Effect Of Propofol On Growth, Invasive,Metastasis Potential Of Human Liver Cancer Cell Line HepG2 And Growth Of Mice Transplantated Tumor

In the human population, liver cancer is the fifth most frequent cancer and the third most common cause of death from cancer. The major strategies for the treatment of patients with liver cancer include surgery, radiotherapy, and chemotherapy. Surgery remains the first-line treatment for patients with solid organ tumors and also plays a role in diagnosis, staging, reconstruction and palliation. A significant proportion of cancer patients will undergo an operation during the course of their disease. Primary excision may be curative in cases of localized disease, and survival can be improved in advanced cancers when used in conjunction with adjuvant or neoadjuvant therapies. Nonetheless, metastatic recurrence remains frustratingly common, even in ostensibly localized disease, and is invariably more difficult to treat. This occurs despite efforts to excise tumors in their entirety by including clear resection margins of normal tissue.There is a growing interest in the impact of anesthesia on outcome following cancer surgery. Anesthetic agents are powerful pharmacological tools able to exert diverse, potent effects on numerous cellular and organ functions. Typically, a number of different drugs and techniques are combined during anesthesia. The potential for anesthetics to directly interfere with cancer cell biology is increasingly recognized.Propofol (2,6-diisopropylphenol), an intravenous anesthetic agent which is characterized by rapid onset and short term of action, has been widely used for clinical anesthesia and intensive care. Previous studies have confirmed that propofol can inhibit cancer cell migration and invasion. Moreover, It has been reported that propofol has a beneficial effect on antitumour immunity via greatly enhancement of cytotoxic T lymphocytes activity in vitro in mice.In addition, propofol does not reduce natural killer activity or increase MADB106 lung tumour retention or lung metastase.However, opposite results suggested that clinically relevant concentrations of propofol are shown to increase migration of MDA-MB-468 breast carcinoma cells by activation of GABAAR. Another study had showed that propofol induces proliferation and promotes invasion of GC cells through activation of NF-E2-related factor 2 (Nrf2).In spite of all of this, to date, few papers have been published specifically addressing a possible correlation between propofol and cancers, and the results are often confused and disputed. Thus, it is surely important to clarify the effects of propofol on the behavior of cancer cells.Growth of tumor cells is a multi-gene and multi-step process. An imbalance between anti-apoptotic gene expression and pro-apoptotic gene expression happens during tumor progression period.Bcl-2 family is involved in the regulation of cell apoptosis. Bcl-2 family consists of a series of anti-apoptotic and pro-apoptotic members. Bcl-2 is an anti-apoptotic member, which prevents apoptosis by inhibiting the release of mitochondrial apoptogenic factors into the cytoplasm.In contrast, Bax is a pro-apoptotic member of this family, which promotes apoptosis by activating caspases.Growth of tumor is also associated with the abnormal regulation of cell cycle. Tumor is a kind of disease that caused by disturbed cell cycle. Development and progress of most tumors are close related to anomalous regulation of cell cycle.Metastasis of tumor cells consists of a series of complex, continuous, and multi-step process. It includes mainly three aspects:(1) Tumor cells adhere with basement membrane and extracellular matrix through adhesion molecule; (2) Tumor cells excrete matrix metalloproteinases (MMPs), invade basement membrane and extracellular matrix; (3) Tumor cells migrate through basement membrane and extracellular matrix, and penetrate the blood vessel walls. Finally, new metastasis is formed at another site. MMP-2 and MMP-9 belong to a family of zinc-dependent proteinases. Their primary function is degradation of proteins in the extracellular matrix, which play an important role in the invasion process of tumor cells.However, there is no available information for the effects of propofol on migration and invasion of liver cancer cells. The aims of the current study were to evaluate effects of propofol on the behavior of HepG2 cells and role of PI3K-AKT in these effects.The alternations of multiple cell signaling pathways were frequently observed in liver cancer, in which the PTEN/PI3K-AKT signaling cascade has been reported to play a crucial role in the regulation of the malignant behaviors including proliferation, survival and invasion.Proliferating cell nuclear antgen (PCNA) was an index of cell proliferation.CD34 was the antigen of hematopoietic stem cell, and had been found expression in normal and newborn vascular endothelial cell, so it had been the most sensitive marker of vessel.In our study,we invest four program:1 To investigate the effect of propofol on proliferation,cell cycle and apoptosis, and related molecules expression in human liver cancer cell line HepG2.2 To investigate the effect of propofol on metastasis potential and related molecules expression in human liver cancer cell line HepG2.3 To investigate the role of PI3K-AKT signaling pathway in propofol inhibiting proliferation,cell cycle,apoptosis, metastasis and invasion potential of liver cancer cell line HepG2.4 To investigate the effects of propofol on growth and expression of PCNA,CD34,pAKT in hepatoma-xenografts BALB/C mice and the Role of PI3K-AKT Pathway.Chapter 1 The effect of propofol on proliferation,cell cycle and apoptosis, and related molecules expression in human liver cancer cell line HepG2Objective To investigate the effect of propofol on proliferation,cell cycle and apoptosis, and related molecules expression in human liver cancer cell line HepG2.Methods HepG2 cell were were seeded in 96-well plates (100μl/hole) with a density of 1×105/ml and randomly divided into five groups(n=6):group Ⅰ control (C), group Ⅱ intralipid (I), group Ⅲ propofol 30 μg/ml (P1), group Ⅳ propofol 60 μg/ml (P2) and group V propofol 120 μg/ml (P3). The proliferation inhibition rates, cell cycle, and apoptosis percentages of HepG2 cells were measured by methyl thiazolyl tetrazolium (MTS) assay, terminal deox -ynucleotidyl transferase dUTP nick end labeling(TUNEL) staining and flow cytometer respectively at 0h,24h,48h or 72h. The mRNA and protein expressions of Fas, Bax, and Bcl-2 in HepG2 cells were determined by Western blotting at 24 h after exposure to propofol.Data analysis was performed by SPSS 13.0 software. Data were expressed as mean±standard deviation (SD). Proliferation rates were assessed by using variance analysis of two-way factorial design. Apoptosis percentages, cell cycle(G1 were assessed by using Kruskal-Wallis Test) and the mRNA and protein expressions of Fas, Bax, and Bcl-2 were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3 test (heterogeneity of variance) was used for post hoc comparisons. P<0.05 was considered statistically significant.Results(1) The comparison of proliferation rates:There was statistical significance among groups of treatment concentration (F=8781.441, P=0.000). There was statistical significance among groups of treatment duration(F=5786.893, P=0.000). There was interaction between treatment concentration and treatment duration (F= 433.681, P=0.000). The proliferation rates in groupPl-3 of each treatment duration were decreased significantly as compared with group C (P< 0.05). The proliferation rates in group I were increased significantly as compared with group C (P=0.41). The proliferation rates in group P2 and group P3 of each treatment duration were decreased significantly as compared with group P1(P<0.05). The proliferation rates in group PI-3 were decreased significantly with treatment duration prolonged (P< 0.05);(2) The comparison of apoptosis percentages:There was no statistical significance among groups of treatment concentration (flow cytometer F= 1.617,P=0.201; TUNEL F=1.316, P=0.291).(3) The comparison of cell cycle percentages:The difference of cell percent in each group on the phase Gl, G2/M and S was no significance(F=8.123, P=0.087; F=0.720, P=0.552; F=14.107, P=0.092).(4) The comparison of Fas, Bax, and Bcl-2 expression:Compared with group C, the expression of Fas, Bax, and Bcl-2 was significantly down-regulated with propofol concentration increased (P<0.05). Compared with group C, the rate of Bax/Bcl-2 was no changed significantly with propofol concentration increased (P>0.05).Conclusion Propofol can inhibit the proliferation of HepG2 cells through up-regulating the expression of Fas and has no effect on cell cycle and apoptosis.Chapter 2 The effect of propofol on invasion, metastasis potential and related molecules expression in in human liver cancer cell line HepG2Objective To investigate the effect of propofol on metastasis potential and related molecules expression in human liver cancer cell line HepG2.Methods HepG2 cell were were seeded in 96-well plates (100μl/hole) with a density of 1 ×105/ml and randomly divided into five groups(n=6), group I control (C), group II intralipid (I), group Ⅲ propofol 30 μg/ml (PI), group IV propofol 60 μg/ml (P2) and group V propofol 120 μg/ml (P3). Cell adhesion rate was assessed by MTS assay at 24h after treatment with propofol.Cell invasion was assessed by Transwell invasion assay at 24 h after exposure to propofol. Cell migration was evaluated by wound healing assay at 24,48 h after exposure to propofol. The expression MMP-2, MMP-9 mRNA or protein in HepG2 cells were determined by RT-PCR or Western blotting at 24 h after exposure to propofol.Data analysis was performed by SPSS 13.0 software. Data were expressed as mean±standard deviation (SD). Cells migration rates was assessed by using variance analysis of two-way factorial design.The number of invasive cells,adhesion rates,expressions of mRNA and protein of MMP-2 and MMP-9 were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3 test (heterogeneity of variance) were used for post hoc comparisons. P<0.05 was considered statistically significant.Results (1) The comparison of cell adhension rate:The cell adhension rates were increased in group I and were significantly lower in group (P1～3) than in group C,and were lower in group P3 than in group P1 or P2 (F=69.00,P=0.00).(2) The comparison of cell invasion:There was statistical significance among groups of treatment concentration(F= 40044.548,P=0.000).There was statistical significance among groups of treatment duration(F=51053.455, P=0.000). There was interaction between treatment concentration and treatment duration (F=5181.341,P= 0.000). There was statistical significance among groups of treatment concentration (F =1210.6, P=0.00). Compared with group C,the simple effect analysis of treatment concentration revealed that the invasive cells number in group P1-3 of each treatment duration were decreased significantly (P<0.05). the invasive cells number in group I were increased significantly (P=0.042).(3) The comparison of cell migration:Compared with group C, the migration rates in group P1-3 were decreased significantly (P<0.05); the migration rates in group I were increased significantly (P<0.05).(4) The comparison of invasion-related molecules expression:Compared with group C, the mRNA and protein expressions of MMP-2 and MMP-9 in group P1-3 were significantly down-regulated with propofol concentration increased (P< 0.05); the mRNA and protein expressions of MMP-2 and MMP-9 in group I were not changed significantly (P=0.753 and P=0.743).Conclusion Propofol can inhibit invasion and migration of human liver cancer cell line HepG2 through down-regulating the mRNA and protein expression of MMP-2 and MMP-9.Chapter 3 The role of PI3K-AKT signaling pathway in propofol inhibiting proliferation,cell cycle, apoptosis, metastasis and invasion potential of liver cancer cell line HepG2Objective To investigate the role of PI3K-AKT signaling pathway in propofol inhibiting proliferation,cell cycle, apoptosis, metastasis and invasion potential of liver cancer cell line HepG2.Methods HepG2 cell were were seeded in 96-well plates (100μl/hole) with a density of 1×105/ml and randomly divided into five groups(n=6):group Ⅰ control (C), group Ⅱ propofol 120 μg/ml (P), group Ⅲ 10 nmol/L IGF-1 (IGF), group Ⅳ 10 nmol/L IGF-1+propofol 120 μg/ml (P+IGF), group Ⅴ 10 μmol LY294002 (LY) and group Ⅵ10 μmol/L LY294002+propofol 120 μg/ml (P+LY). The proliferation inhibition rates, cell cycle, and apoptosis percentages of HepG2 cells were measured by methyl thiazolyl tetrazolium (MTS) assay, terminal deox-ynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometer. Cell adhesion rate was assessed by MTS assay at 24h after treatment.Cell invasion was assessed by Transwell invasion assay. Cell migration was evaluated by wound healing assay. The expression MMP-2, MMP-9, PEG2,AKT, PI3K, pAKTmRNA or protein in HepG2 cells were determined by RT-PCR or Western blotting.Data analysis was performed by SPSS 13.0 software. Data were expressed as mean ± standard deviation (SD). The proliferation inhibition rates, cell cycle, and apoptosis percentages, the number of invasive cells,cells migration rates,the expression MMP-2, MMP-9, PEG2,AKT,PI3K, pAKT mRNA or protein were assessed by using variance analysis of two-way factorial design. LSD test (equal variance) or Dunnett’s T3 test (heterogeneity of variance) were used for post hoc comparisons. P<0.05 was considered statistically significant.Results (1) The comparison of proliferation rates:One way analysis of variance revealed that the cell proliferation rates in group P and LY were decreased and increased in group IGF significantly as compared with group C (P<0.05).The cell proliferation rates in group P+IGF were decreased significantly as compared with group IGF (P=0.000). The cell proliferation rates in group P+LY were decreased significantly as compared with group LY (P=0.000). The cell proliferation rates in group P+LY were lowest in 6 groups (P< 0.05).(2) The comparison of apoptosis percentages:There was no statistical significance among groups of treatment concentration (flow cytometer F= 1.165,P =0.349; TUNELF=2.232, P=0.068).(3) The comparison of cell cycle percentages:The difference of cell percent in each group on the phase G1,G2/M and S was no significance(F=0.533,P=0.750;F=0.543,P=0.742;F=0.811,P=0.551).(4) The comparison of cell adhension rate:One way analysis of variance revealed that the cell adhesion were increased significantly after IGF-1 treatment and decreased after LY294002 treatment as compared with group C(P<0.05). The cell adhesion rates in group P+IGF were decreased significantly as compared with group IGF and those in group P+LY were decreased significantly as compared with group LY(P=0.000). The cell adhesion rates in group P+LY were lowest in 6 groups (P=0.000).(5) The comparison of cell invasion:One way analysis of variance revealed that the invasion cell numbers were increased significantly after IGF-1 treatment and decreased after LY294002 treatment as compared with group C(P< 0.05). The invasion cell numbers in group P+IGF were decreased significantly as compared with group IGF and those in group P+LY were decreased significantly as compared with group LY(P=0.000). The invasion cell numbers in group P+LY were lowest in 6 groups (P=0.000).(6) The comparison of cell migration:One way analysis of variance revealed that the cell migration rates were increased significantly after IGF-1 treatment and decreased after LY294002 treatment as compared with group C(P<0.05). The cell migration rates in group P+IGF were decreased significantly as compared with group IGF and those in group P+LY were decreased significantly as compared with group LY(P=0.000). The cell migration rates in group P+LY were lowest in 6 groups (P=0.000).(7) The comparison of related molecules expression:One way analysis of variance revealed that the expression MMP-2, MMP-9, PEG2,AKT, PI3K, pAKTmRNA or protein in HepG2 cells were increased significantly after IGF-1 treatment and decreased after LY294002 treatment as compared with group C(P< 0.05). The expression MMP-2, MMP-9, PEG2,AKT, PI3K, pAKTmRNA or protein in group P+IGF were decreased significantly as compared with group IGF and those in group P+LY were decreased significantly as compared with group LY(P=0.000). The expression MMP-2, MMP-9, PEG2,AKT, PI3K, pAKTmRNA or protein in group P+LY were lowest in 6 groups (P=0.000).Conclusion The effects of propofol inhibiting proliferation, cell cycle, apoptosis, metastasis and invasion potential of liver cancer cell line HepG2 may be in part through inactivating PI3K-AKT signaling pathway.Chapter 4 Effects of propofol on growth and expression of PCNA、 CD34、pAKT of hepatoma xenografts in BALB/C miceObjective To investigate the effects of propofol on growth and expression of PCNA,CD34,pAKT in hepatoma-xenografts BALB/C mice and the Role of PI3K-AKT Pathway.Methods Part Ⅰ:Effects of propofol dosages on growth and expression of PCNA,CD34,pAKT in hepatoma-xenografts BALB/C miceHuman hepatocellular carcinoma cells SMMC-7721 were inoculated into the subcutaneous in BALB/C mices,to establish the hepatoma-xenograft model of BALB/C mice.Forty successful model mices were randomly divided into five groups (n=8):the control group (C group), intralipid group (I group), low dose (50mg/kg) propofol group (P1 group), medium dose (100mg/kg) propofol group (P2 group) and high dose (150mg/kg) propofol group (P3 group). Before and 3,6,9,12,15,18 days after treatment (T1,T2, T3, T4, T5, T6, T7) to observe the changes of tumor volume,and the growth curve was drown.All mices were sacrificed 19 days after drug withdrawal, Tumors were peeled and weighted,calculated the inhibition rate of propofol. The protein expressions of PCNA,CD34,pAKT in xenograft tumors were analyzed by Immunohistochemistry staining.Part Ⅱ:The mechanism of propofol inhibit growth and expression of PCNA,CD34 and pAKT in hepatoma-xenografts BALB/C miceHuman hepatocellular carcinoma cells SMMC-7721 were inoculated into the subcutaneous in BALB/C mices,to establish the hepatoma-xenograft model of BALB/C mice. Forty eight successful model mices were randomly divided into six groups (n=8):the control group(group C), 100mg/kg propofol group (group P), IGF-1 group(group IGF), 100mg/kg propofol and IGF-1 group(group P+IGF),LY294002 group(groupLY),LY294002 and propofol groups(groupP+LY).Before and 3,6,9,12,15,18 days after treatment (T1,T2, T3, T4, T5, T6, T7) to observe the changes of tumor volume,and the growth curve was drown. All mices were sacrificed 19 days after drug withdrawal, Tumors were peeled and weighted,calculated the inhibition rate of propofol. The protein expressions of CD34 in xenograft tumors were analyzed by Immunohistochemistry staining. The protein expressions of PCNA,pAKT in xenograft tumors were analyzed by Immunofluorescence staining.Data analysis was performed by SPSS 13.0 software. Data were expressed as mean ± standard deviation (SD).The tumor volume in the mice of different groups were assessed by ANOVA for repeated measurement.The tumor weight,tumor inhibited rates and protein expressions of PCNA,CD34 and pAKT were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3 test (heterogeneity of variance) was used for post hoc comparisons. P<0.05 was considered statistically significant.Results Part IThe comparison of tumor volume:There was no statistical significance at T1,among five groups ((F=0.226, P=0.920); Compared with group C, there was no statistical significance at T2-7 in group I (P=0.924);the tumor volume of P1,P2,and P3 groups at T2-7 were significantly decreased with propofol dosages increased (P< 0.05). There was interaction between treatment concentration and treatment duration (F=84927.589, P=0.000).The comparison of tumor weight and tumor inhibited rates:Compared with group C, there was no statistical significance of tumor weight in group I (P=0.997), the tumor weight of P1,P2,and P3 groups were significantly decreased with propofol dosages increased (P< 0.05). Compared with group C, there was no statistical significance of tumor inhibited rates in group I (P=0.817), the tumor inhibited rates of P1,P2,and P3 groups were significantly increased with propofol dosages increased (P < 0.05).The comparison of PCNA,CD34 and pAKT protein expression:Compared with group C, there was no statistical significance of PCNA,CD34 and pAKT protein expression in group I (P> 0.05), the protein expressions of P1,P2,and P3 groups were significantly decreased with propofol dosages increased (P<0.05).Part ⅡThe comparison of tumor volume:There was no statistical significance at T1 among six groups (P>0.05). Compared with group C, at T2-7,the tumor volume of group IGF was increased (P<0.05), the tumor volume of group P,group LY and group P+LY was decreased (P<0.05),the tumor volume of group P+LY was the least one in six groups(P<0.05). There was interaction between treatment concentration and treatment duration (F=85398.671, P=0.000).The comparison of tumor weight:Compared with group C, the tumor weight of group IGF was increased (P<0.05), the tumor weight of group P,group LY and group P+LY was decreased (P<0.05),the tumor weight of group P+LY was the least one in six groups(P<0.05).The comparison of tumor inhibited rates:There was statistical significance among six groups (F=446972.709, P=0.000).Compared with group C, the tumor inhibited rates of group IGF was decreased (P=0.000), the tumor inhibited rates of group P,group LY and group P+LY was increased (P<0.05),the tumor inhibited rates of group P+LY was the highest one in six groups(P<0.05).The comparison of PCNA,CD34 and pAKT protein expression:There was statistical significance among six groups (PCNA F=6535.517,P=0.000;CD34 F=3214.185,P=0.000;pAKT F=13881.71,P=0.000).Compared with group C, the PCNA,CD34 and pAKT protein expression of group IGF was increased (P< 0.05), the PCNA,CD34 and pAKT protein expression of group P,group LY and group P+LY was decreased (P<0.05),the PCNA,CD34 and pAKT protein expression of group P+LY was the least one in six groups(P<0.05).Conclusion Within a certain range, propofol can inhibit growth and PCNA,CD34 and pAKT protein expression in hepatoma-Xenograft BALB/C mice,and the degree is correlated to the dosage of propofol; its mechanism may be related to the inhibition of the PI3K-AKT pathway.