Colorectal Adenocarcinoma HT-29 Cells Research Articles

Probiotic and Postbiotic Potentials of Enterococcus faecalis EF-2001: A Safety Assessment.

Probiotics, which are live microorganisms that, when given in sufficient quantities, promote the host's health, have drawn a lot of interest for their ability to enhance gut health. Enterococcus faecalis, a member of the human gut microbiota, has shown promise as a probiotic candidate due to its functional attributes. However, safety concerns associated with certain strains warrant comprehensive evaluation before therapeutic application. In this study, E. faecalis EF-2001, originally isolated from fecal samples of a healthy human infant, was subjected to a multi-faceted assessment for its safety and probiotic potential. In silico analysis, CAZyme, biosynthetic, and stress-responsive proteins were identified. The genome lacked biogenic amine genes but contained some essential amino acid and vitamin synthetic genes, and carbohydrate-related enzymes essential for probiotic properties. The negligible difference of 0.03% between the 1st and 25th generations indicates that the genetic information of the E. faecalis EF-2001 genome remained stable. The live E. faecalis EF-2001 (E. faecalis EF-2001L) demonstrated low or no virulence potential, minimal D-Lactate production, and susceptibility to most antibiotics except some aminoglycosides. No bile salt deconjugation or biogenic amine production was observed in an in vitro assay. Hemolytic activity assessment showed a β-hemolytic pattern, indicating no red blood cell lysis. Furthermore, the EF-2001L did not produce gelatinase and tolerated simulated gastric and intestinal fluids in an in vitro study. Similarly, heat-killed E. faecalis EF-2001 (E. faecalis EF-2001HK) exhibits tolerance in both acid and base conditions in vitro. Further, no cytotoxicity of postbiotic EF-2001HK was observed in human colorectal adenocarcinoma HT-29 cells. These potential properties suggest that probiotic and postbiotic E. faecalis EF-2001 could be considered safe and retain metabolic activity suitable for human consumption.

The Effects of Photodynamic Therapy with Low-Level Diode Laser Compared with Doxorubicin on HT-29 Colorectal Adenocarcinoma Cells Viability.

Background and Objective: Colorectal adenocarcinoma is considered one of the major causes of cancer-related lethality among other type of malignancies. Given the several limitations and adverse outcomes of conventional therapeutic regimens against colorectal cancer, the focus of many investigations has been attributed to the introduction of a novel combined regimen with harmless agents. The purpose of the present study was to investigate the effect of combined doxorubicin (DOX) treatment and photodynamic therapy (PDT) on colorectal adenocarcinoma cells. Material and Methods: HT-29 cells were exposed to different concentrations of DOX, low-level (630 nm) diode laser, and methylene blue (MB) as a photosensitizer substrate separately and a combination of them. The cytotoxic effect of the DOX, laser, MB, and their combination and the IC50 value for each treatment group were calculated by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT). The malondialdehyde (MDA) content as a biomarker of the lipid peroxidation process and liberated lactate dehydrogenase (LDH) enzyme into supernatant was determined. Results: The results of our study evidenced that a combination of photodynamic light (laser plus MB) and DOX caused a significant reduction in the percentage of HT-29 viable cells compared with control and other treatment groups. In addition, this mentioned combination led to a considerable decrease in IC50 of DOX. Increased cell membrane lipid peroxidation and cell destruction processes in the combination therapy group were proven through significant elevation of MDA content and LDH activity in the medium, respectively. Conclusion: The findings of the present study suggested that DOX combined with PDT had a better therapeutic impact on HT-29 colorectal adenocarcinoma cells. Hence, the simultaneous application of PDT along with antineoplastic drugs improves the chemosensitivity of cancerous cells via the disruption of their membrane and triggering death processes that lead to the decrease of chemotherapeutic agents required doses and undesirable effects.

Sialic acid detection and theranostic activity of phenylboronic acid-based fluorescent probe in human colorectal adenocarcinoma HT-29 cells

A new probe, 4-(((3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)imino)methyl)phenyl)boronic acid (R4B) was prepared by facile condensation of 4-formylphenylboronic acid and rhodamine B hydrazide. R4B was characterized by spectroscopic methods and single crystal X-ray diffraction. The sensor R4B solution turned pink and emitted orange fluorescence only in the presence of sialic acid but remained colorless and non-fluorescent otherwise. The sugar recognition performance was investigated via UV–vis and fluorescence spectroscopic studies. Our results revealed that R4B has good affinity and selectivity for sialic acid over common monosaccharides, with a detection limit as low as 10−7 M. Furthermore, R4B selectively inhibited growth of human colorectal adenocarcinoma HT-29 (IC50 <20 µM) without significant cytotoxicity to normal human colon fibroblasts CCD-18Co. Treatment with R4B suppressed HT-29 colony formation via mitochondrial apoptosis in a time-dependent manner. Cellular imaging studies also revealed the ability of R4B as a fluorescence dye to detect intracellular sialic acid and showed mitochondria-tracking ability in HT-29 cells. In summary, R4B is a potential theranostic for the detection of intracellular sialic acid during the early incubation period, followed by induction of cancer apoptotic cell death at a later treatment point.

Irinotecan-loaded magnetite-silica core-shell systems for colorectal cancer treatment

Colorectal cancer represents a worldwide spread type of cancer and it is regarded as one of the leading death causes, along with lung, breast, and prostate cancers. Since conventional surgical resection and chemotherapy proved limited efficiency, the use of alternative drug delivery systems that ensure the controlled release of cytostatic agents possess immense potential for treatment. In this regard, the present study aimed to develop and evaluate the efficiency of a series of irinotecan-loaded magnetite-silica core–shell systems. The magnetite particles were obtained through a solvothermal treatment, while the silica shell was obtained through the Stöber method directly onto the surface of magnetite particles. Subsequently, the core–shell systems were physico-chemically and morpho-structurally evaluated trough X-ray diffraction (XRD) and (high-resolution) transmission electron microscopy ((HR-)TEM) equipped with a High Annular Angular Dark Field Detector (HAADF) for elemental mapping. After the irinotecan loading, the drug delivery systems were evaluated through Fourier-transform infrared spectroscopy (FT-IR), thermogravimetry and differential scanning calorimetry (TG-DSC), and UV–Vis spectrophotometry. Additionally, the Brunauer–Emmett-Teller (BET) method was employed for determining the surface area and pore volume of the systems. The biological functionality of the core-shells was investigated through the MTT assay performed on both normal and cancer cells. The results of the study confirmed the formation of highly crystalline magnetite particles comprising the core and mesoporous silica layers of sizes varying between 2 and 7 nm as the shell. Additionally, the drug loading and release was dependent on the type of the silica synthesis procedure, since the lack of hexadecyltrimethylammonium bromide (CTAB) resulted in higher drug loading but lower cumulative release. Moreover, the nanostructured systems demonstrated a targeted efficiency towards HT-29 colorectal adenocarcinoma cells, as in the case of normal L929 fibroblast cells, the cell viability was higher than for the pristine drug. In this manner, this study provides the means and procedures for developing drug delivery systems with applicability in the treatment of cancer.

Aminocarbyne-Alkyne Coupling in Diruthenium Complexes: Exploring the Anticancer Potential of the Resulting Vinyliminium Complexes and Comparison with Diiron Homologues.

New diruthenium complexes based on the scaffold Ru2Cp2(CO)2 (Cp = η5-C5H5) and containing a bridging vinyliminium ligand, [2a-d]CF3SO3, were synthesized through regioselective coupling of alkynes with an aminocarbyne precursor (85-90% yields). The reaction involving phenylacetylene proceeded with the formation of a diruthenacyclobutene byproduct, [4]CF3SO3 (10% yield). Complexes [2a-d]+ undergo partial alkyne extrusion in contact with alumina or CDCl3. All products were characterized by elemental analysis, infrared and multinuclear NMR spectroscopy, and single crystal X-ray diffraction in two cases. Complexes [2a-d]+ revealed an outstanding stability in DMEM cell culture medium at 37 °C (<1% degradation over 72 h). These complexes exhibited cytotoxicity in human colon colorectal adenocarcinoma HT-29 cells in the low micromolar range, with lower IC50 values than those obtained with the homologous diiron complexes previously reported. Evaluation of ROS (reactive oxygen species) production and O2 consumption rate (OCR) highlighted the higher potential of Ru2 complexes, compared to the Fe2 counterparts, to impact mitochondrial activity, with the heterometallic Ru2-ferrocenyl complex [2d]+ showing the best performance.

Tumoricidal properties of thymoquinone on human colorectal adenocarcinoma cells via the modulation of autophagy

Colorectal cancer (CRC) is deadly anaplastic changes in the gastrointestinal tract with high-rate mortality. In recent years, the application of phytocompounds has been extended along with different therapeutic protocols. Here, we monitored the effects of Thymoquinone (TQ) on autophagy via mitochondrial function after modulation of the Wnt/β-catenin signaling pathway.Human colorectal adenocarcinoma HT-29 cells were treated with TQ (60 µM) and 15 µM Wnt3a inhibitor (LGK974) for 48 h. The survival rate was evaluated using an MTT assay. The expression of Wnt-related factors (c-Myc, and Axin), angiogenesis (VE-Cadherin), and mitophagy-related factors (PINK1, OPTN) was assessed using real-time PCR assay. Protein levels of autophagy factors (Beclin-1, LC3, and P62) were monitored using western blotting. Using flow cytometry analysis, the intracellular accumulation of Rhodamine 123 was evaluated. The migration properties were analyzed using a scratch wound healing assay.Data indicated that TQ can reduce the viability of HT-29 cells compared to the control cells (p < 0.05). The expression of VE-Cadherin was inhibited while the expression of PINK1 was induced in treated cells (p < 0.05). Both LGK974 and TQ-treated cells exhibited activation of autophagy flux (Beclin-1↑, LC3II/I↑, and p62↓) compared to the control group (p < 0.05). TQ can increase intracellular accumulation of Rhodamine 123, indicating the inhibition of efflux mechanisms in cancer cells. Along with these changes, the migration of cells was also reduced (p < 0.05).TQ is a potential phytocompound to alter the dynamic growth of human colorectal HT-29 cells via the modulation of autophagy, and mitophagy-related mechanisms.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Synthesis of reduced graphene oxide using methanolic extract of Sargassum tenerrimum and its antiproliferative activity against human colorectal adenocarcinoma HT-29 cell lines

Synthesis of reduced graphene oxide using methanolic extract of Sargassum tenerrimum and its antiproliferative activity against human colorectal adenocarcinoma HT-29 cell lines

Phytochemical profile by LC-MS/MS analysis and evaluation of antioxidant, antidiabetic, anti-Alzheimer, and anticancer activity of Onobrychis argyrea leaf extracts

IntroductionStudies on different species of Onobrychis, a member of the Fabaceae family, have revealed a number of biological activities with potential applications in alternative medicine. The phytochemical content of the plant Onobrychis argyrea subsp. argyrea and some of the important biological activities linked to its metabolism are quite limited. Therefore, this study was the first to determine the content of secondary metabolites in this species and to elucidate important biological activities related to metabolism. MethodsLC-MS/MS was used for the quantitative analysis of bioactive compounds in the aerial part of O. argyrea extracts. The antioxidant properties of the extracts were assessed based on their ability to scavenge DPPH radicals and reduce iron ions. The anti-diabetic effect of the extracts was tested by the inhibition of α-amylase and α-glycosidase enzymes, and the anti-Alzheimer's ability was tested by the inhibition of the enzymes AChE and BChE. The cytotoxic effects of the extracts were tested on four different cancer lines, namely A-549, HT-29, MCF-7 and MDA-MB 453 using the XTT assay. The mechanism of the anti-cancer effect was determined by means of flow cytometry analysis on HT-29 cells. ResultsTwenty-nine phytochemicals were identified via the LC-MS/MS analysis in the plant extracts. The most abundant phytochemicals in the extracts were quinic acid, isoquercitrin, epicatechin, and routine, respectively. Antioxidant analysis showed that among all extracts, methanol extract (ME) was most effective in scavenging DPPH (IC50: 23.93 ± 0.96 µg/mL) and in reducing iron ions (53.24 ± 2.14 mgTEAC/g). Enzyme activity studies demonstrated that ME is a potent inhibitor of α-glycosidase (IC50: 15.06±0.64 µg/mL), AChE (IC50: 24.75±0.76 µg/mL), and BChE (IC50: 3.36±0.14 µg/mL). XTT assay results disclosed the strongest anti-proliferative activity of ME against the human colorectal adenocarcinoma HT-29 cell line with an IC50 value of 33.35±0.92 µg/mL. Flow cytometry analysis revealed that ME caused mitochondrial membrane damage in HT-29 cells (**p<0.01) and subsequently induced apoptosis (**p<0.01) by activating the caspase pathway (**p<0.001). ConclusionO. argyrea, rich in bioactive compounds, showed remarkable antioxidant, antidiabetic, Alzheimer's, and anticancer properties in vitro, demonstrating that this plant contains secondary metabolites that may be potential drug candidates for the treatment of metabolic diseases.

Anti-cancer potential of Lactiplantibacillus plantarum NG28 through the induction of intrinsic apoptosis

Introduction: Probiotics and fermented foods have become the focal point of research across different scientific disciplines, with recent findings indicating that Lactiplantibacillus plantarum (L. plantarum) isolated from various origins, holds significant promise in combating numerous diseases, including cancer. Hence, our current study seeks to explore how the L. plantarum NG28 strain might counteract the growth of HT-29 colorectal adenocarcinoma cells, potentially contributing to anti-cancer strategies.&#x0D; &#x0D; Methods: The effect of the ethyl acetate extract of L. plantarum NG28 strain on HT-29 cells was evaluated in several ways. First, the cytotoxicity of HT-29 cells was determined by MTT assay. Additionally, the changes in cell morphology of HT-29 cells through DAPI staining and the induction of apoptosis using Annexin V-FITC/PI staining followed by flow cytometry were investigated. By using the RT-PCR and western blot techniques the expression of pro-apoptotic genes and proteins such as Bax, Caspase-9, and Caspase-3, as well as the anti-apoptotic gene Bcl-2 was examined.&#x0D; &#x0D; Results: The findings revealed that the ethyl acetate extract of L. plantarum NG28 strain had a significant impact on the viability of HT-29 colon cancer cells in a dose-dependent manner, in contrast to its minimal effect on HUVEC cells. DAPI staining revealed the noticeable DNA damage and condensation of treated HT-29 cells and the flow cytometry strengthened the occurrence of apoptosis in the treated HT-29 cells. RT-PCR and western blot techniques investigated the intrinsic apoptosis of HT-29 cells through the increased expression levels of pro-apoptotic genes / proteins and decreased expression levels of anti-apoptotic gene.&#x0D; &#x0D; Conclusion: The study demonstrated the anti-cancer activity of ethyl acetate extract of the L. plantarum NG28 strain against HT-29 colon cancer cells, primarily through the intrinsic apoptotic pathway. This suggests that L. plantarum NG28 strain could be used as a therapeutic tool in the inhibition and medication of colorectal cancer.

Enterococcus Induces MHC-II Expression in the Colonic Epithelium to Aggravate Graft-Versus-Host Disease

Intestinal domination by Enterococcus, a genus of Gram-positive facultative bacteria, has been associated with an increased risk of GVHD mortality in allo-HCT patients and in preclinical models. However, the immunological mechanisms underlying this association remain unclear. Several preclinical studies have demonstrated that MHC-II expression by intestinal epithelial cells (IECs) is involved in allo-antigen presentation and GVHD and can be modulated by the intestinal microbiota. MHC-II genes are constitutively expressed by professional antigen-presenting cells (APCs) but can be induced in non-professional APCs by IFN-γ. Here we reported that Enterococcus could potentially aggravate GVHD through upregulation of MHC-II expression by IECs in an IFN-γ-dependent manner. Using an MHC-disparate mouse model of GVHD (C57BL/6J into BALB/cJ), we profiled the fecal microbiome of GVHD mice by 16S rRNA sequencing and the intestinal epithelium by flow cytometry on day 7 post-transplant. We found that endogenous Enterococcus relative abundance was positively correlated with MHC-II expression by colonic IECs (CD45- CD31- EpCAM+) during GVHD ( Fig. 1a; p=0.0004; 2 replicates; n=19). To determine whether this phenotype occurs in the absence of systemic inflammation, we utilized gnotobiotic mice treated with either E. faecalis, B. producta (a bacteria genus previously associated with improved GVHD outcomes)or PBS via oral gavage. Monocolonization with E. faecalis was sufficient to upregulate MHC-II expression by colonic IECs in non-transplanted (steady-state) animals compared to PBS-treated germfree controls ( Fig. 1b; p=0.0027; 3 replicates; n=8-12/group). On the contrary, monocolonization by B. producta did not induce MHC-II expression by colonic IECs compared to germfree controls ( p=0.96). We also observed that MHC-II upregulation was promoted by specific species within the genus Enterococcus, such that compared to PBS-treated germfree controls, only E. faecalis ( p=0.0007; 3 replicates; n=8-12/group) was able to induce MHC-II expression by colonic IECs, but not E. durans ( p=0.06) , E. gallinarium ( p=0.98) and E. hirae ( p=0.99). Using a Villin-Cre IFN- γ R fl/flmouse model with genetic ablation of the IFN-γ receptor specifically in epithelial cells, we investigated whether MHC-II expression induced by Enterococcus was dependent on IFN-γ signaling. We colonized Villin-Cre pos and Villin-Cre neg littermate controls with E. faecalis via oral gavage after antibiotics decontamination (ampicillin/enrofloxacin) and profiled the intestinal epithelium. We observed that Villin-Cre posmice exhibited a significantly lower MHC-II expression in the small intestine compared to Villin-Cre negmice on day 3 post- Enterococcus colonization ( p=0.012; 2 replicates; n=7/group), suggesting that IFN-γ signaling is necessary to mediate Enterococcus interaction with the intestinal epithelium. Finally, to explore a potential intervention to reduce intestinal inflammation upon Enterococcus domination, we treated GVHD mice with either butyrate-producing B. producta or E. faecalis. Colonization with B. producta reduced fecal Enterococcus burden on day 7 post-transplant ( p=0.007; 2 replicates; n=8/group) and improved survival in GVHD mice, compared to the E. faecalis-colonized group ( p=0.002; 2 replicates; n=18-19/group). We and others have previously shown that butyrate administration can alleviate GVHD through its effects on epithelial homeostasis. To determine whether butyrate could regulate allo-antigen presentation by the intestinal epithelium, we employed the human colorectal adenocarcinoma HT-29 cell culture. We pre-treated the cells with IFNγ (100U/ml) to induce MHC-II expression, then added butyrate (5mM) to the culture for 24 hours. Butyrate treatment was sufficient to suppress MHC-II expression by HT-29 cells, compared to DMSO-treated controls ( p=0.006; 3 replicates; n=6/group). Altogether, our results suggest that one mechanism by which Enterococcus can aggravate GVHD involves the upregulation of MHC-II expression by colonic IECs, and treatment with butyrate might serve as an intervention to prevent intestinal inflammation. Further identification of the upstream mediators involved will provide additional mechanistic insight into the interaction between Enterococcus and the intestinal epithelium at steady state and during GVHD.

7-Methoxyheptaphylline Enhances TRAIL-induced Apoptosis of Colorectal Adenocarcinoma Cell via JNK-mediated DR5 Expression.

A cytokine known as TNF-related apoptosis-inducing ligand (TRAIL) has the ability to precisely cause the death of cancer cells, while normal cells are left undisturbed. Recent studies show that certain cancer cells are sensitive to the apoptotic effect of TRAIL. In this study, HT29 colorectal adenocarcinoma cells exposed to TRAIL were treated with heptaphylline and 7-methoxyheptaphylline from Clausena harmandiana in an effort to comprehend the mechanisms involved behind this activity. The MTT test was utilized to determine cell survival, and phase contrast microscopy was used to examine cell morphology. Through using real-time RT-PCR, Western blotting, and RT-PCR, the molecular mechanisms were investigated. According to the findings, whilst hepataphylline caused cytotoxicity in normal colon FHC cells, in comparison to healthy colon FHC cells, 7-methoxyheptaphylline inhibited cancer cells in a concentration-dependent manner. Heptaphylline alone or in conjunction with TRAIL showed no discernible effect on TRAL-induced HT29 cell death, but 7-methoxyheptaphylline boosted caspase-3 cleavage. The study showed that the JNK pathway was responsible for the 7-methoxyheptaphylline's enhancement of the DR5 (death receptor 5) mRNA, TRAIL receptor, and protein. The results demonstrated that the 7-methoxyheptaphylline of Clausena harmandiana increased the expression of DR5 via the JNK pathway, intensifying TRAIL-induced HT29 cell death.

Metabolomic and Lipidomic Analysis of the Colorectal Adenocarcinoma Cell Line HT29 in Hypoxia and Reoxygenation.

The poor availability of oxygen and nutrients in malignant tumors drives the activation of various molecular responses and metabolic reprogramming in cancer cells. Hypoxic tumor regions often exhibit resistance to chemotherapy and radiotherapy. One approach to enhance cancer therapy is to indirectly increase tumor oxygen availability through targeted metabolic reprogramming. Thus, understanding the underlying metabolic changes occurring during hypoxia and reoxygenation is crucial for improving therapy efficacy. In this study, we utilized the HT29 colorectal adenocarcinoma cell line as a hypoxia-reoxygenation model to investigate central carbon and lipid metabolism. Through quantitative NMR spectroscopy and flow injection analysis - differential mobility spectroscopy-tandem mass spectrometry (FIA-DMS-MS/MS) analysis, we observed alterations in components of mitochondrial metabolism, redox status, specific lipid classes, and structural characteristics of lipids during hypoxia and up to 24 h of reoxygenation. These findings contribute to our understanding of the metabolic changes occurring during reoxygenation and provide the basis for functional studies aimed at metabolic pathways in cancer cells.

Microgels as Platforms for Antibody-Mediated Cytokine Scavenging.

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5µgmL-1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells.

A growth chamber for chronic exposure of mammalian cells to H2S

H2S is a redox-active signaling molecule that exerts an array of cellular and physiological effects. While intracellular H2S concentrations are estimated to be in the low nanomolar range, intestinal luminal concentrations can be significantly higher due to microbial metabolism. Studies assessing H2S effects are typically conducted with a bolus treatment with sulfide salts or slow releasing sulfide donors, which are limited by the volatility of H2S, and by potential off-target effects of the donor molecules. To address these limitations, we describe the design and performance of a mammalian cell culture incubator for sustained exposure to 20–500 ppm H2S (corresponding to a dissolved sulfide concentrations of ∼4–120 μM in the cell culture medium). We report that colorectal adenocarcinoma HT29 cells tolerate prolonged exposure to H2S with no effect on cell viability after 24 h although ≥50 ppm H2S (∼10 μM) restricts cell proliferation. Even the lowest concentration of H2S used in this study (i.e. ∼4 μM) significantly enhanced glucose consumption and lactate production, revealing a much lower threshold for impacting cellular energy metabolism and activating aerobic glycolysis than has been previously appreciated from studies with bolus H2S treatment regimens.

Cupric-ion-promoted fabrication of oxygen-replenishing nanotherapeutics for synergistic chemo and photodynamic therapy against tumor hypoxia.

Mixing a glutathione (GSH)-responsive carboxy zinc(II) phthalocyanine (ZnPc*) and CuSO4·5H2O in water with or without the presence of the anticancer drug SN38 resulted in the formation of self-assembled nanotherapeutics labeled as ZnPc*/Cu/SN38@NP and ZnPc*/Cu@NP, respectively. The Cu2+ ions not only promoted the self-assembly of the carboxy phthalocyanine through metal complexation, but also catalyzed the transformation of H2O2 to oxygen via a catalase-like reaction, rendering an oxygen-replenishing property to the nanosystems. Both nanosystems exhibited high stability in aqueous media, but the nanoparticles disassembled gradually in an acidic or GSH-enriched environment and inside human colorectal adenocarcinoma HT29 cells, releasing the encapsulated therapeutic components. The disassembly process together with the activation by the intracellular GSH led to relaxation of the intrinsic quenching of the nanophotosensitizers and restoration of the photoactivities of ZnPc*. Under a hypoxic condition, ZnPc*/Cu/SN38@NP could attenuate the intracellular hypoxia level and maintain the photodynamic activity due to its Cu2+-promoted oxygen-replenishing ability. The photodynamic effect of ZnPc* and the anticancer effect of SN38 worked cooperatively, causing substantial apoptotic cell death. The dual therapeutic actions could also effectively inhibit the tumor growth in HT29 tumor-bearing nude mice without initiating notable adverse effects to the mice. STATEMENT OF SIGNIFICANCE: The oxygen-dependent nature of photodynamic therapy generally reduces its efficacy against tumor hypoxia, which is a common characteristic of advanced solid tumors and usually leads to resistance toward various anticancer therapies. We report herein a facile approach to assemble a glutathione-responsive carboxy phthalocyanine-based photosensitizer and an anticancer drug in aqueous media, in which Cu(II) ions were used to promote the self-assembly through metal complexation and catalyze the conversion of H2O2 to oxygen through a catalase-like reaction, making the resulting nanoparticles possessing an oxygen-replenishing property that could promote the photodynamic effect against hypoxic cancer cells and tumors. The use of Cu(II) ions to achieve the aforementioned dual functions in the fabrication of advanced nano-photosensitizing systems has not been reported.

Lacticaseibacillus rhamnosus—A Promising Tool for Colorectal Cancer Treatment

Probiotic strains such as Lactobacillus spp. are already known for their beneficial effect on human health and new research supports their role in colon cancer prevention and treatment. The current study reports the effect of different concentrations of Lacticaseibacillus rhamnosus (LGG, 106–109 CFU/mL), alone or in association with 5-fluorouracil (5-FU, 10 μM), tested against normal HaCaT cells, HT-29 colorectal adenocarcinoma and HCT-116 colorectal carcinoma cell lines. The underlying cytotoxic effect was further investigated. LGG treatment of HT-29 and HCT-116 cells caused a variety of apoptotic-related nuclear morphological changes, as revealed by DAPI staining. ELISA studies showed that LGG treatment increased caspase-3 activity and pro-apoptotic BAX protein levels while decreasing anti-apoptotic Bcl-2 protein levels and the proto-oncogene Cyclin D1. A more detailed examination of the mitochondrial function revealed that high concentrations of LGG can impair mitochondrial function in HT-29 and HCT-116 cancer cells. All of these findings suggest that LGG has a pro-apoptotic, mitochondrial-targeted, cytotoxic effect on both colon cancer cell lines studied.

Anticancer properties of curcumin-treated Lactobacillus plantarum against the HT-29 colorectal adenocarcinoma cells

Probiotic bacteria with functions of importance to the health and well-being of the host exhibit various medicinal properties including anti-proliferative properties against cancer cells. There are observations demonstrating probiotic bacteria and their metabolomics can be different in various populations with different eating habits. Here, Lactobacillus plantarum was treated with curcumin (the major compound of turmeric), and its resistance to the curcumin was determined. After then the cell-free supernatants of untreated bacteria (CFS) and bacteria treated with curcumin (cur-CFS) were isolated and their anti-proliferative properties against HT-29 colon cancer cells were compared. The ability of L. plantarum treated with curcumin to combat a variety of pathogenic bacterial species and its ability to survive in acidic conditions were evidence that the probiotic properties of the bacterium were unaffected by the curcumin treatment. L. plantarum treated with curcumin and intact L. plantarum were both able to live in acidic conditions, according to the results of the resistance to low pH test. The MTT result showed that CFS and cur-CFS dose-dependently decreased the growth of HT29 cells with a half-maximal inhibitory concentration of 181.7 and 116.3 µL/mL at 48 h, respectively. Morphological alteration of DAPI-stained cells also exhibited significant fragmentation in the chromatin within the nucleus of cur-CFS-treated cells compared to CFS-treated HT29 cells. Moreover, flow cytometry analyses of apoptosis and cell cycle confirmed DAPI staining and MTT assay results and stipulated the increased occurrence of programmed cell death (apoptosis) in cur-CFS-treated cells (~ 57.65%) compared to CFS-treated cells (~ 47%). These results were more confirmed with qPCR and exhibited the upregulation of Caspase 9–3 and BAX genes, and downregulation of the BCL-2 gene in cur-CFS- and CFS-treated cells. In conclusion, turmeric spice and curcumin may affect the metabolomics of probiotics in intestinal flora which could subsequently influence their anticancer properties.

Reovirus Type 3 Dearing Variants Do Not Induce Necroptosis in RIPK3-Expressing Human Tumor Cell Lines.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

Micro-sized polyethylene particles affect cell viability and oxidative stress responses in human colorectal adenocarcinoma Caco-2 and HT-29 cells

Plastic is a widely utilized material and polyethylene is one of the most used plastic types. Microplastics are plastic particles (size <5 mm) which are primarily a micro-size range or results from degeneration of larger plastic pieces in the environment. Drinking water and food are two main human exposure sources for microplastics and consequently effects of microplastics in gastrointestinal tract are considered important. Still, only little is known how microplastics and plastic associated chemicals affect the human health. The aim of our study was to evaluate the ability of micro-sized polyethylene to cause harmful effects in human intestinal cells. Raw ultra-high molecular-weight polyethylene (size 5–60 μm) was used. In addition, polyethylene particles were extracted with ethanol to determine the effect of extraction process on toxicity of the particles. In the experiments, human colorectal adenocarcinoma Caco-2 and HT-29 cells were exposed to polyethylene (0.25–1.0 mg/ml) or extracts for 48 h. After exposure, cell viability and cytotoxicity were assessed with MTT and lactate dehydrogenase assay. Reactive oxygen species (ROS) production was measured with dichlorofluorescin diacetate and cytoplasmic production of superoxide with dihydroethidium and mitochondrial superoxide production with MitoSOX. The 48-h exposure to polyethylene decreased dose-dependently cell viability and increased oxidative stress, especially mitochondrial superoxide production, in both cell lines. Effects on ROS or cytosolic superoxide production were not observed. Also, exposure to extracts decreased cell viability and increased oxidative stress in cell cultures, but there were differences between cell lines. These effects were most probably caused by the remaining particles rather than the compounds released from the plastic during the extraction. In conclusion, our study shows that micro-sized polyethylene and ethanol-extracted polyethylene in high concentrations decreased cell viability and increased oxidative stress responses in intestinal cells. These results contribute to the existing evidence on potential adverse human health effects of microplastics.