Effect Of Berberine On CXCL12 / CXCR4 Axis In Inhibiting The Invasion And Metastasis Of Colorectal Cancer And Its Possible Mechanism

[Background and Objective]Colorectal cancer(CRC) is the most common malignant tumors, colorectal cancer patients with acquired resistance is an important factor in the failure of chemotherapy leading to tumor recurrence and metastasis. Berberine is an isoquinoline alkaloid, have been used for Chinese traditional medicine many years. Modern medical research has found that berberine has anti-tumor activity in a variety of solid tumors, and its activity through a variety of signaling pathways play a role. CXCL12, also known as stromal cell-derived factor-1, belonging to CXC chemokine subfamily, CXCR4 is a CXCL12 receptor, a G protein-coupled membrane receptors, has been demonstrated that CXCL12/ CXCR4 axis and colorectal cancer lymph node and liver metastases are closely related, and high expression of CXCL12 was considered a poor prognostic factor. Another study confirmed that the chemokine CXCL12 can induce SW480 colon cancer cell signaling proteins activated AKT and NF-κB promote invasion and metastasis of SW480. Berberine can inhibit NF-κB activity to inhibit the growth and invasiveness of primary lymphoma and malignant melanoma.This study sought to determine berberine on SW480 colon cancer cell proliferation, migration, invasion capabilities and depth if it can exert anti-tumor activity by CXCL12/CXCR4axis.[Methods]1. Berberine in vitro inhibition of SW480 cell proliferation, migration, invasion experiments.(1) Analyze growth inhibition rate of SW480 treat with different concentrations of berberine by MTT assay, and calculate half inhibitory concentration (50%) of berberine of colorectal carcinoma cells SW480.(2) Scratch test to determine the effect of colorectal carcinoma cells SW480 cell migration after treat with different concentrations of berberine.(3) Transwell chamber assays for the invasion of colorectal carcinoma cells SW480 after exposure to different concentrations of berberine.2. Studies on the mechanisms of berberine in vitro inhibition the colorectal carcinoma cells SW480 cells.RT-PCR and Western Blot to detect the expression of CXCL12, p-AKT and NF-κB in colorectal carcinoma cells SW480, specifically its anti-tumor mechanism.[Result]1. Berberine in vitro inhibition of colorectal carcinoma cells SW480 proliferation, migration, invasion experiments.(1) Proliferation assay.Different concentrations of berberine (3.125umol/L,6.25umol/L,12.5umol/L、 25umol/L,50umol/L,100umol/L,200umol/L,400umol/L) treated colorectal carcinoma cells SW480 for 24h value-added rate is (91.8±11.5)%, (85.6±9.7) %、(82.1±10.1)%、(79.0±9.0)%、(56.9±7.7)% (49.0±6.5)%、(29.5±8.5) %、(29.5±10.0). Berberine treated colorectal carcinoma cells SW480 for 24h value-added rate is (87.5±9.6)%、(82.9±8.7)%、(76.3±10.1)%、(56.2±8.2) %、(36.9±5.5)% (23.6±3.8)%、(20.5±1.9)%、(22.0±2.0)%. Berberine treated for 24 or 48 hours the proliferation of colon cancer cells was significantly decreased compared with the DMSO.(2) Scratch testDifferent concentrations of berberine (2.5umol/L,5umol/L,1 Oumol/L, 20umol/L、40umol/L) treated colorectal carcinoma cells SW480 for 24h migration rate is (24.4±2.6)%, (18.2±2.4)%, (14.5±1.5)%, (5.4±1.1)%, (5.3±0.8)%, DMSO used to be control that migration rate is (25.5±2.6) %.Berberine treated group compared with the control group colorectal carcinoma cells SW480 migration rate significantly decreased except 2.5umol/L berberine. Berberine treated colorectal carcinoma cells SW480 for 48h migration rate is (50.8±3.4)%, (21.3±2.9)%, (15.7±2.2)%, (7.3±1.2)%, (6.0±1.0) %. DMSO used to be control that migration rate is (55.5±4.1)%. Berberine treated group compared with the control group colorectal carcinoma cells SW480 migration rate significantly decreased.(3) Invasion testDifferent concentrations of berberine (2.5umol/L,5umol/L,10umol/L, 20umol/I、40umol/L) treated colorectal carcinoma cells SW480 for 24h that the number of cells in the lower chamber is (88.2±10.4) cells/field, (62.2±14.3) cells/field, (51.6±11.4)cells/field,(40.8±8.9)cells/field. DMSO used to be control that the number of cells in the lower chamber is (93.4± 12.7) cells/field. Berberine treated group compared with the control group colorectal carcinoma cells SW480 invasiveness significantly decreased.2. Studies on the mechanisms of berberine in vitro inhibition the colorectal carcinoma cells SW480 cells.2.1 Biological Activity(1) Migration and invasiveness after treated with berberine and CXCL12.Colorectal carcinoma cells were treated with 20umol/L berberine; 100ng/ml CXCL12; 20umol/L berberine and 100ng/ml CXCL12 for 24h migration rate is (5.4±1.1)%, (33.4±3.1)%, (26.2±3.9)%. DMSO used to be control that migration rate is (25.6±3.1)%. Colorectal carcinoma cells migration was significantly decreased after treated with berberine. Colorectal carcinoma cells migration was significantly increase after treated with CXCL12. Colorectal carcinoma cells migration was unchanged after treated with berberine and CXCL12,The number of cells in the lower chamber is (40.8±8.9) cells/field, (131.8±16.7) cells/field、(93.4±12.7) cells/field. DMSO used to be control the number of cells in the lower chamber is (97.2±12.7) cells/field. The number of cells in the lower chamber was significantly decreased after treated with berberine. The number of cells in the lower chamber was significantly increase after treated with CXCL12. The number of cells in the lower chamber was unchanged after treated with berberine and CXCL12.(2) Migration and invasiveness after treated with berberine and CXCL12 antibody.Colorectal carcinoma cells were treated with 20umol/L berberine; CXCL12 antibody; 20umol/L berberine and CXCL12 antibody for 24h migration rate is (25.6±3.1)%, (5.4±1.9)%, (4.9.±1.7)%, (4.7±1.5)%,The number of cells in the lower chamber is(98.3±13.1)cells/field、(41.8±9.2)cells/field、(40.7±8.3) cells/field、(38.7±7.9) cells/field.(3) To explore the molecular mechanismRT-PCR and Western Blot to detect the expression of CXCL12, p-AKT and NF-κB in colorectal carcinoma cells SW480 that is decreased.Berberine inhibit colorectal cancer by inhibiting the signaling pathway CXCL12.[Conclusion]1. Berberine can inhibit colorectal carcinoma cells SW480 proliferation, invasion, metastasis, and was dependent metering.2. CXCL12 promotes colorectal carcinoma cells SW480 invasion and metastasis3. Berberine targeted inhibition of CXCL12 expression, deter migration and invasion of cancer cells4. NF-κB and p-AKT signal may be involved in berberine targeting CXCL12, inhibit migration and invasion of cancer.