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Specific sensing and functional tuning of nucleic acid secondary structures remain less explored to date. Herein, we report a thiazole polyamide TPW that binds specifically to c-KIT1 G-quadruplex (G4) with sub-micromolar affinity and â¼1 : 1 stoichiometry and represses c-KIT proto-oncogene expression. TPW shows up to 10-fold increase in fluorescence upon binding with c-KIT1 G4, but shows weak or no quantifiable binding to other G4s and ds26 DNA. TPW can increase the number of G4-specific antibody (BG4) foci and mark G4 structures in cancer cells. Cell-based assays reveal that TPW can efficiently repress c-KIT expression in leukemia cells via a G4-dependent process. Thus, the polyamide can serve as a promising probe for G-quadruplex recognition with the ability to specifically alter c-KIT oncogene expression.
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G-Cuádruplex , Leucemia , Humanos , Leucemia/tratamiento farmacológico , Ligandos , Nylons , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , TiazolesRESUMEN
Herein, a prolinamide-derived peptidomimetic that preferentially binds to c-MYC and BCL2 G-quadruplexes present in the promoter regions of apoptosis-related genes (c-MYC and BCL2) over other DNA quadruplexes are described. Biological assays, such as real-time quantitative reverse transcription, western blot, dual luciferase, and small interfering RNA knockdown assays, indicate that the ligand triggers a synthetic lethal interaction by simultaneously inhibiting the expression of c-MYC and BCL2 genes through their promoter G-quadruplexes. The ligand shows antiproliferative activity in MCF-7 cells that overexpress both MYC and BCL2 genes, in comparison to cells that overexpress either of the two. Moreover, the ligand induces S-phase cell-cycle arrest, DNA damage, and apoptosis in MCF-7 cells.
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Antineoplásicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , G-Cuádruplex , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ligandos , Prolina/análogos & derivados , Prolina/química , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Mutaciones Letales SintéticasRESUMEN
The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5'- and 3'-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.
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G-Cuádruplex/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Neoplasias/patología , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Tiazoles/metabolismo , Células A549 , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Distamicinas/química , Regulación hacia Abajo , Células HeLa , Humanos , Modelos Moleculares , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We herein report a cell-membrane-permeable molecular probe ADG, prepared by conjugating guanosine with anthracene, selectively interacts with c-MYC G-quadruplex over other promoter and telomeric quadruplexes as well as duplex DNA. NMR spectroscopy suggests that ADG interacts with terminal G-quartets as well as with the nearby G-rich tract (G13-G14-G15 and G8-G9-G10) of c-MYC quadruplex. In vitro cellular studies indicate that ADG represses c-MYC expression by stabilizing its promoter G-quadruplex and alters c-MYC-related cellular events. ADG suppresses hTERT and BCL2 gene expressions in a promoter-independent manner, inhibits elongation of telomere length, and activates apoptotic cascades in cancer cells.
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Apoptosis/efectos de los fármacos , G-Cuádruplex , Genes myc/efectos de los fármacos , Sondas Moleculares/química , Homeostasis del Telómero/efectos de los fármacos , Antracenos/química , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Genes myc/genética , Guanosina/química , Humanos , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacología , Neoplasias/patología , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
G-quadruplexes have been considered attractive therapeutic targets for the development of anticancer agents. We herein report synthesis of a series of carbazole derivatives by employing modular one-pot Cu(I) catalyzed cycloaddition. These carbazole derivatives are easily synthesizable, soluble in aqueous media, and able to strongly interact with quadruplexes. FRET based melting assay and fluorescence titration experiments suggest that a carbazole derivative, Cz-1, preferentially binds c-MYC quadruplex DNA over other investigated quadruplex and duplex DNAs. The biological studies revealed that Cz-1 inhibits cancer cell proliferation by inducing apoptosis. Moreover, Cz-1 inhibits the expression of c-MYC at transcriptional as well as translational levels. Exon-specific-assay confirms that the downregulation of MYC expression is mainly driven by the binding of Cz-1 with the promoter G-quadruplex structures. Immunocytochemistry, using quadruplex binding antibody BG4, further suggests that Cz-1 induces and stabilizes G-quadruplexes in a cellular system.
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Carbazoles/síntesis química , Carbazoles/farmacología , G-Cuádruplex , Regulación de la Expresión Génica/efectos de los fármacos , Genes myc , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Apoptosis/efectos de los fármacos , Carbazoles/metabolismo , Proliferación Celular/efectos de los fármacos , Cobre/química , Reacción de Cicloadición , ADN/química , Regulación hacia Abajo , Transferencia Resonante de Energía de Fluorescencia , Células HCT116 , Células HeLa , Humanos , Relación Estructura-ActividadRESUMEN
Small molecules that stabilize G-quadruplex structures in telomeres can prevent telomerase enzyme mediated telomere lengthening and subsequently lead to cell death. We herein report two fluoro-isoquinoline derivatives IQ1 and IQ2 as selective ligands for human telomeric G-quadruplex DNA. IQ1 and IQ2 containing different triazolyl side chains have been synthesized by Cu (I) catalyzed azide-alkyne cycloaddition. Fluorescence Resonance Energy Transfer (FRET) melting assay and fluorescence binding titrations indicate that both these ligands exhibit binding preference for telomeric G-quadruplex DNA ( h-TELO) over other promoter DNA quadruplexes and duplex DNA. However, ligand IQ1, containing pyrrolidine side chains, is capable of discriminating among quadruplexes by showing higher affinity toward h-TELO quadruplex DNA. On the contrary, IQ2, containing benzamide side chains, interacts with all the investigated quadruplexes. NMR analysis suggests that IQ1 interacts strongly with the external G-quartets of h-TELO. Biological studies reveal that IQ1 is more potent than IQ2 in inhibiting telomerase activity by selectively interacting with telomeric DNA G-quadruplex. Moreover, a dual luciferase reporter assay indicates that IQ1 is unable to reduce the cellular expression of c-MYC and BCL2 at transcriptional level. Significantly, IQ1 mostly stains the nucleus, induces cell cycle arrest in G0/G1 phase, triggers apoptotic response in cancer cells, and activates caspases 3/7.
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Apoptosis/efectos de los fármacos , Flúor/química , G-Cuádruplex/efectos de los fármacos , Isoquinolinas/farmacología , Telómero , Disponibilidad Biológica , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Genes myc , Humanos , Isoquinolinas/química , Isoquinolinas/farmacocinética , Luciferasas/genética , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas c-bcl-2/genética , Telomerasa/antagonistas & inhibidoresRESUMEN
A full-field polarization-based technique is presented for quantitative evaluation of the spatial distribution of the refractive index in macro and micro samples. The sample is mounted on a glass-air interface of a prism, illuminated by a linearly polarized collimated light beam, and two intensity frames are digitally recorded with specific orientations of an analyzer. The pair of intensity data frames captured with this simple setup is combined through an algorithm specially developed for the purpose, to yield the phase difference between the transverse electric and transverse magnetic components of the total internally reflected light field. The phase difference is then related to the refractive index of the sample. Experimental results for refractive index variations in a laser-etched glass plate and red blood corpuscles are presented. One of the salient features of the proposed technique is that the depth of measurement is dependent on the penetration depth of the sample's evanescent field, which is typically of the order of a few hundred nanometers, thereby facilitating refractive index measurements along a thin section of the sample.
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Introduction Recognizing the limitations of traditional direct laryngoscopes, particularly in difficult airway situations, video laryngoscopy has emerged as a potentially safer and more effective alternative. This study evaluated the utility of two 3D-printed video laryngoscopes: a standard geometry video laryngoscope (SGVL), resembling the traditional Macintosh blade, and a hyper-angulated video laryngoscope (HAVL) with a more curved design. Their performance was compared to a standard Macintosh direct laryngoscope across various intubation parameters. By leveraging the cost-effectiveness of 3D printing with polylactic acid, the study aimed to assess the potential of this technology to improve airway management across diverse clinical settings and varying levels of physician expertise. Methods This prospective randomized crossover study compared the effectiveness of 3D-printed video laryngoscopes (VL) and a standard direct laryngoscope in intubation. After obtaining IRB approval, physicians from various specialties across multiple centers participated. Participants received training on SGVL, HAVL, and DL intubation using an instructional video and hands-on practice. The training was standardized for all participants. The primary outcome measures were time to successful intubation, number of attempts, and time to visualize vocal cords. Participants were randomized to use all three laryngoscopes on a manikin, with a maximum of two attempts per scope. A 30-minute break separated each laryngoscope evaluation. Successful intubation was defined as the single insertion of each laryngoscope and bougie, followed by endotracheal tube placement and confirmation of lung inflation. Results Ninety-eight doctors, mostly from the EM team (73.5%) and ICU team (23.4%). Teams consist of consultants, residents, and medical officers of the concerned departments. Forty-eight of the participants (49%) were novice operators (<25 intubations). Successful first-attempt intubation in those with <1 year of experience with intubation (n=33) was highest for SGVL (97%) compared to DL (82%) and HAVL (67%). Participants who learned intubation through self-directed learning exhibited a higher acceptance of VL and achieved 100% success on their first attempt. Among those who followed modules or workshops, 97% had successful first-attempt intubation with VL. The average time taken to visualize the vocal cords was lower in SGVL compared to DL (5.6 vs. 7.5 seconds) (p<0.001). The HAVL also had a lower average time compared to the DL (7.1 vs. 7.5 secs) (p<0.001). However, the time taken to intubate using DL (24.2 ±8.7 sec) was similar to SGVL (28.1 ±13 sec). Lastly, the intubation time using HAVL was the longest (49.6 ±35.5 sec). The time to intubate with DL and SGVL had Spearman's rho of 0.64 (p<0.001), and DL and HAVL had 0.59 (p<0.001). Conclusions The ease of use and its cost-effective nature make 3D-printed VLs beneficial in situations where traditional VLs may not be available, especially in simulation and training.
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Background: Rapid screening tools such as the WHO well-being Index (WWBI), Six-item screener (SIS), and the CLOX-1 test can be used to assess overall mental health and cognition, respectively. We sought to evaluate mental health with cognition in individuals with chronic diseases and stable vital signs presenting to the Emergency Department (ED). Methods: An observational study in the ED with 279 participants was conducted. Results: Chronic diseases were more prevalent among 51-70 years (43.4%) and diabetes was most common (58.8%). Fever (22.6%) and GI bleeding (32.6%) presentation were high. Participants with low WWBI had low SIS compared to the ones with higher scores (83.3% vs. 17.7%, p < 0.001) and also had low CLOX-1 compared to ones with high CLOX-1 (67.3% vs. 5%, <0.001). A positive correlation between WWBI with SIS (correlation coefficient = 0.305, p < 0.001) and CLOX-1 (0.441, <0.001). Regression analysis indicates a positive association between WWBI and the SIS (standardized regression coefficient = 0.187, 95%CI = 0.236-1.426, and p = 0.006) and CLOX 1 (0.338, 0.2-0.463, <0.001). Conclusion: In the ED, the evaluation of mental health even among cognitive impaired is feasible and crucial.
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Trastornos del Conocimiento , Disfunción Cognitiva , Humanos , Enfermedad Crónica , Trastornos del Conocimiento/diagnóstico , Servicio de Urgencia en Hospital , Salud Mental , Persona de Mediana Edad , AncianoRESUMEN
The spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by monitoring kinetochore attachment of spindles during mitosis. Proper progression of mitosis depends on orderly ubiquitination and subsequent degradation of various mitotic inhibitors. At the molecular level, upon removal of SAC, Cdc20 activates E3 ubiquitin ligase anaphase-promoting complex/cyclosome that, along with E2 ubiquitin-conjugating enzyme UbcH10, executes this function. Both Cdc20 and UbcH10 are overexpressed in many cancer types and are associated with defective SAC function leading to chromosomal instability. The precise mechanism of correlated overexpression of these two proteins remains elusive. We show that Cdc20 transcriptionally up-regulates UbcH10 expression. The WD40 domain of Cdc20 is required for this activity. Physical interaction between Cdc20 and anaphase-promoting complex/cyclosome-CBP/p300 complex and its subsequent recruitment to the UBCH10 promoter are involved in this transactivation process. This transcriptional regulatory function of Cdc20 was observed to be cell cycle-specific. We hypothesize that this co-regulated overexpression of both proteins contributes to chromosomal instability.
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Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Huso Acromático/metabolismo , Transcripción Genética/fisiología , Enzimas Ubiquitina-Conjugadoras/biosíntesis , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Inestabilidad Cromosómica/fisiología , Segregación Cromosómica/fisiología , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Células HeLa , Células Hep G2 , Humanos , Mitosis/fisiología , Huso Acromático/genética , Enzimas Ubiquitina-Conjugadoras/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Aedes aegypti is the main vector of yellow fever, chikungunya, Zika, and dengue worldwide and is managed by using chemical insecticides. Though effective, their indiscriminate use brings in associated problems on safety to non-target and the environment. This supports the use of plant-based essential oil (EO) formulations as they are safe to use with limited effect on non-target organisms. Quick volatility and degradation of EO are a hurdle in its use; the present study attempts to develop nanoemulsions (NE) of Trachyspermum ammi EO and its constituent thymol using Tween 80 as surfactant by ultrasonication method. The NE of EO had droplet size ranging from 65 ± 0.7 to 83 ± 0.09 nm and a poly dispersity index (PDI) value of 0.18 ± 0.003 to 0.20 ± 0.07 from 1 to 60 days of storage. The NE of thymol showed a droplet size ranging from 167 ± 1 to 230 ± 1 nm and PDI value of 0.30 ± 0.03 to 0.40 ± 0.008 from 1 to 60 days of storage. The droplet shape of both NEs appeared spherical under a transmission electron microscope (TEM). The larvicidal effect of NEs of EO and thymol was better than BEs (Bulk emulsion) of EO and thymol against Ae. aegypti. Among the NEs, thymol (LC50 34.89 ppm) had better larvicidal action than EO (LC50 46.73 ppm). Exposure to NEs of EO and thymol causes the shrinkage of the larval cuticle and inhibited the acetylcholinesterase (AChE) activity in Ae. aegypti. Our findings show the enhanced effect of NEs over BEs which facilitate its use as an alternative control measure for Ae. aegypti.
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Aedes , Ammi , Apiaceae , Insecticidas , Aceites Volátiles , Virus Zika , Acetilcolinesterasa , Ácidos Alcanesulfónicos , Animales , Emulsiones/farmacología , Insecticidas/química , Larva , Mosquitos Vectores , Aceites Volátiles/química , Aceites Volátiles/farmacología , Aceites de Plantas/farmacología , Polisorbatos/farmacología , Tensoactivos/farmacología , Timol/farmacologíaRESUMEN
PURPOSE: Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines. EXPERIMENTAL DESIGN: Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing mice and doxorubicin-resistant sarcoma 180-bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay ex vivo. IFN-gamma and tumor necrosis factor-alpha were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-gamma and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-gamma and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells. RESULTS: CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-gamma and/or tumor necrosis factor-alpha, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-gamma-producing T cells in the ascetic tumor site. CONCLUSION: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.
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Antineoplásicos/farmacología , Apoptosis , Quelantes/farmacología , Cobre/química , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Animales , Carcinoma de Ehrlich/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Doxorrubicina/farmacología , Humanos , Leucocitos Mononucleares/metabolismo , Ganglios Linfáticos/patología , Ratones , Trasplante de Neoplasias , Bazo/metabolismoRESUMEN
The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.
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Alquinos/química , Azidas/química , Reacción de Cicloadición , G-Cuádruplex , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Triazoles/síntesis química , ADN/química , Oro , Nanopartículas de Magnetita , Triazoles/farmacologíaRESUMEN
BACKGROUND: There are about 600 million betel quid chewers in the world. Betal quid chewing is one of the major risk factors of hepatocarcinoma, oropharyngeal and esophagus cancers. Arecoline, the main Areca alkaloid of the betel nut is reported to have cytotoxic, genotoxic and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucosal fibrosis, leukoplakia and oral cancer, and has also been found to impose toxic manifestations in immune, hepatic and other defense systems of the recipient. AIM: The precise molecular mechanisms underlying the toxic effects of arecoline deserve investigation. To clarify the action of arecoline on defense systems, immune, hepatic and detoxification system were studied in mice. METHOD: Cell count and cell cycle of the splenocytes were studied for evaluating cell immunity. Liver function test (LFT) was followed by assaying different enzyme systems from serum (SGPT, SGOT and ALP) and liver (GST for detoxication enzyme, SOD and catalase for antioxidant enzymes and GSH for non-enzymatic antioxidant) and by ultrastructural studies of hepatocytes. RESULTS: Here we report that arecoline arrested splenic lymphocyte cell cycle at lower concentration with induced apoptosis at higher concentration thereby causing immunosuppression in arecoline recipients. Besides, it resulted in hepatotoxicity in arecoline recipient mice by disrupting the hepatocyte ultrastructure, as judged by liver ultrastructural studies that showed decreased nuclear size, RER with profusely inflated cysternae and abundance of lipid droplets, and by up regulating hepatotoxic marker enzymes (SGOT and SGPT) in serum. Arecoline also caused depression of antioxidants, i.e., superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione-S-transferase (GST) that are known to neutralize reactive oxygen species. CONCLUSION: All these above-mentioned results led us to conclude that arecoline attacks multiple targets to finally generate systemic toxicity in mice.
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Antioxidantes/metabolismo , Arecolina/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas , Hígado , Bazo , Animales , Ciclo Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , ADN/metabolismo , Inmunidad Celular/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/ultraestructura , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability.
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Proteínas Cdc20/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Mad2/metabolismo , Huso Acromático/efectos de los fármacos , Witanólidos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Aberraciones Cromosómicas , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HCT116/efectos de los fármacos , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Genetic variations in toll-like receptors and cytokine genes of the innate immune pathways have been implicated in controlling parasite growth and the pathogenesis of Plasmodium falciparum mediated malaria. We previously published genetic association of TLR4 non-synonymous and TNF-α promoter polymorphisms with P.falciparum blood infection level and here we extend the study considerably by (i) investigating genetic dependence of parasite-load on interleukin-12B polymorphisms, (ii) reconstructing gene-gene interactions among candidate TLRs and cytokine loci, (iii) exploring genetic and functional impact of epistatic models and (iv) providing mechanistic insights into functionality of disease-associated regulatory polymorphisms. Our data revealed that carriage of AA (P = 0.0001) and AC (P = 0.01) genotypes of IL12B 3'UTR polymorphism was associated with a significant increase of mean log-parasitemia relative to rare homozygous genotype CC. Presence of IL12B+1188 polymorphism in five of six multifactor models reinforced its strong genetic impact on malaria phenotype. Elevation of genetic risk in two-component models compared to the corresponding single locus and reduction of IL12B (2.2 fold) and lymphotoxin-α (1.7 fold) expressions in patients'peripheral-blood-mononuclear-cells under TLR4Thr399Ile risk genotype background substantiated the role of Multifactor Dimensionality Reduction derived models. Marked reduction of promoter activity of TNF-α risk haplotype (C-C-G-G) compared to wild-type haplotype (T-C-G-G) with (84%) and without (78%) LPS stimulation and the loss of binding of transcription factors detected in-silico supported a causal role of TNF-1031. Significantly lower expression of IL12B+1188 AA (5 fold) and AC (9 fold) genotypes compared to CC and under-representation (P = 0.0048) of allele A in transcripts of patients' PBMCs suggested an Allele-Expression-Imbalance. Allele (A+1188C) dependent differential stability (2 fold) of IL12B-transcripts upon actinomycin-D treatment and observed structural modulation (P = 0.013) of RNA-ensemble were the plausible explanations for AEI. In conclusion, our data provides functional support to the hypothesis that de-regulated receptor-cytokine axis of innate immune pathway influences blood infection level in P. falciparum malaria.