HT-29 Cells Research Articles

Fabrication and Characterization of Andrographolide-loaded Microsponges to Enhance Oral Bioavailability of Drug against Colon Cancer Using HT29 Cells

Background: The current research aimed to determine ways to improve the bioavailability of andrographolide (AGP) for use in colon cancer treatment by developing and evaluating microsponges loaded with the drug. Methods: Utilising the quasi-emulsion solvent diffusion approach, microsponges containing AGP were synthesised. A total of ten formulations were prepared using different concentrations of drug, polymer and other excipients. Particle size, shape, and differential scanning calorimetry (DSC) were used to characterise the microsponges that were created. To find out the rate at which the microsponges would expel their contents, researchers measured their release dynamics. In vitro anticancer activity of formulation was determined using HT29 cells Results: Results showed that the percentage yield of the formulations ranged from 10.85-41.03%. The highest drug concentration was achieved in formulation F8 with a particle size of 33.7 nm. SEM analysis demonstrated that the particles were round and possessed a rough and porous surface. Increasing the ratio of ethyl cellulose to AGP reduces surface roughness. The microsponge's DSC difractogram reveals prominent peaks at 18°, 24°, and 38° (2 θ) with reduced intensity, suggesting that the microsponges' crystalline character has diminished. In vitro drug release study showed 93.85% release upto 12 hours. Mathematical models showed normal release of the formulations with “n” values greater than 0.90 of all the formulations. Formulation F8 decreased the HT-29 cells' ability to survive. The percentage of cell cytotoxicity was 75.54 at 100μg/ml. Since AGP microsponges had a detrimental effect on the survival of colorectal cancer cells. Conclusion: It can be concluded from the study that prepared formulations possess anticancer properties against cancerous cells and can be used as an alternative anticancer drug.

LAT1 expression in colorectal cancer cells is unresponsive to HIF-1/2α accumulation under experimental hypoxia

L-type amino acid transporter 1 (LAT1) is upregulated in various cancer types and contributes to disease progression. Previous studies have demonstrated or suggested that hypoxia-inducible factors (HIFs), the key transcription factors in hypoxic responses, control the expression of LAT1 gene in several types of cancer cells. However, this regulatory relationship has not been investigated yet in colorectal cancer (CRC), one of the cancer types in which the increased LAT1 expression holds prognostic significance. In this study, we found that neither LAT1 mRNA nor protein is induced under hypoxic condition (1% O2) in CRC HT-29 cells in vitro, regardless of the prominent HIF-1/2α accumulation and HIFs-dependent upregulation of glucose transporter 1. The hypoxic treatment generally did not increase either the mRNA or protein expression of LAT1 in eight CRC cell lines tested, in contrast to the pronounced upregulation by amino acid restriction. Interestingly, knockdown of von Hippel-Lindau ubiquitin ligase to inhibit the proteasomal degradation of HIFs caused an accumulation of HIF-2α and increased the LAT1 expression in certain CRC cell lines. This study contributes to delineating the molecular mechanisms responsible for the pathological expression of LAT1 in CRC cells, emphasizing the ambiguity of HIFs-dependent transcriptional upregulation of LAT1 across cancer cells.

Identifying key genes against rutin on human colorectal cancer cells via ROS pathway by integrated bioinformatic analysis and experimental validation

Colorectal cancer (CRC) poses a significant global health challenge, characterized by substantial prevalence variations across regions. This study delves into the therapeutic potential of rutin, a polyphenol abundant in fruits, for treating CRC. The primary objectives encompass identifying molecular targets and pathways influenced by rutin through an integrated approach combining bioinformatic analysis and experimental validation. Employing Gene Set Enrichment Analysis (GSEA), the study focused on identifying potential differentially expressed genes (DEGs) associated with CRC, specifically those involved in regulating reactive oxygen species, metabolic reprogramming, cell cycle regulation, and apoptosis. Utilizing diverse databases such as GEO2R, CTD, and Gene Cards, the investigation revealed a set of 16 targets. A pharmacological network analysis was subsequently conducted using STITCH and Cytoscape, pinpointing six highly upregulated genes within the rutin network, including TP53, PCNA, CDK4, CCNEB1, CDKN1A, and LDHA. Gene Ontology (GO) analysis predicted functional categories, shedding light on rutin's potential impact on antioxidant properties. KEGG pathway analysis enriched crucial pathways like metabolic and ROS signaling pathways, HIF1a, and mTOR signaling. Diagnostic assessments were performed using UALCAN and GEPIA databases, evaluating mRNA expression levels and overall survival for the identified targets. Molecular docking studies confirmed robust binding associations between rutin and biomolecules such as TP53, PCNA, CDK4, CCNEB1, CDKN1A, and LDHA. Experimental validation included inhibiting colorectal cell HT-29 growth and promoting cell growth with NAC through MTT assay. Flow cytometric analysis also observed rutin-induced G1 phase arrest and cell death in HT-29 cells. RT-PCR demonstrated reduced expression levels of target biomolecules in HT-29 cells treated with rutin. This comprehensive study underscores rutin's potential as a promising therapeutic avenue for CRC, combining computational insights with robust experimental evidence to provide a holistic understanding of its efficacy.

Polyphyllin II Induces Apoptosis in Fibrosarcoma Cells via Activating Pyruvate Kinase M2.

Aerobic glycolysis is a metabolic reprogramming of tumor cells that is essential for sustaining their phenotype of fast multiplication by continuously supplying energy and mass. Pyruvate kinase M2 (PKM2) has a vital role in this process, which has given it high interest as a target for anticancer drug development. With potent toxicity to many types of cancer cells, polyphyllin II (PP2), a steroidal saponin isolated from the herbaceous plant Rhizoma paridis, brought to our attention that it might interfere with the PKM2 activity. In this study, we discovered that PP2 was a novel agonist of PKM2. PP2 activated recombinant PKM2 and changed the protein's oligomeric state to activate intracellular PKM2. At the same time, PP2 suppressed its protein kinase function by decreasing the content of nuclear PKM2. The mRNA levels of its downstream genes, such as Glut1, LDHA, and MYC, were inhibited. In addition, PP2 induced oxidative stress by downregulating the expression and activity of antioxidant proteins such as NQO1, TrxR, and Trx in HT-1080 cells, which in turn led to mitochondrial dysfunction and ultimately induced apoptosis. Moreover, PP2 reduced the proliferation and migration of HT-1080 cells. Thus, targeting the glycolysis pathway offers an unprecedented mode of action for comprehending PP2's pharmacological impacts and advances PP2's further development in fibrosarcoma therapy.

The inhibitory effects of the novel Lactobacillus cocktail on colorectal cancer development through modulating BMP signaling pathway: In vitro and in vivo study

This study investigates the impact of a five-strain Lactobacillus cocktail (comprising two strains of L. plantarum, and one strain each of L. brevis, L. reuteri, and L. rhamnosus) on colorectal cancer (CRC) modulation by targeting the bone morphogenetic proteins (BMP) signaling pathway. Both in vitro and in vivo (models were employed. The antiproliferative effects of the Lactobacillus cocktail on HT-29 cells were assessed via the MTT assay. Mice were divided into three groups: a negative control (treated with PBS), a positive control (treated with azoxymethane (AOM)/dextran sulfate sodium (DSS) + PBS), and a test group (treated with AOM/DSS + Lactobacillus cocktail in PBS). The role of the Lactobacillus cocktail in inhibiting the BMP signaling pathway was evaluated using qRT-PCR for gene expression analysis and western blotting for β-catenin protein assessment in both models. The MTT assay results demonstrated a significant, time-dependent reduction in HT-29 cell proliferation. qRT-PCR indicated downregulation of the BMP signaling pathway in treated cells, which subsequently led to decreased expression of the hes1 gene, crucial for cell differentiation and proliferation control. This inhibitory effect was corroborated in the mice model, showing significant downregulation of BMP pathway genes and hes1 in the AOM/DSS/Lactobacillus cocktail-treated group. Additionally, western blotting revealed a marked decrease in β-catenin expression in both in vitro and in vivo experiments. Collectively, these findings suggest that the Lactobacillus cocktail may aid in CRC prevention by downregulating the BMP signaling pathway.

Semisynthesis of novel chalcone hybrid compounds linked by 1,2,3-triazole and evaluation of their cytotoxic effects

A series of novel 1,2,3-triazole-chalcone hybrid compounds were designed and synthesized from thymol. The structural identity of the synthesized products 5a-h was unequivocally confirmed by spectroscopic techniques, including 1H and 13C NMR spectroscopy, and high-resolution mass spectrometry (HRMS), and SC-XRD for 5b. The in vitro anticancer activity of the synthesized compounds was evaluated against a panel of four human cancer cell lines: fibrosarcoma (HT-1080), lung adenocarcinoma (A-549), and breast adenocarcinoma (MCF-7 and MDA-MB-231). Notably, most of the 1,2,3-triazole-chalcone hybrids (5a-h) displayed promising cell growth inhibition with IC50 values ranging from 4.61 to 50 μM. Furthermore, compounds 5a and 5b, bearing a 1-phenyl-1,2,3-triazole moiety and a 1-cyano-1,2,3-triazole moiety, respectively, exhibited particularly significant antiproliferative activity against HT-1080 cells. These compounds displayed impressive IC50 values of approximately 4.61 μM and 9.08 μM, respectively.

Targeting colorectal cancer with Herba Patriniae and Coix seed: Network pharmacology, molecular docking, and in vitro validation

BACKGROUND Herba Patriniae and Coix seed (HC) constitute a widely utilized drug combination in the clinical management of colorectal cancer (CRC) that is known for its diuretic, anti-inflammatory, and swelling-reducing properties. Although its efficacy has been demonstrated in a clinical setting, the active compounds and their mechanisms of action in CRC treatment remain to be fully elucidated. AIM To identify the active, CRC-targeting components of HC and to elucidate the mechanisms of action involved. METHODS Active HC components were identified and screened using databases. Targets for each component were predicted. CRC-related targets were obtained from human gene databases. Interaction targets between HC and CRC were identified. A “drug-ingredient-target” network was created to identify the core components and targets involved. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to elucidate the key pathways involved. Molecular docking between core targets and key components was executed. In vitro experiments validated core monomers. RESULTS Nineteen active components of HC were identified, with acacetin as the primary active compound. The predictive analysis identified 454 targets of the active compounds in HC. Intersection mapping with 2685 CRC-related targets yielded 171 intervention targets, including 30 core targets. GO and KEGG analyses indicated that HC may influence the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Molecular docking showed that acacetin exhibited an optimal interaction with AKT1 , identifying PI3K , AKT , and P53 as key genes likely targeted by HC during CRC treatment. Acacetin inhibited HT-29 cell proliferation and migration, as well as promoted apoptosis, in vitro . Western blotting analysis revealed increased p53 and cleaved caspase-3 expression and decreased levels of p-PI3K , p-Akt , and survivin, which likely contributed to CRC apoptosis. CONCLUSION Acacetin, the principal active compound in the HC pair, inhibited the proliferation and migration of HT-29 cells and promoted apoptosis through the PI3K/Akt/p53 signaling pathway.

Oncofetal morphogenesis similar to embryonic gut formation by a subpopulation of DLD-1 human colon cancer cells

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

A Promising Approach to Target Colorectal Cancer Using Hybrid Triarylmethanes.

Aiming to create an innovative series of anti-colorectal cancer agents, we designed in this work hybrid triarylmethane compounds. Three hybrid triarylmethanes and their corresponding N-oxide analogues were successfully synthesized using an efficient procedure that involved connecting two triarylmethane molecules, through mono-, bi-, and triethylene glycol fragments. In our pursuit to develop more soluble molecules, we synthesized a hybrid triarylmethane featuring a lysine-based spacer through a convergent strategy involving 7 steps. All hybrid compounds were assessed for their antiproliferative activity on human HT-29 and HCT116 colorectal cancer (CRC) cell lines. Three pyridine N-oxide analogs demonstrated notable antiproliferative potential among the set of tested compounds, with IC50 values ranging from 18 to 24 μM on both human CRC cell lines analyzed. A cytotoxicity study conducted on murine fibroblasts revealed that these three active compounds were not toxic at the IC50 values, indicating their suitability for further drug development. A docking study was conducted on two representative compounds, one for each series and protein kinase B (AKT) was identified as a potential target of their in anti-cancer effects. A computational drug-likeness study predicted favourable oral and intestinal absorption efficiency.

Isothiocyanates attenuate heparin-induced proliferation of colon cancer cells invitro.

Isothiocyanates (ITCs), prevalent in cruciferous vegetables, are known for their anticarcinogenic properties. Prior research has indicated that heparin can stimulate the growth of colon cancer cells. However, the implications of ITCs in the diet of cancer patients receiving heparin-based therapies have yet to be fully understood. This exploratory invitro study examines the proliferative effects of low-molecular-weight heparin (LMWH) on human colon cancer cells and assesses the antiproliferative potential of four ITC compounds, exploring possible epidermal growth factor family of receptor tyrosine kinases (Erb-B) related mechanisms. We evaluated cell viability in HCT-116 and HT-29 cell lines following treatment with ITCs alone or combined with LMWH (20 μg/mL) at various concentrations (1-100 μM). Clonogenic and wound-healing assays were performed after 24 h of treatment with 5 μM ITCs. Additionally, messenger RNA (mRNA) and protein expression of Erb-B family genes was measured using quantitative polymerase chain reaction (qPCR) and Western blotting. Statistical analysis was conducted using analysis of variance (ANOVA) with Dunnett's post hoc test. Results indicated that the half-maximal inhibitory concentration (IC50) values for Phenylethyl isothiocyanate (PEITC), Benzyl isothiocyanate (BITC), and Sulforaphane (SFN) were lower than those of Allyl isothiocyanate (AITC) in LMWH-stimulated HCT-116 (20.77, 19.10, and 44.05 μM, respectively) and HT-29 (74.94, 26.77, and 43.49 μM, respectively). PEITC and SFN significantly reduced ErbB1 (epidermal growth factor receptor (EGFR)) and ErbB4 (receptor tyrosine-protein kinase erbB-4) expression, while BITC decreased ErbB2 (receptor tyrosine-protein kinase erbB-2) and transforming growth factor beta (TGF-β) expression in HCT-116 cells (all, p < .05). PEITC, BITC, and SFN also increased proapoptotic Bax expression and decreased the antiapoptotic B-cell lymphoma 2 (Bcl-2) expression (all, p < .05). These findings suggest that specific ITCs may mitigate cancer cell proliferation induced by LMWH in cancer therapies, highlighting their potential therapeutic efficacy.

Protective effects of potential probiotics Lacticaseibacillus rhamnosus SN21-1 and Lactiplantibacillus plantarum SN21-2 against Salmonella typhimurium infection in broilers

This study aimed to explore the probiotic characteristics of Lacticaseibacillus rhamnosus SN21-1 and Lactiplantibacillus plantarum SN21-2 by genotype and phenotype analysis, assess their safety in vitro and in vivo, and investigate the effects of L. rhamnosus SN21-1 and L. plantarum SN21-2 on Salmonella typhimurium-infected broilers in an in vivo experiment. L. rhamnosus SN21-1 and L. plantarum SN21-2 showed antimicrobial activity against pathogens, including S. Typhimurium, resistance to simulated gastrointestinal digestive fluid, and adhesion to HT-29 cells. In addition, L. rhamnosus SN21-1 and L. plantarum SN21-2 showed no resistance to most common antimicrobial agents and no haemolysis in vitro. Whole-genome sequence analyses of L. rhamnosus SN21-1 and L. plantarum SN21-2 provided basic genomic information, functional genes underlying the probiotic characteristics, and evidence of safety. Furthermore, feeding with L. rhamnosus SN21-1 or L. plantarum SN21-2 for 28 d had no significant effect on the growth or blood biochemical parameters of the broilers, and hematoxylin-eosin staining revealed no liver, spleen, heart, or kidney damage. Additionally, L. rhamnosus SN21-1 or L. plantarum SN21-2 did not translocate to the blood, liver, spleen, heart, or kidney of the broilers. Moreover, L. rhamnosus SN21-1 and L. plantarum SN21-2 significantly reduced S. Typhimurium counts in the faeces and caecal contents of S. Typhimurium-infected broilers and reduced small intestinal bleeding in S. Typhimurium-infected broilers. Consequently, L. rhamnosus SN21-1 and L. plantarum SN21-2 have excellent probiotic characteristics and are safe for use as anti-S. typhimurium probiotics in broilers.

Efficacy assessment of a novel pan-RAS inhibitor in KRAS-mutant and wild type colorectal 3D bioprinted organoid tumor tissue.

91 Background: Colorectal cancer (CRC) is the third leading type of cancer worldwide, with ~150,000 new cases in the US annually and a grim 14% 5-year survival for patients diagnosed at a late stage. A lack of treatment options leads to persistently poor prognosis for patients with advanced stage disease. KRAS mutations are well known drivers of CRC and other GI cancers. Multiple KRAS mutations occur in CRC, including G12D (34%), G12V (21%), G13D (20%), G12C (8%), and others (18%). Existing KRAS-targeted therapies have limited use in CRC, underscoring the need for pan-RAS inhibitors in treating CRC and other RAS driven cancers. Objective: Assess activity of ADT-007, our pan-RAS inhibitor, on wild-type (WT) and KRAS-mutant 3D bioprinted organoid tumor (BOT) tissue using our high-throughput ex vivo platform. Methods: Using previously established bioprinting protocols, WT and mutant BOTs were printed with HT29 and HCT116 cells, respectively. HT29 is an established human WT CRC cell line with known sensitivity to proteosome and survivin inhibitors. HCT116 is a KRASG13D mutant human CRC cell line. 3 sets of BOTs were generated and acclimated for 24h. One set was treated for 72h with proteosome inhibitor Bortezomib, another with survivin inhibitor YM155, and the third with our novel pan-RAS inhibitor ADT-007. Dose response curves were generated from both conventional ATP luminescence readouts and high-content imaging. Results: BOT tissue microarchitecture was validated and &gt;200 µm diffusion in BOTs was confirmed using high-content imaging. Differential response was quantified using Cell TiterGlo endpoint assay as well as advanced image processing of high-content live/dead nuclear stained images captured at multiple z-plains. ADT-007 IC50 was found to be substantially lower for mutant HCT116 compared to that for WT HT29 cell line BOTs, which was consistent with separately conducted in vitro and in vivo studies. Conclusions: A pan-RAS inhibitor, such as ADT-007 with high selectivity for cancer cells with activated RAS that is not limited to a specific KRAS mutant allele or RAS isozyme, could have broader use for CRC and other RAS-driven cancers. Further, due to their potential to replicate biophysical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models.

Antioxidant and Anti-Inflammatory Capacities of Three Dry Bean Varieties after Cooking and In Vitro Gastrointestinal Digestion.

The present study delved into the chemical composition, antioxidant, and anti-inflammatory properties of three dry edible beans: Black (BL), Great Northern (GN), and Pinto (PN). The beans were soaked, cooked, and subjected to in vitro gastrointestinal (GI) digestion. BL bean exhibited significantly higher gastric (42%) and intestinal (8%) digestion rates. Comparative assessment of soluble GI-digested fractions (<3 kDa) revealed that the GN bean exhibited the highest abundance of dipeptides (P < 0.05). The BL bean fraction displayed a 4-fold increase in tripeptides (P < 0.05). Both BL and PN bean fractions are high in essential free amino acids, flavonols, and derivatives of hydroxybenzoic acid when compared to the GN bean. All the beans exhibited the ability to mitigate TNF-α-induced pro-inflammatory signaling; however, the BL bean fraction was the most effective at lowering AAPH-induced oxidative stress in HT-29 cells, followed by the GN bean (P < 0.05). In contrast, a low antioxidant effect was observed with PN beans.

Decoration of silver nanoparticles over chitosan-Arabic gum hydrogel: Synthesis, characterization and investigation of its antioxidant and anti-colorectal cancer activities

The present study describes the production of hybrid biopolymer hydrogel supported Ag NPs in a sustainable manner, along with its biological investigations and characterizations. A composite hydrogel of chitosan and Arabic gum (CS-AG) was utilized as the support or template for the green synthesis of nanoparticles. By taking advantage of their electron-rich organo-functions, the biopolymers could function as both an encapsulating stabilizer of the Ag NPs and a green-metric reductant of the incoming Ag ions. The physicochemical properties of the material were evaluated using an array of advanced analytical techniques like X-ray diffraction (XRD), elemental mapping, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), UV–Vis spectroscopy and Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Towards the biological implication, the material was used in anticancer studies against HT-29 and Caco-2 colon cancer cell lines using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)colorimetric method, as well as in the biological evaluation of antioxidant properties by the DPPH (2,2-diphenyl-1-picrylhydrazyl)method. The substance appeared to have strong anticancer properties based on the DPPH assay's noteworthy IC50 value. Remarkably, it was discovered that the percentage of malignant cell lines' cell viability decreased over the Ag NPs@CS-AG nanocomposite in a dose-dependent manner. The MTT assay produced the corresponding IC50 values against the two cell lines, which were 366.1 μg/mL and 264.5 μg/mL, respectively. At 545 nm, the absorbance rate was assessed, showing toxicity on normal cell line (CHO) up to 1927 μg/mL, which was quite insignificant.

Biological and chemical enhancements of Elaeocarpus sylvestris var. ellipticus through fermentation: Implications for therapeutic and industrial applications

This study aimed to evaluate the biological and chemical improvements that take place during the yeast fermentation of hot water-extracted Elaeocarpus sylvestris (HES). HES was diluted with sterile distilled water to a final concentration of 1:200 (v/v), followed by the inoculation of Pichia fermentans (PF) and Saccharomyces cerevisiae (SC) were inoculated as starters. The fermentation by yeast strains proceeded for five days, during which the viable cell count and pH were monitored. The radical scavenging activity was assessed using DPPH and ABTS assays. The impact of HES on proinflammatory cytokines in RAW 264.7 cells and cytotoxicity in HT-29, MCF-7, and INT407 cells was examined. Changes in metabolic profile during fermentation were analyzed using GC-TOF-MS and UPLC-Orbitrap-MS/MS. HES fermented with PF and SC maintained viable cell counts throughout the 5-day fermentation period. The pH levels were consistently within the range of 3.62 ± 0.03 to 3.65 ± 0.15. The expression of TNF-α and IL-6 significantly decreased at all concentrations (31–500 mg/mL) in RAW cells treated with lipopolysaccharide and HES, with cytokine expression levels returning to baseline levels. The cell viability of INT407 significantly increased following HES treatment (p < 0.05). Fermentation of HES with PF or SC led to significant changes in the composition ratios of carboxylic acids and derivatives, and organooxygen compounds. The HES extract demonstrates potential applicability as a raw material with antioxidative, anti-inflammatory, and anticancer properties for applications in the food and cosmetic industries.

Recombinant Methioninase Is Selectively Synergistic With Doxorubicin Against Wild-type Fibrosarcoma Cells Compared to Normal Cells and Overcomes Highly-Doxorubicin-resistant Fibrosarcoma.

Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro. The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope. The IC50 for doxorubicin was 3.3 μM for HT1080 cells, 12.4 μM for DR-HT1080 cells, and 7.25 μM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells. The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.

Berberine suppressed the epithelial–mesenchymal transition (EMT) of colon epithelial cells through the TGF-β1/Smad and NF-κB pathways associated with miRNA-1269a

ObjectiveTo explore the mechanisms of the TGF-β1/Smad and NF-κB pathways in the effect of berberine (BBR) on colon cancer epithelial–mesenchymal transition (EMT) and their regulatory relationships with microRNAs (miRNAs). MethodsTGF-β1 was used to induce EMT in normal colon epithelial HCoEpiC cells and colon cancer HT29 cells in vitro. After BBR intervention, the expression of EMT-related markers and the major molecules involved in the TGF-β1/Smad and NF-κB pathways were detected via western blotting. Cell migration was detected via wound healing assays. SMAD2 and NF-κB p65 were overexpressed and transfected into cells, and the inhibitors SB431542 and BAY 11–7082 were added to block the TGF-β1/Smad and NF-κB pathways, respectively. The mRNA expression levels of related microRNA genes were detected by using RT‒PCR. ResultsTreatment with 10 ng/ml TGF-β1 for 72 h significantly induced EMT in HCoEpiC and HT29 cells, which was repressed by BBR. BBR significantly inhibited the TGF-β1-induced migration of HCoEpiC and HT29 cells and the TGF-β1-promoted expression of p-Smad2/3, NF-κB p65, and p-IκBα. Compared to those in the group treated with TGF-β1, the expression of NF-κB p65 and p-Smad2 in the group treated with NF-κB pathway inhibitor BAY 11–7082 was decreased (P < 0.05), and TGF-β1 signalling inhibitor SB431542 significantly reduced the expression of NF-κB p65 (P < 0.05). Overexpression of NF-κB p65 and SMAD2 in HT29 cells decreased the expression of E-cadherin and caused a relative increase in N-cadherin. BBR mediated the expression profile of microRNAs in TGF-β1-induced HCoEpiC cells, but this pattern differed from that in HT29 cells. SB431542 and BAY 11–7082 significantly reduced the mRNA level of miR-1269a in HCoEpiC and HT29 cells (P < 0.05). Overexpressed NF-κB p65 and SMAD2 increased the mRNA level of miR-1269a in both cell lines; however, this increase was significantly lower than that in the TGF-β1 treatment group (P < 0.05). ConclusionBBR can significantly inhibit TGF-β1-induced EMT in normal and cancerous colon epithelial cells through the inhibition of the TGF-β1/Smad and NF-κB p65 pathways. TGF-β1/Smads can promote the NF-κB p65 pathway, which is a common target of miR-1269a, and can partially regulate the expression of miR-1269a.

Nano-emulsion based on Santolina chamaecyparissus essential oil potentiates the cytotoxic and apoptotic effects of Doxorubicin: an in vitro study

Aim This study was aimed at investigating the cytotoxic effect of a novel combination of doxorubicin (DOX) and nano-formulation of Santolina chamaecyparissus L. essential oil (SCEO-NANO) on hepatic (HepG2) and colon (HT29) cancer cell lines. Methods A nano-emulsion was prepared by high-pressure homogenisation, then analysed by zetasizer and Fourier transform infrared spectroscopy. HepG2 and HT29 cells were used in in vitro tests for apoptosis detection. Results Formulated droplet size increased in DOX@SCEO-NANO/DOX to 11.54 ± 0.02 with uniform distribution (PDI = 0.13 ± 0.01), when compared with SCEO-NANO (size: 8.91 ± 0.02 nm; PDI = 0.1 ± 0.02). In both cells, DOX@SCEO-NANO/DOX led to a considerable reduction in colony formation. Compared to DOX, apoprotein proteins were overexpressed in HepG2 cells, showing increases of 8.66-fold for caspase-3 and 4.24-fold for the Bax/Bcl-2 ratio. In HT29 cells, ROS-dependent necrosis and apoptosis were seen. Comparing DOX@SCEO-NANO/DOX versus DOX, greater levels of caspase-3 and the Bax/Bcl-2 ratio were observed. Conclusion The DOX@SCEO-NANO/DOX formulation showed potential for targeted eradication of colon adenocarcinoma and hepatocellular carcinoma cells.

Knockdown of LRCH4 Remodels Tumor Microenvironment Through Inhibiting YAP and TGF-β/Smad Signaling Pathway in Colorectal Cancer.

Colorectal cancer is one of the most common gastrointestinal malignancies worldwide. LRCH4 is the top 1 gene associated with an unfavorable prognosis in colorectal cancer. Here, we reported that the knockdown of LRCH4 inhibited the proliferation, migration and invasion in HT29 cells. The activity of Yes-Associated Protein (YAP), a transcription factor in the Hppo-YAP signaling pathway, was significantly inhibited by LRCH4-siRNA. LRCH4 knockdown also reversed the EMT and regulated the expression of extracellular matrix (ECM) protein, Fibronectin and Collagen IV in HT29 cells. In addition, the TGF-β/Smad signaling pathway, as the downstream pathway of Yap, was also inhibited by LRCH4 knockdown. Knockdown of LRCH4 involved in the regulation of ECM and EMT and inhibited YAP and the TGF-β/Smad signaling pathway in colorectal cancer cells. Our study provided a mechanism of LRCH4 on colorectal cancer cells, and a new potential target for clinical tumor treatment.

The preventive and therapeutic role of Lactobacillus spp. in in vitro model of inflammation via affecting autophagy signaling pathway.

Intestinal inflammation has various causes and leads to some inflammatory diseases, of which autophagy pathway dysfunction could be considered as one of them. Probiotics could have a positive effect on reducing inflammation by activating the autophagy pathway. To evaluate the precise effects of probiotics as preventive and therapeutic agents to control the symptoms of inflammatory diseases, we aimed to investigate the efficacy of Lactobacillus spp. in regulating the autophagy signaling pathway. A quantitative real-time polymerase chain reaction assay was used to analyze the expression of autophagy genes involved in the formation of phagophores, autophagosomes, and autolysosomes after exposing the HT-29 cell line to sonicated pathogens and adding Lactobacillus spp. before, after, and simultaneously with inflammation. A cytokine assay was also accomplished to evaluate the interleukin(IL)-6 and IL-1β level following the probiotic treatment. Lactobacillus spp. generally increased autophagy gene expression and consumption of Lactobacillus spp. before, simultaneously, and after inflammation, ultimately leading to activate autophagy pathways. The proinflammatory cytokines including IL-6and IL-1β decreased after probiotic treatment. Our native probiotic Lactobacillus spp. showed beneficial effects on HT-29 cells by increasing autophagy gene expression and decreasing the proinflammatory cytokines production in all treatments. Therefore, this novel probiotic cocktail Lactobacillus spp. can prevent and treat inflammation-related diseases.