LNCaP Prostate Cancer Research Articles

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

Knocking down claudin receptors leads to a decrease in prostate cancer cell migration, cell growth, cell viability and clonogenic cell survival

Prostate cancer is the most common solid organ malignancy in the United States, and has the highest probability of all cancers in becoming invasive. New molecular targets are needed to define and impede the growth and progression of advanced prostate cancers. Claudins (Cldns) are transmembrane proteins that regulate paracellular permeability and cell polarity, and their levels are elevated in many human cancers such as breast, ovarian, pancreatic, and prostatic cancers. Previously, we found that Cldn3 and Cldn4 are expressed in aggressive high-grade human prostate cancer specimens. We and others have shown that there are higher levels of Cldn3 and Cldn4 in metastatic human prostate cancer cells than in normal human prostate cells. The result of targeting Cldn3 and Cldn4 expression on the growth and viability of prostate cancer cells has not been elucidated. Human prostate cancer PC3 and LNCaP cells were transfected with Cldn3 or -4 small interfering RNAs (siRNAs). Cldn3/Cldn4 siRNA treatment resulted in a greater than 85% decrease in the protein levels of Cldn3 and Cldn4, which was accompanied by a 30–40% decrease in prostate cancer cell growth and a 60–65% reduction in cell viability. There was decreased cell migration with Cldn3 and Cldn4 siRNA in both PC3 and LNCaP cells and a 60–75% decrease in the number of clones when treated with siCldn3 or siCldn4 compared to control. Knocking down Cldn3/Cldn4 affects prostate cancer cell growth and survival and may have therapeutic implications.

Liquid-Liquid Chromatography Separation of Guaiane-Type Sesquiterpene Lactones from Ferula penninervis Regel & Schmalh. and Evaluation of Their In Vitro Cytotoxic and Melanin Inhibitory Potential.

Ferula penninervis Regel & Schmalh. is a perennial plant used in Kazakh traditional folk medicine to treat epilepsy, neurosis, rheumatism, gastroduodenal ulcers, dyspepsia, wounds, abscesses or tumors. The aim of this work was to isolate series of sesquiterpene lactones from a crude methanolic root extract and investigate their in vitro cytotoxic potential against androgen-dependent prostate cancer LNCaP and epithelial prostate PNT2 cells, as well as to evaluate their melanin production inhibitory effects in murine melanoma B16F10 cells stimulated with α-melanocyte-stimulating hormone (αMSH). Two new (penninervin P and penninervin Q) and five known (olgin, laferin, olgoferin, oferin and daucoguainolactone F) guaiane-type sesquiterpene lactones were isolated with the use of a simple and fast liquid-liquid chromatography method. Olgin and laferin showed the most promising cytotoxic effects in LNCaP cells (IC50 of 31.03 and 23.26 μg/mL, respectively). Additionally, olgin, laferin, olgoferin, and oferin (10 μg/mL) potently impaired melanin release (40.67–65.48% of αMSH + cells) without influencing the viability of B16F10 cells. In summary, our findings might indicate that guaiane-type sesquiterpene lactones from F. penninervis could be regarded as promising candidates for further research in discovering new therapeutic agents with anti-prostate cancer and skin depigmentation properties.

Effect of HDAC9 inhibition on epithelial-mesenchymal transition in CD133+ prostate cancer cell lines

A small fraction of cancer cells known as cancer stem cells (CSCs) are considered to give rise to differentiated cancer cells and have been proposed to predict cancer recurrence and metastasis. There is further evidence that CSCs may act as metastatic precursors of epithelial-mesenchymal transition (EMT). In the present study, we investigated the key molecules involved in maintaining the stability of CSCs by inducing ectopic overexpression of CD133 to characterize EMT in human prostate cancer cell lines, including PC-3, DU145, and LnCaP cells. Additionally, we investigated whether a specific inhibitor of concomitantly expressed metastasis-related genes could alleviate EMT properties in CD133-overexpressing prostate cancer cells. Ectopic overexpression of CD133 in PC-3, DU145, and LnCaP cells led to an increase in the expression of HDAC9. Moreover, HDAC9 inhibition led to a decrease in EMT properties along with increased E-cadherin expression, a narrower wound gap distance, and enhanced cell invasiveness through the suppression of β-catenin activation and its translocation to the nucleus. Overall, these results suggest that HDAC9 inhibition plays a functional role in the modulation of EMT properties in CSC-like prostate cancer cells. Therefore, these findings could facilitate the development of therapeutic strategies for controlling prostate cancer metastasis.

Androprostamine A: a unique antiprostate cancer agent.

The androgen receptor (AR) is an important therapeutic target for all clinical states of prostate cancer. We screened cultured broths of microorganisms for their ability to suppress androgen-dependent growth of human prostate cancer LNCaP and VCaP cells without cytotoxicity. We have already identified androprostamine A (APA) from a Streptomyces culture broth as a functional inhibitor of AR. APA repressed R1881 (the synthetic androgen methyltrienolone)-induced androgen-regulated gene expression and dramatically inhibited R1881-induced prostate-specific antigen levels. However, APA did not act as an AR antagonist and did not inhibit AR transcriptional activity. Moreover, AS2405, an APA derivative, significantly inhibited the growth of VCaP cells in SCID mice upon oral administration.

Predictors of therapeutic efficacy of 5-aminolevulinic acid-based photodynamic therapy in human prostate cancer

BackgroundPhotodynamic therapy (PDT) is a minimally invasive cancer therapy. However, its therapeutic efficacy for prostate cancer is not yet fully understood. In this study, the predictors of therapeutic efficacy of 5-aminolevulinic acid-based PDT (ALA-PDT) on prostate cancer cells are investigated. Materials and methodsThe human prostate cancer cell lines, PC-3, 22Rv1, DU145, and LNCap were used to investigate the effects of ALA-PDT on protoporphyrin IX (PpIX) intracellular accumulation, which was measured by flow cytometry. The cytotoxicity of ALA-PDT was evaluated by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The levels of porphyrin metabolism-related enzyme and transporter mRNA were comprehensively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated by Western blot. A xenograft model was created using PC-3 and 22Rv1, and then, pathological analysis was performed to determine the therapeutic effect of ALA-PDT ResultsPC-3 and LNCap cells showed high accumulation of PpIX and high sensitivity to ALA-PDT, while 22Rv1 and DU145 showed low accumulation of PpIX and low sensitivity to ALA-PDT. ALA-PDT-induced cytotoxicity correlated negatively with PpIX accumulation. The in vitro assays identified the ATP-binding cassette transporter subfamily G2 (ABCG2) transporter dimer as a predictor of treatment response. In vivo immunohistochemical staining of ABCG2 transporter showed low expression in PC-3 cells and high expression in 22Rv1 cells, and ALA-PDT-induced tumor tissue degeneration was greater in PC-3 cells than in 22Rv1 cells. ConclusionThe ABCG2 transporter is a useful predictor of the therapeutic effect of ALA-PDT on human prostate cancer cells.

Abstract 1209: Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract The androgen receptor (AR) is a key driver in the growth of prostate cancer and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients. Here we report the results of a comprehensive in vitro characterization of its mechanism of action. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. Target engagement was measured by a Cellular Thermal Shift Assay (CETSA). Both Nanostring and RNAseq were used to explore the activity of EPI-7386 on the AR transcriptome. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR were carried out to determine the effect of EPI-7386 on AR genomic occupancy. EPI-7386 exhibited potent activity in inhibiting full-length AR (AR-FL) driven transcriptional activity and also strongly impaired the transcriptional activity and the viability of cellular models driven exclusively by truncated AR protein. Using CETSA, we confirmed that EPI-7386 induced a thermal shift of both AR-FL and AR-V7 (lacking the AR LBD) in LNCaP and LNCaP95, respectively, which is an indication of AR target engagement by EPI-7386. ChIP analyses allowed a deeper understanding of epigenetic and transcriptional regulation driven by EPI-7386. It showed EPI-7386 inhibits androgen-activated AR binding to target gene loci such as KLK3. Additionally, EPI-7386 suppresses AR-regulated target gene expression in a comparable manner as lutamides in three human prostate cancer cell lines, LNCaP, 22Rv1, and VCaP, with a few notable exceptions. As a consequence, the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-associated transcriptional activity in both LNCaP and VCaP cells. In AR-V7 driven cell lines, LNCaP95 and 22Rv1, EPI-7386 showed superior activity to enzalutamide in inhibiting AR-regulated genes expression. In conclusion, EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients. The Phase I dose escalation first in human clinical trial of EPI-7386 single agent (NCT04421222) is currently enrolling. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Han-Jie Zhou, Ronan Le Moigne. Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1209.

Abstract 2594: Reactivation of maspin by plant flavone apigenin through inhibition of class I HDACs and increase in p53 transcriptional activity in prostate cancer cells

Abstract Loss of tumor suppressors leads to acquisition of metastatic capability during prostate cancer progression leading to higher mortality. Maspin (SERPINB5) is a unique member of the serpin (serine protease inhibitor) family shown to regulate cell motility, migration and invasiveness. Loss of maspin is frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore novel therapeutic approaches to restore its expression are needed. We recently demonstrated that knockdown of class I HDACs increase maspin expression in prostate cancer cells [Mol Carcinog. 59(8):955-966, 2020]. Apigenin (4', 5, 7-trihydroxyflavone), a plant flavone has shown to possess anticancer properties and alters pathways that regulate tumor cell invasion and metastasis, however, the molecular basis of these effects remains unclear. We investigated whether apigenin has ability to restore maspin expression and contribute to the inhibition of metastasis. Treatment of human prostate cancer LNCaP and 22Rv1 cells, both harboring wild type p53, with 1-40 µM apigenin for 72 h resulted in a dose- and time- dependent decrease in cell invasion and migration with concurrent increase in maspin expression in the nuclear compartment. Since, p53 activates maspin promoter by binding directly to p53 consensus-binding site, we studied the effect of apigenin in potentially restoring p53-mediated maspin levels. Exposure of LNCaP and 22Rv1 cells with 5-20 μM of apigenin resulted in dose-dependent increase in maspin expression and p53 activation through acetylation at the Lys305 residue. These effects were associated with the inhibition of class I HDAC levels. Furthermore, apigenin withdrawal resulted in the loss of maspin expression and p53 acetylation in LNCaP cells. The increased apigenin-mediated p53 acetylation enhanced its binding on maspin promoter, which was associated with decrease in cell invasion and migration. Apigenin treatment also caused accumulation of acetylated histone H3 in total cellular chromatin, increasing accessibility to bind with the promoter sequences of maspin, consistent with the effects elicited by HDAC inhibitor, Tricostatin A. Similar observations of inhibition of class I HDACs and increase in p53 transcriptional activity were noted after feeding apigenin at 20- and 50- µg/day to 22Rv1 tumor xenograft implanted in athymic nude mice. Our results demonstrate that apigenin-mediated increase in maspin expression together with downregulation of class I HDACs, increases p53 activation resulting in decreased invasiveness and migration capabilities in prostate cancer. Citation Format: Eswar Shankar, Albert Lee, Rajnee Kanwal, Sanjay Gupta. Reactivation of maspin by plant flavone apigenin through inhibition of class I HDACs and increase in p53 transcriptional activity in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2594.

Abstract 3163: Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts

Abstract The tumor microenvironment of prostate cancer (PCa) plays an important role in disease progression, responsiveness to therapy, or inversely restraint of tumor growth. PCa cell evolution including loss of androgen receptor dependency is associated with intratumor heterogeneity, generating progressive PCa cell subclones. The tumor stroma of PCa contains multiple functionally different populations of fibroblasts that regulate PCa cell progression, e.g., cancer-promoting or cancer-restraining. Fibroblasts secret a number of growth factors, cytokines, the ECM molecules, and miRNAs in a cellular characteristic-specific manner. Thus, we hypothesize that diversity of fibroblasts may induce heterogenous PCa cell progression. To investigate the heterotypical communications between PCa cells and fibroblasts, we used 3 subclones of androgen-sensitive LNCaP cells and 4 patient-derived fibroblasts. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Commercially available normal human prostate stromal cells (PrSC) was purchased from Lonza Group Ltd. Three original fibroblasts pcPrFs (-M5, -M28, -M31) were isolated from needle biopsy samples of patients with advanced PCa. To examine the effects of fibroblasts on cancer-related gene expression in PCa cells, in vitro co-culture experiment was performed using cell culture insert. In all PCa cells, LNCaP and its subclones, expressions of NFKB1, SRC, and MYC mRNA were not dramatically changed by co-culturing with 3 PCa patient-derived fibroblasts. Only in LNCaP cells, however, they were uniformly down-regulated by co-culturing with PrSC. Only in LNCaP cells, expression of CDH2 mRNA was up-regulated by co-culturing with pcPrF-M5 and pcPrF-M28. In all PCa cells, expression of NKX3-1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M28 and pcPrF-M31. In LNCaP, E9, and F10 cells but not in AIDL cells, expression of GSTP1 mRNA was uniformly up-regulated by co-culturing with pcPrF-M31. Cell growth of LNCaP cells was not remarkably affected by co-culturing with pcPrF-M28 and pcPrF-M31. The heterogeneous stromal compartment in PCa tissues contains multiple functionally different populations of fibroblasts that are also associated with intratumor heterogeneity. Our data showed that up-regulations of NKX3-1 and GSTP1 mRNA expression by co-culturing with pcPrF-M28 and pcPrF-M31 were commonly observed in LNCaP subclones differing in androgen sensitivity. This result suggests that certain fibroblasts may restrain PCa cell progression by up-regulations of tumor suppressor gene expression even in progressive poorly differentiated PCa cells. Citation Format: Kenichiro Ishii, Chise Matsuda, Yasuhisa Nakagawa, Taku Shirai, Yoshifumi Hirokawa, Takeshi Sasaki, Kazuhiro Iguchi, Masatoshi Watanabe. Progressive prostate cancer LNCaP subclones partially retain regressive communications with patient-derived fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3163.

Abstract 1126: Solute carrier (SLC) transporter-mediated metabolic reprogramming during enzalutamide resistance in prostate cancer

Abstract Prostate cancer is the second-leading cause of cancer-related mortality among men in the United States. Androgen receptor (AR) signaling is dominant survival pathway for prostate cancer cells, and androgen-deprivation therapy (ADT) through medical or surgical castration is the mainstay treatment for prostate cancer patients. Enzalutamide (Xtandi®), a second generation antiandrogen, is frequently prescribed for use in this clinical setting. Patient's response to this medication is usually temporary, and prolong treatment results in the emergence of drug resistance. A better understanding of molecular mechanism and its association with enzalutamide resistance will facilitate circumventing this problem. We employed next generation sequencing (NGS) and compared the transcriptomic profile of paired enzalutamide-sensitive and resistant human prostate cancer LNCaP and C4-2B cells for identification of genes involved in drug resistance along with in silico analysis. NGS data identified 9409 and 7757 genes differentially expressed in LNCaP and C4-2B cells compared to their parental counterparts. A subset of differentially expressed genes in both cell lines were validated at transcript level by qRT-PCR. Data analysis performed by IPA/i-pathway revealed metabolic signaling as predominantly overrepresented pathway and membrane transporters including solute carrier proteins, ATP-binding cassette transporters and other metabolizing enzymes as the most prominent genes dysregulated during metabolic reprogramming in both resistant cell lines. RNA-Seq data of resistant LNCaP cells demonstrate predominance of solute carrier genes, in particular, SLC12A5, SLC25A17 and SLC27A6, during metabolic reprogramming and in the development of drug resistance. Upregulation of these genes were associated with an increase in stemness; higher uptake of lactic/citric acid and lower glucose intake by the resistant cells. Our data suggest predominance of solute carrier genes (SLCs) during metabolic reprogramming of prostate cancer cells in an androgen-deprived environment. SLCs are important in cellular uptake of nutrients and drug absorption, thus could play a significant role in the emergence of drug-resistant phenotype, designating SLC genes as potentially attractive therapeutic targets. Citation Format: Shiv Shankar Verma, Eswar Shankar, Ricky Chan, Sanjay Gupta. Solute carrier (SLC) transporter-mediated metabolic reprogramming during enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1126.

Abstract 1421: Targeting cell-adaptive mechanisms of drug resistance in lethal prostate cancer

Abstract Precision oncology therapies are significantly hindered by the emergence of genetically or epigenetically-distinct cellular sub-populations, presenting either pre-existing or acquired drug resistance. For example, patients affected by advanced prostate cancer (PC), which is initially driven by aberrant activation of androgen receptor (AR) signals, often respond to Androgen Deprivation Therapy (ADT). Unfortunately, long-term treatment leads to the emergence of ADT-resistant tumors, termed Castration Resistant Prostate Cancer (CRPC), and poor outcome. Next-generation AR inhibitors extend CRPC patient survival, but frequently lead to the emergence of an even more aggressive Neuroendocrine PC (NEPC) phenotype, with exceedingly poor prognosis. Progression to NEPC is often determined by epigenetic-mediated reprogramming, rather than genomic alterations, resulting in cell-adaptive resistance, driven by aberrant activation of a handful of Master Regulator (MR) proteins. Consequently, better and more mechanistic understanding of cell-adaptive resistance is necessary to develop effective therapeutic strategies for CRPC. To address this challenge, we devised an RNA-based, high-throughput methodology, to systematically elucidate drug resistance mechanisms with single-cell resolution, and we applied it to study diverse, clonal populations that emerge following long-term treatment of PC LnCAP cells with next-generation AR inhibitors. Specifically, we identified Master Regulator (MR) proteins leading to the emergence of drug resistance, as well as clinically-available inhibitors able to reversing their activity, which were experimentally validated. We used PLATE-Seq, a low-cost, high-throughput RNA-Seq technology to profile more than 80 clonal, ADT-resistant populations, with single-cell resolution, and the VIPER algorithm to elucidate the MR proteins that mechanistically regulate their cell-adaptation programs. Our analysis revealed distinct cell-adaptive responses, associated with profound epigenetic reprogramming of prostate cancer cells. We used OncoTreat to prioritize a list of more than 400 FDA-approved and late-stage investigational drugs. Sensitivity to predicted drugs was confirmed in vitro and in vivo studies, in xenograft models derived from ADT- resistant clones are underway. We propose that this framework may represent a relevant advance to the study of cell-adaptive mechanism of drug resistance. Critically, this methodology is not limited to prostate cancer and can be applied to study arbitrary drug-resistance phenotypes. Citation Format: Prem Subramaniam, Alessandro Vasciaveo, Juan Arriaga, Jordan Kesner, Cory Abate-Shen, Andrea Califano. Targeting cell-adaptive mechanisms of drug resistance in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1421.

Kaempferol inhibits benign prostatic hyperplasia by resisting the action of androgen

Kaempferol is a natural compound that inhibits tumor development in androgenic related prostate cancer. However, it is still not clear about its phyto-androgenic activity and whether it suppresses testosterone-induced benign prostatic hyperplasia (BPH) development.In this study, molecular docking, cellular immunofluorescence staining, chromatin immunoprecipitation and dual luciferase reporter assay were performed to investigate the androgenic activity of kaempferol. Dihydrotestosterone-induced gene expression and cell proliferation were further analyzed upon treatment with kaempferol. Testosterone-induced BPH was established in rats and the effect and mechanism of action of kaempferol on BPH development was then assessed.Docking data showed that kaempferol could bind to ASN705 and THR877 residues of androgen receptor which were also the binding sites of dihydrotestosterone. The nuclear translocation of androgen receptor was promoted directly by kaempferol in androgen-dependent prostate cancer LNCaP cells. In addition, the in vivo interaction of androgen receptor with PSA promoter region and the transcriptional activity of androgen receptor were both significantly enhanced after kaempferol stimulation. However, kaempferol pretreatment suppressed dihydrotestosterone-induced effects including the transcriptional activity of androgen receptor, the expressions of PSA and AR genes and cell proliferation of LNCaP, BPH-1 and WPMY-1 cells. Consistently, kaempferol declined the prostate index and improved the pathological properties in BPH rats, and the up-regulated T level in serum from BPH rats was highly decreased after kaempferol administration.Kaempferol exhibited its androgenic-like activity and served as a selective androgen receptor modulator that contributes to androgen-related BPH development.

Preclinical dosimetric studies of 177 Lu-scFvD2B and comparison with 177 Lu-PSMA-617 and 177 Lu-iPSMA endoradiotherapeutic agents.

Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are 177 Lu-DOTA conjugates based on low molecular weight (LMW) Glu-ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA chelating moiety reduces the internalization of the LMW-PSMA agent and its radiation dose to the tumor. Previously, we reported that 177 Lu-scFvD2B, an antibody-based construct, demonstrated statistically significant higher cell uptake and internalization in LNCaP prostate cancer (PCa) cells (PSMA-positive) when compared to the LMW-PSMA agents, 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the endoradiotherapeutic agents which currently are the most used in PCa therapy. The aim of this study is to estimate the preclinical 177 Lu-scFvD2B organ and tumor-absorbed doses, and to compare the values with those of 177 Lu-PSMA-617 and 177 Lu-iPSMA. 177 Lu-scFvD2B, 177 Lu-PSMA-617, and 177 Lu-iPSMA were prepared and their radiochemical purity determined. Biodistribution studies of each radiopharmaceutical were then carried out in healthy mice to define the main source organs (SO) and to calculate the number of disintegrations in each source organs per unit of administered activity (NSO ). Absorbed dose in the main organs were then calculated for each 177 Lu-conjugate by means of OLINDA/EXM 2.1.1 software, using the calculated NSO for both the adult male and the mouse phantoms as program inputs. Images of mice bearing micropulmonary tumors injected with 177 Lu-conjugates were also obtained. Tumor standardized uptake values (SUV) for the different conjugates, obtained from the 3D SPECT image reconstruction of these mice, were used as the number of disintegrations in a tumor site per unit of administered activity (NT ). The tumor-absorbed dose was calculated using the published electron dose S-values for sphere models with diameters ranging from 10µm to 10mm and considering a uniform activity distribution and tumor density equivalent to water density. All 177 Lu-labeled agents were obtained in high yield (98%). Dosimetric studies carried out using mouse phantoms demonstrated that organ absorbed doses of 177 Lu-scFvD2B were from 1.4 to 2.3 times higher than those for 177 Lu-iPSMA and from 1.5 to 2.6 times higher than those for 177 Lu-PSMA-617. However, the 177 Lu-scFvD2B values of tumor-absorbed doses for all investigated tumor sizes were from 2.8 to 3.0 times greater than those calculated for 177 Lu-iPSMA and 177 Lu-PSMA-617, respectively. Moreover, 177 Lu-scFvD2B showed the highest tumor/kidney ratio when compared to those reported for 177 Lu-albumin conjugates. In this preclinical study, we demonstrated the potential of 177 Lu-scFvD2B as a therapeutic agent for PSMA-expressing tumors, due to its higher tumor-absorbed dose when compared with 177 Lu-LMW agents.

Characterization of camptothecin by analytical methods and determination of anticancer potential against prostate cancer

BackgroundObjective of present research work is to develop and validate cost-effective analytical tool for determination of camptothecin (CPT) and determine its anticancer potential against prostate cancer LNCaP cell lines. Structural elucidation has been performed by mass spectrometry, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and MTT assay utilized for in vitro cytotoxicity where spectrometric method was used for estimation of camptothecin.ResultsMass spectra showed peak at 349.2 which matches to standard molecular weight of camptothecin. FTIR and NMR spectra conformed functional moieties and structure of isolated camptothecin which was nearly equal to values mentioned in standard structure of camptothecin. IC50 values of CPT against LNCaP cell lines was found to be 3.561 μg/ml. Lambda max of CPT was found to be at 225 nm and calibration curve found to be linear over the concentration range from 2 to 70 μg/ml of camptothecin. Developed method was found to be linear, accurate, and precise. LOD and LOQ were found to be 0.0524 μg/ml and 0.1614 μg/ml, respectively. Developed method has % relative standard deviation less than one which is reproducible hence % recovery was found to be 99.80%.ConclusionsFTIR, NMR, and mass spectrometry results conforms isolated compound was camptothecin; cytotoxicity study proves it has strong potential in treatment of prostate carcinoma as competent alternative to chemotherapy in the form of herbal medicine and the developed UV method proves to be valid, sensitive, and applicable for rapid, accurate, precise, and economical determination of camptothecin.

In vitro Elucidation of Antiproliferative and Apoptotic Effects of Thymol against Prostate Cancer LNCaP Cells

Recent research suggested the role of plant-derived bioactive compounds as potent anticancer agents. Thymol, a monoterpene phenol, possesses numerous pharmacological properties such as antioxidant, anti-inflammatory, and antitumor effects. However, the inhibitory potential of thymol on prostate cancer cells still elusive. Therefore, the purpose of this study is to explore the antiproliferative and apoptotic effects of thymol against prostate cancer LNCaP cells. Our results indicated dose-dependent growth inhibitory effects of thymol on prostate cancer LNCaP cells. Morphological analysis and DAPI staining revealed that thymol induces marked morphological and nuclear alterations in LNCaP cells. Moreover, thymol could induce significant apoptosis in LNCaP cells through caspase-3 activation and modulation of mRNA expression of apoptotic-related genes. Overall, these findings showed that thymol could offer a novel therapeutic approach against prostate cancer.

Expression of G3BP1 in benign and malignant human prostate tissues.

BackgroundProstate cancer (PCa) is the world’s leading type of cancer in men. GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is overexpressed in a variety of tumors. However, there are limited studies in PCa concerning G3BP1. This present study was to investigates the expression of G3BP1 and the mechanism of action on PCa.MethodsWe explored the G3BP1 expression in PCa using the TCGA database and verified it using clinical samples by immunohistochemistry (IHC) methods. G3BP1 and Androgen receptor (AR) status of 104 human PCa and 50 benign prostate hyperplasia (BPH) samples were analyzed by IHC and the association between G3BP1 expression and biochemical recurrence was determined. Moreover, we generated G3BP1 knockdown cell lines in human PCa LNCaP cell lines, to observe AR changes.ResultsG3BP1 and AR were overexpressed in PCa compared to BPH tissues. The expression of G3BP1 and AR was positively correlated with the malignant degree of the tumor. Higher G3BP1 expression showed a trend toward biochemical recurrence. Western blot showed downregulation of G3BP1 affected AR expression levels.ConclusionsOur study suggested that G3BP1 was frequently upregulated in PCa and closely related to AR expression and tumor metastasis. Besides, G3BP1 might be associated with biochemical recurrence. These results supply potential target for the management of the PCa.

Knockdown of ZEB1 reverses cancer stem cell properties in prostate cancer cells.

Prostate cancer (PCa) is the second most diagnosed type of cancer in men worldwide. Advanced PCa is resistant to conventional therapies and high recurrence has been associated with high rates of metastasis. Cancer stem cells(CSCs) have been proposed to be responsible for this, due to their ability of self‑renewal and differentiation into other cell types. Zinc finger E‑box‑binding homeobox1(ZEB1), a transcription factor involved in the regulation of epithelial‑mesenchymal transition (EMT), has been associated with the activation of several mechanisms that lead to resistance to treatment. As recent evidence has shown that CSCs may originate from non‑CSCs during EMT, it was hypothesized that knocking down ZEB1 expression in PCa cell lines could revert some properties associated with CSCs. Using lentiviraltransduction, ZEB1 expression was silenced in the PCa DU145 and LNCaP cell lines. The mRNA and protein expression levels of key canonical CSC markers (Krüppel‑like factor4, SOX2, CD44 and CD133) were determined using reverse transcription‑-quantitative PCR and western blot analysis, respectively. In addition, the colony forming ability of the ZEB1‑knockdown cells was evaluated, and the type of colonies formed (holoclones, paraclones and meroclones) was also characterized. Finally, the ability to form prostatospheres was evaluated invitro. It was found that in ZEB1‑knockdown DU145 cells, the expression levels of CSC phenotype markers (CD44, CD133 and SOX2) were decreased compared with those in the control group. Furthermore, ZEB1‑knockdown cells exhibited a lower ability to form prostatospheres and to generate colonies. In conclusion, stable silencing of ZEB1 reversed CSC properties in PCa cell lines. Since ZEB1 is associated with malignancy, therapy resistance and a CSC phenotype in PCa cell lines, targeting ZEB1 may be a key factor to eradicate CSCs and improve the prognosis of patients with advanced PCa.

MicroRNA-501-5p Targets PINX1 Gene to Regulate the Proliferation, Migration, and Invasion of Prostatic Carcinoma Cells

The expression of PINX1 is decreased in prostate cancer, and the high level of miRNA-501-5p promotes the proliferation of liver cancer cells. However, there is no relevant research on miRNA-501-5p in prostate cancer. miRNA-501-5p can target the 3’UTR of PINX1 mRNA; however, it is unclear whether they affect the migration, invasion, and proliferation of prostate cancer cells. In this paper, PCR and Western blot were used to detect the expression of miRNA-501-5p and PINX1 in prostate cancer cells PC3, LNCaP, and DU145, and normal prostate epithelial cells RWPE-1. Compared to the normal prostate epithelial cells, miRNA-501-5p expression in prostate cancer cells was increased, and the expression of PINX1 was decreased. The methyl thiazolyl tetrazolium assay was used to detect the migration, proliferation, and invasion of prostate cancer DU145 cells. It was found that suppressing the expression of miRNA-501-5p or overexpressing PINX1 could inhibit the proliferation and other biological behaviors of DU145 cells; at the same time, the level of Cyclin D1, MMP-2, and MMP-14 protein was decreased, and the protein level of P21 was increased. Moreover, inhibition of PINX1 expression could partially reverse miRNA-501-5p’s inhibitory effect on the migration, invasion, and proliferation of prostate cancer cells. Therefore, miRNA-501-5p targeted PINX1 for down-regulation to promote prostate cancer cell migration, invasion, and proliferation.

Circular RNA Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Alpha Affects Prostate Cancer Lymph Node Carcinoma of Prostate-Cell Multiplication and Apoptosis via Regulating MicroRNA-299-3p Expression

Prostate cancer is a common cancer in aging men. This research explores the its molecular mechanisms of the circular RNA (circRNA) circPIP5K1A and its role in the multiplication and apoptosis of prostate cancer LNCaP cells. Cancerous tissue and adjacent non-cancerous tissue samples were selected from 37 prostate cancer patients hospitalized from January 2016 to January 2020. Quantitative reverse transcription PCR (RT-qPCR) was used to amplify circPIP5K1A and detect the miR-299-3p levels. Cells from the LNCaP prostate cancer cell line were divided into the si-circPIP5K1A group, si-negative control (NC) group, miR-299-3p group, miR-NC group, si-circPIP5K1A + anti-miR-299-3p group, and si-circPIP5K1A + anti-miR-NC group. Microculture tetrazolium (MTT) assay was used to assess the cell viability; flow cytometry was used to detect and analyze cell apoptosis; western blot was used to study the protein expression; the dual-luciferase reporter assay was used to determine the targeted regulation of miR-299-3p by circPIP5K1A. CircPIP5K1A expression in prostate cancer tissue increased when miR-299-3p expression was reduced (P < 0.05) compared to those in the adjacent tissue. After inhibition of circPIP5K1A or overexpression of miR-299-3p, the activity of LNCaP cells decreased, the apoptosis rate of LNCaP cells increased, Ki-67 and Bcl-2 expressions in LNCaP decreased, and Bax expression increased (P < 0.05). Due to CircPIP5K1A regulation of miR-299-3p expression, mir-299-3p interference reversed the effect of circPIP5K1A inhibition of LNCaP-cell multiplication and apoptosis. Inhibiting the expression of circPIP5K1A may inhibit LNCaP-cell multiplication and expedite cell apoptosis by up-regulating miR-299-3p.

The Differential Antitumor Activity of 5-Aza-2'-deoxycytidine in Prostate Cancer DU145, 22RV1, and LNCaP Cells.

DNA methylation is a DNA methyltransferase-mediated epigenetic modification affecting gene expression. This process is involved in the initiation and development of malignant disease. 5‐Aza‐2′‐deoxycytidine (5‐Aza), a classic DNA methyltransferase inhibitor, possesses antitumor proliferation activity. However, whether 5-Aza induces cytotoxicity in solid tumors warrants further investigated. In this study, human prostate cancer (CaP) cells were treated with 5-Aza and subjected to cell viability and cytotoxicity analysis. Reverse transcription-polymerase chain reaction and methylation-specific polymerase chain reaction assay were utilized to test the gene expression and methylation status of the p53 and p21 gene promoters. The results showed that 5-Aza differentially inhibited spontaneous proliferation, arrested the cell cycle at S phase in DU145, at G1 phase in 22RV1 and LNCaP cells, and G2 phase in normal RWPE-1 cells, as well as induced the expression of phospho-H2A.X and tumor suppressive mammary serine protease inhibitor (maspin) in all three types of CaP cells. 5-Aza also increased p53 and p21 transcription through promoter demethylation, and decreased the expression of oncogene c-Myc in 22RV1 and LNCaP cells. Western blotting analysis showed that the poly (ADP-ribose) polymerase cleavage was detected in DU145 and 22RV1 cells. Moreover, there were no significant changes in p53, p21 and c-Myc expression in DU145 cells following treatment with 5-Aza. Thus, in responsible for its apoptotic induction and DNA damage, the mechanism of the antitumor activities of 5-Aza may involve in an increase of tumor suppressive maspin, upregulation of wild type p53-mediated p21 expression and a decrease of oncogene c-Myc level in 22RV1 and LNCaP cells, and enhancing the tumor suppressive maspin expression in DU145 cells. These results enriched our understanding of the multifaceted antitumor activity of 5-Aza, and provided the expression basis of biomarkers for its possible clinical application in prostate cancer.