Inhibition Of Cyclooxygenase-2by Tetramethylpyrazine And Its Effects On A549Cell Invasion And Metastasis

1. IntroductionLung cancer is a major cause of malignancy-related deaths worldwide. Non-small cell lung cancers (NSCLC), including adenocarcinomas, constitute the majority of lung cancers (75-80%). Invasion and metastasis are major causes of treatment failure in patients with lung adenocarcinoma. Tumor metastasis is a complex, multistep process that depends on the characteristics of the tumor and its host organs. Understanding the molecular targets involved and their corresponding inhibitors will provide promising new approaches for the treatment of cancer.Cyclooxygenase (COX) is the rate-limiting enzyme for the production of eicosanoid prostaglandins (PGs) and thromboxanes (TXs) from free arachidonic acid. Two isoforms of COX have now been identified in1991. COX-1is constitutively expressed in most cells and tissues, and is important for maintaining homeostatic function. In contrast, COX-2is an inducible isoenzyme that acts as an immediate early gene expressed in response to growth factors, cytokines, and other stimuli. Several studies have demonstrated a constitutive upregulation of COX-2expression in human NSCLC. COX-2levels are significantly higher in adenocarcinomas than in squamous cell carcinomas. Several studies have indicated that COX-2may be a central element in the process of lung cancer invasion and metastasis.Due to the critical role of COX-2in carcinogenesis, considerable interest has been focused on COX-2inhibitors as a chemopreventive strategy. Both isoenzymes can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) including the non-selective COX inhibitors and selective COX-2inhibitors. Although the pharmacological action of NSAIDs have demonstrated efficacy, the adverse effects such as gastrointestinal bleeding or increased cardiovascular risk have limited their widespread usage. Thus, immense effort has been devoted to developing molecules that can potently inhibit COX-2with fewer side effects.Tetramethylpyrazine (TMP), a bioactive constituent of the traditional Chinese medicinal herb Ligusticum Chuanxiong, has been applied in the treatment of neurovascular and cardiovascular diseases in the clinic. The underlying mechanism includes augmentation of organ blood volume, inhibition of platelet aggregation, suppression of apoptosis, and protection against free radicals. In the past decade, TMP has been found to influence some aspects of lung cancer such as invasion and metastasis beyond its traditional roles. However, little information is known about the cellular and molecular mechanisms involved.Wan et al. reported that TMP has the pharmacological effect of inhibiting lipopolysaccharide-induced expression of COX-2in macrophages and the apoptosis of cardiac myocytes in sucking mice, but does not influence the activity of COX-2. Zhang and coworkers reported that TMP can reduce the expression levels of COX-2in rats with acute pulmonary thromboembolism and alleviate the inflammatory reaction. However, whether TMP has any function on COX-2in pulmonary tumors has not yet been reported. We postulate that COX-2might be an important mechanism involved in the effect of TMP in lung cancer. Here, we investigated whether TMP inhibited NSCLC growth, invasion and metastasis by targeting COX-2pathway. For this study, we used the peripheral airway-derived adenocarcinoma cell line, A549, because adenocarcinoma is currently the most common form of lung cancer and COX-2levels are significantly higher in adenocarcinomas than in squamous cell carcinomas. For a positive control we used the nonselective COX inhibitor indomethacin, which has been proven to inhibit COX-2activity and expression in NSCLC cells.The effect of TMP on A549cells has not been reported previously. First, we observed the influence of TMP on the proliferation of A549cells, and select the proper concentrations of TMP for further study. In the second part, we observed the effects of TMP on invasion and metastasis of A549cells from in vitro and in vivo experiments. In the third part, we observed the influences of TMP on COX-2activity, protein expression, PGE2level and the expression level in the metastatic lung nodules, respectively.2. ContentsPart1The influence of TMP on the proliferation of A549cellsObjectives(1) To observe the effect of TMP on the growth of A549cells and the mechanisms involved.(2) To select proper concentrations of TMP for further study.MethodsTMP was obtained in sterile20mg/ml injection ampules from Beijing Yong Kang Pharmaceutical Co.(Beijing, China).(1) We observed the influence of TMP (10various concentration groups from100μg/ml to1000μg/ml) on the proliferation of A549cells using the MTT assay. Based on the data from the MTT assay, we selected three different concentrations of TMP for further investigation:200μg/ml (TMP200),400μg/ml (TMP400) and800μg/ml (TMP800). As a positive control we used the COX inhibitor indomethacin at200μM (IN) according to the data from the MTT assay.(2) To examine whether TMP influenced cell cycle progression, we performed FACS.(3) The expression of p-ERK1/2in A549cells was detected by Western blot.ResultsTMP inhibits the cell viability of A549cells. Our research showed that TMP exhibited a dose-and time-dependent inhibitory effect on A549cell proliferation by suppressing the cell progression from G0-G1phase to S phase. The protein expressions of p-ERK1/2in TMP-treated groups were suppressed, and the effect was dose-dependent, corresponding with the MTT results.Conclusions(1) TMP exhibited a dose-and time-dependent inhibitory effect on A549cell proliferation by suppressing the cell progression from G0-G1phase to S phase.(2) ERK may be an important mechanism involved in the anti-proliferation effect of TMP on A549cells.Part2The influence of TMP on the invasion and metastasis of A549cellsObjectiveTo observe the effect of TMP on the invasion and metastasis of A549cells.Methods(1) The invasion assay was carried out using24-well Transwell cell culture chambers.We selected200μg/ml TMP to investigate the effects of TMP on A549cell invasion. TMP at this concentration has no obvious influence on A549cell viability according to the data from the MTT assay.(2) The activity of MMP-2/TIMP-2was measured by commercially available ELISA assay kits.(3) For experimental lung metastasis assay, A549cells were injected into the lateral tail veins of8-week-old female BALB/c nude mice. Mice in TMP group were given intraperitoneal injections of TMP (100mg/kg/day) daily, and mice in control group received intraperitoneal injections of isovolume physiological saline daily. The diet and mental status of the mice were observed every day, and body weight was measured every4days. The animals were sacrificed at29days after the injection of tumor cells. The number of metastases in the lung surface was counted by visual inspection. H&E histological staining was used to evaluate the presence or absence of tumors. Results(1) TMP inhibits the invasive ability of A549cells. After24h incubation, the number of A549cells adherent to the outer surface of the transwell membrane was significantly decreased in the TMP-treated group.(2) The activity of MMP-2/TIMP-2in the supernatants from the TMP200, TMP400and TMP800groups were decreased compared with control. The inhibitory effect was dose-dependent.(3) TMP suppresses lung metastasis of A549cells in metastatic nude mouse model. TMP-treated mice showed smaller visible metastatic pulmonary nodules compared to the control group. In addition, the average number of visible metastatic pulmonary nodules was significantly lower in TMP-treated mice. H&E stain further confirmed that both the number and the size of the metastatic lung nodules in the TMP group were significantly decreased compared to those in the control group. Except for the quickly released localized stimulus, no significant difference was observed in the diet, mental status, hair color, and luster of the nude mice between the TMP-treated group and the control group.Conclusions(1) TMP exhibited an anti-invasive effect of A549cells at a much lower concentration and much earlier time than its anti-proliferative effect.(2) TMP showed a significant inhibitory effect on tumor metastasis with limited toxicity.(3) MMP-2/TIMP-2is one of the important pathways targeted by TMP in the invasion of A549cells.Part3The effect of TMP on COX-2in A549cellsObjectives(1) To elucidate the effect of TMP on COX-2in A549cells in vitro and in vivo.(2) To investigate the underlying molecular mechanisms involved in the anti-tumorigenie function of TMP. MethodsUsing indomethacin as a known COX inhibitor, the effect of TMP on A549cells was observed following an24h incubation. The COX-2activity was measured with the commercially available COX activity assay kit (Cayman Chemicals). The protein expression level of COX-2was analyzed using Western blot. As an indicator of COX activity, the accumulation of PGE2in the culture medium was measured by specific radioimmunoassay (RIA). COX-2expression level in the metastatic lung nodules was detected by immunohistochemical analysis.Results(1) Similar to the IN group, the COX-2activity in the TMP groups were reduced significantly compared with the control group.(2) There was no significant change of COX-2expression level after treatment for24h with200μg/ml,400μg/ml or800μg/ml of TMP, suggesting that TMP has little influence on the protein expression of COX-2.(3) PGE2levels in the TMP groups were also reduced significantly compared to the control group.(4) Immunohistochemistry analysis of the metastatic lung nodules showed a reduction of COX-2expression level in TMP treated group compared to that in the control group.Conclusions(1) TMP can inhibit COX-2activity in A549cells and the COX-2expression in metastatic lung nodules in nude mice, but had no obvious effect on COX-2expression in A549cells in vitro.(2) The ability of TMP to inhibit COX-2activity may contribute to its anticancer effect in lung adenocarcinoma.3. Summary(1) TMP exhibited outstanding inhibitory effect on A549cells invasion and metastasis at a much lower concentration and much earlier time than its anti-proliferative effect.(2) TMP can inhibit COX-2activity in A549cells and the COX-2expression in metastatic lung nodules in nude mice.(3) The inhibitory effect of TMP on invasion and metastasis of A549cells is mediated, at least in part, through the inhibition of COX-2activity.4. SignificanceThis preclinical study provides the first evidence for the novel anti-tumor effects of TMP as a COX-2pathway inhibitor in human adenocarcinoma cell line A549. These studies suggest that TMP might serve as an effective agent for the treatment and chemoprevention of non-small cell lung cancer with low cytotoxicity.