Replacement of a Quinone by a 5-O-Acetylhydroquinone Abolishes

Jul 22, 2013 - Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, ... of Pharmaceutical Sciences, Meiji Pharmaceutical University, Toky...
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Replacement of a Quinone by a 5‑O‑Acetylhydroquinone Abolishes the Accidental Necrosis Inducing Effect while Preserving the Apoptosis-Inducing Effect of Renieramycin M on Lung Cancer Cells Thaniwan Cheun-Arom,† Pithi Chanvorachote,*,‡ Natchanun Sirimangkalakitti,† Taksina Chuanasa,† Naoki Saito,§ Ikuro Abe,⊥ and Khanit Suwanborirux*,† †

Center of Bioactive Natural Products from Marine Organisms and Endophytic Fungi, Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University, Bangkok 10330, Thailand ‡ Cell-Based Drug and Health Product Development Research Unit and Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand § Department of Pharmaceutical Chemistry, Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University, Tokyo 204-8588, Japan ⊥ Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan S Supporting Information *

ABSTRACT: Renieramycin M (1), a bistetrahydroisoquinolinequinone alkaloid isolated from the marine sponge Xestospongia sp., has been reported to possess promising anticancer effects. However, its accidental necrosis inducing effect has limited further development due to concerns of unwanted toxicity. The presence of two quinone moieties in its structure was demonstrated to induce accidental necrosis and increase reactive oxygen species (ROS) levels. Therefore, one quinone of 1 was modified to produce the 5-O-acetylated hydroquinone derivative (2), and 2 dramatically reduced the accidental necrosis inducing effect while preserving the apoptosisinducing effect of parent 1 on lung cancer H23 cells. Addition of the antioxidant N-acetylcysteine suppressed the accidental necrosis mediated by 1, suggesting that its accidental necrosis inducing effect was ROS-dependent. The fluorescent probe dihydroethidium revealed that the accidental necrosis mediated by 1 was due to its ability to generate intracellular superoxide anions. Interestingly, the remaining quinone in 2 was required for its cytotoxicity, as the 5,8,15,18-O-tetraacetylated bishydroquinone derivative (3) exhibited weak cytotoxicity compared to 1 and 2. The present study demonstrates a simple way to eliminate the undesired accidental necrosis inducing effect of substances that may be developed as improved anticancer drug candidates.

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necrosis.9 Regulated necrosis is mainly mediated by the ligation of death receptors and the activation of the serine-threonine kinase receptor-interacting protein 1 (RIP1), whereas accidental necrosis is characterized by nonspecific cell injury triggered by exposure to severe physical conditions, such as radioisotopes, UV, or high concentrations of pro-oxidants.9−13 The major morphological changes during accidental necrosis include cell swelling, formation of cytoplasmic vacuoles, distended endoplasmic reticulum, disrupted organelle membranes, swollen and ruptured lysosomes, and eventually disruption of cell membranes.14,15 Not only is accidental necrosis nonspecific, it also damages surrounding cells and tissues by releasing cytoplasmic components and induces hyperactive immune response and inflammation.16−18 Thus, even though the

urrently, novel drugs and strategies possessing high efficacy are of the greatest interest in cancer research. In general, anticancer drugs are developed with an eye to killing cancer cells and preserving normal cells.1 Thus, most anticancer drugs are designed to eliminate cancer cells through apoptosis rather than accidental necrosis, as the former is more easily controlled.2 Apoptosis has long been documented as a distinctive model of programmed cell death.3 This type of cell death is the major mechanism for the human body to eliminate unwanted and damaged cells.3,4 The process of apoptosis involves the activation of several signals and proteins in a well-controlled fashion triggered by defined stimuli. Once stimuli reach the cells, caspases will be activated to cause cell death.5−7 At the final step of apoptosis, all cell compartments are wrapped up in vesicles called apoptotic bodies, which will be consumed by immune cells.8 In contrast, necrosis is classified into two types according to morphological and biochemical characteristics: regulated necrosis and accidental © 2013 American Chemical Society and American Society of Pharmacognosy

Received: April 2, 2013 Published: July 22, 2013 1468

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from that of 1 by the presence of one quinone and one hydroquinone. The absence of the unique homoallylic coupling (ca. 2 Hz) between 1-H and 4-Hβ confirmed that the quinone moiety at ring A of 1 was reduced to a hydroquinone ring in 2.34 In addition, the presence of a phenolic hydroxy group and an acetoxy group was supported by the NMR signals at δH 5.81 and δH 2.29/δC 20.2 and 168.6, respectively. The hydroxy group at C-8 was confirmed by HMBC correlations of the hydroxy proton at δH 5.81 to C-7 (δC 143.8) and C-9 (δC 117.1). This information allowed us to readily assign the acetoxy group at C-5. All proton and carbon assignments of 2 were completed after extensive NMR measurements using COSY, HMQC, and HMBC techniques (Supporting Information). Thus, the structure of 2 was confirmed to be 5-Oacetylhydroquinone renieramycin M. The 5-O-acetyl group in 2 prevents the oxidation of the quinone ring, and the 8-hydroxy group is expected to improve the solubility of 2 compared to 1. Cytotoxic Effects of Renieramycin M (1) and the 5-OAcetylated Hydroquinone Derivative (2) on H23 Cells. The cytotoxicities of 1 and 2 to H23 cells were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. H23 cells were incubated in the presence or absence of 1 and 2 at concentrations of 0−20 μM for 24 h. The results indicated that both 1 and 2 significantly decreased cell viability in a concentration-dependent manner (Figure 1A).

cytotoxicity of a particular drug candidate is of interest, the presence of such nonspecific necrotic responses would limit its use for further anticancer drug development. As previously mentioned, cell necrosis is nonspecifically mediated by severe physical conditions. The mediator of cell necrosis that has garnered the most attention in pharmacology is reactive oxygen species (ROS). ROS are implicated in the pharmacological action of many anticancer drugs, such as cisplatin19−21 and doxorubicin.22−25 ROS are important cellular mediators generated continuously via the electron transport chain and include superoxide anion (O2•¯), hydroxyl radical (HO•), and hydrogen peroxide (H2O2).26−30 These ROS were demonstrated to mediate accidental necrosis through the loss of plasma membrane integrity, the alteration of mitochondrial membrane potential, or lysosomal membrane permeabilization.30−32 As part of our continuing investigation of cytotoxic natural products from Thai marine organisms, we have isolated a series of stabilized renieramycin alkaloids from the KCN-pretreated blue sponge Xestospongia sp., distributed around Sichang Island, the Gulf of Thailand.33−35 Renieramycins are a group of bistetrahydroisoquinoline quinone marine alkaloids possessing potent cytotoxicity to several human cancer cell lines.33−36 Renieramycin M (1), the major alkaloid of Xestospongia sp., has been shown to prevent metastasis of and induce apoptosis in lung cancer cells through activation of the p53-dependent pathway.37 It has been hypothesized that the quinone moieties of 1 induce accidental necrosis, thereby hindering the further development of 1 as an effective anticancer drug. To confirm this hypothesis, we tested the 5-O-acetylated hydroquinone derivative 2 in the human non-small-cell lung cancer cell line H23 (hereafter, H23 cells) and demonstrated that 2 significantly reduced the accidental necrosis inducing effect while preserving the apoptosis-inducing effect of parent 1. We also found that the accidental necrosis inducing effect of 1 was ROS-dependent and due to its ability to generate intracellular superoxide anions.

Figure 1. Cytotoxicity and cell death modes of renieramycin M (1) and the 5-O-acetylated hydroquinone derivative (2). H23 cells were treated with various concentrations of each compound for 24 h. (A) Cell viability was analyzed by the MTT assay. (B) Morphologies of apoptotic and accidental necrotic cells were observed by costaining with Hoechst 33342 and PI. (C) Percentages of apoptotic cells and accidental necrotic cells. *p < 0.05 versus nontreated control in each group. #p < 0.05 versus renieramycin M treated groups.

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RESULTS AND DISCUSSION Synthesis of the 5-O-Acetylated Hydroquinone Derivative (2). Hydrogenation of renieramycin M (1) with 10% Pd/C in EtOAc for 4 h gave the bishydroquinone derivative of 1 in a quantitative yield. Stoichiometric acetylation of the bishydroquinone compound with acetic anhydride (1.5 equiv) in dry pyridine gave 2 in 40% yield. Compound 2 was obtained as a pale yellow solid. Its molecular formula, C33H37N3O9, was established by HREIMS. Most of the signals in the 1D and 2D NMR spectra of 2 (Supporting Information) were similar to those of 1 except for the signals assigned to the two quinone carbonyl carbons at δC 186.0 (C-15) and 182.8 (C-18) and the two oxygenated aromatic carbons at δC 143.0 (C-8) and 139.1 (C-5) in 2 instead of the four quinone carbonyl carbons in 1. These data suggested that the chemical structure of 2 differs

In another experiment, the cells were treated with various concentrations (0−20 μM) of 1 and 2 for 24 h, and the modes of cell death, namely, apoptosis and accidental necrosis, were determined by costaining with Hoechst 33342 and propidium iodide (PI). Fluorescence microscopy was used to visualize nuclei and other DNA-containing organelles in apoptotic or necrotic cells. Concentration-dependent increases in the percentage of apoptotic cells (51−68%) and the percentage of accidental necrotic cells (2.5−20%) were noted in the renieramycin M treated cells; however, a concentrationdependent increase in only the percentage of apoptotic cells (46−74%) was observed in the 5-O-acetylated hydroquinone 1469

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Figure 2. Sub-G0 fraction analysis by flow cytometry using PI buffer and cell morphology characterization with trypan blue dye. H23 cells were treated with various concentrations of renieramycin M (1) and the 5-O-acetylated hydroquinone derivative (2) for 24 h. (A) Histogram of DNA content in each population. (B) Relative DNA contents in sub-G0 fractions. (C) Morphologies of treated H23 cells were visualized by an automated cell counter. Viable and apoptotic cells are marked with green circles, and accidental necrotic cells, with red circles. (D) Relative percentage of accidental necrotic cells was measured by an automated cell counter. *p < 0.05 versus nontreated control in each group.

Figure 3. Effects of ROS scavengers on ROS-induced cell deaths by renieramycin M (1) and the 5-O-acetylated hydroquinone derivative (2) in H23 cells. H23 cells were pretreated or not pretreated with 1 mM NAC and then incubated with 20 μM of each compound for 24 h. (A) Morphologies of apoptotic cells and accidental necrotic cells were determined by costaining with Hoechst and PI. (B) Percentage of viable cells. (C) Percentages of apoptotic and accidental necrotic cells. *p < 0.05 versus nontreated control in each group. #p < 0.05 versus renieramycin M treated groups.

similar potency. Structurally related DNA-alkylating alkaloids, saframycins and ecteinascidins, were reported to modify the exocyclic 2-amino group of guanine located in the DNA minor groove via the iminium intermediate generated from αaminonitrile and α-carbinolamine groups, respectively.38,39 Because the equivalent α-aminonitrile functional group is present in the structures of 1 and 2, it is possible that both compounds interact with cellular DNA and mediate apoptosis in a similar manner. In our previous work, we demonstrated that 1 induced lung cancer cell apoptosis through p53 induction, which in turn down-regulated antiapoptotic BCL-2 and MCL-1 proteins.37 As the loss of membrane integrity has long been known to be a marker of accidental necrosis, the trypan blue exclusion assay was performed to quantify accidental necrotic cells with an automated cell counter. Trypan blue did not stain viable cells and apoptotic cells; rather, it traversed only the damaged membranes of cells considered to be accidental necrotic cells. The automated cell counter marked viable cells with green circles and accidental necrotic cells with red circles (Figure 2C). Clearly, the results indicated an accidental necrosis response consistent with that reported above, and a concentrationdependent increase in the percentage of accidental necrotic cells (ranging from 10% to 20% at 5−20 μM 1) was detected in the renieramycin M treated group only. Interestingly, a small

derivative treated cells (Figure 1B and C). Hence, the quinone may play an important role in the accidental necrosis. The apoptotic cells and the accidental necrotic cells exhibiting condensed and/or fragmented nuclei with intense nuclear fluorescence on costaining with Hoechst 33342 and PI are shown in Figure 1B. Surprisingly, the percentage of apoptotic cells in the renieramycin M treated cells and that in the 5-Oacetylated hydroquinone derivative treated cells were comparable at the same concentrations (Figure 1C). Together, these findings suggest that the structural modification of 1 attenuated the accidental necrosis inducing effect of 1. Sub-G0 Fraction and Membrane Integrity Analysis. To confirm apoptosis and accidental necrosis due to the two compounds, specific detection methods for apoptosis and accidental necrosis were utilized. As an increase in the sub-G0 fraction of cells indicates apoptosis, cellular DNA content was analyzed to determine apoptosis responses for both 1 and 2 treatments. Cells treated with 1 and 2 (0−20 μM) for 24 h were stained with PI buffer, and the sub-G0 fraction was quantified by flow cytometry. The histograms confirmed a comparable increase in apoptosis of both renieramycin M and 5-O-acetylated hydroquinone derivative treated cells in a concentration-dependent manner (Figure 2A and B). The results are consistent with the above Hoechst staining results, confirming that 1 and 2 induced the apoptosis of H23 cells with 1470

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percentage of accidental necrotic cells (1−3%) were observed in the same concentration range of 2 (Figure 2D). Renieramycin M (1) Generates ROS To Induce Accidental Necrosis in H23 Cells. Accumulated knowledge of the chemical structures and the related pharmacological activities led us to hypothesize that modification of a quinone in 1 may abolish its accidental necrosis inducing effect. Several studies suggested that the quinone group is responsible for the ROS-inducing action.40−43 Accordingly, many quinone-containing antitumor agents, such as adriamycin, daunorubicin, actinomycin D, mitomycin C, and trenimon, were shown to mediate DNA strand breaks and cell death via an ROSdependent mechanism.40,43−46 Free radicals and highly reactive molecules, including superoxide radicals, hydroxyl radicals, semiquinones, and hydrogen peroxide, were found in cells treated with quinone-containing compounds, and those molecules were proved to induce necrosis.30 Indeed, menadione, a redox-active naphthoquinone, was shown to induce necrosis by damaging plasma membrane integrity via a free radical mediated process.47,48 In addition, furylquinones underwent an activation process via a redox mechanism causing necrosis in TLT hepatoma cells.47,49 As mentioned earlier, ROS are hypothesized as one of the key factors for necrosis in response to several stimuli,30 and the quinone moiety is recognized as a radical generator.44,50−52 To test whether ROS played a role in the renieramycin M and 5-O-acetylated hydroquinone derivative mediated cell deaths, the strong antioxidant N-acetylcysteine (NAC 1 mM) was used. The cells were pretreated with NAC prior to 1 and 2 treatments, and cell viability at 24 h was determined. The results indicated that NAC significantly suppressed cytotoxicity induced by 1 but not by 2 (Figure 3B). Then, the modes of death were clarified by nuclear morphology analysis using Hoechst 33342 and PI (Figure 3A and C). The results indicated that ROS played a role in regulating cell response to the accidental necrosis mediated by 1, as the addition of NAC significantly decreased the percentage of accidental necrotic cells in the renieramycin M treated group, whereas it did not alter the percentages of apoptotic cells in both 1- and 2-treated groups (Figure 3C). These data demonstrate that replacement of a quinone group in 1 by a monoacetylated hydroquinone group decreased its ROSdependent accidental necrosis inducing effect. Renieramycin M (1) Generates Superoxide Anions That Are Responsible for Its Accidental Necrosis Inducing Effect. ROS consist of highly reactive free radicals, such as superoxide anion (O2•¯) and hydroxyl radical (HO•), and nonradicals, particularly hydrogen peroxide (H2O2) and singlet oxygen (O2).26−29 Having shown that ROS played a role in the accidental necrosis inducing effect of 1, we conducted further experiments to identify the specific ROS responsible for the necrosis induction. Cell-specific ROS levels after treatments with 1 and 2 were measured at 0 to 6 h intervals by means of ROS-specific fluorescent probes, which are excellent ROS sensors due to their high sensitivities.53 2′,7′-Dichlorofluorescein diacetate (DCFH2-DA) was used as an oxidative fluorescent probe to show that the increased ROS level induced by 1 was two times higher than that by 2. The ROS levels were dramatically decreased by the addition of NAC, confirming the presence of ROS induced by both compounds (Figure 4A). To identify the presence of H2O2 in those systems, the cells were first treated with sodium pyruvate (SP), a specific H2O2 scavenger, and then treated with 1 and 2. The results showed that SP caused only a slight alteration in the

Figure 4. Characterization of specific intracellular ROS induced by renieramycin M (1) and the 5-O-acetylated hydroquinone derivative (2) treatment of H23 cells. H23 cells were incubated with 20 μM of each compound for different periods. (A) H23 cells were pretreated or not pretreated with NAC. Then, general intracellular ROS levels were measured with DCFH2-DA. (B) H23 cells were pretreated or not pretreated with SP. Then, H2O2 levels were measured with DCFH2DA. (C) Hydroxyl radical levels were measured with HPF. (D) Superoxide anion levels were measured with DHE. Values are means ± standard deviation of triplicate samples. *p < 0.05 versus nontreated control. #p < 0.05 versus treatment in each group.

ROS signals in both treatments, suggesting that H2O2 was not the principal ROS present under such conditions (Figure 4B). Then, the 3′-(p-hydroxyphenyl)fluorescein (HPF) probe was used for hydroxyl radical detection. However, the experiment revealed no significant changes in the intracellular hydroxyl radical levels in all the treated groups, indicating that the hydroxyl radical was not generated in response to both treatments (Figure 4C). Finally, the dihydroethidium (DHE) probe specific for superoxide anion was used. DHE clearly indicated a significant up-regulation of the superoxide anion level in response to the treatment with 1, whereas the superoxide anion level in the 5-O-acetylated hydroquinone derivative treated group was comparable to that of the nontreated control cells. This finding confirmed that replacement of a quinone moiety in 1 diminished its ROSinducing property and its undesired accidental necrosis inducing effect by reducing superoxide anion generation (Figure 4D). Replacement of Both Quinone Moieties Results in Decreased Cytotoxicity. Having shown that the replacement of the quinone in ring A of 1 suppressed the ROS-mediated accidental necrosis, we further investigated whether elimination of the second quinone moiety in ring E of 2 could further reduce ROS levels and suppress necrosis induction. The remaining quinone of 2 was reduced, and the product was acetylated to yield the 5,8,15,18-O-tetraacetylated bishydroquinone derivative (3), as previously reported.34 H23 cells were treated with 1, 2, and 3, intracellular ROS levels were measured after 6 h, and apoptosis, necrosis, and cell viability were determined at 24 h. As expected, the ROS level in the cells treated with 3 was significantly lower than those in the cells treated with 1 and 2 (Figure 5A). The intracellular ROS level in the 5,8,15,18-O-tetraacetylated bishydroquinone derivative treated cells was comparable to that of nontreated control cells. Accidental necrosis was not observed in the cells treated with 3 (Figure 5B), suggesting that the ROS-mediated accidental necrosis was completely suppressed by replacing both quinone moieties. However, the assay for cell viability and the Hoechst 33342 staining experiment indicated that 3 exhibited limited cytotoxicity and apoptosis-inducing activity 1471

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penicillin−streptomycin in a 5% CO2 environment at 37 °C. Nacetylcysteine, sodium pyruvate, propidium iodide, Hoechst 33342, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 2′,7′-dichlorofluorescein diacetate, and dihydroethidium were obtained from Sigma Chemical. 3′-(p-Hydroxyphenyl)fluorescein was obtained from Daiichi Pure Chemicals (Invitrogen). Synthesis of the 5-O-Acetylated Hydroquinone Derivative (2). Compound 2 was synthesized by using renieramycin M (1) (10.2 mg, 0.018 mmol) as the starting material. Hydrogenation of 1 with 10% Pd/C (6 mg) in EtOAc (3 mL) was conducted at 1 atm H2 for 4 h. The catalyst was removed by filtration and washed with EtOAc. The combined filtrates were concentrated in vacuo to obtain a residue, which was used in the next step without further purification. The bishydroquinone derivative of 1 was acetylated with 1.5 equiv of acetic anhydride (2.65 μL) in pyridine (0.5 mL), and the mixture was stirred for 3 h at room temperature under an argon atmosphere. The reaction was quenched by the addition of H2O (5 mL), and the resulting mixture was extracted with CH2Cl2 (3 mL × 3). The combined CH2Cl2 extracts were evaporated to dryness in vacuo and subjected to chromatography on a silica gel column using hexane−EtOAc (3:2) as the eluent. Compound 2 (4.1 mg, 40%) was afforded as a pale yellow solid: [α]20D +35 (c 0.001, MeOH); UV (MeOH) λmax (log ε) 272 (4.40) nm; IR (KBr) νmax 3459, 1714, 1651, 1616, cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.98 (1H, br q, J = 7.2 Hz, H-26), 5.81 (1H, s, OH-8), 4.48 (1H, dd, J = 11.1, 3.0 Hz, H-22a), 4.29 (1H, dd, J = 4.7, 3.0 Hz, H-1), 4.09 (1H, d, J = 2.0 Hz, H-21), 4.03 (1H, dd, J = 11.1, 4.7 Hz, H-22b), 3.95 (1H, overlapped, H-11), 3.94 (3H, s, OCH3-17), 3.74 (3H, s, OCH3-7), 3.34 (1H, br dd, J = 7.3, 2.0 Hz, H-13), 3.17 (1H, br d, J = 12.2 Hz, H-3), 2.72 (1H, dd, J = 20.9, 7.3 Hz, H-14α), 2.53 (1H, br d, J = 13.7 Hz, H-4α), 2.32 (1H, overlapped, H-14β), 2.29 (3H, s, OCOCH3), 2.24 (3H, s, NCH3), 2.06 (3H, s, CH3-6), 1.89 (3H, s, CH3-16), 1.84 (3H, br d, J = 7.2 Hz, H3-27), 1.66 (3H, br s, H3-28), 1.57 (1H, overlapped, H-4β); 13C NMR (CDCl3, 75 MHz) δ 186.0 (C, C-15), 182.8 (C, C-18), 168.6 (C, OCOCH3), 167.1 (C, C-24), 155.4 (C, C-17), 143.8 (C, C-7), 143.0 (C, C-8), 141.8 (C, C20), 139.9 (CH, C-26), 139.1 (C, C-5), 135.6 (C, C-19), 128.9 (C, C16), 126.8 (C, C-25), 124.3 (C, C-10), 122.3 (C, C-6), 117.5 (C, CN21), 117.1 (C, C-9), 64.4 (CH2,C-22), 61.1 (CH3, OCH3-17), 60.6 (CH3, OCH3-7), 59.4 (CH, C-21), 56.5 (CH, C-1), 55.2 (CH, C-3), 54.8 (CH, C-13), 54.6 (CH, C-11), 41.4 (CH3, NCH3), 27.8 (CH2, C4), 21.1 (CH2, C-14), 20.5 (CH3, C-28), 20.2 (CH3, OCOCH3), 15.8 (CH3, C-27), 9.9 (CH3, CH3-16), 8.6 (CH3, CH3-6); HREIMS m/z 619.2529 [M]+ (calcd for C33H37N3O9, 619.2530); HRQTOFMS m/z 620.2609 [M + H]+ (calcd for C33H38N3O9, 620.2608). Assay for Cytotoxicity. Cell viability was examined by the MTT assay. H23 cells (1.5 × 105 cells/mL) were cultured in RPMI 1640 medium in a 96-well plate for 24 h at 37 °C and then further treated with 1 or 2 at various concentrations for 24 h at 37 °C. The treated cells were finally incubated with 500 μg/mL MTT for 4 h at 37 °C. The supernatant was carefully removed, and dimethyl sulfoxide was added to dissolve the formazan product, giving a purple color. The color intensity was spectrophotometrically measured at 570 nm using a microplate reader (Victor X3 multilabel plate reader, Perkin-Elmer). All analyses were repeated for at least three different replicate cultures. Cell viability was calculated from optical density (OD) readings and expressed as percentage of the nontreated control value. Assays for Apoptosis and Accidental Necrosis. Apoptosis and accidental necrosis were determined by costaining with Hoechst 33342 and PI. H23 cells were first treated with 1 and 2 at the indicated concentrations for 24 h at 37 °C and then stained with 10 μM Hoechst 33342 and 10 μM PI for 30 min at 37 °C. Hoechst 33342 stained the nuclei of all the cells. The apoptotic cells had condensed chromatin and/or fragmented nuclei. PI stained only the DNA of membranedamaged cells that were considered as accidental necrotic cells. Fluorescence in the cells was visualized and scored under a fluorescence microscope (Olympus IX51). Sub-G0 Fraction Determination by Flow Cytometry. Relative cellular DNA content and cell distribution were investigated during various phases of the cell cycle. Apoptosis induced by either 1 or 2 was analyzed by flow cytometry (FACSort, BD Biosciences) using PI

Figure 5. Comparative study of cytotoxicity, cell death modes, and ROS production of renieramycin M (1), the 5-O-acetylated hydroquinone derivative (2), and the 5,8,15,18-O-tetraacetylated bishydroquinone derivative (3). (A) ROS levels in H23 cells were measured with DCFH2-DA. H23 cells were treated with 20 μM of each compound for 6 h. (B) Percentages of apoptotic cells and accidental necrotic cells were determined by costaining with Hoechst 33342 and PI. H23 cells were treated with 20 μM of each compound for 24 h. (C) Viability of H23 cells was analyzed by the MTT assay. Cells were treated with 20 μM of each compound for 24 h. (D) Effects of 1, 2, and 3 on accidental necrosis of normal kidney HK-2 cells. Percentages of accidental necrotic cells were determined by PI staining. HK-2 cells were treated with 20 μM of each compound for 24 h. *p < 0.05 versus nontreated control in each group. #p < 0.05 versus renieramycin M treated groups.

(Figure 5B and C). These findings suggest that the quinone moiety in ring E plays a critical role in the cytotoxicity. As accidental necrosis is recognized as a nonspecific event that may be harmful to normal cells, the accidental necrosis mediated by 1, 2, and 3 was evaluated in normal kidney HK-2 cells. Figure 5D shows that treatment with 1 at 20 μM induced approximately 60% necrosis. Interestingly, the modification of renieramycin M (to 2 and 3) dramatically reduced the accidental necrosis inducing effect of 1. In summary, 5-O-acetylated hydroquinone derivative 2, a modified renieramycin M compound, significantly reduced accidental necrosis in the human non-small-cell lung cancer cell line H23. The present study provides an approach to modify the undesired necrotic effects of substances that are candidates for anticancer drug development and offers important inferences for studies of the relationship between structural analogues and biological activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Renieramycin M (1) was isolated from the blue Thai marine sponge Xestospongia sp. as previously described.33,34 Optical rotations were measured on a Horiba-SEPA polarimeter. UV spectra were recorded on a Hitachi 340 UV/vis spectrometer. IR spectra were measured on a Perkin-Elmer 2000 FT-IR spectrometer. NMR spectra were obtained on a Bruker AVANCE DPX-300 FT-NMR spectrometer operating at 300 MHz for 1 H and 75 MHz for 13C nuclei. The chemical shifts were referenced to CHCl3 (δH 7.26) and CDCl3 (δC 77.0). HREI mass spectra were recorded on a JEOL JMS-700 mass spectrometer with a direct inlet system operating at 70 eV. Accurate mass spectra were obtained with an Agilent 6540 UHD Q-TOF LC/MS spectrometer using electrospray ionization and a Zorbax Eclipse Plus C18 HPLC column (3.0 × 100 mm). Cells and Reagents. The human non-small-cell lung cancer cell line H23 was obtained from the American Type Culture Collection, ATCC. The cells were cultured in RPMI 1640 medium containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL 1472

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buffer and by visualization of the peak of sub-G0 cells. Attached H23 cells (3.5 × 105 cells/mL) were collected and resuspended in 1 mL of 3% FBS in 1× phosphate-buffered saline (PBS). The suspended cells were fixed with 2.5 mL of absolute EtOH by vortexing and then kept for 24 h at −20 °C. For staining, the collected cells were suspended in 500 μL of PI buffer (10× PBS, 1 mL; 1 mg/mL RNaseA, 1 mL; Triton X-100, 10 μL; 10 mg/mL PI, 200 μL; FBS, 1 mL; and H2O adjusted to 10 mL) and incubated for 40 min at 37 °C prior to analysis. The DNA histogram indicating cell death through the sub-G0 phase was calculated from the percentages of cells occupying the different phases of the cell cycle. The cells collected for cell cycle analysis were stained together with trypan blue dye and subsequently morphologically visualized under an automated cell counter (TC10 automated cell counter, BIORAD). ROS Detection. The ROS-generated effects of 1 and 2 were determined by using H23 cells pretreated or not pretreated with the antioxidant NAC. Apoptosis and accidental necrosis were visualized by costaining with Hoechst 33342 and PI. H23 cells were grown on a 96well plate for 24 h at 37 °C and further incubated in the presence or absence of 1 mM NAC for 1 h at 37 °C prior to the treatment with 20 μM 1 or 2. After 24 h, fluorescent cells were visualized and scored under a fluorescence microscope (Olympus IX51). In addition, cell viability was examined by the MTT assay. Measurement of Intracellular ROS. Two ROS scavengers were used: NAC as a general ROS scavenger and SP as a specific H2O2 scavenger. Intracellular ROS were examined by using DCFH2-DA as the general oxidative fluorescent probe. H23 cells were seeded onto a 96-well plate for 24 h at 37 °C following culture medium removal. A solution of DCFH2-DA (10 μM final concentration) in PBS was added to the cells with or without 1 mM ROS scavenger (NAC or SP), and incubation was carried out for 30 min at 4 °C. Likewise, two specific fluorescent probes were utilized: HPF as a specific hydroxyl radical probe and DHE as a specific superoxide anion probe. A solution of each fluorescent probe (10 μM final concentration) in PBS was added to the cells for 30 min at 4 °C. Each cell treatment was supplemented with 20 μM 1 or 2, and then incubation was carried out for different periods (0 to 6 h) at 37 °C. Fluorescence intensity was analyzed with a fluorescence microplate reader (DTX 880 multimode detector, Beckman Coulter, Inc.) using a 480 nm excitation beam and a 530 nm band-pass filter for DCFH2-DA, a 490 nm excitation beam and a 515 nm band-pass filter for HPF, and a 488 nm excitation beam and a 610 nm band-pass filter for DHE. Statistical Analysis. All data were determined as means ± standard deviation of three independent experiments. Statistical differences between the means were analyzed by one-way analysis of variance (ANOVA). p < 0.05 was considered as statistically significant.

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(JSPS) Asia-Africa Science Platform Program (2010−2012). The authors wish to thank the Herbal Quality Assurance Center, Medicinal Plant Research Institute, Department of Medical Sciences, for HREIMS. We also thank Mr. N. Sa-ardeiam, Immunology Laboratory, Faculty of Dentistry, Chulalongkorn University, for flow cytometry analysis and Mr. K. Rajprasit, International College for Sustainability Studies, Srinakharinwirot University, for proofreading the manuscript.

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ASSOCIATED CONTENT

S Supporting Information *

Spectroscopic data for 2 are available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Plescia, J.; Salz, W.; Xia, F.; Pennati, M.; Zaffaroni, N.; Daidone, M. G.; Meli, M.; Dohi, T.; Fortugno, P.; Nefedova, Y.; Gabrilovich, D. I.; Colombo, G.; Altieri, D. C. Cancer Cell 2005, 7, 457−468. (2) Kroemer, G.; Dallaporta, B.; Resche-Rigon, M. Annu. Rev. Physiol. 1998, 60, 619−640. (3) Elmore, S. Toxicol. Pathol. 2007, 35, 495−516. (4) Norbury, C. J.; Hickson, I. D. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 367−401. (5) Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. Annu. Rev. Cell Dev. Biol. 1999, 15, 269−290. (6) Ghobrial, I. M.; Witzig, T. E.; Adjei, A. A. CA Cancer J. Clin. 2005, 55, 178−194. (7) Ashkenazi, A. Cytokine Growth Factor Rev. 2008, 19, 325−331. (8) Hacker, G. Cell Tissue Res. 2000, 301, 5−17. (9) Galluzzi, L.; Vitale, I.; Abrams, J. M.; Alnemri, E. S.; Baehrecke, E. H.; Blagosklonny, M. V.; Dawson, T. M.; Dawson, V. L.; EI-Deiry, W. S.; Fulda, S.; Gottlieb, E.; Green, D. R.; Hengartner, M. O.; Kepp, O.; Knight, R. A.; Kumar, S.; Lipton, S. A.; Lu, X.; Madeo, F.; Malorni, W.; Mehlen, P.; Nunez, G.; Peter, M. E.; Piacentini, M.; Rubinsztein, D. C.; Shi, Y.; Simon, H.-U.; Vandenabeele, P.; White, E.; Yuan, J.; Zhivotovsky, B.; Melino, G.; Kroemer, G. Cell Death Differ. 2012, 19, 107−120. (10) Christofferson, D. E.; Yuan, J. Curr. Opin. Cell Biol. 2010, 22, 263−268. (11) Cho, Y. S.; Park, S. Y.; Shin, H. S.; Chan, F. K. Mol. Cell 2010, 29, 327−332. (12) Molquin, D.; Chan, F. K. Trends Biochem. Sci. 2010, 35, 434− 441. (13) Levin, S.; Bucci, T. J.; Cohen, S. M.; Fix, A. S.; Hardisty, J. F.; Legrand, E. K.; Maronpot, R. R.; Trump, B. F. Toxicol. Pathol. 1999, 27, 484−490. (14) Kerr, J. F.; Wyllie, A. H.; Currie, A. R. Br. J. Cancer 1972, 26, 239−257. (15) Majno, G.; Joris, I. Am. J. Pathol. 1995, 146, 3−15. (16) Trump, B. F.; Berezesky, I. K.; Chang, S. H.; Phelps, P. C. Toxicol. Pathol. 1997, 25, 82−88. (17) Kurosaka, K.; Takahashi, M.; Watanabe, N.; Kobayashi, Y. J. Immunol. 2003, 171, 4672−4679. (18) Savill, J.; Fadok, V. Nature 2000, 407, 784−788. (19) Pelicano, H.; Carney, D.; Huang, P. Drug Resist. Update 2004, 7, 97−110. (20) Wang, D.; Lippard, S. J. Nat. Rev. Drug Discovery 2005, 4, 307− 320. (21) Wang, L.; Chanvorachote, P.; Toledo, D.; Stehlik, C.; Mercer, R. R.; Castranova, V.; Rojanasakul, Y. Mol. Pharmacol. 2008, 73, 119− 127. (22) Tsang, W. P.; Chau, S. P. Y.; Kong, S. K.; Fung, K. P.; Kwok, T. T. Life Sci. 2003, 73, 2047−2058. (23) Wang, S.; Konorev, E. A.; Kotamraju, S.; Joseph, J.; Kalivendi, S.; Kalyanaraman, B. J. Biol. Chem. 2004, 279, 25535−25543. (24) Chen, Y.; Jungsuwadee, P.; Vore, M.; Butterfield, D. A.; St. Clair, D. K. Mol. Interventions 2007, 7, 147−156. (25) Luanpitpong, S.; Chanvorachote, P.; Nimmannit, U.; Leonard, S. S.; Stehlik, C.; Wang, L.; Rojanasakul, Y. Biochem. Pharmacol. 2012, 83, 1643−1654. (26) Nishikawa, M. Cancer Lett. 2008, 266, 53−59. (27) Circu, M. L.; Aw, T. Y. Free Radical Biol. Med. 2010, 48, 749− 762.

AUTHOR INFORMATION

Corresponding Author

*(P.C.) Tel: +66 812075039. Fax: +66 22188340. E-mail: pithi. [email protected], [email protected]. (K.S.) Tel: +66 891074069. Fax: +66 22188357. E-mail: khanit.s@pharm. chula.ac.th, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Royal Golden Jubilee Ph.D. Program Grant No. PHD/0248/2550 (T.C.-A.), the Thailand Research Fund Grant No. DBG5280019 (K.S.), and the Thailand Research Fund (P.C.). This work was partially supported by the Japan Society for the Promotion of Science 1473

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(28) Liou, G. Y.; Storz, P. Free Radical Res. 2010, 44, 479−496. (29) Weinberg, F.; Chandel, N. S. Cell. Mol. Life Sci. 2009, 66, 3663− 3673. (30) Berghe, T. V.; Vanlangenakker, N.; Parthoens, E.; Deckers, W.; Devos, M.; Festjens, N.; Guerin, C. J.; Brunk, U. T.; Declercq, W.; Vandenabeele, P. Cell Death Differ. 2010, 17, 922−930. (31) Malorni, W.; Paradisi, S.; Iosi, F.; Santini, M. T. Cell Biol. Toxicol. 1993, 9, 119−130. (32) Silverberg, J. I.; Jaqdeo, J.; Patel, M.; Sieqel, D.; Brody, N. J. Drugs Dermatol. 2011, 10, 1096−1101. (33) Suwanborirux, K.; Amnuoypol, S.; Plubrukarn, A.; Pummangura, S.; Kubo, A.; Tanaka, C.; Saito, N. J. Nat. Prod. 2003, 66, 1441−1446. (34) Amnuoypol, S.; Suwanborirux, K.; Pummangura, S.; Kubo, A.; Tanaka, C.; Saito, N. J. Nat. Prod. 2004, 67, 1023−1028. (35) Saito, N.; Yoshino, M.; Charupant, K.; Suwanborirux, K. Heterocycles 2012, 84, 309−314. (36) Charupant, K.; Daikuhara, N.; Saito, E.; Amnuoypol, S.; Suwanborirux, K.; Owa, T.; Saito, N. Bioorg. Med. Chem. 2009, 17, 4548−4558. (37) Halim, H.; Chunhacha, P.; Suwanborirux, K.; Chanvorachote, P. Cancer Res. 2011, 31, 193−202. (38) Rao, K. E.; Lown, J. W. Chem. Res. Toxicol. 1990, 3, 262−267. (39) Pommier, Y.; Kohlhagen, G.; Bailly, C.; Waring, M.; Mazumder, A.; Kohn, K. W. Biochemistry 1996, 35, 13303−13309. (40) Begleiter, A. Cancer Res. 1983, 43, 481−484. (41) Begleiter, A.; Blair, G. W. Cancer Res. 1984, 44, 78−82. (42) Begleiter, A. Biochem. Pharmacol. 1985, 34, 2629−2636. (43) Lown, J. W.; Begleiter, A.; Johnson, D.; Morgan, A. R. Can. J. Biochem. 1976, 54, 110−119. (44) Lown, J. W.; Sim, S. K.; Majumdar, K. C.; Chang, R. Y. Biochem. Biophys. Res. Commun. 1977, 76, 705−710. (45) Vig, B. K. Mutat. Res. 1977, 49, 189−238. (46) Begleiter, A.; Leith, M. K. Cancer Res. 1990, 50, 2872−2876. (47) Benites, J.; Rojo, L.; Valderrama, J. A.; Taper, H.; Calderon, P. B. Eur. J. Med. Chem. 2007, 43, 1813−1817. (48) Verrax, J.; Beck, R.; Dejeans, N.; Glorieux, C.; Sid, B.; Pedrosa, R. C.; Benites, J.; Vásquez, D.; Valderrama, J. A.; Calderon, P. B. Anticancer Agents Med. Chem. 2011, 11, 213−221. (49) Benites, J.; Valderrama, J. A.; Taper, H.; Calderon, P. B. Invest. New Drugs 2011, 29, 760−767. (50) Cuevas, C.; Francesch, A. Nat. Prod. Rep. 2009, 26, 322−337. (51) Meco, D.; Colombo, T.; Ubezio, P.; Zucchetti, M.; Zaffaroni, M.; Riccardi, A.; Faircloth, G.; Jose, J.; D́ Incalci, M.; Riccardi, R. Cancer Chemother. Pharmacol. 2003, 52, 131−138. (52) Sledge, G. W.; Neuberg, D.; Bernardo, P.; Ingle, J. N.; Martino, S.; Rowinsky, E. K.; Wood, W. C. J. Clin. Oncol. 2003, 21, 588−592. (53) Gomes, A.; Fernandes, E.; Lima, J. J. Biochem. Biophys. Methods 2005, 65, 45−80.

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