| Ovarian cancer is one of the seven most common cancers worldwide and the third most common malignant disease in female reproductive system following uterine cancer and cervical cancer.It is also a leading cause of cancer-related mortality.Due to the atypical symptoms at early stage,most women have advanced ovarian cancer at the time of diagnosis,when it has spread to other organs in addition to ovaries.In addition,there is a likelihood of recurrence and drug resistance after surgery.Advances in surgical skills,chemotherapeutic drugs and treatment contribute to significantly improved5-year survival rate as high as 90% in patients with stage I ovarian cancer,but only 25-30% in those with advanced ovarian cancer.Currently,surgery remains the mainstay for the treatment of ovarian cancer,supplemented with radiotherapy and chemotherapy(CT).As for CT,a combination of paclitaxel with platinum is the first-line treatment program,which,however,brings about CT resistance and recurrence in many cases.The ocurrence and development of ovarian cancer,an extremely complex process which may be related to environmental,psychosocial and genetic factors,is associated with multi-step process,various factors,key molecules and signaling pathways,thereby impacting ovarian cancer cell cycle,proliferation,migration and invasion.The proto-oncogene c-Met(cellular m)is a hepatocyte growth factor(HGF)-specific receptor which is located in the cell membrane.Overexpresson or abnormal activation of c-Met has been reported in tissues of various tumors,such as thyroid cancer,nasopharyngeal carcinoma,breast cancer,lung cancer,gastric cancer,liver cancer,pancreatic cancer,kidney cancer,osteosarcoma and colorectal cancer.C-Met gene mutation or c-Met protein overexpression can activate PI3K/Akt signaling pathway,MAPKsignal pathway,STAT signaling pathway and Notch signaling pathway,thus affecting cell proliferation,movement,apoptosis and cell cycle.Studies have shown that after activation of HGF/c-Met/Akt signaling pathway,endometrial cancer stromal cells can promote epithelial cell proliferation,and that inhibition of HGF/c-Met/Akt signaling pathway leads to decreased proliferation capacity of osteosarcoma cells and uveal melanoma cells.A number of studies indicate that c-Met protein overexpression leads to stronger migration,invasion and anti-apoptotic ability of tumor cells.Studies have reported that inhibition of c-Met protein overexpression or inhibition of c-Met autophosphorylation and downstream pathway can effectively inhibit tumor cell proliferation and migration,and enhance the ability of apoptosis.In terms of c-Met protein as an important anti-tumor target,specific small molecule inhibitors have been developed as anticancer drugs.The Ib-type c-Met inhibitor with a highly selective ability is precisely bound to c-Met at the binding site after conformational changes due to c-Met non-phosphorylation.As a representative inhibitor,Capmatinib(INCB28060,benzazamidine)Is currently used in stage I/II clinical research in a variety of cancers,including advanced liver cancer,colon cancer,non-small cell lung cancer,rectal cancer,malignant glioma and melanoma.To the best of our knowledge,the effect of c-Met inhibitor INCB28060 combined with or wihout paclitaxel on proliferation and apoptosis of ovarian cancer has not been reported.Whether the inhibitor alone or combined with paclitaxel could effectively inhibit the expression of PI3K/Akt/mTOR as well as their effects on biological behavior of ovarian cancer remain incompletely understood.Therefore,in the present study,we investigated ovarian cancer tissues and normal ovarian tissues,ovarian cancer cell line cultured in vitro and nude mice tumor model.Immunohistochemistry,real-time quantitative RT-PCR and Western-blot were used to detect the expression of c-met in ovarian cancer tissues and normal ovarian tissues.After inhibiting c-met protein expression by INCB28060,the effect of c-met protein expression and c-met autophosphorylation on PI3K/Akt/mTOR signaling pathway and ovariancancer cell proliferation and apoptosis was detected by real-time quantitative RT-PCR,Western-blot,MTT and flow cytometry.Nude mice tumor model was established to understand tumor volume changes in nude mice tumor model after the inhibition of c-met protein expression,thus clarifying the role of c-met in ovarian cancer cell proliferation and apoptosis,and providing a theoretical basis for the potential targeted drugs for ovarian cancer treatment.Part One Clinicopathological significance of c-Met expression in ovariancancerObjective: To investigate the expression of c-Met in ovarian cancer and its relationship with clinicopathological features.Methods: A total of 104 patients with pathologically confirmed ovarian cancer presenting to the Fourth Hospital of Hebei Medical University were enrolled from January 2012 to December 2012.In addition,26 patients with pathologically confirmed normal ovarian tissues after a total abdominal hysterectomy with a bilateral adnexectomy served as controls.The differences in expression of c-Met in ovarian cancer tissues and normal ovarian tissues were compared by immunohistochemistry,Real-Time PCR and Western-blot.The median age of patients enrolled was 55,ranging from 25 to 74 years of age.According to the criteria of FIGO staging,there were 32 cases with stage I-II disease and 72 cases with stage III-IV.Based on histological grade,the number of patients with G1 and G2-G3 phase disease was 19 and 85 respectively.Additionally,the number of patients with and without lymph node metastasis was 55 and 49 respectively.The specimens were partially fixed with 10% formaldehyde,and partially put into liquid nitrogen for quick freeze and then transferred to-80? cryogenic refrigerator for storage.Results:1 c-Met was expressed in the cytoplasm and partial cell membrane of epithelial ovarian cancer cells.The positive expression rate of c-Met in ovarian caner tissues and normal ovarian cells of 104 cases was 74.04% and11.54% respectively,and the difference were statistically significant(P<0.05).2 The positive expression rate of c-Met in G1 and G2-G3 phase ovariancancer was 52.63% and 85.88% respectively,showing a significant difference(P<0.05).The positive expression rates were 53.12% and 88.87% in stage I/II and III/IV ovarian cancer tissues respectively(P<0.01).The positive rate of c-Met in groups with or without lymph node metastasis was 92.72% and67.35% respectively.There were significant differences between the two groups(P<0.05).3 Real-Time PCR indicated that the relative expression of c-Met was higher in ovarian cancer tissues(3.0786±0.0900)than in normal ovarian tissues(1.0009±0.0010).Significant differences were found between the two groups(P<0.05).;4 Western-blot results showed relative expression of c-Met protein in ovarian cancer tissues(0.979±0.009)was remarkably higher,as compared with adjacent tissues(0.263±0.003;P<0.05).Conclusion:The expression of c-Met was significantly higher in ovarian cancer tissues than in normal ovarian tissues,suggesting that c-Met may be involved in the development of ovarian cancer.The expression of c-Met protein in ovarian cancer tissues was correlated with clinical stage and histological grade,indicating the involvement of c-Met-Met in the development and progression of ovarian cancer.Part Two Effects of INCB28060 on apoptosis of ovarian cancer cellsin vitroObjective: To investigate the effect of INCB28060 on c-Met,PI3K/Akt,proliferation and apoptosis in ovarian cancer SKOV3 and OVCAR-3 cells and its mechanism.Methods: The expression of c-Met in SKOV3 and OVCAR-3 cells was determined by Real-Time PCR,Wertern-blot,MTT,TUNEL and flow cytometry.The cell lines were added with different concentrations of INCB28060,the effect of which on cell viability was detected by MTT assay.The mitochondrial depolarization and cell cycle were observed by JC-1 probe.The same concentration of INCB28060 was added to the cell lines fordifferent incubation time,and their effect on the cell viability was determined by MTT assay.Paclitaxel at different concentrations were added to the cell lines and the effect of INCB28060+paclitaxel on cell viability was measured using MTT assay.Paclitaxel of the same concentration was added to the cell lines for different incubation time and MTT assay was used to determine their effect on cell viability.The cell line was added with INCB28060,and Wertern-blot method was applied to detect its effect on c-Met and downstream pathway PI3K/Akt.In addition,the expression of pc-Met,p-PI3 K,p-AKT1,mTOR,Mdm2 and p53 were measured.After combination of INCB28060 and paclitaxel,Western-blot method was utilized to determine the expression of?H2AX,a DNA damage repair marker.The cells were incubated with INCB28060 combined with paclitaxel,and the apoptosis was detected by TUNEL.Results:1 INCB28060 was co-cultured with ovarian cancer SKOV3 and OVCAR3 cells.Compared with the control group,cell viability of SKOV3 and OVCAR3 cells treated with INCB28060 at different concentrations(0umol/L,2umol/L,4umol/L,6umol/L and 8umol/L)was decreased with the increase of INCB28060 concentration,suggesting that the effect of INCB28060 on cell viability in a dose-dependent manner,and the optimal concentration of 6umol/L.The cell viability of SKOV3 cells(50.12±2.369%)was significantly different from that in control group(97.23±3.71%)(P<0.05).Additionally,the cell viability of OVCAR-3 cells(65.72±1.996%)was notably higher,as compared with control group(97.62±2.98%)(P <0.05).2 INCB28060 was co-cultured with SKOV3 and OVCAR3 cells.MTT assay was employed to detect cell viability after treatment with INCB28060 for different incubation time(0h,12 h,24h,36 h and 48h).The cell viability of SKOV3 and OVCAR3 cells was decreased with the increase of incubation time,reaching an optimal viability at 24 h.The cell viability of SKOV3 cells(51.92 ± 2.00%)was significantly different from that in control group(98.19±3.69%)(P<0.05).In addition,cell viability of OVCAR3 cells(63.27±3.06%)was remarkably higher than that of control group(96.9±3.84%)(P <0.05).3 INCB28060(6umol/L)alone or in combination with paclitaxel were co-cultured with SKOV3,OVCAR-3 cells.The effects of different concentrations of paclitaxel(0nM,10 nM,20nM,30 nM,and 40nM)on cell viability were investigated by MTT assay.The results suggested that the optimal concentration of paclitaxel was 20 nM,and that cell viability of SKOV3 and OVCAR-3 was reduced with the increase of the dose in a dose-dependent manner.The cell viability of SKOV3 cells was 51.79±2.67%,which was significantly different from 98.16±4.002% in control group(P<0.05).The OVCAR-3 cell viability was 59.72±1.991%,as compared with97.83±3.269% in control group(P<0.05).After treatment with 6umol/LINCB28060 alone and in combination with 20 nm paclitaxel in SKOV3 and OVCAR-3 cells(P<0.05),MTT assay was used to determine the effect of different incubation time(0h,12 h,24h,36 h and 48h)on cell viability.The results demonstrated that the cell viability of SKOV3 and OVCAR3 cells was decreased with the increase of time in a time-dependent manner,reaching the optimal effect at 24 h.Cell viability of SKOV3 cell was 49.91±2.167%,which was significantly different from 96.23±3.99% in control group(P<0.05).By contrast,OVCAR-3 cell viability was 53.17±2.02%,compared with the control group of 96.19±4.10%(P<0.05).4 INCB28060 was co-cultured with SKOV3 and OVCAR3 cells.The expression of c-Met,p-c-Met,p-PI3 k,p-AKT were measured.The results showed that the expression of p-c-Met,p-PI3 k,p-AKT in ovarian cancer SKOV3 and OVCAR3 cells added with inhibitor INCB28060 decreased with the increase of INCB28060 concentration.5 Flow cytometry was used to determine INCB28060 at different concentrations co-cultured with SKOV3 and OVCAR-3 cells,the raito of membrane potential after the mitochondrial depolarization as well as the ratio of depolarization to polarization.The results indicated that the mitochondrial depolarization ratio of the two cell lines increased with the increase of theconcentration,reaching the optimal effect at the concentration of 6umol/L.The depolarization ratio of mitochondrial membrane potential in SKOV3 cells was 65.77±3.26%,compared with 8.41±0.26% in control group(P<0.01).The depolarization ratio of mitochondrial membrane potential in OVCAR-3 cells was 56.81±2.51%,compared with 7.34±0.30% in control group,and the difference was statistically significant(P<0.01).The ratio of depolarization to polarization of SKOV3 cells was 0.83±0.041%,as compared with0.17±0.006% in control group(P<0.01).The ratio of mitochondrial depolarization to polarization in OVCAR-3 cells was 0.77±0.039%,which was significantly different from 0.18±0.006% in control group(P<0.01).6 Effects of INCB28060 on DNA Damage in SKOV3 and OVCAR3CellsThe effects of 6umol/L INCB28060 alone and in combination with 20 nM paclitaxel on ?H2AX in SKOV3 and OVCAR3 cells were observed by Western blot.The findings demonstrated that INCB28060 alone and in combination with paclitaxel enhanced the DNA damage in co-cultured SKOV3 and OVCAR3 cells compared with the control group.Statistical differences were found in two groups(P<0.05).7 The effect of INCB28060 combined with paclitaxel in SKOV3 and OVCAR-3 cells was increased with the increase of time in a time-dependent manner.The G2/M phase cell ratio in SKOV3 cells in INCB28060+paclitaxel group was 23.44±1.026%,which was remarkably lower than that in INCB28060 alone group(29.99±1.391%),and the difference was significant(P<0.05).The G2/M phase cell ratio in OVCAR-3 cells in INCB28060+paclitaxel group was 21.17±1.167%,which was significantly lower than31.73±1.4% in the control group(P<0.05).Sub-G1 phase cell ratio of SKOV3 cells in INCB28060+paclitaxel group was 47.26±1.98%,which was significantly different from that in INCB28060 alone group(21.78 ±1.01%)and in paclitaxel alone group(18.71±0.92%)(P<0.05).Sub-G1 phase cell ratio of OVCAR-3 cells in INCB28060+paclitaxel group was 43.94±2.0016%,which significantly differed from that in INCB28060 alone group(18.73±0.95%)and in paclitaxel alone group(21.16±1.06%)(P <0.05).Conclusion:1 INCB28060 inhibited cell viability of ovarian cancer SKOV3 and OVCAR-3 cells in a dose-dependent and time-dependent manner.2 The expression of c-Met protein in ovarian cancer SKOV3 and OVCAR-3 cells was inhibited by INCB28060.3 INCB28060 inhibited c-Met downstream pathway PI3K/AKt in ovarian cancer SKOV3 and OVCAR-3 cells.4 INCB28060 inhibited tumor cell proliferation and promoted tumor cell apoptosis in ovarian cancer SKOV3 and OVCAR-3 cells.5 INCB28060 increased the sensitivity of tumor cells to chemotherapy drug paclitaxel in ovarian cancer SKOV3 and OVCAR-3 cells.Part Three Effect of INCB28060 combined with paclitaxel on tumormodel of ovarian cancer in nude miceObjective: To construct a tumor model of ovarian cancer in nude mice and to observe the effect of INCB28060 combined with paclitaxel on tumor model.Methods:1 Ovarian cancer SKOV3 and OVCAR-3 cell lines were cultured.2 Nude mice tumor model was constructed.3 INCB28060 combined with paclitaxel was used to inhibit tumor growth in nude mice.4 The expression of c-Met and downstream pathway PI3K/AKt in tumor cells was detected by Western blot.Results:1 Ovarian cancer model in nude mice was successfully constructed.2 INCB28060 combined with paclitaxel was used in the treatment of ovarian cancer in nude mice model.The results revealed the minimal tumor volume in INCB28060+paclitaxel group(540.19±26.11mm3),which was significantly different from that in the control group(930.22±29.10mm3)(P<0.01).In addition,the tumor quality was the lowest in INCB28060+paclitaxel group(900.094±56.099mg),compared with the control group(1630.194±68.031mg)(P<0.01).There was no significant difference in body weight between INCB28060+paclitaxel group(2150.987±0.096mg)and the control group(2070.238±0.079mg)(P>0.05).3 The expression of c-Met,p-c-Met,PI3 K,p-PI3 K,AKt1,p-AKt1,mTOR,p-mTOR,mdm2 and p53 was detected by Western blot.Conclusion:1 Ovarian cancer model in nude mice was successfully constructed.2 Combination of INCB28060 and paclitaxel effectively reduced the tumor volume and tumor quality in nude mice tumor model.3 INCB28060 inhibited c-Met and downstream pathway PI3K/AKt as well as cell proliferation and promoted apoptosis of tumor cells.Conclusions1 c-Met gene of epithelial ovarian cancer tissue was up-regulated and highly expressed..2 INCB28060 down-regulated the expression of PI3K/AKT,p-c-Met,p-PI3 k,p-AKT through inhibition of c-Met expression.Moreover,inhibiting the expression of c-Met as well as expression of mTOR,Mdm2,P53 and?H2AX in downstream pathway can interfere with the proliferation,viability,apoptosis and cell cycle of ovarian cancer cell lines,thereby improving their sensitivity to chemotherapy drug paclitaxel.3 INCB28060 can reduce tumor volume in nude mice,promote cell apoptosis,and increase the sensitivity of tumor cells to chemotherapy drug paclitaxel.4 INCB28060 may be a potential targeted drug for future ovarian cancer treatment,which provides new insight into adjuvant treatment in ovarian cancer. |
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