Cip1 expression, Chk1 and Chk2 activation and leads to increased growth inhibition and death in A2780 ovarian cancer cells

Phytomedicine 35 (2017) 1–10

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Original Article

Haemanthamine alters sodium butyrate-induced histone acetylation, p21WAF1/Cip1 expression, Chk1 and Chk2 activation and leads to increased growth inhibition and death in A2780 ovarian cancer cells

MARK

Martina Seifrtováa,*, Radim Haveleka, Lucie Cahlíkováb, Daniela Hulcováb, Naděžda Mazánkováa, Martina Řezáčováa Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Šimkova 870, Hradec Kralove 500 38, Czech Republic ADINACO Research Group, Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic a

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A R T I C L E I N F O

A B S T R A C T

Keywords: Haemanthamine Sodium butyrate Ovarian carcinoma cells Histone acetylation Cell death

Background: Haemanthamine (HA) and sodium butyrate (NaB) are promising candidates for chemotherapy as a treatment for cancer. Purpose: We aimed to determine the anticancer potential of HA and NaB, alone and in combination, in A2780 ovarian cancer cells and concurrently investigated anticancer potential in contrast to non-cancer human MRC-5 fibroblasts. Methods: Antiproliferative effects were determined by WST-1 assay and by Trypan blue exclusion staining. Cell cycle distributions were studied by flow cytometry and protein levels were determined by Western blotting. Results: The combination of HA and NaB caused a significant decrease in the proliferation of A2780 cells compared to the stand-alone treatment of cells by HA or NaB. This effect was less pronounced in non-cancer MRC-5 fibroblasts. In the later intervals, the number of A2780 living cells was strongly decreased by treatment using a combination of NaB and HA. This simultaneous application had no considerable effect in MRC-5 fibroblasts. The combination of NaB and HA led to the suppression of cells in the G1 phase and caused an accumulation of cells in the S and G2 phase in comparison to those treated with NaB and HA alone. Treatment of cells with NaB alone led to the activation of proteins regulating the cell cycle. Notably, p21WAF1/Cip1 was upregulated in both A2780 and MRC-5 cells, while checkpoint kinases 1 and 2 were activated via phosphorylation only in A2780 cells. Unexpectedly, NaB in combination with HA suppressed the phosphorylation of Chk2 on threonine 68 and Chk1 on serine 345 in A2780 cells and downregulated p21WAF1/Cip1 in both tested cell lines. The sensitization of cells to HA and NaB treatment seems to be accompanied by increased histone acetylation. NaB-induced acetylation of histone H3 and H4 and histone acetylation increased markedly when a combination of NaB and HA was applied. Whereas the most prominent hyperacetylation after HA and NaB treatment was observed in A2780 cells, the acetylation of histones occurred in both cell lines. Conclusion: In summary, we have demonstrated the enhanced activity of HA and NaB against A2780 cancer cells, while eliciting no such effect in non-cancer MRC-5 cells.

Introduction Amaryllidaceae alkaloids represent an enormously large and ever expanding group of compounds and display a wide spectrum of favorable medicinal properties. So far, it has been shown that Amaryllidaceae alkaloids possess cytotoxic, antiproliferative,

antibacterial, antiviral, antimalarial, antifungal and acetylcholinesterase inhibitory activities (Kulhánková et al., 2013; He et al., 2015). At present, large numbers of Amaryllidaceae alkaloids have been chemically classified into diverse structure types based on biosynthetic origin (norbelladine, galanthamine, crinine, narciclasine, haemanthamine, montanine, homolycorine, lycorine and narcissidine type) and/or

Abbreviations: HA, haemanthamine; NaB, sodium butyrate; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitors; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; Chk2_Trh68, phosphorylated chk2 on threonine 68; Chk1_Ser345, phosphorylated Chk1 on serine 345; H3_K9/K14, histone H3 acetylation on lysine 9 and 14; H4_Lys12, histone H4 acetylation on lysine 12; H4_Lys16, histone H4 acetylation on lysine 16 * Corresponding author. E-mail address: [email protected] (M. Seifrtová). http://dx.doi.org/10.1016/j.phymed.2017.08.019 Received 27 February 2017; Received in revised form 3 July 2017; Accepted 18 August 2017 0944-7113/ © 2017 Elsevier GmbH. All rights reserved.

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NaB is a short-chain fatty acid formed naturally in the colon through the fermentation of dietary fibers. NaB exhibits several effects in cells, such as the inhibition of proliferation, induction of differentiation, and/ or apoptosis. In this context it is noteworthy that NaB exhibits growth inhibitory effects because of its ability to induce cell cycle arrest in many cancer cells. Similar to other potent HDAC inhibitors, NaB promotes histone hyperacetylation. It was found to be a non-competitive inhibitor of HDAC, but the exact mechanism by which butyrate inhibits HDAC activity still remains poorly understood (Davie, 2003). Although NaB has been studied in several human cancer cells, its effect in human ovarian cancer cells and in normal human fibroblasts has yet to be complemented. To summarize, HDACi have been demonstrated to have a potent antitumor efficacy and constitute a great potential for clinical application. However, as resistance to HDACi has increased because of natural selection or stable alterations that may hinder the mode of action, many research groups have focused their efforts on discovering therapeutic combinations with improved efficacy. Following our previous studies on HA, in this study we investigate whether the combination of HA and NaB could further enhance the cytostatic and/or cytototoxic effect in human ovarian carcinoma A2780 cells.

ring (skeleton) type (Unver, 2007). However, as the number of Amaryllidaceae alkaloids discovered increases, new core structures have been recognized and proposed for classification. Since Amaryllidaceae family plants are highly praised as a source of precursors with anticancer potential, haemanthamine (HA) has attracted our attention. HA belongs to the α-crinane ring type of alkaloids and represents a promising agent for pharmaceutical development because of its antioxidant, antiviral, antimalarial and anticonvulsant activities. The pronounced antiproliferative and cytotoxic action of HA has attracted serious researcher attention (Nair et al., 2016; Habartová et al., 2016). It is worth pointing out that the anticancer potential of HA has been proved in a number of different types of human cancer cells, such as T lymphoblast leukemia, promyelocytic leukemia, breast adenocarcinoma, colorectal adenocarcinoma, lymphoblastic leukemia, myelogenous leukemia, acute T cell leukemia, hepatocellular carcinoma, lung carcinoma, esophageal squamous carcinoma, brain glioma, melanoma and cervix adenocarcinoma (Antoun et al., 1993; Furusawa et al., 1980; Nair et al., 2012a, 2012b; Van Goietsenoven et al., 2010; Weniger et al., 1995; Havelek et al., 2014; Doskočil et al., 2015). Parallel research efforts to probe the mechanism behind potent cytostatic activity revealed that HA inhibits protein biosynthesis during the elongation stage of translation. Further to this, HA was shown to directly act on ribosomes by blocking the peptide bond formation step on the peptidyl transferase centre of the 60S ribosomal subunit (Nair et al., 2012b; Henry et al., 2016). A deeper understanding of the molecular mechanisms reveals that HA decreased the mitochondrial membrane potential and induced apoptosis via caspases activation in Jurkat cells. Concerning the cell cycle, application of HA decreased the percentage of S phase cells with increased p16 expression and Chk1 Ser345 phosphorylation (Havelek et al., 2014). Recently, the pharmacokinetics of HA was described after administration to rats by means of an intravenous bolus (Hroch et al., 2016). As far as we know, the cytostatic and cytotoxic effects of HA have been reported in numerous cancer cells. However, the current knowledge of how intracellular pathways regulate the fate of the cell in response to HA remains obscure. Histone acetylation and deacetylation are processes that modify the chromatin structure and are of major importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas histone deacetylation is linked to transcriptional silencing. Histone deacetylases (HDAC) remove the acetyl group from the histone, thus making the chromatin structure more compact and suppressing gene expression. An increased HDAC level was found in tumor cells, making HDAC inhibition a promising tool for cancer treatment (Lee et al., 2010). Histone deacetylase inhibitors (HDACi) have been found to cause growth arrest and/or apoptosis of many malignant cells, and the antitumor activity of HDACi has been linked to their ability to induce gene expression through the increased acetylation of histones as well as nonhistone proteins (Marks et al., 2001). HDACi have a high degree of selectivity for killing cancer cells but the mechanisms of the tumor selective action of HDACi is still for the most part unclear. Where contemporary medicine is concerned, successful HDACi have been approved by the Food and Drug Administration (FDA) as anticancer agents. For example, vorinostat and romidepsin have been used to treat patients with cutaneous T cell lymphoma, and panobinostat and vorinostat have been used to treat melanoma (Liu et al., 2016). Interestingly, recent findings suggest that HDAC inhibitors have the potential to enhance the activity of other cancer therapeutics, including radiotherapy. In this regard, Munshi and colleagues have shown the ability of NaB, including other HDACi such as phenyl butyrate, tributyrin, and trichostatin A, to radiosensitize two human melanoma cell lines (A375 and MeWo) (Munshi et al., 2005). Also, Kuribayashi and colleagues have proven that HDACi, scriptaid and trichostatin A, enhanced cell death induced by X-rays exposure in radioresistant human squamous carcinoma cells SQ-20B (Kuribayashi et al., 2010). One of the most studied HDAC inhibitors is sodium butyrate (NaB).

Materials and methods Cell cultures and culture conditions The experiments were carried out with the A2780 (ovarian carcinoma) and MRC-5 (primary human lung fibroblast) cells from the European Collection of Cell Cultures (ECACC, Salisbury, UK). A2780 cells were propagated in a RPMI 1640 medium (Biosera, Nuaille, France) supplemented with 10% (v/v) fetal bovine serum, 2 mM Lglutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). MRC-5 cells were propagated in Dulbecco's Modified Eagle's medium (Biosera, Nuaille, France) supplemented with 10% (v/v) fetal bovine serum, 2 mM Lglutamine, 10 µL/ml MEM Non-Essential Amino Acids, 50 µg/ml penicillin and 50 µg/ml streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). The cell cultures were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CO2 – 95% air. The cultures were split every third day. The cells used were in an exponential growth phase, with 20 passages being the maximum range for the cancer cell line (A2780) and 10 the maximum range of passages for the primary cell line (MRC-5).

Cell treatment Haemanthamine was kindly provided by the ADINACO Research Group from the Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hradec Králové, Charles University. The compound was isolated from fresh bulbs of Zephyranthes robusta (Amaryllidaceae) as described in Kulhánková et al. (2013). The purity of the haemanthamine was > 95% as measured by NMR. A 10 mM stock solution of haemanthamine in dimethyl sulfoxide - DMSO (SigmaAldrich, St.Louis, MO, USA) was prepared and stored at −20 °C until use. For the experiments, the stock solution was diluted with the complete culture medium to reach final concentrations. The sodium butyrate of ≥ 98.5% purity was obtained from a commercial supplier Sigma-Aldrich, St.Louis, MO, USA. The stock solution of sodium butyrate was prepared by dissolving in MiliQ water (Merck Millipore, Darmstadt, Germany) at a concentration of 5 M It was stored at −20 °C in aliquotes until further use. For the experiments, NaB was applied at the desired concentrations. Control cells were sham-treated with a DMSO vehicle only (0.1%; control).

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Cytotoxicity

Activity of caspases

The WST-1 (Roche, Mannheim, Germany) reagent was used to determine the cytostatic effect of tested compounds. WST-1 is designed for the spectrophotometric quantification of cell proliferation, growth, viability and chemosensitivity in cell populations using a 96-well-plate format (Sigma, St.Louis, MO, USA). The principle of WST-1 is based on photometric detection of the reduction of tetrazolium salt to a colored formazan product. The cells were seeded at a concentration of 5 × 103 A2780 cells/well, 3 × 103 MRC-5 cells/well in 100 µl of culture medium and were allowed to reattach overnight. Thereafter, the cells were treated with 100 µl of corresponding HA and NaB stock solutions to obtain the desired concentrations. They were then incubated in 5% CO2 at 37 °C. WST-1 reagent diluted 4-fold with PBS (50 µl) was added 24 or 48 h after treatment. Absorbance was measured after 3 h of incubation with WST-1 at 440 nm. The measurements were performed in a Tecan Infinite M200 spectrometer (Tecan Group, Männedorf, Switzerland). All experiments were performed at least three times with triplicate measurements at each drug concentration per experiment. The viability was quantified as described in Havelek et al. 2012 according to the following formula: (%) viability = (Asample - Ablank) / (Acontrol - Ablank) x 100, where A is the absorbance of the employed WST-1 formazan measured at 440 nm. The viability of the treated cells was normalized to the viability of cells treated with 0.1% DMSO (Sigma-Aldrich, St. Louis, MO, USA) as a vehicle control.

Induction of apoptosis was determined by monitoring the activities of caspase 3/7 by Caspase-Glo Assays (Promega, Madison, WI, USA) at 24 h after treatment. The assays provided proluminogenic substrates in an optimized buffer system. The addition of a Caspase-Glo Reagents resulted in cell lysis, followed by caspase cleavage of the substrate and generation of luminescent signal. The signal generated was proportional to the amount of caspase activity present. A total of 1 × 104 cells were seeded per well using a 96-well-plate format (Sigma, St.Louis, MO, USA). The treated cells were cultivated in culture medium and incubated 24 h in 5% CO2 at 37 °C. Subsequently, Caspase-Glo Assays reagents were added to each well (50 μl/well) and incubated for 30 minutes before measuring the luminescence by means of a Tecan Infinite M200 spectrometer (Tecan Group, Männedorf, Switzerland). Electrophoresis and Western blotting The cells were harvested for the preparation of whole-cell lysates using Cell Lysis Buffer (Cell Signaling Technology, Boston, MA, USA) 24 h after applying the compounds. The protein content was quantified using bicinchoninic acid (BCA) assay (Sigma-Aldrich, St. Louis, MO, USA). The lysates containing an equal amount of protein (20 μg) were loaded into each lane of polyacrylamide gel. After electrophoretic separation, the proteins were transferred to a Polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Prague, Czech Republic). The membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat dry milk and then incubated with a primary antibody (p53 - Exbio, Prague, Czech Republic; β-actin, p21WAF1/Cip1, noxa - Sigma-Aldrich, St. Louis, MO, USA; p53_Ser15 - CalbiochemMerck, Prague, Czech Republic; acetylated-Lys proteins, acetylatedH3_Lys9/14, acetylated-H4_Lys12, acetylated-H4_Lys16, Chk2_Thr68, Chk1_Ser345, puma - Cell Signaling Technology, Boston, MA, USA) at 4 °C overnight. After washing, the blots were incubated with an appropriate horseradish peroxidise (HRP)-conjugated secondary antibody - Polyclonal Goat Anti-Mouse Immunoglobulins or Polyclonal Swine Anti-Rabbit Immunoglobulins (DakoCytomation, Brno, Czech Republic) for one h at room temperature. Antigen-Antibody complexes were detected with a Chemiluminescence Detection Kit (Roche, Mannheim, Germany). The signal was quantitatively detected using a GeneSys image analysis system (Syngene, Cambridge, UK). To confirm equal protein loading each membrane was reprobed and reincubated to detect β-actin. The densities of our protein bands of interest were determined using GeneTools image analysis system (Syngene, Cambridge, UK). Densitometric analysis was performed to quantify the relative gray scale of each band blot and data were normalized to β-actin.

Proliferation and viability Cell proliferation and viability were determined 24, 48 and 72 h after treatment with HA, NaB and their combination. The cells were detached with 0.05% trypsin-EDTA (Life Technologies, Grand Island, NY, USA) for 8 minutes. The trypsin-detached cells were pooled with a medium containing floating cells. The viability and number of proliferating cells was determined using 0.4% Trypan blue (Sigma, St. Louis, MO, USA) exclusion staining. Cell counts were carried out using a Bürker chamber and Nikon Eclipse E200 light microscope (Nikon, Tokyo, Japan). Cell cycle analysis After 24 and 48 h of incubation with HA, NaB and HA+NaB, the cells were harvested and washed with ice cold PBS and fixed with 70% ethanol. For detection of the low molecular-weight fragments of DNA, the cells were incubated for 5 minutes at room temperature in a buffer (192 ml 0.2 M Na2HPO4 + 8 ml of 0.1 M citric acid, pH 7.8) and then labeled with propidium iodide in Vindelov's solution for 1 h at 37 °C (all reagents from Sigma, St. Louis, MO, USA). The DNA content was determined using the flow cytometer Dako CyAn (Beckman Coulter, Miami, FL, USA) with the excitation wave length set at 488 nm. The data were analyzed using Multicycle AV software (Phoenix Flow Systems, San Diego, CA, USA).

Statistics The descriptive statistics of the results was calculated and the charts were made in Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA). In this study, all the values were expressed as arithmetic means with standard deviation (SD) unless otherwise indicated. The significant differences between the groups were analyzed by the Student's t-test. P values ≤ 0.05 were considered significant.

Flow cytometric assay for Annexin V and propidium iodide staining Apoptosis was determined by flow cytometry using an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. The Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit employs the property of Alexa Fluor® 488 conjugated to Annexin V to bind to phosphatidylserine in the presence of Ca2+, and the property of propidium iodide (PI) to enter cells with damaged cell membranes and to bind to DNA. Measurement was performed immediately using a CyAn (Beckman Coulter, Miami, FL, USA) flow cytometer. Listmode data were analysed using Kaluza Analysis 1.3 software (Beckman Coulter, Miami, FL, USA).

Results Haemanthamine and sodium butyrate used in combination exhibited an enhanced cytotoxicity that was more pronounced in A2780 cells At first, the cytotoxicity of HA and NaB was determined by using the cell proliferation reagent WST-1. In this assay, tetrazolium salt WST-1 is reduced by the mitochondrial dehydrogenases present in living cells. Human ovarian carcinoma A2780 and fetal lung fibroblast MRC-5 cells exposed to a broad range of HA (1 – 100 μM) and NaB (1 – 100 mM) 3

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Fig. 1. Cytotoxicity of HA and NaB in a broad concentration range. The percentage of viable A2780 (A) and MRC-5 (B) cells determined by cell proliferation reagent WST-1 24 and 48 h after the application of HA in concentration range of 1 – 100 μM and NaB in concentration range of 1 – 100 mM. Cultures treated with the solvent (0.1% DMSO) were used as controls. Cisplatin at 40 μM was used as a positive control. To calculate cell viability, the value of the signal from the treated culture wells were expressed as a percentage of that of the control wells. Results are shown as mean ± SD from three independent experiments.

Fig. 2. The effect of HA and NaB treatment alone or in combination on cell survival. The A2780 (A) and MRC-5 (B) cells were treated with either 10 μM HA or 5 mM NaB or their combination for 24 h and 48 h and then cell survival was measured by WST-1 test. The vehicle controls contained 0.1% DMSO. Cisplatin at 40 μM was used as a positive control. Values are shown as means from three independent experiments; error bars represent ± SD of triplicate. To calculate cell viability, the value of the signal from the treated culture wells were expressed as a percentage of that of the control wells. * significantly different to HA alone; # - significantly different to NaB alone, (P < 0.05).

concentrations showed a time- and dose-dependent decrease in cell viability. The cytotoxic effect of the compounds was more noticeable after 48 h, when we observed a steep decrease in viable cells, i.e. up to 100 μM of HA and 100 mM of NaB (Fig. 1A, B). The results indicate a dose of HA that resulted in considerable cytotoxicity, reducing the cell viability downwards. The dose of histone deacetylase inhibitor NaB only slightly altered the amount of viable cells used for further evaluations. Thus, in subsequent experiments we studied the effects of HA at 10 μM and NaB at 5 mM first alone and then in combination. In conformity with previous cytotoxicity observations across a broad concentration range, treatment of cells with HA at 10 μM alone caused a marked decrease in viable cells in either cell type in all intervals tested. The effect of HA was more pronounced after 48 h compared to 24 h. At 24-h and 48-h intervals, the application of HA in combination with NaB resulted in significant (P < 0.05) differences in cell viability in A2780 cells when compared with both HA and NaB alone. Results after 24 h were as follows: approximately 60% of A2780 cells treated with HA alone remained viable; 85% viability was observed in cells treated with NaB only; and 42% of cells retained viability when HA and NaB were added together. After 48 h, approximately 32% of HA-treated A2780 cells remained viable, with 46% remaining viable when treated with NaB alone and 8% surviving treatment with HA dosed in combination with NaB (Fig. 2A). When compared to HA stand-alone treatment, HA+NaB combination treatment was found to be less effective in reducing the viability of MRC-5 cells than that of A2780 cells in all tested intervals. In MRC-5 cells treated for 24 h, the HA+NaB combination did not significantly reduce cell viability (42% of cells remained viable) in comparison to HA alone (45% of cells remained viable). Although the effect of HA+NaB

was more pronounced after 48 h of exposure (16% of MRC-5 remained viable), the decrease in the percentage of viable cells was not statistically significant in comparison to HA stand-alone treatment (25% of MRC-5 remained viable). When combined, however, HA+NaB reduced the amount of viable cells more effectively (P < 0.05) than NaB alone (Fig. 2B). The WST-1 assay does not discern the induction of cell death from the inhibition of proliferation, therefore additional tests aimed at clarifying the effects on cell proliferation and viability were performed. The combination of haemanthamine and sodium butyrate caused a significant decrease in the proliferation of A2780 cells First, the numbers of proliferating cells unstained by trypan blue were counted 24, 48 and 72 h after cell exposure to HA at 10 µM, NaB at 5 mM and their combination. After 24 h, the application of HA at 10 µM alone led to a decrease in the relative proliferation of A2780 cells (53% proliferation rate in comparison to the 100% rate of the control). Treatment with NaB at 5 mM for 24 h caused a decrease in proliferation (69%) as well. However, the effect was strongly enhanced when the agents were combined (37%). The response of the cells to HA, NaB and HA+NaB was considerably pronounced after 48 and 72 h After 48 h, we observed 35% proliferation of cells after HA treatment, 25% after NaB treatment and 12% after the application of HA and NaB together. Similarly, the effect was amplified after 72 h, where HA caused a decrease in cell proliferation to a rate of 20%, NaB to 7% and their combination to 3%. 4

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Fig. 4. The effect of HA, NaB and their combination on cell viability. The viability of A2780 (A) and MRC-5 (B) cells was determined by Trypan blue exclusion staining 24, 48 and 72 h following treatment with 10 μM HA and 5 mM NaB alone or in combination. The vehicle controls contained 0.1% DMSO. Cisplatin at 40 μM was used as a positive control. Values are shown as means from three independent experiments; error bar represents ± SD of the triplicate. * - significantly different to HA alone; # - significantly different to NaB alone, (P < 0.05).

Fig. 3. The effect of HA and NaB alone or both agents exposure on cell proliferation. The proliferation of A2780 (A) and MRC-5 (B) cells were determined by Trypan blue exclusion staining 24, 48 and 72 h following treatment with HA at 10 μM and NaB at 5 mM alone or in combination. The vehicle controls contained 0.1% DMSO. Cisplatin at 40 μM was used as a positive control. To calculate relative proliferation, the percentages of the proliferating cells from the treated groups were expressed as a percentage of that of the control group. Values are shown as means from three independent experiments; error bar represents ± SD of triplicate. * - significantly different to HA alone; # - significantly different to NaB alone, (P < 0.05).

cells, the administration of HA at 10 µM and NaB at 5 mM in combination resulted in a significant (P < 0.05) decrease in A2780 cell viability compared to HA (at 48 and 72 h) or NaB (at 72 h) stand-alone treatments. After 48 h of treatment, A2780 cell viability was 97% for HA alone, 79% for NaB alone, and 71% for HA+NaB. At 72 h viability values were 91% for HA, 58% for NaB and 35% for HA+NaB (Fig. 4A). In MRC-5 cells, viability insignificantly varied from about 83% to 100% in the control as well as in HA and NaB-treated cells (either alone or in combination) across all of the evaluated intervals (Fig. 4B). In contrast to A2780 cells, a gradual decrease in MRC-5 cell viability was not observed in HA+NaB exposed cells (83% and 84% cell viability at 24- and 48-h intervals, respectively). Taken together, the proportions of viable MRC-5 cells exposed for 48 h to evaluated compounds were 85% for HA, 95% for NaB, and 83% for HA+NaB. Further treatment for 72 h showed little or no additional enhancement, giving cell viability 92% for HA, 81% for NaB, and 84% for HA+NaB. Notably, although HA treatment alone at 10 µM inhibited the proliferation of both A2780 and MRC-5 cells, cell viability was not seriously affected within the evaluated intervals. In contrast to HA, both the viability and proliferative capacity were reduced by NaB stand-alone treatment in A2780 cells. To further understand the functional mechanisms of HA and NaB in inducing cell death, we explored the caspases −3/7 activity, and Annexin V and PI binding after treatment. Twenty-four hours exposure to HA and NaB resulted in a significant increase in caspases −3/7 activity in both cell lines compared with 0.1% DMSO control (Supplementary Fig. 1). Similarly, the exposure of A2780 and MRC-5 to HA and NaB in combination for 48 h caused considerable decrease in viable cells and increase in apoptotic cells. Moreover, the viability of A2780 cells was slightly reduced after HA single-agent treatment (Supplementary Fig. 2A, B). In view of the further aspects of combination treatments,

Thus, the pairing of HA+NaB led to a significant (P < 0.05) inhibition of A2780 cell proliferation compared to either HA or NaB alone during the entire study period (Fig. 3A). Similarly, where human MRC-5 fibroblasts are concerned, HA in combination with NaB caused a decrease in the relative proliferation within 24, 48 and 72 h (24 h - 54% HA, 64% NaB, 43% HA+NaB; 48 h – 33% HA, 40% NaB, 26% HA+NaB; 72 h - 25% HA, 20% NaB, 11% HA+NaB). Regarding sensitivity to the antiproliferative action, NaB alone inhibited cell growth more strongly in A2780 cancer cells over 24 h and 48 h periods (relative proliferation rates of 25% and 7%, respectively) than in MRC-5 non-cancer cells (relative proliferation rates of 40% and 20%, respectively). The application of HA and NaB together led to a significant (P < 0.05) decrease in proliferation compared to HA alone only 24 h after treatment. The combination of HA+NaB did not significantly change the proliferation in comparison to HA and NaB alone in MRC-5 cells after 48 and 72 h of treatment (Fig. 3B). Overall, MRC-5 cells were less sensitive to HA+NaB combination treatment (in comparison to HA or NaB alone) than A2780 cancer cells. Combined treatment with haemanthamine and sodium butyrate significantly reduced the viability of A2780 cells Plasma membrane integrity is a mandatory condition for cell proliferation. To further explore the mechanisms of HA and NaB singleagent or combination treatment-induced antiproliferative activity, the proportion of viable cells in a cell population was determined by the trypan blue exclusion test. By determining the percentage of viable 5

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Fig. 5. The effect of HA, NaB and their combination on the levels of cell cycle-regulating and epigenetic modifications marker proteins. Protein changes 24 h after application of 10 µM HA and 5 mM NaB alone or in combination in A2780 and MRC-5 cells were measured by Western blotting. The vehicle controls (C) contained 0.1% DMSO as a vehiculum. The amount of proteins in blot bands were analysed by densitometry. The quantitative data are shown as relative intensity of each protein band in arbitrary units that was normalized to β-actin. Representative blots and quantitative analysis of proteins from three independent experiments are shown (n = 3).

after either treatment in both cell types (data not shown). To explore whether HA and NaB modulate histone acetylation levels, the 24-h treated A2780 and MRC-5 cells were examined for panacetylated histone H3 and H4 lysine residues, and for specific K9 (H3), K14 (H3), K12 (H4), and K16 (H4) acetylation by Western blotting. Consistent with previous findings on the HDACi activity of NaB, NaB stand-alone treatment induced histone H3 acetylation on lysine 9 and 14, and histone H4 acetylation on lysine 12 and 16 in A2780 as well as in MRC-5 cells. As a matter of interest, the acetylation of histones was markedly pronounced after combined treatment with NaB and HA. Although Western blot analysis is only semiquantitative, the most prominent hyperacetylation was seen when the NaB+HA-treated A2780 cells were determined. In summary, although exposure to NaB alone resulted in the induction of the p21WAF1/Cip1 either in A2780 or in MRC-5 cells, the levels of Chk2 phosphorylated on threonine 68 (Chk2_Thr68) and Chk1 phosphorylated on serine 345 (Chk1_Ser345) were markedly increased only in A2780 cells. Contrary to the elevated levels of proteins driving the cell cycle transitions found after NaB stand-alone treatment, the combination of HA and NaB apparently downregulated the amount of p21WAF1/Cip1 in both A2780 and MRC-5 cells, whereas Chk2_Thr68 and Chk1_Ser345 were obviously decreased in A2780 cells. In the upcoming analyses, the known inhibitor of HDAC, i.e. NaB, showed increases in histone acetylation in both A2780 and MRC-5 cells. However, surprisingly, we identified an even higher accumulation of acetylated histones by treating either A2780 or MRC-5 cells with HA+NaB together rather than with NaB alone (Fig. 5).

results indicate that HA+NaB enhances cytotoxicity synergistically rather than additively, especially in the later interval of 72 h, while the inhibitory effect of these two tested compounds in combination were more pronounced in A2780 cancer cells in comparison to MRC-5 noncancer cells.

Haemanthamine increased sodium butyrate-induced acetylation of histones, but it attenuated the activation of check-point kinases determined in A2780 and upregulation of p21WAF1/Cip1 determined in both A2780 and MRC-5 cells We focused on the activation of DNA-damage response proteins to further clarify the mechanisms underlying the inhibition of proliferation as well as the downregulation of viability. Changes in protein expression and/or activation were analyzed 24 h after HA, NaB and HA+NaB treatment. We determined the upregulation of cell cycle regulatory proteins such as the inhibitor of cyclin dependent kinases p21WAF1/Cip1 and the activation of check point kinases Chk1 and Chk2. It was previously shown that the acetylation of histones by NaB treatment lead to the chromatin opening and activation of p21WAF1/Cip1. Protein p21WAF1/Cip1 inhibits cyclin-dependent kinases and thereby interrupts the transition of cells to the S phase and arrests cell proliferation (Davie, 2003). In view of this, we sought to investigate the effect of NaB treatment when used in combination with HA. As expected, the amount of p21WAF1/Cip1 increased rapidly in A2780 and MRC-5 cells after treatment with NaB alone. Contrary thereto, HA alone did not cause activation of protein p21WAF1/Cip1. Surprisingly, the level of p21WAF1/Cip1 considerably decreased after treatment with NaB in combination with HA either in A2780 or in MRC-5 cells. We observed a similar trend in the activation of Chk2, which arrests or delays the cell cycle at G1/S or G2/M (Zannini et al., 2014). Unexpectedly, although Chk2 was phosphorylated on threonine 68 after treatment with NaB alone and phosphorylation was suppressed after the application of NaB +HA in A2780 cells, this was not observed in MRC-5 cells. Indeed, Chk1 was phosphorylated following NaB treatment on serine 345 in A2780 cells but not in MRC-5 cells. Corroborating our findings on Chk2, the amount of serine 345-phosporylated checkpoint kinase Chk1 was also obviously downregulated after NaB+HA combination treatment in A2780 cells. On the other hand, upregulation of proteins from the Bcl-2 family, p53 and its phosphorylation on serine 15 was not observed 24 h

Haemanthamine in combination with sodium butyrate increased the percentage of S- and G2-phase cells with a concomitant decrease in the proportion of cells in the G1 phase In view of this, we examined the effect of HA at 10 µM and NaB at 5 mM and their combination on the distribution of cells in the various stages of the cell cycle following 24 and 48 h of treatment. Generally, NaB (and HA to some extent) applied alone for 24 h induced an increase in G1-phase cells accompanied by a decrease of cells in the S phase in both tested cell lines. In parallel experiments, the simultaneous combination of NaB with HA lead to a significant decrease (P < 0.05) in G1phase cells, while such a decrease was accompanied by a statistically 6

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Fig. 6. Cell cycle analysis after 24 h of treatment with HA and NaB alone or in combination. Distribution of cells in the various phases of the cell cycle 24 h after treatment with 10 μM HA, 5 mM NaB or their combination were analysed by flow cytometry. The graphs represent the percentage of cycling cells in the G1, S and G2 phase in A2780 (A) and MRC-5 (B) cells. Results are shown as mean ± SD from three experiments. * - significantly different to HA alone; # - significantly different to NaB alone, (P < 0.05).

Fig. 7. Cell cycle analysis after 48 h of treatment with HA and NaB alone or in combination. Distribution of cells in the various phases of the cell cycle 48 h after treatment with 10 μM HA, 5 mM NaB or their combination were analyzed by flow cytometry. The graphs represent the percentage of the cells in the G1, S and G2 phase in A2780 (A) and MRC-5 (B) cells. Results are shown as mean ± SD from three experiments. * - significantly different to HA alone; # - significantly different to NaB alone, (P < 0.05).

Taken together, the combination of NaB and HA significantly altered cell cycle distribution of A2780 and MRC-5 cells compared to NaB or HA alone. Moreover, dramatic alterations in the cycle-regulatory proteins level detected by Western blot analysis reflect the consequences of cell cycle arrest.

significant accumulation of cells in the S and G2 phase. Notably, 24 h after the application of NaB, 64% of A2780 and 74% of MRC-5 cells were arrested in the G1 phase, whereas the 0.1% DMSO controls maintained 53% of A2780 and 58% of MRC-5 cells in the G1 phase. In this regard, HA treatment alone caused less considerable changes in cell cycle distribution in comparison to sham treated controls. However, the combination of NaB and HA resulted in (P < 0.05) the accumulation of A2780 cells in the S and G2 phase versus NaB or HA dosed alone (HA+NaB 63%; HA 41%; NaB 36%, control 47%) (Fig. 6A, B). Similar results were observed in MRC-5 cells (HA+NaB 61%; HA 38%; NaB 27%, control 42%). In the later interval of 48 h, a minimal treatment effect with NaB alone was seen, redistributing 67% of A2780 (control 70%) and 64% of MRC-5 (control 64%) cells in the G1 phase. Conversely, the combination of NaB and HA caused significant (P < 0.05) changes in cell cycle distribution, accumulating the A2780 cells in the S and G2 phase (HA+NaB 66%; HA 40%; NaB 33%, control 30%). A similar trend was seen in MRC-5 cells (HA+NaB 47%; HA 15%; NaB 36%, control 36%) (Fig. 7A, B).

Discussion HA, a naturally occurring isoquinoline alkaloid of the Amaryllidaceae plant family, has been shown to suppress the growth of a variety of human tumor cell lines and is among the most promising candidates for anticancer therapy. Similarly, histone deacetylase inhibitors (HDACi) represent promising anticancer tools alone or in combination with established chemotherapy agents, and the clinically proven mode of action has drawn significant attention from the scientific community. Among these, NaB, a short-chain fatty acid that functions as HDACi, suppresses proliferation and induces the cell death of cancer cells. With the continuing need for novel therapeutic 7

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population of cells in the G1 phase in A375 and MeWo melanoma cell lines. Contrary to NaB, less diverse changes in the cell cycle were observed in HA-treated A2780 and MRC-5 cells. Although the differences in the percentage of cells were small, they generally correlated with the previous observations on cell cycle perturbation following HA treatment (Havelek et al., 2014). HA alone at 10 µM did not exhibit considerable cytotoxicity in both A2780 and MRC-5 cells up to 72 h. Inversely, NaB at 5 mM showed cytotoxicity only in A2780 cancer cells, which was further enhanced when treated simultaneously with HA. In these evaluations, the administration of HA and NaB in combination showed a clear time-dependent relationship between the decrease in A2780 cell viability and the administration of HA and NaB in combination. In contrast to A2780 cells, a gradual decrease of cell viability was not observed in MRC-5 cells simultaneously exposed to HA+NaB. In fact, the apoptosis-mediated cell death activity of NaB has been shown in many cancer cells, including UCD-Mel-N, A375, and SB2 melanoma cells (Bandyopadhyay et al., 2004), human leukemia lymphoblasts (Bernhard et al., 1999) colorectal (Hague et al., 1995), breast (Mandal and Kumar, 1996), and hepatic cell lines (Wang et al., 1998). In these above mentioned studies, the NaB-induced cell death was closely linked with the down-regulation of protein expression in the Bcl-2 family, which is involved in the regulation of apoptosis in mammalian cells. In the study of Bandyopadhyay et al. (2004), the lowest concentration of NaB with the ability to induce cell death was found to be 1 mM. In this study, the apoptosis of melanoma cells induced by 2 to 8 mM of NaB was detectable 24 h post-treatment. In the study of Xie et al. (2016), NaB suppressed the proliferation of osteosarcoma U2OS and MG63 cells and promoted cell death through apoptosis at a concentration of 64 µM at 48 h post treatment. NaB promoted programmed cell death through changes in the levels of apoptosis-related proteins Bcl-2 and Bax, where U2OS and MG63 cells had a lower expression level of the Bcl-2 protein and a higher expression level of the Bax protein. The study demonstrated that the biochemical mechanism of NaB action depends, at least in part, on the Mdm2–p53 signaling pathway. Both cell types have been significantly decreased in Mdm2 expression and increased in p53 expression (Xie et al., 2016). In our study, the short-term 24 h treatment with NaB at 5 mM did not induce cell death in A2780 and MRC-5 cells; we observed an inhibition of cell growth with no apparent loss of viability. After 24-h incubation, we did not observe changes in the levels of pro-apoptotic proteins from the Bcl2 family nor in p53 levels. The effect of NaB seems to be p53 independent in A2780 and MRC-5 cells, or perhaps p53 up-regulation and/or activation occurs in later intervals of treatment. In our study, p21WAF1/Cip1 protein expression was induced in both tested cell lines treated with NaB. Our results correspond well with Munshi et al. (2005), who observed an increased level of p21WAF1/Cip1 in MeWo and A375 melanoma cell lines and in the normal fibroblasts treated with NaB irrespective of their p53 status. Interestingly, the combination of NaB with HA suppressed the expression of p21WAF1/Cip1 in A2780 as well as in MRC-5 cells. In this context, the suppressed p21WAF1/Cip1 expression in A2780 and MRC-5 after HA+NaB was largely responsible for a decreased percentage of G1-phase and an increased percentage of S- and G2-phase cells. Moreover, NaB treatment alone induced the phosphorylations of Chk2 on threonine 68 and Chk1 on serine 345 in A2780 cells, whereas co-treatment with HA suppressed these phosphorylations. We did not observe any phosphorylations of Chk2 and Chk1 in MRC-5 cells after NaB treatment. These phosphorylated checkpoint kinases are elevated in response to DNA insults. ATR (Ataxia telangiectasia- and Rad3- related) and ATM (Ataxia telangiectasia mutated) kinase substrates Chk1 and Chk2 allow cells to delay not only progression through the cell cycle but also DNA repair and possibly cell death. Upon activation, Chk1 and Chk2 phosphorylate downstream targets such as the Cdc25 phosphatases, thus further propagating checkpoint signaling and leading to S and G2 phase arrest (Ronco et al., 2017). Inhibition of checkpoint kinases activation

approaches to treat cancer, the chemical diversity of molecular actions derived from natural products is becoming increasingly relevant to drug discovery. Thus, the main purpose of this study was to determine the anticancer potential of HA, NaB and their combination in human A2780 ovarian cancer cells and compare it with the reaction of non-cancer human MRC-5 fibroblasts. Furthermore, since the knowledge base of the molecular mechanisms of the growth inhibition or cytotoxicity of HA is still in its infancy, we aim to evaluate the ability of HA to influence the effect of NaB on intracellular signaling pathways. In our previous study, we elucidated the anticancer potential of HA in Jurkat leukemia cells. The results revealed that HA treatment (15 µM) caused the inhibition of proliferation and accumulation of cells preferentially at the G1 and G2 phase as well as causing the induction of apoptosis in leukemia cells (Havelek et al., 2014). Our results correspond with the studies on HA cytotoxicity conducted by other researchers. Although there have been reports of the cytotoxic activity of HA in a number of different types of cancer cells (Antoun et al., 1993; Furusawa et al., 1980; Nair et al., 2012a, 2012b; Van Goietsenoven et al., 2010; Weniger et al., 1995; Havelek et al., 2014; Doskočil et al., 2015), there is scant information on the effect of HA in human ovarian cancer cells. Our results extended these findings and showed that HA at 10 μM inhibited the growth of A2780 cancer cells as well as MRC-5 noncancer cells. NaB is known to inhibit cell growth because of its ability to induce cell cycle arrest. Our data reinforced the well documented antiproliferative activity of NaB. However, our results have more value considering the higher capability of suppressing cell proliferation when NaB is combined with HA. Additionally, our study has demonstrated for the first time the effect of HA+NaB combination treatment compared to HA stand-alone treatment. HA+NaB combination treatment showed a decreased antiproliferative effect in MRC-5 cells compared to A2780 cells in all WST-1-assay tested intervals. Our study also sought to determine whether HA, NaB or their combination cause cell death. For this purpose, viability was determined using Trypan blue staining. Our efforts show that there is a generally higher rate of cell death in A2780 cells treated with HA and NaB in combination compared to HA or NaB stand-alone treatments. What is interesting is that in normal human fibroblast MRC-5 cells, HA+NaB exposure induces death to a lesser extent compared to A2780 cells. Our findings thus are consistent with previous reports on the cancer cell-specificity of some Amaryllidaceae alkaloids (Nair et al., 2016; Doskočil et al., 2015) and illustrate well that the HA+NaB combination exhibits an enhanced cytotoxicity in A2780 cells while eliciting no such effect in non-cancer cells. There is great demand for new potential chemotherapeutic agents and our results suggest that HA may be more valuable in combinatorial therapy with other therapeutic drugs, including NaB. Next, we examined the effect of NaB in combination with HA on cell cycle progression. We observed the differences in the distribution of cells in various phases of the cell cycle in comparison to NaB or HA tested alone. In both cell lines, we observed a clear suppression of cells in the G1 phase and an increase in the population of cells in the G2 phase after NaB and HA combination treatment. Following NaB standalone treatment, we observed an accumulation of cells in the G1 phase, a slight decrease in cells in the S phase accompanied by an increase in cells in the G2 phase in both cell types. As previously reported in many experimental studies, NaB is a potent inducer of growth arrest. NaB exhibits growth inhibitory effects because of its ability to induce cell cycle arrest mostly in the G1 phase. Similar to our results, NaB has been shown to induce growth inhibition in the G1 phase in PC3, TSU-Pr1 and LNCaP prostate cancer cells (Maier et al., 2000) and in L1210 murine leukemia cells (Darzynkiewicz et al., 1981). NaB induces the accumulation of cells in the G1 and G2 phase, with a decrease in cells in the S phase in human MCF-7 breast cancer cells and human HEK293 embryonic kidney non-cancer cells (Li et al., 2015). Our observations on the cell-cycle redistribution of NaB-treated cells are consistent with the experiments of Munshi and colleagues (Munshi et al., 2005), who observed a suppression of cells in the S phase and an increase in the 8

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which works by altering the magnitude of histone acetylation and by inhibiting checkpoint kinase activation. Furthermore, our findings support the synergistic potential of HA and NaB application to selectively impair cancer cell viability and proliferation.

abrogates cell cycle arrest, forcing cells to enter mitosis prior to completion of DNA damage repair (Garrett and Collins, 2011; Isono et al., 2017). Since NaB exerted negligible effects on Chk1_Ser345 and Chk2_Thr68 phosphorylation in MRC-5 cells, Chk1 and Chk2 were markedly phosphorylated in A2780 cells, returning to near control levels after NaB and HA co-treatment. Their diverse activation in cells raises the possibility of the involvement of Chk1 and Chk2 in the HA+NaB course of pronounced bioactivity in A2780 cells. In addition, it seems reasonable to assume that HA+NaB co-treatment-associated down-regulation of Chk1_Ser345 and Chk2_Thr68 might be responsible for pronounced perturbation of cell cycle progression in A2780 ovarian cancer cells. The opposing effect of NaB alone or in combination with HA on signal transduction pathways that control cell cycle arrest provides an important molecular insight into the observed differential efficacy in A2780 and MRC-5 cells. NaB alone induced histone H3 and H4 acetylation in A2780 as well as in MRC-5 cells. Similarly in the study of Bandyopadhyay et al. (2004), NaB at 5 mM increased histone H3 and H4 acetylation within 24 h of exposure in UCD-Mel-N, A375 and SB2 melanoma cells. It is known that HDAC inhibition is associated with the modulation of critical cell cycle regulators, such as p21WAF1/Cip1 and p27KIP1 (Chen et al., 2003). Hyperacetylation of histones by NaB supports chromatin opening and induces the expression of gene encoding p21WAF1/Cip1. Protein p21WAF1/Cip1 inhibits cyclin E-Cdk activity and thereby halts the transition of cells to the S phase of the cell cycle and stops cell proliferation in the G1 phase independently on p53 (Davie, 2003). In accordance with this fact, Munschi et al. (2005) have shown that histone H4 hyperacetylation was accompanied with the upregulation of p21WAF1/Cip1 and Bax in human melanoma cell lines (A375 and MeWo) and normal human lung fibroblasts (MRC-9). In our study, the acetylation of histones H3 and H4 was markedly enhanced after simultaneous application of NaB together with HA compared with those produced by either drug alone. Surprisingly, such increased histone acetylation, which was more pronounced in A2780 cells, did not induce the increase in the levels of p21WAF1/Cip1, regardless of cell type. Considering previous reports showing that excessive histone acetylation is at least partly responsible for NaB-induced cell death, our observations indicate that enhanced histone acetylation after HA+NaB combination treatment may exacerbate a negative effect on A2780 cell viability where increased histone acetylation predominates. The observed epigenetic modulation would seemingly correspond with the recent findings on the potential role of isoquinoline alkaloid berberine in human cells. As Wang and colleagues evidenced, berberine induces histone acetylation and may result in the activation of genes that are responsible for growth suppression and apoptosis in leukemia cells (Wang et al., 2016). Concluding our work, we explored whether HA+NaB co-administration is capable of enhancing the antiproliferative activity and cytotoxicity of NaB or HA in A2780 cancer and MRC-5 non-cancer cells and we looked into the underlying mechanisms. NaB as well as HA alone led to a decrease in relative proliferation, but this effect was significantly enhanced after the application of both agents. Generally, this growthinhibitory action was more obvious in A2780 cells than in MRC-5 cells. Compared to MRC-5 cell stand-alone treatment using either NaB or HA, HA+NaB combination treatment generally led to enhanced MRC-5 cell death, however to a lesser extent (compared to the other tested A2780 cell line), and the increase did not reach any statistical significance. As expected, there was an increase in p21WAF1/Cip1 and histone acetylation after NaB stand-alone treatment. Since Chk1_Ser345 and Chk2_Thr68 were upregulated only in A2780 cells after NaB treatment, NaB failed to activate these DNA-damage response kinases in MRC-5 cells. The combination of HA and NaB caused an increase in NaB-induced histone acetylation (in both A2780 and MRC-5 cells) and a decrease in Chk1_Ser345, Chk2_Thr68 (in A2780 cells) and p21WAF1/Cip1 (in both A2780 and MRC-5 cells). Altogether, our data demonstrated the cooperative cytotoxicity and antiproliferative potency of HA and NaB

Conflict of interest The authors declare that there are no conflicts of interest to reveal. Acknowledgments The skillful technical assistance of Mrs. Božena Janská is greatly acknowledged. The authors are indebted for the financial support offered through the grants Progres Q40, SVV-260397/2017 and grant Nr. 17/2012/UNCE, programs of Charles University. Supplementary materials Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed.2017.08. 019. References Antoun, M.D., Mendoza, N.T., Ríos, Y.R., Proctor, G.R., Wickramaratne, D.B., Pezzuto, J.M., Kinghorn, A.D., 1993. Cytotoxicity of Hymenocallis expansa alkaloids. J. Nat. Prod. 56 (8), 1423–1425. Bandyopadhyay, D., Mishra, A., Medrano, E.E., 2004. Overexpression of histone deacetylase 1 confers resistance to sodium butyrate-mediated apoptosis in melanoma cells through a p53-mediated pathway. Cancer Res. 64 (21), 7706–7710. Bernhard, D., Ausserlechner, M.J., Tonko, M., Löffler, M., Hartmann, B.L., Csordas, A., Kofler, R., 1999. Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts. FASEB J. 13 (14), 1991–2001. Chen, J.S., Faller, D.V., Spanjaard, R.A., 2003. Short-chain fatty acid inhibitors of histone deacetylases: promising anticancer therapeutics? Curr. Cancer Drug Targets. 3 (3), 219–236. Darzynkiewicz, Z., Traganos, F., Xue, S.B., Melamed, M.R., 1981. Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells. Exp. Cell Res. 136 (2), 279–293. Davie, J.R., 2003. Inhibition of histone deacetylase activity by butyrate. J. Nutr. 133, 2485S–2493S (7 Suppl). Doskočil, I., Hošťálková, A., Šafratová, M., Benešová, N., Havlík, J., Havelek, R., Kuneš, J., Královec, K., Chlebek, J., Cahlíková, L., 2015. Cytotoxic activities of Amaryllidaceae alkaloids against gastrointestinal cancer cells. Phytochem. Lett. 13, 394–398. Furusawa, E., Irie, H., Combs, D., Wildman, W.C., 1980. Therapeutic activity of pretazettine on Rauscher leukemia: comparison with the related Amaryllidaceae alkaloids. Chemotherapy. 26 (1), 36–45. Garrett, M.D., Collins, I., 2011. Anticancer therapy with checkpoint inhibitors: what, where and when? Trends Pharmacol. Sci. 32 (5), 308–316. Habartová, K, Cahlíková, L, Řezáčová, M, Havelek, R, 2016. The biological activity of alkaloids from the Amaryllidaceae: from Cholinesterases inhibition to anticancer activity. Nat. Prod. Commun. 11 (10), 1587–1594. Hague, A., Elder, D.J., Hicks, D.J., Paraskeva, C., 1995. Apoptosis in colorectal tumor cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate. Int. J. Cancer. 60 (3), 400–406. Havelek, R., Seifrtova, M., Kralovec, K., Bruckova, L., Cahlikova, L., Dalecka, M., Vavrova, J., Rezacova, M., Opletal, L., Bilkova, Z., 2014. The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53negative human leukemic Jurkat cells. Phytomedicine. 21 (4), 479–490. Havelek, R., Siman, P., Cmielova, J., Stoklasova, A., Vavrova, J., Vinklarek, J., Knizek, J., Rezacova, M., 2012. Differences in vanadocene dichloride and cisplatin effect on MOLT-4 leukemia and human peripheral blood mononuclear cells. Med. Chem. 8 (4), 615–621. He, M., Qu, C., Gao, O., Hu, X., Hong, X., 2015. Biological and pharmacological activities of amaryllidaceae alkaloids. RSC Adv. 21 (5), 16562–16574. Henry, S., Kidner, R., Reisenauer, M.R., Magedov, I.V., Kiss, R., Mathieu, V., Lefranc, F., Dasari, R., Evidente, A., Yu, X., Ma, X., Pertsemlidis, A., Cencic, R., Pelletier, J., Cavazos, D.A., Brenner, A.J., Aksenov, A.V., Rogelj, S., Kornienko, A., Frolova, L.V., 2016. 5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells. Eur. J. Med. Chem. 120, 313–328. Hroch, M., Mičuda, S., Havelek, R., Cermanová, J., Cahlíková, L., Hošťálková, A., Hulcová, D., Řezáčová, M., 2016. LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study. Biomed. Chromatogr. 30 (7), 1083–1091. Isono, M., Hoffmann, M.J., Pinkerneil, M., Sato, A., Michaelis, M., Cinatl Jr., J., Niegisch, G., Schulz, W.A., 2017. Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine. J. Exp. Clin. Cancer Res. 36 (1), 1.

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Phytomedicine 35 (2017) 1–10

M. Seifrtová et al.

Nair, J.J., Rárová, L., Strnad, M., Bastida, J., van Staden, J., 2012a. Apoptosis-inducing effects of distichamine and narciprimine, rare alkaloids of the plant family Amaryllidaceae. Bioorg. Med. Chem. Lett. 22 (19), 6195–6199. Nair, J.J., van Staden, J., Bastida, J., 2016. Apoptosis-inducing effects of Amaryllidaceae alkaloids. Curr. Med. Chem. 23 (2), 161–185. Ronco, C, Martin, AR, Demange, L, Benhida, R, 2017. ATM, ATR, CHK1, CHK2 and WEE1 inhibitors in cancer and cancer stem cells. Med. Chem. Commun. http://dx.doi.org/ 10.1039/C6MD00439C. Unver, N., 2007. New skeletons and new concepts in Amaryllidaceae alkaloids. Phytochem. Rev. 6 (1), 125–135. Van Goietsenoven, G., Andolfi, A., Lallemand, B., Cimmino, A., Lamoral-Theys, D., Gras, T., Abou-Donia, A., Dubois, J., Lefranc, F., Mathieu, V., Kornienko, A., Kiss, R., Evidente, A., 2010. Amaryllidaceae alkaloids belonging to different structural subgroups display activity against apoptosis-resistant cancer cells. J. Nat. Prod. 73 (7), 1223–1227. Wang, X.M., Wang, X., Li, J., Evers, B.M., 1998. Effects of 5-azacytidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines. Ann. Surg. 227 (6), 922–931. Wang, Z., Liu, Y., Xue, Y., Hu, H., Ye, J., Li, X., Lu, Z., Meng, F., Liang, S., 2016. Berberine acts as a putative epigenetic modulator by affecting the histone code. Toxicol. In Vitro. 36, 10–17. Weniger, B., Italiano, L., Beck, J.P., Bastida, J., Bergoñon, S., Codina, C., Lobstein, A., Anton, R., 1995. Cytotoxic activity of Amaryllidaceae alkaloids. Planta. Med. 61 (1), 77–79. Xie, C., Wu, B., Chen, B., Shi, Q., Guo, J., Fan, Z., Huang, Y., 2016. Histone deacetylase inhibitor sodium butyrate suppresses proliferation and promotes apoptosis in osteosarcoma cells by regulation of the MDM2-p53 signaling. Oncol. Targets Ther. 9, 4005–4013. Zannini, L., Delia, D., Buscemi, G., 2014. CHK2 kinase in the DNA damage response and beyond. J. Mol. Cell Biol. 6 (6), 442–457.

Kulhánková, A., Cahlíková, L., Novák, Z., Macáková, K., Kuneš, J., Opletal, L., 2013. Alkaloids from Zephyranthes robusta BAKER and their acetylcholinesterase- and butyrylcholinesterase-inhibitory activity. Chem. Biodivers. 10 (6), 1120–1127. Kuribayashi, T., Ohara, M., Sora, S., Kubota, N., 2010. Scriptaid, a novel histone deacetylase inhibitor, enhances the response of human tumor cells to radiation. Int. J. Mol. Med. 25 (1), 25–29. Lee, J.H., Choy, M.L., Ngo, L., Foster, S.S., Marks, P.A., 2010. Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair. Proc. Natl. Acad. Sci. USA 107 (33), 14639–14644 Aug 17. Li, L., Sun, Y., Liu, J., Wu, X., Chen, L., Ma, L., Wu, P., 2015. Histone deacetylase inhibitor sodium butyrate suppresses DNA double strand break repair induced by etoposide more effectively in MCF-7 cells than in HEK293 cells. BMC Biochem. 16, 2. http://dx. doi.org/10.1186/s12858-014-0030-5. Liu, J., Gu, J., Feng, Z., Yang, Y., Zhu, N., Lu, W., Qi, F., 2016. Both HDAC5 and HDAC6 are required for the proliferation and metastasis of melanoma cells. J. Transl. Med. 14 (1), 7. Maier, S., Reich, E., Martin, R., Bachem, M., Altug, V., Hautmann, R.E., Gschwend, J.E., 2000. Tributyrin induces differentiation, growth arrest and apoptosis in androgensensitive and androgen-resistant human prostate cancer cell lines. Int. J. Cancer 88 (2), 245–251. Mandal, M, Kumar, R, 1996. Bcl-2 expression regulates sodium butyrate-induced apoptosis in human MCF-7 breast cancer cells. Cell Growth Differ. 7 (3), 311–318 Mar. Marks, P., Rifkind, R.A., Richon, V.M., Breslow, R., Miller, T., Kelly, W.K., 2001. Histone deacetylases and cancer: causes and therapies. Nat. Rev. Cancer 1 (3), 194–202. Munshi, A., Kurland, J.F., Nishikawa, T., Tanaka, T., Hobbs, M.L., Tucker, S.L., Ismail, S., Stevens, C., Meyn, R.E., 2005. Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity. Clin. Cancer Res. 11 (13), 4912–4922. Nair, J.J., Bastida, J., Viladomat, F., van Staden, J., 2012b. Cytotoxic agents of the crinane series of amaryllidaceae alkaloids. Nat. Prod. Commun. 7 (12), 1677–6188.

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