HT-29 Colon Cancer Cells Research Articles

Extraction of phenolic compounds from the shells of pecan nuts with cytotoxic activity through apoptosis against the colon cancer cell line HT-29.

The water extraction of phenolic compounds from two varieties ("Mahan" and "Marameck") of pecan nutshells (Carya illinoinensis) without and with sonication, varying the solvent/solid ratio (S), the pH, and the refluxing time (t), was studied. Additionally, the in vitro cytotoxicity and the determination of the cell death mechanism of the extracts against the colon cancer cell line HT-29 were investigated. The content of total phenolic compounds (TPC) of "Marameck" nutshells resulted higher than for the "Mahan" variety, and the pH increase resulted in higher TPC contents for both cultivars. The optimized conditions for TPC extraction without and with sonication resulted: S = 33 ml/g, pH = 12, and t = 9.6 min, and yielded ≈70 and 90 mg/g of TPC for "Mahan" and "Marameck" nutshells, respectively. The optimized extracts of pecan nutshells without sonication from both cultivars presented similar cytotoxicity against HT-29 colon cancer cells (IC50 ≈50 µg/ml), higher than for sonicated extracts (IC50 ≈ 88 and 138 µg/ml for "Mahan" and "Marameck," respectively). Cell death through apoptosis was the main mechanism of cell death induced by the nutshell extracts. PRACTICAL APPLICATION: The extraction of phenolic compounds (TPC) from the residues of two varieties of pecan nutshells ("Mahan" and "Marameck") was studied. An optimal combination of variables within the pH range that minimizes the solvent-to-solid ratio (S) and the time of refluxing (t), saving at the same time, water and energy, was set up. The phenolic compound extracts obtained from the residues of the pecan nuts exhibit cytotoxic effects against colon cancer cells and could be of interest as an alternative treatment of different types of cancer. Additionally, these extracts may be of importance to the food industry as they can be used as antioxidant agents in food formulation. Also, the high levels of anthocyanidins obtained from the pecan nut extracts after proanthocyanidins' strong acid hydrolysis can be purified and employed as natural red dyes.

Real-Time Impedance Monitoring of Alkaline Phosphatase (ALP) Release by Microelectrode Arrays

Alkaline phosphatase (ALP) is a reporter enzyme widely used in point-of-care applications. Real-time monitoring of ALP accelerates understanding of emerging diseases and of cancer progression. This can be achieved through biosensor-based strategies. This paper outlines an approach that facilitates the real-time monitoring of ALP release from the differentiation of cultured human colon cancer cells (Ht-29). Electric cell-substrate impedance sensing was used to generate changes in Ht-29 cells during ALP release and the data were compared using amperometry. The findings indicate a correlation between cell response and shift in impedance magnitude and current. Relevant results had similar attitude during cell monitoring, which influenced cell morphology and cell viability. Our analysis is a clear advance on the current methods such as enzyme-linked immunosorbent assay. The developed method can contribute to the development of microfluidic testing that would facilitate work on small-scale techniques.

Regulation of Butyrate-Induced Resistance through AMPK Signaling Pathway in Human Colon Cancer Cells.

Butyrates inhibit cell growth in colon cancer cells by inhibiting histone deacetylases. However, chronic exposure to butyrates induces butyrate resistance in colon cancer cells. The mechanism underlying the acquisition of resistance is not yet fully understood. Here, butyrate-resistant (BR) colon cancer cells were developed in HCT116, HT29, and SW480 human colon cancer cells and were confirmed by the increase in the inhibitory concentrations of cell growth by 50% (IC50) compared to their respective parental (PT) cells. Chronic exposure to butyrate induced autophagy via higher expression of Beclin-1 and LC3B-II. The AMP-activated protein kinase (AMPK) was downregulated along with the activation of Akt and mammalian target of rapamycin (mTOR) and decrease in acetyl-CoA carboxylase (ACC) in BR colon cancer cells compared to those in their respective PT cells. Activation of AMPK by AICAR treatment in BR colon cancer cells suppressed cell proliferation by inhibiting Akt and mTOR and activating ACC. Taken together, chronic exposure to butyrate increased butyrate resistance in human colon cancer by inducing protective autophagy through the downregulation of AMPK/ACC and activation of Akt/mTOR signaling. Activation of AMPK restored sensitivity to butyrate by the inhibition of Akt/mTOR, suggesting that AMPK could be a therapeutic target for BR colon cancers.

Phenolic content, antioxidant, cytotoxic and antiproliferative effects of fractions of Vigna subterraenea (L.) verdc from Mpumalanga, South Africa

Consistent intake of legumes has been correlated with decreased possibility of developing colorectal cancer (CRC) due to the content of some phytochemicals like polyphenols. Bambara groundnut (BGN) is an underutilized crop with a rich nutritional profile, but have not been exploited for its nutraceutical and medicinal benefits. In this study, total polyphenol, flavonoid (flavonol and flavanol) content, antioxidant activity and cytotoxicity/antiproliferative properties of 70% ethanolic extracts of whole BGN, cotyledon and seed coat on Caco-2 and HT-29 colon cancer cells were evaluated. Seed coat had a significantly (p < 0.05) higher composition of total polyphenol, flavonol and flavan-3-ol (flavanol) compared to whole seed and cotyledon. Antioxidant activity determined with ferric reducing antioxidant power (FRAP), 2,2- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays, showed that seed coat with higher polyphenolic content had significantly (p < 0.05) greater antioxidant activity. BGN fractions demonstrated cytotoxic and antiproliferative effects against HT-29 and Caco-2 colon cancer cells in a dose-dependent manner, with seed coat and whole seed exhibiting greater cytotoxicity and higher antiproliferative activity and colon cancer cell inhibition. Extracts of the cotyledon also showed cytotoxic activity and hindered cancer cell growth/division but to a significantly (p < 0.05) lower magnitude. BGN parts indicated a greater cytotoxic effect and potential to slow down Caco-2 colon cancer cell growth and division over HT-29. This result provides new knowledge on the possible health benefits of BGN, as well as the potential for product development and may influence its consumption and utilisation.

The new andrographolide derivative AGS-30 induces apoptosis in human colon cancer cells by activating a ROS-dependent JNK signalling pathway

Background: The anti-cancer activity of andrographolide (Andro) has been extensively demonstrated in recent years. It is supposed that modifying the chemical structure of Andro can improve its efficacy and reduce its toxicity. Purpose: In this study, the anti-cancer effect of a 14β-(2′-chlorophenoxy) derivative of andrographolide known as AGS-30 was investigated, and its underlying mechanisms were also explored. Study Design/Methods: Different cancer cells were used to evaluate and compare the in vitro anti-cancer effects of Andro and AGS-30. Human colon cancer cells HT-29 and HCT-116 were used to study the underlying anti-cancer mechanisms of AGS-30. HT-29 cells xenografted in nude mouse model was used to compare the in vivo anti-tumour efficacies of Andro and AGS-30. Result:In vitro studies showed that AGS-30 possessed an anti-cancer effect by inhibiting the viability, colony formation and migration of cancer cells. It significantly induced the generation of reactive oxygen species (ROS), caused the loss of mitochondrial membrane potential and triggered the apoptosis in colon cancer cells. These effects of AGS-30 were more potent than those of Andro. In addition, the expression levels of proteins associated with apoptosis, including phospho-JNK1/2 as well as cleaved caspase 9, caspase 3, and poly(ADP ribose) polymerase, were elevated in AGS-30-treated colon cancer cells. Moreover, these elevated levels of the proteins were inhibited by the antioxidant N-acetylcysteine and the JNK inhibitor SP600125, suggesting the involvement of ROS/JNK-dependent mechanisms in AGS-30-induced apoptosis. The in vitro anti-cancer effect could be reproduced in an HT-29 colon cancer cell xenografted nude mouse model. Conclusion: The anti-cancer effect of AGS-30 is stronger than that of Andro. AGS-30 induces apoptosis of colon cancer cells through ROS/JNK-dependent pathway. Our findings may provide insights for the future development of derivatives of Andro as novel chemotherapeutic agents.

Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue\u2212derived cells in vitro

BackgroundThis study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells.MethodsWistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining.ResultsThe results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos.ConclusionsThese findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

A bis indole analog molecule fluorescent probe for living cell staining

In this study, the fluorescent probe property of a bis indole compound (5,14-dimethyl-5,6,7,8,9,14-hexahydroindolo [2 ', 3′: 3,4] cycloocta [1,2-b] indole), a Caulerpin derivative, which is a member of the indole family, was investigated. For this purpose, firstly, the fluorescence character of the molecule was investigated by absorption and fluorescence spectroscopy techniques in both solvent and surfactant environments. Afterwards, the cytotoxicity of the molecule was tested on the human colon cancer HT-29 cells and breast cancer MCF-7 cells as well as mouse fibroblast 3T3-L1 cells. After determining that the molecule has no cytotoxic effect against all the tested cell lines by 100 µM, the cell staining feature of the probe was examined. The obtained data showed us that the molecule, whose confocal microscopy images were taken at 360 nm excitation wavelength, stained the cell cytoplasm. In addition, optical absorption spectra, oscillator strength (f), transition configuration and transition characteristics of the Ars-In in different solvents were investigated at the theoretical level using Time Dependent-Density Functional Theory (TD-DFT). In conclusion, these results confirm that this indole derivative molecule could be evaluated as an effective fluorescence probe for biological systems.

Reversal of ABCG2/BCRP-Mediated Multidrug Resistance by 5,3',5'-Trihydroxy-3,6,7,4'-Tetramethoxyflavone Isolated from the Australian Desert Plant Eremophila galeata Chinnock.

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone (2), which at a concentration of 25 μg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 μM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

Evaluation of Radiosensitive Effect of Tolmetin in Radiotherapy on Human Colonic Carcinoma Cell Line HT-29

Background: Radiotherapy is one of the cancer treatment methods. Although radioresistance and normal tissue toxicity limit the radiation therapy in certain anatomical locations, using some substances can be useful to increase radiosensitivity on cancer cells without cytotoxicity effect on normal cells. Objective: This study aimed to evaluate the radiosensitizing effect of tolmetin in radiotherapy treatment on human colon cancer cell line HT-29. Methods In this study, human colon cancer cells HT-29 in different groups were irradiated with 4 Gy x-ray, and tolmetin was administered in different concentrations (75, 100, and 150 μM). Then, the groups were compared with each other and with the control group by Micronucleus Assay (MN) and Nuclear Division Index (NDI). NDI investigated the cytotoxicity, and MN indicated the genotoxicity. Results: In the group receiving radiation, micronuclei increased significantly compared to the control group. In the group receiving tolmetin with a concentration of 75 and 100 μM, the number of micronuclei also increased compared to the control group. In all groups treated with tolmetin that also received radiation, a significant increase in micronuclei was observed, which was more noticeable at concentrations of 100 and 150 μM. At the same time, tolmetin at the studied concentrations did not change the NDI index. Conclusion: The present study demonstrates that tolmetin has a radiosensitizing effect on HT-29 colon cancer cells, which depends on the tolmetin concentration. In addition, tolmetin has no cytotoxic effect on this cell line.

Recombinant water stress protein 1 (Re-WSP1) suppresses colon cancer cell growth through the miR-539/β-catenin signaling pathway.

Nostoc commune Vauch. is a nitrogen-fixing blue-green algae that expresses a large number of active molecules with medicinal properties. Our previous study found that a water stress protein (WSP1) from N. commune and its recombinant counterpart (Re-WSP1) exhibited significant anti-colon cancer activity both in vitro and in vivo. This study is to investigate the effects of Re-WSP1 on proliferation of colon cancer cells and to elucidate the relevant mechanisms. Real-time quantitative PCR was used to detect the expression of miR-539 in colon cancer HT-29 and DLD1 cells. Colon cancer cells were transfected with miR-539 mimics and negative controls, and cell proliferation were detected by CCK8 and clonogenic assays. The target gene of miR-539 was predicted, and the dual luciferase reporter gene experiment was used to verify the target gene. After colon cancer cells were transfected with miR-539 mimics or inhibitors, the expression of target gene β-catenin was detected by Western blot. miR-539 inhibitor confirmed cell proliferation. Re-WSP1 inhibited colon cancer cell growth in a dose-dependent manner. Re-WSP1 inhibited the expression of β-catenin, which was partly reversed by LiCl treatment. Quantitative PCR analysis showed that the expression of miR-539 was significantly upregulated after Re-WSP1 treatment. Moreover, miR-539 negatively regulated the expression of β-catenin by directly binding to the 3'UTR of β-catenin mRNA. The cell growth inhibition and the decrease in β-catenin expression induced by Re-WSP1 were significantly reversed by miR-539 inhibitor. Re-WSP1 suppresses colon cancer cell growth via the miR-539/β-catenin axis.

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells.

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Synergistic Anti-proliferative Effects of Lenalidomide and Dexamethasone on the HT-29 Cell Line Through Apoptotic Genes

Background: Colorectal cancer (CRC) is the third most common cancer among men and the second most common type of cancer among women worldwide. The resistance of tumor cells to apoptosis is caused by changes in the expression of anti-apoptotic or pro-apoptotic proteins. Histone deacetylase inhibitors (HDACi) are known to cause changes in gene expression. Objectives: The present study aimed at investigating the anti-proliferative effects of lenalidomide (LEN) as HDACi and dexamethasone (DEX) on the human colon cancer HT-29 cell line. Methods: The HT-29 cell line was treated with various concentrations of LEN and DEX individually and in combination for 24, 48, and 72 hours. Cytotoxicity was evaluated by MTT assay. The half-maximal inhibitory concentration (IC50) was measured, and quantitative real-time polymerase chain reaction (qRT-PCR) was also performed to examine the expression of Bcl2, Bax, Fas, and FasL genes. Results: The combination of LEN (1000 µM) with DEX (100 µM) showed potent synergistic anti-proliferative activities in a time- and dose-dependent manner. The combination of these drugs induced cell death by affecting the extrinsic and intrinsic apoptotic gene expression profiles. Conclusions: The combination of LEN with DEX can be proposed as a new therapeutic approach for CRC.

Isolation Set-up and Characterization of cancer stem cells from HT29 colon cancer cell line

Abstract Background: Cancer stem cells (CSCs) have critical roles in tumor initiation, progression, metastasis, resistance to chemoradiotherapy, and recurrence in various cancers such as colon cancer. Therefore, extensive studies are required to understand the CSCs mechanism of action and design novel cancer therapies. Successful isolation and characterization of CSCs from tumor tissues or cancer cell lines provide the proper opportunity for these kinds of studies. This study aimed to isolate and characterize CSCs from HT29 colon cancer cell lines. Methods: Sphere formation assay was used to isolate CSCs from the HT29 cell line. The expression of four stemness genes, including LGR5, SOX2, c-Myc, and Oct4, was assessed by real-time PCR. The CSC markers, CD 44 and CD 24, were also evaluated and compared between the parental HT29 cell line and HT29-derived spheres by flow cytometry. Statistical analysis was performed using GraphPad Prism version 6.0 and the Mann-Whitney U test. Results:HT29-derived sphere cells were successfully formed in serum-free media. The proportion of CD 24 and CD 44-expressing cells and the expression of LGR5, SOX2, c-Myc, and Oct4 stemness genes were significantly higher in CSCs isolated from HT29 spheres compared with parental HT29 cells. Conclusion:Sphere formation assay is a proper method for the isolation of CSCs from the HT29 cell line. The stemness markers LGR5, SOX2, c-Myc, Oct4,CD 44,andCD 24are suitable for the characterization of these cells.

The Regulatory Network of Sturgeon Chondroitin Sulfate on Colorectal Cancer Inhibition by Transcriptomic and Proteomic Analysis.

Chondroitin sulfate (CS) is a food-derived bioactive substance with multiple biological functions, which exists in animal cartilage and/or bone. Sturgeon, a type of cartilaginous fish, is rich in CS. Our recent study demonstrated the effect of sturgeon chondroitin sulfate (SCS) on reducing colorectal cancer cell proliferation and tumor formation. However, the molecular mechanisms of its anticancer activity remain unknown. In this study, the cell proliferation assay and flow cytometric analysis were used to examine the cell viability and apoptosis of colon cancer cell HT-29 cells and normal colonic epithelial cell NCM460 cells. Transcriptomic and proteomic studies were used to identify the main targets of SCS. SCS showed little effect on the genes/proteins expression profile of NCM460 cells but more sensitive to HT-29, in which 188 genes and 10 proteins were differentially expressed after SCS treatment. Enrichment analysis of those genes/proteins showed that the majority of them are involved in DNA replication, cell cycle progression and apoptosis. Quantitative RT-PCR and Western blot were used to determine essential genes/proteins and networks targeted by SCS to exert inhibiting the development of colorectal cancer function. This study provided great insights into developing food-derived novel therapeutics for colorectal cancer treatment.

Biocompatible Eu doped mesoporous calcium silicate nanospheres for pH-responsive drug release

Calcium silicate is a kind of inorganic biomaterial containing Si, Ca, O elements, they have been widely investigated and applied in a variety of medical applications in the past four decades. Herein, Europium (Eu) doped mesoporous calcium silicate nanoparticles (Eu-MCS) with sizes ranging from 60 nm to 110 nm were prepared via a simple hydrothermal synthesis method. The morphology, structure, texture and optical property were characterized by SEM, TEM, XRD, N2 adsorption/desorption and PL spectra, respectively. The prepared material emits strong red luminescence at 613 nm upon UV irradiation and is cytocompatible at a wide range of concentrations, exibiting promising application in bioimaging. The Eu-MCS has a high loading capacity of 356.67 μg/mg for the anticancer drug DOX, and the drug loaded material DOX@Eu-MCS showed pH-controlled drug release behaviour. Moreover, both of the biocompatibility tests on normal human cells HEK293 and human colon cancer cells HT29 indicate a low cytotoxicity of Eu-MCS. The cell cytotoxity assays of the DOX loaded nanomaterial (DOX@Eu-MCS) on Hela cancer cells prove a high cancer cell mortality. Therefore, the as-prepared red emissive nanocarrier Eu-MCS has great potential to be used as an integrated platform for cancer diagnosis and therapy.

Green synthesized monodispersed silver nanoparticles’ characterization and their efficacy against cancer cells

Background: In biomedical research, silver nanoparticles have attracted the attention of many scientists in drug delivery and other applications. This study investigates the characterization of biologically synthesized nanoparticles and their efficacy on cancer cells. Methods: The characterization of biologically produced silver nanoparticles was accomplished using UV:visible absorption spectroscopy, which showed the maximum absorption at 422 nm. The structural morphology was carried out by transmission electron microscope (TEM) and atomic force microscope (AFM), and the anticancer efficacy of the biosynthesized compound was investigated on two cancer cell lines, namely HT-29 (colon cancer) and EAC. Results: The morphological and structural examination revealed spherical, three-dimensional nanoparticles ranging between 5 to 50 nm. The results of the anticancer experiment, which was conducted using an MTT assay, revealed that the prepared silver had excellent anti-cancer activity against both tested cells, that is EAC cancer and HT-29 (colon cancer) cell lines. Conclusion: This study confirms that silver nanoparticles can be biologically synthesized using Rhizopus stolonifer and used as anticancer substances in cancer therapy and drug delivery.

Anticancer Activity Assay of Nano-Fractional Compounds that Purified from Soil Actinomycetes

Background and objectives: Cancer remains a global problem of health, and has been recorded as one of the causes of death after heart disease Natural products from plants, the environment and microorganisms are leveraged for the purpose of fighting cancer. Actinobacteria have been recognized as main sources of bioactive natural products as early as in the 1950s, for which about half of the secondary metabolites revealed, including enzymes, antibiotics, immunosuppressive, and anti-tumour agents Materials and methods:The methods of this study included isolated and identification of bacteria from soil samples and identified by morphology characters and biochemical test. Subjected extract of actinomycetes to HPLC purification then collected purified fractions then analyzed by GC-mass. After the fractions were mixed with liposome nanoparticals which tested activity on HT29 colon cancer cell line. Results: The results of identification of bacterial isolates showed the colonies growing on a SNA medium were morphologically identified where the colonies were well-growth and had a gray color, not producing dyes in the medium. The results of the biochemical tests indicated that isolates were amylase, catalase, and gelatinase producing isolates and non-lipase producing, non H2S production and consuming urea, while the carbon consumption test indicated the isolates' ability to consume starch, glucose and sucrose respectively. While The results of preparative HPLC revealed that 4 fractions analytical HPLC with (50 % HPLC-grade acetonitrile) at 254 nm and cycling up was employed to increase the separation efficiency. The chemical composition of the HPLC fractions using GC-MS showed the identification of many components example ( Hexadeconic acid , Octadecanoic acid,ethyl ester and Fumaric acid). The results of In vitro anti-tumor cytotoxicity showed that all four nano purified fractions were applied on HT 29 colon cancer cells and exhibited significantly differences compared with control.

Identification of the interactome of the DP1 receptor for Prostaglandin D2: Regulation of DP1 receptor signaling and trafficking by IQGAP1

BackgroundMechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D2. MethodsWe performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1. ResultsIn addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD2 treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown. ConclusionsOur findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling. General significanceThe identified putative DP1-interacting proteins open multiple lines of research in DP1 and GPCR biology in various cell compartments.

Metabolite Profiling of Rosemary Cell Lines with Antiproliferative Potential against Human HT-29 Colon Cancer Cells.

Rosemary (Rosmarinus officinalis) is a culinary and medicinal plant used in food and pharmaceutical industry. The wide range of biological activities is mainly related to phenolic and terpenic compounds; like carnosic acid (CA), carnosol (CS) and rosmarinic acid (RA), mainly reported in rosemary leaf extracts, and recently described in rosemary callus extracts. The aim of this work was to investigate the chemical profile of rosemary cell lines and evaluate their antiproliferative potential against human HT-29 colorectal cancer cell lines. For this purpose, rosemary leaf explants were dedifferentiated on MS medium and added with 2, 4-D (2, 4-dichlorophenoxyacetic acid; 2mg/L) and BAP (6-benzylaminopurine; 2mg/L). Cell aggregates were separated according to colour and three rosemary cell lines cultures were established: green (RoG), yellow (RoY) and white (RoW).The chemicalprofile of rosemary cell lines extracts was characterized by combining HPLC and GC platforms coupled to HR-MS/MS. The antiproliferative activityagainst HT-29 cell line was analyzed with MTT assay. A total of 71 compounds, including hydroxycinnamic acid and hydroxybenzoic acid derivatives,flavonoids, phenolic di- and triterpenes, as well as relevant unsaturated fatty acids and their esters, phytosterols, and carotenoids were tentatively identifiedin the extract of the target cell lines. The antiproliferative activity test against HT-29 cell using the MTT assay revealed that the viability of HT-29 coloncancer cells was affected after treatment with the RoW extract (IC50 of 49.63 μg/mL) at 48 h. These results showed that rosemary cell lines can alsoaccumulate other bioactive phytochemicals with demonstrated antiproliferative potential.

Synergistic Growth Inhibition of HT-29 Colon and MCF-7 Breast Cancer Cells with Simultaneous and Sequential Combinations of Antineoplastics and CNS Drugs.

Several central nervous system (CNS) drugs exhibit potent anti-cancer activities. This study aimed to design a novel model of combination that combines different CNS agents and antineoplastic drugs (5-fluorouracil (5-FU) and paclitaxel (PTX)) for colorectal and breast cancer therapy, respectively. Cytotoxic effects of 5-FU and PTX alone and in combination with different CNS agents were evaluated on HT-29 colon and MCF-7 breast cancer cells, respectively. Three antimalarials alone and in combination with 5-FU were also evaluated in HT-29 cells. Different schedules and concentrations in a fixed ratio were added to the cultured cells and incubated for 48 h. Cell viability was evaluated using MTT and SRB assays. Synergism was evaluated using the Chou-Talalay, Bliss Independence and HSA methods. Our results demonstrate that fluphenazine, fluoxetine and benztropine have enhanced anticancer activity when used alone as compared to being used in combination, making them ideal candidates for drug repurposing in colorectal cancer (CRC). Regarding MCF-7 cells, sertraline was the most promising candidate alone for drug repurposing, with the lowest IC50 value. For HT-29 cells, the CNS drugs sertraline and thioridazine in simultaneous combination with 5-FU demonstrated the strongest synergism among all combinations. In MCF-7 breast cancer cells, the combination of fluoxetine, fluphenazine and benztropine with PTX resulted in synergism for all concentrations below IC50. We also found that the antimalarial artesunate administration prior to 5-FU produces better results in reducing HT-29 cell viability than the inverse drug schedule or the simultaneous combination. These results demonstrate that CNS drugs activity differs between the two selected cell lines, both alone and in combination, and support that some CNS agents may be promising candidates for drug repurposing in these types of cancers. Additionally, these results demonstrate that 5-FU or a combination of PTX with CNS drugs should be further evaluated. These results also demonstrate that antimalarial drugs may also be used as antitumor agents in colorectal cancer, besides breast cancer.