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

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.

Selective Toxicity of Non-polar Bioactive Compounds of Sea Cucumber (Holothuria sabra) Extracts on Isolated Mitochondria and Hepatocytes of Induced Hepatocellular Carcinoma Rat Model

“…Given the rise of HCC rates globally coupled with inefficient methods for HCC treatment [23], the authors of this paper sought to investigate novel treatment methods and materials to solve this mentioned problem. Marine animals con¬taining various bioactive compounds present strong anticancer and anti-tumor properties [20,21,26]. In addition, beyond 70% of anti-cancer agents used, are originally sourced with varieties of lead compounds for different cancer activities [25,27].…”

“…After that, Dichlorofluorescin Diacetate (DCFH-DA, at final concentration=10μM) was added then, incubated for 10 minutes at 37°C to measure the ROS level induced by H. sabra extracts. Finally, to determine the intensity of fluorescence induced by dichlorofluorescein (DCF), the Shimadzu RF-5000 U fluorescence spectrophotometer (EX=488 nm and EM=527 nm) was used [21,22].…”

“…Rh 123 (at final concentration=10 μM) was added to the mitochondrial suspensions obtained from HCC and normal groups (1000 μg mitochondrial protein/ml) in the MMP assay buffer including 2 mM of MgCl 2 , 10mM of KCl, 5mM of KH 2 PO 4 , 68 mM of D-mannitol, 50 μM of EGTA, 5 mM of sodium succinate, 10 mM of HEPES, 220 mM of sucrose and 2 μM of rotenone. Then, the amount of uptake was determined by a Shimadzu RF5000U fluorescence spectrophotometer which was set at EX=490 nm and EM=535 nm [20,21,23].…”

Selective Toxicity of Persian Gulf Sea Squirt (Phallusia nigra) Extract on Isolated Mitochondria Obtained from Liver Hepatocytes of Hepatocellular Carcinoma Induced Rat

“…The disease was induced in the rats by intraperitoneal injection of DEN (a single dose of 200 mg/kg body weight). Two weeks following the injection, 2-acetylaminofluorene (2-AAF 0.02%, w/w) was administered 2 times, every 2 weeks, to promote the cancer (18,19).…”

“…The supernatant was kept while the pellet was homogenized for 10 minutes, followed by centrifugation at 760 g for 5 minutes. The supernatants from the two previous steps were combined and centrifuged for 20 min at 8,000 g. The final mitochondria containing pellet was suspended in Tris buffer (0.05 M of Tris-HCl, 0.25 M of sucrose, 20 mM of KCl, 2.0 mM of MgCl 2 , and 1.0 mM of Na 2 HPO 4 ; pH = 7.4) at 4°C (18,20,21).…”