Part 1:echinacoside Induces Apoptotic Cancer Cell Death By Inhibiting The Nucleotide Pool Sanitizing Enzyme MTh1

Contact: joanna.jia@wecistanche.com

Abstract: Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in a vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity ofMTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine), while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents.

Keywords: Echinacoside, MTH1, 8-oxoG, DNA damage, apoptosis, cell cycle arrest

Cistanche extract has anti-cancer health benefits

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Introduction

The cellular and mitochondrial nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pool is a significant target of various reactive oxygen species (ROS).1–3 Because the specificity of DNA polymerases is less than perfect,4,5 oxidized dNTPs can be incorporated into newly synthesized DNA to cause genetic aberrations if not fixed.6 For example, the major oxidized base in the nucleotide pool, 8-oxoguanine (8-oxoG), can base pair with cytosine and adenine. When inserted into opposite adenine during DNA replication, it can potentially cause T: A = G: C transversion,7 which is one of the most common mutations found in human cancers.8,9 Although most of the oxidized free dNTPs are removed by nucleotide pool sanitizing enzymes, studies in the past have revealed that the nucleotide pool is still an important source of oxidized bases in DNA molecules and a significant contributor to oxidative DNA damages.3,10 the most important enzyme for the sanitization of nucleotide pools.15 Nevertheless, even under the surveillance by the nucleotide pool sanitizing enzymes, a significant portion of oxidized dNTPs are incorporated into DNA, and bases in DNA molecules can also be oxidized directly by ROS.6 Cells then rely on more elaborated strategies involving various DNA glycosylases, such as OGG1 and MUTYH, and DNA repair processes, including base excision repair (BER) and mismatch repair (MMR), to fix problematic bases in DNA molecules.10,16,17 BER and MMR normally repair numerous oxidized bases in DNA per day and thus play important roles in protecting the integrity and stability of the genome.

In cells that are under elevated oxidative stress, increased incorporation of oxidized nucleotides into DNA may overwhelm the cellular repair capacity and trigger DNA damage response (DDR).18 Persistent DDR signaling will lead to cell cycle arrest, premature cellular senescence, or apoptosis, thus setting a barrier for tumorigenesis.19 Yet, although cancer cells display highly increased oxidative tension and generate a potentially lethal burden of oxidized nucleotides,20 they can survive the DDR-associated senescence or apoptosis.21 A large number of studies have shown that MTH1, by effectively removing the dangerously abundant 8-oxodGTP and 2-OH-dATP in tumor cells, plays a key role in the development of cancer.22 MTH1 was found to be overexpressed in different types of cancer,23–25 and 8-oxoG levels, and the extent of oxidative DNA lesions was lower in tumors than in the surrounding normal tissues.26–28 Functionally, the overexpression ofMTH1 significantly reduced the number of DNA mutations seen in MMR-deficient mouse embryonic fibroblasts10 and allowed cells to overcome oncogenic Ras-induced premature cellular senescence and apoptosis;29 whereas the suppression of MTH1 prevented the development of cancer in OGG1-deficient mice30 and promoted ROS-induced DDR and cellular senescence in Ras-transformed cells.3,29,31 Thus, although MTH1 plays a protective role by preventing ROS-induced mutations in healthy cells, these studies found that MTH1 is particularly required for the emergence and survival of cancer cells. Recent studies have provided more evidence to support the role of MTH1 in cancer development.31–34 Suppression of MTH1 by RNAi or small molecule inhibitors in various cancer cell lines resulted in markedly increased incorporation of 8-oxo d G into genomic DNA, leading to extensive DNA damages, apoptotic cell death, and reduced cancer cell survival.32,33 In mouse xenograft studies, inhibition of MTH1 efficiently suppressed the growth of cancer explants. Importantly, suppressing MTH1 did not impact the growth and survival of normal cells, probably because normal cells have lower ROS and hence are less dependent on MTH1 for survival.32,33 These findings suggest that inhibition ofMTH1 is a promising novel strategy to fight cancer.

Natural products have been a rich source of medicinal compounds.35 Many modern therapeutics, including anti-cancer drugs, have been derived from herbal or botanical preparations.36–39 Still, a large number of traditional herbs proposed to possess various therapeutic properties to remain to be characterized, largely due to difficulties in controlling their compositions to unambiguously define their efficacy and mechanisms of action.40 New approaches that allow for detection of active compounds and analysis of molecular mechanisms are needed. In this regard, target-guided high-throughput in vitro screening can be instrumental in finding mechanistically defined medicinal compounds from natural resources. In the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro assay to search for natural compounds that can inhibit MTH1. Surprisingly, the screening revealed that Echinacoside, a natural product that is best known for its potent antioxidative activity, is capable of inhibiting MTH1.

Echinacoside in cistanche can anti-apoptosis

Materials and methods

Materials

Natural herbal compounds were purchased from Yuanye Biological Technology Co., Shanghai, People’s Republic of China. The name, catalog number, Chemical Abstracts Service (CAS) registry number, and purity of each compound are listed in Table S1. Stock solutions of the compounds were prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA), and working solutions were prepared in assay buffer or complete cell culture medium. The same solution without the test compound but containing the same amount of DMSO was used as vehicle control. (S)-crizotinib was purchased from Selleck Chemicals, Houston, TX, USA.

in vitro screening

The in vitro enzymatic assay used to screen natural herbal compounds followed the procedures described by Gad et al.32 Briefly, serial dilutions of individual compounds were prepared in the assay buffer consisting of 100 mM Tris-acetate (pH 8.0), 40 mM NaCl, 10 mM Mg acetate, 0.005% Tween 20, and 1 mM DTT. Next, 0.5 nM recombinant humanMTH1 (Abcam, Cambridge, UK), 100 μM dGTP (Thermo Fisher Scientific, Waltham, MA, USA), and 0.2 U/mL inorganic pyrophosphatases (Thermo Fisher Scientific) were added, and the plates were incubated on a plate shaker for 1 hour at room temperature. The final reaction volume was 100 μL in a 96-well plate. At the end of the reaction, malachite green (J&K Scientific, Beijing, People’s Republic of China) was added,41, and the plates were incubated at room temperature for another 15 minutes. Absorbance at 630 nm was measured by a BioRad 680 microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). Inhibitory concentration (IC50) values were determined by nonlinear regression analysis using the GraphPad Prism software.

cell culture

Human MG-63 osteosarcoma, SK-HEP-1hepatocarcinoma, MCF7 breast cancer, SW480 colorectal cancer, HEK293 embryonic kidney, and mouse NIH/3T3 fibroblast cell lines were from American Type Culture Collection, and the human normal liver cell line L-O2 was purchased from KenGen Biotech, Nanjing, People’s Republic of China. These cells were maintained at 37°C in a humidified atmosphere with 5% CO2, following instructions given by the providers. No ethics statement was required from the institutional review board for the use of these cell lines.

Measurement of intracellular 8-oxo Intracellular 8-oxoG levels was measured by staining with Cy3-conjugated avidin.42 A total of 1×104 cells were seeded on a round coverslip in 12-well plates. The next day, the cells were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours and were then fixed with ice-cold methanol for 20 minutes, followed by incubation in Tris-buffered saline (TBS) with 0.1% Triton X-100 (Sigma-Aldrich) for 15 minutes. The samples were blocked in TBS with 0.1% Triton X-100 and 15% fetal bovine serum for 1 hour at room temperature and then stained with Cy3-conjugated avidin (0.5 μg/mL) (Rockland Immunochemicals, Limerick, PA, USA) in blocking solution for 1 hour at 37°C. After washing in phosphate-buffered saline (PBS) 3 times for 5 minutes, the coverslips were sealed on glass slides insect shield Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Images were taken and analyzed by a Zeiss LCM 510 confocal microscope.

Measurement of intracellular Ros Intracellular ROS levels was measured by flow cytometry using a cell-based ROS assay kit (Beyotime Biotechnology, Haimen, People’s Republic of China). Cells grown in six-well plates were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours, washed twice with PBS, and incubated with 10 μM dichlorofluorescein diacetate for 30 minutes at 37°C. The cells were then trypsinized and analyzed by the FACSCaliber flow cytometer (BD Biosciences, San Jose, CA, USA). Intracellular ROS levels were expressed as the average dichlorodihydrofluorescein fluorescence intensity of the cells. The numbers shown were averages of three independent experiments.

Immunofluorescent staining

Cells grown on coverslips were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours and then washed once with PBS, fixed with 4% paraformaldehyde in PBS for 20 minutes, and blocked in TBS with 0.1% Triton X-100 and 15% fetal bovine serum for 1 hour at room temperature. Fixed cells were stained for 2 hours at room temperature with a primary antibody against 8-oxoG (mouse monoclonal anti-8-oxoG, Abcam), 53BP1 (rabbit anti-53BP1; Bethyl Laboratories, Montgomery, TX, USA), or active caspase-3 (rabbit anti-caspase-3, Bioss, Beijing, People’s Republic of China), followed by staining with an Alexa 488-conjugated donkey antimouse (Abcam) or Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibody for 1 hour at room temperature. After washing in PBS 3 times for 5 minutes, the coverslips were sealed on glass slides in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Images were taken by a Zeiss LCM 510 confocal microscope.

colony formation assay

The day before treatment, cells were seeded in a six-well plate at a concentration of 1×104 cells per well. They were then treated with 0 μM, 60 μM, or 80 μM Echinacoside for 7 days. After washing with PBS, cells were fixed with ice-cold methanol and stained with crystal violet solution (Sigma- Aldrich) (0.5% in 25%methanol). Images were photographed after drying the plates overnight. The crystal violet crystals were dissolved by adding 70% ethanol, and absorbance at 595 nm was measured by a BioRad 680 microplate reader. Data were analyzed using the GraphPad Prism software.

MTT assay

One day before treatment, cells were seeded in 96-well plates at a concentration of 1×104 cells per well, followed by treatment with serial dilutions of Echinacoside for 24 hours or 48hours, or with 60 μM Echinacoside for 5hours, 12hours, or 24hours. The medium was removed, and the cells were washed with PBS, and then, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL in PBS, pH 7.2) (Thermo Fisher Scientific) was added to each well. The plates were incubated at 37°C for another 4 hours. In the end, the MTT solution was removed and 150 μL of DMSO was added to each well. The plates were incubated on a plate shaker for 10 minutes, and absorbance at 570 nm was measured by a BioRad 680 microplate reader. Each experiment was conducted twice in triplicate. Data were analyzed using the GraphPad Prism software. IC50 values were determined using nonlinear regression analysis.

cell cycle analysis

Cells in six-well plates were treated with 0 μM, 60 μM, or 80 μM Echinacoside for 24 hours, harvested with trypsin (Life Technologies, Carlsbad, CA, USA), washed twice with PBS, and then fixed with ice-cold ethanol (70%) for 2 hours at −20°C. Fixed cells were washed twice in cold PBS and resuspended in 300 μL of freshly prepared PBS with 0.1% Triton X-100, 0.2 mg/mL DNase-free RNase A (Sigma- Aldrich), 10 μg/mL propidium iodide (PI) (Hoffman-La Roche Ltd., Basel, Switzerland). After incubation at 37°C in the dark for 20 minutes, the cells were filtered through a Falcon nylon mesh (BD Biosciences), loaded onto the FAC- SCalibur flow cytometer, and analyzed using the ModFit software (BD Biosciences).

DNA fragmentation analysis

Cells grown in six-well plates were treated with 0 μM, 60 μM, or 80 μM Echinacoside for 24 hours and fixed in ice-cold 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes. After washing with PBS, cells were sealed in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Nuclear morphology was photographed with an Olympus fluorescent microscope. To detect DNA fragmentation on agarose gels, DNA was extracted using the QIAamp DNA micro kit (Qiagen NV, Venlo, the Netherlands), and electrophoresis was performed on 2% agarose gels containing 0.1 μg/mL ethidium bromide.

Analysis of apoptosis by flow cytometry Apoptotic cells was examined using an Annexin V-FITC Apoptosis detection kit (Bestbio, Shanghai, People’s Republic of China), following the manufacturer’s instructions. Cells grown in 96-well plates were treated with 0 μM, 15 μM, 30 μM, 60 μM, 80 μM, or 160 μM Echinacoside for 2 hours, 5 hours, 12 hours, or 24 hours, washed twice with PBS, and then resuspended in a binding buffer. Next, 5 μL of annexin V-FITC and 5 μL ofPI (Roche) were added sequentially, and the cells were incubated for 15 minutes in the dark at room temperature. The cells were loaded onto the FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using the Cell Quest software (BD Biosciences).

Western blot analysis

Cells grown in six-well plates were treated with 0 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours, washed twice with PBS and scraped off the plate using 100 μL of radioimmunoprecipitation buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris, pH 8.0) (Sigma-Aldrich) containing 1 mM phenylmethane sulfonyl fluoride. Samples were centrifuged at 12,000× g for 20 minutes at 4°C. Protein concentrations in the supernatants were determined by the Bradford reagent (Dingguo, Changchun, People’s Republic of China). Samples were denatured at 95°C for 10 minutes and separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Millipore), followed by blocking in Tris-buffered saline with Tween 20 (10 mM Tris, pH 7.5; 100 mM NaCl; 0.1% Tween 20) containing 5% (w/v) nonfat milk for 1 hour at room temperature. Blots were probed with primary antibodies against p21 (Abcam), poly (ADP-ribose) polymerase (Bioss), or activated caspase-3 (Bioss), followed by horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Signals were developed using the enhanced chemiluminescence Western blotting detection kit (Trans- gen Biotech, Changchun, People’s Republic of China). The experiments were repeated three times.

Echinacoside in cistanche can anti-apoptosis and anti-oxidation

Measurement of mitochondrial

membrane potential

Mitochondrial membrane potential was measured using the JC-1 dye (Biotechnology), following the manufacturer’s instructions. Cells grown in six-well plates were treated with 0 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours, washed twice in PBS and then incubated with JC-1 for 20 minutes. Images were taken using an Olympus fluorescent microscope.

statistical analysis

Statistical analysis was performed using GraphPad Prism Software. Significance was calculated using a one-way analysis of variance, and P0.05 was considered statistically significant. Results were expressed as mean ± standard deviation.

Echinacoside in cistanche can anti-apoptosis and improve immunity