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Icariin displays anticancer activity against human esophageal cancer cells via regulating endoplasmic reticulum stress-mediated apoptotic signaling.

Fan C, Yang Y, Liu Y, Jiang S, Di S, Hu W, Ma Z, Li T, Zhu Y, Xin Z, Wu G, Han J, Li X, Yan X - Sci Rep (2016)

Bottom Line: ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs.The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment.In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, 1 Xinsi Road, Xi'an 710038, China.

ABSTRACT
In this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

No MeSH data available.


Related in: MedlinePlus

Effect of ICA combined with THA on cell viability, Caspase 9 activity, ROS induction, NADPH oxidase activity, and p-eIF2α levels in human ESCC cells.(A) Viability is expressed as OD values. (B) The intracellular Caspase 9 activity levels are shown. (C) ROS concentrations are shown. (D) NADPH oxidase activity is shown. The two indexes in the control group were defined as 100%. (E) Representative Western blot results of p-eIF2α, CHOP, Bcl2, and PUMA are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. The results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the THA 0.5 μM-treated group; cP < 0.05 vs. the ICA 40 μM-treated group.
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f8: Effect of ICA combined with THA on cell viability, Caspase 9 activity, ROS induction, NADPH oxidase activity, and p-eIF2α levels in human ESCC cells.(A) Viability is expressed as OD values. (B) The intracellular Caspase 9 activity levels are shown. (C) ROS concentrations are shown. (D) NADPH oxidase activity is shown. The two indexes in the control group were defined as 100%. (E) Representative Western blot results of p-eIF2α, CHOP, Bcl2, and PUMA are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. The results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the THA 0.5 μM-treated group; cP < 0.05 vs. the ICA 40 μM-treated group.

Mentions: THA, an irreversible inhibitor of sarcoplasmic reticulum/ER calcium ATPase (SERCA), induces calcium signaling and the UPR, thereby significantly decreasing cancer cell viability via apoptosis3334. We combined ICA (40 μM) with THA and determined the cytotoxicity to EC109 and TE1 cells. The concentration and duration of THA treatment (0.5 μM for 24 h) that effectively enhanced ERS activity without affecting cell viability were selected based on our preliminary experiments. As shown in Fig. 8A, the OD value in the ICA treatment group was 0.91 ± 0.07 in EC109 cells and 0.87 ± 0.06 in TE1 cells, and THA treatment alone slightly increased the OD value to 1.16 ± 0.09 in EC109 cells and 0.98 ± 0.07 in TE1 cells. However, when ICA was combined with THA, the OD value was significantly decreased to 0.67 ± 0.08 in EC109 cells and 0.59 ± 0.04 in TE1 cells. The combination index (CI) was calculated according to a previous study35; CI < 1 indicates synergistic activity, CI = 1 indicates additive activity, and CI > 1 indicates antagonistic activity. The CIs in the two ESCC cell lines used in this study were approximately less than 1 (0.86 in EC109 and 0.79 in TE1), indicating that the combination of ICA and THA exerted a synergistic effect. The combination of ICA and THA significantly increased Caspase 9 activity (Fig. 8B), ROS generation (Fig. 8C), and NADPH oxidase activity (Fig. 8D) (P < 0.05 compared with ICA or THA alone). As shown in Fig. 8E, THA significantly increased PUMA expression in ESCC cells (P < 0.05 compared with the control), and treatment with both ICA and THA in EC109 and TE1 cells increased PUMA expression compared with either treatment alone (P < 0.05). Moreover, treatments of ICA and THA upregulated p-eIF2α and CHOP and downregulated Bcl2 (P < 0.05 compared with ICA or THA treatment alone). Additionally, both N-linked glycosylation inhibitor tunicamycin (TM)3637 and strong reducing agent dithiothreitol (DTT)38, another two ERS inducers, further upregulated p-PERK, CHOP, and PUMA and downregulated Bcl2 (P < 0.05 compared with ICA, TM, or DTT treatment alone) (Supplementary Fig. 7).


Icariin displays anticancer activity against human esophageal cancer cells via regulating endoplasmic reticulum stress-mediated apoptotic signaling.

Fan C, Yang Y, Liu Y, Jiang S, Di S, Hu W, Ma Z, Li T, Zhu Y, Xin Z, Wu G, Han J, Li X, Yan X - Sci Rep (2016)

Effect of ICA combined with THA on cell viability, Caspase 9 activity, ROS induction, NADPH oxidase activity, and p-eIF2α levels in human ESCC cells.(A) Viability is expressed as OD values. (B) The intracellular Caspase 9 activity levels are shown. (C) ROS concentrations are shown. (D) NADPH oxidase activity is shown. The two indexes in the control group were defined as 100%. (E) Representative Western blot results of p-eIF2α, CHOP, Bcl2, and PUMA are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. The results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the THA 0.5 μM-treated group; cP < 0.05 vs. the ICA 40 μM-treated group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4759694&req=5

f8: Effect of ICA combined with THA on cell viability, Caspase 9 activity, ROS induction, NADPH oxidase activity, and p-eIF2α levels in human ESCC cells.(A) Viability is expressed as OD values. (B) The intracellular Caspase 9 activity levels are shown. (C) ROS concentrations are shown. (D) NADPH oxidase activity is shown. The two indexes in the control group were defined as 100%. (E) Representative Western blot results of p-eIF2α, CHOP, Bcl2, and PUMA are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. The results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the THA 0.5 μM-treated group; cP < 0.05 vs. the ICA 40 μM-treated group.
Mentions: THA, an irreversible inhibitor of sarcoplasmic reticulum/ER calcium ATPase (SERCA), induces calcium signaling and the UPR, thereby significantly decreasing cancer cell viability via apoptosis3334. We combined ICA (40 μM) with THA and determined the cytotoxicity to EC109 and TE1 cells. The concentration and duration of THA treatment (0.5 μM for 24 h) that effectively enhanced ERS activity without affecting cell viability were selected based on our preliminary experiments. As shown in Fig. 8A, the OD value in the ICA treatment group was 0.91 ± 0.07 in EC109 cells and 0.87 ± 0.06 in TE1 cells, and THA treatment alone slightly increased the OD value to 1.16 ± 0.09 in EC109 cells and 0.98 ± 0.07 in TE1 cells. However, when ICA was combined with THA, the OD value was significantly decreased to 0.67 ± 0.08 in EC109 cells and 0.59 ± 0.04 in TE1 cells. The combination index (CI) was calculated according to a previous study35; CI < 1 indicates synergistic activity, CI = 1 indicates additive activity, and CI > 1 indicates antagonistic activity. The CIs in the two ESCC cell lines used in this study were approximately less than 1 (0.86 in EC109 and 0.79 in TE1), indicating that the combination of ICA and THA exerted a synergistic effect. The combination of ICA and THA significantly increased Caspase 9 activity (Fig. 8B), ROS generation (Fig. 8C), and NADPH oxidase activity (Fig. 8D) (P < 0.05 compared with ICA or THA alone). As shown in Fig. 8E, THA significantly increased PUMA expression in ESCC cells (P < 0.05 compared with the control), and treatment with both ICA and THA in EC109 and TE1 cells increased PUMA expression compared with either treatment alone (P < 0.05). Moreover, treatments of ICA and THA upregulated p-eIF2α and CHOP and downregulated Bcl2 (P < 0.05 compared with ICA or THA treatment alone). Additionally, both N-linked glycosylation inhibitor tunicamycin (TM)3637 and strong reducing agent dithiothreitol (DTT)38, another two ERS inducers, further upregulated p-PERK, CHOP, and PUMA and downregulated Bcl2 (P < 0.05 compared with ICA, TM, or DTT treatment alone) (Supplementary Fig. 7).

Bottom Line: ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs.The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment.In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, 1 Xinsi Road, Xi'an 710038, China.

ABSTRACT
In this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

No MeSH data available.


Related in: MedlinePlus