The EPR effect results from the extravasation of macromolecules or nanoparticles through tumor blood vessels. We here provide a historical review of the EPR effect, including its features, vascular ...mediators found in both cancer and inflamed tissue. In addition, architectural and physiological differences of tumor blood vessels vs that of normal tissue are commented. Furthermore, methods of augmentation of the EPR effect are described, that result in better tumor delivery and improved therapeutic effect, where nitroglycerin, angiotensin I-converting enzyme (ACE) inhibitor, or angiotensin II-induced hypertension are employed. Consequently, better therapeutic effect and reduced systemic toxicity are generally observed. Obviously, the EPR effect based delivery of nanoprobes are also useful for tumor-selective imaging agents with using fluorescent or radio nuclei in nanoprobes. We also commented a key difference between passive tumor targeting and the EPR effect in tumors, particularly as related to drug retention in tumors: passive targeting of low-molecular-weight X-ray contrast agents involves a retention period of less than a few minutes, whereas the EPR effect of nanoparticles involves a prolonged retention time—days to weeks.
Color images in (A) using low molecular weight fluorescent dye rhodamine B is compared with that of high molecular weight in (B) using albumin conjugated with tetraetylrhodamine (67kDa), fluorescent nanoprobe. The latter (B) shows distinct fluorescent tumor image in vivo, reflecting tumor selective accumulation of nanoprobes based on the EPR. Tumor (S-180) is located at both left and right dorsal skin showing intense fluorescence. In contrast no tumor selective drug uptake is seen for the low molecular weight dye, free Rhodamine B in (A). Display omitted
The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors related to their anatomical and pathophysiological differences from normal tissues. For example, ...angiogenesis leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs. This EPR effect served as a basis for development of macromolecular anticancer therapy. We demonstrated methods to enhance this effect artificially in clinical settings. Of great importance was increasing systolic blood pressure via slow angiotensin II infusion. Another strategy involved utilization of NO-releasing agents such as topical nitroglycerin, which releases nitrite. Nitrite is converted to NO more selectively in the tumor tissues, which leads to a significantly increased EPR effect and enhanced antitumor drug effects as well. This review discusses molecular mechanisms of factors related to the EPR effect, the unique anatomy of tumor vessels, limitations and techniques to avoid such limitations, augmenting tumor drug delivery, and experimental and clinical findings.
A major problem with conventional antitumor therapeutics is nonselective delivery of cytotoxic drugs to normal vital organs and tissues but little delivery to tumor tissues.
Here, the authors ...describe the tumor selective delivery of antitumor drugs by taking advantage of nano-sized drugs and the means to augment it further. Based on the enhanced permeability and retention (EPR) effect, the mechanism for more efficient universal tumor delivery using macromolecular drugs to cover wider tumor types than single molecular target is discussed. Unique properties of solid tumor vasculature in the tumor tissue are discussed, especially leakiness of the blood vessels and factors involved and impaired clearance of macromolecular drugs from the tumor interstitium via the lymphatic system. The criteria for such macromolecular drugs or nanomedicines for effective accumulation at tumor sites is commented on as well as the importance of long plasma retention time of such drugs and a need to release active principles from nanoparticles at target sites. Methods to augment the EPR effect and tumor delivery (2 - 3 times) and its application to photodynamic therapy are also discussed.
Tumor selective delivery of antitumor drugs based on the EPR effect can be accomplished and augmented by modulating the tumor environment. This methodology is favorable not only for tumor therapy but also for tumor imaging.
Nucleoporin Nup88, a component of nuclear pore complexes, is known to be overexpressed in several types of tumor tissue. The overexpression of Nup88 has been reported to promote the early step of ...tumorigenesis by inducing multinuclei in both HeLa cells and a mouse model. However, the molecular basis of how Nup88 leads to a multinucleated phenotype remains unclear because of a lack of information concerning its binding partners. In this study, we characterize a novel interaction between Nup88 and vimentin. We also examine the involvement of vimentin in the Nup88-dependent multinucleated phenotype.
Cells overexpressing tagged versions of Nup88, vimentin and their truncations were used in this study. Coprecipitation and GST-pulldown assays were carried out to analyze protein-protein interactions. Vimentin knockdown by siRNA was performed to examine the functional role of the Nup88-vimentin interaction in cells. The phosphorylation status of vimentin was analyzed by immunoblotting using an antibody specific for its phosphorylation site.
Vimentin was identified as a Nup88 interacting partner, although it did not bind to other nucleoporins, such as Nup50, Nup214, and Nup358, in HeLa cell lysates. The N-terminal 541 amino acid residues of Nup88 was found to be responsible for its interaction with vimentin. Recombinant GST-tagged Nup88 bound to recombinant vimentin in a GST-pulldown assay. Although overexpression of Nup88 in HeLa cells was observed mainly at the nuclear rim and in the cytoplasm, colocalization with vimentin was only partially detected at or around the nuclear rim. Disruption of the Nup88-vimentin interaction by vimentin specific siRNA transfection suppressed the Nup88-dependent multinucleated phenotype. An excess amount of Nup88 in cell lysates inhibited the dephosphorylation of a serine residue (Ser83) within the vimentin N-terminal region even in the absence and presence of an exogenous phosphatase. The N-terminal 96 amino acid residues of vimentin interacted with both full-length and the N-terminal 541 residues of Nup88.
Nup88 can affect the phosphorylation status of vimentin, which may contribute to the Nup88-dependent multinucleated phenotype through changing the organization of vimentin.
The enhanced permeability and retention (EPR) effect is a unique pathophysiological phenomenon of solid tumors that sees biocompatible macromolecules (>40 kDa) accumulate selectively in the tumor. ...Various factors have been implicated in this effect. Herein, we report that heme oxygenase‐1 (HO‐1; also known as heat shock protein 32) significantly increases vascular permeability and thus macromolecular drug accumulation in tumors. Intradermal injection of recombinant HO‐1 in mice, followed by i.v. administration of a macromolecular Evans blue–albumin complex, resulted in dose‐dependent extravasation of Evans blue–albumin at the HO‐1 injection site. Almost no extravasation was detected when inactivated HO‐1 or a carbon monoxide (CO) scavenger was injected instead. Because HO‐1 generates CO, these data imply that CO plays a key role in vascular leakage. This is supported by results obtained after intratumoral administration of a CO‐releasing agent (tricarbonyldichlororuthenium(II) dimer) in the same experimental setting, specifically dose‐dependent increases in vascular permeability plus augmented tumor blood flow. In addition, induction of HO‐1 in tumors by the water‐soluble macromolecular HO‐1 inducer pegylated hemin significantly increased tumor blood flow and Evans blue–albumin accumulation in tumors. These findings suggest that HO‐1 and/or CO are important mediators of the EPR effect. Thus, anticancer chemotherapy using macromolecular drugs may be improved by combination with an HO‐1 inducer, such as pegylated hemin, via an enhanced EPR effect. (Cancer Sci 2012; 103: 535–541)
Abstract
We previously demonstrated that expression of a Krüppel-like zinc finger transcription factor, GLIS1, dramatically increases under hypoxic conditions via a transcriptional mechanism induced ...by HIF-2α cooperating with AP-1 members. In this study, we focused on the functional roles of GLIS1 in breast cancer. To uncover its biological function, the effects of altered levels of GLIS1 in breast cancer cell lines on cellular growth, wound-healing and invasion capacities were assessed. Knockdown of GLIS1 using siRNA in BT-474 cells resulted in significant growth stimulation under normoxia, while attenuation was found in the cell invasion assay under hypoxic conditions. In MDA-MB-231 cells expressing exogenous 3xFLAG-tagged GLIS1, GLIS1 attenuated cell proliferation and enhanced cell mobility and invasion capacities under normoxia. In addition, breast cancer cells expressing GLIS1 acquired resistance to irradiation. Whole transcriptome analysis clearly demonstrated that downstream signals of GLIS1 are related to various cellular functions. Among the genes with increased expression, we focused on WNT5A. Knockdown of WNT5A indicated that enhancement of acquired cell motility in the MDA-MB-231 cells expressing GLIS1 was mediated, at least in part, by WNT5A. In an analysis of publicly available data, patients with estrogen receptor-negative breast cancer showing high levels of GLIS1 expression showed much worse prognosis than those with low levels. In summary, hypoxia-induced GLIS1 plays significant roles in breast cancer cells via regulation of gene expression related to cell migration and invasion capacities, resulting in poorer prognosis in patients with advanced breast cancer.
Hypoxia-induced GLIS1 plays significant roles in breast cancer cells via regulation of gene expression related to cell migration and invasion capacities, resulting in poorer prognosis in patients with advanced breast cancer.
Previously, we prepared a pirarubicin (THP)‐encapsulated micellar drug using styrene–maleic acid copolymer (SMA) as the drug carrier, in which active THP was non‐covalently encapsulated. We have now ...developed covalently conjugated SMA‐THP (SMA‐THP conjugate) for further investigation toward clinical development, because covalently linked polymer–drug conjugates are known to be more stable in circulation than drug‐encapsulated micelles. The SMA‐THP conjugate also formed micelles and showed albumin binding capacity in aqueous solution, which suggested that this conjugate behaved as a macromolecule during blood circulation. Consequently, SMA‐THP conjugate showed significantly prolonged circulation time compared to free THP and high tumor‐targeting efficiency by the enhanced permeability and retention (EPR) effect. As a result, remarkable antitumor effect was achieved against two types of tumors in mice without apparent adverse effects. Significantly, metastatic lung tumor also showed the EPR effect, and this conjugate reduced metastatic tumor in the lung almost completely at 30 mg/kg once i.v. (less than one‐fifth of the maximum tolerable dose). Although SMA‐THP conjugate per se has little cytotoxicity in vitro (1/100 of free drug THP), tumor‐targeted accumulation by the EPR effect ensures sufficient drug concentrations in tumor to produce an antitumor effect, whereas toxicity to normal tissues is much less. These findings suggest the potential of SMA‐THP conjugate as a highly favorable candidate for anticancer nanomedicine with good stability and tumor‐targeting properties in vivo.
Covalently amide linked SMA‐THP conjugate showed high stability in circulation and thus prolonged circulation time and tumor targeting property based on the EPR effect. Consequently marked in vivo antitumor effects were achieved, including metastatic lung cancer, with less adverse effects, suggesting the potential of SMA‐THP conjugate as a promising candidate for cancer treatment.
Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are ...asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.
Nanoparticles prepared using human serum albumin (HSA) have emerged as versatile carriers for improving the pharmacokinetic profile of drugs. The desolvation of HSA using ethanol followed by ...stabilization through crosslinking with glutaraldehyde is a common technique for preparing HSA nanoparticles, but our knowledge concerning the characteristics (or functions) of HSA nanoparticles and their efficiency when loaded with drugs is limited. To address this issue in more detail, we prepared anthracycline-loaded HSA nanoparticles. Doxorubicin-loaded HSA nanoparticles with a size similar to doxorubicin-unloaded particles could be prepared by desolvating at a higher pH (8–9), and the size (100–150 nm) was optimum for delivery to tumor tissues. Using this procedure, HSA nanoparticles were loaded with other anthracycline derivatives, and all showed cytotoxicity in cancer cells. However, the efficiency of drug loading and dissolution rate were different among them possibly due to the differences in the type of association of the drugs on nanoparticles (doxorubicin and daunorubicin; covalently bound to nanoparticles, pirarubicin; both covalently bound to and adsorbed on nanoparticles, aclarubicin; adsorbed on nanoparticles). Since the formulation of such drug-loaded HSA nanoparticles should be modified for efficient delivery to tumors, the findings reported herein provide the useful information for optimizing the formulation and the production process for the HSA nanoparticles using a desolvation technique.
Precision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth ...factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism.
We generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient's cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug's efficacies against HER2 E401G.
We confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective.
Afatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.
PDF
