STAT3 Molecular Signaling Pathway In Suppression Of Bisdemethoxycurcumin On Human Ovarian Cancer SKOV-3 Cells

Objective:1. To investigate the effects of Bisdemethoxycurcumin on proliferation of human ovarian cancer cell line SKOV3 in vitro.2. The study of STAT3 family protein expression and matrix metalloproteinase and tumor invasion and metastasis associated protein expression changes in SKOV3 ovarian cancer cell after Bisdemethoxycurcumin.3. The effect of BDMC on ovarian cell adhesion, invasion and metastasis and its mechanism.4. Seek and find the factors associated with tumor metastasis to understand the occurrence and development of ovarian cancer invasion and metastasis of molecular biological changes that occur in order to find a new direction for inhibiting tumor metastasis. Methods:1. The human ovarian cancer cell line SKOV3 was cultured in vitro.2. To detect the difference of the absorb light value between control group and treatment group, treated with different concentration (5μM, 10μM, 15μM) Bisdemethoxycurcumin after 6,12 and 24 hours, and calculate the inhibition rates of SKOV3 with MTT assay.3. To detect the changes of the cell cycle between control group and treatment group treated with different concentration (5μM, 10μM, 15μM) Bisdemethoxycurcumin after 6,12 and 24 hours, with the flow cytometry (FCM).4. To find the morphological changes of ovarian cancer cells, treated with different concentration (5μM, 10μM, 15μM) Bisdemethoxycurcumin after 24 hours, with the inverted microscope.5. To assure the effect of the Bisdemethoxycurcumin between control group and treatment group through the cell scratches experiment.6. Transwell chamber used to observe the BDMC treatment on cell invasion ability.7. By adhesion assay method to detect Bisdemethoxycurcumin inhibited the adhesion of ovarian cancer.8. To detect the expression of STAT3/p-STAT3, VCAM-1/ICAM-1, MMP-2/9, TIMP-1, uPA, CD147 in SKOV3 cell between control group and treatment group with western bolt.9. To detect the the expression of CD147 and uPA and the nuclei changes between control group and treatment group, treated with different concentration (5μM, 10μM, 15μM) Bisdemethoxycurcumin after 24 hours, with immunofluorescence. 10. To detect the expression of STAT3mRNA, MMP-9mRNA and DDK-1mRNA between control group and treatment group, treated with different concentration (5μM, 10μM, 15μM) Bisdemethoxycurcumin, with Real-Time PCR.Results:1. Bisdemethoxycurcumin inhibited the proliferation of SKOV3 cells in a dose-and time-dependent manner.2. The cell growth was arrested at G0/G1 stage and the DNA synthesis was impacted in a dose-and time-dependent manner.3. Along with the increase of Bisdemethoxycurcumin concentration, morphology of the ovarian cancer cell line changed gradually, the number of cancer cells significantly reduced, cytoplasm condensed, some of cell size obviously shrinked, outline is irregular and the cell growth slowly. And cell light transmittance and refractive index decreased, cell shrinkage, cell number reduce, the apoptotic cells increased.4. Most scratches area was covered by the control group cells, in including 10% serum 1640 culture solution, but the distance of the SKOV3 cells moving to the scratches area smaller and smaller with the increase of bisdemethoxycurcumin concentration and the width of the scratches area still exists.5. Transwell chamber results showed that bisdemethoxycurcumin with different concentration can inhibite the invasiveness and metastasis of the SKOV3 cells. The number of the SKOV3 cells treated with bisdemethoxycurcumin through matrigel membrane was much less than control cells in a dose-and-time-dependent manner. 6. Adhesion experiment showed that SKOV3 has the strong adhesion to Fn and Matrigel and the number of cells attach wall growth increased gradually. Bisdemethoxycurcumin can inhibite the adhesive ability of SKOV3 in dose dependent manner under the same time and Fn and Matrigel can enhance the adhesive ability of SKOV3.7. The result of western blot was that the expression of STAT3/p-STAT3, VCAM-1/ICAM-1, MMP-2/9/TIMP-1, uPA, CD147 decreased in SKOV3 cell in dose-dependent manner, but the expression of TIMP-1 increased in SKOV3 cell in dose-dependent manner.8. Immunofluorescence result showed that the expression of CD147 and uPA decreased gradually with the increase of the bisdemethoxycurcumin concentration, nuclear staining obviously becomes shallow, even appeared condensation nuclei fragmentation and apoptotic bodies in dose-dependent manner.9. The result of Real-Time PCR showed that the expression of STAT3mRNA, MMP-2mRNA and DDK-1mRNA decreased with the increase of bisdemethoxycurcumin concentration and had statistical significance.Conclusion:Bisdemethoxycurcumin can inhibited the proliferation, adhesion, invasion and migration, and can induce G1 arrest in SKOV3 cells, by inhibiting cell cycle and reduce the rate of cell proliferation to inhibit tumor cell proliferation; It can also reduce matrix metalloproteinase expression and its downstream factor reduces tumor cell invasion and metastasis, The STAT3 signaling pathway was found inhibited by treatment of BDMC, which was known to mediate the expression of MMPs, uPA, CD147, ICAM-1, and VCAM-1. All these findings strongly suggested that BDMC mediated anti-invasive activity involved modulation of the expression of invasion-associated proteins of STAT3 signaling pathway, which played a very important role in the invasion and metastasis of ovarian cancer. STAT3 signaling pathway to provide a treatment of ovarian cancer new and broad prospects, which is the inbibit ovarian cancer molecular mechanisms of invasion and metastasis.