Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells

<p>Abstract</p> <p>Background</p> <p>Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells.</p> <p>Methods</p> <p>HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed.</p> <p>Results</p> <p>5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21^Cip1and p27^Kip1and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU.</p> <p>Conclusion</p> <p>Our findings suggest that the combination therapy with CQ should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy, possibly by inhibiting autophagy-dependent resistance to chemotherapy.</p

The miR-1-NOTCH3-Asef Pathway Is Important for Colorectal Tumor Cell Migration

<div><p>The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC stimulates the activity of the Cdc42- and Rac1-specific guanine nucleotide exchange factor Asef and promotes the migration and invasion of colorectal tumor cells. Furthermore, Asef is overexpressed in colorectal tumors and is required for colorectal tumorigenesis. It is also known that NOTCH signaling plays critical roles in colorectal tumorigenesis and fate determination of intestinal progenitor cells. Here we show that NOTCH3 up-regulates Asef expression by activating the Asef promoter in colorectal tumor cells. Moreover, we demonstrate that microRNA-1 (miR-1) is down-regulated in colorectal tumors and that miR-1 has the potential to suppress NOTCH3 expression through direct binding to its 3’-UTR region. These results suggest that the miR-1-NOTCH3-Asef pathway is important for colorectal tumor cell migration and may be a promising molecular target for the treatment of colorectal tumors.</p> </div

Involvement of endothelial DLL4 in NOTCH3/Asef-mediated cell migration.

<p>(A) Suppression of DLL4 expression by siRNA. Lysates prepared from HUVECs transfected with an siRNA targeting DLL4 or control siRNA were analysed by immunoblot with antibodies against DLL4. Antibodies against α-tubulin were used as a control. Arrowhead shows DLL4 band; asterisk indicates a non-specific band. (B) HUVECs transfected with an siRNA targeting DLL4 or control siRNA were grown to confluency on polycarbonate filters in the Transwell chambers. DLD-1 cells were subjected to migration assays. (C) DLD-1 cells that had been transfected with Pre-miR-1 were also transfected with either N3ICD or Asef and assayed for migration activity. Results obtained are expressed as the means ±SD of at least three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

NOTCH3/CSL transactivates the Asef promoter.

<p>(A) Schematic representation of the Asef-b promoter region. Predicted CSL-binding sites are indicated by boxes P, the regions corresponding to the primers used for ChIP assays. TSS, Transcription start site. (B) (Left) Schematic diagrams of reporter constructs used for luciferase assays. Fragments of the Asef-b promoter were cloned upstream of the luciferase (Luc) gene. Potential CSL-binding sites are indicated by gray boxes. (Right) Caco-2 cells were transfected with N3ICD or control vector along with reporter constructs containing Asef-b promoter sequences and subjected to luciferase assays. pRL-TK vector was used as an internal control. (C) ChIP assays were performed on DLD-1 cells using anti-CSL antibody or non-specific IgG. The promoter region of <i>Asef-b</i> was enhanced in the immunoprecipitates. The promoter regions of <i>GAPDH</i> and <i>Hes1</i> were used as negative and positive controls, respectively. Results are expressed as the means of the percentage of the input ± SD. *<i>p</i> < 0.05.</p

NOTCH3 is a target of <i>miR-1</i>.

<p>(A) <i>miR-1</i> is downregulated in human colorectal cancer tissues. qRT-PCR was performed with 27 pairs of human normal and tumor colon tissues. (B) DLD-1 cells were transfected with Pre-miR-control or -miR-1, and subjected to immunoblotting analysis with the indicated antibodies (Left) and qRT-PCR analysis of the indicated genes (Right). α-tubulin was used as a control. (C) The predicted <i>miR-1</i>-binding site in the <i>NOTCH3</i> 3’UTR. Sequences of <i>miR-1</i> and wild-type (WT-3’UTR) and mutated (Mut-3’UTR) <i>miR-1</i>-binding sites are shown. Mutated bases in Mut-3’UTR are marked by gray boxes. Bases in WT- and Mut-3’UTR complementary to those in <i>miR-1</i> are indicated by lines. (D) DLD-1 cells were co-transfected with a reporter plasmid and Pre-miR-control or -miR-1, and lysates were subsequently assayed for luciferase. *<i>p</i> < 0.05.</p

Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia

MYU, a Target lncRNA for Wnt/c-Myc Signaling, Mediates Induction of CDK6 to Promote Cell Cycle Progression

Aberrant activation of Wnt/β-catenin signaling is a major driving force in colon cancer. Wnt/β-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU, and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU/hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells