<i>INMAP</i> Overexpression Inhibits Cell Proliferation, Induces Genomic Instability and Functions through p53/p21 Pathways

<div><p>INMAP is a spindle protein that plays essential role for mitosis, by ensuring spindle and centromere integrality. The aim of this study was to investigate the relevant functions of INMAP for genomic stability and its functional pathway. We overexpressed <i>INMAP</i> in HeLa cells, resulting in growth inhibition in monolayer cell cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice. In this system caused micronuclei (MNi) formation, chromosome distortion and <i>γH2AX</i> expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a tumour biochemical marker, lactate dehydrogenase (LDH) isoenzymes were detected to evaluate cell metabolic activity, the results confirming that total activity of LDH, as well as that of its LDH5 isoform, is significantly decreased in <i>INMAP</i>-overexpressing HeLa cells. The levels of p53 and p21 were upregulated, and however, that of PCNA and Bcl-2, downregulated. Indirect immunofluorescence (IIF) and coimmunoprecipitation (CoIP) analyses revealed the interaction between INMAP and p21. These results suggest that INMAP might function through p53/p21 pathways.</p></div

A new compound of thiophenylated pyridazinone IMB5043 showing potent antitumor efficacy through ATM-Chk2 pathway

<div><p>Through cell-based screening models, we have identified a new compound IMB5043, a thiophenylated pyridazinone, which exerted cytotoxicity against cancer cells. In the present study, we evaluated its antitumor efficacy and the possible mechanism. By MTT assay, IMB5043 inhibited the proliferation of various human cancer cells lines, especially hepatocarcinoma SMMC-7721 cells. IMB5043 blocked cell cycle with G₂/M arrest, induced cell apoptosis, and inhibited the migration and invasion of SMMC-7721 cells. As verified by comet assay and γ-H2AX foci formation, IMB5043 caused DNA damage and activated ATM, Chk2 and p53 through phosphorylation. As shown by Gene microarray analysis, the differentially expressed genes in SMMC-7721 cells treated with IMB5043 were highly related to cell death and apoptosis. IMB5043 suppressed the growth of hepatocarcinoma SMMC-7721 xenograft in athymic mice. By histopathological examination, no lesions were found in bone marrow and various organs of the treated mice. Our findings reveal that IMB5043 as an active compound consisting of both pyridazinone and thiophene moieties exerts antitumor efficacy through activation of ATM-Chk2 pathway. IMB5043 may serve as a promising leading compound for the development of antitumor drugs.</p></div

IMB5043 induces DNA damage repair and activated ATM-Chk2 pathway.

<p>SMMC7721 cells were treated with indicated doses of IMB5043 for 24 hours. Damage-associated proteins were detected by Western blot analysis and the quantitative analysis was displayed. Data shown is means ± SD. *P<0.05; **P<0.01.</p

IMB5043 arrests G₂/M phase and decreases the motility and invasion of SMMC-7721 cells.

<p>(A). Cell cycle distribution was determined by flow cytometry after PI staining. Representative pictures from three experiments are shown. (B). Quantification of cell population in different phases of cell cycle treated with IMB5043. SMMC-7721 cells were treated with indicated doses of IMB5043 for 24 hours. Data represents the mean ± SD of three independent experiments. (C) IMB5043 decreases the motility of SMMC-7721 cells by wound closure assay. Representative pictures from three experiments were taken under microscopy after the incision and treated with 24 h, 48 h of 1 μM IMB5043 (x100). (D) Quantification of healing capability of SMMC-7721 cells after treated with IMB5043. *P<0.05. (E) IMB5043 decreases the migration of SMMC-7721 cells by transwell assay. Representative pictures from three experiments were taken under microscopy. SMMC-7721 cells were treated with the indicated concentrations of IMB5043 for 16 h. Cells that migrated through the transwell membrane were stained with hematoxylin (200×). (F) Quantification of migrated cells treated with IMB5043 in SMMC-7721 cells. Data shown is means ± SD of the three independent experiments. **P<0.01. (G) Quantification of invaded cells treated with IMB5043 in SMMC-7721 cells. *P<0.05; **P<0.01. Data represents the means ± SD of the three independent experiments.</p

GO biological process and KEGG pathway analysis of the differentially express genes after IMB5043 treatment.

<p>(A). The top ten down-regulated terms in GO biological process classification. (B). The top ten significant up-regulated pathways in KEGG pathway analysis. (C). The top ten significant down-regulated pathways in KEGG pathway analysis. SMMC-7721 cells were treated with 1 μM IMB5043 for 24 h. The total RNA is isolated and used for gene microarray analysis.</p

<i>INMAP</i> overexpression inhibited tumour growth <i>in vivo</i>.

<p>(A). Control (HeLa and Flag-HeLa) and overexpressing <i>INMAP</i> (Flag-INMAP) cells were injected into Balb/c nude mice (n = 5) in the back (near right axillary). 40 d later, the tumour xenografts were stripped. (B-D). Average tumour weights and sizes and mouse body weights were determined, respectively. No significant differences were detected. (E). Western blot analysis of the tumour tissues with anti-PCNA, anti-Bcl-2 antibodies. GAPDH was used as an internal control. (F). The histopathological analysis of the tumour tissues from different groups with H&E staining. Scale bars, 20 μm (a, c, e) and 5 μm (b, d, f). Larger necrotic areas were found in <i>INMAP</i>-overexpressing tumour tissue. (G). Morphological characteristics of normal (a) and abnormal (b) livers in nude mice. Inflammatory areas of the liver are labelled with arrowheads. (H). The histopathological analysis of normal (a, b) and abnormal (c, d) livers. Multiple necrotic areas, strongly stained nuclei, and infiltrated neutrophils were observed in abnormal livers. Scale bars, 20 μm (a, c) and 5 μm (b, d).</p

IMB5043 induces DNA damage repair and activated ATM-Chk2 pathway.

<p>SMMC7721 cells were treated with indicated doses of IMB5043 for 24 hours. Damage-associated proteins were detected by Western blot analysis and the quantitative analysis was displayed. Data shown is means ± SD. *P<0.05; **P<0.01.</p

Effects of <i>INMAP</i> overexpression on the expression of genes related to proliferation and apoptosis.

<p>(A). Western blot analysis of the three groups of cells (HeLa, Flag-HeLa, and Flag-INMAP) with anti-Bcl-2, anti-p53, anti-p21 and anti-PCNA antibodies. Data shown are representative of three independent experiments. (B). Band quantification was analysed with Image J software. (C). A schematic model illustrating the proposed mechanisms of <i>INMAP</i>-overexpression-induced DNA damage and apoptosis through p53-dependent pathways in transformed cells.</p

<i>In vivo</i> antitumor activity.

<p>(A). Tumor growing curves of human hepatoma cells SMMC7721 xenograft model in athymic mice (n = 6). IMB5043 was given on days 14–18 & 21–24. **P<0.01 (B). Body weight change of SMMC7721 xenograft-bearing mice. (C). Histopathological examination (hematoxylin and eosin stain) of mice treated with IMB5043 (25 mg/kg). No toxicological damage was found in the heart, lung, liver, small intestine, kidney, spleen, and femur bone marrow. (D). γ-H2AX immunofluorescence staining of frozen tumor sections from control groups and 25 mg/kg IMB5043 group. n = 6 mice per group.</p

<i>INMAP</i> overexpression suppressed the growth of HeLa cells.

<p>(A, B). Colony formation of HeLa cells in monolayer cultures. Cells were stained with 10% Giemsa stain. Data are presented as mean ± SD; n = 4. ** and * represent significant differences with P-values under 0.01 and 0.05, respectively (the same below). NS, not significant. (C, D). Anchorage-independent colony growth assay in soft agar. Cells were seeded in 12-well plates containing 0.3% soft agar. After 6 weeks, the number of colonies with more than 50 cells was counted. Frequency of colonies of HeLa cells stably overexpressing <i>INMAP</i> in soft agar was analysed. Scale bars, 50 μm.</p