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In vitro and in vivo antitumor activity of a novel semisynthetic derivative of cucurbitacin B.

Silva IT, Carvalho A, Lang KL, Dudek SE, Masemann D, Durán FJ, Caro MS, Rapp UR, Wixler V, Schenkel EP, Simões CM, Ludwig S - PLoS ONE (2015)

Bottom Line: Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task.DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3.Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; Institute of Molecular Virology (IMV), Center of Molecular Biology of Inflammation (ZMBE), Westfaelische-Wilhelms-University, Muenster, Germany.

ABSTRACT
Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.

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Related in: MedlinePlus

Effects of DACE on cell cycle arrest and apoptosis.(A) A549 cells (5x105) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *p<0.001 and **p<0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *p<0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *p<0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.
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pone.0117794.g003: Effects of DACE on cell cycle arrest and apoptosis.(A) A549 cells (5x105) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *p<0.001 and **p<0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *p<0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *p<0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.

Mentions: To better understand the mechanism by which cell proliferation was suppressed by DACE, we investigated the distribution of cell cycle phases of A549 cells following treatment with DACE by FACS analysis. The untreated control cells showed a typical distribution of G0/G1, G2/M, and S phases, but 24h exposure of cells to DACE caused a significant enrichment of cells in the G2/M phase in a concentration-dependent manner (Fig. 3A). An increase from 10.97±3.04% to 41.92±0.20% and 46.56±6.01% cells in G2/M phase was detected after the treatment with 0.5μM and 1μM of DACE, respectively (p<0.0001 vs. control). This was followed by a reduction in G0/G1-phase cells: from 79.75±5.58% in the controls to 46.23±4.03% and 30.88±3.18% in samples treated with 0.5μM and 1μM of DACE, respectively) (p<0.0001 vs. control). These results indicated that the compound inhibits the growth of A549 cells by arresting them in the G2/M cell cycle phase.


In vitro and in vivo antitumor activity of a novel semisynthetic derivative of cucurbitacin B.

Silva IT, Carvalho A, Lang KL, Dudek SE, Masemann D, Durán FJ, Caro MS, Rapp UR, Wixler V, Schenkel EP, Simões CM, Ludwig S - PLoS ONE (2015)

Effects of DACE on cell cycle arrest and apoptosis.(A) A549 cells (5x105) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *p<0.001 and **p<0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *p<0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *p<0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4326133&req=5

pone.0117794.g003: Effects of DACE on cell cycle arrest and apoptosis.(A) A549 cells (5x105) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *p<0.001 and **p<0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *p<0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *p<0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.
Mentions: To better understand the mechanism by which cell proliferation was suppressed by DACE, we investigated the distribution of cell cycle phases of A549 cells following treatment with DACE by FACS analysis. The untreated control cells showed a typical distribution of G0/G1, G2/M, and S phases, but 24h exposure of cells to DACE caused a significant enrichment of cells in the G2/M phase in a concentration-dependent manner (Fig. 3A). An increase from 10.97±3.04% to 41.92±0.20% and 46.56±6.01% cells in G2/M phase was detected after the treatment with 0.5μM and 1μM of DACE, respectively (p<0.0001 vs. control). This was followed by a reduction in G0/G1-phase cells: from 79.75±5.58% in the controls to 46.23±4.03% and 30.88±3.18% in samples treated with 0.5μM and 1μM of DACE, respectively) (p<0.0001 vs. control). These results indicated that the compound inhibits the growth of A549 cells by arresting them in the G2/M cell cycle phase.

Bottom Line: Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task.DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3.Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; Institute of Molecular Virology (IMV), Center of Molecular Biology of Inflammation (ZMBE), Westfaelische-Wilhelms-University, Muenster, Germany.

ABSTRACT
Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.

Show MeSH
Related in: MedlinePlus