Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum . The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phos-phatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS)


| INTRODUCTION
Topoisomerase II (topo II) and heat shock protein 90 (Hsp 90) complex play important roles in cell mitosis and have been used as potential targets for cancer chemotherapeutic agents, 1,2 including antileukemic drugs. 3 Several topoisomerase II inhibitors including etoposide, teniposide, daunorubicin, doxorubicin, idarubicin, epirubicin, and mitoxantrone, 4 have been commonly used in treating different leukemias such as acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). 5 Currently, multi-agent chemotherapy is the standard therapy for newly diagnosed acute leukemia cases. However, some patients may progress to relapsed or refractory disease, or develop intolerance due to drug toxicity resulting in a poor prognosis. Hsp 90 was also identified as a potential cancer target. 6 Although no drug targeting Hsp 90 has yet been approved by the U.S. Food and Drug Administration, 7 several clinical trials of Hsp 90 inhibitors including ganetespib (STA-9090), are currently ongoing. 8 There is an urgent medical need to develop new agents to treat acute leukemias. The importance of these targets in fighting leukemias encouraged scientists to search for potential inhibitors of topo II and Hsp 90 from natural sources, especially marine organisms. 9,10 Cembranolides belong to cembrane-type diterpenoids and were organically isolated from marine organisms. They are characterized by the presence of a 14-membered carbocyclic ring skeleton with a 5-, 6-, 7-, or 8-membered lactone ring. The cembrane derivatives from the soft coral Lobophytum species demonstrated several pharmacological effects including antiviral, immunostimulant, anti-inflammatory, antitumor, and antibacterial activities. 11 Several compounds were isolated from Lobophytum crassum ( Figure 1A), such as lobocrassins A-E, 14-deoxycrassin, tetrahydrofuran cembranoids, crassumols A-C, and 13-acetoxysarcophytoxide (13-AC) ( Figure 1B). 2 A modest anti-leukemic effect was observed by lobocrassin B and culobophylins A and B. 13-AC and other cembranetype diterpenoids including sarcocrassocolide M and 14-deoxycrassin, were also active against lymphoma cells. 12 The two cembranoids, 13-AC, and 14-deoxycrassin, possessing α-methylene-γ-lactone or α-methylene-δ-lactone functionalities, were the major components of the aquacultural soft coal as demonstrated by HPLC quantitative analysis. Among the isolated cembrane derivatives, 13-AC, isolated from Lobophytum crassum, attracted attention because it exhibited potent cytotoxic activity against several human cancer cell lines including breast, colon, oral, prostate, leukemia, bladder, and gastric cancers. 13 The feasibility of the large-scale aquaculture and a high extraction yield of 13-AC suggested the future potential applications of this technique to produce biologically active compounds on an industrial scale to meet the growing market demands for drug leads. Despite the potent cytotoxic activity of 13-AC against several cancer cell lines, the precise cytotoxic mechanism of action was not explored against leukemia cells. This study aimed to evaluate the cytotoxic potential of 13-AC along with its mechanism of action using in vitro cellular and in vivo xenograft models.   Next, we examined the effect of 13-AC on growth ratio of Sup-T1, U937, K562, and Molt4 cells. These cells were treated with increasing concentrations (1.25, 2.5, and 5 μg/mL) of 13-AC for 24 h and the proliferation was evaluated using MTT cellular proliferation assay. 13-AC at 5 μg/mL significantly suppressed the cellular growth in Molt4 and Sup-T1 cells to 7.75% and 17.63%, respectively, but mildly attenuated the cellular growth to 46.15% in K562 cells and 33.38% in U937 cells (Figure 2A). To confirm the results of the MTT assay, annexin-V/PI staining with flow cytometric analysis was conducted to examine whether the growth inhibition and the cytotoxic activity of 13-AC were elicited by apoptosis of these cancer cells. As shown in Figure 2B, the treatment with 13-AC at 2.5 and 5 μg/mL significantly increased the apoptotic cell population by 11.66% and 43.64% in Sup-T1 cells, and by 39.22% and 61.60% in Molt4 cells, respectively, after 24 h of treatment. The Molt4 leukemia cell was the most susceptible to 13-AC and was therefore selected to investigate the apoptotic mechanism of action of this cembranoid. To evaluate the expression of apoptosis-related proteins, Molt4 cells were treated with 13-AC at 5 μg/mL and the expression of XIAP, cleavage of caspases-3, 7, 8, and -9, as well as PARP, were measured after 0, 6, 12, and 24 h by Western blotting assay. As shown in Figure 2C  Therefore, we sought to determine whether 13-AC exerted similar effects on leukemia Molt4 cells or not. As shown in Figure 4A, the treatment with 13-AC (5 μg/mL) for 24 h resulted in a timedependent increase of the population of mitochondrial membrane potential in Molt4 cells from 10.1% to 12.3%, 47.0%, 48.5%, 81.7%, and 96.9% after 3, 6, 9, 12, and 24 h, respectively, as evaluated by JC-1 staining. In Figure 4B, the treatment with 13-AC (5 μg/mL) for 3, 6, 9, 12, and 24 h resulted in a 1.20-, 1.21-, 1.37-, 1.41-, and 1.13-folds increase in the ROS levels compared with the control, as determined by carboxy-H2DCFDA staining by flow cytometric analysis. ER stress can be induced by oxidative stress leading to mitochondrial-dependent apoptosis. 14 As shown in Figure 4C, an increase in cellular calcium accumulation by 1.38-, 1.32-, 1.33-, 1.97-, and 1.08-folds was observed after the treatment with 13-AC (5 μg/mL) for 3, 6, 9, 12, and 24 h, as determined by a fluorescent calcium T A B L E 1 Antiproliferative activity induced by 13-AC against different cell lines from human hematological malignancy, including Sup-T1 (lymphoblastic lymphoma), U937 (lymphoma), K562 (chronic myeloid leukemia), Molt4 (acute lymphoblastic leukemia), and HEK293 (non-hematological malignancy). indicator Fluo 3. These results indicated that 13-AC treatment disrupted mitochondrial membrane potential, disturbed the homeostasis of calcium, and induced ROS generation leading to apoptosis in Molt4 cells. To further investigate the effect of 13-AC on the interaction of Hsp 90, a fluorescence-based protein thermal shift assay was performed. The thermal shift assay is a method with quantitative realtime PCR to determine the ligand (inhibitor) on the shift of the melting temperature (fluorescence intensity) of the specific protein by incrementally heating samples over-temperature gradient (from 25 to 95 C). 16 As shown in Figure  along with the inhibition of Topo I and II. 10 Clinically, Topo II was proved to be a highly promising target in cancer therapy. 21 In the current study, we performed a molecular docking analysis to determine the molecular interactions between 13-AC and Topo II. The docking results showed that 13-AC interacted with the Topo II binding site. The head structure of 13-AC, which contains a tulipalin A and the acetic acid moiety, extended into the Topo II binding site. The tulipalin A head structure created a hydrogen bond with the Topo II residue Ser149, and the acetic acid moiety formed hydrogen bonds with the residues Ser148 and Asn150 ( Figure 6A).

| 13-AC inhibited the functional activity of Hsp 90 and topoisomerase II α with a cell-free system
Both the tulipalin A and the acetic acid moieties of 13-AC acted as hydrogen acceptors in Topo II ( Figure 6B). The acetic acid also coordinated with the Mg 2+ ion within Topo II ( Figure 6B). The coordination to the Mg 2+ ion was also observed with the ATP cocrystal ligand. 22 It was suggested that the Mg 2+ ion was involved in DNA binding. 23 The interactions with the Mg 2+ ion are important for molecular recognition. The acetic acid moiety formed interactions with a series of residues such as Ile125, Ile141, and Phe142 ( Figure 6B). This carbon ring was also located near the periphery of the Topo II binding site. To confirm the effect of 13-AC on Topo II activity, a cell-free DNA cleavage system using a specific Topo II-mediated negatively supercoiled pHOT1 plasmid DNA was applied. 10 As shown in Figure 6C,

| DISCUSSION
Multiagent chemotherapy remains the standard first-line therapy for newly diagnosed ALL. The overall survival rate is largely influenced by age, with a 5-year survival rate in 80% of patients under the age of 50 but only 35% in patients above 50 years old. 24 The poor prognosis in older patients is likely due to the adverse risks of disease biology, and higher treatment-related complications due to underlying comorbidities. Therefore, there is a medical need to develop novel agents with different mechanisms of action to tailor treatment, to fit the disease and patient profile.
Corals are a rich reservoir of biologically active secondary metabolites such as cembranolides that can act as drug leads for the development of cancer therapeutics. 12   Akt. 29 Moreover, caspase activation occurs with the cleavage of ALK and PLCλ1, receptor tyrosine kinases during apoptosis. 30 Given the characteristic cancer chaperone of Hsp 90, it can act as a hub for many neoplastic signaling pathways involved in the regulation of cell survival and the growth inhibitory activities in cancer cells. 31 The activity of Hsp 90 inhibitors might be dependent on the balance of pro-apoptotic and pro-survival regulatory proteins. 32 The N-terminal inhibitors of Hsp90 failed in the clinic as a single therapy treatment partially because they induce a heat shock response. 33 Moreover, Hsp 90 inhibitors, AT-533, inhibited tumor growth and angiogenesis via the blockage of HIF-1 binding and degradation of HIF-1 in breast cancer. 34 The overexpression of Hsps is heavily dependent in many cancer cells on the upregulation of transcript factor HSF-1 which is the multifaceted crucial regulator of the heat shock response. 35 It is worth to note that Hsp 90 inhibitors competed at ATP-binding site with inhibition of the ATPase activity and ATP-ADP exchange to induce the degradation of clients via the proteasome mechanism and recruited the ligation of ubiquitin and Hsp90.
Mitochondria serve as an important bioenergy hub to regulate the ATP production, ROS generation and apoptotic induction. 36 In this study, the generation of ROS was increased in Molt4 cells after 13-AC treatment.
We found that the pretreatment of NAC, a ROS scavenger, attenuated MMP disruption and apoptosis induction ( Figure 7A,B), as well as

| Determination of ROS generation, calcium accumulation, and MMP disruption
In these assays, we followed previously described protocols. 38

| Molecular docking assay
The docking analysis was performed by LeadIT, a molecular docking software. 39 The Protein Data Bank was used to download the Topo II (PDB ID: 1ZXM) and Hsp 90 (PDB ID: 3VHC) crystal structures. 40 Protein structures were prepared by removing water molecules and the co-crystal ligand using the molecular docking software LeadIT. The binding sites were determined as a radius of 10 Å from the co-crystallized ligands. The docking strategy used an enthalpy and entropy approach.
The maximum number of solutions per iteration and fragmentation was set at 300. All other parameters were used at the default settings.

| Statistics
The results were expressed as mean ± standard deviation (SD). Each experiment was performed using an unpaired Student's t-test. A pvalue of less than 0.05 was considered to be statistically significant.

| CONCLUSIONS
We found that the treatment of 13-acetoxysarcocrassolide, a marine