PC-3 Tumor-bearing Nude Mice Research Articles

Lipid-Based Nanocarrier by Targeting with LHRH Peptide: A Promising Approach for Prostate Cancer Radio-Imaging and Therapy.

Prostate cancer is a prevalently detected malignancy with a dismal prognosis. Luteinizing-hormone-releasing-hormone (LHRH) receptors are overexpressed in such cancer cells, to which the LHRH-decapeptide can specifically bind. A lipid-polyethylene glycol-conjugated new LHRH-decapeptide analogue (D-P-HLH) was synthesized and characterized. D-P-HLH-coated and anticancer drug doxorubicin (DX)-loaded solid lipid nanoparticles (F-DX-SLN) were formulated by the cold homogenization technique and characterized by Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, differential scanning calorimetry, dynamic light scattering, electron microscopy, entrapment efficiency, and drug-release profile studies. F-DX-SLN allows site-specific DX delivery by reducing the side effects of chemotherapy. Cancer cells could precisely take up F-DX-SLN by targeting specific receptors, boosting the cytotoxicity at the tumor site. The efficacy of F-DX-SLN on PC3/SKBR3 cells by the MTT assay revealed that F-DX-SLN was more cytotoxic than DX and/or DX-SLN. Flow cytometry and confocal microscopic studies further support F-DX-SLNs' increased intracellular absorption capability in targeting LHRH overexpressed cancer cells. F-DX-SLN ensured high apoptotic potential, noticeably larger mitochondrial transmembrane depolarization action, as well as the activation of caspases, a longer half-life, and greater plasma concentration. F-DX-SLN/DX-SLN was radiolabeled with technetium-99m; scintigraphic imaging studies established its tumor selectivity in PC3 tumor-bearing nude mice. The efficacy of the formulations in cancer treatment, in vivo therapeutic efficacy tests, and histopathological studies were also conducted. Results clearly indicate that F-DX-SLN exhibits sustained and superior targeted administration of anticancer drugs, thus opening up the possibility of a drug delivery system with precise control and targeting effects. F-DX-SLN could also provide a nanotheranostic approach with improved efficacy for prostate cancer therapy.

Development of 18F-Fluoroglycosylated PSMA-Ligands with Improved Renal Clearance Behavior

The prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is highly expressed in the malignant human prostate epithelium. Therefore, PSMA has emerged as a very attractive target for developing radiopharmaceuticals for the diagnosis, e.g., by positron emission tomography (PET) imaging, and radiotherapy of prostate cancer. The aim of this study was to develop 18F-labeled PSMA ligands bearing different 18F-glycosyl moieties to study the effect on the in vivo clearance behavior of radiotracers in addition to their tumor binding ability. Therefore, we applied click chemistry-based 18F-fluoroglcosylation using 2-deoxy-2-[18F]fluoroglucosyl azide or 6-deoxy-6-[18F]fluoroglucosyl azide as prosthetic groups for the radiosynthesis of the 18F-fluoroglycosylated glutamate-urea-lysine-based PSMA inhibitors 2-[18F]FGlc-PSMA ([18F]7) and 6-[18F]FGlc-PSMA ([18F]8). The PSMA inhibitory potencies were determined by competitive radioligand binding assays using 99mTc-MIP-1404 and PSMA-expressing PC-3 PIP cells, revealing moderate PSMA inhibitory potencies for [18F]7 (IC50 = 234 nM) and [18F]8 (IC50 = 59 nM). Biodistribution and small-animal PET studies were performed using PSMA-positive PC-3 PIP and PSMA-negative PC-3 tumor-bearing nude mice. PSMA inhibitors [18F]7 and [18F]8 were obtained in high radioactivity yields of 19-22% (nondecay-corrected, referred to [18F]fluoride) and with molar activities of 71-136 GBq/μmol. In the biodistribution studies, the uptake levels of [18F]7 and [18F]8 in PC-3 PIP tumors were 13 ± 3%ID/g and 6 ± 5%ID/g at 60 min p.i., respectively. PSMA-negative PC-3 tumors and all other tissues had negligible low uptake values. Interestingly, [18F]7 had high uptake in the kidneys, with remarkable retention from 30 to 60 min p.i. (74 to 72%ID/g). In contrast, [18F]8 revealed a low uptake of 7.5%ID/g in the kidneys at 30 min p.i. and was rapidly cleared through the kidney (0.9%ID/g at 120 min p.i.). In direct comparison to a 68Ga-PSMA-11 PET scan of the same mouse, [18F]7 and [18F]8 showed 2- to 3-fold higher uptake values in PC-3 PIP tumors. Both radiotracers were solely cleared via the kidneys and not via the hepatobiliary pathway. The regional kidney distribution pattern of the tracers in the kidneys revealed that 68Ga-PSMA-11 and 2-[18F]FGlc-PSMA([18F]7) mainly accumulated in the cortex of the kidneys, whereas 6-[18F]FGlc-PSMA([18F]8) showed a 10-fold lower kidney uptake with accumulation in the inner medulla or pelvis of the kidneys. Overall, the developed 6-fluoroglucosyl derivative [18F]8, with its considerably low kidney uptake and fast clearance, demonstrated high uptake in PSMA-positive tumors in vivo. This candidate could, therefore, be valuable for translation into the clinic.

Biological Potential and Mechanism of Prodigiosin from Serratia marcescens Subsp. lawsoniana in Human Choriocarcinoma and Prostate Cancer Cell Lines.

Tripyrrole molecules have received renewed attention due to reports of numerous biological activities, including antifungal, antibacterial, antiprotozoal, antimalarial, immunosuppressive, and anticancer activities. In a screen of bacterial strains with known toxicities to termites, a red pigment-producing strain, HDZK-BYSB107, was isolated from Chamaecyparis lawsoniana, which grows in Oregon, USA. Strain HDZK-BYSB107 was identified as Serratia marcescens subsp. lawsoniana. The red pigment was identified as prodigiosin using ultraviolet absorption, LC-MS, and 1H-NMR spectroscopy. The bacterial prodigiosin had an inhibitory effect on both Gram-negative and Gram-positive bacteria. The main objective of this study was to explore the anticancer activities and mechanism of strain HDZK-BYSB107 prodigiosin by using human choriocarcinoma (JEG3) and prostate cancer cell lines (PC3) in vitro and JEG3 and PC3 tumor-bearing nude mice in vivo. In vitro anticancer activities showed that the bacterial prodigiosin induced apoptosis in JEG3 cells. In vivo anticancer activities indicated that the prodigiosin significantly inhibited the growth of JEG3 and PC3 cells, and the inhibitory activity was dose and time dependent. The anticancer efficacy of the bacterial prodigiosin on JEG3 and PC3 cells, JEG3 and PC3 tumor exhibited a correlation with the down regulation of the inhibitor of IAP family, including XIAP, cIAP-1 and cIAP-2, and the activation of caspase-9 and caspase-3 accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. The expressions of P53 and Bax/Bcl-2 in JEG3 and PC3 cells were significantly higher than in untreated groups. Our results indicated that the bacterial prodigiosin extracted from C. lawsoniana is a promising molecule due to its potential for therapeutic applications.

Multifunctional gold nanorods and docetaxel-encapsulated liposomes for combined thermo- and chemotherapy.

Personalized and precise nanomedicines are highly demanded for today’s medical needs. Liposomes are ideal candidates for the construction of multifunctional drug delivery systems. In this study, a liposome was used to improve the clinical issues of docetaxel (Doc), a potent antimitotic chemotherapy for prostate cancer (PC). RLT, a low-density lipoprotein receptor (LDLR)-binding peptide, and PEG were conjugated to the liposomes, and gold nanorods (GNRs) were also incorporated into the liposomes. The GNRs/Doc-liposome-RLT (GNRs/DocL-R) was tested in PC-3 cells and in PC-3 tumor-bearing nude mice. Results showed that GNRs/DocL-R possessed a diameter approximately 163.15±1.83 nm and a zeta potential approximately −32.8±2.16 mV. GNRs/DocL-R showed enhanced intracellular entrance, increased accumulation in the implanted tumor region, and the highest tumor inhibition in vitro and in vivo. Therefore, the multifunctional GNRs/DocL-R was a potential cancer treatment via combined chemo- and thermotherapy.

Preclinical Study on GRPR-Targeted (68)Ga-Probes for PET Imaging of Prostate Cancer.

Gastrin-releasing peptide receptor (GRPR) targeted positron emission tomography (PET) is a highly promising approach for imaging of prostate cancer (PCa) in small animal models and patients. Developing a GRPR-targeted PET probe with excellent in vivo performance such as high tumor uptake, high contrast, and optimal pharmacokinetics is still very challenging. Herein, a novel bombesin (BBN) analogue (named SCH1) based on JMV594 peptide modified with an 8-amino octanoic acid spacer (AOC) was thus designed and conjugated with the metal chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA). The resulting NODAGA-SCH1 was then radiolabeled with (68)Ga and evaluated for PET imaging of PCa. Compared with (68)Ga-NODAGA-JMV594 probe, (68)Ga-NODAGA-SCH1 exhibited excellent PET/CT imaging properties on PC-3 tumor-bearing nude mice, such as high tumor uptake (5.80 ± 0.42 vs 3.78 ± 0.28%ID/g, 2 h) and high tumor/muscle contrast (16.6 ± 1.50 vs 8.42 ± 0.61%ID/g, 2 h). Importantly, biodistribution data indicated a relatively similar accumulation of (68)Ga-NODAGA-SCH1 was observed in the liver (4.21 ± 0.42%ID/g) and kidney (3.41 ± 0.46%ID/g) suggesting that the clearance is through both the kidney and the liver. Overall, (68)Ga-NODAGA-SCH1 showed promising in vivo properties and is a promising candidate for translation into clinical PET-imaging of PCa patients.

TheMCT4Gene: A Novel, Potential Target for Therapy of Advanced Prostate Cancer

The management of castration-resistant prostate cancer (CRPC) is a major challenge in the clinic. Androgen receptor signaling-directed strategies are not curative in CRPC therapy, and new strategies targeting alternative, key cancer properties are needed. Using reprogrammed glucose metabolism (aerobic glycolysis), cancer cells typically secrete excessive amounts of lactic acid into their microenvironment, promoting cancer development, survival, and progression. Cellular lactic acid secretion is thought to be predominantly mediated by MCT4, a plasma membrane transporter protein. As such, the MCT4 gene provides a unique, potential therapeutic target for cancer. A tissue microarray of various Gleason grade human prostate cancers was stained for MCT4 protein. Specific, MCT4-targeting antisense oligonucleotides (MCT4 ASO) were designed and candidate MCT4 ASOs checked for effects on (i) MCT4 expression, lactic acid secretion/content, glucose consumption, glycolytic gene expression, and proliferation of human CRPC cells and (ii) growth of PC-3 tumors in nude mice. Elevated MCT4 expression was associated with human CRPC and an earlier time to relapse. The treatment of PC-3, DU145, and C4-2 CRPC cultures with candidate MCT4 ASOs led to marked inhibition of MCT4 expression, lactic acid secretion, to increased intracellular lactic acid levels, and markedly reduced aerobic glycolysis and cell proliferation. Treatment of PC-3 tumor-bearing nude mice with the MCT4 ASOs markedly inhibited tumor growth without inducing major host toxicity. MCT4-targeting ASOs that inhibit lactic acid secretion may be useful for therapy of CRPC and other cancers, as they can interfere with reprogrammed energy metabolism of cancers, an emerging hallmark of cancer. Clin Cancer Res; 22(11); 2721-33. ©2016 AACR.

KPT-330, a potent and selective exportin-1 (XPO-1) inhibitor, shows antitumor effects modulating the expression of cyclin D1 and survivin [corrected] in prostate cancer models.

Background and aimsIncreased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.Material and methodsWe evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.Results and conclusionsXPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.

Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells.

Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit.

Enhanced antitumor efficacy by cyclic RGDyK-conjugated and paclitaxel-loaded pH-responsive polymeric micelles

Cyclic RGDyK (cRGDyK)-conjugated pH-sensitive polymeric micelles were fabricated for targeted delivery of paclitaxel to prostate cancer cells based on pH-sensitive copolymer poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) and cRGDyK-PEOz-PLA to enhance antitumor efficacy. The prepared micelles with an average diameter of about 28nm exhibited rapid release behavior at endo/lysosome pH, effectively enhanced the cytotoxicity of paclitaxel to PC-3 cells by increasing the cellular uptake, which was correlated with integrin αvβ3 expression in tumor cells. The active targeting activity of the micelles was further confirmed by in vivo real time near-infrared fluorescence imaging in PC-3 tumor-bearing nude mice. Moreover, the active targeting and pH-sensitivity endowed cRGDyK-conjugated micelles with a higher antitumor effect in PC-3 xenograft-bearing nude mice compared with unmodified micelles and Taxol with negligible systemic toxicity. Therefore, these results suggested that cRGDyK-conjugated pH-sensitive polymeric micelles may be a promising delivery system for efficient delivery of anticancer drugs to treat integrin αvβ3-rich prostate cancers.

Improved radiosynthesis and preliminary in vivo evaluation of a 18F-labeled glycopeptide–peptoid hybrid for PET imaging of neurotensin receptor 2

The neurotensin receptor 2 (NTS2) is an attractive target for cancer imaging, as it is overexpressed in a variety of tumor types including prostate, pancreas and breast carcinoma. The aim of this study was the development of the first NTS2 subtype selective 18F-labeled radioligand for imaging NTS2 expression in vivo by positron emission tomography (PET). The radiosynthesis of glycopeptoid 18F-4 was realized by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), applying the prosthetic group 6-deoxy-6-[18F]fluoroglucosyl azide for 18F-fluoroglycosylation of the alkyne-terminated NT(8–13) analog Pra-N-Me-Arg-Arg-Pro-N-homo-Tyr-Ile-Leu-OH. The binding affinity of the peptide–peptoid 4 for NTS2 was 7nM with excellent subtype selectivity over NTS1 (260-fold). In vitro autoradiography studies of rat brain slices confirmed the high selectivity of 18F-4 for NTS2. Biodistribution experiments using HT29 and PC3 tumor-bearing nude mice revealed high renal and only moderate tumor uptake, while PET imaging experiments revealed specific binding of 18F-4 in NTS2-positive tumors. As 18F-4 displayed high stability in vitro but fast degradation in vivo, future work will focus on the development of metabolically more stable NT(8–13) analogs.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Salvianolic acid A, a novel matrix metalloproteinase-9 inhibitor, reduces microvascular damage induced by hypertension

Salvianolic acid A, a novel matrix metalloproteinase-9 inhibitor, reduces microvascular damage induced by hypertension

Overexpression of oxidored-nitro domain containing protein 1 induces growth inhibition and apoptosis in human prostate cancer PC3 cells.

Previous studies have reported that oxidored-nitro domain containing protein 1 (NOR1) is a novel tumor suppressor gene identified in various types of cancer, such as nasopharyngeal carcinoma and cervical cancer. Recent studies have shown that NOR1 expression is lower in prostate cancer compared with normal prostate tissue. However, the specific function and exact mechanism of NOR1 in prostate cancer remains to be clarified. The present study aimed to investigate the function and mechanism of NOR1 in prostate cancer PC3 cells. DU145 and PC3 cells were transduced with a vector and cell viability, proliferation and apoptosis were determined. As predicted, NOR1 overexpression significantly inhibited growth and apoptosis in PC3 cells. NOR1 overexpression decreased the expression of the anti-apoptotic genes Bcl-2 and Bcl-xl and increased the level of the pro-apoptotic genes Bax and Bak in PC3 cells. Further investigation demonstrated that NOR1 overexpression activates caspase-3. Silencing of NOR1 did not inhibit growth or induce apoptosis in PC3 cells. Moreover, NOR1 inhibited proliferation and induced apoptosis via the activation of MAPK. The overexpression of NOR1 significantly inhibited tumor growth in PC3 tumor-bearing nude mice. The results suggest that the upregulated NOR1 expression was able to inhibit the progression of prostate cancer. Thus, NOR1 may be an ideal target for the treatment of prostate cancer.

Evaluation of 188Re-MAG2-RGD-bombesin for potential prostate cancer therapy

Glu-RGD-bombesin (RGD-BBN) is a heterodimeric peptide that contains motifs recognizing both integrin α(v)β(3) and gastrin releasing peptide receptor (GRPR). We evaluated here (188)Re (t(1/2)=16.9 h) labeled RGD-BBN as a potential agent for radionuclide therapy of prostate cancer. RGD-BBN was conjugated with S-benzoylmercaptoacetylglycylglycyl (MAG(2)), and then labeled with (99m)Tc or (188)Re, respectively. The dual-receptor binding affinity of MAG(2)-RGD-BBN was investigated by a radioligand competition binding assay. Biodistribution study of (188)Re-MAG(2)-RGD-BBN was carried out in normal BALB/c mice and PC-3 human prostate tumor-bearing nude mice. Gamma imaging studies were performed in PC-3 tumor-bearing nude mice. Biodistribution in normal mice showed that both (99m)Tc and (188)Re-labeled MAG(2)-RGD-BBN possessed high pancreas uptake due to the high GRPR expression of this organ. Gamma imaging with both (99m)Tc and (188)Re-labeled RGD-BBN in PC-3 tumor-bearing nude mice demonstrated high tumor uptake. The PC-3 tumors were clearly visible at 1 postinjection, with high contrast to the contralateral background. The use of chelator MAG(2) enables successful and high-yield (99m)Tc and (188)Re radiolabeling of RGD-BBN with favorable tumor targeting specificity. Further optimization may allow potential clinical application of (188)Re-MAG(2)-RGD-BBN for tumor-targeted radionuclide therapy.

Bombesin Antagonist–Based Radioligands for Translational Nuclear Imaging of Gastrin-Releasing Peptide Receptor–Positive Tumors

Bombesin receptors are overexpressed on a variety of human tumors. In particular, the gastrin-releasing peptide receptor (GRPr) has been identified on prostate and breast cancers and on gastrointestinal stromal tumors. The current study aims at developing clinically translatable bombesin antagonist-based radioligands for SPECT and PET of GRPr-positive tumors. A potent bombesin antagonist (PEG(4)-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) [AR]) was synthesized; conjugated to the chelators DOTA, 6-carboxy-1,4,7,11-tetraazaundecane (N4), 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A); and radiolabeled with (111)In, (99m)Tc, (68)Ga, and (64)Cu, respectively. The radioconjugates were evaluated in vitro and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was determined by Ca(2+)-flux measurements and immunofluorescence. All the conjugates showed high binding affinity to GRPr (inhibitory concentration of 50% [IC(50)], 2.5-25 nmol/L). The immunofluorescence and Ca(2+)-flux assays confirmed the antagonist properties of the conjugates. Biodistribution revealed high and specific uptake in PC-3 tumor and in GRPr-positive tissues. Tumor uptake of (64)Cu-CB-TE2A-AR (31.02 ± 3.35 percentage injected activity per gram [%IA/g]) was higher than (99m)Tc-N4-AR (24.98 ± 5.22 %IA/g), (111)In-DOTA-AR (10.56 ± 0.70 %IA/g), and (68)Ga-NODAGA-AR (7.11 ± 3.26 %IA/g) at 1 h after injection. Biodistribution at later time points showed high tumor-to-background ratios because of the fast washout of the radioligand from normal organs, compared with tumor. High tumor-to-background ratios were further illustrated by PET and SPECT images of PC-3 tumor-bearing nude mice acquired at 12 h after injection showing high tumor uptake, clear background, and negligible or no radioactivity in the abdomen. The chelators do influence the affinity, antagonistic potency, and pharmacokinetics of the conjugates. The promising preclinical results warrant clinical translation of these probes for SPECT and PET.

The Pharmacokinetics of Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) are able to penetrate the cell membrane carrying cargoes such as peptides, proteins, oligonucleotides, siRNAs, radioisotopes, liposomes, and nanoparticles. Consequently, many delivery approaches have been developed to use CPPs as tools for drug delivery. However, until now a systematic analysis of their in vivo properties including potential tumor binding specificity for drug targeting purposes has not been conducted. Ten of the most commonly applied CPPs were obtained by solid phase peptide synthesis and labeled with (111)In or (68)Ga. Uptake studies were conducted using a panel of six tumor cell lines of different origin. The stability of the peptides was examined in human serum. Biodistribution experiments were conducted in nude mice bearing human prostate carcinoma. Finally, positron emission tomography (PET) measurements were performed in male Wistar rats. The in vitro uptake studies revealed high cellular uptake values, but no specificity toward any of the cell lines. The biodistribution in PC-3 tumor-bearing nude mice showed a high transient accumulation in well-perfused organs and a rapid clearance from the blood. All of the CPPs revealed a relatively low accumulation rate in the brain. The highest uptake values were observed in the liver (with a maximal uptake of 51 %ID/g observed for oligoarginine (R(9))) and the kidneys (with a maximal uptake of 94 %ID/g observed for NLS). The uptake values in the PC-3 tumor were low at all time points, indicating a lack of tumor specific accumulation for all peptides studied. A micro-PET imaging study with (68)Ga-labeled penetratin, Tat and transportan(10) (TP(10)) confirmed the organ distribution data. These data reveal that CPPs do not show evidence for application in tumor targeting purposes in vivo. However, CPPs readily penetrate into most organs and show rapid clearance from the circulation. The high uptake rates observed in vitro and the relatively low specificity in vivo imply that CPPs would be better suited for topical application in combination with cargoes which show passive targeting and dominate the pharmacokinetic behavior. In conclusion, CPPs are suitable as drug carriers for in vivo application provided that their pharmacokinetic properties are also considered in design of CPP drug delivery systems.

A cell permeable peptide analog as a potential-specific PET imaging probe for prostate cancer detection

Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH(2)GR(11)) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR(9), NHGR(11), and NHGR(13)) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH(2)GR(11) showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR(9), FITC-NHGR(11), and FITC-NHGR(13) performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR(11) in the prostate tissue. Further in vivo evaluation of FITC-NHGR(11) in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with (64)Cu-labeled NH(2)GR(11) showed that the peptide was rapidly cleared from the blood (t(1/2) = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of (64)Cu-labled NH(2)GR(11) was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.

Design, preparation, in vitro and in vivo evaluation of 99mTc-N 2S 2-Tat(49–57)-bombesin: A target-specific hybrid radiopharmaceutical

The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, 99mTc-EDDA/HYNIC-Lys 3-bombesin ( 99mTc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type 99mTc-N 2S 2-Tat(49–57)-Lys 3-bombesin ( 99mTc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of 99mTc-Tat-BN and to compare its cellular internalization with that of 99mTc-BN. Structures of N 2S 2-Tat-BN and Tc(O)N 2S 2-Tat-BN were calculated by an MM procedure. 99mTc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N 2S 2-Tat-BN and 300 kcal/mol for Tc(O)N 2S 2-Tat-BN. 99mTc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of 99mTc-BN ( p < 0.05). The tumor-to-muscle radioactivity ratio was 8.5 for 99mTc-Tat-BN and 7 for 99mTc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.

Celastrol, a Triterpene Extracted from the Chinese “Thunder of God Vine,” Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice

Interest in the use of traditional medicines for cancer prevention and treatment is increasing. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as novel anticancer drugs. Celastrol, an active compound extracted from the root bark of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii Hook F.), was used for years as a natural remedy for inflammatory conditions. Although Celastrol has been shown to induce leukemia cell apoptosis, the molecular target involved has not been identified. Furthermore, whether Celastrol has antitumor activity in vivo has never been conclusively shown. Here, we report, for the first time, that Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome (IC(50) = 2.5 micromol/L) and human prostate cancer cellular 26S proteasome (at 1-5 micromol/L). Inhibition of the proteasome activity by Celastrol in PC-3 (androgen receptor- or AR-negative) or LNCaP (AR-positive) cells results in the accumulation of ubiquitinated proteins and three natural proteasome substrates (IkappaB-alpha, Bax, and p27), accompanied by suppression of AR protein expression (in LNCaP cells) and induction of apoptosis. Treatment of PC-3 tumor-bearing nude mice with Celastrol (1-3 mg/kg/d, i.p., 1-31 days) resulted in significant inhibition (65-93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and end time points showed in vivo inhibition of the proteasomal activity and induction of apoptosis after Celastrol treatment. Our results show that Celastrol is a natural proteasome inhibitor that has a great potential for cancer prevention and treatment.