HT-29 Human Adenocarcinoma Cells Research Articles

Gut Metabolite Indoxyl Sulfate Has Selective Deleterious and Anticancer Effect on Colon Cancer Cells.

There are a number of reports about anticancer activity of indole derivatives. In this study, we investigated the role of indoxyl sulfate (IS) for its selective anticancer activity on colon cancer cells. IS treatment on HCT-116 and HT-29 human epithelial adenocarcinoma cells led to a decrease in cell proliferation, cell viability, and ATP content. Colon cancer cells showed a 10% increase in cell apoptosis in comparison to control. Due to IS treatment, cell morphology got distorted, cell number found decreased, intracellular vesicles formed, and cells were found floating in the media. Cells also showed a loss in membrane integrity and a decrease in colony-forming ability and ceased at the G2/M phase of the cell cycle. No significant change was noted in the level of inflammatory cytokines IL-17A, IL-1β, and TNF-α, histology, length of intestine, and spleen after 100 mM IS treatment to balb/c mice. These observations indicate the selective anticancer effect of IS on colon cancer cells.

The Potential Inhibitory Role of Acetyl-L-Carnitine on Proliferation, Migration, and Gene Expression in HepG2 and HT29 Human Adenocarcinoma Cell Lines.

Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated derivative of carnitine essential for intermediate metabolism in most tissues. This study aimed to investigate the effects of ALC on the proliferation, migration, and gene expression of human liver (HepG2) and colorectal (HT29) adenocarcinoma cell lines. The cell viability and half maximal inhibitory concentration of both cancer cell lines were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing after treatment was assessed using a migration assay. Morphological changes were imaged using brightfield and fluorescence microscopy. Post treatment, apoptotic DNA was detected using a DNA fragmentation assay. The relative mRNA expressions of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) were evaluated using RT-PCR. The results showed that ALC treatment affects the wound-healing ability of HepG2 and HT29 cell lines. Changes in nuclear morphology were detected under fluorescent microscopy. ALC also downregulates the expression levels of MMP9 and VEGF in HepG2 and HT29 cell lines. Our results indicate that the anticancer action of ALC is likely mediated by a decrease in adhesion, migration, and invasion.

Enzymatically Extracted Apple Pectin Possesses Antioxidant and Antitumor Activity.

The biological activity of apple pectin extracted conventionally or enzymatically using endo-xylanase and endo-cellulase, was tested in vitro. The analyses were performerd in tetraplicates and the statistical significance of the differences were assessed using ANOVA, Tukey post hoc and LSD (the least significant difference) tests. Multivariate regression analysis was applied to determine the structural components that have a crucial importance for antioxidant and antitumor properties of pectins. The pectins extracted by enzymes contained up to four times more ferulic acid and showed twice as great ability to neutralize free radicals and Fe(III) reduction. The antiradical potential positively correlated with phenols, fucose and rhamnose content. In the assays performed on HT-29 human adenocarcinoma and B16F10 melanoma cell cultures, the “green” pectins, contrary to acid isolated ones, exhibited remarkable anti-neoplastic potential while being nontoxic to nontransformed L929 cell line. The pectins in the dose of 1 mg/mL were capable of inhibiting adhesion (max 23.1%), proliferation (max 40.4%), invasion (max 76.9%) and anchorage-independent growth (max 90%) of HT-29 cells (significance level p < 0.001). These pectin preparations were slightly less active towards B16F10 cells. The enzyme-isolated apple pectins may be useful as a functional food additive and an ingredient of the ointment formulas for post-surgical melanoma treatment.

The protective effect of stilbenes resveratrol and pterostilbene individually and combined with mycotoxin citrinin in human adenocarcinoma HT-29 cell line in vitro

This study was focused to determine an individual and combined effect of mycotoxin citrinin (CIT) and two compounds of the stilbene family- resveratrol (RES) and his dimethyl ether analogue pterostilbene (PTE) which have many health benefits. As a model the human adenocarcinoma cell line HT-29 was used which may exhibits the properties of small intestine cells. Viability, plasma membrane integrity, lysosomal functionality, intracellular production of superoxide anions and superoxide dismutase activity were examined. The results indicate that concentrations of 50 and 100 μg/mL of the tested compounds were cytotoxic in mostly monitored parameters and probably caused apoptosis. HT-29 cells were more sensitive to PTE than to RES with a higher antioxidant effect of PTE than RES, which may be caused by its chemical structure. Both stilbenes at medium doses act as effective superoxide anions scavengers leading to reduction of oxidative stress and consequent cell damage. The nontoxic concentration of RES (25 µg/mL) protects the HT-29 cell line faced to the toxicity of CIT at 25 µg/mL by increasing viability of cells and by reducing the superoxide production induced by CIT concentrations of 12.5 µg/mL and 25 µg/mL.

Nanostructured Thin Coatings Containing Anthriscus sylvestris Extract with Dual Bioactivity.

Plant extracts are highly valuable pharmaceutical complexes recognized for their biological properties, including antibacterial, antifungal, antiviral, antioxidant, anticancer, and anti-inflammatory properties. However, their use is limited by their low water solubility and physicochemical stability. In order to overcome these limitations, we aimed to develop nanostructured carriers as delivery systems for plant extracts; in particular, we selected the extract of Anthriscus sylvestris (AN) on the basis of its antimicrobial effect and antitumor activity. In this study, AN-extract-functionalized magnetite (Fe3O4@AN) nanoparticles (NPs) were prepared by the co-precipitation method. The purpose of this study was to synthesize and investigate the physicochemical and biological features of composite coatings based on Fe3O4@AN NPs obtained by matrix-assisted pulsed laser evaporation technique. In this respect, laser fluence and drop-casting studies on coatings were performed. The physical and chemical properties of laser-synthesized coatings were investigated by scanning electron microscopy, while Fourier transform infrared spectroscopy comparative analysis was used for determining the chemical structure and functional integrity. Relevant data regarding the presence of magnetic nanoparticles as the only crystalline phase and the size of nanoparticles were obtained by transmission electron microscopy. The in vitro toxicity assessment of the Fe3O4@AN showed significant cytotoxic activity against human adenocarcinoma HT-29 cells after prolonged exposure. Antimicrobial results demonstrated that Fe3O4@AN coatings inhibit microbial colonization and biofilm formation in clinically relevant bacteria species and yeasts. Such coatings are useful, natural, and multifunctional solutions for the development of tailored medical devices and surfaces.

Evaluation of Novel Aspirin Suppository Formulation in HT-29 Human Adenocarcinoma Cells

Evaluation of Novel Aspirin Suppository Formulation in HT-29 Human Adenocarcinoma Cells

Anticancer effects of sodium and potassium quercetin-5'-sulfonates through inhibition of proliferation, induction of apoptosis, and cell cycle arrest in the HT-29 human adenocarcinoma cell line.

In the present study, we compared the anticancer potential of quercetin (3,3',4',5,7-pentahydroxyflavone, I) and its sulfonic derivatives sodium/potassium quercetin-5'-sulfonates (described as II and III) against several human carcinoma cell lines. Quercetin (I) was used as a starting compound for synthesis of II and III. In this work, a modified and more efficient method of synthesizing derivatives II and III has been described. The molecular structures of the compounds were characterized in a solution and in the solid state using 1H NMR, 13C NMR, 2D NMR, and XPS spectroscopy, respectively. The stoichiometry of these complexes was determined by elemental analysis as well as thermogravimetric and X-ray fluorescence methods. The spectral data allowed complete characterization of the investigated compounds in the solution and in the solid state and unambiguous determination of the place of substitution of the sulfonic group in the phenyl ring in the C-5' position. Our in vitro studies revealed that II and III prominently reduced the viability of the HT-29 colon cancer cell line. Additionally, we observed that sulfonic derivatives decreased proliferation of colon (HT-29, LS180), lung (A549), and breast (T47D) cancer cell lines. Moreover, we detected a lower cytotoxic effect of II and III on several normal cell lines (colon epithelial CCD 841 CoTr, mouse subcutaneous connective tissue L-929, and human skin fibroblasts HSF cell lines) than that exerted by pure quercetin. The anticancer properties were especially evident in the HT-29 colon cancer cell line, where cell cycle inhibition in the G2-M phase and prominent apoptosis induced by II and III were observed. In conclusion, the sodium/potassium quercetin-5'-sulfonates prepared from quercetin showed promising anti-proliferative and pro-apoptotic activity against colon cancer cells. Therefore, we support the opinion that sodium/potassium quercetin-5'-sulfonates should be considered as promising organometallic compounds for possible clinical applications.

Cadmium Induces Migration of Colon Cancer Cells: Roles of Reactive Oxygen Species, P38 and Cyclooxygenase-2.

Cadmium (Cd) is a heavy metal contaminant whose toxicity is associated with colorectal cancer (CRC). However, the underlying molecular mechanisms of Cd-induced CRC malignancy remain obscure. A monolayer scratch assay was employed to assess the migration of HT-29 human adenocarcinoma cells. Luciferasereporterassay was used to determine cyclooxygenase-2 (COX-2) transcriptional activity, and Western blotting was used to detect p38 Mitogen Activated Protein Kinase (MAPK) and Akt phosphorylation as well as COX-2 expression. Prostaglandin E2 (PGE2) levels were measured using Enzyme Linked Immunosorbent Assay (ELISA) and reactive oxygen species (ROS) formation was assessed using dihydroethidium (DHE) stain. Here, we show that Cd potentiates the migratory capacity of HT-29 CRC cells. Cd caused a time-dependent increase in COX-2 expression. Celecoxib, a COX-2 selective inhibitor, significantly reduced Cd-induced migration. Cd also increased levels of ROS and phosphorylated p38. Importantly, Cd-induced COX-2 expression and migration were significantly abolished by N-Acetyl-Cysteine (NAC), a ROS scavenger, or SB202190, a specific p38 inhibitor. Furthermore, Cd-induced p38 phosphorylation was inhibited by NAC. Cd (100 nM) also increased PGE2 levels, which was abrogated by NAC, SB202190, or celecoxib. Exogenous PGE2 significantly potentiated cell migration. Cd caused a significant increase in Akt phosphorylation in a ROS-mediated pathway. Moreover, Cd-induced migration was significantly attenuated by LY294 002, a phosphatidylinositol-3-kinase inhibitor. Taken together, our results suggest that exposure to low levels of Cd promotes a more migratory cancer phenotype in a ROS-p38-COX-2-PGE2 pathway as well as ROS-Akt pathway. Therefore, COX-2, PGE2 receptors or Akt represent potential targets in the treatment of CRC, particularly in patients exposed to Cd.

Poly(3-hydroxybutyrate-CO-3-hydroxyvalerate) PHBHV biocompatible nanocarriers for 5-FU delivery targeting colorectal cancer

Aiming to address the issue of poor bioavailability of most anti-tumor medicines against colorectal cancer, we developed a targeted anticancer nanocarrier using biocarriers able to both bind and easily release their load in a controlled manner. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) carriers were obtained via the emulsification-diffusion method, loaded with 5-fluorouracil and then characterized in terms of particle morphology and size (SEM, DLS), drug uptake and release. The cytotoxic potential of the 5-fluorouracil-loaded polymer nanocarriers on human adenocarcinoma cells (HT-29 cell line) was investigated. The in vitro studies clearly demonstrated that while the nanocarriers themselves slightly alter HT-29 cell viability, when loaded with 5-fluorouracil they significantly decrease cell viability, suggesting that the polymer itself exhibits low cytotoxicity and the drug-loaded carrier acts in an efficient manner to kill HT-29 human adenocarcinoma cells.

Abstract A80: Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins

Abstract Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the world. According to the International Agency for Research on Cancer data, Brazil belongs to the group of countries that presented an increase in both incidence and mortality average of the CRC in the last decade. The loss of the apical-basal polarity can lead to cancer progression, since neoplastic cells shows deficiency in the apical junctional complexes (AJC), exhibiting a malign potential. Tight junction (TJ) is one of the junctions found in the AJC. It is a structure that helps to maintain the polarity and also presents a barrier function, which regulates the transit of important molecules through the paracellular flow. Claudin protein family are the main components of the TJ and are composed of transmembrane proteins. These proteins can interact with other TJ proteins such as occludins or scaffold proteins, essentially ZO-1/2. Our group has already showed that patients with CRC overexpress claudin-3 in the tumor tissue. Our goal was to evaluate the importance of the expression and interaction among claudin-3, ZO-1, and occludin proteins during the progression of CRC. For the molecular analysis, samples were obtained from biopsy after patients were submitted to surgery at the Brazilian National Cancer Institute. Adenocarcinoma tissue and normal adjacent region were obtained through colectomy and classified by the TNM staging of tumor. The Immunoblotting essay was performed to quantify the expression of claudin-3 and occludin proteins in those patients in different stages of the disease. Moreover, the interaction between these proteins was identified through immunoprecipitation essay. In vitro assay was performed using transfected HT-29 human adenocarcinoma cells overexpressing claudin-3. In these cells we evaluated the expression levels of claudin-3 and ZO-1, and we also analyzed the subcellular distribution of claudin-3, ZO-1, and occludin by immunofluorescence. This study is being carried out with approval of the INCA Research Ethics Committee (Protocol 84/04). The analysis by immunoblotting of claudin-3 expression showed that the patients can be segregated in two groups: one where the level of this protein in the tumor is low and other group where its levels are high. No alteration in the levels of occludin was observed when comparing the tumor and the normal adjacent tissue. However, the patients in whom the tumor presented high levels of claudin-3 showed less interaction with occludin. In vitro assay using transfected HT-29 cells that overexpress claudin-3 allowed us to observe that there was a reduction in the protein levels of ZO-1 as well as a reduced colocalization between these in the regions of cell-cell contact. Associated with these results, we also observed claudin-3 and occludin colocalization in an intermittent fashion in the regions of cell-cell contact, being more evident in the region of claudin-3 accumulation in HT-29 cells. Altogether, our data suggest that in CRC there are groups of patients that show elevated level of claudin-3 in the tumor and other group that shows low level. Further, it was observed that the increase of claudin-3 expression can be related to the decrease of the ZO-1 expression and a decrease in the interaction between claudin-3 and both, occludin and ZO-1. Our results can help to improve the understanding of the molecular mechanisms involved in the colorectal tumorigenesis, and point out potential prognostic markers aiming a therapeutic design to specific targets. Financial support: FAPERJ, CAPES and MS. Citation Format: Waldemir Fernandes de Souza, Perôny da Silva Nogueira, Maria Teresa dos Santos Guedes, Bruno Kaufmann Robbs, Joao Paulo de Biaso Viola, José Andrés Morgado-Díaz. Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A80.

Korean Ginseng Berry Fermented by Mycotoxin Non-producing Aspergillus niger and Aspergillus oryzae: Ginsenoside Analyses and Anti-proliferative Activities.

To transform ginsenosides, Korean ginseng berry (KGB) was fermented by mycotoxin non-producing Aspergillus niger and Aspergillus oryzae. Changes of ginsenoside profile and anti-proliferative activities were observed. Results showed that A. niger tended to efficiently transform protopanaxadiol (PPD) type ginsenosides such as Rb1, Rb2, Rd to compound K while A. oryzae tended to efficiently transform protopanaxatriol (PPT) type ginsenoside Re to Rh1 via Rg1. Butanol extracts of fermented KGB showed high cytotoxicity on human adenocarcinoma HT-29 cell line and hepatocellular carcinoma HepG2 cell line while that of unfermented KGB showed little. The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB was about 5 µg/mL. As A. niger is more inclined to transform PPD type ginsenosides, niger-fermented KGB showed stronger anti-proliferative activity than oryzae-fermented KGB.

Aquaporin 3 promotes intestinal epithelial proliferation and inhibits cytokine‐induced apoptosis

Aims Aquaporin 3 (AQP3) is involved in cell proliferation, apoptosis and migration. Inflammatory bowel diseases are associated with barrier dysfunction, which includes impaired absorption, decreased secretion and increased apoptosis. Given that AQPs are involved in all of these processes and that AQP3 is expressed throughout the gut, we sought to determine the role of AQP3 in intestinal epithelial cell proliferation and survival. Methods: Stable AQP3 knockdown clones of the HT29 human adenocarcinoma cell line were developed using shRNA and a cell proliferation assay was performed using 10% FBS and serum-free conditions to assess AQP3 knockdown cell growth and replication. To induce apoptosis, cells were treated with 40 ng/mL IFNγ and 10 ng/mL TNFα and cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) products were assessed using western blot. Results: AQP3 knockdown clones exhibited 94-97% decreased AQP3 expression. At 24 and 48 hrs AQP3 knockdown cell proliferation was not impaired, however at 72 hrs the AQP3 knockdown clones exhibited significantly impaired proliferation, reduced by 47-61% under 10% FBS conditions but not in serum free conditions. Western blot results did not show significant differences in cell death markers at 6 hours, however at 24 hours increased levels of cleaved caspase-3 and cleaved PARP were observed in AQP3 knockdown cells compared to scrambled shRNA clones. Conclusion Failure to properly regulate intestinal epithelial cell proliferation and apoptosis can result in inflammation and mucosal injury. Our data highlights a potential functional role of AQP3 in intestinal epithelial cells, and its involvement in maintaining intestinal homeostasis.

Pulsed electric field processing preserves the antiproliferative activity of the milk fat globule membrane on colon carcinoma cells

The present work evaluated the effect of processing on the antiproliferative activities of milk fat globule membrane (MFGM) extracts. The antiproliferative activity on human adenocarcinoma HT-29 cells of untreated MFGM extracts were compared with those extracted from pasteurized cream, thermally treated cream, or cream subjected to pulsed electrical field (PEF) processing. The PEF with a 37 kV/cm field strength applied for 1,705μs at 50 and 65°C was applied to untreated cream collected from a local dairy. Heating at 50 or 65°C for 3min (the passage time in the PEF chamber) was also tested to evaluate the heating effect during PEF treatments. The MFGM extracted from pasteurized cream did not show an antiproliferative activity. On the other hand, isolates from PEF-treated cream showed activity similar to that of untreated samples. It was also shown that PEF induced interactions between β-lactoglobulin and MFGM proteins at 65°C, whereas the phospholipid composition remained unaltered. This work demonstrates the potential of PEF not only a means to produce a microbiologically safe product, but also as a process preserving the biofunctionality of the MFGM.

고추장 제조시 발효가 항돌연변이 및 in vitro 항암효과에 미치는 영향

Increased antimutagenic and in vitro anticancer effects were observed by the fermentation process during Kochujang manufacturing. In order to confirm the increased functionality, wheat grain, first fermented wheat grains (FFWG), second fermented wheat grains (SFWG), final fermented wheat grains (FiFWG), red pepper powder (RPP), and commercial Kochujang (CK) were prepared. Kochujang manufactured with final fermented wheat grains and red pepper powder were further fermented for 15 days and 30 days. The antimutagenic effects were determined by counting the number of revertants in Salmonella Typhimurium TA100 against N-methyl-N'-nitro-nitrosoguanidine (MNNG, 1.0mg/mL). The final fermented wheat grains (52% inhibition) showed higher antimutagenic effects than the wheat grain (34%), and the commercial Kochujang showed the highest antimutagenic effects (55%). We tested the inhibitory effect on the growth of HT-29 human colon carcinoma cells and AGS human gastric adenocarcinoma cells by using MTT (3-(4,5-dimethyl-thiazol)-2,5-diphenyltetrazolium bromide) assay. The results showed that increased fermentation process continually increased the growth inhibitory effect on both cancer cells. The further fermentation for 15 days of the Kochujang product also increased inhibitory growth of the AGS cancer cells. In conclusion, the methanol extract from fermented wheat grains and commercial Kochujang showed sequentially increased antimutagenic and in vitro anticancer activity, and thus the final commercial Kochujang revealed the highest effect.

Two-step sequential supercritical fluid extracts from rosemary with enhanced anti-proliferative activity

Previously, carnosic acid and carnosol have demonstrated anti-proliferative activity against different types of cancer. To obtain extracts enriched in these two key phenolic compounds, two different processes have been developed in the present work based on the use of two-step sequential supercritical fluid extraction (SFE). By removing the interfering, less active fractions in a first step (150 or 300 bar, 40°C, neat CO2, 60 min), suitable enrichment is achieved in the second step (150 bar, 40°C, CO2 + 7% ethanol, 120 min), and this leads to carnosic acid concentrations in the extract as high as 40% of total dry weight, which are among the highest concentrations that have been described with this type of process. The enriched extracts were tested against the HT-29 human adenocarcinoma cell line, showing enhancement of their antiproliferative activity by approximately 3-fold compared to previously reported SFE rosemary extracts and higher inhibitory effects at lower concentrations (30 µg mL−1 of extract). Thus, the proposed two-step SFE process effectively improves the carnosic acid and carnosol recovery in shorter processing times (180 min vs. 300 min). Moreover, the obtained extracts possess higher anti-proliferative activity and consume less solvent.

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria.

Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.

The effect of milk fat globules on adherence and internalization of Salmonella Enteritidis to HT-29 cells

Milk fat globules were extracted from bovine and goat milk and incubated with HT-29 human adenocarcinoma cells to assess the attachment and internalization of Salmonella Enteritidis. Because the expression of bacterial adhesins is highly affected by the presence of antibiotic, the attachment was studied with and without antibiotic in the cell growth medium. Although no inhibitory effect of the fat globules was observed in the presence of the antibiotic, milk fat globules significantly inhibited the binding and internalization of Salmonella in medium free of antibiotic. The fat globules from both bovine and goat milk markedly reduced bacterial binding and invasion compared with controls, and the cells treated with goat milk-derived fat globules demonstrated greater protective properties than those derived from bovine milk. The effect of heat treatment on bovine fat globules was also investigated, and it was shown that the fat globules from heated milk had a higher degree of inhibition than those from unheated milk.

The vascular targeting agent Combretastatin-A4 directly induces autophagy in adenocarcinoma-derived colon cancer cells

Recent clinical data demonstrated that the vascular targeting agent Combretastatin-A4 phosphate (CA-4P) prolonged survival of patients with advanced anaplastic thyroid cancer without any adverse side effects. However, as a single agent CA-4 failed to reduce tumour growth in the murine CT-26 adenocarcinoma colon cancer model. Furthermore, the molecular mechanism of the innate resistance of HT-29 human adenocarcinoma cells to CA-4 is largely unknown. In this report, we demonstrate for the first time that prolonged exposure to CA-4 and an azetidinone cis-restricted analogue, CA-432 (chemical name; 4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one) induced autophagy in adenocarcinoma-derived CT-26, Caco-2 and HT-29 cells but not in fibrosarcoma-derived HT-1080 cells. Autophagy is a fundamental self-catabolic process which can facilitate a prolonged cell survival in spite of adverse stress by generating energy via lysosomal degradation of cytoplasmic constituents. Autophagy was confirmed by acridine orange staining of vesicle formation, electron microscopy and increased expression of LC3-II. Combretastatin-induced autophagy was associated with a loss of mitochondrial membrane potential and elongation of the mitochondria. Furthermore, inhibition of autophagy by the vacuolar H+ATPase inhibitor Bafilomycin-A1 (BAF-A1) significantly enhanced CA-432 induced HT-29 cell death. Both CA-4 and its synthetic derivative, CA-432 induced the formation of large hyperdiploid cells in Caco-2 and CT-26 cells. The formation of these polyploid cells was significantly inhibited by autophagy inhibitor, BAF-A1. Results presented within demonstrate that autophagy is a novel response to combretastatin exposure and may be manipulated to enhance the therapeutic efficacy of this class of vascular targeting agents.

Abstract B76: Synthetic triterpenoids as candidate drugs for cancer prevention and treatment

Abstract Angiogenesis is a limiting step for the switch from a dormant to malignant state in tumor progression. During tumor development tumor cells induce profound changes in the surrounding tissue, leading to the formation of an altered tumor microenvironment that is critical in permitting further tumor growth and tumor progression. Retinoblastoma (Rb) and colorectal cancer (CRC) display the complexity of the microenvironment constituted by different cell types and their extracellular matrix. Here we assessed the efficacy of two synthetic derivates of triterpenoid CDDO, CDDO-Im and CDDO-Me, on endothelial, retinoblastoma and colon cancer cells in order to evaluate their effect on angiogenesis, cancer progression and metastatic dissemination. We observed that CDDO-Im and CDDO-Me inhibited endothelial cells proliferation, migration and tube-formation, hampering the nuclear translocation of NF-kB and affecting the MAPK pathway. In vivo, in a matrigel sponge assay CDDO-Im interfered with angiogenesis when administrated into the pellets or by intraperitoneal injection. To assess whether these compounds could successfully impairs neuroectodermal-derived tumor growth, we performed in vitro experiments on Y79 and Weri-Rb1 retinoblastoma cells. We performed MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and LDH (Lactate Dehydrogenase) assays to evaluate CDDO-Im effect on retinoblastoma cell viability and an Annexin V and 7-Amino-Actinomycin D staining to test apoptosis. We observed that CDDO-Im treatment induced a reduction of cell viability at nanomolar doses and early and late apoptosis at different time points. In order to overcome the side effects of standard chemotherapy drugs, such as vincristine sulphate, we evaluated if CDDO-Im could be used to lower chemotherapy doses. When we combined non-cytotoxic doses of CDDO-Im with vincristine sulphate we observed that after 72 hours of co-treatment, CDDO-Im was able to accomplish the same antiproliferative effect obtained using double doses of vincristine alone. We also tested the in vitro anti-tumour activity of CDDO-Me on human adenocarcinoma HT29 cell line. The triterpenoid was used alone or in combination with 5FU (5 Fluorouracil), the major chemotherapeutic agent used for CRC therapy. We performed an MTT assay to evaluate its effect on cell viability, an apoptosis test and an alkaline comet assay to assess the potential genotoxic damage. Preliminary results demonstrated that CDDO-Me induced an almost immediate inhibitory effect on HT29 cells, reducing cell viability already after 24 hours of treatment at 1000-fold lower concentration then 5FU. When HT29 were co-treated with both CDDO-Me and 5FU, no additive effects were observed. Respect to 5FU, CDDO-Me did not induce apoptosis even after long-term treatment, as well as genotoxicity, suggesting that it is able to block cell growth without exerting toxic effects. These data suggest that the two synthetic triterpenoid CDDO-Im and CDDO-Me could be promising compounds for cancer treatment, acting both on cancer cells and on endothelial cells that support tumor-associated angiogenesis. Our results open the possibility to use chemopreventive molecules in combination with current chemotherapic drugs, to improve their efficacy without cytotoxicity. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B76.

Ku70 and Rad51 vary in their importance for the repair of doxorubicin- versus etoposide-induced DNA damage

For DNA targeting anticancer drugs, cellular DNA repair mechanisms may cause resistance and hamper the therapeutic outcome. DNA damage induced by topoisomerase IIα inhibitors like etoposide and anthracyclines, which are a mainstay of cancer therapy, is also repaired in many cell types, but the impact and precise mechanisms of this repair are still obscure. To investigate the DNA damage response of human adenocarcinoma HT29-cells to doxorubicin and to compare the involvement of Ku70 and Rad51 in the repair of doxorubicin- versus etoposide-induced DNA damage, we assessed cell cycle distribution and cell death, DNA damage, proteins relevant for repair by homologous recombination and non-homologous end-joining, and clonogenicity following exposure to doxorubicin at clinically achievable concentrations. Also, we assessed changes in the repair kinetics after siRNA-mediated attenuation of Ku70 or Rad51 expression. We found that exposure to doxorubicin for 24h induced a substantial amount of DNA damage that was largely repaired when doxorubicin was removed and the cells were maintained in drug-free medium. Nevertheless, a pronounced G(2)/M arrest occurred at times when repair was maximal. This was followed by a distinct increase in cell death and loss of clonogenicity. In this regard, responses to doxorubicin and etoposide were similar. However, distinct differences in the repair process following doxorubicin versus etoposide were seen in concentration dependency, time-course and requirement of Ku70 and Rad51 proteins. In spite of the shared molecular target of doxorubicin and etoposide, DNA lesions induced by these compounds are repaired differently.