Apoptosis-Inducing Activity of Marine Sponge Haliclona sp. Extracts Collected from Kosrae in Nonsmall Cell Lung Cancer A549 Cells

Although various anticancer drugs have been developed for the treatment of nonsmall cell lung cancer, chemotherapeutic efficacy is still limited. Natural products such as phytochemicals have been screened as novel alternative materials, but alternative funds such as marine bioresources remain largely untapped. Of these resources, marine sponges have undergone the most scrutiny for their biological activities, including antiinflammatory, antiviral, and anticancer properties. However, the biological mechanisms of the activities of these marine sponges are still unclear. We investigated the anticancer activity of marine sponges collected from Kosrae in Micronesia and examined their mechanisms of action using nonsmall cell lung cancer A549 cells as a model system. Of 20 specimens, the Haliclona sp. (KO1304-328) showed both dose- and time-dependent cytotoxicity. Further, methanol extracts of Haliclona sp. significantly inhibited cell proliferation and cell viability. A549 cells treated with Haliclona sp. demonstrated induced expression of c-Jun N-terminal kinase (JNK), p53, p21, caspase-8, and caspase-3. The percentage of apoptotic cells significantly increased in A549 cultures treated with Haliclona sp. These results indicate that Haliclona sp. induces apoptosis via the JNK-p53 pathway and caspase-8, suggesting that this marine sponge is a good resource for the development of drugs for treatment of nonsmall cell lung cancer.


Introduction
Complementary medicine has been used for the enhancement of chemotherapeutic efficacy and reduction of adverse effects for several years. Specifically, natural products have been utilized and their active compounds developed as novel anticancer drugs. The major bioresources have largely been phytochemicals, but marine bioresources have been the subject of recent study due to their global abundance. In fact, it has been suggested that more than 3,000,000 such organisms could be good candidates for development of novel drugs. The bioactivities of several biomaterials have been reported, but much remains to be studied. For example, active constituents isolated from soft corals were reported to have anti-inflammatory activity [1,2]. Additionally, various marine sponges are known to have antibacterial, anti-inflammatory, antiangiogenic, and cytotoxic activities [3][4][5][6]. However, the mechanisms of action for these activities are largely unclear. In the present study, we focused on the anticancer effects of marine sponges. In particular, we chose nonsmall cell lung cancer (NSCLC), which has a high mortality due to its resistance to radiation and chemotherapy [7]. Therefore, in an effort to overcome these limitations, we investigated several marine sponge extracts collected from Kosrae, Micronesia, and examined their anticancer activities and mechanisms of action.

Specimen Preparation.
Sponge specimens (KO1304 series) were collected by hand with scuba equipment at Kosrae Island in the Federated States of Micronesia in April 2013. The specimens were immediately washed with sterilized artificial seawater and lyophilized. These specimens extracted with methanol (3 × 3 L) were provided by the Korea Institute of Ocean Science & Technology. Each specimen was dissolved in sterile distilled water (final concentration of 50 mg/mL) as previously described [6]. Aliquots of specimens were stored at −20 ∘ C until use.

Cells and Treatment.
Nonsmall cell lung cancer A549 (CCL-185) cells (ATCC, Manassas, VA) were cultured in Ham's F-12 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (GenDEPOT, Barker, TX) and 1% penicillin/streptomycin (GenDEPOT) in a humidified 5% CO 2 incubator. Cells in the exponential growth phase were used. The samples were added to the medium and treated with extract for 24 h or 48 h.

Cell Cytotoxicity.
Cell cytotoxicity was determined using the Cell Counting Kit-8 (CCK-8, DOJINDO, Japan). Briefly, cells (3 × 10 3 cells/well) were seeded in 96-well plates and incubated for 24 h. After treatment with samples for 24 h or 48 h, CCK-8 reagent (10 L) was added to each well and incubated for 3 h at 37 ∘ C. Absorbance at 450 nm was measured using a microplate reader (Infinite M200 PRO, TECAN, Austria).

Cellular
Morphology. Cells (3 × 10 3 cells/well) were seeded in 96-well plates and incubated for 24 h. Cells were treated with extract for 24 h or 48 h and observed under light microscopy (40x magnification) (Nikon Eclipse TS100, Japan).

Clonogenic
Assay. Cells were seeded in six-well plates at a density of 100-500 cells/well. After 24 h, cells were treated with extract and incubated until colony formation. Colonies consisting of at least 50 cells were fixed and stained with crystal violet (0.5% w/v) in 10% methanol and counted. Plate efficiency (PE) and survival fraction (SF) were calculated using the following [8]: (1) 2.6. Western Blot Analysis. Cells were seeded in a six-well plate at a density of 4-6 × 10 4 cells/well and allowed to incubate for 24 h. Extract was added to each well and incubated for 48 h. Cells were harvested and lysed in RIPA buffer (Gen-DEPOT) with protease inhibitors (Xpert protease inhibitor cocktail solution, GenDEPOT) and phosphatase inhibitors (Xpert phosphatase inhibitor cocktail solution, GenDEPOT).
Each extract (titrated from 6.25 to 100 g/mL) was added to A549 cells for 48 h. IC 30 is the concentration at which 30% inhibition of cell viability is achieved.
Cell lysates were boiled in 5x sample buffer and separated by 10% SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) using a semidry electroblotter (Peqlab, Germany). Membranes were blocked with 5% skim milk in TBS-T (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and sequentially incubated overnight with primary antibodies at 4 ∘ C. Membranes underwent additional incubation at room temperature and were then probed with secondary antibody. Immunoreactive proteins were visualized using ECL reagents and developed with X-ray film.

Screening Test of KO1304
Series. The cytotoxicity of 20 sponge specimens (KO1304 series) was determined in order to determine their use as an anticancer drug candidate resource. Each specimen was serially diluted and applied to A549 cells for 48 h. As shown in Table 1   by more than 30%. KO1304-328 was identified as Haliclona sp. (Figure 1), which has been reported to have cytotoxic [9], antibacterial [10,11], antifungal [11], and anticancer effects in breast, prostate, and colon cancer cells [9,12]. Papuamine and haliclonadiamine isolated from Haliclona sp. were reported as active components [12]. However, the anticancer mechanism of Haliclona sp. is unclear, particularly that against human nonsmall cell lung cancer. and evaluated. Therefore, the mechanism of the anticancer activity of Haliclona sp. extract was investigated in human nonsmall cell lung cancer A549 cells.

Haliclona sp. Suppresses Cell Viability and Cell Proliferation.
To evaluate cytotoxicity, Haliclona sp. extract was serially diluted and applied to A549 cells for 24 h or 48 h. As shown in Figure 2(a), cell viability of A549 cells decreased in a dose-and time-dependent fashion. At 24 h, cytotoxicity was statistically significant for extracts of 50 and 100 g/mL, but maximal cell viability inhibition was only 27.5 ± 2%. In A549 cells treated with Haliclona sp. extract for 48 h, a significant difference in cell viability was shown at 12.5, 25, 50, and 100 g/mL (Figure 2(a)). Further, the maximum dose (100 g/mL) inhibited cell viability by 51.6 ± 4.7%. Concurrently, we treated A549 cells with a control or Haliclona sp. extract and observed the decrease of cell density at 24 or 48 h (Figure 2(b)). Furthermore, the cytotoxicity of Haliclona sp. extract showed only in A549 cells, but not in RAW264. We also investigated the effect of Haliclona sp. extract on cell proliferation. Single, untreated A549 cells proliferated and formed colonies, but those cells treated with Haliclona sp. extract were suppressed in colony formation ability ( Figure 3). This result indicates that Haliclona sp. extract dose-dependently inhibits cell proliferation.
Inhibition of cell proliferation was commonly induced by the cell cycle arrest [13,14]. Radiation is well known for inducing the permanent G1 arrest and suppressing the cell proliferation [13]. So, we investigated if Haliclona sp. extracts affected the cell cycle as radiation. For analysis of cell cycle, A549 cells treated with Haliclona sp. extracts were stained with PI solution and detected the phases of the cycle (Figure 4). Haliclona sp. showed slight and temporary G1 phase arrest (Figures 4(b) and 4(c)). The results suggested that Haliclona sp. could delay the cell proliferation.

Haliclona sp. Induces Apoptosis via the JNK and Extrinsic
Pathway. To investigate the cellular mechanism of Haliclona sp. extract, protein levels were analyzed using Western blots. Specifically, we examined the apoptosis-inducing factors of JNK, p53, Bax, caspase-3, caspase-8, and caspase-9 which activate the mitochondrial or intrinsic apoptotic pathway [15]. JNK phosphorylates and regulates the activity of p53 and its stability [16][17][18]. The p53 tumor suppressor gene plays an important role in cell cycle, DNA repair, replicative senescence, and cell death [19]. As shown in Figure 5, A549 cells treated with Haliclona sp. extracts displayed significantly increased levels of JNK and p53 proteins, as well as p21 a downstream gene upregulated by p53. In several studies, the JNK-p53 pathway was also reported as one of the apoptotic pathways induced by natural products [16,20]. However, levels of     [21][22][23]. TRAIL death receptor activates caspase-8 and also JNK [21][22][23]. It may influence the anticancer effect of Haliclona sp. extract.
To verify the induction of apoptosis, cells treated with Haliclona sp. extract were stained with Annexin V and PI. As shown in Figure 6, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that Haliclona sp. extracts have apoptosis-inducing activity via the JNK and extrinsic apoptotic pathway.

Conclusions
In this study, we examined the chemotherapeutic effects of marine sponges against A549 nonsmall cell lung cancer cells. We found that a single isolate, Haliclona sp., had significant anticancer activity and investigated its mechanism.
Our results indicate that Haliclona sp. extracts suppress cell viability and proliferation. Eventually, Haliclona sp. extract could induce apoptosis via activation of JNK and caspase-8 ( Figure 7). Thus, the apoptosis-inducing activity of Haliclona sp. extract could be utilized for future development of chemotherapeutic drugs against nonsmall cell lung cancer.