The ability to accurately measure drug-target interaction is critical for the discovery of new therapeutics. Classical pharmacological bioassays such as radioligand or fluorescent ligand binding assays can define the affinity or Kd of a ligand for a receptor with the lower the Kd, the stronger the binding and the higher the affinity. However, in many drug discovery laboratories today, the target of interest if often artificially upregulated by means of transfection to modify the host cell\'s genetic makeup. This then potentially invalidates the assumptions of classical pharmacology affinity calculations as the receptor of interest is no longer at normal physiological densities. The CXCR4 receptor is expressed on many different cancer cell types and is associated with metastasis and poor prognosis. Therefore, the CXCR4 receptor is a desirable target for novel therapeutics. In this study, we explore the applicability of the newly developed fluorescently tagged CXCR4 antagonists, IS4-FAM as an investigative tool to study CXCR4 affinity and competitive antagonism in native, non-transfected cancer cells using confocal microscopy and flow cytometry. IS4-FAM directly labels CXCR4 in several cell lines including high CXCR4 expressing SK-MEL-28 (malignant melanoma) and PC3 (metastatic prostate cancer) and lower CXCR4 expressing THP-1 (acute monocytic leukemia) and was competitive with the established CXCR4 antagonist, AMD3100. This highlights the potential of IS4-FAM as a pharmacological tool for drug discovery in native cells lines and tissues.

Clinically, prostate cancer is infamous for its histological and molecular heterogeneity, which causes great challenges to pinpoint therapy and pharmaceutical development. To overcome these difficulties, researchers are focusing on modulating tumor microenvironment and immune responses in addition to genetic alteration and epigenetic regulation. Here, we aimed to identify potential biomarkers or modulators of prostate cancer by investigating genes specifically altered in prostate cancer cells treated with established anti-cancer agents. Glycerol kinase 1 (GK1) is phosphotransferase encoded on the X chromosome, is associated with the synthesis of triglycerides and glycerophospholipids, and has been mainly studied for X-linked metabolic disorder GK deficiency (GKD). Interestingly, our DNA microarray analysis showed that several anti-cancer agents highly induced the expression of GK1, especially GK1a and GK1b isoforms, in human prostate cancer PC-3 cells. To elucidate the relationship between GK1 and cancer cell death, a human GK1b-specific expression vector was constructed and transfected into the PC-3 cells. Surprisingly, GK1b overexpression dramatically reduced cell viability and significantly accelerated apoptotic cell death. These findings suggest that GK1b may serve as a promising modulator and biomarker of cell death in prostate cancer, offering potential avenues for therapeutic intervention.

BACKGROUND: Natural variations in the abundance of the stable isotopes of nitrogen (δ15N) and carbon (δ13C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ15N and δ13C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions.
METHODS: Prostate cancer cells (PC3) were cultured in different conditions and their δ15N and δ13C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia.
RESULTS: Successive cell culture passages generally resulted in reproducible δ15N and δ13C values. The impact of culture medium composition on δ15N and δ13C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ13C in bulk cell samples, with only the former affecting δ15N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided.
CONCLUSIONS: Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ13C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach.

OBJECTIVE: To evaluate phytochemicals and in vitro biological potential of flowers, leaves and stem extracts of Rosa arvensis.
METHODS: Presence of twenty secondary metabolites was confirmed and then phenolic and flavonoid contents were quantified spectrophotometrically. Fourier Transform Infrared spectroscopy was conducted to ascertain functional groups and antioxidant potential was examined using 2,2-diphenyl-1-picrylhydrazyl scavenging activity, total antioxidant capacity and total reducing power assays. Human erythrocytes were used to assess anti-hemolytic activity and five bacterial strains were examined to determine antibacterial potential of plant extracts. Radish seeds were used to perform phytotoxic activity and cytotoxic potential was evaluated via brine shrimps and PC3 cell lines.
RESULTS: Highest phenolic contents were detected in the methanolic extract of Rosa arvensis flower (RAFM) [(151.635 ± 0.005) gallic acid equivalent mg/g] and highest flavonoid contents in the chloroform leaf extract (RALC) [(108.228 ± 0.004) quercetin equivalent mg/g]. Fourier-transform infrared spectroscopy analysis showed the presence of wide range of functional groups. The antioxidant assays indicated highest DPPH scavenging activity [IC50 (23.5 ± 0.6) μg/mL] in the methanolic stem extract (RASM), highest total antioxidant capacity [(265.1 ± 0.9) μg/mL] in RAFM and highest reducing potential [(209.9 ± 0.6) μg/mL] in leaf extract (RALM). Highest anti-hemolytic activity [(90.0 ± 0.5) μg/mL] was recorded in RAFM and brine shrimp cytotoxicity potential [(52.3 ± 0.3) μg/mL] in RASM. The antimicrobial activity was detected highest [(21.1 ± 0.5) mm inhibition zones] in RALM against Streptococcus aureus. In the end, anti-inflammatory and anti-cancer activity results depicted less than 50 % inhibition in the methanolic extracts. CONCLUSIONS: Our findings will be helpful in designing pharmaceutical regimens and therefore, more studies can be recommended to isolate and characterize compounds associated with the biological activities of Rosa arvensis.

For many years, it has been speculated that elevated testosterone levels may be critically involved in the genesis and proliferation of prostate cancer.
METHODS: The effect of testosterone on the metabolic activity of hormone-independent [PC-3] and hormone-dependent [LNCAP] cancer cells was investigated in vitro. Additionally, the impact of testosterone nanoemulsion [nanocare®] on cell viability was accessed by flow cytometry.
RESULTS: Despite the dependency of the normal prostate and of most prostatic cancers upon androgens, the obtained results indicate that, contrary to prevailing opinion, the supplementation of testosterone with higher doses in nanoemulsion was able to lower the metabolic activity and viability of prostate cancer cells.
CONCLUSIONS: We conclude that the growth of hormone-independent and hormone-dependent prostate cancer cells was reduced by the exposure of a nanoemulsion of bioidentical testostosterone in vitro. To the best of our knowledge, this is the first time that the potential effect of a testosterone nanoemulsion on the metabolic activity of prostate cancer cells has been shown. Such tests suggest that the growth of hormone-independent and hormone-dependent prostate cancer cells was reduced by the administration of bioidentical testostosterone, and this might be an interesting strategy for prostate cancer treatment in diagnosed patients.

Anticancer theranostic nanocarriers have the potential to enhance the efficacy of pharmaceutical evaluation of drugs. Semiconductor nanocrystals, also known as quantum dots (QDs), are particularly promising components of drug carrier systems due to their small sizes and robust photoluminescence properties. Herein, bright CdZnSeS quantum dots were synthesized in a single step via the hot injection method. The particles have a quasi-core/shell structure as evident from the high quantum yield (85 %), which decreased to 41 % after water solubilization. These water solubilized QDs were encapsulated into gallic acid / alginate (GA-Alg) matrices to fabricate imaging QDs@mod-PAA/GA-Alg particles with enhanced stability in aqueous media. Cell viability assessments demonstrated that these nanocarriers exhibited viability ranging from 63 % to 83 % across all tested cell lines. Furthermore, the QDs@mod-PAA/GA-Alg particles were loaded with betulinic acid (BA) and ceranib-2 (C2) for in vitro drug release studies against HL-60 leukemia and PC-3 prostate cancer cells. The BA loaded QDs@mod-PAA/GA-Alg had a half-maximal inhibitory concentration (IC50) of 8.76 μg/mL against HL-60 leukemia cells, which is 3-fold lower than that of free BA (IC50 = 26.55 μg/mL). Similar enhancements were observed with nanocarriers loaded with C2 and simultaneously with both BA and C2. Additionally, BA:C2 loaded QDs@mod-PAA/GA-Alg nanocarriers displayed a similar enhancement (IC50 = 3.37 μg/mL compared against IC50 = 11.68 μg/mL for free BA:C2). The C2 loaded QDs@mod-PAA/GA-Alg nanocarriers had an IC50 = 2.24 μg/mL against HL-60 cells. C2 and BA loaded QDs@mod-PAA/GA-Alg NCr had IC50 values of 7.37 μg/mL and 24.55 μg/mL against PC-3 cells, respectively.

Active targeting to cancer involves exploiting specific interactions between receptors on the surface of cancer cells and targeting moieties conjugated to the surface of vectors such that site-specific delivery is achieved. Prostate specific membrane antigen (PSMA) has proved to be an excellent target for active targeting to prostate cancer. We report the synthesis and use of a PSMA-specific ligand (Glu-NH-CO-NH-Lys) for the site-specific delivery of brusatol- and docetaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles to prostate cancer. The PSMA targeting ligand covalently linked to PLGA-PEG3400 was blended with methoxyPEG-PLGA to prepare brusatol- and docetaxel-loaded nanoparticles with different surface densities of the targeting ligand. Flow cytometry was used to evaluate the impact of different surface densities of the PSMA targeting ligand in LNCaP prostate cancer cells at 15 min and 2 h. Cytotoxicity evaluations of the targeted nanoparticles reveal differences based on PSMA expression in PC-3 and LNCaP cells. In addition, levels of reactive oxygen species (ROS) were measured using the fluorescent indicator, H2DCFDA, by flow cytometry. PSMA-targeted nanoparticles loaded with docetaxel and brusatol showed increased ROS generation in LNCaP cells compared to PC-3 at different time points. Furthermore, the targeted nanoparticles were evaluated in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors. Evaluation of the percent relative tumor volume show that brusatol-containing nanoparticles show great promise in inhibiting tumor growth. Our data also suggest that the dual drug-loaded targeted nanoparticle platform improves the efficacy of docetaxel in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-β-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.

In this study, a new series of cis and trans 5-substituted-3-(dibenzyloxyphosphoryl)isoxazolidines 16a-g were synthesized by the 1,3-dipolar cycloaddition reaction of N-benzyl-C-(dibenzyloxyphosphoryl)nitrone and selected N1-allyl-N3-benzylquinazoline-2,4-diones. All the obtained trans-isoxazolidines 16a-g and the samples enriched in respective cis-isomers were evaluated for anticancer activity against three tumor cell lines. All the tested compounds exhibited high activity against the prostate cancer cell line (PC-3). Isoxazolidines trans-16a and trans-16b and diastereoisomeric mixtures of isoxazolidines enriched in cis-isomer using HPLC, namely cis-16a/trans-16a (97:3) and cis-16b/trans-16b (90:10), showed the highest antiproliferative properties towards the PC-3 cell line (IC50 = 9.84 ± 3.69-12.67 ± 3.45 μM). For the most active compounds, induction apoptosis tests and an evaluation of toxicity were conducted. Isoxazolidine trans-16b showed the highest induction of apoptosis. Moreover, the most active compounds turned out safe in vitro as none affected the cell viability in the HEK293, HepG2, and HSF cellular models at all the tested concentrations. The results indicated isoxazolidine trans-16b as a promising new lead structure in the search for effective anticancer drugs.

Here, we revealed the reversibility of cabazitaxel (CBZ)-induced apoptosis in PC-3 castration resistant metastatic prostate cancer cells (mCRPC) through the hallmarks of apoptosis. The recovery of PC-3 cells from apoptosis upon removal of CBZ at different recovery periods was evaluated by Annexin V, DNA damage, oxidative damage, mitochondrial membrane depolarization, and caspase activation. Our results showed that the administration of CBZ caused apoptosis for 72 h in PC-3 cells. However, recovered cells exhibited decreased nuclear damage, plasma membrane disruption, ROS level, release cytochrome c level and caspase-3 activation with upregulation of Bcl-2 expression upon removal of especially 1 nM CBZ for 24 h recovery period in PC-3 cells. Our study indicates that CBZ treated PC-3 cells can recover after apoptotic cell death. However, advanced molecular analysis should elucidate the relationship between the molecular mechanisms of recovery and taxane response or resistance in PC-3 mCRPC cells.