The Study Of Inhibitory Effect On Proliferation And Mechanism Of Curcumin Associated With Cytokine-induced Killer Cells Against Ovarian Carcinoma Cells

Ovarian cancer occurs with all lifetime incidence in approximately 1 in 58 women and it is the fifth leading cause of cancer death in women and is the leading cause of death among gynecologic cancers. Ovarian cancer has no obvious syndrome and most patients are diagonosed at late stages due to ovary lies in inner part of the body. Surgery and chemotherapy of Cisplatin(CDDP) combined with paclitaxol are mainly be used. The effect of survival rate upon patients at late stages be improved with cytoreductive surgery are limited, and chemotherapy has much side-effect, some patients at late stages can not tolerant chemotherapy due to poor physical. So it is urgently needed to find a more efficient adjunctive therpy to delay recurrenc and enhance survival rate of ovarian cancer patients.Cytokine-induced killer(CIK) cells are immune effector cells which are induced by various cytokines such as monoclonal antibody(mAb), interleukin-2 (IL-2), IL-1, and interferon-gamma (IFN-y) in vitro. A large number of experiment research indicated that CIK cells can effectively restrain and kill tumor cells, and can prevent tumor from spreading and recurance when combined with surgery, radiotherapy and chemotherapy. But the study on antitumor of CIK cells combined with traditional Chinese medicine is rarely reported at home and abroad.Curcumin (Cur) is one of effective ingredient which is abstracted from the curcuma rhizome of curcuma genus in ginger family, which has broad-spectrum pharmaceutic value, and anti-tumor is one of the major effect.Adoptive immunity therapy act as one kind of biotherapy mode on tumor, by means of transfusing immunocompetent cell to enhance tumor patients immunologic function, provide a new treatment way for late stage ovarian carcinoma patients,especially recurrent ovarian carcinoma patients and those who has poor physical fitness failure to tolerance chemotherapy.This study was designed to observe inhibitory effects on the proliferation of Curcumin associated with CIK cells on ovarian carcinoma SKOV3 cells in vitro and to investigate wheather they have synergetic anti-tumor effects if they are used together thereby strengthen CIK cells superiority on adoptive immunity therapy and to discuss the possible mechanism, so that provide experimental study for traditional Chinese medicine and immunotherapy on ovarian cancer, provide a new remedy thingking for comprehensive treatment on ovarian cancer, to lay the foundation for more clinical pratice.Methods:CIK cells were induced from umbilicus cord blood firstly, and the SKOV3 cells were randomly divided into Cur group, CIK group, Cur associated with CIK group. The inbition ratio on the proliferation of each group was checked with MTT methods, and the effect of joint use was calculated. The apoptosis morphology of each group cells was observed under electron microscope. Flowcytometric analysis was used to dectect cell apoptosis. The mRNA level of Fas, FADD, Caspase3 of each group of drug administration was measured by RT-PCR. The Fas level on ovarian cells surface and the FasL level on CIK cells surface after Curcumin function on them were detected with Weston blot. The level of inbition ratio on proliferation of CIK cells and CIK associated with Cur after FasL monoclonal antibody being administrated was checked with MTT methods.Experimental result:(1) The lower concentration of CIK and Cur were choosed in the study, the inhibition ratio of each group was lower when they functioned on SKOV3 cells alone, but the inhibitory effect on proliferation was promoted obviously when they were used together, and inhibition ratio was also gradually enhanced with the rise of medicine concentration and the ratio of effector contrast to target and time, that is they present time and dose depence relation. The inhibition ratio of SKOV3 cells was respectively 48.7% and 41.9% when they act alone for 48 hours by the concentration of CIK of effector to target ratio of 12.5:1 and Cur at 20μmol·L-1, but it approached 76.2% when they were used together, was 1.6 times and 1.8 times compared to that when been applied alone.And the value of Q of joint use effect reveal that the value of Q all greater than or equal to 1.2 after Cur and CIK were used together on SKOV3 cells, more to move forward to prove they have synergetic anti-tumor effect when they were used together.(2) Transmission electron microscope show that the ovarian cancer cell size was unequal in the control group,a little of microvilli was visible on the cell surface,cell division photo and multinuclear giant cells were visible, but each group been drug administrated for 24h without exception appeared a change on apoptosis morphology, the change of apoptosis morphology in the group of Cur associated with CIK was most obvious, the cell size was unequal in the group of Cur, electron densification matter was mostly seen, part of cell mitochondria and asperities endoplasmic reticulum became vacuole shape, vacuole structure was visible in the cytoplasm, exceptional chromatin rise in the nucleus, in bulk shape; the cell size was unequal in the group of CIK, cell membrane microvilli reduced,a small amount of cell membrane damaged, much vacuole structure and electron densification were visible in the cytoplasm, mitochondria became vacuole shape; the cell size was unequal in the group of Cur associated with CIK, cell count obviously decreased, cell membrane damaged, polymorphism nucleus giant cell was scarce, a mass of vacuole structure or electron densification were visible in the cytoplasm,mitochondria ridge ruptured or became vacuole shape, nucleus pyknosis.It make clear that Cur combined with CIK can induce SKOV3 cells apoptosis obviously.(3) The mRNA level of Fas, FADD, Caspase3 of SKOV3 cells after CIK, Cur, CIK associated with Cur act on SKOV3 cells was measured by RT-PCR. The result show that the mRNA level on Fas of DMSO and Cur group had no obvious change, the mRNA level on Fas was promoted in the group of CIK and CIK associated with Cur compared with control group (P<0.05), the positive rate was respectively 120% and 186% upon control group. the mRNA level on fas was increased sharply in the group of Cur associated with CIK, was 1.55(186/120) times upon CIK cells group. The mRNA level of FADD in each group of drug administration had no obvious change compared to control group. Compared to control group, the mRNA level of Caspase3 of DMSO group had no obvious change, but the group of Cur, CIK and Cur associated with CIK could promote the mRNA level of Caspase3 (P<0.05), the positive rate was respectively 115%,205%,302% upon the control group, and the mRNA level of the group in Cur associated with CIK obviously increased, was respectively 2.62 times(302/115) upon the group of Cur and 1.47 times (302/205) upon the group of CIK.(4) The Fas level on ovarian cells surface after Curcumin function on them was detected with Weston blot.The result show that the level of Fas protein in the group of DMSO had no obvious change compared with the control group, but Cur of 5,10 and 20μmol·L-1 could promote the level of Fas of SKOV3 cells (P<0.05). The positive rate was respectively 168%,253%, and 249% upon the control group.(5) the FasL level on CIK cells surface after Curcumin function on them was detected with Weston blot. The result show that the level of FasL protein in the group of DMSO had no obvious change compared with the control group, but Cur of 5,10 and 20μmol·L-1 could promote the level of FasL protein of cell membrance of CIK cells (P<0.05), the positive rate was respectively 139%, 142% and 138%.(6) The level of inhibition ratio on proliferation of CIK cells and CIK associated with Cur after addition of anti-FasmAb was checked with MTT methods. The pathway of Fas/FasL was blocked after addition of anti-FasmAb, The group of CIK and Cur associated with CIK could be restrained obviously the inhibitory effect on proliferation on SKOV3 cells after addition of anti-FasmAb. The inhibition ratio respectively drop from 31.4% to 21.1%, 69.8% to 51.6%.Conclusion:(1) CIK cells extracted from umbilicus cord blood proliferate fast in vitro, extraction ratio of mononuclear cells is high, the killing activity on ovarian cancer SKOV3 cells is powerful. It can be expediently used to clinic treatment.(2) Cur associated CIK has synergic effect of anti-tumor on SKOV3 cells when they are used together in vitro. It bring about hope for tumour sufferer, especially later period tumour patients who is recurrent and failure to tolerate chemotherapy as an adjuvant therapy means.(3) The effects of inducing apoptosis of SKOV3 cells are increased when Cur and CIK are used together, it make clear that they have preferable therapeutic effect when be used together, and it is prominsing to become favourable prospect comprehensive trerapies.(4) The mechanism of inducing apoptosis of Cur and CIK maybe that Cur promote the expression of Fas on SKOV3 cells surface and FasL on CIK cells surface, so that raise the expression of Fas in SKOV3 cells, finally the expression of Caspase3 rise. It offers theory proof for they further clinical practice.The study strengthen the superiority of CIK cells as an adoptive immunity therapeutic by make use of Cur and CIK cells together on ovarian carcinoma SKOV3 cells, which enhance the inhibitory effect on SKOV3 cells. It can provide a new remedy thinking for comprehensive treatment on ovarian cancer: the comprehensive treatment of various means being used jointly should be adopted on specific project design of ovarian cancer therapy.