Seydi E et al. (2015), Selective Toxicity of Persian Gulf Sea Cucumber...

ECB-ART-44567

Hepat Mon 2015 Dec 01;1512:e33073. doi: 10.5812/hepatmon.33073.

Show Gene links Show Anatomy links

Selective Toxicity of Persian Gulf Sea Cucumber (Holothuria parva) and Sponge (Haliclona oculata) Methanolic Extracts on Liver Mitochondria Isolated from an Animal Model of Hepatocellular Carcinoma.

Seydi E , Motallebi A , Dastbaz M , Dehghan S , Salimi A , Nazemi M , Pourahmad J .

???displayArticle.abstract???
BACKGROUND: Natural products isolated from marine environments are well known for their pharmacodynamic potential in diverse disease treatments, such as for cancer or inflammatory conditions. Sea cucumbers are marine animals of the phylum Echinoderm and the class Holothuroidea, with leathery skin and gelatinous bodies. Sponges are important components of Persian Gulf animal communities, and the marine sponges of the genus Haliclona have been known to display broad-spectrum biological activity. Many studies have shown that sea cucumbers and sponges contain antioxidants and anti-cancer compounds. OBJECTIVES: This study was designed to determine the selective toxicity of Persian Gulf sea cucumber (Holothuria parva) and sponge (Haliclona oculata) methanolic extracts on liver mitochondria isolated from an animal model of hepatocellular carcinoma, as part of a national project that hopes to identify novel potential anticancer candidates among Iranian Persian Gulf flora and fauna. MATERIALS AND METHODS: To induce hepatocarcinogenesis, rats were given diethylnitrosamine (DEN) injections (200 mg/kg i.p. by a single dose), and then the cancer was promoted with 2-acetylaminofluorene (2-AAF) (0.02 w/w) for two weeks. Histopathological evaluations were performed, and levels of liver injury markers and a specific liver cancer marker (alpha-fetoprotein), were determined for confirmation of hepatocellular carcinoma induction. Finally, mitochondria were isolated from cancerous and non-cancerous hepatocytes. RESULTS: Our results showed that H. parva methanolic extracts (250, 500, and 1000 µg/mL) and H. oculata methanolic extracts (200, 400, and 800 µg/mL) increased reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), mitochondrial swelling, and cytochrome c release in the mitochondria obtained from cancerous hepatocytes, but not in mitochondria obtained from non-cancerous liver hepatocytes. These extracts also induced caspase-3 activation, which is known as a final mediator of apoptosis, in the hepatocytes obtained only from cancerous, not non-cancerous, rat livers. CONCLUSIONS: Our results suggest that H. parva and H. oculata may be promising therapeutic candidates for the treatment of HCC, following further confirmatory in vivo experiments and clinical trials.

???displayArticle.pubmedLink??? 26977167
???displayArticle.pmcLink??? PMC4774342
???displayArticle.link??? Hepat Mon

Genes referenced: LOC100887844 LOC577219 LOC587800 mmp7 ros1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Histopathological Analysis and Complex II ActivityA, liver section from the control group shows normal cellular architecture (H and E; 40 × magnification); B and C, liver sections from the HCC group show areas of aberrant hepatocellular phenotype with variation in nuclear size, hyperchromatism, binucleation, and irregular sinusoids (H and E, 40 × magnification). The effect of H. parva concentrations on complex II (succinate dehydrogenase) activity in the liver mitochondria obtained from hepatocytes of untreated control D, and HCC groups E, values are represented as mean ± SD (n = 3). and * indicate significant differences in comparison with the corresponding control mitochondria (P < 0.01 and P < 0.001, respectively).
	Figure 2. Complex II Activity and ROS MeasurementThe effect of H. oculata concentrations on complex II (succinate dehydrogenase) activity in the liver mitochondria obtained from hepatocytes of both the untreated control A, and HCC B, groups. Values are represented as mean ± SD (n = 3). and * indicate a significant difference in comparison with the corresponding control mitochondria (P < 0.01 and P < 0.001, respectively). Measurement of mitochondrial ROS formation showing increases after addition of various concentrations of C, H. parva (250, 500, and 1000 µg/mL) and D, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation, in the mitochondria obtained from hepatocytes of the HCC group but not the control group. Values are presented as mean ± SD (n = 3). , , and ** indicate significant differences between the control and HCC groups (P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively).
	Figure 3. Determination of the Mitochondrial Membrane Potential (MMP) and Mitochondrial SwellingDetermination of the collapse of mitochondrial membrane potential (MMP). Decreased MMP after the addition of various concentrations of A, H. parva (250, 500, and 1000 µg/mL) and B, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation in the mitochondria obtained from hepatocytes of the HCC group but not the control group. Values are presented as mean ± SD (n = 3). , , and indicate significant differences in the comparison with the control group (P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively). Determination of mitochondrial swelling showed an increase after the addition of various concentrations of C, H. parva (250, 500, and 1000 µg/mL) and D, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation in the mitochondria obtained from hepatocytes of the HCC group but not of the control group. Values are presented as mean ± SD (n = 3). and **** indicate significant differences in the comparison with the control group (P < 0.01 and P < 0.0001).
	Figure 4. Measurement of Cytochrome c Expulsion and Caspase 3 ActivityMeasurement of cytochrome c expulsion. Increased cytochrome c release after addition of A, H. parva (500 µg/mL) and B, H. oculata (400 µg/mL) to the mitochondria obtained from hepatocytes of the HCC group but not from the control group. Pretreatment with BHT or CsA significantly inhibited cytochrome c release in the HCC liver mitochondria. The amount of expelled cytochrome c from the mitochondrial fraction into the suspension buffer was determined using a rat/mouse cytochrome c ELISA kit. Values are presented as mean ± SD (n = 3). *** indicates significant difference in comparison with the untreated group (P < 0.001). * and indicate significant differences in comparison with H. oculata (400 µg/mL) and H. parva (500 µg/mL)-treated HCC group (P < 0.05). C, Determination of caspase-3 activity. Caspase-3 activation was measured in the HCC and control hepatocytes following exposure to H. parva (500 µg/mL) and H. oculata (400 µg/mL) extracts, using a Sigma-Aldrich kit. The kit measures pNA released from the interaction between caspase-3 and AC-DEVD-pNA (peptide substrate). Values are expressed as mean ± SD from three separate experiments (n = 3). * indicates a significant difference in comparison with the untreated HCC group (P < 0.001).

References [+] :

Abdel-Hamid, Can methanolic extract of Nigella sativa seed affect glyco-regulatory enzymes in experimental hepatocellular carcinoma? 2013, Pubmed

Abdel-Hamid, Can methanolic extract of Nigella sativa seed affect glyco-regulatory enzymes in experimental hepatocellular carcinoma? 2013, Pubmed
Al Marzouqi, Frondoside A inhibits human breast cancer cell survival, migration, invasion and the growth of breast tumor xenografts. 2011, Pubmed , Echinobase
Aminin, Anticancer activity of sea cucumber triterpene glycosides. 2015, Pubmed , Echinobase
Barogi, Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing. 1995, Pubmed
Beedessee, Cytotoxic activities of hexane, ethyl acetate and butanol extracts of marine sponges from Mauritian Waters on human cancer cell lines. 2012, Pubmed
Bishayee, Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: inhibition of cell proliferation and induction of apoptosis. 2009, Pubmed
Bordbar, High-value components and bioactives from sea cucumbers for functional foods--a review. 2011, Pubmed , Echinobase
Burroughs, Systemic treatment and liver transplantation for hepatocellular carcinoma: two ends of the therapeutic spectrum. 2004, Pubmed
Circu, Reactive oxygen species, cellular redox systems, and apoptosis. 2010, Pubmed
Coello, 1,5-diazacyclohenicosane, a new cytotoxic metabolite from the marine sponge Mycale sp. 2009, Pubmed
Dembitsky, Novel antitumor agents: marine sponge alkaloids, their synthetic analogs and derivatives. 2005, Pubmed
El Mesallamy, The chemopreventive effect of Ginkgo biloba and Silybum marianum extracts on hepatocarcinogenesis in rats. 2011, Pubmed
Essack, Recently confirmed apoptosis-inducing lead compounds isolated from marine sponge of potential relevance in cancer treatment. 2011, Pubmed
Flammang, Biomechanics of adhesion in sea cucumber cuvierian tubules (echinodermata, holothuroidea). 2002, Pubmed , Echinobase
Gupta, Antifilarial activity of marine sponge Haliclona oculata against experimental Brugia malayi infection. 2012, Pubmed
Guzmán, A novel activity from an old compound: Manzamine A reduces the metastatic potential of AsPC-1 pancreatic cancer cells and sensitizes them to TRAIL-induced apoptosis. 2011, Pubmed
Han, Cytotoxic holostane-type triterpene glycosides from the sea cucumber Pentacta quadrangularis. 2010, Pubmed , Echinobase
Hosseini, Toxicity of vanadium on isolated rat liver mitochondria: a new mechanistic approach. 2013, Pubmed
Hussain, Evaluation of chemopreventive effect of Fumaria indica against N-nitrosodiethylamine and CCl4-induced hepatocellular carcinoma in Wistar rats. 2012, Pubmed
Jin, AFP gene expression after acute diethylnitrosamine intoxication is not Afr2 regulated. 2005, Pubmed
Khan, Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats. 2011, Pubmed
Kowaltowski, Mitochondrial permeability transition and oxidative stress. 2001, Pubmed
Liao, Apigenin induces the apoptosis and regulates MAPK signaling pathways in mouse macrophage ANA-1 cells. 2014, Pubmed
Liu, Argusides B and C, two new cytotoxic triterpene glycosides from the sea cucumber Bohadschia argus Jaeger. 2008, Pubmed , Echinobase
Modica-Napolitano, Mitochondrial dysfunction in cancer. 2004, Pubmed
Pourahmad, Involvement of mitochondrial/lysosomal toxic cross-talk in ecstasy induced liver toxicity under hyperthermic condition. 2010, Pubmed
Pourahmad, Involvement of Lysosomal Labilisation and Lysosomal/mitochondrial Cross-Talk in Diclofenac Induced Hepatotoxicity. 2011, Pubmed
Seglen, Preparation of isolated rat liver cells. 1976, Pubmed
Shaki, Toxicity of depleted uranium on isolated rat kidney mitochondria. 2012, Pubmed
Singh, Potential chemoprevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from Acacia nilotica bark. 2009, Pubmed
Sreepriya, Chemopreventive effects of embelin and curcumin against N-nitrosodiethylamine/phenobarbital-induced hepatocarcinogenesis in Wistar rats. 2005, Pubmed
Taha, Potential chemoprevention of diethylnitrosamine-initiated and 2-acetylaminofluorene-promoted hepatocarcinogenesis by zerumbone from the rhizomes of the subtropical ginger (Zingiber zerumbet). 2010, Pubmed
Talari, Dracocephalum: novel anticancer plant acting on liver cancer cell mitochondria. 2014, Pubmed
Thomas, Marine drugs from sponge-microbe association--a review. 2010, Pubmed
Tohme, A journey under the sea: the quest for marine anti-cancer alkaloids. 2011, Pubmed
Toogood, Mitochondrial drugs. 2008, Pubmed
Trisciuoglio, Induction of apoptosis in human cancer cells by candidaspongiolide, a novel sponge polyketide. 2008, Pubmed
Van Dyck, Qualitative and quantitative saponin contents in five sea cucumbers from the Indian ocean. 2010, Pubmed , Echinobase
Wang, Gene expression profiles reveal significant differences between rat liver cancer and liver regeneration. 2012, Pubmed
Wijesinghe, Anticancer activity and mediation of apoptosis in human HL-60 leukaemia cells by edible sea cucumber (Holothuria edulis) extract. 2013, Pubmed , Echinobase
Zhang, SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in hepatocellular carcinoma. 2015, Pubmed

	Figure 1. Histopathological Analysis and Complex II ActivityA, liver section from the control group shows normal cellular architecture (H and E; 40 × magnification); B and C, liver sections from the HCC group show areas of aberrant hepatocellular phenotype with variation in nuclear size, hyperchromatism, binucleation, and irregular sinusoids (H and E, 40 × magnification). The effect of H. parva concentrations on complex II (succinate dehydrogenase) activity in the liver mitochondria obtained from hepatocytes of untreated control D, and HCC groups E, values are represented as mean ± SD (n = 3). and * indicate significant differences in comparison with the corresponding control mitochondria (P < 0.01 and P < 0.001, respectively).
	Figure 2. Complex II Activity and ROS MeasurementThe effect of H. oculata concentrations on complex II (succinate dehydrogenase) activity in the liver mitochondria obtained from hepatocytes of both the untreated control A, and HCC B, groups. Values are represented as mean ± SD (n = 3). and * indicate a significant difference in comparison with the corresponding control mitochondria (P < 0.01 and P < 0.001, respectively). Measurement of mitochondrial ROS formation showing increases after addition of various concentrations of C, H. parva (250, 500, and 1000 µg/mL) and D, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation, in the mitochondria obtained from hepatocytes of the HCC group but not the control group. Values are presented as mean ± SD (n = 3). , , and ** indicate significant differences between the control and HCC groups (P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively).
	Figure 3. Determination of the Mitochondrial Membrane Potential (MMP) and Mitochondrial SwellingDetermination of the collapse of mitochondrial membrane potential (MMP). Decreased MMP after the addition of various concentrations of A, H. parva (250, 500, and 1000 µg/mL) and B, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation in the mitochondria obtained from hepatocytes of the HCC group but not the control group. Values are presented as mean ± SD (n = 3). , , and indicate significant differences in the comparison with the control group (P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively). Determination of mitochondrial swelling showed an increase after the addition of various concentrations of C, H. parva (250, 500, and 1000 µg/mL) and D, H. oculata (200, 400, and 800 µg/mL) extracts at different time intervals within 60 min of incubation in the mitochondria obtained from hepatocytes of the HCC group but not of the control group. Values are presented as mean ± SD (n = 3). and **** indicate significant differences in the comparison with the control group (P < 0.01 and P < 0.0001).
	Figure 4. Measurement of Cytochrome c Expulsion and Caspase 3 ActivityMeasurement of cytochrome c expulsion. Increased cytochrome c release after addition of A, H. parva (500 µg/mL) and B, H. oculata (400 µg/mL) to the mitochondria obtained from hepatocytes of the HCC group but not from the control group. Pretreatment with BHT or CsA significantly inhibited cytochrome c release in the HCC liver mitochondria. The amount of expelled cytochrome c from the mitochondrial fraction into the suspension buffer was determined using a rat/mouse cytochrome c ELISA kit. Values are presented as mean ± SD (n = 3). *** indicates significant difference in comparison with the untreated group (P < 0.001). * and indicate significant differences in comparison with H. oculata (400 µg/mL) and H. parva (500 µg/mL)-treated HCC group (P < 0.05). C, Determination of caspase-3 activity. Caspase-3 activation was measured in the HCC and control hepatocytes following exposure to H. parva (500 µg/mL) and H. oculata (400 µg/mL) extracts, using a Sigma-Aldrich kit. The kit measures pNA released from the interaction between caspase-3 and AC-DEVD-pNA (peptide substrate). Values are expressed as mean ± SD from three separate experiments (n = 3). * indicates a significant difference in comparison with the untreated HCC group (P < 0.001).