LNCaP Proliferation Research Articles

1,2,3-Triazole-totarol conjugates as potent PIP5K1α lipid kinase inhibitors

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a key role in the development of prostate cancer. In this work, seventeen derivatives of the natural diterpene totarol were prepared by copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of the correspondingO-propargylated totarol with aryl or alkyl azides and screened for their inhibitory activities toward hPIP5K1α. Five compounds, 3a, 3e, 3f, 3i, and 3r, strongly inhibited the enzyme activity with IC50 values of 1.44, 0.46, 1.02, 0.79, and 3.65 µM, respectively, with the most potent inhibitor 3e 13-[(1-(3-nitrophenyl)triazol-4yl)methoxy]-totara-8,11,13-triene). These compounds were evaluated on their antiproliferative effects in a panel of prostate cancer cell lines. Compound 3r inhibited the proliferation of LNCaP, PC3 and DU145 cells at 20 µM, strongly, but also has strong cytotoxic effects on all tested cells.

The KDM5 inhibitor PBIT reduces proliferation of castration-resistant prostate cancer cells via cell cycle arrest and the induction of senescence

The compound 2–4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT) is an inhibitor of the KDM5 family of lysine-specific histone demethylases that has been suggested as a lead compound for cancer therapy. The goal of this study was to explore the effects of PBIT within human prostate cancers. Micromolar concentrations of PBIT altered proliferation of castration-sensitive LNCaP and castration-resistant C4–2B, LNCaP-MDV3100 and PC-3 human prostate cancer cell lines. We then characterized the mechanism underlying the anti-proliferative effects of PBIT within the C4–2B and PC-3 cell lines. Data from Cell Death ELISAs suggest that PBIT does not induce apoptosis within C4–2B or PC-3 cells. However, PBIT did increase the amount of senescence associated beta-galactosidase. PBIT also altered cell cycle progression and increased protein levels of the cell cycle protein p21. PC-3 and C4–2B cells express varying amounts of KDM5A, KDM5B, and KDM5C, the therapeutic targets of PBIT. siRNA-mediated knockdown studies suggest that inhibition of multiple KDM5 isoforms contribute to the anti-proliferative effect of PBIT. Furthermore, combination treatments involving PBIT and the PPARγ agonist 15-deoxy-Δ-12, 14 -prostaglandin J2 (15d-PGJ₂) also reduced PC-3 cell proliferation. Together, these data strongly suggest that PBIT significantly reduces the proliferation of prostate cancers via a mechanism that involves cell cycle arrest and senescence.

MDM2 regulates the stability of AR, AR-V7, and TM4SF3 proteins in prostate cancer.

Androgen receptor (AR) and its constitutively active splice variant, AR Variant 7 (AR-V7), regulate genes essential for the development and progression of prostate cancer. Degradation of AR and AR-V7 by the ubiquitination proteasomal pathway is important for the regulation of both their protein stability. Our published results demonstrate that the interaction of TM4SF3 with either AR or AR-V7 leads to mutual stabilization due to a reduction in their ubiquitination and proteasomal degradation. These results led us to search for a common E3 ligase for AR, AR-V7, and TM4SF3. Depletion by siRNA of several E3 ligases identified MDM2 as the common E3 ligase. MDM2 inhibition by siRNA depletion or using a pharmacological inhibitor (MDM2i) of its E3 ligase activity led to elevated levels of endogenous AR, AR-V7, and TM4SF3 in prostate cancer cells. MDM2 knockdown in PC-3 cells, which do not express AR, also increased TM4SF3, demonstrating that MDM2 affects the TM4SF3 protein independent of AR. We further demonstrate that MDM2i treatment reduced the ubiquitination of AR and TM4SF3, suggesting that MDM2 can induce the ubiquitination of these proteins. Increased AR and AR-V7 protein levels induced by MDM2i treatment resulted in the expected increased expression of AR-regulated genes and enhanced proliferation and migration of both LNCaP and Enzalutamide-resistant CWR-22Rv1 prostate cancer cells. Thus, our study expands the known roles of MDM2 in prostate cancer to include its potential involvement in the important mutual stabilization that TM4SF3 exhibits when interacting with either AR or AR-V7.

Peroxisomal β-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer

BackgroundPeroxisomes are central metabolic organelles that have key roles in fatty acid homoeostasis. As prostate cancer (PCa) is particularly reliant on fatty acid metabolism, we explored the contribution of peroxisomal β-oxidation (perFAO) to PCa viability and therapy response.MethodsBioinformatic analysis was performed on clinical transcriptomic datasets to identify the perFAO enzyme, 2,4-dienoyl CoA reductase 2 (DECR2) as a target gene of interest. Impact of DECR2 and perFAO inhibition via thioridazine was examined in vitro, in vivo, and in clinical prostate tumours cultured ex vivo. Transcriptomic and lipidomic profiling was used to determine the functional consequences of DECR2 inhibition in PCa.ResultsDECR2 is upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa (CRPC). Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and therapy-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. DECR2 influences cell cycle progression and lipid metabolism to support tumour cell proliferation. Further, co-targeting of perFAO and standard-of-care androgen receptor inhibition enhanced suppression of PCa cell proliferation.ConclusionOur findings support a focus on perFAO, specifically DECR2, as a promising therapeutic target for CRPC and as a novel strategy to overcome lethal treatment resistance.

Chemical Composition of Essential Oil of Cymbopogon schoenanthus (L.) Spreng from Burkina Faso, and Effects against Prostate and Cervical Cancer Cell Lines.

The aim of this research was to evaluate the essential oil of Cymbopogon schoenanthus (L.) Spreng. (C. schoenanthus) from Burkina Faso in terms of cytotoxic activity against LNCaP cells, derived from prostate cancer, and HeLa cells, derived from cervical cancer. Antioxidant activities were evaluated in vitro. Essential oil (EO) was extracted by hydrodistillation and analyzed by GC/FID and GC/MS. Thirty-seven compounds were identified, the major compounds being piperitone (49.9%), δ-2-carene (24.02%), elemol (5.79%) and limonene (4.31%). EO exhibited a poor antioxidant activity, as shown by the inhibition of DPPH radicals (IC50 = 1730 ± 80 µg/mL) and ABTS+. (IC50 = 2890 ± 26.9 µg/mL). Conversely, EO decreased the proliferation of LNCaP and HeLa cells with respective IC50 values of 135.53 ± 5.27 µg/mL and 146.17 ± 11 µg/mL. EO also prevented LNCaP cell migration and led to the arrest of their cell cycle in the G2/M phase. Altogether, this work points out for the first time that EO of C. schoenanthus from Burkina Faso could be an effective natural anticancer agent.

AMPK targets a proto-oncogene TPD52 (isoform 3) expression and its interaction with LKB1 suppress AMPK-GSK3β signaling axis in prostate cancer.

Tumor protein D52 (TPD52) is a proto-oncogene overexpressed in prostate cancer (PCa) due to gene amplification and it is involved in the cancer progression of many cancers including PCa. However, the molecular mechanisms underlying the role of TPD52 in cancer progression are still under investigation. In this study, we report that the activation of AMP-activated protein kinase (AMPK) by AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) inhibited the LNCaP and VCaP cells growth by silencing TPD52 expression. Activation of AMPK inhibited the proliferation and migration of LNCaP and VCaP cells. Interestingly, AICAR treatment to LNCaP and VCaP cells led to the downregulation of TPD52 via activation of GSK3β by a decrease of inactive phosphorylation at Ser9. Moreover, in AICAR treated LNCaP cells, inhibition of GSK3β by LiCl attenuated downregulation of TPD52 indicating that AICAR acts via GSK3β. Furthermore, we found that TPD52 interacts with serine/threonine kinase 11 or Liver kinase B1 (LKB1) a known tumor suppressor and an upstream kinase for AMPK. The molecular modeling and MD simulations indicates that the interaction between TPD52 and LKB1 leads to inhibition of the kinase activity of LKB1 as its auto-phosphorylation sites were masked in the complex. Consequently, TPD52-LKB1 interaction may lead to inactivation of AMPK. Moreover, overexpression of TPD52 is found to be responsible for the reduction of pLKB1 (Ser428) and pAMPK (Thr172). Therefore, TPD52 may be playing its oncogenic role via suppressing the AMPK activation. Altogether, our results revealed a new mechanism of PCa progression in which TPD52 overexpression inhibits AMPK activation by interacting with LKB1. These results support that the use of AMPK activators and/or small molecules that could disrupt the TPD52-LKB1 interaction might be useful to suppress PCa cell growth. TPD52 interacts LKB1 and interfere with activation of AMPK in PCa cells.

Plasmacytoma variant translocation 1 inhibits miR-515-5p function and modulates high mobility group B3 to promote the growth of prostate cancer cells.

This study is performed to analyze the role of long non-coding RNA plasmacytoma variant translocation 1 in prostate cancer. Plasmacytoma variant translocation 1, miR-515-5p, and high mobility group B3 mRNA expressions were examined using quantitative real-time polymerase chain reaction and immunohistochemistry. After gain-of-function and loss-of-function models were established, the changes in cell proliferation, migration, and invasion were evaluated using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and Transwell experiments. Validation of the targeting relationships between plasmacytoma variant translocation 1 and miR-515-5p, and between miR-515-5p and high mobility group B3 was conducted using bioinformatics prediction, a dual-luciferase reporter assay, and an RNA immunoprecipitation experiment. Moreover, the effects of plasmacytoma variant translocation 1 and miR-515-5p on high mobility group B3 protein expression were examined using Western blot. Plasmacytoma variant translocation 1 expression and high mobility group B3 expression were up-regulated in prostate cancer tissues and cell lines while miR-515-5p expression was down-regulated. Plasmacytoma variant translocation 1 knockdown restrained the proliferation, migration, and invasion of LNCaP and DU145 cells in vitro, and the transfection with miR-515-5p inhibitors reversed these effects. Mechanistically, plasmacytoma variant translocation 1 could repress the function of miR-515; high mobility group B3 was proved to be a target gene of miR-515-5p, and its expression could be indirectly positively modulated by plasmacytoma variant translocation 1. Plasmacytoma variant translocation 1 accelerates prostate cancer progression by repressing miR-515-5p's function to upregulate high mobility group B3 expression.

Target Protein for Xklp2 Functions as Coactivator of Androgen Receptor and Promotes the Proliferation of Prostate Carcinoma Cells.

The activation of the androgen receptor (AR) pathway is crucial in the progression of human prostate cancer. Results of the present study indicated that the target protein xenopus kinesin-like protein (TPX2) enhanced the transcription activation of AR and promoted the proliferation of LNCaP (ligand-dependent prostate carcinoma) cells. The protein-protein interaction between AR and TPX2 was investigated using coimmunoprecipitation assays. Results of the present study further demonstrated that TPX2 enhanced the transcription factor activation of AR and enhanced the expression levels of the downstream gene prostate-specific antigen (PSA). TPX2 did this by promoting the accumulation of AR in the nucleus and also promoting the recruitment of AR to the androgen response element, located in the promoter region of the PSA gene. Overexpression of TPX2 enhanced both the in vitro and in vivo proliferation of LNCaP cells. By revealing a novel role of TPX2 in the AR signaling pathway, the present study indicated that TPX2 may be an activator of AR and thus exhibits potential as a novel target for prostate carcinoma treatment.

Comprehensive Characterization of Ageing-Relevant Subtypes Associated With Different Tumorigenesis and Tumor Microenvironment in Prostate Cancer.

Objective: Accumulated evidence demonstrates that ageing is a robust risk factor of prostate cancer prognosis. Herein, we conducted a systematic analysis about ageing-relevant molecules and relevant tumor microenvironment features in prostate cancer. Methods: Transcriptome data, clinical information, and mutational data of prostate cancer patients were retrospectively collected from the Cancer Genome Atlas cohort. In accordance with the expression of specific ageing-relevant genes, prostate cancer patients were clustered with consensus clustering analyses. WGCNA was adopted for determination of subtype-associated co-expression modules and genes. Thereafter, characteristic genes were further screened with random forest algorithm and a prognostic model was conducted with multivariate cox regression analyses. Tumor microenvironment-infiltrating immune cells were estimated with ssGSEA and ESTIMATE. Activities of the cancer immunity cycle and expressions of HLA and immune checkpoint molecules were then quantified across prostate cancer cases. A serious experiment was conducted to investigate the roles of EIF2S2 in prostate tumorigenesis. Results: This study characterized three ageing-relevant subtypes (C1, C2, and C3) with diverse clinical prognosis. Subtype C1 presented the features of low mutational frequency and immune activation; C2 was characterized by stromal and immune activation; and C3 showed immune suppression. An ageing-derived gene signature was conducted, which independently and robustly predicted patients’ prognosis. Additionally, this signature was in relation to immune inactivation. Among the genes in the signature, EIF2S2 triggered proliferation, invasion, and migration of LNCaP and PC-3 cells. Conclusion: Collectively, ageing-relevant molecular subtypes and gene signature might be of great significance to determine clinical outcomes and tumor microenvironment features and immunotherapeutic responses in prostate cancer.

Conjugates of Pyropheophorbide a with 17-Substituted Steroidal Androgens. Synthesis, Molecular Modeling, Interaction with Some Cancer Cells

Five new bifunctional conjugates of pyropheophorbide a with 17-substituted testosterone, dihydrotestosterone and epitestosterone differing in the length of linker (1 � 5) and two new complex conjugates 6 and 7 (containing three functional units: pyropheophorbide a, 17?-substituted testosterone, and lipophylic hexadecyl chain, connected with L-lysine joining block) were synthesized. Mutual influence of steroidal and macrocyclic fragments in conjugates (1 � 7) was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates 1 � 5 with prostate carcinoma cells revealed that their uptake and internalization were dependent on the structure of conjugates, particularly on the stereochemical configuration of 17-hydroxyl group in steroidal moiety, and the length of linker connecting pyropheophorbide a with steroid fragments. Conjugates 1 � 5 significantly decreased the growth and proliferation of LNCaP and PC-3 cells. The highest anti-proliferative activity demonstrated by epitestosterone derivative 3, comprising short linker. Irradiation of labeled cells with light (? = 660 nm) was significantly increased cytotoxicity. Trifunctional conjugates 6 and 7 easily formed mixed micells with phosphatidyl choline and pluronic F68; these mixed micelles efficiently internalized by human hepatocarcinoma Hep G2 cells. The binding of conjugates 6 and 7 in the form of mixed micelles to Hep G2 cells depended on the conjugate structure, rather than on the method of solubilization.

Co-administration of tyrosine kinase inhibitors with rottlerin in metastatic prostate cancer cells.

After prostatectomy due to prostate carcinoma, patients often develop metastases. Although prostate cancer is susceptible to hormonal manipulation, many patients become castration-resistant. Therefore, new therapies are the focus of investigations. We analyzed the effect of the tyrosine kinase inhibitors (TKIs), sorafenib and sunitinib, in combination with rottlerin, a PKCδ inhibitor, on metastatic mechanisms in prostate carcinoma cells. LNCaP and PC-3 prostate carcinoma cells were treated with sorafenib or sunitinib alone at various concentrations (1-20 µM) or in combination with rottlerin (10 µM) for 24 h. Then, cell toxicity (MTT test) and cell proliferation (BrdU incorporation assay) were quantified. The study demonstrated a dose-dependent inhibitory effect of sorafenib and sunitinib on PC-3 and LNCaP cell activity and proliferation. Both agents showed significantly stronger cytotoxic effects in LNCaP cells. At the highest concentrations, sorafenib and sunitinib inhibited the viability of LNCaP cells up to 2 % and 31 %, respectively, and the viability of PC-3 cell line up to 20 % and 43 %, respectively. The proliferation of both cell lines was significantly stronger inhibited by sorafenib than by sunitinib. In LNCaP cells, sorafenib and sunitinib at the highest concentrations inhibited cell proliferation up to 46 % and 49 %, respectively, and the proliferation of PC-3 line up to 40 % and 47 %, respectively. Rottlerin reduced the viability and proliferation of PC3 cells to 81 % and 42 %, whereas the viability and proliferation of LNCaP cells were reduced to 25 % and 57 %, respectively. Sorafenib and sunitinib at low concentrations partly neutralized the inhibitory effect of rottlerin on cell viability and proliferation. On the other hand, in PC-3 cells, rottlerin reduced the inhibitory effects of sorafenib and sunitinib at the highest concentrations on cell viability from 20 % to 30 % and from 43 % to 61 %, respectively. An additive effect on cell activity was observed after treating LNCaP cells with both sunitinib at high concentrations and rottlerin. This combination increased the cytotoxic effect from 31 % to 13 % at the highest sunitinib concentration. Our results showed that monotherapy with sorafenib was the most efficient in both PCa cell lines. A marginally additive effect of rottlerin was only observed in LNCaP cells treated with sunitinib at a high concentration. Sorafenib and sunitinib reduced cell migration in PC-3 cells to 10 % and 32 % of untreated cells, respectively. Co-treatment with sorafenib/sunitinib and rottlerin did not result in a significantly stronger anti-migratory effect than the treatment with each TKI alone. Given the strong cytotoxic effect of TKIs, especially sorafenib, on LNCaP cells, the results of the migration assay in this line were severely biased and not considered in the analysis. Unlike in other malignancies, combination therapy with TKI and rottlerin seems not beneficial in prostate cancer. More promising seems to be monotherapy with rottlerin, but further studies are needed to confirm this observation.

Identification of evodiamine as a suppressor of prostate cancer progression by reducing AR transcriptional activity via targeting Src.

Evodiamine (EVO) is a bioactive alkaloid that exerts antitumor activity in various cancers, including prostate cancer (PCa). In this paper, we further investigated the molecular mechanisms underlying the anti-PCa effect of evodiamine. In the present study, cell proliferation, colony formation, migration, and invasion were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and transwell assays, respectively. Animal studies were used to evaluate the effect of evodiamine on the tumorigenicity of LNCaP cells in vivo. The expression levels of steroid receptor coactivator (Src), androgene receptor (AR), and prostate-specific antigen (PSA) were detected by western blot, quantitative real-time PCR (qRT-PCR) or ELISA assay. Association between Src and AR was examined by Co-Immunoprecipitation (CoIP). The impact of evodiamine on AR-mediated transcriptional activity was confirmed by dual-luciferase reporter assay. The results showed that evodiamine reduced LNCaP and 22Rv1 cell proliferation, colony formation, migration, and invasion induced by dihydrotestosterone (DHT) in vitro, as well as diminished tumor growth in vivo. Mechanistically, evodiamine directly targeted Src and reduced DHT-induced Src activation. Moreover, the restoration of Src activation abolished evodiamine-mediated suppression of proliferation, migration, and invasion of DHT-treated LNCaP and 22Rv1 cells. Furthermore, evodiamine inhibited DHT-induced AR transcriptional activity through targeting Src. As a conclusion, our findings demonstrate the antitumor property of evodiamine in PCa by blocking AR transcriptional activity through targeting Src and provide a rationale for developing evodiamine as a promising antitumor agent against PCa.

Effects of <italic>Lycium ruthenicum</italic> Murray anthocyanin Pt3G on the proliferation and apoptosis of prostate cancer LNCaP andPC-3 cells

<p indent="0mm"> <italic>Lycium ruthenicum</italic> Murray ( <italic>L</italic>. <italic>ruthenicum</italic>), known as “black Goji”, has been widely distributed in northwest of China. The fruit of <italic>L</italic>. <italic>ruthenicum</italic> has been used as a traditional Chinese medicine for a long time. Many modern studies indicated that the fruit of <italic>L</italic>. <italic>ruthenicum</italic> has a variety of biological activities such as antioxidant, anti-inflammatory, anti-cancer, neuroprotective, and potential prevention of cardiovascular disease. The fruit of <italic>L</italic>. <italic>ruthenicum</italic>is black in color and rich in anthocyanins. It has been reported that petunidin 3- <italic>O</italic>-[6- <italic>O</italic>-(4- <italic>O</italic>-( <italic>trans-p</italic>-coumaroyl)-α-l-rhamnopyranosyl)-β-D-glucopyranoside]-5- <italic>O</italic>-[β-D-glucopyranoside] (Pt3G) is the main constituent of anthocyanins in fully ripe fruits of <italic>L</italic>. <italic>ruthenicum</italic>. In our previous study, we found that the Pt3G from the fruit of <italic>L</italic>. <italic>ruthenicum</italic> could inhibit the proliferation of prostate cancer DU145 cells through ROS/PTEN/PI3K/Akt pathway. To further explore the mechanisms underlying the inhibitory effect of Pt3G on proliferation of prostate cancer cells, the prostate cancer LNCaP and PC-3 cells were used in this study. MTT assay was used to determine the proliferation rate of cells treated with different concentrations of Pt3G solution. To verify whether the inhibitory effect of Pt3G on the proliferation of prostate cancer cell is due to apoptosis, flow cytometric analysis and TUNEL assay were used to detect cell apoptosis, and the Western Blot analysis was used to detect the expression of apoptosis related proteins. The mitochondrial membrane potential in LNCaP and PC-3 cells was analyzed with JC-1 fluorescent dye. Our results showed that Pt3G inhibited the proliferation of LNCaP and PC-3 cells in a concentration dependent manner. The half inhibitory concentrations (IC50) of Pt3G against LNCaP and PC-3 cells were <sc>601.97</sc> and <sc>445.79 μg/mL</sc> at <sc>24 h</sc> after treatment, respectively. Flow cytometry and TUNEL results showed that the mean percentages of apoptotic cells were significantly increased in the Pt3G treated LNCaP and PC-3 cells as compared to the control group. Furthermore, Pt3G could lead to excess reactive oxygen species (ROS) generation, decreased the mitochondrial membrane permeability and was able to induce prostate cancer cells arrest in S phase after <sc>24 h</sc> treatment. Western Blot analysis showed that the expression levels of tumor suppressor PTEN in prostate cancer cells were significantly increased after Pt3G treatment. Moreover, Pt3G treatment significantly up-regulated the expression levels antiapoptotic factors PI3K, <italic>p</italic>-PI3K, Akt and <italic>p</italic>-Akt and down-regulated the expression levels proapoptotic factors Bax, cytochrome c, caspase 3 and cleaved caspase 3 in prostate cancer cells treated with Pt3G for <sc>24 h.</sc> Taken together, these findings suggest that the Pt3G could inhibit cell proliferation and induce apoptosis of prostate cancer cells line, and its anti-tumor mechanisms may be through ROS/PTEN/PI3K/Akt signal pathway. Although the anthocyanin Pt3G from the fruit of <italic>L</italic>. <italic>ruthenicum</italic> has a chemopreventive activity against prostate cancer cells lines, its anti-tumor mechanisms needs to be further clarified in the future.

Identification of a Hypoxia-Related Gene Signature for Predicting Systemic Metastasis in Prostate Cancer.

Background: Systemic metastasis is the main cause of death in patients with prostate cancer. It is necessary to establish a more accurate model to distinguish and predict patients with a high risk of metastasis to optimize individualized treatment.Methods: In this study, it was determined that hypoxia could affect the metastasis-free survival of patients with prostate cancer, and a hypoxia-related gene signature composed of seven genes for predicting metastasis was established and verified in different cohorts. The study further evaluated the effects of ALDOB expression on the proliferation and invasion of the LNCaP and DU145 cell lines under hypoxia and finally constructed a nomogram containing specific clinical characteristics of prostate cancer combined with the hypoxia gene signature to quantify the metastasis risk of individual patients.Results: The hypoxia-related gene signature was identified as an independent risk factor for metastasis-free survival in patients with prostate cancer. The expression of ALDOB increased under hypoxia and promoted the proliferation and invasion of LNCaP and DU145 cells. In addition, patients with a high risk score showed therapeutic resistance and immunosuppression. Compared with other parameters, the nomogram had the strongest predictive power and net clinical benefit.Conclusion: The study established a hypoxia-related gene signature and a nomogram to distinguish and predict patients with a high risk of prostate cancer metastasis, which may help to optimize individualized treatment and explore possible therapeutic targets.

Induction of apoptosis in human hormone‐refractory prostate cancer cell lines by using resveratrol in combination with AT‐101

The aim of this study was to determine the appropriate doses of AT-101 and resveratrol combination in the in vitro hormone-refractory prostate cancer (PC) cell lines, in order to evaluate the cytotoxic and genotoxic effects of this combination on the proliferation of cancer cells, namely PC-3, DU-145 and LNCAP. Cytotoxicity in PC cell lines was analysed by using the XTT Cell Proliferation Assay. DNA damage was performed with the cell death assay. Apoptotic protein levels were performed by Roche Human Apoptosis Array. IC50 values were determined by XTT analysis. The strongest combined doses (100µM resveratrol+5µM AT-101) were found to have the strongest synergistic apoptotic and cytotoxic effects on DU-145 cells at 72hr. While the combined use of resveratrol and AT-101 increased the expression of markers in apoptotic cell pathways on cells, a decrease in the expression of anti-apoptotic markers was detected (p˂0.05). Combined applications of these compounds showed an important synergism in the hormone-refractory PC cell lines, and it was determined that after the post-translational modification, they were significantly effective on the apoptotic pathway. These results have revealed that the combination of resveratrol and AT-101 holds great expectation as a new chemotherapeutic application in the treatment of human prostate cancer.

MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION Xiaobo Wu, Christina Sharkey, Zongwei Wang, and Aria Olumi Xiaobo WuXiaobo Wu More articles by this author , Christina SharkeyChristina Sharkey More articles by this author , Zongwei WangZongwei Wang More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies revealed that obesity is significantly associated with a higher incidence of prostate cancer (PCa). Meanwhile, obesity-associated inflammation plays an important role in tumorigenesis. Here we wished to identify the downstream signaling pathways of obesity-associated proinflammatory cytokines IL-6 and TNF-α’s effects on prostate cancer cell proliferation and migration. METHODS: Adipocytes and THP-1 macrophages were differentiated and treated with myristic acid (MA) to induce inflammatory cytokines, and conditioned media (CM) were collected to treat prostate epithelial cancer, androgen-dependent LNCaP and androgen-independent PC-3 cell lines. Cells were also treated with interleukin-6 (IL-6) 10ng/ml, tumor necrosis factor-alpha (TNF-α) 10ng/ml, oncometabolite R-2-hydroxyglutarate 50µM (R-2-HG) and Dutasteride 10µM (SRD5A1 inhibitor). Prostate cancer cell migration and proliferation were assessed by scratch assay (0h, 24h, 48h) and MTT assay (0h, 24h, 48h, 72h) and analyzed by 2-way ANOVA. Enzymes regulating steroid hormone metabolism SRD5A1, AKR1C1, AKR1C3, HSD3B2, CYP17A1 and UGT2B15 were determined by immunocytochemistry and qPCR. The protein level of SRD5A1 was measured by western blot. RESULTS: The proliferation of LNCaP and PC-3 cells was significantly stimulated by adding of CM for 24h, 48h, and 72h. The migration of PC-3 cells was also promoted by CM. Addition of testosterone did not change the effect of CM. Administration of IL-6, TNF-α and R-2-HG significantly increased the proliferation and migration of LNCaP and PC-3 cells. IL-6 also increased the expression of SRD5A1.. The expression of AKR1C1 and HSD3B2 was downregulated, and UGT2B15 was upregulated by TNF-α. SRD5A1 was upregulated either by R-2-HG alone or combining with IL-6 and TNF-α, but downregulated by Dutasteride alone. CONCLUSIONS: Our study suggests that obesity-associated proinflammatory cytokines, IL-6 and TNF-α, affect prostate cancer cells’ proliferation and migration in an androgen-independent manner. Synergistic targeting oncometabolite R-2-HG and proinflammatory cytokines may improve the management of androgen-independent prostate cancer. Source of Funding: NIH/NIDDK © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e610-e611 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xiaobo Wu More articles by this author Christina Sharkey More articles by this author Zongwei Wang More articles by this author Aria Olumi More articles by this author Expand All Advertisement Loading ...

Specific Targeting of Recombinant Human Pancreatic Ribonuclease 1 using Gonadotropin-Releasing Hormone Targeting Peptide toward Gonadotropin-Releasing Hormone Receptor-Positive Cancer Cells.

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells.Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal–Wallis tests with Dunn’s post hoc tests were performed to determine the significant differences between the study groups.Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction.Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R.

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.

Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter.

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.

CASC11 promotes aggressiveness of prostate cancer cells through miR-145/IGF1R axis.

Prostate cancer (PCa) is the most common malignancy diagnosed among men after lung cancerin developed countries. Investigation of the underlying molecular mechanisms of PCa is urgently needed in order to develop better therapeutic strategies and to reveal more effective therapeutic targets. In this study, we aimed at exploring the potential functions of CASC11 in association with miR-145 and IGF1R during the malignant progression of PCa cells. We initially investigated the oncogenic potential of noncoding members of CASC gene family and analyzed the effects of CASC11 overexpression on proliferation, migration, and colony formation ability of DU145, LNCaP, and PC3 PCa cells. We, then, exprlored the association of CASC11, miR-145, and IGF1R expression and their impacts on PI3K/AKT/mTOR signaling pathway in in vitro models. In silico analysis revealed that of the CASC family only CASC11 showed consistent results considering its differential expression as well as its association with the overall survival of patients. We demonstrated that ectopic overexpression of CASC11 significantly increased the proliferation, colony formation, and migration capacity in all three cell lines. CASC11 overexpression caused suppression of miR-145 and overexpression of IGF1R, leading to activation of PI3K/AKT/mTOR signaling pathway. In summary, we found that CASC11 is upregulated in PCa cells and clinical tumor samples in comparison to corresponding controls and revealed that ectopic CASC11 overexpression promotes cellular phenotypes associated with PCa progression through CASC11/miR-145/IGF1R axis.