Paeonol suppresses invasion, migration, and epithelial-to-mesenchymal transition in colorectal cancer cells through inhibition of COX-2 and PGE2


 Purpose: To observe the effects of paeonol on the invasion and migration of LoVo colorectal cancer cells, and investigate its possible mechanisms. Materials and Methods: Cell transwell assay and wound-healing assay were applied, and the results suggested that paeonol could significantly inhibit the invasion and migration abilities of LoVo cells, which was associated with a reduction in COX-2 expression and PGE2 synthesis. Treatment with the selective COX-2 inhibitor, celecoxib, or transient transfection of colorectal cancer cells with COX-2 siRNA, also inhibited cell invasion and migration. Results: The invasion and migration capacity were evaluated in LoVo cells by transwell assay and wound-healing in vitro. Compared with the control group, the invasion cells through Matrigel and the wound-healing rate were significantly decreased after treated with paeonol for 24 h. Paeonol treatment downregulated the expression of MMP-9 and downregulated the COX-2 expression and PGE2 synthesis in LoVo cells. Paeonol could up-regulate the expression of epithelial marker E-cadherin while down-regulate the expressions of mesenchymal markers, Fibronectin and Vimentin. Paeonol inhibited PI3K-Akt and MAPK-ERK pathways in LoVo cells. Celecoxib treatment significantly decreased the cells penetrating the matrigel in a dose-dependent manner. siRNA knockdown of COX-2 leaded to inhibition of cell invasion in LoVo cells. Knockdown of COX-2 increased the expression of E-cadherin, whereas the expressions of Fibronectin and Vimentin were downregulated. Conclusion: Paeonol may inhibit PI3K-Akt and MAPK-ERK pathways through suppressing of the COX-2 expression and PGE2 synthesis, thus inhibiting the cell invasion, migration, and EMT in LoVo cells.


Background
Colorectal cancer (CRC), a common malignancy of the digestive system, is a serious public health problem. CRC is the second leading cause of death from cancer in the developed world. What's worse, the CRC death rate is still increasing in many developing countries. 1− 3 Although a rapid progress has been achieved about the treatment and diagnosis in recent years, the prognosis for CRC remain poor for patients with metastasis.
Therefore, the development of novel agents to block metastasis is an important area of study for addressing CRC.
Previous studies have demonstrated that cyclooxygenase-2 (COX-2) is constitutively overexpressed in a variety of malignancies, including colorectal cancer, breast cancer, gastric cancer, bladder cancer and non-small cell lung cancer (NSCLC). 4,5 Deregulation of COX-2 expression leads to an increased abundance of prostaglandin E 2 (PGE 2 ), through which COX-2 contributes to carcinogenesis, progression, invasion, metastasis. 6,7 It has been confirmed that COX-2 inhibitors could effectively decrease the incidences of gastric, colon, lung, breast and prostate cancer to some extent. 8,9 Therefore, COX-2 is considered as a promising target for cancer therapy.
Paeonol (2-hydroxy-4-methoxyacetophenone), a major active extract from the root bark of Paeonia suffruticosa Andrews, possesses a wide range of biologic and pharmacologic activities, including anti-inflammatory 10,11 anti-allergic, 12 Anti-oxidative, 13 and antiplatelet aggregation. 14 The dosage forms of paeonol for clinical application approved by China Food and Drug Administration include injection, tablet and ointment. It can be used in treating inflammation/pain-related indications such as fever, headache, neuralgia, muscle pain and rheumatoid arthritis, as well as various skin diseases such as dermatitis, 4 eczema, mosquito and bedbug bites. 15 Recently, the antitumor effect of paeonol has garnered considerable attention. Unlike cytotoxic drugs, paeonol has shown cardioprotective, 16 hepatic-protective, 17− 19 renal protective 20 and neuroprotective effects. 21 Paeonol has been shown to exhibit antitumor activities on gastric, 22,23 prostate, 24 breast, 25 hepar, 26 ovarian 27 and esophageal cancer cells. 28 Our previous study also revealed that paeonol exhibited the inhibiting effects on colorectal cancer lines (HCT116, SW620 and LoVo), especially in the LoVo cells. 29 Here, the present study aimed to further investigate the effects of paeonol on the invasion and migration, and explore the underlying mechanisms in colorectal cancer cells.

Materials And Methods
Cell culture.  Western blot assay.
The cells were lysed with RIPA buffer supplemented with protease inhibitor. Total protein extract was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and then transferred to nitrocellulose membranes using a wet transfer system. The membrane, after blocked by 5% fat-free milk, was incubated with primary antibodies: Technology) at 4 ℃ for 12 hours. Following that, PVDF was then rinsed with TBST solution and incubated at room temperature for 1 hour with the secondary antibodies (Cell Signaling Technology) conjugated to HRP. The automatic developing instrument (ChemiDocXRS imaging system) was adopted to develop and the gray value was calculated.
Statistical analysis.
All data were presented as Mean ± SD. GraphPad Prism 7.0 was adopted for statistical analysis of data. Student's t-test was used for comparison of the values between two groups. P < 0.05 indicated statistical significance.

Results
Paeonol inhibited cell invasion and migration in LoVo cells.
The invasion and migration capacity were evaluated in LoVo cells by transwell assay and wound-healing in vitro. Compared with the control group, the invasion cells through Matrigel and the wound-healing rate were significantly decreased after treated with paeonol for 24 h (Fig. 1A-D). Meanwhile, paeonol treatment downregulated the expression of MMP-9, which could degrade extracellular matrix and destroy basilar membrane, therefore promotes tumor cell metastasis (Fig. 1E-F).
Paeonol downregulated the COX-2 expression and PGE2 synthesis in LoVo cells.
To investigate whether COX-2 and PGE 2 are involved in the antitumor effect of paeonol, 7 the protein level of COX-2 was assessed by western blot, and the level of PGE 2 in the cell culture supernatant was performed using the PGE 2 ELISA kit. The results showed that Paeonol treatment decreased COX-2 expression and PGE2 synthesis in LoVo cells in a dose-dependent manner (Fig. 2). Moreover, downregulation of COX-2 significantly decreased the expressions of p-Akt and p-ERK in LoVo cells (Fig. 7B).

Discussion
A majority of CRC patients are diagnosed with advanced stage, resulting in poor prognosis. 30 Metastatic dissemination is the leading cause of colorectal cancer-related deaths. Therefore, the development of novel, efficacious therapeutic agents to block metastasis is especially urgent.
In the present study, paeonol elicited dramatic inhibition of invasion and migration in LoVo cells as demonstrated by the transwell assay and wound-healing assay. Meanwhile, paeonol treatment decreased expression of MMP-9, which could degrade extracellular matrix and destroy basilar membrane, therefore promotes tumor cell metastasis.
Epithelial-to-mesenchymal transition (EMT), a biological process of epithelial phenotype transforming to interstitial phenotype because of extracellular factor stimulation, is the essential mechanism for initiating cancer invation and metastasis. 31