Structure Determinants of Lagunamide A for Anticancer Activity and Its

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Structure Determinants of Lagunamide A for Anticancer Activity and Its Molecular Mechanism of Mitochondrial Apoptosis Xiaoxing Huang, Wei Huang, Li Li, Xihuan Sun, Siyang Song, Qingyan Xu, Lianru Zhang, Bang-Guo Wei, and Xianming Deng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00564 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Molecular Pharmaceutics

Structure Determinants of Lagunamide A for Anticancer Activity and Its Molecular Mechanism of Mitochondrial Apoptosis Xiaoxing Huang,†,‡,

⊥

Wei Huang,†,‡, §,

⊥

Li Li,†,‡ Xihuan Sun,†,‡ Siyang Song,†,‡

Qingyan Xu,†,‡ Lianru Zhang,†,‡ Bang-Guo Wei, § and Xianming Deng†,‡,* †

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. State-province Joint Engineering Laboratory of Targeted Drugs from Natural

‡

Products, Xiamen University, Xiamen, Fujian 361102, China. Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. §

1

ACS Paragon Plus Environment


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GRAPHIC ABSTRACT

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ABSTRACT

Marine natural products are served as attractive source of anticancer therapeutics, with the great success of “first-in-class” drugs, such as Yondelis, Halaven, and Brentuximab vendotin. Lagunamides A-C from marine cyanobacterium, Lyngbya majuscula, exhibit exquisite growth inhibitory activities against cancer cells. In this study, we have systematically investigated the structure-activity relationships (SAR) of a concise collection of lagunamide A and its analogs constructed by total chemical synthesis against a broad panel of cancer cells derived from various tissues or organs, including A549, HeLa, U2OS, HepG2, BEL-7404, BGC-823, HCT116, MCR-7, HL-60, and A375. The R configuration of lagunamide A at C-39 position was found to be the structure determinant for anticancer activity. Further molecular mechanism study in A549 cells revealed that lagunamide A induced caspase-mediated mitochondrial apoptosis. Accompanied with the dissipation of mitochondrial membrane potential (∆φm) and overproduction of reactive oxygen species (ROS), lagunamide A led to mitochondrial dysfunction and finally caused cell death. Moreover, both anti- and pro-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins participated in lagunamide A-induced mitochondrial apoptosis, especially myeloid cell leukemia-1 (Mcl-1). Over-expression of Mcl-1 partly rescued A549 cells from lagunamide A-induced apoptosis. This study suggests that lagunamide A may exert anticancer property through mitochondrial apoptosis. Together, our findings would provide insightful information for the design of new anticancer drugs derived from lagunamides. KEYWORDS: marine natural products; lagunamide A; apoptosis; mitochondria;

Bcl-2 family.

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INTRODUCTION

Cancer is one of the world’s deadliest diseases. Although a great progress has been made in developing chemo-preventive and therapeutic remedies in the past decade, the search of new drugs for combating cancer is still urgently needed. Marine natural products from diverse organisms having distinct physiology and adaptation capability have long been an attractive source for identifying new drug leads.1 Beginning in 1951, the isolation of unusual nucleosides from marine sponges led to the great success of discovery of anticancer drugs, ara-A (vidarabine) and ara-C (cytarabine).2-4 Until now, eight marine drugs have been approved by US Food and Drug Administration

(FDA)

or

European

Medicines

Agency

(EMA),

most as

“first-in-class” drugs, such as Yondelis, Halaven, and Brentuximab vendotin.5 Additionally, five out of eight are anticancer drugs, representing the great potential of marine natural products in developing as anticancer therapeutics. To explore the drug-like properties and to elucidate the mechanisms of action are key steps for the success of developing new anticancer therapeutics from marine natural products, which remain great challenges. In current cancer research, the apoptotic pathway, a major programmed cell death (PCD) to counteract tumor growth, has been proven as an effective anticancer drug target. The intrinsic apoptosis triggered by intracellular stress signals centers on mitochondrial membrane permeability (MMP),6 which arises a series prominent consequences including dismission of ∆φm, generation of ROS and release of cytotoxic proteins.7 Mitochondrial cytotoxic protein cytochrome c releases into the cytosol, activates caspases and forms apoptosome, finally proceed apoptosis following the cleavage of multiple protein substrates such as PARP.8-10 Localizing to mitochondria, Bcl-2 family proteins play important role in apoptosis through regulation of MMP and activation of caspases.11-16 Dysregulation of Bcl-2 family such as the overexpression of anti-apoptotic proteins is common in diverse cancers, thus developing bioactive agent that modulates the level of antiapoptotic Bcl-2 family proteins became an efficient strategy for anti-cancer therapy. Many cytotoxic agents are identified to have potent anticancer activity for therapy through apoptotic pathways screening.17 Lagunamides A-C, cyclic depsipeptides, were first isolated from the marine cyanobacterium, Lyngbya majuscule, exhibiting exquisite growth inhibitory activities against cancer cells.18 These preliminary results imply the potential of lagunamides as anticancer agents. The asymmetric total synthesis of lagunamide A provide the possibility of further SAR and molecular 4


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mechanism research.19 In this study, we first performed SAR study of lagunamide A and its analogs. These compounds exhibited differential activities profiles against the tested cancer cells. The key structure features for having anticancer activity were identified to be the chirality of key substitutents, especially the R configuration of C-39. Further investigation on the mode of action of lagunamide A in lung cancer cells A549 revealed that it induced mitochondrion-mediated apoptosis representing by the loss of ∆φm and overproduction of ROS, resulted in mitochondrial dysfunction and finally led to cell apoptosis. Moreover, lagunamides A down-regulated anti-apoptotic Bcl-2 family proteins whereas up-regulated pro-apoptotic Bcl-2 family proteins to accelerate cell death. These results would provide critical information for further development of anticancer therapeutics from this class of compounds. MATERIAL AND METHODS Cell Culture. Human non-small cell lung cancer cell line A549, human uterine

cervix carcinoma cell line HeLa, human liver carcinoma cell line HepG2 and BEL-7404, human colorectal carcinoma cell line HCT116, human osteosarcoma cell line U2OS, gastric adenocarcinoma cell line BGC-823, human acute promyelocytic leukemia HL-60, breast adenocarcinoma MCF-7, and malignant melanoma cell line A375 were cultured in Dulbecco's modified eagle medium (DMEM) or Roswell Park Memorial Institute medium (RPMI1640) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 U/mL penicillin. Compounds and Antibodies. In present work, compounds were dissolved in

DMSO. Antibody against β-actin, Flag, HSP60 and α-tubulin were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against PARP and Bad were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mcl-1 antibody was purchased from AbCam (Cambridge, UK). Other antibodies were purchased from Cell Signaling Technology (Boston, MA). Cell Viability Assay. Cells proliferation was determined using a commercially

proliferation assay kit (CellTiter 96® AQueous One Solution Reagent, Promega, US). Cells were seeded in 96-well plates at an appropriate density in culture medium and allowed to attach overnight. After treatment of vehicle (0.1% DMSO) or lagunamides A for indicated times and concentrations, 20 µL of MTS reaction solution (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS (a) and 100 µg/mL phenazine 5



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methosulfate; PES) was added to each well. The absorbance values were read at 490 nm wavelength with a spectrophotometer (Varioskan Flash, Thermo, US) after 1 to 4 hours incubation. The cell viability was calculated as: cell survival = (ODcompd.ODblank)/(ODcontrol- ODblank)*100%. DMSO was used as control. IC50 was calculated using software GraphPad Prism 5. DAPI Staining. The A549 cells were seeded in 6-well plate containing sterile

coverslips (20 mm*20 mm). After treatment of DMSO or lagunamides A for indicated times and concentrations, cells were washed twice by phosphate buffer (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then stained in 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) dye for 15 min at room temperature. Unbound DAPI was washed and cell-containing coverslips were fixed on object slips by resin. The resins were observed using a confocal laser scanning microscope (CLSM) (LSM 780, Carl Zeiss, Germany). Transmission Electron Microscope (TEM). After treatment, cells were scraped

gently and harvested, successively followed by fixing in 2.5% glutaraldehyde, 2% paraformaldehyde, 2.5% glutaraldehyde, 0.05% calcium chloride, 1% osmic acid at 4 ˚C. After dehydration by ethyl alcohol and acetone, the cells were embedded in resin

and cut into 60-80 nm thickness. The section was dyed with 3% uranium acetate-lead citrate, examined in a TEM (JEM-2100, JEOL, Japan). Cell Cycle Detection. After the treatment with DMSO or lagunamide A for the

indicated times and concentrations, the cells were harvested and fixed with 70% (v/v) alcohol at -20 ˚C overnight. Cells were incubated in 50 µg/mL propidium iodide (PI) solution containing 0.1% sodium citrate, 0.3% NP-40 and 100 U/mL RNase for 30 min at 37 ˚C. DNA content was measured using a flow cytometer (EPICS XL, Beckman, US). Annexin V and PI Staining. After treatment, the cells were harvested and stained

using a commercial assay kit (Annexin-V-Fluors staining kit, Roche, Switzerland) according

to

manufacturer’s

instructions

and

further

analyzed

by

a

fluorescence-activated cell sorting (FACScan) flow cytometer (LSRFortessa, BD, NJ).

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Caspase 3 Activity Assay. After treatment, cells were lysed and total proteins were

quantified by BCA analysis (Beyotime, China). Caspase 3 activity was determined according to manufacturer’s instructions of caspase 3 activity assay kit (Beyotime, China). Briefly, caspase 3 activity (with DEVD as substrate) was measured at 405 nm wavelength by a spectrophotometer (Varioskan Flash, Thermo, US) after 2 hours incubation. Measurement of ∆φm. After the treatment with DMSO or lagunamides A for the

indicated time, cells were harvested and incubated with JC-1 (Beyotime, China) for mitochondrial membrane potential assay. Fluorescence-activated cells were observed in an optical microscopy (AX20 OBSERVER A1, Zeiss, Germany) and detected by FACScan flow cytometer. Measurement of ROS. Using 0.02% (v/v) 30% H2O2 as a positive control, ROS

production was detected on a FACScan flow cytometer with a commercial Reactive Oxygen Species Assay Kit (Beyotime, China). Mitochondria Isolation. The mitochondria of tested cells were isolated using a

commercial mitochondria isolation kit (Thermo, US). The eventual protein extractive was quantified by BCA analysis (Beyotime, China) for subsequent western blot. Western Blot. After compounds treatment, cells were harvested and lysed in cell

lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% (v/v) Triton X-100, 5% glycerol, 5 µg/mL leupeptin, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM PMSF. Total protein concentrations were quantified by BCA analysis (Beyotime, China). Equal amounts of protein were loaded for separation by electrophoresis in 10% to 15% SDS-PAGE followed by transferring to PVDF membranes. Proteins were then immune-reacted with specific primary antibodies and secondary antibodies, finally detected by an enhanced chemiluminescence reagent and imaged using a chemiluminescence imaging system (CHEMIDOC, Bio-rad, US). Constructs of Exogenetic Mcl-1 Expression. The full-length human Mcl-1 cDNA

was amplified by PCR using PrimerSTAR DNA Polymerase (PrimerSTAR HS DNA Polymerase, TAKARA, China). The forward and reverse primers for Mcl-1(FL) were as

follows:

5’-AAAAGAATTCGGTTTGGCCTCAAAAGAAACG-3’ 7


and


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5’-AAAAGATCTCTATCTTATTAGATATGCCAAACC-3’. Then the PCR product was double digested using restriction endonucleases EcoR I and Bgl II and cloned into the pCMV5–FLAG expression vector. Transient Transfection. When A549 cells reach about 70% confluent, Mcl-1

plasmid or vector was transiently transfected into A549 cells using Lipofectamine reagent (Lipofectamine® 3000, Invitrogen, US) according to the manufacturer’s instructions. After incubation for 48 hours, transfected cells were used in subsequent experiments. Data Analysis. All data are presented as mean±standard deviation (s.d.) for three or

more separate experiments. RESULTS SAR Study Identifies Critical Elements in the Lagunamide A

Lagunamide A, a cyclic depsipeptide, was first isolated from the marine cyanobacterium Lyngbya majuscule, exhibiting significant anti-malarial properties against Plasmodium falciparum and growth inhibitory activity against P388 murine leukemia cell lines.18 To systematically explore the SAR of lagunamide A (1), a concise collection of its analogs was designed and synthesized via diverse synthetic approach (Figure 1).19 We evaluated the antiproliferative activities of these analogs against a broad panel of cancer cells, including lung cancer cells A549, cervical cancer cells HeLa, osteosarcoma cells U2OS, liver cancer cells HepG2 and BEL-7404, stomach cancer cells BGC-823, colon cancer cells HCT116, breast cancer cells MCF-7, leukemia cells HL-60 and melanoma cells A375 (Table 1). The results revealed that lagunamide A (1) exhibited the best cytotoxicity with the IC50 less than 20 nM against all the tested cancer cells. Compound 2 with the chirality change at C-2 position, 2-epi-lagunamide A, had more than 3-folds dropped in activities compared to 1 with IC50 values ranging from 40 to 150 nM. However, compound 3, with the only switch of the chirality at C-39 position, from the configuration of R to S, dramatically decreased its activities more than 20-folds comparing with 1. 7, 39-epi-lagunamide A (4) and 7, 37, 39-epi-lagunamide A (5) with additional changes of the chirality at C-7 and C-37 positions further lost their anticancer activities. Compound 6, dehydrated from 5, was inactive with the IC50 higher than 10 µM. Together, this SAR study identifies the key structure features of lagunamide A, the R configuration at C-39 position, which would guide the further structure optimization. 8


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Figure 1. Chemical structures of lagunamide A and its analogs (1-6). Table 1. IC50 values of lagunamide A and its analogs against diverse human cancer cellsa IC50 (nM)

Lagunamide A (1)

2

3

4

5

6

A549

8.7±0.3

62.3±11.5

273.9±38.3

3400.3±567.3

>10000

>10000

HeLa

17.0±1.7

67.3±20.3

191.6±63.7

1377.7±313.5

>10000

>10000

U2OS

8.6±1.0

59.3±9.8

437.9±58.0

4036.7±400.7

>10000

>10000

HepG2

11.4±1.9

67.4±1.2

246.6±9.7

2537. 3±486.3

>10000

>10000

BEL-7404

11.0±0.7

59.9±8.3

725.0±153.5

5859. 3±384.4

>10000

>10000

BGC-823

4.7±0.4

42.5±1.8

231.7±25.3

3430.3±577.1

>10000

>10000

HCT116

8.2±1.2

52.4±5.1

200.0±19.5

3799.7±332.7

>10000

>10000

MCF-7

18.7±2.2

145.8±21.1

946.4±206.8

839.8±407.8

>10000

>10000

HL-60

19.8±0.9

92.6±1.9

713.9±97.6

8630.3±515.1

>10000

>10000

A375

8.3±1.8

56.3±6.6

212.6±42.8

970.2±178.7

>10000

>10000

a

Cell viability was detected using MTS assay after 48 h of treatment with serial dilutions of lagunamide A and its derivatives. IC50 values were calculated by GraphPad Prism 5 and displayed as mean±s.d..

Lagunamide A Induces Apoptosis in Multiple Human Cancer Cells

With potent cytotoxicity of lagunamide A against various human cancer cells in hand, we strived to dissect the molecular mechanism of lagunamide A. First, we checked the morphology change of cancer cells under lagunamide A treatment. In A549 cells, pseudopodia retraction, shrinkage (Figure 2A (a)), chromatin condensation (Figure 2A (b)) and karyopyknosis (Figure 2A (c)) were observed with the treatment of lagunamide A at 10 nM for 24 hours. Moreover, nuclear cracking, DNA releasing into cytoplasm (Figure 2B (b)) and lysosome packing damaged 9



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organelle (Figure 2B (c)) were also observed using TEM. These typical apoptotic morphologies indicate that lagunamide A treated cells underwent apoptosis pathway. To further confirm cell apoptosis induced by lagunamide A, Annexin-V-Fluors/PI double staining was applied to detect the translocation of phosphatidylserine (PS), whose exposure on the plasma membrane surface serves as an early event in apoptosis17. After incubation with 10 nM lagunamide A for 48 hours, up to 80% cells were stained with Annexin V-positive, which suggested lagunamides A resulted in an extremely high apoptosis rate in A549 cells (Figure 2C). The occurrence of apoptosis in Hela, HepG2 and HCT116 cells were also increased to 81%, 50%, and 40% respectively, upon 20 nM or 40 nM lagunamide A treatment (Figure S1). Collectively, lagunamide A induces apoptosis in various cancer cells, which leads to blockage of cell proliferation.

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Figure 2. Lagunamide A induces apoptosis in A549 cells. (A) Morphological variation of lagunamide A treated A549 cells was visualized using optical microscope (OM). Chromatin of conditional treated cells stained by DAPI dye was detected on confocal laser scanning microscopy (DAPI). Karyomorphism of conditional treated cells was visualized on transmission electron microscope (TEM). The scale bar rpresents 2 µm. (B) Karyomorphism and organelle were visualized on TEM. The left scale bars present 2 µm and the right ones present 0.5 µm. Number× means magnification times of cell area. The red, orange and blue arrows indicate normal mitochondria, endoplasmic reticulum and autolysosome respectively. (C) After the treatment with 10 nM lagunamide A for indicated times, conditional A549 cells stained with PI/ Annexin V were analyzed by a FACScan flow cytometer. Apoptotic cells were presented as mean±s.d. of three independent experiments.

Lagunamide A Induces Caspase-dependent Apoptosis Caspases are activated during early apoptosis, which leads to cleave critical cellular substrates including PARP, thereby mediating the dramatic apoptosis events.20-23 Therefore, the apoptosis-related proteins were assessed and quantified by immunoblot after the treatment of A549 cells with DMSO or 10 nM lagunamide A for indicated times. As expected, caspase 7 and PARP were all rendered by lagunamide A treatment while the cleavage form of caspase 7 and PARP increased in a time-dependent manner (Figure 3A). Moreover, lagunamide A increased the caspase 3 activity in a dose-dependent manner (Figure 3B). With the additional pre-treatment of pan-caspase inhibitor z-VAD-fmk, cleavages of caspase 7 and PARP induced by lagunamide A were markedly eliminated (Figure 3C) and the enzymatic activity of caspase 3 was back to normal level (Figure 3D). Collectively, these results demonstrate that lagunamide A could induce caspases-dependent apoptosis in A549 cells.

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Figure 3. Lagunamide A induces caspase-dependent apoptosis in A549 cells. (A) After the treatment with DMSO or 10 nM lagunamide A for the indicated times, the A549 cells were harvested and lysed. PARP, cleaved PARP, caspase 7, cleaved caspase 7 and β-actin were detected by immunoblot analysis using appropriate antibodies. (B) After the treatment of A549 cells with lagunamide A at the indicated concentration for 12 h, caspase 3 activation was detected using caspase 3 activity assay kit. β-actin was used as total proteins control. Data were shown as mean±s.d. of three independent experiments. (C and D) A549 cells were pretreated with 25 µM pan caspase inhibitor z-VAD-fmk or not for 1 h and then followed by 10 nM lagunamide A incubation for another 18 h. The levels of PARP, cleaved PARP, caspase 7, and cleaved caspase 7 were detected by immunoblot analysis using appropriate antibodies. (C). Activity of caspase 3 was measured by a commercial assay kit. β-actin was used as total proteins control (D). Data were shown as mean±s.d. of three independent experiments. *, p

Structure Determinants of Lagunamide A for Anticancer Activity and Its

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