Prostate cancer is the second most common cancer in males, with reports stating that over 80% of cases are detected after age 65 [1
]. If prostate cancer is detected in an early stage, it is considered curable with any of several treatments, including prostatectomy and androgen deprivation therapy (ADT) [2
]. However, prostate cancer patients recurrently treated with ADT can develop an incurable disease state called castration-resistant prostate cancer (CRPC). Although majority of metastatic disease patients are responsive to hormone deprivation therapy, CRPC can grow without testosterone, and most cases of CRPC develop into metastatic prostate cancer, which is responsible for high mortality rates in prostate cancer patients [3
]. Metastatic prostate cancer migrates primarily to the lymph nodes and the skeleton. When cancer cells initiate metastasis, small populations of the primary tumor invade the surrounding tissues and then intravasate the circulatory system. Migrating cancer cells eventually colonize distant organs by interacting with the extracellular matrix (ECM). Since most chemotherapeutic agents for cancer patients have been developed to attenuate cancer cell proliferation and/or induce apoptosis, relatively high doses of agents are often used for cancer treatment, and this fact could induce cytotoxicity in normal cells, resulting in severe side effects. Therefore, the development of anticancer therapeutics with lower cytotoxicity and that target the inhibition of metastatic events such as cell migration and invasion could lead to an effective cure for prostate cancer, particularly metastatic CRPC (mCRPC).
Integrin is a well-known heterodimeric transmembrane receptor responsible for cell-to-ECM interactions. They are composed of α and β subunits that make up 24 unique αβ complexes possessing distinct ligand-binding properties [5
]. When an integrin interacts with its ligand, such as collagen or fibronectin, it undergoes a conformational change to produce the ligand-binding specificity [7
] and activates various intracellular signaling, such as focal adhesion kinase (FAK) molecules. Such signaling is known to contribute to lamellipodium formation, cell adhesion, and cell migration on the ECM [8
]. Integrins have been a focus of research as a potential target for the development of anticancer therapeutics because it has been reported that the expression of integrins correlates with stages of human cancers [9
]. Although there are several kinds of integrin expression in prostate cancer, α2β1, a receptor for collagen I, seems to be most abundantly expressed in prostate cancer cells [11
] and appears to have a more important role in the invasion process of PC3 cells than other kinds of integrin [12
]. In addition, Bonkhoff et al. reported that the expression of α2β1 integrin is upregulated in lymph node metastases as compared to primary prostate tumors [14
], suggesting that α2β1 integrin is a potential therapeutic target for prostate tumorigenesis, especially for mCRPC.
Roxb. (SGR) is a traditional folk medicine that has been used in the treatment of hyperglycemia and for detoxication [15
]. SGR has been reported to have various bioactivities, including antiviral [17
], anti-inflammatory [18
], and immunomodulatory activities [18
]. Moreover, an anticancer activity of SGR has been suggested against hepatocarcinomas [20
], and a glycoprotein, SGF2, which is isolated from SGR has been reported to have antiproliferative effects on MCF-7 breast cancer cells [22
]. However, these reports have focused on the antiproliferative or apoptotic effects of SGR on cancer cell lines. In our study, we estimated the regulatory function and molecular mechanisms of SGR, particularly in regard to adhesion and migration of prostate cancer cells. In addition, we estimated the single components of WESGR through HPLC-MS/MS analysis.
For the development of effective antiprostate cancer agents that are able to attenuate metastatic steps, our group screened potential natural agents that might attenuate the interaction between collagen and prostate cancer cells and also investigated the potential target molecules responsible for their inhibition. Water extracts of Gleditsia sinensis
thorns (WEGST) was defined in our previous study [28
] as a potential antiprostate cancer agent that can attenuate the adhesion of prostate cancer cells to collagen, and the antiprostate cancer activity of water extracts of Smilax glabra
Roxb. (WESGR) was characterized in this study.
Prostate cancer causes the second highest mortality level from cancer in men, and it has been reported that prostate cancer metastasis leads to bony lesions can be easily found in men who die from prostate cancer [29
]. Although several anticancer drugs such as paclitaxel, vinblastine, and docetaxel, which aim to inhibit cell proliferation by attenuating mitotic spindle formation, have been developed, resistance to these drugs may occur in a portion of prostate cancer cells, resulting in the development of mCRPC [30
]. Furthermore, there is a limitation to using high doses of anticancer drugs to reach lethal toxicity because such doses could affect the viability of normal cells and result in severe side effects. Therefore, it seems very difficult to completely eliminate prostate cancer with only antiprostate cancer drugs that target attenuation of proliferation.
Because adhesion and migration are among the critical steps of prostate cancer progression, we hypothesized that antiprostate cancer agents that target specific steps, such as adhesion and migration, might be an effective alternative treatment to cure patients suffering from prostate cancer as well as mCRPC. So, we investigated whether nontoxicological levels of WESGR could suppress collagen-mediated adhesion and migration of PC3 and LNCaP prostate cancer cells. Interestingly, pretreatment with WESGR (25 and 50 μg/mL, respectively) dose dependently inhibited PC3 and LNCaP cell migration toward collagen but not to serum.
Integrin is a well-known heterodimeric receptor for ECM proteins and is reported to play important roles in cell migration and attachment. Among the several integrins, α2β1 integrin, which is a receptor for collagen, is abundantly expressed in prostate cancer cells [11
], and the invasiveness of PC3 cells could be attenuated by administration of α2 and β1 neutralizing antibodies, but not α1 and α6 neutralizing antibodies [12
], indicating that prostate cancer cells may primarily use α2β1 integrin during migration and adhesion. Therefore, α2β1 integrin could be a potential molecular target for inhibiting the progression of prostate cancer. After finding that WESGR could significantly attenuate collagen-mediated migration and adhesion, we investigated whether WESGR might inhibit the expression of α2β1 integrin. Interestingly, ectopic administration of WESGR did not affect the expression of α2β1 integrin on PC3 and LNCaP cells. However, pretreatment with WESGR inhibited collagen-induced expression of β1 integrin when PC3 and LNCaP cells were placed on a collagen-coated plate, suggesting that WESGR may attenuate the collagen-mediated intracellular signaling for activating β1 integrin expression during the adhesion process. If the expression of β1 integrin is downregulated, the heterodimerization of α2β1 should be disrupted, and subsequently, integrin-mediated intracellular signaling, such as with FAK, should be inactivated. Interestingly, pretreatment with WESGR attenuated the collagen-mediated expression of β1 integrin as well as the phosphorylation of FAK. In addition, we also found that pretreatment of WESGR inhibited the collagen-mediated actin formation on PC3 and LNCaP cells which is important in lamellipodium formation (Supplementary Figure S3
). These data suggested that WESGR could effectively inhibit the collagen-mediated prostate cancer cells migration and adhesion.
In our previous study, WEGST was defined as a potential antiprostate cancer agent that can inhibit the adhesion of prostate cancer cells to collagen through attenuation of the expression of α2 integrin, but not that of β1 integrin [12
]. Interestingly, WESGR showed a similar activity but had different target specificity. WESGR attenuated the collagen-mediated migration and adhesion, but the major target molecule of WESGR was β1 integrin. We think that different target specificity of both WEGST and WESGR may come from different active principles of these extracts. Lupine acid, ethyl gallate, and stigmasterol were suggested as active component of WEGST [12
], however caffeoylquinic acids were proposed as major components of WESGR in this study. These results suggest that if a new agent was developed that comprised both WEGST and WESGR, it might inhibit the migration and adhesion of prostate cancer cells more effectively than a single treatment with either WEGST or WESGR because it could attenuate the expression of both α2 and β1 integrin during prostate cancer cell migration toward and adhesion to the ECM. A functional analysis of a new agent with both WEGST and WESGR will be performed in our further study.
To determine the major constituents of WESGR, HPLC/MS/MS analysis was performed. By referencing to the reported data, 5-O
-caffeoylquinic acid, 4-O
-caffeoylquinic acid, and 3-O
-caffeoylquinic acid may be major constituents of WESGR. Caffeoylquinic acids are composed of quinic acid core and one or more caffeoyl groups. The proliferation of cancer cells seems to be attenuated by caffeoylquinic acids directly. For examples, it was reported that 5-O
-caffeoylquinic acid has antiproliferative effects on MBA-MB-231 breast cancer cells through modulating the Ras-dependent signaling [25
], and caffeoylquinic acid derivatives seems to have cell cycle arrest and apoptosis induction properties in gastric adenocarcinoma (AGS) cells [34
]. In addition, it was reported that mitogen-stimulated invasion but not proliferation of non-small cell lung cancer (NSCLC) could be attenuated by administration of 5-O
-caffeoylquinic acid [35
]. Interestingly, we found that pretreatment of 5-O
-caffeoylquinic acid effectively attenuated collagen-mediated PC3 cell adhesion. In addition, collagen-induced β1 integrin expression and FAK activation were also attenuated by pretreatment of 5-O
-caffeoylquinic acid. Therefore, taken together, there is possibility that 5-O
-caffeoylquinic acid could be the active component which accounts for anti-tumor effects of WESGR.
In conclusion, our results strongly suggest that nontoxicological levels of WESGR could be used for attenuating the progression of particular steps in prostate cancer metastasis, such as migration and adhesion, through restricting collagen-mediated β1 integrin expression. More detailed functional analysis of 5-O-caffeoylquinic acid on the progress of prostate cancer by using in vivo animal model will be helpful for understanding the more detailed mechanism of WESGR-mediated antiprostate cancer effects and will be performed in our future study.
4. Materials and Methods
4.1. Preparation of WESGR
Smilax glabra Roxb. (SGR) was purchased from Kyungdong market in Seoul, South Korea and 200 g of SGR was extracted with 2 L of hot water. The supernatant was harvested, filtered, and concentrated using rotary evaporation system (Heidolph Instruments GmbH & Co., Schwabach, Germany). Final concentrates were lyophilized and kept at −80 °C in a refrigerator until use. The yield of the dried extract was approximately 6.8 g/L.
4.2. Cytotoxicity Assay of WESGR
PC3 and LNCaP cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were seeded in a 96-well plate (5 × 103 cells/well for PC3 cells and 4 × 104 cells/well for LNCaP cells) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT, USA) and 1% penicillin–streptomycin solution (BioWhittaker Inc., Walkersville, MD, USA) for 24 h. After washing with RPMI 1640 medium, WESGR was added to each well at various concentrations (50–400 μg/mL for PC3 cells, and 10–500 μg/mL for LNCaP cells) and incubated at 37 °C in a CO2 incubator for 24 h. Subsequently, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) solution (Dozen, Seoul, Korea) was added to each well, and the cells were incubated for a further 2 h. The absorbance was estimated at 450 nm with a microplate reader (Model 680 Microplate Reader, Bio-Rad, Hercules, CA, USA).
4.3. Cell Adhesion Assay
Prostate cancer cells (PC3 and LNCaP) were pretreated with and without WESGR (50 μg/mL) for 6 h, and then, those were loaded on collagen I (10 μg/mL) or 20% FBS-coated 96-well plates (2 × 105 cells/well for PC3 cells and 1 × 105 cells/well for LNCaP cells). After further incubation for 15 min (PC3 cells) and 3 h (LNCaP cells) at room temperature, the plates were washed once with PBS. The remaining cells were fixed with paraformaldehyde solution (4% w/v) and then stained with crystal violet solution (5% w/v). The number of adhered cells was then counted under a microscope.
4.4. Collagen Against Migration Assay
The antimigratory effects of WESGR on the collagen-dependent migration of PC3 and LNCaP cells were measured using Transwell Permeable Supports (BD Biosciences, San Jose, A, USA). The bottom area of the Transwell membrane was coated with serum (20% w/v) or collagen I (10 μg/mL, Sigma-Aldrich Co., St. Louis, MO, USA) overnight in PBS at 4 °C. The PC3 cells (5 × 104 cells/well) and LNCaP cells (2 × 105 cells/well), respectively, were pretreated with 25 and 50 μg/mL of WESGR for 6 h and then added to the upper side of a Transwell chamber (8 μm pore size). The PC3 cells were further incubated for 6 h (serum-coated Transwell) or 2 h (collagen-coated Transwell) at 37 °C in a CO2 incubator, whereas the LNCaP cells were further incubated for 18 h (serum-coated Transwell) or 6 h (collagen-coated Transwell) at 37 °C in a CO2 incubator. Any PC3 and LNCaP cells that migrated to the bottom layer were fixed with paraformaldehyde (4% w/v) and then stained with crystal violet (0.5% w/v).
4.5. Analysis of Collagen for Expression of α2β1 Integrin and Focal Adhesion Kinase (FAK)
About 70% confluent PC3 and LNCaP cells were washed twice with PBS and separated into single cells by treatment with a trypsin/EDTA solution (HyClone Laboratories, Logan, UT, USA). The activity of trypsin was deactivated by treatment with a trypsin inhibitor from glycine max soybean (Sigma-Aldrich Co., St. Louis, MO, USA). PC3 and LNCaP cells were either not treated or were pretreated with WEGST (100 μg/mL for PC3 and 50 μg/mL for LNCaP) for 30 min (PC3 cells) or 2 h (LNCaP cells) and then seeded on collagen I (10 μg/mL)-coated plates. Cells were harvested at various time points, as indicated, and total proteins were solubilized with a lysis buffer composed of 20 mM Tris-HCl (pH 7.4), Nonidet P-40 (1% w/v), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Sigma-Aldrich Co., St. Louis, MO, USA). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated separately with an antiphospho-FAK antibody (BD Biosciences, San Jose, CA, USA), mouse anti-FAK antibody (BD Biosciences, San Jose, CA, USA), rabbit anti-α2 integrin antibody (Merck Millipore, Gibbstown, NJ, USA), anti-β1 integrin antibody (sc-374429) (Santa Cruz, CA, USA), and mouse anti-β-actin antibody (Santa Cruz, CA, USA), followed by horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin G (IgG). ECL reagents (Bio-Rad Co., Hercules, CA, USA) were used to activate signals.
4.6. HPLC-MS/MS Analysis
HPLC-MS/MS analysis was performed to determine the major constituents of WESGR. The analysis was carried out on an Ultimate 3000 RS system (Thermo Fisher Scientific, San Jose, CA, USA) coupled with LTQ Ion Trap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). WESRG was separated on a Phenomenex Kinetex C18 column (150 mm, 2.10 mm, 1.7 um, Phenomenex Inc., USA) by using a flow rate of 0.2 mL/min at 40 °C. The mobile phase of eluent A was aqueous formic acid solution, 0.1% v/v and that of eluent B was methanol with formic acid, 0.1%, v/v. A gradient program was used for elution: 0–30 min, A from 99% to 0%, and B from 1% to 100%. Ion Trap MS and spray chamber conditions were capillary temperature of 300 °C and source voltage of 3.5 kV.
4.7. Statistical Analysis
Statistical analysis was performed using Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). The statistical significance between two samples was analyzed using unpaired student’s t-test. The results are presented as mean ± standard deviation (SD). A p value of < 0.05 was considered to be significant.