Granatin B and punicalagin from Chinese herbal medicine pomegranate peels elicit reactive oxygen species-mediated apoptosis and cell cycle arrest in colorectal cancer cells.

BACKGROUND: Colorectal cancer ranks among the most common cancers. 5-Fluorouracil (5-FU) based first-line chemotherapy for colorectal cancer treatment often leads to chemoresistance and gastrointestinal mucositis. PURPOSE: This study aimed to find potential therapeutic agents from herbal medicine with anti-colorectal cancer and anti-mucositis activities. METHODS: Chinese medicine theory, network pharmacology analyses, and antioxidant activity coupled with liquid chromatography tandem mass spectrometry analyses were used to identify potential bioactive compounds. HT-29 human colorectal cancer cell culture and xenograft tumor models were employed to study anti-colorectal cancer efficacy. Lipopolysaccharide-induced RAW 264.7 and 5-FU treated Dark Agouti rats were used to evaluate anti-inflammatory and anti-mucositis activities. Histological staining, immunofluorescence imaging, western blots, and flow cytometric analyses were employed to explore the underlying mechanisms. RESULTS: Both Chinese medicine theory and network pharmacology analyses indicated pomegranate peels as a potential anti-colorectal cancer and anti-mucositis agent. Antioxidant activity coupled with liquid chromatography tandem mass spectrometry analyses revealed granatin B and punicalagin as the most potent antioxidant compounds in pomegranate peels. Granatin B and punicalagin demonstrated superior anti-colorectal cancer activities in both cell culture and xenograft tumor models. Granatin B and punicalagin also exhibited strong anti-inflammatory activities in lipopolysaccharide-induced RAW264.7 cells and anti-mucositis activities in 5-FU-treated rats. Mechanistic studies revealed that granatin B and punicalagin induced reactive oxygen species-mediated S-phase cell cycle arrest and apoptosis in HT-29 cells. Moreover, these compounds sensitized HT-29 cells to 5-FU-induced cell death and S-phase cell cycle arrest. CONCLUSION: We report that granatin B and punicalagin exhibit superior anti-colorectal cancer and anti-mucositis activities. To the best of our knowledge, these results are novel and suggest that utilizing phenols from herbal medicine, such as granatin B and punicalagin, to target reactive oxygen species may be an innovative therapy to treat colorectal cancer and intestinal mucositis.

Unravelling the Anti-Inflammatory and Antioxidant Potential of the Marine Sponge Cliona celata from the Portuguese Coastline.

Inflammation is a double-edged sword, as it can have both protective effects and harmful consequences, which, combined with oxidative stress (OS), can lead to the development of deathly chronic inflammatory conditions. Over the years, research has evidenced the potential of marine sponges as a source of effective anti-inflammatory therapeutic agents. Within this framework, the purpose of this study was to evaluate the antioxidant and the anti-inflammatory potential of the marine sponge Cliona celata. For this purpose, their organic extracts (C1-C5) and fractions were evaluated concerning their radical scavenging activity through 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and anti-inflammatory activity through a (lipopolysaccharides (LPS)-induced inflammation on RAW 264.7 cells) model. Compounds present in the two most active fractions (F5 and F13) of C4 were tentatively identified by gas chromatography coupled to mass spectrometry (GC-MS). Even though samples displayed low antioxidant activity, they presented a high anti-inflammatory capacity in the studied cellular inflammatory model when compared to the anti-inflammatory standard, dexamethasone. GC-MS analysis led to the identification of n-hexadecanoic acid, cis-9-hexadecenal, and 13-octadecenal in fraction F5, while two major compounds, octadecanoic acid and cholesterol, were identified in fraction F13. The developed studies demonstrated the high anti-inflammatory activity of the marine sponge C. celata extracts and fractions, highlighting its potential for further therapeutic applications.

Dual-modified PCL-PEG nanoparticles for improved targeting and therapeutic efficacy of docetaxel against colorectal cancer.

Polycaprolactone-poly (ethylene glycol) block copolymer (PCL-PEG) based nanoparticles were prepared for the intravenous administration of docetaxel (DTX). PCL-PEG-Tyr and PCL-PEG-Ang were synthesized by using tyrosine (Tyr) and angiopep-2 (Ang) as coupling ligands, and dual-modified PCL-PEG-based nanoparticles (PCL-PEG-Tyr/Ang) were prepared. The physicochemical properties, in vitro drug release, in vitro cytotoxicity, in vitro cellular uptake efficiency, in vivo biodistribution and in vivo antitumor efficacy of PCL-PEG-based nanoparticles were investigated. The PCL-PEG-based nanoparticles were spherical with a mean diameter of 100 nm and high encapsulation efficiencies (> 85%). The results of in vitro drug release showed that the PCL-PEG-based nanoparticles loaded with DTX had sustained-release characteristics. For in vitro cytotoxicity tests, the dual-modified PCL-PEG-based nanoparticles (PCL-PEG-Tyr/Ang) demonstrated the minimum IC50 value (2.94 µg/mL) compared with other PCL-PEG-based nanoparticles. In addition, the cellular uptake of coumarin-6 (C6) in HT29 cells was observed and determined in the PCL-PEG-Tyr/Ang nanoparticles group, which was significantly higher than that in the other PCL-PEG-based groups and C6 solution group. The results of in vivo imaging showed that dual-modified PCL-PEG nanoparticles had better tumor targeting than the other PCL-PEG-based nanoparticles. In the HT29 tumor-xenografted nude mice model, DTX-loaded PCL-PEG-Tyr/Ang nanoparticles also had a significantly higher inhibitory efficacy on tumor growth than Taxotere®-treated group. These results indicated that the dual-modified PCL-PEG-based nanoparticles (PCL-PEG-Tyr/Ang) could be a promising anticancer drug delivery system.

In-vitro evaluation of dendrimeric formulation of oxaliplatin.

The aim of this study is, preparing various dendrimeric formulations of oxaliplatin and investigating their properties. First of all, the solubility enhancement capabilities of polyamidoamine (PAMAM) G3.5 and PAMAM G4.5 dendrimers were investigated. The results showed that oxaliplatin solubility mostly increasing linearly with dendrimer concentration. Additionally, the increase was more notable in PAMAM G4.5 dendrimers. Then, drug-dendrimer complexes were prepared in different mediums, since the medium used can affect the amount of drug-loaded to dendrimers. Prepared complexes were examined for loading capacity and loading efficiency. It was found that PAMAM G4.5 dendrimers can complex with 2- to 5-fold more oxaliplatin than PAMAM G3.5. Finally, oxaliplatin was modified to a platinum (IV) compound to prepare chemical drug-dendrimer conjugates. Ester bonds were established by Steglich esterification through the hydroxyl group of modified oxaliplatin and the carboxyl groups of the dendrimers. The formulations were characterized by UV, IR, NMR spectroscopy, and dynamic light scattering techniques. PAMAM G3.5 conjugate was further evaluated for the cytotoxicity test. The IC50 value of PAMAM G3.5 conjugate was found as 0.72 µM. For unmodified oxaliplatin, this value was 14.03 µM. As a result, a dendrimer-based drug delivery system that has been found promising for further improvement has been developed successfully.

LC-DAD-ESI-MS/MS analysis and cytotoxic and antiproliferative effects of chlorogenic acid derivative rich extract from Nerium oleander L. pink flowers.

Nerium oleander L. is a widely used medicinal plant for pharmaceutical purposes. In this work, an extract of the pink flowers of this plant (FE) was characterized in terms of phenolic composition by LC-DAD-ESI-MS/MS and bioactivity, namely, antioxidant and antiproliferative effects. A total of 20 compounds from different classes, including derivatives of phenolic acids and flavonoid glycosylated derivatives, were identified in FE. Chlorogenic acid was the dominant phenolic compound in the extract (62.28 ± 1.74 µg mg-1 of dry extract). The antioxidant activity was assessed by ORAC assay, and FE showed an ability to reduce peroxyl radicals (ORAC value of 791.26 µmol TEAC per g DE). Additionally, the FE inhibited the proliferation of a colorectal cancer cell line (HT29 cells, EC50 = 11.72 ± 0.02 µg mL-1) and showed no cytotoxicity to confluent Caco-2 cells, a model of human intestinal epithelium. These results provide new information about the phenolic composition of Nerium oleander pink flowers and the bioactivity of the extracts.

Salidroside induces cell apoptosis and inhibits the invasiveness of HT29 colorectal cells by regulating protein kinase R, NF-κB and STAT3.

BACKGROUND: Protein kinase R (PKR) can suppress various types of solid tumors by inducing cellular oxidative stress and apoptosis. Likewise, Slaidorside, a plant flavonoid, was shown to have anti-tumorigenesis in many solid tumors. OBJECTIVE: This study evaluated anti-tumorigenesis of Salidroside in HT29 colorectal cancer and investigated if the underlying mechanism involves activation of PKR. METHODS: Control or PKR deficient cells were cultured in DMEM media treated with 100 µM Salidroside and cell survival, apoptosis, and other biochemical-related markers were evaluated. RESULTS: Salidroside significantly reduced cell survival and proliferation and increased the release of lactate dehydrogenase (LDH) and levels of single-stranded DNA (ssDNA). It also increased the protein levels of caspases 3 and 8. Concomitantly, Salidroside increased the protein level and activity of PKR and increased the expression of its downstream targets, p-eIF2α (Ser51), p53 MAPK, and p53. On the contrary, it inhibited the nuclear activation of STAT-3 and NF-κB p65. In PKR deficient cells, the partial effects of Salidroside on cell survival, proliferation, and apoptotic markers were observed coincided with no effects on the expression of eIF-2α, and JNK, p53, p38 MAPK, and caspase 8 but with a significant decrease in the nuclear activities of STAT3 and NF-κB. CONCLUSION: Salidroside suppresses the tumorigenesis of HT29 CRC by increasing activation of eIF-2α and JNK and upregulation of p53, p38 MAPK, and caspase-8 through upregulating and activation of PKR. However, the tumor suppressor effect of Salidroside requires also inhibition of STAT3 and NF-κB in a PKR-independent mechanism.

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition.

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.

Fucoidan Induces Apoptosis of HT-29 Cells via the Activation of DR4 and Mitochondrial Pathway.

Fucoidan has a variety of pharmacological activities, but the understanding of the mechanism of fucoidan-induced apoptosis of colorectal cancer cells remains limited. The results of the present study demonstrated that the JNK signaling pathway is involved in the activation of apoptosis in colorectal cancer-derived HT-29 cells, and fucoidan induces apoptosis by activation of the DR4 at the transcriptional and protein levels. The survival rate of HT-29 cells was approximately 40% in the presence of 800 µg/mL of fucoidan, but was increased to 70% after DR4 was silenced by siRNA. Additionally, fucoidan has been shown to reduce the mitochondrial membrane potential and destroy the integrity of mitochondrial membrane. In the presence of an inhibitor of cytochrome C inhibitor and DR4 siRNA or the presence of cytochrome C inhibitor only, the cell survival rate was significantly higher than when cells were treated with DR4 siRNA only. These data indicate that both the DR4 and the mitochondrial pathways contribute to fucoidan-induced apoptosis of HT-29 cells, and the extrinsic pathway is upstream of the intrinsic pathway. In conclusion, the current work identified the mechanism of fucoidan-induced apoptosis and provided a novel theoretical basis for the future development of clinical applications of fucoidan as a drug.

Efficient extraction of cucurbitacins from Diplocyclos palmatus (L.) C. Jeffrey: Optimization using response surface methodology, extraction methods and study of some important bioactivities.

Diplocyclos palmatus (L.) C. Jeffrey is an important medicinal plant used in several reproductive medicines. It serves as a wide source of tetracyclic triterpens called cucurbitacins. Response surface methodology (RSM) with Box-Behnken design (BBD) was studied to optimize the production of cucurbitacins. RSM put forth the ideal conditions such as 1:30 SS ratio (g/mL), 80 rpm (mixing extraction speed), 150 µm mean particle size, 30 min extraction time and 50 °C using chloroform in continuous shaking extraction (CSE) and showed the highest cucurbitacin I (CUI) content (2.345 ± 0.1686 mg/g DW). Similarly, the highest yield of cucurbitacin B (CUB) (1.584 ± 0.15 mg/g DW) was recorded at ideal conditions (1:40 g/mL SS ratio and 60 min time and others similar to CUI). Among the tested extraction methods, the highest CUI, CUB, and CUI + B yield (1.437 ± 0.03, 0.782 ± 0.10, 2.17 ± 0.35 mg/g DW, respectively) as well as promising DPPH radical scavenging activity (25.06 ± 0.1 µgAAE/g DW) were recorded from the SBAE (steam bath assisted extraction). In addition, MAE and UAE revealed the highest inhibition of α-amylase (68.68%) and α-glucosidase (56.27%) enzymes, respectively. Fruit extracts showed potent anticancer activity against breast (MCF-7) and colon (HT-29) cancer cell lines (LC50 - 44.27 and 46.88 µg/mL, respectively). Our study proved that SS ratio, particle size and temperature were the most positively influencing variables and served to be the most efficient for the highest recovery of CUI and CUB. Based on the present study, the fruits of D. palmatus were revealed as a potent antioxidant, anti-diabetic and anticancer bio-resource that could be explored further to develop novel drug to manage diabetes, cancer and oxidative stress related disorders.

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells.

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum-chloride colorimetric and Folin-Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125 ng/mL of DOX and 0.625 mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p < 0.05) and down-regulation of BCL2 (p < 0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

Toxicological impact of acute exposure to E171 food additive and TiO2 nanoparticles on a co-culture of Caco-2 and HT29-MTX intestinal cells.

TiO2 particles are widely used in products for everyday consumption, such as cosmetics and food; their possible adverse effects on human health must therefore be investigated. The aim of this study was to document in vitro impact of the food additive E171, i.e. TiO2, and of TiO2 nanoparticles, on a co-culture of Caco-2 and HT29-MTX cells, which is an in vitro model for human intestine. Cells were exposed to TiO2 particles three days after seeding, i.e. while they were not fully differentiated. Cell viability, reactive oxygen species (ROS) levels and DNA integrity were assessed, by MTT assay, DCFH-DA assay, alkaline and Fpg-modified comet assay and 8-oxo-dGuo measurement by HPLC-MS/MS. The mRNA expression of genes involved in ROS regulation, DNA repair via base-excision repair, and endoplasmic reticulum stress was assessed by RT-qPCR. Exposure to TiO2 particles resulted in increased intracellular ROS levels, but did not impair cell viability and did not cause any oxidative damage to DNA. Only minor changes in mRNA expression were detected. Altogether, this shows that E171 food additive and TiO2 nanoparticles only produce minor effects to this in vitro intestinal cell model.

Arctigenin Inhibits Etoposide Resistance in HT-29 Colon Cancer Cells during Microenvironmental Stress.

Microenvironmental stress, which is naturally observed in solid tumors, has been implicated in anticancer drug resistance. This tumor-specific stress causes the degradation of topoisomerase IIα, rendering cells resistant to topoisomerase IIα-targeted anticancer agents. In addition, microenvironmental stress can induce the overexpression of 78kDa glucose regulated protein (GRP78), which can subsequently block the activation of apoptosis induced by treatment with anticancer agents. Therefore, inhibition of topoisomerase IIα degradation and reduction in GRP78 expression may be effective strategies for inhibiting anticancer drug resistance. In this study, we investigated the active compound arctigenin, which inhibited microenvironmental stress-induced etoposide resistance in HT-29 cells. Arctigenin was also highly toxic to etoposide-resistant HT-29 cells, with an IC50 value of 10 µM for colony formation. We further showed that arctigenin inhibited the degradation of topoisomerase IIα and reduced the expression of GRP78. Thus, these results suggest that arctigenin is a novel therapeutic agent that inhibits resistance to etoposide associated with microenvironmental stress conditions.

Thermally-responsive Virus-like Particle for Targeted Delivery of Cancer Drug.

Multifunctional nanocarriers displaying specific ligands and simultaneously response to stimuli offer great potentials for targeted and controlled drug delivery. Several synthetic thermally-responsive nanocarriers have been studied extensively for hyperthermia incorporated chemotherapy. However, no information is available on the application of virus-like particle (VLP) in thermally-controlled drug delivery systems. Here, we describe the development of a novel multifunctional nanovehicle based on the VLP of Macrobrachium rosenbergii nodavirus (MrNVLP). Folic acid (FA) was covalently conjugated to lysine residues located on the surface of MrNVLP, while doxorubicin (Dox) was loaded inside the VLP using an infusion method. This thermally-responsive nanovehicle, namely FA-MrNVLP-Dox, released Dox in a sustained manner and the rate of drug release increased in response to a hyperthermia temperature at 43 °C. The FA-MrNVLP-Dox enhanced the delivery of Dox to HT29 cancer cells expressing high level of folate receptor (FR) as compared to CCD841CoN normal cells and HepG2 cancer cells, which express low levels of FR. As a result, FA-MrNVLP-Dox increased the cytotoxicity of Dox on HT29 cells, and decreased the drug's cytotoxicity on CCD841CoN and HepG2 cells. This study demonstrated the potential of FA-MrNVLP-Dox as a thermally-responsive nanovehicle for targeted delivery of Dox to cancer cells rich in FR.

Nonlinear ultrasound parameter to monitor cell death in cancer cell samples.

A scaling subtraction method was proposed to analyze the radio frequency data from cancer cell samples exposed to an anti-cancer drug and to estimate a nonlinear parameter. The nonlinear parameter was found to be well correlated (R2 = 0.62) to the percentage of dead cells in apoptosis and necrosis. The origin of the nonlinearity may be related to a change in contacts between cells, since the nonlinear parameter was well correlated to the average total coordination number of binary packings (R2 ≥ 0.77). These results suggest that the scaling subtraction method may be used to early quantify chemotherapeutic treatment efficiency.

lincRNA-p21 Mediates the Anti-Cancer Effect of Ginkgo Biloba Extract EGb 761 by Stabilizing E-Cadherin Protein in Colon Cancer.

BACKGROUND Ginkgo biloba extract EGb 761 is a putative antioxidant and has been used for thousands of years to treat a variety of ailments, including cancer. While it is known that cell behavior can be modulated by long non-coding RNAs (lncRNAs), the contributions of lncRNAs in EGb 761-induced anti-cancer effects are largely unknown. MATERIAL AND METHODS Colon cancer cell lines HT29 and HCT116 were used in this study. RT-qPCR was used to detect the relative expression of lincRNA-p21 in colon cancer cells. Wound-healing assay and Matrigel Transwell assay were performed to investigate the migration and invasion of colon cancer cells. Immunoprecipitation and Western blot experiments were used to verify ubiquitination and the interaction between lincRNA-p21 and E-cadherin, or E-cadherin and b-transducin repeat containing (BTRC) E3 ubiquitin protein ligase. RESULTS Cell function assay verified that treatment with EGb 761 suppressed the migratory and invasive abilities of colon cancer cells in a dose-dependent manner via the suppression of E-cadherin expression level. lincRNA-p21 was upregulated in colon cancer cells after treatment with EGb 761, and knockdown of lincRNA-p21 reversed the EGb 761-induced anti-metastatic effect. Furthermore, lincRNA-p21 was localized in cytoplasm of colon cells and regulated E-cadherin expression at a post-transcriptional level. Specifically, lincRNA-p21 promotes E-cadherin stability by preventing the interaction between BTRC and E-cadherin, which leads to the inhibition of E-cadherin ubiquitination. CONCLUSIONS We demonstrated that lincRNA-p21 mediates the anti-cancer effect of Ginkgo biloba extract EGb 761 by stabilizing E-cadherin protein in colon cancer, which may help define the functional role of EGb 761 in cancer treatment.

Biological studies of synthesized silver nanoparticles using Prosopis farcta.

The evaluation of cytotoxic and apoptotic activities of silver nanoparticles (Ag-NPs) synthesized by aqueous extract of Prosopis farcta was investigated against lung (A549) and colon (HT-29) cell lines. The cytotoxic activity of nanoparticles was performed using MTT assay, while their apoptotic activity was tested using TUNEL method. The obtained results of MTT showed that the cell viability of A549 was dependent on the nanoparticles concentration and incubation time. Therefore, although the cytotoxic effect increased as the Ag-NPs concentration and incubation time heightened, yet the viability of HT-29 cells seems to be dependent only on the incubation time. The apoptotic results of the nanoparticles showed more than 50% of apoptosis on A549 and HT-29 cell lines, which in this case, HT-29 demonstrated 100% apoptosis at concentrations of more than 400 µg/ml. It seems that Ag-NPs synthesized using P. farcta extract can serve as anti-cancer agent in the treatment many cancers through creating or discovering new drug forms.

Nisin, a potent bacteriocin and anti-bacterial peptide, attenuates expression of metastatic genes in colorectal cancer cell lines.

Colorectal cancer is the third most common cause of cancer-related death in the world which genetic and environmental agents are responsible for cancer. When cells detach from the tumor and invade surrounding tissues, the tumor is malignant and may form secondary tumors at other locations in a process called metastasis. Probiotics are the largest group of inhabitation bacteria in the colon. Gut microbiota has a central role in prevented the risk colon cancer. Probiotics are beneficial microorganisms, like Lactic acid bacteria and Lactobacilli bacteria which are using in the dairy industry. Probiotics nisin are having the most important category of safe usage. In this study LS180, SW48, HT29 and Caco2 was cultured and treated with different dose of nisin. Cell proliferation was assayed with MTT. The expression of CEA, CEAM6 and MMP2F genes was analyzed with Real-time PCR. Protein expression of CEA was evacuated with ELISA. Our result was shown that the 40-50 IU/mL nisin could suppress proliferation of LS180. Cell proliferation of SW48, HT29, Caco2 cells was decreased in 250-350 IU/mL concentration of nisin. The gene expression of CEA, CEAM6, MMP2F was significantly down-regulated with nisin treatment (p < 0.001, p < 0.01). Also, after cells treated with nisin, CEA protein expression was down regulated (p < 0.01). In conclusion, nisin could suppressed metastatic process via down-regulation of CEA, CEAM6, MMP2F, MMP9F genes. We suggested the new treatment strategies beyond Probiotics, which play a role in the prevention local tumor invasion, metastasis and recurrence.

Induced cortical tension restores functional junctions in adhesion-defective carcinoma cells.

Normal epithelial cells are stably connected to each other via the apical junctional complex (AJC). AJCs, however, tend to be disrupted during tumor progression, and this process is implicated in cancer dissemination. Here, using colon carcinoma cells that fail to form AJCs, we investigated molecular defects behind this failure through a search for chemical compounds that could restore AJCs, and found that microtubule-polymerization inhibitors (MTIs) were effective. MTIs activated GEF-H1/RhoA signaling, causing actomyosin contraction at the apical cortex. This contraction transmitted force to the cadherin-catenin complex, resulting in a mechanosensitive recruitment of vinculin to cell junctions. This process, in turn, recruited PDZ-RhoGEF to the junctions, leading to the RhoA/ROCK/LIM kinase/cofilin-dependent stabilization of the junctions. RhoGAP depletion mimicked these MTI-mediated processes. Cells that normally organize AJCs did not show such MTI/RhoA sensitivity. Thus, advanced carcinoma cells require elevated RhoA activity for establishing robust junctions, which triggers tension-sensitive reorganization of actin/adhesion regulators.

The Cytolethal Distending Toxin Subunit CdtB of Helicobacter hepaticus Promotes Senescence and Endoreplication in Xenograft Mouse Models of Hepatic and Intestinal Cell Lines.

Cytolethal distending toxins (CDTs) are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB). For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3) was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

SL2B aptamer and folic acid dual-targeting DNA nanostructures for synergic biological effect with chemotherapy to combat colorectal cancer.

DNA nanostructures prepared by self-assembly possess good stability, high biocompatibility, and low immunogenicity as drug delivery vehicles. In this work, DNA tetrahedron (TD) was constructed and modified with SL2B aptamer (S) and folic acid (F). TD possessed a small diameter (~6 nm) and entered into the nucleus quickly. SL2B aptamer can inhibit cancer cell growth by disturbing vascular endothelial growth factor/Notch signaling pathways. To explore the effect of SL2B number on colorectal cancer inhibition, SL2B multimers (dimer, trimer, and tetramer) were constructed by functionalization of TD with different numbers of SL2B. One SL2B per TD was the most efficient anticancer strategy and showed significantly better anticancer efficacy than SL2B, probably due to the enhanced stability of SL2B by TD. Doxorubicin (DOX) is a potent anticancer agent that can intercalate into DNA double strands. Results showed that TD could facilitate DOX entrance into the nucleus and the intracellular delivery of DOX was further enhanced by functionalization of SL2B and F. DOX-intercalated TD modified with two F and two S (DOX@TD-2F2S) could cause sufficient HT-29 cell inhibition at a much lower DOX concentration. In sum, DOX@TD-2F2S exhibited a synergic anticancer biological effect with chemotherapy and can be a promising strategy for treating colorectal cancer.