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. 2012 Jul 11:12:93.
doi: 10.1186/1472-6882-12-93.

NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death

Chuan Bian Lim  1 , Pan You Fu, Nung Ky, Hong Shuang Zhu, XiaoLing Feng, Jinming Li, Kandhadayar Gopalan Srinivasan, Mohamed Sabry Hamza, Yan Zhao
Affiliations

NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death

Chuan Bian Lim et al. BMC Complement Altern Med. .

Abstract (VSports最新版本)

Background: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy VSports手机版. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained. .

Methods: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers V体育安卓版. .

Results: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers V体育ios版. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death. .

Conclusions: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway. VSports最新版本.

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"VSports" Figures

Figure 1
Figure 1
Helenalin induces cytotoxicity in A2780 human ovarian cancer cell line Phase contrast micrographs of A2780 cells treated with increasing concentrations of helenalin of 0.5, 1.0 and 2uM for 24 h. Changes in cell number and morphology were observed when compared to DMSO control. (B) Percent of viable cells after treatment with increasing concentrations of helenalin (serial dilution for drug concentration ranging from 10uM to 0.001uM) using the MTT assay. (C) Clonogenic survival assay. A2780 cells were treated with increasing concentrations of helenalin and then incubated for 2 weeks. The cells were stained with crystal violet for 16 h. Representative dishes are shown. All results represent data from three independent experiments.
Figure 2
Figure 2
Helenalin induces cell cycle arrest in G1 phase and modulates apoptosis in a dose-dependent manner. (A) 1.85 × 105 cells/ml were seeded onto 6-well plates and incubated for 24 h. Various concentrations of helenalin were added to the culture medium and incubated for an additional 24 h. Cells were then harvested and analyzed by flow cytometry. The cell cycle phase distribution was determined using CellQuest software. (B) Percent of cells in Sub-G1 after drug treatment representing three independent experiments. (C) Measurement of apoptotic cell death by staining with FITC-Annexin V and Propidium Iodide. Lower right hand quadrant for each dose treatment represents percent of apoptosis, while upper right hand quadrant represents early necrotic cells. (D) Percent of cells that are either apoptotic, necrotic or total cell death as measured by staining cells with FITC-Annexin V and Propidium Iodide. Percentages are the average of three independent biological replicates.
Figure 3
Figure 3
Helenalin induces cell cycle arrest in G1 phase and modulates apoptosis in a time-dependent manner. (A) 1.85 × 105 cells/ml were seeded onto 6-well plates and incubated for 24 h. 2uM of helenalin was added to the culture medium and incubated for 4,8,12 and 24 h. Cells were harvested and analyzed by flow cytometry. The cell cycle phase distribution was determined using CellQuest software. Figures represent data from three independent experiments. (B) Percent of cells in Sub-G1 after the indicated timepoints representing three biological experiments.
Figure 4
Figure 4
Helenalin activates caspase cleavage. A2750 cells were treated with increasing concentrations of helenalin for 24 h (A) or with 2uM helenalin for the indicated timepoints (B), following which cells were harvested and cell lysates prepared and subjected to immunoblot analysis for cleaved PARP, caspase 3 and 9. Actin was used as a loading control.A2750 cells were subjected to caspase inhibitor, Z-VAD-fmk treatment two hours before addition of 2uM helenalin for 24 h, following which the cells were lysed and subjected to (C) immunoblot analysis for cleaved caspases and PARP or (D) harvested and analyzed for cell cycle flow distribution using FACS analysis. (E) Percent of cells in Sub-G1 in DMSO, helenalin or Z-VAD-fmk plus helenalin treatment. All figures represent data from three independent experiments.
Figure 5
Figure 5
Helenalin induces autophagy cell death. A2750 cells were treated with increasing concentrations of helenalin for 24 h, following which cells were harvested and cell lysates prepared and subjected to immunoblot analysis for (A) BCL2, BAX and Bid or (B) Atg12 and LC3-B. Actin was used as the loading control. A2750 cells were treated with DMSO, 0.5uM, 1uM, 2uM helenalin or pretreated with 200nM bafilomycin A1 for 45 min before treatment with 2uM helenalin for 24 h. Following which, cells were stained with Acridine Orange Solution and photographed using a florescence microscope (positive AVO staining in red) (C) or (D) trypsinized and harvested for FACS analysis to quantitate levels of cell staining representing autophagy. Figures represent data from three independent experiments.
Figure 6
Figure 6
Helenalin induced caspase cleavage and autophagy is dependent on Atg12 and LC3-B. A2780 cells were transfected with either non-targeting siRNA (siRNA Neg) or Atg12 or LC3-B-specific siRNA for 24 h and then treated with either DMSO or helenalin for 24 h. Cells were harvested and (A and B) cell lysates were subjected to immunoblot analysis for Atg12, LC3-B, cleaved PARP, caspase 3 or 9 or (C and D) analyzed for cell cycle phase distribution by FACS. All experiments were performed in biological triplicates. Actin was used as the loading control. One way ANOVA was performed between control and treatment groups, and significant differences were observed between the groups as indicated in Figure 6C and D. Categories significant after multiple comparison are marked as “Yes”.(p-value <0.05; Tukey’s multiple comparison test).
Figure 7
Figure 7
Repression of NF-κB p65 expression by Helenalin is necessary for caspase cleavage and autophagy. (A) A2750 cells were treated with increasing concentrations of helenalin for 24 h, following which cells were harvested and cell lysates prepared and subjected to immunoblot analysis for NF-κB. (B) A2780 cells were transfected with an empty vector or vector over-expressing RelA p65. 24 h post transfection, cells were treated with DMSO or 2uM helenalin for 24 h,and harvested for immunoblot analysis for cleaved caspase 3 and 9, LC3-B or NF-κB p65 or (C) trypsined and analyzed by FACS for precent of cells in sub-G1. One way ANOVA was performed for sub-G1 cells between control (DMSO) and treatment groups, and significant differences were observed between the groups as indicated in Figure 7C. Categories significant after multiple comparison are marked as “Yes”.(p-value <0.05; Tukey’s multiple comparison test). (D) A2780 cells were transfected either with a non-targeting siRNA (siRNA Neg) or siRNA targeting RelA p65. 24 h post transfection, cells were treated with DMSO or 2uM helenalin for 24 h and harvested for immunoblot analysis for cleaved caspase 3 and 9, LC3-B or NF-κB p65 or (E) trypsined and analyzed by FACS for percent of cells in sub-G1. One way ANOVA was performed for sub-G1 cells between control (DMSO) and treatment groups, and significant differences were observed between the groups as indicated in Figure 7E. Categories significant after multiple comparison are marked as “Yes”.(p-value <0.05; Tukey’s multiple comparison test). All experiments were performed in biological triplicates and actin was used as a loading control for immunoblot analysis.
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