Viability Of PC-3 Cells Research Articles

The inhibition effect of psoralen on prostate cancer PC3 cells via down-regulation of long non-coding RNA ENST00000510619.

New medications are needed to improve outcomes of castration-resistant prostate cancer (CRPC). Psoralen has been reported to have anti-cancer properties for various tumors, but there are limited reports about psoralen treatment in prostate cancer (PCa). This study aimed to investigate the effect of psoralen on PC3 cells and to investigate potential underlying mechanisms of action. The effect of psoralen on the proliferation and cell cycle progression of PC3 cells was determined using Cell Counting Kit-8 (CCK-8) test and flow cytometry, respectively. The differential gene profiles in PC3 cells treated with psoralen were determined with microarray analyses. The effect of psoralen on long non-coding RNA (lncRNA) ENST00000510619 expression in PC3 cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The effect of psoralen and transfection of small interfering lnc-RNA (si-lncRNA) ENST00000510619 on cell viability, invasion ability, and migratory activity of PC3 cells were evaluated using the CCK-8 test, transwell assay, and wound healing, respectively. Psoralen significantly inhibited PC3 cells in a concentration- and time-dependent manner and caused G1 phase and G2/M phase cycle arrests. When screened with a fold change (FC) of ≥2 and a P value of <0.05, 1,716 lncRNAs and 1,160 messenger RNAs (mRNAs) were significantly up-regulated, whereas 3,269 lncRNAs and 3,263 mRNAs were significantly down-regulated in PC3 cells after psoralen treatment. Among the differentially down-regulated lncRNAs in which the signal of the probe showed significant differences compared to the background, lncRNA ENST00000510619 had the highest FC. The expression of lncRNA ENST00000510619 was shown to be down-regulated by psoralen in a concentration-dependent manner. CCK-8 assay, wound healing, and transwell assay showed that both psoralen and si-lncRNA ENST00000510619 transfection significantly inhibited the activity, invasion, and migration of PC3 cells (P<0.01 for all). Psoralen was confirmed to inhibit proliferation and block the cell cycle in PC3 cells in this in vitro study. The molecular mechanism involves multiple differentially expressed lncRNAs and mRNAs and is related to the down-regulation of lncRNA ENST000000510619 expression. This study provides the experimental basis for the development of psoralen as a novel anti-CRPC drug and for the consideration of lncRNA ENST00000510619 as a potential clinical target for CRPC.

Phytochemical composition, antioxidant activity, and cytotoxicity of the aqueous extracts of Dracaena arborea and Bridelia ferruginea: In vitro and in silico studies

Dracaena arborea (DA) and Bridelia ferruginea (BF) are medicinal plants used in the treatment of various ailments such as diabetes, infertility, and sexual dysfunctions. The aim of this work was to characterize these plants chemically and investigate their antioxidant and cytotoxicity in vitro and in silico. The cytotoxicity was determined in PC3, NIH3T3 and BT474 cells, and through hemolysis using erythrocytes. Furthermore, selected compounds from DA and BF were docked against some oxidative stress (Keap1: Kelch-like ECH-associated protein 1 and GST: glutathione S-transferase) and cytotoxicity (PARP10: poly(ADP-ribose) polymerase family member 10 and p90 RSK: p90 ribosomal S6 kinases) proteins to predict the ligand-protein interaction. We found that the total phenolic and flavonoid content in BF was significantly (p < 0.001) higher than that of DA. 16 and 32 phytocompounds were identified in DA and BF, respectively. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) free radical scavenging activity, and cupric and ferric reducing power of BF were significantly (p < 0.05–0.001) higher than those of DA. Moreover, the ability of BF to inhibit lipid peroxidation was higher than that of DA. DA dose-dependently decreased the viability of PC3, NIH3T3, and BT474 cells, while BF tended to feed the cells. Drug-Likeness and toxicity prediction of selected compounds found in DA and BF were within the acceptable standards, according to Lipinski's rule. The majority of compounds found in DA and BF exhibited a strong binding affinity with Keap1, GST, PARP10 and p90 RSK, which supports the in vitro results. For instance, Cis-cyclohexane-1,3-diamine from DA showed a binding affinity of -6.73 kcal/mol and interacted with Keap1 through five conventional hydrogen bonds, while Nrf2 (a Keap1 inhibitor) exhibited a binding affinity of -11.82 kcal/mol and interacted with Keap1 via two conventional hydrogen bonds. 1,3-propanediamine, n-methyl identified in BF exhibited a high stability with Keap1 (-5.95 kcal/mol) due to the presence of 6 conventional hydrogen bonds. Among the compounds identified in DA, the best stable ligand interacting with PARP10 was 2-deoxy-alpha-d-ribopyranose (-7.62 kcal/mol), as it was well-matched in the PARP10 pocket through five conventional hydrogen bonds. In parallel, among the compounds present in BF, 2-[(methylamino)methyl]cyclohexanol showed the best binding affinity with PARP10 (-9.80 kcal/mol), which was higher than that of veliparib (-8.26 kcal/mol), used as a reference inhibitor. In conclusion, BF has the possibility to be exploited in the prevention/treatment of oxidative stress-related diseases, while DA could be a potential anticancer agent. This could justify their ethnomedicinal uses.

Hispidin Increases Cell Apoptosis and Ferroptosis in Prostate Cancer Cells Through Phosphatidylinositol-3-Kinase and Mitogen-activated Protein Kinase Signaling Pathway.

Chemotherapy is mainly used in the clinical treatment of prostate cancer. Different anticancer mechanisms can induce cell death in various cancers. Reactive oxygen species (ROS) play crucial roles in cell proliferation, differentiation, apoptosis, and signal transduction. It is widely accepted that ROS accumulation is closely related to chemical drug-induced cancer cell death. We utilized the MTT assay to detect changes in cell proliferation. Additionally, colony formation and wound healing assay were conducted to investigate the effect of hispidin on cell colony formation and migration ability. Fluorescence microscopy was used to detect intracellular and mitochondrial ROS levels, while western blot was used for detection of cell apoptosis. Hispidin treatment significantly decreased viability of PC3 and DU145 cancer cells but exhibited no cytotoxicity in WPMY-1 cells. Furthermore, hispidin treatment inhibited cell migration and colony formation and triggered cellular and mitochondrial ROS accumulation, leading to mitochondrial dysfunction and mitochondrion-dependent apoptosis. Moreover, hispidin treatment induced ferroptosis in PC3 cells. Scavenging of ROS with N-acetyl cysteine significantly inhibited hispidin-induced apoptosis by altering the expression of apoptosis-related proteins, such as cleaved caspase-3, 9, Bax, and Bcl2. Furthermore, hispidin treatment dramatically up-regulated MAPK (involving p38, ERK, and JNK proteins) and NF-kB signaling pathways while down-regulating AKT phosphorylation. Hispidin treatment also inhibited ferroptosis signaling pathways (involving P53, Nrf-2, and HO-1 proteins) in PC3 cells. In addition, inhibiting these signaling pathways via treatment with specific inhibitors significantly reversed hispidin-induced apoptosis, cellular ROS levels, mitochondrial dysfunction, and ferroptosis. Hispidin may represent a potential candidate for treating prostate cancer.

Exploring the Antitumor Efficacy of PEGylated Liposomes Loaded with Licorice Extract for Cancer Therapy.

Glycyrrhizic Acid (GA), a compound derived from licorice, has exhibited promising anticancer properties against several cancer types, including Prostate Cancer (PCa) and Gastric Cancer (GCa). This study has introduced a novel approach involving the encapsulation of GA and Licorice extract (Lic) into Polyethylene Glycol Liposomes (PEG-Lip) and assessed their efficacy against AGS (human gastric cancer) and PC-3 (human prostate cancer) cells, marking the first report of this endeavor. We synthesized GA-loaded PEG-Lip (GA PEG-Lip) and Lic-loaded PEG-Lip (Lic PEG-Lip) through the reverse-phase evaporation method. Characterization of these liposomal formulations revealed their size, drug encapsulation, and loading efficiencies to be 110 ± 2.05 nm, 117 ± 1.24 nm; 61 ± 0.81%, 34 ± 0.47%; and 8 ± 0.41% and 4.6 ± 0.21%, respectively. Importantly, the process has retained the chemical structure of both GA and Lic. Furthermore, GA and Lic have been released from the PEG-Lip formulations in a controlled manner. In our experiments, both nanoformulations exhibited enhanced cytotoxic effects against AGS and PC-3 cells. Notably, GA PEG-Lip outperformed Lic PEG-Lip, reducing the viability of PC-3 and AGS cells by 12.5% and 15.9%, respectively. These results have been corroborated by apoptosis assays, which have demonstrated GA PEG-Lip and Lic PEG-Lip to induce stronger apoptotic effects compared to free GA and Lic on both PC-3 and AGS cells. This study has underscored the potential of encapsulating GA and Lic in PEG-Lip as a promising strategy to augment their anticancer efficacy against prostate and gastric cancers.

Hybridization-based discovery of novel quinazoline-2-indolinone derivatives as potent and selective PI3Kα inhibitors

IntroductionPhosphatidylinositol 3-kinases (PI3Ks) overexpression can elicit cellular homeostatic dysregulation, which further contributes to tumorigenesis, with PI3Kα emerging as the most prevalent mutant isoform kinase among PI3Ks. Therefore, selective inhibitors targeting PI3Kα have attracted considerable interest in recent years. Molecular hybridization, with the advantage of simplified pharmacokinetics and drug-drug interactions, emerged as one of the important avenues for discovering potential drugs. ObjectivesThis study aimed to construct PI3Kα inhibitors by hybridization and investigate their antitumor activity and mechanism. Methods26 quinazoline-2-indolinone derivatives were obtained by molecular hybridization, and their structure–activity relationship was analyzed by MTT, in vitro kinase activity and molecular docking. The biological evaluation of compound 8 was performed by transwell, flow cytometry, laser scanning confocal microscopy, Western blot, CTESA and immunohistochemistry. ResultsHere, we employed molecular hybridization methods to construct a series of quinazoline-2-indolinone derivatives as PI3Kα selective inhibitors. Encouragingly, representative compound 8 exhibited a PI3Kα enzymatic IC50 value of 9.11 nM and 10.41/16.99/37.53-fold relative to the biochemical selectivity for PI3Kβ/γ/δ, respectively. Moreover, compound 8 effectively suppressed the viability of B16, HCT116, MCF-7, H22, PC-3, and A549 cells (IC50 values: 0.2 μM ∼ 0.98 μM), and dramatically inhibited the proliferation and migration of NSCLC cells, as well as induced mitochondrial apoptosis through the PI3K/Akt/mTOR pathway. Importantly, compound 8 demonstrated potent in vivo anti-tumor activity in non-small cell lung cancer mouse models without visible toxicity. ConclusionsThis study presented a new avenue for the development of PI3Kα inhibitors and provided a solid foundation for novel QHIDs as potential future therapies for the treatment of NSCLC.

Anticancer and Antimicrobial Activity of Some New 2,3-Dihydro-1,5-benzodiazepine Derivatives

A series of 2,3-dihydro-1,5-benzodiazepine derivatives have been synthesized and characterized using IR, NMR, GC-MS, single crystal XRD, and microanalysis. The results of their antibacterial activity against methicillin-resistance Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, and Streptococcus pyrogens indicated that most of the compounds were bacteriostatic (0.125−4 mg/mL) and also exhibited good biofilm inhibition (0.21−72.69%). The compounds were found to be synergistic when used in combination with other antibiotics. The antiproliferative and cytotoxic effects were also investigated against PC-3 prostate cancer and RAW 264.7 macrophage cell lines, respectively, using the MTT assay. Apart from compounds 6 and 7, a good number of the compounds (1, 2, 3, 4, 5, and 8) were selectively toxic to the prostate cancer cells at 20 µM, whilst sparing the normal cells. Compound 3 demonstrated the highest antiprostate cancer effect by reducing the viability of PC-3 cells to (13.75%), which was followed by compounds 1 (47.72%), 2 (48.18%), 4 (62.61%), 5 (66.70%), and 8 (69.55%).

Implications of albumin in cell culture media on the biological action of vanadates(V)

In this article, the implications of binding competition of vanadates(V) with dodecyl sulfates for bovine serum albumin on cytotoxicity of vanadium(V) species against prostate cancer cells have been investigated. The pH- and SDS-dependent vanadate(V)-BSA interactions were observed. At pH 5, there is only one site capable of binding ten vanadates(V) ions (logK(ITC)1 = 4.96 ± 0.06; ΔH(ITC)1 = −1.04 ± 0.03 kcal mol−1), whereas at pH 7 two distinctive binding sites on protein were found, saturated with two and seven V(V) ions, respectively (logK(ITC)1 = 6.11 ± 0.06; ΔH(ITC)1 = 0.78 ± 0.12 kcal mol−1; logK(ITC)2 = 4.80 ± 0.02; ΔH(ITC)2 = − 4.95 ± 0.14 kcal mol−1). SDS influences the stoichiometry and the stability of the resulting V(V)-BSA complexes. Finally, the cytotoxicity of vanadates(V) against prostate cancer cells (PC3 line) was examined in the presence and absence of SDS in the culture medium. In the case of a 24-h incubation with 100 μM vanadate(V), a ca. 20 % reduction in viability of PC3 cells was observed in the presence of SDS. However, in other considered cases (various concentrations and time of incubation) SDS does not affect the dose-dependent action of vanadates(V) on the investigated prostate cancer cells.

Antiproliferative and Apoptosis Effects of Hybrid Varieties of Vitis vinifera L. Sweet Sapphire and Sweet Surprise on Human Prostate Cancer Cells Using In Vitro and In Silico Approaches.

Grape hybrids are characterized by different chemical compositions; often with high hybrids are characterized by different chemical compositions, often with a high phenolic content and a specific profile of anthocyanins. The aim of study was to characterize the constituents of hybrid Vitis vinifera L. varieties Sweet Sapphire (SA) and Sweet Surprise (SU) extracts and their influence on apoptosis induction and antiproliferative effects on human prostate cancer cells. We used the MTT assay to evaluate the cytotoxic effect of extracts of SA and SU, on the prostate adenocarcinoma cell lines PC-3 and DU-145. To analyze the inhibiting impact by flow cytometry, used 24 and 48 hours. Anthocyanins were quantified by liquid chromatography and analysed by their absorption rate, hepatotoxicity, blood concentration, blood-brain barrier passage ability and maximum recommended dose by in silico approaches. Our results showed that malvidin derivatives present the highest content in both cultivars. We identified 14.46mg/100g malvidin-3-O-glycoside in SA and 2.76 mg/100 g in SU. A reduction in cell viability of DU-145 (45 and 65%) and PC-3 (63 and 67%) cells after 48h treatment with SA and SU, respectively, was found via MTT assay. Flow cytometry showed that the treatment with extracts from SA and SU had an inhibitory impact on cell development due to G2/M arrest and caused a rise in apoptotic cells compared to control group. None of the anthocyanin presented hepatotoxicity as well as blood-brain barrier passage ability. Peonidin 3-O-glucoside had the lower maximum recommended dose as well as the highest intestinal absorption rate. However, delphinidin 3-O-glucoside had the highest blood concentration values. The findings of this study highlight the potential of hybrid Vitis vinifera L. varieties as an important source of natural antioxidants and their protective effect against prostate cancer cells as well as elucidate in part their anthocyanin's metabolism.

In Vitro Assessment of Antiviral, Antimicrobial, Antioxidant and Anticancer Activities of Ulvan Extracted from the Green Seaweed Ulva lactuca

Marine macroalgae contain a variety of bioactive substances that have many different uses. Ulvan, a sulfated hetero-polysaccharide isolated from Ulva lactuca, has been described and investigated for its antioxidant, antiviral, antibacterial, and cytotoxic action against the cell lines PC-3 and A-549. The yield of polysaccharides was 26.53 ± 0.32. (Algal dry weight) with 2.23% uronic acid. According to HPLC estimates, the polysaccharide isolated from Ulva lactuca contains the neutral sugars glucose, rhamnose and galactose, as well as fructose, and melibiose. The FT-IR spectrum of ulvan revealed bands at 1227.73 cm− 1 and 1333.97 cm− 1, respectively, indicative of sulfate esters and uronic acids. This study came to the conclusion that Ulva lactuca ulvan (ULU) contains beneficial elements that can stop the development of cancer cells. The lowest viability of PC-3 cells, which occur at the highest concentration of ULU (500 µg/ml), was 17.58% with an IC50 of 237.02 ± 6.84 µg/ml, while the lowest viability of lung carcinoma cells was 13.91% with an IC50 of 124.38 ± 4.78 µg/ml. This indicates that the cytotoxicity against the prostate cancer cell line was lower than that against the lung carcinoma cell line. Although ULU has no activity against adenovirus, it had antiviral efficacy against HAV-10. ULU has been shown to have antibacterial efficacy against pathogenic bacteria, including Staphylococcus aureus ATCC25923 Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29,212, Pseudomonas aeruginosa ATCC 9027, Klebsiella pneumonia ATCC 13,883, Pseudomonas fluorescent ATCC13525 and Streptococcus agaloctiae ATCC13813. The polysaccharide, meanwhile, has limited antioxidant action.

Anticancer, Antioxidant, and Catalytic Activities of Green Synthesized Gold Nanoparticles Using Avocado Seed Aqueous Extract.

Disposal of agricultural waste has a negative impact on the environment and human health and may contribute to the greenhouse effect. The field of nanotechnology could provide alternative solutions to upcycle agricultural wastes in a safer manner into high-end value products. Organic waste from plants contain biomaterials that could serve as reducing and capping agents in the synthesis of nanomaterials with enhanced activities for use in biomedical and environmental applications. Persea americana (avocado) is a fruit with a high nutritional value; however, despite its rich phytochemical profile, its seed is often discarded as waste. Therefore, this study aimed to upcycle avocado seeds through the synthesis of gold nanoparticles (AuNPs) and evaluate their anticancer, antioxidant, and catalytic activities. The biosynthesis of avocado seed extract (AvoSE)-mediated AuNPs (AvoSE-AuNPs) was achieved following the optimization of various reaction parameters, including pH, temperature, extract, and gold salt concentrations. The AvoSE-AuNPs were poly-dispersed and anisotropic, with average core and hydrodynamic sizes of 14 ± 3.7 and 101.39 ± 1.4 nm, respectively. The AvoSE-AuNPs showed excellent antioxidant potential in terms of ferric reducing antioxidant power (343.88 ± 0.001 μmolAAE/L), 2,2-diphenyl-1-picrylhydrazyl (128.80 ± 0.0159 μmolTE/L), and oxygen radical absorbance capacity (1822.02 ± 12.6338 μmolTE/L); significantly reduced the viability of Caco-2 and PC-3 cells in a dose-dependent manner; and efficiently reduced 4-nitrophenol (4-NP) to 4-aminophenol. This study demonstrated how avocado seeds, an agricultural waste, can be used as sources of new bioactive materials for the synthesis of AuNPs, which have excellent antioxidant, anticancer, and catalytic activities, showing AvoSE-AuNPs' versatility in various applications. In addition, the AvoSE-AuNPs exhibited good stability and recyclability during the catalytic activity, which is significant because some of the primary issues with the use of metallic NPs as catalysts are around the cost-effectiveness, recovery, and reusability of the catalyst.

Paclitaxel nanocrystals carrying miR-200c inhibit prostate cancer in rats by regulating forkhead box protein O (FOXO) activity

Prostate cancer is a malignant tumor in the male reproductive organs. Its incidence is increasing year by year, and therefore, this study investigated regulatory effect of paclitaxel nanocrystals on forkhead box protein O (FOXO) activity in rats with prostate cancer. Firstly, paclitaxel nanocrystals carrying miRNA-200c were synthesized. Nine rats in the model control group were used to establish a tumor-bearing model. The rats were intervened with miRNA-200c in the miRNA-200c group on the basis of model group, and treated with paclitaxel nanosuspension in the paclitaxel nano group. The rats were intervened with paclitaxel nano and miRNA-200c in the paclitaxel nano+miRNA-200c group. Tumor inhibition rates were measured, and tumor cell morphology was assessed by hematoxylin-eosin (HE) staining, while cell behaviors were detected by CCK-8, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), AnnexinV-Fluorescein isothiocyanate (FITC), and Transwell methods, which were used to analyze miRNA-200c and FOXO levels. Paclitaxel nanocrystals carrying miR-200c (PN-miR-200c) significantly slowed down the growth rate of prostate cancer. In addition, it also reduced the viability, proliferation and invasion of rat PC3 cells, and increased cell apoptosis. On the other hand, PN-miR-200c further increased FoxO mRNA expression by activating FoxO pathway. The bioinformatics software RegRNA 2.0 predicted that, the specific target gene that may be regulated by miR-200c is FoxO, which confirmed that, the miR-200c has a targeting relationship with FoxO. PN-miR-200c can activate FOXO pathway, up-regulate FoxO, reduce the biological behaviors of rat PC3 cells, increase apoptosis, and ameliorate histopathological damage, providing new ideas for clinical treatment of prostate cancer.

Emodin-Induced Necroptosis in Prostate Cancer Cells via the Mitochondrial Fission HSP90/MLKL/PGAM Pathway.

Currently, prostate cancer is one of the major malignant tumors in males. Recurrence and metastasis are the main obstacles that prevent the effective treatment of prostate cancer. In the present study, we aimed to evaluate emodin (EG) against human prostate cancer PC3 and DU145 cells. Our study showed that EG significantly decreased the cell viability of PC3 and DU145 cells and strikingly induced non-apoptotic cell death via necroptosis that was visualized through colony formation assay, Hoechst 33258 staining, and TEM analysis. Furthermore, RNA-sequencing and KEGG functional enrichment analysis revealed that the necroptosis-related pathway was activated upon EG treatment in PC3 cells. mRNA and protein expression of necroptosis markers were analyzed by qPCR and immunoblotting, which implied that EG-induced cell necroptosis via enhancing the expression of MLKL and HSP90AA1 activating PGAM pathway which is considered as a key mediator of mitochondrial fission and leading to ROS generation in PC3 and DU145 cells. Thus, our findings suggested that EG is a new small molecule agonist that induced necroptosis in prostate cancer cells via the mitochondrial fission HSP90/MLKL/PGAM pathway.

Bioactive compounds from roasted and green coffee obtained by supercritical fluid extraction decrease survival and induce apoptosis in prostate cancer cells

Prostate cancer is the most frequent and lethal neoplastic disease among men worldwide. There is growing interest in the biologically active compounds in coffee for their potential chemopreventive. The present study aimed to analyze the content of bioactive compounds, the antioxidant and antiproliferative activity of green arabica coffee and different roasts extracted by supercritical fluid. The concentration of caffeine, chlorogenic acid and caffeic acid were evaluated. Additionally, the colorimetric profile, antioxidant capacity, effect on cell viability, cell cycle and apoptosis of the extracts. Caffeine, caffeic acid and chlorogenic acid content were higher in green coffee extract than in roasted coffee (80.0mg/100g, 16.76 and 19.37mg/mL, respectively). The green coffee extract showed higher antioxidant activity and total phenolic content compared to the other coffee extracts by all methods tested; and among the roasted coffee extracts, light roast coffee stands out with the highest antioxidant capacity. After 48 hours of treatment, coffee extracts caused a reduction in the viability of PC-3 cells, with light roast coffee being more effective. Green and light roast coffee extracts induced a cell cycle arrest in the S phase and increased apoptosis. The present results strongly suggest that coffee extracts have an antiproliferative effect on prostate adenocarcinoma cells.&#x0D;

Newly developed MEIS inhibitor selectively blocks MEISHigh prostate cancer growth and induces apoptosis

Prostate cancer (PCa) is the second most diagnosed cancer in males. Understanding the molecular mechanism and investigation of novel ways to block PCa growth or metastasis are vital and a medical necessity. In this study, we examined differential expression of MEIS1/2/3 and its associated factors in PCa cell lines. MEIS1/2/3 content, reactive oxygen species, and cell cycle status were analyzed in PCa cells post MEIS inhibitor (MEISi) treatments, which is developed in our laboratory as a first-in-class small molecule inhibitor. A correlation was detected between MEIS content and MEISi IC50 values of PCa cells. MEISi decreased the viability of PC-3, DU145, 22Rv-1 and LNCaP cells, and significantly increased apoptosis in parallel with the increased cellular ROS content. The efficacy of MEISi was shown to positively correlate with the levels of MEIS1/2/3 proteins and the long term exposure to MEISi elevated MEIS1/2/3 protein content in PCa cells. Our findings suggest that MEISi could be used to target PCa with high MEIS expression in order to reduce PCa viability and growth; however, more research is needed before this can be translated into clinical settings.

Abstract 1239: Intracellular effects of blocking MDM2 and HMG-CoA reductase in metastatic prostate cancer cells

Abstract Our study aimed to analyze the effects of MDM2 inhibitor RG7388 and HMGCR inhibitor simvastatin, on EMT (epithelial to mesenchymal transition) and cancer stemness. Prostate Cancer (PCa) is the second leading cause of cancer death among men, ranked next to lung cancer in the United States of America. Aggressive growth, metastatic abilities, resistance, and reoccurrence are the main reasons for the most number of deaths among PCa patients. The proto-oncogene MDM2 controls multiple signaling pathways that are involved in the regulation of the cell cycle, proliferation, and survival of cancer cells. MDM2 also has a major role in imparting stemness and EMT abilities to PCa cells that are inherently less aggressive. During cancer growth, lipid metabolism is also known to play a pivotal role in cellular reprogramming, which eventually leads to the induction of EMT and the acquisition of stemness. It has been well documented that the intermediates of mevalonate metabolism can significantly affect gene expressions and influence the tumor microenvironment to support cancer growth and survival. Also, there is a lack of comprehensive knowledge about the use of RG7388 in cancers with p53 mutation since it is believed to be most effective in cancers with functional p53. Therefore, our study was designed to investigate the individual and combined effects of RG7388 and simvastatin in p53 mutant PCa cells. Western blot analysis technique was used to analyze the expression levels of mesenchymal and stem cell markers in PC3 and DU145 PCa cells, before and after they were treated with RG7388, simvastatin, or their combination. The MTT assay method was used to assess the cell viability of the PCa cells after 24 or 48 hours of treatment with different concentrations of RG7388 or simvastatin. Spheroid formation assay with stem cell marker analysis was performed following drug treatments. A scratch assay was also conducted to determine the inhibition of cancer cell migration by the above-mentioned drugs. Our experimental results showed a decrease in the viability of PC3 and DU145 cells, in a time and dose-dependent manner, after treatment with RG7388 or simvastatin. The mesenchymal and stem cell markers were also downregulated along with the disintegration of spheroids causing a reduction in the total number of spheroids and colonies after RG7388, simvastatin, and combination treatments. Immunofluorescence staining of spheroids revealed a significant downregulation of cancer stem cell markers among all treated groups. Furthermore, the scratch assay showed inhibition of migration of the PCa cells, particularly after the combination treatments. Our results have confirmed that RG7388, simvastatin, and their combination treatments can reverse EMT, inhibit migration, and reduce cancer stemness in p53 mutant prostate cancer cells. (This research was supported by the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida). Citation Format: Samia Alsubhi, Umamaheswari Natarajan, Amal Alzahrani, Ahmed Alsarrani, Appu Rathinavelu. Intracellular effects of blocking MDM2 and HMG-CoA reductase in metastatic prostate cancer cells [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 1239.

Inhibition Effect of Cell-Free Supernatants of Environmental Acanthamoeba Strains on the Viability of Metastatic Cell-Lines

It is known that some of the therapeutic agents against cancer cells are isolated from natural sources such as plants and animals. However, due to increasing drug resistance, studies on the discovery of new sources are needed. In this study, it was aimed to investigate the inhibition effects of four native Acanthamoeba strains on different cancer cell lines (MDA-MB-231, PC3, MAT-LyLu). 3T3 cells were used as normal cell line. All strains were recultured by using non-nutrient agar spread by heat-inactivated Escherichia coli ATCC 25922. A.castellanii ATCC 50373 was used as the standard strain. Molecular identification of the native Acanthamoeba isolates was done by polymerase chain reaction (PCR) and DNA sequence analysis using specific primer pairs (P-FLA-F, P-FLA-R, JDP-F, JDP-R). Axenic cultures of all strains were obtained in 25 cm2 tissue culture flasks and in peptone yeast extract glucose (PYG) medium. In order to investigate the effect of cell-free supernatants obtained from axenic cultures on cancer cell lines and 3T3 cell viability, MTT method was applied using different concentrations of cell-free supernatants (1%, 2%, 5%, 10%, 15%). It was determined that the viability of 3T3 cells was not affected by any Acanthamoeba cell-free supernatants (p≤ 0.05). All of the samples tested were found to have a significant inhibitory effect (p<0.05) on the viability of PC3 and MAT-LyLu cells (human and rat prostate cancer cell line). However, none of the samples had an inhibitory effect on the viability of MDA-MB-231 (breast cancer cell-line). Two native Acanthamoeba cell-free supernatants showed higher inhibitory potency (28% and 21.9%) at 2% concentration against PC3 cells compared to the reference strain (16%). Similarly, the same Acanthamoeba samples were also shown to have a better inhibition potential on the viability of MAT-LyLu cells than the reference strain. It was found that the inhibitory potential of Acanthamoeba cell-free supernatants may not be related to proteins and proteases. The results obtained from this study showed that Acanthamoeba species living in the aquatic environment isolated from our country have a potential inhibitory effect against the tested cancer cell lines. In addition to plants and animals, Acanthamoeba cell-free supernatants can also be a source for natural therapeutic substances that act against cancer cells. However, it is necessary to carry out new studies using more strains in order to detect strains with higher inhibitory effects.

4-Nerolidylcatechol (4-NC) and Docetaxel Synergize in Controlling Androgen- independent Prostate Cancer Cells.

Effective cancer treatment still challenges medicine since the strategies employed so far are not sufficiently safe and capable of specifically eliminating tumor cells. Prostate cancer (PCa) is a highly incident malignant neoplasm, and the outcome of patients, especially those with advanced castration-resistant PCa (CRPC), depends directly on the efficacy of the therapeutic agents, such as docetaxel (DOC). This study investigated the synergistic potentiation of 4-nerolidylcatechol (4-NC) with DOC in inhibiting androgen-independent PCa cells. The cytotoxic effect of 4-NC was evaluated against non-tumorigenic (RWPE-01) and PCa cell lines (LNCaP and PC-3), and the antiproliferative potential of 4-NC was assessed by flow cytometry and colony formation. The Chou-Talalay method was applied to detect the synergistic effect of 4-NC and DOC, and the mechanism of anticancer activities of this combination was investigated by analyzing players in epithelial-mesenchymal transition (EMT). 4-NC significantly reduced the viability of PC-3 cells in a dose-dependent manner, decreasing colony formation and proliferation. The combination of 4-NC and DOC was synergistic in the androgen-independent cells and allowed the reduction of DOC concentration, with increased cytotoxicity and induction of apoptosis when compared to compounds alone. Furthermore, when 4- NC was co-administered with DOC, higher expression levels of proteins associated with the epithelial phenotype were observed, controlling EMT in PC-3 cells. Collectively, these data demonstrated, for the first time, that the combination of 4-NC with reduced doses of DOC could be especially valuable in the suppression of oncogenic mechanisms of androgen-independent PCa cells.

Narirutin. A flavonoid found in citrus fruits modulates cell cycle phases and inhibits the proliferation of hormone-refractory prostate cancer cells by targeting hyaluronidase.

Narirutin is a dietary flavanone found in lemons, oranges, passion fruit, bergamot and grapefruit. It possesses anti-allergic, cardioprotective, neuroprotective, hepatoprotective potential, and its enriched fraction suppresses the growth of prostate cancer cells; however, there is currently no information on the chemopreventive potential of narirutin alone against hormone-refractory prostate cancer cells (PC-3) and its mode of action. Thus, the chemopreventive possibility of narirutin was investigated in PC-3cells by utilising cytotoxicity assays. Further, a mechanism was deduced targeting hyaluronidase, an early-stage diagnosis marker, by cell-free, cell-based and in silico studies. The results indicate that narirutin reduced the viability of PC-3cells with the inhibitory concentration range of 66.87-59.80μM. It induced G0/G1 phase arrest with a fold change of 1.12. Besides, it increased the generation of reactive oxygen species (ROS) with a fold change of 1.34at 100μM. Narirutin inhibited hyaluronidase's activity in cell-free (11.17μM) and cell-based assays (67.23μM) and showed a strong binding interaction with hyaluronidase. Finally, the MD simulation analysis supported the idea that narirutin binding enhanced compactness and stability and created a stable complex with hyaluronidase. In addition, ADMET prediction indicates that it is a non-toxic, non-CYPs inhibitor and thus didn't alter the metabolism. The results reveal that narirutin may be a potential chemopreventive agent for hormone-resistant prostate cancer cells in addition to offering data for supporting diet-based nutraceutical agents to prevent prostate cancer.

The effect of renalase-derived peptides on viability of HepG₂ and PC3 cells.

Renalase (RNLS) is a recently discovered protein, which plays different roles inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5), while extracellular RNLS lacks its N-terminal peptide, FAD cofactor, and exhibits various protective effects in a non-catalytic manner. Certain evidence exists, that plasma/serum RNLS is not an intact protein secreted into the extracellular space, and exogenous recombinant RNLS is effectively degraded during short-term incubation with human plasma samples. Some synthetic analogues of the RNLS sequence (e.g. the Desir's peptide RP-220, a 20-mer peptide corresponding to the RNLS sequence 220-239) have effects on cell survival. This suggests that RNLS-derived peptides, formed during proteolytic processing, may have own biological activity. Based on results of a recent bioinformatics analysis of potential cleavage sites of RNLS (Fedchenko et al., Medical Hypotheses, 2022) we have investigated the effect of four RNLS-derived peptides as well as RP-220 and its fragment (RP-224) on the viability of two cancer cell lines: HepG₂ (human hepatoma) and PC3 (prostate cancer). Two RNLS-derived peptides (RP-207 and RP-220) decreased the viability of HepG₂ cells in a concentration dependent manner. The most pronounced and statistically significant effect (30-40% inhibition of cell growth) was observed at 50 μM concentration of each peptide. In the experiments with PC3 cells five of six RNLS-derived peptides had a significant impact on the cell viability. RP-220 and RP-224 decreased cell viability; however, no concentration dependence of this effect was observed in the range of concentrations studied (1-50 μM). Three other RNLS-derived peptides (RP-207, RP-233, and RP-265) increased viability of PC3 cells by 20-30%, but no concentration-dependence of this effect was found. Data obtained suggest that some RNLS-derived peptides may influence the viability of various cells and manifestation and direction of the effect (increase of decrease of the cell viability) is cell-type-specific.

Anticancer activity and metabolic alteration in colon and prostate cancer cells by novel moxifloxacin conjugates with fatty acids

The positive and pro-economic trend in the management of cancer treatment is the search for the antineoplastic potential of known, widely used and safe drugs with a different clinical purpose. A good candidate seems to be moxifloxacin with broad-spectrum antibacterial activity, which as the member of the fourth generation fluoroquinolone is known to affect not only bacterial but also eukaryotic DNA topoisomerases, however at high concentration. Due to the fact that the modification of parent drug with lipid component can improve anticancer potential by increasing of bioavailability, selectivity, and cytotoxic efficiency, we evaluated the mechanisms of cytotoxic activity of novel moxifloxacin conjugates with fatty acids and verified metabolic profile in SW480, SW620 and PC3 cell lines.Our study revealed that cytotoxic potential of moxifloxacin conjugates was stronger than free moxifloxacin, moreover, they remained non-toxic to normal HaCaT cells. PC3 were more sensitive to MXF conjugates than colon cancer cells. The most promising cytotoxic activity exhibited conjugate 4m and 16m with oleic and stearic acid reducing viability of PC3 and SW620 cells. Tested conjugates activated caspases 3/7 and induced late-apoptosis, mainly in PC3 and SW620 cells. However, the most pronounced inhibition of NF-κB activation and IL-6 secretion was observed in SW480. Metabolomic analysis indicated influence of the moxifloxacin conjugates on intensity of lipid derivatives with the most successful metabolite profile in PC3. Our findings suggested the cytotoxic potential of moxifloxacin conjugates, especially with oleic and stearic acid can be beneficial in oncological therapy, including their possible anti-inflammatory and known antibacterial effect.