Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.


Introduction
Palytoxin (Figure 1) was initially found in soft corals from tropical areas of the Pacific Ocean, for example, Hawaii; it was first discovered in the tropical soft coral Palythoa species, a zoanthid [1]. Palytoxin was first characterized by the chemist Moore in 1981. The molecular action of palytoxin leads to the inhibition of the Na /K adenosine tri-phosphatase (ATPase) channel [8,9]. This transmembrane protein exchanges three Na + ions and two K + ions using ATP hydrolysis as the driving force. The electrochemical gradient generated by the sodium pump for maintaining cell homeostasis is then inhibited by palytoxin. Palytoxin binds to the extracellular domain of the Na + /K + -ATPase to transform the pump into a non-specific, permanently open ion channel causing membrane depolarization, voltage-gated ion channel opening, and a massive increase of calcium ions (Ca 2+ ) into the cytosol [10] strongly interfering with vital functions of the cell. An alteration of intracellular cation concentration, in particular, a calcium increase, is associated with cell death as a modification in the distribution of these ions across the plasma membrane plays a crucial role in palytoxin-induced cell-type specific cytotoxic response [11]. Further characterization of the anticancer mode of action of this compound would allow a better understanding of cell death mechanisms triggered by palytoxin at the cellular level.
Here, we first focused on assessing differential cell toxicity and mechanisms involved in palytoxin-induced cell death in human leukemia cell lines. Palytoxin activates intrinsic and extrinsic apoptotic pathways triggered by alterations in the expression levels of the pro-and antiapoptotic Bcl-2 family proteins. Moreover, we provide evidence that palytoxin inhibits 3D tumor growth in colony formation assays and xenograft models. Altogether our results show that palytoxin acts as a potent and promising anti-leukemic agent at picomolar concentrations.  The molecular action of palytoxin leads to the inhibition of the Na + /K + adenosine triphosphatase (ATPase) channel [8,9]. This transmembrane protein exchanges three Na + ions and two K + ions using ATP hydrolysis as the driving force. The electrochemical gradient generated by the sodium pump for maintaining cell homeostasis is then inhibited by palytoxin. Palytoxin binds to the extracellular domain of the Na + /K + -ATPase to transform the pump into a non-specific, permanently open ion channel causing membrane depolarization, voltage-gated ion channel opening, and a massive increase of calcium ions (Ca 2+ ) into the cytosol [10] strongly interfering with vital functions of the cell. An alteration of intracellular cation concentration, in particular, a calcium increase, is associated with cell death as a modification in the distribution of these ions across the plasma membrane plays a crucial role in palytoxin-induced cell-type specific cytotoxic response [11]. Further characterization of the anticancer mode of action of this compound would allow a better understanding of cell death mechanisms triggered by palytoxin at the cellular level.
Here, we first focused on assessing differential cell toxicity and mechanisms involved in palytoxin-induced cell death in human leukemia cell lines. Palytoxin activates intrinsic and extrinsic apoptotic pathways triggered by alterations in the expression levels of the pro-and antiapoptotic Bcl-2 family proteins. Moreover, we provide evidence that palytoxin inhibits 3D tumor growth in colony formation assays and xenograft models. Altogether our results show that palytoxin acts as a potent and promising anti-leukemic agent at picomolar concentrations.

Cytotoxic Effect of Palytoxin on Human Cancer Cells
Before investigating the cell death mechanisms, we first evaluated the antiproliferative and cytotoxic effects of palytoxin on various leukemia ( Figure S1). Cells were treated for 2, 4, 6, and 8 h at different concentrations of palytoxin, and viability was assessed by a Trypan Blue exclusion assay. Our results showed that solid tumor cell lines were more resistant to palytoxin under our experimental conditions than leukemia cell types. We also used PBMCs from healthy donors ( Figure 2D) to evaluate the differential toxicity of palytoxin. We treated cells with palytoxin at various concentrations and conducted Trypan Blue staining to assess cell integrity and viability. At concentrations of 100 pM, palytoxin did not generate levels of toxicity comparable to cancer cells.

Cytotoxic Effect of Palytoxin on Human Cancer Cells
Before investigating the cell death mechanisms, we first evaluated the antiproliferative and cytotoxic effects of palytoxin on various leukemia (Figure 2A-C) (Supplementary Figure S1) and solid tumor cell lines (Supplementary Figure S1). Cells were treated for 2, 4, 6, and 8 h at different concentrations of palytoxin, and viability was assessed by a Trypan Blue exclusion assay. Our results showed that solid tumor cell lines were more resistant to palytoxin under our experimental conditions than leukemia cell types. We also used PBMCs from healthy donors ( Figure 2D) to evaluate the differential toxicity of palytoxin. We treated cells with palytoxin at various concentrations and conducted Trypan Blue staining to assess cell integrity and viability. At concentrations of 100 pM, palytoxin did not generate levels of toxicity comparable to cancer cells. Data are the mean of SD ± of three independent experiments. Statistical significance was assessed as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Effect of Palytoxin on Healthy Zebrafish Embryos
Palytoxin was used at various concentrations to treat zebrafish embryos and larvae for 24 h. Interestingly, we did not observe any toxicity nor alterations of the morphology of the yolk-sac nor axial/tail malformations ( Figure 3A-C). Altogether this compound demonstrates a remarkable differential toxicity and selectively targets cancer cells. human T-cell leukemia Jurkat cell viability and proliferation. (C) Effects of Palytoxin on human histiocytic lymphoma U937 cell lines. (D) Time and concentration-dependent effect of palytoxin on the viability of healthy PBMCs from three buffy coats. Data are the mean of SD ± of three independent experiments. Statistical significance was assessed as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Effect of Palytoxin on Healthy Zebrafish Embryos
Palytoxin was used at various concentrations to treat zebrafish embryos and larvae for 24 h. Interestingly, we did not observe any toxicity nor alterations of the morphology of the yolk-sac nor axial/tail malformations ( Figure 3A-C). Altogether this compound demonstrates a remarkable differential toxicity and selectively targets cancer cells.

Palytoxin-Induced Cell Death Leads to Caspase Activation in U937 Cells
We subsequently investigated whether palytoxin could trigger caspase-dependent apoptotic cell death. Palytoxin-induced cell death was identified by changes in the nuclear morphology typical of apoptotic cell death after staining with Hoechst and PI ( Figure 4A). As caspase activation is sensitive to pan-caspase inhibitor zVAD, we concluded that palytoxin induces apoptosis ( Figure 4B). Palytoxin induced the cleavage of the pro-caspases-8 and -9 and executor pro-caspases-3 and -7 after 6 h of treatment in a dose-dependent manner ( Figure 4C). These results were further confirmed by luminescent assays where palytoxin induced the enzymatic activity of caspase-3/7 in U937 cells ( Figure 4D).

Palytoxin-Induced Cell Death Leads to Caspase Activation in U937 Cells
We subsequently investigated whether palytoxin could trigger caspase-dependent apoptotic cell death. Palytoxin-induced cell death was identified by changes in the nuclear morphology typical of apoptotic cell death after staining with Hoechst and PI ( Figure 4A). As caspase activation is sensitive to pan-caspase inhibitor zVAD, we concluded that palytoxin induces apoptosis ( Figure 4B). Palytoxin induced the cleavage of the pro-caspases-8 and -9 and executor pro-caspases-3 and -7 after 6 h of treatment in a dose-dependent manner ( Figure 4C). These results were further confirmed by luminescent assays where palytoxin induced the enzymatic activity of caspase-3/7 in U937 cells ( Figure 4D). Mar. Drugs 2023, 21, x FOR PEER REVIEW 5 of 18

Palytoxin Downregulates Expression of Antiapoptotic Bcl-2 Family Proteins
As the induction of apoptosis can be triggered by the inhibition of antiapoptotic proteins or activation of proapoptotic mechanisms, we investigated the overall expression levels of the Bcl-2 family proteins after palytoxin treatment ( Figure 5A). Myeloid cell leukemia-1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xL) expression are downregulated in a dose-dependent manner. In contrast, B-cell lymphoma 2 (Bcl-2) is affected to a lesser extent. Moreover, proapoptotic Bcl-2 interacting domain death agonist (BID) was truncated by palytoxin treatment (Figure 5B), which implied that the amplification of intrinsic and extrinsic cell death pathways could be expected [12]. We generalized our finding by investigating the effect of palytoxin on a range of leukemia cell types ( Figure   The percentage of apoptotic cells was evaluated by counting cells corresponding to apoptosis, late apoptosis, and necrosis. At least 100 cells in three random fields were counted by fluorescence microscopy. (C) Palytoxin activation of pro-caspase-8, -9, -7, and -3 was investigated by Western blot analysis (left; one of three independent experiments). Enzymatic activity of caspases-3 and -7 was upregulated in a dose-dependent manner. (right; data representative of three independent experiments (mean ± SD). (D) Etoposide-treated cells (100 µM, 4 h) were positive controls for the induction of apoptosis. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001 versus control.

Palytoxin Downregulates Expression of Antiapoptotic Bcl-2 Family Proteins
As the induction of apoptosis can be triggered by the inhibition of antiapoptotic proteins or activation of proapoptotic mechanisms, we investigated the overall expression levels of the Bcl-2 family proteins after palytoxin treatment ( Figure 5A). Myeloid cell leukemia-1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xL) expression are downregulated in a dose-dependent manner. In contrast, B-cell lymphoma 2 (Bcl-2) is affected to a lesser extent. Moreover, proapoptotic Bcl-2 interacting domain death agonist (BID) was truncated by palytoxin treatment ( Figure 5B), which implied that the amplification of intrinsic and extrinsic cell death pathways could be expected [12]. We generalized our finding by investigating the effect of palytoxin on a range of leukemia cell types ( Figure

Mcl-1 Is Downregulated by Palytoxin in a Proteasome-Dependent Manner
It is well established that antiapoptotic Mcl-1 can be downregulated by several mechanisms [13], including caspase-mediated-or proteasome-dependent degradation [14]. Accordingly, we assessed palytoxin-induced Mcl-1 degradation in the presence of two prototypical inhibitors, zVAD and MG-132. Our results show that palytoxin-induced Mcl-1 degradation is completely abolished in the presence of MG-132, underlining the essential role of proteasome degradation in the effect of palytoxin ( Figure 6a). Unexpectedly, the three major proteasomal enzymatic activities, including trypsin, chymotrypsin, and caspase-like, were upregulated after palytoxin treatment, most likely further exacerbating the degradation of Mcl-1 ( Figure 6B).
To get a deeper insight into the chronology of the molecular events triggered by palytoxin, we attempted to identify drivers of cell death rather than investigate the consequence of ongoing cellular demise. We used a kinetic approach to compare proteasomal activation, Mcl-1 degradation, and caspase activation. We obtained a rapid and significant activation of proteasomal activities before Mcl-1 degradation and caspase cleavage (Figure 6C). In parallel, ATP levels were quantified in the cells. We also observed a rapid decline in ATP levels ( Figure 6D). BID was cleaved at early steps, which is in line with our hypothesis, as BID requires polyubiquitination and proteasomal activation for its processing ( Figure 6D). Altogether, we concluded that palytoxin selectively targets essential cell death regulators before activation of the executioner caspase 3 and overall cellular degradation.

Mcl-1 Is Downregulated by Palytoxin in a Proteasome-Dependent Manner
It is well established that antiapoptotic Mcl-1 can be downregulated by several mechanisms [13], including caspase-mediated-or proteasome-dependent degradation [14]. Accordingly, we assessed palytoxin-induced Mcl-1 degradation in the presence of two prototypical inhibitors, zVAD and MG-132. Our results show that palytoxin-induced Mcl-1 degradation is completely abolished in the presence of MG-132, underlining the essential role of proteasome degradation in the effect of palytoxin ( Figure 6A). Unexpectedly, the three major proteasomal enzymatic activities, including trypsin, chymotrypsin, and caspase-like, were upregulated after palytoxin treatment, most likely further exacerbating the degradation of Mcl-1 ( Figure 6B).
To get a deeper insight into the chronology of the molecular events triggered by palytoxin, we attempted to identify drivers of cell death rather than investigate the consequence of ongoing cellular demise. We used a kinetic approach to compare proteasomal activation, Mcl-1 degradation, and caspase activation. We obtained a rapid and significant activation of proteasomal activities before Mcl-1 degradation and caspase cleavage ( Figure 6C). In parallel, ATP levels were quantified in the cells. We also observed a rapid decline in ATP levels ( Figure 6D). BID was cleaved at early steps, which is in line with our hypothesis, as BID requires polyubiquitination and proteasomal activation for its processing ( Figure 6D). Altogether, we concluded that palytoxin selectively targets essential cell death regulators before activation of the executioner caspase 3 and overall cellular degradation. The three major proteolytic activities of the 26S proteasome were assessed under the same conditions as the Western blots. MG-132 at 5 µM was used as a positive control for proteasomal inhibition in U937 cells. The asterisk indicates significant differences between treated versus control, **** p < 0.001. (C) Kinetic analysis of the assessment of three major proteasome activities in U937 cells. Cell viability was determined to normalize the three major proteasome activities induced by palytoxin. (D) Expression of Mcl-1 and Bcl-xL degradation, BID truncation, and Caspase 3 cleavage was evaluated by Western blot analysis under the same conditions. Data representative of three independent experiments is shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control.

Bcl-2 Serine 70 Dephosphorylation Induced by Palytoxin Is Mediated through Protein Phosphatase 2A Activation
Our results also show that palytoxin affects Bcl-2 expression and phosphorylation levels witnessed by a discrete upshift of the band [15] ( Figure 7A). We investigated phosphorylation levels of Bcl-2 as it is well known that serine 70 phosphorylation is required for the pro-survival function of this protein [16]. Our results show a dose-dependent dephosphorylation of Bcl-2, potentially contributing to a loss of its pro-survival function Our results also show that palytoxin affects Bcl-2 expression and phosphorylation levels witnessed by a discrete upshift of the band [15] (Figure 7A). We investigated phosphorylation levels of Bcl-2 as it is well known that serine 70 phosphorylation is required for the pro-survival function of this protein [16]. Our results show a dose-dependent dephosphorylation of Bcl-2, potentially contributing to a loss of its pro-survival function ( Figure 7A). Serine 70 phosphorylation of Bcl-2 can be abrogated by phosphatase activation, mainly by protein phosphatase 2A (PP2A) activity [17]. Accordingly, in the next step, we used chemical inhibitors of selected cell signaling pathways, including okadaic acid used at a concentration inhibiting specifically PP2A. Our results show that PP2A inhibition partially rescues U937 cells from palytoxin-induced cell death, whereas other kinase pathways do not seem to be involved. (Figure 7B).
Mar. Drugs 2023, 21, x FOR PEER REVIEW ( Figure 7A). Serine 70 phosphorylation of Bcl-2 can be abrogated by phosphatase tion, mainly by protein phosphatase 2A (PP2A) activity [17]. Accordingly, in the nex we used chemical inhibitors of selected cell signaling pathways, including okada used at a concentration inhibiting specifically PP2A. Our results show that PP2A tion partially rescues U937 cells from palytoxin-induced cell death, whereas other pathways do not seem to be involved. ( Figure 7B).
Altogether, we concluded that palytoxin-induced dephosphorylation of Bc PP2A exacerbates the proapoptotic effect of Mcl-1 and Bcl-xL degradation and tha daic acid could rescue in part palytoxin-induced cell death.  Hydroquinone (10 µM, 24 h) was used as a positive control for PP2A activation. Cells were stained with Hoechst to analyze the nuclear morphology merged with phase contrast images using ImageJ. The percentage of cell death was evaluated by counting the number of cells undergoing various types of cellular death. Three independent fields of 100 cells were quantified. SB202190 and PD98059 were used as MAP kinase inhibitors. Data representative of three independent experiments is shown as mean ± SD. * p < 0.05.
Altogether, we concluded that palytoxin-induced dephosphorylation of Bcl-2 by PP2A exacerbates the proapoptotic effect of Mcl-1 and Bcl-xL degradation and that okadaic acid could rescue in part palytoxin-induced cell death.

Anticancer Effect of Palytoxin in 3D Cell Culture and Zebrafish Xenografts
We investigated the colony formation ability of cancer cells in the presence of increasing concentrations of palytoxin to confirm its potential to impair the replicative capacity of cancer cells. After 10 days of treatment, colony formation in AML cell lines was strongly reduced by palytoxin at concentrations from 0.4 pM. Concentrations beyond 1 pM completely abrogated colony formation capacity ( Figure 8A-D). Besides AML cells, palytoxin significantly inhibited colony formation in CML cell lines, K562 and imatinib-resistant K562 (K562IR) (Figure 8E,F). Moreover, we generalized our findings with the solid prostate cancer cell line PC3. Concentrations between 0.5 and 1 pM abrogated colony formation (Supplementary Figure S3) and prevented spheroid formation in a dose-dependent manner (Supplementary Figure S4).

Anticancer Effect of Palytoxin in 3D Cell Culture and Zebrafish Xenografts
We investigated the colony formation ability of cancer cells in the presence of increasing concentrations of palytoxin to confirm its potential to impair the replicative capacity of cancer cells. After 10 days of treatment, colony formation in AML cell lines was strongly reduced by palytoxin at concentrations from 0.4 pM. Concentrations beyond 1 pM completely abrogated colony formation capacity ( Figure 8A-D). Besides AML cells, palytoxin significantly inhibited colony formation in CML cell lines, K562 and imatinib-resistant K562 (K562IR) (Figure 8E,F). Moreover, we generalized our findings with the solid prostate cancer cell line PC3. Concentrations between 0.5 and 1 pM abrogated colony formation (Supplementary Figure S3) and prevented spheroid formation in a dose-dependent manner (Supplementary Figure S4).
To further extend our evaluation of the anticancer potential of palytoxin, we examined the ability of palytoxin to abrogate tumor development in an ex vivo zebrafish xenotransplantation model. Fluorescently labeled leukemia cells were pre-treated for 6 hours with palytoxin at 5, 10, and 30 pM and then injected into the yolk sac of zebrafish embryos. Results revealed that the tumor-associated fluorescence intensity signal was drastically lowered in the palytoxin-treated zebrafish group compared to the untreated controls as early as 5 pM ( Figure 9A-F for a representative overview; Supplementary Figure S5 shows all nine fish for each group). , and K562IR (F). All data represent the mean ± SD. of three independent experiments. Statistical significance was assessed as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control. To further extend our evaluation of the anticancer potential of palytoxin, we examined the ability of palytoxin to abrogate tumor development in an ex vivo zebrafish xenotrans-plantation model. Fluorescently labeled leukemia cells were pre-treated for 6 hours with palytoxin at 5, 10, and 30 pM and then injected into the yolk sac of zebrafish embryos. Results revealed that the tumor-associated fluorescence intensity signal was drastically lowered in the palytoxin-treated zebrafish group compared to the untreated controls as early as 5 pM (Figure 9A-F Figure S5). Statistical significance was assessed as *** p < 0.001, **** p < 0.0001.

Discussion
We demonstrated that palytoxin, one of the most toxic marine biotoxins known [18], can significantly induce apoptotic cell death at low picomolar concentrations in different cancer cell types. We focused on cell death mechanisms, especially in acute myeloid leukemia (AML) U937 cells.
So far, palytoxins have essentially been investigated at high nanomolar doses. Palytoxins act as a skin tumor promoter when combined with a carcinogenic agent. It can modulate critical signal transduction pathways involved in carcinogenesis and inflammation [19]. It has been shown that palytoxin stimulates prostaglandin production from arachidonic acid and activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-acti-  Figure S5). Statistical significance was assessed as *** p < 0.001, **** p < 0.0001.

Discussion
We demonstrated that palytoxin, one of the most toxic marine biotoxins known [18], can significantly induce apoptotic cell death at low picomolar concentrations in different cancer cell types. We focused on cell death mechanisms, especially in acute myeloid leukemia (AML) U937 cells.
So far, palytoxins have essentially been investigated at high nanomolar doses. Palytoxins act as a skin tumor promoter when combined with a carcinogenic agent. It can modulate critical signal transduction pathways involved in carcinogenesis and inflammation [19].
In contrast, this manuscript shows that palytoxin induced apoptosis at lower pM concentrations in a caspase-dependent manner. Apoptosis is the most common and welldefined form of programmed cell death, a physiological process of cellular suicide [21]. However, as we observed a small measurable amount of non-apoptotic cell death after caspase inhibition by zVAD, it is essential to determine in the future which type of secondary cell death mechanism the cell activates in the absence of apoptotic capacity [22]. In that sense, investigating the activation of non-caspase proteases inducing calpains or cathepsins could be interesting [23,24].
Among Bcl-2 family proteins, Mcl-1 and Bcl-xL were found to be strongly downregulated by palytoxin, even at low picomolar concentrations. As we observed a consistent and early onset of BID truncation, the differential effect of extrinsic versus intrinsic cell death pathway is interesting and will be investigated by pathway-specific caspase inhibitors [12].
It will be interesting to investigate the overexpression of Mcl-1 with plasmid constructs, specifically wild-type and proteasome-resistant isoforms of Mcl-1 [29]. Overexpression of these proteins will further allow the strengthening of the hypothesis of proteasomal degradation. Similarly, stably-transfected Jurkat T cells expressing mutated Bcl-2 (serine70, serine87, and threonine 69) will allow us to understand the role of Bcl-2 expression regulated by palytoxin in various models [30,31].
This study also showed that inhibition of PP2A activity rescued palytoxin-induced cell death, which implies that phosphatases play a significant role in cellular mechanisms activated by palytoxin [32]. PP2A has been suggested as a therapeutic target in Philadelphia chromosome-positive chronic or acute myeloid leukemia, where PP2A inactivation is a recurrent event [32]. Palytoxin could restore PP2A or related phosphatases to induce caspase-dependent apoptosis mediated through AKT and ERK1/2 pathways [33]. PP2A exerts inhibitory and stimulatory effects on MAP kinase signaling pathways [34]. Previous findings suggested that palytoxin activates the mitogen-activated protein kinase (MAPK) cascade (including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinase (JNK) and p38 protein kinase) in response to heat shock protein 27 (HSP27) phosphorylation in human breast adenocarcinoma (MCF-7) cells [35]. In monocyte-derived human macrophages, palytoxin phosphorylates p38 MAPK. It activates nuclear factor kappa B (NF-κB) pathways by increasing the mRNA levels of inflammation-related genes like interleukin 8 (IL-8), an inhibitor of kappa alpha (IκB-α), leading to NF-κB nuclear translocation [6]. These observations allow us to hypothesize that palytoxin may activate a proinflammatory signaling cascade in leukemia cell lines by regulating kinases and phosphatases [36]. This manuscript is the first step in the preclinical evaluation of palytoxin. We are aware that there might be a long way to derive this high molecular weight compound into a druggable formulation. However, the potential of Na + /K + ATPase modulating agents was clinically investigated and exploited by cardiac glycosides so that the molecular target of palytoxin is of clear clinical relevance. In the future, the potent cytotoxicity, combined with the described proinflammatory potential, could find clinical applications in targeted antibody-drug conjugate (ADC) compounds like brentuximab vedotin or trastuzumab emtansine. Despite the limited success of first-generation ADCs, palytoxin could provide a cytotoxic, likely immunogenic warhead able to kill difficult-to-target cancer cells.
One of the future outcomes of this study is to validate the Na + /K + -ATPase inhibition by palytoxin as the possible cancer target. In this context, functional studies (i.e., silencing or overexpressing the pump subunits) and the future design of simplified analogs based on essential pharmacophores of palytoxin might be considered. Palytoxin inhibited tumor formation in a zebrafish xenograft model at concentrations between 10 and 30 pM. We plan to extend our investigation to animal models other than zebrafish to exclude any intrinsic evolutionary resistance developed by aquatic animal models. Furthermore, studies on primary cells will consolidate the documented differential cytotoxicity.

Compounds
Palytoxin was extracted from Palythoa Aff. Clavata following an established procedure (patent publication EP3087172B1). The compound, with a molecular mass of 2680.14 g mol −1 , was received as a powder, solubilized in DMSO (Sigma-Aldrich, Saint Louis, MO, USA), and further diluted to get working aliquots at 1 mM. Stocks and aliquots were stored at 4 • C for up to 2 years without loss of activity and protected from light and were used directly before the experiments. Etoposide was purchased from Sigma-Aldrich, Saint Louis, MO, USA, and dissolved in DMSO at a stock concentration of 50 mM. Pancaspase inhibitor, z-VAD FMK, was purchased from Calbiochem (San Diego, CA, USA), dissolved in DMSO, and added 1 h before at a concentration of 50 µM. Protein phosphatase 2A inhibitor okadaic acid was purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO at a stock concentration of 1 mM. Proteasome inhibitor MG-132 was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in DMSO at a stock concentration of 10 mM. MAP kinase inhibitors, SB202190 and PD98059, were purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO at a stock concentration of 100 mM. Hydroquinone was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in DMSO at 10 mM. Inhibitors were used for 1h before palytoxin treatment at the indicated working concentrations.

Cell Viability Assessment
Cell vitality was assessed in three leukemia cell lines, K562, Jurkat, and U937. Each cell line was treated with various concentrations of palytoxin for 2, 4, 6, and 8 h in a dose-dependent manner. The percentage of viability was evaluated using the Trypan Blue exclusion test (0.2% Trypan Blue), and the percentage of viability of cells without palytoxin treatment was considered to be 100%.

Caspase 3/7 Activity Assay
3 × 10 5 U937 cells/mL were seeded in 96-well plates in triplicate in 75 µL for palytoxin treatment. The enzymatic activities of caspases-3 and -7 were determined using a luminescent Caspase-glo®3/7 Assay (Promega, Cosmogenetech, Seoul, Republic of Korea). The assay was performed per the manufacturer's instruction by adding 75 µL of the caspase-3/7 substrate to U937 cells in suspension. After one hour of incubation with DEVD-aminoluciferin substrate and luciferase, the luminescent signal was measured using a Centro LB 960 microplate luminometer (Berthold Technologies, JCBio Co. Ltd., Seoul, Republic of Korea).

Proteasome Activity Assay
The Proteasome-Glo®cell-based assay (Promega, Cosmogenetech, Seoul, Korea) was used to evaluate the three major proteolytic enzyme activities (chymotrypsin-like, caspaselike, trypsin-like). The assays were performed as indicated in the manufacturer's protocol. U937 cells were treated at a concentration of 10 6 cells/mL in RPMI 1640 medium containing 0.1% FCS at indicated concentrations of palytoxin. After an incubation period, 50 µL of U937 cellular suspension was mixed with 50 µL of the assay reagent. The luminescence signal was measured on the Centro LB 960 Microplate Luminometer (Berthold Technologies, JCBio Co., Ltd., Seoul, Republic of Korea). The proteasome signal was normalized to the number of viable cells performed in parallel using CellTiter-Glo®(Promega, Cosmogenetech, Seoul, Republic of Korea). MG-132 at 5 µM (Sigma, USA) was used as a control for proteasomal inhibition.

Fluorescent Microscopy Analysis
3 × 10 5 U937 cells/mL were seeded in 24-well plates before palytoxin treatment. Then, 300 µL of treated cells were transferred into another 24-well plate for double staining with (1) the DNA-specific dye Hoechst 33342 (Sigma, USA) at 1 µL/mg for 30 min of incubation and (2) propidium iodide (Sigma, USA) at 1.5 µL/mg, diluted in PBS for 15 min of incubation. Induction of apoptosis was assessed by fluorescent microscopy (Nikon TI-U, Seoul, Republic of Korea) and expressed as the percentage of cells presenting fragmented/condensed nuclei. Different stages of nuclear fragmentation were considered, and at least 300 cells were counted in three independent fields as previously described [37][38][39][40][41][42]. Microscopy images were analyzed using the ImageJ 1.54 software (http://rsb.info.nih.gov/ij/docs/index.html, accessed on 4 March 2023).

Systemic Toxicity in Zebrafish
Zebrafish (Danio rerio) were obtained from the "Zebrafish International Resource Center" (ZIRC) (University of Oregon, Eugene, OR, USA) and maintained according to the zebrafish guidelines [43]. Adult fish were kept at 28.5 • C on a 14-h light/10-h dark cycle comparable to natural conditions, and all embryos were collected from natural mating as a unit of hours-post fertilization (HPF). Then, 0.003% phenylthiourea (PTU) was added 14 h before palytoxin treatment to remove the pigmentation of zebrafish models. Embryos were then treated with the indicated concentration of palytoxin in 24 HPF. Photographs were taken under light microscopy (Carl Zeiss Stereo microscope DV4, Seoul, Republic of Korea) to quantify viable zebrafish.

Differential Toxicity Effects on Healthy Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMC) were purified from three freshly collected buffy coats from healthy adult human donors (Red Cross, Luxembourg, Luxembourg) after ethical approval (see informed consent statement) and informed written consent from all the donors, using the standard Ficoll-Hypaque (GE Healthcare, Roosendaal, The Netherlands) density separation method as previously described [40]. After isolation and three washes in Dulbecco's Phosphate Buffered Saline (DPBS) (Lonza, Rockland, ME, USA), cells were counted and re-suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 1% antibiotics-antimycotic at a cell density of 2 × 10 6 cells/mL. The day after, cell concentration was adjusted to 1 × 10 6 cells/mL, and PBMCs were treated with palytoxin at indicated concentrations. After 8 hours of incubation, a Trypan Blue assay was used to analyze PBMC cell viability.

Statistical Analysis
Data are presented as the mean of three independent experiments with standard deviations. All statistical analyses were done with GraphPad Prism 9.0. p-values below 0.05 were considered significant. See figure legends for details.

Conclusions
We have demonstrated that the marine compound palytoxin rapidly induces cell death through apoptosis in human leukemia cell lines. While downregulating the expression of Mcl-1, palytoxin activates BID truncation, eventually leading to intrinsic apoptotic pathways are a caspase-dependent cell death mechanism. While additional analysis is required to elucidate the role of phosphatases that could be involved, these findings show for the first time that palytoxin acts as an apoptosis inducer affecting leukemia cells able to prevent tumor formation in vivo and makes palytoxin a promising candidate for new potential anticancer drug development.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/md21040233/s1, Figure S1: Calculated IC50 values of different human cancer cell lines incubated with palytoxin at various concentrations between 2 and 8 h. Data are the mean of SD ± of three independent experiments; Figure S2: Palytoxin down-regulates the expression of anti-apoptotic Bcl-2 family proteins in solid tumor cell lines. Western blot analysis of anti-apoptotic Bcl-2 family protein expression levels in A549 (lung cancer), PC3 (prostate cancer), MCF7 (breast cancer), and SH-SY5Y (neuroblastoma) cells; Figure S3: Palytoxin reduces the colony-forming ability in PC3 prostate cancer cells. Palytoxin showed an inhibitory effect on colony formation in a dose-dependent manner. All data represent the mean ± SD. of three independent experiments. Statistical significance was assessed as ** p < 0.01 versus control; Figure S4: Palytoxin inhibited PC3 prostate cancer spheroid formation in a dose-dependent manner. Statistical significance was assessed as * p < 0.05 and *** p < 0.0001 versus control; Figure S5: Palytoxin inhibits tumor-forming ability in in vivo zebrafish xenografts. Fluorescently-labeled leukemia cells were treated with palytoxin at 5, 10, and 30 Pm for 6 h and then injected into the zebrafish yolk sac. A total of nine fish were obtained per condition. Top: bright field, middle: CM-Dil, bottom: merge. PBS injection was used as a control for injection toxicity.
Researchers Program through Seoul National University (SNU) (Funding number: 370C-20160062). Research at SNU is also supported by the National Research Foundation (NRF) (Grant Number 2022R1A2C101314111). Support from the BrainKorea21 FOUR program is acknowledged. Barbora Orlikova-Boyer, Claudia Cerella, Florian Muller, and Marc Diederich are supported by the "Recherche Cancer et Sang" foundation, the "Recherches Scientifiques Luxembourg" association, the "Een Häerz fir kriibskrank Kanner" association, the Action LIONS "Vaincre le Cancer" association, and by Télévie Luxembourg. Claudia Cerella also thanks the 'Waxweiler grant for cancer prevention research' (Action LIONS 'Vaincre le Cancer').

Institutional Review Board Statement:
The study was conducted in agreement with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-191218-5-1; date of approval 29 June 2022).
Informed Consent Statement: PBMCs were obtained from Red Cross Luxembourg with informed consent obtained from all subjects (donators of PBMC blood products) involved in the study (LBMCC-2019-0001: Assessment of differential toxicity of new drugs or drug combinations in preclinical development in ex-vivo proliferating peripheral blood mononuclear cells vs. proliferating cancer cells; LBMCC-2019-0002: Assessment of toxicity of new drugs or drug combinations in preclinical development in non-proliferating peripheral blood mononuclear cells (acute systemic toxicity)).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.