- Open Access
Hirsutanol A, a novel sesquiterpene compound from fungus Chondrostereum sp., induces apoptosis and inhibits tumor growth through mitochondrial-independent ROS production: Hirsutanol A inhibits tumor growth through ROS production
- Fen Yang†1,
- Wen-Dan Chen†1,
- Rong Deng1,
- Hui Zhang3,
- Jun Tang1,
- Ke-Wei Wu1,
- Dan-Dan Li1,
- Gong-Kan Feng1,
- Wen-Jian Lan4,
- Hou-Jin Li2Email author and
- Xiao-Feng Zhu1Email author
© Yang et al.; licensee BioMed Central Ltd. 2013
Received: 9 August 2012
Accepted: 21 January 2013
Published: 8 February 2013
Hirsutanol A is a novel sesquiterpene compound purified from fungus Chondrostereum sp. in Sarcophyton tortuosum. Our previous studies had demonstrated that hirsutanol A exhibited potent cytotoxic effect on many kinds of cancer cell lines. In the current study, the antitumor activity of hirsutanol A and its molecular mechanisms were investigated.
Hirsutanol A induced growth inhibition and apoptotic cell death of human colon cancer SW620 cells and human breast cancer MDA-MB-231cells were determined using MTT assay and flow cytometry assay, respectively. The effect of hirsutanol A on intrinsic ROS level and change in mitochondrial membrane potential (△ψm) of different cell lines were also measured by flow cytometry assay. The function of JNK was compromised by JNK siRNA or JNK inhibitor SP600125. The expression of cytochrome c, p-JNK, p-c-Jun after treatment with hirsutanol A were detected by Western blot analysis. Finally, the in vivo anti-tumor effect of hirsutanol A was examined in human cancer cell SW620 xenograft model.
The results showed that hirsutanol A significantly induced apoptosis, mitochondrial-independent increase of Reactive Oxygen Species (ROS) level, change of mitochondrial membrane potential, release of cytochrome c in human cancer cells. Preventing increase of ROS level using the potent antioxidant N-acetyl-L-cysteine (NAC) markedly decreased hirsutanol A-induced apoptosis. In addition, JNK signaling pathway was activated by hirsutanol A through elevating ROS level. Blockade of JNK signaling pathway by JNK specific inhibitor SP600125 enhanced apoptosis and hirsutanol A-induced ROS accumulation. Also, hirsutanol A exhibited antitumor activity in human cancer cell SW620 xenograft model.
These data suggested that hirsutanol A inhibited tumor growth through triggering ROS production and apoptosis.
ROS (Reactive Oxygen Species) is a collective term for oxygen derived species, including superoxide anion radical O2.-, hydroxyl radicals .OH etc. . The basic level of intracellular ROS is considered to be important to promote cell proliferation and differentiation. However, excessive amounts of ROS can contribute to carcinogenesis and cancer progression [2, 3]. Therefore, maintaining ROS homeostasis is crucial for normal cell growth and survival . Compared to normal cells, cancer cells with increasing intrinsic ROS are more vulnerable to damage by further ROS insults induced by exogenous agents . Manipulating ROS levels by redox modulation is one way to selectively kill cancer cells sparing normal cells. Hence, the redox system is considered as a new target for anticancer drugs [6, 7].
Apoptosis is a highly regulated and organized cell death process, which controls the development and homeostasis of multicellular organisms . Death receptor signaling pathway and mitochondrial pathway are two major pathways mediating apoptosis triggered by different apoptotic stimuli [9, 10]. Alterations in the cellular ROS status have been proven to play an important role in apoptotic cell death . Excessive ROS will attack lipids and proteins of mitochondria membrance, leading to severe and irreversible oxidative damage, dysfunction of mitochondria, and release of cytochrome c, which in turn activates the caspase-3 initiating mitochondria / cytochrome c –mediated apoptosis [12, 13].
C-Jun NH2-terminal kinases (JNKs) are strongly activated by oxidative stress, which can induce apoptosis or regulate cellular ROS level by activating its downstream molecule c-Jun . C-Jun is fisrt phosphorylated by JNK and then translocates to the nucleus for further regulating the transcription of target genes including some pro-apoptotic or antiapoptotic proteins such as Bax and Bcl-2 and some redox proteins such as NOX, SOD [15, 16].
Hirsutanol A is a novel sesquiterpene compound purified from fungus Chondrostereum sp. in Sarcophyton tortuosum. Our previous studies showed that Hirsutanol A exerted potent cytotoxic effect on many kinds of human cancer cell lines [18, 19]. In this study, we examined the molecular mechanism of Hirsutanol A-induced apoptosis and its antitumor activity in human cancer cell SW620 xenograft model. We demonstrated that Hirsutanol A could induce apoptosis in SW620 and MDA-MB-231 cells and significantly inhibit tumor growth in vivo. Futhermore, we found that hirsutanol A could elevate intrinsic ROS level, and activate mitochondria/ cytochrome c signaliing pathway to trigger apoptosis.
Drugs and reagents
Cell lines and cell culture
Human colon cancer cell line SW620 and human breast cancer cell line MDA-MB-231 were cultured in RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum, penicillin (50U/ml) and streptomycin (50 μg/ml) at 37°C in 5% (v/v) CO2. All experiments were carried out with cells in logarithmic growth phase.
SW620 and MDA-MB-231 cells were first seeded in 96-well plate at a density of 8,000 cells per well, then treated with different concentrations of hirsutanol A for indicated times or pre-incubated with 1mmol/L antioxidant NAC for 1 h, then cultivated for 72 h at 37°C. 10 μL of 5 mg/mL MTT was added into each well before the termination of experiment. The plates were incubated at 37°C, 5% (v/v) CO2 for 4 h. After complete removal of the medium, 100 μL of DMSO was added into each well to dissolve the insoluble purple formazan product. Absorbance values were obtained with a test wavelength of 570 nm. The rates of cell growth inhibition were calculated based on the absorbance values. The 50% inhibitory rates (%) were calculated by the Bliss method: Inhibitory rate = (1-the average OD value of treatment group/ the average OD value of the control group) × 100% .
Annexin V/Propidium Idodide (PI) double-staining assay
Annexin V/PI staining was performed using the Annexin V-fluorescein isothiocyanate apoptosis detection kit. Cells (3.0 × 105 per mL) were seeded into six-well plate with 2 mL in each well, then treated with different concentrations of hirsutanol A for 72 h or pretreated with 1mmol/L NAC or 10 μmol/L SP600125 followed by hirsutanol A for 72 h. Both floating and attached cells were collected, washed with ice-cold PBS twice, then incubated at room temperature in the presence of media binding reagent and Annexin V-FITC for 15min in the dark. After washing with PBS, the cells were re-suspended in ice-cold 1 × binding buffer and treated with 10 μL propidium iodide (30 μg/ml) on ice in the dark. Apoptosis was quantified by flow cytometry (Becton Dickinson) at the wavelength of 488 nm immediately and analyzed by the Cell-Quest software .
Flow cytometry assay and measurement of ROS
Cells were diluted to 3.0 × 105 per mL, seeded into six-well plate with 2 mL in each well. SW620 cells and MDA-MB-231 cells were treated with hirsutanol A for indicated times or treated with various concentrations of hirsutanol A for 24 h, or pre-incubated with 1 mmol/L NAC or 1 μmol/L SP600125 followed by hirsutanol A for 24 h, then incubated with 1 μmol/L (final concentration) CM-H2DCF-DA or DHE florescent dye in dark for 1 h at 37°C. After washing twice with ice cold PBS, cells were centrifuged and re-suspended in PBS. The level of intracellular ROS was detected by flow cytometry with FACS Calibur system and CellQuestPro analysis software .
Cells were seeded into six-well plate, treated with hirsutanol A for indicated times or pre-incubated with NAC for 1 h followed by hirsutanol A for 24 h. Cells were harvested and washed twice with PBS and lysed in lysis buffer. The cell lysates were clarified by centrifugation at 12,000 g for 10 min at 4°C and the protein concentration was determined using the Bio-Rad protein assay (Bio-Rad laboratories). SDS-PAGE sample buffer was added to cell lysates. Then the cell lysates were heated at 100°C for 5 min, and cell lysates containing 20–40 μg protein was loaded in each well of 8% (w/v) and 15% (w/v) SDS-PAGE gel. Resolved proteins were electrophoretically transferred to PVDF membrane, which was incubated sequentially with primary antibody and horseradish peroxidase–conjugated second antibody. After washing, the bound antibody complex was detected using an ECL chemiluminescence reagent and XAR film (Kodak) as described by the manufactures [23, 24].
The target sequence for JNK-specific siRNA was 5′-AAAAAGAAUGUCCUAC CUUCU-3′, and control siRNA (no silencing) were synthesized by GenChem Co. One day before transfection, cells were plated in six-well plates with antibiotic-free growth medium at a density of 1.5 × 105 cells per well. When cells grew to a confluency of 30-50% on the second day, transfection was performed by using Opti-MEM media, lipofectamine 2000 and JNK siRNA according to manufacturer’s recommendations. The final concentration of JNK siRNA was 100 nM. After 6 h, the Opti-MEM media was replaced with the antibiotic free growth media and cells were treated with 20 μmol/L hirsutanol A for 3 h.Cells transfected with lipofectamine 2000 were used as control .
Mitochondrial /cytosol fractionation
The isolation of cell mitochondrial and cytosolic fractions was performed using mitochondria/cytosol fractionation kit according to the following protocol. Cells previously treated with 20 μmol/L hirsutanol A for 24 h were harvested at ~850 × g for 2 min and 800 μl of mitochondria isolation reagent A and 10 μl of mitochondria isolation reagent B were added. After 5 min incubation on ice, 800 μl of mitochondria isolation reagent C was added and the mixture was centrifuged at 700 × g for 10 min at 4°C. The supernatant was further centrifuged at 12,000 × g for 15 min at 4°C in order to pellet the crude mitochondria. 500 μl mitochondria isolation reagent C was then added to the pellet before the final centrifugation at 12,000 × g for 5 min at 4°C. The resulting mitochondrial pellet and cytosol fraction were lysed by lysis buffer before further processing .
In vivo antitumor studies
BALB/c nude mice (4 to 6 week-old) were obtained from Guangzhou University of Chinese Medicine. All manipulation was done under sterile conditions. The procedures involving mice and their care were in accordance with National Institutes of Health Guide for the care and use of Laboratory Animals and with the United Kingdom Coordinating Committee on Cancer Research. Tumor xenografts were established by injecting 1 × 106 SW620 cells into the subcutaneous tissue in both flank of nude mice. Mice were randomly divided into six groups and each group contained 6 mice. Treatment was initiated on day 6 after inoculation, by which time the volume of the tumor had reached approximately 50mm3. Different concentration of hirsutanol A (20 mg/kg/d, 10 mg/kg/d, 5 mg/kg/d), DMSO, 0.9% (w/v) NaCl Saline (N.S.) and HCPT were administered i.p. for 28 days for the assigned group. Tumor volumes and body weight of the mice were observed. Tumor volumes were calculated by the formula: 0.5 × a × b2 in millimeters, where a is the length and b is the width. On day 28 after administration, the mice were sacrificed. The tumor tissues were excised and weighed. Tumor growth inhibition was determined as the ratio of the average tumor weight of the treated group (T) to the average tumor weight of the control group (C) .
Data were analyzed by student’s t test with SPSS 11.0 analysis software, and results were considered statistically significant at p < 0.05. Results are presents as mean and standard deviation (±SD).
Hirsutanol A inhibited proliferation and induced apoptosis in SW620 and MDA-MB-231 cells
Hirsutanol A induced mitochondrial-independent accumulation of intrinsic ROS
Preventing ROS accumulation by antioxidant agent NAC reduced hirsutanol A-induced apoptosis
Hirsutanol A activated mitochondria/cytochrome c signaling pathway
Hirsutanol A activated JNK signaling pathway and blockade of JNK signal pathway increased ROS level and cell apoptosis
We further investigated the effect of activation of JNK signaling pathway on cellular ROS levels. Cellular ROS levels were remarkably increased in SW620 cells by JNK inhibitor SP600125 or JNK-siRNA (Figure 6E).These results suggested that activation of JNK could be one response to oxidant stress which protects cells from death via regulation of ROS in a negative feedback manner. It was not a classic mechanism involved in apoptosis.
In vivo antitumor effect of hirsutanol A on human colon cancer cell SW620 xenografts
Hirsutanol A is a novel sesquiterpene compound purified from fungus Chondrostereum sp. in Sarcophyton tortuosum. Our previous studies had demonstrated that hirsutanol A exhibited potent cytotoxic effect on some human cancer cell lines. In addition, we also confirmed that hirsutanol A could induce autophagical cell death by causing accumulation of ROS level in human hepatocellular carcinoma cells . ROS inducer as an anticancer drug has received a lot of attention due to its selective effect on cancer cells but sparing normal cells . To date, there are some ROS inducers targeting ROS generating system or ROS scavenging system [7, 36]. However, most of them cannot enter clinical trials because of the high toxicity or poor bioavailability. Here, we reported that hirsutanol A could significant induce cell growth inhibition and apoptosis, elevate the level of ROS in both SW620 and MDA-MB-231 cells and suppress tumor growth in SW620 xenografts (Figures 2, 3 and 7). Some evidences supported that ROS as a potent oxidant agent could damage mitochondrial membrane to result in mitochondrial membrane potential disorder and release of cytochrome c from mitochondria which could further activate caspase-3, leading to mitochondria/cytochrome c –mediated apoptosis . We had examined the mitochondrial membrane potential and the expression of cytochrome c in mitochondria and cytosol. The results showed that hirsutanol A could trigger the dysfunction of mitochondrial membrane potential and release of cytochrome c from mitochondria (Figure 5). Furthermore, we evaluated whether hirsutanol A-induced growth inhibition and apoptosis were evoked by accumulation of ROS. After treatment with NAC, a potent antioxidant agent that could prevent hirsutanol A-induced ROS accumulation , we found that cell growth inhibition and apoptosis remarkably decreased (Figure 4). As our data has clearly demonstrated that hirsutanol A could elevate intrinsic ROS level, and activate mitochondria/cytochrome c signaliing pathway to trigger apoptosis, further studies are required to elucidate if the release of cytochrome c is due to the elevated ROS induced by hirsutanol A.
ROS, which serves as a second messenger, can modulate several signaling pathways including JNK, Akt, NF-κB etc. [32, 33]. In this study, we showed that hirsutanol A enhanced the phosphorylation levels of JNK and c-Jun dose-and time-dependently in SW620 cells (Figure 6A). Moreover, prevention of hirsutanol A-induced ROS accumulation by NAC could reverse the phosphorylation of JNK and c-Jun (Figure 6B). These data indicated that hirsutanol A-induced production of ROS activated JNK signaling pathway. JNK signaling pathway is involved in both stress-induced and chemotherapeutical drugs-induced apoptosis. However, inhibition of JNK signaling pathway by a special inhibitor SP600125 promoted the hirsutanol A-induced cell growth inhibition and apoptosis. Mass evidences verified that JNK signaling pathway is responsible for regulation of ROS level by activating c-Jun, a transcription factor, which further regulates the transcription of some target genes involved in redox such as NOX and SOD, etc. [39, 40]. In our studies, we found that blockade of JNK signaling pathway by SP600125 and siRNA-JNK could significantly enhance hirsutanol A-induced ROS production, suggesting that hirsutanol A-induced activation of JNK signaling pathway regulated ROS level in a negative feedback manner. These evidences point us in the direction that treatment with hirsutanol A in combination with inhibitor of JNK may produce synergistic effect.
In summary, hirsutanol A is a ROS generating agent which exerts anticancer effect via up-regulation of ROS level and activation of mitochondria/cytochrome c signaling pathway. Moreover, hirsutanol A could activate JNK signaling pathway. Activation of JNK signaling pathway did not mediate apoptosis; however, it could regulate ROS level in a negative feedback fashion which protects cells against oxidant stress induced cell death. Our results revealed that hirsutanol A may be a promising lead compound in future anticancer treatments.
This work was supported by Major science and technology project of the National Basic Research Program (973 Program) of China (2012CB967004); National Natural Science Foundation of China [81001446,81272199]; Natural Science Foundation of Guangdong in China (S2012010008761, S2011020002759); Major science and technology project of Guangzhou (2010U1-E00531-5, 2011Y1-00036).
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