near-infrared (NIR) fluorescent dye, were conducted to assess the imaging ability of this device. Tumor-bearing mice with systematically injected tumor-targeted NIR fluorescent probes were scanned to further demonstrate the ability of this MEMS-based FMT for imaging small animals.
The spread of cancer cells between organs, a process known as metastasis, is the cause of most cancer deaths1,2. Detecting circulating tumour cells—a common marker for the development of metastasis3,4—is difficult because ex vivo methods are not sensitive enough owing to limited blood sample volume and in vivo diagnosis is time-consuming as large volumes of blood must be analysed5–7. Here, we show a way to magnetically capture circulating tumour cells in the bloodstream of mice followed by rapid photoacoustic detection. Magnetic nanoparticles, which were functionalized to target a receptor commonly found in breast cancer cells, bound and captured circulating tumour cells under a magnet. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. By integrating in vivo multiplex targeting, magnetic enrichment, signal amplification and multicolour recognition, our approach allows circulating tumour cells to be concentrated from a large volume of blood in the vessels of tumour-bearing mice, and this could have potential for the early diagnosis of cancer and the prevention of metastasis in humans.
Background & Aims
Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target pancreatic cancer holds great promise for the development of novel approaches for the detection and treatment of pancreatic cancer. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging of pancreatic cancer using multifunctional nanoparticles.
The uPAR-targeted dual-modality molecular imaging nanoparticle probe is designed and prepared by conjugating a near-infrared dye-labeled amino-terminal fragment of the receptor binding domain of urokinase plasminogen activator to the surface of functionalized magnetic iron oxide nanoparticles.
We have shown that the systemic delivery of uPAR-targeted nanoparticles leads to their selective accumulation within tumors of orthotopically xenografted human pancreatic cancer in nude mice. The uPAR-targeted nanoparticle probe binds to and is subsequently internalized by uPAR-expressing tumor cells and tumor-associated stromal cells, which facilitates the intratumoral distribution of the nanoparticles and increases the amount and retention of the nanoparticles in a tumor mass. Imaging properties of the nanoparticles enable in vivo optical and magnetic resonance imaging of uPAR-elevated pancreatic cancer lesions.
Targeting uPAR using biodegradable multifunctional nanoparticles allows for the selective delivery of the nanoparticles into primary and metastatic pancreatic cancer lesions. This novel receptor-targeted nanoparticle is a potential molecular imaging agent for the detection of pancreatic cancer.
Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single-chain anti-EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR-expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR-QDs or ScFvEGFR-IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular-targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR-targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.
antibodies; drug delivery; nanoparticles; proteins; quantum dots
Combining ground breaking research and developments in cancer biomarkers, nanotechnology and molecular targeted medicine, a new realm of therapy is possible: personalized and predictive medicine. Developing a method to detect the overexpression of several tumor marker genes simultaneously, knowing that a single cell generally expresses more than one altered gene, should have a high predictive value for identifying cancer cells amidst the normal cellular background. Theoretically, a cancer’s unique molecular profile can be used to predict its invasive and metastatic potential, its ability to evade immune surveillance, and its potential response to treatment. Fluorescent probes have been developed to detect the levels of expression of various biomarkers in tumor cells and tissues. Expression of biomarker messenger RNAs (mRNAs) or the presence of a specific mutation in an oncogene in cancer cells can be detected using molecular beacons (MBs) that only emit fluorescent signals after binding to its specific target mRNAs. Antibodies or ligands labeled with fluorophores or fluorescent quantum dots (QDs) have been successfully used to identify specific proteins expressed in cells. Furthermore, multiplex imaging using both MBs and antibodies labeled with a fluorescent probe on the same sample may provide important information correlating the level of mRNA expression and the subsequent level of protein production for a given biomarker. This technology will be useful in research investigating cancer biology, molecular imaging and molecular profiling. With the identification of biomarkers that are related to aggressive tumor types, we may be able to predict within certain patient populations who will develop invasive cancers, and what their prognosis will be given different treatment modalities, ultimately delivering medical care and treatment strategies that are specifically tailored to each individual patient, making personalized and predictive medicine a reality.
Biomarkers; molecular beacons; molecular profiling; multiplexed imaging; nanotechnology; personalized medicine; predictive medicine; quantum dots
We describe a novel dual-modality imaging approach that integrates diffuse optical tomography (DOT) and photoacoustic imaging (PAI) through a miniaturized handheld probe based on microelectromechanical systems (MEMS) scanning mirror. We validate this dual-modal DOT/PAI approach using extensive phantom experiments, and demonstrate its application for tumor imaging using tumor-bearing mice systematically injected with targeted contrast agents.
(110.6960) Tomography; (170.0110) Imaging systems; (170.5120) Photoacoustic imaging
A highly efficient process using iron oxide magnetic nanoparticles (IO)-based immunomagnetic separation of tumor cells from fresh whole blood has been developed. The process involved polymer coated 30 nm IO that was modified with antibodies (Ab) against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) forming IO-Ab. HER2 is a cell membrane protein that is over expressed in several types of human cancer cells. Using a HER2/neu over expressing human breast cancer cell line, SK-BR3, as a model cell, the IO-Ab was used to separate 73.6 % (with a maximum capture of 84%) of SK-BR3 cells that were spiked in 1 mL of fresh human whole blood. The IO-Ab preferentially bound to SK-BR3 cells over normal cells found in blood due to the high level of HER2/neu receptor on the cancer cells unlike the normal cell surfaces. The results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field (with gradient of 100 T/m) through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells. This research holds promise for efficient separation of circulating cancer cells in fresh whole blood.
magnetic nanoparticles; iron oxide; cancer cells; cell sorting; immunomagnetic separation
apoptosis; caspase-3; enhanced green fluorescent proteins; protease sensors; ratiometric measurements
Obese breast cancer patients exhibit a higher risk for larger tumor burden and increased metastasis. Molecular effects of obesity on carcinogenesis are mediated by autocrine and paracrine effects of adipocytokine leptin. Leptin participates in tumor progression and metastasis of human breast. We show that leptin induces clonogenicity and migration potential of breast cancer cells. We found that survivin expression is induced in response to leptin. In this study, we examine the role and leptin-mediated regulation of survivin. Leptin treatment leads to survivin upregulation, due in part to the activation of Notch1 and release of transcriptionally active Notch1-intracellular-domain (NICD). ChIP analysis show that NICD gets recruited to survivin promoter at CSL-binding-site in response to leptin treatment. Inhibition of Notch1 activity inhibits leptin-induced survivin upregulation. Leptin-induced transactivation of EGFR is involved in leptin-mediated Notch1 and survivin upregulation showing a novel upstream role of leptin-EGFR-Notch1 axis. We further show that leptin-induced migration of breast cancer cells requires survivin, as overexpression of survivin further increases, whereas silencing survivin abrogates leptin-induced migration. Using a pharmacological approach to inhibit survivin, we show that 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors (HRIs), lovastatin, can effectively inhibit leptin-induced survivin expression and migration. Importantly, leptin increased breast tumor growth in nude mice. These data show a novel role for survivin in leptin-induced migration and put forth pharmacological survivin inhibition as a potential novel therapeutic target. This conclusion is supported by in vivo data showing overexpression of leptin and survivin in epithelial cells of high grade ductal carcinoma in situ and high grade invasive carcinoma.
Breast Cancer cell migration; Leptin; Notch; EGFR; Survivin
Optical imaging is an inexpensive, fast, and sensitive imaging approach for the non-invasive detection of human cancers in locations that are accessible by an optical imaging device. Light is used to probe cellular and molecular function in the context of cancer in the living body. Recent advances in the development of optical instrumentation make it possible to detect optical signals produced at a tissue depth of several centimeters. The optical signals can be endogenous contrasts that capture the heterogeneity and biological status of different tissues including tumors, or extrinsic optical contrasts that selectively accumulate in tumors to be imaged after local or systemic delivery. The use of optical imaging is now being applied in the clinic and operating room for the localization and resection of malignant tumors in addition to screening for cancer.
Optical Imaging; Cancer; Glioblastoma; Breast Cancer; Bladder Cancer; Near Infrared; Nanoparticles
Engineering and functionalizing magnetic nanoparticles have been an area of the extensive research and development in the biomedical and nanomedicine fields. Because their biocompatibility and toxicity are well investigated and better understood, magnetic nanoparticles, especially iron oxide nanoparticles, are better suited materials as contrast agents for magnetic resonance imaging (MRI) and for image-directed delivery of therapeutics. Given tunable magnetic properties and various surface chemistries from the coating materials, most applications of engineered magnetic nanoparticles take advantages of their superb MRI contrast enhancing capability as well as surface functionalities. It has been found that MRI contrast enhancement by magnetic nanoparticles is highly dependent on the composition, size and surface properties as well as the degree of aggregation of the nanoparticles. Therefore, understanding the relationships between these intrinsic parameters and the relaxivities that contribute to MRI contrast can lead to establishing essential guidance that may direct the design of engineered magnetic nanoparticles for theranostics applications. On the other hand, new contrast mechanism and imaging strategy can be developed based on the novel properties of engineered magnetic nanoparticles. This review will focus on discussing the recent findings on some chemical and physical properties of engineered magnetic nanoparticles affecting the relaxivities as well as the impact on MRI contrast. Furthermore, MRI methods for imaging magnetic nanoparticles including several newly developed MRI approaches aiming at improving the detection and quantification of the engineered magnetic nanoparticles are described.
magnetic nanoparticles; engineering; functionalizing; magnetic resonance imaging
We have developed a fluorescence imaging-based approach to detect expression of tumor marker genes in pancreatic cancer cells using molecular beacons (MBs). MBs are short hairpin oligonucleotide probes that bind to specific oligonucleotide sequences and produce fluorescent signals. MBs targeting transcripts of two tumor marker genes, mutant K-ras and survivin, were synthesized and their specificity in detection of the expression of those genes in pancreatic cancer cells was examined. We found that K-ras MBs differentially bind to mutant K-ras mRNAs, resulting in strong fluorescent signals in pancreatic cancer cells with specific mutant K-ras genes but not in normal cells or cancer cells expressing either wild type or a different mutation of the K-ras gene. Additionally, MBs targeting survivin mRNA produced a bright fluorescent signal specifically in pancreatic cancer cells. We also demonstrated that MBs labeled with different fluorophores could detect survivin and mutant K-ras mRNAs simultaneously in single cancer cells. Furthermore, we showed that survivin and K-ras MBs have a high specificity in identifying cancer cells on frozen sections of pancreatic cancer tissues. In conclusion, molecular beacon-based imaging of expression of tumor marker genes has potential for the development of novel approaches for the detection of pancreatic cancer cells.
molecular imaging; molecular beacon; K-ras mutation; survivin; in situ gene expression detection and pancreatic cancer cells
Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.
The identification of differentially regulated apoptotic signals in normal and tumor cells allows the development of cancer cell-selective therapies. Increasing evidence shows that the inhibitor of apoptosis (IAP) proteins survivin and XIAP are highly expressed in tumor cells but are absent or have very low levels of expression in normal adult tissues. We found that inhibiting AKT activity with 10 to 100 nM deguelin, a small molecule derived from natural products, markedly reduced the levels of both survivin and XIAP, inducing apoptosis in human breast cancer cells but not in normal cells. It is noteworthy that we detected an elevated level of cleaved poly(ADP-ribose) polymerase, a signature of caspase activation, without a significant increase in caspase activity in deguelin-treated cancer cells. Our results suggest that severe down-regulation of the IAPs by deguelin releases their inhibitory activity over pre-existing active caspases present in cancer cells, inducing apoptosis without the need for further caspase activation. Because normal cells have very low levels of p-AKT, XIAP, survivin, and preexisting caspase activity, deguelin had little effect on those cells. In addition, we found that combining deguelin with chemotherapy drugs enhanced drug-induced apoptosis selectively in human tumor cells, which suggests that deguelin has great potential for chemosensitization and could represent a new therapeutic agent for treatment of breast cancer.
One of the major limitations impeding the sensitivity and specificity of biomarker targeted nanoparticles is non-specific binding by biomolecules and uptake by the reticuloendothelial system (RES). We report the development of an antibiofouling polysiloxane containing amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS), for coating and functionalizing high quality hydrophobic nanocrystals such as iron oxide nanoparticles and quantum dots. These PEO-b-PγMPS coated nanocrystals were colloidally stable in biological medium and showed low non-specific binding by macromolecules after incubation with 100% fetal bovine serum. Both in vitro experiments with macrophages and in vivo biodistribution studies in mice revealed that PEO-b-PγMPS copolymer coated nanocrystals have an antibiofouling effect that reduces non-specific cell and RES uptake. Surface functionalization with amine groups was accomplished through co-crosslinking the polysiloxane coating layer and (3-Aminopropyl) trimethoxysilane in aqueous solution. Tumor integrin αvβ3 targeting peptide cyclo-RGD ligands were conjugated on the nanoparticles through a heterobifunctional linker. The resulting integrin αvβ3 targeting nanoparticle conjugates showed improved cancer cell targeting with a stronger affinity to U87MG glioma cells, which have a high expression of αvβ3 integrins, but minimal binding to MCF-7 (low expression of αvβ3 integrins).
Nanoparticles; Copolymer; Antifouling; Non-specific binding; Reticuloendothelial system; Cancer targeting
Nanotechnology is a multidisciplinary scientific field undergoing explosive development. Nanometer-sized particles offer novel structural, optical and electronic properties that are not attainable with individual molecules or bulk solids. Advances in nanomedicine can be made by engineering biodegradable nanoparticles such as magnetic iron oxide nanoparticles, polymers, dendrimers and liposomes that are capable of targeted delivery of both imaging agents and anticancer drugs. This leads toward the concept and possibility of personalized medicine for the potential of early detection of cancer lesions, determination of molecular signatures of the tumor by non-invasive imaging and, most importantly, molecular targeted cancer therapy. Increasing evidence suggests that the nanoparticles, whose surface contains a targeting molecule that binds to receptors highly expressed in tumor cells, can serve as cancer image contrast agents to increase sensitivity and specificity in tumor detection. In comparison with other small molecule contrast agents, the advantage of using nanoparticles is their large surface area and the possibility of surface modifications for further conjugation or encapsulation of large amounts of therapeutic agents. Targeted nanoparticles ferry large doses of therapeutic agents into malignant cells while sparing the normal healthy cells. Such multifunctional nanodevices hold the promise of significant improvement of current clinical management of cancer patients. This review explores the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents, the potential of nanomedicine, and the development of novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.
Nanotechnology; nanomedicine; multifunctional nanoparticles; molecular diagnosis; targeted therapy; drug delivery; nanotherapeutics; tumor imaging
Cell surface receptor-targeted magnetic iron oxide (IO) nanoparticles provide molecular magnetic resonance imaging (MRI) contrast agents for improving specificity of the detection of human cancer.
The present study reports the development of a novel targeted IO nanoparticle using a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator conjugated to IO nanoparticles (ATF-IO). This nanoparticle targets urokinase plasminogen activator receptor (uPAR), which is overexpressed in breast cancer tissues.
ATF-IO nanoparticles are able to specifically bind to and be internalized by uPAR-expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing subcutaneous and intraperitoneal mammary tumors leads to the accumulation of the particles in tumors, generating a strong MRI contrast detectable by a clinical MRI scanner at a field strength of 3 Tesla. Target specificity of ATF-IO nanoparticles demonstrated by in vivo MRI is further confirmed by near infrared (NIR) fluorescence imaging of the mammary tumors using NIR dye-labeled ATF peptides conjugated to IO nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared to those receiving non-targeted IO nanoparticles.
Our results suggest that uPAR-targeted ATF-IO nanoparticles have potential as molecularly-targeted, dual modality imaging agents for in vivo imaging of breast cancer.
magnetic resonance imaging; near infrared optical imaging; molecular imaging; magnetic iron oxide nanoparticle; urokinase plasminogen receptor; breast cancer
Great efforts have been made to develop novel and efficacious therapeutics against pancreatic cancer to improve the treatment outcomes. Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) is such a therapeutic cytokine with selective killing effect toward malignant cells. However, some human pancreatic cancers are intrinsically resistant to TRAIL-mediated apoptosis or therapy. In this study, we have shown that the histone deacetylase inhibitor LBH589 can synergize with TRAIL to augment apoptosis even in TRAIL-resistant cells. LBH589 decreased c-FLIP levels in every tested cell line and survivin levels in some of the tested cell lines. Enforced expression of ectopic c-FLIP, but not survivin, abolished the cooperative induction of apoptosis by the combination of LBH589 and TRAIL, indicating that c-FLIP downregulation plays a critical role in LBH589 sensitization of pancreatic cancer cells to TRAIL. Moreover, LBH589 decreased c-FLIP stability and the presence of the proteasome inhibitor MG132 prevented c-FLIP from reduction by LBH589. Correspondingly, we detected increased levels of ubiqutinated c-FLIP in LBH589-treated cells. These data thus indicate that LBH589 promotes ubiqutin/proteasome-mediated degradation of c-FLIP, leading to downregulation of c-FLIP. Collectively, LBH589 induces c-FLIP degradation and accordingly sensitizes pancreatic cancer cells to TRAIL-induced apoptosis, highlighting a novel therapeutic regimen against pancreatic cancer.
Breast tumors are the second major cause of cancer-related death in women worldwide. These tumors are aggressive, leading to metastatic cancers that are heterogeneous in nature, with numerous subtypes. The basal-like tumor subtype invariably shows unfavorable prognosis and is often characterized by the lack of estrogen, progesterone and HER2 receptors. These cancer types do not respond to the current targeted therapies. Therefore, the need for the discovery of novel diagnostic markers/therapeutic targets is of paramount importance. Immortalization of breast tumor cells leading to advanced stage cancer is one of the pivotal steps in breast cancer and telomeres/telomerase play a critical role in this process. Using single telomere length analysis, cell lines with a basal-like phenotype encompassing immortalized/non-tumorigenic MCF10A and invasive/metastatic MCF10CA1 along with the MCF-7 cell line were examined for the presence of a unique class of telomere t-stumps. Telomerase activity, protein levels of telomerase and bulk telomere lengths were assessed in the above-mentioned cell lines. This is the first study describing the existence of a distinct class of extremely short telomeres termed ‘t-stumps’ in breast cancer cell lines. The cell lines MCF10A and MCF10CA1 showed distinct telomeric bands in the molecular size range of 100–1,000 bp, whereas the MCF-7 cell line showed very low levels of t-stumps. Of note is that only the highly invasive/metastatic cancer cell line MCF10CA1 exhibited an abundance of a cluster of t-stumps with a size distribution range of 500–700 bp. These unique t-stumps observed in the advanced breast cancer cell line may serve as a novel diagnostic marker and also form a key molecular target for novel anticancer therapy.
telomere t-stumps; isogenic breast cancer cell lines; immortalized cell line; invasive/metastatic cell line; telomerase reverse transcriptase; cluster of t-stumps
Although it was observed that inhibition of the antiapoptotic protein survivin expression in lung cancer cells induces apoptosis, the expression and role of survivin variants (survivin-2B and survivin-ΔEx3) in lung cancer have not yet been characterized. We analyzed 24 non-small-cell lung cancer (NSCLC) samples by semi-quantitative RT-PCR. Surprisingly, our results revealed that high-level expression of survivin-2B is significantly associated with the patient category of “no relapse and alive” (p-value < 0.0001). In contrast, high-level expression of survivin-ΔEx3 is highly associated with the patient category of “relapse and dead” (p-value < 0.0001). Consistent with this observation, exogenous expression of survivin-2B in A549 lung cancer cells inhibited cell growth, disrupted the mitochondria potential, and induced apoptotic cell death, while expression of survivin-ΔEx3 protected the mitochondria potential and facilitated cell survival. These findings provide evidence that survivin-2B and survivin-ΔEx3 play opposite roles in disease relapse and NSCLC cell survival, which is likely through the differential modulation of mitochondrial potential. Thus, controlling the differential expression of survivin-2B and survivin-ΔEx3 may represent novel approaches for cancer therapeutics in NSCLC.
Survivin-2B; Survivin-ΔEx3; Non-small-cell lung cancer
PI3K pathway exerts its function through its downstream molecule AKT in regulating various cell functions including cell proliferation, cell transformation, cell apoptosis, tumor growth and angiogenesis. PTEN is an inhibitor of PI3K, and its loss or mutation is common in human prostate cancer. But the direct role and mechanism of PI3K/PTEN signaling in regulating angiogenesis and tumor growth in vivo remain to be elucidated. In this study, by using chicken chorioallantoic membrane (CAM) and in nude mice models, we demonstrated that inhibition of PI3K activity by LY294002 decreased PC-3 cells-induced angiogenesis. Reconstitution of PTEN, the molecular inhibitor of PI3K in PC-3 cells inhibited angiogenesis and tumor growth. Immunohistochemical staining indicated that PTEN expression suppressed HIF-1α, VEGF and PCNA expression in the tumor xenographs. Similarly, expression of AKT dominant negative mutant also inhibited angiogenesis and tumor growth, and decreased the expression of HIF-1α and VEGF in the tumor xenographs. These results suggest that Inhibition of PI3K signaling pathway by PTEN inhibits tumor angiogenesis and tumor growth. In addition, we found that AKT is the downstream target of PI3K in controlling angiogenesis and tumor growth, and PTEN could inhibit angiogenesis by regulating the expression of HIF-1 and VEGF expression through AKT activation in PC-3 cells.
PTEN; AKT; Hypoxia inducible factor 1; Vascular endothelial growth factor
Hypoxia-inducible factor-1α (HIF-1α) primarily mediates the hypoxic response. HIF-1α induction by various stimuli contributes to cell proliferation and survival. To investigate the effect of HIF-1α, we used small interfering RNA (siRNA), and expected that cell apoptosis and sensitivity to chemotherapeutic drug increase, when we blocked the HIF-1α gene. Thus we performed in vitro and in vivo experiment to clarify the effect of hypoxia-inducible factor-1α on tumor growth.
Materials and Methods
We made control and HIF-1α
siRNA using vector plasmid and then transfected Mia-paca cell lines with these RNAs. After selection with geneticin, two new cell lines were made, confirmed via immunoblotting. After treating with gemcitabine, each cell line was assayed to confirm the effect of HIF-1α siRNA using the cell proliferation assay and capase-3 assay. And then in vivo study was performed using female athymic nude mice. After subcutaneously injecting each new cell lines, intraperitoneal gemicitabine chemotherapy was performed for 3 weeks. During that period, we analyzed the difference of tumor growth rate.
tumor growth of HIF-1α siRNA-transfected group was slower than that of the control group both in vitro and in vivo experiment.
The suppression of HIF-1α results in decrease of cell proliferation and increase of chemosensitivity of pancreatic cancer cell line. Therefore, targeting the HIF-1α may be useful treatment modality for some pancreatic cancers.
Hypoxia-Inducible Factor 1 alpha; small interfering RNA-pancreatic neoplasms
We have previously shown that hedamycin, a GC-rich DNA-binding antitumor agent, downregulates survivin transcription (Wu et al. (2005) Molecular mechanism of inhibition of survivin transcription by the GC-rich sequence selective DNA-binding antitumor agent, hedamycin: evidence of survivin downregulation associated with drug sensitivity. J. Biol. Chem., 280, 9745–9751). Here, we report that treatment of cancer cells with Hoechst33342, an AT-rich DNA-binding ligand, upregulated survivin protein, mRNA and promoter activity. Functional analysis of survivin promoter-luciferase constructs followed by in vivo footprinting experiments identified a 28-bp AT-rich DNA element (−908 to −881, designated as H369W) that mediates a major effect of Hoechst33342 on the upregulation of survivin promoter activity. Electrophoresis mobility shift assay (EMSA) experiments showed that Hoechst33342 binds to H369W and abrogates H369W–protein interactions. Intriguingly, there is a highly conserved DNA-binding motif for growth factor independence 1 (Gfi-1), a transcriptional repressor protein, in the H369W DNA element. Accordingly, EMSA experiments demonstrated that either the cold canonical Gfi-1-binding DNA oligonucleotide or the cold H369W specifically competes with H369W–protein complexes. Consistently, anti-Gfi-1 antibody is able to supershift the H369W–protein complex on the EMSA gel. Lastly, our data reveal that upregulation of survivin by Hoechst33342 is involved in cancer drug resistance. We propose that hindrance of H369W–Gfi-1 interactions in the survivin promoter, initiated by Hoechst33342, contributes to upregulation of survivin transcription, and as a consequence, hampers Hoechst33342's cytotoxicity.