Identification of Vaccinia-H1 related phosphatase as an anticancer target for 1,2,3,4,6-pentagalloyl glucose
Authors: Sang Jeon Chung, Sun-Young Yoon, Do-Hwi Kim, Kyung Min Roh, Dohee Ahn, and Hyo Jin Kang
ABSTRACT
Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia-H1 related phosphatase (VHR) has been shown to arrest at the G1-S and G2-M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anti-cancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR-inhibitory compounds, 1,2,3,4,6- pentagalloyl glucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (Ki = 53 nM) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl-2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.
Keywords: Vaccinia-H1 related phosphatase; 1,2,3,4,6-pentagalloyl glucose; anticancer; HeLa cervical cancer cells; apoptosis
INTRODUCTION
Protein tyrosine phosphatases (PTPs) act in coordination with the protein tyrosine kinases to control signalling pathways relevant to cell growth, proliferation, and differentiation.[1, 2] PTPs are known to be involved in diverse human diseases, including cancer, diabetes and inflammatory disorders.[3] Vaccinia H1-related phosphatase (VHR) is classified as a dual specificity phosphatase and dephosphorylates the mitogen-activated protein kinases such as extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK).[4, 5] VHR has been shown to be upregulated in various cervical cancer cell lines such as CaSki, HeLa, and SiHa cells.[6] Moreover, VHR is overexpressed in human prostate cancer and VHR knockdown increases cell death of LNCaP prostate cancer cells, as determined by TUNEL assay.[7, 8] Small- molecule VHR inhibitors have been reported to exert antiproliferative effects on HeLa cervical cancer cells, suggesting that VHR could be a potential target for anticancer therapy.[5, 6] There has been an elevated interest in the discovery and development of natural products as new anticancer agents.[9] In this study, we screened a library of 658 natural products to identify the VHR inhibitors. Among the VHR-inhibitory compounds, 1,2,3,4,6-pentagalloyl glucose (PGG) was identified as an anti-cancer drugs for treating cervical cancer. PGG, a polyphenolic compound, is found in a wide variety of herbals, including Punica granatum, Mangifera indica, and Elaeocarpus sylvestris. PGG has been reported to lead to cell cycle arrest in MDA-MB- 231 and BT474 breast cancer cells through downregulation of Skp2 Protein, and to exert antioxidative effects against 1,1-diphenyl-2-picrylhydrazyl radical.[10, 11] It has been reported previously that PGG enhanced apoptosis of leukemia and breast cancer cells as well as prostate cancer cells.[12-14] Furthermore, PGG has also been shown to inhibit MDA-MB231 breast cancer xenograft tumor growth and lung metastasis in nude mice and suppress DU145 prostate cancer xenograft tumor growth via inhibition of STAT3 oncogenic signaling.[14, 15]
Nevertheless, the direct target for PGG has not been identified. In this study, PGG inhibited the enzymatic activity of VHR through measurement of catalytic activities in vitro. After PGG treatment, we assessed the extent of apoptosis via cell viability assay and examined the expression of proteins relevant to cell death signaling pathways in HeLa cervical cancer cells. Overall, our cell-based studies indicated that PGG as an inhibitor of VHR could be a potential new therapeutic candidate for the treatment of cervical cancer.
RESULTS AND DISCUSSION
PGG inhibited the catalytic activity of VHR
Loss of VHR has been shown to block the cell cycle at the G1-S and G2-M transitions, indicating that VHR inhibition may be a helpful strategy to stop tumor growth.[5] Moreover, it has been reported previously that VHR knockdown increases cell death of LNCaP prostate cancer cells through JNK activation.[8] Small-molecule inhibitors of VHR display apoptotic effects on cervix cancer cells, suggesting that VHR can be considered as an anti-cancer target.[6] In this study, 658 natural compounds purified from traditional oriental medicinal plants were screened to identify VHR inhibitors. The VHR was overexpressed and purified using a cobalt affinity resin (Fig. 1a). Kinetic constants of VHR were determined using DiFMUP as a fluorogenic PTP substrate (Fig.1b, c and Table 1). VHR was added to 20 μM of each natural product with DiFMUP and the increase in fluorescence was observed for 10 min. Based on the inhibition potency of the compounds, PGG was chosen for further study as a potential anti- cancer drug candidate for treating cervical cancer (Table 2). Furthermore, PGG showed better inhibition of DiFMUP hydrolysis by VHR than other PTPs such as MKP6 and PTPN3 (Table 3). The half-inhibitory concentration (IC50) of PGG against VHR was determined to be 104nM (Fig. 2a). Dixon plot showed that PGG acted as a competitive inhibitor of VHR and its inhibition constant (Ki) was estimated to be 53 nM (Fig. 2b).
PGG enhanced apoptosis in HeLa cervical cancer cells
We next investigated whether PGG, as a VHR inhibitor, might induce apoptosis of HeLa cells. To this end, HeLa cells were incubated with various concentrations of PGG for 2 days and cell viability was assessed using an EZ-Cytox assay kit. Treatment with 20 or 40 μM PGG significantly reduced viability of HeLa cells in a dose-dependent manner, indicating that PGG led to cell death in HeLa cervical cancer cells (Fig. 3a). PGG, a phenolic compound, is not highly soluble but soluble in dimethyl sulfoxide (DMSO). In this study, 10% DMSO was used for enzyme kinetics, and only 0.2% DMSO was used for cell viability test. In addition, high molar mass of PGG (940.6 g/mol) causes low cell permeability. For these reasons, there was a significant difference between the in vitro IC50 (104 nM) and IC50 for HeLa cells (around 20 μM). In agreement with these results, treatment with 20 μM potent VHR inhibitors (IC50 = 18~78 nM in vitro) has been reported previously to have apoptotic effects on HeLa cells.[6] Previously, VHR has been shown to lead to cell cycle progression and knockdown of VHR by RNA interference results in cell cycle arrest in G1/S and G2/M through downregulation of cyclin D1 expression.[5, 16] Consistent with these findings, we found that incubation of HeLa cells with PGG significantly decreased the protein levels of cyclin D1 (Fig. 3b, c). To next examine whether PGG induces increased expression levels of proteins relevant to apoptosis, HeLa cells were incubated with 20 or 40 μM PGG for 2 days. The cells were lysed, and Western blotting was performed using antibodies for cleaved PARP or Bcl2 because cleavage of PARP by caspases has been considered as a hallmark of apoptosis[17] and Bcl-2 has been shown to promote cell survival.[18] Furthermore, Signal transducer and activator of transcription 3 (STAT3) is a potential carcinogenic protein and its activation is associated with oncogenic signaling pathway.[14, 15] We found that PGG treatment markedly increased the protein levels of the cleaved PARP compared with that in the control (Fig. 3b, d). In agreement to this result, incubation of HeLa cells with PGG significantly decreased the protein levels of Bcl-2 and STAT3 phosphorylation compared with that in control (Fig. 3b, e, f). However, incubation of 3T3-L1 adipocytes with PGG had no major effects on the cell viability (Fig. 3g). In addition, PGG treatment in 3T3-L1 adipocytes did not showed significant differences in protein levels of cleaved PARP or Bcl2 compared with the control, indicating that PGG induced apoptosis of cancer cells, but not adipocytes (Fig. 3h). Furthermore, we investigated the cytotoxicity of PGG in normal human skin fibroblast Detroit 551 cells. When we cultured Detroit 551 normal cells with 40 µM PGG, the cell viability was 74%, whereas 40 µM PGG treatment in HeLa cervical cancer cells showed 31% cell viability (Fig. 3i). These results indicate that PGG induces cell death by increasing PARP cleavage and suppressing protein expressions of Bcl-2 expression, cyclin D1 and STAT3 phosphorylation in HeLa cells.
CONCLUSIONS
Some previous reports have demonstrated that PGG suppresses prostate xenograft tumor growth in vivo through activation of p53 pathway, and reduces breast cancer xenograft growth via inhibition of JAK1-STAT3 inhibition.[14, 15] In this study, PGG inhibited the catalytic activity of VHR which is considered as an anti-cancer target. In addition, PGG dramatically promoted cell death of HeLa cervical cancer cells in a dose-dependent manner, as determined by cell viability assay. Furthermore, PGG markedly induced apoptosis of HeLa cells by increasing the levels of cleaved PARP and decreasing the protein expressions of cyclin D1, Bcl-2 and STAT3 phosphorylation, as determined by Western blotting. However, incubation of 3T3-L1 adipocytes with PGG did not showed significant differences of protein levels associated with apoptosis, as compared to control, indicating that PGG had apoptotic effects only on cancer cells, but not on healthy normal cells. Taken together, these results suggest that PGG could be a potential therapeutic candidate for cervical cancer treatment through VHR inhibition.
Experimental Section
Cell culture
The methods used for culturing HeLa cervical cancer cells obtained from the Korean Cell Line Bank (Seoul, Korea) have been described previously.[19] HeLa cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Welgene Inc., Gyeongsan-si, Korea) containing 10% fetal bovine serum (FBS; Welgene Inc.) and antibiotic-antimycotic solution (Gibco BRL, Middlesex, UK). 3T3-L1 preadipocytes obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in high glucose DMEM (Welgene Inc.) supplemented with 10% bovine calf serum (BCS; Thermo Fisher Scientific) and antibiotic- antimycotic solution (Gibco BRL). Normal human skin fibroblast Detroit 551 cells were purchased from the Korean Cell Line Bank. Detroit 551 normal cells were grown in MEM (Gibco BRL) containing10% FBS (Welgene Inc.) and antibiotic-antimycotic solution (Gibco BRL).
Cell differentiation
The methods used for differentiating 3T3-L1 preadipocytes have been described previously.[20] When 3T3-L1 preadipocytes reached 100% confluence, they were cultured in DMEM containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), antibiotic-antimycotic solution, 0.5 mM isobutylmethylxanthine (IBMX; Merck KGaA, Darmstadt, Germany), 1 μM dexamethasone (Sigma-Aldrich, Saint Louis, MO, USA), and 5 μg/mL insulin (Merck KGaA) for 2 days. Cells were then maintained in DMEM supplemented with 10% FBS, antibiotic- antimycotic solution, and 5 μg/mL insulin for an additional 2 days followed by culture in DMEM containing 10% FBS and antibiotic-antimycotic solution for an additional 4 days.
Overexpression and purification of recombinant VHR
The human gene of VHR with both an N-terminal maltose-binding protein (MBP) and a C- terminal His6-tag was transformed into E. coli Rosetta (DE3) (Merck Millipore, Darmstadt, Germany). Expression of the recombinant MBP/His6 fused VHR was induced by the addition of 1 mM IPTG, and the cells were grown at 18 °C for 16 h. The cells were then harvested by centrifugation (3,570 g at 4 °C for 15 min), washed with buffer A (50 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 0.025% 2-mercaptoethanol, and 1 mM phenyl-methylsulfonyl fluoride), and then lysed by ultrasonication. After centrifugation (29,820 g at 4 °C for 30 min), the supernatant was incubated with a cobalt affinity resin (TALON®, Takara Korea, Seoul, Korea) on a rocker at 4 °C for 1 h. The resin was then washed with buffer A containing 10 mM imidazole. VHR was next eluted with buffer A supplemented with 100 mM imidazole, and stored at -70 °C.
Measurement of enzymatic activities and inhibition constant (Ki)
The enzymatic activities of purified VHR were measured using 6,8-difluoro-4- methylumbelliferyl phosphate (DiFMUP) (100 M), the generally used protein tyrosine phosphatase (PTP) substrate, as described previously.[21] To obtain the KM values, VHR was added to reaction buffer (20 mM Bis-tris pH 6.0, 150 mM NaCl, 2.5 mM dithiothreitol (DTT), 0.01% Triton X-100) containing DiFMUP (800, 400, 200, 100, 50, 25, 12.5, 6.25 M) to a final volume of 100 l in a 96 well-plate. Fluorescence intensities were assessed continuously for 10 min (Excitation/Emission = 355/460 nm) using a VictorTM X4 multi label plate reader (Perkin Elmer, Waltham, MA, USA), and KM values were obtained by Lineweaver-Burk plots. To evaluate VHR inhibition by 658 natural compounds, VHR was added to reaction buffer containing each of the compounds (20 M) and DiFMUP (2 ⨉ KM). To assess IC50 values of PGG, PGG was added to DiFMUP (KM) for VHR in reaction buffer. After addition of 10 nM VHR, enzyme inhibition was determined by monitoring fluorescence intensities. The curves were plotted against inhibition (%) for various concentrations of PGG (0.0012 – 5 M) and IC50 values were calculated by KaleidaGraph. To determine the Ki values of PGG to VHR, PGG was added to DiFMUP (95, 190, 380 M) in reaction buffer. After the addition of 10 nM VHR, enzyme activity was determined by assessing the change of fluorescence intensities and Ki values were obtained using Dixon plots. VHR inhibition was determined at a substrate concentration of 2 ⨉ KM and PGG concentration range of 0 – 80 nM.
Cell viability assay
To assess cell viability, EX-Cytox (DOGEN Bio., Seoul, Korea) was used according to the manufacturer’s instructions. HeLa cells were incubated with various concentrations of PGG for 48 h and then the absorbance was read at 450 nm using a microplate reader (VictorTM X4, PerkinElmer, Waltham, MA, USA).
Western blotting
Proteins were extracted using a buffer containing 25 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, and protease inhibitor cocktail (Roche Korea, Seoul, Korea). Proteins were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C using the following primary antibodies: anti-Poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Inc., Massachusetts, USA), anti-Bcl2 (Cell Signaling Technology), anti-phosphorylated STAT3 (Cell Signaling Technology), anti-total-STAT3(Cell Signaling Technology), anti-Cyclin D1 (Cell Signaling Technology) and anti-β-actin (AbFrontier, Seoul, Korea). Membranes were then probed with the anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Antibody–antigen complexes were detected by enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Korea). To quantify the levels of cleaved PARP and Bcl2, protein levels were normalized 1,2,3,4,6-O-Pentagalloylglucose to β-actin levels using ATTO image analysis software (CS analyzer 4, Tokyo, Japan).
Statistical analysis
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