Chemical Profiles and Bioactivities of Brazilian Amazonian Solanum Species: Antioxidant, Cytotoxic, and Anti-Metastatic Properties.

Background: Signaling by the insulin-like growth factor 1 receptor (IGF-1R) promotes cell growth, migration and survival in several human tumor types. IGF-1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF-1R/IR TKI, OSI-906, indicated that overexpression of the mitogen-activated protein kinase (MAPK) pathway conferred resistance to OSI-906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI-906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI-906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty-eight CRC cell lines were exposed to either OSI-906 (0–5µM), or U0126 (0–20µM). In reference to OSI-906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 < 5µM were regarded as S to U0126 and cell lines with IC50 > 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI-906 but R to U0126, and 4) R to OSI-906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI-906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI-906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI-906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI-906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI-906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI-906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI-906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

10527 Background: MSI is a phenomenon found in tumor DNA of individuals with dysfunction of the mismatch repair system (MMR). Epigenetic inactivation by promoter hypermethylation of hMLH1 causes 15 to 20% of sporadic CRC. In vitro studies have suggested an increased sensitivity to CPT-11 of MMR-deficient human CRC cell lines. Chemosensitivity evaluation in preclinical models of human CRC cell lines according to the MMR status could help in the design of specific studies in the clinical setting. Methods: We have performed drug cytotoxicity assays to compare sensitivity to CPT-11 in several human CRC cell lines with different MMR gene status that resemble the most common clinical situations in CRC patients. HCT116, HCT15, SW48 and RKO are MSI-High (MSI-H), being HCT116 due to a homozygous nonsense mutation of hMLH1 gene, HCT15 due to MSH6 mutation (both of them similar to hereditary cases) and both SW48 and RKO due to methylation of hMLH1 promoter (as MSI-H sporadic CRC cases). HT29 expresses normal levels of MMR proteins (as microsatellite stable (MSS) CRC cases). Drug concentrations resulting in 50% growth inhibition (IC50) were determined by a curve-fitting analysis and cell cycle analyses in order to characterize the cytotoxicity of cell lines were performed. Results: IC50 values and 95% confidence intervals (CI) are show in Table 1 . hMLH1-deficient cell lines due to either epigenetic silencing or mutation showed very similar IC50 and were 5- to 8-fold more sensitive to CPT-11 than the MSS line. hMSH6- deficient cell line HCT15 has sensitivity closer to MSS than MSI cell lines. Treatment with CPT-11 induced a G2/M arrest. Conclusions: Lack of hMLH1 protein due to either genetic alteration or epigenetic silencing correlates with increased sensitivity to CPT-11. MSI-H CRC cell lines are more sensitive to CPT-11 than MSS. Future clinical trials tailoring chemotherapy regimens based on microsatellite status are warranted. [Table: see text] No significant financial relationships to disclose.

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway

Herbal compounds are important resources that used in the treatment of many diseases, and are inspiring in drug design and development. Rheum ribes, a perennial native plant, has potentially diverse medicinal effects, including antioxidant, antibacterial and anti-tumor activity. The key molecules associated with anticancer activity of R. ribes in tumor formation still remains unclear. The present study was aimed to elucidate the antiproliferative effects of two purified compounds, emodin and aloe-emodin, from R. ribes. In our study, chromatographic separation was used to purification of compounds. Experimental 1D and 2D 1H and 13C-NMR spectra helped us to confirm the chemical structures of emodin and aloe-emodin. The viability of human glioblastoma cell line (U373), human breast carcinoma cells (MCF-7), and human colorectal cancer cell line (HT-29) were detected by WST-1 proliferation assay as dose- and time-dependent. The isolated emodin and aloe-emodin from R. ribes, reduced viability of human glioblastoma, breast and colorectal cancer cell lines in dose- and time-dependent manner significantly. These antiproliferative effects may contribute as a possible strategy to the development of new therapeutic approaches in the treatment of cancer.

BackgroundIndividual responses to oxaliplatin (L-OHP)-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC) cell lines. We performed expression difference mapping (EDM) analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF).ResultsOf the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations) (P < 0.001, R2 = 0.80). We identified this protein as Protein S100-A10 (S100A10) by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells.ConclusionsBy proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

Colorectal cancer(CRC) cell lines are ideal in vitro models for colorectal cancer study. Although the number of colorectal cell lines increases hence provides diversified choices for research, several problems occur including the uneven quality control, insufficient attention to the origin and biological characteristics of CRC cell lines, resulting in inappropriate selection of CRC cell lines for study. In this paper, we classify the current CRC cell lines for study, review the biological characteristics, current problems of experiment choices, and discuss the exact choice of cell lines for CRC study.

BackgroundThevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines.MethodsThe cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry.ResultsThe T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides.ConclusionT. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines.

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the β1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to β1 integrin. Function-blocking antibodies to α2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in α2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of α2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type α2 integrin or a non-signaling chimeric α2 integrin. Overexpression of wild-type α2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric α2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor α2β1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

BackgroundLactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated.MethodsPostbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses.ResultsUL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis.ConclusionIn conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.

A novel series of 4-anilinoquinazoline analogues, DW (1–10), were evaluated for anticancer efficacy in human breast cancer (BT-20) and human colorectal cancer (CRC) cell lines (HCT116, HT29, and SW620). The compound, DW-8, had the highest anticancer efficacy and selectivity in the colorectal cancer cell lines, HCT116, HT29, and SW620, with IC50 values of 8.50 ± 2.53 µM, 5.80 ± 0.92 µM, and 6.15 ± 0.37 µM, respectively, compared to the non-cancerous colon cell line, CRL1459, with an IC50 of 14.05 ± 0.37 µM. The selectivity index of DW-8 was >2-fold in colon cancer cells incubated with vehicle. We further determined the mechanisms of cell death induced by DW-8 in SW620 CRC cancer cells. DW-8 (10 and 30 µM) induced apoptosis by (1) producing cell cycle arrest at the G2 phase; (2) activating the intrinsic apoptotic pathway, as indicated by the activation of caspase-9 and the executioner caspases-3 and 7; (3) nuclear fragmentation and (4) increasing the levels of reactive oxygen species (ROS). Overall, our results suggest that DW-8 may represent a suitable lead for developing novel compounds to treat CRC.

It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts. Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels. The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05). Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did.

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing

The pattern of reactivity of 10 monoclonal antibodies (MCA) developed against human lymphoid leukemia cells and tested with human T-cells, B-cells, as well T-lymphoma and B-lymphoma cell lines suggested that six of them detect la-like determinants, three detect HLA-like determinants, and the remaining one detects a non-Ia B-cell antigen. With the use of three binding assays, the six MCA that appeared to detect Ia-like determinants reacted strongly with the human colorectal cancer cell (CCC) line LoVo, and none of the three MCA that reacted with HLA-like determinants reacted with this cell line. Immunoprecipitation and polyacrylamide gel electrophoresis analysis confirmed the apparent specificities of the MCA for Ia-like and HLA molecules, demonstrated the presence of Ia-like molecules in LoVo, and failed to detect HLA in these cells. The cellular enzyme-linked immunosorbent assay was used for the testing of our anti-Ia and anti-HLA MCA with 15 other CCC lines. Marked heterogeneity was found in the expression of different Ia-like and HLA determinants defined by different or overlapping subsets of MCA, which suggested that these determinants might be present on different molecules or that different conformations of the same molecules exist in various CCC lines. Analysis of the surface phenotype of subclones of LoVo cells revealed the presence of stable variant cell subpopulations, which lost reactivity with four out of six of the anti-Ia-like MCA but retained at least one Ia-like molecule recognized by two of our MCA. All of the subclones maintained the HLA-negative phenotype. The possible immunologic and diagnostic consequences of the presence or absence of Ia-like or HLA markers on nonlymphoid tumor cells are discussed.