Protein In Colon Cancer Cells Research Articles

Nonsense Mediated Decay Resistant Mutations Are a Source of Expressed Mutant Proteins in Colon Cancer Cell Lines with Microsatellite Instability

BackgroundFrameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours.MethodsUsing antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines.ResultsMutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested.ConclusionNMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours.

Analysis of Nuclear High Mobility Group Box 1 (HMGB1)-Binding Proteins in Colon Cancer Cells: Clustering with Proteins Involved in Secretion and Extranuclear Function

HMGB1 is a nuclear protein that is overexpressed and secreted in cancer cells. However, little is known about the roles of HMGB1 in the cytoplasm and secretory pathway in cancer cells. To clarify this aspect of HMGB1 function, we fractionated the cytoplasm of HCT116 colon cancer cells and used a proteomic approach to analyze cytoplasmic HMGB1-binding proteins. Pull-down experiments using recombinant HMGB1 protein as bait, followed by mass spectrometry analysis identified 162 interacting proteins. Among them were 74 proteins known to be localized exclusively to the extra-nuclear region, and 60 proteins known to be localized to both nuclear and extranuclear regions. The functions of these binding proteins include involvement in cell-cycle progression, cell proliferation, anti-apoptosis, and angiogenesis. In addition, nine of the identified proteins are related to protein translocation and secretion. These include annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10, which are involved in unconventional protein secretion. Cytoplasmic HMGB1 was primarily associated with the lysosomal cytosol fraction and was colocalized with the lysosomal marker LAMP1. Our findings suggest that cytoplasmic HMGB1 binds to a number of molecules related to cancer progression and the unconventional secretory pathway.

Triterpenes From Ganoderma Lucidum Induce Autophagy in Colon Cancer Through the Inhibition of p38 Mitogen-Activated Kinase (p38 MAPK)

Medicinal mushroom Ganoderma lucidum is one of the most esteemed natural products that have been used in the traditional Chinese medicine. In this article, we demonstrate that G. lucidum triterpene extract (GLT) suppresses proliferation of human colon cancer cells HT-29 and inhibits tumor growth in a xenograft model of colon cancer. These effects of GLT are associated with the cell cycle arrest at G0/G1 and the induction of the programmed cell death Type II–autophagy in colon cancer cells. Here, we show that GLT induces formation of autophagic vacuoles and upregulates expression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase) proteins in colon cancer cells and in tumors in a xenograft model (Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy is mediated through the inhibition of p38 mitogen-activated protein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190, induces autophagy and expression of Beclin-1 (1.2-fold increase) and LC-3 (7.4-fold increase), and GLT suppresses phosphorylation of p38 MAPK (≈60% inhibition) in colon cancer cells. Taken together, our data demonstrate a novel mechanism responsible for the inhibition of colon cancer cells by G. lucidum and suggest GLT as natural product for the treatment of colon cancer.

Abstract 1218: SMAC modulates gemcitabine-induced apoptosis in head and neck cancer cells

Abstract Apoptosis induction is an important anti-tumor mechanism of many conventional chemotherapeutic agents. Biochemical data indicate that SMAC promotes caspase activation by antagonizing IAPs. However, SMAC-knockout mice do not exhibit obvious apoptotic deficiencies in response to a wide range of stimuli. Work for us and others demonstrate that SMAC plays an essential role in apoptosis induced by Nonsteroidal Anti-inflammatory Drug (NSAID), TRAIL or BH3 only proteins in colon cancer cells. In this study we documented a critical role of SMAC in mediating apoptosis induced by chemotherapeutic agent gemcitabine in head and neck squamous cell carcinoma (HNSCC) cells. Using RNA interference, we found that that SMAC mediates gemcitabine-induced apoptosis through the intrinsic/mitochondrial pathway. SMAC Knockdown led to impairment in caspase-3 activation, mitochondrial membrane depolarization and release of cytochrome c. Small-molecule SMAC mimetics at nanomolar concentrations significantly sensitized HNSCC cells to gemcitabine-induced apoptosis, and overcame gemcitabine resistance in SMAC-knockdown cells by restoring caspase-3 activation and cytochrome c release. Furthermore, in xenograft models, SMAC knockdown significantly blocked the therapeutic responses of HNSCC tumors to gemcitabine by inhibiting apoptosis, while SMAC overexpression dramatically enhanced that. Together, these results suggest that SMAC mediates apoptosis in cancer cells, and provide a strong rationale for combining SMAC mimetics with other anticancer agents to treat apoptosis-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1218.

Erythropoietin increases Epo and EpoR expression in DLD-1 cells

Introduction. Supplementation of recombinant human erythropoietin (rHuEpo) is one of the methods for the treatment of anemia for patients with colon cancer. However, the results of in vitro studies investigating the influence of rHuEpo on cancer cells are contradictory. Aim. The aim of the present study was an assessment of the effect of rHuEpo on proliferation, as well as Epo and EpoR protein expressions in normoxia and hypoxia conditions on human colon adenocarcinoma cells (DLD-1). Materials and methods. The cells were cultured in medium with rHuEpo in con centrations of 1 and 3 IU without (normoxia) or with (hypoxia) cadmium chloride for 48 hours. Cell viability was counted using a haematocytometer and trypan blue 0.4% (w/v) dye. Expression of Epo and EpoR protein was assessed by western blot. Results and Discussion. We observed a decrease in the number of colon cancer cells in hypoxia. Addition of rHuEpo did not modify cell numbers in normoxia and hypoxia. We found a significant increase of EpoR expression in all cells growing in medium with cobalt chloride in comparison with respective normoxic cells. We also noted that rHuEpo in concentration of 3 IU significantly increased expression of Epo and EpoR protein in colon cancer cells in normoxia and hypoxia conditions. Conclusions. We concluded that Epo and EpoR are constitutively expressed in DLD-1 cells. In hypoxia as well as in the presence of rHuEpo the increase of Epo and EpoR protein was found. However, the expression of Epo and EpoR protein in these cells does not seem essential to their growth.

Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours

The mutation cluster region (MCR) of adenomatous polyposis coli (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the beta-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any beta-catenin binding activity. Similarly, we also show that the tumour-associated truncations abolish the interaction of beta-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to beta-catenin binding.

Expression and nucleus translocation of calcyclin binding protein in colon cancer cells

Expression and nucleus translocation of calcyclin binding protein in colon cancer cells

Lysophosphatidic Acid Facilitates Proliferation of Colon Cancer Cells via Induction of Krüppel-like Factor 5

Among the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of beta-catenin. However, the majority of colon cancer cells have deregulation of the Wnt/beta-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA(2) and LPA(3). Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-delta. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol 12-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA.