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Abstract

Background:

We evaluated the effect of cabazitaxel (CAB) as a third-line taxane on Toll-like receptor 4 (TLR4)-mediated signaling pathways, especially NF-κB activity, in metastatic castration-resistant prostate cancer (mCRPC) cells.

Methods:

CAB cytotoxicity was determined by WST-1 assay. To assess the relationship between CAB efficacy and TLR4 signaling pathways, RT-PCR, western blot and immunofluorescence analysis were performed. Additionally, CAB-mediated apoptotic cell death was assessed by Annexin V and RT-PCR analysis.

Results:

Our results demonstrated that CAB exerted considerably cytotoxic and apoptotic effects on PC-3 mCRPC cells (p < 0.05). CAB treatment altered TLR4 expression level in a dose-dependent manner. Furthermore, 1 nM CAB treatment significantly induced NF-κB activity through p65 nuclear localization and increased the expression level of caspase-3, Bax and p53. Interestingly, total apoptotic cell death and IRF3 protein levels were increased at 5 nM concentration of CAB despite a decrease in the levels of both NF-κB and pro-apoptotic genes.

Conclusions:

Therefore, NF-κB activity may be a potential target for the efficacy of CAB in mCRPC cells.

Keywords

Metastatic castration-resistant prostate cancer cabazitaxel toll-like receptor 4 NF-κB activity apoptosis

Introduction

Toll-like receptor-4 (TLR4), expressed in mainly macrophages, induces inflammation after exposure to bacteria-produced lipopolysaccharide (LPS) under normal conditions. Activation of TLR4 promotes cell proliferation, survival, invasion and migration for providing host defense and tissue repair. However, the overexpression of TLR4 induces proliferation of cancer cells and metastasis through alteration of cytokine levels in the tumor microenvironment. Therefore, TLR4 overexpression is observed in many types of cancer including breast, ovarian, pancreatic, prostate, melanoma, glioblastoma and hematopoietic malignancies and leads to poor prognosis, chemoresistance, an increase in tumor size and stage, lymph node metastasis and recurrence.^1
–3

TLR4 signaling complexes activate two pathways including myeloid differentiation primary response 88 (MyD88)-dependent and MyD88-independent TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling. These complexes also induce the transcription of inflammatory cytokines and type I interferons, particularly IFN-β, through the canonical Nuclear Factor kappa B (NF-κB) pathway. In the MyD88-dependent pathway, NF-κB activation is based on sequential phosphorylation of Interleukin-1 receptor-associated kinase 1 (IRAK1) and Interleukin-1 receptor-associated kinase 4 (IRAK4) and then interaction with TNF Receptor-Associated Factor 6 (TRAF6). After ubiquitination of TRAF6, transforming growth factor-β-activated kinase 1 (TAK1) and TAK1 binding proteins (TABs) are recruited and activate the IkB kinase (IKK) complex by catalyzing the phosphorylation of IkB proteins and the mitogen-activated protein kinases (MAPK) (ERK, JNK, p38). The degradation of IkB can result in the translocation of NF-κB into the nucleus. Additionally, the activation of MAPK pathway leads to activation of activator protein 1 (AP-1). Both NF-κB and AP-1 mediates the inflammatory cytokines level. In the TRIF-dependent pathway, TRIF interacts with TANK-binding kinase 1 (TBK1) and IKKi kinases and phosphorylates Interferon regulatory factor 3 (IRF3) for translocation into the nucleus. TRIF also regulates NF-κB through TRAF6 and receptor-interacting serine/threonine-protein kinase 1 (RIP1) interaction. Therefore, the role of TLR4 on each signaling pathway mediating NF-κB in immune response is further elucidated.^4

–8 NF-κB is a transcriptional factor and induces cancer cell proliferation and survival. When the heterodimer forms p65 (RelA) and p50 of NF-κB in the cytoplasm is activated by multiple signaling pathways, IkB kinases phosphorylate IkBα, degrade IkBβ and result in an increase in nuclear NK-kB. Therefore, nuclear NF-κB level is associated with cancer cell growth and chemoresistance.^9
–11 However, NF-κB also exerts a pro-apoptotic role in cancer cells through the activation of different targets (caspase 3, apoptosis inhibitors, Bcl-2 and p53).^12

–15 Furthermore, the NF-κB activation plays a crucial role in the progression of castration-resistant prostate cancer (CRPC). CRPC patients exhibit worse prognosis due to limited treatment options.^16,17 Thus, it is important to identify the effects of chemotherapeutic drugs on the pro-apoptotic or anti-apoptotic role of NF-κB in CRPC for a better therapeutic response. Previously, some studies have determined that the anti-mitotic drugs stimulate NF-κB and the effects of docetaxel and 2-methoxyestradiol on the pro-apoptotic or anti-apoptotic role of NF-kB changes in the cell type.^14,18

–21 Furthermore, the study of Hua et al.²² note that metastatic human prostate cancer cell lines express a higher level of TLR4, whereas several studies have reported that the downregulation or upregulation of TLR4 depends on the cancer type (colon cancer, breast cancer, melanoma, pancreatic cancer, ovarian) as well as the selection of chemotherapy drugs and concentrations (sodium butyrate, paclitaxel, 5-FU, oxaliplatin, TAK-242).^23

–27

In this report, we aimed to clarify the effects of cabazitaxel (CAB), as a third-generation taxane and used for the treatment of patients with mCRPC, on TLR4 associated signaling pathways, and to identify its role in the NF-κB activity in terms of the induction or inhibition of cell death in castration-resistant prostate cancer cells.

Materials and methods

Cell culture method

PC-3 mCRPC cells were obtained from ATCC (Rockville, MD, USA). The cells were cultured in Roswell Park Memorial Institute (RPMI 1640, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium, including 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 0.1% antibiotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37°C with 5% CO₂.

Determination of cell viability and cell death upon CAB treatment

To determine the most effective CAB concentration and the incubation time, the cells (2 × 10⁴ cells/well) were exposed to different ranges of CAB concentrations (0.5, 1, 5 and 10 nM) for 24, 48 and 72 h. After incubation until the relevant time, WST-1 reagent (BioVision, San Francisco, CA, USA) was added and the cells were incubated at 37°C for 30 min in the dark. Then, absorbance obtained from the wells was analyzed by an Elisa reader (Allsheng, China) at 450 nm. According to the results, the most effective concentrations and exposure time were selected for further experiments. To analyze the apoptotic effects of CAB on the cells, Muse™ Annexin V & Dead Cell Assay (Millipore, Germany) was conducted according to the instructions and the cells analyzed by using a Muse Cell Analyzer (Millipore, Germany).

RT-PCR assay

To determine the mRNA expression levels of TLR4 and NF-κB, RT-PCR assay was used. The cells were incubated with 1 and 5 nM CAB for 72 h and total RNA was obtained from the cells by using a RNA extraction kit (Omega Bio-Tek, Norcross, GA) according to manufacturer’s instructions. After the extraction step, the purity and the concentration of RNA samples were examined by a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was converted to cDNA with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression mRNA levels of TLR4 and NF-κB were measured by using a StepOnePlus™ Real Time PCR System (Applied Biosystems, Foster City, CA). β-actin was used as an endogenous reference gene. Furthermore, the expression of Bcl-2, Bax, Caspase 3, and p53 levels were analyzed to support the apoptotic effects of CAB on PC-3 cells.

Western blot

To detect the effects of CAB on TLR4 and NF-κB, PC-3 cells were treated with 1 and 5 nM CAB for 72 h. Then the cells were lysed with RIPA lysis buffer (Santa Cruz, California, USA). After the extraction of the total proteins, cell lysates were separated at 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, California, USA) for blotting. After the transfer the membranes were blocked with 5% nonfat milk in tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h and washed three times and then treated with the primary antibodies of TLR4, and NF-κB p65 and β-actin (Abcam, Cambridge, UK) overnight. The membranes were then treated with HRP-conjugated anti-mouse IgG for TLR-4 and β-actin, and anti-rabbit IgG for NF-κB p65 (Abcam, Cambridge, UK). Following incubation, ECL substrate (BioVision, San Francisco, CA, USA) was added to the membranes to observe indicated blotting protein bands for 5 min. Then membranes were photographed and the band intensity levels were analyzed with the G-Box software (Syngene, Cambridge, UK).

Immunofluorescence assay

The cells (5 × 10⁵ cells/well) were cultured on slides and then treated with 1 and 5 nM CAB for 72 h. After incubation, the cells were treated with 4% paraformaldehyde (PFA) for fixation and permeabilized with 5% goat serum. Following fixation and permeabilization, the cells were incubated with primary antibodies of TLR4, IRF-3, NF-κB and p-NF-κB, and then treated with goat anti-mouse Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 594 seconder antibodies (Abcam, Cambridge, UK) to determine the subcellular localization of indicated proteins. Finally, DAPI (Sigma Aldrich, St. Louis, Missouri, USA) staining was conducted to visualize the nucleus of cells. Afterward, the cells were examined and florescent photographs were obtained by EVOS FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analysis

To perform the statistical analysis and create the graphs, GraphPad Prism 6 (La Jolla, CA) was used. For multiple comparisons, One-way ANOVA analysis of variance Tukey’s test was used. Additionally, the software of Qiagen (https://www.qiagen.com/tr/shop/genes-andpathways/data-analysis-center-overview-page/other-real-time-pcrprobes-or-primersdataanalysis-center/) was used for evaluating the fold changes in the indicated gene expressions. A level of p < 0.05 was considered statistically significant in all tests.

Results

Evaluation of the cytotoxic and apoptotic effects of CAB on PC-3 cells

According to WST-1 results, CAB exerted a significant cytotoxic effect on PC-3 cells in a dose and time dependent manner (p < 0.05) as shown in Figure 1. However, the cytotoxicity of CAB was more prominent for 72 h. After 72 h of treatment with 0.5, 1, 5 and 10 nM CAB, the growth rate of PC-3 cells remarkably reduced to 90.8%, 52.9%, 33.7% and 33.2%, respectively (p < 0.01; Figure 1(a)). Therefore, 1 and 5 nM CAB treatments for 72 h were selected for further analysis. Furthermore, Annexin V results demonstrated that CAB caused a remarkable apoptotic cell death in PC-3 cells. After 72 h treatment with 1 and 5 nM CAB, the percentage of late apoptotic cells considerably increased to 43.5% and 55.4%, respectively (p < 0.01; Figure 1(b), (c)) compared with control cells. Therefore, CAB was shown to have cytotoxic and apoptotic effects on PC-3 cells.

Figure 1.

CAB exhibits significant cytotoxic and apoptotic effects on PC-3 cells. (a) The results of WST-1 assay. (b) Histograms of Annexin V analysis in mCRPC cells (a′) Control, (b′) 1 nM CAB and (c′) 5 nM CAB, respectively. (c) Statistical comparison of total apoptotic cells in the cells. (p < 0.05*, p < 0.01**).

Gene expression analysis in PC-3 cells following CAB treatment

After 1 and 5 nM CAB treatment, the expression of TLR4 level significantly down-regulated (p < 0.001) compared with control group (Figure 2(a)). However, NF-κB level was significantly upregulated 5.27 (p = 0.003) and 2.43-fold (p = 0.018) at 1 and 5 nM CAB, respectively, in PC-3 cells. To evaluate the association of NF-κB activity with apoptosis, as NF-κB target, the expression levels of Bcl-2, Bax, Caspase-3 and p53 were also analyzed. Caspase-3 and Bax expression levels reached to 8.15 (p < 0.001) and 2.78-fold (p < 0.001), respectively, in the presence of 1 nM CAB for 72 h. However, 5 nM CAB treatment caused less increase in caspase-3 (4.94; p < 0.001) and Bax (2.31; p = 0.011) levels. Therefore, alterations in the levels of caspase-3 and Bax were more prominent in the cells after treatment with 1 nM CAB. Additionally, the relative expression level of Bcl-2 was significantly down-regulated in the cells treated with 1 and 5 nM CAB. CAB treatment also altered p53 expression level (Figure 2(b)).

Figure 2.

(a) RT-PCR and western blot results of TLR4 and NF-κB expression in mCRPC cells. (b) The

Changes in the TLR4 and NF-κB protein levels

To assess TLR4 signaling pathways in the response of CAB treatment in the cells, the protein expressions of TLR4, and total p65 NF-κB were analyzed by western blot as shown in Figure 2(a). 1 nM CAB did not affect the expression of TLR4, whereas decreased TLR4 and total p65 protein levels were detected in mCRPC cells upon treatment with 5 nM CAB. Additionally, both p-p65 and p65 levels were increased at 1 nM CAB. Therefore, TLR4 and NF-κB activity were higher in the cells upon 1 nM CAB treatment.

The subcellular localization of TLR4, IRF3 and NF-κB proteins in the cell

After treatment with CAB, the cytoplasmic expression of TLR4 was lower in the cells. However, higher expression of nuclear and cytoplasmic IRF3 was observed in CAB-treated cells (Figure 3(a)). Additionally, p-NF-κB p65 or NF-κB expressions in the nucleus and cytoplasm increased following incubation with CAB at especially 1 nM CAB due to increased NF-κB activity. However, the nuclear localization of p65 decreased at 5 nM CAB in the cells (Figure 3(b)). Therefore, our findings demonstrated that CAB increased NF-κB translocation as well as activity and induced apoptosis due to the characteristic changes in nuclear morphology. These findings were consistent with western blot results.

Figure 3.

Immunofluorescence analysis of TLR4 signaling pathways in mCRPC cells. (a) The subcellular localization of (a′) TLR4 and (b′) IRF-3 proteins. (b) The distribution of (a′) p-NF-kB and (b′) total NF-kB p65 proteins in the cells after treatment with 1 and 5 nM CAB at two different scales (100 and 50 μm).

Discussion

Herein, we demonstrated that higher concentration of CAB treatment inhibited the level of TLR4 expression and the pro-apoptotic activity of CAB was mediated by NF-κB activity, a downstream signaling molecule of the TLR4, in mCRPC cells, in vitro.

The activation of TLR4 promotes metastasis, migration and drug resistance in cancer cell through two signaling pathways. Moreover, some taxane based chemotherapeutics such as paclitaxel (PTX) and docetaxel (Doc) stimulate TLR4 expression and NF-κB activation and lead to drug resistance in cancer cells including breast, ovarian, prostate, lung and melanoma.^1,28,29 Therefore, the molecular interaction between chemotherapeutic drugs and TLRs and associated signaling pathways activation may be vital for overcoming drug resistance.

CAB, the major natural taxane, exhibits considerable anticancer activity in mCRPC cells through binding microtubules and inhibiting cellular mitosis. Therefore, CAB treatment increases progression-free survival and overall survival in mCRPC patients.^29,30 However, there is no clear evidence evaluating the relationship between the efficacy of CAB and TLR4 mediated signaling pathways in the literature. In this context, we assessed the activation of TLR4 based signaling pathways, especially NF-κB stimulation upon CAB treatment in mCRPC cells, for the first time. Our findings indicated that particularly 1 nM CAB increased both mRNA and protein expression levels of TLR4 and NF-κB in PC-3 cells. Furthermore, higher concentration of CAB treatment resulted in the downregulation of TLR4 and NF-κB activity. The study of Taxman et al.³¹ state that the upregulation of pro-survival and inflammatory genes is detected after 6 h PTX post-treatment by transcriptional analysis in different cancer cells. PTX mediated TLR4 as well as NF-κB activation eliminate the secondary effects of the cell death results. Therefore, higher concentrations of CAB treatment may surpass resistance due to leading to the downregulation of TLR4 and nucleolar localization of p65 and induce a higher apoptotic cell death in mCRPC cells. Furthermore, the study of Rezania et al.³² note that TLR4 expression is not changed upon treatment with lipopolysaccharide (LPS) which modulates TLR4 expression in PC-3 cells. However, LPS could significantly increase adhesion of PC-3 cells. In our study, we only analyzed the interaction of CAB treatment with TLR4 activity in PC-3 cells. However, the effects of LPS stimulation on TLR4 activity upon CAB treatment should be investigated in further studies. Additionally, TLR3 activates the transcription factor NF-κB and IRF-3 through TRIF as in TLR4. Several studies have shown that TLR3 exerts apoptotic effects through the secretion of interferons and cytokines in prostate cancer cells upon infection or stimulation with synthetic agonist Poli I:C.^33,34 According to Gambara et al.,³⁴ IRF-3 plays a crucial role in TLR3-mediated apoptosis in prostate cancer cells due to increased nuclear localization of IRF-3 and the anticancer effect of Poli I:C is more proficient in LNCaP cells than PC-3 cells. Therefore, the relationship between TLR3 mediated cell death and CAB efficacy is further elucidated at both mRNA and protein levels due to increased nuclear and cytoplasmic expression of IRF-3 in CAB-treated PC-3 cells.

Furthermore, the study of Parrondo et al.¹⁴ demonstrate that docetaxel (Doc) and 2-methoxyestradiol (2ME2) effectiveness is based on the levels of NF-κB activity. Particularly, the combination of NF-κB activator (betulinic acid) with Doc and 2ME2 can lead to an increase in apoptosis in LNCaP and caspase independent cell death in mCRPC cells. Therefore, Doc or 2ME2 based cell death is mediated through increased NF-κB activity. In the present study, we found that 1 nM CAB stimulated NF-κB activity as well as upregulated the level of caspase-3, Bax and p53 expressions. Interestingly, 5 nM CAB still increased total apoptotic cell death and IRF3 protein levels despite a decrease in both NF-κB and pro-apoptotic genes (Bax, caspase-3 and p53) expression. Thus, further studies are needed to investigate the underlying molecular mechanism of the pro-apoptotic or anti-apoptotic role of NF-κB activity through NF-κB activators or inhibitors in CAB-treated mCRPC cells.

Conclusions

Our findings suggest that CAB exerts remarkably cytotoxic and apoptotic effects on PC-3 mCRPC cells through the upregulation of pro-apoptotic genes as well as NF-κB activity and the downregulation of the TLR4 and Bcl-2 levels. Additionally, higher CAB concentration changes the expression pattern of TLR4 mediating signaling pathways and pro-apoptotic genes despite an increase in apoptotic cell death. Therefore, further investigations should be performed to assess the molecular mechanism of apoptotic cell death, TLRs activation or inhibition and the pro-apoptotic and anti-apoptotic role of NF-κB activity in mCRPC, in vitro and in vivo.

Footnotes

Author contributions

GGE, ADO and IEE originated and designed the research. UE and GC advised on experimental design. GGE, ADO and IEE conducted experiments. GGE, UE and GC analyzed and interpreted the data. All authors read carefully and approved the final version of the manuscript to be published.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the (Scientific Research Projects Foundation of the Bursa Uludag University) under Grant (Number BUAP(T)-2015/4).

ORCID iDs

Gamze Guney Eskiler

Unal Egeli

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Association between the anticancer efficacy of cabazitaxel and toll-like receptor 4 mediating signaling pathways in metastatic castration-resistant prostate cancer cells