Cytotoxic Constituents from the Sclerotia of Poria cocos against Human Lung Adenocarcinoma Cells by Inducing Mitochondrial Apoptosis

Previous studies have revealed the antitumor potential of Poria cocos Wolf against a broad spectrum of cancers. However, the biological activity of P. cocos against lung cancer, which is known as the leading cause of cancer mortality worldwide, and its underlying chemical and molecular basis, remain to be investigated. We aimed to evaluate the in vitro cytotoxicity of P. cocos toward human lung adenocarcinoma cells with different p53 statuses, to identify the bioactive constituents of P. cocos, and explicate the molecular mechanisms underlying the cytotoxicity of these constituents in human lung adenocarcinoma cells. An EtOH extract of the sclerotia of P. cocos exhibited cytotoxicity toward four human lung cancer cell lines: A549, H1264, H1299, and Calu-6, regardless of their p53 status. Chemical investigation of the extract resulted in the isolation of two triterpenoids, dehydroeburicoic acid monoacetate (1) and acetyl eburicoic acid (4); a sterol, 9,11-dehydroergosterol peroxide (2); and a diterpenoid, dehydroabietic acid (3). All of the isolated compounds were cytotoxic to the lung adenocarcinoma cell lines, exhibiting IC50 values ranging from 63.6 μM to 171.0 μM at 48 h of treatment. The cytotoxicity of the extract and the isolated compounds were found to be mediated by apoptosis, and accompanied by elevated Bax expression and/or Bcl-2 phosphorylation along with caspase-3 activation. Our data demonstrate that the sclerotium of P. cocos and its four bioactive constituents (1–4) exert cytotoxicity against human lung adenocarcinoma cells, regardless of their p53 status, by inducing apoptosis associated with mitochondrial perturbation, and proposing the potential to employ P. cocos in the treatment of lung cancer.


Introduction
Lung cancer is known as the most prevalently diagnosed cancer worldwide [1]. Despite considerable advances in cancer diagnosis and management, the prognosis of lung cancer patients continues to be unsatisfactory, as the five-year survival rate is less than 16%. Thus, lung cancer predominantly accounts for the leading cause of cancer-associated death, and was reported to cause approximately 1.69 million deaths in 2015 [1,2]. with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA, USA), 2 mM of L-glutamine, 50 U/mL penicillin, and 50 µg/mL of streptomycin (WelGENE).

Cell Viability Analysis
A549, H1264, H1299 (5 × 10 3 cells per well), and Calu-6 (7.5 × 10 3 cells per well) cells were plated in triplicate in 96-well tissue culture plates (Thermo Scientific, Waltham, MA, USA) and grown overnight. Cells were then treated with the EtOH extract of the sclerotia of P. cocos, and the compounds isolated from it at various concentrations. Cells were also incubated in growth medium containing dimethyl sulfoxide (DMSO) at concentrations ranging from 0 to 0.5% and 0 to 1.25% as vehicle controls for the treatments with the EtOH extract and the isolated compounds, respectively. At 48 h after the treatment, cell viability was assessed through a WST-1 cell proliferation assay with an EZ-Cytox Enhanced Cell Viability Assay kit (Daeil Lab Service, Seoul, Korea) in accordance with the manufacturer's instructions, as previously described [22]. Cell viability was determined as a percentage of that of the corresponding vehicle control. The IC 50 values of the EtOH extract and the isolated compounds were estimated by a non-linear regression analysis of the dose-response curve in GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

TUNEL Assay
A549, H1264, H1299 (7.5 × 10 3 cells), and Calu-6 (1.0 × 10 4 cells) were seeded in triplicate on 12-mm glass coverslips (Marienfeld GmbH, Lauda-Königshofen, Germany) and grown overnight. The cells were then treated with the EtOH extract of the sclerotia of P. cocos and the isolated compounds. Cells were also treated with growth medium containing DMSO as a vehicle control. After 48 h of treatment, apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining with a Dead-End labeling kit (Promega, Madison, WI, USA) according to the manufacturer's protocol, as previously described [22]. The cells were also counterstained with 0.5 µg/mL of 4 ,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) so that the nuclei could be visualized. The stained cells were then examined under a fluorescence microscope (Carl Zeiss, Jena, Germany), and the percentage of apoptotic cells was calculated as the ratio of TUNEL-positive cells to the total number of cell nuclei counted in six randomly selected high-power fields (400×) on each slide.

Statistical Analysis
Two-tailed unpaired Student's t tests were employed to determine the statistical significance of differences between the treated and control groups. All of the data are presented as the mean ± standard error of the mean (SEM), and p values less than 0.05 were considered statistically significant.

Cytotoxicity of the EtOH Extract of the Sclerotia of P. cocos toward Human Lung Adenocarcinoma Cells with Different p53 Statuses
The status of p53 in cancer cells has been shown to correlate closely with their chemoresistance phenotype [18]. Therefore, in order to verify the previous findings on the cytotoxicity of P. cocos toward human lung cancer cells [17] and determine its relationship to the p53 status of the cells, we prepared an EtOH extract of the sclerotia of P. cocos, and examined its effects on cell viability in four human lung adenocarcinoma cell lines accompanying different p53 statuses: A549 (p53-wild-type), H1264 (p53-mutated), H1299 (p53-null), and Calu-6 (p53-null) [23,24] (Figure 1 and Table 1).
Consistent with the previous study [15], a WST-1 cell viability assay showed that the EtOH extract exhibited cytotoxicity toward A549 cells after 48 h of treatment in a dose-dependent manner, with an IC 50 value of 301.1 µg/mL ( Figure 1A and Table 1). In addition, the EtOH extract significantly and dose-dependently reduced cell viability in H1264, H1299, and Calu-6 cells, with IC 50 values ranging from 284.0 µg/mL to 372.7 µg/mL ( Figure 1A and Table 1). However, no notable correlation was examined between the cytotoxic effect of the EtOH extract and the p53 status of the human lung cancer cells, implying the cytotoxicity of P. cocos in human lung cancer cells, irrespective of their p53 status. standard error of the mean (SEM), and p values less than 0.05 were considered statistically significant.

Cytotoxicity of the EtOH Extract of the Sclerotia of P. cocos toward Human Lung Adenocarcinoma Cells with Different p53 Statuses
The status of p53 in cancer cells has been shown to correlate closely with their chemoresistance phenotype [18]. Therefore, in order to verify the previous findings on the cytotoxicity of P. cocos toward human lung cancer cells [17] and determine its relationship to the p53 status of the cells, we prepared an EtOH extract of the sclerotia of P. cocos, and examined its effects on cell viability in four human lung adenocarcinoma cell lines accompanying different p53 statuses: A549 (p53-wild-type), H1264 (p53-mutated), H1299 (p53-null), and Calu-6 (p53-null) [23,24] (Figure 1 and Table 1).
Consistent with the previous study [15], a WST-1 cell viability assay showed that the EtOH extract exhibited cytotoxicity toward A549 cells after 48 h of treatment in a dose-dependent manner, with an IC50 value of 301.1 μg/mL ( Figure 1A and Table 1). In addition, the EtOH extract significantly and dose-dependently reduced cell viability in H1264, H1299, and Calu-6 cells, with IC50 values ranging from 284.0 μg/mL to 372.7 μg/mL ( Figure 1A and Table 1). However, no notable correlation was examined between the cytotoxic effect of the EtOH extract and the p53 status of the human lung cancer cells, implying the cytotoxicity of P. cocos in human lung cancer cells, irrespective of their p53 status.   P. cocos and its constituents, including lanostane-type triterpenoids and polysaccharides, have been demonstrated to promote apoptosis in a variety of human cancer cells [7,17]. In parallel with these previous findings, all of the human lung cancer cells tested in the current study were found to undergo morphological changes typical of apoptosis, which include cell rounding, cell shrinkage, cell membrane blebbing, and the detachment of the cell from the substratum [25], after treatment with the EtOH extract of the sclerotia of P. cocos ( Figure 1B). This suggests that the cytotoxicity of the EtOH extract in the examined lung cancer cells is mediated by pro-apoptotic activities. To further confirm the promotion of apoptosis in human lung cancer cells, we performed TUNEL staining on A549, H1264, H1299, and Calu-6 cells that are treated with the EtOH extract for 48 h ( Figure 2). As predicted, the proportion of TUNEL-positive cells was significantly greater following EtOH extract treatment than it was following DMSO treatment (the vehicle control) in all of the cancer cell lines tested (Figure 2A-D). These observations not only demonstrate that the cytotoxicity of the EtOH extract toward the cancer cells is attributable to its pro-apoptotic activity, but also suggest that apoptosis induced by the extract in the cancer cells is mediated by p53-independent pathways.
Cells 2018, 7, x FOR PEER REVIEW 5 of 13 P. cocos and its constituents, including lanostane-type triterpenoids and polysaccharides, have been demonstrated to promote apoptosis in a variety of human cancer cells [7,17]. In parallel with these previous findings, all of the human lung cancer cells tested in the current study were found to undergo morphological changes typical of apoptosis, which include cell rounding, cell shrinkage, cell membrane blebbing, and the detachment of the cell from the substratum [25], after treatment with the EtOH extract of the sclerotia of P. cocos ( Figure 1B). This suggests that the cytotoxicity of the EtOH extract in the examined lung cancer cells is mediated by pro-apoptotic activities. To further confirm the promotion of apoptosis in human lung cancer cells, we performed TUNEL staining on A549, H1264, H1299, and Calu-6 cells that are treated with the EtOH extract for 48 h ( Figure 2). As predicted, the proportion of TUNEL-positive cells was significantly greater following EtOH extract treatment than it was following DMSO treatment (the vehicle control) in all of the cancer cell lines tested (Figure 2A-D). These observations not only demonstrate that the cytotoxicity of the EtOH extract toward the cancer cells is attributable to its pro-apoptotic activity, but also suggest that apoptosis induced by the extract in the cancer cells is mediated by p53-independent pathways.
Collectively, our results reveal that P. cocos exerts cytotoxicity by inducing apoptosis in human lung cancer cells, regardless of the p53 status of the cancer cells, and thus further confirms the potential of P. cocos to be applied in the treatment of lung cancer.

Chemical Analysis of Bioactive Compounds from the EtOH Extract of the Sclerotia of P. cocos
To determine the major components of the sclerotium of P. cocos contributable to its cytotoxicity against human lung cancer cells, the EtOH extract was fractionated into four different fractions (hexane-soluble, CH2Cl2-soluble, EtOAc-soluble, and n-BuOH-soluble fractions), and sequentially analyzed by LC/MS. The hexane-soluble fraction was found to contain the main constituents. This fraction was chemically investigated by repeated column chromatography and HPLC techniques (see Supplementary Materials), where two triterpenoids (1 and 4), a sterol (2), and a diterpenoid (3) were successfully isolated ( Figure 3). Through comparison of spectroscopic and physical data of the Collectively, our results reveal that P. cocos exerts cytotoxicity by inducing apoptosis in human lung cancer cells, regardless of the p53 status of the cancer cells, and thus further confirms the potential of P. cocos to be applied in the treatment of lung cancer.

Chemical Analysis of Bioactive Compounds from the EtOH Extract of the Sclerotia of P. cocos
To determine the major components of the sclerotium of P. cocos contributable to its cytotoxicity against human lung cancer cells, the EtOH extract was fractionated into four different fractions (hexane-soluble, CH 2 Cl 2 -soluble, EtOAc-soluble, and n-BuOH-soluble fractions), and sequentially analyzed by LC/MS. The hexane-soluble fraction was found to contain the main constituents.

Cytotoxicity of Isolated Compounds 1-4 toward Human Lung Adenocarcinoma Cells
Compounds 1 and 4 were previously identified in the sclerotia of P. cocos [30,31]. However, their biological effects in cancer cells, including lung cancer cells, have not yet been examined. In addition, to the best of our knowledge, compounds 2 and 3 were identified in P. cocos for the first time in this study. To determine whether these compounds, which were isolated as the main constituents of the EtOH extract of the sclerotia of P. cocos, contributed to the cytotoxicity of the extract against human lung cancer cells in vitro, we investigated their effects on the cell viability of the same human lung adenocarcinoma cell lines ( Figure 4 and Table 1). WST-1 assay was carried out and revealed that after 48 h of treatment, compounds 1, 2, 3, and 4 showed significant dose-dependent cytotoxicity toward all of the cancer cell lines tested, with IC50 values ranging from 63.6 to 108.0 M, 50.6 to 121.9 M, 122.5 to 171.0 M, and 75.8 to 141.7 M, respectively (Figure 4A-D and Table 1). In addition, their cytotoxic effects did not correlate notably with the p53 status of the cancer cells. These observations imply that all of the isolated compounds contributed to the cytotoxicity of the EtOH extract of the sclerotia of P. cocos toward human lung cancer cell lines in vitro.
Similar to the EtOH extract, all four isolates were observed to increase the cell population exhibiting an apoptotic morphology in all of the human lung cancer cells tested ( Figure 4E). To validate that the isolated compounds triggered apoptosis in the cancer cells, we performed TUNEL staining on A549, H1264, H1299, and Calu-6 cells treated with each isolated compound for 48 h ( Figure 5). As predicted, all of the compounds showed a significant increase in the TUNEL-positive cell populations of all of the tested cancer cell lines, compared to DMSO (the vehicle control) ( Figure  5A-D), demonstrating that all of the isolated compounds exerted cytotoxicity in human lung cancer cells through the promotion of apoptosis.
Taken together, these results support our notion that the isolated compounds 1-4 are the main bioactive components contributing to both the cytotoxic and pro-apoptotic effects of the EtOH extract of the sclerotia of P. cocos examined in human lung cancer cells in vitro.

Cytotoxicity of Isolated Compounds 1-4 toward Human Lung Adenocarcinoma Cells
Compounds 1 and 4 were previously identified in the sclerotia of P. cocos [30,31]. However, their biological effects in cancer cells, including lung cancer cells, have not yet been examined. In addition, to the best of our knowledge, compounds 2 and 3 were identified in P. cocos for the first time in this study. To determine whether these compounds, which were isolated as the main constituents of the EtOH extract of the sclerotia of P. cocos, contributed to the cytotoxicity of the extract against human lung cancer cells in vitro, we investigated their effects on the cell viability of the same human lung adenocarcinoma cell lines ( Figure 4 and Table 1). WST-1 assay was carried out and revealed that after 48 h of treatment, compounds 1, 2, 3, and 4 showed significant dose-dependent cytotoxicity toward all of the cancer cell lines tested, with IC 50 values ranging from 63.6 to 108.0 µM, 50.6 to 121.9 µM, 122.5 to 171.0 µM, and 75.8 to 141.7 µM, respectively (Figure 4A-D and Table 1). In addition, their cytotoxic effects did not correlate notably with the p53 status of the cancer cells. These observations imply that all of the isolated compounds contributed to the cytotoxicity of the EtOH extract of the sclerotia of P. cocos toward human lung cancer cell lines in vitro.
Similar to the EtOH extract, all four isolates were observed to increase the cell population exhibiting an apoptotic morphology in all of the human lung cancer cells tested ( Figure 4E). To validate that the isolated compounds triggered apoptosis in the cancer cells, we performed TUNEL staining on A549, H1264, H1299, and Calu-6 cells treated with each isolated compound for 48 h ( Figure 5). As predicted, all of the compounds showed a significant increase in the TUNEL-positive cell populations of all of the tested cancer cell lines, compared to DMSO (the vehicle control) ( Figure 5A-D), demonstrating that all of the isolated compounds exerted cytotoxicity in human lung cancer cells through the promotion of apoptosis.
Taken together, these results support our notion that the isolated compounds 1-4 are the main bioactive components contributing to both the cytotoxic and pro-apoptotic effects of the EtOH extract of the sclerotia of P. cocos examined in human lung cancer cells in vitro.

Induction of the Mitochondrial Apoptotic Pathway by the EtOH Extract of the Sclerotia of P. cocos and Its Cytotoxic Constituents in Human Lung Adenocarcinoma Cells
To elucidate the molecular mechanisms underlying the pro-apoptotic activities of the EtOH extract of the sclerotia of P. cocos and its constituents in human lung cancer cells, we first explored their effects on the activation of caspase-3 (a major downstream effector caspase in the apoptotic pathway [32]) and the cleavage of its substrate PARP [33] in Calu-6 cells ( Figure 6). Upon treatment of Calu-6 cells with the EtOH extract, a dramatic increase in the cleaved forms of both caspase-3 and PARP compared to their levels in cells treated with DMSO ( Figure 6A) was observed. Furthermore, compounds 1-4 were all found to induce the cleavage of both caspase-3 and PARP in these cells ( Figure 6B). These data suggest that the EtOH extract and the constituents isolated from it induced apoptosis in human lung cancer cells through a caspase-3-dependent mechanism.
It is well known that the upregulation of Bax and the inactivation of Bcl-2 (through downregulation or hyperphosphorylation) reduce the mitochondrial membrane potential, leading to caspase-dependent apoptosis [34,35]. Therefore, to further delineate the upstream pathway leading to caspase-3 activation by the EtOH extract and the isolated compounds, we performed an immunoblot assay to probe lysates of Calu-6 cells treated with the extract and compounds for Bax and Bcl-2 proteins (Figure 6). Calu-6 cells were also treated with doxorubicin, which is an anticancer drug known to increase Bax protein expression and induce Bcl-2 protein hyperphosphorylation

Induction of the Mitochondrial Apoptotic Pathway by the EtOH Extract of the Sclerotia of P. cocos and Its Cytotoxic Constituents in Human Lung Adenocarcinoma Cells
To elucidate the molecular mechanisms underlying the pro-apoptotic activities of the EtOH extract of the sclerotia of P. cocos and its constituents in human lung cancer cells, we first explored their effects on the activation of caspase-3 (a major downstream effector caspase in the apoptotic pathway [32]) and the cleavage of its substrate PARP [33] in Calu-6 cells ( Figure 6). Upon treatment of Calu-6 cells with the EtOH extract, a dramatic increase in the cleaved forms of both caspase-3 and PARP compared to their levels in cells treated with DMSO ( Figure 6A) was observed. Furthermore, compounds 1-4 were all found to induce the cleavage of both caspase-3 and PARP in these cells ( Figure 6B). These data suggest that the EtOH extract and the constituents isolated from it induced apoptosis in human lung cancer cells through a caspase-3-dependent mechanism.
It is well known that the upregulation of Bax and the inactivation of Bcl-2 (through downregulation or hyperphosphorylation) reduce the mitochondrial membrane potential, leading to caspase-dependent apoptosis [34,35]. Therefore, to further delineate the upstream pathway leading to caspase-3 activation by the EtOH extract and the isolated compounds, we performed an immunoblot assay to probe lysates of Calu-6 cells treated with the extract and compounds for Bax and Bcl-2 proteins ( Figure 6). Calu-6 cells were also treated with doxorubicin, which is an anticancer drug known to increase Bax protein expression and induce Bcl-2 protein hyperphosphorylation [36,37], as a positive control. Of note, Bax protein expression and Bcl-2 protein phosphorylation were significantly greater in Calu-6 cells treated with the EtOH extract than in those treated with the vehicle (Figure 6A). In addition, compounds 1, 2, and 3 increased the protein levels of both Bax and hyperphosphorylated Bcl-2 in these cells compared to the vehicle control ( Figure 6B). Although compound 4 did not upregulate Bax, it did increase the hyperphosphorylated form of Bcl-2 compared to DMSO treatment in Calu-6 cells ( Figure 6B). These observations imply that the EtOH extract and isolated compounds 1-4 promoted apoptosis in human lung cancer cells by perturbing the mitochondria.
Correspondingly, our data strongly suggest that the sclerotia of P. cocos exert cytotoxicity against human lung cancer cells by the promotion of apoptosis mediated by the caspase-3-dependent mitochondrial pathway, and further support our notion that the isolated compounds, which are considered as the main constituents of the sclerotia of P. cocos, are responsible for the cytotoxicity.
Cells 2018, 7, x FOR PEER REVIEW 9 of 13 [36,37], as a positive control. Of note, Bax protein expression and Bcl-2 protein phosphorylation were significantly greater in Calu-6 cells treated with the EtOH extract than in those treated with the vehicle ( Figure 6A). In addition, compounds 1, 2, and 3 increased the protein levels of both Bax and hyperphosphorylated Bcl-2 in these cells compared to the vehicle control ( Figure 6B). Although compound 4 did not upregulate Bax, it did increase the hyperphosphorylated form of Bcl-2 compared to DMSO treatment in Calu-6 cells ( Figure 6B). These observations imply that the EtOH extract and isolated compounds 1-4 promoted apoptosis in human lung cancer cells by perturbing the mitochondria. Correspondingly, our data strongly suggest that the sclerotia of P. cocos exert cytotoxicity against human lung cancer cells by the promotion of apoptosis mediated by the caspase-3-dependent mitochondrial pathway, and further support our notion that the isolated compounds, which are considered as the main constituents of the sclerotia of P. cocos, are responsible for the cytotoxicity.

Discussion
In the last 20 years, P. cocos and its constituents, especially triterpenoids such as pachymic acid and polysaccharides such as β-glucan, have been found to exhibit a broad spectrum of antitumor activities both in vitro and in vivo, demonstrating the potential use of P. cocos as a functional food and a natural source of novel lead compounds for cancer management [7]. However, the anticancer potential of this mushroom and its constituents against human lung cancer required further investigation. In addition, the biological activity of ingredients other than triterpenoids and polysaccharides remained poorly studied.
In this study, an EtOH extract of the sclerotia of P. cocos exhibited significant cytotoxicity toward human lung cancer cells in vitro. This cytotoxicity was found to be independent of the p53 status of the cancer cells, and was mediated by the caspase-3-dependent mitochondrial apoptosis pathway. These observations not only support the results from previously published studies on the cytotoxicity of P. cocos toward A549 cells [12,15,17], they also provide further insight into the molecular mechanisms underlying this biological activity in human lung cancer cells. Considering that p53, a pivotal tumor suppressor gene, is mutated in more than 50% of human cancers, and that its status correlates closely with the clinical outcomes of cancer patients receiving chemotherapy and with the drug responsiveness of cancer cells in vitro [18], our results also broaden the potential application of P. cocos in lung cancer treatment.

Discussion
In the last 20 years, P. cocos and its constituents, especially triterpenoids such as pachymic acid and polysaccharides such as β-glucan, have been found to exhibit a broad spectrum of antitumor activities both in vitro and in vivo, demonstrating the potential use of P. cocos as a functional food and a natural source of novel lead compounds for cancer management [7]. However, the anticancer potential of this mushroom and its constituents against human lung cancer required further investigation. In addition, the biological activity of ingredients other than triterpenoids and polysaccharides remained poorly studied.
In this study, an EtOH extract of the sclerotia of P. cocos exhibited significant cytotoxicity toward human lung cancer cells in vitro. This cytotoxicity was found to be independent of the p53 status of the cancer cells, and was mediated by the caspase-3-dependent mitochondrial apoptosis pathway. These observations not only support the results from previously published studies on the cytotoxicity of P. cocos toward A549 cells [12,15,17], they also provide further insight into the molecular mechanisms underlying this biological activity in human lung cancer cells. Considering that p53, a pivotal tumor suppressor gene, is mutated in more than 50% of human cancers, and that its status correlates closely with the clinical outcomes of cancer patients receiving chemotherapy and with the drug responsiveness of cancer cells in vitro [18], our results also broaden the potential application of P. cocos in lung cancer treatment.
Our chemical investigation of the sclerotia of P. cocos revealed two triterpenoids, dehydroeburicoic acid monoacetate (1) and acetyl eburicoic acid (4); a sterol, 9,11-dehydroergosterol peroxide (2); and a diterpenoid, dehydroabietic acid (3), as the main constituents. All four isolated compounds were found to exert cytotoxicity by inducing apoptosis along with the activation of caspase-3 in human lung cancer cells. Compounds 1-3 stimulated both the expression of a pro-apoptotic protein, Bax, and the hyperphosphorylation of an anti-apoptotic regulator, Bcl-2, in human lung cancer cells. Compound 4 did not upregulate the Bax protein, but it did increase the level of hyperphosphorylated Bcl-2 in these cells. As with increased Bax expression, Bcl-2 hyperphosphorylation perturbs the mitochondrial membrane integrity by interfering with the interaction between Bcl-2 and pro-apoptotic partners such as Bax, leading to caspase-3 activation [34,35,38]. Hence, our data suggest that all four isolated compounds induce apoptosis through the mitochondrial pathway, similar to the EtOH extract of the sclerotia of P. cocos, and thus are the main bioactive constituents responsible for the cytotoxicity of P. cocos toward human lung cancer cells.
Triterpenoids derived from natural sources including mushrooms are well known for their anticancer potential, as they exert antioxidant, anti-inflammatory, and immunomodulatory activities and cytotoxicity against a broad spectrum of cancer cells [10,39]. P. cocos is a plentiful source of triterpenoid derivatives [7], suggesting its potential application in cancer prevention and treatment. So far, more than 50 triterpenoid derivatives have been identified from P. cocos, and their biological activities toward various cancer types have been evaluated in vitro and in vivo [7]. In this study, we identified two triterpenoid derivatives, dehydroeburicoic acid monoacetate (1) and acetyl eburicoic acid (4), as the main constituents of the sclerotia of P. cocos with cytotoxicity toward human lung cancer cells. Dehydroeburicoic acid monoacetate (1), which was identified in P. cocos for the first time in our previous study [30], had not previously been examined for its biological activity in cancer cells, and the present study is the first to demonstrate its cytotoxicity against cancer cells and the underlying mechanism. Acetyl eburicoic acid isolated from Laetiporus sulphureus was previously shown to exert cytotoxicity against HL-60 cells, which is a human myeloid leukemia cell line, by inducing caspase-3-dependent apoptosis [27]. In parallel with this finding, we found that acetyl eburicoic acid (4) derived from P. cocos exerted cytotoxic effects by triggering apoptosis accompanied by caspase-3 activation and mitochondrial destabilization in human lung cancer cells. Taken together, our findings provide additional experimental evidence for the anticancer potential of triterpenoid derivatives isolated from P. cocos, and thus further support the potential application of P. cocos for the treatment of lung cancer in particular.
Dehydroabietic acid (a naturally occurring diterpene rosin acid) and its synthetic derivatives have exhibited cytotoxicity toward various types of human cancer cells, including lung cancer cells [40,41]. Although the methyl ester derivative of dehydroabietic acid was previously identified in P. cocos and was shown to inhibit the tumor-promoting activity of 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro [42], dehydroabietic acid (3) was isolated from P. cocos and evaluated for its biological effects on human lung cancer cells for the first time in the present study. In addition, compound 2, a sterol identified as 9,11-dehydroergosterol peroxide, was isolated from P. cocos for the first time in this study. Although 9,11-dehydroergosterol peroxide isolated from the mycelium of Ganoderma lucidum has been evaluated for its cytotoxicity against human hepatocellular carcinoma and melanoma cells [28,43], its biological effects have not been examined in lung cancer cells. In the present study, we demonstrated that 9,11-dehydroergosterol peroxide exerts cytotoxicity against human cancer cells by inducing apoptosis. This finding is supported by previous reports that the apoptosis induced by this sterol was caspase-dependent and mediated through the mitochondrial pathway [43]. Taken together, our findings indicate that constituents of P. cocos other than triterpenoids and polysaccharides also contribute to its in vitro cytotoxic activity against human cancer cells, especially lung cancer cells.
Overall, we demonstrated that the cytotoxicity of P. cocos against human lung cancer cells with different p53 statuses in vitro and elucidated the underlying chemical and molecular basis for this activity. Although the antitumor activity of P. cocos needs to be validated in vivo in lung tumor models, our findings suggest the potential application of P. cocos and the isolated compounds (1)(2)(3)(4) in lung cancer therapy.

Conclusions
Our study demonstrates that P. cocos and its four main bioactive compounds, including two triterpenoids (1 and 4), a sterol (2), and a diterpenoid (3), are cytotoxic to human lung cancer cells with different p53 statuses. In terms of the molecular mechanism, our study indicates that P. cocos and these isolated compounds exert cytotoxicity against human lung cancer cells by inducing apoptosis accompanied by caspase-3 activation and mitochondrial perturbation. These findings provide experimental evidence and a molecular explanation for the anticancer properties of P. cocos, especially against lung cancer, and support the previous notion that P. cocos can be applied for cancer treatment.