MMP-1 Over-expression Promotes Malignancy and Stem-Like Properties of Human Osteosarcoma MG-63 Cells In Vitro.

Osteosarcoma is the most common primary malignant bone tumor in childhood, and it maintains a high level of recurrence. Matrix metalloproteinase-1 (MMP-1) was found to contribute to cancer progression. The present study was to investigate the in vitro effects of MMP-1 over-expression on the proliferation, invasion, metastasis and stem-like properties of osteosarcoma MG-63 cells. The MG-63 cells were cultured and had a full length MMP-1 cDNA inserted by the lentiviral vector (MG-63MMP-1+). MG-63 negative control and MG-63 blank control groups were established as well. MMP-1 expression was detected in MG-63MMP-1+, MG-63 negative control and MG-63 blank control cells using qPCR, Western blotting and immunofluorescence after 24 h of culture. The cell proliferation assay was performed with a camera attached to a bioreactor, which was programmed to photograph five regions of each well every 10 min over a period of 48 h. The cell invasion assay was conducted with Matrigel to assess the invasive potential of MG-63 cells over 24 h, the qPCR analysis to measure stem cell markers, including Oct4, Sox-2, Nanog, and Pax-7, and Western blot analysis to detect invasive and metastatic potential markers TIMP-1, VEGF and BMP2/4, after 24 h of culture. Immunofluorescence was used to investigate the presence of the stem cell marker Pax-7 after 24-h culture. The results showed that over-expression of MMP-1 after transfection could significantly increase tumor cell proliferation and invasion (P<0.05, MG-63MMP-1+versus controls). Pax-7 was highly expressed in MG-63MMP-1+ cells, with no significant changes of Oct-4, Sox-2, and Nanog observed (P<0.05). MG-63MMP-1+ cells showed higher expression of VEGF and BMP 2/4 proteins and lower expression of TIMP-1 protein than controls (P<0.05). It was concluded that MMP-1 over-expression in MG-63 cells contributed to the proliferation, invasion, metastasis and stem-like properties of osteosarcoma cells. Future studies should focus on in vivo effects of MMP-1 over-expression and the application of MMP-1 and Pax-7 inhibition in vivo to osteosarcoma therapies.

[Acute toxicity of CCR5 antagonist C25P polypeptide in mice and its carcinogenicity in vitro].

OBJECTIVE: To study the acute toxicity of C25P polypeptide, a CCR5 antagonist, in mice and its carcinogenic effect in vitro. METHODS: The acute toxicity of C25P polypeptide in mice was assessed by determining the maximum tolerated dose (MTD). The mice were given C25P at the dose of 3.64 g/kg by tail vein injection, and the control mice received saline (40 ml/kg) injection. The mice were continuously observed for 14 days after the administration and sacrificed on day 14 for routine blood test, examination of the blood biochemistry and pathological examination. The carcinogenicity of C25P polypeptide in vitro was evaluated in cultured cell lines by chromosome aberration test, cell transformation test and non-anchorage dependent growth test. RESULTS: No mice died following administration of the drug, but 3 mice showed mild adverse reactions. The rats in both groups showed an increase in the body weight at a comparable rate. GPT increased and ALP decreased significantly in C25P polypeptide group (P<0.05). Most of the organs of the rats treated with in C25P polypeptide remained normal, but 3 mice showed pathologies in the lung, spleen and liver. Chromosome aberration test, cell transformation test and non-anchorage-dependent growth test all yielded negative results for C25P polypeptide. CONCLUSION: C25P polypeptide is a low-toxicity drug that produces no apparent acute toxicity in mice or obvious carcinogenicity in vitro.

Urokinase-type plasminogen activator receptor induces conformational changes in the integrin alphaMbeta2 headpiece and reorientation of its transmembrane domains.

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin.

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.

Down-regulation of integrin alpha M beta 2 ligand-binding function by the urokinase-type plasminogen activator receptor.

The cell adhesion molecule integrin alphaMbeta2 associates with the urokinase-type plasminogen activator receptor (uPAR) on monocytes and neutrophils. uPAR also associates with members of the beta1 and beta3 integrins, and it modulates the ligand-binding function of these integrins. In this study, we showed that co-expressing uPAR with alphaMbeta2 in 293 transfectants down-regulated the ligand-binding capacity of alphaMbeta2 to denatured protein, fibrinogen, and intercellular adhesion molecule 1 (ICAM-1). Migration of transfectants on fibrinogen mediated by alphaMbeta2 was reduced in the presence of uPAR. In addition, the constitutive ligand-binding property of an alphaMbeta2 mutant was attenuated by its association with uPAR. Co-immunoprecipitation analyses using a panel of alphaMbeta2-specific mAbs suggest shielding of the ligand-recognition site of alphaMbeta2 by uPAR.