Intensive exploration of the marine ecosystem has provided an immense source of diverse bioactive compounds. Recently, a purple ink extract released by the sea hare Bursatella (B.) leachii has been studied and found to be endowed with anti-HIV and anti-inflammatory activities [16, 17], two prominent properties for a potential liver cancer treatment. To widen the biological activities of this B. leachii purple ink-derived extract as a promising natural neo-adjuvant for the treatment of liver cancer, we have investigated its potential cytotoxic effects against HCC HepG2 cells and established molecular target and pharmacokinetic predictions of identified metabolites-derived B. leachii purple ink-derived extract.
In this present study, human HCC cell line HepG2 exposed to the B. leachii purple ink-derived extract led to an inhibition of the cell proliferation in a dose- and time-dependent manner. An induction of apoptosis in B. leachii extract-treated HepG2 cells was observed at intermediate concentrations (100 and 400 µg/mL) of B. leachii extract after 24-h exposure. Among the studied time periods, B. leachii extract added to the HepG2 cells for 72-h treatment resulted in the lowest IC50 value of 242.9 µg/mL compared with IC50 values determined after 24- and 48-h exposure. A previous study conducted by Suntornchashwej and colleagues [18], reported the cytotoxicity exhibited by the ethyl acetate-derived extract of B. leachii against the human small cell lung cancer (NCI-H187), oral human epidermoid carcinoma (KB), and breast cancer (BC) cell lines with an effective dose (ED50) value of 16.2, 7.2, and 6.6 µg/mL, respectively. A protective effect of B. leachii extracts against neuroblastoma cell line SH-SY5Y pre-treated with hydrogen peroxide and against microglia cells stimulated by bacterial lipopolysaccharide was studied [19]. B. leachii extracts were stated to be effective only against microglia cells by decreasing intracellular nitric oxide production and exhibiting an IC50 value of 5.74 µg/mL while the SH-SY5Y cells have no cell response to the studied concentration of BL extracts [20]. Thus, the determination of different IC50 values reflecting the anti-proliferative activity of B. leachii extracts indicates the specificity of the extract to exert cytotoxicity against various cancer cell lines.
Similarly, the anticancer potential of B. leachii extract at 100 and 400 µg/mL through apoptosis induction in HepG2 cells treated for 24 h was confirmed using Western blot technology by qualitatively detecting the expression of the most important pro-apoptotic proteins: the initiator caspase-8 and the effector/executioner caspase 3. Apoptosis is initiated and achieved by the cleavage of initiator and executioner pro-caspases into cleaved-caspases, the activated form of the enzymes. In this present study, the cleavage of caspase-8 and caspase-3 was observed in HepG2 cells after 24 h of cell exposure to B. leachii extract. Equal level of cleaved-caspase-3 was observed at both B. leachii extract concentrations (100 and 400 µg/mL), confirming the slight decrease of HepG2 cell growth noticed at similar conditions. Tested at the same intermediate concentrations, a concomitant cleavage of pro-caspase-3 induced by B. leachii extract would be expected after 72-h exposure, due to the important decrease of the B. leachii extract-treated HepG2 cell growth. However, the degree of pro-caspase-8 cleavage increased with B. leachii extract concentrations, confirming the role of caspase-8 as initiator of apoptosis. In addition, the expression of cleaved-caspase-8 and cleaved-caspase-3 detected in B. leachii extract-treated HepG2 cells may reveal that the induction of apoptosis occurs via the activation of the extrinsic death receptor pathway by transmitting the death signal from the cell surface to the intracellular signaling pathways through tumor necrosis factor (TNF) receptor gene family, for instance [21]. Active caspase-8 either initiates apoptosis directly by cleaving pro-caspase-3 into activated cleaved-caspase-3 or through mitochondria by the cleavage of BID to induce cell death [22]. Thus, an investigation of the involvement of the mitochondrial-dependent intrinsic apoptosis pathway in B. leachii extract-treated HepG2 cells would be of interest.
Consecutively, the quantitative expression of cell cycle and apoptotic regulatory genes in HepG2 cells treated with 400 µg/mL of B. leachii after 48-h exposure was analyzed using RT-qPCR. The expression of pro-apoptotic BAX, TP53, and Cyclin D1 genes were significantly up-regulated while the anti-apoptotic BCL-xL gene was up-regulated at a lesser extent. The gene expression of BCL-2, the main regulator of apoptosis that is endowed with pro- and anti-apoptotic activities, was not modulated even after HepG2 cell exposure to B. leachii extract. The tumor suppressor p53 is a transcriptional protein activated by a variety of oncogenic/hyperproliferative stimuli including DNA damage or chemotherapeutic drugs [23]. The protein p53 located in the cytosol induces the activation of pro-apoptotic Bax by protein-protein interactions and with Bcl-xL and Bcl-2 by p300/CBP binding [24]. Therefore, the elevated expression levels of TP53 and BAX in B. leachii extract-treated HepG2 cells endorses the cell death might be due to p53-dependent apoptosis.
The chemical analysis in this study was performed using high-resolution Q-TOF analysis, which enabled to tentatively identify the chemicals with more accuracy and compare them with previous structure identification studies related to the B. leachii ink extract biomolecules. For example, the appeared m/z value at retention time (0.164 - 0.661) were correlated with the parent compound hectochlorin, [18] with m/z [M+K]+ 703.5708 daltons and molecular formula of [C27H34Cl2N2O9S2]+, in positive ion mode [M+H]+ m/z 666.236, and [M-H]- with m/z 664.163 daltons in negative mode, indicating that the compound has a molecular weight of 665.603 g mol-1. The appeared m/z value at retention time (0.164 - 0.661) were correlated with the parent compound malyngamide X, [19] with m/z [M+K]+ 646.5302 daltons and molecular formula of [C33H57N3O7]+, in positive ion mode [M+H]+ m/z 607.420, and [M-H]- with m/z 606.822 daltons in negative mode, indicating that the compound has a molecular weight of 607.420 g mol-1.The appeared m/z value at retention time (3.398 - 4.559) were correlated with the parent compound bursatellin [25] with m/z [M+2H]+ 266.9169 daltons and molecular formula of [C13H16N2O4]+, in positive ion mode [M+H]+ m/z 264.111, and [M-H]- with m/z 263.210 daltons in negative mode, indicating that the compound has a molecular weight of 264.277 g mol-1. The appeared m/z value at retention time (1.176 - 2.071) were correlated with the parent compound malyngamide S [26] with m/z [M+K]+ 522.4386 daltons and molecular formula of [C26H42ClNO5]+, in positive ion mode [M+H]+ m/z 483.279, and [M-H]- with m/z 482.109 daltons in negative mode, indicating that the compound has a molecular weight of 484.069 g mol-1. The appeared m/z value at retention time (9.634 - 13.382) were correlated with the parent compound lyngbyatoxin A [27] with m/z [M-CH3]+ 426.4968 daltons and molecular formula of [C27H39N3O2]+, in positive ion mode 437.304, and [M-H]- with m/z 437.304 daltons in negative mode, indicating that the compound has a molecular weight of 437.617 g mol-1.
The analysis of the bioactivity predictions revealed that among the five identified compounds only hectochlorin and malyngamide S demonstrated the highest scores as anti-neoplastic agents. This is of great importance for lead optimization and further lead development. Additionally, these predictions support the observed anticancer activity of the B. leachii extract that could contribute to the presence of hectochlorin and malyngamide S. Additionally, the molecular target predictions established from B. leachii extract suggested the involvement of proteases and kinase inhibitors as potential targets that could explain the observed up-regulation of several apoptotic markers. Several studies have reported the modulation of proteases and kinases by marine bioactive molecules, which induced apoptosis in cancer cells [27–30].
The in silico ADME predictions for the identified molecules are useful for the potential use of these compounds as a lead for the discovery of a novel anticancer therapy. The ADME properties data showed that most of the bioactive molecules have acceptable pharmaceutical properties and follow Lipinski’s rule of five of drugability related to absorption/permeation, molecular weight, and solubility [31] except for hectochlorine that demonstrated two violations for this rule. Moreover, the CYP enzymes inhibition profile suggests that some of the compounds could inhibit CYP2C19 and CYP3A4 activities. However, this inhibition profile could be overcome for future optimization of the lead compound.