| Liver and lung cancer are malignant diseases that threaten health and life of human, and their incidence and mortality keep very high nowadays, according to epidemiology data from cancer. Though many kinds of chemotherapies and surgical resections are used in clinical, there is no sensitive and efficiency method to overcome the problems. Therefore, it is very urgent to find some appropriate ways for the patients.Recent studies have found that, cancer development had close relationship with disorders of cPLA2 and COX-2, the two key enzymes in the AA metabolic pathway. The membrane phospholipids are catalyzed into AA by cPLA2, and then AA is converted into PGE2, which has the activities of inhibiting apoptosis, stimulating division and angiogenesis, promoting invasion and metastasis on tumor cells. It was reported that COX-2 and cPLA2 were over-expressed in cancers, such as colorectal cancer, liver cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer etc. These findings suggested that COX-2 and cPLA2 played a very important part in the development and differentiation of liver cancer and lung cancer.In addition, PPARγ/NF-kB pathway also had very close relationship with tumorigenesis and development. The PPARs are some clasess of nuclear transcription factors, and activated by their ligands. After being activated, the PPARs will bind to the specific sequences of DNA in heterodimer forms and regulate the transcription of the NF-κB. NF-κB activation can inhibit cell apoptosis and promote cell proliferation. The expressions of PPARγand NF-kB in tumor tissue were higher than those in adjacent tissues, depending on tumor histological type, cell differentiation and postoperative TNM staging. The agonists of PPARγpioglitazone and blocking NF-kB could inhibit tumor proliferation and promote apoptosis of tumor cells. Pathw.The mitochondria is an important place for apoptosis. With the stimulations of apoptosis signal external or internal in tumor cells, the mitochondrial membrane permeability was changed, and then cytochrome c, AIF and other apoptotic factors were released into the cytoplasm. The cytochrome c would lead to cell apoptosis after forming apoptosome with the Apaf-1 and caspase-9, activating caspase-3, caspase-6 or caspase-7 in the downstream. The Bcl-2 family proteins, including anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, are some important factors that regulated the changes of the mitochondrial membrane permeability, and play a very important role in the mitochondrial apoptosis pathway.Celecoxib belongs to the new generation of non-steroidal anti-inflammatory drugs, and a large number of studies declare the potential inhibition effects of celecoxib on occurrence, development and transfer of tumor. Celecoxib is a kind of selective inhibitors of COX-2. It promoted tumor cell apoptosis by inhibiting the expression of COX-2 and decreasing the level of PGE2 in traditional theory. But, in recent years, some COX-2 independent pathways were also found a number of studies. Those showed that its anti-tumor mechanisms were not yet fully understood. In the present study, we attempted to elucidate the inhibition effects of celecoxib on the H22 hepatoma and Lewis lung cancer and their mechanisms by COX-2 dependent and -independent Pathway in vivo and in vitro. Moreover, we tried to explain the preventive and therapeutic effect of celecoxib on cancer through its regulations on the AA metabolic pathway, the PPARγ/NF-kB pathway and the mitochondrial apoptosis pathway. These findings will provide some theoretical basis to its clinical application as antitumor drugs in the future. 1 Inhibition Effects of Celecoxib on H22 Hepatoma and its MechanismForty BALB/c mice receiving tumor implantation were divided randomly into control group and celecoxib groups at low, middle, high dosage (100,200,400mg/kg). All the groups were gavaged continuously with normal saline or celecoxib on the third day after implantation. The mice were killed on the 15th day, and the volume and weight of the tumor tissues were calculated. The protein expressions of cPLA2 and COX-2 were examined in the tumor tissues by Western blot analysis. The proliferation of H22 Hepatoma cells, treated with different doses (0, 25, 50, 100, 200μM) of celecoxib for 12, 24, 48 and 72 h, was measured by MTT assay. After being treated with celecoxib of different doses (0, 50, 100, 200 and 400μM) for 24h, the H22 cells were measured as followed: The apoptosis ratio and the mitochondrial membrane potential were detected by flow cytometry; The protein expressions of cPLA2, COX-2, Bax, Bcl-2, AIF, cytochrome c, caspase-3 and caspase-9 were examined by Western blot analysis, and the mRNA levels of cPLA2 and COX-2; The concentrations of arachidonic acid and prostaglandin E2 in the culture supernatants for 24h were measured by the methods of RP-HPLC and PGE2-specific ELISA respectively.The results in vivo showed that: The solid tumor volume and weight of treatment groups were lower than those of control group, and there was significant difference among the groups (P<0.05). The tumor inhibitory ratios of treatment groups were more than 30%, especially 75% in high dose group. The protein expressions of cPLA2 and COX-2 in treatment groups were lower than control group in a dose-dependent manner.The results in vitro showed that: Celecoxib exposure significantly reduced the viability of H22 cells in a dose- and time-dependent manner. IC50 values of celecoxib were 134.7±0.5μM, 112.4±0.3μM, 78.0±0.6μM and 56.9±0.8μM at 12, 24, 48, and 72h; Compared with controlgroup, the apoptosis ratio of tumor cell in treated groups notably raised and mitochondrial membrane potential decreased; The mRNA and protein expression of COX-2 were not altered in H22 cells treated with celecoxib at 50 and 100μM, however the COX-2 mRNA and protein levels were significantly decreased in H22 cells at 200 and 400μM dosage of celecoxib when compared to untreated H22 cells. Exposure to celecoxib caused an enhancement of the mRNA and protein levels of cPLA2 at dosages of 50 and 100μM, but resulted in an inhibition on the expression of cPLA2 at dosages of 200 and 400μM; The concentration of AA was elevated in the H22 cells after incubation with celecoxib at a dose of 50-400μM for 24h, and AA concentration was the highest at 100μM celecoxib, and the PGE2 production in H22 cells was not significantly influenced by treatment with 50 and 100μM celecoxib, but he levels of PGE2 production were significantly decreased when the cells were treated with 200 and 400μM celecoxib; The ratio of AA to PGE2 concentration was increased by celecoxib treatment in a dose-dependent manner in H22 cells; Compared with control group, Bax,caspase-3,caspase-9 were up-regulated and Bcl-2 was down-regulated; The protein levels of cytochrome c and AIF were up-regulated in the cytoplasmic, and down-regulated in the mitochondrias of the cells.2 Inhibition Effects of Celecoxib on Lewis lung cancer and its MechanismTwenty C57BL/6 mice receiving tumor implantation were divided randomly into control group and treatment (200 mg/kg) groups. All the groups were gavaged continuously with normal saline or celecoxib on the third day after implantation. The mice were killed on the 15th day, and the volume and weight of the tumor tissues were calculated. The proliferation of Lewis lung carcinoma cells, treated with different doses (50, 100, 200μM) of celecoxib for 12, 24, 48 and 72 h, was measured by MTT assay. The apoptosis and mitochondrial membrane potential were detected by flow cytometry. The protein expression of cPLA2, COX-2, PPARγand NF-κB was examined using Western blot analysis. The concentrations of AA and PGE2 in culture supernatants were measured by the methods of RP-HPLC and PGE2-specific ELISA respectively. The results in vivo showed that: The solid tumor volume and weight of treatment groups were lower than those of control group, and there was significant difference among the groups (P<0.05). The tumor inhibitory ratio of treatment groups was 50%.The results in vitro showed that: The proliferation of Lewis lung carcinoma cells was inhibited by celecoxib in a dose and time-dependent manner. The IC50 value of 72 h was 134.06±2.97μM; the apoptosis ratio of tumor cell in treated groups notably raised; The expressions of cPLA2 and PPARγwere up-regulated, but COX-2 and NF-κB were down-regulated in the Lewis cells exposed to celecoxib. The amount of AA was increased and PGE2 was decreased in the culture supernatant respectively. The ratio of AA to PGE2 was increased in a dose-dependent manner.In summary, our findings suggest that celecoxib could adjust the productions of AA and PGE2 by regulating on cPLA2 and COX-2, the two key enzymes in AA metabolic pathway. Although the regulations were different in H22 hepatoma and Lewis lung cancer cells, the ratio of AA and PGE2 was increased in both cell lines treated by celecoxib. The ratio of AA and PGE2 maybe was the a key factor in tumor development and reflecting the anti-tumor effect of celecoxib and other NSAIDs. Celecoxib could significantly inhibit and Lewis lung cancer in vivo and in vitro. Celecoxib may inhibited the H22 hepatoma by COX-2 dependent, non-dependent pathway, and activating mitochondrial apoptosis pathway. Celecoxib may inhibited the Lewis lung cancer by COX-2 dependent pathway, and PPARγ/ NF-κB channels.
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