Anti-viral and Anti-cancer Effect of Sea Cucumber Extracts

Anti-viral and Anti-cancer Effect of Sea Cucumber Extracts The Anti-viral and Anti-cancer effect of secondary metabolite extracts from sea cucumber (Holothuria leucospilota) in vitro Abstract Sea cucumber is used as food purposes and traditional medicine in Asia and Middle East society. In this scientific study we try to examine antiviral effect of organic extracts, obtained from sea cucumber Holothuria leucospilota species against HIV-1. For this reason, sea cucumber collected from 10-30 meters depths, around Larak Island. In order to extract, were used from methanol and diethyl ether solvents. All obtained extracts concentrated by rotary evaporator in 40  ° C and then changed to lyophilized powder by vacuum freeze dryer. After that, potential antiviral effect of each extracts on HIV-1 was investigated. The results of this experiments showed that all extracts in some concentrations were able to inhibit the replication of HIV-1. IC50 for their was variable between 5.03  ± 1.90  µg/ml until 337.60  ± 1.34  µg/ml . but cytotoxic effect of all extracts in host cell were also many and CC50 for their was variable between 5.11  ± 1.89  µg/ml until 56.27  ± 1.54  µg/ml. results shown detail ether body wall extract have highest antiviral effect and also it was relatively less cytotoxic effect. 2.79 TI for this extract was shown, it has potential to inhibit HIV-1 after identify and extract effective substances. For survey anti-cancer probably effect have used XTT assay. The results of this experiment showed, all different extracts could be able to prevent Human carcinoma oral epidermoid cells (KB) in some concentration. But also, they had strong cytotoxic effect on normal cell line (HEK293T). Totaly between all different extracts, body wall diethyl ether extract had less cytotoxic effect on normal cells and with 2.46 TI index, showed rather anticancer activitythan other extract. Introduction In recent years, many bioactive compounds identify and derived from various marine organisms. Searches for discover new metabolites led to isolated 10,000 new combination from marine animals. Many of these compounds are related to medicine and pharmacy. This compounds and natural products have been source of materials that have medicine effects (Faulkner, 1996). These bioactive compounds isolated from various marine organisms, including Corals, crabs, seaweeds, Echinoderms, fishes, sponges and etc. Sea cucumbers belong to the phylum Echinodermata, meaning that, they are spiny-skinned, under the class Holothuridea. They are found throughout the nearshore coral reef environment and are also found in the deepest parts of the ocean. Sea cucumbers play an important role in reef recycling, gathering organic detritus and bacteria from the water or sand for food. These particles are digested by the animals in order to extract nutrients, a process that helps turn over sediments to maintain an environment that supports other marine life. Other animals, including fish, crustaceans, and molluscs, eat sea cucumber eggs, larvae and juveniles making them an important member of the food web. Many species eject Cuvierian tubules when threatened. These are very sticky and can be toxic or irritating to predators. They are a diverse group of flexible, elongated, worm-like organ- isms, with a leathery skin and gelatinous body, resembling cucumber (Bordbar, Anwar, Saari, 2011) Sea cucumbers are one of the marine animals which are important as human food source, particularly in some parts of Asia . Sea cucumbers, informally named as bà ªche-de-mer, or gamat, have long been used for food and folk medicine in the communities of Asia and Middle East. In Holothuria leucospilota live specimens have reddish-purple until black and their color is converted to brownish-pink in alcohol. they have Cylindrical body and their abdomen is a little flat. The body wall of the sea cucumber lacks the rigidity found in other echinoderms because the calcareous plates (ossicles) that compose the skeletal system are very small and widely isolated. These ossicles are secreted by special cells called sclerocytes and are embedded in the outer layers of the skin. Ossicles are species-specific in structure and complexity, and can be used to identify species ( Lambert., 2005). Many bioactive compounds have been reported from different species of sea cucumber. A number of these compounds possess biological activity (Bryan et al., 1992; Villasin and Pomory, 2000) Some of sea cucumber species in Malaysia water are being used in traditional medicine to treat wound, eczema, arthritis or hypertension (Farouk et al., 2007). Sea cucumbers have been well recognized as a tonic and traditional remedy in Chinese and Malaysian literature for their effectiveness against hypertension, asthma, rheumatism, cuts and burns, impotence and constipation [18–23]. Several unique biological and pharmacological activities namely anti-angiogenic [24], anticancer [25], anticoagulant [26,27], anti-hypertension [28], anti-inflammatory [29–31], antimicrobial [32,33], antioxidant [34], antithrombotic [35,36], antitumor [37,38], and wound healing [39] have been ascribed to chemical compounds extracted from different sea cucumber species (Bordbar et al., 2011). These medicinal benefits and health functions of sea cucumbers can be attributed to the presence of appreciable amounts of bioactive compounds, especially the triterpene glycosides (saponins) [40–42], chondroitin sulfates [43], glycosaminoglycan [26,36], sulfated polysaccharides [44], sterols (glycosides and sulfates) [45], phenolics [46], peptides [47], cerberosides [48] and lectins [49–51]. Unlike bacteria, fungi and parasites, viruses have no cellular structure. when Viruses are outside live cells they behave like organic compounds and they are not able to replicate and clone independently. They do not have inner cytoplasmic organs such as ribosomes, mitochondria and lysosome. HIV-1 virus or human immunodeficiency virus is an RNA virus of the retrovirus that causes acquired immunodeficiency disease (AIDS) in humans. According to the World Health Organization, 60 million people worldwide are infected with HIV and each day 5,700 lose their live because of this disease.( UNAIDS, 2010) HIV tends to infect and kill T lymphocytes that cause reduction and losing host cellular immunity and will make susceptibility to opportunistic infections. The presence of various materials, such as Liouvillosides A and B which are trisulfated triterpene glycosides and fucosylated chondroitin sulfates (FCS), are causing appear anti-viral effects in extracts of these animals. The body is made up of trillions of living cells. Normal body cells grow, divide into new cells, and die in an orderly fashion. During the early years of a persons life, normal cells divide faster to allow the person to grow. After the person becomes an adult, most cells divide only to replace worn-out or dying cells or to repair injuries. Cancer begins when cells in a part of the body start to grow out of control. There are many kinds of cancer, but they all start because of out-of-control growth of abnormal cells. Cancer cell growth is different from normal cell growth. Instead of dying, cancer cells continue to grow and form new, abnormal cells. Cancer cells can also invade (grow into) other tissues, something that normal cells cannot do. Growing out of control and invading other tissues are what makes a cell a cancer cell. Cells become cancer cells because of damage to DNA. DNA is in every cell and directs all its actions. In a normal cell, when DNA gets damaged the cell either repairs the damage or the cell dies. In cancer cells, the damaged DNA is not repaired, but the cell doesn’t die like it should. Instead, this cell goes on making new cells that the body does not need. These new cells will all have the same damaged DNA as the first cell does. People can inherit damaged DNA, but most DNA damage is caused by mistakes that happen while the normal cell is reproducing or by something in our environment. Sometimes the cause of the DNA damage is something obvious, like cigarette smoking. But often no clear cause is found. Material and methods Sample collection All samples collected from 10-30 meters depth around Larak Island and they had transferred to shore with ice. Upon reaching the shore, samples were frozen using dry ice and transported to the laboratory. Samples kept in separated labeled plastic bags in frozen at -20 C until extraction. Extraction Sampels thawed with water and then mud, or sand, foreign particles remaining from the body surface and were washed away with tap water. Samples were cuted from both sides of the midline of the body. internal organs separated from body wall and they cleaned with tap water before extraction. Extracts were prepared following Naik et al. at first, fresh holothurians were rinsed and cut into small pieces. Then cut samples moved to Erlenmeyer with 1000 cc diethyl ether solvent. The sample was collected in diethyletter about 24 hours, the semi- polar and non- poplar extraction was produced. After solution filtered and evaporating diethyletter to dryness, at low pressure at 35- 40C by using Rota vapor. Then the sample put in methanol for 72 hours, The polar extraction was produced The polar compounds in the phase of methanol- aqueous extracts were separated. The concentrated methanol extracts was then dried to obtain crude semi-solid extracts. The crude extract was then weighted and percentages of extraction from sea cucumber were calculated. After 72 hours evaporating methanol to dryness, at low pressure at 40-45 C by using Rota vapor and at the end , extract changed to lyophilized powder by vacuum freeze dryer. Production of Pseudotyped Single-Cycle Replicable HIV Virions Single-cycle replicable HIV-1 (SCR HIV-1) virions were constructed by deleting a 2-kb segment within the Pol region of the HIV-1 genome from the pNL4-3 strain (provided by Dr. Navid Madani). Pseudotyped SCR HIV- 1 virions were produced by co-transfection of HEK293T cells with pmzNL4-3 (containing the mutated genome), psPAX2, and pMD2G plasmids obtained from Addgene (www.addgene.org) (10, 11). The pmzNL4-3 plasmid encodes the HIV-1 full-length RNA, with packaging ability containing the above-mentioned deletion in the Pol region; the psPAX2 plasmid encodes HIV Gag and Gag-Pro-Pol polyproteins, in addition to all the viral accessory proteins; and the pMD2G plasmid encodes the vesicular stomatitis virus surface glycoprotein (VSVG), which is necessary for virion assembly and the budding process. These pseudotyped virions are able to infect a broad spectrum of cells, even without the CD4 receptor. After co-transfection of the HEK293T cells with the  above-mentioned plasmids b y using the Polyfect reagent (Qiagen, Germany), supernatant containing the  virions was harvested at 24, 48, and 72 h. Virus stock was concentrated 20 times by ultracentrifugation, p24 load was quantified (HIV p24 ELISA, Biomerieux, France), and the stock was stored at -70 °C (10, 11) Cell lines The human immunodeficiency virus (HIV-1)–infected cell line Hela and human embryonic kidney (HEK) 293T cells and Human carcinoma oral epidermoid cells (KB) (American Type Culture Collection) were cultured at 37 °C with 5% CO2 in RPMI1640 medium (Biosera, England) and DMEM (Biosera, England), respectively. The media were supplemented with 10% fetal bovine serum (Biosera, England), 200 units/mL of penicillin G, and 80 µg/mL of streptomycin (Sigma, USA). XTT-Based Cytotoxicity Assay The cellular toxicity of methanol and diethyl ether extracts in HEK293T, Hela and KB cells were assessed using a cell proliferation XTT kit (Roche Diagnostics, Germany), as described previously (9). Briefly, cells were plated in triplicate in 96-well plates in the presence or absence of various concentrations of methanol and diethyl ether extracts. After incubation at 37 °C with 5% CO2 for 3 days, 50 ÃŽ ¼L of prepared XTT mixture was added to each well. The cells were incubated for an additional 4 h to allow the production of XTT formazan. Absorbance was measured using an ELISA plate reader (BioTek ELx800) at a test wavelength of 450 nm and a reference wavelength of 690 nm. Percent inhibition was calculated using the following formula: Inhibition (%) = [100 – (At/ As)] Ãâ€" 100, where As is the absorbance of the solvent and At, of the test sample, respectively. The cytotoxic concentration that resulted in a reduction of the number of viable cells by 50% (CC50) was calculate d from doseresponse curves. Replication assay Vesicular stomatitis virus glyco ­protein (VSVG)-SCR virions can infect Hela cells and complete their replication cycle by assembling of inactive virions. Hela cells (6Ãâ€"104) were seeded in each well of 24 well plates containing 250 ÃŽ ¼l of complete medium and infected with 400 ng P24 VSVG-SCR virions. Cells and virions were incubated together overnight and cells were then washed two times with pre-warmed 5% FBS supplemented DMEM. Complete medium (400 ÃŽ ¼l) was added into each well and cell supernatants were analyzed for p24 load after 48 hrs (HIV P24 ELISA, BIOMERIEUX). Result Antitumor effect of the sea cucumber extracts Evaluations of sea cucumber H. leucospilota extracts for potential anticancer activity on growth human cancer cell lines, Human carcinoma oral epidermoid cells (KB) and human embryonic kidney cells (HEK293T) were evaluated by XTT assay. Methanol digestive organs extract with 2.46 TI index (table 1), showed better anti-cancer effect than other extracts obtained from sea cucumber. Compared to untreated control was detected. The dose dependent decreasing in the percentage of viability of treated cancer cells comparing to controls was represented in the (Fig. 1A to D). As shown in (Fig. 1B), among the other extracts, only methanol digestive organs exhibited antiproliferative effects against the cancer cells. In addition this extract was rather less cytotoxic against HEK293T compared with other extracts. Bars represent means of triplicate determinations, and error bar indicate SD. Results were accepted to be significant at p Table-1- Inhibitory effects of sea cucumbers extracts on growth growth human cancer cell lines, Human carcinoma oral epidermoid cells (KB) and human embryonic kidney cells (HEK293T) Extracts IC50 CC50 TI (CC50/ IC50) KB HEK Methanol body wall 224.9  ± 1.33 281.0  ± 1.18 1.24 Methanol digestive organs 152.5  ± 1.29 375.4  ± 1.11 2.46 Methanol gonad 500 360.1  ± 1.75 0.72> Diethyl ether body wall 279.0  ± 1.17 284.9  ± 1.19 1.02 Diethyl ether digestive organs ~ 449.4  ± 2.51 444.2  ± 1.55 ~1.01 Diethyl ether gonad 367.1  ± 1.29 302.2  ± 1.12 0.82 The IC50 (inhibition concentration 50% of extract that caused inhibition cancerous KB cell line), CC50 (50% cytotoxic concentration of the extracts on HEK) and TI (therapeutic index) of different extracts of sea cucumber by using XTT assay, mean  ± SD . Comparison of the cytotoxic effects of sea cucumber extracts on cancerous (KB) and normal (HEK293T) cells, was determined using the XTT assay (un- treated control (1  µl DMSO) cells) Effect of sea cucumber H. leucospilota extracts on KB and HEK293T cells. Internal organs extracts (50 mg/ml) highest antiproliferative effects against KB cells and don’t significant cytotoxic effects than other concentration. Values are mean  ± SD of three determinations. Anti-HIV activity of the sea cucumber extracts In this study first we evaluated the cytotoxic activity on hella cells of different concentration of each extracts with XTT assay. XTT assay appeared all extracts have substantial cytotoxic effect on host cell line. The antiviral activities of each extract are summarized in Table 2. We further determined whether the inhibitory effects on HIV-1 replication of these extracts were dose-dependent. We infected hella cells with HIV-1 viruses and then treated the cells with each of the extracts at a concentration of 10  µg/ml to 1000  µg/ml fig 2A to F. None of the extracts showed significant inhibition of HIV-1 replication but the concentration of 100 µ g/ml methanol digestive organs, inhibit HIV-1 replication with less cytotoxic effect compared to other extract fig 2A.in addition body wall extract, with 2.79 TI index has rather better antiviral activity than other extracts(table 2). We also included 0.1% DMSO as a negative control and nevirapine as a positive control in these experi ments. Bars represent means of triplicate determinations, and error bar indicate SD. Results were accepted to be significant at p Table-2- The IC50 (inhibition concentration 50% of extract that caused inhibition of viral replication in HIV-1), CC50 (50% cytotoxic concentration of the extracts on host cells (Hela)) and TI (therapeutic index) of different extracts of sea cucumber Extracts IC50 CC50 TI (CC50/ IC50) Methanol body wall 35.89  ± 1.21 19.15  ± 1.45 0.53 Methanol digestive organs 57.61  ± 3.02 23.79 ± 1.67 0.41 Methanol gonad 337.60  ± 1.34 9.084  ± 1.15 0.02 Diethyl ether body wall 20.14  ± 1.16 56.27  ± 1.54 2.79 Diethyl ether digestive organs 37.01  ± 1.19 49.65  ± 1.53 1.34 Diethyl ether gonad 5.03  ± 1.90 5.11  ± 1.89 1.01 The average percentage of HIV-1 replication with extracts treatments based on three independent experiments. The percentage was considered as 100% when HIV-1 replication of DMSO sample reached to the peak. Instead, the rest of other samples with extracts treatments were calculated and converted into percentages based on DMSO and nevirapine used as a positive control in these experiments. The data were mean  ± SEM of three independent experiments.