Curcumin inhibits the proliferation and invasion of MG-63 cells through inactivation of the p-JAK2/p-STAT3 pathway

Purpose The aims of this study were to determine the effect of curcumin on osteosarcoma (OS) cells due to inactivation of the p-JAK2/p-STAT3 pathway and evaluate the prognostic value of this pathway in OS. Materials and methods We exposed a human OS cell line (MG-63) to different concentrations of curcumin. Then, we characterized the effects on MG-63 cells using assays (cell viability, colony formation, cell cycle, wound healing, invasion), flow cytometry, Western blot, immunohistochemical analyses, and tumor xenograft. Results The half-maximal inhibitory of curcumin for MG-63 cells at 24 hours was 27.6 µM. The number of colonies of MG-63 cells was decreased obviously upon curcumin (10 and 20 µM) treatment. We also found increased accumulation of MG-63 cells in the G2/M phase upon curcumin (10 and 20 µM) treatment. Apoptosis was increased in 10 and 20 µM curcumin-treated MG-63 cells. After incubation of physically wounded cells for 24 hours, the percentage wound width increased upon curcumin exposure. Curcumin obviously decreased the expression of pJAK-2 and pSTAT-3 in MG-63 cells in a dose-dependent manner. Curcumin dose-dependently inhibited the proliferation, migration, and invasion of MG-63 cells and induced arrest of the G0/G1 phase and apoptosis by inhibiting the p-JAK2/p-STAT3 pathway. The linear correlativity between expression of p-JAK2 and STAT3 was very prominent, and both were closely associated with lung metastasis. In vivo study suggested that curcumin suppressed tumor growth through JAK2/STAT3 signaling. Conclusion Curcumin-mediated inhibition of the proliferation and migration of MG-63 cells was associated with inactivation of JAK/STAT signaling.


Introduction
Osteosarcoma (OS) is the most prevalent primary cancer of the bones. Standard treatment comprises multiagent neoadjuvant chemotherapy (eg, doxorubicin, cisplatin, high dose of methotrexate or ifosfamide) followed by surgery and adjuvant chemotherapy with the same agents. This widely applied treatment has improved 5-year survival from 25% in the early 1970s to ~70% in the last decade. 1,2 However, outcomes for OS remain unsatisfactory for patients with metastasis. 3 Moreover, highdose chemotherapy also induces multidrug resistance and cachexia. 4,5 Meanwhile, a high dose of currently used drugs is limited by their side effects: nephrotoxicity, cardiomyopathy, hemorrhagic cystitis, and nephrotoxicity. 6,7 Therefore, development of novel, safe, efficacious therapeutic agents for late-stage OS is especially urgent. Curcumin is a phenolic, yellow compound found in Curcuma longa. It has been reported to have a wide range of biologic and pharmacologic activities: anti-inflammatory, antidiabetes mellitus, and antioxidant. 8 Recently, the anticancer effect of curcumin has garnered considerable attention. Unlike cytotoxic drugs, curcumin has shown minimal toxicity and high safety at high doses in clinical trials. 9,10 Studies have shown curcumin's activities against cancer of the breast, 11,12 pancreas, 13 colon, 14 prostate gland, 12 as well as melanoma 15,16 and OS. [17][18][19][20] Lee et al 17 reported that curcumin caused the death of OS cells by blocking cells successively in G(1)/S and G(2)/M phases and activating the caspase-3 pathway. Leow et al 18 found that curcumin exhibited anti-invasive and antimetastatic effects in OS cells though activation of the Wnt/βcatenin pathway. Moreover, curcumin has been reported to inhibit the proliferation and invasion of OS cells by regulating miRNA-125a and miRNA-138. 19,20 However, how curcumin works against OS is not known. We explored a pathway to explain the inhibitory property of curcumin on OS cells.

Materials and methods cell culture and reagents
A human OS cell line (MG-63) was obtained from the Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, People's Republic of China). Cells were grown in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin and streptomycin (100 mg/mL of each) in a humidified atmosphere of 5% CO 2 at 37°C. Curcumin (99% purity) was purchased from Sigma-Aldrich Co. (St Louis, MO, USA), and 100 mM of it was stored in 99.9% dimethyl sulfoxide (Sangon Biotech, Shanghai, People's Republic of China). Curcumin at 5,10,15,20,25,30,35,40, and 80 µM was used to treat MG-63 cells.
colony-formation assay MG-63 cells (5×10 4 /dish) were seeded in 100 mm dishes with curcumin (0, 10, and 20 µM). Two weeks later, cells were washed twice with PBS, fixed with 10% formaldehyde for 5 minutes, and then stained with 1% crystal violet for 30 seconds. Each clone with 30 cells was counted using a dissection microscope.

cell-cycle assay
MG-63 cells treated with 0, 10, or 20 µM of curcumin for 48 hours were harvested through trypsinization. Then, they were fixed in 70% (v/v) ethanol at -20°C for 24 hours. Before detection, cells were resuspended in a solution of RNase (0.2 mg) and 1 mL of propidium iodide (PI)/Triton X-100 (20 µg [PI]/0.1% Triton X-100) in the dark for 15 minutes at room temperature. After washing twice, cell-cycle distribution was analyzed using a FACSCalibur™ flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Apoptosis detection by flow cytometry
Cells treated as stated earlier were trypsinized and harvested after washing with ice-cold PBS. Cells (5×10 5 ) were stained with Annexin V-fluorescein isothiocyanate/PI (Thermo Fisher Scientific) according to manufacturer's instructions and resuspended in 500 µL of binding buffer. Flow cytometry was undertaken using a flow cytometer (FACSCalibur).

Wound-healing assay
Cell mobility was assessed by a wound-healing assay. MG-63 cells (1×10 6 ) were seeded in six-well plates until 80% confluence had been reached. A sterile yellow 100 µL pipette tip was used to make a straight scratch. Detached cells were removed by washing with PBS. Then, cells were treated with curcumin (0, 10, or 20 µM) in the serum-free medium. Phase-contrast images were taken in the same field at 0 and 24 hours. Experiments were carried out in triplicate.

invasion assay in vitro
For the cell-invasion test, 24-well transwells (pore size =8 mm; Corning Incorporated, Corning, NY, USA) were coated with Matrigel (1 mg/mL) according to the manufacturer's (Becton Dickinson) instructions. Briefly, 1×10 4 cells were seeded into the upper chamber containing 0, 10, or 20 µM of curcumin. Then, 24 hours later, noninvasive cells in the upper chamber were removed with a cotton swab. After fixation with 4% paraformaldehyde for 20 minutes, the upper chamber was stained with Giemsa solution for 5 minutes. Cells in five random fields were counted at a high power to obtain the mean value. Each experiment was carried out in triplicate on separate occasions.

immunohistochemical (ih) analysis
OS tissue samples in paraffin from 80 patients who had been followed up for 3 years were collected. The baseline characteristics of patients are given in Table 1. Expression of p-JAK2 (BM4839; Boster Biological Technology, Wuhan, People's Republic of China) and STAT3 (BM3922; Boster Biological Technology) was measured using a SV0004 two-step immunohistochemistry kit according to manufacturer's instructions. The ethics committee of Shanghai Sixth People's Hospital approved the research protocol on human tissues. All patients provided written informed consent.

Tumor xenograft model
A total of six BALB/c nude mice were divided into two groups (three per group). The animal experimental operations were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. MG-63 cells (1×10 6 ) were injected subcutaneously into the right thigh of each mouse. As soon as the xenografts were 0.5 cm in diameter, curcumin (Sigma-Aldrich Co.) was administered orally to them at a dose of 50 mg/kg QD for four consecutive weeks. The tumor, pJAK2, and STAT3 levels were evaluated. All of the in vivo experimental protocols were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. The procedures were performed according to the guidelines and regulations of Shanghai Jiao Tong University Affiliated Sixth People's Hospital (Shanghai, People's Republic of China).

statistical analyses
Data are the mean ± SD of triplicates. Statistical analyses of data were undertaken using Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined by one-way or two-way ANOVA. P0.05 was considered as significant.         Western blot. Curcumin treatment decreased expression of p-JAK2 and p-STAT3 in MG-63 cells obviously in a dosedependent manner (Figure 7).

JaK2/sTaT3 signaling is closely related to metastasis
The importance of JAK2 and STAT3 in OS is uncertain. We measured expression of p-JAK2 and STAT3 by the IH method in the tissue of OS patients with or without lung metastasis ( Figure 8). For lung-metastasis samples, median expression of p-JAK2 and STAT3 was 9.23 and 14.82, respectively, which was much higher than that in patients without metastasis (2.43 and 5.06, respectively). There was a positive correlation between expression of p-JAK2 and STAT3 (r=0.715, P=0.002).

curcumin inhibited Os cells in vivo
We established OS xenografts to evaluate the effect of curcumin on tumor growth in vivo. After curcumin treatment for 4 weeks, the tumor sizes were significantly smaller than those in the control group ( Figure 9A and B). The IH analysis also demonstrated the expression of p-JAK2 and STAT3 ( Figure 9C and D), confirming that curcumin suppressed OS tumor growth through JAK2/STAT3 signaling.

Discussion
Curcumin has been shown to possess potent activity in vitro and in vivo against cancer of the prostate gland, 21 breast, 22 colon, 23 as well as OS. 24 In the present study, curcumin elicited dramatic inhibition of proliferation and arrest of the G2⁄M cycle in MG-63 OS cells as demonstrated by the CCK-8 assay and flow cytometry, respectively. These results are consistent with data published by other research teams. [25][26][27][28] Curcumin undertakes anticancer activities through nuclear factor-kappa B, 29 AKT/mTOR, 30 Wnt, 31 and JAK/STAT 32 signaling pathways. Whether curcumin exerts an antitumor effect in OS cells via JAK/STAT signaling has yet to be determined. We found that the phosphorylation levels of STAT3 in curcumin-treated MG-63 cells were decreased. JAK/STAT signaling has a central role in tumorigenesis; p-STAT3 activation contributes to cell proliferation by regulating expression of various genes required for cell proliferation. After activation by cytokine-mediated transphosphorylation, JAK2 triggers the phosphorylation of STAT3 proteins. JAK2 is involved in epithelial-mesenchymal transition and promotes the metastasis of several types of cancer cell. JAK2 phosphorylation was decreased significantly in curcumin-treated groups, especially in the high-concentration groups (10 and 20 µM), similar to STAT3 phosphorylation. This observation suggests that curcumin exerts its effects by inhibiting JAK2 phosphorylation (and thus inhibiting STAT3 phosphorylation) without affecting expression of both proteins in MG-63 cells.
Aberrant activation of JAK/STAT signaling has been documented in cancers of the head and neck, 33 breast, 34 and prostate gland 35 and in OS. 36,37 Few reports have focused on the role of JAK/STAT signaling in OS. Gerland et al 38 found that growth hormone induced expression of the JAK/STAT negative-regulatory proteins, which was inhibited by 1-alpha, 25-dihydroxyvitamin D. Sandoval-Usme et al reported that imvastatin decreases the proliferation, migration, and invasion of OS cells in a time-and dose-dependent manner through the JAK/STAT pathway. 36 We found a positive correlation between expression of p-JAK2 and STAT3, and both were closely related to lung metastasis.

Conclusion
We showed curcumin to be an antitumor agent. Curcuminmediated inhibition of the proliferation and migration of MG-63 cells was associated with inactivation of JAK/STAT signaling. Expression of JAK2 and STAT3 was much higher in OS patients with lung metastasis than in OS patients without metastasis. Downregulation of JAK/STAT signaling by curcumin could be a new strategy for OS treatment.