GLC-MS profiling of non-polar extracts from Phlomis bucharica and P. salicifolia and their cytotoxicity

Phlomis species (Phlomis bucharica Regel and P. salicifolia Regel) have been traditionally used by Uzbek people as stimulant, tonic, diuretic, and in the treatment of ulcers, hemorrhoids, wounds and gynecological problems. In the present study, we characterized the chemical composition of non-polar extracts from P. bucharica and P. salicifolia by high resolution GLC-MS and evaluated their cytotoxicity. Concentrations of hexadecanoic acid in hexane and chloroform extracts were higher in P. bucharica than in P. salicifolia. 1,8- Cineol, camphor, borneol, α-terpinol, thymol, and isobornyl acetate were detected in P. bucharica but not in P. salicifolia. About 45 components were identified in P. bucharica and 40 in P. salicifolia. The chloroform extract from P. bucharica showed cytotoxicity in HeLa and HL-60 cells, with IC50 values of 26.07 and 29.42 μg/ml, respectively

Avaliação da capacidade citotóxica de macroalgas da costa de Peniche (Portugal) em células tumorais humanas

Dissertação de Mestrado em Biotecnologia dos Recursos Marinhos apresentada à ESTM - Escola Superior de Turismo e Tecnologia do Mar do Instituto Politécnico de LeiriaOs tumores malignos são uma das maiores causas de morte em humanos, podendo tornar-se na primeira causa de morte nos países desenvolvidos em 2015. Os organismos marinhos produzem moléculas bioativas, normalmente como metabolitos secundários em resposta a pressões ecológicas. Estudos anteriores têm demonstrado que estes organismos são uma fonte importante de novas moléculas com potencial farmacológico contra o cancro. Neste trabalho foi avaliado o potencial antitumoral de doze algas da costa de Peniche em células do adenocarcinoma colorectal humano (Caco-2) e do carcinoma hepatocelular humano (HepG-2). As algas estudadas foram Fucus spiralis Halopteris filicina, Saccorhiza polyschides, Stypocaulon scoparium, Asparagopsis armata, Plocamium cartilagineum, Ceramium ciliatum, Sphaerococcus coronopifolius, Codium adhaerens, Codium tomentosum, Codium vermilara e Ulva compressa. A extração dos compostos bioativos foi realizada com solventes de diferente polaridade (metanol, diclorometano e n-Hexano). O potencial antitumoral das frações metanólica e diclorometano foi testado nas linhas celulares Caco-2 e HepG-2 através de ensaios de citotoxicidade e proliferação celular. Os resultados foram obtidos pela realização de ensaios espectrofotométricos, método do 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), e por ensaios fluorimétricos, método de calceína-AM. As algas que exibiram maior potencial citotóxico pela redução da viabilidade celular (1mg.ml-1, 24h) foram as mesmas para linha celular Caco-2 e HepG-2, a alga A. armata, F. spiralis e S. coronopifolius, com a exceção da fração diclorometano da P. cartilagineum que exibiu maior efeito nas células Caco-2 (9,77 ± 2,28% células viáveis). Na fração metanólica observaram-se efeitos citotóxicos marcados para as algas F. spiralis (23,68 ± 2,93% de células Caco-2 viáveis; 44,04 ± 5,44% de células HepG-2 viáveis), A. armata (7,32 ± 1,17% de células Caco-2 viáveis; 11,22 ± 2,98% de células HepG-2 viáveis) e S. coronopifolius (3,53 ± 1,26% de células Caco-2 viáveis; 14,04 ± 2,62% de células HepG-2 viáveis). Na fração diclorometano as algas A. armata (1,04 ± 0,39% de células Caco-2 viáveis; 1,51 ± 0,38% de células HepG-2 viáveis) e S. coronopifolius (1,92 ± 0,89% de células Caco-2 viáveis; 12,84 ± 3,82% de células HepG-2 viáveis) foram as mais citotóxicas. Todos os efeitos observados foram dependentes da concentração. A fração diclorometano da alga S. coronopifolius demonstrou ser a mais potente na redução da viabilidade das celulas Caco-2 e HepG-2, com IC50 de 21,3 e 14,1 ƒÊg ml-1, respetivamente. Na proliferacao celular, as fracoes metanolicas (1mg m-1, 24h) que apresentaram maiores efeitos de inibicao nas linhas tumorais utilizadas foram a A. armata (24,32 } 7,56% de celulas Caco-2; 43,42 } 7,69% de celulas HepG-2) e S. coronopifolius (34,84 } 4,45% de celulas Caco-2; 44,87 } 3,64% de celulas HepG-2) para ambas as linhas celulares e a F. spiralis (44,60 } 6,75% de celulas), apenas para as celulas HepG-2. Na fracao diclorometano (1mg m-1, 24h) a A. armata (0 } 0,48% de celulas Caco-2; 1,44 } 0,81% de celulas HepG-2), P. cartilagineum (4,95 } 1,19% de celulas Caco-2; 14,87 } 1,04% de celulas HepG-2) e S. coronopifolius (0,96 } 0,51 de celulas Caco-2; 0,39 } 0,27% de celulas HepG-2) exibiriam o maior efeito de inibicao na proliferacao celular das celulas Caco-2 e HepG-2. Todos os efeitos observados foram dependentes da concentracao. A fracao diclorometano da S. coronopifolius exibiu o efeito mais potente sobre a proliferacao das celulas Caco-2 e HepG-2, com um IC50 de 36,5 e 32,3 ƒÊg ml-1, respetivamente. Conclui-se que as algas Asparagopsis armata, Fucus spiralis, Plocamium cartilagineum e Sphaerococcus coronopifolius produzem moleculas bioativas com elevado potencial antitumoral contra as celulas Caco-2 e HepG-2, sendo portantouma fonte promissora de novos compostos com aplicacao terapeutica no cancro

<i>HAMP</i> Downregulation Contributes to Aggressive Hepatocellular Carcinoma via Mechanism Mediated by Cyclin4-Dependent Kinase-1/STAT3 Pathway

Background: Hepcidin encoded by HAMP is vital to regulating proliferation, metastasis, and migration. Hepcidin is secreted specifically by the liver. This study sought to examine the functional role of hepcidin in hepatocellular carcinoma (HCC). Methods: Data in the Cancer Genome Atlas database was used to analyze HAMP expression as it relates to HCC prognosis. We then used the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell assay, and flow cytometric analysis, respectively, to assess proliferation, migration, and the cell cycle. Gene set enrichment analysis (GSEA) was used to find pathways affected by HAMP. Results: HAMP expression was lower in hepatocellular carcinoma samples compared with adjacent normal tissue controls. Low HAMP expression was linked with a higher rate of metastasis and poor disease-free status. Downregulation of HAMP induced SMMC-7721 and HepG-2 cell proliferation and promoted their migration. HAMP could affect the cell cycle pathway and Western blotting, confirming that reduced HAMP levels activated cyclin-dependent kinase-1/stat 3 pathway. Conclusion: Our findings indicate that HAMP functions as a tumor suppressor gene. The role of HAMP in cellular proliferation and metastasis is related to cell cycle checkpoints. HAMP could be considered as a diagnostic biomarker and targeted therapy in HCC

The Effect and Mechanism of Tamoxifen-Induced Hepatocyte Steatosis in Vitro

The aim of this study was to determine the effect and mechanism of tamoxifen (TAM)-induced steatosis in vitro. HepG 2 (Human hepatocellular liver carcinoma cell line) cells were treated with different concentrations of TAM for 72 h. Steatosis of hepatocytes was determined after Oil Red O staining and measurement of triglyceride (TG) concentration. The expressions of genes in the TG homeostasis pathway, including sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD), carnitine palmitoyltransferase 1 (CPT1) and microsomal triglyceride transfer protein (MTP), were examined using quantitative real-time PCR and Western blot analysis. Cell proliferation was examined using the cell counting kit-8 (CCK-8) assay. We found that hepatocytes treated with TAM had: (1) induced hepatocyte steatosis and increased hepatocyte TG; (2) upregulation of SREBP-1c, FAS, ACC, SCD and MTP mRNA expressions (300%, 600%, 70%, 130% and 160%, respectively); (3) corresponding upregulation of protein expression; and (4) no difference in HepG 2 cell proliferation. Our results suggest that TAM can induce hepatocyte steatosis in vitro and that the enhancement of fatty acid synthesis through the upregulations of SREBP-1c and its downstream target genes (FAS, ACC and SCD) may be the key mechanism of TAM-induced hepatocyte steatosis

β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells

Objective. To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. Methods. After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. Results. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. Conclusion. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα