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Mansonella perstans infections are widespread in Sub-Saharan Africa and Central and South America and thus can be considered as the most prevalent parasite of man in tropical Africa. In contrast to the high prevalence, knowledge about the biology of this filarial nematode is restricted and no effective treatment regimens of this ivermectin-resistant parasite is lacking. An obstacle for the research is that M. perstans resides in body cavities and thus have been only rarely recovered during surgery or autopsy. Therefore, alternative methods like in vitro culture systems need to be implemented to decipher the nature of mansonellosis and effective drugs. Previously, we have established a monkey kidney epithelial cell-based in vitro culture for the maintenance of M. perstans infective larvae (L3) up to 77 days. However, no alternative for this culture system have been postulated to allow longer survival rates and development of adult worms in vitro. Thus, we aim to establish an alternative in vitro culture system for M. perstans L3. M. perstans L3 were isolated from engorged and laboratory reared Culicoides midges. The larvae were then cultured in Dulbecco's Modified Eagle Medium supplemented with either 10% foetal bovine serum (FBS), 10% newborn calf serum (NCS) or 1% bovine serum albumin (BSA) together with human colon carcinoma cells (HCT-8) as feeder cells. Survival and growth were recorded. We obtained that the 10% NCS culture condition was superior allowing long-term maintenance of M. perstans L3 for up to 100 days and boosted growth of the parasites for up to 5-folds compared to the initial size at culture inception. Although no moulting of the L3 into L4 or adult worms could be overserved, the human colon carcinoma cell-based in vitro culture provides an alternative platform to analyse M. perstans biology and screen for novel drugs against M. perstans.
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BACKGROUND: Despite recent advances in drug discovery, cancer is still one of the most lethal health problems worldwide. In most cases, standard therapy methods and multi-modal treatments fail, and new therapeutic approaches are required. Ion channels are essential in multiple cellular processes regulating cell division, differentiation, and death. Recent studies on ion-channel modulators emphasize their potential to suppress tumor growth. In that regard, we reasoned that an underinvestigated potassium channel modulator, Hydroquinidine (HQ), may exhibit an anti-carcinogenic activity. METHODS AND RESULTS: HQ's potential as an anti-neoplastic compound was examined using colony formation assay, wound healing assay, soft agar assay, and Annexin-V assay in the colon, pancreatic, and hepatocellular carcinomas. Our findings unveiled a remarkable anti-cancer activity of HQ by decreasing colony-forming ability, migration capacity, tumorigenicity, and proliferation and stimulating cellular death. HQ significantly reduced the formed colonies and tumorigenicity for all cells. It displayed a significant anti-migrative effect on hepatocellular carcinoma cells and promoted apoptosis in pancreatic and liver cancer cells. The altered gene expression profile upon HQ treatment was in accordance with observed cellular effects. Cells incubated with HQ downregulated the genes acting in cell division and survival, whereas the expression level of genes functioning in cell cycle arrest and apoptosis was elevated. CONCLUSION: Our data indicate HQ's competency to limit cancer growth and suggest its utilization as a novel potent anti-carcinogenic agent. Future studies are necessary to provide new insights into the HQ action mechanism and to evaluate its capacity in in-vivo.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Neoplasias Pancreáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Quinidina/farmacologia , Quinidina/uso terapêutico , Apoptose , Carcinogênese , Colo/metabolismo , Proliferação de Células , Linhagem Celular Tumoral , Neoplasias Pancreáticas/metabolismoRESUMO
Hericium erinaceus (HE), a widely utilized natural remedy and dietary source, has garnered significant attention for its therapeutic potential in various diseases. In this study, we employed supercritical fluid extraction (SFE) technology to isolate the bioactive compounds from HE's fruiting body. Comprehensive assessments of the antioxidant and antibacterial activities were conducted, along with in vitro investigations on the human colon cancer cell line (HCT-8). The SFE rate served as the evaluation metric, while the variables of extraction time, pressure, and temperature were systematically examined. By integrating the response surface center composite design, we successfully optimized the extraction process, yielding optimal parameters of 80 min, 30 MPa, and 35 °C, thus resulting in an extraction rate of 2.51%. These optimized conditions exhibited considerable antioxidant capacity, anticancer activity, and antibacterial potential. Furthermore, we employed graded alcohol extraction to refine the crude extracts, thereby confirming superior anticancer effects under a 70% alcohol precipitation. To elucidate the composition, Fourier-transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectrometry (GC-MS) were employed to analyze the crude extracts and isolates of HE, facilitating a comparative analysis of six HE varieties. Our findings suggest that sterol derivatives hold promise as the active component against the colon cancer HCT-8 cell line. In conclusion, this study underscores the potential of HE SFE in the development of functional foods or alternative drugs for colon cancer treatment, thus opening new avenues for therapeutic interventions.
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Neoplasias do Colo , Humanos , Antioxidantes/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Neoplasias do Colo/tratamento farmacológico , Antibacterianos/farmacologiaRESUMO
Type I interferon (IFN) signaling in neoplastic cells has a chemo-sensitizing effect in cancer therapy. Toll-like receptor 3 (TLR3) activation promotes IFN-ß production, which induces apoptosis and impairs proliferation in some cancer cells. Herein, we tested whether the TLR3 agonist polyinosinic: polycytidylic acid (poly I:C) can improve chemotherapeutic efficacy in paclitaxel (PTX) resistant cell lines. Human colon cancer cell lines HCT116, SW620, HCT-8 (sensitive to PTX), and HCT-8/PTX (resistant to PTX) were treated with poly I:C and the cell viability was measured. Results showed that poly I:C specifically impaired the cell viability of HCT-8/PTX by simultaneously promoting cell apoptosis and inhibiting cell proliferation. In addition, when TLR3 was overexpressed in HCT-8/PTX cells, we found that TLR3 contributed to the production of IFN-ß that reduced cell viability, and poly I:C preferentially activated the TLR3-UNC93B1 signaling pathway to mediate this effect. Moreover, cotreatment of poly I:C and PTX acted synergistically to induce cell apoptosis of HCT-8/PTX via upregulating the expression of TLR3 and its molecular chaperone UNC93B1, assisting in the secretion of IFN-ß. Notably, a combination of poly I:C and PTX synergistically inhibited the PTX-resistant tumor growth in vivo without side effects. In conclusion, our studies demonstrate that poly I:C reinforces the potency of cytotoxic chemotherapeutics in PTX-resistant cell line through the TLR3-UNC93B1-IFN-ß signaling pathway, which supplies a novel mechanism of poly I:C for the chemotherapy sensitizing effect in a PTX-resistant tumor.
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Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interferon beta/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Paclitaxel/farmacologia , Poli I-C/farmacologia , Receptor 3 Toll-Like/agonistas , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Interferon beta/genética , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Escin, a triterpene saponin isolated from horse chestnut seed, has been used to treat encephaledema, tissue swelling and chronic venous insufficiency. Recent studies show that escin induces cell cycle arrest, tumor proliferation inhibition and tumor cell apoptosis. But the relationship between escin-induced DNA damage and cell apoptosis in tumor cells remains unclear. In this study, we investigated whether and how escin-induced DNA damage contributed to escin-induced apoptosis in human colorectal cancer cells. Escin (5-80 µg/mL) dose-dependently inhibited the cell viability and colony formation in HCT116 and HCT8 cells. Escin treatment induced DNA damage, leading to p-ATM and γH2AX upregulation. Meanwhile, escin treatment increased the expression of p62, an adaptor protein, which played a crucial role in controlling cell survival and tumorigenesis, and had a protective effect against escin-induced DNA damage: knockdown of p62 apparently enhanced escin-induced DNA damage, whereas overexpression of p62 reduced escin-induced DNA damage. In addition, escin treatment induced concentration- and time-dependent apoptosis. Similarly, knockdown of p62 significantly increased escin-induced apoptosis in vitro and produced en escin-like antitumor effect in vivo. Overexpression of p62 decreased the rate of apoptosis. Further studies revealed that the functions of p62 in escin-induced DNA damage were associated with escin-induced apoptosis, and p62 knockdown combined with the ATM inhibitor KU55933 augmented escin-induced DNA damage and further increased escin-induced apoptosis. In conclusion, our results demonstrate that p62 regulates ATM/γH2AX pathway-mediated escin-induced DNA damage and apoptosis.
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Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Escina/uso terapêutico , Proteína Sequestossoma-1/metabolismo , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Escina/farmacologia , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos Nus , Proteína Sequestossoma-1/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
Cryptosporidiosis is a zoonotic disease caused by species in the genus Cryptosporidium. In young ruminants, Cryptosporidium parvum causes economically significant disease with mild to severe clinical signs and occasional death. The typical clinical course in animals aged 1-3 weeks old is acute diarrhoea. Currently there are no available treatments that are fully effective against cryptosporidiosis in either humans or animals. Therefore there is a critical need for the development of new therapeutic agents. We adapted two in vitro culture systems (HCT-8 and Caco-2â¯cell lines) for C. parvum infection to investigate the "anticryptosporidial" activity of two chitosans; Chitosan NAG and Chitosan Mix. Chitosan-a naturally-occurring polysaccharide compound-has been found to be active against a variety of diseases, possessing both antimicrobial and anticancer properties. We investigated both chitosan's toxicity and effects on C. parvum in the two in vitro models. To evaluate chitosan's effects on oocyst shedding in vivo, CD-1 neonate mice were orally inoculated with C. parvum oocysts (Iowa strain), treated with chitosan, and compared to infected non-treated animals. Paromomycin, a classical drug used in veterinary medicine, was used as a reference compound. Immunofluorescence techniques were used to analyse the parasites. Our results showed significant reductions in Cryptosporidium oocyst viability (>95%) after oocyst pre-incubation with either paromomycin (Pâ¯<â¯0.001), Chitosan Mix or Chitosan NAG (Pâ¯<â¯0.001), for 24â¯hâ¯at 37⯰C. Additionally, paromomycin, Chitosan Mix, and Chitosan NAG significantly inhibited C. parvum multiplication in HCT-8 and Caco-2â¯cell lines (Pâ¯<â¯0.005). These effects were dose-dependent. In in vivo studies, treatment with both chitosans (Chitosan NAG, Chitosan Mix) or paromomycin sulfate significantly reduced parasite shedding in infected treated newborn mice (-56%, -34.5% and -58%, respectively). In conclusion, these findings provide the first in vitro and in vivo evidence of the anticryptosporidial activities of this natural polysaccharide.
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Antiprotozoários/farmacologia , Quitosana/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Células CACO-2 , Bovinos , Linhagem Celular Tumoral , Quitosana/uso terapêutico , Quitosana/toxicidade , Modelos Animais de Doenças , Humanos , Enteropatias Parasitárias/tratamento farmacológico , Enteropatias Parasitárias/parasitologia , Camundongos , Paromomicina/farmacologia , Paromomicina/uso terapêutico , Paromomicina/toxicidadeRESUMO
Cryptosporidium parvum is a parasitic protist and a causative agent of mild-to-severe diarrheal diseases in humans and animals. Despite its globally recognized importance, knowledge on the mechanism of parasite invasion and molecular interactions between host cells and the parasite is limited. Here, we report the establishment of 43 mutant cell lines derived from HCT-8 cells by UV-induced mutagenesis and the characterization of three mutants with significantly reduced susceptibility to cryptosporidial infection. Based on qRT-PCR assay performed at 18 h postinfection time, the parasite loads could be reduced by ~45%, ~35%, and ~20% in mutants A05, B08, and B12, respectively (p < 0.001 in all three mutants vs. HCT-8 cells). The mutagenesis mainly affected the attachment of parasite in A05 (i.e. ~30% reduction, p < 0.001 vs. HCT-8), and intracellular development in B08 and B12. The three cell mutants may serve as valuable reagents to further investigate the mechanism of parasite invasion and intracellular development by identifying the gene mutations associated with the parasite attachment (A05) and intracellular development (B08 and B12).
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Cryptosporidium parvum/imunologia , Resistência à Doença , Células Epiteliais/parasitologia , Interações Hospedeiro-Patógeno , Mutação , Adesão Celular , Linhagem Celular , Humanos , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.
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Butiratos/metabolismo , Fibras na Dieta/farmacologia , Suscetibilidade a Doenças/microbiologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/fisiologia , Trato Gastrointestinal/microbiologia , Análise de Variância , Animais , Linhagem Celular , Primers do DNA/genética , Escherichia coli O157/metabolismo , Citometria de Fluxo , Imunofluorescência , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Imuno-Histoquímica , Camundongos , Toxina Shiga/metabolismo , Especificidade da EspécieRESUMO
Cryptosporidium is an important waterborne pathogen for which no treatment or vaccination is available. This study set out to quantify DNA replication of Cryptosporidium parvum in vitro. Cryptosporidium DNA could be detected at up to 60 % of input level in both host-cell-free and host cell containing cultures 6 days after infection with living sporozoites, but was lost within 2 days in cultures inoculated with UV-inactivated sporozoites. Total DNA increased between days 2 and 6, evidence of successful DNA replication in both cell-free and host-cell-containing cultures. Overall however, only a small fraction (up to 5 %) of parasite DNA could be found associated with host cells or bound to plastic of the cell-free cultures, and the majority of parasite DNA was present in the cell culture medium, separable by simple decantation. After 2 days, in host-cell-containing cultures, the parasite DNA could be concentrated by slow centrifugation, suggesting that it was associated with intact parasite cells, but at 6 days, the majority could not be centrifuged and is therefore thought to have represented copies associated with dead and degraded parasites. In cell-free cultures and in larger plates, the majority of DNA was in this form. Performance of the parasite was best in small culture plates, and least in the largest plate sizes. We interpret these results as suggesting that Cryptosporidium sporozoites first bind to the host cell monolayer or to the plasticware, but then by 2 days, there has been a substantial release of parasites back into the medium. Host-cell-free cultures also supported modest replication and may have represented DNA synthesis in cells beginning merogony. The role of the host cells is unclear, as so much of the parasite DNA is released into the medium. Host cells may provide a feeder role, conditioning the medium for Cryptosporidium development.
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Cryptosporidium parvum/crescimento & desenvolvimento , Replicação do DNA , DNA de Protozoário/análise , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Cryptosporidium parvum/efeitos da radiação , Meios de Cultura , DNA de Protozoário/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/efeitos da radiação , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Background: Remimazolam is a new ultrashort-acting benzodiazepine for sedation and anesthesia. The effects of remimazolam and the mechanism by which it functions in cancer cells have not been determined. This research aimed to explore the mechanism of remimazolam action in colon cancer treatment, using bioinformatics analysis and in vitro experiments. Methods: Cell cycle progression, colony formation, self-renewal capacity, and apoptosis detection were performed in HCT8 cells treated with or without remimazolam. Transcriptome sequencing, Gene Ontology, Kyoto Encyclopedia of Genes and Genome, Protein-Protein Interaction, Gene Set Enrichment Analysis, Western blotting, and qPCR were performed to investigate the mechanism of action of remimazolam in HCT8 colon cancer cells. Results: Remimazolam promoted proliferation and cell-cycle progression of HCT8 cells. After remimazolam treatment, a total of 1,096 differentially expressed genes (DEGs) were identified: 673 genes were downregulated, and 423 genes were upregulated. The DEGs were enriched mainly in "DNA replication", "cell cycle", and "G1/S transition" related pathways. There were 15 DEGs verified by qPCR, and representative biomarkers were detected by Western Bloting. The remimazolam-mediated promotion of cell proliferation and cell cycle was reversed by G1T28, a CDK4/6 inhibitor. Conclusion: Remimazolam promoted cell-cycle progression and proliferation in HCT8 colon cancer cells, indicating that the long-term use of remimazolam has potential adverse effects in the anesthesia of patients with colon cancer.
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Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.
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Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Biflavonoides/toxicidade , Catequina/análogos & derivados , Extrato de Sementes de Uva/toxicidade , Proantocianidinas/toxicidade , Antineoplásicos Fitogênicos/química , Biflavonoides/química , Células CACO-2 , Catequina/química , Catequina/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Extrato de Sementes de Uva/química , Humanos , Proantocianidinas/química , Vitis/química , Vitis/metabolismoRESUMO
In this study, we found that it is possible to screen Lactobacillus strains that enhance the immune function of mice using HCT-8 cells. Lactobacillus were co-incubated with intestinal epithelial HCT-8 cells to detect and screen the strains that induced more interleukin-6 (IL-6) in the culture supernatant. Simultaneously, a mouse model of low immunity was established to administer the screened lactobacilli by gavage. After 4 weeks of continuous gavage, related cytokines in blood and immune cell indexes in organs were detected to comprehensively evaluate the feasibility of in vitro cell culture model for screening immune-enhancing strains. The content of IL-6 in the culture supernatant of HCT-8 cells induced by the three tested strains increased approximately 5, 8 and 15 fold compared with that of the control group. IL-6 content in serum of mice was significantly higher than that of the control group provided with cyclophosphamide (CTX). Lactobacillus paracasei ZLPC01 presented a higher ability to protect against the immune damage of CTX by decreasing the serum IgG level, increasing the transformation of mouse splenocytes, and the activity of NK cells. Furthermore, L. paracasei ZLPC01 increased cytokine content in serum (IL-6, IL-2, TNF-α and IFN-γ) and colon (IL-6 and TNF-α) in CTX-treated mice. Screening strains that enhance immunity via an in vitro cell-line is simple in operation, and the results are well correlated with those of animal experiments, which is feasible and effective in practice. In addition, L. paracasei ZLPC01 could have the potential to enhance the immunity of mice effectively through inducing intestinal cells to produce IL-6, TNF-α and other cytokines.
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Meios de Cultivo Condicionados/química , Citocinas/sangue , Interleucina-6/biossíntese , Lacticaseibacillus paracasei/classificação , Lacticaseibacillus paracasei/imunologia , Animais , Linhagem Celular , Ciclofosfamida/farmacologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Agentes de Imunomodulação/metabolismo , Interleucina-6/análise , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Neonatal diarrhoea in calves is one of the major health problems in the cattle industry. Although co-infections are often associated with greater severity of disease, there is limited information on any impact on the pathogens themselves. Herein, we studied Cryptosporidium parvum and bovine coronavirus (BCoV) in human HCT-8 cells, inoculated either sequentially or simultaneously, to investigate any influence from the co-infections. Quantitative results from (RT)-qPCR showed that prior inoculation with either of the two pathogens had no influence on the other. However, the results from simultaneous co-inoculation showed that entry of viral particles was higher when C. parvum sporozoites were present, although elevated virus copy numbers were no longer evident after 24 h. The attachment of BCoV to the sporozoites was probably due to specific binding, as investigations with bovine norovirus or equine herpes virus-1 showed no attachment between sporozoites and these viruses. Flow cytometry results at 72 h post inoculation revealed that C. parvum and BCoV infected 1-11% and 10-20% of the HCT-8 cells, respectively, with only 0.04% of individual cells showing double infections. The results from confocal microscopy corroborated those results, showing an increase in foci of infection from 24 to 72 h post inoculation for both pathogens, but with few double infected cells.
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Doenças dos Bovinos , Coinfecção , Coronavirus Bovino , Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Vírus , Animais , Bovinos , Linhagem Celular Tumoral , Fezes , Cavalos , HumanosRESUMO
Cryptosporidium spp. can cause diarrhea and even death in humans and animals. Host microRNAs (miRNAs) play an important role in the post-transcriptional regulation of the innate immune response to Cryptosporidium infection. To study host miRNA activity in the innate immune response to C. parvum infection, we examined the expression of miR-181d in HCT-8 cells infected with C. parvum and found that it was significantly downregulated, while TLR2, TLR4, NF-κB, and myD88 involved in the TLR/NF-κB signaling pathway were significantly upregulated at the early stages of C. parvum infection. We transfected cells with short-interfering RNAs (siRNA) as TLR2, TLR4, and NF-κB inhibitors. Analysis by quantitative real-time polymerase chain reaction (qPCR) and western blot confirmed that C. parvum downregulates miR-181d expression via the p50 subunit-dependent TLR2/TLR4-NF-κB signaling pathway in HCT-8 cells. This study provides a new theoretical foundation to elucidate the regulatory mechanism of host miRNAs against Cryptosporidium infection.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , MicroRNAs , Animais , Cryptosporidium/genética , Cryptosporidium parvum/genética , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. METHODS: The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. RESULTS: After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. CONCLUSIONS: The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , MicroRNAs , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Circular/genética , Cryptosporidium parvum/genética , Cryptosporidium parvum/metabolismo , Criptosporidiose/genética , Redes Reguladoras de Genes , Regulação Neoplásica da Expressão Gênica , Cryptosporidium/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Perfilação da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs) are involved in the regulation of both the innate and adaptive immune response to Cryptosporidium parvum infection. We previously reported that C. parvum upregulated miR9425p expression in HCT8 cells via TLR2/TLR4NFκB signaling. In the present study, the role of miRNA-942-5p in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated HCT-8 cell apoptosis induced by C. parvum was investigated. METHODS: Quantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis. RESULTS: Forced expression of miRNA-942-5p resulted in decreased apoptosis and an increased C. parvum burden in HCT-8 cells. The opposite results were observed using the suppressed expression of miRNA-942-5p. The miRNA-942-5p led to the translational suppression of IFI27 gene through targeting the 3'-untranslated region of the IFI27 gene. Moreover, overexpression of the IFI27 gene produced a high apoptotic ratio and low C. parvum burden. In contrast, a low apoptotic ratio and a high C. parvum burden were observed following downregulation of the IFI27 gene. Both miR-942-5p and the IFI27 gene influenced TRAIL and caspase-8 expression induced by C. parvum in HCT-8 cells. Moreover, TRAIL promoted HCT-8 cell apoptosis in a concentration-dependent manner. CONCLUSIONS: These data suggested that C. parvum induced the downregulation of IFI27 via relief of miR-942-5p-mediated translational suppression. IFI27 downregulation was affected the burden of C. parvum by regulating HCT-8 cell apoptosis through TRAIL-dependent pathways. Future studies should determine the mechanisms by which C. parvum infection increases miR-942-5p expression and the role of miR-942-5p in hosts' anti-C. parvum immunity in vivo.
Assuntos
Apoptose , Criptosporidiose , Proteínas de Membrana , MicroRNAs , Ligante Indutor de Apoptose Relacionado a TNF , Regiões 3' não Traduzidas , Proliferação de Células , Criptosporidiose/genética , Cryptosporidium parvum , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Tumor metastasis is the major cause of cancer mortality; therefore, it is imperative to discover effective therapeutic drugs for anti-metastasis therapy. In the current study, we investigated whether ivermectin (IVM), an FDA-approved antiparasitic drug, could prevent cancer metastasis. Colorectal and breast cancer cell lines and a cancer cell-derived xenograft tumor metastasis model were used to investigate the anti-metastasis effect of IVM. Our results showed that IVM significantly inhibited the motility of cancer cells in vitro and tumor metastasis in vivo. Mechanistically, IVM suppressed the expressions of the migration-related proteins via inhibiting the activation of Wnt/ß-catenin/integrin ß1/FAK and the downstream signaling cascades. Our findings indicated that IVM was capable of suppressing tumor metastasis, which provided the rationale on exploring the potential clinical application of IVM in the prevention and treatment of cancer metastasis.
RESUMO
Resveratrol (RES) and Vitamin E (VE) are anti-cancer active ingredients with relatively high content in peanut seeds and sprouts. This study aimed to determine the synergistic inhibitory effect of RES and VE on colorectal cancer. Using 5-FU as a positive drug control, the effect of RES combined with VE on HCT-8 cells was determined, and cell viability was detected using the cell-counting kit 8 (CCK8) method. Cell morphology changes were observed using optical microscopy. Cell migration ability was evaluated by the scratch test, while cell colonies were determined by the cloning test formation ability. Apoptosis status was assessed by flow cytometry and nuclear staining by DAPI, and the expression level of apoptosis-related proteins was determined by western blotting. Compared with the single component group, the RES combined with VE group significantly inhibited the growth and proliferation of HCT-8 intestinal cancer cells in vitro. The RES combined with VE group had a greater impact on cell morphology changes and cell colony formation and significantly reduced cell migration ability and intestinal cancer cell apoptosis (p < 0.05). Additionally, combined treatment with RES and VE significantly upregulated the expression of pro-apoptotic proteins BAX, caspase-3, caspase-8, and caspase-9, and downregulated the expression of anti-apoptotic protein BCL-2, compared to the single component treatment. RES combined with VE is effective in promoting intestinal cancer cell apoptosis. This study demonstrated the significant positive synergy of RES and VE on HCT-8 cells, providing a new perspective for more effective use of RES.
RESUMO
BACKGROUND: Micro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection. METHODS: In vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition. RESULTS: HCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling. CONCLUSIONS: miR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.
Assuntos
Cryptosporidium parvum/imunologia , Imunidade Inata , MicroRNAs/metabolismo , Linhagem Celular , Cryptosporidium parvum/metabolismo , Humanos , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.