RESUMO
Cancer associated drug resistance is a major cause for cancer aggravation, particularly as conventional therapies have presented limited efficiency, low specificity, resulting in long term deleterious side effects. Peptide based drugs have emerged as potential alternative cancer treatment tools due to their selectivity, ease of design and synthesis, safety profile, and low cost of manufacturing. In this study, we utilized the Red Sea metagenomics database, generated during AUC/KAUST Red Sea microbiome project, to derive a viable anticancer peptide (ACP). We generated a set of peptide hits from our library that shared similar composition to ACPs. A peptide with a homeodomain was selected, modified to improve its anticancer properties, verified to maintain high anticancer properties, and processed for further in-silico prediction of structure and function. The peptide's anticancer properties were then assessed in vitro on osteosarcoma U2OS cells, through cytotoxicity assay (MTT assay), scratch-wound healing assay, apoptosis/necrosis detection assay (Annexin/PI assay), RNA expression analysis of Caspase 3, KI67 and Survivin, and protein expression of PARP1. L929 mouse fibroblasts were also assessed for cytotoxicity treatment. In addition, the antimicrobial activity of the peptide was also examined on E coli and S. aureus, as sample representative species of the human bacterial microbiome, by examining viability, disk diffusion, morphological assessment, and hemolytic analysis. We observed a dose dependent cytotoxic response from peptide treatment of U2OS, with a higher tolerance in L929s. Wound closure was debilitated in cells exposed to the peptide, while annexin fluorescent imaging suggested peptide treatment caused apoptosis as a major mode of cell death. Caspase 3 gene expression was not altered, while KI67 and Survivin were both downregulated in peptide treated cells. Additionally, PARP-1 protein analysis showed a decrease in expression with peptide exposure. The peptide exhibited minimal antimicrobial activity on critical human microbiome species E. coli and S. aureus, with a low inhibition rate, maintenance of structural morphology and minimal hemolytic impact. These findings suggest our novel peptide displayed preliminary ACP properties against U2OS cells, through limited specificity, while triggering apoptosis as a primary mode of cell death and while having minimal impact on the microbiological species E. coli and S. aureus.
Assuntos
Anti-Infecciosos , Antineoplásicos , Sais , Animais , Camundongos , Humanos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 3/farmacologia , Survivina/genética , Survivina/metabolismo , Survivina/farmacologia , Escherichia coli/metabolismo , Peptídeos Antimicrobianos , Linhagem Celular Tumoral , Oceano Índico , Antígeno Ki-67/metabolismo , Staphylococcus aureus , Apoptose , Peptídeos/farmacologia , Peptídeos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Anti-Infecciosos/farmacologia , Anexinas/farmacologiaRESUMO
AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Humanos , Survivina/genética , Survivina/metabolismo , Survivina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Sensibilidade Colateral a Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Antineoplásicos/uso terapêutico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/farmacologiaRESUMO
AIMS: Cognitive impairment is associated with reduced hippocampal neurogenesis; however, the causes of decreased hippocampal neurogenesis remain highly controversial. Here, we investigated the role of survivin in the modulation of hippocampal neurogenesis in AD. METHODS: To investigate the effect of survivin on neurogenesis in neural stem cells (NSCs), we treated mouse embryonic NSCs with a survivin inhibitor (YM155) and adeno-associated viral survivin (AAV-Survivin). To explore the potential role of survivin expression in AD, AAV9-Survivin or AAV9-GFP were injected into the dentate gyrus (DG) of hippocampus of 7-month-old wild-type and 5XFAD mice. Cognitive function was measured by the Y maze and Morris water maze. Neurogenesis was investigated by BrdU staining, immature, and mature neuron markers. RESULTS: Our results indicate that suppression of survivin expression resulted in decreased neurogenesis. Conversely, overexpression of survivin using AAV-Survivin restored neurogenesis in NSCs that had been suppressed by YM155 treatment. Furthermore, the expression level of survivin decreased in the 9-month-old 5XFAD compared with that in wild-type mice. AAV-Survivin-mediated overexpression of survivin in the DG in 5XFAD mice enhanced neurogenesis and cognitive function. CONCLUSION: Hippocampal neurogenesis can be enhanced by survivin overexpression, suggesting that survivin could serve as a promising therapeutic target for the treatment of AD.
Assuntos
Doença de Alzheimer , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Survivina/farmacologia , Survivina/uso terapêutico , Hipocampo , Neurogênese/fisiologia , Cognição , Modelos Animais de Doenças , Camundongos TransgênicosRESUMO
Background and Objectives: Cancer is the second-most-important deadly disease in the world, leading to severe socioeconomic consequences and posing a public threat. Consequently, breast and colorectal cancers are significant cancer types that affect women and men more commonly, respectively. Treatment failure or recurrent diseases frequently occur due to resistance, in addition to the side effects of the currently available anticancer agents. Therefore, in this study, herbal melanin anticancer activity was investigated against human breast adenocarcinoma (MDA-MB-231) and human colorectal (HCT 116) cell proliferation and the expression of downregulated anti-apoptotic proteins and upregulated pro-apoptotic p53. Materials and Methods: MDA-MB-231 and HCT 116 cells were monitored for their real-time proliferation properties using Xcelligence. Herbal melanin of various concentrations significantly inhibited MDA-MB-231 and HCT 116 cell proliferation. Then, the expression of proapoptotic and anti-apoptotic proteins such as p53, Bcl-2 and Bcl-xl was studied using Western blotting. Results: The Bcl-2 and Bcl-xl expressions were downregulated, while the p53 expression was upregulated after treatment with herbal melanin. Similarly, the expression of apoptotic proteins such as Bcl-2, Bcl-xl, XIAP, Survivin, Bid, Bax, p53, Cytochrome C, PARP genes and mRNA was studied after herbal melanin treatment using real-time PCR, which revealed the downregulation of Bcl-2, Bcl-xl, XIAP and Survivin and the upregulation of Bid, Bax, p53, Cytochrome C and PARP apoptotic protein expression. Also, caspase 3 and 9 expressions were monitored after the treatment with herbal melanin, which revealed the upregulation of both the MDA-MB-231 and HCT 116 cell types. Conclusions: Overall, herbal melanin can be used as an alternative anticancer agent against the MDA-MB-231 and HCT 116 cell types.
Assuntos
Antineoplásicos , Neoplasias da Mama , Feminino , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Reguladoras de Apoptose/uso terapêutico , Células HCT116 , Proteína Supressora de Tumor p53/genética , Survivina/metabolismo , Survivina/farmacologia , Survivina/uso terapêutico , Melaninas/metabolismo , Melaninas/farmacologia , Melaninas/uso terapêutico , Apoptose , Proteína X Associada a bcl-2/genética , Citocromos c/metabolismo , Citocromos c/farmacologia , Citocromos c/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proliferação de Células , Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular TumoralRESUMO
The oncoprotein survivin plays a pivotal role in controlling cell division and preventing apoptosis by inhibiting caspase activation. Its significant contribution to tumorigenesis and therapeutic resistance has been well established. Isoliquiritigenin (ISL), a natural compound, has been recognized for its powerful inhibitory effects against various tumors. However, whether ISL exerts regulatory effects on survivin and its underlying mechanism in oral squamous cell carcinoma (OSCC) remains unclear. Here, we found that ISL inhibited the viability and colony formation of OSCC, and promoted their apoptosis. The immunoblotting data showed that ISL treatment significantly decreased survivin expression. Mechanistically, ISL suppressed survivin phosphorylation on Thr34 by deregulating Akt-Wee1-CDK1 signaling, which facilitated survivin for ubiquitination degradation. ISL inhibited CAL27 tumor growth and decreased p-Akt and survivin expression in vivo. Meanwhile, survivin overexpression caused cisplatin resistance of OSCC cells. ISL alone or combined with cisplatin overcame chemoresistance in OSCC cells. Overall, our results revealed that ISL exerted potent inhibitory effects via inducing Akt-dependent survivin ubiquitination in OSCC cells.
Assuntos
Carcinoma de Células Escamosas , Chalconas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Survivina/farmacologia , Survivina/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Apoptose , Chalconas/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin-induced cell death in human oral squamous cell carcinoma (OSCC). The growth-inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit-8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4',6-diamidino-2-phenylindole staining, measurement of the sub-G1 population, and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real-time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase-dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p-STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia-1 (Mcl-1) expression, leading to apoptotic cell death. The genipin-mediated reduction in survivin and Mcl-1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/patologia , Survivina/metabolismo , Survivina/farmacologia , Survivina/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Neoplasias Bucais/patologia , Apoptose , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Prostate cancer is the second prevalent cancer in men. While the anti-cancer effect of Hesperidin and (Aprepitant) AP on prostate cancer cells is well documented, their combined effect and their mechanism of action are not fully investigated. Therefore, this study aimed to investigate the anti-cancer effects of Hesperidin and AP alone and in combination on prostate cancer cells. PC3 and LNCaP cell lines were treated with Hesperidin and AP alone and in combination. The Resazurin test was used for assessing cell viability. The ROS (reactive oxygen Species) level, P53, P21, Bcl-2, and Survivin gene expression were assessed. Also, a trypan blue assay was done. Hesperidin and AP reduced cell viability and increased apoptosis in PC3 and LNCaP cells. The ROS level reduced after treating the PC3 and LNCaP cells with AP with or without Hesperidin. P53 and P21 gene expression increased after treatment with Hesperidin with or without AP compared to the untreated group in the PC3 cell line. Bcl-2 and Survivin gene expression decreased with AP with or without Hesperidin in the PC3 and LNCaP cells. The current study showed the synergic anti-cancer effect of Hesperidin and AP in both PC3 and LNCaP cell lines.
Assuntos
Hesperidina , Neoplasias da Próstata , Masculino , Humanos , Hesperidina/farmacologia , Survivina/metabolismo , Survivina/farmacologia , Aprepitanto/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Linhagem Celular Tumoral , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , OxirreduçãoRESUMO
Although all-trans retinoic acid (ATRA) is an efficient pattern in acute promyelocytic leukemia (APL) therapy, further studies are required due to the extant clinical limitations of ATRA. It has been reported that Silymarin, an anti-cancer herbal substance extracted from milk thistle (Silybum marianum), is able to regulate apoptosis in various types of cancer cells through different mechanisms of action. This study investigated the apoptosis-inducing effect of Silymarin (SM) alone and in combination with ATRA on human acute promyelocytic NB4 cells. Examination using MTT assay indicated that SM treatment leads to growth inhibition in NB4 cells in a dose-dependent manner. The IC50 values of SM and ATRA were calculated 90 µM and 2 µM, respectively. Cell cycle analysis by flow cytometry revealed that a more increase in the sub-G1 phase (a sign of apoptosis) when cells were exposed to SM in combination with ATRA. The incidence of apoptosis was confirmed through Hoechst 33258 staining and Annexin V-FITC analysis. The results showed that Silymarin enhances ATRA-induced apoptosis. The flow cytometric analysis also indicated an enhancement in levels of ROS in the treated cells with both compounds. The real-time PCR illustrated that SM targets apoptosis by down-regulation in Survivin and Bcl-2 while up-regulation in Bax. The findings showed that the combination of the two compounds is more effective in the induction of apoptosis in NB4 cells. Molecular docking studies indicated that Sylibin, as a primary compound of the SM, binds to the BH3 domain of Bcl-2 and the BIR domain of Survivin with various affinities. Based on the findings, it seems that SM used alone and in combination with ATRA may be beneficial for inducing apoptosis in APL cells.
Assuntos
Leucemia Promielocítica Aguda , Silimarina , Humanos , Survivina/farmacologia , Silimarina/farmacologia , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Diferenciação Celular , Tretinoína/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Objective: LncRNAs are closely correlated with chronic obstructive pulmonary disease (COPD). We investigated the molecular mechanism of lncRNA RP11-521C20.3, which targets the action of the Bcl-2 modifying factor (BMF) signaling pathway in the apoptosis of cigarette smoke extract (CSE)-treated A549 cells. Methods: Lung tissues derived from cigarette smoke exposed rats (COPD group) and controls were examined using TUNEL assay for apoptotic cells and using immunohistochemistry for BMF expression levels. Overexpression and knockdown of BMF by lentiviral vector transfection were used to explore the role of BMF on the apoptosis of CSE-treated A549 cells. Overexpression and knockdown of RP11-521C20.3 were used to assess the effect of RP11-521C20.3 on the expression levels of BMF and apoptosis in CSE-treated A549 cells. Cell proliferation, mitochondrial morphology, and apoptosis were assessed in A549 cells. Real-time quantitative polymerase chain reactions and Western blotting detected the expression of apoptosis-related molecules. Results: The number of apoptotic cells and the level of BMF protein were significantly increased in lung tissues of the COPD group compared to the control group. Overexpression of BMF or knockdown of RP11-521C20.3 in CSE-treated A549 cells increased apoptosis, inhibited cell proliferation, and exacerbated mitochondrial damage. There were also increased protein levels of p53, cleaved caspase-3, and cleaved caspase-7, and decreased protein levels of Bcl-2 and survivin. Knockdown of BMF or overexpression of RP11-521C20.3 in CSE-treated A549 cells attenuated apoptosis, promoted cell proliferation, and alleviated mitochondrial damage. Observed effects also included decreased protein levels of p53, cleaved caspase-3, and cleaved caspase-7, and increased protein levels of Bcl-2 and survivin. In CSE-treated A549 cells, overexpression of RP11-521C20.3 suppressed the expression of BMF mRNA and protein. Conclusion: In CSE-treated A549 cells, BMF promoted apoptosis and RP11-521C20.3 might target the BMF signaling axis to protect CSE-treated A549 cells from apoptosis.
Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , RNA Longo não Codificante , Ratos , Animais , Humanos , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA Longo não Codificante/genética , Células A549 , Survivina/genética , Survivina/metabolismo , Survivina/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Caspase 7/farmacologia , Fumar Cigarros/efeitos adversos , Proteína Supressora de Tumor p53 , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Transdução de Sinais , Nicotiana , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologiaRESUMO
In the present study, we investigated the antitumor effect and associated molecular mechanisms of the copper (II) complex of salicylate phenanthroline [Cu(sal)(phen)] against hepatocellular carcinoma (HCC). Cu(sal)(phen) inhibited the proliferation of HCC cells (HepG2 and HCC-LM9) and induced apoptosis of HCC cells in a dose-dependent manner by upregulating mitochondrial reactive oxygen species (ROS) production. The expression of the antiapoptotic proteins survivin and Bcl-2 was decreased, while the expression of the DNA damage marker γ-H2 AX and the apoptotic marker cleaved PARP was upregulated with Cu(sal)(phen) treatment. In vivo, the growth of HepG2 subcutaneous xenograft tumors was greatly attenuated by Cu(sal)(phen) treatment. Immunohistochemistry staining showed that the expression of survivin, Bcl-2, and Ki67 in the tumor was downregulated by Cu(sal)(phen). Toxicity experiments with BALB/c mice revealed that Cu(sal)(phen) is a relatively safe drug. Our results indicate that Cu(sal)(phen) possesses great potential as a therapeutic drug for HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Humanos , Carcinoma Hepatocelular/patologia , Survivina/farmacologia , Survivina/uso terapêutico , Cobre/toxicidade , Cobre/química , Fenantrolinas/farmacologia , Fenantrolinas/química , Fenantrolinas/uso terapêutico , Neoplasias Hepáticas/patologia , Salicilatos/farmacologia , Salicilatos/química , Salicilatos/uso terapêutico , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Proliferação de Células , Linhagem Celular Tumoral , Antineoplásicos/uso terapêutico , Células Hep G2RESUMO
This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.
Assuntos
Neoplasias do Colo , Fluoruracila , Metformina , Humanos , Apoptose , Proteína X Associada a bcl-2/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Citocromos/metabolismo , Citocromos/farmacologia , Fluoruracila/farmacologia , Metformina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Survivina/metabolismo , Survivina/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
OBJECTIVE: To investigate the effects of mTOR inhibitors everolimus (EVE) and gemcitabine (GEM) on the proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma (DLBCL) cell line U2932, and further explore the molecular mechanisms, so as to provide new ideas and experimental basis for the clinical treatment of DLBCL. METHODS: The effect of EVE and GEM on the proliferation of U2932 cells was detected by CCK-8 assay, the IC50 of the two drugs was calculated, and the combination index (CI=) of the two drugs was calculated by CompuSyn software. The effect of EVE and GEM on apoptosis of U2932 cells was detected by flow cytometry with AnnexinV-FITC/PI staining. Flow cytometry with propidium iodide (PI) staining was used to detect the effect of EVE and GEM on the cell cycle of U2932 cells. Western blot assay was used to detect the effects of EVE and GEM on the channel proteins p-mTOR and p-4EBP1, the anti-apoptotic proteins MCL-1 and Survivin, and the cell cycle protein Cyclin D1. RESULTS: Both EVE and GEM could significantly inhitbit the proliferation of U2932 cells in a time- and dose-dependent manner (r=0.465, 0.848; 0.555, 0.796). According to the calculation of CompuSyn software, EVE combined with GEM inhibited the proliferation of U2932 cells at 24, 48 and 72 h with CI=<1, which had a synergistic effect. After treated U2932 cells with 10 nmol/L EVE, 250 nmol/L GEM alone and in combination for 48 h, both EVE and GEM induced apoptosis, and the difference was statistically significant compared with the control group (P<0.05). The apoptosis rate was significantly enhanced after EVE in combination with GEM compared with single-agent (P<0.05). Both EVE and GEM alone and in combination significantly increased the proportion of cells in G1 phase compared with the control group (P<0.05). The proportion of cells in G1 phase was significantly increased when the two drugs were combined (P<0.05). The expression of p-mTOR and effector protein p-4EBP1 was significantly downregulated in the EVE combined with GEM group, the expression of anti-apoptotic proteins MCL-1, Survivin and cell cycle protein cyclin D1 was downregulated too (P<0.05). CONCLUSION: EVE combined with GEM can synergistically inhibit the proliferation of U2932 cells, and the mechanism may be that they can synergistically induce apoptosis by downregulating the expression of MCL-1 and Survivin proteins and block the cell cycle progression by downregulating the expression of Cyclin D1.
Assuntos
Gencitabina , Linfoma Difuso de Grandes Células B , Humanos , Everolimo/farmacologia , Survivina/farmacologia , Ciclina D1/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Linhagem Celular Tumoral , Proliferação de Células , Serina-Treonina Quinases TOR , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas de Ciclo CelularRESUMO
The main strategy of cancer cells for survival is uncontrolled cell division and escape from apoptosis. The use of anticancer agents inducing the production of reactive oxygen species (ROS) and controlling cell division might be a therapeutic approach to eradicate cancer cells. Herein, we examined the therapeutic effects of Auraptene on CT26 cells as well as on a mouse model of colorectal cancer (CRC). The spheroid assay was also conducted to analyze the anti-proliferative activity of Auraptene. We also assessed the in vitro analysis of ROS generation. The impact of Auraptene on oxidant/antioxidant markers, as well as the mRNA expression of Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin genes, was evaluated by qPCR in tumor samples. As a result, Auraptene significantly reduced the size of CT26 spheroids at a dose of 200 µM. After 12 h, ROS levels were significantly elevated in CT26 cells. The administration of Auraptene induced apoptosis and the cell cycle arrest by modulating Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin mRNA levels. Furthermore, our results demonstrated that Auraptene suppressed CAT, GSH (reduced Glutathione), and FRAP while increasing MDA in tissue homogenates which in turn could raise oxidative stress and stimulate apoptosis. Therefore, Auraptene may act as a powerful adjuvant therapy in CRC since it triggers apoptosis and cell cycle.
Assuntos
Apoptose , Neoplasias Colorretais , Cumarínicos , Ciclina D1 , Estresse Oxidativo , Animais , Camundongos , Apoptose/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Ciclina D1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Survivina/metabolismo , Survivina/farmacologia , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Modelos Animais de DoençasRESUMO
Colorectal cancer is the third most common cause of cancer-associated deaths due to a high recurrence rate and an increasing occurrence of resistance to established therapies. This highlights the importance of developing new chemotherapeutic agents. The current study focuses on cancer-specific targets such as apoptosis-inhibiting survivin, which distinguishes cancer cells from healthy tissue. A combination of pharmacophores of established anticancer agents to afford chimeric pleiotropic chemotherapeutic agents was tested on this cancer entity. We analysed the effects of the dual mode anticancer agents, animthioxam, brimbam, troxbam, and troxham, as well as their structural congeners suberoylanilide hydroxamic acid and combretastatin A-4 on human cancer cell lines. Their cytotoxicity was determined using the MTT assay, further techniques for detecting apoptotic events, cell cycle analyses, clonogenic and wound healing assays, immunostaining, histone deacetylase (HDAC) activity measurements, and Western blot analysis for the detection of survivin expression in HCT116 colon cancer cells. Molecular docking studies were conducted to assess potential molecular targets of the test compounds. The test compounds were found selectively cytotoxic toward cancer cells by inducing apoptosis. The metastatic potential was effectively reduced by disruption of the microtubular cytoskeleton. The test compounds were also proven to be general HDAC inhibitors and to lead to reduced survivin expression.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Inibidores de Histona Desacetilases , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Ácidos Hidroxâmicos/farmacologia , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Survivina/farmacologia , Neoplasias Colorretais/tratamento farmacológicoRESUMO
Survivin is an important member of the antiapoptotic protein family and controls the cell's life cycle. Overexpression of survivin in tumor cells leads to inhibition of apoptosis, thus contributing to cancer cell proliferation. The largest binding pocket in the survivin dimer was located in the BIR domain. The key to the efficacy of 3-cyanopyridines was their surface interaction with the survivin amino acid Ile74. METHODS: Through the optimization of the 3-cyanopyridine, 29 new compounds with a 3- Cyanopyridine structure were designed, synthesized, and characterized by NMR, IR, and mass spectrometry. The antitumor activity of the compounds in vitro was detected by the MTT method. RESULTS: In vitro anti-tumor experiments showed that some compounds exhibited good anti-cancer effects. The IC50 values of the compound 2-amino-6-(2,4-difluorophenyl)-4-(4-hydroxyphenyl) nicotinonitrile (10n) against human liver cancer (Huh7), human glioma (U251), and human melanoma (A375) cells were 5.9, 6.0 and 7.2 µM, respectively. The IC50 values of the compound 6-(2,4-difluorophenyl)- 4-(4-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (9o) against Huh7, U251 and A375 cells were 2.4, 17.5 and 7.2 µM, respectively, which were better than those of 10- hydroxycamptothecin and 5-fluorouracil. Analysis of the results of molecular dynamics simulation established that the BIR domain is the optimal binding site on the survivin protein, and the fingerprints of the eight most active compounds and the molecular docking to the survivin protein are analyzed. CONCLUSION: 3-Cyanopyridine is an excellent backbone for antitumor lead compounds, 10n and 9o, as derivatives of 3-Cyanopyridine are excellent survivin protein-targeting inhibitors worthy of further study. The key factor in inhibiting survivin protein through the action of amino acid Ile74.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Simulação de Acoplamento Molecular , Survivina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Antineoplásicos/química , Proliferação de Células , Aminoácidos , Estrutura Molecular , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Desenho de FármacosRESUMO
BACKGROUND: The mechanism of Heat Shock Protein 90 (HSP90) in Ulcerative Colitis (UC) has been studied, and mitogenic-activated protein kinases (MAPK) also contribute to the pathogenesis of UC. However, the effect of the HSP90/MAPK pathway in UC is still unclear. Therefore, the mainstay of this research is to explore the mechanism of action of this pathway in UC. Compound sophorae decoction (CSD), as a Chinese herbal decoction, can synergistically affect the above process. OBJECTIVE: This study aimed to uncover the synergistic effects of HSP90 inhibitors regulating the MAPK pathway for treating DSS-induced colitis in mice and the synergistic effects of CSD. METHODS: This experiment used oral administration of standard diets containing 3% dextran sodium sulfate (DSS) to establish an experimental colitis model in mice. The model was treated with HSP90 inhibitor, CSD, or dexamethasone. Mouse feces, mobility, body weight, colon length, and colon histopathology scores were recorded daily to assess the degree of colitis inflammation. Expression levels of HSP90 and MAPK pathway-related genes and proteins were evaluated by Western blot and qPCR. The evaluation of intestinal mucosal permeability was measured by enzyme-linked immunosorbent assay (ELISA), which could detect the protein level of D-Amino Acid Oxidase (DAO) and D-lactic acid (D-LA). The same went for downstream molecules AFT-2, p53, and apoptosis-related proteins BAX, BCL-2, Caspase3, and survivin in the MAPK pathway. Immunohistochemical measured p-38, p-JNK, and p-ERK expressions. JAM-A and claudin-1 connexin were tested by immunofluorescence staining. The TUNEL method was for measuring the apoptosis rate of colonic epithelial cells. CBA kit determined the level of inflammatory factors of colons. RESULTS: HSP90 inhibitor can improve the degree of pathological damage in the colon of mice treated with DSS, increase the mice's weight and the length of the colon, and significantly reduce the disease activity index (DAI) score. Intraperitoneal injection of HSP90 inhibitor can reduce the expression of MAPK pathway markers P38, JNK, ERK, and their phosphorylation and decrease the content of AFT-2 and p53, which is downstream of the MAPK pathway. In addition, treatment of the HSP90 inhibitor up-regulated the expression of anti-apoptotic proteins BCL-2 and survivin, as well as down-regulated apoptotic protein caspase3, BAX in the colon of mice with colitis. Lower levels of inflammatory factors such as IL-6, MCP-1, IFN-γ, TNF, IL-12p70, and increased IL-10 were observed after HSP90 inhibitor therapy. Furthermore, the combination treatment of CSD can enhance the effect of the single HSP90 inhibitor treatment and play a synergistic effect. CONCLUSION: These data suggest that an HSP90 inhibitor is available to treat UC by inhibiting the MAPK signaling pathway. This axis can restore the intestinal mucosa barrier's function by reducing intestinal mucosa's permeability and inhibiting apoptosis of intestinal epithelial cells. The specific mechanism is that HSP90 inhibitor can reduce the pathological damage and inflammation levels of colitis mice, and reduce the apoptosis rate of colonic epithelial cells and the mucosal permeability, thereby restoring the mucosal barrier function. During this process, CSD works synergistically to improve the therapeutic effect of the HSP90 inhibitor.
Assuntos
Colite Ulcerativa , Colite , Sophora , Animais , Camundongos , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia , Proteína X Associada a bcl-2/uso terapêutico , Colite/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colo/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sophora/metabolismo , Survivina/metabolismo , Survivina/farmacologia , Survivina/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/farmacologia , Proteína Supressora de Tumor p53/uso terapêutico , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.
Assuntos
Neoplasias da Mama , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Inativação Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , RNA Interferente Pequeno/genética , Survivina/genética , Survivina/farmacologia , TransfecçãoRESUMO
In this study, it is aimed to investigate the effect of radiofrequency radiation (RFR) on apoptotic and antiapoptotic factors under different exposure conditions in human colonic adenocarcinoma cells (Caco-2). We analyzed the effects of 2.5 GHz continuous wave and 3 GPP modulated radiofrequency radiation exposure (15 min on, 15 min off) for 1 h and (1 h on, 1 h off) for 3 hours on Caco-2 cell lines. The cell viability of Caco-2 cells was determined by XTT method. Then, the cells were analyzed by flow cytometry to determine the effects on apoptosis staining with AnnexinV-FITC and PI. Protein expression levels of Bcl-2, Bax, Caspase-3 and Survivin were subsequently analyzed by using flow cytometric methods. Bax, Caspase 8, and Survivin protein levels were also analyzed by western blot. The cell viability rates were not significantly different after 2.5 GHz of RFR exposure for 1 h, but RFR exposure for 3 h at 2.5 GHz frequencies caused a decrease on cell viability of Caco-2 cells. RFR exposure for 1 and 3 hours at 2.5 GHz frequencies resulted in an apoptotic response. Protein analyses of Bcl-2, Bax, Survivin, Caspase-3, and Caspase-8 showed that RFR led to increase the levels of proapoptotic Bax, Caspase-3, and Caspase 8 in Caco-2 cells under different exposure conditions. However, 3-h exposure caused a decrease in antiapoptotic survivin levels. The results of our study indicate that RFR exposure affects the cell death mechanism due to apoptotic pathway.
Assuntos
Apoptose , Neoplasias Colorretais , Células CACO-2 , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 8/farmacologia , Neoplasias Colorretais/radioterapia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2 , Survivina/metabolismo , Survivina/farmacologia , Proteína X Associada a bcl-2RESUMO
Glyphosate is a universal herbicide with genital toxicity, but the effect of glyphosate on oocytes has not been reported. This study aimed to evaluate the effect of glyphosate (0, 10, 20, 50 and 100 mM) on bovine oocyte in vitro maturation. We showed that 50 mM glyphosate adversely affects the development of bovine oocytes. Exposure of oocytes to 50 mM glyphosate caused an abnormal reduction in oxidative (redox) levels compared with that in the control group, with a significantly higher reactive oxide species level (P < 0.05) and significantly lower glutathione (GSH) expression (P < 0.05). Additionally, the mRNA levels of antioxidant genes (SOD1, SOD2, SIRT2, SIRT3) and the mitochondrial membrane potential (MMP) were significantly reduced (P < 0.05). Furthermore, treatment with 50 mM glyphosate-induced apoptosis, and the mRNA levels of the apoptotic genes Caspase-3 and Caspase-4 were significantly higher than those in the control group (P < 0.05); however, the mRNA level of BAX was significantly higher than that in the control group (P < 0.01). Additionally, the mRNA levels of the anti-apoptotic genes Survivin and BCL-XL were significantly lower than those in the control group (P < 0.05), and oocyte quality was adversely affected. Together, our results confirmed that glyphosate impairs the quality of oocytes by promoting abnormal oocyte redox levels and apoptosis.
Assuntos
Herbicidas , Sirtuína 3 , Animais , Antioxidantes/metabolismo , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Bovinos , Glutationa/metabolismo , Glicina/análogos & derivados , Herbicidas/metabolismo , Herbicidas/toxicidade , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos , Estresse Oxidativo , Óxidos/metabolismo , Óxidos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 2/metabolismo , Sirtuína 2/farmacologia , Sirtuína 3/metabolismo , Sirtuína 3/farmacologia , Superóxido Dismutase-1 , Survivina/metabolismo , Survivina/farmacologia , Proteína X Associada a bcl-2 , GlifosatoRESUMO
The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.