RESUMO
The lack of treatment options for high-grade brain tumors has led to searches for alternative therapeutic modalities. Electrical field therapy is one such area. The Optune™ system is an FDA-approved novel device that delivers continuous alternating electric fields (tumor treating fields-TTFields) to the patient for the treatment of primary and recurrent Glioblastoma multiforme (GBM). Various mechanisms have been proposed to explain the effects of TTFields and other electrical therapies. Here, we present the first study of genome-wide expression of electrotherapy (delivered via TTFields or Deep Brain Stimulation (DBS)) on brain tumor cell lines. The effects of electric fields were assessed through gene expression arrays and combinational effects with chemotherapies. We observed that both DBS and TTFields significantly affected brain tumor cell line viability, with DBS promoting G0-phase accumulation and TTFields promoting G2-phase accumulation. Both treatments may be used to augment the efficacy of chemotherapy in vitro. Genome-wide expression assessment demonstrated significant overlap between the different electrical treatments, suggesting novel interactions with mitochondrial functioning and promoting endoplasmic reticulum stress. We demonstrate the in vitro efficacy of electric fields against adult and pediatric high-grade brain tumors and elucidate potential mechanisms of action for future study.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Encéfalo/patologia , Proliferação de Células/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Criança , Terapia Combinada/métodos , Terapia por Estimulação Elétrica/métodos , Estresse do Retículo Endoplasmático/genética , Fase G2/genética , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Mitocôndrias/genética , Fase de Repouso do Ciclo Celular/genéticaRESUMO
Warburgia ugandensis Sprague (W. ugandensis), widely distributed in Africa, is a traditional medicinal plant used for the treatment of various diseases including cancer. We intended to evaluate the anticolorectal cancer (CRC) activities of the crude extract from W. ugandensis (WUD) and reveal the underlying molecular mechanisms of its action. We found that WUD inhibited the proliferation of HT-29 and HCT116 cells in a time- and dose-dependent manner and induced intracellular ROS generation. The inhibitory effect of WUD on the proliferation of HT-29 and HCT116 cells could be attenuated by NAC (a ROS scavenger) in a dose-dependent manner. WUD induced G0/G1 phase arrest, down-regulated the protein expression of Cyclin D1 via ROS accumulation in HT-29 cells. In search of the molecular mechanism involved in WUD-induced Cyclin D1 down-regulation, it was found that WUD can suppress PI3K/Akt/GSK3ß signaling pathway in HT-29 cells. Next, it was found that WUD also activated apoptosis, poly-ADP ribose polymerase 1 (PARP1) cleavage and down-regulated pro-caspase 3 in HT-29 and HCT116 cells. Besides, WUD decreased the growth of colon tumors in vivo in the xenograft mouse model. We demonstrated for the first time that ROS and their modulation in the corresponding intracellular signaling could play a significant role in the potential activity of WUD against CRC cells.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Neoplasias do Colo/genética , Fase G1/genética , Extratos Vegetais/química , Fase de Repouso do Ciclo Celular/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/patologia , Feminino , Células HT29 , Humanos , Camundongos , Espécies Reativas de OxigênioRESUMO
18ß-Glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicine, Glycyrrhrzae Radix et Rhizoma. Here, we explored the effects of GA on hepatocellular carcinoma (HCC) in vitro and in vivo and the underlying molecular mechanisms. We confirmed that GA suppressed proliferation of various HCC cell lines. Treatment of GA caused G0/G1 arrest, apoptosis and autophagy in HCC cells. GA-induced apoptosis and autophagy were mainly due to the unfolded protein response. We compared the roles of the ATF4/CHOP and IRE1α/XBP1s UPR pathways, which were both induced by GA. The ATF4/CHOP cascade induced autophagy and was indispensable for the induction of apoptosis in GA-treated HCC cells. In contrast, the IRE1α/XBP1s cascade protected HCC cells from apoptosis in vitro and in vivo induced by GA. Despite this, activation of autophagy protected HCC cells from apoptosis induced by GA. We concluded that pharmacological inhibition of autophagy or IRE1α may be of benefit to enhance the antitumor activity of GA.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Ácido Glicirretínico/análogos & derivados , Neoplasias Hepáticas/metabolismo , Resposta a Proteínas não Dobradas/genética , Apoptose/genética , Autofagia/genética , Carcinoma Hepatocelular/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/genética , Ácido Glicirretínico/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is among the most common lethal cancers of the digestive system with poor prognosis rates and ineffective therapeutic options. Matrine, a traditional Chinese medicine found in the roots of sophora species, has been used in the clinical treatment of liver fibrosis, chronic hepatitis B and other diseases. We have synthesized a matrine derivatives named WM622 (C26H35ON3S2) with a significant inhibitory effect on transplanted tumors in vivo. The half inhibitory concentration (IC50) of WM622 is 34 µM, which is much lower than matrine. WM622 inhibited the proliferation and promoted apoptosis of hepatocellular carcinoma cells significantly, and the cell cycle was blocked in G0/G1 phase. The protein phosphorylation levels of EGFR, AKT, PI3K and GSK3ß (p-EGFR, p-AKT, p-PI3K, and p-GSK3ß) were also decreased by WM622 treatment dose dependently. In tumor-bearing mice, WM622 could reduce the tumor volumes. In conclusion, the study demonstrated that WM622 could inhibit the proliferation of the hepatocellular carcinoma both in vivo and in vitro by inducing apoptosis, blocking cell cycle in G0/G1 phase and inhibiting the PI3K/AKT signal pathways.
Assuntos
Alcaloides , Carcinoma Hepatocelular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolizinas , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Alcaloides/química , Alcaloides/farmacologia , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinolizinas/química , Quinolizinas/farmacologia , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/genética , MatrinasRESUMO
Solanum lyratum (SLEC) Thunberg (Solanaceae) has been used as a traditional herbal medicine in China for centuries. Numerous studies have shown that SLEC Thunberg (Solanaceae) extract inhibited cancer cell growth in vitro. Herein, we investigated cell death-induced by EcoAc, water, chloroform, butanol extract of SLEC in human oral cancer cell lines (HSC-3, SAS, and CAL-27) in vitro. Different SLEC extract induced cytotoxic effects in human oral cancer cells were examined by contrast phase microscopy. We selected the chloroform extract of SLEC to examine the cytotoxic effects by using DAPI staining, comet assays, flow cytometric assay, Western blotting and examination of confocal laser microscopy. SLEC decreased the percentage of viable cells, induced G0/G1 arrest and apoptosis. These effects were concentration- and time-dependent manners. SLEC increased protein levels of p21, p16, CDK2, and cyclin D1 in HSC-3, SAS, and CAL-27 cells. Also, SLEC increased CDK6 in HSC-3 and CAL-27 cells, but inhibited CDK6 in SAS cells. Cyclin E in HSC-3 and SAS cells was increased by SLEC, but it was inhibited in CAL-27 cells. SLEC suppressed the anti-apoptotic proteins Bcl-2 and Bcl-xl, but increased the pro-apoptotic proteins Bax and Bad in HSC-3, SAS, and CAL-27 cells. SLEC promoted the production of reactive oxygen species (ROS) and Ca²âº, decreased the mitochondrial membrane potential (Δψm) and stimulated NO production in HSC-3, SAS, and CAL-27 cells. Specific caspase inhibitors (caspase-8 inhibitor: Z-IETD-FMK; caspase-9 inhibitor: Z-LEHD-FMK and caspase-3 inhibitor: Z-DEVD-FMK) for caspase-8, -9, and -3 blocked SLE-activated caspase-8, -9, and -3 activities which were associated with an increase in the percentage of viable cells. Taken together, SLE induced G0/G1 arrest and apoptosis via extrinsic- and intrinsic-dependent pathways in HSC-3, SAS, and CAL-27 cells.
Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Caspases/metabolismo , Clorofórmio , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Bucais/patologia , Proteínas de Neoplasias/metabolismo , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Solanum/química , Antineoplásicos Fitogênicos/isolamento & purificação , Cálcio/metabolismo , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias Bucais/metabolismo , Extratos Vegetais/isolamento & purificação , Fase de Repouso do Ciclo Celular/genéticaRESUMO
Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). ß-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Marsdenia/química , Pregnanos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Senescência Celular/genética , Fase G1/efeitos dos fármacos , Fase G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Espectrometria de Massas , Pregnanos/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Tristetraprolin (TTP, Zfp36, Nup475, Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements in their 3' untranslated regions. Through this mechanism, TTP functions as a rheostatic, temporal regulator of gene expression. TTP knockout (KO) mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell compartment in TTP KO mice is also altered. Although no change was detected in long-term hematopoietic stem cell (HSC) frequency or function, as assayed by immunophenotypic markers or limiting dilution transplants, we observed increases in the frequencies and numbers of short-term HSCs, multipotent progenitors, and granulocyte-monocyte progenitors. This pattern is consistent with "reactive granulopoiesis," in which committed myeloid progenitors and more primitive progenitors cycle more actively to increase production of mature granulocytes in response to infection or adjuvant. We created reverse chimeras by transplanting wild-type bone marrow into TTP KO mice and found the "reactive granulopoiesis" phenocopied, indicating a non-hematopoietic stem-progenitor cell-autonomous mechanism. Correspondingly, we found elevated levels of the granulopoietic TTP targets IL-1ß, TNF-α, and IL-6 in the plasma of TTP KO mice. Consistent with the non-cell-autonomous nature of the phenotype, we found elevated levels of IL-1ß, TNF-α, and IL-6 transcripts in the livers of TTP KO mice and no detectable difference in the bone marrows. These findings demonstrate the importance of TTP in inflammatory homeostasis and highlight the ability of the hematopoietic system to respond to stress without significant numbers of quiescent HSCs entering the cell cycle.
Assuntos
Granulócitos/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucopoese/imunologia , Fase de Repouso do Ciclo Celular/imunologia , Tristetraprolina/deficiência , Tristetraprolina/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Ciclo Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/metabolismo , Feminino , Granulócitos/metabolismo , Granulócitos/patologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Homeostase/imunologia , Imunofenotipagem , Leucopoese/genética , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fase de Repouso do Ciclo Celular/genética , Tristetraprolina/fisiologia , Regulação para Cima/imunologiaRESUMO
The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Glioblastoma/tratamento farmacológico , Anidridos Ftálicos/farmacologia , Angelica sinensis , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/fisiopatologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Glioblastoma/enzimologia , Glioblastoma/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Anidridos Ftálicos/uso terapêutico , Ratos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The putative G0/G1 switch regulatory gene G0S30/EGR1/NFGI-A show increased expression shortly after adding concanavalin-A (ConA) to cultured T lymphocytes. However, it is reported that lymphocytes from mice in which the gene has been deleted proliferate normally in response to ConA. This suggests that G0S30 expression is not critical for the response. Paradoxically, others report that proliferation of ConA-stimulated rat lymphocytes is inhibited by an antisense oligonucleotide complementary to G0S30. Because the G0S30 sequence is highly conserved between species, we used a similar oligonucleotide (differing by 1 base) to show for humans that the response to ConA is also inhibited. However, no oligonucleotide-induced changes in the concentrations of G0S30 protein or mRNA are detectable. This suggests that the oligonucleotide is not acting by influencing the expression of G0S30, and may be targeting another gene. The phosphorothioated oligonucleotide was maximally inhibitory at a 50 nM concentration, which is near to the "physiological" concentration found with CpG-containing oligonucleotides to activate mouse B lymphocytes. In the present work, increasing the concentration above 50 nM, or adding further quantities of control oligonucleotides, decreased the inhibition. It is suggested that by using low oligonucleotide concentrations (the "stealth" approach), one may avoid "tripping" an endogenous defense system directed against exogenous oligonucleotides, yet still get sufficient uptake to inhibit lymphocyte activation.