LNCaP Human Prostate Cancer Cells Research Articles

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 55: TT-702, a selective and potent A2B receptor antagonist for the treatment of cancer

Abstract Adenosine signaling via the A2A and A2B receptors is emerging as an important regulatory mechanism of immune responses. While A2AR has high affinity for adenosine, A2BR is a low-affinity receptor that becomes activated only under pathological conditions when adenosine concentration reaches micromolar ranges. Besides high levels of adenosine (up to 100 μM), the tumor microenvironment (TME) is characterized by hypoxia. Hypoxia upregulates A2BR on tumors and on immune cells. TT-702 is a novel, selective and highly potent, orally active A2BR antagonist. Here we show that TT-702 inhibits tumor growth through multiple mechanisms including enhancement of dendritic cell (DC) function and T-cell activation, reduction of MDSCs and Tregs, and suppression of cancer cell growth, fibrosis and angiogenesis. The cellular potency of TT-702 was assessed in NECA induced cAMP production. The ability of TT-702 to reverse NECA-mediated immune suppression was evaluated using standard human DC differentiation and MLR assays. The effects of TT-702 on tumor growth were determined in B16F10 melanoma and MC38 and CT26 colorectal cancers in mice treated with TT-702 at 0.3, 1, 3 or 10 mg/kg (BID), anti-PD-1 (RMP1-14, 5 mg/kg, Q2D) or TT-702+RMP1-14 intraperitoneally. TT-702 binds to human A2BR with Ki value of 8.3 nM and exhibits greater than 100x selectivity for A1, A2A and A3 receptors. It potently inhibited NECA-stimulated cAMP production (IC50=2.8 nM). TT-702 dose-dependently inhibited the growth of human prostate cancer cells LNCaP and PC-3 (IC50=1.4 μM). Combination of TT-702 with enzalutamide showed an additive and synergistic effects to inhibit LNCaP and PC-3 growth, respectively. TT-702 prevented NECA-induced suppression of human DC differentiation and completely reversed NECA-induced T cell inactivation (EC50=0.45 μM). By contrast, preladenant, an A2AR antagonist, had no effect on cancer cell growth and DC differentiation. Moreover, TT-702 increased MC38 tumor-infiltrating immune stimulatory cells (DCs, CD4+ and CD8+ T cells) and reduced immunosuppressive cells (MDSCs and Tregs). TT-702 dose-dependently inhibited the growth of mouse MC38 and CT26 colorectal cancers. Interestingly, TT-702 also inhibited the growth of mouse B16F10 melanoma cold tumor. Furthermore, combination of TT-702 with anti-PD-1 synergistically inhibited both B16F10 and CT26 tumor growth. Most importantly, TT-702 significantly reduced tumor fibrosis and angiogenesis, suggesting that TT-702 may improve the TME for T cell penetration, cancer killing and prevention of metastasis. TT-702, a novel, selective and potent A2B receptor antagonist, has the potential to treat a diverse range of hard-to-treat and aggressive tumors (including cold ones) as a monotherapy, and in combination with anti-PD-1, enzalutamide and other anticancer therapies. TT-702 exhibits excellent safety and PK profiles in animals and is slated to enter clinical development in 2021. Citation Format: Peidong Fan, Filomena Housley, Helena Haberstock, Ken Horne, Jim Liu, Elfatih Elzein, Lina Yao. TT-702, a selective and potent A2B receptor antagonist for the treatment of 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 55.

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 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.

Sodium bicarbonate transporter NBCe1 regulates proliferation and viability of human prostate cancer cells LNCaP and PC3.

Studies on cultured cancer cells or cell lines have revealed multiple acid extrusion mechanisms and their involvement in cancer cell growth and progression. In the present study, the role of the sodium bicarbonate transporters (NBCs) in prostate cancer cell proliferation and viability was examined. qPCR revealed heterogeneous expression of five NBC isoforms in human prostate cancer cell lines LNCaP, PC3, 22RV1, C4-2, DU145, and the prostate cell line RWPE-1. In fluorescence pH measurement of LNCaP cells, which predominantly express NBCe1, Na+ and HCO3–-mediated acid extrusion was identified by bath ion replacement and sensitivity to the NBC inhibitor S0859. NBCe1 knockdown using siRNA oligonucleotides decreased the number of viable cells, and pharmacological inhibition with S0859 (50 µM) resulted in a similar decrease. NBCe1 knockdown and inhibition also increased cell death, but this effect was small and slow. In PC3 cells, which express all NBC isoforms, NBCe1 knockdown decreased viable cell number and increased cell death. The effects of NBCe1 knockdown were comparable to those by S0859, indicating that NBCe1 among NBCs primarily contributes to PC3 cell proliferation and viability. S0859 inhibition also decreased the formation of cell spheres in 3D cultures. Immunohistochemistry of human prostate cancer tissue microarrays revealed NBCe1 localization to the glandular epithelial cells in prostate tissue and robust expression in acinar and duct adenocarcinoma. In conclusion, our study demonstrates that NBCe1 regulates acid extrusion in prostate cancer cells and inhibiting or abolishing this transporter decreases cancer cell proliferation.

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.

Circulating prostate cancer cells have differential resistance to fluid shear stress-induced cell death.

ABSTRACTCirculating tumor cells (CTCs) are exposed to fluid shear stress (FSS) of greater than 1000 dyn/cm2 (100 Pa) in circulation. Normally, CTCs that are exposed to FSS of this magnitude die. However, some CTCs develop resistance to this FSS, allowing them to colonize distant organs. We explored how prostate CTCs can resist cell death in response to forces of this magnitude. The DU145, PC3 and LNCaP human prostate cancer cell lines were used to represent cells of different metastatic origins. The cell lines were briefly treated with an average FSS of 3950 dyn/cm2 (395 Pa) using a 30 G needle and a syringe pump. DU145 cells had no change in cell viability, PC3 cells had some cell death and LNCaP cells exhibited significant cell death. These cell death responses correlated with increased cell membrane damage, less efficient membrane repair and increased stiffness. Additionally, FSS treatment prevented the LNCaP FSS-sensitive cell line from forming a growing tumor in vivo. This suggests that these properties play a role in FSS resistance and could represent potential targets for disrupting blood-borne metastasis.

Antitumor Activity of Monasnicotinic Acid Isolated from the Fungus Aspergillus cavernicola

It has been found that monasnicotinic acid (MNA) isolated from the fungus Aspergillus cavernicola VKM F-906 reduces the proliferation and migration of human prostate cancer cells LnCaP and inhibits the AKT–mTORC1 and FAK–Src signaling pathways. However, MNA also increases the level of HSP60 and the phosphorylation of c-Jun, factors that stimulate the vital activity of cancer cells. MNA-induced cellular responses appear only after 48 h, which indicates the involvement of either the products of MNA metabolism or tautomers arising during the MNA protonation due to the growth of cell cultures and utilization of the compound by human cells. These results suggest that MNA is a promising compound for the production of various derivatives that would inhibit oncogenic signaling pathways without stimulating the factors providing the survival of tumor cells.

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7

Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.

Inhibition of lipogenesis and induction of apoptosis by valproic acid in prostate cancer cells via the C/EBPα/SREBP-1 pathway.

Lipid metabolism reprogramming is now accepted as a new hallmark of cancer. Hence, targeting the lipogenesis pathway may be a potential avenue for cancer treatment. Valproic acid (VPA) emerges as a promising drug for cancer therapy; however, the underlying mechanisms are not yet fully understood. In this study, we aimed to investigate the effects and mechanisms of VPA on cell viability, lipogenesis, and apoptosis in human prostate cancer PC-3 and LNCaP cells. The results showed that VPA significantly reduced lipid accumulation and induced apoptosis of PC-3 and LNCaP cells. Moreover, the expression of CCAAT/enhancer-binding protein α (C/EBPα), as well as sterol regulatory element-binding protein 1 (SREBP-1) and its downstream effectors, including fatty acid synthase (FASN), acetyl CoA carboxylase 1 (ACC1), and anti-apoptotic B-cell lymphoma 2 (Bcl-2), was markedly decreased in PC-3 and LNCaP cells after VPA administration. Mechanistically, the overexpression of C/EBPα rescued the levels of SREBP-1, FASN, ACC1, and Bcl-2, enhanced lipid accumulation, and attenuated apoptosis of VPA-treated PC-3 cells. Conversely, knockdown of C/EBPα by siRNA further decreased lipid accumulation, enhanced apoptosis, and reduced the levels of SREBP-1, FASN, ACC1, and Bcl-2. In addition, SREBP-1a and 1c enhanced the expressions of FASN and ACC1, but only SREBP-1a had a significant effect on Bcl-2 expression in VPA-treated PC-3 cells. Based on the results, we concluded that VPA significantly inhibits cell viability via decreasing lipogenesis and inducing apoptosis via the C/EBPα/SREBP-1 pathway in prostate cancer cells. Therefore, VPA that targets lipid metabolism and apoptosis is a promising candidate for PCa chemotherapy.

Abstract 6550: WD105, a novel decursin derivative, showed greater growth inhibition and androgen receptor suppression in LNCaP human prostate cancer cells

Abstract Androgen receptor (AR) is a primary target for treatment of prostate cancer (PCa). AR signaling plays a critical function in all stages of PCa. Our previous research demonstrated that decursin and its isomer decursinol angelate, a pair of natural compounds from Angelica gigas Nakai, inhibit AR signaling and suppressed the growth of prostate cancer cells in vitro and in vivo. In this study, we have designed a series of pro-drug conjugates of decursin, based on acid-esterified modification, in order to achieve higher water solubility, stronger cytocidal effect and better AR suppression activity. Twelve novel esterified derivatives of decursin were synthesized from decursinol and characterized by 1H-, 13C-NMR and HR-MS techniques. Among them, WD105 displayed the best solubility. Initial structure-activity relationship (SAR) studies were carried out with a cell growth (CCK8) assay and identifıed WD105 and WD106 as lead compounds with more potency at suppressing LNCaP cell growth than decursin judged by IC50 value. In addition, both WD105 and WD106 induced caspase-mediated cell apoptosis (48h), evidenced by cleavage of PARP, in a concentration-dependent manner. Furthermore, WD105 decreased the transcriptional activity of AR and androgen-induced expression of PSA better than WD106 and decursin when assessed by Western Blot and ELISA. Taken together, these data suggest that WD105 has greater in vitro activity than decursin as an AR targeting lead compound. Future studies will characterize the PK metrics and, if warranted, determine the in vivo antitumor effect of WD105 on the growth of LNCaP-AR xenografts. Citation Format: Wei Wu, Huaiwei Ding, Huiyong Zhang. WD105, a novel decursin derivative, showed greater growth inhibition and androgen receptor suppression in LNCaP human prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6550.

Abstract 5344: Treatment efficacy of 212Pb-RM2 targeted alpha therapy in human prostate cancer cell lines: An in vitro investigation

Abstract Introduction: Receptor targeted alpha therapy (TAT) has garnered attention with isotopes such as Pb-212, Bi-213, and Ac-225 due to the receptor specific and potentially lethal damage caused by the in vivo alpha decay of these isotopes when linked to small peptides. We are investigating Pb-212-RM2, a BB2 receptor (BB2r) antagonist as a potential TAT agent for use in BB2r positive prostate cancer. This in vitro study evaluates Pb-212-RM2 at 3 dose levels in a panel of human prostate cancer cell lines assessing cell viability (MTT), DNA damage (53BP1), and clonogenic potential. Methods: Pb-212-RM2 was evaluated in the PC3, LNCAP, C42, C42B, DU-145, VCAP, and 22Rv1 human prostate cancer cell lines. Cells were plated the day before treatment. Pb-212-RM2 was prepared using an automated radiosynthesizer and QC was performed by HPLC. Cells were exposed to 3, 6, 13, or 25uCi/mL of Pb-212 RM2. Cell viability was evaluated at 24, 48, 72, 96, 120, and 144hrs after treatment. DNA damage was evaluated at 24hrs post treatment using the 53BP1 marker. Media in clonogenic plates was refreshed every 5-7 days until colonies of ≥50 cells were present. All assays were conducted in triplicate on different days. Results: LNCAP, C42, 22Rv1, and C42B cell lines were ≤50% viable at all dose levels by 72hrs. PC3 and DU-145 did not show a decrease to 50% viability until 96-144hrs post administration. At 144hrs post treatment, only the 25uCi/mL treated VCAP cells showed less than 50% viability (43.9%±4.1%). Observed DNA damage was highest in the PC3 and LNCAP cell lines with a range of 5.4±0.7 to 26.6±1.7 and 2.4±0.5 to 19.7±1.2 avg 53BP1 foci/cell at 3 and 25uCi/mL, respectively. Clonogenic potential was lowest in PC3 and LNCAP lines with <20% surviving fraction (SF) at 3uCi/mL and decreasing to <5% SF at 6uCi/mL. DU-145, C42B, 22Rv1, and C42 showed a SF of 71.4±11.8%, 57.1±8.8%, 55.5±2.8%, and 41.5±6.5% at 3uCi/mL. Conclusions: Targeted alpha therapy (TAT), in the form of 212Pb-RM2, was evaluated in the PC3, LNCAP, C42, C42B, DU-145, VCAP, and 22Rv1 human prostate cancer cell lines representing the spectrum of human prostate cancer in relation to androgen dependence and sensitivity. At dose levels of 3, 6, 13, or 25uCi/mL, treatment response does not appear to be related to androgen dependence or sensitivity. Cell viability decreased with increasing time and dose level in all cell lines treated with 212Pb-RM2. The VCAP cell line demonstrated the most resistance to Pb-212-TAT. DNA damage, using the 53BP1 marker, was highest in the PC3 and LNCAP cell lines. As expected, this sensitivity was also demonstrated in clonogenic response with the PC3 and LNCAP cell lines exhibiting the lowest surviving fraction. These studies provide the foundation for further exploration of the therapeutic efficacy of 212Pb-RM2 for the treatment of BB2r positive prostate cancer. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Treatment efficacy of 212Pb-RM2 targeted alpha therapy in human prostate cancer cell lines: An in vitro investigation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5344.

Abstract 6219: Combined inhibition of androgen signaling and apoptosis pathways in hormone sensitive prostate cancer

Abstract In the United States, where prostate cancer (PC) screening is common, over 90% of patients present initially with localized or locally advanced disease. Over the past decade, research has confirmed that PC tumor cells' response to the presence of androgen (testosterone) is a principal driver of PC. Men with PC who recur after surgery or radiation frequently undergo androgen deprivation therapy (ADT), wherein androgen exposure is chemically diminished. However, it is frequently followed by resistance, androgen-independent growth, and eventually the development of metastatic castration-resistant prostate cancer (mCRPC), the most lethal form of the disease. For those cancers that become mCRPC, many respond to other forms of ADT or chemotherapy. Therefore, it is vital to investigate additional therapeutic strategy to delay or prevent the transition of hormone sensitive cancer to mCRPC. In response to stress signals from anti-cancer therapies, malignant cells may express pro-apoptotic activator proteins, such as the expression of the Bcl2 family of proteins. The block in apoptosis (Bcl2 family) that keeps cancer cells alive in response to anti-cancer agents is therefore an attractive candidate for targeted therapies. Previous studies have documented that overexpression of Bcl2 family proteins are associated with therapeutic resistance of PC, disease recurrence and shortened survival in CRPC. However, association between androgen signaling pathways and Bcl2 family proteins is not clearly understood. In our current study we observed that treatment with androgen inhibits the expression of Bcl2 family proteins (Bcl2, Bcl-w, Bcl-xL) in hormone sensitive human PC LNCaP cell lines. Importantly, we also observed that treatment with the androgen receptor (AR) inhibitor enzalutamide restores their (Bcl2, Bcl-w, Bcl-xL) expression even when treated with a very low concentration. This data indicates that there is direct negative-regulation of the Bcl2 family of proteins by the AR-signaling pathway. We also observed that there is synergistic effect of enzalutamide and the Bcl2-inhibitor venetoclax on growth inhibition of LNCaP cells. Our current study develops a rationale for combining enzalutamide or other androgen signaling agent inhibitors with Bcl2 targeted therapies for hormone sensitive prostate cancer. We believe this combinatorial therapeutic approach will show great potential for future clinical trials of high-risk hormone sensitive PC patients and may block the ADT-induced shift of hormone sensitive PC to mCRPC. Citation Format: Goutam Chakraborty, Rahim Hirani, Lina E. Jehane, Ying Z. Mazzu, Yuki Yoshikawa, Sai Harisha Rajanala, Gwo-Shu M. Lee, Philip W. Kantoff. Combined inhibition of androgen signaling and apoptosis pathways in hormone sensitive prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6219.

Eupatilin Inhibits the Proliferation and Migration of Prostate Cancer Cells through Modulation of PTEN and NF-κB Signaling.

Despite advances in the treatment of prostate cancer, side effects and the risks of developing drug resistance require new therapeutic agents. Eupatilin is a secondary metabolite of Artemisia asiatica and has shown potential anti-tumor activity in some cancers, but its potential in prostate cancer treatment has not yet been evaluated. The aim of the study was to investigate the effectiveness of eupatilin on prostate cancer cell proliferation and migration. Human prostate cancer PC3 and LNCaP cells were exposed to eupatilin and its efficacy on cell survival was determined by the MTT test. Apoptosis and cell cycle phases were evaluated by an image-based cytometer. Cell migration and invasion were evaluated by wound healing and matrigel migration assays; the expression of mRNA and protein was assessed by RT-qPCR and Western blot, respectively. Eupatilin time- and dose-dependently reduced the viability of prostate cancer cells. Exposure of PC3 cells to 12.5μM-50μM eupatilin resulted in apoptosis by upregulating the expression of caspase 3, Bax and cytochrome c. Annexin V assessment also confirmed that eupatilin causes apoptosis. The treatment significantly upregulated the mRNA expression of p53, p21, and p27, causing cell cycle arrest in the G1 phase. Administration of eupatilin inhibited migration and invasion of the cells by downregulating the expression of Twist, Slug and MMP-2, -7. In addition, the agent increased protein expression of tumor suppressor PTEN, while transcription factor NF-κB expression was reduced. Eupatilin strongly prevents the proliferation of prostate cancer cells, and suppresses migration and invasion. Due to its therapeutic potential, the clinical use of eupatilin in prostate cancer should also be supported by in vivo studies.

Pyruvate-lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET.

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐13C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐13C]pyruvate to lactate conversion was followed by 13C MRS. Subsequently [18F]FDG uptake was investigated by static and dynamic PET measurements.Standard uptake values (SUVs) for [18F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k pl of 13C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [18F]FDG PET measurements and k pl values for the xenografts of both tumor types. The k pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [18F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐13C]pyruvate.

Abstract B089: Kaiso as a novel therapeutic target in castration-resistant prostate cancer

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10μM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCa, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. Citation Format: Huixian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjaminal Adu-Addai, William Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B089.

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Influence of human adipose stem cells on prostate cancer cell growth.

ABSTRACTIn a novel regenerative cell-based treatment developed by us for the patients with stress urinary incontinence, autologous adipose-derived stem cells (ASCs) are injected into the periurethral region and the external urethral sphincter. Since the candidates for this treatment included prostate cancer patients after radical prostatectomy, we investigated the effects of ASCs on prostate cancer cell proliferation in vitro and in vivo to confirm the feasibility of our therapeutic approach. The LNCaP (human prostate cancer cell line) cells and ASCs were co-cultured, and prostate-specific antigen (PSA) concentration in their culture medium supernatant was measured at 48 and 96 h. The PSA concentration significantly decreased in the coculture medium supernatant as compared to the culture medium with LNCaP cells alone. On the contrary, PSA concentrations in the culture medium of LNCaP cells were not affected by supplementation with ASC culture supernatant. After subcutaneous transplantation of LNCaP cells, with or without ASCs, in immunodeficient mice, tumor growth was compared. The growth of LNCaP xenograft tumor in immunodeficient mice was significantly suppressed by ASC addition. These results indicated that ASCs inhibit prostate cancer cell growth, without no proliferative effect on prostate cancer cells.

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer. Eupatorin, a flavonoid with a wide range of phytomedical activities, suppresses proliferation of and induces apoptosis of multiple cancer cell lines. However, low solubility, poor bioavailability, and rapid degradation limit its efficacy. The aim of our study was to evaluate whether the use of mPEG-b-poly (lactic-co-glycolic) acid (PLGA) coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines. Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology, surface charge, particle size, drug loading, encapsulation efficiency and in vitro drug-release profile. The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test. Apoptosis was then determined by Hoechest staining, cell cycle analysis, NO production, annexin/propidium iodide (PI) assay, and Western blotting. The results indicated that eupatorin was successfully entrapped in Fe3O4@mPEG-b-PLGA nanoparticles with an efficacy of (90.99 ± 2.1)%. The nanoparticle’s size was around (58.5 ± 4) nm with a negative surface charge [(−34.16 ± 1.3) mV]. In vitro release investigation showed a 30% initial burst release of eupatorin in 24 h, followed by sustained release over 200 h. The MTT assay indicated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 μM and 75 μM, respectively. Next, apoptosis was confirmed by nuclear condensation, enhancement of cell population in the sub-G1 phase and increased NO level. Annexin/PI analysis demonstrated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency. Finally, Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles. Encapsulation of eupatorin in Fe3O4@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin. Based on these results, this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose, making it a suitable candidate for pharmacological uses.