Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of DACE on TNFα-mediated activation of signaling pathways.(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4326133&req=5

pone.0117794.g004: Effects of DACE on TNFα-mediated activation of signaling pathways.(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.

Mentions: To detect signaling pathways that might be affected by DACE, human non-small-cell lung cancer A549 cells were treated with DACE for 24h followed by stimulation with TNFα (30ng/mL) for an additional 15 min. TNF activates a broad spectrum of different signaling cascades, thus allowing narrowing down the affected pathways in the same experimental setting. As PI3K/AKT and RAS/RAF/ERK pathways are key regulators of cell survival and proliferation, and their deregulation is commonly found in tumors [4], special attention was paid to these cascades. As shown in Fig. 4A, the TNFα-mediated activation/phosphorylation of both AKT and ERK was inhibited when A549 cells were treated with either 0.5μM or 1.0μM of the compound. In contrast, the activation state of key signaling proteins after TNF stimulation, such as p38, JNK, and NF-kappaB p65 as well as the degradation of IκBα, was not significantly changed upon treatment with DACE (data not shown). To reveal whether DACE affects the AKT kinase directly, A549 cells were transiently transfected with either empty vector or a wild-type form of AKT and exposed to 0.5μM and 1.0μM of DACE for 24h. The expression of recombinant AKT kinase caused increased AKT phosphorylation in non-stimulated cells and, interestingly, this basal phosphorylation was not inhibited by DACE. In contrast, phosphorylation of endogenous AKT was completely inhibited by DACE (Fig. 4B). Overexpression of AKT also had no effect on inhibition of ERK phosphorylation by DACE. These data indicate that upstream components of the AKT signaling cascade, rather than AKT itself, are affected by DACE. However, while phosphorylation of AKT and PI3K were strongly inhibited by both 0.5μM and 1.0μM of DACE, the phosphorylation of upstream components of PI3K/AKT signaling pathway such as PTEN and PDK1 remained unaffected (Fig. 4C).


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 TNFα-mediated activation of signaling pathways.(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117794.g004: Effects of DACE on TNFα-mediated activation of signaling pathways.(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.
Mentions: To detect signaling pathways that might be affected by DACE, human non-small-cell lung cancer A549 cells were treated with DACE for 24h followed by stimulation with TNFα (30ng/mL) for an additional 15 min. TNF activates a broad spectrum of different signaling cascades, thus allowing narrowing down the affected pathways in the same experimental setting. As PI3K/AKT and RAS/RAF/ERK pathways are key regulators of cell survival and proliferation, and their deregulation is commonly found in tumors [4], special attention was paid to these cascades. As shown in Fig. 4A, the TNFα-mediated activation/phosphorylation of both AKT and ERK was inhibited when A549 cells were treated with either 0.5μM or 1.0μM of the compound. In contrast, the activation state of key signaling proteins after TNF stimulation, such as p38, JNK, and NF-kappaB p65 as well as the degradation of IκBα, was not significantly changed upon treatment with DACE (data not shown). To reveal whether DACE affects the AKT kinase directly, A549 cells were transiently transfected with either empty vector or a wild-type form of AKT and exposed to 0.5μM and 1.0μM of DACE for 24h. The expression of recombinant AKT kinase caused increased AKT phosphorylation in non-stimulated cells and, interestingly, this basal phosphorylation was not inhibited by DACE. In contrast, phosphorylation of endogenous AKT was completely inhibited by DACE (Fig. 4B). Overexpression of AKT also had no effect on inhibition of ERK phosphorylation by DACE. These data indicate that upstream components of the AKT signaling cascade, rather than AKT itself, are affected by DACE. However, while phosphorylation of AKT and PI3K were strongly inhibited by both 0.5μM and 1.0μM of DACE, the phosphorylation of upstream components of PI3K/AKT signaling pathway such as PTEN and PDK1 remained unaffected (Fig. 4C).

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