LNCaP Xenograft Mice Research Articles

Identification of Oral Bioavailable Coumarin Derivatives as Potential AR Antagonists Targeting Prostate Cancer.

Androgen receptor (AR) is a crucial driver of prostate cancer (PCa), but acquired resistance to AR antagonists significantly undermines their clinical efficacy. We previously discovered coumarin derivative 1, which is capable of disrupting AR ligand-binding domain dimers, offering the potential for overcoming resistance. However, its poor oral bioavailability limited further development. In this study, comprehensive structure optimizations led to compound 4a (IC50 = 0.051 μM), which exhibited comparable AR antagonistic activity to enzalutamide (IC50 = 0.060 μM) and demonstrated excellent selectivity over other nuclear receptors in vitro. Especially, 4a showed superior efficacy against ARF876L/T877A and ARW741C mutants compared to darolutamide and enzalutamide. Moreover, 4a exhibited favorable pharmacokinetic profiles (F = 66.24%) in vivo and significant tumor growth inhibition in an LNCaP xenograft mouse model upon oral administration. These results highlight the potential of 4a as a promising oral AR antagonist for overcoming drug resistance in PCa.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti-Hook Effect for Tumor Therapy.

Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target induced unintended tissue effects and intrinsic "hook effect" hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano-PROTACs) modality with a center-spoke degradation network for achieving efficient dose-dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self-assemble into Nano-PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano-PROTACs can form effective polynary complexes and E3 ligases degradation network with multi-binding sites, achieving dose-dependent protein degradation with "anti-hook effect". The generality and efficacy of Nano-PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide-range dose-dependent manner with a 95 % degradation rate and long-lasting potency up to 72 h in vitro. Significantly, Nano-PROTACs achieve in vivo dose-dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self-assembly strategy, the Nano-PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti‐Hook Effect for Tumor Therapy

AbstractProteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post‐translational protein degradation capabilities. However, off‐target induced unintended tissue effects and intrinsic “hook effect” hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano‐PROTACs) modality with a center‐spoke degradation network for achieving efficient dose‐dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self‐assemble into Nano‐PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano‐PROTACs can form effective polynary complexes and E3 ligases degradation network with multi‐binding sites, achieving dose‐dependent protein degradation with “anti‐hook effect”. The generality and efficacy of Nano‐PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide‐range dose‐dependent manner with a 95 % degradation rate and long‐lasting potency up to 72 h in vitro. Significantly, Nano‐PROTACs achieve in vivo dose‐dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self‐assembly strategy, the Nano‐PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Targeted α-therapy using astatine (211At)-labeled PSMA1, 5, and 6: a preclinical evaluation as a novel compound

PurposeTargeted α-therapy (TAT) for prostate-specific membrane antigen (PSMA) is a promising treatment for metastatic castration-resistant prostate cancer (CRPC). Astatine is an α-emitter (half-life=7.2 h) that can be produced by a 30-MeV cyclotron. This study evaluated the treatment effect of 211At-labeled PSMA compounds in mouse xenograft models.MethodsTumor xenograft models were established by subcutaneous transplantation of human prostate cancer cells (LNCaP) in NOD/SCID mouse. [211At]PSMA1, [211At]PSMA5, or [211At]PSMA6 was administered to LNCaP xenograft mice to evaluate biodistribution at 3 and 24 h. The treatment effect was evaluated by administering [211At]PSMA1 (0.40 ± 0.07 MBq), [211At]PSMA5 (0.39 ± 0.03 MBq), or saline. Histopathological evaluation was performed for the at-risk organs at 3 and 6 weeks after administration.Results[211At]PSMA5 resulted in higher tumor retention compared to [211At]PSMA1 and [211At]PSMA6 (30.6 ± 17.8, 12.4 ± 4.8, and 19.1 ± 4.5 %ID/g at 3 h versus 40.7 ± 2.6, 8.7 ± 3.5, and 18.1 ± 2.2%ID/g at 24 h, respectively), whereas kidney excretion was superior in [211At]PSMA1 compared to [211At]PSMA5 and [211At]PSMA6. An excellent treatment effect on tumor growth was observed after [211At]PSMA5 administration. [211At]PSMA1 also showed a substantial treatment effect; however, the tumor size was relatively larger compared to that with [211At]PSMA5. In the histopathological evaluation, regenerated tubules were detected in the kidneys at 3 and 6 weeks after the administration of [211At]PSMA5.ConclusionTAT using [211At]PSMA5 resulted in excellent tumor growth suppression with minimal side effects in the normal organs. [211At]PSMA5 should be considered a new possible TAT for metastatic CRPC, and translational prospective trials are warranted.

Establishment of an orthotopic prostate cancer xenograft mouse model using microscope-guided orthotopic injection of LNCaP cells into the dorsal lobe of the mouse prostate

BackgroundOrthotopic LNCaP xenograft mouse models closely mimic the progression of androgen-dependent prostate cancer in humans; however, orthotopic injection of LNCaP cells into the mouse prostate remains a challenge.MethodsUnder the guidance of a stereoscopic microscope, the anatomy of the individual prostate lobes in male Balb/c athymic nude mice was investigated, and LNCaP cells were inoculated into the mouse dorsal prostate (DP) to generate orthotopic tumors that mimicked the pathophysiological process of prostate cancer in humans. Real-time ultrasound imaging was used to monitor orthotopic prostate tumorigenesis, contrast-enhanced ultrasonography (CEUS) was used to characterize tumor angiogenesis, and macroscopic and microscopic characteristics of tumors were described.ResultsThe DP had a trigonal bipyramid-shape and were located at the base of the seminal vesicles. After orthotopic inoculation, gray scale ultrasound imaging showed progressive changes in tumor echotexture, shape and location, and tumors tended to protrude into the bladder. After 8 weeks, the tumor take rate was 65% (n = 13/20 mice). On CEUS, signal intensity increased rapidly, peaked, and decreased gradually. Observations of gross specimens showed orthotopic prostate tumors were well circumscribed, round, dark brown, and soft, with a smooth outer surface and a glossy appearance. Microscopically, tumor cells were arranged in acini encircled by fibrous septa with variably thickened walls, mimicking human adenocarcinoma.ConclusionsThis study describes a successful approach to establishing an orthotopic LNCaP xenograft Balb/c athymic nude mouse model. The model requires a thorough understanding of mouse prostate anatomy and proper technique. The model represents a valuable tool for the in vivo study of the biological processes involved in angiogenesis in prostate cancer and preclinical evaluations of novel anti-angiogenic therapies.

CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer

Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Cai2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Cai2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.

Gli2 mediates the development of castration‑resistant prostate cancer.

Glioma-associated oncogene family zinc finger 2 (Gli2), a key component of the hedgehog signaling pathway, has been previously demonstrated to promote the malignant properties of prostate cancer in vitro. However, the role of Gli2 in the development of castration-resistant prostate cancer (CRPC) has yet to be fully elucidated. In the present study, Gli2 expression was knocked down in androgen-responsive prostate cancer cells using an inducible Gli2 short hairpin RNA. Suppression of Gli2 expression resulted in significant reduction of cell viability, increased the proportion of cells in the G0/G1 phases of the cell cycle and reduced the expression of genes associated with cell cycle progression. Gli2 knockdown sensitized both androgen-dependent and -independent prostate cancer cells to the antiandrogen drug Casodex and prevented the outgrowth of LNCaP prostate cancer cells. In addition, Gli2 knockdown significantly suppressed the development of CRPC in a LNCaP xenograft mouse model, which was reversed by the re-expression of Gli2. In conclusion, to the best of our knowledge, the present study was the first occasion in which the essential role of Gli2 in the development of CRPC was demonstrated, providing a potential therapeutic target for the intervention of CRPC.

Abstract B011: Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Current available treatments include chemical castration and therapies targeting androgen receptor (AR) signaling pathways. Although initial responses are observed, 30% of patients have primary resistance to both forms of treatment and the majority of patients progress from androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC). Studies on mechanisms of resistance suggest that AR signaling remains a key driver in PCa even after treatment with AR-targeted therapies. Co-treatment with therapies that modulate alternative signaling pathways involved in oncogenesis in PCa including epigenetic modifiers, have been proposed as a modality to overcome resistance. Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 and is one of the most highly upregulated genes in CRPC compared to localized PCa. Several reports suggest that EZH2 plays an oncogenic role both in ADPC and CRPC.Tazemetostat is a potent, selective, orally bioavailable, investigative first-in-class small molecule inhibitor of EZH2. Phase 2 clinical trials have demonstrated objective clinical responses in patients with B-cell lymphomas and molecularly defined solid tumors, including epithelioid sarcoma and other INI1-negative tumors. Given the implication of EZH2 in PCa, we explored the combination potential of tazemetostat with current second-line therapies, the androgen signaling inhibitors (ASIs) abiraterone and enzalutamide. Tazemetostat displayed both time- and dose-dependent antiproliferative activity in PCa cell lines dependent on AR signaling or those of the neuroendocrine subtype with IC50 values at or below 1 μM in 14-day assays. Synergistic activity was observed in vitro in a subset of cell lines when tazemetostast was combined with ASIs in a 7-day co-treatment model and this activity was further enhanced when cells were pretreated with tazemetostat prior to the cotreatment. To further validate these findings, we conducted in vivo studies in PCa cell line-derived xenograft models. Tazemetostat and enzalutamide showed single agent antitumor activity in a subcutaneous LNCaP xenograft mouse model. Co-administration of enzalutamide and tazemetostat markedly reduced tumor growth rate compared to either of the single agent(s). Similar results were observed with the combination of tazemetostat and abiraterone. Interestingly in the 22Rv1-derived in vivo xenograft model, a cell line that expresses AR-V7, a splice variant implicated in resistance to ASIs in CRPC, modest tumor growth inhibition was observed with tazemetostat and no significant antitumor activity was observed when given in combination with abiraterone or enzalutamide. A significant decrease in intratumoral H3K27me3 levels were observed in all treatment groups containing tazemetostat in both in vivo models, indicating an on-target activity of EZH2 inhibition. Current efforts are underway to investigate the mechanism of action and cellular inhibitory pathways for these combinations. In summary, our preclinical data demonstrates the importance of EZH2 inhibition in CRPC and provides a rationale for combination of tazemetostat with abiraterone and enzalutamide in the clinic. Citation Format: Vinny Motwani, Dorothy Brach, Chloe Pantano, Vania Estanek, Jeffrey A. Keats, Daniel T. Dransfield, Alejandra Raimondi. Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B011. doi:10.1158/1535-7163.TARG-19-B011

An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy.

The 18-membered macrocycle H2 macropa was investigated for 225 Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225 Ac (26 kBq) in 5 min at RT. [225 Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa-NCS, was conjugated to trastuzumab as well as to the prostate-specific membrane antigen-targeting compound RPS-070. Both constructs rapidly radiolabeled 225 Ac in just minutes at RT, and macropa-Tmab retained >99 % of its 225 Ac in human serum after 7 days. In LNCaP xenograft mice, 225 Ac-macropa-RPS-070 was selectively targeted to tumors and did not release free 225 Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225 Ac chelation that will facilitate the clinical development of 225 Ac TAT for the treatment of soft-tissue metastases.

An Eighteen‐Membered Macrocyclic Ligand for Actinium‐225 Targeted Alpha Therapy

AbstractThe 18‐membered macrocycle H2macropa was investigated for 225Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225Ac (26 kBq) in 5 min at RT. [225Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa‐NCS, was conjugated to trastuzumab as well as to the prostate‐specific membrane antigen‐targeting compound RPS‐070. Both constructs rapidly radiolabeled 225Ac in just minutes at RT, and macropa‐Tmab retained >99 % of its 225Ac in human serum after 7 days. In LNCaP xenograft mice, 225Ac‐macropa‐RPS‐070 was selectively targeted to tumors and did not release free 225Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225Ac chelation that will facilitate the clinical development of 225Ac TAT for the treatment of soft‐tissue metastases.

A PSMA Ligand Labeled with Cobalt-55 for PET Imaging of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) comprises a recognized target for molecular imaging of prostate cancer. As such, radiolabeled PSMA inhibitors are of great value for diagnosis and staging of this disease. Herein, we disclose the preclinical characterization of [55Co]PSMA-617 for positron emission tomography (PET)/x-ray computed tomography (CT) imaging of prostate cancer lesions. By the application of microwave heating, PSMA-617 in acetate buffer (0.4M, pH4.4) was labeled with the radioisotopes cobalt-55/57. The extents of internalization and dissociation constants (K D) were determined against 2-(phosphonomethyl)-pentanedioic acid in two PSMA-positive cell lines, LNCaP, and PC3-PIP, with [57Co]PSMA-617 as a surrogate for [55Co]PSMA-617 (T½ 17.5h, β max 1.5MeV, Iβ 76%). The biodistribution in LNCaP xenograft mice was investigated using [57Co]PSMA-617 and [55Co]PSMA-617 was employed for PET/CT imaging at 1, 4, and 24h and compared to PET/CT scans using [68Ga]PSMA-617. The radiolabeling with cobalt-55/57 was performed in yields greater than 99.5 and 99.8% and radiochemical purities of 99.7 and 98.9%, respectively. The molar-specific activities were 18.2MBq/nmol and 3.3MBq/nmol. The cellular K D were determined to be 4.7nM for LNCaP and 9.8nM for PC3-PIP, correspondingly. Internalization of 76 and 71% of the cell-associated radioactivity was found for LNCaP and PC3-PIP cells after incubation up to 240min, respectively. In regard to the biodistribution in LNCaP xenograft mice, [57Co]PSMA-617 displayed a high and relatively constant uptake in the tumor (12.9%IA/g at 1h to 10.5%IA/g at 24h) with an initial but transient high uptake in the kidneys, adrenals, and spleen. Tumor-to-background ratios improved over time as normal tissue cleared of the radioligand (tumor-to-blood: 26, 258, and 3013; tumor-to-kidney: 0.11, 0.28, and 4.3 at 1, 4, and 24h). PET/CT imaging with [55Co]PSMA-617 in xenograft mice confirmed the high tumor uptake and fast clearance of normal tissues over time and was found superior to imaging with [68Ga]PSMA-617. Radiolabeling of PSMA-617 was achieved in excellent yields and radiochemical purities. Favorable in vitro data comprising low K D values and high extent of internalization was determined for two PSMA-positive cell lines. In xenograft mice, high tumor accumulation and excellent tumor-to-normal tissues ratios were established by biodistribution experiments and PET/CT imaging and, hence, confirm the potential of [55Co]PSMA-617 for delayed clinical imaging of prostate cancer.

Diet-induced alteration of fatty acid synthase in prostate cancer progression.

Fatty acid synthase (FASN) is a cytosolic metabolic enzyme that catalyzes de novo fatty acid synthesis. A high-fat diet (HFD) is attributed to prostate cancer (PCa) progression, but the role FASN on HFD-mediated PCa progression remains unclear. We investigated the role of FASN on PCa progression in LNCaP xenograft mice fed with HFD or low-fat diet (LFD), in PCa cells, and in clinical PCa. The HFD promoted tumour growth and FASN expression in the LNCaP xenograft mice. HFD resulted in AKT and extracellular signal-regulated kinase (ERK) activation and 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation. Serum FASN levels were significantly lower in the HFD group (P=0.026) and correlated inversely with tumour volume (P=0.022). Extracellular FASN release was enhanced in the PCa cells with phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinase (MAPK) inhibition and AMPK signalling activation. FASN inhibition resulted in decrease of PCa cell proliferation through PI3K/MAPK downregulation and AMPK activation. Furthermore, AMPK activation was associated with FASN downregulation and PI3K/MAPK inactivation. Clinically, high FASN expression was significantly associated with high Gleason scores and advanced pathological T stage. Moreover, FASN expression was markedly decreased in the PCa response to androgen deprivation therapy and chemotherapy. HFD modulates FASN expression, which may be an important mechanism in HFD-associated PCa progression. Furthermore, a critical stimulatory loop exists between FASN and the PI3K/MAPK system, whereas AMPK signalling was associated with suppression. These may offer appropriate targets for chemoprevention and cancer therapy in HFD-induced PCa.

Abstract 1727: DNA-binding domain as an alternative target site on androgen receptor for prostate cancer therapy: Pre-clinical in vivo study of a novel small molecule for drug development

Abstract Androgen receptor (AR) is a key regulatory transcription factor in the development of prostate cancer (PCa). Common treatments for PCa include hormone therapy, radiotherapy and surgery. However, many PCa patients develop resistance towards hormone therapies in later stages of PCa. There is growing evidence suggesting that AR splice variants render PCa treatments that target the LBD, such as enzalutimide, ineffective. Our objective is to develop a drug to treat recurrent and metastatic PCa by targeting the DNA Binding Domain (DBD). Through in silico docking prediction and in vitro bioassays, we have identified a novel small molecule - VPC-14449 (4-(4-(4,5-bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine) - that may inhibit AR activation through targeting DBD. In order to bring VPC -14449 into the clinic, we conducted preclinical in vivo studies to determine both toxicity and efficacy. CD1 mice were treated with 25, 50, and 100 mg/kg of VPC-14449 I.P. twice daily for four weeks to determine the maximum tolerable dose. During this time, we assessed the signs of physical toxicity, behavioral changes, and gross manifestations of stress. At the end of the experiment, the mice were euthanized and organs were collected for further immunohistochemical analysis. Furthermore, we assessed the in vivo therapeutic effects of VPC-14449 in different nude mouse xenograft models including LNCaP, MR49-F, and C4-2. Body weight, tumor volume (π/6 x length x width x height) and PSA were measured weekly. LNCaP xenograft mice were castrated when PSA level reached 25 ng/ml. Mice were randomly assigned to one of the three treatment groups (VPC-14449 (100mg/kg), enzalutimide (10 mg/kg), or vehicle) when PSA level returned to 25 ng/ml (for LNCaP xenografts) or when the tumor sizes reached 100mm3(for MR49-F and C4-2 xenografts). The mice were treated I.P. twice daily for four weeks before euthanization. Toxicity studies revealed no signs of physical toxicity, behavioral changes, and gross manifestations of stress in animals treated with up to 100 mg/kg dose of VPC-14449. Efficacy studies revealed that VPC-14449 effectively suppresses tumor growth in LNCaP, MR49 and C4-2 xenografts with P value <0.01, <0.05 and < 0.001 respectively, when compared to control. VPC-14449 also significantly reduced PSA levels in LNCaP xenografts (p value <0.01). The results suggest that VPC-14449 is effectively suppressing PCa tumor growth even in androgen insensitive and MDV resistant PCa cell lines in vivo. Citation Format: Mohamed DH Hassona, Leslie G. Chan, Gray R. Meckling, Huifang Li, Eric LeBlanc, Fuqiang Ban, Artem Cherkasov, Paul S. Rennie, Emma Guns. DNA-binding domain as an alternative target site on androgen receptor for prostate cancer therapy: Pre-clinical in vivo study of a novel small molecule for drug development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2015-1727

In vivo assessment of the therapeutic effects of lyophilized leech saliva extract from (Huridinaria manillensis) on LNCaP tumor xenograft model in nude mice.

271 Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. A large number of peptides and proteins have been identified and characterized in leech saliva extract (LSE), including anti-thrombotic agents, cancer metastasis inhibitors and anti-microbials Currently, leech therapy is established as an important tool in microsurgery and reconstructive operations having demonstrated superior clinical outcomes for the optimal salvage of grafted tissues. Methods: In the current study, we have determined the in vivo efficacy of LSE from (Huridinaria manillensis) on castration resistant LNCaP xenograft mouse model. Mice were divided into three groups of six, mice were subcutaneously injected with either LSE (5 mg/kg), docetaxel (10 mg/kg), or vehicle once a week. PSA and tumor volume were measured weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and immunohistochemical (IHC) analysis. Results: There was a significant decrease in the tumor volume and PSA with either docetaxel (10mg/kg) or LSE (5 mg/kg) treated groups compared to the control. While there was no significant difference between the anti-tumor activity of docetaxel (10mg/kg) and LSE (5 mg/kg). IHC showed significant increase in caspase-3 and significant decrease in Ki-67 and PCNA expression in the LSE treated mice compared to the control group. Interestingly, transcriptome analysis of tumor samples showed that LSE modulated cytokine production, monocyte adhesion, steroidogenesis, and P38 MAPK signaling pathways. Conclusions: LSE has significant anti-tumor activity in LNCaP tumor xenograft models with no apparent side effects. This can be attributed, at least partly, to its inhibition of cellular proliferation, induction of apoptosis, modulation of immunity and steroidogenesis.

RNA aptamer-conjugated liposome as an efficient anticancer drug delivery vehicle targeting cancer cells in vivo

To minimize the systemic toxicity prevalent to chemotherapeutics, we designed a novel anticancer drug-encapsulating liposome conjugated with an RNA aptamer specific to the prostate specific membrane antigen (PSMA), which is expressed on the surface of prostate cancer cells. The RNA aptamer-conjugated liposome, termed an aptamosome, was prepared by the post-insertion method, in which RNA aptamer-conjugated micelles were inserted into a liposome. These nanosized (90–100nm) aptamer-conjugated liposomes specifically bind to LNCaP prostate epithelial cells that express PSMA and thus cause the nanoparticles to have significantly enhanced in vitro cellular binding and uptake as compared with nontargeted nanoparticles that lack the PSMA aptamer. Aptamosomes encapsulated with the anticancer drug doxorubicin (Dox) were significantly more toxic to the targeted LNCaP cells than to nontargeted cancer cells. Dox-encapsulating aptamosomes administered to LNCaP xenograft nude mice were selectively retained in tumor tissue. We also demonstrated in vivo anticancer efficacy of the Dox-encapsulating PSMA-aptamosomes on tumor size regression in LNCaP xenograft mice. We suggest that the encapsulation of toxic chemicals with aptamer-conjugated liposomes will enable the use of these bioconjugates in clinical practice with fewer side effects.

Abstract 4590: Cisplatin-RNAi nanotherapeutics for synergistic anti-tumor activity

Abstract Cisplatin and other DNA-damaging chemotherapeutics are widely used to treat a broad spectrum of malignancies. However, the development of acquired chemoresistance is a persistent clinical problem limiting the successful treatment of malignancies and considerable work has been done to identify the molecular mechanisms involved. Many possible mechanisms have been suggested for platinum resistance emergence, such as drug efflux, apoptosis inhibition among others. Recent studies have shown that the suppression of crucial gene products (e.g. REV1, REV3L) involved in the error-prone translesion DNA synthesis pathway can sensitize intrinsically resistant tumors to chemotherapy. Due to the lack of effective inhibitors and the undruggable feature of some targets, it is not easy to screen and validate the target combinations, especially in vivo. Since synthetic siRNA has emerged as a class of attractive therapeutics for treatment of various cancers by silencing genes that cause diseases. Given the ability to target and silence nearly any gene of interest, specific siRNA can be constructed to target genes encoding proteins involved in DNA repair and the acquisition of multidrug resistance. Although a powerful means to silence the expression of target genes, the safe and effective systemic delivery of siRNA therapeutics remains an important challenge. Herein we have developed an innovative chemo-RNAi nanotherapeutic strategy that can simultaneously deliver platinum drugs and multiple siRNAs. We hypothesize that the NP-mediated delivery of multiple siRNAs against different platinum resistance mechanisms will allow the validation of key targets. We aim to improve the efficacy of chemotherapy through additive or synergistic effects coupled by platinum drug and siRNAs targeting different drug resistance mechanisms. As proof-of-concept, we have used poly(lactide-co-glycolide)-b-poly(ethylene glycol)-lipid hybrid nanocarriers as a delivery platform, translesion DNA polymerases Rev1/3L as model target proteins responsible for platinum resistance, and prostate cancer as a model disease. We have successfully created polymer-lipid NPs that exhibited simultaneous entrapment of siRNA (up to 99%) and cisplatin (around 10%). The size of the NPs ranges from 180 to 220 nm and the drug release profile shows a controlled release of cisplatin and siRNA over 10 days. We demonstrated the potency of the siRNA-containing NPs to efficiently knockdown target genes both in vitro and in vivo. The therapeutic efficacy of NPs containing both cisplatin prodrug and REV1/REV3L specific siRNAs was further investigated in vitro and in vivo. qRT-PCR results showed that the NPs exhibited a significant and sustained suppression of both genes in tumors for up to 3 days after a single dose. Administering these NPs revealed a synergistic effect on tumor inhibition in an LNCaP xenograft mouse model that was strikingly more effective than platinum monotherapy. Note: This abstract was not presented at the meeting. Citation Format: Xiaoyang Xu, Xueqing Zhang, Kun Xie, Graham Walker, Omid Farokhzad. Cisplatin-RNAi nanotherapeutics for synergistic anti-tumor activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4590. doi:10.1158/1538-7445.AM2014-4590

MP31-06 DIET INDUCED ALTERATION OF FATTY ACID SYNTHASE IS INVOLVED IN PROSTATE CANCER PROGRESSION

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-06 DIET INDUCED ALTERATION OF FATTY ACID SYNTHASE IS INVOLVED IN PROSTATE CANCER PROGRESSION Mingguo Huang, Shintaro Narita, Norihiko Tsuchiya, Takamitsu Inoue, Shigeru Satoh, Hiroshi Nanjo, Takehiko Sasaki, and Tomonori Habuchi Mingguo HuangMingguo Huang More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , Hiroshi NanjoHiroshi Nanjo More articles by this author , Takehiko SasakiTakehiko Sasaki More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.915AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High-fat diet (HFD) or obesity is strongly suggested to influence prostate cancer (PCa) progression, but the underlying mechanism is not well defined. Fatty acid synthase (FASN) is a cytosolic metabolic enzyme catalyzing de novo biosynthesis of fatty acid. METHODS In the present study, we investigated the role of FASN on PCa progression in the LNCaP xenograft mouse fed HFD or low-fat diet (LFD). RESULTS The tumor growth was increased in the HFD group than LFD group (p = 0.025). The mRNA expression of FASN and SREBP-1, which encodes Sterol regulatory element-binding protein-1 (SREBP-1) were increased in the xenograft tumor by 1.8- and 2.1-fold, respectively (p < 0.05). The serum level of FASN was significantly lower in the HFD group than in the LFD group (p = 0.026) and correlated inversely with the tumor volumes (total, r = 0.642, p = 0.022; HFD group, r = −0.618, p = 0.024; LFD group, r = −0.439, p = 0.154; respectively). Upregulation of P-AKT and P-ERK, and downregulation of P-AMPK were observed in xenograft tumors of the HFD group than LFD group. The extracellular release of FASN protein in vitro and AMPK activation was induced in the PCa cells by inhibition of the PI3K and MAPK signaling. In addition, pharmacological activation of AMPK stimulated the extracellular FASN release along with the downregulation of FASN and SREBP-1 in the PCa cells in dose-dependent manner. Cell proliferation was significantly decreased in the PCa cells by inhibition of FASN, and the decreased effect was rescued by addition of palmitic acid. Clinically, imuunohistological evaluation of surgical specimens showed that the expression of FASN was markedly decreased in the PCa response to hormone and chemotherapy. CONCLUSIONS In conclusion, HFD increased the FASN expression and inhibited the extracellular release of FASN, which may be one of important mechanism in HFD or obesity associated PCa progression. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e324 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Mingguo Huang More articles by this author Shintaro Narita More articles by this author Norihiko Tsuchiya More articles by this author Takamitsu Inoue More articles by this author Shigeru Satoh More articles by this author Hiroshi Nanjo More articles by this author Takehiko Sasaki More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy. This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.

Small molecule inhibitors of PSMA incorporating technetium-99m for imaging prostate cancer: Effects of chelate design on pharmacokinetics

Single amino acid chelate (SAAC) systems for the complexation of the M(CO)3 moiety (M=Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation of 99mTc(CO)3 complexes has resulted in substantial hepatobiliary uptake when examined either as lysine derivatives or integrated into biologically active small molecules and peptides. Here, we designed, synthesized, and evaluated novel polar functionalized imidazole derived SAAC systems (SAAC II) which have been chemically modified to promote overall 99mTc(CO)3L3 complex hydrophilicity with the intent of reducing non-target effects and enhancing renal clearance of prostate specific membrane antigen (PSMA) targeting small molecules. The 99mTc-labeled compounds were prepared, purified, and evaluated for stability, lipophilicity, and tissue distribution in LNCaP xenograft mice. The Glu-urea-Lys-C11 analogs were prepared with a variety of chelators to form (19R,23S)-1-(X)-2-((Y)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylic acid where X=Y=(methyl)pyridin-2-yl (6), X=Y=(methyl)-1H-imidazol-2-yl (7), X=(methyl)pyridin-2-yl, Y=carboxymethyl (8), X=Y=1-(carboxymethyl)-1H-imidazol-2-yl (9), X=1-(carboxymethyl)-1H-imidazol-2-yl, Y=carboxymethyl (10), and X=Y=1-(1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl) (11). 99mTc labeling was achieved at ligand concentrations as low as 10−6M and the complexes were stable (>90%) for 24h. These new SAAC II chelators were evaluated for their influence on binding of the Glu-urea-Lys-C11 analogs to PSMA-positive LNCaP cells and compared to pyridine- and N-methylimidazole-containing SAAC ligands. Tissue distribution of the 99mTc-complexes containing the more polar chelators, 9 and 11, demonstrated decreased liver (<2% ID/g) and increased LNCaP tumor (>11% ID/g) accumulation at 1h post-injection.

Image‐Guided Prostate Cancer Therapy Using Aptamer‐Functionalized Thermally Cross‐Linked Superparamagnetic Iron Oxide Nanoparticles

CG-rich duplex containing prostate-specific membrane antigen (PSMA) aptamer-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPIONs) is reported as prostate cancer-specific nanotheranostic agents. These agents are capable of prostate tumor detection in vivo by magnetic resonance imaging (MRI) and selective delivery of drugs to the tumor tissue, simultaneously. The prepared PSMA-functionalized TCL-SPION via a hybridization method (Apt-hybr-TCL-SPION) exhibited preferential binding towards target prostate-cancer cells (LNCaP, PSMA+) in both in vitro and in vivo when analyzed by T(2) -weighted MRI. After Dox molecules were loaded onto the Apt-hybr-TCL-SPION through the intercalation of Dox to the CG-rich duplex containing PSMA aptamer as well as electrostatic interaction between the Dox-and-polymer coating layer of the nanoparticles, the resulting Dox@Apt-hybr-TCL-SPION showed selective drug-delivery efficacy in the LNCaP xenograft mouse model. These results suggest that Dox@Apt-hybr-TCL-SPION has potential for use as novel prostate cancer-specific nanotheranostics.