Cultured Colon Cancer Cells Research Articles

Ginger Root Bioactive Compounds Specifically Inhibits Growth of Colon Cancer Cells in Culture.

Colon cancer is affluent among many people, and having cancer greatly impacts the lives of many. Ginger is a common food, particularly in Asian cuisine. However, the health benefits of ginger as a whole food and 6-gingerol, its bioactive compound in prevention of colon cancer have not been fully addressed. This experiment investigated effects of ginger juice and 6-gingerol on colon cancer cell growth and death. Fresh ginger roots were homogenized for juice preparation. Total phenolic contents of ginger juice were measured using Folin-C assay. Colon cancer SW480 cells and normal colon epithelial cells CCD-18Co were treated with ginger juice and/or 6-gingerol. Cell metabolic activity was assessed by MTT assay. Cell apoptosis and cell cycle arrest were accessed by immunoblotting. Data were analyzed by 2-way ANOVA with a Tukey post-hoc test and statistical significance was set at P < .05. The results showed that ginger juice selectively inhibited SW480 cell growth at 25 µL/mL for 40 hours. High doses of ginger juice (at 50 and 100 µL/mL for 40 hours) inhibited the growth of both cell types. This was independent of caspase-3 activation. Six-gingerol specifically inhibited SW480 cell growth starting at 0.5 µmoL/L (P < .01). More than 1 µmoL/L 6-gingerol did not give more power to inhibit SW480 cell growth. The results also showed that CCD-18Co cell growth rates were not changed after 6-gingerol treatments (up to 10 µmoL/L, P > .1). Immunoblotting results revealed that the elevation of Myt1 levels and decreases in CDK1, p21 Wafl/Cip1 and pSer642-Wee1 only occurred in SW480 but not CCD-18Co cells when treated with 1 and/or 2.5 µmoL/L 6-gingerol for 40 hours. 6-gingerol can specifically inhibit SW480 cancer cells without killing normal CCd-18Co cells, through cell cycle arrest. Ginger juice can selectively inhibit colon cancer cell growth in a narrow window at ~25 µL/mL.

Lipidomics Profiling Reveals Differential Alterations after FAS Inhibition in 3D Colon Cancer Cell Culture Models.

Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat) is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes a decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.

Cellular Effects of Selected Unsymmetrical Bisacridines on the Multicellular Tumor Spheroids of HCT116 Colon and A549 Lung Cancer Cells in Comparison to Monolayer Cultures.

Multicellular tumor spheroids are a good tool for testing new anticancer drugs, including those that may target cancer stem cells (CSCs), which are responsible for cancer progression, metastasis, and recurrence. Therefore, we applied this model in our studies of highly active antitumor unsymmetrical bisacridines (UAs). We investigated the cellular response induced by UAs in 2D and 3D cultures of HCT116 colon and A549 lung cancer cells, with an additional focus on their impact on the CSC-like population. We showed that UAs affected the viability of the studied cells, as well as their spherogenic potential in the 2D and 3D cultures. Furthermore, we proved that the most promising UAs (C-2045 and C-2053) induced apoptosis in the HCT116 and A549 spheres to a similar, or even higher, extent than what was found in monolayer conditions. Next, we identified the population of the CSC-like cells in the 2D and 3D cultures of the studied cell lines by determining the levels of CD166, CD133, CD44, and EpCAM markers. We showed that the selected UAs affected the CSC-like population in both of the cell lines, and that A549 was affected more profoundly in 3D than in 2D cultures. Thus, the UAs exhibited high antitumor properties in both the 2D and 3D conditions, which makes them promising candidates for future therapeutic applications.

Muscarinic receptor agonist-induced βPix binding to β-catenin promotes colon neoplasia

M3 muscarinic receptors (M3R) modulate β-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.

The effect of chitosan hydrogel containing gold nanoparticle complex with paclitaxel on colon cancer cell line

Colon cancer is a significant global health issue, and its primary treatment, chemotherapy, is limited by toxicity and drug resistance. This has led researchers to explore alternative therapeutic approaches. One such approach is the use of chitosan, a natural biopolymer with anti-cancer properties, and paclitaxel, a potent chemotherapeutic agent with promising activity against many types of cancer. In this study, the effectiveness of a chitosan hydrogel that contains a complex of gold nanoparticles with paclitaxel in treating LS174T colon cancer cell line was investigated. The synthesized chitosan hydrogel was characterized and used to treat the colon cancer cells in cell culture. MTT assay and apoptotic gene expression analysis were conducted to evaluate the complex's effectiveness. The results showed that the chitosan hydrogel-loaded gold nanoparticle-paclitaxel complex exhibited a potent cytotoxic effect against the cancer cells. Moreover, the treatment resulted in a significant increase in pro-apoptotic BAX and BAD expression and a decrease in anti-apoptotic BCL2 expression, indicating a pro-apoptotic effect. These findings suggest that utilizing a chitosan hydrogel that contains a complex of gold nanoparticles with paclitaxel shows promise as a viable treatment option for colon cancer. Further research is needed to determine the potential efficacy and safety of this treatment approach in clinical settings.

Protein–Mineral Composite Particles with Logarithmic Dependence of Anticancer Cytotoxicity on Concentration of Montmorillonite Nanoplates with Adsorbed Cytochrome c

Montmorillonite (MM) colloid nanoplates have high adsorption capacity due to their large size/thickness ratio, which allows them to be used as carriers for drug delivery. Upon adsorption of the mitochondrial protein cytochrome c (cytC) onto MM plates, the composite cytC-MM particles acquire anticancer properties because of the ability of cancer cells to phagocytize submicron particles (in contrast to the normal cells). In this way, exogenous cytC can be introduced into tumor cells, thereby triggering apoptosis-an irreversible cascade of biochemical reactions leading to cell death. In the present study, we investigated the physicochemical properties of cytC-MM particles as a function of the cytC concentration in the suspension, namely, the electrophoretic mobility, the mass increment of MM monoplates upon cytC adsorption, the ratio of the adsorbed to the free cytC in the bulk, the protein density on the MM's surface, the number of cytC globules adsorbed on an MM monoplate, the concentration of cytC-MM composite particles in the suspension, and the dependence of cytotoxicity on the cytC-MM particle concentration. For this purpose, we used microelectrophoresis, static and electric light scattering, and a colon cancer cell culture to test the cytotoxic effects of the cytC-MM suspensions. The results show that the cytotoxicity depends linearly on the logarithm of the particle concentration in the cytC-MM suspension reaching 97%.

Supplementation of quinoa peptides alleviates colorectal cancer and restores gut microbiota in AOM/DSS-treated mice

Quinoa protein hydrolysate has been previously reported to exert anti-cancer effects in cultured colon cancer cells. Here, we investigated the effect of quinoa protein and its hydrolysate on an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced mouse model of colorectal cancer (CRC) and examined its underlying mechanism using gut microbiota analysis and short chain fatty acids (SCFAs) production analysis. Our results showed that quinoa protein or its hydrolysate mitigated the clinical symptoms of CRC and increased SCFAs contents in colon tissues. Moreover, administration of quinoa protein or its hydrolysate partially alleviated gut microbiota dysbiosis in CRC mice by decreasing the abundance of pathogenic bacteria and increasing the abundance of probiotics. Additionally, PICRUSt analysis revealed that the functional profile of gut microbiota in the quinoa protein treated groups was more similar to that of the control group. These findings indicated that the modulation of gut microbiota by quinoa protein diet intervention may ameliorate AOM/DSS-induced CRC.

Improving the Effect of Cancer Cells Irradiation with X-rays and High-Energy Protons Using Bimetallic Palladium-Platinum Nanoparticles with Various Nanostructures

Nano-sized radiosensitizers can be used to increase the effectiveness of radiation-based anticancer therapies. In this study, bimetallic, ~30 nm palladium-platinum nanoparticles (PdPt NPs) with different nanostructures (random nano-alloy NPs and ordered core-shell NPs) were prepared. Scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), energy-dispersive X-ray spectroscopy (EDS), zeta potential measurements, and nanoparticle tracking analysis (NTA) were used to provide the physicochemical characteristics of PdPt NPs. Then, PdPt NPs were added to the cultures of colon cancer cells and normal colon epithelium cells in individually established non-toxic concentrations and irradiated with the non-harmful dose of X-rays/protons. Cell viability before and after PdPt NPs-(non) assisted X-ray/proton irradiation was evaluated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Flow cytometry was used to assess cell apoptosis. The results showed that PdPt NPs significantly enhanced the effect of irradiation on cancer cells. It was noticed that nano-alloy PdPt NPs possess better radiosensitizing properties compared to PtPd core-shell NPs, and the combined effect against cancer cells was c.a. 10% stronger for X-ray than for proton irradiation. Thus, the radio-enhancing features of differently structured PdPt NPs indicate their potential application for the improvement of the effectiveness of radiation-based anticancer therapies.

Anticancer Activity of Calcium Channel Blockers in Colon Cancer Cell Culture

Objective: Repurposing non-cancer drugs for cancer treatment has many advantages. We can access to a new cancer drug easily, quickly and cheaply. In addition, we generally know safety prifile of repurped drugs. There are few studies in the literature investige the anticancer effects of Calcium channel blockers (CCBs). We planned to investigate the anticancer effects of CCBs on colon cancer cell line.&#x0D; Material and Method: We adminestered different doses of T-type CCB NNC-55-0396 and L-type CCB amlodipine on colon cancer cell line HT-29. MTT analysis was performed at 48 hours to measure cell viability. The dose-response curve was constructed using GraphPad Prism.8 programme.&#x0D; Results: Amlodipine caused more than 90% cytotoxicity at all concentrations of 500, 250, 100, 50, 10 µg/ml in MTT analysis at 48 hours. Similarly, NNC-55-0396 caused more than 90% cytotoxicity at all 80, 40, 20, 10.5 µM concentrations.&#x0D; Discussion: In our study, NNC-55-0396 and amlodipine molecules showed severe cytotoxicity on HT-29 cells. There are publications indicating that it may have other anticancer effects other than cytotoxicity. They are promising molecules as anti-cancer drugs. They should be investigated in clinical studies alone or in combination with other cancer drugs.

Hypoxia-inducible factors, mTOR, and astrin constitute an integrative regulatory network in colon cancer cells

Astrin/SPAG5 is a mitotic spindle protein found to be overexpressed in several human cancers, functioning as an oncogene. The expression of Astrin has not been reported so far in colon cancer, nor has it been related to HIFs expression or action. Since mTOR, Astrin, and hypoxia-inducible factors (HIFs) are involved in promoting the growth and survival of cancer cells, we investigated the possible interaction between them in cultured colon cancer cells. Both Astrin and HIF-1α and HIF-2α protein levels were found only expressed in colon cancer cells compared with nonmalignant cells. Our data indicate that mTOR stimulates both Astrin and HIFs expression, but notably, mTORC activity seems to be independent of Astrin expression levels. However, while HIF-1α or HIF-2α stable knockdown increased Astrin expression, mTOR activity was affected in an opposite way by HIF-1α or HIF-2α silencing, indicating that HIF-1α inhibits mTOR while HIF-2α stimulates its activity. These data suggest that mTOR, Astrin, and HIFs compose an integrative network interacting to activate positive or negative regulatory loops probably to coordinate cancer cell growth, metabolism, and survival under oncogenic stress.

The differential cancer growth associated with anaesthetics in a cancer xenograft model of mice: mechanisms and implications of postoperative cancer recurrence.

Anaesthetics may modify colorectal cancer cell biology which potentially affects long-term survival. This study aims to compare propofol and sevoflurane regarding with the direct anaesthetic effects on cancer malignancy and the indirect effects on host immunity in a cancer xenograft mode of mice. Cultured colon cancer cell (Caco-2) was injected subcutaneously to nude mice (day 1). Mice were exposed to either 1.5% sevoflurane for 1.5h or propofol (20μgg-1; ip injection) with or without 4μgg-1 lipopolysaccharide (LPS; ip) from days 15 to 17, compared with those without anaesthetic exposure as controls. The clinical endpoints including tumour volumes over 70 mm3 were closely monitored up to day 28. Tumour samples from the other cohorts were collected on day 18 for PCR array, qRT-PCR, western blotting and immunofluorescent assessment. Propofol treatment reduced tumour size (mean ± SD; 23.0 ± 6.2mm3) when compared to sevoflurane (36.0 ± 0.3mm3) (p = 0.008) or control (23.6 ± 4.7mm3). Propofol decreased hypoxia inducible factor 1α (HIF1α), interleukin 1β (IL1β), and hepatocyte growth factor (HGF) gene expressions and increased tissue inhibitor of metalloproteinases 2 (TIMP-2) gene and protein expression in comparison to sevoflurane in the tumour tissue. LPS suppressed tumour growth in any conditions whilst increased TIMP-2 and anti-cancer neutrophil marker expressions and decreased macrophage marker expressions compared to those in the LPS-untreated groups. Our data indicated that sevoflurane increased cancer development when compared with propofol in vivo under non-surgical condition. Anaesthetics tested in this study did not alter the effects of LPS as an immune modulator in changing immunocyte phenotype and suppressing cancer development.

Lipidomic comparison of 2D and 3D colon cancer cell culture models.

Altered lipid metabolism is one of the hallmarks of cancer. Cellular proliferation and de novo synthesis of lipids are related to cancer progression. In this study, we evaluated the lipidomic profile of two‐dimensional (2D) monolayer and multicellular tumor spheroids from the HCT 116 colon carcinoma cell line. We utilized serial trypsinization on the spheroid samples to generate three cellular populations representing the proliferative, quiescent, and necrotic regions of the spheroid. This analysis enabled a comprehensive identification and quantification of lipids produced in each of the spheroid layer and 2D cultures. We show that lipid subclasses associated with lipid droplets form in oxygen‐restricted and acidic regions of spheroids and are produced at higher levels than in 2D cultures. Additionally, sphingolipid production, which is implicated in cell death and survival pathways, is higher in spheroids relative to 2D cells. Finally, we show that increased numbers of lipids composed of polyunsaturated fatty acids (PUFAs) are produced in the quiescent and necrotic regions of the spheroid. The lipidomic signature for each region and cell culture type highlights the importance of understanding the spatial aspects of cancer biology. These results provide additional lipid biomarkers in colon cancer cells that can be further studied to target pivotal lipid production pathways.

Lipidomic comparison of 2D and 3D colon cancer cell culture models By Fernando Tobias and Amanda B. Hummon

Lipidomic comparison of 2D and 3D colon cancer cell culture models By Fernando Tobias and Amanda B. Hummon

6-Gingerol, but Not Whole Ginger Juice, Specifically Inhibits Growth of Colon Cancer Cells in Culture

ObjectivesColon cancer is affluent among many people, and having cancer greatly impacts their lives. Ginger is a common food, particularly in Asian cuisine. However, the health benefits of ginger and 6-gingerol, as its bioactive component in the prevention of colon cancer, have not been fully addressed. This experiment investigated the effects of ginger juice and 6-gingerol on colon cancer cell growth and death. MethodsColon cancer SW480 cells and CCD-18Co normal colon epithelial cells were purchased from ATCC.SW480 cells were grown in DMEM with 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin, and normal colon CCD-18Co cells were maintained in EMEM with 4.5 g/L glucose and glutamine with 10% FBS. 6-gingerol was dissolved in DMSO in a stock of 100 mmol/L. Ginger roots were homogenized and the juice was collected through 3 layers of cheesecloth filtering, followed by centrifugation and 0.2 μm filter sterilization. About 5000 cells were seeded in a 24-well plate and treated with various amounts of ginger juice and/or 6-gingerol for up to 72 hours. Cell growth was examined using Trypan blue stain, and cell cycle arrest was determined by immunoblotting using antibodies against key proteins in cell cycles. Data were analyzed by two-way ANOVA with a Tukey posthoc test and statistical significance was set at P < 0.05. ResultsTime course and dosage curve experiments showed that 6-gingerol significantly inhibited SW480 cell numbers starting at 0.5 μM (P < 0.005). More than 1 μM 6-gingerol did not give more power to inhibit SW480 cell growth. The results also showed that CCD-18Co cell numbers were not changed after 6-gingerol treatments (P > 0.1). Low dosages of ginger juice (2,000 x- to 100 x- fold dilutions) didn’t affect the growth of both cell types. Immunoblotting results revealed that the elevation of pSer10-CDC2 levels and decreases in p21 Wafl/Cip1 and pSer642-Wee1 only occurred in SW480 but not CCD-18Co cells when treated with 1 μM 6-gingerol for 40 hrs. ConclusionsThrough this experiment, it can be concluded that 6-gingerol can kill SW480 cancer cells without killing normal CCD-18Co cells through cell cycle arrest. Further experiments could be run to discover more properties of 6-gingerol, how it works, and how it could be used in the medical world. Funding SourcesOklahoma State University internal funds to support high school student research.

Inhibitory Effect of Lotus Leaf-Enriched Flavonoid Extract on the Growth of HT-29 Colon Cancer Cells through the Expression of PI3K-Related Molecules.

Objectives Lotus leaf is rich in flavonoids, and this study is aimed at examining the inhibitory effect of lotus leaf-enriched flavonoid extract (LLEFE) on HT-29 colon cancer cells through phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) expression regulation. Methods Lotus leaves were extracted by ethanol and purified using FL-3 macroporous resin to create the LLEFE. HT-29 colon cancer cells were tested using various methods: their proliferation was observed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, their survival status was observed by fluorescence staining, their oxidative stress level was observed by biochemical kits, and their mRNA expression was determined by quantitative polymerase chain reaction (qPCR) assay. Additionally, the composition of the flavonoids in lotus leaf was determined by HPLC. Results The results showed that the proliferation of NCM460 normal human colon cells was not affected by 0–500 μg/mL LLEFE but the proliferation of HT-29 human colon cancer cells decreased. LLEFE increased the LDH level in an HT-29 colon cancer cell culture medium; also increased the superoxide dismutase (SOD), catalase (CAT) activities, and glutathione (GSH) level in HT-29 cells; and decreased the malondialdehyde (MDA) level. Further experimental results showed that LLEFE upregulated the expression of SOD1, CAT, and GSH mRNA and downregulated the expression of PI3K, Akt, and mammalian target of rapamycin (mTOR) in HT-29 cells. The high-performance liquid chromatography (HPLC) results showed that kaempferin, hyperoside, astragaloside, phloridzin, and quercetin were the main chemical constituents of lotus leaf. Conclusion Lotus leaves contain functional flavonoids that inhibit the proliferation of HT-29 colon cancer cells and regulate the expression of PI3K/Akt through five important chemicals.

CFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIPL expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIPL knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.

Fabrication of Antheraea pernyi Silk Fibroin-Based Thermoresponsive Hydrogel Nanofibers for Colon Cancer Cell Culture.

Antheraea pernyi silk fibroin (ASF)-based nanofibers have wide potential for biomaterial applications due to superior biocompatibility. It is not clear whether the ASF-based nanofibers scaffold can be used as an in vitro cancer cell culture platform. In the current study, we fabricated novel ASF-based thermoresponsive hydrogel nanofibers by aqueous electrospinning for colon cancer (LoVo) cells culture. ASF was reacted with allyl glycidyl ether (AGE) for the preparation of allyl silk fibroin (ASF-AGE), which provided the possibility of copolymerization with allyl monomer. The investigation of ASF-AGE structure by 1H NMR revealed that reactive allyl groups were successfully linked with ASF. ASF-based thermoresponsive hydrogel nanofibers (p (ASF-AGE-NIPAAm)) were successfully manufactured by aqueous electrospinning with the polymerization of ASF and N-isopropylacrylamide (NIPAAm). The p (ASF-AGE-NIPAAm) spinning solution showed good spinnability with the increase of polymerization time, and uniform nanofibers were formed at the polymerization time of 360 min. The obtained hydrogel nanofibers exhibited good thermoresponsive that the LCST was similar with PNIPAAm at about 32 °C, and good degradability in protease XIV PBS solution. In addition, the cytocompatibility of colon cancer (LoVo) cells cultured in hydrogel nanofibers was assessed. It was demonstrated that LoVo cells grown on hydrogel nanofibers showed improved cell adhesion, proliferation, and viability than those on hydrogel. The results suggest that the p (ASF-AGE-NIPAAm) hydrogel nanofibers have potential application in LoVo cells culture in vitro. This study demonstrates the feasibility of fabricating ASF-based nanofibers to culture LoVo cancer cells that can potentially be used as an in vitro cancer cell culture platform.

Venom of the desert black snakeWalterinnesia aegyptiaenhances anti-tumor immunity via its beneficial modulatory effects on pro- and anti-tumorigenic inflammatory mediators in cultured colon cancer cells

The role of inflammation in colon cancer is understood as a well-accepted factor that has the tendency to release multiple pro- and anti-tumorigenic inflammatory mediators. Inflammation-induced increased expression of anti-tumorigenic inflammatory mediators and decreased expression of pro-tumorigenic inflammatory mediators encourage beneficial inflammatory effects in terms of powerful anti-tumor immunity. The present study aims to screen the beneficial inflammatory effects of Walterinnesia aegyptia venom via determining its modulatory tendency on the expression of 40 pro- and anti-tumorigenic inflammatory mediators (cytokines/growth factors/chemokines) in LoVo human colon cancer cell line. LoVo-cells were treated with varying doses of crude venom of W. aegyptia. Cell viability was checked utilizing flow cytometry, and IC50 of venom was determined. Venom-induced inflammatory effects were evaluated on the expression of 40 different inflammatory mediators (12 anti-tumorigenic cytokines, 11 pro-tumorigenic cytokines, 7 pro-tumorigenic growth factors, 9 pro-tumorigenic chemokines and 1 anti-tumorigenic chemokine) in treated LoVo-cells [utilizing enzyme-linked immunosorbent assay (ELISA)] and compared with controls. Treatment of venom induced significant cytotoxic effects on inflamed LoVo-cells. IC50 treatment of venom caused significant modulations on the expression of 22 inflammatory mediators in treated LoVo-cells. The beneficial modulatory effects of venom were screened via its capability to significantly increase the expression of five powerful anti-tumorigenic mediators (IL-9, IL-12p40, IL-15, IL-1RA and Fractalkine) and decrease the expression of four major pro-tumorigenic mediators (IL-1β, VEGF, MCP-1 and MCP-3). Walterinnesia aegyptia venom-induced beneficial modulations on the expression of nine crucial pro/anti-tumorigenic inflammatory mediators can be effectively used to enhance powerful anti-tumor immunity against colon cancer.

Montmorillonite colloid plates with adsorbed cytochrome c: in vitro cytotoxic effect on colon cancer cell culture

BackgroundThe apoptosis (a cascade of biochemical reactions leading to suicide of damaged biological cells) is blocked in the cancer cells because of impossibility of cytochrome c (cytC) go out from the mitochondria. However, the apoptosis can be started by introducing of exogenous cytC into cytoplasm using colloid particles as a protein carrier due to ability of the cancer cells to phagocytize extracellular particles with submicron size.ResultsThe clay mineral montmorillonite (MM) were used to prepare aqueous suspension of protein/mineral composite particles by electrostatic adsorption of the positively charged cytC globules on the negatively charged MM colloid plates, and then added to colon cancel culture. The results shows out that separately cytC and MM have no effect but the composite cytC-MM particles kill 95% of the cancer cells after 96 h treatment using equine cytC which is 97% structurally identical with the human cytC. To reach this high cytotoxicity we have formulated requirements to: (a) bare colloid particles (electric charge, form and size), (b) conditions for protein adsorption (concentrations, pH, ionic strength), and (c) suspension with the composite particles (positive total charge and optimal concentration). Due to satisfying these requirements we have reached cytotoxicity which is 1/3 higher than the reached by other authors using different artificial particles. The cytotoxicity rapidly increases with concentration of the cytC-MM particles but further it shows tendency to saturation.MethodsThe optimal pH 6.5 and the 10:3 mg/mg cytC/MM concentration ratio at adsorption were found out by employing computer (protein electrostatics) and physicochemical methods (microelectrophoresis and colloid electrooptics) to prepare cytC-MM suspension. The anticancer capability of cytC-MM nanoplates were investigated using cell culture of metastasizing colon cancer.ConclusionThe in vitro experiments with colon cancer cell culture disclose that cytC-MM composite particles have potential for application in anticancer therapy of superficial neoplasms of the skin and the alimentary system (mouth cavity, esophagus, stomach, jejunum and colon).Graphic abstract

Multi-Layer Nanofibrous PCL Scaffold-Based Colon Cancer Cell Cultures to Mimic Hypoxic Tumor Microenvironment for Bioassay.

Simple SummaryMulti-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based colon cancer cell cultures mimic the hypoxic tumor microenvironment. The simple procedure generates a 3D hypoxic tumor microenvironment comprising defined numbers and densities of colon cancer cells with easily controllable lateral dimensions and a thickness defined by pNFS. This pNFS-based multi-layered colon cancer cell culture system is useful for bioassays, for drug screening, and as a replacement for small animals in testing the effects of a hypoxic tumor microenvironment.Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.