Stomach Cancer Cell Lines Research Articles

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background:Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over- expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study.Methods:Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method.Results:The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both.Conclusions:The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.

Design, Synthesis and Antiproliferative Activity of Novel Phenothiazine-1,2,3-Triazole Analogues

A series of seven novel 1-aryl-1,2,3-triazole derivatives bearing a 4-(phenothiazin-10-ylmethyl) moiety were designed using a molecular hybridisation approach and synthesised by alkyne/azide click chemistry. Most of the synthesised compounds exhibited moderate to good antiproliferative activity (IC50 values: 0.5 to 6.7 μM) against stomach, oesophagus, prostate, breast and liver cancer cell lines, but a compound containing a 4-chlorophenyl moiety showed better activity against all cell lines than 5-fluorouracil.

Synthesis of Nano-Metric Gold Complexes with New Schiff Bases Derived from 4-Aminoantipyrene, Their Structures and Anticancer Activity

Two new Schiff bases derived from combination of 4-aminoantipyrine with ethylenediamine (L1) or benzaldehyde (L2), gave Au(III) complexes. Their structures were elucidated from microanalytical, magnetic, conductance, and FT-IR, UV-Vis, Mass, and 1H and 13C NMR spectral data. High conductance values indicated electrolytic nature of the complexes. Magnetic moments and electronic spectral data indicated that two synthesized Au(III) Schiff base complexes had a square planar geometry. FT-IR spectroscopic data demonstrated that the Schiff bases were coordinated to Au(III) ions in a tetradentate manner with NNNN donor sites of two 4-amino antipyrine and two azomethine (L1), while L2 Schiff base ligand coordinated to Au(III) ions via its four azomethine nitrogen, which was further supported by the appearance of new bands in IR spectra due to ν(M–N). Activation thermodynamic parameters (E*, ΔH*, ΔS*, and ΔG*) were calculated on the basis of TG curves. Crystalline structures of Schiff bases and their Au(III) complexes were characterized by X-ray diffraction (XRD), their morphology was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The Schiff base ligands and their Au(III) chelates were screened for their antimicrobial activity. Cytotoxic activity of those was tested against the human breast cancer (MCF-7) and human hepatocellular carcinoma (HepG-2) tumor cell lines.

Sclerostin induced tumor growth, bone metastasis and osteolysis in breast cancer

Breast cancer is the second leading cause of cancer-related deaths among women worldwide. Many patients suffer from bone metastasis. Sclerostin, a key regulator of normal bone remodeling, is critically involved in osteolytic bone diseases. However, its role in breast cancer bone metastasis remains unknown. Here, we found that sclerostin was overexpressed in breast cancer tumor tissues and cell lines. Inhibition of sclerostin by antibody (Scl-Ab) significantly reduced migration and invasion of MDA-MB-231 and MCF-7 cells in a time- and dose-dependent manner. In xenograft model, sclerostin inhibition improved survival of nude mice and prevented osteolytic lesions resulting from tumor metastasis. Taken together, sclerostin promotes breast cancer cell migration, invasion and bone osteolysis. Inhibition of sclerostin may serve as an efficient strategy for interventions against breast cancer bone metastasis or osteolytic bone diseases.

Potent Anticancer Activity with High Selectivity of a Chiral Palladium N-Heterocyclic Carbene Complex.

Five enantiomeric pairs of palladium complexes of 1,2,4-triazole-derived chiral N-heterocyclic carbene ligands were investigated to probe the influence of chirality on the compound’s anticancer activity. Although no chirality-related influence was observed for any of the enantiomeric pair, strong anticancer activity was seen for a particular pair, (1S,2S,5R)-1c and (1R,2R,5S)-1c, which was significantly more active than the benchmark drug cisplatin for human breast cancer cells, MCF-7 (ca. 24–27-fold), and human cervical cancer cells, HeLa (ca. three- to fourfold). Broadening its scope of application, (1R,2R,5S)-1c also exhibited antiproliferative activity against lung cancer (A549), skin cancer (B16F10), and multidrug-resistant mammary tumor (EMT6/AR1) cell lines. Interestingly, (1R,2R,5S)-1c displayed 8- and 16-fold stronger antiproliferative activity toward B16F10 and MCF-7 relative to their respective noncancerous counterparts, L929 (fibroblast skin cells) and MCF10A (epithelial breast cells), thereby upholding the potential of these complexes for further development as anticancer agents. (1R,2R,5S)-1c inhibited tumor-cell proliferation by blocking the cells at the G2 phase. (1R,2R,5S)-1c caused DNA damage in MCF-7 cells, leading to mitochondrial reactive oxygen species production and subsequently cell death. We also present evidence indicating that (1R,2R,5S)-1c induced p53-dependent programmed cell death in MCF-7 cells.

CD44v8-10 as a potential theranostic biomarker for targeting disseminated cancer cells in advanced gastric cancer

Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage IV advanced gastric cancer (AGC) patients with distant metastasis is very low. Thus, the detection and eradication of disseminated cancer cells by targeting cell surface molecules in AGC would improve patient survival. The hyaluronic acid receptor, CD44, has various isoforms generated by alternative splicing, and some isoforms are known to be correlated to gastric cancer. In this study, to find out the most appropriate CD44v for targeting AGC, we analysed the expression differences of CD44 isoforms at the mRNA level in stomach cancer cell lines as well as in 74 patients with AGC by using exon-specific qRT-PCR. Among the CD44v isoforms, CD44v8-10 was determined as the most promising biomarker for the development of theranostic agents of gastric cancer. Next, we synthesised the conjugate of anti-CD44v9 antibody with near-infrared fluorophore or photosensitiser, and then demonstrated its feasibility for target cell-specific imaging and photoimmunotherapy in gastric cancer. As a result, these conjugates have clearly demarcated the surface of CD44v8-10 expressing cancer cells and showed efficient phototoxic effects. Therefore, this study revealed that CD44v8-10 is the efficient theranostic biomarker to target disseminated cancer cells in AGC.

Abstract 4524: The effect of acute intraoperative hypoxia in breast cancer

Abstract Background: Hypoxia is defined as oxygen levels in tumor microenvironment of less than that in blood (90-100 mm Hg) and influences many aspects of tumour biology. During surgery tumour vasculature is cut off gradually leading to induction of acute hypoxia.The present study aims to experimentally test the genotypic and phenotypic effects of surgically induced acute hypoxia in breast cancer tumor samples and cell lines. Methodology: Core biopsy samples were collected from breast tumors (N=8 patients) at three time points during their curative surgery: prior (pre), mid-way (intra) and at the end (post). The samples were subjected to RNA-Seq and a list of differentially expressed genes (DEG) was prepared. A set of 26 DEG (‘pre’ Vs ‘intra’ Vs ‘post’) obtained from RNA-Seq analysis and additional 17 genes involved in inflammation, EMT and hypoxia pathways were chosen for validation in tumor samples. These genes were validated using a customized qPCR Array (Qiagen). A gene was considered validated if it was significantly deregulated in at least 4 out of 8 patients. In another experiment, MCF-7 cells were exposed to varying levels of oxygen concentrations (0.1-20%) for varying time periods ranging from 30 minutes to 72 hours, to study time and dose dependent effects of hypoxia on following functional characteristics: proliferation, invasion and cell cycle changes. Results: Concordant, statistically significant up-regulation of FOS, DUSP1, JUNB, FOSB, ZFP36, RGS1, S100A4, CXCL8 and CCL2 were observed in RNA-Seq and qPCR experiments while MMP13, HIF1A and VEGFA were up-regulated only in qPCR. However, 7 protein markers of inflammation, EMT and hypoxia did not show any significant change between pre, intra and post-operative samples. In MCF-7 cells, a dose and time dependent decrease in cell viability was observed with increasing severity of hypoxia as well as decrease in invasiveness, but there was no significant impact on cell cycle phases. When hypoxic cells were re-incubated under normoxic conditions an increase in cell proliferation and accumulation of cells in S phase (with a reduction in G2-M fraction) were observed, compared to cells grown under only normoxic conditions. Conclusion: Acute intra-operative hypoxia up-regulates expression of genes related to cell survival, chemoresistance, invasiveness, inflammation and angiogenesis in breast tumors. Breast cancer cells exposed to acute severe hypoxia followed by normoxia show increased proliferation. These effects may have implications for tumor cells that disseminate during surgery. Citation Format: Dimple R. Bhatia, Shalaka Joshi, Rohan Chaubal, Poonam Gera, Prajakta Kalkar, Farheen Naeem, Nisanth Mathew Raju, Nilesh Gardi, Nita Nair, Vaibhav Vanmali, Rohini W. Hawaldar, Amit Dutt, Rajendra A. Badwe, Sudeep Gupta. The effect of acute intraoperative hypoxia in breast 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 4524. doi:10.1158/1538-7445.AM2017-4524

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent-kaurane diterpenoid, 11β,16α-dihydroxy-ent-kauran-19-oic acid (1), together with eight known analogues 2 - 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid (3), was converted to seven derivatives, 10 - 16. All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG-2), and stomach (SGC-7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 - 16, with 15-keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC50 value ranging from 0.05 to 3.71 μm. Compounds 10 - 12, with oxime on C(15) showed moderate inhibitory effects and compounds 1 - 9 showed no cytotoxicities on them. Structure-activity relationships were also discussed based on the experimental data obtained. The known derivative, 15-oxokaurenoic acid 4-piperdin-1-ylbutyl ester (17), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis-inducing activity by flow-cytometric analysis.

Induction of cathepsin B by the CXCR3 chemokines CXCL9 and CXCL10 in human breast cancer cells.

Cathepsin B (CTSB) is a lysosomal cysteine protease that has been linked to the progression of breast cancer, for example by activation of other proteases and tumor-promoting cytokines, thereby supporting tumor invasion and metastasis. Previously, it was shown that CTSB cleaves and inactivates C-X-C motif chemokine receptor 3 (CXCR3) chemokines. As CXCR3 ligands have been demonstrated to induce proteases in cancer cells, the present study hypothesized that they may also affect CTSB in breast cancer cells. The results demonstrated that the human breast cancer tumor cell lines MCF-7 and MDA-MB-231 express the CXCR3 splice variants A and B and CTSB. Upon binding to CXCR3, the two chemokine ligands C-X-C motif chemokine ligand (CXCL) 9 and CXCL10 trigger upregulation of CTSB in these breast cancer cells, whereas the CXCR3-B-specific ligand CXCL4 has no such effect, suggesting the involvement of CXCR3-A in the regulation of CTSB. In early-stage human breast cancer specimens (n=81), overexpression of CXCR3 is associated with statistically significant poorer overall survival, independent of lymph node status, tumor size and nuclear grading (hazard ratio=1.99; 95% confidence interval=1.00–3.97; P=0.050). In conclusion, the data from the current study propose a so far unknown mechanism by which breast cancer cells may exploit tumor-suppressive chemokines to enhance their invasiveness and reduce immune cell infiltration by the degradation of these chemokines. This mechanism may support the established unfavorable prognostic feature of CXCR3 expression in breast cancer.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain ‘mini-myoferlin’. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate ‘mini-myoferlin’ can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Copper-catalyzed chalcogenation of imidazoheterocycles with sulfur/selenium powder and coumarinyl triflates.

An efficient and convenient copper-catalyzed chalcogenation of imidazoheterocycles with sulfur/selenium powder and coumarinyl triflates has been described. This procedure provides a wide range of structurally diverse coumarinylthio-/coumarinylseleno-substituted imidazoheterocycles in good yields and with good functional group tolerance. Biological evaluation showed that the newly synthesized compound 6d possesses significant in vitro antiproliferative activities against human-derived esophageal, breast, stomach, and prostate cancer cell lines compared with the positive control, 5-fluorouracil.

Bioactivity-guided isolation of biphenanthrenes from Liparis nervosa

Three new biphenanthrenes, Liparisphenanthrenes A–C (1–3), along with three known ones were obtained from the ethanolic extract of Liparis nervosa (Orchidaceae) by bioactivity-guided fractionation. Their structures were elucidated on the basis of extensive spectroscopic analysis. All the compounds obtained were tested in vitro for cytotoxic activities against stomach (HGC-27) and colon (HT-29) cancer cell lines. 1, 4 and 5 showed potent cytotoxicities to HGC-27 cell line with IC50 values of 8.21–9.95μmol/L, and 1 and 5 also exhibited potent cytotoxic activities to HT-29 cell line with IC50 values of 8.53–9.27μmol/L.

Palladium-catalyzed external-oxidant-free coupling reactions between isoquinoline/quinoline N-oxides with olefins

A convenient and efficient approach for the synthesis of 1-alkenylisoquinolines and 2-alkenylquinolines was developed via palladium-catalyzed coupling reactions between isoquinoline/quinoline N-oxides with olefins under external-oxidant-free conditions. Biological evaluation revealed that some of the obtained 1-alkenylisoquinolines exhibited in vitro antiproliferative activities on human-derived prostate, breast, esophageal, and stomach cancer cell lines.

Abstract 3015: Plitidepsin targets the GTP-bound form of eEF1A2 in cancer cells

Abstract Plitidepsin (APL), an antitumor agent originally isolated from the marine tunicate Aplidium albicans, is being tested in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone. eEF1A2 is one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold pro-oncogenic activities (5). Here we analyze the interaction of plitidepsin with its target, eEF1A2. DARTS assays, either with whole cell extracts or with purified eEF1A2 protein, indicate that plitidepsin binds to eEF1A2 and protects it from digestion by subtilisin (EC 3.4.21.62). Moreover, a [14C]-APL binding-guided fractionation of K562 cell extracts through differential centrifugation and three chromatographic steps demonstrated that eEF1A2 is the only cellular protein that can be retrieved through specific binding to plitidepsin. A saturation binding experiment with [14C]-APL and purified GTP-bound eEF1A2 (from rabbit muscle) allowed us to calculate a Kd of around 80 nM for the interaction, while a dissociation experiment revealed a residence time of around 9 minutes. Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2. HeLa-APL-R cells, ≥1000 fold more resistant to plitidepsin than parental HeLa wt cells (6), are now shown to have lower eEF1A2 mRNA and protein levels than parental cells. Furthermore, when eEF1A2 levels were restored to normal in HeLa-APL-R cells through ectopic expression of an eEF1A2-GFP construct, they were rendered partially sensitive to plitidepsin. Interestingly, transfected cells recovered most of the signaling events typically induced by the drug in HeLa wt cells. NCI-H460 (lung) and HGC-27 (stomach) cancer cell lines were rendered resistant to plitidepsin following the same procedure described in (6) for HeLa-APL-R cells. When we analyzed the levels of eEF1A2 in this two new cell lines we observed that both of them were lacking eEF1A2. Altogether, our results demonstrate that plitidepsin targets the pro-oncogenic eEF1A2 protein, a new druggable target for anticancer therapy. (1)Sun et al, 2014, Biochem Biophys Res Commun 450:1-6 (2)Xu et al, 2013, Clin Exp Metastasis 30:933-44 (3)Anand et al, 2002, Nat Genet 31:301-5 (4)Li et al, 2010, PLOS One 5, e10755 (5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93 (6)Losada et al., 2004, Br J Cancer 91:1405-13 Citation Format: Alejandro Losada, Maria J. Munoz-Alonso, Juan F. Martínez-Leal, Juan M. Dominguez, Carlos M. Galmarini. Plitidepsin targets the GTP-bound form of eEF1A2 in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3015.

Abstract 1462: Non-inflammatory role of ASC-dependent inflammasomes in promoting gastric tumourigenesis via IL-18

Abstract Introduction Gastric cancer is the third most lethal cancer worldwide and represents a growing number of cancers linked with inflammation. However, the identity of innate immune regulators with oncogenic potential in the host gastric mucosal epithelium remains obscure. We aim to identify the molecular basis by which specific pattern recognition receptors belonging to the inflammasome family promotes gastric tumorigenesis. Experimental procedures We have employed the gp130F/F gastric cancer mouse model coupled with mice lacking specific inflammasome-related genes. In addition, human gastric cancer tumor (and matched non-tumor) biopsies and cell lines were used. Immunohistochemistry on gastric tissue sections was used to assess the extent of cellular proliferation, apoptosis, inflammation and angiogenesis, and quantitative real-time PCR was used to measure the expression of genes of interest. Immunoblotting and ELISA were used to measure the activation and expression levels of inflammasome-related proteins. Results A causal role for ASC-dependent inflammasomes in gastric tumorigenesis was confirmed upon the genetic ablation of ASC in gp130F/F:Asc-/- mice, which resulted in a ∼50% reduction in tumor burden compared to parental gp130F/F mice. The suppressed gastric tumorigenesis in gp130F/F:Asc-/- mice was associated with increased gastric epithelial (tumor) cell apoptosis, as determined by elevated numbers of TUNEL and caspase-8 positive cells. Surprisingly however, no changes in the number and activation status of inflammatory cells were observed. The suppressed gastric tumorigenesis in gp130F/F:Asc-/- mice was also characterized by a gene signature comprising apoptotic-related genes. In gastric tumor tissue, IL-18, but not IL-1β, protein levels were augmented compared to matched non-tumour tissue, and genetic ablation of IL-18 in gp130F/F:IL-18-/- mice suppressed gastric tumorigenesis comparable to that observed upon ASC deficiency. The treatment of human gastric cancer cells with IL-18 also potentiated their growth compared to IL-1β. Conclusions Collectively, these data reveal that ASC-dependent inflammasomes and their downstream mediator IL-18 represent a novel oncogenic mechanism in gastric cancer. Citation Format: Brendan Jenkins, Virginie Deswaerte, Alison West, Paul Nguyen, Tracy Putoczki. Non-inflammatory role of ASC-dependent inflammasomes in promoting gastric tumourigenesis via IL-18. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1462.

Abstract 385: mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer

Abstract BACKGROUND Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States. Several key mutations in CRC include APC (80%), TP53 (50%), and PIK3CA (20-30%). Mutations in the PIK3CA gene, resulting in a constitutively active PI3K, often occur concomitantly with loss of the APC gene in human CRCs. Our lab has developed a murine model system where a constitutively active PI3K and loss of APC occur simultaneously in the colon (AK3K) as well as concomitant loss of p53 (AK3KTO). Colon tumors from these models are cultured as three-dimensional spheroids and treatment studies. METHODS AK3K and AK3KTO spheroids were treated with a dual PI3K/mTOR inhibitor, NVP-BEZ235, or a mTORC1/2 inhibitor, MLN0128. Images were taken both pre- and post-treatment and changes in spheroid diameter were measured. Parallel treatment studies were performed on a primary human colon cancer tumor cell line, SW48, which carries a mutant FBXW7 and also on a cell line transfectedwith a PIK3CA mutation (SW48PK). RESULTS Treatment with NVP-BEZ235 and MLN0128 resulted in a significant treatment response as measured by marked change in spheroid diameter in the AK3K spheroids at 100, 200 and 400nM (p<0.01), with MLN0128 eliciting a greater treatment response. There were no significant differences between drugs in the AK3KTO spheroids. Comparing across cell lines, there were highly significant differences when treated with NVP-BEZ235, as the treatment was much more effective in the AK3KTO cell line at 100, 200, and 400nM (p<0.01). There were no significant differences between cell lines for MLN0128. Immunoblotting further demonstrated a dramatic inhibition of the PI3K/mTOR pathway in both spheroids following treatment with 200nM MLN0128 and 200nM NVP-BEZ235. Treatment of SW48 and SW48PK cells with NVP-BEZ235 and MLN0128 showed highly significant differences between the drugs in both cell lines, with MLN0128 being more effective at 1, 10, and 50nM (p<0.01). At higher drug concentrations (100, 200, and 400nM), there were highly significant differences between drugs for the SW48PK cell line (p<0.01) but no significant differences in the SW48 cells. Comparing across cell lines, there were highly significant differences when treated with MLN0128 at 100, 200, and 400nM (p<0.01), with MLN0128 being more effective in the SW48PK cell line. Immunoblotting demonstrated a reduction in activation of the PI3K/mTOR pathway following treatment with 10nM MLN0128 but this inhibition was not seen when treated with 10nM NVP-BEZ235. CONCLUSIONS mTOR inhibition is sufficient to induce responses in PIK3CA and APC mutated CRCs and concomitant loss of TP53 does not lead to resistance and potentially increases sensitivity. In a cell line predisposed to PI3K/mTOR signaling sensitivity, mTOR inhibition remains sufficient and the addition of a PIK3CA mutation further increases sensitivity Citation Format: Stephanie Fricke, Cheri Pasch, Susan Payne, Alexander Yueh, Tyler Foley, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 385.

The tumor suppressor gene 5 in the study of differences in regulatory mechanisms and the molecular level in the process of human gastric cancer

Objective To explore the role of tumor suppresser gene tumor suppressor gene 5 (ST5) in the occurrence of gastric adenocarcinoma. Methods DNA methylation chip and bioinformatic analysis were applied to deep the different genes between diffused gastric adenocarcinoma and normal gastric tissues. The mRNA and protein expression level of ST5 gene were detected by reverse transcription-polymerase chain reaction (RT-PCR) and westen-blot in gastric adenocarcinoma tissues and gastric cancer cell lines. Results The ST5 gene showed significant high methylation in diffused gastric adenocarcinoma; the mRNA expression level of ST5 gene was lower in poorly differentiated SGC-7901 stomach cancer cell lines (fold change=0.61, P<0.01); ST5 gene was demonstrated lower protein expression level in diffused gastric adenocarcinoma tissue and cell lines like BGC-823, MGC-803 and SGC-7901. Conclusion The ST5 gene represented as a tumor suppressor gene, playing an important role in gastric cancerization via regulation of DNA methylation. Key words: Human gastric cancer; DNA methylation chip; Tumor suppressor gene 5

Isolation and Identification of an Antiproliferative Compound from Fructose-Tryptophan Maillard Reaction Products.

This study was performed to isolate and identify a compound with antiproliferative activity against human stomach cancer cell lines, from fructose-tryptophan Maillard reaction products (MRPs). The MRPs, prepared from a fructose-tryptophan solution heated at 130 °C for 2 h, were fractionated into five solvent fractions: n-hexane, chloroform, ethyl acetate, butanol, and water. The highest antiproliferative activity was found in the chloroform fraction (85.93% at 200 μg/mL), and the active compound from this chloroform fraction was purified by silica gel column chromatography, TLC, and preparative HPLC. The antiproliferative activity (IC50) of the active compound was 42.24 μg/mL, and the active compound was identified as perlolyrine (C16H10N2O2) by (1)H/(13)C NMR, DEPT, HMBC, and LC-ESI-MS. Therefore, this research may be useful in developing perlolyrine as a functional therapeutic agent.

Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells.

BackgroundAn abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered.MethodsWe experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5′- and 3′-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells.ResultsWe found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells.ConclusionsAn interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0200-y) contains supplementary material, which is available to authorized users.

Abstract P2-05-26: Focal adhesion kinase is required for efficient tumor-induced osteoclastogenesis via control of macrophage colony stimulating factor expression

Abstract Breast cancer most commonly metastasizes to bone where metastases cause the potentiation of the vicious cycle. During this process, breast cancer bone metastases inhibit osteoblasts from forming new bone, while also activating osteoclasts to degrade bone. This in turn causes release of bone-matrix bound growth factors which propagate tumor growth. The overall net bone destruction can lead to significant adverse clinical consequences for patients including fractures or substantial pain. While agents such as bisphosphonates or the monoclonal antibody to RANKL denosumab, both of which inhibit the osteolytic activity of osteoclasts, are currently used in breast cancer bone metastatic patients to alleviate these adverse clinical outcomes, they have not been associated with increased patient survival. Given the lack of successful treatments for bone metastases providing survival advantages, we sought to evaluate the role of focal adhesion kinase (FAK), a novel therapeutic target, in breast cancer mediated osteolysis. FAK is a non-receptor tyrosine kinase that regulates many pathways that contribute to enhanced tumor progression and metastasis. FAK is also expressed in all the cell types involved in the vicious cycle. We thus hypothesized that FAK plays an important role in breast tumor-induced osteolysis and that its inhibition would lead to restoration of bone homeostasis, in addition to inhibition of tumor progression. Using in vitro siRNA-targeted depletion of FAK in breast cancer tumor cell lines, we found that production of numerous soluble growth factors, many of which are known contributors to osteoclastogenesis, was inhibited. We confirmed that FAK regulates the expression of the osteoclastogenic factor macrophage colony stimulating factor in breast cancer cell lines. Further, using conditioned media from FAK expressing versus depleted breast cancer tumor cells in osteoclastogenesis assays, we show that FAK-depletion results in impaired osteoclastogenesis. These data suggest that in addition to its proven direct anti-tumor effects, inhibition of FAK may also result in therapeutic blockade of bone degradation. Citation Format: Landon K, Howe GA, Zhao H, Addison CL. Focal adhesion kinase is required for efficient tumor-induced osteoclastogenesis via control of macrophage colony stimulating factor expression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-26.