Role of Resveratrol as Radiosensitizer by Targeting Cancer Stem Cells in Radioresistant Prostate Cancer Cells (PC-3).

AIM OF WORK: Here, we examined the role of resveratrol as a radiosensitizer by targeting cancer stem cells in radioresistant prostate cancer cells (PC-3) using stem cell markers CD44, CD49b and CD29, SOX2, OCT4, CXCR4, DCLK1 and EMT markers such as VIM and E-cadherin. MATERIAL AND METHODS: This study was an in vitro study involving PC-3 cell line which was dividing into four groups. Group I (CO): Control group composed of cells grown in the same medium without treatment with ionizing radiation or resveratrol. Group II (IR): Cells were treated with ionizing radiation alone. Group III (RV): Cells were treated with resveratrol alone. Group VI (IR&RV): The cells were treated with ionizing radiation and resveratrol in combination. The viability of cells was assessed by MTT assay. Genes of interest were measured by RT-PCR and the radiosensitizing efficacy of RV on proliferating cancer cells was determined by clonogenic assay. RESULTS: Ionizing radiation significantly reduced PC-3 viability, lowered stem cell markers and affected epithelial to mesenchymal transition (EMT) genes expression at all doses (2, 4, 6 and 8 Gray). Resveratrol significantly decreased PC-3 viability and lowered stem cell markers and EMT genes expression at concentrations 35, 70 and 140 µM. Combining resveratrol treatment with ionizing radiation leads to significant reduction in cell viability and stem cell markers genes which was noticed with increasing the radiation dose when compared to ionizing radiation alone treated group. CONCLUSION: Resveratrol has a radiosensitizing effect, that ability is triggered by reducing the expression of cancer stem cell markers and affecting EMT markers. Resveratrol showed to be a good candidate for further studies as anticancer drug in the treatment of human prostate cancer.

Antiproliferative activity of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine thione (THTT) derivatives and evaluation as potential prodrug.

Four series of tetrahydro-2H-1,3,5-thiadiazine thione derivatives were screened for their in vitro antiproliferative activities against two human cancerous PC3 and HeLa cell lines. The cytotoxicity of all the compounds (series A-D) was also determined on mammalian mouse fibroblast 3T3 cells. Most of the compounds showed significant anticancer potential against both cancer cell lines within the range of IC50 = 6.4-29.9 and 2.4-23.8 M respectively when compared with standard doxorubicin (IC50 = 0.3 M). All compounds demonstrated a notable selectivity for Hela cells and found either non-toxic or relatively less toxic for 3T3 cell lines model. The structure-activity relationship indicated that antiproliferative activity mainly influenced by the nature and position of substituents at thidiazine nucleus. In general, the presence of aryl groups for example 3,4-(OMe) 2.Bzl and CH(Ph)Me at N-3 position resulted in a significant activity. Under enzymatic hydrolysis, complete conversion (100%) of ester derivative of thiadiazine thione (10a) into its acidic counterpart (7c) was achieved during 20 min which indicated that these types of THTT ester derivatives can be a possible lead for future investigations as prodrug anticancer probes.

Sensitizing the cytotoxic action of Docetaxel induced by Pentoxifylline in a PC3 prostate cancer cell line.

BACKGROUND: Prostate cancer is one of the most frequently diagnosed types of cancers worldwide. In its initial period, the tumor is hormone-sensitive, but in advanced states, it evolves into a metastatic castration-resistant tumor. In this state, chemotherapy with taxanes such as Docetaxel (DTX) comprises the first line of treatment. However, the response is poor due to chemoresistance and toxicity. On the other hand, Pentoxifylline (PTX) is an unspecific inhibitor of phosphodiesterases; experimental, and clinically it has been described as sensitizing tumor cells to chemotherapy, increasing apoptosis and decreasing senescence. We study whether the PTX sensitizes prostate cancer cells to DTX for greater effectiveness. METHODS: PC3 human prostate cancer cells were treated in vitro at different doses and times with PTX, DTX, or their combination. Viability was determined by the WST-1 assay by spectrophotometry, cell cycle progression, apoptosis, generic caspase activation and senescence by flow cytometry, DNA fragmentation and caspases-3, -8, and -9 activity by ELISA. RESULTS: We found that PTX in PC3 human prostate cancer cells induces significant apoptosis per se and increases that generated by DTX, while at the same time it reduces the senescence caused by the chemotherapy and increases caspases-3,-8, and -9 activity in PTX + DTX-treated cells. Both treatments blocked the PC3 cell in the G1 phase. CONCLUSIONS: Our results show that PTX sensitizes prostate tumor cells to apoptosis induced by DTX. Taken together, the results support the concept of chemotherapy with rational molecular bases.

Highly Oxygenated Constituents from a Marine Alga-Derived Fungus Aspergillus giganteus NTU967.

Agar-based disc diffusion antimicrobial assay has shown that the ethyl acetate extract of the fermented broth of Aspergillus giganteus NTU967 isolated from Ulva lactuca exhibited significant antimicrobial activity in our preliminary screening of bioactive fungal strains. Therefore, column chromatography of the active principles from liquid- and solid-state fermented products of the fungal strain was carried out, and which had led to isolation of eleven compounds. Their structures were determined by spectral analysis to be seven new highly oxygenated polyketides, namely aspergilsmins A-G (1-7), along with previously reported patulin, deoxytryptoquivaline, tryptoquivaline and quinadoline B. Among these, aspergilsmin C (3) and patulin displayed promising anticancer activities against human hepatocellular carcinoma SK-Hep-1 cells and prostate cancer PC-3 cells with IC50 values between 2.7-7.3 µM. Furthermore, aspergilsmin C (3) and patulin exhibited significant anti-angiogenic functions by impeding cell growth and tube formation of human endothelial progenitor cells without any cytotoxicity.

Stevia eupatoria and Stevia pilosa Extracts Inhibit the Proliferation and Migration of Prostate Cancer Cells.

Background and Objectives: Prostate cancer is the second most harmful disease in men worldwide and the number of cases is increasing. Therefore, new natural agents with anticancer potential should be examined and the response of existing therapeutic drugs must be enhanced. Stevia pilosa and Stevia eupatoria are two species that have been widely used in traditional medicine, but their effectiveness on cancer cells and their interaction with antineoplastic drugs have not been studied. The aim of this study was to evaluate the anticancer activity of Stevia pilosa methanolic root extract (SPME) and Stevia eupatoria methanolic root extract (SEME) and their effect, combined with enzalutamide, on prostate cancer cells. Materials and Methods: The study was conducted on a human fibroblast cell line, and on androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. The cell viability was evaluated using a Trypan Blue exclusion test for 48 h, and the migration by a wound-healing assay for 24, 48, and 72 h. Results: The results indicate that SPME and SEME were not cytotoxic at concentrations less than 1000 µg/mL in the human fibroblasts. SPME and SEME significantly reduced the viability and migration of prostate cancer cells in all concentrations evaluated. The antiproliferative effect of the Stevia extracts was higher in cancer cells than in normal cells. The enzalutamide decreased the cell viability in all concentrations tested (10-50 µM). The combination of the Stevia extracts and enzalutamide produced a greater effect on the inhibition of the proliferation and migration of cancer cells than the Stevia extracts alone, but not of the enzalutamide alone. Conclusion: The results indicate that SPME and SEME have an inhibitory effect on the viability and migration of prostate cancer cells and do not interfere with the enzalutamide anticancer effect. The data suggest that Stevia extracts may be a potential source of molecules for cancer treatment.

3,6-diazabicyclo[3.3.1]heptanes Induces Apoptosis and Arrests Cell Cycle in Prostate Cancer Cells.

BACKGROUND Prostate cancer, non-cutaneous malignant tumor, is the second common cause of cancer related mortalities in American men and is responsible for 13% of deaths related to cancer. The present study investigated the anti-cancer effects of 3,6-diazabicyclo[3.3.1]heptane on LNCaP and PC3 prostate cancer cells in vitro and on tumor growth in vivo in BALB/C nude mice. MATERIAL AND METHODS Reduction of cell viability by 3,6-diazabicyclo[3.3.1]heptane was evaluated by sulphorhodamine-B staining and apoptosis onset using annexin V and propidium iodide (PI) staining. The 2',7'-dichlorofluorescein-diacetate stain was used for assessment of reactive oxygen species (ROS) formation while as western blotting for analysis of protein expression. RESULTS The viability of LNCaP and PC3 cells was reduced significantly (P<0.05) by 3,6-diazabicyclo[3.3.1]heptane in dose-based manner. At 30 µM of 3,6-diazabicyclo[3.3.1]heptane the viability of LNCaP and PC3 cells was reduced to 32 and 28%, respectively. The 3,6-diazabicyclo[3.3.1]heptane treatment increased apoptosis in LNCaP cells to 43.31% at 30 µM. The cell cycle in LNCaP cells was arrested in G1 phase on treatment with 3,6-diazabicyclo[3.3.1]heptane. The expression of cyclin D1 and p21 proteins was significantly increased by 3,6-diazabicyclo[3.3.1]heptane in LNCaP and PC3 cells. The growth of prostate tumor was also suppressed in vivo in mice by 3,6-diazabicyclo[3.3.1]heptane treatment. CONCLUSIONS In summary, the study demonstrated that LNCaP and PC3 prostate cancer cell viability is suppressed by 3,6-diazabicyclo[3.3.1]heptane treatment. The suppression of prostate cancer cell viability by 3,6-diazabicyclo[3.3.1]heptane involves apoptosis induction, cell cycle arrest and upregulation of p21 expression. Therefore, 3,6-diazabicyclo[3.3.1]heptane can be a potential chemotherapeutic agent for prostate cancer.

A new phenolic compound from Phedimus middendorffianus with antiproliferative activity.

A new phenolic compound (1) and together with 12 known compounds-eight flavonoids (2 ∼ 9), two phenolic compounds (10 and 11) and two benzoic acid (12 and 13)-were isolated from Phedimus middendorffianus (Maxim.). The structures of all compound were determined on the basis of spectroscopic (MS and NMR) analyses. Compounds 4, 5, 7 and 11 ∼ 13 were showed anti-proliferative activities against MCF-7 than PC-3 cell line. Also compound 12 and 13 showed the significant cytotoxic activities against two cancer cell lines, PC-3 and MCF-7.

Topological properties and in vitro identification of essential nodes of the Paclitaxel and Vincristine interactomes in PC-3 cells.

BACKGROUND: Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby inducing apoptosis via mitochondrial pathway activation through the modulation in the expression of the Bcl-2 family. METHODS: To describe topological features of the MTAs networks associated to intrinsic apoptosis induction in p53-null prostate cancer cells, we predicted and compared the interactomes and topological properties of Paclitaxel and Vincristine, and thus, the essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics were subjected to experimental analysis in PC-3 cell line. RESULTS: The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX, and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells exposed to both treatments. Immunoprecipitation analysis showed that treatments induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the role of BimEL as a de-repressor from sequestering complexes, in addition, new protein complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing data to the Vincristine network for p53-null cells in response to MTAs. CONCLUSION: The differences in sensitivities, protein profiles, and protein complex kinetics observed between the drugs confirmed that the selectivity and stimulation of the apoptotic system vary depending on the cell's genotype, the drug used and its exposure period.

Photoirradiation after aminolevulinic acid treatment suppresses cancer cell proliferation through the HO-1/p21 pathway.

BACKGROUND: Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to control the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. Although various studies have been published regarding cell death analysis after photoirradiation by ALA-PDT, the changes in gene expressions induced by it are yet unclear. Here, we focused on studying gene expression and cell proliferation changes in cancer cells that survive photoirradiation. METHODS: HEK293 human embryonic kidney cells, MKN45 human gastric cells, and PC-3 human prostate cancer cells were selected for this research. Cell viability was measured using trypan blue and MTT assays. ALA-PDT experiments were performed using a calibrated LED irradiation module. Furthermore, mRNA and protein gene expression analysis were performed using our previously reported methods. RESULTS: mRNAs of PAI-1, HO-1, and p21 were upregulated after photoirradiation of HEK293, which was suppressed by N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger. Primer array results in PC-3 cells and p21 and Ki-67 expression results in both PC-3 and MKN45 cells suggested that photoirradiation suppressed cell proliferation. Cell numbers post-photoirradiation revealed that the proliferation of surviving cells was suppressed in PC-3 and MKN45 cells. CONCLUSION: ALA-PDD or ALA-PDT can result in rapid ROS-induced cell death and may decrease long-term recurrence rates through several pathways including the HO-1/p21 pathway.

Silymarin Enriched Extract (Silybum marianum) Additive Effect on Doxorubicin-Mediated Cytotoxicity in PC-3 Prostate Cancer Cells.

Silymarin-enriched extract (SEE) is obtained from Silybum marianum (Asteraceae). Doxorubicin (DXR) is a widely used chemotherapeutical yet with severe side effects. The goal of the present study was to assess the pharmacologic effect of SEE and its bioactive components silibinin and silychristine when administrated alone or in combination with DXR in the human prostate cancer cells (PC-3). PC-3 cells were treated with SEE, silibinin (silybins A and B), silychristine, alone, and in combination with DXR, and cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis, and autophagy rate were assessed by flow cytometry. Expression levels of autophagy-related genes were quantified by qRT-PCR, ELISA and western blot while transmission electron microscopy was performed to reveal autophagic structures. Finally, NMR spectrometry was used to identify specific metabolites related to autophagy. SEE inhibited PC-3 cell proliferation in a dose-dependent manner while the co-treatment (DXR-SEE) revealed an additive cytotoxic effect. Cell cycle, apoptosis, and autophagy variations were observed in addition to altered expression levels of autophagy related genes (LC3, p62, NBR1, Beclin1, ULK1, AMBRA1), while several modifications in autophagic structures were identified after DXR-SEE co-treatment. Furthermore, treated cells showed a different metabolic profile, with significant alterations in autophagy-related metabolites such as branched-chain amino acids. In conclusion, the DXR-SEE co-treatment provokes perturbations in the autophagic mechanism of prostate cancer cells (PC-3) compared to DXR treatment alone, causing an excessive cell death. These findings propose the putative use of SEE as an adjuvant cytotoxic agent.

Metformin may offer no protective effect in men undergoing external beam radiation therapy for prostate cancer.

OBJECTIVES: To assess whether metformin reduces radio-resistance and increases survival in men undergoing external beam radiation therapy (EBRT) for prostate cancer (PCa), and to determine its effect on hypoxia inducible factor 1-α (HIF1α). PATIENTS AND METHODS: All patients treated with curative intent with EBRT for PCa at a major cancer centre between 2000 and 2007 were included in this study. The outcome measures of time to biochemical failure (BF), metastasis, PCa-specific mortality and overall survival (OS) were analysed in those taking metformin vs those not, using competing risk and Cox regression models. To determine metformin's effect on HIF1α expression and survival in vitro, PC3 cells with high basal HIF1α levels were subjected to increasing doses of metformin after H2 O2 -induced oxidative stress. RESULTS: A total of 2055 eligible cases, including 113 who were on metformin, were identified, with a median follow-up of 95.7 months. There were no differences in age, initial prostate-specific antigen level, Gleason score, T-stage, D'Amico risk class or duration of androgen deprivation therapy (ADT) between patients who were or were not on metformin. Treatment with metformin did not result in any apparent improvement in time to BF, time to metastasis detection or OS, but there was a 1.5-fold increase in PCa-specific deaths (P = 0.045) in patients on metformin and ADT when adjusted for cancer risk and comorbidities. When comparing patients on high-dose metformin (>1 g/d) with those on low-dose metformin (≤1 g), there was no difference in either time to metastases or time to BF. In vitro metformin at a high concentration of 100 µM did not reduce HIF1α expression, nor did metformin affect the PC3 cell survival when exposed to oxidative stress (H2 O2 ). CONCLUSIONS: No association was found between the use of metformin and time to metastasis detection, time to BF or OS in patients undergoing radiation therapy with or without ADT for PCa. In vitro, low therapeutic concentrations of metformin had no effect on HIF1α, and this observation could explain the conflicting evidence for the effectiveness of metformin in men undergoing EBRT for PCa. Higher, more toxic doses of metformin may be required to inhibit the mammalian target of rapamycin-HIF1α pathway in this patient group.

Dopamine receptor antagonists induce differentiation of PC-3 human prostate cancer cell-derived cancer stem cell-like cells.

BACKGROUND: The objective of this study was to determine whether PC-3 human prostate cancer cell-derived cancer stem cells (CSC)-like cells grown in a regular cell culture plate not coated with a matrix molecule might be useful for finding differentiation-inducing agents that could alter properties of prostate CSC. METHODS: Monolayer cells prepared from sphere culture of PC-3 cells were characterized for the presence of pluripotency and tumorigenicity. They were then applied to screen a compound library to find compounds that could induce morphology changes of cells. Mechanisms of action of compounds selected from the chemical library that induced the loss of pluripotency of cells were also investigated. RESULTS: C5A cells prepared from PC-3 cell-derived sphere culture expressed pluripotency markers such as Oct4, Sox2, and Klf4. C5A cells were highly proliferative. They were invasive in vitro and tumorigenic in vivo. Some dopamine receptor antagonists such as thioridazine caused reduction of pluripotency markers and tumorigenicity. Thioridazine, unlike promazine, inhibited phosphorylation of AMPK in a dose dependent manner. BML-275, an AMPK inhibitor, also induced differentiation of C5A cells as seen with thioridazine whereas A769663, an AMPK activator, blocked its differentiation-inducing ability. Transfection of C5A cells with siRNAs of dopamine receptor subtypes revealed that knockdown of DRD2 or DRD4 induced morphology changes of C5A cells. CONCLUSIONS: Some dopamine receptor antagonists such as thioridazine can induce differentiation of CSC-like cells by inhibiting phosphorylation of AMPK. Binding to DRD2 or DRD4 might have mediated the action of thioridazine involved in the differentiation of CSC-like cells.

Exposure to bisphenol A: current levels from food intake are toxic to human cells.

In the present work, cell lines of different origin were exposed to BPA levels from food intake reported elsewhere. Specifically, we used an in vitro assay to determine cytotoxicity of BPA in three cell lines: MCF7 (breast cancer), PC3 (prostate cancer) and 3T3-L1 (mouse fibroblast). Cytotoxic effects were observed at concentrations higher than 50 µg/mL which is above the involuntary exposure level of BPA described before in fresh, canned and frozen foods and beverages. Furthermore, medial inhibitory concentrations (IC50) of 85.17 µg/mL and 88.48 µg/mL were observed for PC3 and 3T3-L1, respectively, and a slightly lower IC50 of 64.67 µg/mL for MCF7. These results highlight BPA's toxicity potential at current levels from food intake. The cell line-dependent divergent response to BPA reported herein is discussed.

Cytotoxic Prenylated Xanthones from the Leaves of Garcinia bracteata.

Six new prenylated xanthones (1: -6: ) and seventeen known xanthones were isolated from extracts of Garcinia bracteata leaves. Their structures were determined by extensive NMR and MS spectroscopic data analysis. The inhibitory activities of the isolates were assayed on HeLa, A549, PC-3, HT-29, and WPMY-1 cell lines. Compounds 1: and 15: -17: showed moderate inhibitory effects on tumor cell growth, with IC50s ranging from 3.7 to 14.7 µM.

Effect of glucose and palmitate environment on proliferation and migration of PC3-prostate cancer cells.

Recent studies have been trying to find out how diet and metabolic changes such as dyslipidaemia, hyperglycaemia, and hyperinsulinaemia can stimulate cancer progression. This investigation aimed to evaluate the effect of high concentrations of fatty acids and/or glucose in tumour prostate cells, focusing on the proliferation/migration profile and oxidative stress. PC3 cells were treated with high concentration of saturated fatty acid (palmitate, 100 µM), glucose (220 mg/dL), or both for 24 or 48 h. Results demonstrated that PC3 cells showed a significant increase in proliferation after 48 h of treatment with glucose and palmitate+glucose. Cell proliferation was associated with reduced levels of AMPK phosphorylation in glucose group at 24 and 48 h of treatment, while palmitate group presented this result only after 48 h of treatment. Also, there was a significant increase in cell migration between time 0 and 48 h after all treatments, except in the control. Catalase activity was increased by palmitate in the beginning of treatment, while glucose presented a later effect. Also, nitrite production was increased by glucose only after 48 h, and the total antioxidant activity was enhanced by palmitate in the initial hours. Thus, we conclude that the high concentration of the saturated fatty acid palmitate and glucose in vitro influences PC3 cells and stimulates cellular activities related to carcinogenesis such as cell proliferation, migration, and oxidative stress in different ways. Palmitate presents a rapid and initial effect, while a glucose environment stimulates cells later on, maintaining high levels of cell proliferation.