BackgroundChloroquine (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.MethodsHT-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.Results5-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 p21Cip1 and p27Kip1 and 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.ConclusionOur 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.
This study suggests that lateral node dissection is not necessary in terms of curability for patients with advanced carcinoma of the lower rectum who undergo preoperative radiotherapy.
The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).
AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy.
Familial adenomatous polyposis (FAP) of the colon is characterized by multiple polyps in the intestine and extra-colonic manifestations. Most FAP cases are caused by a germline mutation in the tumor-suppressor gene APC, but some cases of adenomatous polyposis result from germline mutations in MUTYH, POLD1 or POLE. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic FAP patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. Whole-genome sequencing and subsequent deep sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in ~12% of his peripheral leukocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data implied that genetic analysis by next-generation sequencing is an effective strategy to identify genetic mosaicism in hereditary diseases.
HER2 is a receptor tyrosine kinase, which is amplified and overexpressed in a subset of human cancers including breast and gastric cancers, and is indicated in its involvement in progression of cancer. Although its specific ligand(s) has not been detected, HER2 homodimerization, which is critical for its activation, is considered to be dependent on its expression levels. Here, we demonstrate a significant role of HER2 methylation by protein lysine methyltransferase SMYD3 in HER2 homodimerization. We found that SMYD3 trimethylates HER2 protein at lysine 175. HER2 homodimerization was enhanced in the presence of SMYD3, and substitution of lysine 175 of HER2 with alanine (HER2‐K175A) reduced the formation of HER2 homodimers. Furthermore, HER2‐K175A revealed lower level of autophosphorylation than wild‐type HER2. We also identified that knockdown of SMYD3 attenuated this autophosphorylation in breast cancer cells. Our results imply that SMYD3‐mediated methylation of HER2 at Lysine 175 may regulate the formation of HER2 homodimer and subsequent autophosphorylation and suggest that the SMYD3‐mediated methylation pathway seems to be a good target for development of novel anti‐cancer therapy.
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