RESUMO
PURPOSE: Although the effect of lysosome-associated protein transmembrane 4 beta (LAPTM4B) on the proliferation, migration, and invasion of breast cancer (BC) cells has already been studied, its specific role in BC progression is still elusive. Here, we evaluated the effect of different levels of LAPTM4B expression on the proliferation, invasion, adhesion, and tumor formation abilities of BC cells in vitro, as well as on breast tumor progression in vivo. METHODS: We investigated the influence of LAPTM4B expression on MCF-7 cell proliferation, invasion, adhesion, and tube formation abilities in vitro through its overexpression or knockdown and on breast tumor progression in vivo. RESULTS: Cell growth curves and colony formation assays showed that LAPTM4B promoted the proliferation of breast tumor cells. Cell cycle analysis results revealed that LAPTM4B promoted the entry of cells from the G1 into the S phase. Transwell invasion and cell extracellular matrix adhesion assays showed that LAPTM4B overexpression increased the invasion and adhesion capabilities of MCF-7 cells. More branches were observed in MCF-7 cells overexpressing LAPTM4B under an electron microscope. In comparison with LAPTM4B overexpression, LAPTM4B knockdown decreased the expression of vascular endothelial growth factor-A and significantly inhibited the vasculogenic tube formation ability of tumors. These results were also verified with western blot analysis. CONCLUSION: LAPTM4B promoted the proliferation of MCF-7 cells through the downregulation of p21 (WAF1/CIP1) and caspase-3, and induced cell invasion, adhesion, and angiogenesis through the upregulation of hypoxia-inducible factor 1 alpha, matrix metalloproteinase 2 (MMP2), and MMP9 expression. This specific role deems LAPTM4B as a potential therapeutic target for BC treatment.
Assuntos
Western Blotting , Neoplasias da Mama , Mama , Caspase 3 , Ciclo Celular , Progressão da Doença , Regulação para Baixo , Matriz Extracelular , Fator 1 Induzível por Hipóxia , Técnicas In Vitro , Metaloproteinase 2 da Matriz , Células MCF-7 , Fase S , Regulação para Cima , Fator A de Crescimento do Endotélio VascularRESUMO
PURPOSE: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells. MATERIALS AND METHODS: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. RESULTS: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secretion in mouse Leydig cells. CONCLUSIONS: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.
Assuntos
Animais , Humanos , Masculino , Camundongos , Apoptose , Western Blotting , Contagem de Células , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células Germinativas , Guanosina Monofosfato , Infertilidade Masculina , Lentivirus , Células Intersticiais do Testículo , Peptídeo Natriurético Tipo C , Reação em Cadeia da Polimerase , Receptores de Peptídeos , Reprodução , Transcrição Reversa , Interferência de RNA , Fase S , Doenças Testiculares , Testículo , TestosteronaRESUMO
Gallbladder cancer (GBC) is the most common malignancy in the biliary tract. Without effective treatment, its prognosis is notoriously poor. Tea polyphenols (TPs) have many pharmacological and health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-thrombotic, antibacterial, and vasodilatory properties. However, the anti-cancer effect of TPs in human gallbladder cancer has not yet been determined. Cell viability and colony formation assay were used to investigate the cell growth. Cell cycle and apoptosis were evaluated by flow cytometry analysis. Western blot assay was used to detect the expression of proteins related to cell cycle and apoptosis. Human tumor xenografts were used to examine the effect of TPs on gallbladder cancer cells in vivo. TPs significantly inhibited cell growth of gallbladder cancer cell lines in a dose- and time-dependent manner. Cell cycle progression in GBC cells was blocked at the S phase by TPs. TPs also induced mitochondrial-related apoptosis in GBC cells by upregulating Bax, cleaved caspase-3, and cleaved PARP expressions and downregulating Bcl-2, cyclin A, and Cdk2 expressions. The effects of TPs on GBC were further proven in vivo in a mouse xenograft model. Our study is the first to report that TPs inhibit GBC cell growth and these compounds may have potential as novel therapeutic agents for treating gallbladder cancer.
Assuntos
Humanos , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Camellia sinensis/química , Neoplasias da Vesícula Biliar/patologia , Polifenóis/farmacologia , Fase S/efeitos dos fármacos , Chá/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias da Vesícula Biliar/tratamento farmacológico , Xenoenxertos , Polifenóis/isolamento & purificaçãoRESUMO
Chemotherapy-induced cancer cell secretomes promote resistance due, in part, to a predominant glycolytic energy metabolism, which drives aggressive cancer cell proliferation. However, the characterization of these secretomes and the molecular events that associate them with acquired drug resistance remain poorly understood. In this study, we show that secretomes of cancer cells with high-level paclitaxel resistance stimulated cell proliferation and suppressed drug-induced apoptosis of drug-sensitive cells. We also found that drug (docetaxel)-stimulated induction of interferon-α (IFN-α), IFN-λ and tumor necrosis factor-α (TNF-α) release in drug-sensitive cells was lowered by these secretomes. The promotion of cell proliferation by paclitaxel-resistant (PacR) cancer cell secretomes was associated, in part, with an increase in S phase of the cell cycle and downregulation of the cell death pathway that supports escape from apoptosis. In addition, we also found that the regulation of targeted glycolysis in PacR cancer cells alters the effects of the secretomes on cell growth, apoptosis, ATP generation and acquired drug resistance. Further study revealed that the deletion of FOXO3a transcription exacerbates glycolytic shift-induced apoptosis by rescuing TRAIL expression. By generating a docetaxel–cross-resistant PacR cancer cell line (PacR/DCT), we further clarified the role of FOXO3a in glycolysis-associated mediation of P-glycoprotein/ABCB1 hyperactivity that induces docetaxel cross-resistance. These findings suggest that suppression of the cellular energy supply by targeting glycolysis may inhibit the multiplicity of acquired chemotherapy resistance. Therefore, the therapeutic inhibition of FOXO3a might direct glycolysis to induce apoptosis and overcome multidrug resistance in cancer cells.
Assuntos
Trifosfato de Adenosina , Apoptose , Ciclo Celular , Morte Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Resistência a Medicamentos , Resistência a Múltiplos Medicamentos , Tratamento Farmacológico , Metabolismo Energético , Glicólise , Necrose , Negociação , Paclitaxel , Fase S , Nações UnidasRESUMO
BACKGROUND/OBJECTIVES: Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. MATERIAL/METHOD: HaCaT keratinocyte cells were exposed to 0.25-1 µg/mL of ASX. Proliferation of keratinocytes was analyzed by using MTT assays and flow cytometry. Keratinocyte migration was determined by using a scratch wound-healing assay. A mechanism for regulation of migration was explored via immunocytochemistry and western blot analysis. RESULTS: Our results suggest that ASX produces no significant toxicity in human keratinocyte cells. Cell-cycle analysis on ASX-treated keratinocytes demonstrated a significant increase in keratinocyte cell proliferation at the S phase. In addition, ASX increased keratinocyte motility across the wound space in a time-dependent manner. The mechanism by which ASX increased keratinocyte migration was associated with induction of filopodia and formation of lamellipodia, as well as with increased Cdc42 and Rac1 activation and decreased RhoA activation. CONCLUSIONS: ASX stimulates the migration of keratinocytes through Cdc42, Rac1 activation and RhoA inhibition. ASX has a positive role in the re-epithelialization of wounds. Our results may encourage further in vivo and clinical study into the development of ASX as a potential agent for wound repair.
Assuntos
Humanos , Western Blotting , Carotenoides , Movimento Celular , Proliferação de Células , Estudo Clínico , Citometria de Fluxo , Imuno-Histoquímica , Queratinócitos , Pseudópodes , Reepitelização , Fase S , Salmão , Pele , Cicatrização , Ferimentos e LesõesRESUMO
PURPOSE: The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates. RESULTS: The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells. CONCLUSION: Alkyl cinnamates specifically target cancer cells through induction of PKC translocation and the mitochondrial pathway of apoptosis, and could be promising anticancer drugs.
Assuntos
Humanos , Antineoplásicos , Apoptose , Neoplasias da Mama , Mama , Caspase 3 , Caspase 9 , Caspases , Ciclo Celular , Morte Celular , Membrana Celular , Cinamatos , Curcumina , Citocromos c , Citosol , Fragmentação do DNA , Citometria de Fluxo , Imunofluorescência , Immunoblotting , Potencial da Membrana Mitocondrial , Mitocôndrias , Estresse Oxidativo , Proteína Quinase C , Proteínas Quinases , Proteínas Serina-Treonina Quinases , Fase SRESUMO
We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.
Assuntos
Humanos , Canais de Cálcio , Cromatografia Líquida de Alta Pressão , Crotalus , Eletroforese em Gel de Poliacrilamida , Membranas , Neutrófilos , Fosfodiesterase I , Fosfolipases A2 , Fase S , PeçonhasRESUMO
Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.
Assuntos
Animais , Ratos , Contagem de Células , Ciclo Celular , Linhagem Celular , Proliferação de Células , Exossomos , Citometria de Fluxo , Análise em Microsséries , MicroRNAs , Microscopia Confocal , Mioblastos , Fase S , Toxoplasma , Toxoplasmose , Azul TripanoRESUMO
MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that suppress protein expression by binding to the 3′ untranslated regions of their target genes. Many studies have shown that miRNAs have important roles in congenital heart diseases (CHDs) by regulating gene expression and signaling pathways. We previously found that miR-30c was highly expressed in the heart tissues of aborted embryos with ventricular septal defects. Therefore, this study aimed to explore the effects of miR-30c in CHDs. miR-30c was overexpressed or knocked down in P19 cells, a myocardial cell model that is widely used to study cardiogenesis. We found that miR-30c overexpression not only increased cell proliferation by promoting cell entry into S phase but also suppressed cell apoptosis. In addition, we found that miR-30c inhibited dimethyl sulfoxide-induced differentiation of P19 cells. miR-30c knockdown, in contrast, inhibited cell proliferation and increased apoptosis and differentiation. The Sonic hedgehog (Shh) signaling pathway is essential for normal embryonic development. Western blotting and luciferase assays revealed that Gli2, a transcriptional factor that has essential roles in the Shh signaling pathway, was a potential target gene of miR-30c. Ptch1, another important player in the Shh signaling pathway and a transcriptional target of Gli2, was downregulated by miR-30c overexpression and upregulated by miR-30c knockdown. Collectively, our study revealed that miR-30c suppressed P19 cell differentiation by inhibiting the Shh signaling pathway and altered the balance between cell proliferation and apoptosis, which may result in embryonic cardiac malfunctions.
Assuntos
Feminino , Gravidez , Feto Abortado , Apoptose , Western Blotting , Diferenciação Celular , Proliferação de Células , Desenvolvimento Embrionário , Expressão Gênica , Coração , Cardiopatias , Comunicação Interventricular , Ouriços , Luciferases , MicroRNAs , RNA , Fase S , Regiões não TraduzidasRESUMO
<p><b>OBJECTIVE</b>To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.</p><p><b>METHODS</b>Eca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot.</p><p><b>RESULTS</b>The half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group.</p><p><b>CONCLUSIONS</b>TLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.</p>
Assuntos
Humanos , Western Blotting , Contagem de Células , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Medicamentos de Ervas Chinesas , Farmacologia , Neoplasias Esofágicas , Metabolismo , Patologia , Citometria de Fluxo , Concentração Inibidora 50 , NF-kappa B , Metabolismo , Fase S , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. METHODS: The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. RESULTS: The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27(kip-1) increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. CONCLUSIONS: Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27(kip-1).
Assuntos
Humanos , Adenocarcinoma , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Immunoblotting , Fosforilação , Fosfotransferases , Fase S , Neoplasias GástricasRESUMO
<p><b>OBJECTIVE</b>To investigate the effect of solanine on the growth of human prostate cancer cell xenograft in nude mice.</p><p><b>METHODS</b>Human prostate cancer Du145 cells were injected into the subcutaneous layers on the back of nude mice. After a week, the mice bearing subcutaneous tumor graft were randomly divided into solanine treatment group and saline control group for treatment for 3 weeks. The tumor grafts were then harvested to evaluate the inhibition rate. The mRNA and protein expressions of cell cycle-related genes in the tumors were detected by qRT-PCR and Western blotting, respectively, and tumor cell apoptosis was detected using TUNEL method.</p><p><b>RESULTS</b>The tumor growth rate in solanine-treated group was significantly slower than that in the control group (P<0.01). The mRNA and protein expressions of C-myc, cyclin D1, cyclin E1, CDK2, CDK4 and CDK6 were significantly inhibited by solanine. Solanine significantly up-regulated p21 mRNA and protein expression in the tumors and induced a higher apoptosis rate of the tumor cells than saline (P<0.01).</p><p><b>CONCLUSION</b>The tumor-inhibition effect of solanine is probably mediated by regulating the expressions of genes related with G1/S cell cycle arrest and cell apoptosis.</p>
Assuntos
Animais , Humanos , Masculino , Camundongos , Apoptose , Quinases Ciclina-Dependentes , Metabolismo , Ciclinas , Metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Camundongos Nus , Transplante de Neoplasias , Patologia , Neoplasias da Próstata , Tratamento Farmacológico , Patologia , Fase S , Solanina , FarmacologiaRESUMO
<p><b>OBJECTIVE</b>To study the pattern of DNA double-strand break (DSB) formation in S-phase cells after thermal damage and explore the mechanisms behind heat sensitivity of S-phase cells and delayed DSBs.</p><p><b>METHODS</b>Flow cytometry was used to analyze the cell cycle arrest in H1299 cells exposed to thermal damage, and EdU incorporation assay was employed to evaluate the DNA replication capacity of the cells. The cells synchronized in S phase were obtained by serum starvation and DSBs were observed dynamically using neutral comet assay. Trypan blue dye exclusion technique was used to analyze the cell viability after thermal damage. Western blotting (WB) was used to detect the phosphorylation of ATM and DNA binding RAD18.</p><p><b>RESULTS</b>The percentage of S-phase cells increased significantly after exposure of the cells to 45 degrees celsius; for 1 h (P<0.01). The time-dependent variation pattern of EdU incorporation was similar to that of S-phase cell fraction. The comet tail began to appear only after incubation of the cells at 37 degrees celsius; for some time and the Olive tail moment (OTM) increased with prolonged incubation. Cell death remained low until 7.5 h after heat exposure of the S-phase cells and then increased rapidly. The phosphorylation of ATM first increased but then decreased drastically. In cells with heat exposure, DNA binding RAD18 was attenuated obviously compared that in non-exposed cells.</p><p><b>CONCLUSION</b>Thermal damage causes cell cycle arrest in S phase, and delayed fatal DSBs occur in the arrested cells due to persistent replication and DNA damage repair suppression, which are the possible cause of heat sensitivity of S-phase cells.</p>
Assuntos
Humanos , Proteínas Mutadas de Ataxia Telangiectasia , Metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , Metabolismo , Temperatura Alta , Fosforilação , Fase S , Ubiquitina-Proteína LigasesRESUMO
MicroRNAs (miRNAs) modulate the expression of tumorigenesis-related genes and play important roles in the development of various types of cancers. It has been reported that miR-144 is dysregulated and involved in multiple malignant tumors, but its role in renal cell carcinoma (RCC) remains elusive. In this study, we demonstrated miR-144 was significantly downregulated in human RCC. The decreased miR-144 correlated with tumor size and TNM stage. Moreover, overexpression of miR-144 in vitro suppressed RCC cell proliferation and G2 transition, which were reversed by inhibition of miR-144. Bioinformatic analysis predicted that mTOR was a potential target of miR-144, which was further confirmed by dual luciferase reporter assay. Additionally, the examination of clinical RCC specimens revealed that miR-144 was inversely related to mTOR. Furthermore, knocking down mTOR with siRNA had the same biological effects as those of miR-144 overexpression in RCC cells, including cell proliferation inhibition and S/G2 cell cycle arrest. In conclusion, our results indicate that miR-144 affects RCC progression by inhibiting mTOR expression, and targeting miR-144 may act as a novel strategy for RCC treatment.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma de Células Renais , Genética , Metabolismo , Patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Fase G2 , Neoplasias Renais , Genética , Metabolismo , Patologia , MicroRNAs , Genética , Metabolismo , Fase S , Serina-Treonina Quinases TOR , Genética , MetabolismoRESUMO
Upper urinary tract-derived urine stem cells (USCs) are considered a valuable mesenchymal stem cell source for autologous cell therapy. However, the reported culture condition for USCs is not appropriate for large-quantity production, because cells can show limited replicativity, senescence, and undesirable differentiation during cultivation. These drawbacks led us to reconstitute a culture condition that mimics the natural stem cell niche. We selected extracellular matrix protein and oxygen tension to optimize the ex vivo expansion of USCs, and compared cell adhesion, proliferation, gene expression, chromosomal stability, differentiation capacity, immunity and safety. Culture on collagen type I (ColI) supported highly enhanced USC proliferation and retention of stem cell properties. In the oxygen tension analysis (with ColI), 5% O₂ hypoxia showed a higher cell proliferation rate, a greater proportion of cells in the S phase of the cell cycle, and normal stem cell properties compared to those observed in cells cultured under 20% O₂ normoxia. The established reconstituted condition (ColI/hypoxia, USCs(recon)) was compared to the control condition. The expanded USCs(recon) showed highly increased cell proliferation and colony forming ability, maintained transcription factors, chromosomal stability, and multi-lineage differentiation capacity (neuron, osteoblast, and adipocyte) compared to the control. In addition, USCs(recon) retained their immune-privileged potential and non-tumorigenicity with in vivo testing at week 8. Therefore, reconstituted condition allows for expanded uUSC cell preparations that are safe and useful for application in stem cell therapy.
Assuntos
Envelhecimento , Hipóxia , Adesão Celular , Ciclo Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos , Instabilidade Cromossômica , Colágeno Tipo I , Matriz Extracelular , Expressão Gênica , Células-Tronco Mesenquimais , Osteoblastos , Oxigênio , Fase S , Nicho de Células-Tronco , Células-Tronco , Fatores de TranscriçãoRESUMO
OBJECTIVE: Glycogen synthase kinase 3β (GSK3β) is a pluripotent protein kinase involved in the development of cancers through regulation of numerous oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of target proteins that GSK3β regulate. Our objective was to assess the expression of GSK3β and cyclin D1 in cervical neoplasm of different histologic grades and to identify their correlation in cervical carcinogenesis. METHODS: Immunohistochemical analysis of GSK3β and cyclin D1 was performed in a total of 137 patients with 12 normal, 62 cervical intraepithelial neoplasia (CIN) (31 CIN1 and 31 CIN3) and 63 invasive cancers including 56 squamous cell carcinomas and 7 adenocarcinomas. RESULTS: The expression of GSK3β increased in parallel with the lesion grade, while that of cyclin D1 decreased with severity of the lesion (P<0.001). There was a significant inverse correlation between GSK3β and cyclin D1 expression in overall cervical neoplasia (Φ=-0.413, P<0.001). GSK3β expression was higher in squamous cell carcinoma than in adenocarcinoma (P=0.049). CONCLUSION: These results suggest that the expressional increase in GSK3β plays a role in cervical carcinogenesis and has inverse correlation with cyclin D1 expression in this process. In addition, GSK3β expression appears to be associated with the histologic type of cervical cancer, especially squamous cell carcinoma.
Assuntos
Humanos , Adenocarcinoma , Carcinogênese , Carcinoma de Células Escamosas , Displasia do Colo do Útero , Ciclina D1 , Ciclinas , Quinases da Glicogênio Sintase , Glicogênio Sintase , Glicogênio , Imuno-Histoquímica , Proteínas Quinases , Fase S , Neoplasias do Colo do ÚteroRESUMO
The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [3H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.
Assuntos
Angioplastia Coronária com Balão , Aterosclerose , Contagem de Células , Ciclo Celular , Ciclina D1 , Ciclina E , Ciclinas , Regulação para Baixo , Músculo Liso Vascular , Fosforilação , Fosfotransferases , Fator de Crescimento Derivado de Plaquetas , Antígeno Nuclear de Célula em Proliferação , Proteína do Retinoblastoma , Rutaceae , Fase SRESUMO
In addition to well-known process of proteasome-mediated degradation of polyubiquitinated proteins, monoubiquitination of proteins is also an important post-translational modification that regulates various non-degradative cellular processes like protein trafficking, cellular signalling, DNA replication and DNA repair. We have previously characterized a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani, which binds specifically to a conserved CAUAGAAG octamer containing RNAs via its uniquely arranged CCCH type Zn-fingers and degrades them using its Smr endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Remarkably, its riboendonuclease activity is inhibited due to the incorporation of a monoubiquitin residue in the ZnF domain, though the target Lys residue remains unknown. Here, we report through systematic mutation of Lys residue to Ala that Lys-413 in LdCSBP is the site of monoubiquitination. However, the amino acid motif around the target Lys in LdCSBP is not consensus with any previously known monoubiquitination site, though partial homology is observed with a subset of recently identified mammalian ubiquitination target sites. Interestingly, Lys-413 of LdCSBP is conserved in the homologous annotated proteins from the related kinetoplastida parasites, suggesting similar monoubiquitination-mediated regulation of RNA endonuclease activity in the organisms.
Assuntos
Sequência de Aminoácidos , Sítios de Ligação , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Leishmania donovani/citologia , Leishmania donovani/fisiologia , Lisina/química , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA , Fase S/fisiologia , Relação Estrutura-Atividade , Ubiquitinação , Dedos de ZincoRESUMO
PURPOSE: Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes. METHODS: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expression level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells. RESULTS: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG 60 microM and PGlc 200 microg/m compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of PPARgamma, C/EBPalpha, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2. CONCLUSION: Combination treatment of EGCG and PGlc inhibit-ed adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.
Assuntos
Células 3T3-L1 , Adipócitos , Adipogenia , Western Blotting , Catequina , Pontos de Checagem do Ciclo Celular , Ciclo Celular , Sobrevivência Celular , Ciclina A , Lipólise , Transição de Fase , PPAR gama , Fase S , Chá , TriglicerídeosRESUMO
PURPOSE: This study was conducted to investigate the small double-stranded RNA (dsRNA) mediated anti-tumor effects of p21(WAF1/ClP1) (p21) transcriptional activation in vitro in the human glioma SHG-44 cell line. MATERIALS AND METHODS: Human glioma SHG-44 cells were transfected with dsRNA using LipofectAMINE 2000 transfection reagent. Real-time PCR and Western blot analysis were conducted to detect p21 and survivin mRNA and protein levels, respectively. Cell proliferation was examined by MTT assay. Cell cycle distribution and apoptosis were detected by flow-cytometric analysis. RESULTS: We found that dsRNA targeting p21 promoter (dsP21) significantly induced the expression of p21 at transcription and protein levels, and reduced the expression of survivin. AS well, dsP21 transcription significantly inhibited human glioma SHG-44 cell proliferation. Analysis of cell cycle distribution revealed that dsP21 transfection increased accumulation of cells in the G0/G1 phase and reduced accumulation of cells in the S phase. Further analysis revealed that dsP21 transcription led to an increase in both early and late stages of apoptosis in human glioma SHG-44 cells. CONCLUSION: In the present study, P21 activation by RNA-induced gene activation (RNAa) induced anti-tumor activity in vitro in a human glioma SHG-44 cell line. The results suggested that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.