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. 2010 Dec;101(12):2612-20.
doi: 10.1111/j.1349-7006.2010.01701.x.

Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints

Affiliations

Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints

Hui-Min Wang et al. Cancer Sci. 2010 Dec.

Abstract

Several compounds from Cinnamomum kotoense show anticancer activities. However, the detailed mechanisms of most compounds from C. kotoense remain unknown. In this study, we investigated the anticancer activity of obtusilactone A (OA) and (-)-sesamin in lung cancer. Our results show that human Lon is upregulated in non-small-cell lung cancer (NSCLC) cell lines, and downregulation of Lon triggers caspase-3 mediated apoptosis. Through enzyme-based screening, we identified two small-molecule compounds, obtusilactone A (OA) and (-)-sesamin from C. kotoense, as potent Lon protease inhibitors. Obtusilactone A and (-)-sesamin interact with Ser855 and Lys898 residues in the active site of the Lon protease according to molecular docking analysis. Thus, we suggest that cancer cytotoxicity of the compounds is partly due to the inhibitory effects on Lon protease. In addition, the compounds are able to cause DNA double-strand breaks and activate checkpoints. Treatment with OA and (-)-sesamin induced p53-independent DNA damage responses in NSCLC cells, including G(1) /S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G(1) accumulation. In conclusion, OA and (-)-sesamin act as both inhibitors of human mitochondrial Lon protease and DNA damage agents to activate the DNA damage checkpoints as well induce apoptosis in NSCLC cells. These dual functions open a bright avenue to develop more selective chemotherapy agents to overcome chemoresistance and sensitize cancer cells to other chemotherapeutics.

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Figures

Figure 1
Figure 1
Level of Lon determines cell fate. (A) Abundance of Lon protease in normal and lung cancer cell lines. Whole cell lysates from normal lung fibroblasts, MRC‐5 and HEL299, and non‐small‐cell lung cancer cell lines, H1299, A549, and H1437, were immunoblotted with an antibody to Lon and antibody to actin as a loading control. (B) Downregulation of Lon induces caspase‐3‐dependent apoptosis. The expression of Lon protease was suppressed in cells by expressing two different shRNAs (Lon‐1 and Lon‐2) from the retroviral vector. Cell lysates were prepared from H1299 cells expressing shLon‐1, shLon‐2 or vector and immunoblotted with antibodies to Lon, anti‐cleaved caspase‐3 and actin as a loading control. (C) Downregulation of Lon suppresses cell proliferation and induces cytotoxicity in a dose‐dependent manner. The expression of Lon was inhibited in H1299 cells by expressing shRNA from the retroviral vector. LonshRNA and LonshRNA(1/2) represent cells that were infected by full and half amounts of the retrovirus expressing shRNA to get complete and half inhibition of Lon expression, respectively. Cell growth was measured under the different extent of Lon inhibition at the indicated time. Cell numbers were calculated using hemocytometers. Each value represents the mean ± SD.
Figure 2
Figure 2
(A) Analysis of recombinant human Lon by SDS‐PAGE. Crude lysate from Escherichia coli Rosetta cells containing human Lon expression plasmid with (lane 1) or without (lane 2) isopropyl‐β‐D‐thiogalactoside induction was incubated with a Ni‐NTA affinity agarose gel. After binding, bound fractions were eluted by imidazole (lane 4, 200 mM; lane 5, 250 mM) from the column. Affinity purified human Lon was concentrated (lane 6). The arrow shows the recombinant human Lon. M, molecular mass markers. (B) Substrate saturation curve for the recombinant human Lon, incubated with indicated concentrations of fluorogenic substrate (Glt‐AAF‐MNA,[S]) in a real‐time manner (0–30 min). The velocity (Y‐axis) of the reaction was determined at various Glt‐AAF‐MNA concentrations (X‐axis). The values of K m and k cat were 9.15 ± 1.95 μM and 68.3/min, respectively, by fitting the data with the Michaelis–Menten equation using the KaleidaGraph program.
Figure 3
Figure 3
Obtusilactone A (OA) and (−)‐sesamin inhibit human Lon protease in vitro and in vivo. (A) Chemical structure of OA and (−)‐sesamin. (B) Dose‐dependent inhibition of Lon protease by OA. The inhibitory effect of OA on Lon protease was measured using FITC‐α‐casein as a substrate. Where v i is the initial velocity in the presence of inhibitor at concentration [I] and v 0 is the initial velocity in the absence of inhibitor. (C) Dose‐dependent inhibition of Lon protease by (−)‐sesamin. The same as (B) except OA is replaced by (−)‐sesamin. (D) Time‐dependent inhibition of Lon protease by OA in vivo. H1299 cells were treated with OA for the indicated time at the concentration of 40 μM. Whole cell lysates from the treated H1299 cells were immunoblotted with an antibody to aconitase and anti‐actin as a loading control. (E) Dose‐dependent inhibition of Lon protease by OA in vivo. H1299 cells were treated with OA for 12 h at the indicated concentration. Whole cell lysates from the treated H1299 cells were immunoblotted with an antibody to aconitase and anti‐actin as a loading control.
Figure 4
Figure 4
Modeling obtusilactone A (OA) or (−)‐sesamin in the active site of human Lon protease. (A) Multiple sequence alignment of Lon proteins. The sequences are taken from the GenBank database, including Homo sapiens Lon (Accession No.: NM_004793) and Escherichia coli Lon (Accession No.: P08177). Amino acid residues 744–959 in human Lon (Human Lon744–959) and 585–784 in E. coli Lon (E. coli Lon585–784) are used to align the sequences. The identical residues are highlighted in a green frame. Serine 855 (*) and lysine 898 (•), acting as the active site, are shown. (B) Homology modeling of human Lon proteolytic (P) domain and binding mode of inhibitors in the active site. The model of the P domain was used as the target for ducking simulation. Obtusilactone A (a) or (−)‐sesamin (b) was docked into the protease catalytic site.
Figure 5
Figure 5
Obtusilactone A (OA) and (−)‐sesamin cause DNA double‐strand breaks (DSBs) and DNA damage response‐induced apoptosis. (A) Detection of DSBs in H1299 cells after treatment by comet assay. H1299 cells were treated with control (DMSO) or the indicated concentration of OA or (−)‐sesamin for 2 h. The circular spots in red are nuclei and the comet tails indicate the cells carrying the DNA damaged DSBs. Tail moment of cells with a comet tail is plotted. (B) Obtusilactone A activates the ATM‐CHK2 mediated checkpoint and JNK‐mediated apoptosis pathways. H1299 cells were treated with DMSO or the indicated concentration of OA for 4 h. Whole cell lysates from treated H1299 cells were immunoblotted with phosphospecific antibodies to Ser343 of Nbs1 (NBS1‐pS343), Ser317 of Chk1 (CHK1‐pS317), Thr68 of Chk2 (CHK2‐pT68), Ser139 of H2AX (γ‐H2AX‐S139), JNK (p‐JNK), or antibody to cleaved caspase‐3. Anti‐actin is a loading control. (C) Obtusilactone A induces G1/S cell cycle arrest and DNA damage‐induced apoptosis. H1299 cells were treated with DMSO or the indicated concentration of OA for 12 h. The determination of G1, S, G2/M, or sub‐G1 DNA content in H1299 cells by FACS analysis. The percentages of the cell cycle phase are shown at the bottom. Mean ± SD of three experiments. (D) Obtusilactone A activates JNK‐mediated apoptosis signaling. H1299 cells were treated with OA for the indicated time at the concentration of 40 μM. Whole cell lysates from the treated H1299 cells were immunoblotted with a phosphospecific antibody to JNK (p‐JNK) or antibody to cleaved caspase‐3 and anti‐actin as a loading control. (E) H1299 is a p53‐deficient cell line. Cell lysates were prepared from H1299 and 293T cells after mock treatment or exposure to UV (50 J/m2, 1 h after) and immunoblotted with anti‐p53 antibody. Actin was used as a loading control.
Figure 6
Figure 6
Obtusilactone A (OA) suppresses proliferation and induces selective cytotoxicity in lung cancer cell lines. (A) Cytotoxicity in H1299 cells treated with increasing concentrations of OA at the indicated incubation times. Cell images were captured using a Nikon Eclipse TE2000‐U inverted microscope. (B) Cell growth was measured under the different concentrations of OA at 12 and 24 h. Cell numbers were calculated using hemocytometers. Each value represents the mean ± SD. (C) Cytotoxicity in MRC‐5, A549, or H1299 cells treated with various concentrations of OA. The indicated cells were incubated with concentrations of OA (from 0, 20, 40, and 60 μM) for 12 h. Mean ± SD of three experiments.

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