Effects Its Mechanisms Of TQ On A549 Lung Cancer Cell Proliferation, Migration And Invasion

Lung cancer is one of the most common cancers and leading cause of tumour-related death worldwide. The two major histological types of lung cancer are non-small cell lung cancer(NSCLC) and small cell lung cancer(SCLC), accounting for about 85% and 15% of cases, respectively. Despite improvements in surveillance and clinical treatment strategies, progressive stages and metastasis of NSCLC still remains the most common cause for the high NSCLC lethality. Tumour progression is a multistep process, in which cancer cells uncontrolled growth, detach from the primary tumor, invade surrounding tissues, and intravasate into blood and/or lymphatic systems. Finally, cancer cells settle and colonize at the target organs. Matrix metalloproteinases(MMPs) play important roles in tumor metastasis. They are widely considered to be a secreted, zinc-dependent endopeptidase which can degrade extracellular matrix(ECM) components such as collagen, fibronectin, proteoglycan, laminin, and elastin in both physiological and pathological processes. And it has been reported that the expression of MMPs is regulated by mitogen- activated protein kinases(MAPKs) pathways, which are involved in regulating cell proliferation, migration and invasion. Although the researchers gradually understand the mechanisms of cell migration and invasion, efficacy of the present drugs designed to block tumor progression by modulating these mechanisms is very limited.There have been many reports focus on the effectiveness of chemopreventive or therapeutic drugs from natural products. The Black Caraway seed, also named Nigella Sativa, which belonging to Ranunculaceae family, is an annual herbaceous plant that grows in countries bordering Mediterranean Sea, Pakistan and India. It commonly used traditionally as a natural treatment for numerous diseases for more than 2000 years. Thymoquinone(TQ) was the primary bioactive component of Nigella sativa Linn seed oil and used as anti-inflammatory, anti-oxidant, and anti-neoplastic agent. In the last decade, multiple papers have reported that TQ was able to inhibit a variation of carcinomas including breast, prostate, ovarian, liver, colorectal carcinoma, and so on. And previous studies have shown that TQ exhibits inhibitory effects on multiple process of cancer, including proliferation, apoptosis, migration, invasion, and angiogenesis. In addition, TQ synergistically augments conventional medicine inhibition of cancer cells, such as NCI-H460 non-small cell lung cancer cells and U266 multiple myeloma cells.However, the detailed molecular mechanisms of the antineoplastic effects of TQ are are not entirely elucidated yet, and the potential therapeutic effects of TQ in lung cancer also remains enigmatic.Based on the above information, in order to explore effects of TQ on lung cancer cell proliferation, migration, invasion, and its molecular mechanisms, we investigated:(1) Effects of TQ on A549 cell proliferation, migration, and invasion in A549 cells.(2) Effects of TQ on activity and expression of MMPs in A549 cells.(3) Effects of TQ on the signal transductions of MAPKs in A549 cells.(4) TQ inhibited proliferation, migration and invasion of A549 cells through ERK1/2 pathway. Part 1 Effects of TQ on A549 cell proliferation, migration and invasionObjective: To investigate the effects of TQ on A549 cell proliferation, migration and invasion.Methods: A549 cells were purchased from Cell Resource Center of Life Sciences(Shanghai, China) and grown in RPMI 1640 medium containing 10% FBS, 100 units/m L penicillin, and 100 mg/m L streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37℃ and were passaged three times a week by treating with 0.25% trypsin containing 0.02% EDTA.After treatment with various concentrations(0, 5, 10, 20, 40, 80, 160 μmol/L) of TQ for 24, 48 or 72 h, cell proliferation activity was determined by MTT and cell count assays. After treatment with different concentrations(0, 5, 10, 20, 40, 80, and 160 μmol/L) of TQ for 48 h or treatment with 40 μmol/L TQ for 24, 48 and 72 h, the effect of TQ on the m RNA and protein expression of growth marker gene PCNA in A549 cells we re detected using quantitative real-time PCR and western-blot assays, respectively.The effect of TQ on A549 cells migration was detected by the wound-healing assay. A549 cells were seeded(5×104cells/well) into 12-well plates and grown to 80~90% confluence for the experiment. Monolayer cells were wounded by scratching. After treatment with different concentrations(0, 10, 20, and 40μmol/L) of TQ for 48 h, or treatment with 40 μmol/L TQ for 24, 48 and 72 h, the cell migration activity was expressed as the number of cells migrating into the wound.After treatment with different concentrations TQ(0, 10, 20, or 40 μmol/L) for 48 h, or treatment with TQ(40 μmol/L) for 24, 48 and 72 h, the cell invasion ability was detected by transwell invasion assay.Results:1 TQ had a dramatic inhibitory effect on A549 cell proliferation with a dose- and time-dependent manner. However, proliferation inhibition rate were not significantly altered after A549 cells treated by 5 μmol/L TQ. In addition, TQ inhibited the m RNA and protein expression level of growth marker gene PCNA in a dose- and time-dependent manner, especially at 10, 20, 40 μmol/L concentrations(P<0.01). The results suggested that TQ played a role in inhibiting the proliferation of lung cancer cells.2 TQ had a dramatic inhibitory effect on migration of A549 cells with a dose- and time-dependent manner(P<0.01). These results suggested that TQ played a role in inhibiting the migration of lung cancer cells.3 TQ had a dramatic inhibitory effect on invasion of A549 cells with a dose- and time-dependent manner(P<0.01). These results suggested that TQ could inhibit the invasion of lung cancer cells.Conclusions: TQ could inhibit A549 cell proliferation, migration, and invasion. These results suggested TQ had inhibited effects on tumour progression and metastasis of lung cancer. Part 2 Effects of TQ on activity and expression of MMPs in A549 cellsPart 2 Effects of TQ on A549 cell proliferation, migration and invasionObjective: To investigate the effects of TQ on activity and expression of MMPs and the degree of degradation of extracellular matrix(ECM) in A549 cells.Methods: A549 cells were treated with TQ(0, 10, 20 or 40 μmol/L)for 48 h. The expression of MMP2, MMP9, TIMP1, and TIMP2 in A549 cells were detected using western-blot and quantitative real-time PCR assays. The effect of TQ on MMP2 and MMP9 gelatinases activities was assessed using gelatin zymography.Results:1 The protein expression of MMP2 and MMP9 were inhibited by TQ treatment in a dose-dependent manner(P<0.01).2 The m RNA expression of MMP2 and MMP9 were inhibited by TQ treatment in a dose-dependent manner(P<0.01).3 TQ has no effect on the expression of TIMP1 and TIMP2.4 TQ suppressed the MMP2 and MMP9 activity dose-dependent ma nner in A549 cells(P<0.01).Conclusions: TQ could inhibit MMP2 and MMP9 expression and activities in A549 cells. These results suggested TQ may play major roles in degradation of extracellular matrix, and then affect the cell migration and invasion. Part 3 Effects of TQ on the signal transductions of MAPKsObjective: To investigate the effect of TQ on the signal transductions of MAPKs.Methods: A549 cells were treated with 40 μmol/L of TQ for 0, 4, 8, 12, 24 or 48 h, and then total protein lysates of each sample were collected and then subjected to western-blot with phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-p38 MAPKs and p38 MAPKs antibodies.Results: TQ inhibiteded phosphorylation of ERK1/2 in a time-dependent manner, whereas TQ had no obvious effects on p38 and JNK1/2 protein and its phosphorylation levels.Conclusions: TQ could inhibite ERK1/2 signaling pathway of A549 cell. These results suggested the inhibition of TQ on cell proliferation, migration and invasion may rely on its inhibitory effects of ERK1/2 pathway.Part 4 TQ inhibited proliferation, migration and invasion of A549 cells through ERK1/2 pathwayObjective: To investigate the effect of ERK1/2 inhibitor(PD98059) in combination with TQ on proliferation, migration and invasion for A549 cells, so that we can confirm whether the inhibitory action of TQ rely on its suppression of ERK1/2 pathway.Methods: A549 cells was divided into four groups as follows: 1 group(TQ-, PD98059-); 2 group(TQ+, PD98059-); 3 group(TQ-, PD98059+); 2 group(TQ+, PD98059+). A549 cells were pretreated with 20 μmol/L PD98059 for 2h and incubated with TQ(40 μmol/L) for 48 h. Cells were collected and subjected to western-blot assay to detecte the expression of MMP2, MMP9, ERK1/2 and p-ERK1/2. Meanwhile, the media was collected for gelatin zymography assay. Cell proliferation activity was measured by MTT and cell count assays. Cell migration and invasion activities were detected by wound healing and transwell assays, respectively.Results:1 TQ decreased the activity and expression of MMP2 and MMP9, however, the inhibitory effect was blocked by ERK1/2 inhibitor PD98059(P<0.01).2 TQ decreased phosphorylation of ERK1/2 in A549 cells(P<0.01). In addition, TQ has no effect on ERK1/2 protein expression.3 TQ decreased A549 cell proliferation activities. However, the inhibitory effect was blocked by PD98059(P<0.01).4 TQ decreased A549 cell migration activities. However, the inhibitory effect was blocked by PD98059(P<0.01).5 TQ decreased A549 cell invasion activities. However, the inhibitory effect was blocked by PD98059(P<0.01).Conclusions: TQ could inhibit phosphorylation of ERK1/2, and further inhibit A549 cell proliferation, migration and invasion; and inhibit the expression and activities of MMP2 and MMP9. However, these effects were blocked by ERK1/2 inhibitor PD98059. The study suggested that down-regulation of tumour progression and metastasis may rely on ERK1/2 pathway.