HT-29 Colon Adenocarcinoma Cells Research Articles

Monitoring Quantitative Ultrasound Parameter Changes in a Cell Pellet Model of Cell Starvation

Although it has previously been shown that the spectral analysis of ultrasound backscatter data is sensitive to the cellular changes caused by apoptosis, the sensitivity of spectral analysis to oncosis or ischemic cell death had not previously been studied. Whereas many anticancer treatments induce apoptosis, others induce cell starvation, or oncosis. HT-29 colon adenocarcinoma cells were formed into pellets and covered in phosphate-buffered saline at room temperature for 56 h. The pellets were imaged every 8 h with high-frequency (55 MHz) ultrasound and the raw radio-frequency data processed. The lack of nutrients available to the cells induced cell death due to oncosis. The attenuation slope, speed of sound, spectral slope, and midband fit were estimated at each of the eight time points to identify changes as the cells died due to starvation. The spectral slope decreased monotonically over the 56 h, whereas the attenuation slope showed an increase between 1 and 48 h, followed by a slight decrease between 48 and 56 h. The midband fit did not vary over time. The speed of sound increased from 1514 to 1532 m/s over the first 24 h, after which time it plateaued. These in vitro results indicate different trends in ultrasound parameter changes from those of in vitro apoptotic cells, suggesting that these different methods of cell death can be identified not only by morphological markers, but also by specific ultrasound signatures.

Microbiota source impact in vitro metabolite colonic production and anti-proliferative effect of spent coffee grounds on human colon cancer cells (HT-29)

Human gut flora-mediated non-digestible fraction of spent coffee grounds (hgf-NDSCG) was evaluated for its chemopreventive effect and molecular mechanisms involved on human colon adenocarcinoma HT-29 cell survival using two different microbiota source [lean (L) and overweight (OW)]. The source of human gut flora (hgf) (L or OW) affected the pH of hgf-NDSCG only minimally, but linearly reduced those of hgf-inulin. The variability between lean and overweight microbiota was characterized by the metabolism and/or bioaccessibility of different phenolic metabolites, their intermediate and end products as well as by variable time courses. Apoptosis of colon cancer HT-29 cells depended on the microbiota source with the lean microbiota expressing a low lethal concentration 50 (LC50/L-hgf-NDSCG=13.5%). We demonstrate that NDSCG and its colonic metabolite from lean microbiota induced HT-29 cell apoptosis by reducing catalase and 8-iso-prostaglandin F2α as biomarkers of in vivo oxidative stress as the primary mechanism underlying its overall chemoprotection against colon cancer.

16 - Identification of Aberrant Splicing Events in Myelodysplastic Syndrome Patients with Splicing Factor Gene Mutations

The aim of this study was to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells.The antioxidant capability of L. fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the oxygen radical absorbance capacity method.The analysis revealed that the interaction of probiotic strain cells supplemented with a prebiotic exerts a remarkable antioxidant capacity.The L. fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.

ROS-mediated induction of apoptosis by benzoquinone embelin in human colon adenocarcinoma cells HT-29.

Background Embelin is a benzoquinone reported to possess anticancer activity in several in vivo and in vitro models of carcinogenesis, especially hematopoietic and prostate malignancy. A detailed investigation on the influence of embelin on epithelial malignancy model system, especially colon adenocarcinoma, is lacking. The objective of the current study is to investigate the antiproliferative, antiinvasive and proapoptotic potential of embelin on colon adenocarcinoma cell line HT-29. Methods The effect of embelin (35 µg/mL for 24 h) on cell proliferation was assessed by Sulforhodamine B assay and bromodeoxyuridine incorporation test, antiinvasive effect by Boyden chamber assay and scratch assay. Proapoptotic effects of embelin were determined by studies on DNA fragmentation, annexin V-FITC labeling, TUNEL assay, COMET assay and assay of caspase-3 activity. Influence of embelin on the expression of genes regulating apoptosis (caspase 3 and 9, Bcl-2, Bax, cytochrome C and X-linked inhibitor of apoptosis protein) and migration/invasion (matrix metalloproteinase [MMP]-2 and MMP-9) was investigated by reverse transcription polymerase chain reaction (PCR). Further, the effect of embelin on the levels of reactive oxygen species (ROS), lipid peroxides, nitric oxide, mitochondrial membrane potential and antioxidant status (total reduced glutathione [GSH] and GSH-S-transferase) was evaluated. Results Results implicated that embelin treatment inhibited proliferation (IC50 35 µg/mL), induced DNA fragmentation, phosphatidyl serine externalization, increased caspase expression, decreased cell migration and expression of MMPs in HT-29 cells. Interestingly, embelin exhibited prooxidant effect on HT-29 cells and induced excessive ROS generation resulting in apoptotic cell death. Conclusions To conclude, embelin treatment could be a promising strategy for the chemotherapy of colon cancer.

Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1\u03b1 stabilization

BackgroundSolid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia.MethodsThe human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses.ResultsHere we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism.ConclusionsWe show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.

Isolation and Characterization of Lectin from the Artist's Conk Medicinal Mushroom, Ganoderma applanatum (Agaricomycetes), and Evaluation of Its Antiproliferative Activity in HT-29 Colon Cancer Cells.

The growth and lectin production of Ganoderma applanatum, a white rot fungus, was optimized in broth cultures. The fungus was found to have a higher growth rate and higher lectin activity when grown in a medium adjusted to pH 6.5 at 26°C under stationary conditions. Expression of lectin activity started in 5-day-old mycelial culture; maximum activity was expressed after the 15th day of incubation. Among the various carbon and nitrogen sources tested, the carbon source sucrose and the nitrogen source yeast extract support maximum growth and lectin production. Lectin from G. applanatum was purified by ammonium sulfate precipitation and ion exchange chromatography. The purified fraction revealed a single band with a molecular weight of 35.0 kDa. Moreover, carbohydrates such as mannitol, glucose, sucrose, maltose, mannose, galactose, sorbose, and fructose were found to inhibit the hemagglutinating activity of the lectin. The purified lectins from G. applanatum contain cytotoxic and proapoptotic activities against HT-29 colon adenocarcinoma cells.

Rutamarin, an Active Constituent from Ruta angustifolia Pers., Induced Apoptotic Cell Death in the HT29 Colon Adenocarcinoma Cell Line.

Background:Ruta angustifolia Pers. is a perennial herb that is cultivated worldwide, including Southeast Asia, for the treatment of various diseases as traditional medicine.Objective:The purpose of the study was to identify an active principle of R. angustifolia and to investigate its effect on the HT29 cell death.Materials and Methods:The methanol and fractionated extracts (hexane, chloroform, ethyl acetate, and water) of R. angustifolia Pers. were initially investigated for their cytotoxic activity against two human carcinoma cell lines (MCF7 and HT29) and a normal human colon fibroblast cell line (CCD-18Co) using sulforhodamine B cytotoxicity assay. Eight compounds including rutamarin were isolated from the active chloroform extract and evaluated for their cytotoxic activity against HT29 human colon carcinoma cell line and CCD-18Co noncancer cells. Further studies on the induction of apoptosis such as morphological examinations, biochemical analyses, cell cycle analysis, and caspase activation assay were conducted in rutamarin-treated HT29 cells.Results:Rutamarin exhibited remarkable cytotoxic activity against HT29 cells (IC50 value of 5.6 μM) but was not toxic to CCD-18Co cells. The morphological and biochemical hallmarks of apoptosis including activation of caspases 3, 8, and 9 were observed in rutamarin-treated HT29 cells. These may be associated with cell cycle arrest at the G0/G1 and G2/M checkpoints, which was also observed in HT29 cells.Conclusions:The present study describes rutamarin-induced apoptosis in the HT29 cell line for the first time and suggests that rutamarin has the potential to be developed as an anticancer agent.SUMMARY Rutamarin was cytotoxic to HT29 colon cancer cells but exerted no damage to normal colon cellsRutamarin induced morphological and biochemical hallmarks of apoptosis in HT29 cellsRutamarin induced cell cycle arrest at the G0/G1 and G2/M checkpoints in a dose-dependent manner in HT29 cellsRutamarin activated caspases 3, 8, and 9 in a dose-dependent manner in HT29 cells. Abbreviations used: ACN: Acetonitrile, ANOVA: One-way analysis of variance, BrdU: Bromodeoxyuridine, 13C-NMR: Carbon-13 Nuclear magnetic resonance, CAD: Caspase-activated endonuclease, CCD-18Co: Human colon normal, DLD1: Human Duke's type C colorectal adenocarcinoma, DMRT: Duncan's multiple range test, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4/5: Death receptor 4/5 protein, EMEM: Eagle's minimum essential media, FBS: Fetal bovine serum, FITC Annexin V: Annexin V conjugated with fluorescein isothiocyanate, FITC-DEVD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone, FITC-IETD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Ile-Glu-Thr-Asp-fluoromethyl ketone, FITC-LEHD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Leu-Glu-His-Asp-fluoromethyl ketone, G0: Quiescent phase of cell cycle, G1: Gap 1 phase of cell cycle, G2: Gap 2 phase of cell cycle, GC-MS: Gas chromatography-mass spectrometry, HeLa: Human cervical adenocarcinoma, HPLC: High performance liquid chromatography, HT29: Human colon adenocarcinoma, Huh7.5: Human hepatocellular carcinoma, IC50: Half maximal inhibitory concentration, KSHV: Kaposi's sarcoma-associated herpesvirus, M phase: Mitotic phase of cell cycle, MCF7: Human breast adenocarcinoma, NMR: Nuclear magnetic resonance, PBS: Phosphate-buffered saline, PI: Propidium iodide, RNase: Ribonuclease, rt: Retention time, S phase: Synthesis phase of cell cycle, SD: Standard deviation, SRB: Sulforhodamine B, TCA: Trichloroacetic acid, TLC: Thin layer chromatography, TNF-R1: Tumor necrosis factor receptor 1 protein, TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling, UV: Ultraviolet.

Sorbus rufopilosa Extract Exhibits Antioxidant and Anticancer Activities by Inducing Cell Cycle Arrest and Apoptosis in Human Colon Adenocarcinoma HT29 Cells.

BackgroundSorbus rufopilosa, a tsema rowan, is a species of the small ornamental trees in the genus Sorbus and the family Rosaceae found in East Asia. The bioactivities of S. rufopilosa have not yet been fully determined. The objective of this study is to evaluate the antioxidant and anticancer effects of ethanol extract of S. rufopilosa (EESR) and to determine the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells.MethodsTo examine the antioxidant activity of EESR, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay was performed. Inhibitory effect of EESR on cancer cell growth and proliferation was determined by water-soluble tetrazolium salt assay. To investigate the mechanism of EESR-mediated cytotoxicity, HT29 cells were treated with various concentrations of EESR and the induction of cell cycle arrest and apoptosis was analyzed by flow cytometry, 4,6-diamidino-2-phenylindole staining, and Western blot analysis.ResultsEESR showed significant antioxidant activity and inhibitory effect on HT29 cell growth in a dose-dependent manner. EESR induced cell cycle arrest at G2/M phase in a dose-dependent manner by modulating cyclin B, cyclin-dependent kinase 1 (CDK1), and CDK inhibitor p21 expression. EESR-induced apoptosis was associated with the upregulation of p53, a death receptor Fas, and a pro-apoptotic protein Bax and the activation of caspase 3, 8, and 9, resulting in the degradation of PARP.ConclusionsEESR possessing antioxidant activity efficiently inhibits proliferation of HT29 cells by inducing both cell cycle arrest and apoptosis. EESR may be a possible candidate for the anticancer drug development.

Cytotoxic Activity of Microalgal-derived Oxylipins against Human Cancer Cell lines and their Impact on ATP Levels

Oxylipins are metabolites derived from lipid peroxidation. The plant oxylipin methyl jasmonate (MJ) shows cytotoxic activity against cancer cell lines of various origins, with ATP-depletion being one of the mechanisms responsible for this effect. The cytotoxic activity of oxylipins (OXLs) isolated from the microalgae Chlamydomonas debaryana (13-HOTE) and Nannochloropsis gaditana (15-HEPE) was higher against UACC-62 (melanoma) than towards HT-29 (colon adenocarcinoma) cells. OXLs lowered the ATP levels of HT-29 and UACC-62 cells, but the effect was higher on the second cell line, which had higher basal ATP. This result proves a link between the cytotoxicity and the capability of these compounds to deplete ATP. In addition, the combination of 13-HOTE with the anticancer drug 5-fluorouracil (5-FU) induced a synergistic toxicity against HT-29 cells. These results highlight the therapeutic potential of oxylipins derived from microalgae.

Abstract B057: The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to mobilization of antitumor immunity and synergistic activity

Abstract Background: NKTR-214 is a novel immunostimulatory cytokine which provides a sustained and biased activation signal through the heterodimeric IL-2R receptor (IL2Rbg or CD122), resulting in significant expansion of CD8+ T and NK cells relative to regulatory T cells in the tumor microenvironment. As a single agent in preclinical tumor models, NKTR-214 significantly increases the intratumoral CD8T/Treg ratio (>400). Here we examine activity and pharmacodynamic effects of NKTR-214 alone and combined with immune checkpoint blockade in both a murine tumor model and in a human in vitro co-culture system. Methods: Mice bearing subcutaneous CT26 colon tumors were treated with single agent NKTR-214 (q9d), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Serum cytokines were assessed by multiplex ELISA (Quansys). T cell clonality and infiltration were assessed in tumors 7 days after treatment initiation, with TCR V and J beta usage determined by ImmunoSEQ (Adaptive Biotechnologies). Effects on human cytokine and chemokine expression were examined by the BioMAP Combo ELECT (DiscoverX) in vitro system utilizing human fibroblasts or endothelial cells, PBMCs and either HT29 colon adenocarcinoma or HT1299 NSCLC cell lines. 1-PEG-IL2, the most active species of the NKTR-214 prodrug, or Keytruda (anti-PD-1) was added over a range of concentrations alone or in combination, with biomarkers assessed 48 hours later. Results: In tumor-bearing mice, NKTR-214 led to increases in serum IFNg as well as increases in the chemokine MCP-1 (CCL2), a chemoattractant of CD4 T, CD8 T and NK cells. Single-agent NKTR-214 led to significant tumor growth inhibition and 10% tumor free animals, while single-agents anti-CTLA-4 or anti-PD-1 lacked efficacy. Combination of NKTR-214 with checkpoint blockade was synergistic and led to greater tumor-free animals (67% in combination with anti-CTLA-4, 90% with anti-PD-1). NKTR-214 combined with anti-PD-1 was also more efficacious than the combination of anti-CTLA-4 and anti-PD-1. TCR repertoire analysis demonstrated superior increases in TIL clonality and infiltration with NKTR-214 compared to either anti-CTLA-4 or anti-PD-1 alone. NKTR-214 combined with either mode of checkpoint inhibition led to significant increases in both parameters, with the greatest effect occurring when combined with anti-PD-1. In the human BioMAP system, NKTR-214 active species led to significant increases in granzyme B, IFNg, IL-6, IL-17A and TNFa, while these markers were only modestly induced by Keytruda alone. When NKTR-214 was combined with Keytruda, additive and synergistic increases in these factors were found. In addition, the combination significantly decreased uPA in the fibroblast-HT29 system and MDC in the endothelial-HT1299 system, both factors associated with tumor progression. Conclusions: NKTR-214 delivers sustained signaling of the IL-2 pathway. Analysis of multiple immune markers in mice and human tumor-immune cell cultures provide mechanistic rationale for the increased T and NK cell activation in the tumor microenvironment after treatment with NKTR-214. When combined with anti-PD-1, significant increases in T cell receptor clonality, tumor infiltration and immune activation biomarkers are achieved - a significant finding, given that concomitant increase in clonality and infiltration has been correlated with favorable responses in the clinic. In vitro studies with human cells demonstrate the potentiation of immune activation markers associated with anti-tumor effects in combination with anti-PD-1, further suggesting these findings may be translatable from mouse to human. NKTR-214 is currently in a Phase 1 clinical trial to evaluate the pharmacokinetics, pharmacodynamics and activity in an outpatient setting. Citation Format: John L. Langowski, Murali Addepalli, Laurie VanderVeen, Rhoneil Pena, Ravikumar Nutakki, Yolanda Kirksey, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to mobilization of antitumor immunity and synergistic activity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B057.

JNK1 Inhibition Attenuates Hypoxia-Induced Autophagy and Sensitizes to Chemotherapy.

Inhibition of hypoxia-induced stress signaling through JNK potentiates the effects of oxaliplatin. The JNK pathway plays a role in both autophagy and apoptosis; therefore, it was determined how much of the effect of JNK inhibition on oxaliplatin sensitivity is dependent on its effect on autophagy. We studied the impact of JNK isoform downregulation in the HT29 colon adenocarcinoma cell line on hypoxia- and oxaliplatin-induced responses. Electron microscopic analyses demonstrated that both oxaliplatin- and hypoxia-induced formations of autophagosomes were reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. The role of specific JNK isoforms was defined using HT29-derived cell lines stably expressing dominant-negative constructs for JNK1 and JNK2 (HTJ1.3 and HTJ2.2, respectively). These cell lines demonstrated that functional JNK1 is required for hypoxia-induced autophagy and that JNK2 does not substitute for it. Inhibition of autophagy in HTJ1.3 cells also coincided with enhancement of intrinsic apoptosis. Analysis of Bcl2-family proteins revealed hyperphosphorylation of Bcl-XL in the HTJ1.3 cell line, but this did not lead to the expected dissociation from Beclin 1. Consistent with this, knockdown of Bcl-XL in HT29 cells did not significantly affect the induction of autophagy, but abrogated hypoxic resistance to oxaliplatin due to the faster and more robust activation of apoptosis. These data suggest that balance between autophagy and apoptosis is shifted toward apoptosis by downregulation of JNK1, contributing to oxaliplatin sensitization. These findings further support the investigation of JNK inhibition in colorectal cancer treatment. Mol Cancer Res; 14(8); 753-63. ©2016 AACR.

Abstract 1597: Claudin-1 expression promote TNF-a-induced epithelial-mesenchymal transition and growth in colorectal adenocarcinoma cells

Abstract Epithelial-mesenchymal transition (EMT) is an important mechanism in colorectal cancer progression and malignancy. Importantly, inflammatory mediators are critical constituents of the local tumor environment and an intimate link between colon cancer progression and inflammation in now validated. Notably, we and others have previously reported key role of the deregulated claudin-1, a tight junction protein, in colon carcinogenesis including colitis-associated colon cancer (CAC). Of interest, the increase in claudin-1 expression in CAC was found to be associated with the inflammatory epithelium. We have further demonstrated that inflammation induced by DSS (Dextran Sodium Sulfate) administration promotes colon cancer in APCmin/Claudin-1 transgenic mice. However, what role claudin-1 plays in inflammation-induced colon cancer remains unclear. In current study, we tested the causal significance of claudin-1 expression in inflammation-associated colon cancer using colon cancer cells subjected to TNF-αα, a pro-inflammatory cytokine, treatment. HT29 colon adenocarcinoma cells cultured in the presence of TNF-α (10ng/ml), demonstrated a sharp decrease in E-cadherin expression and an increase in Vimentin expression versus control cells suggesting TNF-αα treatment induced EMT. Also, significant increase in cell proliferation and wound healing was observed in TNF-α treated cells at 24 and 48 hours of TNF-αα treatment (versus control cells). Interestingly, TNF-αα treatment also upregulated (and delocalized) claudin-1 expression in a time-dependent manner. An increase in the phosphorylation of Erk1/2 and Src, signaling associated with colon cancer survival and transformation, in TNF-αα treated cells further correlated with our published observations that claudin-1 expression promotes these signaling pathways in colon cancer cells. In support, shRNA-mediated inhibition of claudin-1 expression in HT-29 cells largely abrogated the effects of TNF-αα treatment including proliferation, p-erk and p-Src expression. Also, TNF-αα induced EMT was largely suppressed in the absence of claudin-1 expression. Taken together, our data confirm the previously described key role of claudin-1 in regulating tumorigenic abilities of colon cancer cells and further highlights a key role of claudin-1 in inflammation-induced colorectal cancer growth and progression. We predict claudin-1 expression can serve as a biomarker to predict CAC progression and malignancy. Citation Format: Ajaz Ahmad Bhat, Rizwan Ahmad, Amar B. Singh, Punita Dhawan. Claudin-1 expression promote TNF-a-induced epithelial-mesenchymal transition and growth in colorectal adenocarcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1597.

Photodynamic action of palmatine hydrochloride on colon adenocarcinoma HT-29 cells

Palmatine hydrochloride (PaH) is a natural active compound from a traditional Chinese medicine (TCM). The present study aims to evaluate the effect of PaH as a new photosensitizer on colon adenocarcinoma HT-29 cells upon light irradiation. Firstly, the absorption and fluorescence spectra of PaH were measured using a UV–vis spectrophotometer and RF-1500PC spectrophotometer, respectively. Singlet oxygen (1O2) production of PaH was determined using 1, 3-diphenylisobenzofuran (DPBF). Dark toxicity of PaH was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake of PaH in HT-29 cells was detected at different time intervals. Subellular localization of PaH in HT-29 cells was observed using confocal laser fluorescence microscopy. For photodynamic treatment, HT-29 cells were incubated with PaH and then irradiated by visible light (470nm) from a LED light source. Photocytotoxicity was investigated 24h after photodynamic treatment using MTT assay. Cell apoptosis was observed 18h after photodynamic treatment using a flow cytometry with Annexin V/PI staining. Results showed that PaH has an absorption peak in the visible region from 400nm to 500nm and a fluorescence emission peak at 406nm with an excitation wavelength of 365nm. PaH was activated by the 470nm visible light from a LED light source to produce 1O2. Dark toxicity showed that PaH alone treatment had no cytotoxicity to HT-29 cancer cells and NIH-3T3 normal cells after incubation for 24h. After incubation for 40min, the cellular uptake of PaH reached to the maximum and PaH was located in mitochondria. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on HT-29 cells. The rate of cell death increased significantly in a PaH concentration-dependent and light dose-dependent manner. Further evaluation revealed that the early and late apoptotic rate of HT-29 cells increased remarkably up to 21.54% and 5.39% after photodynamic treatment of PaH at the concentration of 5μM and energy density of 10.8J/cm2. Our findings demonstrated that PaH as a naturally occurring photosensitizer has potential in photodynamic therapy on colon adenocarcinoma.

Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4.

Magnetic solid lipid nanoparticles in hyperthermia against colon cancer

A reproducible double emulsion/solvent evaporation procedure is developed to formulate magnetic solid lipid nanoparticles (average size≈180nm) made of iron oxide cores embedded within a glyceryl trimyristate solid matrix. The physicochemical characterization of the nanocomposites ascertained the efficacy of the preparation conditions in their production, i.e. surface properties (electrokinetic and thermodynamic data) were almost indistinguishable from those of the solid lipid nanomatrix, while electron microscopy characterizations and X-ray diffraction patterns confirmed the satisfactory coverage of the magnetite nuclei. Hemocompatibility of the particles was established in vitro. Hysteresis cycle determinations defined the appropriate magnetic responsiveness of the nanocomposites, and their heating characteristics were investigated in a high frequency alternating gradient of magnetic field: a constant maximum temperature of 46°C was obtained within 40min. Finally, in vitro tests performed on human HT29 colon adenocarcinoma cells demonstrated a promising decrease in cell viability after treatment with the nanocomposites and exposure to that alternating electromagnetic field. To the best of our knowledge, this is the first time that such type of nanoformulation with very promising hyperthermia characteristics has been developed for therapeutic aims.

N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells.

BackgroundN-Hydroxyphthalimide (NHPI), an important chemical raw material, was found to have potent and selective anti-proliferative effect on human breast carcinoma BT-20 cells, human colon adenocarcinoma LoVo and HT-29 cells during our screening for anticancer compounds. The purpose of this study is to assess the antitumor efficacy of NHPI in vitro and in vivo and to explore the underlying antitumor mechanism.MethodsCell cytotoxicity of NHPI was evaluated using MTS assay and cell morphological analysis. After NHPI treatment, cell cycle, apoptosis and mitochondrial membrane potential were analyzed using flow cytometer. The subcellular localization of eukaryotic initiation factor 4E (eIF4E) was analyzed by immunofluorescence assay. The antitumor efficacy of NHPI in vivo was tested in BT-20 xenografts. The underlying antitumor mechanisms of NHPI in vitro and in vivo were investigated with western blot analysis in NHPI-treated cancer cells and tumor tissues. Statistical significance was determined using Student’s t-test.ResultsIn vitro, NHPI selectively inhibited the proliferation and induced G2/M phase arrest in BT-20 and LoVo cells, which was attributed to the inhibition of cyclin B1 and cdc2 expressions. Furthermore, NHPI induced apoptosis via mitochondrial pathway. Of note, NHPI effectively inhibited mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling, and overcame the feedback activation of Akt and extracellular signal-regulated kinase (ERK) caused by mTORC1 inhibition in BT-20 and LoVo cells. In vivo, NHPI inhibited tumor growth and suppressed mTORC1 and mTORC2 signaling in BT-20 xenografts with no obvious toxicity.ConclusionsWe found for the first time that NHPI displayed antitumor activity which is associated with the inhibition of mTOR signaling pathway. Our findings suggest that NHPI may be developed as a promising candidate for cancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

Evaluation of cellular retinoic acid binding protein 2 gene expression through the retinoic acid pathway by co-incubation of Blastocystis ST-1 with HT29 cells in vitro.

Blastocystis is a parasitic protist with a worldwide distribution that is commonly found in patients with colon and gastrointestinal pathological symptoms. Blastocystis infection has also commonly been reported in colorectal cancer and HIV/AIDS patients with gastrointestinal symptoms. To understand the pathway networks of gene regulation and the probable mechanisms influencing functions of HT-29 host cells in response to parasite infection, we examined the expression of 163 human oncogenes and kinases in human colon adenocarcinoma HT-29 cells co-incubated with Blastocystis by in-house cDNA microarray and PCR analysis. At least 10 genes were shown to be modified following Blastocystis co-incubation, including those with immunological, tumorigenesis, and antitumorigenesis functions. The expression of genes encoding cellular retinoic acid binding protein 2 (CRABP2) and proliferating cell nuclear antigen (PCNA) was markedly upregulated and downregulated, respectively. Reverse transcriptase-PCR validated the modified transcript expression of CRABP2 and other associated genes such as retinoic acid (RA)-related nuclear-receptor (RARα). Together, our data indicate that CRABP2, RARα, and PCNA expressions are involved in RA signaling regulatory networks that affect the growth, proliferation, and inflammation of HT-29 cells.

Potential antimicrobial and antiproliferative activities of autochthonous starter cultures and protease EPg222 in dry-fermented sausages.

This work studied the presence of nitrogen compounds with bioactive properties in Iberian pork sausages that were manufactured using different autochthonous starter cultures (Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34) and protease EPg222. Nitrogen compounds were extracted and evaluated for their antimicrobial effect against spoilage and pathogenic bacteria, such as Bacillus cereus, Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus and Listeria monocytogenes, and antiproliferative activity on the HT-29 colon adenocarcinoma cell line. Dry-fermented sausages elaborated with starter cultures P200S34 and protease EPg222 generate extracts that cause inhibition of the growth of pathogens reaching 25% inhibition of Bacillus cereus, making this a promising tool for biocontrol in the meat industry. On the other hand, the inoculation of well-adapted starter cultures with high proteolytic activity also increased the antiproliferative activity of these extracts, around 45% inhibition at 72 h, mainly due to an increase in free amino acids, such as Lys and Pro, but also small peptides.

Radiosensitizing potential of rutin against human colon adenocarcinoma HT-29 cells.

The present study was focused at evaluating the potential of rutin to improve the radiotherapeutic index and thereby the cytotoxicity towards colon cancer cells. HT-29 cells were pre-treated with rutin and the effect on cell proliferation was determined by MTT assay followed by exposure to radiation. After irradiation, experimental groups were sham control, rutin alone, radiation alone, rutin along with radiation-treated HT-29 cells. Cytotoxicity study illustrated that treatment of HT-29 cells with different concentrations of rutin reduced cell proliferation in a dose- and time-dependent manner. After irradiation, the HT-29 cells revealed that the combined effect of 4 Gy radiation and rutin at 80 µM concentration showed a decrease in cell viability as compared to rutin‑alone treated and 4 Gy‑alone irradiated HT-29 cells. Furthermore, the increase in apoptotic cells, change from normal nuclei to abnormal nuclei, alterations in mitochondrial membrane potential, increase in DNA damage, increase in levels of lipid peroxidative markers, and decrease in antioxidant status were observed in 4 Gy‑ and rutin-treated group as compared to the other treated groups. Combined effect of rutin and radiation in HT-29 cells leads to a more pronounced cell death rate. Thus, rutin exhibits radiosensitizing effects on HT-29 cells (Tab. 4, Fig. 8, Ref. 42).