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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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Effects of DACE on c-RAF-1-induced lung tumor growth in mice.(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (n = 4) and DACE-treated mice (n = 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*p<0.05, t test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (n = 4) and DACE-treated mice (n = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*p<0.05, t test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (n = 4) and DACE-treated mice (n = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by t test. (p>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).
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pone.0117794.g005: Effects of DACE on c-RAF-1-induced lung tumor growth in mice.(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (n = 4) and DACE-treated mice (n = 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*p<0.05, t test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (n = 4) and DACE-treated mice (n = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*p<0.05, t test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (n = 4) and DACE-treated mice (n = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by t test. (p>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).

Mentions: To study the in vivo relevance of DACE in preventing tumor growth, we injected the compound into the peritoneal cavity of c-RAF-1-BxB mice. These transgenic mice develop multiple adenomas of type II pneumocyte origin within 4 months after birth. Similar to NIH3T3(v-RAF) cells, these mice contain a truncated oncogenic version of the human RAF kinase, the expression of which, however, is restricted to type II lung epithelial cells, as the c-RAF-1-BxB transgene is driven by the surfactant protein-C (SP-C) promoter [28]. Thus, the development of NSCLC–like tumors by these mice is driven mainly by the oncogenic human RAF kinase. Histological analyses of H&E stained lung tissue of 4-month-old mice that have been treated with 1mg/kg of DACE every second day for 21 days revealed a slight decrease in the number and size of tumor foci (16% tumor reduction was measured, data not shown). In addition, immunohistochemical study of lung sections with an antibody recognizing the human c-RAF-1 kinase confirmed that tumor but not normal lung tissue was affected and that the DMSO control did not show any effect (Fig. 5A). At this dose and scheme of treatment, DACE was well tolerated (data not shown). However, when DACE was administrated at the same dose (1mg/kg) and for the same time period (21 days), but every single day, which was still tolerated by the animals, the size of c-RAF-BxB-1-positive tumor foci was significantly reduced compared to the control-treated mice (Fig. 5A-C). As the development of lung adenomas in these mice mainly depends on expression of the oncogenic human c-RAF-1-BxB [28], it is conceivable that the amount of c-RAF-1-BxB protein or mRNA in lungs will be directly proportional to the amount of tumor tissue. Indeed, not only the number of tumor colonies and their size were reduced after treatment of mice with DACE, but also the amount of c-RAF-BxB protein and mRNA (Fig. 5D-G) and the measured amounts of tumor tissue correlated very well with the amounts of expressed human c-RAF-1-BxB protein and mRNA (Fig. 5F-5H).


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 c-RAF-1-induced lung tumor growth in mice.(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (n = 4) and DACE-treated mice (n = 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*p<0.05, t test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (n = 4) and DACE-treated mice (n = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*p<0.05, t test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (n = 4) and DACE-treated mice (n = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by t test. (p>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).
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Related In: Results  -  Collection

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

pone.0117794.g005: Effects of DACE on c-RAF-1-induced lung tumor growth in mice.(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (n = 4) and DACE-treated mice (n = 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*p<0.05, t test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (n = 4) and DACE-treated mice (n = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*p<0.05, t test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (n = 4) and DACE-treated mice (n = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by t test. (p>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).
Mentions: To study the in vivo relevance of DACE in preventing tumor growth, we injected the compound into the peritoneal cavity of c-RAF-1-BxB mice. These transgenic mice develop multiple adenomas of type II pneumocyte origin within 4 months after birth. Similar to NIH3T3(v-RAF) cells, these mice contain a truncated oncogenic version of the human RAF kinase, the expression of which, however, is restricted to type II lung epithelial cells, as the c-RAF-1-BxB transgene is driven by the surfactant protein-C (SP-C) promoter [28]. Thus, the development of NSCLC–like tumors by these mice is driven mainly by the oncogenic human RAF kinase. Histological analyses of H&E stained lung tissue of 4-month-old mice that have been treated with 1mg/kg of DACE every second day for 21 days revealed a slight decrease in the number and size of tumor foci (16% tumor reduction was measured, data not shown). In addition, immunohistochemical study of lung sections with an antibody recognizing the human c-RAF-1 kinase confirmed that tumor but not normal lung tissue was affected and that the DMSO control did not show any effect (Fig. 5A). At this dose and scheme of treatment, DACE was well tolerated (data not shown). However, when DACE was administrated at the same dose (1mg/kg) and for the same time period (21 days), but every single day, which was still tolerated by the animals, the size of c-RAF-BxB-1-positive tumor foci was significantly reduced compared to the control-treated mice (Fig. 5A-C). As the development of lung adenomas in these mice mainly depends on expression of the oncogenic human c-RAF-1-BxB [28], it is conceivable that the amount of c-RAF-1-BxB protein or mRNA in lungs will be directly proportional to the amount of tumor tissue. Indeed, not only the number of tumor colonies and their size were reduced after treatment of mice with DACE, but also the amount of c-RAF-BxB protein and mRNA (Fig. 5D-G) and the measured amounts of tumor tissue correlated very well with the amounts of expressed human c-RAF-1-BxB protein and mRNA (Fig. 5F-5H).

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