Introduction
Prostate cancer is one of the most common cancer types and the second leading cause of cancer related death in men in the world [
1,
2]. The androgen deprivation therapy (ADT), which suppresses or reduces androgens binding to the androgen receptor (AR), is a well-known treatment strategy for advanced, recurrent and even metastatic prostate cancer; however, the long term therapeutic outcomes of ADT on prostate cancer remain uncertain, and are associated with considerable known adverse effects that affect the quality of life in prostate cancer patients [
3]. Also, development of resistance to ADT is a major obstacle for the management of advanced prostate cancer [
4]. Therapies with AR antagonists such as bicalutamide (Casodex) and androgen withdrawal initially regress tumors but development of various compensatory mechanisms including AR bypass signaling leading to tumor growth, and eventually develop more aggressive castration resistant prostate cancer (CRPC) [
5]. Therefore, search for the novel therapeutic approaches based on various combinations of anticancer drugs and procedures to minimize the resistance in order to enhance the therapeutic efficacy are strongly required. Curcumin, an active natural polyphenol derived from the root of Curcuma longa, has been shown great potential as a novel therapeutic agent due to its pharmacological safety and efficacy in treating a wide variety of cancers [
6,
7]. These facts, tested and confirmed in many different cancer types [
8], have paved the way for research aimed at elucidating the potential beneficial effects of combining curcumin and various anti-cancer drugs in order to establish more efficient and less toxic cancer treatment modalities.
Cell surface-associated mucin 1 (MUC1) (previously known as PUM, MCKD1), a highly glycosylated transmembrane heterodimeric protein and a transmembrane member of the mucin family, is highly expressed in various human malignant tumors including prostate cancers and is correlated with a poor prognosis [
9,
10]. MUC1 cytoplasmic tail can interact with many signaling pathways, and act as a co-transcription factor to activate various genes involved in tumor progression and metastasis. In many tumor types, expression of MUC1 correlates with aggressive, metastatic phenotype, limited response to therapy and poor survival [
10,
11]. The MUC-1 C-terminal subunit (MUC1-C) is a single-pass transmembrane protein that interacts with receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and others, at the cell membrane and contributes to activation of other kinase signaling pathways that induce proliferation and tumor growth [
12‐
14]. MUC1-C also binds directly to nuclear factor NF-kappaB (NF-κB) p65 and promotes NF-κB-mediated gene transcription [
9]. Thus, mucins including MUC1-C are considered important markers for early diagnosis and targeted therapy due to their unique expression pattern during cancer progression. Studies have provided substantial evidence for the involvement of transmembrane MUC1-C in altered cell signaling, tumor growth, and metastasis [
15,
16].
In this study, we explored the potential mechanism by which combination of curcumin with bicalutamide in the inhibition growth of androgen-independent prostate cancer cells.
Materials and methods
Cell culture and chemicals
The prostate cancer cell lines (PC3, DU145, LNCaP) were obtained from the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. All cell lines have been tested and authenticated for absence of Mycoplasma, genotypes, drug response, and morphology in the Laboratory. Cells were grown in F-12K or DMEM (1:1) medium (obtained from GIBCO, Life Technologies, Grand Island, NY, USA) with supplemented 10 % fetal bovine serum. The polyclonal antibody against both MUC1 and MUC1-C (Cat No. ab109185) was obtained from Abcam (Cambridge, MA, USA). The antibodies against p65 (Cat No. 8242), total extracellular signal-regulated kinase1/2 (ERK1/2) (Cat No. 4695), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) (Cat No. 9258) and the phosphor-forms (Cat No. 4370 and 4668) were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). Curcumin, PD98059 [(MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK inhibitor] and SP600125 (SAPK/JNK inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Western blot analysis
The detailed procedure was reported previously [
17,
18]. Briefly, protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in 5 × SDS-sample buffers and separated on 12 % SDS polyacrylamide gels. Membranes were incubated with antibodies against MUC1-C, p65, the phosphor and total ERK1/2, and SAPK/JNK. The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millpore, Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio-Rad, Hercules, CA, USA) and documenting the results.
Cell viability assay
Cell viability was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay [
18]. Briefly, prostate cancer cells were harvested, counted and seeded in a 96-well microtitre plate. The cells were treated with increasing concentrations of curcumin for up to 72 hrs. After incubation, 20 μl MTT solution (5 g/L) was added to each well and prostate cancer cells were incubated at 37 °C for additional 4 h. Supernatant was removed, then 200 μL solvent dimethyl sulfoxide was added to each well and oscillated for 10 min. Absorbance at 490 nm was determined through the use of ELISA reader (Perkin Elmer, Victor × 5, Waltham, MA, USA). Cell viability (% of control) was calculated as (absorbance of test sample/absorbance of control) × 100 %.
Transient transfection assay
The cells were seeded in 6-well dishes and grown to 50–60 % confluence. The control or MUC1-C and p65 overexpression vectors (pCMV6-AC-MUC1, pCMV6-AC-p65) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). Briefly, cells were seeded in 6-well dishes and grown to 50–60 % confluence. For each well, 2 μg of control (pCMV6-AC) and MUC1-C and p65 plasmid DNA constructs were transfected into the cells using Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY, USA) for up to 30 h based on the instruction from the provider, followed by treating with curcumin for an additional 24 or 48 h for other experiments.
Statistical analysis
Data are presented as mean ± SD from three independent experiments with triplicates. Statistical significance was determined with Student's t test (two-tailed) comparison between two groups of data set. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05, see figure legends).
Discussion
Majority of advanced prostate cancers are sensitive to androgen deprivation therapy in the first beginning, then subsequently progress to the CRPC. The reasons remain unknown. Because of the limitations of current therapeutic approaches, many patients die of recurrent and metastatic diseases. The combination with anti-inflammatory and other adjuvant therapies present a very promising treatment approach for this malignancy. Curcumin is a promising anticancer agent for various cancer types including prostate cancer cells, and involves in multiple signaling and potential targets [
6,
7,
19]. However, the detailed molecular mechanisms underlining suppression of androgen-independent prostate cancer cell growth still remain to be elucidated. In this study, we demonstrated not only a significant inhibition of growth of prostate cancer cells by curcumin, but more importantly, a synergistic effect observed in combination of curcumin with bicalutamide, an androgen receptor antagonist, in androgen-independent prostate cancer cells. These findings implied a potential new mechanism for this synergy that was AR-unrelated. The molecular mechanisms by which curcumin inhibited growth of androgen-dependent and -independent prostate cancer cells were reported in other studies demonstrating that pathways and transcription factors other than AR-mediated and -regulated downstream genes were involved in this process [
20‐
22]. However, more experiments are needed to further determine this.
In this study, we demonstrated the role of SAPK/JNK and MEK/ERK1/2 signaling pathways in mediating the effect of curcumin and bicalutamide on controlling growth of androgen-independent prostate cancer cells. The activation of these kinases by curcumin or/and bicalutamide has been shown in other studies implicating in the regulation of other gene expression and subsequent cellular responses [
23‐
28]. Consistent with ours, one report showed that activation of SAPK/JNK was involved in the curcumin-triggered intrinsic apoptotic pathway in cardiac myoblasts [
23]. Also, activation of MEK/ERK1/2 pathway mediated in curcumin-induced cell cycle arrest and apoptosis in human gastric cancer cells [
25]. We noticed that opposite results were also reported in other cell system [
29]. It was possible that the different environment contexts, cell lines used, and other unknown factors were account for this discrepancy. Thus, more studies are required to further confirm this. Findings from other studies including gene deletion data indicated the conflicting results in essence that turn MEK/ERK1/2 and SAPK/JNK functions from oncogene to tumor suppressor, which could suggest the possible dual functions of these kinases (pro-oncogene or tumor suppressor) [
30‐
32]. Therefore, the insight true role of SAPK/JNK and MEK/ERK1/2 signaling pathways in triggering cancer cell differentiation, senescence, apoptosis, and survival appeared to be context dependent and more complicated, which needs to be clarified in the future.
To further explore the potential mechanism underlining the aforementioned, we tested the involvement of MUC1-C. Our results indicated the critical role of MUC1-C in mediating the effect of curcumin on inhibition of growth of androgen-independent prostate cancer cells. Consistent with this, other studies were observed the similar findings and suggested that MUC1 including its subunits (e.g., MUC1-C) could be a potential target of curcumin in the treatment of prostate and breast cancer cells [
33,
34]. Furthermore, we demonstrated an important role of NF-κB/p65 that may involve in the effects of curcumin on MUC1-C expression and prostate cancer cell growth. Curcumin was shown to be a strong inhibitor of NF-κB activity and its inhibitory effect on NF-κB related pathways led to enhance the cytotoxicity of chemotherapeutic agents in prostate cancer cells [
35]. Moreover, reported data have demonstrated the link of MUC1-C and NF-κB signaling in other studies [
36,
37]. Cancer cells are dependent on intact MUC1-C function for constitutive activation of the canonical NF-κB pathway and cancer cell growth, and survival [
36,
37]. MUC1-C is a direct activator of NF-κB/p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-κB pathway in nonmalignant epithelial cells [
36,
37]. Our results implied that MUC1-C was downstream of NF-κB. Consistent with this, early study showed that MUC1 promoter region contained NF-κB/p65 binding site that mediated the proinflammatory cytokines-induced MUC1 promoter activity and gene expression in normal breast epithelia and breast cancer cells [
38]. Thus, the function and regulation of NF-κB/p65-MUC1-C complexes are more complicated than we could think. Nevertheless, we reasoned that regulation of p65 played a crucial role in mediating curcumin-inhibited MUC1-C expression in androgen-independent prostate cancer cells.
We also observed the involvement of activation of MEK/ERK1/2 and SAPK/JNK signaling pathways in the regulation of p65 and MUC1-C affected by curcumin. The links of these kinases to the regulation of NF-κB and MUC1-C have been shown in other studies, suggesting that blockade of these kinases could reduce expression of MUC1 in several cell systems [
12,
39‐
41]. More interestingly, we also showed the negative feedback regulation of ERK1/2 and SAPK/JNK by MUC1-C. These signaling axes formed a bidirectional feedback loop to mediate curcumin-inhibited prostate cancer proliferation. The feedback regulation circuit of kinases with other genes/proteins was reported in other studies demonstrating relative common autocrine or paracrine physiopathological phenomena [
42,
43]. One study found that sustained activation of SAPK/JNK inhibited NF-κB signaling via a feedback loop mechanism that led to an alteration in the transcription of the NF-κB-induced apoptotic gene in immortalized renal proximal tubular epithelial cells [
44]. More experiments are warranted to better elucidate the in-depth mechanism for these unexplored complicated regulatory circuit.
More importantly, we demonstrated a synergy of combination of curcumin and bicalutamide, the androgen receptor antagonist, in the inhibition of p65, MUC1-C and androgen-independent prostate cancer cell growth, implying that signaling and mechanisms other than through the AR-mediated regulatory pathways and genes contributed to the overall enhanced effects. On the other hand, this also suggested the potential new mechanism of bicalutamide in controlling androgen-independent prostate cancer cell growth. Bicalutamide was found to inhibit androgen-independent prostate cancer cell growth appeared through AR-independent pathways [
45,
46]. One study showed that combination of bicalutamide with other therapeutic agents enhanced the CRPC growth inhibition via up-regulation of insulin-like growth factor-binding protein 3 (IGFBP3) [
47]. Also, previous study indicated that MUC1-C subunit suppressed AR expression through a posttranscriptional mechanism and resisted to bicalutamide treatment in androgen-dependent prostate cancer cells, this implied that inhibition of AR expression by MUC1-C led to develop more aggressive androgen-independent phenotype in prostate cancer cells that was sensitive to MUC1-C inhibition [
10]. Consequently, this may facilitate the therapeutic effects of bicalutamide. We reasoned that MUC1-C could be a potential target of curcumin in suppression of androgen-independent prostate cancer cell growth, and inhibition of MUC1-C by curcumin sensitized the therapeutic effect of bicalutamide in prostate cancer cell growth. A cell based morphology experiment showed that curcumin analogs or curcumin-anti-androgen conjugates demonstrated more potent than bicalutamide alone in the cytotoxic effects on LNCaP and PC-3 cells through suppression of pseudopodia formation, which was highly related to cell migration and tumor metastasis, other than targeting AR [
48]. Also, note that studies implicated the dissected mechanisms by which using different anti-androgen receptor compounds on affecting prostate cancer invasion and metastasis, resulting in opposite effects [
49]; the potential risks of using these agents, such as bicalutamide, among others, on prostate cancer metastasis require to be carefully evaluated. Thus, additional studies are warranted to further explore the combination (curcumin and bicalutamide) effects on invasion and metastasis of androgen-independent prostate cancer cells.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SSH is fully responsible for the study designing, experiment adjustment, drafting and finalizing the manuscript. JL, STX performed most of the experiments involved. QHZ, LJY carried out transfection assays and some protein measurement by Western blot and statistical analysis. JJW, QT, JFZ conducted the densitometry, statistical analysis and participated in coordination manuscript. ZQC coordinated and provided important suggestions including some reagents, and critical read the manuscript. All authors read and approved the final manuscript.