Background
The cytotoxicity of chemotherapeutic agents is attributed to apoptosis. One feature that cytotoxic treatments of cancer have in common is their activation of the transcription factor NFκB, which regulates cell survival, suppresses the apoptotic potential of chemotherapeutic agents and contributes to drug resistance [
1]. Acquired resistance to the effects of chemotherapy has emerged as a significant impediment to effective cancer therapy. As such, it is believed that inhibitors of NFκB might promote apoptosis in cancer cells and can be helpful to overcome resistance to chemotherapeutic agents.
Nuclear factor kappa B (NFκB) is a family of transcription factors that play important roles in regulating cell differentiation, proliferation, immune response and blocking apoptosis [
2,
3]. In mammalian cells, the NFκB/Rel family consists of five members: RelA (p65), RelB, c-Rel, p105/p50 (NFκB1), and p100/p52 (NFκB2). Each family member has a conserved Rel homology domain specifying DNA binding, protein dimerization, and nuclear localization. In most cells, NFκB is composed of a heterodimer of p65 and p50, where the p65 protein is responsible for the transactivation potential. In unstimulated cells, NFκB is sequestered predominantly in the cytoplasm in an inactive complex through interaction with IκB inhibitor proteins. In response to stimulation by a variety of potent activators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, phorbol ester (PMA) or lipopolysaccharide [
4] and genotoxic agents (doxorubicin, radiation) [
5,
6], IκBα is rapidly phosphorylated at two conserved NH
2-terminal serines (Ser-32 and Ser-36) and degraded through a ubiquitin-dependent proteolysis, resulting in the release of NFκB, its translocation into the nucleus and induction of gene transcription. The NFκB has a role in oncogenesis and regulation of cancer therapy sensitivity. Overexpression, amplification, and rearrangements of different genes related to NFκB have been observed in tumors [
7]. NFκB is activated in response to various inflammatory stimuli including cytokines, mitogens, bacterial products, viral proteins, and apoptosis-inducing agents [
8,
9]. Constitutive expression of NFκB leads to activation of several factors involved in cell cycle progression and cell differentiation for cancer metastasis. Inhibition of NFκB activity in tumor cells dramatically reduces cell growth
in vitro and
in vivo [
10]. NFκB, possibly through the activation of the antiapoptotic genes, plays a key role in the protection of cells against inducers of apoptosis including chemotherapeutic drugs [
11]. Several mechanisms including increased expression of NFκB proteins, mutations and/or deletions in IκBα gene, and increased IκBα turnover, are involved in NFκB hyperactivation in tumor cells [
7,
12]. As such, various therapeutic strategies aim to decrease chronic NFκB hyperactivation by pharmacological as well as phytomedicinal approaches in cancer [
13‐
17]. NFκB-regulated genes are involved in cell death, invasiveness, proliferation, angiogenesis, inflammation and multidrug resistance (MDR). One of the most important mechanisms by which tumor cells resist to cytotoxic effects of a variety of chemotherapeutic drugs (including vinblastine, doxorubicine, etoposide and teniposide, as well as many other cytotoxic agents) is overexpression of the
mdr1 gene and its product, P-glycoprotein (P-gp) [
18].
P-gp is a 180 kDa protein which belongs to the ATP-binding cassette (ABC) superfamily of membrane transporter proteins [
19,
20]. It is expressed in various tissues, such as kidney tubules, colon, pancreas and adrenal gland, and tumors derived from these tissues are often resistant to chemotherapeutic drugs. Furthermore,
mdr1 expression is also increased in many relapsing cancers. P-gp is an energy-dependent drug efflux pump that maintains intracellular drug concentrations below cytotoxic levels, thereby decreasing the cytotoxic effects of a variety of chemotherapeutic agents, including anthracyclines, vinca alkaloids, and epipodophyllotoxins [
18,
21]. P-gp also plays a role in inhibition of drug accumulation and caspase activation in the MDR tumor [
22‐
24]. Of special note, NFκB-mediated drug resistance was found to depend on the regulation of P-gp [
25]. In addition, NFκB-dependent regulation of P-gp expression has also been demonstrated in renal tubules or liver [
26,
27]. By upregulation of P-gp expression, NFκB was found to control drug efflux in cancer cells.
Cancer cells contain multiple signal transduction pathways whose activities are frequently increased due to cell transformation, and these pathways are often activated following cell exposure to established cytotoxic therapies, including ionizing radiation and chemical DNA-damaging agents. Many pathways activated in response to transformation or cytotoxic agents promote cell growth and invasion, which counteract the processes of cell death. As a result of these findings, many drugs with varying specificities have been developed to block the signaling by these cell survival pathways in the hope of killing tumor cells and sensitizing them to toxic therapies [
28]. Unfortunately, due to the plasticity of signaling processes within a tumor cell, inhibition of a single growth factor receptor or signaling pathway frequently has only modest long-term effects on cancer cell viability, tumor growth, and patient survival. As a result of this observation, a greater emphasis has begun to be put on multi-target natural compounds, such as polyphenols, withanolides, xanthones, indanones, curcuminoids, which simultaneously inhibit multiple inter-linked signal transduction/survival pathways [
14,
28‐
33]. Hopefully, this could limit the ability of tumor cells to adapt and survive, because the activity within multiple parallel survival signaling pathways has been reduced [
34]. As such, over the past decades, the efforts of researchers in looking for the new drugs to use in oncology have refocused on natural products [
1,
34].
One of the expanding directions of modern medicine is based on using of natural phytochemical compounds. Polyphenols or phenolic compounds encompass molecules that possess an aromatic ring bearing one or more hydroxyl substituents. Natural polyphenols can range from simple molecules, such as phenolic acids and flavonoids, to large highly polymerized compounds, such as tannins [
35]. This class of phytochemicals can be found in high concentrations in wide varieties of higher plants and their products, such as wine and tea. They were also demonstrated to exert a wide range of biological activities including antioxidant, anticarcinogenic, antiproliferative, antimicrobial anti-inflammatory and apoptosis-inducing activities [
36‐
40].
Various polyphenols have been characterized with respect to their anti-invasive potential. Because invasion is, either directly or via metastasis formation, the main cause of death in cancer patients, development of efficient anti-invasive agents is an important research challenge [
31]. Vanden Berghe
et al. showed that phytoestrogenic soy isoflavones can selectively block nuclear NFκB transactivation of specific NFκB target genes independently of their estrogenic activity in highly metastatic breast cancer cells [
16]. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tumor, the oligomeric and polymeric polyphenols decreased TPA-induced cell proliferation by attenuating the activation of signaling kinases [c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 protein kinase and Akt], transcription factors [activator protein-1 (AP1) and NFκB] and inflammatory protein [cyclooxygenase-2 (Cox-2)] [
41,
42]. The NFκB and Akt kinase pathways, which play critical roles in inflammation, vascular homeostasis and angiogenesis, were repressed by the polyphenolic compound deguelin in human vascular endothelial cells,
HT1080 fibrosarcoma cells and chronic lymphocytic leukemia cells [
43‐
45]. Nitric oxide production was reduced by the green tea polyphenols (-)-Epigallocatechin-3-gallate (EGCG) and black tea theaflavins by suppressing inducible nitric oxide synthase in a breast cancer cell line [
46]. The latter treatment blocks nuclear translocation of the transcription factor NFκB as a result of decreased IκB kinase activity. However, anti-cancer effects of polyphenols may also indirectly also involve effects on immune cells at the cancer-inflammation interface. Several studies demonstrated that polyphenolic compounds exhibit anti-inflammatory activity in activated macrophages by inhibiting the NFκB signaling pathway [
39,
47,
48]. Dijsselbloem
et al. demonstrated that genistein inhibits IL6 gene expression by modulating the transcription factor NFκB in TLR4-stimulated dendritic cells [
49]. Pycnogenol inhibits TNFα-induced NFκB activation and adhesion molecule expression in human vascular endothelial cells [
50]. Red wine polyphenols, delphinidin and cyanidin inhibit platelet-derived growth factor
AB (PDGF
AB)-induced VEGF release in vascular smooth muscle cells by preventing activation of p38 MAPK and JNK [
51]. Olive oil polyphenols exert rapid inhibition of p38 and CREB phosphorylation leading to a downstream reduction in COX-2 expression in human colonic adenocarcinoma, Caco-2 cells [
52].
Previously, we have already reported the significant anti-cancer activities of quercetin, Siamois 1 and Siamois 2 polyphenols and the withasteroid withaferin A, which hold promise as dietary supplements in nutrition-based intervention in cancer treatment [
32,
53]. In this study we wanted to further investigate whether interference of Siamois polyphenols and withasteroids with NFκB-dependent apoptosis and inflammatory pathways can sensitize doxorubicin-resistant P-gp-overexpressing K562 erythroleukemic cells for cell death.
Materials and methods
Reagents and Chemicals
Quercetin, Kaempferol, and Eriodictyol were from Extrasynthèse (Genay, France), Withaferin A from Chromadex (Irvine, US), whereas home-purified 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (WP283) has been described elsewhere [
54]. These compounds were stored as 100 mM solutions in DMSO at -20°C. Doxorubicin hydrochloride was kindly provided by Dr. F. Offner (University Hospital UGent). Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma Chemical Company (St Louis, MO, USA) and stored as 1 mg/ml solution in DMSO at -20°C. Recombinant murine TNF, produced in
Escherichia coli and purified in our laboratory to at least 99% homogeneity, had a specific biological activity of 8.58 × 10
7 IU/ml of protein as determined in a standard TNF cytolysis assay. Reference TNF (code 88/532) was obtained from the National Institute of Biological Standards and Control (Potters Bar, UK). Anti-IκBα, anti-p65 (C20), anti-p50 (NLS), anti-cRel (N), anti-RelB (C19), anti-Fra1(H50), anti-Nrf2 (C10), anti-Bax antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-p38, anti-p44/42, anti-cfos, anti-cjun, anti-junB, anti-junD from Active Motif, anti-Sirt1 from Biomol, anti-Stat3 from Upstate, anti-histone-H3 antibodies from Abcam and anti-tubulin were from Sigma (Bornem, Belgium). The phospho-specific antibodies directed against p65 Ser536, p38 and p44/42 MAPK, cjun, Akt, MEK were from Cell Signaling (Beverly, CA). Anti-Bcl-2, anti-Bim, anti-Bad, anti-P-Bad antibodies were purchased from Cell Signaling (Boston, MA).
Cell culture and Cytotoxicity assay
Murine fibrosarcoma L929sA cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum, 5% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Twenty-four hours before induction, cells were seeded in multiwell dishes such that they were confluent at the time of the experiment.
Doxorubicin-sensitive erythroleukemic cells (K562) and doxorubicin-resistant erythroleukemic cells (K562/Adr) which overexpress P-gp were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin, in an incubator at 37°C, 95% humidified, 5% CO2. Cultures initiated at a density of 105cells/ml grew exponentially to about 106 cells/ml in 3 days. K562/Adr cell line was cultured in RPMI 1640 medium in the presence of 100 nM doxorubicin for 72 h, after that the cells were grown in RPMI 1640 medium without doxorubicin for 2 weeks before the experiments. For the assays and in order to have cells in the exponential growth phase, cultures were initiated at 5 × 105 cells/ml and used 24 h later, reaching a density of about 8-10 × 105 cells/ml.
The cytotoxicity assay was performed as described previously [
55]. Cells (5 × 10
4 cells/ml) were incubated in the presence of various concentrations of compounds tested. The viability of cells was determined by MTT reduction. The concentration of compound required for 50% inhibition of the proliferation of cells (IC50) was determined by plotting the percentage of cell growth inhibition (%IC) versus the compound concentration when measured at 72 h. Alternatively, cell cytotoxicity assays were performed by the ToxiLight Assay (Lonza) according to manufacturer's instructions.
Apoptosis assay
Cells were washed with ice-cold phosphate-buffered saline (PBS) after treatment, and 5 × 105 cells were stained with annexin V (AnnV)-FITC during 15 min in the dark followed by propidium iodide (PI) staining (human AnnexinV-FITC Detection kit, Bender MedSystems Diagnostics, Vienna, Austria). The stained cells (104 cells) were measured by flow cytometry (Cytomics FC500 1 laser, Beckman Coulter, Fullerton, USA) and results were expressed as percentage of living (AnnV-, PI-), early apoptotic (AnnV+, PI-), and late apoptotic/dead cells (AnnV+, PI+). The percent of living cells was normalized to 100% living cells incubated in control medium with 0.1% DMSO. All measurements were made in duplicate and averaged.
IL6 ELISA
IL6 cytokine levels in cell-free culture supernatants were determined using Hu-IL6 Cytoset ELISA kit (Biosource International Inc. Camarillo USA) with detection limits of 15 pg/ml, according to the manufacturer's instructions. Three independent experiments were done, each in triplicate.
Measurement of caspase-3/7 activity
After appropriate induction, cells were washed with ice-cold PBS and the cytosolic cell lysate was prepared as described previously [
56]. Measurement of caspase-3/7 activity was carried out by the incubation of cytosolic cell lysate with fluorogenic substrates, Ac-DEVD-AMC. The release of fluorescent AMC was monitored for 1 h at 37°C at 2-min time intervals in a fluorescence microplate reader (Packard Instrument Co.) using a filter with an excitation wavelength of 360 nm and a filter with an emission wavelength of 460 nm [
57,
58]. Data are expressed as the increase in fluorescence as a function of time (Δfluorescence/min) normalized with that of cells incubated in control medium with 0.1% DMSO.
Reporter Gene Analysis
The recombinant plasmid p(IL6κB)
350 hu.IL6P-luc+ was described previously [
59,
60]. Stable transfection of L929sA cells was performed by the calcium phosphate precipitation procedure according to standard protocols [
59]. Luciferase and galactosidase reporter assays were carried out according to the manufacturer's instructions (Promega) and have been described previously [
59]. Normalization of luciferase activity was performed by measurement of β-galactosidase levels in a chemiluminescent reporter assay Galacto-Light kit (Tropix, Bedford, MA). Light emission was measured in a luminescence microplate reader (Packard Instrument Co.). Luciferase activity, expressed in arbitrary light units, was corrected for the protein concentration in the sample by normalization to the co-expressed β-galactosidase levels. β-Galactosidase protein levels were quantified with a chemiluminescent reporter assay Galacto-Light kit (Tropix).
Western blot analysis
For the western blot analysis of total cell lysates, cells were washed with ice-cold PBS before lysis in catenine lysis buffer (1% Triton X-100, 1% NP-40, 10 mg/l leupeptine, 10 mg/l aprotinin, 2 mM PMSF, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate). Protein concentration in lysates was measured using BCA™ Protein Assay Kit (Thermo Scientific, Rockford, USA) according to the manufacturer instructions. Lysates were stored at -20°C until assayed.
Before analysis, lysates were diluted to reach equal protein concentration in each sample, and SDS sample buffer was added (0.5 mM Tris-HCl, pH 6.8, 40% glycerol, 9.2% SDS, 5% mercaptoethanol, and 0.05% w/v bromophenol blue), one part of buffer for three parts of diluted lysate. To shear DNA and reduce sample viscosity, samples were heated to 95°C for 5 min, after which they were immediately cooled on ice and microcentrifuged for 5 min. For the western blot analysis of nuclear extract, the nuclear proteins were suspended in SDS sample buffer at the same concentration. The protein samples were separated by 12% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Blots were probed using the appropriate antibodies and the immunoreactive protein was detected using enhanced chemiluminescence reagents on an Odyssey imaging system (LI-COR Biosciences, Lincoln, USA).
Electrophoretic Mobility Shift Assay (EMSA)
After treatment, cells were washed with ice-cold PBS and pelleted in 1 ml PBS by centrifugation for 10 min at 2600 rpm (4°C). Preparation of nuclear extracts has been described previously [
60]. For EMSA, equal amounts of protein were incubated for 25 min with an NFκB-specific
32P-labeled oligonucleotide and binding mix as described previously [
32]. For supershift assay, antibodies were preincubated to the sample of interest for 10 minutes prior to incubation with radiolabeled probe [
61]. Labeling of the oligonucleotides was performed with [α-
32P]-dCTP by using Klenow enzyme (Boehringer Mannheim). For EMSA competition assays, 100 fold excess of unlabeled NFκB oligonucleotide was added to the binding mix. The NFκB oligonucleotide comprises the sequence: 5'-AGCTATGT
GGGTTTTCCC ATGAGC-3', in which the single IL6 promoter-derived NFκB motif is bold and italicized. Samples were loaded on a 6% polyacrylamide gel run in 0.5 × TBE buffer (pH 8) and complexes formed were analyzed using Phosphor Imager Technology.
RNA isolation and real-time Q-PCR analysis
Total RNA was extracted with the acid guanidinium thiocyanate-phenol-chloroform method using the Trizol reagent (Invitrogen, Merelbeke, Belgium). Reverse transcription was performed on 500 ng of total RNA in a 30 μl total volume. For normalization, cDNA concentrations in each sample were determined prior to quantitative real-time PCR (Q-RT-PCR). The Q-RT-PCR was performed on 5 μl of each condition using Invitrogen Sybr green platinum Supermix-UDG on a iCycler apparatus (Bio-Rad, Eke, Belgium). All amplifications were performed in duplicate or triplicate, and data were analyzedanalyzed using Genex software (Bio-Rad, Eke, Belgium). Data were expressed as mRNA expression normalized with that of cells incubated in control medium with 0.1% DMSO. Q-PCR primers are summarized in Table
1.
Table 1
Primers sequences used in the real-time Q-PCR. Note: the abbreviations used in the table: FW indicates forward; REV, reverse.
IL6 REV
|
AGTGCCTCTTTGCTGCTTTC
|
IL8 FW
|
GCTCTCTTGGCAGCCTTCCTGA
|
IL8 REV
|
ACAATAATTTCTGTGTTGGCGC
|
A/BFL-1 FW
|
GATTTCATATTTTGTTGCGGAGTTC
|
A/BFL-1 REV
|
TTTCTGGTCAACAGTATTGCTTCAG
|
MCP1 FW
|
ACTCTCGCCTCCAGCATG
|
MCP1 REV
|
TTGATTGCATCTGGCTGAGC
|
A20 FW
|
CCTTGCTTTGAGTCAGGCTGT
|
A20 REV
|
TAAGGAGAAGCACGAAACATCGA
|
CYCLIND1 FW
|
CGCCCCACCCCTCCAG
|
CYCLIND1 REV
|
CCGCCCAGACCCTCAGACT
|
VEGF FW
|
GCCTCCCTCAGGGTTTCG
|
VEGF REV
|
GCGGCAGCGTGGTTTC
|
MDR1 FW
|
CTGCTTGATGGCAAAGAAATAAAG
|
MDR1 REV
|
GGCTGTTGTCTCCATAGGCAAT
|
Discussion
Extensive studies indicate that both hyperactivation of NFκB and overexpression of multidrug transporters play important roles in cancer chemoresistance [
7,
18,
25,
27,
62,
68]. Since expression of the multidrug transporter P-gp was found to be NFκB-dependent, it is believed that NFκB inhibitors can decrease P-gp expression and restore chemosensitivity [
1,
14]. However, our studies have shown that the picture is more complicated. Previously, we have already demonstrated apoptosis of MDA-MB435 cells in presence of Siamois polyphenols in a xenograft model in vivo [
53]. Furthermore, the NFκB inhibitor withaferin A has been described as a promising drug for cancer chemotherapy and radiosensitization [
32,
79,
80]. Now, we further analyzed whether withaferin A or Siamois polyphenols quercetin, kaempferol, eriodictyiol, and WP283 hold therapeutic promise as NFκB-inhibitors for chemosensitization of doxorubicin resistant K562/Adr erythromyelogenous leukemia cells. In NFκB reporter gene studies, we compared dose-dependent repression of luciferase gene expression in response to Siamois polyphenols quercetin, kaempferol, eriodictyiol, and WP283 with IC50 values in the range of 0.1-50 μM respectively. Furthermore, upon comparing endogenous gene transcription and protein expression of specific NFκB target genes, we observed comparable potencies in NFκB-dependent gene repression by Siamois polyphenols in K562 and K562/Adr cell types. Of special note, both cell types express different subsets of NFκB target genes. More particularly, K562 cells reveal a predominant inflammatory gene expression profile (i.e. strong expression of IL6, IL8, MCP1, and A1/Bfl1), whereas K562/Adr cells demonstrate a more tumorigenic pattern (i.e. strong expression of A20, cyclin D1, VEGF, and mdr1). As such, we further studied NFκB signaling mechanisms and coregulatory pathways which may be responsible for differential NFκB target gene expression/inhibition and apoptosis sensitivity for withaferin A and Siamois polyphenols. Upon characterization of the major NFκB activation and transactivation pathways, we found differential regulation of NFκB activity by withaferin A and quercetin, kaempferol, eriodictyol and WP283. Interestingly, IκB degradation and NFκB/DNA binding was significantly reduced by all compounds tested in both cell types, among which withaferin A, quercetin and eriodictyol showing the most potent inhibition, and kaempferol and WP283 much weaker and variable inhibition. Remarkably, increased levels of basal NFκB binding in K562/Adr cells cannot be inhibited by Siamois polyphenols in contrast to inhibition of inducible NFκB/DNA-binding. Furthermore, relative composition of NFκB/DNA binding complexes reveals that K562 cells contain much higher levels of p65-p65 homodimers. Of particular interest, the inflammatory cytokine IL8 was found to preferentially bind p65-p65 homodimers instead of p50-p50 and p50-p65 dimers [
81], which could explain strong expression of inflammatory cytokines in K562 cells. From another perspective, NFκB dimer composition may also depend on the repertoire posttranslational modifications present on NFκB [
63,
70,
71,
82,
83]. More specifically, we have detected variable and compound-specific effects on p38 MAPK, MEK1, Akt kinase pathways, which may also interfere with NFκB transcription factor composition and/or activity. Finally, besides phosphoregulation of transcription factors, acetylation by cofactors (CBP, HDAC, Sirtuin) and DNA methylation have recently added an additional epigenetic control of inducible NFκB transcription [
84‐
87]. Of special note, as doxorubicin was found to increase Sirt1 HDAC levels [
68], we compared nuclear Sirt1 levels in both cell types and observed a significant increase in Sirt1 protein in K562/Adr. As such, we cannot exclude that in addition to kinases also Sirt HDACs may contribute in cell-specific phosphoacetylation control of TF/DNA binding and transcriptional activity and may prevent NFκB p65 homodimer formation. In addition to cell specific regulation of NFκB, it can be observed from Fig.
5 that also AP1 members (i.e. Fra1, cjun and junD) and Nrf2 are differentially expressed in both cell types. As such, we can also neither exclude compound-specific kinase effects on these transcription factor families, since various NFκB target genes involved in inflammation, metastasis, angiogenesis and drug resistance are also coregulated by AP1 and Nrf2 [
66,
67,
88,
89].
Most surprisingly, although inhibition of NFκB activity in general contributes in chemosensitization of cancer cells [
5,
62], caspase activation is delayed and apoptosis is attenuated in K562/Adr cells treated with Siamois polyphenols, although efficacy of NFκB inhibition and initiation of early apoptosis by Siamois polyphenols is similar in doxorubicin-sensitive and resistant cell types. This is in line with previous reports on drug resistance, which describe that P-glycoprotein inhibits cytochrome c release and caspase-3/8 activation, but not formation of the death-inducing signal complex [
23,
24,
90]. Along the same line, impaired activation of caspase 3,6,7,8,9,10 has been described in doxorubicin-resistant breast cancer cells [
91]. The fact that Siamois polyphenols are able to completely ablate NFκB target gene expression, hyperactivate MEK1 and trigger early apoptosis in K562/Adr cells argues against the hypothesis that Siamois polyphenols may not be uptaken or are secreted out of the cell because of hyperactivated P-gp activity in K562/Adr cells. As such, P-gp overexpression confers resistance to a wide range of caspase-dependent apoptotic agents not only by removing drugs from the cell, but also by inhibiting the activation of proteases involved in apoptotic signaling [
92]. Only a few drugs are reported to overcome this P-gp/Mdr phenotype and most of them are molecules that induce cell death in a caspase-independent manner [
93]. Interestingly, in analogy to some specific glutathione S transferase inhibitors (NBDHEX) and mitochondria-targeting drugs (oligomycin) [
94‐
96], withaferin A was found to bypass the P-gp resistance and to overcome attenuation of late apoptosis in K562/Adr cells. Unfortunately, we could not detect major differences in regulation of intracellular regulators of mitochondrial apoptosis of the Bcl2, BH123 or BH3 family proteins in K562 and K562/Adr cells treated with withaferin A or quercetin: both treatments trigger time-dependent decrease of Bcl2, Bim and P-Bad protein levels in K562 cells (albeit delayed in K562/Adr cells). However, upon investigation of cytoskeletal proteins, we observed that withaferin A is able to decrease tubulin protein levels, whereas Bcl
XL and Bax protein levels remain unaffected. Interestingly, various chemoresistant tumors, including doxorubicin resistant cancers reveal therapy induced cytoskeletal changes in microtubules and intermediate filaments [
97,
98]. In analogy to other microtubule-targeted anti-cancer drugs, withaferin A could restore therapy sensitivity in P-gp-overexpressing cells by targeting the cytoskeletal organization. Further support for this mechanism has recently been provided by other groups, describing involvement of withaferin A-dependent actin and vimentin microfilament aggregation in cancer cell apoptosis and suppression of angiogenesis via a direct thiol oxidation mechanism [
77,
99,
100]. Along the same line, we were able to block withaferin A-induced effects upon competition with excess amounts of the cysteine donor molecule DTT. Alternatively, it cannot be excluded that thiol-reactivity of withaferin A interferes with cysteine-sensitive P-gp protein folding steps and/or P-gp protein function [
101,
102]. Further investigation is needed to map cysteine target proteins of withaferin A which allow to bypass P-gp chemoresistance and restore apoptosis sensitivity.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WS carried out the molecular studies and drafted the manuscript. AP, SZ and SG assisted in molecular studies. SM, GH participated in the design of the study. WP has purified WP283. WVB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.