HT29 Colorectal Carcinoma Cells Research Articles

In Vitro Anticancer and Proapoptotic Activities of Steroidal Glycosides from the Starfish Anthenea aspera.

New marine glycoconjugates—the steroidal glycosides designated as anthenosides V–X (1–3)—and the seven previously known anthenosides E (4), G (5), J (6), K (7), S1 (8), S4 (9), and S6 (10) were isolated from the extract of the tropical starfish Anthenea aspera. The structures of 1–3 were elucidated by extensive NMR and ESIMS techniques. Glycoside 1 contains a rare 5α-cholest-8(14)-ene-3α,7β,16α-hydroxysteroidal nucleus. Compounds 2 and 3 were isolated as inseparable mixtures of epimers. All investigated compounds (1–10) at nontoxic concentrations inhibited colony formation of human melanoma RPMI-7951, breast cancer T-47D, and colorectal carcinoma HT-29 cells to a variable degree. The mixture of 6 and 7 possessed significant anticancer activity and induced apoptosis of HT-29 cells. The molecular mechanism of the proapoptotic action of this mixture was shown to be associated with the regulation of anti- and proapoptotic protein expression followed by the activation of initiator and effector caspases.

LC-MS guided isolation of sinodamines A and B: Chimonanthine-type alkaloids from the endangered ornamental plant Sinocalycanthus chinensis

Two previously undescribed chimonanthine-type [sinodamines A and B] and five related known dimeric tryptamine-derived alkaloids were isolated and characterized from the leaves of the endangered ornamental plant Sinocalycanthus chinensis under the guidance of LC-MS detection and dereplication analyses, along with conventional isolation procedures. Their structures were established on the basis of spectroscopic methods and chemical transformations. Sinodamine A can be regarded as the naturally occurring N-oxide derivative of its pseudo-mesomer sinodamine B. An acid-catalyzed Meisenheimer rearrangement from sinodamine A to its oxazine-form with a final equilibrium of 1:2 was observed by monitoring their NMR spectra. (−)-Folicanthine showed significant cytotoxicity against human lung carcinoma A549 and colorectal carcinoma HT29 cells, with IC50 values of 7.76 and 6.16 μM, respectively.

Anti-Proliferative and Apoptotic Effects of Polygonum cuspidatum and its Bioactive Compound, Emodin, in Colorectal Carcinoma HT-29 cells

Antiproliferative and apoptotic effects of ethyl acetate from the root of Polygonum cuspidatum (EAPC) and its bioactive compound, emodin, in colorectal adenocarcinoma HT-29 cells were investigated. HPLC analysis of major compounds, cell viability by MTT assay, the Comet assay, determination of reactive oxygen species (ROS) levels, apoptosis assay by flow cytometry and Western blot analysis were conducted. The results show significant antiproliferative effects of EAPC, IC50 = 141.4 μg/mL, and emodin isolated from EAPC, IC50 = 73.0 μg/mL, on HT-29 cells. HT-29 cells which were exposed to EAPC and emodin exerted apoptosis, after annexin V FITC/PI staining. EAPC (at 200 μg/mL) and emodin (at 100 μg/mL) increase DNA damage in HT-29 cells by 75.6% and 67.8%, respectively, supporting that apoptosis occurred when the cells were treated with EAPC and emodin. Cells treated with EAPC and emodin generated intracellular ROS. In addition, emodin at tested doses of 25-100 μg/mL suppressed nuclear factor κB (NFκB) and enhanced activator protein 1 (AP-1), which are involved in both proliferation and apoptosis events. EAPC and emodin demonstrated significant anti-proliferation of HT-29 cells through their pro-oxidant activity and contribution to oxidative stress, which in turn leads to cell death. EPAC and emodin inhibited HT-29 cell growth via apoptosis and could be considered as potential candidates for anti-colorectal cancer treatment.

Opposing effects of low versus high concentrations of water soluble vitamins/dietary ingredients Vitamin C and niacin on colon cancer stem cells (CSCs).

Colorectal cancer is one of the global causes of cancer deaths. Cancer stem cells (CSCs) inside the tumour niche responsible for metastasis and relapses, and hence need to be targeted for cancer therapeutics. Although dietary fibre and lifestyle changes have been recommended as measures for colorectal cancer prevention, no such recommendations are available for using water soluble vitamins as prophylaxis measure for colorectal cancers. High dose of Vitamin C has been proven to selectively kill colon cancer cells having BRAF and KRAS mutations by inducing oxidative stress. In this study, we show for the first time the opposing effects of the low and high dose of Vitamin C and vitamin B3 on colon CSCs isolated from HT-29 and HCT-15 colorectal carcinoma cell lines. At small doses, both of these vitamins exerted a cell proliferative effect only on CSCs, while there was no change in the proliferation status of non-stem cancer cells and wild-type (WT) populations. On the other hand, the death effects induced by high doses of Vitamin C and B3 were of the order of 50-60% and ∼30% on CSCs from HT-29 and HCT15, respectively. Interestingly, the control fibroblast cell line (NIH3T3) was highly refractory all the tested concentrations of Vitamin C and B3, except for the highest dose - 10,000 μg of Vitamin C that induced only 15% of cell death. Hence, these results indicate the future scope of use of therapeutic doses of Vitamin C and B3 especially in patients with advanced colorectal cancer.

Microenvironment and Dose-Delivery-Dependent Response after Exposure to Ionizing Radiation in Human Colorectal Cancer Cell Lines.

A significant body of knowledge about radiobiology is based on studies of single dose cellular irradiation, despite the fact that conventional clinical applications using dose fractionation. In addition, cellular radiation response strongly depends on cell-cell and cell-extracellular matrix (ECM) interactions, which are poorly established in cancer cells grown under standard 2D cell culture conditions. In this study, we investigated the response of human colorectal carcinoma (CRC) DLD1 and HT29 cell lines, bearing distinct p53 mutations, to a single 2 or 10 Gy dose or fractionated 5 × 2 Gy doses of radiation using global transcriptomics analysis. To examine cellular response to radiation in a cell-ECM-interaction-dependent manner, CRC cells were grown under laminin-rich ECM 3D cell culture conditions. Microarray data analysis revealed that, overall, a total of 1,573 and 935 genes were differentially expressed (fold change >1.5; P < 0.05) in DLD1 and HT29 cells, respectively, at 4 h postirradiation. However, compared to a single dose of radiation, fractionated doses resulted in significantly different transcriptomic response in both CRC cell lines. Furthermore, pathway enrichment analysis indicated that p53 pathway and cell cycle/DNA damage repair or immune response functional categories were most significantly altered in DLD1 or HT29 cells, respectively, after fractionated irradiations. Novel observations of radiation-response-mediated activation of pro-survival pathways in CRC cells grown under lr-ECM 3D cell culture conditions using fractionated doses provide new directions for the development of more efficient radiotherapy strategies. Our results also indicated that cell line specific radiation response with or without activation of the conventional p53 pathway is ECM dependent, suggesting that the ECM is a key component in cellular radiation response.

Spermidine/spermine N1-acetyltransferase regulates cell growth and metastasis via AKT/β-catenin signaling pathways in hepatocellular and colorectal carcinoma cells.

Hepatocellular carcinoma (HCC) and colorectal cancer (CRC) are among the most common cancers across the world. Therefore, identifying the potential molecular mechanisms that promote HCC and CRC progression and metastasis are urgently needed. Spermidine/spermine N1-acetyltransferase (SSAT) is a catabolic enzyme that acetylates the high-order polyamines spermine and spermidine, thus decreasing the cellular content of polyamines. Several publications have suggested that depletion of intracellular polyamines inhibited tumor progression and metastasis in various cancer cells. However, whether and how SSAT regulates cell growth, migration and invasion in hepatocellular and colorectal carcinoma cells remains unclear. In this study, depletion of polyamines mediated by SSAT not only attenuated the tumor cell proliferation but also dramatically inhibited cell migration and invasion in hepatocellular and colorectal carcinoma cells. Subsequent investigations revealed introduction of SSAT into HepG2, SMMC7721 hepatocellular carcinoma cells and HCT116 colorectal carcinoma cells significantly suppressed p-AKT, p-GSK3β expression as well as β-catenin nuclear translocation, while inhibition of GSK3β activity or exogenous polyamines could restore SSAT-induced decreases in the protein expression of p-AKT, p-GSK3β and β-catenin. Conversely, knockdown of SSAT in Bel7402 hepatocellular carcinoma cells and HT-29 colorectal carcinoma cells which expressed high levels of SSAT endogenously significantly promoted the expression of p-AKT, p-GSK3β as well as β-catenin nuclear translocation. Taken together, our results indicated depletion of polyamines by SSAT significantly inhibited cell proliferation, migration and invasion through AKT/GSK3β/β-catenin signaling pathway in hepatocellular carcinoma and colorectal cancer cells.

Targeting Enox1 in tumor stroma increases the efficacy of fractionated radiotherapy.

The goal of this investigation was to clarify the question of whether targeting Enox1 in tumor stroma would synergistically enhance the survival of tumor-bearing mice treated with fractionated radiotherapy. Enox1, a NADH oxidase, is expressed in tumor vasculature and stroma. However, it is not expressed in many tumor types, including HT-29 colorectal carcinoma cells. Pharmacological inhibition of Enox1 in endothelial cells inhibited repair of DNA double strand breaks, as measured by γH2AX and 53BP1 foci formation, as well as neutral comet assays. For 4 consecutive days athymic mice bearing HT-29 hindlimb xenografts were injected with a small molecule inhibitor of Enox1 or solvent control. Tumors were then administered 2 Gy of x-rays. On day 5 tumors were administered a single ‘top-up’ fraction of 30 Gy, the purpose of which was to amplify intrinsic differences in the radiation fractionation regimen produced by Enox1 targeting. Pharmacological targeting of Enox1 resulted in 80% of the tumor-bearing mice surviving at 90 days compared to only 40% of tumor-bearing mice treated with solvent control. The increase in survival was not a consequence of reoxygenation, as measured by pimonidazole immunostaining. These results are interpreted to indicate that targeting of Enox1 in tumor stroma significantly enhances the effectiveness of 2 Gy fractionated radiotherapy and identifies Enox1 as a potential therapeutic target.

Extracellular Matrix-dependent Pathways in Colorectal Cancer Cell Lines Reveal Potential Targets for Anticancer Therapies.

Cancer cells grown in a 3D culture are more resistant to anticancer therapy treatment compared to those in a monolayer 2D culture. Emerging evidence has suggested that the key reasons for increased cell survival could be gene expression changes in cell-extracellular matrix (ECM) interaction-dependent manner. Global gene-expression changes were obtained in human colorectal carcinoma HT29 and DLD1 cell lines between 2D and laminin-rich (lr) ECM 3D growth conditions by gene-expression microarray analysis. The most significantly altered functional categories were revealed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The microarray data revealed that 841 and 1190 genes were differentially expressed in colorectal carcinoma DLD1 and HT29 cells. KEGG analysis indicated that the most significantly altered categories were cell adhesion, mitogen-activated protein kinase and immune response. Our results indicate altered pathways related to cancer development and progression and suggest potential ECM-regulated targets for the development of anticancer therapies.

Enhanced ADCC and NK Cell Activation of an Anticarcinoma Bispecific Antibody by Genetic Insertion of a Modified IL-15 Cross-linker.

Previously, we constructed a bispecific NK-cell-engager (BiKE) bearing single-chain variable fragments (scFv) against CD16 on NK cells and EpCAM on tumor cells. This BiKE facilitated antigen-specific antibody-dependent cell-mediated cytotoxicity (ADCC) but did not induce NK cell expansion. We incorporated a modified interleukin-15 cross-linker to create a trispecific construct (TriKE) in order to improve activation, proliferation, and survival of NK cells. Synthesis and assembly of hybrid genes encoding the TriKE was accomplished using DNA-shuffling and DNA-ligation techniques. The TriKE was tested for specificity, efficacy, proliferative capability, and cytokine profile using functional assays. The molecular modifications improved yield without compromising binding to EpCAM(+) HT-29 colorectal carcinoma cells. (51)Chromium-release and degranulation assays showed better killing rates with TriKE compared to BiKE. TriKE was more active in a variety of different carcinoma cell lines. TriKE showed the ability to stimulate expansion of CD56(+)CD3(-) NK cells. BiKE and TriKE showed enhanced but not supraphysiologic levels of cytokine secretion. 1615EpCAM TriKE drives enhanced ADCC while significantly improving proliferation, activation, and survival of NK cell effectors. The TriKE provides a selectively delivered self-sustaining signal at the NK/tumor cell synapse. Targeted cytokine stimulation, rather than systemic cytokine administration, may impact toxicity in patients rendering the TriKE a promising new off-the-shelf carcinoma therapy.

Comprehensive and Holistic Analysis of HT-29 Colorectal Cancer Cells and Tumor-Bearing Nude Mouse Model: Interactions Among Fractions Derived From the Chinese Medicine Formula Tian Xian Liquid in Effects on Human Colorectal Carcinoma.

The Chinese medicine formula Tian Xian Liquid (TXL) has been used clinically for cancer therapy in China for more than 25 years. However, the comprehensive and holistic effects of its bioactive fractions for various antitumor therapeutic effects have not been unraveled. This is the first study to scientifically elucidate the holistic effect of Chinese medicine formula for treating colon cancer, hence allowing a better understanding of the essence of Chinese medicine formula, through the comparison of the actions of TXL and its functional constituent fractions, including ethyl acetate (EA), butanol (BU), and aqueous (WA) fractions. Tissue-specific proliferative/antiproliferative effects of these fractions on human colorectal carcinoma HT-29 cells and splenocytes were studied by using the MTT assay. Their modulations on the expression of markers of antiproliferation, antimetastasis, reversion of multidrug resistance in treated HT-29 cells were examined with real-time polymerase chain reaction and Western blot analysis, and their modulations in a xenografted nude mouse model were examined by Western blot analysis. Results revealed that EA fraction slightly inhibited the proliferation of HT-29 cells, but tissue-specifically exerted the most potent antiproliferative effect on splenocytes. On the contrary, only TXL and BU fraction tissue-specifically contributed to the proliferation of splenocytes, but inhibited the proliferation of HT-29 cells. WA fraction exerted the most potent antiproliferative effect on HT-29 cells and also the strongest inhibitory action on tumor size in the nude mouse model in our previous study. In the HT-29 model, TXL and WA fraction exerted the most pronounced effect on upregulation of p21 mRNA and protein; TXL, and EA and WA fractions exerted the effect on downregulation of G1 phase cell cycle protein, cyclin D1 mRNA and protein; EA and BU fractions exerted the most prominent anti-invasive effect on anti-invasion via downregulation of MMP-1 mRNA; TXL potently reversed most multidrug resistance via downregulation of MDR-1 protein. In conclusion, the comprehensive and holistic effects of TXL were demonstrated with (a) mutual accentuation and mutual enhancement, (b) mutual counteraction and mutual suppression, and (c) mutual antagonism among the 3 constituent fractions. Moreover, the design of the present study may lead to further development of more tissue-specific effective drugs with minimal side effects for clinical use in combating carcinoma.

Insights into molecular mechanism of action of salan titanium(IV) complex with in vitro and in vivo anticancer activity

Titanium compounds, in particular, Ti(IV) based diaminobis(phenolato) “salan” complexes demonstrate high cytotoxicity towards a wide range of cancer cell lines in vitro, and still, very little is known on their mode of action. A representative salan Ti(IV) complex was tested both in vitro and in vivo on human HT-29 colorectal adenocarcinoma and A2780 ovarian carcinoma cells. Both cell lines were sensitive in vitro with A2780 demonstrating an enhanced rate of uptake and intracellular accumulation and thus an earlier response to the drug. HT-29 cells responded in vivo by impaired tumor development in nude mice. Both cell lines responded in vitro (but to a different extent) by upregulation of p53 with no apparent effect on p21 followed by cell cycle arrest, apoptosis and necrosis as demonstrated by sub-G1 cell accumulation and staining by Annexin-V and propidium iodide. Furthermore, time dependent activation of cysteine-aspartic proteases9 (caspase9) as well as some minor activation of cysteine-aspartic proteases3 (caspase3) support a direct effect on the apoptotic pathway. The differential response of the two cell lines to the salan titanium(IV) complex suggests that more than one pathway is involved in their growth regulation and thus could inhibit development of drug resistant variants.

Folates Induce Colorectal Carcinoma HT29 Cell Line Proliferation Through Notch1 Signaling

Folic acid (FA) consumption at high levels has been associated with colon cancer risk. Several mechanisms have been proposed to explain this association. The Notch signal pathway has been implicated in the regulation of cellular proliferation. Our aim was to demonstrate that high concentrations of FA or its reduced form, 5-methyltetrahydrofolic acid (5-MTHF), increase colorectal carcinoma HT29 cell proliferation through an increase of Notch1 activation and to prove if the inhibition of Notch1 activation by gamma secretase inhibitor, reduce the effect of folic acid. HT29 cells were cultured in high (400 nM), low (20 nM), or 0 nM FA or 5-MTHF concentrations during 96 h with or without DAPT (gamma secretase inhibitor). Cell proliferation was determined by the methylthiazole tetrazolium method, and Notch1-intracellular domain (NICD) was analyzed by flow cytometry. HT29 cells exposed to 400 nM FA or 5-MTHF showed higher proliferation rate than those exposed to 20 nM of FA or 5-MTHF (P < 0.01) during 96 h. NICD expression increased at higher FA or 5-MTHF concentrations compared with lower concentrations (P < 0.01). This effect on proliferation was partially reversible when we blocked Notch1 activation with the inhibitor of γ-secretase (P < 0.05).These data suggest that high concentration of FA and 5-MTHF induce HT29 cell proliferation activating Notch1 pathway.

Expression and effect of CXCL14 in colorectal carcinoma.

Chemokines are important in the proliferation and metastasis of tumors. CXCL14 is a member of the CXCL chemokine family and exhibits various expression patterns in different types of tumor, even those tumors that occur in the same type of tissue. The expression of CXCL14 and its clinical significance in colorectal carcinoma are unclear. In the present study, the expression levels of CXCL14 in colorectal carcinoma and adjacent normal tissues were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Kaplan‑Meier survival curves and the Cox regression model were applied to evaluate the clinical significance of the expression levels of CXCL14 in colorectal carcinoma compared with those in normal tissues. To investigate the effects at a cellular level, a replication‑defective lentivirus overexpressing CXCL14 was constructed and transfected into HT29 colorectal carcinoma cells. The effect of CXCL14 on the proliferation of colorectal carcinoma cells and the change in cell cycle distributions were investigated using a cell counting kit‑8 assay and flow cytometry, respectively. Results of the current study indicated that the expression levels of CXCL14 mRNA and protein in colorectal carcinoma were markedly reduced compared with levels in normal tissues (P<0.05). The clinical correlation analysis suggested that downregulation of CXCL14 expression in tumors was associated with lymph metastasis, tumor location, and clinicopathological stage (P<0.05). Kaplan‑Meier survival analysis revealed that downregulation of CXCL14 expression was correlated with a poor prognosis (P<0.01). Overexpression of CXCL14 by lentiviral transfection produced an inhibitory effect on cell proliferation by arresting the cell cycle in the G1 stage. The data of the current study suggest that CXCL14 may be involved in the development and progression of colorectal carcinoma, and may act directly as a potential cancer suppressor gene. The level of CXCL14 expression may be a valuable adjuvant parameter in predicting the prognosis of colorectal carcinoma and may be a potential therapeutic target.

Targeting carbonic anhydrase IX by nitroimidazole based sulfamides enhances the therapeutic effect of tumor irradiation: A new concept of dual targeting drugs

Background and purposeCarbonic anhydrase IX (CAIX) plays an important role in pH regulation processes critical for tumor cell growth and metastasis. We hypothesize that a dual targeting bioreductive nitroimidazole based anti-CAIX sulfamide drug (DH348) will reduce tumor growth and sensitize tumors to irradiation in a CAIX dependent manner. Material and methodsThe effect of the dual targeting anti-CAIX (DH348) and its single targeting control drugs on extracellular acidification and radiosensitivity was examined in HT-29 colorectal carcinoma cells. Tumor growth and time to reach 4× start volume (T4×SV) was monitored for animals receiving DH348 (10mg/kg) combined with tumor single dose irradiation (10Gy). ResultsIn vitro, DH348 reduced hypoxia-induced extracellular acidosis, but did not change hypoxic radiosensitivity. In vivo, DH348 monotherapy decreased tumor growth rate and sensitized tumors to radiation (enhancement ratio 1.50) without systemic toxicity only for CAIX expressing tumors. ConclusionsA newly designed nitroimidazole and sulfamide dual targeting drug reduces hypoxic extracellular acidification, slows down tumor growth at nontoxic doses and sensitizes tumors to irradiation all in a CAIX dependent manner, suggesting no “off-target” effects. Our data therefore indicate the potential utility of a dual drug approach as a new strategy for tumor-specific targeting.

Antitumor effect of capsaicin on colorectal carcinoma xenograft in nude mice

To evaluate the effect of capsaicin on nude mice xenografted with colorectal carcinoma cells, and to explore its mechanism of action. A nude mouse model of colorectal cancer was established by subcutaneous inoculation of human colorectal carcinoma HT-29 cells. Terminal deoxynucleotidyl transferase-mediated nicked labeling assay (TUNEL) was undertaken to detect the cell proliferation and apoptosis in the xenograft tissue in nude mice. Immunohistochemical (IHC) staining and Western blot were used to detect the expression of HSP27, Cyt-C and active caspase-3. The tumor growth of the groups C10 and C20 was significantly slower than that of the group NS. The integrated optical density (IOD) of both the group C5 (2532.14 ± 578.11) and group C10 (6364.03 ± 1137.98) was significantly higher than that of the group NS (760.12 ± 238.05), (P < 0.05). The integrated optical density (IOD) of the group C20 was (15743.96 ± 1855.95), significantly higher than that of the groups C10, C5 and NS (all were P < 0.01). Immunohistochemistry showed that the cytoplasmic expression of HSP27 was strongly positive in the group NS, and significantly reduced with the increasing dose of capsaicin in the treated groups. The expression of active caspase-3 and Cyt-C in the group NS was weakly positive, and was significantly increased with the increasing dose of capsaicin in the groups C5 and C10 (P < 0.05), and the expression of active caspase-3 and Cyt-C of the group C20 was significantly higher than that of the groups C5, C10 and NS (P < 0.01). Western blot analysis showed that both the expressions of HSP27 of the group C5 (0.73 ± 0.05) and the group C10 (0.41 ± 0.03) were significantly lower than that of the group NS (P < 0.05). The expression of HSP27 of the group C20 (0.22 ± 0.06) was significantly lower than that of the groups C5, C10 and NS (P < 0.01). The expressions of active-caspase-3 and Cyt-C in the group C5 were (2.57 ± 0.34) and (2.03 ± 0.38), significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C10 were (4.23 ± 0.45) and (3.13 ± 0.44), also significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C20 were (5.78 ± 0.48) and (4.92 ± 0.52), significantly higher than those of the group C5, C10 and NS (P < 0.01). TUNEL analysis showed that there was a significant difference of cell apoptosis in comparison of each two groups. The higher dose of capsaicin was used, the more apoptosis was observed. Capsaicin can significantly inhibit the tumor growth and induce cell apoptosis in the colorectal carcinoma xenograft in nude mice. Its mechanism of action is possibly related with the down-regulation of HSP27 expression and up-regulation of expression of active caspase-3 and Cyt-C in the colorectal carcinoma xenograft in nude mice.

Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of 125I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of 125I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.

The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: Evidence of hypoxia-induced potentially lethal damage

Purpose: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill.Materials and methods: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N2 gas containing 0, 0.1 or 0.5% O2. Cell survival was determined by colony formation assay immediately after 0–96 h hypoxia. To reoxygenate medium were replaced fresh following 48 or 72 h in hypoxia and cells were incubated in aerobic environment for 2–24 h before survival assay.Results: When incubated in hypoxia, cells lost reproductive capability ∼ exponentially as a function of time in hypoxia, and depending on the O2 concentration. DNKu70 rendered cells more prone to hypoxia-induced cell-kill. Following reoxygenation cell survival increased rapidly but without detectable cell proliferation during first 24 hours. This evinced hypoxia-induced potentially lethal damage (PLD) that was repairable upon reoxygenation. DNKu70 did not significantly inhibit this repair.Conclusion: Hypoxia-induced cell lethality was facilitated by DNKu70, but substantially repaired upon reoxygenation. This may have negative impact on the effect of reoxygenation in cancer therapy.

The role of COX‐2/PGE2 in gossypol‐induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.

Synthesis of Novel Tadalafil Analogues and their Evaluation as Phosphodiesterase Inhibitors and Anticancer Agents

Two closely related series of novel beta-carboline derivatives, electronically similar to tadalafil (CAS 171596-29-5), were synthesized and evaluated for their inhibitory effects upon phosphodiesterase 5 (PDE5) and phosphodiesterase 11 (PDE11) and their in vitro tumor cell growth inhibitory activity versus HT29 colorectal carcinoma cell line. Interestingly, some of the synthesized compounds showed growth inhibitory properties that appear to be associated with their ability to inhibit PDE5. Moreover, the PDE5 inhibition seems relevant to the stereochemical aspects of the compounds.

Abstract C34: The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Abstract Our previous study has shown that gossypol (GOS) exhibited the potent cytotoxic effect via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase 2 (COX-2)/prostaglandin E2 on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma cells COLO205 in according with inducing COX-2 protein/PGE2 production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase C (PKC) inhibitors including ST, GF, and H7, characterized by an occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE2 production in COLO205 cells. Addition of COX activity inhibitors including NS398, aspirin (AS), diclofenac (DI), and indomethacin (IN) suppressed TPA protection of GOS-induced apoptosis with decreased PGE2 production in COLO205 cells. Application of PGE2, but not it analogues PGD2, PGJ2, and PGF2, protected COLO205 cells from GOS-induced DNA ladders, and the EP1 receptor agonist 17PT-PGE2 mimicked the protection by PGE2, whereas the selective EP2 receptor agonist butaprostol (BUT), EP3 receptor agonist sulprostol (SUL), and EP4 receptor agonist PGE1 alcohol (PGE1) showed no significant effect on GOS-induced apoptosis in COLO205 cells. PGE2 protection of GOS-induced apoptosis was reversed by adding a selective EP1 receptor antagonist SC-19220. Furthermore, GOS had less apoptotic effect on HT-29 colorectal carcinoma cells which expressed higher level of endogenous COX-2 protein than COLO205 cells, and decreased COX-2 protein level via COX-2 siRNA significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection enhanced GOS-induced cell death by MTT assay. The notion of COX-2/PGE2 activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and combination of GOS and COX-2 inhibitors to treat colon carcinoma reserved clinical potential for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C34.