Bursatella leachii purple ink secretion extract exerts cytotoxic properties against human hepatocarcinoma cell line (HepG2): In vitro and in silico studies

Liver cancer is the third leading cause of cancer death worldwide. Marine mollusc-derived extracts have gained attention as new potential natural-based anticancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. We evaluated the cytotoxic effects of a crude extract from the purple-ink released by the sea hare named Bursatella leachii (B. leachii) against human hepatocarcinoma cell line (HepG2) and explored the underlying mechanisms causing the programmed cell death (i.e., apoptosis). Expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to B. leachii extract. Gene expression levels of pro-apoptotic BAX, tumour suppressor TP53 and Cyclin D1 were increased after treatment with B. leachii. Using liquid chromatography-mass spectrometry, the main biomolecules in the B. leachii extract were identied as hectochlorin, malyngamide X, malyngamide S, bursatellin, and lyngbyatoxin A. Applying in silico approaches, the high scores predicted bioactivities for the ve compounds were protease and kinase inhibitors. The ADME and cytochrome proles for the compounds were also predicted. Altogether, the cytotoxic B. leachii extract presents high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.


Introduction
Liver cancer is the sixth most commonly diagnosed cancer and the third leading cause of cancer death worldwide in 2020 [1]. Liver cancer has a variable geographical distribution, which predominantly overlays with the geographic incidence of viral hepatitis (i.e., hepatitis B virus and hepatitis C virus) and of human immunode ciency virus [2]. This viral infection to the patients leads to liver cancer onset and progression from chronic hepatitis, liver cirrhosis to heterogeneous hepatocellular carcinoma (HCC) [3].
Liver cancer onset can also be due to aging, exposure to toxic compounds, autoimmunity, and metabolic diseases [4]. Conventional treatments such as surgery, radiotherapy and chemotherapy including gene and immune-based therapeutic drugs are currently used [5]. Cytotoxic chemotherapy is not the rst-line treatment (i.e., protein kinase inhibitor, Sorafenib) for HCC, the main type of primary liver cancer, which is considered as a chemotherapy-refractory tumor [6]. However, there is an intensive discovery of natural bioactive compounds as neo-adjuvant agents inhibiting liver cancer cell growth, which would enhance liver cancer prevention, overcome hepatotoxicity side effects from conventional therapy and liver cancer recurrence [7].
So far, an alternative treatment for liver cancer prior to transplantation is highly on demand. Natural product extracts have been widely studied for their bioactive compounds endowed with anti-proliferative activity and pro-apoptotic effects, revealed by tyrosine kinase inhibition [8] and by caspase activation, cell cycle-and apoptosis-related gene up-regulation [9]. The main advantage of the induction of apoptosis is the absence of in ammation reaction, well known to be triggered by necrotic cells [10]. Hence, some natural bioactive compounds have been discovered for their anti-in ammatory and anti-angiogenic effects, which prevent cancer progression [11].
Molluscs are the second largest animal phylum on earth and provide a rich source of medicinal natural bioactive molecules [12]. Opisthobranch molluscs are a subclass of Gastropoda family Aplysiidae, order Anaspide genus and species Bursatella (B.) leachii commonly known as sea hares [13]. Sea hare-derived bioactive compounds, including soblidotin (dolastatin-10 derivative), synthadotin/ ILX 651 , cemadotin and kahalalide F, have been discovered for their anticancer activity and are currently in clinical trials [14]. The sea hares release a purple ink like that of squid to fend off predators [15]. We previously reported B. leachii purple ink-derived anti-HIV protein [16], 7,9-di-tert-butyl-1-oxaspiro [4.5]deca-6,9-diene-2,8-dione, and dioxigenin acetate as potent anti-in ammatory compounds [17]. However, so far, B. leachii purple inkderived extracts, including the presence of these recently identi ed anti-in ammatory compounds, have not been studied for their potential anticancer activity. Therefore, we evaluated the potential cytotoxic effects of a crude extract of species B. leachii against the growth of the human hepatocellular carcinoma (HCC) cell line HepG2. Protein and gene expression levels of apoptosis and cell cycle regulatory markers in the B. leachii extract-treated HepG2 cells were assessed. The chemical analysis of B. leachii extract and several biological target predictions were performed as well.

Cytotoxic effect of the B. leachii extract
The effect of B. leachii extract on HepG2 cell proliferation was tested at different concentrations and time periods. The increasing concentrations (from 10 to 1,000.0 µg/mL) of the B. leachii extract and the elongation of exposure time periods (from 24 to 72 h) resulted in a decrease of the viability of HepG2 cells based on ATP generated by living cells, compared to the control cells, which described a dose-and time-dependent effect ( Figure 1). The IC 50 values of B. leachii extracts required to inhibit 50% of HepG2 cell growth were determined at each exposure time. The treatment of HepG2 cells with B. leachii extract for 72 h of exposure displayed the lowest IC 50 value of 242.9 µg/mL while the highest IC 50 value was recorded after 24-h exposure with 465.4 µg/mL followed by 48-h exposure with an IC 50 value of 447.5 µg/mL of B. leachii extract.

Induction of apoptosis by B. leachii extract
As one of the cell death mechanisms, the potential induction of apoptosis in HepG2 cells treated with 100 µg/mL and 400 µg/mL of the B. leachii extract was investigated using Western blot analysis. After 24 h of exposure, the expression levels of pro-apoptotic proteins, such as cleaved caspase-8 (key enzyme prompting extrinsic apoptotic pathway) and cleaved-caspase-3 (key enzyme resulting in the completion of apoptosis), were evaluated in B. leachii-treated cells in comparison with untreated cells. Used as a positive control, staurosporine (STS) led to a cleavage of both caspase-8 and caspase-3 in HepG2 cells while, as expected, no cleaved-caspase-8 and cleaved-caspase-3 expression was detected in untreated cells ( Figure 2). A cleavage of both caspase-8 and caspase-3 was observed in B. leachii extract-treated HepG2 cells (Figure 2). Higher expression levels of cleaved-caspase-8 was observed in HepG2 cells treated with 400 µg/mL of B. leachii extract as compared with that detected in cells treated with 100 µg/mL of B. leachii extract (Figure 2). A similar expression level of cleaved-caspase-3 was noticed in HepG2 cells exposed to B. leachii extract concentrations ( Figure 2).

Modulation of gene expression levels of apoptotic and cell cycle regulatory genes by B. leachii extract
The pro-apoptotic effect of B. leachii extract on HepG2 cells was evaluated by monitoring the expression levels of apoptosis and cell cycle-related genes, including BCL-2, BCL-xL, TP53, BAX, CDKN1A, Cyclin A, Cyclin D1 and Survivin using RT-qPCR. The treatment of HepG2 cells with 400 µg/mL of B. leachii extract signi cantly enhanced the expression level of pro-apoptotic genes BAX (2.6-fold, p = 0.000012), TP53

Chemical identi cation of B. leachii extract using LC-QTOF
The crude extract of the B. leachii was subjected to total ion current spectra (TIC) raw data (See Figure 4). The data-analysis program Mass Hunter (Agilent Technologies) qualitative and quantitative analysis software were also used. After conducting a mass screening on the below spectrum (

B. leachii extract bioactivity predictions
In addition to exploring the pro-apoptotic effects of B. leachii extract, we sought to determine the bioactivity score of each bioactive metabolite identi ed in B. leachii extract. These bioactivity predictions gave more information about which molecule in the extract could contribute to the observed anticancer activity. Thus, the bioactivity score of the ve bioactive molecules was investigated using the PASS online webserver. Our results showed that hectochlorin and malyngamide S exhibited the highest bioactivity score (Table 1) with Pa 0,933 and Pa 0,747 respectively, suggesting a promising anti-neoplastic activity for these two molecules. Additionally, the anticancer activity of B. leachii extract could contribute to the presence of hectochlorin and malyngamide S metabolites. The remaining molecules exhibited a lower bioactivity score, and no predicted score was identi ed for bursatellin. To investigate the possible molecular targets that could mediate the observed and predicted anticancer activity, Molinspiration was utilized for this evaluation. Each bioactive molecule was evaluated as a G protein-coupled receptors (GPCR) ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease, and enzyme inhibitors. Interestingly, most of the ve molecules exhibited a positive bioactivity score as protease inhibitors with malyngamide X, malyngamide S, and lyngbyatoxin A possessing the highest scores (0.46, 0.32, and 0.36 respectively). Moreover, malyngamide, malyngamide S, and lyngbyatoxin A demonstrated high bioactivity scores as Enzyme inhibitors with values of 0.32, 0.32, and 0.35, respectively. Other possible targets such as ion channels, GPCR, and kinase inhibition were seen with malyngamide X, malyngamide S, and lyngbyatoxin A, suggesting that these bioactive molecules could regulate several molecular targets. Additional target mapping was conducted using SWISS target prediction and the results were comparable with Molinspiration webserver with a high probability of targeting proteases and kinases, as summarized and shown in Table 2.

Pharmacokinetics ADME predictions and CYP P450 enzymes inhibition pro ling
To assess the potential pharmaceutical properties of these B. leachii extract-derived bioactive molecules, SWISS ADME was utilized to evaluate several parameters important for drug discovery. Among the ve bioactive metabolites, malyngamide S, bursatellin, and lyngbyatoxin A demonstrated a molecular weight of fewer than 500 Daltons. Moreover, all the compounds that demonstrated high lipophilicity except for bursatellin exhibited a low Log P value. The solubility of the compounds was poor with exception of bursatellin that demonstrated excellent solubility (Log S -2.39). Most of the compounds were predicted to have a peripheral effect (no blood-brain barrier (BBB) penetration except for lyngbyatoxin A) and have high gastrointestinal (GI) absorption as summarized in Table 3. Additionally, we sought to determine and qualitatively predict the possibility of cytochrome P450 (CYP) enzymes inhibition that could be associated with these bioactive molecules, thus SWISS webserver was utilized, and ve compounds were subjected for this prediction. Our results showed that hectochlorine and bursatellin did not exhibit any CYP enzymes inhibition for all the ve enzymes. malyngamide X demonstrated only two inhibitions for CYP2C19 and CYP3A4. Moreover, malyngamide S was predicted to inhibit CYP2C19, CYP2D6, and CYP3A4 enzymes and lyngbyatoxin A inhibited three CYP enzymes including CYP2C19, CYP2C9, and CYP3A4 as shown in Table 4.

Discussion
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-in ammatory 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 identi ed 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.  [20]. Thus, the determination of different IC 50 values re ecting the anti-proliferative activity of B. leachii extracts indicates the speci city 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 con rmed 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), con rming 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, con rming 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 signi cantly 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 p53dependent 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 identi cation studies related to the B. leachii ink extract biomolecules. indicating that the compound has a molecular weight of 437.617 g mol -1 .
The analysis of the bioactivity predictions revealed that among the ve identi ed 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][28][29][30].
The in silico ADME predictions for the identi ed 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 ve 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 pro le suggests that some of the compounds could inhibit CYP2C19 and CYP3A4 activities. However, this inhibition pro le could be overcome for future optimization of the lead compound.

Collection of B. leachii Extract
The adult B. leachii sea hares were collected from intertidal waters of the Pulicat lake in the position Lat. 13.452523° N Long. 80.319133°E+/-0.03°N/E and brought to the laboratory in live condition. The accession number was M-1697, obtained from Zoological Survey of India, Marine Biology Regional Centre (MBRC), Chennai, India and dated on 23 July 2015. Obtained by disturbing the B. leachii, the purple uid ink (extract) was ltered through Whatman® lter paper. All aqueous ink-derived samples were centrifuged at 15,000×g for 15 min as described by Vennila and colleagues [33] and the supernatant was kept and lyophilized to extract a purple reside using a freeze dryer then stored at 4°C for further use.

Chemicals and Reagents
Dulbecco

Gene Expression Analysis
HepG2 cells (1.5 × 10 6 ) were seeded in Nunc™ 6-well plates. After 24 h of incubation, the cells were treated in triplicate with or without 400 µg/mL of B. leachii extract and incubated for a further 48-h incubation. From total RNA extraction to reverse-transcribed cDNA, RT-qPCR was carried out as previously described in [36]. Relative quanti cation of the mRNA expression level for the target genes are listed in the Table 1.

Chemical Analysis
The ngerprinting of B. leachii secretion aqueous extract was performed using the Agilent (Santa Clara, CA, USA) 1260 In nity high performance liquid chromatography (

Activity Prediction using PASS online Webserver
The antineoplastic activity of the bioactive metabolites identi ed from B. leachii extract was assessed using Pass online webserver [37]. For each compound, the SMILES (Simpli ed Molecular Input Line Entry System) was generated and entered in the webserver to perform the assessment. The generated results are classi ed based on the compound probability of being active (Pa) and inactive (Pi) at the speci ed activity. The pharmacokinetics concerning the absorption, distribution, metabolism, excretion (ADME) of the identi ed bioactive metabolites from B. leachii extract were explored using the SWISS ADME web server that provided a detailed fast in silico predictions of the pharmaceutical pro les of bioactive compounds [40]. The selected ADME parameters for the analysis were molecular weight, lipid solubility (Log P), water solubility (Log S), blood-brain barrier (BBB) penetration, and gastrointestinal (GI) absorption. After data generation, results were compared to the established drug-likeness properties (rule-of-ve, ROF) considered important for drug discovery [41].

Target Predictions using Molinspiration and SWISS Target Prediction Tools
Additional investigations were conducted to assess the cytochrome P450 (CYP) inhibition pro le of the identi ed bioactive molecules using the SWISS web server. Each compound was evaluated against the following CYP enzymes including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4. The CYP enzymes inhibition pro le was important for the early identi cation of possible signi cant drug interactions.

Statistical analysis
All the data are expressed as mean ± SD of three independent experiments.

Conclusion
Brusatella leachii ink extract exerts anti-proliferative and pro-apoptotic activities in human liver cancer HepG2 cell line, suggesting B. leachii extract as a promising safe natural-based neo-adjuvant drug for liver cancer treatment. Our computational predictions for the B. leachii extract-derived identi ed bioactive molecules suggest that these compounds have promising anticancer properties with acceptable druglikeness pro le and minimal CYP enzymes inhibition that warrants further lead optimization and development to discover novel drug entities from marine-derived natural resources. Further chemical isolation and in vivo studies are still needed.

Declarations
Ethical approval statement   Figure 1 Effects of B. leachii extract on HepG2 cell viability. The cell viability was determined using CellTiter-glo® kit and expressed as percentage of the control, the untreated cell viability, corresponding to 100%.