Antitumor Activity Of Hirsutanol A, A Novel Sesquiterpene Compound And Its Mechanisms

Background and objectiveCancer has become an increasing public health problem because of its high rates of morbidity and mortality. About 7 million people die from cancer each year all over the world. Recently, cancer significantly showed an increasing trend in mortality. Since chemotherapy is still one of the key therapeutic strategies against cancer, developing the novel anticancer drugs is implicated in cancer therapy. By exploiting the genetic differences between normal cells and cancer cells, targeting therapeutic strategies have acquired progress. However, it still faces significant challenges owing to acquired drug resistance and the genomic instability of cancer cells. Therefore, depending on the events involved in carcinogenesis and cancer progression, it is important to exploit new therapeutic target of anticancer drugs. Recently, targeting unique biochemical alternation has got extensively attentions as a new target of anticancer drugsReactive oxygen species(ROS) are a collective term of oxygen derived species, including hydrogen peroxide H2O2, hydroxyl radicals OH, superoxide anion radical O2, singlet oxygen, ozone, hypochloric acid etc. A basic level of intracellular ROS is essential for regulating cell proliferation and differentiation, whereas excessive amounts of ROS can contribute to the carcinogenesis and cancer progression. Compared to normal cells, cancer cells with increased intrinsic ROS are more vulnerable to damage by further ROS insults and more difficult to scavenge the increasing ROS induced by exogenous agents. Therefore, induction of ROS levels by redox modulation is a way to selectively kill cancer cells without significant toxicity to normal cells. So the redox system would be a new target of anticancer drugs.As the biologic resources in the land are decreasing, researchers focus on marine resources. Since the particular living environments (high pressure, hypoxia and low salty), thousands of peculiar metabolites with physiological activities are found in marine lives, and have evoked great interests to find some natural drugs with high efficacy and low toxicity. Hirsutanol A is a novel sesquiterpene compound purified from Sarcophyton tortuosum. The pharmacologic effect of this compound is still unkown, though the chemcial structure has been reported. We found that Hirsutanol A showed potent cytotoxic effect via crude screen assay from six natural compounds extracted from Fungus Chondrostereum sp in Sarcophyton tortuosum. In this study we investigated the anticancer activity of Hirsutanol A and its mechanism.MethodsMTT assay was used to screen compounds and evaluate the growth inhibition of various cancer cell lines induced by Hirsutanol A. Cells were stained with DHE or CM-H2DCF-DA to detect cellular superoxide or hydrogen peroxide level measured by flow cytometry. Annexin V-PI staining was used to assess the apoptosis. The transiently transfected GFP-LC3 cells were established by lipofectamine 2000 transfection. The punctuate dots of YFP-LC3 were detected by confocal microscopy. Western blot analysis was used to detect the expression of JNK and Akt signaling pathway -related proteins, cleavage fraction of caspase-3 and PARP, and conversion of LC3 I to LC3-II. Akt kinase activity assay in cell-free system was used to evaluate effect of Hirsutanol A on Akt kinase activity in vitro. Isolation of Mitochondrial and cytosol fraction was obtained by using Mitochondria isolation kit, then expression of cytochrome c in Mitochondria or cytosol was determined by Western blot analysis.SP600125,a small-molecule JNK inhibitor, and specific siRNA against JNK were used to block JNK activity or expression.3-MA and Bafilomycin A1 or siRNA against Beclinl and siRNA against Atg7 were employed to inhibit autophagy. Hep3B cells were stably transinfected with myr-Aktl plasmid to establish Hep3B/myr-Aktl which stably expressed constitutively activated Aktl. MTT assay was used to analysis the growth inhibition of Hep3B/myr-Aktl and Hep3B-vector cells induced by Hirsutanol A.Tumor xenografts were established by SW620 cells injected s.c into nude mice, the growth inhibitory effect was finally calculated after Hirsutanol A treatment.Results1 Screening compounds with cytotoxic activity and investigating the cytotoxic effect of Hirsutanol A on various cancer cell lines. To explore compounds with anticancer activity,we detected the cytotoxic effect of six compounds by MTT assay, the results showed that Hirsutanol A exhibits potent cytotoxic effect on various human cancer cell lines in a dose-and time-dependent manner.2 The anticancer activity of Hirsutanol A and its molecular mechanisms.(1) Effect of Hirsutanol A on cellular ROS level in cancer cells:To investigate the Effect of Hirsutanol A on cellular superoxide and hydrogen peroxide level in cancer cells, we analyzed the level of superoxide and hydrogen peroxide in cancer cells using flow cytometry. There was no significant change in DHE fluorescence but markedly increase in DCF-DA fluorescence after treatment with Hirsutanol A in a dose-and time-dependent fashion, suggesting that ROS induced by Hirsutanol A were mainly hydrogen peroxide but not superoxide.(2) Hirsutanol A induces apoptosis via a ROS-mediated mechanism①Hirsutanol A induces apoptosis in SW620, MDA-MB-231, MCF-7 cancer cellsTo confirm that Hirsutanol A induced apoptosis, SW620,MDA-MB-231 and MCF-7 cells exposed to various concentrations of Hirsutanol A for 72h. The cells were analyzed by Annexin V-FITC/PI staining assay. The ratio of AnnexinV-positive cells was 2.8%,12.1%,45.0%,65.6% in SW620,2.6%,10%,22.7%,30.6% in MDA-MB-231, and 1.7%,10%,20.8%,31.3% in MCF-7. The results suggested that Hirsutanol A could induce apoptosis in a dose-dependent manner. Using immuno-blotting analysis, the cleavage of caspase-3 and PARP in both cancer cells was detected following treatment with Hirsutanol A. The results showed that pro-caspase-3 was cleaved to yield a 17KDa fragmentation and PARP was cleaved into 89KD fragmentation. These results revealed that Hirsutanol A remarkably induced apoptosis in SW620, MDA-MB-231 and MCF-7 cells.②Hirsutanol A induced apoptosis through activation of mitochondria/ cytochrome c signaling pathwayTo study whether Hirsutanol A induced apoptosis via activation of mitochondria/ cytochrome c signaliing pathway, we examined the change of mitochondrial membrane potential and the release of cytochrome c from mitochondria. mitochondrial membrane potential was elevated after treatment with various concentrations of Hirsutanol A. The expression of cytochrome c in mitochondrial was downregulated, whereas that in cytosol was upregulated after treatment with Hirsutanol A for 24h. These data revealed that Hirsutanol A induced apoptosis through activation of mitochondria/cytochrome c signaling pathway. ③Induction of apoptosis by Hirsutanol A associated with increased ROS generationThe evidences of apoptosis and upregulation of ROS levels by Hirsutanol A prompted us to investigate whether upregulation of ROS resulted in apoptosis. The increase of ROS levels induced by Hirsutanol A in cancer cells was prevented by preincubation with NAC, then growth inhibition and AnnexinV-positive cells were analyzed using MTT and AnnexinV/PI assay. The results showed that the induction of apotosis by Hirsutanol A was blocked when the cells were pretreated with NAC.④Hirsutanol A activated JNK signaling pathway through upregulation of ROSIt has been reported that ROS can modulate several signaling pathways including JNK, Akt, NF-κB and so on. Therefore, we explored the effect of increasing ROS induced by Hirsutanol A on JNK signaling pathway. JNK and c-Jun phosphorylation were significantly activated in SW620 cells after treatment with Hirsutanol A for 24 h. Moreover, NAC prevented the activation of JNK induced by Hirsutanol A. It suggested that JNK may be a downstream target of increased ROS following Hirsutanol A treatment.⑤The role of JNK signaling pathway in Hirsutanol A induced ROS increaseIn order to explore the contribution of JNK signaling pathway to Hirsutanol A induced ROS, JNK signaling pathway was blocked using the small-molecule JNK inhibitor SP600125. Treated with Hirsutanol A only, the ratio of AnnexinV-positive cells was 35.6%, whereas in parallel treatment in combination with SP600125, the ratio of AnnexinV-positive cells was up to 48.3%, suggesting that blocking JNK signaling pathway promoted apoptosis induced by Hirsutanol A.We further investigated the effect of activation of JNK signaling pathway on cellular ROS levels. Cellular ROS levels were remarkly increased in SW620 cells by SP600125 or JNK-siRNA to block JNK signaling pathway.These results suggest that activation of JNK is an oxidant stress which protect cell from death via regulation of ROS in a negative feedback manner but not a classic mechanism involved in apoptosis.(3) Hirsutanol A induced autophagy through increasing ROS production①Hirsutanol A induced autophagy via increase of ROS levelThe lipidation of the protein LC3 during the process of autophagy can be used as a marker. In order to detect the autophagy induced by Hirsutanol A, we observed GFP-tagged LC3 puncta distribution in Hep3B cells. Hep3B cells showed diffuse distribuction of GFP-LC3 in the absence of Hirsutanol A, whereas Hirsutanol A-treated cells showed a marked increase in number and frequency of GFP-LC3 dots.To futher validate autophagy induced by Hirsutanol A, we measured the modification of LC3-Ⅰto LC3-Ⅱby Western blot. LC3 I to LC3-II conversion was remarkedly increased in a dose-and time-dependent manner indicating induction of autophagy by Hirsutanol A.Since Hirsutanol A can increase cellular ROS levels and induce autophagy, we investigated the relationship between autophagy and upregulation of ROS induced by Hirsutaanol A. The increase of ROS level induced by Hirsutanol A was prevented by preincubation with NAC in Hep3B cells. NAC inhibited the LC3 I to LC3-II conversion and reduced the number of GFP-LC3 dots. These results demonstrated that activation of autophagy depended on the upregulation of ROS levels.②Hirsutanol A inhibited Akt signaling pathway via increase of ROS levelsThe above observations suggested that Hirsutanol A can induce autophagy and accumulation of cellular ROS. Since Akt/mTOR is a key modulator for autophagy, we futher detected the effect of Hirsutanol A on Akt signaling pathway. The results showed that the phosphorylated Akt and FKHR were inhibited by Hirsutanol A, suggesting Hirsutanol A could block Akt signaling pathway.In order to futher study whether upregulation of ROS resulted in inhibition of Akt signaling pathway. We used Akt kinase assay kit to evalutate the effect of Hirsutanol A on the Akt kinase activity in vitro. Western blot analysis showed that Hirsutanol A could not inhibit phosphorylation of GSK-3a/β. However, NAC can prevent the inhibition of Akt and FKHR phosphorylation induced by Hirsutanol A. These results suggested that Hirsutanol A inhibited Akt signaling pathway not through targeting Akt kinase directly but through upregulation of ROS levels.③Hirsutanol A induced no-apoptotic cell death in Hep3B cellsWe detected the apoptosis in Hep3B cells after treated with 5,10,20μmol/L Hirsutanol A for 48 h using AnnexinV/PI assay.The AnnexinV positive cells were only 0.5%, whereas PI positive cells were up to 5.5%,15.9%%,49.7%respectively. Using immunoblotting analysis, the cleavage of caspase-3 in both cancer cells was not observed following treatment with Hirsutanol A. The cleavage of PARP was also undetectable. These data suggested that Hirsutanol A induced no-apoptotic cell death in Hep3B cells.④Hirsutanol A induced autophagic cell death in Hep3B cellsIn order to certify that Hirsutanol A induced autophagic cell death in Hep3B cells, we used the autophagy inhibitor 3-MA or Beclin-siRNA to inhibit Hirsutanol A-induced autophagy in Hep3B cells. Compared with control cells, cells treated with 3-MA or Beclin-siRNA reduced cell death induced by Hirsutanol A, indicating that Hirsutanol A induced autophagic cell death in Hep3B cells.⑤Upregulation of ROS and inhibition of AKT pathway involved in autophagic cell death induced by Hirsutanol ATo investigate whether autophagic cell death induced by Hirsutanol A depended on increase of ROS in Hep3B cells, NAC was used to inhibit the ROS upregulation. The results showed that NAC reduced the cell death induced by Hirsutanol A. Furthermore, we established Hep3b/myr-Aktl cell line which stably expressed constitutively activated Akt1. Expressing constitutively active Akt increased growth inhibition in Hep3B cells after treatment with Hirsutanol A for 72h.These data together clarified that Hirsutanol A induced autophagic cell death via increase of ROS and inhibition of Akt signaling pathway.⑥Autophagy protected cells from death induced by Hirsutanol ATo validate the contribution of autophagy in cell death induced by Hirsutanol A, we used autophagy inhibitor Bafilomycin A1 or Atg-7-siRNA to block autophagy induced by Hirsutanol A in MCF-7 cells. Blocking autophagy could remarkedly increase cell death induced by Hirsutanol A. These evidences suggested that Hirsutanol A-treated autophagy protected cells from death.3 Effect of Hirsutanol A on tumor growth in mice bearing human colon cancer cells SW620To detect antitumor activity of Hirsutanol A in vivo, we established SW620 xenografts. The results showed that Hirsutanol A at 20 mg/kg/d potently inhibited tumor growth and the tumor growth inhibition(T/C) was about 31.5%.Conclusions1 Hirsutanol A induced apoptosis via change of mitochondrial membrane potential and releasing of cytochrome c from mitochondria to cytosol modulated by increasing ROS production in SW620 and MDA-MB-231 cells.2 Hirsutanol A induces autophagical programmed cell death through blockage of Akt signaling pathway and upregulation of ROS in Hep3B cells.3 Hirsutanol A induces both autophagy and apoptosis in MCF-7 cells. Autophagy induced by Hirsutanol A protected MCF-7 cells from apoptosis.4 Accumulation of ROS induced by Hirsutanol A resulted in activation of JNK signaling pathway. Blockage of JNK signaling pathway promoted apoptosis 5 Hirsutanol A significantly inhibited tumor growth of xenografts in nude mice.