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Currently, there is still a lack of effective carriers with minimal side effects to deliver therapeutic miRNA. Thus, it is crucial to optimize novel drug delivery systems. MiR-375 has proven superior therapeutic potency in Hepatocellular carcinoma (HCC). The purpose of this study was to fabricate 2 novel and smart nano-carriers for the transportation efficiency of miR-375 in HCC cells and enhance its anti-tumor effects. We established the miR-375 construct through the pEGP- miR expression vector. Two nano-carriers of solid/liquid lipids and chitosan (CS) were strategically selected, prepared by high-speed homogenization, and optimized by varying nano-formulation factors. Thus, the two best nano-formulations were designated as F1 (0.5% CS) and F2 (1.5% CS) and were evaluated for miR-375 conjugation efficiency by gel electrophoresis and nanodrop assessment. Then, physio-chemical characteristics and stability tests for the miR-375 nano-plexes were all studied. Next, its efficiencies as replacement therapy in HepG2 cells have been assessed by fluorescence microscopy, flow cytometry, and cytotoxicity assay. The obtained data showed that two cationic nanostructured solid/liquid lipid carriers (NSLCs); F1 and F2 typically had the best physio-chemical parameters and long-term stability. Moreover, both F1 and F2 could form nano-plexes with the anionic miR-375 construct at weight ratios 250/1 and 50/1 via electrostatic interactions. In addition, these nano-plexes exhibited physical stability after three months and protected miR-375 from degradation in the presence of 50% fetal bovine serum (FBS). Furthermore, both nano-plexes could simultaneously deliver miR-375 into HepG2 cells and they ensure miR re-expression even in the presence of 50% FBS compared to free miR-375 (p-value < 0.001). Moreover, both F1 and F2 alone significantly exhibited minimal cytotoxicity in treated cells. In contrast, the nano-plexes significantly inhibited cell growth compared to free miR-375 or doxorubicin (DOX), respectively. More importantly, F2/miR-375 nano-plex exhibited more anti-proliferative activity in treated cells although its IC50 value was 55 times lower than DOX (p-value < 0.001). Collectively, our findings clearly emphasized the multifunctionality of the two CS-coated NSLCs in terms of their enhanced biocompatibility, biostability, conjugation, and transfection efficiency of therapeutic miR-375. Therefore, the NSLCs/miR-375 nano-plexes could serve as a novel and promising therapeutic strategy for HCC.
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BACKGROUND: Chia seed is an oil seed with multiple biological activities. Doxorubicin is effective chemotherapy for liver cancer. Resistance and adverse effects are doxorubicin limitations. OBJECTIVE: This study aimed to investigate the effect of chia seeds oil (CSO) on the resistance of HepG2 cells to liposomal-doxorubicin (DOX). METHODS: The objective were investigated through measuring cytotoxicity, doxorubicin-metabolizing enzyme Cytochrome P450 3A4 (CYP-3A4), multidrug resistance-associated protein (MRP1), and the expression of multiple tumor suppressor microRNAs. RESULTS: The findings indicated that low concentration of CSO increased HepG2 cells' sensitivity to DOX, as concluded from its higher cytotoxicity. DOX-induced mRNAs of CYP-3A4 and MRP1 and their protein levels. CSO inhibited both in DOX-treated cells. CSO-induced tumor suppressor miRNAs. Doxorubicin inhibited miR-122 and let-7/b/e expression, while it led to overexpression of let- 7a. CSO/DOX upregulated let-7/b/e, miR-34a, and miR-122 (which inhibits MRP1) and downregulated let-7a, which may lead to increased apoptosis. CONCLUSION: CSO effectively re-sensitized HepG2 cells to liposomal-doxorubicin via inhibiting MRP1 and CYP-3A4, which may increase in vivo doxorubicin bioavailability and decrease its therapeutic dose to diminish its adverse effects.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Resistencia a Medicamentos Antineoplásicos , Doxorrubicina/farmacologia , Linhagem Celular TumoralRESUMO
BACKGROUND: miR-210, a key hypoxamiR, regulates hypoxia and inflammation-linked hypoxia. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is responsible for many pathological disorders, including photosensitivity. OBJECTIVE: This study aimed to find the correlation between circulating miR-210/HIF-1α levels and photosensitivity in SLE patients and other SLE-associated pathological complications in a single-center case-control study. METHODS: The study population comprised 104 SLE Egyptian patients with photosensitivity, 32 SLE patients without photosensitivity, and 32 healthy subjects. SLE activity was assessed for all patients using the SLE Disease Activity Index (SLEDAI). Clinical complications/manifestations and hematological/serological analyses were recorded. HIF-α concentration was investigated by ELISA, and miR-210 expression was analyzed by qRT-PCR. RESULTS: The results revealed that circulating miR-210 was significantly increased in the SLE/photosensitivity group versus the SLE and control groups. The additional occurrence of malar rash, oral ulcers, renal disorders, or hypertension resulted in a higher expression of miR-210. SLEDAI activity status showed no effect on miR-210. Erythrocyte sedimentation rate, white blood cells, hemoglobin, platelets, patient age, and disease duration were positively correlated with circulatory miR-210. HIF-α concentration was significantly induced in the SLE/photosensitivity group versus the SLE and control groups. In SLE/photosensitivity, the presence of renal disorders and hypertension resulted in the highest HIF-α concentrations. A strong positive correlation was recorded between HIF-α concentration and circulatory miR-210 in SLE/photosensitivity patients (r = 0.886). CONCLUSION: The dysregulation of circulating miR-210/HIF-1α levels in SLE/ photosensitivity patients is controlled by the presence of additional pathological complications, and results suggest that the hypoxia pathway might interact positively with the pathogenesis and disease progression of SLE.
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Lúpus Eritematoso Sistêmico , MicroRNAs , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estudos de Casos e Controles , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/genética , Hipóxia/complicações , Hipóxia/genética , MicroRNAs/genéticaRESUMO
OBJECTIVE: MicroRNAs are fine regulators for gene expression during the post-transcriptional stage in many autoimmune diseases. HypoxamiRs (miR-210 and miR-21) play an important role in hypoxia and in inflammation-associated hypoxia. Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that would potentiate many pathological complications, including hemolytic anemia. This study aimed to investigate the role of hypoxamiRs in SLE/hemolytic anemia patients. METHODS: This work was designed to analyze the circulating levels ofâ± the miR-210 and miR-21 expressions and hypoxia-inducible factor-1α (HIF-α) in SLE/hemolytic anemia patients. SLE activity was evaluated for all patients by SLE Disease Activity Index (SLEDAI). Clinical manifestations/complications and serological/hematological investigations were reported. HIF-α concentration was assayed by ELISA and expression of miR-21 and miR-210 was analyzed by qRT-PCR. RESULTS: The results indicated that the fold change of the miR-210/miR-21 expressions in plasma was significantly elevated in SLE/hemolytic anemia patients. A strong positive correlation between the miR-210 and miR-21 expression levels was also recorded. Among the associated-disease complications, hypertension, arthritis, oral ulcers, and serositis were associated with a high circulating miR-210 expression, while the occurrence of renal disorders was associated with the increased miR-21 expression. Furthermore, the HIF-α level was remarkably elevated in SLE/hemolytic anemia patients. A high positive correlation was recorded between the HIF-α concentration and miR-210/miR-21 expression levels. The occurrence of oral ulcers, arthritis, and hypertension was associated with the increased HIF-α concentration. On the other hand, SLEDAI and white blood cell count were positively correlated with miR-21/ miR-210. The erythrocyte sedimentation rate was positively correlated with miR-21. CONCLUSION: The dysregulation of the circulating miR-210/miR-210/HIF-1α levels in SLE/hemolytic anemia patients advocated that the hypoxia pathway might have an essential role in the pathogenesis and complications of these diseases.
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Anemia Hemolítica , Artrite , Hipertensão , Lúpus Eritematoso Sistêmico , MicroRNAs , Úlceras Orais , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Úlceras Orais/complicações , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/genética , Anemia Hemolítica/complicações , Artrite/complicações , Hipertensão/complicaçõesRESUMO
Cyclophosphamide (CP) is a mutagen that is used in cancer chemotherapy, due to its genotoxicity and as an immunosuppressive agent. Thalidomide (TH) is another cancer chemotherapeutic drug. In this study, the cytogenotoxicity and hypoxia modulatory activities of two phthalimide analogs of TH have been evaluated with/without CP. Both analogs have increased CP-stimulated chromosomal aberrations than those induced by TH, including gaps, breaks/fragments, deletions, multiple aberrations, and tetraploidy. The analogs have elevated the cytotoxic effect of CP by inhibiting the mitotic activity, in which analog 2 showed higher mitosis inhibition. CP has induced binucleated and polynucleated bone marrow cells (BMCs), while micronuclei (MN) are absent. TH and analogs have elevated the CP-stimulated binucleated BMCs, while only analogs have increased the CP-induced polynucleated BMCs and inhibited the mononucleated BMCs. MN-BMCs were shown together with mononucleated, binucleated, and polynucleated cells in the CP group. Both analogs have elevated mononucleated and polynucleated MN-BMCs, whereas in presence of CP, TH and analogs have enhanced mononucleated and binucleated MN-BMCs. The analogs significantly induce DNA fragmentation in a comet assay, where analog 1 is the strongest inducer. The treatment of mice with CP has resulted in a high hypoxia status as indicated by high pimonidazole adducts and high HIF-1α and HIF-2α concentrations in lymphocytes. Analogs/CP-treated mice showed low pimonidazole adducts. Both analogs have inhibited HIF-1α concentration but not HIF-2α. Taken together, the study findings suggest that both analogs have a higher potential to induce CP-genotoxicity than TH and that both analogs inhibit CP-hypoxia via the HIF-1α-dependent mechanism, in which analog 1 is a more potent anti-hypoxic agent than analog 2. Analog 1 is suggested as an adjacent CP-complementary agent to induce CP-genotoxicity and to inhibit CP-associated hypoxia.
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Perftoran® (perfluorodecalin) is an oxygen carrier, and carboplatin is a common chemotherapy drug used worldwide for lung cancer treatment. Hypoxia is one of the factors that induce resistance of lung cancer cells to carboplatin. This study explored the role of Perftoran®, as an oxygen carrier, in lowering the resistance of lung cancer cells to carboplatin through suppression of hypoxia pathway mediators. The effect of Perftoran® on the resistance of human lung cancer A549 cells to carboplatin was investigated through the evaluation of cytotoxicity by MTT, cell death mode by dual DNA staining, DNA damage by comet assay, DNA platination (DNA/carboplatin adducts) by atomic absorption spectroscopy, hypoxia degree by pimonidazole, HIF-1α/HIF-2α concentrations by ELISA, expression of miRNAs (hypoxamiRs miR-210, miR-21, and miR-181a) by qRT-PCR, and the content of drug resistance transporter MRP-2 by immunocytochemical staining. Results indicated that compared to carboplatin, Perftoran®/carboplatin decreased cell resistance to carboplatin by potentiating its cytotoxicity using only 45% of carboplatin IC50 and inducing apoptosis. Perftoran® induced DNA platination and DNA damage index in cells compared to carboplatin alone. Moreover, compared to treatment with carboplatin alone, co-treatment of cells with Perftoran® and carboplatin inhibited cellular pimonidazole hypoxia adducts, diminished HIF-1α/HIF-2α concentrations, suppressed hypoxamiR expression, and decreased MRP-2. In conclusion, Perftoran® inhibited resistance of lung cancer cells to carboplatin through the inhibition of both hypoxia pathway mediators and the drug resistance transporter MRP-2 and through the induction of DNA/carboplatin adduct formation.
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Indocyanine green (ICG) is a nontoxic registered photosensitizer used as a diagnostic tool and for photodynamic therapy (PDT). Hypoxia is one the main factors affecting PDT efficacy. Perfluorodecalin emulsion (Perftoran®) is a known oxygen carrier. This study investigated the effect of Perftoran® on ICG/PDT efficacy in presence and absence of Perftoran® via evaluation of phototoxicity by MTT; hypoxia estimation by pimonidazole, HIF-1α/ß by ELISA, and 17 miRNAs (tumor suppressors, oncomiRs, and hypoxamiRs) were analyzed by qPCR. Compared to ICG/PDT, Perftoran®/ICG/PDT led to higher photocytotoxicity, inhibited pimonidazole hypoxia adducts, inhibited HIF-1α/ß concentrations, induced the expression of tumor-suppressing miRNAs let-7b/d/f/g, and strongly inhibited the pro-hypoxia miRNA let-7i. Additionally, Perftoran®/ICG/PDT suppressed the expression of the oncomiRs miR-155, miR-30c, and miR-181a and the hypoxamiRs miR-210 and miR-21 compared to ICG/PDT. In conclusion, Perftoran® induced the phototoxicity of ICG/PDT and inhibited ICG/PDT-hypoxia via suppressing HIF-α/ß, miR-210, miR-21, let-7i, miR-15a, miR-30c, and miR-181a and by inducing the expression of let-7d/f and miR-15b.
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Sargassum dentifolium, (Turner) C. Agarth, 1820, is an edible brown alga collected from red seashores, Egypt. Oral tongue squamous cell carcinoma (OTSCC) is an aggressive malignancy. Hypoxia leads to chemotherapeutic resistance. This work aimed to explore the anti-hypoxia effect of water-soluble polysaccharide fractions of S. dentifolium (SD1-SD3) in CAL-27 OTSCC cells. Cell cytotoxicity assay (MTT); cell death mode (DNA staining); total hypoxia (pimonidazole), HIF-1α (ELISA and immunocytochemistry), HIF-1ß (ELISA), and hsa-miRNA-21-5p and hsa-miRNA-210-3p (qRT-PCR) were investigated. SD1 and SD2 showed a cytotoxic effect due to apoptosis. SD2 and SD3 decreased total cell hypoxia, inhibited miR-210 (p < 0.001 and p < 0.01), miR-21 (p < 0.01 and p < 0.05), and HIF-1α (p < 0.01 and p < 0.05), respectively. However, only SD3 suppressed HIF-1ß (p < 0.05). In conclusion, SD2 showed a potential anti-hypoxia effect through amelioration of HIF-1α regulators, which may help in decreasing hypoxia-induced therapeutic resistance.
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BACKGROUND: Interferon-gamma (IFN-γ) is a chief proinflammatory cytokine with a significant role in the immune response against viral infections. Today there is increasing evidence about the association between individual genetic polymorphisms and cytokines in predicting HBV infection susceptibility. AIM: This study aimed to investigate the association between IFN-γ gene polymorphisms and susceptibility to hepatitis B viral infection (HBV), and the impact of these genetic polymorphisms on IFN-γ production. IFN-γ (+874A/T, rs2430561, and +2109A/G, rs1861494) was genotyped by single-stranded polymorphism-polymerase chain reaction (SSP-PCR) in 126 Egyptians with chronic HBV infection and in 100 healthy control subjects. The plasma levels of IFN-γ were measured by Enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to the control subjects there was a slight increase in +874TT genotype frequency in HBV patients. However, no statistical significance in IFN-γ (+874A/T and +2109A/G) genotype/allele distribution was demonstrated, indicating the lack of association between these SNPs and susceptibility to HBV infection. In +2109A/G, only AG genotype was observed with a complete abrogation of GG and AA genotypes. Haplotypes between different loci on selected genes showed insignificant changes in their frequency in patients and control subjects. HBV patients had a significantly higher level of IFN-γ (P < 0.001) compared to controls. The maximum significant increase in IFN-γ production was observed in subjects harboring the +874TA genotype. CONCLUSIONS: As no association could be characterized between the polymorphism in IFN-γ (+874A/T and +2109A/G) and susceptibility to chronic HBV infection, our data support the concept that IFN-γ gene polymorphisms are not predictors of HBV susceptibility in this segment of the Egyptian population.
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Vírus da Hepatite B , Hepatite B , Predisposição Genética para Doença , Hepatite B/genética , Humanos , Interferon gama/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
BACKGROUND: Shortage of oxygen is a common condition for residents of high-altitude (HA) areas. In mammals, hemoglobin (Hb) has four derivatives: oxyhemoglobin (Hb-O2), carboxyhemoglobin (Hb-CO), sulfhemoglobin (Hb-S), and methemoglobin (Met-Hb). In HA areas, aberrant physiological performance of blood hemoglobin is well-established. OBJECTIVES: The study aimed to investigate the influence of 30 days of HA residence on rabbits' total Hb, Hb derivatives, Hb autooxidation rate, and antioxidant enzymes in comparison to low-altitude control rabbits. Further, the study aimed to investigate the effect of antioxidant-rich Angelica archangelica and/or Ginkgo biloba extracts on the same parameters in HA-resident rabbits. METHODS: Rabbits subjected to 30 days of HA residence were compared to low-altitude control rabbits. HA-residence rabbits were then orally administered 0.11 g/kg b.wt. of Angelica archangelica and/or Ginkgo biloba extract for 14 days. Hb derivatives and Hb autooxidation rate were measured spectrophotometrically. Antioxidant enzymes were estimated using specialized kits. RESULTS: Compared to low-altitude rabbits, 30-day HA-residence rabbits showed a noticeable increase (p<0.05) in Hb-O2 and Hb-CO concentration. In addition, Met-Hb concentration, autooxidation rate of Hb molecules, and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) exhibited a remarkable increase in HA-residence rabbits (p<0.01), reflective of rapid ROS generation. In HA-residence rabbits, both individual and combined treatment with antioxidant-rich extracts for 14 days resulted in recovery to near-normal functional levels of Hb-O2 and Met-Hb, Hb autooxidation rate, and activities of SOD and GPx, while only combined treatment led to Hb-O2 recovery. CONCLUSION: The findings suggest that functional Hb levels may be recovered by oral administration of A. archangelica, G. biloba, or combined treatments. In conclusion, oxidative stress due to living in HA areas may be avoided by supplementation with natural antioxidants.
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Angelica archangelica , Altitude , Animais , Antioxidantes/farmacologia , Ginkgo biloba , Hemoglobinas , Mamíferos , Extratos Vegetais/farmacologia , Coelhos , Superóxido DismutaseRESUMO
BACKGROUND: α-Solanine is a natural toxic glycoalkaloid produced in some species of the Solanaceae family with antiproliferative activity in various cancers. OBJECTIVE: This study aimed to investigate the effect of α-solanine on the oxidative stress status in human hepatocellular carcinoma HepG2 cells and to evaluate its influence on microRNAs (miRNAs) associated with oxidative stress and NF-κB regulation. METHODS: The prooxidant effect of α-solanine was tested by the decay rate of the fluorescent probe, ß-phycoerythrin, and by measuring malondialdehyde, reduced Glutathione, catalase, and superoxide dismutase following treatment of HepG2 cells with low doses of α-solanine. Immunocytochemical techniques were used to detect mitochondrial membrane potential (ΔΨm) and NF-κB protein. The gene expression of NF-κB and miRNAs was evaluated by real-time PCR. RESULTS: α-Solanine is a prooxidant that causes a rapid decay in the fluorescence intensity of ß-phycoerythrin. It induces oxidative stress-related alterations such as increased lipid peroxidation and reduced antioxidant markers. Oxidative stress induced by α-solanine was mediated by decreased ΔΨm, increased NF-κB expression, upregulation of miRNAs that control oxidative stress by regulating the NF-κB pathway, and downregulation of oncogenic miRNAs that inhibit the NF-κB pathway. CONCLUSION: α-Solanine-induced oxidative stress is mediated by alterations in the NF-κB pathway with a detected crosstalk between α-solanine treatment and the expression of oxidative stress-responsive miRNAs.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Estresse Oxidativo , Apoptose , Carcinoma Hepatocelular/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , MicroRNAs/genética , NF-kappa B/genética , NF-kappa B/metabolismo , SolaninaRESUMO
BACKGROUND: Abelmoschus esculentus (AE) (okra), is an edible plant used in many food applications. OBJECTIVE: This study explored whether sulfated AE (SAE) has promising cancer chemopreventive activities that may recommend it as a functional food supplement instead of (or in addition to) AE for the population at risk of cancer and in the health food industry. METHODS: Cytochrome P450-1A (CYP1A) was estimated by fluorescence enzymatic reaction, using ß-naphthoflavone-treated cells (CYP1A inducer). Peroxyl and hydroxyl radical scavenging was assayed by oxygen radical absorbance capacity assay. Flow cytometry was used to analyze apoptosis/necrosis in MCF-7 cells, cell cycle phases in MCF-7 cells, and macrophage binding to fluorescein isothiocyanate-lipopolysaccharide (FITC-LPS). Nitric oxide was determined by Griess assay in LPS-stimulated macrophages, and cytotoxicity was determined by MTT assay. Diethylnitrosamine (DEN) was used to induce hepatic tumor initiation in rats. Placental glutathione-S-transferase (GSTP; an initiation marker) was stained in a fluorescence immunohistochemical analysis of liver sections, and histopathological changes were examined. RESULTS: SAE exhibited strong antitumor initiation and antitumor promotion activities. It suppressed CYP1A, scavenged peroxyl and hydroxyl radicals, induced macrophage proliferation, suppressed macrophage binding to FITC-LPS, inhibited nitric oxide generation, showed specific cytotoxicity to human breast MCF-7 adenocarcinoma cells, and disturbed the cell cycle phases (S and G2/M phases) in association with an increased percentage of apoptotic/necrotic MCF-7 cells. Over a short time period, DEN stimulated liver cancer initiation, but SAE treatment reduced the DEN-induced histopathological alterations and inhibited CYP1A and GSTP. CONCLUSION: SAE extract has the potential for use as an alternative to AE in health foods to provide cancer chemoprevention in populations at risk for cancer.
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Abelmoschus , Neoplasias , Abelmoschus/química , Animais , Apoptose , Feminino , Fluoresceína-5-Isotiocianato/química , Humanos , Lipopolissacarídeos/química , Óxido Nítrico/química , Placenta , Extratos Vegetais/farmacologia , Gravidez , Ratos , Sulfatos/química , Sulfatos/isolamento & purificação , Sulfatos/farmacologiaRESUMO
Enterolobium cyclocarpum (EC) is an edible plant and a gum source for food industries. Its sulfated polysaccharide extract (SEC) was examined for cancer chemopreventive properties to estimate its anti-tumor activity. The modulation of carcinogen metabolism and the antioxidant activity revealed that SEC is a potent tumor anti-initiator since it inhibited cytochrome P450-1A (CYP1A) and induced carcinogen detoxification enzyme glutathione-S-transferase. SEC is also a weak scavenger for hydroxyl and peroxyl radicals. SEC was found to modulate macrophage functions into an anti-inflammatory pattern, where it enhanced macrophage proliferation and phagocytosis of fluorescein isothiocyanate-lipopolysaccharide (FITC-LPS). In addition, SEC strongly inhibited the nitric oxide (NO) generation in LPS-stimulated macrophages and induced the binding affinity of FITC-LPS to macrophages. SEC exhibited specific cytotoxicity against human hepatocellular Hep G2 carcinoma cells. SEC disturbed the cell cycle phase, as indicated by the concomitant arrest in S- and G2/M-phases that was associated with necrosis induction. A short-term initiation model for liver cancer was prepared using diethylnitrosamine (DEN) in rats. SEC inhibited the DEN-histopathological findings and reduced both CYP1A and the tumor initiation marker placental glutathione S-transferase (GSTP). Taken together, SEC could be used as an alternative gum in health food industries to provide cancer prevention in high-risk populations.
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Fabaceae , Neoplasias , Animais , Antioxidantes , Dietilnitrosamina , Feminino , Humanos , Placenta , Extratos Vegetais/farmacologia , Gravidez , Ratos , SulfatosRESUMO
Epigallocatechin gallate (EGCG), major constituent of green tea, possesses antioxidant, antiviral, and anticancer activities. Gold nanoparticles (AuNPs) play an important role in drug delivery due to their stability, ease of surface functionalization, and unique optical properties. This study aimed to investigate the influence of EGCG-capped AuNPs on tumor suppressor miRNAs (miR-34a and let-7a) and their targeted cell death mediators in HepG2 cells, compared with celastrol. EGCG-AuNPs were prepared and characterized. antioxidant activity was estimated by DPPH scavenging assay; cytotoxicity was assessed by MTT assay; let-7a and miR-34a expression was analyzed by qPCR; and miRNAs targets (c-Myc and caspase-3) were assessed by ELISA and immunocytofluorescence, respectively. The average size of EGCG-AuNPs was 35 nm, with a λmax of ~535 nm. EGCG-AuNPs exerted cytotoxicity on HepG2 cells stronger than that of EGCG alone. EGCG-AuNPs and EGCG presented half-maximal radical scavenging concentrations (SC50) of 539 µg/ml and 45 µg/ml, respectively. The expression levels of let-7a and miR-34a were significantly elevated in HepG2 cells after EGCG-AuNP treatment for 72 h. c-Myc protein expression was reduced, whereas caspase-3 expression was increased following treatment with EGCG-AuNPs. In conclusion, Au-NPs are effective carrier for EGCG, and EGCG-AuNPs are promising anti-cancer agent.
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Carcinoma Hepatocelular , Catequina , Neoplasias Hepáticas , Nanopartículas Metálicas , MicroRNAs , Carcinoma Hepatocelular/tratamento farmacológico , Catequina/análogos & derivados , Catequina/farmacologia , Ouro , Humanos , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/genéticaRESUMO
Indole-3-carbinol (I3C) is a naturally occurring glucosinolate found in Brassica vegetables that is usually converted in gastric acidic environment to the efficient metabolite 3,3'-diindolylmethane (DIM). Both indoles (I3C and DIM) are known chemopreventive agents for various cancers including breast cancer. This study aimed to investigate the influence of both indoles on the tumor suppressor miRNAs (let-7a-e, miR-15a, miR-16, miR-17-5p, miR-19a, and miR-20a) and oncomiRs (miR-181a, miR-181b, miR-210, miR-221, and miR-106a), which are controlling the cell cycle key regulators: cyclin-dependent kinases (CDKs), CDK inhibitor p27Kip1, and cyclin D1. Our results indicated that both indoles generally elevated the expression of the tumor suppressor miRNAs let-7a-e, miR-19a, miR-17-5p, and miR-20a and decreased the expression of the oncomiR list. Both indoles were able to significantly suppress the expression of CDK4 and CDK6 as well as the apoptotic markers Bcl-2 and survivin. Both indoles decreased cyclin-D1 protein, where I3C decreased cytoplasmic and nuclear cyclin-D1 significantly. Cytoplasmic and nuclear P27Kip1 showed overexpression following treatment with I3C higher than that detected following DIM treatment. This study provides a mechanistic elucidation of the previously reported cell cycle arrest by I3C and DIM in breast cancer cells suggesting that this effect could be through modulation of miRNAs expression that, in turn, regulates the genetic network controlling the G1/S phase in cell cycle progression.
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Neoplasias da Mama/patologia , Fase G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , MicroRNAs/genética , Fase S/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Fase G1/efeitos dos fármacos , Humanos , Fase S/efeitos dos fármacosRESUMO
BACKGROUND: TNFRSF13B, TACI, is a member of the TNF receptor superfamily; it plays a key role in cancer cell proliferation and progression. METHOD: Influence of silencing of human cytokine receptors on cell viability was screened by Luminescent Cell Viability Assay, after transfection of the siRNA library to find the maximum cell death superhits in both triple-negative MDA-MB-231 and double-positive MCF7 breast cells. The mode of cell death was investigated by dual DNA fluorescence staining. The expression of mRNAs of TACI, BAFF, BAFF-R, and APRIL was explored by qPCR. Immunocytofluorescence analysis was used to evaluate changes in TACI, Bcl-2, TNFR2, cyclin-D2, and PCNA. NF-kB p65, cell cycle, and necrosis/apoptosis (late and early) were analyzed by flow cytometry. RESULTS: TACI is the most potent cytotoxic superhit resulted from high-throughput screening of the siRNA library, in both types of cells. Our findings indicated that silencing receptor TACI in both types of breast cancer cells led to significant cell death, after different intervals from siRNA transfection. Cell death mediators (TNFR2, Bcl-2, and NF-κB) were significantly decreased after TACI silencing. The key factors for cell division (Cyclin-D2 and PCNA) were significantly increased in silenced cells of both types but the cell cycle was arrested before the completion of mitosis. Expression of BAFF, BAFF-R and APRIL mRNA in TACI-silenced cells showed significant upregulation in MDA-MB-231 cells, while only BAFF-R and APRIL showed significant downregulation in MCF7 cells. CONCLUSION: TACI silencing can be a new and promising therapeutic target for mesenchymal-stem like triple-negative breast cancer subtype.
Assuntos
Apoptose/genética , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/metabolismo , Sobrevivência Celular/genética , Ciclina D2/genética , Ciclina D2/metabolismo , Regulação para Baixo , Feminino , Citometria de Fluxo , Imunofluorescência , Inativação Gênica , Ensaios de Triagem em Larga Escala , Humanos , Células MCF-7 , NF-kappa B/genética , NF-kappa B/metabolismo , Necrose/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Neoplasias de Mama Triplo Negativas/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
BACKGROUND: MicroRNA modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC), however Efficient tissue-specific and safe delivery remains a major challenge. OBJECTIVE: We sought to develop an inorganic-organic hybrid vehicle for the systemic delivery of the tumor suppressor miR-34a, and to investigate the efficiency of the delivered miR-34a in the treatment of HCC in vitro and in vivo. METHODS: In the present study, pEGP-miR cloning and expression vector, expressing miR-34a, was electrostatically bound to polyethyleneimine (PEI), and then loaded onto ZSM-5 zeolite nanoparticles (ZNP). Qualitative and quantitative assessment of the transfection efficiency of miR-34a construct in HepG2 cells was applied by GFP screening and qRT-PCR, respectively. The expression of miR-34a target genes was investigated by qRT-PCR in vitro and in vivo. RESULTS: ZNP/PEI/miR-34a nano-formulation could efficiently deliver into HepG2 cells with low cytotoxicity, indicating good biocompatibility of generated nanozeolite. Furthermore, five injected doses of ZNP/PEI/miR-34a nano-formulation in HCC induced male Balb-c mice, significantly inhibited tumor growth, and demonstrated improved cell structure, in addition to a significant decrease in alphafetoprotein level and liver enzymes activities, as compared to the positive control group. Moreover, injected ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels. Results also showed that ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo. CONCLUSION: Our results suggested that miR-34a is a powerful candidate in HCC treatment and that AEG-1 and SOX-9 are novel oncotargets of miR-34a in HCC. Results also demonstrated that our nano-formulation may serve as a candidate approach for miR-34a restoration for HCC therapy, and generally for safe gene delivery.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Genes Supressores de Tumor , Terapia Genética , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , MicroRNAs/farmacologia , Nanopartículas/metabolismo , Compostos Organometálicos/farmacologia , Polietilenoimina/farmacologia , Piridinas/farmacologiaRESUMO
AIM: The clinical application of cisplatin is limited by severe side effects associated with high applied doses. The synergistic effect of a combination treatment of a low dose of cisplatin with the natural alkaloid α-solanine on human hepatocellular carcinoma cells was evaluated. METHODS: HepG2 cells were exposed to low doses of α-solanine and cisplatin, either independently or in combination. The efficiency of this treatment modality was evaluated by investigating cell growth inhibition, cell cycle arrest, and apoptosis enhancement. RESULTS: α-solanine synergistically potentiated the effect of cisplatin on cell growth inhibition and significantly induced apoptosis. This synergistic effect was mediated by inducing cell cycle arrest at the G2/M phase, enhancing DNA fragmentation and increasing apoptosis through the activation of caspase 3/7 and/or elevating the expression of the death receptors DR4 and DR5. The induced apoptosis from this combination treatment was also mediated by reducing the expression of the anti-apoptotic mediators Bcl-2 and survivin, as well as by modulating the miR-21 expression. CONCLUSION: Our study provides strong evidence that a combination treatment of low doses of α-solanine and cisplatin exerts a synergistic anticancer effect and provides an effective treatment strategy against hepatocellular carcinoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Cisplatino/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Solanina/farmacologia , Antineoplásicos/química , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Solanina/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Lapatinib, one of the tyrosine kinase inhibitors (TKIs), is used to reduce epidermal growth factor family proteins overexpression. This study aims to assess the cytotoxic and genotoxic effects of lapatinib on the triple negative breast cancer cell line "MDA-MB-231". We investigated the cytotoxicity of lapatinib by MTT assay, mode of cell death using apoptosis-necrosis assay, DNA damage using micronucleus test, EGFR protein expression by immunocytochemistry, and assessed its effect on EGFR (7p11.2 locus) and TP53 (17p13 locus) genes using interphase-FISH technique. Lapatinib induced cytotoxicity on MDA-MB-231 cell line by elevating the concentration and its IC50 value was 32.5 µM after 24â¯h. Lapatinib increased apoptotic cells and micronuclei in binucleated cells gradually by increasing the concentration for 24â¯h. The EGFR protein expression was reduced by double fold that expressed in non-treated cells. Lapatinib enhanced deletion of EGFR gene signals highly significantly from the lowest concentration. Alternatively, lapatinib amplified signals of TP53 gene effectively by raising the concentration. In conclusion, lapatinib induced cytotoxic and genotoxic effects on MDA-MB-231 cell line. However, laptinib reduced the EGFR protein expression and EGFR signals, it raised the apoptotic cells and TP53 gene signals, which triggered extensive DNA damage. Therefore, lapatinib is an effective TKI in triple negative breast cancer cells as elucidated by its mode of cell death.