Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells

Background Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Methods Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. Results In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-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 shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. Conclusions In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria.


Background
Cancer develops because of abnormal cellular proliferation or defective apoptosis that leads to uncontrolled growth [1]. Therefore, new treatments that target the proliferation and apoptosis of cancer cells are necessary. Under normal conditions, programmed cell death occurs after exposure to pathological factors. Apoptosis involves cell shrinkage, condensation of nuclei and chromatin, and DNA fragmentation, all of which result in unmistakable cellular morphology. Apoptosis is initiated by external signals through a series of cysteine acid proteases, including important regulatory factors such as caspases. Cytochrome c-mediated Casp3 activation may be utilized by a specific and restricted set of external apoptosis stimuli. Defective signaling during the regulation of cell death can result in the abnormal proliferation of cells and can cause cancer. Therefore, repairing defective cell death mechanisms or developing drugs or food components that induce cell differentiation may be a promising approach for the generation of anticancer agents [2,3]. In particular, many studies are being performed to identify natural products that can be used as anticancer drugs and that do not have the toxicity and adverse effects associated with chemotherapeutic drugs. Several biologically active ingredients that show effective anticancer activity have been derived from edible or medicinal mushrooms [4][5][6], and the anticancer effects of Ganoderma lucidum have been described in various studies [7][8][9][10]. Additionally, Polyporus umbellatus induces G2/M cell cycle arrest and apoptosis in HepG2 cells, thereby causing growth suppression [11].
Khz-cp is an extract mixture from the mycelia of a G. lucidum and P. umbellatus nuclear fusion ( Figure 1A). The anticancer effect of the fusion of G. lucidum and P. umbellatus has been previously demonstrated [12,13]. In this study, we investigated the mechanism underlying Khz-induced cell death in gastric cells.
Oxidative stress is widely implicated in apoptotic and non-apoptotic cell death [14][15][16]. The major sources of intracellular reactive oxygen species (ROS) include NADPH oxidase and the mitochondrial electron transport chain (ETC.). When ROS production, either by the mitochondria or by NADPH oxidase, becomes excessive, the natural cellular antioxidant defense system is overwhelmed, which results in oxidative stress. Cancer cells are more susceptible to oxidative stress than healthy cells, and some anticancer agents such as cisplatin, arsenic trioxide (As 2 O 3 ), and 2-methoxyestradiol exert their effects by inducing ROS production [17][18][19]. Much of the available data indicate that the ROS that accumulate during cell death are generated by the mitochondria in response to impairment of the mitochondrial respiratory chain [20][21][22][23]. Although mitochondrial ROS production is regarded as an integral component of the apoptotic program, the role of NADPH oxidase as a primary source of ROS during the induction of apoptosis has also been reported [24][25][26]. Sustained elevation of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) is associated with the induction of apoptosis [27]. When the cytoplasmic [Ca 2+ ] i increases, the mitochondria take up Ca 2+ and function as a Ca 2+ buffer; however, excessive accumulation of mitochondrial Ca 2+ triggers apoptosis, at least in part by inducing ROS generation via the mitochondrial ETC. An increase in cytoplasmic [Ca 2+ ] i can also activate NADPH oxidase, which has been well documented in neutrophils [28]. In some cell types, the activation of protein kinase C via intracellular Ca 2+ leads to the phosphorylation of the p47 phox subunit and subsequent enzyme assembly [29].
In the present study, we investigated the role of Khzcp in cellular apoptosis and found that Khz-cp induced a sustained increase in [Ca 2+ ] i that resulted in ROS generation by NADPH oxidase via P38 and, finally, cellular apoptosis.

Cell lines and Khz-cp treatment
The BEAS-2B (normal immortalized), 1799 (non-transformed), 1198 (transformed but non-tumorigenic), and 1170-I (tumorigenic) cell lines that compose the in vivo lung carcinogenesis model used in this study have been previously described [30,31]. The human gastric cancer cell line SNU-1 was maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B. Unless otherwise indicated, all the cells were treated with Khz-cp diluted 1:100 in the media.
Extraction of Khz-cp (crude polysaccharide extract obtained from the fusion of G. lucidum and P. umbellatus mycelia) First, 1 kg of powder was added to 8.5 L of clean water, heated to 115°C, and extraction was performed for 60 min under pressure. This was followed by a 60-min maturation period. Next, the remaining water from the first extraction was added to 7.5 L of clean water and heated to 115°C; extraction was performed under pressure for 60 min, followed by maturation for a further 60 min. The first and second extracts were then mixed, boiled, and placed in bottles after 5 min.
We purified Khz-cp from Khz by using the Sevag method for deproteinization. The Sevag reagent, which is a 4:1 mixture of chloroform and n-butanol, was added with shaking; the volume of the reagent added was onefourth that of the sample solution. After the mixture was allowed to stand and separate, the water layer and the solvent layer at the junction of the denatured protein were removed. This step was repeated several times until the denatured protein content was minimized. Preliminary experiments showed that a fourfold excess of 95% ethanol with respect to the sample volume was appropriate for precipitation. Therefore, after the removal of the denatured protein, 95% ethanol (4 times the sample volume) was added slowly until additional precipitation did not occur. The mixture was centrifuged and the supernatant was removed. The sediment collected was a brown precipitate and represented the total bacterial crude polysaccharide fraction. The polysaccharides were then dissolved and filtered by membrane ultrafiltration (molecular weight cutoff: 100,000 Da). Khz-cp was obtained from BrainGroup (Seoul, South Korea).

Western blot analysis
Cells were lysed in an extraction buffer (31.25 mM Tris-HCl [pH 6.8], 1% sodium dodecyl sulfate [SDS], 10% glycerol, and 2.5% mercaptoethanol), and the whole cell lysate was subjected to 10% SDS-polyacrylamide gel electrophoresis. Size-fractionated proteins on the gel were transferred onto a nitrocellulose membrane. The membrane was blocked in 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 and was incubated with a primary antibody. After washing, the membrane was incubated with the peroxidase-conjugated secondary antibody. The protein band of interest was detected using enhanced chemiluminescence reagents (Amersham).

Apoptosis assay
Cells treated with Khz-cp were washed twice in cold phosphate-buffered saline (PBS) and stained with annexin-V-FITC (A13199; Invitrogen) and propidium iodide (PI) according to the manufacturer's instructions. Briefly, annexin-V-FITC (5 μL) was added to the cells, which were resuspended in 100 μL of binding buffer (10 mM HEPES, 140 mM NaCl, and 2 mM CaCl 2 ; pH 7.4). The cells were then incubated at room temperature for 15 min, and PI was added before flow cytometry or fluorescence microscopy analysis. Apoptosis were determined using a FACS calibur (Becton and Dickson) and analysed using Cell Quest pro software. Images were analyzed using NIS Elements software (Nikon).

Assessment of cytoplasmic and mitochondrial ROS levels
The levels of cytoplasmic ROS were estimated using the oxidation-sensitive fluorescent dye H 2 DCF-DA (2′,7′dichlorodihydrofluorescein diacetate; Invitrogen) or the Amplex Red hydrogen peroxide assay kit (Invitrogen). For DCF staining, the cells were loaded with H 2 DCF-DA (100 nM) for 1 h at 37°C and washed once with PBS. After treatment with Khz-cp, ROS levels were analyzed using a flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA, USA) or a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan). The Amplex Red hydrogen peroxide assay was performed according to the manufacturer's protocol. In brief, the cells were lysed in 50 μM Amplex Red solution supplemented with 0.1 U/mL horseradish peroxidase and incubated in the dark for 30 min. Fluorescence was measured using a plate reader (Victor 2; Perkin-Elmer Life Sciences, Boston, MA, USA) with an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
Mitochondrial superoxide anion levels were analyzed by staining with MitoSOX™ Red (Invitrogen). Cells were loaded with MitoSOX Red (5 μM) for 30 min at 37°C and then treated with Khz-cp. The fluorescence was then analyzed by flow cytometry or fluorescence microscopy.

Preparation of subcellular fractions
To prepare the mitochondrial and cytosolic fractions, the cells (1 × 10 7 ) were washed once in PBS and disrupted by passing them through a glass homogenizer 80 times in ice-cold isolation buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, and 0.1 mM PMSF). Nuclei and non-disrupted cells were removed by centrifugation at 750 × g for 20 min at 4°C. The supernatant was further centrifuged at 10,000 × g for 15 min at 4°C to obtain a mitochondrion-enriched pellet and a cytoplasm-enriched supernatant.
Membrane and cytosolic fractions were prepared using the Compartmental Protein Extraction kit (Millipore, Temecula, CA, USA) according to the manufacturer's instructions.

Ca 2+ imaging
Digital imaging of the intracellular free Ca 2+ was performed using the fura-2 AM dye (Invitrogen). When fura-2 binds to Ca 2+ , its maximal absorption wavelength shifts from 363 to 335 nm. SNU-1 cells (1 × 10 4 ) were cultured in 35-mm glass-bottomed dishes and loaded with fura-2 AM (2 μM) for 30 min at 37°C. Fluorescence images of fura-2 were digitally captured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm with an IX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a digital cooled charge-coupled device camera. Paired 340/380 ratiometric images were analyzed using the Metafluor software (Molecular Devices, Sunnyvale, CA, USA). Confocal images of intracellular free Ca 2+ were obtained using the fluo-4 AM Ca 2+ -sensitive fluorescent dye (Invitrogen). The cells were loaded with fluo-4 AM (1 μM) for 30 min at 37°C, and Ca 2+ imaging was performed using a confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany).

Khz-cp induces apoptosis in transformed cells
Khz-cp is shown in Figure 1A. The aim of the present study was to examine whether Khz-cp causes apoptosis in human cancer cells and, if so, to identify the signaling mechanisms involved. As shown in Figure 1b and c, staining with annexin-V-FITC and PI showed that Khzcp triggered apoptosis in SNU-1 cells (annexin-V singlepositive or annexin-V/PI double-positive cells) as early as 60 min after treatment. As apoptosis progressed, the population of cells stained with annexin-V alone declined, whereas that of cells stained with both annexin-V and PI increased (Figure 1c). The population of cells positive for PI alone may represent necrotic cells. The induction of preferential apoptosis by Khz-cp in transformed cells was then evaluated in a series of cell lines that comprise an in vivo lung epithelial carcinogenesis model. BEAS-2B is an immortalized normal human bronchial epithelial cell line, and 1198 and 1170-I are transformed cell lines derived from BEAS-2B cells exposed in vivo to beeswax pellets containing cigarette smoke condensate (CSC). The 1799 cell line is a nontransformed line derived from BEAS-2B cells exposed to beeswax alone. Khz-cp induced apoptosis in the transformed 1198 and 1170-I cells but not in nontransformed BEAS-2B and 1799 cells (Figure 1d). These data indicate that Khz-cp induces apoptosis preferentially in cancer cells, which suggests that it has potential as a therapeutic agent against cancer.

Khz-cp-induced apoptosis is caspase dependent and occurs through a mitochondrial pathway
To determine whether the Khz-cp-induced apoptosis was caspase dependent, caspase activation was analyzed after Khz-cp treatment. Cleavage of caspase 3 and PARP (indicating their activation) increased in SNU-1 cells after Khz-cp treatment (Figure 2a). Moreover, pretreatment of these cells with the pan-caspase inhibitor z-VAD-fmk completely blocked Khz-cp-induced apoptosis (Figure 2b), which indicates that Khz-cp induces caspase-dependent apoptosis.
Natural products or cytotoxic chemicals often induce apoptosis through a mitochondrial pathway; therefore, the release of cytochrome c from the mitochondria into the cytosol was analyzed to determine whether Khz-cpinduced apoptosis also occurred via a mitochondrial pathway. As shown in Figure 2c, cytochrome c levels in the cytosol of SNU-1 cells increased after Khz-cp treatment, whereas cytochrome c levels in the mitochondria concurrently decreased, which indicates the release of mitochondrial cytochrome c. Ectopic expression of the protective Bcl-2 protein prevented Khz-cp-induced apoptosis in SNU-1 cells (Figure 2d). Furthermore, treatment with CsA (which blocks PTP opening by binding to cyclophilin D) abrogated Khz-cp-induced apoptosis, which suggests that mitochondrial permeability transition is required for apoptosis (Figure 2e).

Oxidative stress mediates Khz-cp-induced apoptosis
ROS production after Khz-cp treatment was analyzed because oxidative stress is typically involved in apoptosis.     Figure 3c shows that DPI (flavoprotein inhibitor) and apocynin (p47 phox inhibitor) prevented ROS production. Moreover, silencing of Nox2 and Nox4 by using specific siRNAs almost completely abrogated ROS production (Figure 3d, e). Transfection with Nox2 or Nox4 siRNA alone partially blocked ROS production, which suggests that Nox2 and Nox4 contribute to Khz-cp-induced ROS generation in combination. Activation of NADPH oxidase by Khz-cp was also shown by the translocation of the cytosolic subunits of NADPH oxidase p47 phox and p67 phox to the cell membrane 15 min after Khz-cp treatment (Figure 3f). Taken together, these data indicate that NADPH oxidase produces ROS upon Khz-cp treatment.
Because the mitochondrial respiratory chain is another major source of cellular ROS, mitochondrial ROS production was evaluated using MitoSOX Red staining. Mitochondrial ROS levels in SNU-1 cells increased 60 min after Khz-cp treatment ( Figure 3g); therefore, it appears that ROS production by mitochondria occurs later than that by NADPH oxidase. Mitochondrial ROS generation by Khz-cp was prevented by pretreatment with DPI or apocynin, which suggests that NADPH oxidase plays a critical role in mitochondrial ROS production ( Figure 3h). Furthermore, the mitochondria-targeting antioxidant MitoQ did not significantly block cytoplasmic ROS generation at a concentration that completely blocked mitochondrial ROS production (Figure 3i). TPP, which is the lipophilic moiety of MitoQ, was used as a control. Taken together, these data show that mitochondrial ROS generation induced by Khz-cp was mediated indirectly by NADPH oxidase-derived ROS.
We further examined whether ROS generation through NADPH oxidase and/or mitochondria was necessary for Khz-cp-induced apoptosis. Pretreatment of cells with DPI or apocynin suppressed mitochondrial cytochrome c release and apoptosis induced by Khz-cp treatment, which indicates that ROS generation through NADPH oxidase was required for the induction of apoptosis (Figure 4a, b). Furthermore, pretreatment with MitoQ, but not TPP, prevented Khz-cp-induced apoptosis, which suggests that mitochondrial ROS generation was also necessary for the induction of apoptosis (Figure 4c). Therefore, NADPH oxidase-derived ROS appear to trigger apoptosis via mitochondrial ROS generation.

Khz-cp induces a rapid and sustained increase in [Ca 2+ ] i
Because increased [Ca 2+ ] i has been widely implicated in mitochondria-mediated apoptosis, we evaluated whether Khz-cp affected [Ca 2+ ] i and, if so, whether it played a role in the activation of NADPH oxidase and ROS generation. Figure 5a shows that Khz-cp induced an immediate and sustained increase in [Ca 2+ ] i in SNU-1 cells. The effects of the increase in [Ca 2+ ] i on the activation of NADPH oxidase were then examined. EGTA and BAPTA-AM, which are extracellular and intracellular Ca 2+ chelators, respectively, prevented NADPH oxidase activation as measured by the membrane translocation of the p47 phox and p67 phox subunits (Figure 5b). Furthermore, both of these chelators inhibited ROS generation ( Figure 5c) and blocked Khz-cp-induced mitochondrial cytochrome c release and apoptosis (Figure 5d, e). These results indicate that Khz-cp triggers a rapid and sustained increase in [Ca 2+ ] i that activates NADPH oxidase to induce ROS generation and, finally, apoptosis.

P38 mediates Ca 2+ -dependent activation of NADPH oxidase
The mitogen-activated protein kinase P38 is activated in response to oxidative stress [32,33]. Because Khz-cp induced ROS generation, the activation of P38 and its possible role in Khz-cp-induced apoptosis were examined. As shown in Figure 6a, P38 was activated as early as 15 min after Khz-cp treatment. Considering that ROS generation was observed 30 min after Khz-cp treatment (Figure 4b), it is unlikely that the activation of P38 was caused by oxidative stress. Nonetheless, P38 activity was required for the induction of apoptosis because a chemical inhibitor of P38 (SB203580) blocked mitochondrial cytochrome c release and apoptosis induced by Khz-cp treatment, but not an inactive analog of SB202474 ( Figure 6b, c). Inhibition of P38 by siRNA transfection or pretreatment with chemical inhibitors suppressed Khz-cpinduced ROS production, which indicates that the activation of P38 caused ROS generation rather than the opposite (Figure 6d-f). In agreement with these findings, pretreatment of SNU-1 cells with SB203580 also suppressed the activation of NADPH oxidase, as assessed by the membrane translocation of p47 phox and p67 phox (Figure 6g). Therefore, we examined whether P38 activation was dependent on [Ca 2+ ] i . Collectively, these results strongly indicate that P38 is activated by Khz-cp via an increase in [Ca 2+ ] i, thereby triggering ROS generation by NADPH oxidase and the induction of apoptosis.

Discussion
In the present study, Khz-cp triggered caspase-dependent and mitochondria-mediated apoptotic death in human cancer cells. Khz-cp-induced apoptosis was selective for transformed cells, and Khz-cp only minimally affected non-transformed cells, which suggests that it is a potential anticancer therapeutic agent. Intrinsic oxidative stress in transformed cells may render them more susceptible to apoptosis induced by Khz-cp. Alternatively, cancer cells may have defective restoration of glutathione (GSH) reserves. However, the GSH levels in tumor tissues are higher than those in normal tissues [34], and depleting GSH reserves often sensitizes cancer cells to ROS-induced cell death, which suggests that the GSH present in cancer cells protects them from oxidative stress.
Khz-cp-induced apoptosis was observed to be caspase dependent, as indicated by the activation of caspases and inhibition of apoptosis by pretreatment with the pancaspase inhibitor z-VAD-fmk (Figure 2a, b). The involvement of a mitochondria-mediated pathway was confirmed by the release of mitochondrial cytochrome c, and inhibition of apoptosis was confirmed by the overexpression of Bcl-2 (Figure 2c, d). Oxidative stress is associated with apoptotic and non-apoptotic cell death, although prooxidative conditions are not a prerequisite for apoptosis [35]. The present study shows that the induction of apoptosis by Khz-cp required ROS generation by both NADPH oxidase and mitochondria. Several studies have implicated ROS generated from mitochondria in the induction of apoptosis; however, the results of the present study indicate that NADPH oxidase-derived ROS were critical for Khz-cp-induced apoptosis (Figure 4a, b). Although mitochondrial ROS generation was also necessary for Khz-cpinduced apoptosis (Figure 4c), its effect was secondary to that of the initial ROS production by NADPH oxidase because the generation of mitochondrial ROS occurred after that of cytoplasmic ROS and was prevented by pretreatment with NADPH oxidase inhibitors (Figure 3g, h). Of the 7 members of the human NADPH oxidase family, Nox2 and Nox4 were found to be responsible for the ROS generation induced by Khz-cp treatment in SNU-1 cells (Figure 3d, e).
It is widely accepted that calcium signaling plays an important role in apoptosis [29,36]. Cross-talk between ROS and calcium signaling pathways may lead to synergistic effects on mitochondrial permeabilization and cell death. The present study showed that an increase in the [Ca 2+ ] i induced by Khz-cp treatment resulted in ROS generation by NADPH oxidase. The [Ca 2+ ] i increased before the generation of ROS, and Ca 2+ chelators such as EGTA and BAPTA-AM abrogated the activation of NADPH oxidase and ROS generation (Figure 5b, c). However, the inhibitors of NADPH oxidase did not affect the increase in [Ca 2+ ] i induced by Khz-cp treatment (data not shown). The activation of NADPH oxidase by elevated [Ca 2+ ] i in neutrophils is well known and has also been reported for other cell types [37,38].
P38 was identified as the mediator of Ca 2+ -dependent NADPH oxidase activation. P38 was activated within 15 min of Khz-cp treatment in a Ca 2+ -dependent manner (Figure 6a) Furthermore, treatment with P38 siRNA or chemical inhibitors prevented Khz-cp-induced ROS generation (Figure 6d-g). Most studies place ROS upstream of P38 activation [39,40] however, in this study, ROS generation by NADPH oxidase was found to be mediated by the activation of P38.

Conclusions
In this study, we investigated that Khz induces apoptosis by increasing intracellular calcium levels and activating JNK and NADPH oxidase-dependent generation of ROS Figure 7 Summary of the signaling events leading to Khz-cp-induced apoptosis. [12]. We showed that Khz-cp induces mitochondrionmediated apoptosis preferentially in transformed cells, and the signaling pathway of Khz-cp-induced apoptosis involves an increase in [Ca 2+ ] i , activation of P38, and ROS generation through NADPH oxidase and mitochondria (Figure 7).