The Effect Of Dalteparin On Lung Adenocarcinoma A549 Cell Line In Vitro And The Potential Mechanism

Backgrounds and objectiveUnfractionated heparin (UFH) and low molecular weight heparin (LMWH) play their anticoagulant roles by activating the physiological coagulation inhibitor antithrombin, which neutralizes many of the serine proteases involved in the coagulation system, particularly thrombin and activated factor X.Besides their anticoagulant effects, a wide variety of biological activities of UFH and LMWH have been discovered. Recently, their beneficial roles on cancer progression have been reported. The results of the malignancy and low-molecular-weight heparin therapy (MALT) which were conducted on patients with advanced malignancy without venous thromboembolism indicated that LMWH therapy may favorably prolong survival. The fragmin advanced malignancy outcome study (FAMOUS) showed the similar results. In addition, LMWH can strengthen the efficacy of chemotherapeutic drugs.Several underlying mechanisms by which heparins and LMWH affect tumor growth and/or metastasis have been discovered. According to the previous results, the beneficial role of UFH and LMWH were attributed to their influences on angiogenesis and the effects on the binding of selectins with their natural ligands. Additionally, the roles could also be related to the host immune response which was affected by UFH and LMWH. UFH can also inhibit heparinase which are involved in degrading basement membranes, modulating various heparin-binding growth factors and regulating integrin functions in cell adhesion.UFH and LMWH have the direct inhibitive effects to the growth of different cell types. For example, UFH and LMWH can decrease the mitotic activity of vascular smooth muscle cell both in vivo and in vitro. This efficacy has also been found in glomerular mesangial cells and smooth muscles of airway and intestine. UFH can induce the lymphoblasts, neutrophils, and mononuclear cells from children with acute lymphoblastic leukemia to apoptosis. However, there is few data about the effects of UFH or LMWH to pulmonary adenocarcinoma (ACA) cell line and it is unknown whether the growth and proliferation of A549 cell could be inhibited or delayed by UFH or LMWH directly.Cell and tissue homeostasis are dependent on a tightly coordinated network of signaling pathways, resulting in a spatial and temporal balance of proliferation, growth arrest, differentiation, senescence, and apoptosis. Aberrant regulation of any of these biological processes can contribute to the onset and progression of tumorigenesis. In another word, the inactivation or down-regulation of some negative regulators of cell growth is involved in the formation of tumor cells characteristics of high aggressiveness and proliferation.The cell cycle progression is regulated by cyclin, cyclin-dependent-kinase (CDK) and CDK-inhibitor (CDKI). The CDK-cyclin complexes promote the progression of cell cycle. CDKI are modulators which negatively regulate the activity of CDK-cyclin complexes. There two families of CDKI, KIP family and INK4 family. KIP family, which exercises broad-acting inhibition of CDK involved in the G1/S transition, includes p21^WAF1, p27^KIP1, and P57^KIP2. The INK4 family, which specifically inhibits CDK4 and CDK6, include p16^ink4a, p15^INK4b, p18^INK4c,andp19^INK4d.There are one restriction point (R-point) and two check-points which are critical during cell cycle progression. Cell cycle progression through the G₁ phase into S is a major checkpoint for proliferating cells, and is under multiple levels of control by p21^WAF1.In the absence of growth factors or other mitogenic stimuli, cells finishing mitosis are arrested in G₀, an early, reversible stage of G₁. During sustained mitogenic stimulation, expression of D-type cyclins is induced and gives rise to the formation of cyclin D/ CDK4 and cyclin D/ CDK6 complexes. The active cyclin D complexes rapidly phosphorylate pRb, allowing for subsequent phosphorylation of pRb by cyclin E/ CDK2 complexes. These cyclin E complexes must also be phosphorylated by CAK to be active and further phosphorylate pRb. Once pRb becomes hyperphosphorylated by the sequential activities of CDK4 and CDK2, E2F is released from inhibition by pRb, and the expression of genes required for the S phase is induced. The cell will past the checkpoint in late G₁ phase and enter S phase where mitogenic stimulation is no longer required.Both p21^WAF1 and p27^KIP1 have a role in carcinogenesis. In many human cancers, including lung cancer, the level of p27^KIP1 is frequently decreased (13). Thus, the physiologic balance between cell proliferation and death is disrupted. It is unclear whether UFH or LMWH exert their anti-cancer efficacy on A549 cell line throughp21^WAF1 and p27^KIP1 proteins.Materials and methodsIn this study, we interfered with the growth of A549 cells with dalteparin, a kindof LMWH, observed the alteration of some biological characteristics.First, the viability of A549 cells in the absence or presence of dalteparin (OIU/mL, 2IU/mL, 10IU/mL, 50IU/mL, 100IU/mL and 500IU/mL) was examined by MTT assay at different time points (6h, 12h, 24h and 36h). Dalteparin at the concentration of 50 IU/mL was employed in the following tests.Secondly, the cells were cultured in the medium in the absence or presence of dalteparin (50IU/mL) for 24h incubation and stained with PI. Distribution of cell cycle phase was quantified and analyzed by flow cytometry.Thirdly, in order to observe the influences of dalteparin at 50IU/mL on apoptotic status, the A549 cells were stained with AnnexinV-FITC and PI. The ratio of early apoptotic cells was detected and analyzed by flow cytometry.To elucidate whether the changes in apoptosis and cell cycle distribution correlate to p21^WAF1 and p27^KIP1 genes which are regarded as main regulators of cell cycle, the levels of p21^WAF1 and p27^KIP1 proteins and mRNA were examined by western blotting and RT-PCR assay respectively.To illuminate further mechanisms by which dalteparin exerts its effects on A549 cell line, the expression of p21^WAF1 and p27^KIP1 were silenced by siRNA which specifically targets p21^WAF1 and p27^KIP1 mRNA. The A549 cells transfected with random 21-mer siRNA were considered as the control. Western blotting analysis was performed to confirm the gene silencing by siRNA. The A549 cells were cultured in the presence of dalteparin for 24h and subjected to flow cytometry analysis for the cell cycle distribution and apoptotic status.Data were analyzed by SPSS 11.0 software. Either /-test or ANOVA was used. P<0.05 was considered as statistically significant.Results1. Dalteparin decreases the A549 cell viability dose-dependently and time-dependently. Dalteparin at the concentration of 50IU/mL have a moderate inhibitory effect on A549 cell viability.2. Dalteparin delays the cell cycle progression. A significant increase in the G₁ phase of the cell cycle (P<0.05) together with significant decrease in the S phase (P<0.05) (in comparison with the corresponding values for cultured A549 cells without dalteparin) occurred after dalteparin treatment. Although the cells proportion in G2/M phase was elevated, this difference has no statistical significance (P>0.05).3. Flow cytometry revealed a statistically significant increase in early apoptosis in group incubated with 50 IU/mL of dalteparin in comparison with the control (14.3±3.8% vs 6.5±2.2%, P<0.05). Additionally, dalteparin at the concentration of 50 IU/mL did not increase the late apoptotic cell after 24h treatment.4. Dalteparin enhances the expressions of p21^WAF1 and p27^KIP1 proteins, but does not affect the mRNA of p21^WAF1 and p27^KIP1.5. Genes silencing by introduction of p21^WAF1 siRNA to A549 cells attenuated dalteparin-dependent G₁ phase arrest (P<0.05) and apoptosis induction (P<0.05). The results caused by p27^KIP1 transfection on cell cycle and apoptosis are similar with those by p21^WAF1. Conclusions and significances1. Dalteparin can decrease the A549 cell viability.2. The effects of dalterparin on cell viability were caused by the cell cycle arrest in G₁ phase and the induction to early apoptosis.3. The changes in cell cycle distribution and apoptosis status partly result from the alteration of expression of p21^WAF1 and p27^KIP1 proteins.4. A549 cells express a low to moderate levels of p21^WAF1 and p27^KIP1 proteins.5. The altered expression of p21^WAF1 and p27^KIP1 proteins caused by dalteparin occurred at posttranscriptional levels.6. The enhanced p21^WAF1 and p27^KIP1 proteins which occurred at posttranscriptionallevels ultimately contributed to the changes of cell viability, cell cycle distribution and apoptosis in the A549 cells.