Prostate Cancer Epithelial Cells Research Articles

THE CYTOTOXIC EFFECT OF BENZOIC ACID ON TEN DIFFERENT CANCER CELL LINES

Cancer, having numerous types, is among the most dangerous and complex chronic diseases in the world affecting the wellbeing of humans, society and economy. The exploration and reassessment of effective chemicals, compounds, and natural products as potential agents for alleviating the adverse effects of cancer and its related symptoms continue on a global scale. This process involves an initial evaluation of the cytotoxic activities of potential drug candidates or treatment regimens on diverse cancer cell types in an ex vivo context. Benzoic acid (BA), an aromatic carboxylic acid that is widely available and used in the food industry, is one of the phenolic acids that may bear considerable anti-cancer potential. It is useful to find out the comparable effect of BA on various cancer types. Therefore, in this study, we tested the cytotoxicity of BA using MTT assay, on a number of ten different cancer cell lines and one normal cell type, namely prostate cancer (PC3), cervical cancer (HeLA), liver cancer (HUH7), colon cancer (CaCO2, HT29, SW48), bone cancer (MG63 and A673), pharyngeal cancer (2A3), lung cancer (CRM612) and kidney epithelial control cell line (Phoenix), respectively. IC50 (µg/ml) values after 48 and 72-hour exposure to BA were found to differ between 85.54±3.17 to 670.6±43.26, while the IC values for the control cell line Phoenix were 410.54±32.29 and 231.16±25.25, respectively. Taking into account of statistical evaluation of the IC50 values for BA on 11 cell types, we suggest that the molecular and omics approaches can be implemented in more details in order to find cellular and biochemical targets of BA as well as elucidating molecular mode of action, especially starting with the cancer cell lines of MG63, CRM612 and A673, in which the IC50 levels are relatively the lowest compared to those of the control cell line.

LMNB1 targets FOXD1 to promote progression of prostate cancer.

Forkhead box D1 (FOXD1) expression is upregulated in various types of human cancer. To the best of our knowledge, the roles of FOXD1 in prostate cancer (PC) remain largely unknown. The Cancer Genome Atlas dataset was used for the bioinformatics analysis of FOXD1 in PC. FOXD1 expression levels in normal immortalized human prostate epithelial cells (RWPE-1) and prostate cancer cells were detected by reverse transcription-quantitative PCR. PC cell viability was detected using Cell Counting Kit-8 assay. Transwell assays were performed to assess the migration and invasion of PC cells. Luciferase reporter gene assay was used to validate the association between FOXD1 and lamin (LMN)B1. LMNB1 is an important part of the cytoskeleton, which serves an important role in the process of tumor occurrence and development, regulating apoptosis and DNA repair. FOXD1 expression was upregulated in PC tissues, with its high expression being associated with clinical stage and survival in PC. Knockdown of FOXD1 inhibited viability, migration and invasion of PC cells. FOXD1 positively regulated LMNB1 expression. The effect of FOXD1 knockdown on PC cells was reversed by LMNB1 overexpression. In conclusion, FOXD1, positively regulated by LMNB1, served as an oncogene in PC and may be a potential biomarker and treatment target for PC.

A comprehensive study on microstructure, in-vitro biodegradability, bacterial sensitivity, and cellular interactions of novel ternary Zn-Cu-xAg alloys for urological applications

Novel ternary Zn-Cu-xAg alloys are designed and fabricated by the casting method under a vacuum atmosphere and then homogenized at 350 °C for 15 h. The effect of Ag concentration (x: 0, 1, 2, 3, and 4 wt%) on microstructure, hardness, in vitro corrosion, biodegradability, and bacterial sensitivity was studied systematically. The structural analysis of the alloys was investigated by using a scanning electron microscope (SEM-EDS), and an optical microscope (OM). Besides, phase characterization of the samples was conducted using X-ray diffraction (XRD). The degradation behaviors of the alloys were determined under in vitro conditions, electrochemical polarization, and immersion tests (up to 21 days) in artificial urine (AU) solution. The maximum hardness value for Zn-1Cu-4Ag alloy was about 2.38 times higher than pure Zn due to solid-solution strengthening and precipitation hardening of (Ag, Cu)Zn4 phases. The highest and lowest Icorr values, reference also corrosion rate, were calculated in Zn-1Cu and Zn-1Cu-3Ag alloys as 17.13 and 1.318 mA·cm−2, respectively. The alloys' bacterial sensitivities were evaluated with the disc diffusion test for E. coli and S. aureus. The inhibition ratio of the Zn-1Cu-1Ag was higher than the inhibition rates of the other alloys on E. coli and S. aureus. In addition, the biological properties of the generated material were promising by altering cell survival/death ratio in both prostate epithelial and bladder cancer cells related to their use potential in urology.

Synthesis of lactones from fatty acids by ring-closing metathesis and their biological evaluation

The present study involves the synthesis of macrocyclic lactones by the esterification of unsaturated fatty acids (oleic acid, undecenoic acid, and erucic acid) with unsaturated alcohols (allyl alcohol, prop-2-ene-1-ol, oleyl alcohol, and undecenol) followed by a ring closing metathesis reaction employing Grubbs' second generation catalyst (1.0-1.5 mmol). The structure of the compounds was confirmed by 1H NMR, 13C NMR, FT-IR, and ESI-Mass spectral studies. The antibacterial activity of the synthesised lactones was evaluated. The larger ring-sized lactone, namely, erucic acid lactone, exhibited excellent antibacterial activity against three bacterial cell lines, namely, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis. Undecenoic acid-based lactones exhibited excellent antibacterial activity selectively against only Staphylococcus epidermidis. The assay of macrolactones for their in vitro anticancer activity was carried out by MTT for different cancer cell lines, namely, human prostate epithelial cancer cells (ATCC HTB-81), HepG2 derived from hepatic cancer cells (ATCC HB-8065), SKOV3 derived from human ovarian cancer cells (ATCC HTB-77), MDAMB-231 derived from human breast cancer cells (ATCC HTB-26) and Chinese hamster ovarian (CHO-K1) cell lines. The molecules selectively exhibited anticancer activity against Chinese hamster ovarian (CHO-K1) cell lines. Among macrolactones, (E)-oxacyclotridec-11-en-2-one (MALUN) was more active and its activity was much higher compared to others and on par with the reference standard Mitomycin C. This was followed by (E)-oxacyclotricos-14-en-2-one (MOLER) and (E)-oxacyclononadec-10-en-2-one (MOLOH). The fatty acid-based cyclic lactones with selective antibacterial and anticancer activities can be further explored for a variety of pharmaceutical formulations.

Abstract B024: Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression

Abstract Very small nuclear (vsn) circulating tumor cells (CTCs) are detected in a subgroup of metastatic, castration resistant prostate cancer (mCRPC) patients who develop visceral metastasis following intensive androgen receptor suppression. In addition, we have previously observed that nuclear shape instability (characterized by the loss and/or mis-localization of the nuclear envelop protein emerin) is present in human prostate cancer tissues, and in CTCs from patients with metastatic disease. Herein, we investigated the biological and clinical implications of the vsn phenotype in prostate CTCs. Patients with vsnCTCs had poor overall survival and low PSA response to androgen receptor suppression compared with non-vsnCTC patients. The vsnCTCs exhibited a loss/downregulation of emerin (EMD) and we found a direct correlation between emerin loss and nuclear size reduction in CTC from mCRPC patients. Further, in vitro studies revealed that the vsn phenotype was characterized by high invasive capacity, enhanced cellular plasticity, and resistance to AR target therapy. Finally, depletion of EMD in prostate tumor cells resulted in the vsn phenotype. Together, these findings demonstrate that the vsnCTC phenotype is characterized by deregulation of EMD expression, and the presence of these cells result in poor clinical outcomes in prostate cancer. Citation Format: Karen Cavassani, Edwin Posadas, Pai-Chi Teng, Sadaf Pustchi Pustchi, Minhyung Kim, Neil Bhowmick, Sungyong You, Michael Freeman, Leigh Ellis, Dolores Di Vizio, Hsian-Rong Tseng, Leland W. Chung, Susan Yao, Catherine Grasso. Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B024.

Cell-autonomous complement C3 maintains prostate epithelial cell homeostasis and prevents malignant transformation

Abstract Intracellularly active complement C3 and C3a emerged as central regulators of normal immune cell function and proliferation via control of mTOR and glycolysis. Because increased glycolysis is a hallmark of many cancer cells, we hypothesized that augmented cell-intrinsic C3 activity may contribute to epithelial cell malignancy. However, while benign prostate epithelial cells (PECs) harbored expected intracellular C3/C3a storages, cancerous PECs, surprisingly, exhibited loss of C3/C3a. Further, C3/C3a reduction levels in PECs correlated inversely with worsening Gleason scores in patients, silencing of C3 in healthy PECs induced uncontrolled proliferation, and reconstitution of intracellular C3a in prostate cancer cell line DU145 normalized their hyper-proliferation. In line with an unexpected anti-proliferative role for C3a in PECs, C3ar−/−mice developed spontaneous hyperplasia of prostate tubular cells with age, treatment of DU145 tumors, implanted subcutaneously into the flanks of athymic mice, with C3a-expressing adenovirus significantly reduced tumor growth in vivo, and augmented C3 levels are associated with a better prognosis in patients with prostate and other types of epithelial cell cancers. RNA-sequencing of C3or C3aR-deficient human PECs identified intrinsic C3a as novel controller of several known PEC oncogenes, likely via up-stream impact on the c-MYC pathway. In sum, while intracellular C3 drives proliferation in immune cells, in (prostate) epithelial cells, cell-autonomous C3/C3a is a negative regulator of growths and, thus, represses oncogenic transformation. Such cell-specific intracellular C3/C3a activities should be taken into consideration when targeting this system therapeutically.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

Toll-like receptors (TLRs) are the key sensors of innate immunity for triggering immune responses against infections. TLRs are well known to be expressed and activated in innate immune cells, such as macrophage and dendritic cells, but we and others have found that some TLRs are also functional in epithelial cells. However, the role of an epithelial TLR in prostate cancer remains elusive. TLR5 expression in messenger RNA and protein level in prostate cancer was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The activation of TLR5 signaling in epithelial cells was detected upon nuclear factor-κB activation by luciferase assay and western blot analysis, and proinflammatory cytokine activation by RT-qPCR. Distinguishing between the TLR5 and NLRC4 pathways, both recognizing flagellin, is determined by small interfering RNA and proinflammatory cytokine activation. The role of TLR5 in prostate cancer was analyzed by IHC and bioinformatics using a general and single-cell database. In the present study, we show that TLR5, among other TLRs, is exceedingly expressed in human prostate cancer cells. This cancer epithelial cell TLR5 functions to activate the TLR5 signaling pathway in human prostate cancer cells, as it does with innate immune cell TLR5. The bacterial protein flagellin induces a robust immune response in prostate cancer cells in a TLR5-dependent but NLRC4-independent manner. TLR5 is highly expressed in prostate cancer patient specimens, and high TLR5 expression in prostate cancer patients indicates a favorable prognosis. TLR5, as an innate immunity receptor, is a functional TLR in human prostate cancer epithelial cells. TLR5 plays an important role in prostate cancer development and is a new potential prognosis biomarker. TLR5 may represent a novel immunotherapy target against prostate cancer.

IL-1β Is an Androgen-Responsive Target in Macrophages for Immunotherapy of Prostate Cancer.

Great attention is paid to the role of androgen receptor (AR) as a central transcriptional factor in driving the growth of prostate cancer (PCa) epithelial cells. However, the understanding of the role of androgen in PCa-infiltrated immune cells and the impact of androgen deprivation therapy (ADT), the first-line treatment for advanced PCa, on the PCa immune microenvironment remains limited. On the other hand, immune checkpoint blockade has revolutionized the treatment of certain cancer types, but fails to achieve any benefit in advanced PCa, due to an immune suppressive environment. In this study, it is reported that AR signaling pathway is evidently activated in tumor-associated macrophages (TAMs) of PCa both in mice and humans. AR acts as a transcriptional repressor for IL1B in TAMs. ADT releases the restraint of AR on IL1B and therefore leads to an excessive expression and secretion of IL-1β in TAMs. IL-1β induces myeloid-derived suppressor cells (MDSCs) accumulation that inhibits the activation of cytotoxic T cells, leading to the immune suppressive microenvironment. Critically, anti-IL-1β antibody coupled with ADT and the immune checkpoint inhibitor anti-PD-1 antibody exerts a stronger anticancer effect on PCa following castration. Together, IL-1β is an important androgen-responsive immunotherapeutic target for advanced PCa.

Abstract 3082: Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer

Abstract SerpinA6 is a main transport protein for cortisol binding, which helps in cortisol tissue delivery particularly at inflammatory sites. Cortisol plays a vital role in adaptation to stress, modulating fuel metabolism, and inflammation. SerpinA6 releases cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. Castration induce inflammation in the prostate increases glucocorticoids (GCs) level and elevates pro-inflammatory cytokine IL-6 levels. European and Asian populations represented association between single nucleotide polymorphisms (SNPs) in CYP17A1 and SERPINA6/A1 and circulating glucocorticoid concentrations. We propose increasing IL-6 levels after castration alters SerpinA6 levels in prostate may results in prostate cancer disease progression. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6 (5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Silencing IL-6 in PC-3 cells increased SerpinA6 expression. Moreover, silencing SerpinA6 in metastatic variant of PC-3 cells increased phospho-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45µg/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36µg/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. In-vivo data represented altered SerpinA6 levels in castrated TRansgenic Adenocarcinoma Mouse Prostate model after dexamethasone (GR agonist) and Mifepristone (GR antagonist) treatment. We suggest decreasing levels of SerpinA6 in prostate cancer patients may have potential role in disease progression. We need to have an effective agent, to prevent maintain SerpinA6 level to inhibit the progression of prostate cancer. Citation Format: Mohammed Al-Toubat, Jatinder Kumar, Shiva Gautam, Kethandapatti Balaji, Sanjeev Shukla. Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3082.

Agave mapisaga aqueous extract shows in vitro and in vivo activity on murine prostate cancer cells

Plants are a source of multiple antineoplastic treatments. However, the effect of many species used in traditional medicine has yet to be demonstrated. In this work, the taxonomic identification of Agave mapisagawas made and a high-performance liquid chromatography-mass spectrometry (HPLC-MS) study suggested the presence of the aglycone hecogenin, which is part of compounds such as agavoside C and cantalasaponin 4. The antineoplastic activity of an aqueous extract was tested in vitroand in vivoon PEC-Src epithelial murine prostate cancer cells. In vitrostudy revelead a significant chemosensivity at 0.125mg/100μL(p=0.0001). Also, in in vivo, using an isotransplantation model with 1x106cells subcutaneously, it was observed that the group treated with 50 mg/kg presented a lower tumor implantation compared with the control without treatment (p=0.04).

Apoptotic Induction in Human Cancer Cell Lines by Antimicrobial Compounds from Antarctic Streptomyces fildesensis (INACH3013)

The Antarctic Streptomyces fildesensis has been recognized for its production of antimicrobial compounds with interesting biological activities against foodborne bacteria and multi-resistant strains, but not for its potential antiproliferative activity and mechanisms involved. Two bioactive ethyl acetate extract (EAE) fractions were purified via thin-layer chromatography and High-Performance Liquid Chromatography (HPLC), showing that orange-colored compounds displayed antimicrobial activity against pathogenic bacteria even after shock thermal treatment. The UV–VIS features of the active compounds, the TLC assay with actinomycin-D pure standard, Fourier transform infrared (FTIR) spectra and the ANTISMASH analysis support the presence of actinomycin-like compounds. We demonstrated that S. fildesensis displays antiproliferative activity against human tumor cell lines, including human breast cancer (MCF-7), prostate cancer (PC-3), colon cancer (HT-29) and non-tumoral colon epithelial cells (CoN). The half-maximal effective concentrations (EC50) ranged from 3.98 µg/mL to 0.1 µg/mL. Our results reveal that actinomycin-like compounds of S. fildesensis induced apoptosis mediated by caspase activation, decreasing the mitochondrial membrane potential and altering the cell morphology in all tumoral and non-tumoral cell lines analyzed. These findings confirm the potential of the psychrotolerant Antarctic S. fildesensis species as a promising source for obtaining potential novel anticancer compounds.

Nonmalignant AR-positive prostate epithelial cells and cancer cells respond differently to androgen.

Prostate cancer research suffers from the lack of suitable models to study the role of normal cells in prostate carcinogenesis. To address this challenge, we developed a cell line model mimicking luminal prostate epithelial cells by modifying the immortalized prostate epithelial cell line RWPE-1 to constitutively express the androgen receptor (AR). RWPE-1-AR cells express known AR target genes, and exhibit coexpression of luminal and basal markers characteristic of transient amplifying cells, and an RNA signature resembling prostate luminal progenitor cells. Under unstimulated conditions, constitutive AR expression does not have a biologically significant effect on the proliferation of RWPE-1 cells, but when stimulated by androgens, growth is retarded. The transcriptional response of RWPE-1-AR cells to androgen stimulation involves suppression of the growth-related KRAS pathway and is thus markedly different from that of the prostate cancer cell line LNCaP and its derivative AR-overexpressing LNCaP-ARhi cells, in which growth- and cancer-related pathways are upregulated. Hence, the nonmalignant AR-positive RWPE-1-AR cell line model could be used to study the transformation of the prostate epithelium.

Chemical Composition and Cytotoxic Activity of Extracts from Carpesium divaricatum: In Vitro- versus Field-Grown Plants.

Carpesium divaricatum Sieb. & Zucc. is a plant species rich in terpenoids of anti-inflammatory and cytotoxic activity, especially germacranolides of potential medicinal value. The present study describes in vitro multiplication of C. divaricatum, analysis of active constituents in the multiple shoots, and assessment of cytotoxic activities of extracts prepared from in vitro- and field-grown plants. The plant extracts were evaluated for cytotoxicity using two melanoma cell lines (HTB140 and A375); human keratinocytes (HaCaT); two colon cancer cell lines (Caco2 and HT29); human hepatocellular carcinoma cells (HepG2); two lines of prostate cancer cells (DU145 and PC3) and prostate epithelial cells (PNT2). Chemical compositions of the assayed extracts were analyzed by HPLC/DAD, in reference to isolated compounds. Maximum of 4.07 ± 1.61 shoots regenerated from a nodal explant of C. divaricatum, cultivated in a liquid MS medium supplemented with thidiazuron (1 μM). In vitro grown shoots and plantlets of C. divaricatum accumulated terpenoids that are known as active constituents of the intact plant. Cytotoxic activity of the extracts prepared from the in vitro cultured plants was like that demonstrated by the extracts prepared from field-grown plants and seemed to be more selective than cytotoxicities of the individual germacranolides.

Investigation of Antimicrobial and Anti Proliferative Properties of Prostate Epithelial Cancer Cells Resistance to Centipeda Minima Parts

Investigation of Antimicrobial and Anti Proliferative Properties of Prostate Epithelial Cancer Cells Resistance to Centipeda Minima Parts

Gene Expression Markers of Prognostic Importance for Prostate Cancer Risk in Patients with Benign Prostate Hyperplasia.

Comparative analyses utilizing publicly available big data have the potential to generate novel hypotheses and knowledge. However, this approach is underutilized in the realm of cancer research, particularly for prostate cancer. While the general progression of prostate cancer is now well understood, how individual cell types transition from healthy, to pre-cancerous, to cancerous cell types, remains to be further elucidated. To address this, we re-analyzed two publicly available single-cell RNA-seq datasets of prostate cancer and benign prostate hyperplasia cell types. The differential expression analysis of 15,505 epithelial cell profiles across 18,638 genes revealed 791 genes that were up regulated in prostate cancer epithelial cells. Here we report six markers that show significant upregulation in prostate cancer cells relative to BPH epithelial cells: HPN (5.62X), RAC3 (3.51X), CD24 (2.18X), HOXC6 (1.77X), AGR2 (1.71X), and IGFBP2 (1.28X). In particular, the significant differential expression of AGR2 further supports its clinical relevance in supplementing prostate-specific antigen screening for detecting prostate cancer. These findings have the potential to further advance our knowledge of genes governing the development of cancer in prostate epithelial cells. Clinical Relevance- Our results establish the importance of 6 prostate cancer markers (HPN, RAC3, CD24, HOXC6, AGR2, and IGFBP3) in distinguishing between prostate cancer epithelial cells and benign prostate hyperplasia epithelial cells.

Abstract 425: Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant prostate cancer

Abstract Introduction: The primary driver of castration-resistant prostate cancer (CRPC) is the Androgen receptor (AR). Drugs such as Abiraterone or Enzalutamide that target the Ligand Binding Domain (LBD) of AR are largely ineffective on AR splice variants (AR-SVs) that lack LBD and retain the N-terminal Domain (NTD) of AR. AR continues to function through other stimuli (ex: growth factors), leading to disease relapse and progression. Moreover, AR-SVs are known to heterodimerize with AR full length (AR-FL). Hence, treatment strategies that target both AR-FL and AR-NTD are needed for the complete remission of CRPC. Through structure-activity relationship (SAR) studies based on naturally occurring Urolithin A (UroA) structure, we have developed a novel small molecule UroBoc that directly binds to the NTD and inhibits CRPC growth. Methods: To analyze the binding of UroBoc to AR-NTD, we performed differential scanning fluorimetry and measured saturation transfer difference (STD) in NMR spectroscopic studies. In addition, we investigated the effect of UroBoc on CRPC by western blotting, immunofluorescence, Immunoprecipitation, and performed Xenograft studies in castrated/non-castrated mice models. Results: UroBoc treatment shifted the melt temperature (ΔTm) of purified NTD by 12-15 °C (median of 13.5 °C) in differential scanning fluorimetry. Moreover, STD effects observed were 30-18%, indicating a significant binding to NTD in our NMR studies. UroBoc inhibits the growth of AR+ CRPC, including enzalutamide resistant cells, at 800nM concentration, which is 40 times lower than that of the parent compound, UroA. At this concentration, UroBoc is non-toxic to normal prostate epithelial cells or other cancer cells. Interestingly, UroBoc also inhibited AR, AR-SV and PSA expressions in AR-FL (LNCaP, C42B) and AR-V7 (22-RV1) CRPC cell lines. Molecular studies suggested that UroBoc selectively promotes ubiquitination and subsequent degradation of AR and AR-V7 by proteasome activation. Remarkably, UroBoc failed to inhibit AKT, mTOR, PTEN and hormonal receptors such as estrogen and progesterone receptors suggesting AR could be a direct target for UroBoc. Further, in vivo studies revealed that oral administration of UroBoc inhibits tumor growth in CRPC in both castrated and non-castrated mice models. Conclusion: Our results suggest that the novel UroBoc selectively degrades AR, AR-SV and overcomes enzalutamide resistance in preclinical CRPC models. We believe performing PK/PD studies will confirm the translation potential of UroBoc as a therapy for CRPC. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Uttara Saran, Murali K. Ankem, Nikolay Dokholyan, Jyh M. Lin, Arun K. Sharma, Chendil Damodaran. Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 425.

Abstract 3983: Dysregulated SerpinA6 levels and progression of prostate cancer

Abstract SerpinA6 is a major cortisol binding transport protein, which helps in cortisol tissue delivery particularly at inflammatory sites. Moreover, SerpinA6 has been reported to participate in stress response by releasing cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. In castrate resistant prostate cancer (CRPC) patients, reports suggest castration induces inflammation in the prostate, which increases glucocorticoids (GCs) levels and elevates pro-inflammatory cytokine IL-6 levels. We propose declining SerpinA6 levels in the prostate may play a role in prostate cancer disease progression. This could be due to increasing IL-6 levels after castration, which may inhibit or degrade SerpinA6. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6(5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Additionally we observed increased FOXO3a and SerpinA6 expression, whereas decreased GR and HNF3α levels. However, silencing IL-6 in PC-3 cells increased SerpinA6 expression. To further support our findings, silencing SerpinA6 in metastatic variant of PC-3 cells increased phosphor-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45ug/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36ug/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. These findings suggest the SerpinA6 context dependent role. We suggest that decreasing levels of SerpinA6 and FOXO3a expressions in the prostate cancer patients may have potential role in disease progression. Moreover, we need to have an effective agent, which could prevent the decreasing levels of SerpinA6 and FOXO3a in prostate to inhibit the progression of prostate cancer. Citation Format: Carlos Riveros, Victor Chalfant, Jatinder Kumar, Sanjeev Shukla. Dysregulated SerpinA6 levels and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3983.

Phytoene Is More Efficiently Absorbed Than Lycopene by In Vitro Intestinal Cells but Is Absorbed and Cleared Similarly to Lycopene by In Vitro Prostate Cancer Cells

ObjectivesConsuming tomatoes and the tomato carotenoid lycopene (LYC) is epidemiologically associated with reduced prostate cancer risk. Tomato carotenoids, including LYC, phytoene (PE), and beta-carotene (BC), are found in human prostate tissue, with greater PE: LYC ratios found in the blood and prostate tissue relative to the diet. We tested the hypothesis that PE is more efficiently absorbed by prostate cells and by absorptive intestinal cells than LYC, explaining the enrichment of PE in blood and prostate tissue. MethodsIn vitro prostate cancer (LNCaP, PC-3) and non-cancerous prostate epithelial (RWPE-1) cells were treated with human serum enriched with PE, LYC, BC, or control (final media concentration 1 uM) for 6 hours. Clearance of PE, LYC, and BC from LNCaP cells was monitored for 48 hours after a 16 hour pre-incubation with carotenoids (n = 5 trials). Differentiated in vitroabsorptive intestinal cells (Caco-2 HTB-37 cultured 21 d on a permeable support) were treated with mixed lipid micelles and either PE, LYC, BC, or no carotenoid (final media carotenoid concentration 2 uM) for 4 or 24 h (n = 5 trials). Cell and media carotenoid concentrations were measured by HPLC-PDA. ResultsPC-3s absorbed 3.8%, 1.9%, and 2.5% (not significantly different, ns) and LNCaP cells absorbed 1.5%, 2.6%, and 2.0% of the LYC, PE, and BC (ns) doses, respectively, while RWPE-1 cells absorbed more of the BC dose (8.7%) than the PE dose (0.4%, P < 0.05), and LYC dose absorption was intermediate and similar to both PE and BC (2.5%). LNCaP cells cleared PE at the same rate as BC and LYC over 48 h. Intestinal Caco-2 cells absorbed more PE dose at 4 and 24 h (16.3%, 10.8%) than LYC (0.6%, 0.9%, respectively) or BC (5.8%, 7.5% respectively) (P < 0.05), while the amount of carotenoid recovered in the basolateral media showed a similar, though non-significantly different, pattern. ConclusionsThese results align with whole body pharmacokinetic findings suggesting that differences in intestinal absorption may be a key determinant in relative tissue carotenoid accumulation, and reveal that cancer versus non-cancer cell types may differ in carotenoid absorption characteristics. Funding SourcesNIH NCCIH/ODS R01 AT008576.

TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells.

Simple SummaryProstate cancer is the second leading cause of cancer death among men. Its poor prognosis is mainly due to metastases, and therefore, it is crucial to understand the mechanisms by which cancer cells can spread throughout the body. In this context, among others, we demonstrated the TRPM8 channel to be a major player due to its ability to impair prostate cancer cell motility. The present work elucidates the molecular mechanism through which TRPM8 exerts its inhibitory effect on cell migration. We found that this effect is mediated by a direct interaction between residues E207 and Y240 in the N-terminal TRPM8 tail and residue Y32 in the Switch I region of the Rap1-GDP bound inactive form. Since these results have also been validated in breast and cervical cancer cells, the TRPM8-Rap1 interaction could constitute a therapeutic target against metastatic progression in several types of tumors.Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

Fraxetin down-regulates polo-like kinase 4 (PLK4) to inhibit proliferation, migration and invasion of prostate cancer cells through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway

ABSTRACT Fraxetin, a natural product isolated from herb Cortex Fraxini, has been demonstrated to exhibit anti-cancer effects on various cancers. The aim of this work is to investigate the anti-tumor effect of Fraxetin in prostate cancer and the potential mechanisms. In this study, the prostatic epithelial cell RWPE-1 and prostate cancer cell DU145 were exposed to Fraxetin (10, 20, 40, and 80 μM) to detect the changes in cell viability using cell counting kit-8 (CCK-8) assay. Fraxetin (10, 20, and 40 μM) was utilized to treat DU145 cell, then the changes in cell proliferation, apoptosis, migration, and invasion were assessed. Western blot assay was employed to detect the expression of proteins that participate in the above cellular processes as well as Polo-like kinase 4 (PLK4), phosphatidylinositol 3-kinase (PI3K). In addition to 40 μM Fraxetin treatment, DU145 cells were overexpressed with PLK4, and then the above experiments were repeated. Results revealed that Fraxetin markedly decreased DU145 cell viability, but didn’t affect the cell viability of RWPE-1. Fraxetin suppressed cell proliferation, migration, invasion, and induced apoptosis of DU145 cells in a concentration-dependent manner. Furthermore, the expression of PLK4 and phosphorylated PI3K and protein kinase B (Akt) were reduced upon Fraxetin treatment. Finally, PLK4 overexpression significantly reversed all the effects of Fraxetin on DU145 cells. Collectively, Fraxetin acted as a cancer suppressor in prostate cancer through inhibiting PLK4 expression thereby inactivating PI3K/Akt signaling.