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.

Keywords: Carcinoma, Hepatocellular; Haliclona oculata; Hepatocytes; Holothuria parva; Mitochondria.

Figure 1.. Histopathological Analysis and Complex II Activity

A, 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 Measurement

The 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 Swelling

Determination 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 Activity

Measurement 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).