The Facilitated Effect Of Profilin1 On Apoptosis Of Breast Cancer Cells And Its Molecular Mechanism

(?)rofilinl (Pfn1), among the first actin-binding proteins (ABPs) to be highly conserved during evolution, leads to the dynamic transformation of F-actin and remodeling of actin skeleton by regulating the polymerization and depolymerization of G-actin, with the major role in cell adhesion, movement, and growth, et al. It is believed that when malignant change happens, ABPs vary along with the enhancement of cells migration and invasion. In ABPs, Pfnl was found down-regulated in several cell lines of breast cancer, liver cancer, pancreatic cancer and nasopharyngeal cancer compared with their corresponding normal cell ones. Besides, overexpression of Pfnl resulted in morphological change following the cytoskeleton remodeling, and the suppressed tumorigenicity of breast cancer cells in both ectopic and orthotopic model systems of nude mice. This correlation of loss of Pfnl with tumor progression has led to Pfnl being considered a tumor suppressor. The molecular mechanisms underlying Pfnl's tumor-suppressive action, however, remain to be elucidated.In the previous work of our lab, we analyzed differential expressed proteins of all-trans retinoic acid (atRA)-treated and -control cells using two-dimensional electrophoresis (2-DE) in hepatocellular carcinoma cell line, and identified the proteins with mass spectrometry (MS) and database searching, showing that Pfnl is one kind of up regulated proteins. The further researches confirmed that Pfnl was related with atRA inhibitory effects on tumor cells proliferation and migration, and the inhibitions were partially through upregulating of the Pfnl expression. Thus it is believed that some of Pfnl may become the target in tumor metastasis blocking. Due to the fact that one of the critical mechanisms of chemical drugs and plants extracts is to induce tumor cells to apoptosis, it is hinted that Pfnl might have a role in the apoptotic process. Our researches showed that the natural agent Staurosporine (STS) could also upregulate the protein level of Pfnl, indicating that Pfnl may play an important part in the STS apoptotic model. STS is a kind of broad-spectrum protein kinase inhibitor which can induce the apoptosis in various cancer cell lines thus it has been used to produce the apoptotic model. In this study, we identified that Pfnl could facilitate the apoptosis of breast cancer cells under STS action, and performed in-depth research on the molecular mechanism of Pfnl tumor suppression in a certain degree.We chose carcinoma of large intestine (including carcinoma of colon and rectum) as subject and immunohistochemical tests were performed on non-necrotic tumor tissues and normal tissues of paracarcinoma or of distal (3cm afar). It was exposed that Pfnl was down-regulated in tumor compared to normal tissues, with accordance to the reports in mammary and hepatic tissues. Besides, a comparison between several available different histogenetic cancer cells revealed that breast cancer cell lines presented a lower level of Pfnl, and especially breast cancer cell MDA-MB-468 showed a relatively least amount of Pfnl protein and a lower level than that in normal mammary epithelium cells. Purposely, we set up and characterized stably-transfected cloned strain of MDA-MB-468 with Pfnl overexpression, Pfnl-468 cells and its control cloned strain, Mock cells. We found that Pfn1 could enhance the apoptotic sensitivity induced by STS. Under STS treatment, Pfnl-468 cells had more sub-G1 cells and dramatically lower viability, displaying the nuclear apoptotic features ahead of time, compared to Mock. It is well recognized that the change of mitochondrial inner transmembrane potential (△ψm) is one of key features in the early phage of apoptosis. Pfn1 overexpression promoted the loss of△ψm and release of cyto.c, which could further trigger the caspase3,9 activation and PARP cleavage in the intrinsic pathway. All these data illustrated that Pfnl could enhance the apoptosis of mitochondrial pathway induced by STS.Caspase activation requires mitochondria release apoptotic activators. We found that ectopic-Pfnl expression upregulated the p53 protein level as an apoptotic activator. Actually, approximately half of breast cancers contain inactivating mutations of p53, commonly whose mutant points locating at the central DNA-binding domain, abrogating the ability to transactivate downstream genes. MDA-MB-468 just posseses the mutant p53. Further studies showed that mutant p53 was involved in apoptosis induced by STS via transcription-independent mitochondrial functions since we observed (i) the increased cytosolic localization of p53, (ii) the activation of phosphorylation at Ser15, (iii) its mitochondrial localization; and Pfnl acted as a positive regulator of this process. Intriguingly, we found that Pfnl interacted with p53 and thus facilitated it to exert the transcription-independent activity in the cytoplasm during STS action. And the mutant p53 abrogating DNA binding activity was found to play a major role in the Pfnl-sensitized apoptosis through a transactivation-independent and cytosolic activity.Pfn1, as the actin-binding protein is crucial for the adhesion of cells to extracellular matrices, cell growth and survival, as well as signaling transduction, as it involves in the linkage of integrins to the cortical actin cytoskeleton. In our research, we found that Pfnl overexpression could induce the upregulation of integrinα5β1, already known to inhibit the transformed phenotype for cancer cells in some previous reports. Further, the Pfnl-facilitated apoptosis induced by staurosporin was blocked by the cells attached to a supplementary fibronectin substrate as a ligand of integrinα5β1. Thus, the findings demonstrated that integrinα5β1 upregulation by Pfnl attached to insufficient fibronectin activated a signaling pathway leading to enhanced cellular apoptosis and that one of the likely mechanisms for Pfnl's cancer suppression. Moreover, Pfnl that primarily functioned to promote local superstructure formation involving actin filaments and integrinβ1 notably contributed to its auxo-action on apoptosis.Previously we reported that overexpressed integrinβ1 subunit in the hepatocellular carcinoma cell line SMMC-7721 imposed a growth inhibitory effect through the upregulation of p21cipl and p27kipl. In this study, we confirmed the growth inhibitory effect ofβ1 subunit overexpression in different cancer cell lines. The upregulated CDK inhibitors induced byβ1 integrin overexpression were essential for this integrin-mediated growth arrest. Reduced c-Jun level after integrinβ1 overexpression plays an important role in the transcriptional activation of p21 through the Sp1 sites. Solely overexpressedβ1 subunit could induce the expression of diverse a subunit in different cell lines, among whichα5 subunit was found to be correlated with integrinβ1-mediated growth arrest. Relative lack of ECM-integrin interaction might be a reason for integrinβ1 overexpression-mediated growth arrest. These results helped us understand more about the mechanisms that integrins regulate cell growth.