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

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

Chemical structure of IMB5043 and its effect on cancer cell lines.

<p>(A) Chemical structure of IMB5043. (B) IC₅₀ of IMB5043 in various cancer cells. Cells were treated with different concentrations of IMB5043 for 24 h, and IC₅₀ is calculated. Data shown are means ± SD. (C). Effect of IMB5043 on the morphology of SMMC-7721 cells were observed by bright field microscopy (100×). (D). Effect of IMB5043 on the nucleus of SMMC-7721 cells were observed by fluorescent microscopy (200×). The nucleus was staining by Hoest33342. (E) Effect of IMB5043 on the nucleus of SMMC-7721 cells were observed by Electron Microscopy (6000×). SMMC-7721 cells were incubated with indicated concentration of IMB5043 for 24 h. The representative picture is shown.</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

Synthesis, Biological Evaluation, and Autophagy Mechanism of 12<i>N</i>‑Substituted Sophoridinamines as Novel Anticancer Agents

A series of 12<i>N</i>-substituted sophoridinamine derivatives were synthesized and evaluated for their cytotoxic activities in human HepG2 hepatoma cells. Structure–activity relationship revealed that introduction of a suitable arylidene or arylethyl at the <i>N</i>′-end could greatly enhance antiproliferation potency. Among them, compound <b>6b</b> possessing a <i>N</i>′-trimethoxyphenyl methylene exhibited potent antiproliferation effect against three human tumor cell lines including HepG2, leukemia (K562), and breast cancer (HMLE), with IC₅₀ between 0.55 and 1.7 μM. The underlying mechanism of <b>6b</b> against tumor cells is to block autophagic flux, mainly through neutralizing lysosomal acidity. Our results indicated that compound <b>6b</b> is a potent lysosomal deacidification agent and is accordingly able to block autophagic flux and inhibit tumor cell growth

IMB5043 induces apoptosis of in SMMC-7721 cells.

<p>(A). Flow cytometry analysis of apoptosis in SMMC-7721 cells treated with IMB5043. Representative pictures from three experiments is shown. (B). Quantification of apoptosis rate in SMMC-7721 cells treated with IMB5043. Cells were stained with FITC-Annexin V/PI. Apoptosis is determined by the percentages of FITC-Annexin V⁺ cells. The lower-right quadrant shows the early apoptotic cells, the upper-right quadrant shows necrotic or late apoptotic cells, and the upper-left quadrant shows necrotic cells or nuclear debris, and the lower-left quadrant shows healthy, viable cells. *P<0.05; **P<0.01. (C). Western blot showed the levels of apoptosis-related proteins including in SMMC-7721 cells. SMMC-7721cells were treated with IMB5043 at indicated concentrations for 24 h, then caspase-3, 7, and PARP was detected. Representative images of three independent experiments are shown. (D) The histogram shows the relative density of proteins shown in (C).Data represents the mean ± SD of three independent experiments. Results were derived from three independent experiments. *P<0.05; **P<0.01.</p

IMB5043 leads to cell DNA breakage, γ-H2AX foci formation.

<p>(A). p-H2AX was up-regulated by IMB5043 detected by Western blot. SMMC-7721 cells were treated with indicated doses of IMB5043 for 24 hours. (B). Quantification of p-H2AX expression treated with IMB5043 in SMMC-7721 cells. Data shown is means ± SD. **P<0.01. (C). Comet electrophoresis assays were performed to detect the DNA double strand breaks. SMMC-7721 cells were treated with indicated doses of IMB5043 for 6 hours. (D). Quantitative analysis of relative tail moment was performed with the comet analysis software. Data shown is means ± SD. **P<0.01. (E). γ-H2AX immunofluorescence foci was tested with γ-H2AX antibody conjugated with FITC. SMMC-7721 cells were treated with indicated doses of IMB5043 for 24 hours. (F). Quantification of γ-H2AX foci. Data shown is means ± SD. **P<0.01.</p