Antiapoptotic Effect Of PKC On Mechanism Of X-ray Radiation-induced Apoptosis In Hepatocarcinoma Cells

BACKGROUND: Hepatocellular carcinoma (HCC) is a common, extremely aggressive malignancy and the mortality of this disease is very high worldwide. More than half of these patients are in China and the number is increasing. Five year mortality of HCC exceeds 95%. Surgical resection is generally accepted as the first choice treatment of HCC. HCC very often develops in the cirrhotic liver. It occurs on a background of chronic liver disease or viral hepatitis in the vast majority of cases. Surgical treatment of cirrhotic patients is associated with considerable risk. Even partial resection of the liver carries the risk of liver failure in such patients. Thus these are available or appropriate of surgical resection in only a minority of patients. Treatment options are constrained not only by the characteristics of the tumor but also by hepatocellular reserve, severity of portal hypertension, and the general condition of the host. Many non-surgical techniques have been developed and used for the treatment of inoperable HCC, and radiotherapy is one of them.Historically, radiotherapy for HCC has yielded poor survival because of dose limited by enormous side effects. Recent advances in several technologies have opened a new era in radiation oncology. Recent studies have shown that three-dimensional conformal radiotherapy can be an effective component of thetreatment for HCC. While hepatologists are beginning to show less reluctance for applying radiotherapy to the treatment of HCC, the radioresistance is another block to cure HCC. To further improve therapeutic efficacy, use of drugs that can beneficially interact with radiation has been suggested. Most combined multimodal interventional therapies reveal their enormous advantages as compared with any single therapeutic regimen alone, and play more important roles in treating unresectable HCC.Radiation-induced cellular lesions include both DNA and membrane damage and lead to a coordinate network of fatal and non-fatal signal transduction pathways, thus resulting into radiation-induced biology changes. It is well known that apoptotic cell death is the main form of ionizing radiation-induced cell death. The multigene family of protein kinase C (PKC) codes for serine/threonine kinases and distributes in mammalian cells extensively. PKC acts as an intracellular mediator in a wide variety of cellular processes including growth factor activation, apoptosis, cell cycle regulation, proliferation, differentiation, and tumor promotion et al. Recent studies suggested that PKC is an important participant in radiation-induced signaling cascades and is involved in regulating radiation-induced apoptosis/survival pathway. Conflicting observations on PKC involved in apoptosis point to a great variability depending on cell types, stimuli causing apoptosis, phases of cell cycle and intracellular signaling pathways. Role of PKC are still controversial since it has been reported that PKC activation induces apoptosis in some cases and promotes cell survival in others. Development of radiation resistance is one of the major reasons that cancer cells do not respond to radiotherapy and the mechanism of radioresistance is still not clear. Whether PKC is involved in radioresistance or not? Further, novel strategies to abrogate the "induced radioresistance" leading to enhanced therapeutic efficacy of ionizing radiation have been proposed. The complete understanding of the molecular pathways leading to apoptosis/survival of cells following ionizing radiation will help in tailoring more effective novel strategies and treatment modalities for complete eradication of cancer.In order to elucidate the role of PKC in radiation-induced apoptosis, this study was performed to obtain further information about the function of PKC in the regulationof radiation-induced apoptosis in human hepaticarcinoma line HepG2 cells. The specific PKC isoforms involved in radiated HepG2 cells and their distribution were also investigated. The experimental results will be used to understand the mechanism of PKC involvement in radiation-induced tumor cell apoptosis and development of radiosensitizer.Part I X-ray radiation-induced apoptosis in human hepatocarcinoma HepG2 cellsOBJECTIVE To establish models of X-ray radiation-induced apoptosis in HepG2 cells and investigate cellular morphological damages in X-ray radiated HepG2 cells. Role of PI3-K/Akt, ERK, p38 MAPK, JNK pathways and PKC were detected in cellular levels.METHODS Cells were cultured as usual method; HepG2 cells were divided into seven groups: Control group; radiation group(Cells were radiated at a dose of 6Gy); PMA group(Cell were pretreated by lOOnmol/L PMA, a PKC activator, for about 30min and radiated); SP group(Cell were pretreated by lOOnmol/L SP, a PKC inhibitor, for about 30min and radiated); PD98059 group(Cell were pretreated by 20umol/L PD98059, a MEK1/2 inhibitor, for about 30min and radiated); SB2O358O group(Cell were pretreated by 20umol/L SB203580, a p38 pathway inhibitor, for about 30min and radiated); LY294002 group(Cell were pretreated by 20umol/L LY294002, a PI3-K inhibitor, for about 30min and radiated). Cells were radiated to a dose of 6Gy with 6MV X-ray using Varian 2100 linear accelerator. Hoechst 33258 staining to reveal the nuclear morphology of HepG2 cells; Ultrastructure of HepG2 cells was observed by transmission electron microscope; Survival rates were detected by MTT colorimetric assay; Detection of apoptosis rate by flow cytometry (FCM).RESULTS 1. Radiation-induced nuclear changes were evaluated under a fluorescence microscope using Hoechst33258 staining. In control group, the nuclei showed uniform staining, indicating that cells were healthy and nuclei were intact. In radiation group, some nuclei exhibited typical apoptotic characteristics, such as condensation of nuclear chromatin and aggregation at the nuclear membrane. Cellularnuclei in PMA group and PD98059 group were similar to those in control group. Cellular nuclei in SB203580 group and LY294002 group were similar to that in radiation group. Cellular nuclei in SP group were more than those in radiation group. 2. Ultrastructure of apoptotic HepG2 cells was observed by transmission electron microscope. In control group, ultrastructure of HepG2 cell was normal. In radiation group, classic morphologic change of apoptosis was found, for example, cells shrinkage, cell sizes dwindling, membrane blebbing, mitochondria swelling, formation of apoptotic bodies, higher electron density, chromatin digestion and condensation. Cellular ultrastructure in PMA group and PD98059 group were similar to those in control group. Cellular ultrastructure in SB203580 group, LY294002 group and SP group were similar to that in radiation group. 3. Survival rates detected by MTT in control, radiation group, PMA group, SP group, PD98059 group, SB203580 group and LY294002 group were 97.90%, 79.17%, 96.26%, 59.57%, 97.25%, 76.63% and 77.07% respectively. 4, Apoptosis rates detected by FCM in control, radiation group, PMA group, SP group, PD98059 group, SB203580 group and LY294002 group were 1.73%, 20.90%, 3.20%, 40.57%, 2.43%, 22.83% and 22.67% respectively. The apoptosis rate in radiation group was higher than control group (P<0.01). The survival rate in radiation group was lower than control group (P<0.01). The survival rates in PMA and PD98059 groups were higher compared to that in control group and apoptosis rates in PMA and PD98059 groups were lower compared to that in control group (/><0.01).On the other hand, the survival rate in SP group was lower compared to that in control group and apoptosis rate in SP group was higher compared to that in control group (P<0.01). SB203580 and LY294002 pretreatment had no effect on radiation-induced apoptosis in HepG2 cells.Part II Signal transduction mechanism of X-ray radiation-induced apoptosis and role of PKC in HepG2 cellsOBJECTIVE To investigate roles of PI3-K/Akt, ERK, p38 MAPK and JNK pathways, NF-kB in radiated HepG2 cells in molecular level. To further dectect regulating role of PKC in mechanism of X-ray radiation-induced apoptosis/survivalin HepG2 cells.METHODS Cells were cultured as usual method; HepG2 cells were divided into four groups: Control group; radiation group(Cells were radiated at a dose of 6Gy); PMA group(Cell were pretreated by lOOnmol/L PMA, a PKC activator, for about 30min and radiated); SP group(Cell were pretreated by lOOnmol/L SP, a PKC inhibitor, for about 30min and radiated); Cells were radiated to a dose of 6Gy with 6MV X-ray using Varian 2100 linear accelerator; Western blotting was used to assess expressions of phospho-c-Jun, phospho-ERK, phospho-p38, phospho-Akt and phospho-lKB; Laser confocal microscopy was applied to observe cellular distribution of ERK1/2 in HepG2 cells; Immunocytochemistry technique was applied to assay cellular distribution of NF- kB in HepG2 cells; EMS A technique was used to detect activation of NF- kB in HepG2 cells.RESULTS 1. Phospho-c-Jun and phospho-Akt expressions were not changed at various time points after radiation compared to non-radiated in HepG2 cells; phospho-p38 was undetected. 2. Phospho-ERK 1/2 expression assayed by western blotting was increased at various time points after radiation in a sustained manner in HepG2 cells. PKC activator PMA and inhibitor SP had no significant effect on phospho-ERKl/2 expression assayed by western blotting and cellular distribution assayed by laser focal microscopy. 3. Activation of NF-kB was detected by EMS A. The result showed that NF-kB activation in control was slight and was increased gradually after X-ray radiated. Preincubation with lactacystin, an inhibitor of NF-kB, increased apoptosis rate induced by X-ray radiation in HepG2 cells. PMA promoted NF-kB activation and translocation induced by X-ray radiation. On the contrary, SP played an opposite role in NF-kB activation and translocation. Moreover, degradation of cytoplasm IkB was showed by western blotting and PMA upregulated IkB degradation, SP downregulated it.Partm PKC specific isoforms expression and subdistribution of antiapoptotic effect on mechanism of X-ray radiation-induced apoptosis in HepG2 cellsOBJECTIVE To investigate PKC specific isoforms expression and subdistributionin X-ray radiated HepG2 cells and demonstrate the PKC isoform that induced radioresistance in HepG2 cells.METHODS Cells were cultured as usual methods; HepG2 cells were divided into four groups: Control group; radiation group(Cells were radiated at a dose of 6Gy); PKC412 group(Cell were pretreated by lOOnmol/L PKC412, a cPKC(a, p, y) inhibitor, for about 30min and radiated); rottlerin group(Cell were pretreated by 200nmol/L rottlerin, a PKC5 inhibitor, for about 30min and radiated); Cells were radiated to a dose of 6Gy with 6MV X-ray using Varian 2100 linear accelerator; PKC activity was assayed by measuring the incorporation of 32P from [y-32P]-ATP into peptide substrates. Fluorescence intensity (FI) and percentage of positive cells were determined by FCM; Western blotting was used to assess expressions of PKC isoforms and phospho-ERK; EMS A was used to determine activation of NF-kB.RESULTS 1. X-ray radiation at a dose of 6Gy induced PKC activation in a time-dependent manner in HepG2 cells. At the time point 30min after radiation PKC activity was increased to a highest level and then declined rapidly. PKC activity was declined to basic level at lh after radiation; PKC activity in control HepG2 cells was in basal level and fluctuated slightly. 2. Percentage of PKC activity of cytoplasm fraction in control HepG2 cells was 75% and higher than that in radiation group. On the other hand, Percentage of PKC activity of membrane fraction in radiation group was 78% and higher than that in control HepG2 cells. 3. FT of PKCa and PKC6 were 2.28 and 5.05 respectively in radiation group, higher than those in control group cells(P<0.05). Percentages of PKCa and PKC8 positive cells were higher in radiation groups than those in control cells(P<0.05). FI and Percentages of PKC ï¿¡, y, ï¿¡, 8 positive cells were petty in control and radiation groups and have no significant difference (P>0.05). The FT and Percentage of PKC0 positive cells were zero. 4. Radiation increased expressions of PKCa and PKC5 and induced translocation from cytoplasm to membrane, whereas radiation had no effect on PKC^, s. Expression and translocation of PKC5 was higher than that of PKCa. No Expressions of PKCp, y, 0 were detected. Apoptosis rates of control group, radiation group, PKC412 group, rottlerin group were 1.73%, 20.90%, 22.23%, 36.57% respectively. There was nostatistical difference of apoptosis rate between control and PKC412 group (P>0.05). But apoptosis rate in rottlerin group was higher than that in radiation group (P<0.01). 5. PKC412 or rottlerin preincubation had on effect on ERKl/2 activation induced by X-ray radiation. X-ray radiation induced activation of NF-kB was not influenced by PKC412 but was influenced by rottlerin.CONCLUSIONS 1. X-ray radiation at a dose of 6Gy induced apoptosis in human hepatocarcinoma HepG2 cells. 2. p38 MAPK, c-Jun and PI3-K/Akt might not be involved in X-ray radiation-induced apoptosis in HepG2 cells. 3. X-ray radiation induced apoptosis in HepG2 cells mediated through sustained activation of ERKl/2. 4. Activation of NF-kB was involved in radiation-induced apoptosis/survival in HepG2 cells and promoted survival in radiated HepG2 cells. 5. PKC was involved in protecting mechanism in radiation-induced apoptosis in HepG2 cells and was playing an antiapoptic role in X-ray radiated HepG2 cells. PKC8 was centered in protecting HepG2 cells from radiation-induced apoptosis. 6. Activation of ERKl/2 induced by radiation was PKC-independent. and activation of NF-kB was PKC5-dependent. The balance between ERKl/2 activation and PKC8/NF-kB activation determined the fate of HepG2 cells.