Khz (fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating JNK and NADPH oxidase-dependent generation of reactive oxygen species.

Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47(phox) and p67(phox) to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca(2+)](i), which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47(phox) and p67(phox) subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca(2+)](i), JNK activation, and ROS generation via NADPH oxidase and mitochondria

Khz induces apoptosis through a mitochondrial pathway.

<p>(A) HepG2 cells were treated with a 1∶2 dilution of Khz, and caspase or PARP activation was analyzed by immunoblotting. (B) Cells were pretreated with z-VAD-fmk (20 µM) for 1 h and treated with Khz at the same concentrations as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(B)</a>. After 1 h, cells were stained with propidium iodide (PI) and annexin-V-FITC (An) for fluorescent microscopy. (C) Immunoblot analysis of cytochrome c levels in cytosolic and mitochondrial fractions of HepG2 cells treated with Khz. (D) HepG2 cells stably transfected with Bcl-2 cDNA or an empty vector (pcDNA3) were treated with Khz for 1 h. Apoptosis was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(C)</a>. (E) Cells were pretreated with CsA (10 µM) for 1 h. Khz treatment and analysis of apoptosis were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(B)</a>. After 1 h, the cells were stained with PI and An for fluorescent microscopy.</p

JNK mediates the increase in [Ca²⁺]<i>_i</i> to induce NADPH oxidase-derived ROS generation.

<p>(A) Activation of JNK was analyzed in Khz-treated HepG2 cells by immunoblot analysis of phosphorylated JNK. (B) Cells were pretreated with 20 µM SP600125 (SP) for 1 h. Apoptosis was analyzed 1 h after Khz treatment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(C)</a>. (C) HepG2 cells were pretreated with JNK inhibitors as in (B), and cytochrome c levels in the cytosol were analyzed by immunoblotting 2 h after Khz treatment. (D, E) HepG2 cells were transfected with control or JNK-1 siRNA. After 48 h, ROS generation was analyzed 30 min after Khz treatment using the Amplex Red hydrogen peroxide assay. Data represent mean ± SD (D). JNK protein expression was analyzed by immunoblotting 48 h after transfection (E). (F, G) HepG2 cells were pretreated with 20 µM SP200125 (SP) for 1 h, and ROS generation induced by Khz treatment was measured using the Amplex Red hydrogen peroxide assay. Data represent the mean ± SD (F). Levels of p47^phox and p67^phox protein in the membrane fractions of HepG2 cells were analyzed by immunoblotting 15 min after Khz treatment (G). (H) HepG2 cells were pretreated with NAC (5 mM), BAPTA-AM (10 µM), and SP600125 (20 µM), and JNK phosphorylation was analyzed by immunoblotting 15 min after Khz treatment.</p

Summary of the signaling events leading to Khz-induced apoptosis.

<p>Summary of the signaling events leading to Khz-induced apoptosis.</p

An increase of [Ca²⁺]<i>_i</i> is necessary for Khz-induced ROS generation and apoptosis.

<p>(A) HepG2 cells were loaded with fura-2 AM for 30 min and changes in [Ca²⁺]<i>_i</i> after Khz treatment were analyzed by digital imaging microscopy. (B) HepG2 cells were pretreated with EGTA (10 µM) or BAPTA-AM (10 µM) for 30 min, and membrane fractions were prepared for immunoblot analysis 5 min after Khz treatment. (C) Cells were pretreated with EGTA or BAPTA-AM for 30 min and treated with Khz for 30 min. ROS generation in HepG2 cells was then measured using the Amplex Red hydrogen peroxide assay. Data represent mean ± SD. (D) Cells were pretreated with EGTA or BAPTA-AM for 30 min and treated with Khz for 30 min. (E) Apoptosis was analyzed 1 h after Khz treatment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(C)</a>.</p

ROS generation by NADPH oxidase and mitochondria is required for Khz-induced apoptosis.

<p>(A) HepG2 cells were pretreated with DPI or apocynin for 1 h, and cytochrome c levels in the cytosol were analyzed 2 h after Khz treatment by immunoblot analysis. (B) Cells were pretreated with DPI (10 µM), apocynin (300 µM), or NAC (5 mM) for 30 min, and apoptosis was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(C)</a>. (C) HepG2 cells were pretreated with 0.5 µM MitoQ or TPP for 30 min, and apoptosis was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046208#pone-0046208-g002" target="_blank">Fig. 2(C)</a>.</p

Khz induces apoptosis in transformed cells.

<p>(A) (A-a) The shape and type of fused fruiting bodies. (A-b) Hyphae isolated from a <i>Ganoderma lucidum</i> mushroom on a Petri dish. (A-c) Shape of <i>G. lucidum</i>. (A-d) Shape and type of fused fruiting bodies and hyphae from <i>Polyporus umbellatus</i>. (A-e) Fusion of <i>G. lucidum</i> and <i>P. umbellatus</i>. (A-f) The fused hyphae of <i>G. lucidum</i> and <i>P. umbellatus</i>. (A-g) Agar-cultured fusion fungi. (A-h) DNA from fused hyphae (Khz). (A-i) Cultivation conditions for Khz. (B) Analysis of apoptosis using annexin-V-FITC (An) and propidium iodide (PI) staining. HepG2 cells were treated with a 1∶2 dilution of Khz, and apoptosis was analyzed after 0.5, 1, and 2 h by flow cytometry. Data are representative of more than 3 experiments. (C) HepG2 cells were treated with a 1∶2 dilution of Khz and stained with An and PI for flow cytometric analysis. Data represent mean ± SD. (D) BEAS-2B, 1799, 1198, and 1170-I cells were treated with Khz (1∶2 dilution), and apoptosis was examined by PI staining followed by flow cytometric analysis. Data are representative of more than 3 experiments.</p

Khz triggers cytoplasmic and mitochondrial ROS generation.

<p>(A) HepG2 cells were loaded with H₂DCF-DA and treated with Khz (diluted 1∶2), and the cytoplasmic ROS levels were assessed by flow cytometry. Data are representative of more than 3 experiments. (B) Intracellular ROS levels in HepG2 cells treated with Khz (diluted 1∶2) were analyzed using an Amplex Red hydrogen peroxide assay. Data represent mean ± SD. (C) Cells were pretreated with NAC (5 mM), DPI (10 µM), or apocynin (Apo; 300 µM) for 1 h. Intracellular ROS levels in HepG2 cells were analyzed 30 min after Khz treatment using the Amplex Red hydrogen peroxide assay. (D–E) HepG2 cells were transfected with siRNAs targeting Nox2 and Nox4. After 48 h, the cells were treated with Khz, and ROS generation was measured after 60 min using the Amplex Red hydrogen peroxide assay. Data represent mean ± SD. (E). Silencing of Nox2 and Nox4 mRNA expression by siRNA transfection was assessed by RT-PCR. (F) HepG2 cells were treated with Khz, and the membrane and cytosol fractions were separated using the Compartmental Protein Extraction kit (Millipore). The expression of the p47^phox and p67^phox proteins in the membrane and cytosol fractions was analyzed by immunoblotting. (G) HepG2 cells were loaded with MitoSOX Red for 30 min and treated with Khz. Mitochondrial ROS generation was then assessed by fluorescent microscopy at the indicated time points. Data are representative of more than 3 experiments. (H) HepG2 cells were pretreated with DPI or apocynin for 1 h and mitochondrial ROS generation was analyzed 60 min after Khz treatment as in (G). (I) HepG2 cells were pretreated with 0.5 µM MitoQ or TPP for 30 min. Right panel: Cytoplasmic ROS generation was measured 30 min after Khz treatment by DCF staining and flow cytometry. Left panel: Mitochondrial ROS generation was assessed 60 min after Khz treatment by MitoSOX Red staining and flow cytometry. Data represent mean ± SD.</p