Plumbagin Increases Paclitaxel-Induced Cell Death and Overcomes

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Plumbagin Increases Paclitaxel-Induced Cell Death and Overcomes Paclitaxel Resistance in Breast Cancer Cells through ERK-Mediated Apoptosis Induction Anna Kawiak,* Anna Domachowska, and Ewa Lojkowska

J. Nat. Prod. Downloaded from pubs.acs.org by WASHINGTON UNIV on 02/28/19. For personal use only.

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307, Gdansk, Poland ABSTRACT: ERK is a component of mitogen-activated protein kinases that controls a range of cellular processes including cell proliferation and survival. The upregulation of ERK has been associated with apoptosis inhibition in response to various stimuli including chemotherapeutic agents. Research has suggested that the upregulation of ERK signaling by the anticancer agent paclitaxel leads to acquired resistance of cells to this compound. The presented research focused on determining the role of plumbagin, a naturally derived naphthoquinone, in the sensitization of breast cancer cells to paclitaxel-induced cell death and the involvement of ERK signaling in this process. The results of the study indicated that plumbagin increases the sensitivity of breast cancer cells to paclitaxel. Moreover, a synergistic effect between plumbagin and paclitaxel was observed. Plumbagin was shown to decrease levels of phosphorylated ERK in breast cancer cells and abrogated paclitaxel-induced ERK phosphorylation. The role of ERK in plumbagin-mediated sensitization of breast cancer cells to paclitaxel was shown through the enhancement of the synergistic effect between compounds in cells with decreased ERK expression. Furthermore, plumbagin reduced p-ERK levels in paclitaxelresistant breast cancer cells and resensitized paclitaxel-resistant cells to this compound. These results imply that plumbagin inhibits ERK activation in breast cancer cells, which plays a role in the sensitization of cells to paclitaxel-induced cell death.

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significantly reduce tumor formation in mouse tumor xenograft models.6 Significant advances have been made in the development of kinase inhibitors targeting the MAPK/ERK pathway, mainly including BRAF and MEK inhibitors.7 Their successful use has however been hindered due to side effects and development of drug resistance. The molecular mechanism underlying the resistance to these inhibitors has been linked with ERK reactivation.8 Combination treatment with ERK1/2 inhibitors could be a promising strategy to reduce drug resistance and increase treatment efficacy. Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently in use in clinical treatment and others under preclinical evaluation. Moreover, a significant rise in the study of natural products in targeted therapy has been observed.9 The naturally occurring naphthoquinone plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is gaining increasing interest due to its potential anticancer activity. The antiproliferative properties of plumbagin have been previously described in in vitro studies,10−12 and in vivo studies have demonstrated the ability of plumbagin to reduce the formation of tumors in mouse xenograft models.13,14 The studies of Kuo et al.15 demonstrated the

reast cancer is one of the leading causes of cancer-related deaths among women. Despite advances in breast cancer treatment, a major drawback in treatment remains resistance acquired toward therapy. Research has shown that one of the factors contributing to drug-induced resistance is the activation of the MAPK/ERK signaling pathway. The ERK1/2 (extracellular signal-regulated kinase 1 and 2) signaling pathway is a prosurvival pathway consisting of a cascade of protein kinases in which RAF (ARAF, BRAF, or CRAF) phosphorylation activates mitogen-activated kinases MEK1/2, followed by p42/44 MAPK (ERK1/2).1 Active ERK1/2 phosphorylate various cellular substrates such as the apoptotic regulators, Bcl-2 family proteins and enhance cell survival.2 ERK1/2 promote cell invasiveness and migration through controlling the expression of cytokines and matrix metalloproteases.3 Others have published that anticancer agents such as paclitaxel activate the ERK signaling pathway, which increases cell survival and proliferation and can compromise the efficacy of this compound.4 The concomitant use of a MAPK/ERK inhibitor was shown to abrogate ERK activation and restore the activity of paclitaxel.5 Preclinical studies have also demonstrated that the use of small-molecule inhibitors of the MAPK/ERK pathway significantly reduces cancer cell survival and induces apoptosis.6 Furthermore, in vivo studies support these findings and report that MAPK/ERK inhibitors © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 15, 2018

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DOI: 10.1021/acs.jnatprod.8b00964 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Plumbagin increases paclitaxel-induced cell death. (A) Effects of plumbagin and paclitaxel on the viability of breast cancer cells. MCF-7, BT474, and MDA-MB-468 cells were treated with combination doses of plumbagin and paclitaxel for 24 h, and cell viability was assessed with the MTT assay. (B) Effects of plumbagin on the induction of apoptosis by paclitaxel. Cells were treated with plumbagin and/or paclitaxel for 24 h. Cells were stained with annexin V-PE7/AAD and analyzed with flow cytometry. Data were analyzed by one-way ANOVA with Tukey’s post hoc test [p < 0.05 (*), p < 0.01 (**), p < 0.005 (***)].

ability of plumbagin to significantly reduce the growth of breast tumors in vivo without accompanying toxicity. Studies have shown that plumbagin induces apoptosis in breast cancer cells,16−18 and the ability of plumbagin to inhibit MAP kinase signaling in cancer cells was also reported.19−21 In regard to breast cancer, plumbagin suppressed NF-κB/MAPK signaling and inhibited breast-cancer-induced osteoclastogenesis and tumorigenesis.12,22 Plumbagin has also been shown to increase the cytotoxic activity of paclitaxel in myelogenous leukemia KBM-5 cells; however, the mechanism of action was not evaluated.23 These findings compelled us to further elucidate the involvement of plumbagin in paclitaxel-induced cell death in breast cancer cells and determine the mechanism of these interactions. The influence of combination treatment with

plumbagin and paclitaxel on the inhibition of ERK activity and ERK-mediated cell death induction was evaluated. Furthermore, the activity of plumbagin toward paclitaxel-resistant breast cancer cells was determined.

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RESULTS AND DISCUSSION Plumbagin Increases the Sensitivity of Breast Cancer Cells to Paclitaxel-Induced Cell Death. Previous research has shown the antiproliferative activity of plumbagin toward breast cancer cells. In the present study, the ability of plumbagin to increase the sensitivity of breast cancer cells to paclitaxel was evaluated. Interactions between plumbagin and paclitaxel were screened with the MTT assay, and the combined effects of plumbagin and paclitaxel were evaluated B



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Figure 2. Effects of plumbagin on p-ERK levels in breast cancer cells. (A) Effects of plumbagin on ERK1/2 expression levels. Cells were treated with the indicated concentrations of plumbagin for 24 h, followed by a 30 min incubation with EGF (50 ng/mL). Levels of p-ERK1/2, ERK, and βactin were assessed by Western blot analysis. Densitometric analysis represents p-ERK levels normalized to total ERK1/2 levels. (B) Quantitative analysis of the effects of plumbagin on the levels of p-ERK1/2 (Thr202/Tyr204). Cells were treated with the indicated concentrations of plumbagin for 1 h followed by a 30 min incubation with EGF (50 ng/mL). Protein levels were determined with the AlphaScreen assay after the incubation of cell lysates with biotin-conjugated anti-pERK1/2 antibodies and donor/acceptor beads. (C) Quantitative analysis of the effects of plumbagin on the levels of paclitaxel-induced p-ERK1/2 (Thr202/Tyr204). Cells were treated with the indicated concentrations of plumbagin for 1 h followed by a 30 min incubation with paclitaxel. Protein levels were analyzed with AlphaScreen assay. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Differences between plumbagin-treated and control (EGF-stimulated) cells are indicated [p < 0.05 (*), p < 0.01 (**), p < 0.005 (***)].

with the use of the median-effect method.24 Breast cancer cells were treated with a fixed ratio of five plumbagin and paclitaxel combination doses ranging from 0.5 to 5 μM (plumbagin) and 0.1 to 20 nM (paclitaxel). Alongside combination treatment, cells were treated with single agents, plumbagin and paclitaxel. Results depicted in Figure 1A show the effects of combination treatments on breast cancer cell viability. For all the examined cell lines, the CI (combination index) values were below 1 and ranged from 0.5 for the MDA-MB-468 cell line to 0.6 and 0.9 for BT474 and MCF-7 cells, respectively. These results point to a synergistic effect in the activity of plumbagin and paclitaxel toward breast cancer cells.

To further examine the effects of plumbagin on the activity of paclitaxel toward breast cancer cells, the ability of plumbagin to increase paclitaxel-induced apoptosis was examined. Cells were treated with combination doses of plumbagin and paclitaxel at which synergistic effects were observed. Cells were treated for 24 h with combination and/or single agents, and cell death was analyzed with annexin V-PE/7AAD staining. The treatment of cells with paclitaxel increased the percentage of apoptotic cells by 10−15%. Combination treatment with plumbagin increased the apoptotic population of cells by 10% and 15% in MCF-7 and BT474 cells, respectively. The highest increase in apoptosis was observed in C



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Figure 3. Effects of ERK expression on apoptosis induction in breast cancer cells by combination treatment with plumbagin and paclitaxel. (A) Effects of ERK siRNA on ERK expression levels in breast cancer cells. Cells were transiently transfected with ERK1/2 siRNA for 24 h, after which protein levels were determined with Western blot analysis. Densitometric analysis represents ERK1/2 levels normalized to β-actin levels. (B) Influence of ERK1/2 silencing on apoptosis induction by plumbagin and paclitaxel. Twenty-four hours post siRNA transfection, cells were treated for 24 h with plumbagin and paclitaxel. Cells were stained with annexin V-PE7/AAD and analyzed with flow cytometry. Data were analyzed by oneway ANOVA with Tukey’s post hoc test [p < 0.05 (*), p < 0.01 (**), p < 0.005 (***)].

transfer from donor beads following the capture of p-ERK1/2 between a donor and acceptor bead sandwich. The donor beads used were conjugated with a biotinylated peptide substrate and acceptor beads with an anti-phosphotyrosine antibody. The AlphaScreen assay was performed following the pretreatment of cells with plumbagin for 1 h followed by the induction of p-ERK1/2 with EGF stimulation for 30 min. As presented in Figure 2B, the stimulation of phosphorylated ERK1/2 by EGF was induced 13-, 20-, and 30-fold in MCF7, BT474, and MDA-MB-468 cells, respectively. The pretreatment of cells with plumbagin significantly reduced the levels of phosphorylated ERK1/2 induced by EGF. A marked reduction in ERK1/2 phosphorylation was observed at the lowest plumbagin concentrations tested, ranging from 0.2 to 0.5 μM (Figure 2B). Since paclitaxel has been previously shown to induce pERK1/2 levels in breast cancer cells, the ability of plumbagin

MDA-MB-468 cells, where combination treatment increased apoptosis by 20% and 35% at the plumbagin concentrations of 0.2 and 0.5 μM, respectively (Figure 1B). Plumbagin Inhibits ERK1/2 Activation and Decreases p-ERK1/2 Levels Induced by Paclitaxel. In order to determine the effects of plumbagin on the activity of ERK in breast cancer cells, Western blot analysis was performed. Breast cancer cells were pretreated with plumbagin for 24 h, after which p-ERK1/2 was stimulated for 30 min with EGF, and the levels of p-ERK1/2 in breast cancer cells were evaluated. As shown in Figure 2A, plumbagin reduced the levels p-ERK1/2 induced by EGF in all of the examined breast cancer cell lines. In order to perform a quantitative analysis of the effects of plumbagin on ERK phosphorylation in breast cancer cells, a bead-based proximity luminescent assay was employed (AlphaScreen). Phosphorylated ERK1/2 was detected in this assay based on a signal induced upon a cascade of energy D



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Figure 4. Influence of plumbagin on paclitaxel-resistant breast cancer cells. (A) Effects of paclitaxel on MCF-7 and MCF-7/PTX cell viability. Cells were treated with paclitaxel for 24 h, and cell viability was assessed with the MTT assay. (B) Effects of plumbagin and paclitaxel on apoptosis induction in MCF-7 and MCF-7/PTX. Cells were treated with plumbagin or paclitaxel for 24 h, then stained with annexin V-PE/7AAD and analyzed with flow cytometry. (C) Effects of combination treatment with plumbagin and paclitaxel on the induction of apoptosis in MCF-7 and MCF-7/PTX cells. Cells were treated with plumbagin and paclitaxel for 24 h, then stained with annexin V-PE/7AAD and analyzed with flow cytometry. (D) Quantitative analysis of the effects of plumbagin on the levels of p-ERK1/2 (Thr202/Tyr204) in MCF-7 and MCF-7/PTX cells. Cells were treated with the indicated concentrations of plumbagin for 1 h. Cell lysates were incubated with biotin-conjugated anti-pERK1/2 antibodies and donor/acceptor beads, and protein levels were analyzed with AlphaScreen assay. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Differences between plumbagin- and/or paclitaxel-treated cells and control cells are indicated [p < 0.01 (**), p < 0.005 (***)].

transfection of cells, transfection efficacy was examined by determining ERK1/2 levels in breast cancer cells with Western blot. As shown in Figure 3A siRNA transfection significantly decreased the levels of ERK1/2 expression in all of the examined cells lines in comparison with cells transfected with control siRNA. The role of ERK expression in plumbaginmediated sensitization of breast cancer cells to paclitaxel was examined by comparing the percentage of apoptotic cell populations induced by combination treatment in control and siRNA transfected cells. Breast cancer cells were transiently transfected for 24 h, after which cells were treated with a combination of agents for 24 h, and apoptosis induction was determined with annexin V-PE/7AAD staining. The silencing of ERK1/2 expression in breast cancer cells sensitized cells to apoptosis induced by plumbagin and paclitaxel. In comparison with control cells transfected with control siRNA, in cells with ERK1/2 downregulation, the percentage of apoptotic cells increased significantly. The highest increase in apoptosis was observed in MDA-MB-468 cells, where a 25% increase in the apoptotic population of cells was observed in cells with ERK1/ 2 downregulation (Figure 3B). These results point to the involvement of ERK in the sensitization of breast cancer cells

to prevent paclitaxel-induced ERK1/2 activation was determined with the AlphaScreen assay. Cells were pretreated for 1 h with plumbagin, after which p-ERK1/2 was stimulated with paclitaxel for 30 min. As determined by the AlphaScreen assay the levels of p-ERK1/2 increased after paclitaxel treatment 2-, 2.5-, or 4-fold in MCF-7, BT474, or MDA-MB-468 cells, respectively. Pretreatment of cells with plumbagin significantly reduced paclitaxel-induced p-ERK1/2 levels. At the plumbagin concentrations of 0.2 and 0.5 μM, the levels of p-ERK1/2 reached basal values in MDA-MB-468 and BT474 cells, respectively. In MCF-7 cells 1 μM plumbagin reduced pERK1/2 levels to basal values (Figure 2C). Collectively these results indicate that plumbagin reduces ERK1/2 activation and prevents paclitaxel-induced p-ERK1/2 in breast cancer cells. Plumbagin Sensitizes Breast Cancer Cells to Paclitaxel through ERK1/2 Inhibition. The involvement of ERK1/2 phosphorylation inhibition in plumbagin-mediated sensitization of breast cancer cells to paclitaxel was examined by silencing ERK1/2 expression in breast cancer cells and determining the influence of combination treatment on apoptosis induction. MCF-7, BT474, and MDA-MB-468 cells were transiently transfected with ERK1 and ERK2 siRNA or control, scrambled siRNA. Twenty-four hours following E



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transiently or extendedly activated, which results in cell death inhibition. This pro-survival mechanism can be abrogated after prolonged stimulation through the downregulation of ERK activity through its dephosphorylation or degradation of ERK.29 Paclitaxel has been reported to induce ERK1/2 activation in various cancer cells. Seidman et al.27 showed that paclitaxel induces ERK levels in ovarian cancer cells during short treatments, and incubations longer than 6 h result in a decrease in ERK phosphorylation. Okano and Rustgi30 reported prolonged activation of ERK in esophageal cancer cells upon paclitaxel treatment over a period of 72 h. In breast cancer cells paclitaxel was also shown to activate ERK.4 In agreement with these findings our research showed that paclitaxel activates ERK1/2 in HER2-overexpressing, ER-positive and triple negative breast cancer cells. The level of p-ERK induction by paclitaxel corresponded with activation levels of the ERK pathway in cells. In cells with the lowest ERK activation levels, MCF-7 cells, the lowest increase in p-ERK was induced with EGF and paclitaxel, whereas in MDA-MB-468 cells, high ERK activation levels corresponded with the highest induction of pERK upon EGF and paclitaxel stimulation. These findings agree with those of McDaid and Horwitz,31 who found that paclitaxel induced ERK1/2 levels in cells with increased endogenous levels of ERK1/2. The association between ERK1/2 activation by paclitaxel and cell death has been examined in previous studies. Seidman et al.27 showed that with the decrease in ERK activation after prolonged paclitaxel treatment, cell proliferation inhibition as well as apoptosis induction was observed. McDaid and Horwitz31 reported that inhibition of the ERK pathway by MEK inhibitors increased paclitaxel-induced apoptosis. On the other hand, the studies of Okano and Rustgi30 showed that ERK activation occurred independent of cell death induced by paclitaxel, and pretreatment with a MEK inhibitor did not alter apoptosis induction. Research has shown that ERK can colocalize with microtubules;32 it is therefore probable that when bound to microtubules, paclitaxel can activate ERK1/2 directly, independent of MEK activation.30 Another study supporting the association between ERK1/2 inhibition and paclitaxel-induced cell death was reported by MacKeigen et al.4 This study showed that inhibition of MEK/ERK signaling with a MEK inhibitor, U0126, and the concomitant treatment with paclitaxel markedly increased apoptosis induced by paclitaxel in breast, ovarian, and lung cancer cells. In accordance with these findings, our results showed that decreasing ERK1/2 phosphorylation with plumbagin resulted in increased cell death and apoptosis induction by paclitaxel. The involvement of ERK inhibition in combination treatment with plumbagin and paclitaxel was shown by an increase in the apoptotic population of cells with ERK1/2 downregulation. Furthermore, the highest CI value for plumbagin and paclitaxel interactions was obtained in cells with the highest sensitivity to ERK induction. The mechanism through which ERK1/2 signaling controls apoptosis induction has been associated with the regulation of the Bcl-2 family proteins.2 ERK activation can inhibit apoptosis by increasing the activity or transcriptional expression of Bcl-2, Bcl-XL, and Mcl-1 and increase survival through enhancing Bcl2 phosphorylation and blocking its proteasomal degradation.2,33 Accordingly ERK increases proliferation through phosphorylating the pro-apoptotic Bim-EL protein and enhancing its degradation in proteasomes.34 The role of Bcl-

to apoptosis by combination treatment with plumbagin and paclitaxel. Plumbagin Induces Apoptosis in Paclitaxel-Resistant Breast Cancer Cells. Since plumbagin inhibited p-ERK induced by paclitaxel and sensitized breast cancer cells to paclitaxel-induced cell death, the ability of plumbagin to induce cell death in paclitaxel-resistant breast cancer cells was examined. Paclitaxel-resistant MCF-7 (MCF-7/PTX) cells were obtained by the maintenance of MCF-7 cells in the presence of increasing concentrations of paclitaxel. The MTT assay revealed a 30% increase in viability of MCF-7/PTX cells upon paclitaxel treatment in comparison with parental MCF-7 cells (Figure 4A). Treatment of MCF-7/PTX with plumbagin had a similar effect on the viability of these cells in comparison with MCF-7 cells, with IC50 values of 3.5 μM for both cell lines. The ability of plumbagin to induce apoptosis in MCF-7/ PTX cells was examined. MCF/PTX cells were resistant to paclitaxel-induced apoptosis. At the concentration of 3 nM, paclitaxel increased the percentage of apoptotic cells by 15%, whereas in MCF-7/PTX cells the percentage of apoptotic cells did not increase upon paclitaxel treatment. Plumbagin induced apoptosis to a similar extent in MCF-7/PTX as in MCF-7 cells. At the plumbagin concentrations of 3 μM, a 20% increase in the population of apoptotic cells was observed in MCF-7/PTX and MCF-7 cells (Figure 4B). Furthermore, the ability of plumbagin to resensitize MCF-7/PTX cells to paclitaxel was evaluated. Combination treatment of plumbagin and paclitaxel induced apoptosis to the same extent in both MCF-7 and MCF7/PTX cells, indicating the ability of plumbagin to overcome paclitaxel resistance in breast cancer cells (Figure 4C). To further evaluate whether the effects of plumbagin on paclitaxel-resistant cells are associated with the ability of plumbagin to downregulate p-ERK1/2 in breast cancer cells, the effects of plumbagin on p-ERK1/2 levels in MCF-7/PTX were examined with the AlphaScreen assay. The AlphaScreen analysis revealed that the basal levels of p-ERK1/2 were elevated in MCF-7/PTX in comparison to MCF-7 cells. Similarly to the effects of plumbagin on the reduction of paclitaxel-induced p-ERK1/2 levels in breast cancer cells, a decrease in the elevated levels of p-ERK1/2 in MCF-7/PTX was also observed upon plumbagin treatment. The levels of pERK1/2 in MCF-7/PTX reached basal values in plumbagintreated cells (Figure 4D). Paclitaxel (Taxol) is a chemotherapeutic agent used in the first-line treatment of various cancers, including breast cancer.25 Paclitaxel exerts its effects through promoting tubulin dimerization and microtubule stabilization.26 Recent research has also reported a broader activity of paclitaxel in cells. Studies have shown that paclitaxel has an influence on cell signaling pathways through the phosphorylation of tyrosine on proteins.27 Paclitaxel activates mediators of the mitogenactivated protein kinase (MAPK) signaling pathway, including extracellular signal-regulated kinases (ERK1/2) and p38 and Jun-terminal kinases (JNK).27 The MAPK/ERK signaling pathway has been shown to play a central role in the regulation of cell survival.28 ERK1/2 activation leads to the phosphorylation of an estimated 150 cellular substrates, including kinases and phosphatases, transcription factors, cytoskeletal proteins, and apoptosis-related proteins.1 A wide range of stimuli has been shown to activate ERK, such as growth factor withdrawal, matrix detachment, hypoxia, and chemotherapeutic agents.29 In response to stimulation, ERK is either F



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of drug 1 and 2 required to induce the same percentage effect. CI values less than 1 indicate synergism, whereas CI values equal to 1 and greater than 1 are indicative of an additive and antagonistic effect, respectively. Apoptosis Determination. Apoptosis induction was examined with an annexin V-PE apoptosis detection kit I (BD Biosciences, Belgium). The procedures were carried out according to the manufacturer’s instructions. MCF-7, BT474, or MDA-MB-468 cells were treated with plumbagin and/or paclitaxel at the indicated concentrations for 24 h. Following incubation, cells were collected, washed with annexin-binding buffer, and stained with annexin Vphycoerythrin (PE) and 7-amino-actinomycin (7-AAD). After a 30 min incubation at 15 °C in the dark, apoptosis induction was determined by flow cytometry (BD FACSCalibur). Western Blot Analysis. MCF-7, BT474, and MDA-MB-468 cells were pretreated with plumbagin (0.2−1 μM) for 24 h and subsequently with EGF (50 ng/mL). Western blot analysis was performed according to a previously published procedure.39 Specific primary antibodiesanti-ERK1/2, anti-p-ERK1/2 (1:1000) (Cell Signaling, Danvers, MA, USA), anti-β-actin (1:1000) (Cell Signaling)were incubated with membranes overnight at 4 °C. Membranes were further incubated at room temperature for 1 h with HRPconjugated secondary antibodies (1:2000) (Cell Signaling), and proteins were detected by chemiluminescence (ChemiDoc, Bio-Rad, Hercules, CA, USA) with an HRP substrate (Thermo Scientific, Waltham, MA, USA). Analysis was performed in triplicate. ERK1/2 Silencing. The expression of ERK1/2 was silenced in breast cancer cells using ERK1 and ERK2 siRNA or control, scrambled siRNA (Santa Cruz, Heidelberg, Germany). The siRNA transfection reagent (Santa Cruz) was used to transiently silence cells with siRNA (0.5 μg) according to the manufacturer’s instructions. Transfection was carried out for 24 h, after which ERK1/2 silencing was analyzed with Western blot. Apoptosis induction was determined following ERK1/2 silencing after a 24 h transfection and a further 24 h treatment with plumbagin and paclitaxel. Cells were stained with annexin V-PE and 7-AAD, and apoptosis induction was analyzed by flow cytometry. Alphascreen Analysis. ERK1/2 inhibition was examined using the bead-based amplified luminescent proximity homogeneous assay (AlphaScreen), to detect phosphorylated ERK (SureFire p-ERK 1/2 (Thr202/Tyr204), PerkinElmer, Rodgau, Germany). Cells were serum-starved overnight, then pretreated with plumbagin for 1 h, after which cells were stimulated with EGF (50 ng/mL) or paclitaxel (1nM) for 30 min in 5% CO2 at 37 °C. The medium was discarded, 50 μL of SureFire lysis buffer was added, and plates were agitated on a plate shaker for 10 min (∼350 rpm) at room temperature. The levels of phospho-ERK were determined in cell lysates according to the manufacturer’s instructions. Briefly, 4 μL of lysate was transferred to a 384-well plate (Proxiplate, PerkinElmer). A 7 μL amount of reaction mix containing the reaction buffer, activation buffer, and acceptor and donor beads was added to the plate followed by a 2 h incubation at room temperature. The fluorescent signals were read with an Envision multilabel reader (PerkinElmer) with standard AlphaScreen settings. Statistical Analysis. Values are expressed as means ± SE of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad software). Differences between control and treated samples were analyzed by one-way ANOVA with Tukey’s post hoc tests. A p value of 95% purity from Sigma-Aldrich (St. Louis, MO, USA). Compounds were dissolved in 100% DMSO and used at the final concentration of 0.5%. All cell culture materials and other chemicals, if not indicated otherwise, were obtained from the same company. Cell Culture. The MCF-7, BT474, and MDA-MB-468 breast cancer cell lines were purchased from Cell Lines Service (Eppelheim, Germany). MCF-7 and MDA-MB-468 cells were cultured in RPMI medium, whereas BT474 cells were cultured in DMEM/F12 medium. Media were supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 10 mg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C in an incubator (Heraeus, HERAcell, Hanau, Germany). Development of Paclitaxel-Resistant Cells. MCF-7 cells were cultured in the presence of increasing concentrations of paclitaxel ranging from 0.4 to 6.0 nM for a period of three months according to a previously published procedure.38 Cytotoxicity Assay. Cell viability upon plumbagin and/or paclitaxel treatment was determined using the MTT [(3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cells were treated with plumbagin and/or paclitaxel at the indicated concentrations for 24 h. Analysis was performed according to the previously published procedure.39 Evaluation of Synergistic Activity. The median effect method described by Chou and Talalay (1984)24 was used to determine the effects of combined treatment with plumbagin and paclitaxel. Cells were treated with five fixed ratios of plumbagin (μM) and paclitaxel (nM) in the combination of 1/0.5, 2/1, 3/3, 4/5, 5/10 for MCF-7 cells and 0.5/0,05; 1/0.1; 2/1; 3/10, 5/10 and 0.1/0.1; 0.2/1; 0.5/5; 1/10; 2/20 for BT474 and MDA-MB-468 cells, respectively. The CI values were calculated at different dose and effect levels with the following formula: (D1/Dx1) + (D2/Dx2) + (D1D2/DxlDx2), where Dx1 and Dx2 are the doses of drug 1 and drug 2 required to induce an effect of x percentage, whereas D1 and D2 are the combination doses

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Corresponding Author

*Tel: +48 (58) 5236308. E-mail: [email protected]. pl. ORCID

Anna Kawiak: 0000-0001-8105-2555 Notes

The authors declare no competing financial interest. G



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(32) Reszka, A. A.; Seger, R.; Diltz, C. D.; Krebs, E. G.; Fischer, E. H. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 8881−8885. (33) Dimmeler, B. S.; Breitschopf, K.; Haendeler, J.; Zeiher, A. M. J. Exp. Med. 1999, 189, 1815−1822. (34) Luciano, F.; Jacquel, A.; Colosetti, P.; Herrant, M.; Cagnol, S.; Pages, G.; Auberger, P. Oncogene 2003, 22, 6785−6793. (35) Haldar, S.; Chintapalli, J.; Croce, C. M. Cancer Res. 1996, 56, 1253−1255. (36) Tan, T. T.; Degenhardt, K.; Nelson, D. A.; Beaudoin, B.; Nieves-Neira, W.; Bouillet, P.; Villunger, A.; Adams, J. M.; White, E. Cancer Cell 2005, 7, 227−238. (37) Miller, A. V.; Hicks, M. A.; Nakajima, W.; Richardson, A. C.; Windle, J. J.; Harada, H. PLoS One 2013, 8, e60685. (38) Ajabnoor, G. M. A.; Crook, T.; Coley, H. M. Cell Death Dis. 2012, 3 (1), e260−9. (39) Kawiak, A.; Zawacka-Pankau, J.; Wasilewska, A.; Stasilojc, G.; Bigda, J.; Lojkowska, E. J. Nat. Prod. 2012, 75, 9−14.

ACKNOWLEDGMENTS This work was supported by grant no. Lider/15/217/L-3/11/ NCBR/2012 from the National Centre for Research and Development (A.K.).

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REFERENCES

(1) Yoon, S.; Seger, R. Growth Factors 2006, 24, 21−44. (2) Balmanno, K.; Cook, S. J. Cell Death Differ. 2009, 16, 368−377. (3) Schulze, A.; Lehmann, K.; Jefferies, H. B. J.; McMahon, M.; Downward, J. Genes Dev. 2001, 15, 981−94. (4) MacKeigan, J. P.; Collins, T. S.; Ting, J. P. Y. J. Biol. Chem. 2000, 275, 38953−38956. (5) Seidman, R.; Gitelman, I.; Sagi, O.; Horwitz, S. B.; Wolfson, M. Exp. Cell Res. 2001, 268, 84−92. (6) Frémin, C.; Meloche, S. J. Hematol. Oncol. 2010, 3, 1−11. (7) Herrero, A.; Pinto, A.; Colón-Bolea, P.; Casar, B.; Jones, M.; Agudo-Ibáñez, L.; Vidal, R.; Tenbaum, S. P.; Nuciforo, P.; Valdizán, E. M.; et al. Cancer Cell 2015, 28 (2), 170−182. (8) Little, A. S.; Smith, P. D.; Cook, S. J. Oncogene 2013, 32, 1207− 1215. (9) Atanasov, A. G.; Yeung, A. W. K.; Banach, M. Biotechnol. Adv. 2018, 36, 1559−1562. (10) Kawiak, A.; Piosik, J.; Stasilojc, G.; Gwizdek-Wisniewska, A.; Marczak, L.; Stobiecki, M.; Bigda, J.; Lojkowska, E. Toxicol. Appl. Pharmacol. 2007, 223, 267−276. (11) McKallip, R. J.; Lombard, C.; Sun, J.; Ramakrishnan, R. Toxicol. Appl. Pharmacol. 2010, 247, 41−52. (12) Li, Z.; Xiao, J.; Wu, X.; Li, W.; Yang, Z.; Xie, J.; Xu, L.; Cai, X.; Lin, Z.; Guo, W.; Luo, J.; Liu, M. Curr. Mol. Med. 2012, 12, 967−81. (13) Cao, Y. Y.; Yu, J.; Liu, T. T.; Yang, K. X.; Yang, L. Y.; Chen, Q.; Shi, F.; Hao, J. J.; Cai, Y.; Wang, M. R.; Lu, W. H.; Zhang, Y. Cell Death Dis. 2018, 9, 17. (14) Sinha, S.; Pal, K.; Elkhanany, A.; Dutta, S.; Cao, Y.; Mondal, G.; Iyer, S.; Somasundaram, V.; Couch, F. J.; Shridhar, V.; Bhattacharya, R.; Mukhopadhyay, D.; Srinivas, P. Int. J. Cancer 2013, 132, 1201− 1212. (15) Kuo, P.-L.; Hsu, Y.-L.; Cho, C.-Y. Mol. Cancer Ther. 2006, 5 (12), 3209−3221. (16) Kawiak, A.; Zawacka-Pankau, J.; Lojkowska, E. J. Nat. Prod. 2012, 75 (4), 747−751. (17) Kawiak, A.; Domachowska, A.; Jaworska, A.; Lojkowska, E. Sci. Rep. 2017, 7, 1−9. (18) Nair, R. S.; Kumar, J. M.; Jose, J.; Somasundaram, V.; Hemalatha, S. K.; Sengodan, S. K.; Nadhan, R.; Anilkumar, T. V.; Srinivas, P. Sci. Rep. 2016, 6, 26631. (19) Checker, R.; Gambhir, L.; Sharma, D.; Kumar, M.; Sandur, S. K. Cancer Lett. 2015, 357, 265−278. (20) Chen, H.; Wang, H.; An, J.; Shang, Q.; Ma, J. BMC Complementary Altern. Med. 2018, 18, 89. (21) Pan, S. T.; Qin, Y.; Zhou, Z. W.; He, Z. X.; Zhang, X.; Yang, T.; Yang, Y. X.; Wang, D.; Qiu, J. X.; Zhou, S. F. Drug Des., Dev. Ther. 2015, 9, 1601−1626. (22) Qiao, H.; Wang, T.; Yu, Z.; Han, X.; Liu, X.; Wang, Y.; Fan, Q.; Qin, A.; Tang, T. Cell Death Dis. 2016, 7, e2094. (23) Sandur, S. K.; Ichikawa, H.; Sethi, G.; Kwang, S. A.; Aggarwal, B. B. J. Biol. Chem. 2006, 281, 17023−17033. (24) Chou, T. C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27−55. (25) Perez, E. A. Oncologist 1998, 3 (6), 373−389. (26) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. Journal of the National Cancer Institute. 1990, 82, 1247−59. (27) Seidman, R.; Gitelman, I.; Sagi, O.; Horwitz, S. B.; Wolfson, M. Exp. Cell Res. 2001, 268 (1), 84−92. (28) Downward, J. Nat. Rev. Cancer 2003, 3, 11−22. (29) Lu, Z.; Xu, S. IUBMB Life 2006, 58 (11), 621−631. (30) Okano, J. I.; Rustgi, A. K. J. Biol. Chem. 2001, 276, 19555− 19564. (31) McDaid, H. M.; Horwitz, S. B. Mol. Pharmacol. 2001, 60 (2), 290−301. H


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