SW480 Colon Carcinoma Research Articles

Synthesis and Chemopreventive Potential of 5-FU/Genistein Hybrids on Colorectal Cancer Cells.

A series of 5-FU-Genistein hybrids were synthesized and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated in human colon adenocarcinoma cells (SW480 and SW620) and non-malignant cell lines (HaCaT and CHO-K1). Hybrid 4a displayed cytotoxicity against SW480 and SW620 cells with IC50 values of 62.73 ± 7.26 µM and 50.58 ± 1.33 µM, respectively; compound 4g induced cytotoxicity in SW620 cells with an IC50 value of 36.84 ± 0.71 µM. These compounds were even more selective than genistein alone, the reference drug (5-FU) and the equimolar mixture of genistein plus 5-FU. In addition, hybrids 4a and 4g induced time- and concentration-dependent antiproliferative activity and cell cycle arrest at the S-phase and G2/M. It was also observed that hybrid 4a induced apoptosis in SW620 cells probably triggered by the extrinsic pathway in response to the activation of p53, as evidenced by the increase in the levels of caspases 3/8 and the tumor suppressor protein (Tp53). Molecular docking studies suggest that the most active compound 4a would bind efficiently to proapoptotic human caspases 3/8 and human Tp53, which in turn could provide valuable information on the biochemical mechanism for the in vitro cytotoxic response of this compound in SW620 colon carcinoma cell lines. On the other hand, molecular dynamics (MD) studies provided strong evidence of the conformational stability of the complex between caspase-3 and hybrid 4a obtained throughout 100 ns all-atom MD simulation. Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) analyses of the complex with caspase-3 showed that the interaction between the ligand and the target protein is stable. Altogether, the results suggest that the active hybrids, mainly compound 4a, might act by modulating caspase-3 activity in a colorectal cancer model, making it a privileged scaffold that could be used in future investigations.

Chemopreventive Effect on Human Colon Adenocarcinoma Cells of Styrylquinolines: Synthesis, Cytotoxicity, Proapoptotic Effect and Molecular Docking Analysis.

Seven styrylquinolines were synthesized in this study. Two of these styrylquinolines are new and were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, and normal cells (HaCaT). According to the results, compounds 3a and 3d showed antiproliferative activity in SW480 and SW620 cells, but their effect seemed to be caused by different mechanisms of action. Compound 3a induced apoptosis independent of ROS production, as evidenced by increased levels of caspase 3, and had an immunomodulatory effect, positively regulating the production of different immunological markers in malignant cell lines. In contrast, compound 3d generated a pro-oxidant response and inhibited the growth of cancer cells, probably by another type of cell death other than apoptosis. Molecular docking studies indicated that the most active compound, 3a, could efficiently bind to the proapoptotic human caspases-3 protein, a result that could provide valuable information on the biochemical mechanism for the in vitro cytotoxic response of this compound in SW620 colon carcinoma cell lines. The obtained results suggest that these compounds have chemopreventive potential against CRC, but more studies should be carried out to elucidate the molecular mechanisms of action of each of them in depth.

Chemical Composition and Cytotoxic Activity of Eucalyptus torquata Luehm. and Eucalyptus salmonophloia F. Muell. Trunk Bark Essential Oils against Human SW620 and MDA-MB-231 Cancer Cell Lines.

In recent years, there has been a growing interest in the screening of natural active ingredients from Eucalyptus essential oils because of their evident importance in practical utility and their undeniable therapeutic properties. Based on this, the aim of the present study was to investigate the chemical profile of the essential oils of the trunk bark of Eucalyptus torquata Luehm. (ETEO), and E. salmonophloia F. Muell. (ESEO), growing in Tunisia. The in vitro cytotoxic properties of the extracted EOs were also evaluated against two human cancer cell lines: breast carcinoma cell lines MDA-MB-231 and colorectal cancer cell lines SW620. The analysis by gas chromatography coupled with mass spectrometry (GC/MS) led to the identification of 32 compounds from the ETEO, with the dominant constituents being the monoterpenes trans-myrtanol (73.4 %) and myrtenol (4.7 %), and the apocarotene (E)-β-ionone (3.9 %). In the case of ESEO, 29 compounds were identified with trans-myrtanol (25.0 %), decanoic acid (22.1 %), nonanoic acid (9.8 %), γ-elemene (6.5 %), γ-maaliene (5.5 %), and α-terpineol (5.3 %) as the main components. The cytotoxicity of EOs against the two chosen cell lines was tested using Crystal Violet Staining (CVS) assay and 5-fluorouracil as a reference drug. The two EOs exhibited a significant dose-dependent inhibition against the viability of the used cell lines. Their inhibitory effects were particularly observed towards SW620 colon carcinoma cells with IC50 values of 26.71±1.22 and 22.21±0.85 μg/mL, respectively, indicating that both oils were more cytotoxic for SW620 cells compared to MDA-MB-231 one.

IGF-1R depletion sensitizes colon cancer cell lines to radiotherapy.

Insulin like growth factor receptor 1 (IGF-1R) has been documented to play a key role in radiation response, thereby offering an attractive drug target to enhance tumor sensitivity to radiotherapy. Here, we investigated wether knockdown of IGF-1R can sensitize colorectal cancer (CRC) cell lines to radiation. Human colon carcinoma SW480 and HT-29 cells were transfected with specific small interference RNA (siRNA) to mediate IGF-1R depletion. The expression of IGF-1R mRNA and protein among transfected and untransfected cells was detected by Western blot analysis. Changes in cell proliferation and radiosensitivity were evaluated by the clonogenic survival assay. NVP-ADW742, an IGF-1R inhibitor, in combination with radiation was studied. RAD51, a measure for homologous recombination repair, and 53BP1, a maker for non-homologous end-joining (NHEJ), were determined by immunofluorescence for double-strand breaks (DSB) repair pathways. Cell cycle was also examined in the IGF-1R knockdown and IGF-1R-inhibited cells. CRC cell lines were selectively sensitized to radiation after siRNA-mediated IGF-1R depletion. NVP-ADW742 efficiently increases cancer cell response to radiation. Furthermore, initial formation of RAD51 foci after IR, and 53BP1 foci were significantly reduced in IGF-1R-depleted or with IGF-1R Inhibitor CRC cell lines. Lastly, IGF-1R-depleted or with IGF-1R Inhibitor caused more G2 phase cell arrest. Our findings demonstrate that depletion of IGF-1R lead to an increase in radiosensitivity in CRC.

Assessing Suitability of Co@Au Core/Shell Nanoparticle Geometry for Improved Theranostics in Colon Carcinoma.

The interactions between cells and nanomaterials at the nanoscale play a pivotal role in controlling cellular behavior and ample evidence links cell intercommunication to nanomaterial size. However, little is known about the effect of nanomaterial geometry on cell behavior. To elucidate this and to extend the application in cancer theranostics, we have engineered core–shell cobalt–gold nanoparticles with spherical (Co@Au NPs) and elliptical morphology (Co@Au NEs). Our results show that owing to superparamagnetism, Co@Au NPs can generate hyperthermia upon magnetic field stimulation. In contrast, due to the geometric difference, Co@Au NEs can be optically excited to generate hyperthermia upon photostimulation and elevate the medium temperature to 45 °C. Both nanomaterial geometries can be employed as prospective contrast agents; however, at identical concentration, Co@Au NPs exhibited 4-fold higher cytotoxicity to L929 fibroblasts as compared to Co@Au NEs, confirming the effect of nanomaterial geometry on cell fate. Furthermore, photostimulation-generated hyperthermia prompted detachment of anti-cancer drug, Methotrexate (MTX), from Co@Au NEs-MTX complex and which triggered 90% decrease in SW620 colon carcinoma cell viability, confirming their application in cancer theranostics. The geometry-based perturbation of cell fate can have a profound impact on our understanding of interactions at nano-bio interface which can be exploited for engineering materials with optimized geometries for superior theranostic applications.

PRODUCTION OF VINEGAR FROM AN ALCOHOLIC 1 BEVERAGE OF 2 ANDEAN BERRY (Vaccinium meridionale SW), MEASUREMENT OF 3 ANTIOXIDANT ACTIVITY AND EVALUATION OF CYTOTOXIC EFFECT ON 4 COLON CANCER CELLS SW480

BACKGROUND: Vaccinium meridionale presents fruits with a high content of phenolic compounds and anthocyanins and therefore a high antioxidant capacity.
 OBJECTIVE: The aim was the production of vinegar from three treatments of berries alcohol beverage, to evaluate its antioxidant and cytotoxic activity.
 METHODS: Vinegars were prepared by acetic fermentation from alcoholic beverages of Andean berries by three treatments: mechanical maceration, heating, and mixing. The titratable acidity and acetic acid content were evaluated in the vinegar; antioxidant potential (DPPH, FRAP, and ORAC); content of antioxidant metabolites (total polyphenols, total anthocyanins, and phenolic hydroxycinnamic acids) were evaluated in the juice, alcoholic beverages, and vinegar; the antiproliferative capacity in colon cancer cell SW480 was determined in vinegar.
 RESULTS: Alcoholic beverages presented the main antioxidant activity. However, after the acetic fermentation, it was observed a fall in the antioxidant potential in all techniques evaluated in three treatments. The vinegar obtained by heated and mixed treatments were presented the highest values in antioxidant activity, phenols, and anthocyanins. Antiproliferative activity inhibitions of vinegars in cell viability were 6.2% (20 μg/mL) and 22.5% (200 μg/mL).
 CONCLUSIONS: Production of acetic acid is independent of the treatment. The antioxidant activity is favored in alcoholic beverages with the treatment of hot maceration. Andean berry CAL vinegar has antiproliferative activity, inhibiting a colon carcinoma SW480 line although in a low amount compared with reports in the literature.

TSP-1 as a novel biological marker of tumor vasculature normalization in colon carcinoma induced by Endostar.

Blood vessels in tumors often exhibit abnormal morphology and function, which promotes the growth, metastasis and resistance of tumors to conventional therapies. Therefore, vascular normalization is an emerging strategy to enhance the effectiveness of radiotherapy and chemotherapy when used in combination; however, there is a lack of evidence regarding the optimal schedule for the co-administration of anti-angiogenic and chemotherapeutic drugs. Scheduling treatment is important as the period for normalization is transient, also known as the ‘time window’; however, no biomarker has been identified to detect this window. In the present study, recombinant human endostatin (rhES) was employed as an anti-angiogenic agent in xenograft tumor tissue in mice. Following rhES or control (saline) treatment, the density and integrity of tumor vessels were detected by immunofluorescence staining for cluster of differentiation 31 and α-smooth muscle actin; the level of hypoxia in tumor tissue was examined by immunohistochemistry with pimonidazole; the necrotic area was evaluated by hematoxylin and eosin staining; and the level of thrombospondin-1 (TSP-1) in plasma was tested by ELISA. The Cell Counting Kit-8 assay was also used to evaluate the effect of rhES on the proliferation of colon carcinoma SW620 cells. A ‘time window’ normalized vasculature was determined between day 4 and 6 following rhES treatment, and accompanied by a decrease in hypoxia in tumor tissue. Decreasing plasma TSP-1 levels were consistent with changes in vascular morphology and hypoxia, which exhibited features of normalization. In addition, rhES had no effect on the proliferation of SW620 cells, suggesting that the reduction in TSP-1 was associated with increased oxygen content during vascular normalization, rather than inhibited cell proliferation. In conclusion, TSP-1 may be a potential biomarker for predicting the normalization window of colon cancer vessels.

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2.

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.

Effects and Mechanism of Tanshinone II A in Proliferation, Apoptosis, and Migration of Human Colon Cancer Cells.

BackgroundThe aim of this study was to explore the effect and mechanism of tanshinone II A on proliferation, apoptosis, and migration of human colon cancer cells.Material/MethodsCCK-8 approach was carried out to evaluate proliferation after applying various levels of tanshinone II A to SW620 colon carcinoma cells. Flow cytometry (FC) was used to assess apoptosis. Transwell assay was performed to assess invasion in vitro, and the wound-healing assay was applied to assess migration. Western blot analysis was performed to evaluate translation of mTOR, while RT-PCR was carried out to assess transcription of VEGF.ResultsCCK-8 assay showed that tanshinone II A inhibited SW620 proliferation in comparison to the control group subsequent to 24 h, 48 h, and 72 h (P<0.001). FC revealed that tanshinone II A promoted SW620 apoptosis (P<0.001). The cell migration test revealed that the migration index of cells receiving tanshinone II A decreased. mTOR translation as well as VEGE transcription in cells receiving tanshinone II A was noticeably prohibited compared to control group (P<0.001).ConclusionsTanshinone II A is able to inhibit proliferation and migration of human colon cancer SW620 cells and promoted cell death. Its mechanism may be by downregulation of mTOR protein and VEGF mRNA.

Corrigendum to the author’s birth year of the article entitled “Involvement of G1/S checkpoint regulators during photodynamic-therapy-mediated cell cycle arrest in human colon carcinoma SW480 cells” published in Shijie Huaren Xiaohua Zazhi 2004; 12(5): 1048-1052

Corrigendum to the author’s birth year of the article entitled “Involvement of G1/S checkpoint regulators during photodynamic-therapy-mediated cell cycle arrest in human colon carcinoma SW480 cells” published in Shijie Huaren Xiaohua Zazhi 2004; 12(5): 1048-1052

Cytotoxic and Apoptotic Activities of Prunus spinosa Trigno Ecotype Extract on Human Cancer Cells.

The aim of this work was to demonstrate that a natural compound, not-toxic to normal cells, has cytotoxic and sensitizing effects on carcinoma cells, with the final goal of combining it with chemotherapeutic drugs to reduce the overall dose. Prunus spinosa Trigno ecotype (PsT) drupe extract with a nutraceutical activator complex (NAC) made of amino acids, vitamins and mineral salt blends, has shown in vitro anticancer activity. The cytotoxic effect of (PsT + NAC)® has been evaluated on human cancer cells, with an initial screening with colorectal, uterine cervical, and bronchoalveolar cells, and a subsequent focus on colon carcinoma cells HCT116 and SW480. The viability reduction of HCT116 and SW480 after treatment with (PsT 10 mg/mL + NAC)® was about 40% (p < 0.05), compared to control cells. The cell’s survival reduction was ineffective when the drug vehicle (NAC) was replaced with a phosphate buffer saline (PBS) or physiological solution (PS). The flow cytometry evaluation of cancer cells’ mitochondrial membrane potential showed an increase of 20% depolarized mitochondria. Cell cycle analysis showed a sub G1 (Gap 1 phase) peak appearance (HCT116: 35.1%; SW480: 11.6%), indicating apoptotic cell death induction that was confirmed by Annexin V assay (HCT116: 86%; SW480: 96%). Normal cells were not altered by (PsT + NAC)® treatments.

Abstract 182: A new anticancer agent derived from decenoic acid for the treatment of colon cancer

Abstract The final goal of the current study is to create a promising lead compound that targets human colon carcinoma. After discovering in vitro antiproliferative activity of a naturally occurring compound, 10-hydroxy-2-decenoic acid, we designed and synthesized over 150 derivatives. The initial screening resulted in the discovery of three primary lead compounds, we then proceeded with modifications in side chain and eventually obtained a second-generation lead compound, named PPI (patented in 2014), whose IC50 was 0.3 μM. We focused on the transcription factor STAT3 that is able to drive carcinogenesis, apoptosis, and angiogenesis-related genes. In silico docking simulation exhibited that PPI can bind to SH2 domain of STAT3, suggesting that this drug blocks dimerization of STAT3, thereby PPI inactivates function of STAT3. PPI inhibited transcriptional activity of STAT3 when SW480, SW837, and HT29 human colon carcinoma cells were treated with 3.7 to 5 μM PPI for 24h. Flowcytometry analysis indicated that when HT29 cells were treated with 1 μM PPI, the percentage of cells in G1 increased by 5% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. The results indicate that PPI causes carcinoma cells to arrest in the G1 phase. When HT29 cells were treated with 10 μM PPI, subG1 fraction and DNA fragmentation were seen, indicating induction of apoptosis. We then performed western blot analysis to determine whether treatment of carcinoma cells with PPI alters cellular levels of the G1 cell cycle control protein cyclin D1 and apoptosis/angiogenesis-related molecules. When HT29 cells were treated with PPI for 24 to 48h, there was a marked decrease in the levels of expression of the Bcl-2, Bcl-xL, and VEGF proteins and a marked increase in the levels of expression of the cleaved caspase 3, 7, 8, 9, and PARP proteins. PPI also inhibited expression levels and nuclear tanslocation of pSTAT3. To see if PPI exerts preventive effect on tumor promotion, F344 rats received sc injections of a carcinogen azoxymethane and then treated with soybean oil or PPI. There was a dose dependent decrease in multiplicity of aberrant crypt foci (ACF) in the colon. PPI inhibited the occurrence of larger ACF consisting of more than 4 aberrant crypts, indicating inhibitory effect of PPI on tumor promotion. In a mouse xenograft model, PPI decreased the size and number of blood vessels in the tumor. All mice survived without causing significant body weight loss during experiment. No histopathological abnormality was found in any of organ site. To confirm the effects of PPI on angiogenesis, we performed chorioallantoic membrane assays and found a dose-dependent decrease in average number of blood vessels. Inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis, and cell cycle progression and eventually contributes to PPI-induced growth inhibition. Citation Format: Masumi Suzui, Saeko Ando, Harutoshi Matsumoto, Katsumi Fukamachi, Mitsuru Futakuchi, Naoki Yoshimi. A new anticancer agent derived from decenoic acid for the treatment of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2017-182

Abstract LB-151: The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizer inducing tumor cell death through the spindle assembly checkpoint. BAL101553 has anti-cancer activity in diverse tumor models refractory to standard therapies, and is currently undergoing Phase 1/2a evaluation in advanced cancer patients with IV (weekly) and oral (daily) dosing schedules. Here, the vascular disruption activity of BAL101553 was evaluated in the context of administration route and implications for combinations with VEGF-targeted therapies. Methods: MTD IV (day[d] 1) and oral (d1 &amp; d1 - 4) dosing regimens were tested in H460 NSCLC xenografts. Tumors were analyzed by IHC for proliferation (Ki67) and vascularization (CD31, lectin perfusion). Drug combinations were performed in Pgp-overexpressing, colon carcinoma SW480 xenografts using BAL101553 IV 1xweek (21.3 mg/kg) or oral daily (15 mg/kg) and bevacizumab IP d1 &amp; 4 (5 mg/kg). Results: H460 tumor-bearing mice received once weekly (IV 25 mg/kg; oral 60 mg/kg; high Cmax regimens) or daily (oral 25 mg/kg; low Cmax, high AUC) BAL101553 regimens which elicited equipotent anti-tumor effects (ΔT/Cs = 26-34%). Although all treatments elicited a similar anti-proliferative response (% reduction of mean Ki67 signal+/-SD=10+/-1.2; p&amp;lt;0.05 vs. controls), tumor vessel density was most profoundly reduced by high Cmax regimens. Interestingly, daily-oral treatment (low Cmax, high AUC) had reduced tumor anti-vascular activity, while presenting equivalent anti-proliferative and anti-tumor efficacy. In vitro assays against tumor cell lines showed that sub-cytotoxic BAL27862 concentrations reduced HIF-1α protein levels in normoxic and hypoxic conditions, diminishing VEGF-expression and secretion, indicating direct interference of the drug on hypoxic adaptation and endothelial tube formation. Based on these data, combination of the anti-VEGF antibody bevacizumab with BAL101553 was assessed in the paclitaxel refractory SW480 tumor model, using MTD BAL101553 dosing (IV weekly &amp; oral daily). All monotherapies induced an anti-tumor response (ΔT/Cs bevacizumab: 35%; IV BAL101553 22%; oral BAL101553: 53%). However, bevacizumab combined with IV or oral BAL101553 was superior to single agents (ΔT/C: 6% and 17%, resp., p&amp;lt;0.05 vs. control) with IV combination associated with the least functional tumor vasculature. Conclusions: BAL101553 targets tumor cell proliferation, tumor hypoxic adaptation and vascularization. The latter is Cmax-driven and can be attenuated by daily oral dosing, providing an alternative pharmacodynamic endpoint with implications for clinical treatment strategies. IV and orally administered BAL101553 both elicit superior anti-tumor responses in combination with anti-VEGF therapy. Citation Format: Ashish Sharma, Felix Bachmann, Angela Broggini-Tenzer, Matthias Guckenberger, Heidi Lane, Martin N. Pruschy. The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2017-LB-151

Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established.

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.

(3′R)-hydroxytabernaelegantine C: A bisindole alkaloid with potent apoptosis inducing activity in colon (HCT116, SW620) and liver (HepG2) cancer cells

Ethnopharmacological relevanceTabernaemontana elegans Stapf. (Apocynaceae) is a medicinal plant traditionally used in African countries to treat cancer. Aims of the studyTo discover new apoptosis inducing lead compounds from T. elegans and provide scientific validation of the ethnopharmacological use of this plant. Materials and methodsThrough fractionation, (3′R)-hydroxytaberanelegantine C (1), a vobasinyl-iboga bisindole alkaloid, was isolated from a cytotoxic alkaloid fraction of the methanol extract of T. elegans roots. Its structure was identified by spectroscopic methods, mainly 1D and 2D NMR experiments. Compound 1 was evaluated for its ability to induce apoptosis in HCT116 and SW620 colon and HepG2 liver carcinoma cells. The cell viability of compound 1 was evaluated by the MTS and lactate dehydrogenase (LDH) assays. Induction of apoptosis was analyzed through Guava ViaCount assay, by flow cytometry, caspase-3/7 activity assays and evaluation of nuclear morphology by Hoechst staining. To determine the molecular pathways elicited by 1 exposure, immunoblot analysis was also performed. Results(3′R)-hydroxytaberanelegantine C (1) displayed strong apoptosis induction activity as compared to 5-fluorouracil (5-FU), the most used anticancer agent in colorectal cancer treatment. In the MTS assay, compound 1 exhibited IC50 values similar or lower than 5-FU in the three cell lines tested. The IC50 value of 1 was also calculated in CCD18co normal human colon fibroblasts. The lactate dehydrogenase assay showed increased LDH release by compound 1, and the Guava ViaCount assay revealed that 1 significantly increased the incidence of apoptosis to a further extent than 5-FU. Moreover, the induction of apoptosis was corroborated by evaluation of nuclear morphology by Hoechst staining and caspase-3/7 activity assays of 1 treated cells. As expected, in immunoblot analysis, compound 1 treatment led to poly(ADP-ribose) polymerase cleavage. This was accompanied by decreased anti-apoptotic proteins Bcl-2 and XIAP steady state levels in all three cancer cell lines tested. ConclusionsCompound 1 showed remarkable induction of apoptosis in HCT116, SW620 and HepG2 cells. Together, the results suggest that compound 1 is a promising lead structure for inducing apoptosis.

Co-expression of autophagic markers following photodynamic therapy in SW620 human colon adenocarcinoma cells

Photodynamic therapy (PDT) is a minimally invasive cancer treatment. It involves the combination of a photosensitizer and light of a specific wavelength to generate singlet oxygen and other reactive oxygen species that lead to tumor cell death. Autophagy is one of the pathways that tumor cells undergo during photodamage and it is common in photodynamic therapy. The aim of this study was to examine the effect of in vitro PDT on the expression of autophagy-related proteins, autophagy related 7 (Atg7), light chain 3 (LC3) and Beclin-1. Human SW620 colon carcinoma cells were treated with 5-aminolevulinic acid (ALA)-based PDT at a dose of 3 mM. The irradiation was performed using 4.5 J/cm2 total light and a fluence rate of 60 mW/cm2. Autophagy was evaluated by immunocytochemistry using specific antibodies to Atg7, Beclin-1 and LC3. The evaluation was repeated at several time points (0, 4, 8 and 24 h) following irradiation. The induction of autophagy was observed directly following the 5-ALA-mediated PDT procedure with the strongest expression of autophagy-related proteins at 4 and 8 h after irradiation as demonstrated using immunocytochemistry. It was characterized by significantly increased expression of Beclin-1, Atg7 and LC3. To the best of our knowledge this is the first study to analyze Beclin-1, Atg7 and LC3 expression in a PDT-related experiment. This study enhances the understanding of the role of autophagy in PDT, which may contribute to better and more effective tumor responses to this therapy.

Vimentin, colon cancer progression and resistance to butyrate and other HDACis.

Dietary fibre protects against colorectal cancer (CRC) most likely through the activity of its fermentation product, butyrate. Butyrate functions as a histone deacetylase inhibitor (HDACi) that hyperactivates Wnt signalling and induces apoptosis of CRC cells. However, individuals who consume a high‐fibre diet may still develop CRC; therefore, butyrate resistance may develop over time. Furthermore, CRC cells that are resistant to butyrate are cross‐resistant to clinically relevant therapeutic HDACis, suggesting that the development of butyrate resistance in vivo can result in HDACi‐resistant CRCs. Butyrate/HDACi‐resistant CRC cells differ from their butyrate/HDACi‐sensitive counterparts in the expression of many genes, including the gene encoding vimentin (VIM) that is usually expressed in normal mesenchymal cells and is involved in cancer metastasis. Interestingly, vimentin is overexpressed in butyrate/HDACi‐resistant CRC cells although Wnt signalling is suppressed in such cells and that VIM is a Wnt activity‐targeted gene. The expression of vimentin in colonic neoplastic cells could be correlated with the stage of neoplastic progression. For example, comparative analyses of LT97 microadenoma cells and SW620 colon carcinoma cells revealed that although vimentin is not detectable in LT97 cells, it is highly expressed in SW620 cells. Based upon these observations, we propose that the differential expression of vimentin contributes to the phenotypic differences between butyrate‐resistant and butyrate‐sensitive CRC cells, as well as to the differences between early‐stage and metastatic colorectal neoplastic cells. We discuss the hypothesis that vimentin is a key factor integrating epithelial to mesenchymal transition, colonic neoplastic progression and resistance to HDACis.

C-MET inhibition enhances the response of the colorectal cancer cells to irradiation in vitro and in vivo.

The aim of the present study was to investigate the effect of hepatocyte growth factor receptor (c-MET) inhibition on the viability of colon cancer cells and xenografts exposed to irradiation using short hairpin (sh)RNA or the c-MET inhibitor PHA665752. The underlying mechanisms were also investigated. Human colorectal adenocarcinoma HT-29 cells were infected with a lentivirus expressing shRNAs against c-MET and were irradiated at 0, 2, 4, 6 and 8 Gy. The viability of the cells was assessed by alamarBlue® assays. Mice bearing human colon carcinoma SW620 xenografts were randomly selected to receive 2.5% dimethyl sulfoxide (DMSO), 25 mg/kg PHA665752 intraperitoneally once every 2 days for 3 weeks, irradiation at 10 Gy, or 25 mg/kg PHA665752 intraperitoneally once every 2 days for 3 weeks followed 24 h later by irradiation at 10 Gy. The mean tumor volume (MTV) was measured. The apoptotic rate of cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays, and double stranded break marker antibody γ-H2AX and hypoxia inducible factor (HIF)-1α expression was examined by immunohistochemistry. alamarBlue assays revealed that c-MET downregulation by shRNA markedly accentuated the irradiation-induced reduction in the viability of HT-29 cells compared with HT-29 cells irradiated at the same doses (P<0.05). A combination of irradiation and PHA665752 caused an additional reduction in the MTV (382.8±42.4 mm3; P<0.01 vs. irradiation and PHA665752, 998.0±180.6 and 844.8±190.0 mm3, respectively). TUNEL assays revealed that irradiation and PHA665752 alone caused significant apoptosis of the SW620 cells in the tumor xenografts (P<0.01 vs. DMSO). The apoptotic index in the tumor xenografts of mice treated with a combination of irradiation and PHA665752 was significantly increased compared with mice treated with either agent alone (P<0.01). The combination of irradiation and PHA665752 was also associated with a marked increase in γ-H2AX levels and a significant decrease in HIF-1α expression in the xenografts (P<0.01). In conclusion, c-MET inhibition sensitizes colorectal cancer cells to irradiation by enhancing the formation of DNA double strand breaks and possibly alleviating tumor hypoxia.

In situ targeting TEM8 via immune response and polypeptide recognition by wavelength-modulated surface plasmon resonance biosensor.

There is an increasing interest in real-time and in situ monitoring of living cell activities in life science and medicine. This paper reports a whole cell sensing protocol over the interface of Au film coupled in a wavelength-modulated surface plasmon resonance (WMSPR) biosensor. With dual parabolic mirrors integrated in the sensor, the compact and miniaturized instrument shows satisfactory refractive index sensitivity (2220 nm/RIU) and a high resolution of resonance wavelength shift of 0.3 nm to liquid samples. The affinity interactions between the biomarker of human tumor endothelial marker 8 (TEM8) and antibody (Ab) or specific polypeptide (PEP) were firstly introduced to WMSPR biosensor analysis. Both the interaction events of Ab-cell and PEP-cell over the Au film interface can be recognized by the sensor and the balance time of interactions is about 20 min. The concentration range of Ab for quantitative monitoring of the TEM8 expression on human colon carcinoma SW620 cells was investigated. The present low-cost and time-saving method provides a time resolution of binding specificity between Ab/PEP and TEM8 for real-time analysis of antigen on living tumor cell surface.

Abstract 5461: Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance

Abstract Due to their enhanced proliferation rate, tumor cells are highly susceptible for ribonucleotide pool disruption. Therefore, several thiosembicarbazone-based ribonucleotide reductase inhibitors have been developed, out of which Triapine is the most promising candidate. Triapine is currently tested in clinical phase I and II studies and shows promising effects in haematological diseases. Unfortunately, triapine is rather ineffective in solid cancer types. However, the mechanism underlying this failure is yet not fully understood. One possible theory could be development of rapid acquired resistance against the chemotherapeutic drug. To investigate this issue we generated a triapine-resistant cell line (SW480/tria) by stepwise selection of human colon carcinoma SW480 cells. SW480/tria cells displayed a broad cross-resistance especially against several well-known ABCB1 substrates (e.g. vincristine). In accordance, strong ABCB1 expression was detected in SW480 Tria cells. The induction of ABCB1 was not based on gene amplification but on hypomethylation of the ABCB1 promoter. As a next step, rhodamine-123 and ATPase assays were performed to investigate if triapine does interact with the ABCB1 transport function. However, no ABCB1 inhibitory potential of triapine could be found. Further on, combined treatment in SW480/tria cells with triapine and the known ABCB1 inhibitors cyclosporine A (CSA) and verapamil did not restore triapine sensitivity. In addition, the intracellular triapine levels were comparable between parental and triapine-selected SW480 cell lines. Moreover, increased ABCB1 expression was found to be a consequence of triapine stress-induced PKC activation. This suggests that the strong ABCB1 expression of SW480/tria cells might rather be a consequence of triapine-induced cell stress than a major driver for specific triapine resistance. Taken together our data reveal that, although triapine is only a weak substrate for ABCB1, strong ABCB1 expression is induced by short- and long-term triapine exposure resulting in distinct cross resistance against other anticancer drugs. This definitely has to be considered in the selection of combination schemes and in second line therapy following triapine failure. Citation Format: Walter Miklos, Karla Pelivan, Christian Kowol, Rita Dornetshuber-Fleiss, Melanie Spitzwieser, Margit Cichna-Markl, Gunda Köllensperger, Bernhard Keppler, Walter Berger, Petra Heffeter. Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5461. doi:10.1158/1538-7445.AM2015-5461