AMD3100 Group Research Articles

Role of CXCR4-FAK signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells towards damaged tissues resulting from spinal cord ischemia-reperfusion injury in rats

Objective To investigate the role of CXC chemokine receptor 4-focal adhesion kinase (CXCR4-FAK) signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) towards damaged tissues resulting from spinal cord ischemia-reperfusion (I/R) injury in rats.Methods Part I Rat BMSCs transfected with recombinant adenovirus-mediated green fluorescent protein gene 3 were seeded in 24-well plates and randomly divided into 5 groups (n =18 wells each):control group (group C),normoxia-incubated group (group N),HP group (group H),HP + CXCR4 antagonist AMD3100 group (group HA) and HP + FAK inhibitor FAK-related nonkinase group (group HF).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to 21％ O2-74％ N2-5％ CO2 for 36 h.In group H,BMSCs were exposed to 0.5％O2-94.5％ N2-5.0％CO2 for 24 h followed by 12 h exposure to normoxia.In groups HA and HF,5 μg/ml AMD3100 and 10 μg/ml FAK-related nonkinase were added to the culture medium before HP,respectively.The expression of stromal derived factor-1α (SDF-1α),CXCR4 and phosphorylated FAK (p-FAK) in BMSCs was determined by Western blot.The migratory capability and adhesive ability of BMSCs were measured by Transwell invasive assay and fibronectin adhesive assay,respectively.Part Ⅱ Two hundred and sixteen male Sprague-Dawley rats weighing 300-350 g were used and 210 out of the 216 rats underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Thirty-six rats were chosen and sacrificed before spinal cord I/R and at 12 h and 1,3,5 and 7 days of reperfusion (T0-5) and the lumbar segment of spinal cord was removed for detection of the content of SDF-1α.The left 180 rats with spinal cord I/R were randomly divided into 5 groups (n =36 each).IT DMEM medium 300 μl was injected in group C and BMSC suspension 300 μl (1 × 106/ml) was injected in groups N,H,HA and HF immediately after onset of reperfusion.Neurological function was scored at T0-5.The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the degree of BMSC aggregation.Results There was no significant difference in the expression of SDF-1α,CXCR4 and p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation between groups C and N (P ＞ 0.05).Compared with group N,the expression of SDF-1α,CXCR4 and p-FAK was significantly up-regulated,and migratory capability and adhesive ability of BMSCs,neurological function scores and the degree of BMSC aggregation were increased in group H,while no significant change was found in the expression of p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation in HA and HF groups (P ＞ 0.05).Compared with group H,the expression of p-FAK was down-regulated and the migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation were decreased in groups HA and HF (P ＜0.05).The content of SDF-1α was significantly higher at T2,3 than at T0.Conclusion HP can promote migration and adhesion of BMSCs towards damaged tissues resulting from spinal cord I/R injury through CXCR4-FAK signaling pathway in rats; thus CXCR4-FAK signal pathway provides the protective effect on spinal cord. Key words: Receptors, CXCR4; Focal adhesion protein-tyrosine kinases; Mesenchymal stem cell transplantation ; Cell Hypoxia ; Ischemic preconditioning ; Cell movement; Cell adhesion; Spinal cord ; Reperfusion injury

Effects of stromal cell-derived factor-1 on the proliferation and apoptosis of articular chondrocytes in rabbits

Objective To observe the effect of stromal cell-derived factor-1 (SDF-1) on the proliferation of articular chondrocytes in rabbits and discuss the role of SDF-1 in chondrocyte apoptosis.Methods The chondrocytes were isolated and cutured,then divided into 4 groups:control group,50 μg/L SDF-1 group,50 μg/L SDF-1 + 5 mg/L CXC chemokine receptor-4 specific antagonist (AMD3100) group,and 5 mg/L AMD3100 group.The MTT assay and methods for cell cycle were used to observe the proliferation of chondrocytes.In addition,each group was exposured to the condition of 250 μmol/L hydrogen peroxide.Then cell viability was detected by using trypan blue exclusion assay,apoptosis rate of chondrocytes was detected by using flow cytometry,and the dynamic changes of Caspase-3 were measured in each group.Furthermore,the apoptosis of chondrocytes was evaluated by TdT-mediated dUTP nick end labeling (TUNEL) assay.Results The absorbance value of SDF-1 set (0.504 ±0.024) was higher than other groups,and the S +G2/M cell ratio in SDF-1 group was higher than other groups with the differnce bing statistically significant (all P ＜0.05).In terms of apoptosis,the viable rate in SDF-1 group was higher than other groups (P ＜ 0.01),while the Annexin V-FITC (+) proportion of cartilage cells in the SDF-1 group (4.00 ± 0.92) ％ was lower than other groups (P ＜ 0.05).Caspase-3 activity in SDF-1 group [(40.03 ± 2.56) nmolpNA/(h·mg) protein] was lower than other groups (P ＜0.01),and in addition,the number of apoptotic cells in SDF-1 group was less than other groups (P ＜ 0.05),but there was no significant difference among control group,SDF-1 + AMD3100 group and AMD3100 group in all experiments.Conclusion SDF-1 could contribute to the proliferation of rabbit chondrocytes in vitro,and in 50 μg/L group the Proliferation activity is reasonable,meanwhile SDF-1 can protect the chondrocytes from apoptosis induced by hydrogen peroxide. Key words: Stromal cell-derived factor-1 ; Apoptosis; Proliferation; Chondrocyte; Rabbit

Role of stromal cell derived factor/CXC chemokine receptor axis in regulation of expression of CD133 in gastric cancer cells via PI3K/Akt pathway in vitro

Objective To observe the effects of stromal cell derived factor/CXC chemokine recep- tor （SDF-1α/CXCR4） axis on the expression of CD133 via PI3K/Akt signaling pathway in gastric cancer （GC） in vitro. Methods The morphological expression of CXCR4, Akt, p-Akt and CD133 in KATO-1α cells were detected by using immunocytochemistry. SDF-1α, AMD3100 and LY294002 were respectively used to stimulate/inhibit KATO-m ceils. The semi-quantitative enzyme-linked polymerization assay for CXCR4 and CD133 mRNA expression was applied. The expression levels of CXCR4, Akt, p-Akt and CD133 proteins were detected by using Western blotting. Results Positive staining of CXCR4, Akt, p-Akt and CD133 was observed in the cultured cells. The expression levels of CXCR4 protein （0. 980 ±0. 083）, p-Akt protein （0. 770 ± 0. 071 ）, and CD133 protein （0. 890 ±0. 078 ） were significantly higher （ P 〈 0. 05） in SDF-1α group than in control group （0. 750 ±0. 042, 0. 680 ±0. 038, 0. 720 ±0. 034 respectively）. The expression levels of CD133 mRNA （0. 890 ±0. 061 ） were significantly higher （P 〈 0. 05 ） in SDF-1α group. In AMD3100 group, the expression levels of CXCR4 protein （0. 430 ±0. 055）, p-Akt protein （0. 350 ±0. 050） and CD133 protein （0. 110 ±0. 060） were decreased significantly as compared with those in control group （0. 750 ±0. 042, 0. 680 .SO. 038, and 0. 720 ±0. 034 respectively, P 〈0. 05）. In LY294002 group, the expression levels of p-Akt protein （ 0. 100 ± 0. 033 ） and CD133 protein （0. 440 ±0. 055 ） were decreased significantly as compared with those in control group （0. 680 ±0. 038 and 0. 720 ± 0. 034 respectively, P 〈0. 05） ; In comparison with that in control group （0. 540 ±0. 036）, CD133 mRNA （0. 310 ±0. 021 ） was decreased significantly （P 〈0. 05）. Conclusion The axis of SDF-la/CXCR4 stimulated or inhibited can up-regulate or down-regulate the CD133 expression via PI3K/Akt signaling pathway in GC in vitro. Key words: Gastric cancer; Stromal cell derived factor/CXC chemokine receptor; CD133; PI3K/Akt pathway

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

IntroductionThis study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.MethodsOA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.ResultsSDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.ConclusionsThe binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.

Effect of chemokine stromal cell derived factor-1 and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer

To investigate the effect of chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 on the peritoneal carcinometastasis of gastric cancer. Human gastric cancer cells of the lie NUGC4 and mesothelial cells of the line HMrSV were cultured. RT-PCR was used to detect the expression of CXCR4 and SDF-1 mRNA in the NUGC4 and HMrSV cells. The proliferation of NUGC4 cells was detected by MTT method. In mesothelial cell adhesion teat NUGC4 cells were cultured with confluent HMrSV mesothelial cells in 24-well plate and then divided into 3 groups: chemokine group, added with SDF-1, antibody blocking group, in which the NUGC4 cells were pre-incubated with CXCR4 monoclonal antibody for 2 h and then SDF-1 was added, and control group added with only culture fluid. Microscopy was used to calculate the number of gastric cancer cells adhered with mesothelial cells. In the mesothelial cell migration test HMrSV cells were put in the upper chamber of a Transwell chamber so as to cover the infiltration membrane. This Transwell chamber was put into a culture plate, NUGC4 cells, divided into 3 groups as mentioned above were put into the upper chamber, 24 h later HE staining and microscopy were performed to calculate the number of the NUGC4 cells that penetrated the membrane. BALB/c nu/nu female nude mice underwent intraperitoneal injection of NUGC4 cells, and then with PBS or AMD3100, small molecular specific antagonist, one day after the cancer cell injection once a day for 2 weeks. Then the mice were killed to observe the intraperitoneal tumorigenesis. CXCR4 mRNA was highly expressed in the NUGC4 cells but only very weekly expressed in the HMrSV cells. SDF-1 mRNA expression was seen in the HMrSV cells but in the NUGC4 cells. Anti-CXCR4 monoclonal antibody (McAb) inhibited the proliferation of NUGC4 cells significantly (P < 0.05). In mesothelial cell adhesion test, the number of the NUGC4 cells adhered with HMrSV cells after SDF-1 stimulation was 84.4 +/- 21.2, significantly higher than that of the control group (43.6 +/- 12.4, P < 0.05). The number of migrating NUGC4 cells in the chemokine group was 170.8 +/- 24.2, significantly higher than hat of the control group (102.8 +/- 18.2, P < 0.05); and the number of migrating NUGC4 cells in the antibody blocking group was 114.7 +/- 20.3, significantly lower than that of the chemokine group (P < 0.05). The survival time of the mice injected with both NUGC4 cells and AMD3100 was (43.8 +/- 2.8) days, significantly longer than that of the control group [(28.2 +/- 2.5) days, P < 0.01]. The tumor number of the AMD3100 group was (64.6 +/- 8.2), significantly lower than that of the control group [(103 +/- 12.4), P < 0.01]. SDF-1 and its receptor CXCR4 play an important role in the development of peritoneal carcinometastasis from gastric cancer. Interfering with the SDF-1/CXCR4 biological axis may become a potential strategy in the prevention and treatment of peritoneal carcinometastasis from gastric cancer.

AMD3100 Affects Autograft Lymphocyte Collection and Progression-Free Survival After Autologous Stem Cell Transplantion in Non-Hodgkin Lymphoma

Purpose:Autograft absolute lymphocyte count (A-ALC) affects survival after autologous stem cell transplantation (ASCT) in non- Hodgkin lymphoma (NHL). AMD3100, a CXCR4 antagonist, mobilizes CD34+ stem cells in patients with NHL undergoing ASCT. We sought to study the impact of AMD3100 on A-ALC collection in patients with NHL undergoing ASCT. Patients and Methods:The primary endpoint of the study was to assess the association between AMD3100 and A-ALC collection. We compared 7 consecutive patients with NHL mobilized with AMD3100 and granulocyte colony-stimulating factor with 29 control patients with NHL mobilized with granulocyte colony-stimulating factor alone. Results:Higher median A-ALCs were observed in the AMD3100 group compared with the control group (4.16 × 109 lymphocytes/kg vs. 0.288 × 109 lymphocytes/kg; P < 0.0001). With a median follow-up of 20 months (range, 4-24 months), no relapses were reported in the AMD3100 group compared with 15 of 29 in the control group (P < 0.02). Conclusion:Our data suggest that AMD3100 affects A-ALC and clinical outcome in patients with NHL undergoing ASCT.