Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18INK4c , p21 waf1/cip1 , and p27 Kip1 all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18INK4c and p27 Kip1 expression positively correlate with growth arrest, whereas p21 waf1/cip1 is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18INK4c is found in increasing association with CDK6, whereas p27Kip1 associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growthconducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) conditions and apparatus:10 μl sample was injected into ultra-performance liquid chromatography (UPLC) instrument equipped with reverse phase C-18 column (ACQUITY UPLC -BEH C18, 1.7 µm particle size -2.1 × 50 mm Column). Sample mobile phase was prepared by 0.2 μm filter membrane disc and degassed by sonication before injection. Mobile phase elution was made using gradient mobile phase comprising two eluents: eluent A is 0.1% formic acid in water and eluent B is 0.1% formic acid in methanol. Elution was performed using the following gradient: 0-2 min (90%) A: (10%) B, 2-5 min (70%) A: (30%) B, 5-10 min. (30%) A: (70%) B, 15-25 min (10%) A: (90%) B, 25-29 min (100%) B, finally, 29-32 min (90%) A: (10%) B. The flow rate of the mobile phase was 0.2 ml/min, and the injection volume was 10 𝜇l.

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Novel halogenated phenoxychalcones 2a–f and their corresponding N -acetylpyrazolines 3a–f were synthesised and evaluated for their anticancer activities against breast cancer cell line (MCF-7) and normal breast cell line (MCF-10a), compared with staurosporine. All compounds showed moderate to good cytotoxic activity when compared to control. Compound 2c was the most active, with IC 50 = 1.52 µM and selectivity index = 15.24. Also, chalcone 2f showed significant cytotoxic activity with IC 50 = 1.87 µM and selectivity index = 11.03. Compound 2c decreased both total mitogen activated protein kinase (p38α MAPK) and phosphorylated enzyme in MCF-7 cells, suggesting its ability to decrease cell proliferation and survival. It also showed the ability to induce ROS in MCF-7 treated cells. Compound 2c exhibited apoptotic behaviour in MCF-7 cells due to cell accumulation in G2/M phase and elevation in late apoptosis 57.78-fold more than control. Docking studies showed that compounds 2c and 2f interact with p38alpha MAPK active sites.

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The main objective of this work was to synthesize novel compounds with a benzo[de][1,2,4]triazolo[5,1-a]isoquinoline scaffold by employing (dioxo-benzo[de]isoquinolin-2-yl) thiourea as a building block. Molecular docking was conducted in the COX-2 active site to predict the plausible binding mode and rationalize the structure–activity relationship of the synthesized compounds. The structures of the synthesized compounds were confirmed by HREI-MS, and NMR spectra along with X-ray diffraction were collected for products 1 and 5. Thereafter, anti-inflammatory effect of molecules 1–20 was evaluated in vivo using carrageenan-induced paw edema method, revealing significant inhibition potency in albino rats with an activity comparable to that of the standard drugs indomethacin. Compounds 8, 9, 15 and 16 showed the highest anti-inflammatory activity. However, thermal sensitivity-hot plat test, a radiological examination and motor coordination assessment were performed to test the activity against rheumatoid arthritis. The obtained results indicate promising anti-arthritic activity for compounds 9 and 15 as significant reduction of the serum level of interleukin-1β [IL-1β], cyclooxygenase-2 [COX-2] and prostaglandin E2 [PGE2] was observed in CFA rats.Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-017-0321-1) contains supplementary material, which is available to authorized users.

The aim of the present study was designed to evaluate the hepatoprotective and antioxidant potentials of GSE (100 and 200 mg/kg) and/or silymarin against TAA-induced liver fibrosis in rats. This study was designed to investigate the protective effect of grape seed extract (GSE) and/or silymarin against thioacetamide (TAA)-induced hepatic fibrosis in Sprague-Dawley rats. Mature male Sprague-Dawley rats were divided into 7 equal groups (8 rats each) and treated as follows: Group 1, kept as control group and orally given saline; groups 2-7 were injected intraperitoneally (i.p.) with TAA (100 mg/Kg) twice weekly for 6 weeks to induce hepatic fibrosis. Group 2, kept as control positive; groups 3-5 were administered daily oral doses of silymarin (50 mg/kg), GSE (100 mg/kg) and GSE (200 mg/kg), respectively. While groups 6-7 were administered combined treatments of silymarin and GSE (100 mg/kg) or GSE (200 mg/kg), respectively. Our results indicated that TAA caused significant elevation of hydroxyproline (Hyp), malondialdehyde (MDA) and nitric oxide (NO) contents in liver homogenate and increased serum levels of: aminotransferases (AST and ALT), alkaline phosphatase (ALP) and total bilirubin. While, TAA-treatment alone significantly decreased serum total protein and reduced glutathione (GSH) content in liver homogenate. Administration of GSE (100 and 200 mg/kg) and/or silymarin attenuated TAA-induced hepatic fibrosis, improved enzymes and reduced the oxidative stress in dose dependant manner Histopathological study showed disruption of the hepatic architecture and collagen fibers deposition in the portal tract of TAA-injected group. Concomitant treatment with GSE (100 and 200 mg/kg) and/or silymarin significantly improved histopathological structure of liver tissue in variable degrees. In conclusion, the combined effect of GSE (200 mg/kg) with silymarin (50 mg/kg) had powerful hepatoprotective effect than any other studied doses.

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