Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC₅₀ doses within the range of 0.5–10 mM. IC₅₀ doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.     

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy × 10, 1 Gy × 10, and 2 Gy × 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 × 10^-73, 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 × 10^-30) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.     

Enhanced G2/M Arrest,Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133^+high/CD44^+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC₅₀ dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.     

Semi-synthesis and anti-tumor activity of novel 25-OCH3-PPD derivatives incorporating aromatic moiety

Previously we have reported that 25-OCH₃-PPD could suppress the reproduction of cancer cells and cause apoptosis without obvious toxicity. Herein, we aimed to enhance its bioactivity by introducing aromatic groups to its dammarane-type skeleton. These synthesized derivatives were tested for their inhibitory activities against five cancer cell lines. Of them, compounds 3a, 14a and 18a had the strongest antiproliferative activities against tumor cells (IC₅₀?<?15?µM, 5-fold to 10-fold increases than 25-OCH₃-PPD). Especially compound 14a displayed the most potent activity against DU145, MCF-7 and HepG2 cells (IC₅₀?=?6.7?±?0.8, 4.3?±?0.8 and 5.8?±?0.6?µM, respectively). Structure-activity relationships demonstrated that having aromatic ester at the C3 position could improve the bioactivity. The data provided new insights into exploring novel antiproliferative lead compounds.     

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU‐145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU‐145 and LNCaP cells. The IC₅₀ values decreased 1.5‐fold and 1.3‐fold in the DU‐145 and LNCaP cells respectively. In the DU‐145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl‐2 expression. Treatment with both drugs in DU‐145 cells led to an increase in sub‐G₁ arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G₂/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU‐145 through apoptosis and in LNCaP cells through cell cycle arrest (G₂/M arrest).     

Liposomes modulate docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells

The aim of this study was to investigate the effect of liposomes on docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells. Cytotoxicity of free docetaxel and docetaxel-containing liposomes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in human hepatoma cell lines HepG2 and SMMC-7721. To the cell lines, blank liposomes prepared with soybean phosphatidylcholine (SPC), dimyristoylphosphocholine (DMPC), and dioleoylphosphocholine (DOPC) did not show any significant toxicity below a 0.02-mg/mL phospholipid concentration. On the other hand, free docetaxel showed IC₅₀ values of 9.13?×?10^?6?±?1.54?×?10^?5 and 1.58?×?10^?2?±?2.71?×?10^?2 mg/mL in HepG2 cells and SMMC-7721 cells, respectively, after of 24 hours of incubation. IC₅₀ values of docetaxel-encapsulating liposomes, measured in terms of total docetaxel concentration, were at least 1.5-fold higher than those of free docetaxel. SPC liposomes reduced cellular damage caused by free docetaxel, as evidenced by the attenuation of docetaxel-induced lactate dehydrogenase (LDH) leakage by over 11% after liposome encapsulation at each dosage. Docetaxel-induced oxidative membrane damage was monitored by the formation of the lipid peroxidation product, malondialdehyde (MDA), and the antioxidative property of SPC liposome was monitored by the suppression of superoxide dismutase (SOD). These data demonstrated that free docetaxel facilitated MDA formation and suppressed SOD, and that these membrane-damaging effects were reduced by SPC liposomes.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

We investigated the effects of KML001 (NaAsO₂, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-_L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-_L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.     

Mitostatin is down-regulated in human prostate cancer and suppresses the invasive phenotype of prostate cancer cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Biphasic Alteration of Butyrylcholinesterase (BChE) During Prostate Cancer Development

Butyrylcholinesterase (BChE) is a plasma enzyme that hydrolyzes ghrelin and bioactive esters, suggesting a role in modulating metabolism. Serum BChE is reduced in cancer patients. In prostate cancer (PC), the down-regulation is associated with disease recurrence. Nonetheless, how BChE is expressed in PC and its impact on PC remain unclear. We report here the biphasic changes of BChE expression in PC. In vitro, BChE expression was decreased in more tumorigenic PC stem-like cells (PCSLCs), DU145, and PC3 cells compared to less tumorigenic non-stem PCs and LNCaP cells. On the other hand, BChE was expressed at a higher level in LNCaP cells than immortalized but non-tumorigenic prostate epithelial BPH-1 cells. In vivo, BChE expression was up-regulated in DU145 xenografts compared to LNCaP xenografts; DU145 cell-derived lung metastases displayed comparable levels of BChE as subcutaneous tumors. Furthermore, LNCaP xenografts produced in castrated mice exhibited a significant increase of BChE expression compared to xenografts generated in intact mice. In patients, BChE expression was down-regulated in PCs (n = 340) compared to prostate tissues (n = 86). In two independent PC populations MSKCC (n = 130) and TCGA Provisional (n = 490), BChE mRNA levels were reduced from World Health Organization grade group 1 (WHOGG 1) PCs to WHOGG 3 PCs, followed by a significant increase in WHOGG 5 PCs. The up-regulation was associated with a reduction in disease-free survival (P = .008). Collectively, we demonstrated for the first time a biphasic alteration of BChE, its down-regulation at early stage of PC and its up-regulation at advanced PC.     

Development of selective inhibitors for the treatment of rheumatoid arthritis: (R)-3-(3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor

A series of 3(R)-aminopyrrolidine derivatives were designed and synthesized for JAK1-selective inhibitors through the modification of tofacitinib’s core structure, (3R,4R)-3-amino-4-methylpiperidine. From the new core structures, we selected (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a scaffold for further SAR studies. From biochemical enzyme assays and liver microsomal stability tests, (R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) was chosen for further in vivo test through oral administration. Compound 6 showed improved selectivity for JAK1 compared to that of tofacitinib (IC₅₀ 11, 2.4?×?10², 2.8?×?10³, and 1.1?×?10²?nM for JAK1, JAK2, JAK3, and TYK2, respectively). In CIA and AIA model tests, compound 6 exhibited similar efficacy to tofacitinib citrate.     

Two-stage association study in Chinese Han identifies two independent associations in CCR1/CCR3 locus as candidate for Beh?et’s disease susceptibility

Previous GWAS studies from Turkey suggested a potential risk locus at CCR1/CCR3 for Beh?et’s disease. However, this locus did not reach the GWAS significance threshold and has not yet been examined in other ethnic populations. The current study aimed to explore whether this locus was associated with Beh?et’s disease in Chinese Han and the functional role of the identified variants. A two-stage association study was performed in 653 patients and 1,685 controls using the iPLEX system. Real-time PCR was performed to examine the expression level of CCR1 and CCR3 genes. Haplotype analysis was used to construct the haplotype block. Logistic regression analysis was applied to calculate the independence of multiple associations. Bonferroni correction was applied to account for multiple testing. First stage analysis showed that ten SNPs, located in 3′UTR, 5′UTR in CCR1 or 5′UTR in CCR3, were significantly associated with Beh?et’s disease (P _c?=?0.018 to 1.3?×?10^?3). The associations of six SNPs within this locus are independent after control for the genetic effect of rs17282391 using logistic regression analysis. Haplotype analysis identified three associated haplotypes: H3 (GTGAC), H6 (CCATTA) and H9 (CGA) (P _c?=?0.04 to 7.79?×?10^?4). Three SNPs rs13084057, rs13092160 and rs13075270 showed consistent association in replication and combining studies (replication P _c?=?5.31?×?10^?5 to 1.44?×?10^?5; combining P _c?=?2.76?×?10^?7 to 6.50?×?10^?8). Interestingly, eQTLs database reveals that SNP rs13092160 is eQTLs SNP, suggesting that this SNP is likely to be functional SNP that directly affects gene expression. The expression of CCR1 and CCR3 was increased in individuals with the CT genotype of rs13092160 (P?<?0.05). No significant difference was found for the mRNA level of CCR1 and CCR3 between Beh?et’s patients and controls. These findings strongly indicate CCR1/CCR3 as a novel locus underlying Beh?et’s disease.     

Investigation of microRNA expression changes in HepG2 cell line in presence of URG4/URGCP and in absence of URG4/URGCP suppressed by RNA interference

Hepatocellular carcinoma (HCC) originates from liver cells and is one of the most common malignant cancers in the world. microRNAs (miRNA), are single strand non-coding RNA molecules with the length of 18–25 nucleotides. miRNAs play an important role in the development of HCC, i.e., miRNAs have a significant impact on multistep hepatocellular carcinogenesis including cellular migration and invasion. URG4/URGCP (up-regulated gene-4/upregulator of cell proliferation) is up-regulated in the presence of HBxAg and has been identified and characterized by Satiroglu-Tufan et al. The full-length URG4/URGCP is 3.607?kb. Overexpression of URG4/URGCP in the presence of HBV X protein may function as a putative oncogene that significantly contributes to multi-step hepatocarcinogenesis. In this study, we aimed to investigate potential miRNA expression changes in HepG2 cell line model system in the presence of URG4/URGCP and in the absence of URG4/URGCP, which was suppressed by RNA interference. To functionally characterize URG4/URGCP, independent cultures of HepG2 cells were stably transfected with pcDNA3 or pcDNA3-URG4/URGCP. Relative quantification of whole genome miRNAs was analyzed by RT-PCR using human whole genome miRNA qPCR profiling kits. Among the 1,034 human miRNAs investigated by the arrays, 77 miRNAs were up-regulated and nine miRNAs were down-regulated in the presence of URG4/URGCP. In conclusion, we have analyzed miRNA profiles in HepG2 cells in presence or absence of URG4/URGCP gene using RNA interference. Some of these miRNAs may play roles in URG4/URGCP gene related disease development through the regulation of different signaling pathways.     

Analysis of HLA–ABC locus-specific transcription in normal tissues

We developed a novel human leukocyte antigen HLA–ABC locus-specific quantitative real-time polymerase chain reaction (PCR) to determine the locus-specific gene expression of HLA–ABC in peripheral blood leukocytes (PBLs, n?=?53), colon mucosa (n?=?15), and larynx mucosa (n?=?15). Laser-assisted tissue microdissection allowed us to study the selected cells without interference from surrounding stroma. We report evidence on the specificity of the technique, describing the HLA–ABC locus-specific gene expression patterns found in the PBLs and two solid tissues studied. PBLs showed a higher gene expression of HLA-B than of HLA-A or HLA-C (p?=?4.7?×?10^?10 and p?=?1.6?×?10^?6, respectively). In solid tissue, HLA-A and HLA-B gene expressions were similar and HLA-C expression lower. In particular, in larynx mucosa, significant differences were found between HLA-A and HLA-C expressions and between HLA-B and HLA-C expressions (p?=?6.5?×?10^?4 and p?=?8.1?×?10^?4, respectively). The same differences were observed in colon mucosa, but significance was not reached (p?=?0.08 and p?=?0.06, respectively). Differences in locus-specific regulation may be related to the control of cytotoxic responses of NK and CD8 positive T cells. Gene expression of HLA–ABC specific locus showed no intra-individual variability, but there was a high inter-individual variability. This may result from differences in the expression of common regulatory factors that control HLA–ABC constitutive expression.     

Synthesis,biological evaluation and SAR of naftopidil-based arylpiperazine derivatives

For the development of potential anti-prostate cancer agents, 24 kinds of novel naftopidil-based arylpiperazine derivatives have been synthesized and characterized by spectroscopic methods. Their antitumor activities were evaluated against several classical prostate cancer cell lines including PC-3, LNCaP, and DU145. Among all the compounds, 9, 13, 17, 21 and 27 showed strong cytotoxic activities against DU145 cells (IC₅₀?<?1?μM). Further testing confirmed that compound 17 inhibited the growth of DU145 cells by inducing cell cycle arrest at G0/G1 phase. Besides, antagonistic activities of compounds (9, 13, 17, 21 and 27) towards a₁-ARs (α_1A, α_1B, and α_1D) were further evaluated using dual-luciferase reporter assays, and the compounds 13 and 17 exhibited better a₁-ARs subtype selectivity. The structure–activity relationship (SAR) of these developed arylpiperazine derivatives was rationally discussed. Taken together, these results suggested that further development of such compounds may be of great interest.     

Synthesis and anticancer activity of novel 9,13-disubstituted berberine derivatives

Novel berberine derivatives with disubstituents on positions C9 and C13 were synthesized and evaluated for antiproliferative activities against human prostate cancer cell lines (PC3 and DU145), breast cancer cell line (MDA-MB-231) and human colon cancer cell lines (HT29 and HCT116). All compounds showed significantly enhanced antiproliferative activities compared with berberine. Notably, compound 18e exhibited the strongest cytotoxicity against PC3 cells with an IC₅₀ value of 0.19 μM, and the highest selectivity index (SI^PC3 > 20). Further studies showed that 18e could arrest the cell cycle at G1 phase, and significantly inhibit tumor cell colony forming and migration even at low concentrations. Interestingly, 18e could significantly induce cytoplasmic vacuolation, suggesting a different mode of action from berberine.     

mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN^?/? LNCaP and androgen independent (AR?), PTEN^+/? DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (?) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.     

Rational design,molecular docking and synthesis of novel homopiperazine linked imidazo[1,2-a]pyrimidine derivatives as potent cytotoxic and antimicrobial agents

Designed and synthesized novel homopiperazine linked imidazo[1,2-a]pyrimidine derivatives (10a–i, 11a–g, 12), and evaluated them for their in vitro cytotoxicity against HeLa cells (cervical cancer), A549 cells (lung cancer) cells, by MTT assay. Compound 12 (IC₅₀ = 4.14 µM) and compound 10c (IC₅₀ = 5.98 µM) were found to be 2.5 fold, and 1.74 fold more potent when compared with standard Etoposide (IC₅₀ = 10.44 µM), against A549 (lung cancer cells). Compound 12 also found to be 1.57 and 1.13 fold potent against DU145 (IC₅₀ = 6.24 µM) and HeLa (IC₅₀ = 6.54 µM), respectively when compared with Etoposide (DU145, IC₅₀ = 9.8 µM; HeLa, IC₅₀ = 7.43 µM). Compound 10f (IC₅₀ = 6.12 µM) was found to be 1.31 fold more potent than Etoposide (IC₅₀ = 7.43 µM) against HeLa cell lines.Moreover compounds 10a and 11a showed cytotoxicity at low micro-molar concentrations against A549 cells. Synthesized compounds were also evaluated for their antimicrobial activity by Cup plate diffusion method. Compounds 10c, 11b, 11d and 11f displayed remarkable antimicrobial activity relating to their standard drugs Gentamycin, Amphotericin B and Ampicillin. Significantly, compound 10c showed broad spectrum activity against tested microbial strains. All the designed compounds were well occupied the binding site of the colchicine and interacted with both α- and β-tubuline interface (PDB ID: 3E22), which demonstrates that synthesized compounds are promising tubulin inhibitors. Also, the synthesized compounds occupied the catalytic triad and adenine-binding site, in the active site of β-ketoacyl-acyl carrier protein synthase III enzyme (PDB ID: 1MZS). The molecular docking results provided the useful information for the future design of more potent inhibitors. These preliminary results convinced further investigation and modifications on synthesized compounds aiming towards the development of potential cytotoxic as well as antimicrobial agents.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.