Peloruside A-Induced Cell Death in Hypoxia Is p53 Dependent in

Note Cite This: J. Nat. Prod. 2018, 81, 634−640

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Peloruside A‑Induced Cell Death in Hypoxia Is p53 Dependent in HCT116 Colorectal Cancer Cells Jiří Ř ehulka,†,§ Narendran Annadurai,†,§ Ivo Frydrych,† Petr Džubák,† John H. Miller,‡ Marián Hajdúch,† and Viswanath Das*,† †

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 5, 77900 Olomouc, Czech Republic ‡ School of Biological Sciences and Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand ABSTRACT: HCT116 colorectal cancer cell sensitivity to peloruside A (PLA) in normoxia is not altered by hypoxia preconditioning of the cells. We examined whether the PLA effects were altered in hypoxia and whether the activity was dependent on p53. The cytotoxicity of PLA in wild-type HCT116 cells was largely unaffected by hypoxia; however, cells in which p53 was knocked out showed resistance. Knockout of the p21 gene had little effect on the activity of PLA in hypoxia. It was concluded that the response of cells to the microtubule-stabilizing agent PLA under hypoxic conditions is a p53-dependent process.

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arrest, rather than their typical mitotic arrest and other MSAs can cause a p53-p21-dependent interphase arrest, rather than their typical mitotic arrest.9,10 These interphase effects are possibly the cause of antitumor effects of MSAs in hypoxic tumors, where the majority of cells are proliferation-arrested (reviewed in ref 11). Loss of p53 (or p21) in human colorectal carcinoma HCT116 cells results in altered levels of apoptosis-regulating proteins, including BAX and BCl-2, that decrease the sensitivity of cells to paclitaxel.9,12 The loss of functional p53 provides a clonal advantage, facilitating the development of paclitaxelresistant phenotypes.8 Conversely, loss of p53 function results in a defective G1/S checkpoint that may increase the number of cells entering mitotic phase, increasing the antimitotic effects of paclitaxel or other MSAs.9 In addition to regulating p21 and apoptosis-related genes, p53 regulates the expression of microtubule-associated proteins, such as stathmin and MAP4, that alter binding of MSAs to microtubules.13−15 Interestingly, both HIF-1α and p53 have been reported to reciprocally regulate each other’s activity and/or expression, depending on the level of hypoxic stress (reviewed in ref 3). Our previous study revealed that hypoxia (1% oxygen) preconditioning does not affect the sensitivity of HCT116 cells to peloruside A (PLA), an MSA from the marine sponge Mycale hentscheli.16 Although PLA is less cytotoxic than paclitaxel in normoxia, the ability of PLA to induce mitotic arrest of cervical cancer HeLa cells and apoptosis in HCT116 cells is not altered by hypoxia conditioning of cells. Additionally, PLA induces p53

ypoxia is a prominent feature of solid tumors and develops as a result of high interstitial pressure and poor blood supply leading to low oxygen levels.1 Cellular responses to hypoxia are regulated by hypoxia-inducible factor-1 (HIF-1), a key transcription factor that induces the expression of a number of adaptive genes for the survival of cancer cells under hypoxic stress.2 HIF-1 is a dimeric protein made up of two subunits, HIF-1α and HIF-1β. Although HIF-1β shows a constitutive level of expression, the basal level of HIF-1α is maintained low in normoxia by proteasomal degradation in cells. Hypoxia increases the expression and stabilizes HIF-1α, resulting in its dimerization with HIF-1β and nuclear translocation and binding to cofactors to facilitate the hypoxic adaptive pathways in cells.3 Overexpression of HIF-1α is associated with the stage of the tumor, overall survival, and metastases in colorectal cancer and other solid tumors and also correlates with a poor outcome after chemotherapy and radiotherapy.4 The effectiveness of many anticancer drugs, including microtubule-stabilizing agents (MSAs) that are widely used in the treatment of various solid tumor types, is reduced under hypoxic conditions.2 Similar to HIF-1, p53 tumor suppressor protein allows a cell to adapt to various genotoxic stresses. By induction of the cyclin-dependent kinase inhibitor p21Cip1 (p21), p53 plays a key role in the response to genotoxic stress or aneuploidy.5 The basal level of p53 is maintained low in the cells by ubiquitination and proteasomal degradation by the murine double minute-2 protein.6 Although the role of p53 in resistance to MSAs, such as taxanes, remains debatable, a number of reports show that p53 increases the sensitivity of different cancer cell lines to MSAs in different experimental settings.7,8 Depending on cell type and concentration, paclitaxel and other MSAs can cause a p53-p21-dependent interphase © 2018 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Susan Horwitz Received: November 14, 2017 Published: February 5, 2018 634

DOI: 10.1021/acs.jnatprod.7b00961 J. Nat. Prod. 2018, 81, 634−640

Journal of Natural Products

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Figure 1. Absence of p53 decreases sensitivity of HCT116 cells to PLA in hypoxia. (A) Hypoxia for 32 h decreases the percent of surviving druguntreated WT cells. Data are the mean ± SEM of 3 or 4 independent experiments, **p < 0.01 comparing cells growing in 16−32 h hypoxia to those in 16−32 h normoxia set to a hypothetical mean of 100% (shown by dashed lines), one-sample t-test. (B) Effect of PLA treatment on the percent of surviving WT, p53−/−, p21−/−, and A549 cells following treatment in normoxia (0 h hypoxia) for 24 h and 16−32 h in hypoxia measured by crystal violet assay. Data are the mean ± SEM of 4 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05 comparing PLA-treated to untreated set to a hypothetical mean of 100% (shown by dashed lines), one-sample t-test. (C) Mean (±SEM) doubling time of four cell lines under normoxia; n = 3. (D) Western blot showing the increase in the protein levels of HIF-1α following hypoxia culturing of cells.

in breast cancer MCF-7 cells17 and activates the expression of p21 in PLA-sensitive and PLA-resistant ovarian 1A9 cancer cells.18 Similarly, HIF-1α-expressing HCT116 cells show an induction of p53 following PLA treatment.16 Given the relation between HIF-1α and p53, and since the expression of p21 is regulated by both p53 and HIF-1α, we sought to determine if the effects of PLA in hypoxic (1% oxygen) cells are p53- and/or p21-dependent. We, therefore, examined PLA efficacy in HCT116 cells with wild-type p53 and p21 (WT cells) and two isogenic HCT116 cell lines with either knocked out p53 (p53−/− cells) or p21 (p21−/− cells) under normoxic and hypoxic conditions. We also used human lung carcinoma A549 cells harboring wild-type p53 to determine if there are differences between cell types in p53-dependent responses to PLA under hypoxia. Unlike our previous study in which hypoxia-preconditioned cells were treated with drugs in normoxia,16 in the present study, HCT116 cells, preconditioned to hypoxia for 4 h, were treated with PLA under hypoxic conditions for a further 16−32 h. The reason for this preconditioning was to allow resistance mechanisms to develop and be consistent with our earlier paper.16 We first determined the effect of hypoxia alone on the proliferation of cells cultured under hypoxic conditions for 0− 32 h using a crystal violet staining (CVS) cell proliferation assay.19 For the CVS assay, we focused on cells that remained adherent following the end of the experimental period. However, it is to be noted that hypoxia is known to facilitate cell detachment by altering the expression of cell adhesion

molecules without affecting cell viability.20 As evident in Figure 1A, 32 h hypoxia reduced the percent of surviving WT cells compared to cells in normoxia for 32 h (0 h hypoxia). There was no effect of other hypoxic conditions on WT, p53−/−, and A549 cells (Figure 1A). Likewise, there was no effect of hypoxia on p21−/− cells in our study, unlike El-Khatib et al., who reported a reduced viability of p21−/− cells after 24 h of hypoxia.21 This difference in our study in which p21 knockout had no effect on the percent of surviving viable cells presumably resulted from differences in the viability assays used. These data indicate that p53 partially contributes to hypoxia-induced responses in line with a previous report that described the relation between HIF-1α and p53 by a network modeling study.22 To determine if hypoxia altered the PLA effect on cell growth, we measured the percent of surviving cells after PLA treatment in hypoxia. First, PLA treatment of WT, p53−/−, and A549 cells for 24 h under normoxic conditions resulted in a concentration-dependent decrease in cell number (Figure 1B). Cells lacking p21 have been shown to be more sensitive to antitumor agents,9,17 and the p21−/− cells in our study were also more sensitive to PLA at all tested concentrations. Moreover, the p21−/− cells displayed a differential cell growth rate compared to other cell lines. The doubling time of p21−/− cells was increased relative to WT cells (Figure 1C). Next, cells were preconditioned for 4 h in hypoxia (1% O2) and then treated with PLA under continued hypoxic conditions for 16− 32 h. Similar to the PLA effect in normoxic cells, there was a 635



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Figure 2. Hypoxia effect on colony formation by PLA-treated cells. (A) Images showing the cell colonies formed by untreated and (B) PLA-treated cultures following a clonogenic survival assay as described in the Experimental Section. Each colony is formed from a single viable cell. (C) Graphs showing the number of colonies formed by WT, p53−/−, p21−/−, and A549 cells after 7 days of culture in normoxia conditions (5% CO2/ atmospheric air) following PLA treatment for 16−32 h under normoxia (0 h hypoxia) or hypoxia. Data are the mean ± SEM of at least 2 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05 comparing 0 nM to 50 nM and 250 nM PLA, one-way ANOVA with Dunnett’s post hoc test; ##p < 0.01, #p < 0.05 comparing untreated normoxic cells (0 h hypoxia) to untreated cells growing in hypoxia for 16−32 h (white bars). (D) Comparison of the clonogenicity of WT and p53−/− cells following treatment with 50 nM and 250 nM PLA as indicated in C. Data are the mean ± SEM of 2 independent experiments, **p < 0.01, *p < 0.05 comparing WT to p53−/− cells, Student’s t-test, unpaired.

significant reduction in the percentage of surviving WT, p21−/−, and A549 cells under 16−32 h hypoxia. Although PLA reduced the proliferation of p53−/− cells under 16 h hypoxia, 24 h and 32 h hypoxia abrogated the sensitivity to PLA at all the tested concentrations (Figure 1B), indicating that the sensitivity to PLA under hypoxic conditions is partly p53-dependent. There is, however, no further increase in the proliferation of 24 h and 32 h PLA-treated cells under hypoxia (Figure 1B). It is known that p21−/− cells contain a functional p53; however, the lack of p21-mediated G1 arrest increases the number of cells entering mitosis, offering no protection against antimitotic effects of MSAs.9 This is also evident in our study from increased sensitivity of p21−/− cells to PLA under all treated conditions (Figure 1B). To determine if the differences observed were due to hypoxia, we analyzed the level of HIF-1α induction following growth of cells in hypoxia. Compared to cells in normoxia (0 h hypoxia), 16−24 h hypoxia increased the level of HIF-1α protein in cells (Figure 1D). Overall, the data indicate that the cytotoxic action of PLA is largely unaffected by hypoxia. While the loss of p53 function reduces the cytotoxic effects of PLA,

loss of p21 activity does not affect PLA cytotoxicity in normoxia or hypoxia. To determine the proliferative ability under hypoxic conditions of the surviving cells after PLA treatment, a clonogenic cell survival assay was performed (Figure 2). A comparison of untreated HCT116 cells showed a hypoxiadependent sharp increase in the clonogenic potential of p53−/− cells (Figure 2A,C; see white bars in C), confirming previous findings that hypoxia selects for cells carrying nonfunctional p53.23 Although a prolonged hypoxia decreased the colonyforming ability of WT and p21−/− cells, hypoxia increased the clonogenic potential of A549 cells harboring a functional p53 (Figure 2A,C; see white bars in G). This difference shows that hypoxia results in cell-specific changes in proliferation and metastatic potential of different tumor cell types.24 In contrast to the untreated cells, PLA treatment abolished the clonogenicity of WT, p21−/−, and A549 cells (Figure 2B,C). However, PLA-treated p53−/− cells, in part, retained good colony-forming ability (Figure 2C). This may have been partly due to the large population of viable p53−/− cells following 636



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Figure 3. Hypoxia does not alter PLA-induced changes in the cell cycle profile of p53-deficient cells. (A) Histograms showing an increase in the accumulation of WT and A549 cells in the G1 phase of the cell cycle with an increase in hypoxia time in the absence of PLA treatment. (B) Bar graphs showing the percentage of G2/M-arrested cells following treatment of WT, p53−/−, p21−/−, and A549 cells with 50 nM and 250 nM PLA for 16−32 h under normoxic and hypoxic conditions. Data are the mean ± SEM of at least 2 independent experiments. (C) Representative histograms of cell cycle distributions following treatment with 50 nM and 250 nM PLA for 32 h under normoxia (NRX) or hypoxia (HPX).

replication without cell division and were arrested with 8N DNA content (Figure 3C). Similarly, p21−/− cells displayed multinucleation, and a significant proportion of cells were distributed in the 4N to 8N DNA content range. These data indicate that p53- and p21-deficient HCT116 cells undergo mitotic slippage and tolerate polyploidy following PLA treatment in comparison to parental HCT116 and A549 cells with a functional p53 signaling pathway. Hypoxia for 16 and 24 h did not induce the expression of p53 in untreated WT and A549 cells (Figure 4A). The slight increase in p53 levels in p21−/− cells potentially resulted from a compensation for the loss of p21.9 Paclitaxel has been reported to induce p53 up-regulation to a different extent in HCT116 and A549 cells;9 however, the level of p53 induced by PLA was comparable between the two cell lines in our study (Figure 4A). Additionally, the Western blots also revealed that the induction of p53 and p21 in response to PLA was not modulated by hypoxia (Figure 4A). In p53−/− cells, the lack of p53 resulted in p53-independent expression of p21 following PLA treatment under hypoxic conditions. The clonogenic survival data of p21−/− cells (Figure 2), however, indicate that a low p21 level is presumably not the causative factor for the lowered sensitivity of p53−/− cells to PLA. Acetylation is a marker of long-lived microtubules, and the level of acetylated tubulin is proportional to the microtubule

treatment with 50 nM and 250 nM PLA (Figure 1B). A comparison of WT and p53−/− cells further shows the absence of p53 decreases the effect of PLA on clonogenicity of these cells (Figure 2D). In addition to p53, other factors, including cell doubling time, have been suggested to influence the response of cancer cells to paclitaxel.25 Indeed, the doubling time of p53−/− cells was 20.1 h, which represents a reduction of the period required for cell division, in comparison with WT and p21−/− cells with doubling times 21.9 and 25.0 h, respectively (Figure 1C). To assess the effect of hypoxia on PLA-induced cell cycle arrest, we analyzed cell cycle distribution by flow cytometry. Hypoxia resulted in an expected increase in G1-arrested WT and A549 cells (Figure 3A). However, hypoxia for 16−32 h did not result in the G1 arrest of either p53−/− or p21−/− cells. This lack of a G1 arrest in p53- and p21-deficient cells shows the classical p53-p21-dependent inhibition of cell cycle progression by hypoxia.3 PLA treatment resulted in the expected dosedependent increase in the number of G2/M-arrested cells in normoxia. PLA treatment (50 nM and 250 nM) of 4 h hypoxiapreconditioned cells for 16−32 h under continuous hypoxia did not affect 2/M arrest in all cell types (Figure 3B). Interestingly, however, there was a distinctive p53- and p21-dependent effect of PLA following 24 and 32 h of treatment. While WT and A549 cells remained diploid, p53−/− cells underwent DNA 637



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Figure 4. Expression of p53, microtubule stabilization, and cell death by PLA in hypoxia. (A) Western blots of p53 and p21 in untreated and PLAtreated cells following either 24 h of incubation in normoxia or 16−32 h of incubation in hypoxic conditions. The arrows on the left in A indicate the position of p53 bands. Note that the bands above the p53 position are bands from the previous staining of the blots with Ac-α-Tubulin. (B) Western blots showing no effect of hypoxia on Ac-α-Tubulin or total tubulin (α-Tubulin) levels. Although PLA (250 nM) stabilizes microtubules in all four cell lines, there is an increased stabilization of microtubules in p53−/− and p21−/− cells under hypoxic conditions. The Ac-α-Tubulin and α-tubulin are from the same batch of protein lysates but run on different gels. Blots in A and B are representative of at least 2 independent experiments. (C−F) Bar graphs showing the percentage of cell death measured by propidium iodide staining and flow cytometry following PLA treatment of WT, p53−/−, and p21−/− cells for 16 h (C), 24 h (D), and 32 h (E), and A549 cells for 16−32 h (F) under normoxic and hypoxic conditions. Data are the mean ± SEM, **p < 0.01, *p < 0.05 of 3 independent experiments, one-way ANOVA with Bonferroni’s post hoc test. In A549 graphs (F), asterisks indicate a comparison between 0 nM vs 50 nM and 250 nM PLA, **p < 0.01, *p < 0.05 of 3 independent experiments, one-way ANOVA with Bonferroni’s post hoc test.

stability.26 To determine whether PLA stabilizes cellular microtubules following treatment under hypoxic conditions, we analyzed the levels of acetylated-α-Tubulin (Ac-α-Tubulin) as a marker of the extent of microtubule stabilization.26 Although there was no major difference in the effect of PLA on Ac-α-Tubulin levels under hypoxic and normoxic conditions, the level of stabilized microtubules was increased by PLA treatment under 24 h hypoxia in WT and A549 cells (Figure 4B). Intriguingly, in comparison to WT cells, there was a timedependent increase in Ac-α-Tubulin levels in p53−/− and p21−/− following PLA treatment under hypoxic conditions. Hypoxia alters microtubule configuration, which can potentially affect the MSA−tubulin interaction.27,28 These results indicate that tubulin stabilization by PLA is unaffected by hypoxia or by p53/p21 knockout. Hypoxia can cause apoptosis-induced cell death independent of p53.29 However, there was no apparent cell death in PLAuntreated WT or p53-deficient cells due to hypoxia in our study (Figure 4C−E), although the increase in the percentage of cell death in p21−/− cell cultures after 16 h hypoxia (Figure 4C) is

noteworthy. We have previously shown that PLA activates caspases and cell death by apoptosis in hypoxia-preconditioned WT HCT116 cells under normoxic conditions.16 Similarly, compared to the untreated cells, there was a dose- and timedependent increase in the percentage of cell death following PLA treatment of all four cell lines under hypoxia (Figure 4C− E). However, p53−/− cells were less sensitive to PLA-induced cell death than the WT cells (Figure 4C−E). Although p21−/− cells also appeared to be less susceptible than WT cells to PLA-induced cell death under both normoxia and hypoxia (at 24 and 32 h), the difference was not statistically significant (Figure 4D,E). Hence, the response to PLA was presumably dependent mainly on the presence of p53 but not p21. A549 cells with a wild-type p53 were more susceptible to PLA than WT HCT116 cells, but neither of these cells showed a reduced response to PLA under hypoxic conditions (Figure 4F). The data suggest that the underlying factor responsible for the differential response of p53−/− cells to PLA is ineffective control of genomic integrity that allows endoreduplication and escape from apoptosis. 638



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ice-cold ethanol overnight at −20 °C. The following day, cells were stained with staining solution containing 50 μg/mL propidium iodide and 0.5 mg/mL RNase in PBS for 30 min at 37 °C. Cell cycle and cell death were then immediately analyzed by flow cytometry as described previously.31 Immunoblotting. Cells were harvested and lysed in RIPA buffer [150 mM NaCl, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.0)] supplemented with Complete Protease Inhibitor Cocktail (Roche Holding AG, Basel, Switzerland) by sonication on ice. Protein lysates were electrophoresed, transferred onto a PVDF membrane (MerckMillipore, Billerica, MA, USA) and probed with antibodies as described previously.32 Fluorescence of blots was detected using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Primary antibodies against Ac-α-tubulin (cat. no. 5335, 1:1000 dilution) and p21 (cat. no. 2947, 1:1000 dilution) were purchased from Cell-Signaling Technologies (Danvers, MA, USA), p53 (cat. no. ab131442; 0.02 μg/mL dilution) from Abcam (Cambridge, UK), and α-tubulin (cat. no. T5168, 1:8000 dilution) and β-actin (cat. no. A2228, 1:4000 dilution) from Sigma-Aldrich. Secondary anti-mouse and anti-rabbit Alexa Fluor 488-conjugated antibodies were obtained from Thermo Fisher Scientific and used at 1:2000 dilution. Statistical Analysis. All statistical analyses were performed using GraphPad Prism (version 7; GraphPad Software, San Diego, CA, USA). Results were considered significant at p ≤ 0.05. Descriptions of statistical tests are described in the figure legends.

In conclusion, the present study showed that hypoxia did not alter PLA effects in HCT116 and A549 cells harboring wildtype p53. Loss of p53 led to a decreased sensitivity of HCT116 cells to PLA presumably as a result of multinucleation and perturbation of p53-dependent cell death. Although PLA stimulates p21 expression by p53 stabilization, p21 is not required for the response to PLA.

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EXPERIMENTAL SECTION

Compounds and Chemicals. PLA was supplied by Dr. Peter Northcote, Victoria University of Wellington, NZ, after isolation and purification as described previously.30 Unless otherwise mentioned, all chemicals, reagents, and cell culture supplements were purchased from Sigma-Aldrich (Prague, Czech Republic). Cell Culture, Hypoxia, and Drug Treatment. HCT116 cell lines (WT, p53−/−, and p21−/−) were purchased from Horizon Discovery Ltd. (Cambridge, UK) and cultured in McCoy’s 5A medium. A549 cells were obtained from ATCC (Middlesex, UK) and cultured in Dulbecco’s modified Eagle’s medium. All cell culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1× penicillin−streptomycin solution. Cells were cultured in a standard humidified incubator in 5% CO2/atmospheric air at 37 °C. Hypoxic conditions were established by culturing cells in a Heracell 150i humidified incubator (Thermo Fisher Scientific) under a 1% O2, 94% N2, and 5% CO2 atmosphere at 37 °C. For all drug exposure experiments under normoxic conditions, cells were treated with PLA in a standard CO2 incubator for 16−32 h. For drug treatment under hypoxia, the cells were first preconditioned for 4 h in hypoxia in the Heracell 150i incubator. Following this preconditioning, PLA was added and the cells were exposed to 1% O2 for 16−32 h. Cell doubling time was determined by counting the number of viable cells every 24 h for 3 days using Vi-CELL XR cell counter (Beckman Coulter, Brea, CA, USA). Cell Survival and Clonogenic Assays. The effect of PLA on cell proliferation under hypoxia was determined by a crystal violet cell survival assay as described previously.19 Briefly, cells were seeded in 100 μL of medium per well at a density of 25 000 cell/mL in 96-well plates and allowed to attach overnight in a normoxic incubator. Cells were then processed for drug treatment as described above. Following PLA treatment, the medium was aspirated off, and the cells were placed on ice and washed 2× in cold phosphate-buffered saline (PBS). Cells were then stained with 50 μL of crystal violet staining solution (0.5% crystal violet in 20% methanol) by gentle shaking on a benchtop rocker for 20 min at room temperature. The staining solution was removed, and the stained cells were washed 3× with distilled water. Plates were then dried overnight at room temperature in the dark. The following day, the crystal violet content in the wells was dissolved in 100 μL of 100% methanol using a benchtop rocker, and the absorbance was measured at 570 nm in an EnVision Multilabel plate reader (PerkinElmer). The absorbance of blank wells was subtracted from the absorbance of control and treated wells, and the data were analyzed as a percentage of control absorbance. For the clonogenic assay, cells were treated with 50 nM and 250 nM PLA for 24 h under normoxia conditions and 16−32 h in hypoxia. Cells were then harvested by trypsinization and replated as a single-cell suspension into six-well plates in a total of 2 mL of drug-free growth medium at a density of 500 cells/well. Cells were allowed to grow for an additional 7 days under normoxia. After 7 days, cells were washed 2× with PBS and stained with 2 mL of crystal violet staining solution (0.5% crystal violet in 20% methanol) for 2 h at room temperature. The crystal violet solution was aspirated off, and the plates were washed with distilled water to remove excess staining solution from the wells. Images of crystal-violet-stained plates were acquired against a white background using a standard Epson Perfection V750 Pro photo scanner (Suwa, Nagano Prefecture, Japan). The acquired images were analyzed using NIH ImageJ freeware to quantify the number of viable cell colonies. Cell Cycle and Cell Death. Attached cells were harvested and collected together with the floating cells (detached) and fixed in 70%

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AUTHOR INFORMATION

Corresponding Author

*Tel: +420 585 632 111. Fax: +420 585 632 180. E-mail: [email protected] (V. Das). ORCID

Viswanath Das: 0000-0001-5973-5990 Author Contributions

J. Ř ehulka and N. Annadurai contributed equally to this work.

§

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants of the Czech Ministry of Education, Youth and Sports (LO1304, LM2011024), Cancer Society of New Zealand (E1807), and Wellington Medical Research Foundation (E1707).

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DEDICATION Dedicated to Dr. Susan Band Horwitz, of Albert Einstein College of Medicine, Bronx, NY, for her pioneering work on bioactive natural products.

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REFERENCES

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Peloruside A-Induced Cell Death in Hypoxia Is p53 Dependent in

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