Calpain and Caspase Orchestrated Death Signal to Accomplish Apoptosis Induced by Resveratrol and Its Novel Analog Hydroxstilbene-1 in Cancer Cells

Stomach ulceration is a major side effect of most chemopreventive drugs. We have established that although resveratrol is a promising chemopreventive compound, it delays the ulcer healing process. However, its analog hydroxystilbene-1 (HST-1) was devoid of such an ulcerogenic side effect. Consequently, here we tried to explore the chemopreventive efficacy of HST-1 compared with resveratrol in different cancer cell lines and identified the probable signaling pathways responsible for cell death. Our cell viability study established that HST-1, compared with resveratrol, showed better chemopreventive potential in all of the cell lines tested, with U937 and MCF-7 being the cells most affected. Furthermore, in U937 and MCF-7 cell lines, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, cell cycle analysis, and nuclear fragmentation by confocal microscopy established that both HST-1 and resveratrol switched on the apoptotic death cascade to execute cell death. The initiator signal was Fas-independent but synchronized in terms of cytosolic Ca influx, dissipation of mitochondrial membrane potential, and oxidative burst. It is noteworthy that the executioner signal was cell-specific as in U937 cells; HST-1 and resveratrol treatment induced mitochondrial permealization followed by cardiolipin depletion and cytochrome c release, which eventually activated downstream caspases 9 and 3 to execute the death process. In contrast, in MCF-7 cells the death process was executed in a caspase-independent but calpain-dependent manner as calpain activation induced cleavage of cytosolic -fodrin, stimulated mitochondrial release of apoptotic inducing factor and endonuclease G, and thus harmonized cytosolic and mitochondrial death signals to accomplish apoptosis. Resveratrol (Resv), a naturally occurring dietary compound, had already established its tremendous anticancer potential on different types of cancers by interfering with different cellular events associated with initiation, promotion, and progression of multistage carcinogenesis (Jang et al., 1997). We have previously reported that the use of Resv as an anticancer drug was severely constrained because of its tendency to prolong preexisting gastric ulceration (Guha et al., 2009). Furthermore, we showed that This work was supported by the Life Science Research Board, Defense Research and Development Organization, Government of India and the Department of Science and Technology, Government of India. P.G. and A.D. received fellowships from the Life Sciences Research Board, Defense Research and Development Organization, and Department of Science and Technology, Government of India. P.G. and A.D. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.110.167668. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: HST-1, hydroxstilbene-1; Resv, resveratrol; AIF, apoptotic inducing factor; Endo G, endonuclease G; cyt c, cytochrome c; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; PD150606, (2S)-3-(4-iodophenyl)-2-sulfanylpropanoic acid; PARP, poly(ADP-ribose) polymerase; IMM, inner mitochondrial membrane; MPTP, mitochondrial transition pore; NAC, N-acetyl-cysteine; ER, endoplasmic reticulum; PEG, polyethylene glycol; SOD, superoxide dismutase; PBMC, peripheral blood mononuclear cell; BAPTA-AM, 1,2-bis(oaminophenoxy)ethane-N,N,N ,N -tetraacetic acid tetra(acetoxymethyl) ester; CM-H2DCFDA, 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate acetyl ester; TG, thapsigargin; MMP ( m), mitochondrial transmembrane potential; BSA, bovine serum albumin; NHEK, normal human embryonic kidney; DMSO, dimethyl sulfoxide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; Ru360, ( )[(HCO2)(NH3)4Ru]2OCl3; JC-1, 5,5 6,6 -tetracholoro-1,1 ,3,3 -tetraethyl-benzimidazolylcarbo cyanine iodide; PBS, phosphate-buffered saline; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; Fluo-4 AM, fluo-4-acetoxymethyl ester; AFC, 7-amino-4-trifluoromethyl coumarin; RFU, relative fluorescent unit; FMK, fluoromethylketone; FACS, fluorescence-activated cell sorting. 0022-3565/10/3342-381–394$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 334, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 167668/3608725 JPET 334:381–394, 2010 Printed in U.S.A. 381 http://jpet.aspetjournals.org/content/suppl/2010/05/18/jpet.110.167668.DC1 Supplemental material to this article can be found at: at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from HST-1 (3,5,3 ,5 -tetrahydroxy stilben), a novel congener of Resv, was devoid of such an ulcerogenic side effect (Guha et al., 2009). Therefore, for the first time, we have attempted to explore the chemopreventive efficacy of HST-1 compared with Resv and enumerate the activation of the probable signaling cascade to delineate the mode of action of HST-1. Materials and Methods Chemicals and Reagents. RPMI medium 1640, Dulbecco’s modified Eagle’s medium, fetal bovine serum, antibiotics (penicillin-G, sterptomycin, Gentamycin), Hoechst, Alexa Fluor 488-AnnexinV/PI kit, CM-H2DCFH-DA, 10-N-nonyl-acridin orange, a MitoProbe transition pore assay kit, fluo-4-acetoxymethyl ester (Fluo-4 AM), and calcium calibration buffer kits were from Invitrogen (Carlsbad, CA). Primary antibodies (PARP, -fodrin, AIF, Endo G, -actin, and cytochrome c oxidase subunit IV) and polyclonal secondary antibody were obtained from Cell Signaling Technology (Danvers, MA). A Cycle Test Plus DNA reagent kit and JC-1 kit were from BD Biosciences (San Jose, CA). Apo-direct TUNEL assay kit, caspase protease assay kit (caspases 3, 8, and 9), and an anticytochrome c ELISA kit were from Millipore Bioscience Research Reagents (Temecula, CA). BAPTA-AM, thapsigargin, (2S)-3-(4-iodophenyl)-2-sulfanylpropanoic acid (PD150606), and ( )[(HCO2)(NH3)4Ru]2OCl3 (Ru360) were from Calbiochem (San Diego, CA). Trans-resveratrol, a mammalian cell lysis kit, protease inhibitor cocktail, BSA, Tween 20, Tris-HCl, DMSO, caspase inhibitors Z-VAD-FMK (Pan caspase inhibitor), Z-DQMD-FMK (caspase 3-specific inhibitor), Z-LEHD-FMK (caspase 9-specific inhibitor), Z-IETD-FMK (caspase 8-specific Inhibitor), NAC, PEG-SOD, and PEG-catalase, and BSA were procured from Sigma-Aldrich (St. Louis, MO). One-step NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) was from Pierce Biotechnology (Rockford, IL). A mitochondria/cytosol fractionation kit and calapin activation assay kit were from BioVision (Mountain View, CA), and a CellTiter-Glo luminescent cell viability assay kit was from Promega (Madison, WI). Culture of Cell Lines. Cell lines studied included U937 (human leukemic monocyte lymphoma), K562 (human myelogenous leukemia), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), and NHEK (normal human embryonic kidney) that were obtained from the National Centre for Cell Science, Pune, India. Human peripheral blood mononuclear cells (PBMCs) were harvested from healthy donors. MCF-7, HepG2, and NHEK cells were cultured in Dulbecco’s modified Eagle’s medium, pH 7.4, supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin-G, 100 g/ml-streptomycin, and 6 g/ml Gentamycin). RPMI 1640 medium was used for U937, K562, and PBMCs. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. CellTiter-Glo Luminescent Cell Viability Assay. The CellTiter-Glo luminescent cell viability assay (Promega) was used in a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. In brief, the cells (5 104 cells in 100 l of medium/well) were plated in 0.02 to 0.07% DMSO in media as control in 96-well plates. The cells as such or in the presence of Resv or HST-1 (dissolved in 0.02– 0.07% DMSO in media) were incubated for 48 h. At the end of the treatments, each well was treated with a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well (e.g., 100 l of reagent added to 100 l of medium containing cells for a 96-well plate). Then contents were mixed for 2 min on an orbital shaker to induce cell lysis, and the plate was incubated at room temperature for 10 min in the dark to stabilize the luminescent signal. At the end cellular luminescence was reordered in a luminometer (FLX800; BioTek Instruments, Winooski, VT). Cell Cycle Analysis. U937 and MCF-7 cells (1 10) were incubated for 48 h with HST-1 and Resv after which cell cycle distribution was studied with a Cycle Test Plus DNA reagent kit and acquired on a linear scale in a flow cytometer (FACS Calibur; BD Biosciences, San Diego, CA) equipped with a fluorescence detector (488-nm argon laser light source and 623-nm band pass filter), and analyzed by using CellQuest software (BD Biosciences). A total of 10,000 events were acquired and analyzed. Cellular and Nuclear Morphology Analysis by Confocal Microscopy. A TCS-SP-2 confocal microscope (Leica, Wetzlar, Germany) was used for all microscopic imaging for nuclear morphology with Hoechst staining, as described previously (Guha et al., 2009). TUNEL Assay. To confirm the nature of tumor killing by different concentrations of HST-1 and Resv on U937 and MCF-7 cells, the cells were fixed, permeabilized, and incubated with terminal deoxynucleotidyl transferase enzyme and fluorescein isothiocyanate-dUTP. Cells were washed, incubated with RNAase solution, and analyzed by FACS (BD Biosciences). The fragmented DNA of apoptotic cells was labeled by using an Apo-Direct TUNEL assay kit (Millipore Corporation, Billerica, MA). The cells were then analyzed by a FACS cytometer (equipped with a 488-nm argon laser light source; 515-nm band pass filter, FL1-H; and 623-nm band pass filter, FL2-H) using CellQuest software. A total of 10,000 events were acquired and analyzed. Measurement of Mitochondrial Membrane Potential. MMP was measured with a JC-1 kit following the protocol submitted by the supplier. The mitochondrial membrane potential ( m) detection kit used a unique fluorescent cationic dye, JC-1 (5,5 6,6 tetracholoro-1,1 ,3,3 -tetraethyl-benzimidazolylcarbo cyanine iodide) (excitation, 488 nm and emission, 525 nm), to signal the loss of MMP. Cells were harvested after HST-1 and Resv treatment. Then mitochondrial permeability transition was determined by staining the cells with JC-1 as described. In brief, equal numbers of cells (1 10) were incubated with JC-1 at 2.5 g/ml in 1 ml of PBS for 30 min at 37°C with moderate shaking. Cells were then centrifuged at 1000g at 4°C for 5 min, washed twice with ice-cold PBS, and resuspended in 200 l of PBS. Mitochondrial permeability transition was subsequently quantified on a spectrofluorimeter (FP6300; Jasco, Tokyo, Japan). Data are given in a ratio of 590/ 530. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a mitochondrial uncoupler. Measurement of Mitochondrial Cardiolipin Depletion. The binding of 10-N-nonyl-acridin orange to mitochondria-specific cardiolipin was measured spectrofluorimetrically at an excitation of 485 nm and an emission of 530 nm as described previously (Asumendi et al., 2002). Flow Cytometric Measurement of the Mitochondrial Permeability Transition Pore. The MitoProbe transition pore assay kit (Invitrogen) was used to study the opening of the mitochondrial transition pore (MPTP) in HST-1and Resv-treated cells. This kit provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. In brief, 1 10 cells were loaded with the acetoxymethyl ester of calcein dye, calcein AM, which passively diffused into the cells and accumulated in cytosolic compartments, including the mitochondria. Once inside cells, intracellular esterases cleaved the acetoxymethyl esters to liberate the very polar fluorescent dye calcein. The fluorescence from cytosolic calcein was quenched by the addition of CoCl2 (cobalt chloride), while the fluorescence from the mitochondrial calcein was maintained. However, opening the MPTP instigated the release of mitochondrial calcein to the cytosol where CoCl2 was quenched, leading to the dramatic reduction of calcein fluorescence. Determination of Cytosolic Release of Mitochondrial Cytochrome c. MCF-7 and U937 cells were harvested after treatment with HST-1 and Resv for 24 and 48 h. Isolation of a highly enriched mitochondrial fraction and cytosolic fraction of cells was performed by using a mitochondria/cytosol fractionation kit (BioVision, Moun382 Guha et al. at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from tain View, CA). In brief, cells (5 10) were centrifuged at 600g for 5 min at 4°C, resuspended in ice-cold PBS, and centrifuged at 600g for 5 min at 4°C. Then the cells were resuspended in 1.0 ml of cytosol extraction buffer mix containing dithiothreitol and protease inhibitors and incubated on ice for 10 min. The cells were homogenized on ice. The homogenate was centrifuged at 700g for 10 min at 4°C, and the supernatant was collected and centrifuged at 10,000g for 30 min at 4°C. Then the supernatant was collected as the cytosolic fraction. The pellet was resuspended in 0.1 ml of mitochondrial extraction buffer mix containing dithiothreitol and protease inhibitors, vortexed for 10 s, and saved as the mitochondrial fraction. These isolated cytosolic and mitochondrial fractions were subjected to colorimetric ELISA for cytochrome c (cyt c). Activity of Caspases. Caspases 3, 9, and 8 were assayed by using a caspase fluorimetric assay kit (Millipore Corporation, Billerica, MA) per the manufacturer’s instructions. Western Blot Analysis. Cell lysates were prepared by using a mammalian cell lysis kit; in brief, cells were immersed in freshly prepared lysis buffer with protease inhibitor cocktail, sonicated, and centrifuged (12,000 g for 10 min), and the protein concentration of the supernatant was measured (Bradford, 1976). The proteins (50 g) were resolved by 10% nonreducing SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked for 2 h at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween 20 containing 3% BSA followed by overnight incubation at 4°C in 1:500 dilution of the respective antibodies for PARP, -fodrin, AIF, and Endo G in 3% BSA. The membrane was washed three times with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween 20 containing 3% BSA and incubated with alkaline phosphatase-conjugated secondary antibody. The bands were visualized by using a 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium substrate. Intracellular Ca Measurement. Intracellular Ca was measured by using the fluorescent probe Fluo 4-AM spectrofluorimetrically at an excitation of 488 nm and an emission of 516 nm. In brief, the cells were preloaded with Fluo 4-AM in loading media for 30 min at 37°C after which the cells were washed in loading media devoid of Fluo 4-AM. The cells were resuspended in the same media, and spectrofluorimetric analysis were carried out followed by HST-1 and Resv treatment. Response calibration was carried out with calcium calibration buffer kits. The following equation was used to determine cytosolic calcium concentration: Ca free Kd F Fmin Fmax F (1) where Fmin is the fluorescence intensity of the indicator in the absence of calcium, Fmax is the fluorescence of the calcium-saturated indicator (after 1 M ionomycin treatment), and F is the fluorescence at intermediate calcium levels in the cell. The Kd value of Fluo 4-AM was 345 nM. Calpain Activation Assay. Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). A calapin activation assay kit (BioVision) was used for measuring intracellular calpain activity quantitatively. This fluorometric assay was based on the detection of cleavage of the calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light ( max 400 nm) upon cleavage of the substrate by calpain, and free AFC emits a yellowgreen fluorescence ( max 505 nm), which was quantified by using a fluorimeter. In brief, 1 to 2 10 cells were suspended in 100 l of extraction buffer (provided in the kit), and samples were incubated on ice for 20 min. The samples were gently mixed by tapping several times during incubation. They were centrifuged for 1 min in a microcentrifuge (10,000g), and supernatant was transferred to a fresh tube and put on ice. Then protein concentration was assayed, and the cell lysate ( 50–200 g) was diluted to 85 l of extraction buffer. For positive control, 1 to 2 l of active calpain (provided in the kit) was added to 85 l of extraction buffer. Then, 10 l of 10 reaction buffer (provided in the kit) was added followed by 5 l of calpain substrate to each assay. The reaction mixture was incubated at 37C for 1 h in the dark. The samples were read in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. The changes in calpain activity were expressed as relative fluorescent unit (RFU) per milligram protein of each sample. To explore the probable effects of calpain on HST-1and Resvinduced cell death the cells were pretreated for 3 h with the calpainspecific inhibitor PD150606 at a dose of 50 M. Detection of Reactive Oxygen Species. Reactive oxygen species (ROS) was measured by CM-H2DCFDA (excitation 490 nm; emission 527 nm). The cells were preloaded with these dyes, and their reactivity with ROS was analyzed spectrofluorimeterically. Statistical Analysis. Data are expressed as mean S.D. unless otherwise stated. Comparisons were made between different treatments (analysis of variance) by using the software InStat (GraphPad Software Inc., San Diego, CA), where an error protecting the multiple comparison procedure, namely Tukey-Kramer multiple comparison tests, was applied for the analysis of significance of all of the data.