The growth suppressing effects of girinimbine on HepG2 involve induction of apoptosis and cell cycle arrest

Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G(0)/G(1) peak (hypodiploid) and caused G(0)/G(1)-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.     

Dielectrophoretic studies of the activation of human T lymphocytes using a newly developed cell profiling system

Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty-four hours post-activation the cells were harvested for DNA content and for measurements using a newly developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (+/- 4.72) mF/m(2) to 10.62 (+/- 5.13) mF/m(2). This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0-fold decrease of cells in S-phase, and a 1.5-fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.     

Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.     

Changes in expression of cell cycle regulators after G1 progression upon repetitive thioacetamide treatment in rat liver

Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).     

Enhanced delivery of gamma-secretase inhibitor DAPT into the brain via an ascorbic acid mediated strategy

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce pathogenic Abeta peptides, is an attractive approach for the treatment of Alzheimer's disease. We designed a gamma-secretase inhibitor bearing an ascorbic acid moiety which allows a specific delivery of the drug to the brain. Through, on the one hand, Abeta peptide production measurements by specific in vitro assays (gamma-secretase cell free assay and cell based assay on HEK 293 APP transfected cells) and on the other hand through pharmacokinetic studies on animal models, the new inhibitor shows a good pharmacokinetic profile as well as a potent gamma-secretase inhibitory activity in vitro. From the obtained results, it is expected that drug will be mainly delivered to the CNS with a low diffusion in the peripheral tissues. Consequently the side effects of this gamma-secretase inhibitor on the immune cells could be reduced.     

Activation of heat shock protein 70 promoter with meso-tetrahydroxyphenyl chlorin photodynamic therapy reported by green fluorescent protein in vitro and in vivo

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT-mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue-exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/ GFP-transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.     

Novel Bradykinin Receptor Inhibitors Inhibit Proliferation and Promote the Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the ERK Pathway

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Studies have shown that bradykinin (BK) is highly expressed in liver cancer. We designed the novel BK receptor inhibitors J051-71 and J051-105, which reduced the viability of liver cancer cells and inhibited the formation of cancer cell colonies. J051-71 and J051-105 reduced cell proliferation and induced apoptosis in HepG2 and BEL-7402 cells, which may be due to the inhibition of the extracellular regulated protein kinase (ERK) signaling pathway. In addition, these BK receptor inhibitors reversed the cell proliferation induced by BK in HepG2 and BEL-7402 cells by downregulating B1 receptor expression. Inhibiting B1 receptor expression decreased the protein levels of p-ERK and reduced the malignant progression of HCC, providing a potential target for HCC therapy.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.     

Trichostatin A induces apoptosis in lung cancer cells via simultaneous activation of the death receptor-mediated and mitochondrial pathway?

Trichostatin A (TSA), originally developed as an antifungal agent, is one of potent histone deacetylase (HDAC) inhibitors, which are known to cause growth arrest and apoptosis induction of transformed cells, including urinary bladder, breast, prostate, ovary, and colon cancers. However, the effect of HDAC inhibitors on human non-small cell lung cancer cells is not clearly known yet. Herein, we demonstrated that treatment of TSA resulted in a significant decrease of the viability of H157 cells in a dose-dependent manner, which was revealed as apoptosis accompanying with nuclear fragmentation and an increase in sub-G0/G1 fraction. In addition, it induced the expression of Fas/FasL, which further triggered the activation of caspase-8. Catalytic activation of caspase-9 and decreased expression of anti-apoptotic Bcl-2 and Bcl-XL proteins were observed in TSA-treated cells. Catalytic activation of caspase-3 by TSA was further confirmed by cleavage of pro-caspase-3 and intracellular substrates, including poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). In addition, a characteristic phenomenon of mitochondrial dysfunction, including mitochondrial membrane potential transition and release of mitochondrial cytochrome c into the cytosol was apparent in TSA-treated cells. Taken together, our data indicate that inhibition of HDAC by TSA induces the apoptosis of H157 cells through signaling cascade of Fas/FasL-mediated extrinsic and mitochondria-mediated intrinsic caspases pathway.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

An automated method for in vitro anticancer drug efficacy monitoring based on cell viability measurement using a portable photodiode array chip

An integrated circuit (IC) bipolar semiconductor photodiode array (PDA) microchip system coupled with light emitting diodes (LEDs) was used for rapid, automated cell viability measurements and high-throughput drug efficacy monitoring. Using the absorption property of trypan blue dye against the red light emitted by LEDs, we determined the effect of three anticancer drugs, viz., camptothecin (CAM), sodium salicylate (Na-Sal) and naringenin (Nar) on the cell viability of human promyelocytic leukemia cells (HL-60) and human embryonic kidney cells (HEK-293). Cell viabilities were measured based on the relative reduction in the photo responses of the photodiodes, covered with known concentration of trypan blue-stained cells. The developed method offers greater sensitivity and hence an excellent estimation of cell viability, but without all the hassle of conventional methods. Flow cytometric measurement and confocal microscopy were applied as complementary techniques for further validation of the results. The work presented here has important implications with regard to high-throughput measurement of optimal concentrations of different drugs against different cell lines in vitro.     

Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a–2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI₅₀ concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.     

Antiproliferation and Induction of Apoptosis in Ca9-22 Oral Cancer Cells by Ethanolic Extract of <em>Gracilaria tenuistipitata</em>

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.     

Brassinin inhibits proliferation and induces cell cycle arrest and apoptosis in nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging.ObjectiveHere, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells.MethodologyThe in vitro free radical scavenging property of the brassinin was assessed by various free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1–50 µM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apoptosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits.ResultsThe brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not disturbed the Vero cell viability. The 30 µM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with ‘S’ and ‘G2/M’ phase. The Bax, caspase-3 and ?9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells.ConclusionOur findings discovered that the brassinin has the capacity to prevent the proliferation and stimulate the apoptotic cell death C666‐1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future.     

The Polyphenols α-Mangostin and Nordihydroguaiaretic Acid Induce Oxidative Stress,Cell Cycle Arrest,and Apoptosis in a Cellular Model of Medulloblastoma

Medulloblastoma is a common malignant brain tumor in the pediatric age. The current therapeutics present serious collateral effects. Polyphenols α-mangostin and nordihydroguaiaretic acid (NDGA) exert potent antitumoral activity in different cancer models, although their antitumoral effects have not been described in medulloblastoma cells yet. This study aimed to examine the proapoptotic effects of these polyphenols on human medulloblastoma cells. Medulloblastoma cell line Daoy was incubated with increasing concentrations of α-mangostin or NDGA for 24 h. The cell viability was analyzed using crystal violet and trypan blue dyes. Determination of the glutathione (GSH)/glutathione disulfide (GSSG) ratio and levels of carbonylated proteins was performed to evaluate the oxidative stress. Cell cycle progression and induction of cell death by fluorochrome-couple and TUNEL assays were evaluated using flow cytometry assays. Individual treatments with α-mangostin or NDGA decreased the viability of Daoy cells in a dose-dependent manner, inducing G2/M and S-G2/M cell cycle arrest, respectively. Both polyphenols induced cell death and increased oxidative stress. Very interestingly, α-mangostin showed more potent effects than NDGA. Our results indicate that α-mangostin and NDGA exert important cytostatic and cytotoxic effects in the Daoy cell line. These data highlight the potential usefulness of these compounds as an alternative strategy in medulloblastoma treatment.     

Two-dimensional database of a Burkitt lymphoma cell line (DG 75) proteins: protein pattern changes following treatment with 5'-azycytidine.

Hypermethylation is an important mechanism for repression of tumor gene suppressor in cancer. The drug 5'-azacytidine (AZC) has been used as demethylating agent to induce the expression of previously silencing genes. In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of an Epstein Barr virus (EBV)-negative Burkitt lymphoma (BL) cell line DG 75. The effects of the treatment in terms of cell viability and growth were first examined. The following observations were made: AZC treatment led to (i) a decrease in cell growth with an arrest of the cell at G0/G1 phase of the cell cycle, (ii) the expression of p16, a tumor-suppressor gene whose expression was dependent on its promoter demethylation. Proteomic study evidenced that AZC treatment affected protein expression in two different ways. Twenty-one polypeptides were down-expressed, while 14 showed an increased expression. Some of the upregulated proteins appeared related to the energy metabolism, to organization of cytoskeletal structures, and to cell viability and protein synthesis. We also established a reference map for proteins in DG 75 cell line, comprising 74 different polypeptides corresponding to 67 proteins. This map will be accessible via Internet as a resource for proteome analyses of B-cells. Taken together, the results presented here highlight new insights into lymphoma cell gene regulations following a treatment of lymphoma cells with AZC and illustrate a use of proteomics to evidence the direct and indirect effects of a drug and the pathways it possibly regulates.     

Stereoselective synthesis of photoreactive peptidomimetic gamma-secretase inhibitors

The first asymmetric synthesis of novel, potent photoreactive gamma-secretase inhibitors 2 and 3 has been accomplished. Two stereoselective methods for the preparation of lactone 9 are described. Protected benzophenone intermediate 19 is prepared via an aldol-elimination reaction followed by a PtO(2)-catalyzed asymmetric hydrogenation. Two routes leading from 19 to compounds 2 and 3 are evaluated. The application of 3 as an activity-based probe has been demonstrated by localizing gamma-secretase activity in the plasma membrane of intact cells.     

High-throughput cell cycle synchronization using inertial forces in spiral microchannels

Efficient synchronization and selection of cells at different stages of the cell replication cycle facilitates both fundamental research and development of cell cycle-targeted therapies. Current chemical-based synchronization methods are unfavorable as these can disrupt cell physiology and metabolism. Microfluidic systems developed for physical cell separation offer a potential alternative over conventional cell synchronization approaches. Here we introduce a spiral microfluidic device for cell cycle synchronization, using the combined effects of inertial forces and Dean drag force. By exploiting the relationship between cell diameter and cell cycle (DNA content/ploidy), we have successfully fractionated several asynchronous mammalian cell lines, as well as primary cells comprising bone marrow-derived human mesenchymal stem cells (hMSCs), into enriched subpopulations of G0/G1 (>85%), S, and G2/M phases. This level of cell cycle enrichment is comparable to existing microfluidic systems, but the throughput (～ 15 × 10(6) cells per h) and viability (～ 95%) of cells thus synchronized are significantly greater. Further, this platform provides rapid collection of synchronized cells or of diameter-sorted cells post-separation, to enable diverse applications in the study and manipulation of cell proliferation.     

Solution-phase parallel synthesis of carbamates as gamma-secretase inhibitors

A novel methodology for parallel liquid-phase synthesis of carbamates suitable for the preparation of sterically hindered molecules is disclosed. The alcohols are converted to 4-nitrophenylcarbonates, followed by the reaction with amines. Side product 4-nitrophenol and the unreacted excess amines are scavenged by appropriately chosen cleanup resins, selected among Amberlyst A26 (hydroxide form) and macroporous sulfonic acid (MP-TsOH) or polystyrene isocyanate (PS-NCO) and polystyrene benzaldehyde (PS-PhCHO) resins. As a part of a medicinal chemistry program directed toward finding gamma-secretase inhibitors as prospective drug candidates for Alzheimer's disease, a 6 x 24 library of carbamates was prepared. Out of 144 library members, 133 had a purity for the targeted compound of 80% or better. The prepared compounds were assessed in the gamma-secretase inhibition assay and demonstrated activity with IC 50 values in the range from 1 microM to 5 nM, with the activity of 7 compounds being better than 10 nM.