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A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells.

Chu B, Liu F, Li L, Ding C, Chen K, Sun Q, Shen Z, Tan Y, Tan C, Jiang Y - Cell Death Dis (2015)

Bottom Line: We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis.The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo.On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Tsinghua University, Beijing 100084, People's Republic of China [2] The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, People's Republic of China.

ABSTRACT
Aberrant expression or function of epidermal growth factor receptor (EGFR) or the closely related human epidermal growth factor receptor 2 (HER2) can promote cell proliferation and survival, thereby contributing to tumorigenesis. Specific antibodies and low-molecular-weight tyrosine kinase inhibitors of both proteins are currently in clinical trials for cancer treatment. Benzimidazole derivatives possess diverse biological activities, including antitumor activity. However, the anticancer mechanism of 5a (a 2-aryl benzimidazole compound; 2-chloro-N-(2-p-tolyl-1H-benzo[d]imidazol-5-yl)acetamide, C(16)H(14)ClN(3)O, MW299), a novel 2-aryl benzimidazole derivative, toward breast cancer is largely unknown. Here, we demonstrate that 5a potently inhibited both EGFR and HER2 activity by reducing EGFR and HER2 tyrosine phosphorylation and preventing downstream activation of PI3K/Akt and MEK/Erk pathways in vitro and in vivo. We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis. Moreover, 5a potently induced apoptosis via the c-Jun N-terminal kinase (JNK)-mediated death receptor 5 upregulation in breast cancer cells. The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo. Analysis of the primary breast cancer cell lines with HER2 overexpression further confirmed that 5a significantly inhibited Akt Ser473 and Bad Ser136 phosphorylation and reduced cyclin D3 expression. On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

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5A induced G1 arrest and apoptosis by inhibiting EGFR and HER2 activity. (a) MDA-MB-453 and HCC1937 cells were treated with 5 μM 5a or dimethylsulfoxide (DMSO) for 24 h. The fractions of cells in G1, S and G2/M were determined by flow cytometry. (b) BT-474 and MDA-MB-468 cells were treated with 5a for 8 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (c) BT-474 and MDA-MB-468 cells were treated with 5a for 16 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (d) MDA-MB-453 cells were treated with 10 μM 5a for 24, 30 or 36 h. Cytosolic and mitochondrial fractions were isolated to examine the distribution of cytochrome c. β-Actin and COX-IV (cytochrome c oxidase subunit 4) were used as the cytosolic and mitochondrial markers, respectively. (e) BT-474 cells were transfected with control or HER2 siRNA for 48 h and then treated with 10 μM 5a for 14 h, cell lysates were analyzed by immunoblotting with the antibodies indicated
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fig3: 5A induced G1 arrest and apoptosis by inhibiting EGFR and HER2 activity. (a) MDA-MB-453 and HCC1937 cells were treated with 5 μM 5a or dimethylsulfoxide (DMSO) for 24 h. The fractions of cells in G1, S and G2/M were determined by flow cytometry. (b) BT-474 and MDA-MB-468 cells were treated with 5a for 8 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (c) BT-474 and MDA-MB-468 cells were treated with 5a for 16 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (d) MDA-MB-453 cells were treated with 10 μM 5a for 24, 30 or 36 h. Cytosolic and mitochondrial fractions were isolated to examine the distribution of cytochrome c. β-Actin and COX-IV (cytochrome c oxidase subunit 4) were used as the cytosolic and mitochondrial markers, respectively. (e) BT-474 cells were transfected with control or HER2 siRNA for 48 h and then treated with 10 μM 5a for 14 h, cell lysates were analyzed by immunoblotting with the antibodies indicated

Mentions: Following 5a treatment, two types of breast cancer cells (HER2-positive and EGFR-positive) were noticeably arrested in the G1 phase of the cell cycle, with a concomitant loss of the S- and G2/M-phase cell populations (Figure 3a). The effects of 5a on protein expression of INK4 (p15 and p18) and CIP/KIP (p21 and p27) families of cyclin-dependent kinase (CDK) inhibitors were studied, as they are negative regulators of the G1/S-phase progression. Interestingly, it was demonstrated that 5a could significantly upregulate protein expression of p27 and p21, which control the cell cycle progression at G1, but could not alter the expression of p18 and p15 in breast cancer cells. Furthermore, E2F1, CDK4 and cyclin D, which are also important for cell cycle progression, decreased in response to 5a treatment. However, 5a showed no obvious effect on the protein levels of CDK1 and CDK2 (Figure 3b and Supplementary Figure S3). Therefore, the downregulation of E2F1, CDK4, cyclin D1 and cyclin D3 and upregulation of p27 and p21 in breast cancer cells likely contributed to the G1 cell cycle arrest induced by 5a.


A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells.

Chu B, Liu F, Li L, Ding C, Chen K, Sun Q, Shen Z, Tan Y, Tan C, Jiang Y - Cell Death Dis (2015)

5A induced G1 arrest and apoptosis by inhibiting EGFR and HER2 activity. (a) MDA-MB-453 and HCC1937 cells were treated with 5 μM 5a or dimethylsulfoxide (DMSO) for 24 h. The fractions of cells in G1, S and G2/M were determined by flow cytometry. (b) BT-474 and MDA-MB-468 cells were treated with 5a for 8 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (c) BT-474 and MDA-MB-468 cells were treated with 5a for 16 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (d) MDA-MB-453 cells were treated with 10 μM 5a for 24, 30 or 36 h. Cytosolic and mitochondrial fractions were isolated to examine the distribution of cytochrome c. β-Actin and COX-IV (cytochrome c oxidase subunit 4) were used as the cytosolic and mitochondrial markers, respectively. (e) BT-474 cells were transfected with control or HER2 siRNA for 48 h and then treated with 10 μM 5a for 14 h, cell lysates were analyzed by immunoblotting with the antibodies indicated
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fig3: 5A induced G1 arrest and apoptosis by inhibiting EGFR and HER2 activity. (a) MDA-MB-453 and HCC1937 cells were treated with 5 μM 5a or dimethylsulfoxide (DMSO) for 24 h. The fractions of cells in G1, S and G2/M were determined by flow cytometry. (b) BT-474 and MDA-MB-468 cells were treated with 5a for 8 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (c) BT-474 and MDA-MB-468 cells were treated with 5a for 16 h and 12 h, respectively, with different concentrations as indicated; cell lysates were analyzed by immunoblotting with the antibodies indicated. (d) MDA-MB-453 cells were treated with 10 μM 5a for 24, 30 or 36 h. Cytosolic and mitochondrial fractions were isolated to examine the distribution of cytochrome c. β-Actin and COX-IV (cytochrome c oxidase subunit 4) were used as the cytosolic and mitochondrial markers, respectively. (e) BT-474 cells were transfected with control or HER2 siRNA for 48 h and then treated with 10 μM 5a for 14 h, cell lysates were analyzed by immunoblotting with the antibodies indicated
Mentions: Following 5a treatment, two types of breast cancer cells (HER2-positive and EGFR-positive) were noticeably arrested in the G1 phase of the cell cycle, with a concomitant loss of the S- and G2/M-phase cell populations (Figure 3a). The effects of 5a on protein expression of INK4 (p15 and p18) and CIP/KIP (p21 and p27) families of cyclin-dependent kinase (CDK) inhibitors were studied, as they are negative regulators of the G1/S-phase progression. Interestingly, it was demonstrated that 5a could significantly upregulate protein expression of p27 and p21, which control the cell cycle progression at G1, but could not alter the expression of p18 and p15 in breast cancer cells. Furthermore, E2F1, CDK4 and cyclin D, which are also important for cell cycle progression, decreased in response to 5a treatment. However, 5a showed no obvious effect on the protein levels of CDK1 and CDK2 (Figure 3b and Supplementary Figure S3). Therefore, the downregulation of E2F1, CDK4, cyclin D1 and cyclin D3 and upregulation of p27 and p21 in breast cancer cells likely contributed to the G1 cell cycle arrest induced by 5a.

Bottom Line: We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis.The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo.On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Tsinghua University, Beijing 100084, People's Republic of China [2] The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, People's Republic of China.

ABSTRACT
Aberrant expression or function of epidermal growth factor receptor (EGFR) or the closely related human epidermal growth factor receptor 2 (HER2) can promote cell proliferation and survival, thereby contributing to tumorigenesis. Specific antibodies and low-molecular-weight tyrosine kinase inhibitors of both proteins are currently in clinical trials for cancer treatment. Benzimidazole derivatives possess diverse biological activities, including antitumor activity. However, the anticancer mechanism of 5a (a 2-aryl benzimidazole compound; 2-chloro-N-(2-p-tolyl-1H-benzo[d]imidazol-5-yl)acetamide, C(16)H(14)ClN(3)O, MW299), a novel 2-aryl benzimidazole derivative, toward breast cancer is largely unknown. Here, we demonstrate that 5a potently inhibited both EGFR and HER2 activity by reducing EGFR and HER2 tyrosine phosphorylation and preventing downstream activation of PI3K/Akt and MEK/Erk pathways in vitro and in vivo. We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis. Moreover, 5a potently induced apoptosis via the c-Jun N-terminal kinase (JNK)-mediated death receptor 5 upregulation in breast cancer cells. The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo. Analysis of the primary breast cancer cell lines with HER2 overexpression further confirmed that 5a significantly inhibited Akt Ser473 and Bad Ser136 phosphorylation and reduced cyclin D3 expression. On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

Show MeSH
Related in: MedlinePlus