Mechanism Of Proliferation Inhibition Of Stem Cell-like Cells In Ovarian Cancer Based On HMSN Compound Drug Delivery System

[Objective]Ovarian cancer represents the leading cause of deaths by women’s malignancy,for primary ovarian cancer patients who have received cytoreductive surgery and platinum-based combination chemotherapy,a long period of complete remission is expected.However,almost 70%patients will have the trouble of recurrence.The proliferation of cancer stem cells directly contribute to tumor recurrence,which associated with the Insulin-like Growth Factor(IGF)pathway.Activation of IGF-1 pathway was involved in malignant transformation and exerts an anti-apoptotic effect.The combination of nanocarriers and chemotherapy drugs becomes a hot spot,by using an ideal nanocarriers,the chemotherapy drugs can release almost only in the tumor tissue,which can enhance the anti-tumor effect and reduce the adverse reactions at the same time.In this study the Hollow mesoporous silica nanoparticles(HMSN)was chosen as the nanocarriers due to its larger aperture,good histocompatibility,stable chemical property and suitable diameter(50 nm)In this experiment,a co-delivery system based on Hollow mesoporous silica nanoparticles(HMSN)was developed and characterized,IGF-1R was chosen as the drug target,apoptosis rate and expression of cyclinB1,Bax,Bcl-xl,P-akt were used to evaluate the inhibiting proliferation and anti-apoptosis in cancer stem like cells of HMSN-COOH@DOX fluorescence NVP.[Methods]The expression of IGF-1,IGF-2 and IGF-1R in the CD117+CD44+A2780 were detectedby immunohistochemical method.Different concentrations of IGF-1(10ng/ml,25ng/ml,40ng/ml,55ng/ml)were incubated with cells to select the suitable concentration.According to the different processing modes,the experiment was divided into three groups:the IGF-1 stimulating group,NVP inhibiting group and the blank control group.The expression of IGF-1R,distribution of cell cycle and apoptosis rate in cells of these three groups were detected by flow cytometry,and the expression of CyclinB1 after cells incubated with drugs for 24 hours,was detected by Western Blot.The physical properties of HMSN-COOH@DOX fluorescence NVP were determined by infrared spectrometer,high-resolution transmission electron microscope(TEM),scan electron microscope(SEM)and laser particle size analyzer.Calculated the entrapment efficiency and loading efficiency of DOX and fluorescence NVP,the release rate of them in PBS at PH 7.4,6.5,5.5 and 4.5 respectively.Three groups were set up,HMSN-COOH@DOX fluorescence NVP group,HMSN-COOH@DOX group and the free control group.The intracellular accumulation process of this drug delivery system was observed by confocal microscope.Detected the cell apoptosis by flow cytometry and inhibition rate of these three groups.The expression of apoptosis related proteins Bax,Bcl-xl,P-akt were detected by Western Blot.Statistical methods:Data were evaluated using spss19.0 statistical software,data were expressed as mean ± standard deviation.The significance difference was determined using analysis of t test.P≤0.05 was considered significant.[Results]Brown granules were shown in the cell membrane and cytoplasm in CD117+CD44+A2780 cells can prove the protein of IGF-1,IGF-2 and IGF-1R were expressed in this cell line.40ng/ml was chosen as the suitable concentration of IGF-1 by cells count.The expression of IGF-1R was observed by flow cytometry,and a significant high level of IGF-1R was found in the IGF-1 stimulating group than that in NVP inhibiting group and the blank control group(P≤0.05).Compared with the blank group,the cells in the G2 phase were significantly decreased in the IGF-1 stimulating group(P≤0.05),and the cells in S phase were significantly increased(P≤0.05).More cells were blocked in the G2/M phase in NVP inhibiting group than the other two group(P≤0.05).The apoptosis rate of IGF-1 stimulating group and blank control group were respectively(6.53±0.36)%,(8.38 ±0.25)(P≤0.05).While the cells apoptosis rate of NVP inhibiting group(32.79 ± 0.34)%was significant difference with the blank control group and IGF-1 stimulating group.The expression of CyclinBl in IGF-1 stimulating group was significant higher than that of the blank control group and NVP inhibiting group,which was lowest in the three groups.Infrared spectrometer proved the carboxylic acid was modified on the HMSN.The charge of HMSN-COOH was reversal with the gradually reduced PH of PBS.The form and size were not changed after loaded drugs.The entrapment efficiency and loading efficiency of were 37%and 6.17%for DOX,and 44%and 2.10%for the fluorescence NVP.The release rates of this two drugs were increased according to the decreased PH value of PBS.According to the confocal microscope,the co-delivery system gradually gathered in the intracellular with extension of time.The apoptosis rate was significantly increased in the HMSN-COOH@DOX fluorescence NVP group than that of HMSN-COOH@DOX group(P≤0.05).Compared with the free control group,DOX and NVP that loaded in the HMSN have more stronger effect than free drugs(P≤0.05).The inhibition rate of HMSN-COOH@DOX fluorescence NVP group was higher than that of the other two groups(P≤0.05),the rate in free control group was significantly lower than that of the HMSN-COOH@DOX fluorescence NVP group(P≤0.05).The result of Western Blot showed that the expression of Bax was significantly increased in the HMSN-COOH@DOX fluorescence NVP group than that of the free control group(P≤0.05),while the expression of Bcl-xl and P-akt were significantly decreased(P≤0.05).[Conclusion]1.IGF-1,IGF-2 and IGF-1R are expressed on CD117+CD44+A2780 cells,IGF-1 can effectively stimulate the proliferation in CD117+CD44+A2780 cell and enhance the anti-apoptosis effect.While the NVP can block cells in G2/M phase,promote apoptosis of tumor cells.2.HMSN-COOH can become protonation when the PH value gradually reduced,the negative charge changed into positive charge.DOX and fluorescence NVP have high entrapment efficiency in HMSN,and the release rate were increased as PH value decreased.3.HMSN-COOH@DOX fluorescence NVP shows the effect of inhibiting proliferation and anti-apoptosis in tumor cells,and enhance the sensitization of chemotherapy by actively targeting IGF-1R.4.The nanoparticles drug delivery system HMSN-COOH@DOX fluorescence NVP may become a new type of chemotherapy drug which can improve the effect of chemotherapy and reduce the side effects after further in vitro experiment.