Cyclosporin A inhibits prostate cancer growth through suppression of E2F8 transcription factor in a MELK­dependent manner.

The treatment of advanced prostate cancer remains a formidable challenge due to the limited availability of effective treatment options. Therefore, it is imperative to identify promising druggable targets that provide substantial clinical benefits and to develop effective treatment strategies to overcome therapeutic resistance. Cyclosporin A (CsA) showed an anticancer effect on prostate cancer in cultured cell and xenograft models. E2F8 was identified as a master transcription factor that regulated a clinically significant CsA specific gene signature. The expression of E2F8 increased during prostate cancer progression and high levels of E2F8 expression are associated with a poor prognosis in patients with prostate cancer. MELK was identified as a crucial upstream regulator of E2F8 expression through the transcriptional regulatory network and Bayesian network analyses. Knockdown of E2F8 or MELK inhibited cell growth and colony formation in prostate cancer cells. High expression levels of E2F8 and androgen receptor (AR) are associated with a worse prognosis in patients with prostate cancer compared with low levels of both genes. The inhibition of E2F8 improved the response to AR blockade therapy. These results suggested that CsA has potential as an effective anticancer treatment for prostate cancer, while also revealing the oncogenic role of E2F8 and its association with clinical outcomes in prostate cancer. These results provided valuable insight into the development of therapeutic and diagnostic approaches for prostate cancer.

Duodenal Dual-Wavelength Photobiomodulation Improves Hyperglycemia and Hepatic Parameters with Alteration of Gut Microbiome in Type 2 Diabetes Animal Model.

BACKGROUND: Recently, the duodenum has garnered interest for its role in treating metabolic diseases, including type 2 diabetes (T2DM). Multiple sessions of external photobiomodulation (PBM) in previous animal studies suggested it resulted in improved hyperglycemia, glucose intolerance, and insulin resistance with a multifactorial mechanism of action, despite the target organ of PBM not being clearly proven. This study aimed to determine whether a single session of a duodenal light-emitting diode (LED) PBM may impact the T2DM treatment in an animal model. METHODS: Goto-Kakizaki rats as T2DM models were subjected to PBM through duodenal lumen irradiation, sham procedure, or control in 1-week pilot (630 nm, 850 nm, or 630/850 nm) and 4-week follow-up (630 nm or 630/850 nm) studies. Oral glucose tolerance tests; serum glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide, and insulin levels; liver chemistry and histology; and gut microbiome in the PBM, sham control, and control groups were evaluated. RESULTS: In the 1-week study, duodenal dual-wavelength (D, 630/850 nm) LED PBM showed improved glucose intolerance, alkaline phosphatase and cholesterol levels, and weight gain than other groups. The D-LED PBM group in the 4-week study also showed improved hyperglycemia and liver enzyme levels, with relatively preserved pancreatic islets and increased serum insulin and GLP-1 levels. Five genera (Bacteroides, Escherichia, Parabacteroides, Allobaculum, and Faecalibaculum) were significantly enriched 1 week after the D-LED PBM. Bacteroides acidifaciens significantly increased, while Lachnospiraceae significantly decreased after 1 week. CONCLUSION: A single session of D-LED PBM improved hyperglycemia and hepatic parameters through the change of serum insulin, insulin resistance, insulin expression in the pancreatic ß-cells, and gut microbiome in T2DM animal models.

Crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in non-small cell lung cancer cells.

Crizotinib is a clinically approved tyrosine kinase inhibitor for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring EML4-ALK fusion. Crizotinib was originally developed as an inhibitor of MET (HGF receptor), which is involved in the metastatic cascade. However, little is known about whether crizotinib inhibits tumor metastasis in NSCLC cells. In this study, we found that crizotinib suppressed TGFß signaling by blocking Smad phosphorylation in an ALK/MET/RON/ROS1-independent manner in NSCLC cells. Molecular docking and in vitro enzyme activity assays showed that crizotinib directly inhibited the kinase activity of TGFß receptor I through a competitive inhibition mode. Cell tracking, scratch wound, and transwell migration assays showed that crizotinib simultaneously inhibited TGFß- and HGF-mediated NSCLC cell migration and invasion. In addition, in vivo bioluminescence imaging analysis showed that crizotinib suppressed the metastatic capacity of NSCLC cells. Our results demonstrate that crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in NSCLC cells. Therefore, our findings will help to advance our understanding of the anticancer action of crizotinib and provide insight into future clinical investigations.

Designed ferritin nanocages displaying trimeric TRAIL and tumor-targeting peptides confer superior anti-tumor efficacy.

TRAIL is considered a promising target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors, DR4 or DR5. Although recombinant human TRAIL has shown high potency and specificity for killing cancer cells in preclinical studies, it has failed in multiple clinical trials for several reasons, including a very short half-life mainly caused by instability of the monomeric form of TRAIL and rapid renal clearance of the off-targeted TRAIL. To overcome such obstacles, we developed a TRAIL-active trimer nanocage (TRAIL-ATNC) that presents the TRAIL ligand in its trimer-like conformation by connecting it to a triple helix sequence that links to the threefold axis of the ferritin nanocage. We also ligated the tumor-targeting peptide, IL4rP, to TRAIL-ATNC to enhance tumor targeting. The developed TRAIL-ATNCIL4rP showed enhanced agonistic activity compared with monomeric TRAIL. The in vivo serum half-life of TRAIL-ATNCIL4rP was ~ 16-times longer than that of native TRAIL. As a consequence of these properties, TRAIL-ATNCIL4rP exhibited efficacy as an anti-tumor agent in vivo against xenograft breast cancer as well as orthotopic pancreatic cancer models, highlighting the promise of this system for development as novel therapeutics against cancer.

EW-7197, a Transforming Growth Factor-Beta Type I Receptor Kinase Inhibitor, Ameliorates Acquired Lymphedema in a Mouse Tail Model.

Background: Acquired lymphedema is a common consequence of cancer surgery. Fibrosis is one of the main causes of chronic lymphedema since it hinders lymphatic regeneration and this causes a significant decrease in lymphatic flow and accumulation of excessive protein-rich fluid. The transforming growth factor-ß1 (TGF-ß1) signaling pathway is known in a process of wound repair and fibrosis. In our study, the purpose was to evaluate the efficacy of EW-7197, a peroral TGF-ß type I receptor kinase inhibitor, in treating acquired lymphedema. Methods and Results: For lymphedema mouse tail model, we used 10- to 12-week-old female C57BL/6 mice. The skin was circumferentially excised, making a circular band followed by cauterization of lymphatic collecting vessels. Two groups were made in this study: control and treatment. The treatment group (n = 12) received a solution consisting of 0.1 mL of artificial gastric juice and 20 mg/kg EW-7197 by gavage once daily. For evaluation, tail diameter measurement, fluorescence lymphography, and immunofluorescence images were used. EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing lymphangiogenesis and interstitial flow of the lymphatics by inhibition of the fibrosis. The differences in maximal tail thicknesses between the control and treatment groups were statistically significant from 2 to 4 weeks after surgery. The treatment group showed a greater number of lymphatic vessels at the surgery site than the control group. The treatment group also showed more FITC coverage area at the surgery site. Conclusion: EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing lymphangiogenesis and interstitial flow.

Altered expression of fucosylation pathway genes is associated with poor prognosis and tumor metastasis in non­small cell lung cancer.

Fucosylation is a post­translational modification that attaches fucose residues to protein­ or lipid­bound oligosaccharides. Certain fucosylation pathway genes are aberrantly expressed in several types of cancer, including non­small cell lung cancer (NSCLC), and this aberrant expression is associated with poor prognosis in patients with cancer. However, the molecular mechanism by which these fucosylation pathway genes promote tumor progression has not been well­characterized. The present study analyzed public microarray data obtained from NSCLC samples. Multivariate analysis revealed that altered expression of fucosylation pathway genes, including fucosyltransferase 1 (FUT1), FUT2, FUT3, FUT6, FUT8 and GDP­L­fucose synthase (TSTA3), correlated with poor survival in patients with NSCLC. Inhibition of FUTs by 2F­peracetyl­fucose (2F­PAF) suppressed transforming growth factor ß (TGFß)­mediated Smad3 phosphorylation and nuclear translocation in NSCLC cells. In addition, wound­healing and Transwell migration assays demonstrated that 2F­PAF inhibited TGFß­induced NSCLC cell migration and invasion. Furthermore, in vivo bioluminescence imaging analysis revealed that 2F­PAF attenuated the metastatic capacity of NSCLC cells. These results may help characterize the oncogenic role of fucosylation in NSCLC biology and highlight its potential for developing cancer therapeutics.

Whole-Body Physiologically Based Pharmacokinetic Modeling of Trastuzumab and Prediction of Human Pharmacokinetics.

In the present study, we evaluated the pharmacokinetics (PK) of trastuzumab and sought to predict human PK based on animal studies, through the use of optical imaging and a whole-body physiologically based pharmacokinetic (WB-PBPK) modeling approach. The PK study was conducted in 24 mice, where serial blood samples were withdrawn and major organs were isolated after the last blood withdrawal. The drug concentrations in blood and major organs were measured via optical imaging. The WB-PBPK model was constructed using known physiological values including the volumes of major organs and blood/lymphatic flow. The NONMEM software (version 7.3) was used to determine tissue partition coefficients. Using the WB-PBPK model, a clinical trial simulation was performed with reference to human physiological values acquired from the literature. The simulated human PK was then compared with the actual PK observed in the previous study in which healthy male subjects received 6 mg/kg trastuzumab (Herceptin®) via intravenous route. The ratio of the simulated versus observed area under the concentration-time curve was 1.02 and that of maximal concentration was 0.72. The current study describes the potential synergistic applications of WB-PBPK and optical imaging in human PK prediction based on preclinical data obtained in early-stage drug development.

Multi-Spectral Fluorescence Imaging of Colon Dysplasia InVivo Using a Multi-Spectral Endoscopy System.

BACKGROUND AND STUDY AIM: To develop a molecular imaging endoscopic system that eliminates tissue autofluorescence and distinguishes multiple fluorescent markers specifically on the cancerous lesions. METHODS: Newly developed multi-spectral fluorescence endoscope device has the potential to eliminate signal interference due to autofluorescence and multiplex fluorophores in fluorescent probes. The multiplexing capability of the multi-spectral endoscope device was demonstrated in the phantom studies and multi-spectral imaging with endoscopy and macroscopy was performed to analyze fluorescence signals after administration of fluorescent probe that targets cancer in the colon. Because of the limitations in the clinical application using rigid-type small animal endoscope, we developed a flexible channel insert-type fluorescence endoscope, which was validated on the colonoscopy of dummy and porcine model. RESULTS: We measured multiple fluorescent signals simultaneously, and the fluorescence spectra were unmixed to separate the fluorescent signals of each probe, in which multiple fluorescent probes clearly revealed spectral deconvolution at the specific targeting area in the mouse colon. The positive area of fluorescence signal for each probe over the whole polyp was segmented with analyzing software, and showed distinctive patterns and significantly distinguishable values: 0.46 ±â€¯0.04, 0.39 ±â€¯0.08 and 0.73 ±â€¯0.12 for HMRG, CET-553 and TRA-675 probes, respectively. The spectral unmixing was finally demonstrated in the dummy and porcine model, corroborating the targeted multi-spectral fluorescence imaging of colon dysplasia. CONCLUSION: The multi-spectral endoscopy system may allow endoscopists to clearly identify cancerous lesion that has different patterns of various target expression using multiple fluorescent probes.

Endoscopic Transplantation of Mesenchymal Stem Cell Sheets in Experimental Colitis in Rats.

Owing to the recent progress in regenerative medicine technology, clinical trials that harnessed the regeneration and immune modulation potentiality of stem cells for treating IBD have shown promising results. We investigated the feasibility and utility of intraluminal endoscopic transplantation of rat MSC sheets in murine models of experimental colitis for targeted delivery of stem cells to lesions. We isolated adipose-derived mesenchymal stem cells (AD-MSC) and bone marrow-derived mesenchymal stem cells (BM-MSC) from EGFP-transgenic rats and fabricated the cells in sheet forms using temperature-responsive culture dishes. The MSC sheets were endoscopically transplanted to the inflamed area in electrocoagulation and DNBS colitis model. The effect of the transplantation was verified using endoscopic scoring and histological analysis. In the electrocoagulation model, the AD-MSC group showed significantly decreased ulcer size in the transplanted regions. In the DNBS colitis model, the AD-MSC group showed decreased inflammation and colitis in the transplanted regions. Histologic analysis showed that the MSC sheets had successfully attached to the inflamed mucosa in both the electrocoagulation and DNBS colitis model. Our results show that endoscopic transplantation of MSC sheets could be a new effective mode of stem cell therapy for IBD treatment.

PSP1, a Phosphatidylserine-Recognizing Peptide, Is Useful for Visualizing Radiation-Induced Apoptosis in Colorectal Cancer In Vitro and In Vivo.

Accurate and timely visualization of apoptotic status in response to radiation is necessary for deciding whether to continue radiation or change to another mode of treatment. This is especially critical in patients with colorectal cancer, which requires a delicate combination of surgery, radiation, and chemotherapy in order to achieve optimal outcome. In this study, we investigated the potential of phosphatidylserine-recognizing peptide 1 (PSP1) as an apoptosis-targeting probe, which identifies phosphatidylserine on cell surfaces. We first screened colon cancer cell lines for their sensitivity to radiation and selected two cell lines: HCT116 and HT29. Cell binding assay using fluorescence-activated cell sorting and optical imaging showed that HCT116 cells had better binding to PSP1 than HT29 cells. Thus, mouse xenograft model using HCT116 cells was generated and was topically irradiated with either single or fractionated dose of radiation followed by systemic administration of PSP1 for subsequent molecular optical imaging. We confirmed that the PSP1 probe was selectively bound to apoptosis-induced tumor in a radiation dose-dependent manner. We also observed that fractionated radiation regimen, which is recently being used in clinical situation, was more effective in inducing tumor apoptosis than corresponding single-dose radiation treatment. We then evaluated the correlation between tumor targeting of PSP1 and suppression effect of tumor development and found that tumor volume and fluorescence intensity were correlated before (correlation coefficient r2 = 0.534) and after (r2 = 0.848) radiation therapy. Our study shows that PSP1 peptide is an efficient index probe for deciding "go or no-go" for radiation therapy in colorectal cancer.

Molecular Imaging of Colorectal Tumors by Targeting Colon Cancer Secreted Protein-2 (CCSP-2).

A versatile biomarker for detecting colonic adenoma and colon cancer has yet to be developed. Colon cancer secreted protein-2 (CCSP-2) is a protein specifically expressed and secreted in colon adenomas and cancers. We developed a fluorescent imaging method based on CCSP-2 targeting for a more sensitive and specific detection of colorectal tumors. CCSP-2 expression was evaluated in human colon adenoma and colorectal specimens. Anti-CCSP-2 antibody was labeled with a near-infrared fluorescent dye, FPR-675, and molecular imaging of surgical human colorectal tumors was performed. Immunohistochemistry identified CCSP-2 expression in 87.0% of colorectal cancer specimens and 89.5% of colon adenoma specimens. Fluorescence imaging of surgical human colon specimens after spraying treatment with the probe permitted a clear distinction of cancer from paired normal colon tissue (target-to-background ratio, 4.09±0.42; P<.001). CCSP-2 targeting imaging was also evaluated in patient-derived colon cancer xenograft mouse and liver metastasis murine models. CCSP-2-positive colon cancer xenografts and liver metastases were visualized by near-infrared fluorescence imaging after intravenous injection of the probe, which showed significantly higher fluorescence. Our results show that CCSP-2 is a promising marker for colorectal tumor detection in clinical settings and that a CCSP-2-targeting molecular imaging strategy might improve the diagnosis of colorectal tumors in metastatic or recurrent cancers and aid in early colonoscopic detection of premalignant lesions.

Interleukin-4 receptor-targeted delivery of Bcl-xL siRNA sensitizes tumors to chemotherapy and inhibits tumor growth.

IL-4 receptor (IL-4R) is commonly up-regulated on tumor cells, and interactions between the receptor and Interleukin-4 (IL-4) can induce the expression of anti-apoptotic proteins, including Bcl-xL. This contributes to tumor cell survival and their resistance to chemotherapy. In this study, we exploited IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumor cells in order to sensitize them to chemotherapy. To target IL-4R, an IL-4R-binding peptide, IL4RPep-1, was attached to branched polyethyleneimine-superparamagnetic iron oxide nanoparticles (BPEI-SPION). These nanoparticles were then complexed with Bcl-xL-targeting siRNA. IL-4R-targeted BPEI-SPION/Bcl-xL siRNA more efficiently reduced Bcl-xL gene expression and enhanced cytotoxicity of doxorubicin in MDA-MB231 breast tumor cells compared to untargeted BPEI-SPION/Bcl-xL siRNA. The siRNA was released from the complexes after 15 h of incubation at pH 5.5 and was stable in the complexes up to 72 h in the serum. The IL-4R-targeted BPEI-SPION/siRNA was internalized by cells through IL-4R, successfully escaped the endosomes, and was dispersed into the cytoplasm. Near-infrared fluorescence and magnetic resonance imaging demonstrated that in vivo tumor homing and accumulation of IL-4R-targeted BPEI-SPION/siRNA were both higher than untargeted BPEI-SPION/siRNA. The IL-4R-targeted BPEI-SPION/Bcl-xL siRNA, in combination with doxorubicin, significantly inhibited tumor growth in mice compared to untargeted BPEI-SPION/Bcl-xL siRNA. These results suggest that the IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumors can sensitize tumors to chemotherapy and enhance the efficacy of anti-tumor therapeutics.

Sulfasalazine attenuates evading anticancer response of CD133-positive hepatocellular carcinoma cells.

BACKGROUND: CD133-positive cells in hepatocellular carcinoma (HCC) exhibit cancer stem cell (CSC)-like properties as well as resistance to chemotherapeutic agents and ionizing radiation; however, their function remains unknown. In this paper, we identified a hitherto unknown mechanism to overcome CD133-induced resistance to anticancer therapy. METHODS: We applied an alternative approach to enrich the CD133-positive HCC population by manipulating 3D culture conditions. Defense mechanisms against reactive oxygen species (ROS) in CSC spheroids were evaluated by fluorescence image-based phenotypic screening system. Further, we studied the effect of sulfasalazine on ROS defense system and synergistic therapeutic efficacy of anticancer therapies both in culture and in vivo HCC xenograft mouse model. RESULTS: Here, we found that oxidative stress increase CD133 expression in HCC and increased CD133 expression enhanced the capacity of the defense system against ROS, and thereby play a central role in resistance to liver cancer therapy. Moreover, ablation of CD133 attenuated not only the capacity for defense against ROS, but also chemoresistance, in HCC through decreasing glutathione (GSH) levels in vitro. Sulfasalazine, a potent xCT inhibitor that plays an important role in maintaining GSH levels, impaired the ROS defense system and increased the therapeutic efficacy of anticancer therapies in CD133-positive HCC but not CD133-negative HCC in vivo and in vitro. CONCLUSION: These results strongly indicate functional roles for CD133 in ROS defense and in evading anticancer therapies in HCC, and suggest that sulfasalazine, administered in combination with conventional chemotherapy, might be an effective strategy against CD133-positive HCC cells.

Decursin from Angelica gigas Nakai Inhibits B16F10 Melanoma Growth Through Induction of Apoptosis.

Decursin, a bioactive phytochemical isolated from Angelica gigas Nakai (danggwi), has shown preclinical anticancer efficacy in various cancer models. However, the antitumor effect of decursin in melanoma models remains undefined. The antitumor activities of decursin were investigated in B16F10 cells in vitro and in vivo. In this study, we show that treatment with decursin inhibited cell proliferation in a dose-dependent manner in B16F10 cells, but not in normal cells. Decursin also induced apoptosis in B16F10 cells, as determined by annexin V-staining assay and transferase-mediated nick-end labeling (TUNEL) staining assay. Decursin increased the phosphorylation of p38 as well as the expression of Bax while decreasing the phosphorylation of extracellular signaling-regulated kinase (ERK) and the expression of Bcl-2 in B16F10 cells. Moreover, decursin activated caspase-3 in B16F10 cells and xenograft tumor tissue. Together, these findings support further investigations into the potential use of decursin in the treatment of melanoma cells.

Advantages of the phosphatidylserine-recognizing peptide PSP1 for molecular imaging of tumor apoptosis compared with annexin V.

A number of peptide-based indicators have been identified and reported as potential apoptosis probes, offering great promise for early assessment of therapeutic efficacy in several types of cancer. Direct comparison of the newly developed probes with previously used ones would be an important step in assessing possible applications. Here, we compared the newly identified peptide-based phosphatidylserine (PS) indicator PSP1 (CLSYYPSYC) with annexin V, a common probe for molecular imaging of apoptotic cells, with respect to PS binding kinetics, apoptotic cell-targeting ability, and the efficacy of homing to apoptotic tumor cells in a mouse model after treatment with the anticancer agent camptothecin. Our results indicate that PSP1 efficiently targeted apoptotic cells and generated apoptosis/tumor-specific signals after cancer treatment in the animal model, whereas a similar dose of annexin V showed weak signals. The formation of a stable complex of PSP1 with PS might be one reason for the efficient in vivo targeting. We suggest that PSP1 has potential advantages for in vivo apoptotic cell imaging and could serve as a platform for the development of de novo peptide-based probes for apoptosis.

LHT7, a chemically modified heparin, inhibits multiple stages of angiogenesis by blocking VEGF, FGF2 and PDGF-B signaling pathways.

Despite the therapeutic benefits of the angiogenesis inhibitors shown in the clinics, they have encountered an unexpected limitation by the occurrence of acquired resistance. Although the mechanism of the resistance is not clear so far, the upregulation of alternative angiogenic pathways and stabilization of endothelium by mural cells were reported to be responsible. Therefore, blocking multiple angiogenic pathways that are crucial in tumor angiogenesis has been highlighted to overcome such limitations. To develop an angiogenesis inhibitor that could block multiple angiogenic factors, heparin is an excellent lead compound since wide array of angiogenic factors are heparin-binding proteins. In previous study, we reported a heparin-derived angiogenesis inhibitor, LHT7, as a potent angiogenesis inhibitor and showed that it blocked VEGF signaling pathway. Here we show that LHT7 could block the fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) in addition to VEGF. Simultaneous blockade of these angiogenic factors resulted in inhibition of multiple stages of the angiogenic process, including initial angiogenic response to maturation of the endothelium by pericyte coverage in vitro. In addition, the treatment of LHT7 in vivo did not show any sign of vascular normalization and directly led to decreased blood perfusion throughout the tumor. Our findings show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of the angiogenesis, and could potentially be used to overcome the resistance.

Spraying quantum dot conjugates in the colon of live animals enabled rapid and multiplex cancer diagnosis using endoscopy.

The detection of colon cancer using endoscopy is widely used, but the interpretation of the diagnosis is based on the clinician's naked eye. This is subjective and can lead to false detection. Here we developed a rapid and accurate molecular fluorescence imaging technique using antibody-coated quantum dots (Ab-QDs) sprayed and washed simultaneously on colon tumor tissues inside live animals, subsequently excited and imaged by endoscopy. QDs were conjugated to matrix metalloproteinases (MMP) 9, MMP 14, or carcinoembryonic antigen (CEA) Abs with zwitterionic surface coating to reduce nonspecific bindings. The Ab-QD probes can diagnose tumors on sectioned mouse tissues, fresh mouse colons stained ex vivo and also in vivo as well as fresh human colon adenoma tissues in 30 min and can be imaged with a depth of 100 µm. The probes successfully detected not only cancers that are readily discernible by bare eyes but also hyperplasia and adenoma regions. Sum and cross signal operations provided postprocessed images that can show complementary information or regions of high priority. This multiplexed quantum dot, spray-and-wash, and endoscopy approach provides a significant advantage for detecting small or flat tumors that may be missed by conventional endoscopic examinations and bestows a strategy for the improvement of cancer diagnosis.

Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

An apoptosis-homing peptide-conjugated low molecular weight heparin-taurocholate conjugate with antitumor properties.

Various angiogenesis inhibitors and apoptosis-targeting agents have been therapeutically applied in preclinical cancer models, some of which have been tested in clinical trials. In a previous study, we demonstrated that LHT7, a low molecular weight heparin (LMWH)-taurocholate conjugate, has strong antiangiogenic and tumor-suppressive activity and diminished anticoagulant properties. In this study, we developed LHT7-ApoPep-1, an apoptosis-homing peptide-conjugated variant of LHT7. LHT7-ApoPep-1 exhibited antiangiogenic activity in endothelial cell tube-formation assays and apoptotic cell-targeting ability in tumor cell binding assays; it also showed little toxicity toward healthy cells. Administration of LHT7-ApoPep-1 in mouse xenograft models of breast carcinoma delayed tumor growth compared to LHT7-only, and histological evaluations revealed decreased vessel formation and increased apoptotic area in tumor tissues. Moreover, an examination of LHT7-ApoPep-1-Cy7.5 localization within the body using in vivo live imaging showed accumulation at the tumor site of tumor-bearing mice, with a more prolonged circulation time and enhanced intensity compared to LHT7-Cy7.5. Inspection of the tumor microenvironment revealed that Cy5.5-labeled LHT7-ApoPep-1 was located on and near CD31-positive vessels in tumor tissue. We conclude that LHT7-ApoPep-1 has antiangiogenic and apoptosis-targeting properties and exerts antitumor effects by suppressing tumor vessel growth and homing to apoptotic cells within the tumor.