Structure Elucidation and Anti-Tumor Activities of Trichothecenes from Endophytic Fungus Fusarium sporotrichioides

The secondary metabolites of Fusarium sporotrichioides, an endophytic fungus with anti-tumor activity isolated from Rauvolfia yunnanensis Tsiang, were investigated. Five trichothecenes, including one previously undescribed metabolite, were isolated and identified. Their structures were elucidated by means of extensive spectroscopic methods; the absolute configuration of compound 1 was determined by the ECD method. Surprisingly, 8-n-butyrylneosolaniol (3) exhibited stronger anti-tumor activity than T-2 toxin against Huh-7 cell line, with an IC50 value of 265.9 nM. 8-n-butyrylneosolaniol (3) promoted apoptosis induction in Huh-7 cells. Moreover, cell cycle analysis showed that cell cycle arrest caused by 8-n-butyrylneosolaniol (3) at the G2/M phase resulted in cell proliferation inhibition and pro-apoptotic activity. Further studies showed a significant decrease in mitochondrial membrane permeabilization and a significant increase in ROS generation, which led to the activation of caspase cascades and subsequent cleavage of PARP fragments. In conclusion, 8-n-butyrylneosolaniol (3) induced cell apoptosis in Huh-7 cells via the mitochondria-mediated apoptotic signaling pathway, which could be a leading compound for anti-tumor agents.


Introduction
Endophytic microorganisms have been found in virtually every medicinal plant and contribute to their host plant by producing a plethora of substances that provide protection to the plant [1,2]. Endophytic fungi have been viewed as an outstanding source of novel and bioactive natural products because they have produced a number of famous and important compounds [3][4][5]. As our going research on anti-tumor secondary metabolites from endophytic fungi of medicinal plants, one strain Fusarium sporotrichioides isolated from Rauvolfia yunnanensis Tsiang has attracted our attention because its mycelial extracts showed strong cytotoxicity against A549 cell line (IC 50 = 15.4 µg/mL). Then, the crude extract of F. sporotrichioides in solid rice culture was chemically investigated, which led to the isolation of five trichothecenes (1)(2)(3)(4)(5), including one previously undescribed trichothecene named 8-(2-methylbutyryl)-neoso-laniol (1).
It is well known that trichothecenes are a large family of mycotoxins produced by several fungal genera, including Fusarium [6,7]. Currently, approximately 200 members of the trichothecene family have been identified [8]. Trichothecenes are also a source of contamination in food, which could induce a series of acute and chronic symptoms, such as hypotension, anemia and so on [9]. Nevertheless, some trichothecenes have also been reported to exhibit antifungal, antimalarial, antiviral and phytotoxic activities [9][10][11]. The biological investigation of the five compounds suggested that all of them exhibited cytotoxicities against Huh-7 cells and MRMT-1 cells. Among them, 8-n-butyrylneosolaniol (3) exhibited stronger cytotoxic activity against the Huh-7 cell line than the T-2 toxin, with an IC 50

Strain Isolation and Cultivation
The fungal strain was isolated from the inner tissue of the surface-sterilized fruit of RauwoIfia yunnanensis Tsiang, which was collected in Yunnan Province in China in 2017. It was identified as Fusarium sporotrichioides by its rRNA gene sequence and given the Genbank accession number MT649476.1. A voucher specien (No.luo4-fruit-2i-b) was deposited at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing. The strain was fermented on autoclaved rice solid-substrate medium (eighty 500 mL Erlenmeyer flasks, each containing 40 g rice and 60 mL water) for 12 days at 28 • C.

Cell Lines and Cell Culture
The human hepatoma Huh-7 cells and rat mammary tumor MRMT-1 cells were purchased from ATCC (Manassas, VA, USA). DMEM medium was purchased from ATCC (Manassas, VA, USA). Penicillin-streptomycin, 0.25% trypsin and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere at 37 • C with 5% CO 2 .

Cell Cycle Assay
The cell cycle was measured by PI staining and flow cytometry analysis. Approximately 1 × 10 5 cells/mL of Huh-7 cells were seeded into 24-well plates and incubated for 24 h. The cells were treated with 40, 400 and 800 nM of compound 3 for 24 h, harvested and washed once with PBS. Then the cells were incubated with 70% cold ethanol at −20 • C overnight. After fixation, cells were washed twice with cold PBS and centrifuged (12,000 rpm) to discard the supernatant. The pellet was resuspended with PBS and stained with PBS containing 50 µg/mL PI and 100 µg/mL RNase A for 30 min in the dark. The cell cycle phase distribution was analyzed using flow cytometry (Beckman coulter, CA, USA).

Cell Apoptosis Analysis
The apoptosis induction was detected by the annexin-V/PI staining method. Huh-7 cells (2 × 10 5 /well) were cultured in 6-well plates for 24 h, then treated with 3 of various concentrations (0, 40, 400 and 800 nM) for 36 h. The test procedure followed the manufacturer's instructions. Then cells were harvested, washed, and suspended in 500 µL annexin V binding buffer; after that, annexin V-FITC (5 µL) and PI (5 µL) were added. After being stained in the dark for 15 min at room temperature, the cells were analyzed by use of flow cytometry (Beckman coulter, CA, USA).

Mitochondrial Membrane Potential Analysis
The mitochondrial membrane potential (MMP) was determined by JC-10 dye staining. Huh-7 cells were incubated with different concentrations of compound 3 (0, 40, 400 and 800 nM) for 32 h. After removing the culture, the cells were collected by trypsinization, centrifuged (12,000 rpm), and then suspended in a DMEM medium at the concentration of 1 × 10 5 cells/mL. The cells were kept in the incubator (5% CO 2 ) for 20 min after being mixed with JC-10 stain liquid. Finally, the cells were imaged by means of a fluorescence microscope after washing.

Measurement of ROS Generation
The production of ROS was detected by flow cytometry. Briefly, Huh-7 cells were plated in six-well plates and incubated for 24 h. Then cells were treated with various concentrations of 3 (0, 40, 400 and 800 nM) for 32 h. After exposure, DCFH-DA (10 mmol/L) was added and incubated at 37 • C for 30 min in the dark. Then, cells were washed with 3 at doses of 40, 400 and 800 nM for 32 h. Cells were analyzed by flow cytometer (Beckmancoulter, CA, USA).

Western Blot Analysis
Huh-7 cells were incubated with 3 for whole-cell lysates or lysis buffer NP-40 for cytoplasmic lysates. Then, cells were harvested in lysis buffer RIPA for whole-cell lysates and then incubated for 15 min at 4 • C. After centrifugation (12,000 rpm), the supernatant was obtained for analysis. The protein concentration of the supernatant was determined by BCA (Thermo Fisher, Carlsbad, CA, USA) assay. Supernatants were then mixed with SDS-PAGE sample buffer and boiled for 5 min. Samples were subsequently loaded into each lane of a 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Philadelphia, PA, USA). Membranes were blocked for 1 h in 5% (w/v) non-fat milk and then incubated with a primary antibody at room temperature for 1 h. The following antibodies were used: cytochrome C, caspase-9, cleaved caspase-3 and PARP. Having been washed 3 times in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbit IgG (diluted 1:10,000) for 1 h at room temperature and then washed 3 times. Finally, proteins were detected using SuperSignal West Dura ECL substrates (Thermo Fisher, Carlsbad, CA, USA). The load protein was normalized to GAPDH. All experiments were performed in triplicate.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD). Unpaired t-tests were performed for comparison between the two groups. One-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test was used to compare more than two groups. Differences were considered significant at p < 0.05.
ferences were considered significant at p < 0.05.

Structure Elucidation of Compound 1
Compound 1 (Figure 1) was obtained as a white powder with a molecular formula of C24H34O9 based on its HRESIMS data at m/z 489.2089 [M+Na] + (calcd for C24H34O9Na, 489.2095), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands at 3432 and 1733 cm -1 , assignable to hydroxyl and ester carbonyl groups. The 1 Table 1). The 13 C-NMR and HSQC results revealed the presence of 24 carbon resonances attributable to six methyls, four methylenes, seven methines and seven quaternary carbons (including an oxygenated quaternary carbon at δC 65.3 (C-12), one olefinic carbon at δC 137.1 (C-9), three carbonyls at δC 172.3 (C-17), 172.1 (C-19) and 177.3 (C-1') and two carbons at δC 49.8 (C-5) and 44.0 (C-6). A comparison of the NMR data of 1 with those reported suggested that 1 belonged to a group of trichothecenes sesquiterpenoids [12][13][14], except that the ester side chain at C-8 changed (    ferences were considered significant at p < 0.05.

Structure Elucidation of Compound 1
Compound 1 (Figure 1) was obtained as a white powder with a molecular formula of C24H34O9 based on its HRESIMS data at m/z 489.2089 [M+Na] + (calcd for C24H34O9Na, 489.2095), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands at 3432 and 1733 cm -1 , assignable to hydroxyl and ester carbonyl groups. The 1 Table 1). The 13 C-NMR and HSQC results revealed the presence of 24 carbon resonances attributable to six methyls, four methylenes, seven methines and seven quaternary carbons (including an oxygenated quaternary carbon at δC 65.3 (C-12), one olefinic carbon at δC 137.1 (C-9), three carbonyls at δC 172.3 (C-17), 172.1 (C-19) and 177.3 (C-1') and two carbons at δC 49.8 (C-5) and 44.0 (C-6). A comparison of the NMR data of 1 with those reported suggested that 1 belonged to a group of trichothecenes sesquiterpenoids [12][13][14], except that the ester side chain at C-8 changed (    The relative configuration of 1 was established by analysis of NOESY correlations ( Figure 3A) 0.72) positioned these protons on the other side. It is known that oxidations at C-8 in trichothecenes were stereospecific in Fusarium species [13]. The oxygen function at C-8 in the trichothecene ring normally resulted in an oxygen function with an 8α-configuration. Thus, the relative configuration of 1 was determined, which was consistent with known trichothecenes reported previously.
the H-11, H-4 and H-15 were in the α orientation. Furthermore, the NOESY cross of H-7β (δH 2.43) / H-13 (δH 3.02, 2.85), H-2 (δH 3.54) / H-3 (δH 4.22) / Me-14 (δH 0.72 tioned these protons on the other side. It is known that oxidations at C-8 in trichoth were stereospecific in Fusarium species [13]. The oxygen function at C-8 in the tric cene ring normally resulted in an oxygen function with an 8α-configuration. Thu relative configuration of 1 was determined, which was consistent with known tric cenes reported previously. Moreover, the absolute configuration of 5 was previously confirmed on the b single-crystal X-ray analysis [14]. Therefore, to determine the stereochemistry of 1, sorted to the ECD analysis by comparison with 5 ( Figure 3B). The ECD spectrum of nm, Δε −1.86) displayed a similar negative cotton effect to that of 5 (211 nm, Δε Therefore, the absolute stereochemistry of 1 is assumed to be the same as 5. As far know, the trichothecenes that have been reported all had the same configuration [ It was given the name 8-

Anti-Tumor Activities of Compounds 1-5
The anti-tumor effects of isolated compounds 1-5 on Huh-7 and MRMT-1 wer uated by CCK-8 assay. The results indicated all compounds showed strong inhibi the growth of Huh-7 and MRMT-1 cells compared to the T-2 toxin. Surprisingly, 3 icantly decreased the cell viability of Huh-7 cells and MRMT-1 cells with the IC50 of 265.9 nM and 279.4 nM, respectively ( Table 2). These data indicated that 3 exhib more destructive effect in Huh-7 cells even than T-2 toxin. Due to the strong reduc Huh-7 cells viability even at a very low concentration, the mechanism of action o Huh-7 cells was further studied.  Moreover, the absolute configuration of 5 was previously confirmed on the basis of single-crystal X-ray analysis [14]. Therefore, to determine the stereochemistry of 1, we resorted to the ECD analysis by comparison with 5 ( Figure 3B). The ECD spectrum of 1 (220 nm, ∆ε −1.86) displayed a similar negative cotton effect to that of 5 (211 nm, ∆ε −1.00). Therefore, the absolute stereochemistry of 1 is assumed to be the same as 5.
As far as we know, the trichothecenes that have been reported all had the same configuration [15,16]. It was given the name 8-

Anti-Tumor Activities of Compounds 1-5
The anti-tumor effects of isolated compounds 1-5 on Huh-7 and MRMT-1 were evaluated by CCK-8 assay. The results indicated all compounds showed strong inhibition of the growth of Huh-7 and MRMT-1 cells compared to the T-2 toxin. Surprisingly, 3 significantly decreased the cell viability of Huh-7 cells and MRMT-1 cells with the IC 50 values of 265.9 nM and 279.4 nM, respectively ( Table 2). These data indicated that 3 exhibited a more destructive effect in Huh-7 cells even than T-2 toxin. Due to the strong reduction in Huh-7 cells viability even at a very low concentration, the mechanism of action of 3 on Huh-7 cells was further studied.

Effects of Compound 3 Inducing Cell Cycle Arrest in Huh-7 Cells
We investigated the changes in the cell cycle phase distribution on the basis of DNA content in Huh-7 cells by flow cytometry analysis using PI staining. A cell-cycle assay was performed by treating Huh-7 cells at various concentrations of 3 (0, 40, 400 and 800 nM) for 24 h, and the observed FACS histogram statistics are shown in Figure 4. In the untreated control group, the G2/M phase cells percentage was only 18.79%, which increased to 23.93%, 27.95% and 32.09% in groups treated with different concentrations of 3. Therefore, the result suggested that 3 had a conspicuous effect on the cell cycle and compelled the cells to get arrested in the G2/M phase in a dose-dependent manner. content in Huh-7 cells by flow cytometry analysis using PI staining. A cell-cycle assay was performed by treating Huh-7 cells at various concentrations of 3 (0, 40, 400 and 800 nM) for 24 h, and the observed FACS histogram statistics are shown in Figure 4. In the untreated control group, the G2/M phase cells percentage was only 18.79%, which increased to 23.93%, 27.95% and 32.09% in groups treated with different concentrations of 3. Therefore, the result suggested that 3 had a conspicuous effect on the cell cycle and compelled the cells to get arrested in the G2/M phase in a dose-dependent manner.

Effects of Compound 3 Inducing Apoptosis of Huh-7 Cells
Next, we checked whether the anti-tumor activity of 3 was due to the induction of apoptosis in Huh-7 cells. It is well known that cells that were undergoing apoptosis would shift from the viable quadrant (Q1-LL) to the early apoptosis quadrant (Q1-LR) and eventually end up in the late apoptosis quadrant (Q1-UR). On the other hand, cells that underwent necrosis would shift from the viable quadrant (Q1-LR) to the late necrosis quadrant (Q1-UL). Cell apoptosis assay was performed with an Annexin V-FITC and PI double staining. After treatment with 3 for 36 h at different concentrations (0, 40, 400 and 800 nM), the distribution of cells undergoing apoptosis or necrosis was analyzed by flow cytometry assay. The results showed that the cells incubated with different concentrations of 3 for 36 h underwent apoptosis ( Figure 5). As concentration increased, the rate of cell apoptosis significantly increased (62.47% for 800 nM treated with 3 and 1.02% for blank control), suggesting that 3 could significantly induce cell apoptosis in a dose-dependent manner. All data are the mean ± SD from three independent experiments. *** p < 0.001 vs. control group.

Effects of Compound 3 Inducing Apoptosis of Huh-7 Cells
Next, we checked whether the anti-tumor activity of 3 was due to the induction of apoptosis in Huh-7 cells. It is well known that cells that were undergoing apoptosis would shift from the viable quadrant (Q1-LL) to the early apoptosis quadrant (Q1-LR) and eventually end up in the late apoptosis quadrant (Q1-UR). On the other hand, cells that underwent necrosis would shift from the viable quadrant (Q1-LR) to the late necrosis quadrant (Q1-UL). Cell apoptosis assay was performed with an Annexin V-FITC and PI double staining. After treatment with 3 for 36 h at different concentrations (0, 40, 400 and 800 nM), the distribution of cells undergoing apoptosis or necrosis was analyzed by flow cytometry assay. The results showed that the cells incubated with different concentrations of 3 for 36 h underwent apoptosis ( Figure 5). As concentration increased, the rate of cell apoptosis significantly increased (62.47% for 800 nM treated with 3 and 1.02% for blank control), suggesting that 3 could significantly induce cell apoptosis in a dose-dependent manner.

Effects of Compound 3 on Inducing Depolarization of Mitochondrial Membrane Potential and Generation of ROS
To verify whether the mitochondria-mediated signaling pathway was involved in the induced apoptosis of 3, MMP was measured by use of an MMP assay kit with JC-10 after treatment with 3. In the normal mitochondria, the JC-10 can be aggregated and emit red fluorescence. With the depolarization of 3, JC-10 would form monomers that could produce green fluorescence. A significant increase was observed in the intensity of the green fluorescence in Huh-7 cells, indicating that the MMP decreased after treatment with 3 ( Figure 6A). The color change from red to green accompanied by increased doses of 3 indicated the increased mitochondrial injury.
It has been shown in previous reports that ROS is involved in the mitochondriamediated apoptosis pathway induced by multiple toxins [18]. Mitochondrial damage led to impaired oxidative phosphorylation and electron transport chain, which resulted in excessive ROS production and release from mitochondria. To determine whether 3 triggered ROS generation in Huh-7 cells, the ROS level was measured using 2',7'-dichlorodihydrofluoresce in diacetate (DCFH-DA). When compared with the control group, the concentration assay of ROS levels showed a significant increase by treatment with 3 in a dose-dependent manner ( Figure 6B).

Effects of Compound 3 on Inducing Depolarization of Mitochondrial Membrane Potential and Generation of ROS
To verify whether the mitochondria-mediated signaling pathway was involved in the induced apoptosis of 3, MMP was measured by use of an MMP assay kit with JC-10 after treatment with 3. In the normal mitochondria, the JC-10 can be aggregated and emit red fluorescence. With the depolarization of 3, JC-10 would form monomers that could produce green fluorescence. A significant increase was observed in the intensity of the green fluorescence in Huh-7 cells, indicating that the MMP decreased after treatment with 3 ( Figure 6A). The color change from red to green accompanied by increased doses of 3 indicated the increased mitochondrial injury.
It has been shown in previous reports that ROS is involved in the mitochondria-mediated apoptosis pathway induced by multiple toxins [18]. Mitochondrial damage led to impaired oxidative phosphorylation and electron transport chain, which resulted in excessive ROS production and release from mitochondria. To determine whether 3 triggered ROS generation in Huh-7 cells, the ROS level was measured using 2',7'-dichlorodihydrofluoresce in diacetate (DCFH-DA). When compared with the control group, the concentration assay of ROS levels showed a significant increase by treatment with 3 in a dosedependent manner ( Figure 6B).

Effect of Compound 3 on the Expression of Apoptosis-Related Proteins
To further evaluate if 3 induced cell apoptosis by activating the mitochondrial pathway, Western blot analysis was used to detect the expression levels of mitochondrial apoptosis pathway-related proteins, as shown in Figure 7. To assess if the equilibrium of mitochondrial fusion was disturbed and caused cellular apoptosis, cytochrome C release was tested. As expected, the protein expression was higher in compound 3 groups. In addition, the expression level of cleaved caspase 9 was markedly increased as well. Moreover, as the concentration of 3 increased, the protein expression levels of cleaved caspase-3 and cleaved PARP were significantly increased. Altogether, our results indicated that 3 induced apoptosis via a caspase-dependent mechanism.

Effect of Compound 3 on the Expression of Apoptosis-Related Proteins
To further evaluate if 3 induced cell apoptosis by activating the mitochondrial pathway, Western blot analysis was used to detect the expression levels of mitochondrial apoptosis pathway-related proteins, as shown in Figure 7. To assess if the equilibrium of mitochondrial fusion was disturbed and caused cellular apoptosis, cytochrome C release was tested. As expected, the protein expression was higher in compound 3 groups. In addition, the expression level of cleaved caspase 9 was markedly increased as well. Moreover, as the concentration of 3 increased, the protein expression levels of cleaved caspase-3 and cleaved PARP were significantly increased. Altogether, our results indicated that 3 induced apoptosis via a caspase-dependent mechanism. apoptosis pathway-related proteins, as shown in Figure 7. To assess if the equilibrium of mitochondrial fusion was disturbed and caused cellular apoptosis, cytochrome C release was tested. As expected, the protein expression was higher in compound 3 groups. In addition, the expression level of cleaved caspase 9 was markedly increased as well. Moreover, as the concentration of 3 increased, the protein expression levels of cleaved caspase-3 and cleaved PARP were significantly increased. Altogether, our results indicated that 3 induced apoptosis via a caspase-dependent mechanism.

Discussion
During our research on anti-tumor secondary metabolites from endophytic fungi, one novel and four known trichothecene metabolites belonged to the type A trichothecene mycotoxin family [8]. T-2 toxin was the earliest investigated member of type A trichothecene, which was one of the most cytotoxicity trichothecenes up to now [18]. However, its antitumor activity has not been reported in Huh-7 cells. In this study, surprisingly, compound 3 exhibited lower IC 50 values compared to T-2 toxin against Huh-7 cells, which was reported for the first time. Therefore, further mechanism experiments investigating the effects of compound 3 on Huh-7 cell are important.
Cell cycle progression is tightly linked with cell proliferation [19]. The cell cycle is mainly adjusted by checkpoints which are activated by DNA damage to assure the exact progress of the cell cycle. In this condition, the growth arrest due to checkpoints allows cellular DNA damage repair; otherwise, the cell is eliminated by apoptosis [20,21]. When DNA is damaged, the G2/M checkpoint is a barrier that inhibits the cell's progress entry into the M phase [22]. When cells possess an unrepaired DNA from S or G1 phases or partially replicated chromatin from the S phase. In this case, it is necessary to inhibit cell progress before mitotic entry [23]. Due to DNA damage, the cells activate an apoptotic program and halt mitotic during the metaphase [24]. In this study, our result demonstrated that the growth inhibitory effect of the 3 on Huh-7 cells was associated with a G2/M arrest and cell cycle progression.
Cell cycle could help to regulate cell growth, which played an important role in apoptosis. To investigate if the cell death caused by 3 was induced by apoptotic activity, the Annexin V-FITC/PI assay was used. This technique detects early apoptosis by means of monitoring the translocation of phosphatidylserine from the inner side of the cell membrane to the outer layer [25,26]. The quantification means of apoptotic cells are measured Annexin V-FITC binding to the externalized phosphatidylserine. Propidium iodide (PI) was added to distinguish between viable, early apoptotic, late apoptotic and necrotic cells [26,27]. After treating the Huh-7 cells with different concentrations of 3 for 36 h, the early apoptotic rate was significantly increased compared to the control group. The analysis of Huh-7 cells by flow cytometry revealed that treatment with the 3 induced a shift in the cell population toward apoptosis.
Apoptosis was often accompanied by a decrease in mitochondrial membrane permeabilization [28]. The decrease in mitochondrial membrane permeabilization was widely regarded as one of the earliest events of apoptosis [29]. It is crucial to maintain the mitochondrial membrane potential for mitochondrial function [30,31]. A mitochondrial membrane potential measurement with the membrane potential-sensitive dye JC-10 was conducted to confirm whether the mitochondria-mediated signaling pathway was involved in 3-induced apoptosis. In our study, increased exposure to 3 led to a significant decrease in mitochondrial transmembrane potential, confirming that apoptosis was triggered through the mitochondrial pathway.
Mitochondria were the major cellular organelles that produced ROS. Furthermore, excessive ROS was involved in the damage of the mitochondrial membrane and the initiation of apoptotic tissue damage [32,33]. Thus, the effects of 3 on the level of ROS were investigated by the DCFH-DA method [34]. A significant dose-dependent increase in intracellular ROS in Huh-7 cells treated by 3 gave evidence that 3 could inhibit mitochondrial dysfunction.
Subsequently, we explored the molecular mechanisms underlying the regulation of cell apoptosis processes. Apoptosis was a form of normal programmed cell death and a complex process that could be triggered by many different factors [35,36]. Numerous studies have shown that the apoptotic pathway was categorized into the extrinsic apoptotic and intrinsic apoptotic pathways [37]. The mitochondria-initiated intrinsic pathway required the release of cytochrome C and promoted the caspase-activating apoptosome, a complex that induced the activation of caspase-9 and initiated the apoptotic caspase cascades [35]. Caspases are both the initiators and executors of cell death, which are the center of the apoptosis mechanism [38]. Moreover, activation of caspase was regarded as a typical indication of apoptosis [39]. Caspase-3 is regarded as the apoptosis promoter [40]. Thus, cytochrome C could activate caspase-9 and caspase-3 and stimulate the proteolytic cleavage of PARP, resulting in cell apoptosis. Taken together, our results indicate that 3 induced apoptosis via a caspase-dependent mechanism. Eventually, these studies offer vital information on the apoptosis and anti-tumor of trichothecenes, which could lead to further studies dedicated to this topic. Additionally, this research work may give an insight into the discovery of trichothecenes-derived new drug leads. In the future, we will further investigate if compound 3 could be developed as an anti-tumor agent through more in vivo experiments.

Conclusions
In summary, five secondary metabolites were isolated from F. sporotrichioides, including one new trichothecene. Among these compounds, 3 displayed significantly cytotoxic activity against Huh-7 cells by cell viability assay. The mechanism responsible for the cytotoxicity of 3 was further studied. Flow cytometry revealed that 3 could induce G2/M arrest and apoptosis. Further studies showed a significant decrease in mitochondrial membrane permeabilization and a significant increase in ROS generation, which led to the activation of caspase cascades (caspase-3 and caspase-9) and subsequent cleavage of PARP fragment. These results indicated that 3 induced cell apoptosis in Huh-7 cells via the mitochondria-mediated apoptotic signaling pathway.