Cytotoxic Activities, Determining Toxin, and Molecular Docking of Ovary Pufferfish ( Tetraodon leiurus ) in Singkarak Lake as Cancer Chemoprevention Candidate

The primary toxin class discovered in freshwater pufferfish is a category of neurotoxins called PSTs (Paralytic shellfish toxins) and pufferfish toxin has been observed to have biological, biochemical, and cytotoxic effects on cancer cell lines. Therefore, it is crucial to determine the cytotoxic activity, toxins present in the ovary of T. leiurus, and interaction between ligand (toxin compound) and receptors test . This study used the MTT method in the T47D cell lines, liquid chromatograph-tandem mass spectrometry (LC-MS/MS), and analysis of the molecular interaction using molecular docking. The ovary of T. leiurus had cytotoxicity on the T47D cell, having an IC50 value of 229.535 μg/ml, and generated a chromatogram with a retention duration of 1.25 min that was similar to the Decarbamoylneosaxitoxin (dcNEO) standard solution. In molecular interactions between the dcNEO ligand to receptors, the lowest ΔG value was -9.29 kcal/mol at the Na v 1.7 receptor, and the lowest KI value was 1.23 µM at the Mcl-1 receptor. These findings indicate that the ovary of T. leiurus is cytotoxic to the T47D cell line and contains dcNEO toxin. It is more stable for the dcNEO ligand to engage with the Na v 1.7 receptor than with other receptors, and it inhibits the Mcl-1 receptor more potently than with other receptors. These findings indicate that the ovary of T. leiurus may be chemotherapy for the prevention of cancer strategy.


Introduction
Chemoprevention refers to the reversal, inhibition, or prevention of a premalignant lesion's transformation into a malignant form using synthetic or natural substances 1 .Natural cancer treatment is effective in preventing malignancies, which can be found in terrestrial or marine sources, as well as in animals, plants, and microbes 2 .Several researchers have researched the use of natural substances as anticancer, the leaf extracts (Anchusa strigosa) have shown potential in suppressing the proliferation against various cancer cell lines including MCF-7, T47D, MDA-MB-231, and Caco-2 3 .Peptides from the skin of Iraqi frogs (Rana ridibunda) exert remarkably selective effects on the cell cultures of leukemia patients and have low toxic effects on human red blood cells 4 .
In the Mekong River, in pufferfish (T.leiurus), STX (saxitoxin) is the dominant toxin, dcSTX (decarbamoylsaxitoxin) and NeoSTX (neosaxitoxin) are secondary, and TTX is absent 10 .Furthermore, experimental evidence for STX's apoptosis-inducing effect elevated pro-apoptotic and reduced anti-apoptotic gene expression.Additionally, STX had negative impacts on zebrafish embryonic development that may have been brought on by oxidative stress 11 .
Neurotoxins that are present in pufferfish include TTX and STX 12 .STX can affect muscle and nerve conduction and selectively block voltage-gated sodium (NaV) channels, it may cause death and respiratory paralysis.In terms of molecular weight, toxicity, and intoxication mechanism, it is comparable to TTX 13 .STX is a member of the PST subgroup, the carbamate PST group.PST is divided into four subgroups from a collection of chemically related tetrahydropurines: decarbamoyl, deoxydecarbamoyl, carbamate, and Nsulfocarbamoyl.Decarbamoyl analogs include dcNEO, and Deoxydecarbamoyl analogs include doGTX2.Carbamate includes NeoSTX, GTX1-4, and STX.The N-sulfocarbamoyl includes C1-C4 14 .
The freshwater pufferfish naturally harbors PSTs and selectively accumulates PSTs.Marine pufferfish naturally harbor TTX and selectively accumulate TTX 15 .PSTs and TTX are produced by the food that pufferfish consume both marine and freshwater.Marine dinoflagellates produce TTX, PSTs whereas freshwater cyanobacteria produce PSTs 16 .Pufferfish toxins may vary between individuals.Geographical niches, bacterial buildup, seasonal fluctuations, and biological food chains all have an impact on these variances 17 .
To our knowledge, cytotoxic activity in the T47D cell line, the discovery of a toxin, and molecular docking in the ovary of T. leiurus, a pufferfish in Singkarak Lake, have not yet been confirmed and published.Thus, using the ovary of T. leiurus as a breast cancer treatment and the evaluation of the effect of the toxin on proteins that play a role in apoptosis (sodium channel and B-cell lymphoma-2 protein family).

Collection of pufferfish samples
Pufferfish (T.leiurus) samples were taken using nets in August 2021 in Singkarak Lake, located in the Tanah Datar Regency of West Sumatra.After dissecting the pufferfish, the collected ovary and liver were stored in film bottles, briefly placed in liquid nitrogen tubes, and then transferred to a -20°C freezer.

Reagent and standard toxins
Analysis of toxins pufferfish used standard solutions of STX, NeoSTX (National Research Council Canada), dcNEO, GTX1&4 (Cifga Laboratory).

Toxin extraction of the ovary pufferfish (T. leiurus)
Toxin extraction from the pufferfish 18 .Accurately weigh 5 g of pufferfish ovary and liver, respectively and 0.1 N hydrochloric acid (HCl), 5 ml, was used to homogenize the mixture, for every 500 μl of extract sample, add 25 μl of 30% trichloroacetic acid (TCA), vortex until evenly mixed and centrifuged for five min at 7000×g.After that, add 20 μl of 1.0 M sodium hydroxide (NaOH), and vortex until evenly mixed and centrifuged.Next, the solution was filtered using an Advancetec 0.2 m syringe filter.The filtered solution was heated for 10 min and centrifuged at 4 o C for 10 min, 7000×g.In the final stage by adding 0.1% acetic acid to the supernatant up to a 10 ml capacity, then, a 2 ml sample was put through a syringe filter for analysis.

Cytotoxicity test
Cytotoxicity testing was carried out using the MTT method.The ovary and liver extract were plated in 96-well plates containing T47D cells (10000 cells) at six different concentrations (25; 50; 100; 150; 200; and 250 μg/ml) in DMEM media with three replications and incubated for 24 and 48 h.After discarding the medium in each well, 20 μl of MTT is added and incubated for 4-6 h, then adding 100 μl of DMSO, and the MTT reaction was stopped.
Finally, the absorbance of each well is then calculated.

Liquid chromatograph-tandem mass spectrometry (LC-MS/ MS)
At the National Police Criminal Investigation Agency, Forensic Laboratory Center, Bogor, Indonesia the LC-MS/MS test was conducted.Liquid Chromatography coupled to a triplequadrupole mass spectrometry (Waters Alliance 2695 Quatro Premiere) was used for detection and quantification and set to condition: column XTerra® MS C18 100 mm x 2,1 mm ID, flow rate 0.3 ml/min, particle size 5 μm, injection volume 5 μL.Acquisition mode: positive polarity, resolution analyzer mode, extended dynamic range.Mobile phases with gradient concentrations are summarized in Table 1.

Cytotoxic effect of the ovary and liver of T. leiurus
The cytotoxic test objective is to calculate the IC50 value using the linear regression equation.The ovary and liver of T. leiurus elevated cytotoxicity in T47D for 24 and 48 h in a concentration-and timedependent manner.Treatment with 250 μg/ml ovary extract reduced the number of cells by 17.988% and 56.764% in 24 and 48 h of incubation, respectively, in Fig. 1(a).At these time points, a liver extract treatment with the same concentration resulted in 17.304% and 33.91%, respectively, in Fig. 1(b).
The morphological differences between control cells and cell induced by the ovary of T. leiurus in Fig. 2 The ovary of T. leiurus at 48 h of incubation had the highest cytotoxicity on the T47D cell with an IC50 value of 229.535 μg/ml.The classification of compounds as cytotoxic included three categories: active (IC50 < 100 μg/ml), moderately active (100 μg/mL < IC50 < 1000 μg/ml), and nontoxic (IC50 > 1000 μg/ml) 19 .To reduce the negative effects of the chemotherapeutic treatment, extracts with moderate activity might be helpful in combination with chemotherapy medications.Moreover, it might stop the carcinogenesis route 20 .
Based on several evaluated variables, the ovary of T. leiurus may be used in chemoprevention that can stop the T47D cell from growing and inhibit it.

Molecular interaction of the ligand and receptors
As a result of the identification of toxin in the ovary of T. leiurus, obtained dcNEO as a ligand for analysis of molecular interaction against receptors test.The receptors used in this study are sodium channel proteins (Nav 1.1; Nav 1.All the molecular interaction has RMSD values <2Å.In molecular interactions between the dcNEO ligand to receptors, the lowest ΔG value was -9.29 kcal/mol at the Nav1.7 receptor in Table 2 and Fig. 5.According to the ΔG value, the dcNEO ligand's interaction with the Nav1.7 receptor is more stable than that of other receptors.At the Mcl-1 receptor, the lowest KI value was 1.23 M in Table 2.The Mcl-1 receptor was more effectively inhibited by the interaction of the dcNEO ligand in a small concentration than the other receptors, according to the KI value.Proapoptotic protein interaction with dcNEO ligand, Bak receptor has five conventional hydrogen bonds (Asp30, Gln66, Ser69, and Pro67) and one alkyl hydrophobic interaction (Met71).The Bax receptor has two alkyl hydrophobic interactions (Ala183 and Val180) and seven conventional hydrogen bonds (Asp98; 102 and Ser184).
Based on the ligand-receptor interaction, the dcNEO ligand has the greatest hydrogen bonds with the Nav 1.5 and Nav 1.7 receptors, making this association more stable than that with other receptors.
PSTs can be structurally altered by different biological agents, leading to the production of brand-new PST that cannot be produced by cyanobacteria or dinoflagellates by themselves.Moreover, PSTs that are less harmful may be changed into harmful analogs or the other way around 22 .On the other hand, it has been discovered that Placopecten magellanicus (homogenate of the scallop) converts the GTXs and neoSTX to STX by reducing the O22-sulfate and N1-hydroxyl groups, respectively 23 and STX is the most common toxin in freshwater pufferfish, followed by dcSTX and NeoSTX 10 .If the selective absorption includes internal component conversion of the toxin and/or pufferfish tissues, or whether the composition of the toxin corresponds to that of harmful prey organisms, more research is required to answer these questions 12 .
Pufferfish toxin content varies between individuals and tissues, and the underlying molecular pathways are still under investigation 12 .A more thorough examination of the expression, distribution, and use of PSTBP/TBT-bp2 isoforms in conjunction with TTX/STX distribution patterns is required 10 .PSTBP and its analogs are caused for the accumulation of TTX in toxic pufferfish 24 , and suggests that in freshwater pufferfish, TBT-bp2 is involved in STX accumulation 12 .
Guanidinium alkaloids are a class of neurotoxins that includes STX and TTX analogs that have a strong affinity for the NaV channel and ion flux blockage abilities 25 .Inhibition of NaV channel expression in astrocytoma cells caused a reduced Na + concentration 26 and an elevator in the Ca 2+ concentration from intracellular storage 27 .Ca 2+ has a crucial part in intrinsic apoptosis, hence an excessive accumulation in the mitochondria can result in apoptosis.It was discovered that many proteins regulate the Ca 2+ by controlling Ca 2+ flux between the endoplasmic reticulum (ER) and the mitochondria 28 .
Pufferfish toxin (TTX/STX) is a cytotoxic compound 6 that can trigger the development of apoptosis mechanisms and its application in the treatment of cancer 29 .STX increases reactive oxygen species (ROS) 11 , which also activates P53 and JNK, and the promoter of apoptosis such as Bax, Noxa, and Puma, and can interfere with the activity of the inhibitor of apoptosis including Bcl-2 and Bcl-XL proteins.In addition, cardiolipin oxidation, which is a result of ROS, releases and allows cytochrome c to enter the cytosol.Procaspase 9, cytochrome c, and APAF-1 are the cause of caspase 9 activations 30 .Following the activation of the initiator caspase 9, the executioner's caspases are triggered, which causes a feedback stimulation of the initiator caspases and direct substrate proteolysis, both of which end in cell death (apoptosis) 31 .

Conclusion
The ovary of T. leiurus is cytotoxic to the T47D cell line and contains decarbamoylneosaxitoxin (dcNEO) toxin.The molecular interaction of the dcNEO ligand with the Nav 1.7 receptor is more stable than other receptors and was more effective in inhibiting the Mcl-1 receptor than other receptors.

Figure 1 .Figure 2 .
Figure 1.Cytotoxicity in the T47D cell line induced by (a) ovary extract, (b) liver extract of T. leiurus with various concentrations for 24 and 48 h

Figure 5 .
Figure 5. Structure of Nav1.7 receptor (blue) and its binding site for the dcNEO ligand (yellow)