Human Colon Carcinoma SW480 Cells Research Articles

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

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

Structural behaviour and gene delivery in complexes formed between DNA and arginine-containing peptide amphiphiles.

We describe in depth the structure of complexes formed between DNA and two classes of arginine-containing peptide amphiphiles, namely, the lipopeptide PRW-C16 (P = proline, R = arginine, W = tryptophan, C16 = C16 : 0 alkyl chain) and the bolaamphiphile RFL4FR (R = arginine, F = phenylalanine, L = leucine). A combination of X-ray and neutron scattering provided unprecedented insights into the local structure of these complexes. Lipopeptide-based complexes self-assembled into layered structures with large-scale fractal features, hosting DNA in the interstices. Bola-amphiphile scaffolds were characterized by planar structures with DNA strands presumably sandwiched in-between peptide nanotapes. Importantly, complexation did not affect the structural integrity of DNA in either of the two complexes. The bolaamphiphile conjugates displayed high levels of molecular ordering in contrast to the liquid-crystalline features observed in lipopeptide assemblies. Peptide-DNA complexes were assessed for their potential as a means to deliver the reporter vector pEGFP-N1 into SW480 human colon carcinoma cells. Successfully transfected cells expressed green fluorescent protein. The potentiating effect of PRW-C16 on the cellular uptake of ectopic DNA was found to be much greater than that observed with RFL4FR. In contrast to the bolaamphiphile-based conjugate, the liquid-crystalline nature of the lipopeptide complex is likely to play a key role in DNA release and transfection efficiency since these weakly bound structures require lower energy expenditure during disassembly and load release.

Dequalinium blocks macrophage-induced metastasis following local radiation

A major therapeutic obstacle in clinical oncology is intrinsic or acquired resistance to therapy, leading to subsequent relapse. We have previously shown that systemic administration of different cytotoxic drugs can induce a host response that contributes to tumor angiogenesis, regrowth and metastasis. Here we characterize the host response to a single dose of local radiation, and its contribution to tumor progression and metastasis. We show that plasma from locally irradiated mice increases the migratory and invasive properties of colon carcinoma cells. Furthermore, locally irradiated mice intravenously injected with CT26 colon carcinoma cells succumb to pulmonary metastasis earlier than their respective controls. Consequently, orthotopically implanted SW480 human colon carcinoma cells in mice that underwent radiation, exhibited increased metastasis to the lungs and liver compared to their control tumors. The irradiated tumors exhibited an increase in the colonization of macrophages compared to their respective controls; and macrophage depletion in irradiated tumor-bearing mice reduces the number of metastatic lesions. Finally, the anti-tumor agent, dequalinium-14, in addition to its anti-tumor effect, reduces macrophage motility, inhibits macrophage infiltration of irradiated tumors and reduces the extent of metastasis in locally irradiated mice. Overall, this study demonstrates the adverse effects of local radiation on the host that result in macrophage-induced metastasis.

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

Upregulation of Toll-like receptor 2 expression in colorectal cancer infected by human cytomegalovirus.

The aim of the present study was to investigate the association between Toll-like receptor 2 (TLR2) expression and human cytomegalovirus (HCMV) in colorectal carcinoma by detecting the expression of IE1–72, TLR2, TLR4 and tumor necrosis factor (TNF)-α in colorectal carcinoma and colon adenoma samples, as well as by analyzing the mRNA levels of the proteins in colon cancer cell lines, following HCMV infection. For this study, 56 colorectal cancer and 36 colon adenoma samples were collected, and normal mucosal tissue adjacent to the tumor was used as the control. The expression of the IE1–72, TLR2, TLR4, nuclear factor (NF)-κB and TNF-α protein was detected by immunohistochemistry. Cells from the SW480 human colon carcinoma cell line were infected with HCMV. The expression of IE1–72, TLR2, TLR4, NF-κB and TNF-α mRNA was quantified at different time points prior to and following infection. The positive expression rate of IE1–72 was 44.6% (25/56) in colorectal cancer and 41.7% (15/36) in colon adenoma. These rates were significantly higher when compared with the 12.5% (7/56) observed in the normal tissues adjacent to the cancer tissues (P<0.05). The expression levels of TLR2, TLR4, NF-κB and TNF-α in colorectal cancer and adenoma were also higher than those in the control tissues. Furthermore, the expression of IE1–72 in colorectal cancer tissues was found to correlate with TLR2 and TLR4, and the correlation coefficients were 0.515 and 0.462, respectively. Following the infection of SW480 cells, the mRNA levels of TLR2 and TNF-α increased gradually from 6 h, peaked at 48 h, and then decreased gradually. No significant differences in TLR4 and NF-κB expression were identified. The results of the present study indicated that there is a specific association between HCMV and the occurrence and development of colorectal cancer, which may be facilitated by the TLR2 signaling pathway.

Periostin as a promising target of therapeutical intervention for colorectal cancer

The expression of periostin in the tissue of colorectal cancer patients and its correlation with clinical features were studied. Periostin expression was ~4 fold up-regulated in cancer tissues compared to adjacent non-cancerous tissues. Serum levels of periostin in patients were significantly elevated to 32.6 ± 10.8 ng/mL vs 20.4 ± 11.1 ng/mL in healthy volunteers. Higher preoperative serum periostin levels in patients were closely related to advanced-stage disease (stage III/IV), distant and lymph nodes metastasis. High level of periostin expression was detected in the SW480 human colon carcinoma cells, and could be down-regulated by small interfering RNAs (siRNA). The siRNA-mediated knockdown of periostin arrested the cell cycle at G0/G1 phase and induced apoptotic cell death in the SW480 cells. In conclusion, periostin is a promising prognostic and therapeutic target for colorectal cancer.

Silencing of the hTERT gene by shRNA inhibits colon cancer SW480 cell growth in vitro and in vivo.

Human telomerase reverse transcriptase (hTERT) is the key enzyme responsible for synthesizing and maintaining the telomeres on the ends of chromosomes, and it is essential for cell proliferation. This has made hTERT a focus of oncology research and an attractive target for anticancer drug development. In this study, we designed a small interfering RNA (siRNA) targeting the catalytic subunit of hTERT and tested its effects on the growth of telomerase-positive human colon carcinoma SW480 cells in vitro, as well as on the tumorigenicity of these cells in nude mice. Transient and stable transfection of hTERT siRNA into colon cancer SW480 cells suppressed hTERT expression, reduced telomerase activity and inhibited cell growth and proliferation. Knocking down hTERT expression in SW480 tumors xenografted into nude mice significantly slowed tumor growth and promoted tumor cell apoptosis. Our results suggest that hTERT is involved in carcinogenesis of human colon carcinoma, and they highlight the therapeutic potential of a hTERT knock-down approach.

Biotransformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis

Microbial transformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis CICC 3043 yielded seven metabolites. Their structures were elucidated on the basis of extensive spectroscopic analyses. R. chinensis could catalyze hydroxylation and further dehydrogenation at C-24 of 20(R)-panaxadiol, as well as hydroxylation at C-7, C-15, C-16, and C-29. Three of these compounds at 10μM could moderately inhibit growth of HepG2 human hepatocellular carcinoma cells with an inhibition rate of about 40%. Three compounds (also at 10μM) showed approximately 30% inhibition on NF-κB transcriptional activity in SW480 human colon carcinoma cells stably transfected with NF-κB luciferase reporter and induced by LPS.

Increasing the Bioavailability of RuIIIAnticancer Complexes through Hydrophobic Albumin Interactions

A series of pyridine-based derivatives of the clinically successful Ru(III)-based complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (KP1339) have been synthesized to probe the effect of hydrophobic interactions with human serum albumin (hsA) on anticancer activity. The solution behavior and protein interactions of the new compounds were characterized by using electron paramagnetic resonance (EPR) and UV/Vis spectroscopy. These studies have revealed that incorporation of hydrophobic substituents at the 4'-position of the axial pyridine ligand stabilizes non-coordinate interactions with hsA. As a consequence, direct coordination to the protein is inhibited, which is expected to increase the bioavailability of the complexes, thus potentially leading to improved anticancer activity. By using this approach, the lifetimes of hydrophobic protein interactions were extended from 2 h for the unsubstituted pyridine complex, to more than 24 h for several derivatives. Free complexes were tested for their anticancer activity against the SW480 human colon carcinoma cell line, exhibiting low cytotoxicity. Pre-treatment with hsA improved the solubility of every compound and led to some changes in activity. Particularly notable was the difference in activity between the methyl- and dibenzyl-functionalized complexes. The former shows reduced activity after incubation with hsA, indicating reduced bioavailability due to protein coordination. The latter exhibits little activity on its own but, following treatment with hsA, exhibited significant cytotoxicity, which is consistent with its ability to form non-coordinate interactions with the protein. Overall, our studies demonstrate that non-coordinate interactions with hsA are a viable target for enhancing the activity of Ru(III)-based complexes in vivo.

Expression of set is downregulated by rapamycin in human colorectal cancer cells

The purpose of this study was to determine the mechanism through which rapamycin treatment affects the expression of the set gene in human colorectal adenocarcinoma cells. The effect of rapamycin treatment on set expression was evaluated by assessing the mRNA and protein expression of set in the SW480 and LoVo human colon carcinoma cell lines following treatment with rapamycin by quantitative polymerase chain reaction (qPCR) and western blot analysis, respectively. Our results demonstrated that the mRNA and protein levels of set were significantly decreased subsequent to rapamycin treatment in the two cell lines, indicating that set expression may be downregulated by rapamycin in human colorectal adenocarcinoma cells. Our findings suggested that the mammalian target of rapamycin signaling pathway may play a role in tumorigenesis through the regulation of the set gene.

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Runt‐related transcription factor 2 in human colon carcinoma: A potent prognostic factor associated with estrogen receptor

Runt-related transcription factor 2 (RUNX2) belongs to the RUNX family of heterodimeric transcription factors, and is mainly associated with osteogenesis. Previous in vitro studies demonstrated that RUNX2 increased the cell proliferation of mouse and rat colon carcinoma cells but the status of RUNX2 has remained unknown in human colon carcinoma. Therefore, we examined clinical significance and biological functions of RUNX2 in colon carcinoma. RUNX2 immunoreactivity was examined in 157 colon carcinoma tissues using immunohistochemistry. RUNX2 immunoreactivity was evaluated as percentage of positive carcinoma cells [i.e., labeling index (LI)]. We used SW480 and DLD-1 human colon carcinoma cells, expressing estrogen receptor-β (ER) in subsequent in vitro studies. RUNX2 immunoreactivity was detected in colon carcinoma cells, and the median value of RUNX2 LI was 67%. RUNX2 LI was significantly associated with Dukes' stage, liver metastasis and ERβ status. In addition, RUNX2 LI was significantly associated with adverse clinical outcome of the colon carcinoma patients, and turned out an independent prognostic factor following multivariate analysis. Results of in vitro studies demonstrated that both SW480 and DLD-1 cells transfected with small interfering RNA against RUNX2 significantly decreased their cell proliferation, migration and invasive properties. In addition, RUNX2 mRNA level was significantly decreased by ER antagonist in these two cells. These findings all suggest that RUNX2 is a potent prognostic factor in human colon carcinoma patients through the promotion of cell proliferation and invasion properties, and is at least partly upregulated by estrogen signals through ERβ of carcinoma cells.

Modulation of Human Serotonin Transporter Expression by 5-HTTLPR in Colon Cells

Serotonin (5-HT) is a monoamine neurotransmitter and plays important roles in several of the human body’s systems. Known as a primary target for psychoactive drug development, the 5-HT transporter (5-HTT, SERT) plays a critical role in the regulation of serotonergic function by reuptaking 5-HT. The allelic variation of 5-HTT expression is caused by functional gene promoter polymorphism with two principal variant alleles, 5-HTT gene-linked polymorphic region (5-HTTLPR). It has been demonstrated that 5-HTTLPR is associated with numerous neuropsychiatric disorders. The functional roles of 5-HTTLPR have been reported in human choriocarcinoma (JAR), lymphoblast and raphe cells. To date, the significance of 5-HTTLPR in gastrointestinal tract-derived cells has never been elucidated. Thus, the impact of 5-HTTLPR on 5-HTT transcription was studied in SW480 human colon carcinoma cells, which were shown to express 5-HTT. We found 42-bp fragment in long (L) allele as compared to short (S) allele, and this allelic difference resulted in 2-fold higher transcriptional efficiency of L allele (P < 0.05) as demonstrated using a functional reporter gene assay. Nevertheless, the transcriptional effect of estrogen and glucocorticoid on 5-HTT expression via 5-HTTLPR was not found in this cell line. Our study was the first to demonstrate the molecular role of this allelic variation in gastrointestinal tract cells.

Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

BackgroundInactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells.MethodsHigh resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction.ResultsWe identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization.ConclusionBased on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.

A Role of the Aryl Hydrocarbon Receptor in Attenuation of Colitis

The aryl hydrocarbon receptor (AhR), which is a member of the basic helix-loop-helix/Per-Arnt-Sim homology superfamily, plays an important role in multiple biological functions, and AhR knockout (AhR KO) animals suffer from a variety of organ disorders including a decline in the efficacy of their immune system. In addition, AhR activation is known to aid the maintenance of homeostasis in vivo. In this study, we investigated whether AhR is functionally associated with intestinal immunity. In in vivo experiments, it was found that dextran sodium sulfate (DSS)-evoked colitis was more severe in AhR KO mice than in C57BL/6J wild type mice. It was also revealed that the administration of DSS increased the expression levels of AhR and CYP1A1 mRNA in the colon epithelium. In addition, oral administration of β-naphthoflavone (βNF), a non-toxic agonist of AhR, suppressed the pathogenesis of DSS-induced colitis. βNF also attenuated DSS-induced colitis. In cell culture experiments, downregulation of AhR in human colon carcinoma SW480 cells enhanced the inflammatory responses evoked by lipopolysaccharide (LPS), and furthermore, AhR activation attenuated LPS-induced inflammatory responses, suggesting that AhR expressing intestinal epithelial cells are involved in the prevention of colitis. Our findings about the potential role of AhR activators in epithelial immune regulation aid our understanding of mucosal homeostasis and inflammatory bowl disease (IBD) and suggest that AhR activation has therapeutic value for the treatment of IBD.

Peptides from Lactobacillus Hydrolysates of Bovine Milk Caseins Inhibit Prolyl-peptidases of Human Colon Cells

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.

Abstract C233: BAL27862: A unique microtubule-targeted agent with a potential for the treatment of human brain tumors

Abstract Background: BAL27862 is a synthetic small molecule, potently inducing apoptosis in cancer cells through tubulin depolymerization characterized by a unique microtubule phenotype. BAL27862 has demonstrated a broad in vitro anti-proliferative activity against a range of human tumor histotypes (low nM IC50s). Significant antitumor responses occur in animal models of human cancer after oral or intravenous (i.v.) administration, including tumors refractory to conventional treatments. The purpose of this study was to investigate the tissue distribution of BAL27862 in tumor-bearing mice. Tumor and brain penetration, together with anti-proliferative activity in glioblastoma (GBM) cell lines, is described. Materials and Methods: Anti-proliferative activity and induction of tumor cell death in vitro were analyzed using the YO-PRO® assay (48h incubation). CD1 nu/nu female mice were implanted with human colon carcinoma SW480 cells. For pharmacokinetic studies, tumor-bearing (approx. 150 mm3) mice were administered i.v. with either 2 mg/kg 14C-BAL27862 (n=6, single dose) or 8 mg/kg cold BAL27862 (n=33, once-weekly during 4 weeks). Animals were culled at serial time points. Radioactivity was measured in slices, with Bio-Imaging Analyzer read-outs of the exposed phosphor imaging plates quantified with Aida software. Cold BAL27862 was determined in plasma, brain and tumors using a specific LC-MS/MS method after sample homogenization and protein precipitation. Results: BAL27862 elicited a potent anti-proliferative activity in 6 GBM cell lines (IC50 range: 10–20nM), which was independent of PTEN status. Strikingly, at optimal concentrations, a dramatic loss of cell viability was observed (% cell death at 50nM BAL27862: &amp;gt;15% in 5 lines, &amp;gt;30% in 2 lines), demonstrating that BAL27862 potently drives GBM cells into a cell death program..In mice, after i.v. administration of 14C-BAL27862, radioactivity was distributed to all organs: notably brain and tumor. A delayed peak was observed in slowly perfused organs such as skin, tumor and fat-tissue. There was no tissue-specific retention of radioactivity, as halflives were comparable between tissues and blood. 48h after administration, radioactivity was almost undetectable in most tissues. Following administration of 8 mg/kg i.v. cold BAL27862 to mice, the excellent brain and tumor penetration was confirmed. Specifically, similar levels of BAL27862 were found in brain and plasma (Cmax of 6.86 µg/g and 7.34 µg/mL, respectively). The ratio tumor/plasma was also around 1. There was no brain or tumor accumulation over time, as concentrations paralleled those in plasma. Conclusions: BAL27862 is efficiently distributed to tissues and tumor in a mouse model of human cancer. Significant brain penetration, coupled with cytotoxic activity in GBM tumor cell lines, would support further evaluation of BAL27862 for the treatment of human brain cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C233.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

Short hairpin RNA-mediated silencing of APRIL gene inhibits proliferation and metastasis of human colon carcinoma SW480 cells in vitro

Objective To study the influence of short hairpin RNA-mediated inhibition of APRIL gene on proliferation,invasion and metastasis of human colon carcinoma SW480 ceils in vitro. Methods After treatment with shRNA, the expression level of APRIL protein in human colon carcinoma SW480 cells was detected by western blotting. And cell adhesion, cell migration and cell invasion of SW480 cells transfected with sh637 were analyzed respectively as compared with non-transfected SW480 cells and mocktransfectant. Results The expression of APRIL protein in SW480 transfected with sh637 were significantly lower than mock-plasmid groups and untransfected ones (F=42.15, P=0.00). Cell proliferation was markedly inhibited, compared with untransfected groups and negative control ones (F=24.76, P<0.05).And the average absorption of cell adhesion in transfected groups, mock-plasmid and non-transfected ones were 0.343, 0.409, 0.412, respectively. Cell adhesion decreased 39% compared with untransfected groups. Similarly, there was significant difference for cell migration (F=65.53, P<0.01) between transfected groups (The average ceil number in exposed area to migrate was 47.89±13.16) and nontransfected (98.78±23.26) as well as mock-plasmid ones (108.01±39.11). Then, in view of the average absorption of cell invasion between transfected groups(0.58±0.10) and non-transfected (0.94±0.23) as well as mock-plasmid ones(1.11±0.21), a prominently difference for cell invasion was also found between them (F=5.771, P<0.05). Conclusion The results suggest APRIL has a proliferation-inducing effect and probably be involved in cell invasion and cell metastasis of colon carcinoma and it could enhance capacity of invasion and metastasis of SW480 cells. Key words: Colorectal neoplasms; Membrane proteins; Tumor necrosis factors; RNA interference; Cell proliferation; Neoplasm metastasis