Exploration Of The Effect And Mechanism Of Radiosensitization Of Isoegomaketone On Hepatocellular Carcinoma Cells

Backgroud and ObjectionAs the novel statistical data from American Society of Clinical Oncology in2012suggested that Hepatic carcinoma is the fifth most common cause of deaths from cancer for male and the ninth for female. Hepatic carcinoma is one of the ten malignant carcinoma announced by World Health Organization,about0.7million people are died of hepatic carcinoma each year. Primary hepatocellular carcinoma is the most common malignant tumor in clinic, the symptom is alway masked and the progression is very quick, thus most patients is diagnosed advanced HCC while they feel uncomfortable. So HCC is a big killer to people in the world. Radiation therapy is a major modality for HCC treatment, especially for advanced HCC. But radiotherapy with high dose is harmful for patient and bring them many side effects and complications. Some HCC cells scarcely response to radiotherapy and even exhibit an resistance to radiotherapy. Looking for a new radio sensitizer with high efficacy in blocking the proliferation of cancer cells and radiosensitizing seems very urgent for clinical treatment.HCC is very special for some patients are come from HBV infection. Patients with HBx sometimes not so sensitive to chemotherapy and radiotherapy. Some radiosensitizer like Paclitaxel, are not so effective in treatment for HBx-positive HCC cells. In China, HBV infection is the key risk factors for HCC. About30million subjects infected with HBV were diagnosed as active chronic hepatitis B, and15-40%of these subjects will develop to hepatic cirrhosis or HCC. Thus the HBV positive patients of HCC should be treated with antivirus therapy and liver-protect agents in chemo/radiotherapy. Keeping individual clinical regimens will help to get better curative effect and prognosis. HBV belongs to hepadnavividae. Its genome is an3.2Kb DNA double chain which is not intact. It contains4regions:S, C, P and X region. The DNA in X region can product an HBx protein. The second structure of HBx is consist of4a-helix and4P-sheet. A Leucine Zipper is formed of the105-148aa, and it is a typical activating domain in trans-acting factor. It is the base of HBx trans-acting activity. HBx regulates the expression of so many genes in virus and cells genome, such as c-myc, c-fos, c-jun, the long terminal repeats of rous sarcoma virus, SV40early promoter and p53promoter, to inducing disturbance of cell cycle and proliferation and interferes the expression of normal genes, which contribute to tumorigenesis. Some reports suggested that HBx protect tumor cells from apoptosis and DNA damage though activating complement regulatory molecules, inducing anti-apoptosis signals, blocking DNA damage and so on. Whereas HCC cells present a multidrug resistance and resistance to harmful stimulus. After treated with Paclitaxel, HBx down-regulated p53and p21via blocking the interaction between ribosomal protein L11and MDM2to help HCC cells escaping from apoptosis, keep HCC cells proliferating well and resisting the drug treatment. HBx up-regulated the expression of complement regulatory protein CD46and CD59, and resistant the damage from complement dependent cytotoxicity to HCC cells. HBx plays a pivotal role in the proliferation and apoptosis signal network and it is a target site in cancer therapy. Thus we detected the differences after IK combing with IR treatment between HBx positive cells and HBx negative cells to emphasize the importance of HBx in HCC therapy. The investigation is beneficial to develop novel effective radio sensitizer and determine an individual therapeutic strategy to reducing the potential resistance to radiotherapy.Recent years thousands of documents showed that the abnormality of signal transduction is the dominant internal factor for tumorigenesis and tumor development. Large quantity of signal pathways are involved in tumorigenesis. Radiotherapy is one of the important modality in cancer treatment. It is very useful in killing tumor cells and it can directly treat target tumor tissue. Radiotherapy are suitable for inhibiting the fast proliferation of cancer cells because of its high energy capable of breaking the chromosome in cancer cell. Radiotherapy is always applied as major treatment or adjunctive treatment for cancer patients. However, there was many side effects or complications after radiotherapy, for example, local bone marrow suppression, general immunity reduction, radiodermatitis, serious gastrointestinal responses and so on, even threating to life. Because some tumor is not sensitive to radiotherapy and exhibit some resistance to irradiation, and HCC is very special which can not be treated with irradiation of high dose, the development of some new radio sensitizer is very important. Liver and peripheral organ exhibit an intolerance to radiotherapy, so the irradiation to HCC is very difficult because high dose is forbidden and HCC cells are not cleared at a low dose irradiation. Radio sensitization may reduce the injury to liver of high dose radiation and enhance the efficacy of the radiotherapy. Whereas radio sensitizers(such as taxotere, cisplatin) applied clinical recently have many side effects such as hepatotoxicity, these agents has no hepatic-protect function and reduce the life quality of the patients. Thus how to choose a suitable radio sensitizer with killer effects on tumor cells and sensitizing effects to IR for a perfect clinical regimen has been a pivotal problem in tumor treatment.In the researches for radio sensitizer, the traditional herbs have been focused on in these years. In thousands of herbs, perilla frutescens attracts more attentions from scholars for its mild nature and better antitumor effects. Many reports suggested that the perilla frutescens extracts exhibit efficacy on blocking proliferation and inducing apoptosis of tumor cells. The essential components of the perilla frutescens extracts are rosmarinic acid, caffeic acid, isoegomaketone, Luteolin and so on. Isoegomaketone(IK) is effective in inhibiting tumor cell proliferation. IK promotes the cleavage of Bid, translocation of Bax, release of cytochrome C and AIF nuclear translocation, enhances the activity of apoptosis-related enzyme such as Caspase-8, Caspase-9and Caspase-3. IK is capable of inducing apoptosis though caspase-dependent apoptosis pathway and caspase-independent apoptosis pathway simultaneously. Apart from this, IK also inhibits the phosphorylation of STAT1and activation of NF-κB. NF-κB is an pleiotropic nuclear transcription regulating factor, and its activation enhances the expression of the anti-apoptosis molecules(such as TRAF1, c-IAP1, Al/Bfl-1, Bcl-xL) and inhibits the cleavage of apoptosis downstream molecules-Caspase-8. On the whole NF-κB blocks the apoptosis caspase cascade reaction and protect tumor cells from death. The inhibiting effects of IK on STAT1and NF-κB just interrupted this escape of tumor cells from apoptosis. Lin and his members found that the perilla frutescens leaves extracts inhibited the proliferation of HepG2cells through upregulating of Fos-B, Jun-B and Caspase-8to induce Fos/Jun mediated apoptosis and activating of TNF-a. Kwak et al applied perilla frutescens leaves extracts to treat HL-60cells and found that this extract inhibited the cell proliferation in a dose dependent manner. After treatment of perilla frutescens leaves extracts there was a G0/G1arrest of HL-60cells, the activation of Caspase-8and Caspase-9, the upregulation of GRP78, p-TIF2a, p21.In a word the perilla frutescens leaves extracts initiating apoptosis and inhibiting cell proliferation through mitochondria mediated apoptosis, death receptor mediated apoptosis, endoplasmic reticulum mediated apoptosis and p21mediated G1phase arrest. Osakabe et al showed that Perilla frutescens extracts presented marked reduction on tumorigenesis in a murine, two-stage skin carcinogenesis model. In this model, cancer is initiated by application of7,12-dimethylbenz anthracene (DMBA) and promoted by application of12-tetradecanoylphorbol13-acetate (TPA). Myeloperoxidase activity, a marker of neutrophil recruitment, was also increased in TPA-challenged mice skin and was significantly decreased in the Perilla frutescens extracts treated groups. The mRNA expression levels of intercellular adhesion molecule1and vascular cell adhesion molecule-1were reduced by pre-treatment with Perilla frutescens extracts. TPA-induced increases in synthesis of the chemokines KC and macrophage inflammatory protein-2were significantly decreased by pre-treatment with Perilla frutescens extracts. These levels were only numerically decreased in the Perilla frutescens extracts treated groups. However, induction of mRNA expression of cyclooxygenase-2was obviously reduced by pre-treatment with Perilla frutescens extracts. Reactive oxygen radical production, detected by thiobarbituric acid reactive substance and lipid peroxide, was reduced by pre-treatment with Perilla frutescens extracts. Production of8-hydroxy-2’deoxyguanosine, which was detected immunohistochemically, was barely visible in Perilla frutescens extracts treated mice. Thus, we conclude that part of the anticarcinogenic effects of Perilla frutescens extracts is via two independent mechanisms:inhibition of the inflammatory response and scavenging of reactive oxygen radicals. Ueda et al manipulated a similar research. They investigated the effects of the perilla leaves extracts on7,12-dimethylbenz anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin papillomas in mice. Topical application of perilla leaves extracts prior to TPA treatment in DMBA initiated mouse skin resulted in a notable reduction in tumor incidence and multiplicity. Perilla leaves extracts was dissolved in the drinking water at a0.05%dose and all mice ingested it ad libitum; no significant differences was observed in tumor incidence/multiplicity but there was a significant decrease in tumor volume between the Perilla leaves extracts treated and untreated groups. These results suggest that PLE has potent anti tumorigenesis activity and ingesting it as a daily food may provide people a beneficial chemopreventive effect. Besides these evidence, caffeic acid and rosmarinic acid in perilla frutescens extracts were verified a hepatoprotective effects. Caffeic acid remarkably reduced the oxidative damage than rosmarinic acid in vitro study. Oral intubation(SD rats) with caffeic acid or rosmarinic acid alone for five days was conducted prior to treatment with tert-butyl hydroperoxide, which led to a significant reduction of some indicators of hepatic toxicity, such as aspartate aminotransferase, oxidized glutathione, alanine aminotransferase, lipid peroxidation and antioxidant enzyme activities such as catalase, and superoxide dismutase,glutathione peroxidase. These results suggest that extracts from perilla leaves plays a role in the increased hepatic GSH concentration, and shows an additive hepatic protection against oxidative hepatic damage.According to the anticancer and hepatic protecting activity of perilla frutescens extracts, we choose one of the extracts-IK to investigate. IK may conquer the hepatotoxicity of the former radio sensitizer and has the potential to be a novel HCC radiosensitizer with high quality. All the researches about IK implied from different aspect that it act as a new agent extracted from the natural antitumor plant and green vegetable-perilla frutescens, may exhibit its powerful attractiveness on the big stage of clinical therapy. In summary, this study prompt to confirm the effects of IK on the proliferation and radiosensitivity of HCC cells, explore a better radio sensitizer with low hepatotoxicity, observe the differences of proliferation and apoptosis between Huh7cells and Huh7-HBx cells with treatment of IK combing IR, study the potential mechanism of IK sensitizing HCC cells to radiotherapy. These investigation will provide theoretic evidences for the establishment of individual radio therapeutic regimens for HCC.Methods and materialsA.The effects of IK on the proliferation of HCC cells1.HCC cells(HepG2cells、Huh7cells and Huh7-HBx cells) and Human normal hepatic cells L02were treated with isoegomaketone(IK)(0μmol/mL,10μmol/mL,20μmol/mL,30μmol/mL,40μmol/mL,50μmol/mL) for24h,48h,72h. CCK-8assay were done to detect the effect of IK on the proliferation of HCC cells.2. The plated Huh-7cells and Huh7-HBx cells were treated with IK(0μmol/mL,15μmol/mL,30μmol/mL,45μmol/mL), and the colony were observed14d latter. The effects of IK on colony formation of HCC cells were detected and the further consequence of IK treatment were confirmed.3.Huh-7cells and Huh7-HBx cells after treatment of IK were collected, fixed and stained, then examined on flow cytometer. The single-labeled sample were used for determine the suitable cells.The cell cycle and cell apoptosis rate were recorded.B.The radiosensitization of IK on HCC cells1.Huh7cells and Huh7-HBx cells were treated with IK(Oμmol/mL,15μmol/mL,30/umol/mL,45μmol/mL) combining IR(0Gy,2Gy,4Gy,6Gy) for24h,48h and72h. The effects of proliferation inhibition and radiosensitization of IK on HCC cells were measured. LC50and LD50were calculated. 2.The plated Huh7cells and Huh7-HBx cells were treated with IK (3μmol/mL) combing with IR(0Gy,2Gy, AGy,6Gy), and colony were observed14d latter. The effects of IK combining IR on colony formation of HCC cells were detected and the further consequence of IK&IR treatment were confirmed.3.The plated Huh7cells and Huh7-HBx cells were treated with IK (34μmol/mL) combing with IR(4Gy). When it reached the time point, cells were collected, clearaged and added with substrate, then the Caspase-3activity were measured to evaluate the apoptosis state.4.Huh-7cells and Huh7-HBx cells after treatment of IK&IR were collected, fixed and stained, then examined on flow cytometer. The single-labeled sample were used for determine the suitable cells.The cell cycle and cell apoptosis rate were recorded.C.The potential mechanism of radiosensitization of IK on HCC cells1.Huh7cells and Huh7-HBx cells were treated with IK&IR. Then the mRNA expression level of pro-proliferation molecules, anti-proliferation molecules, pro-apoptosis molecules and anti-proliferation molecules were detected using RT-PCR.2.Huh7cells and Huh7-HBx cells were treated with IK&IR. Then the protein expression level of CyclinDl, CyclinEl, Bax, Bcl-2and Cleaved Caspase-3were detected using Western Blotting assay.D.Statistical methodsMeasurement Data can be expressed as Mean±SD. Use univariate analysis of variance to analyze multi-groups data. Use2-independent-samples T test to analyze difference between two groups; Use t’ test with heterogeneity of variance. Multiple comparisons can be used on the premise that there are significant differences among groups. The difference (P<0.05) was considered statistically significant. The soft of SPSS16.0for windows was used to analyze all the data.ResultsA.The effects of IK on the proliferation of HCC cells1.In contrast to Human normal hepatic cells L02(control), IK inhibits the proliferation of HepG2、Huh7and Huh7-HBx cells markedly in a dose/time-dependent manner. After treated with50μmol/mL IK for48h, HepG2cells viability is34.12±3.91%; After treated with50μmol/mL IK for48h, Huh7-HBx cells viability is41.95±4.05%; After treated with50μmol/mL IK for48h, Huh7cells viability is35.01±3.35%. The effects of IK with same concentration in different time point on HepG2and Huh7cells is stronger than that in Huh7-HBx cells, and the differences is significant.2.IK inhibits the colony formation of Huh7cells and Huh7-HBx cells in an concentration manner. After treated with30μmol/mL IK, the plating efficiency of Huh7cells is14.27%, and the plating efficiency of Huh7-HBx is17.33%.3.IK induces a G0/G1arrest and apoptosis of Huh7cells and Huh7-HBx cells. The apoptosis rate of Huh7cells and Huh7-HBx cells is19.51%and16.96%.B.The radiosensitization of IK on HCC cells1.The cell viability of HepG2cells, Huh7cells and Huh7-HBx cells decrease markedly after IK combing with IR in a dose/time dependent manner. There is a notable synergistic sensitizing effect of IK to radiotherapy on HCC cells. Average LC50of HCC cells after treatment is IK34μmol/mL+IR4Gy. The sensitivity of HBx negative cells Huh7to IK and IR is higher than that of Huh7-HBx cells.3.The plating efficiency of Huh7cells and Huh7-HBx cells is8.93%and12.4%after treatment of IK combing IR, while the control is20.93%and23.53%, and differences are significant(P<0.05). The SF of Huh7cells and Huh7-HBx cells after treatment of IK combing IR decrease significantly. The treatment of IK increase the sensitivity of Huh7cells and Huh7-HBx cells to IR markedly.4.The caspase-3activity of Huh7cells and Huh7-HBx cells after treatment of IK combing with IR are higher than that in IR group, and the differences is significant(P<0.05). The caspase-3activity of Huh7cells are higher than that of Huh7-HBx cells.5.The cell apoptosis rate of Huh7cells and Huh7-HBx cells after treatment of IK combing with IR are higher than that in IK/IR alone group, and the differences is significant(P<0.05).C.The potential mechanism of radiosensitization of IK on HCC cells1.The mRNA relative expression level of p15, p18, p21, p27and p53in Huh7cells and Huh7-HBx cells after treatment of IK combing with IR increased notably, while p18, p21and p53increased in IK alone group. The mRNA level of Cyclins and Cdk2in Huh7cells and Huh7-HBx cells after treatment of IK combing with IR decreased notably, while they also decreased in IK alone group. The mRNA level of Fas, TRAILR1, Bax in Huh7cells and Huh7-HBx cells after treatment of IK combing with IR increase notably, and anti-apoptosis molecules Bcl-2and FLIP decrease. In contrast to IK/IR alone group, The inducing effect on these molecules stronger in IK and IR group.2.The protein level of Cyclin D1and Cyclin E1in Huh7cells and Huh7-HBx cells after IK treatment reduce significantly. The change of Cyclin D1expression is not obviously after IR treatment, and Cyclin D1expression decreases. The expression levels of Cyclins are much lower in Huh7cells and Huh7-HBx cells treated with IK and IR. Bax, Cleaved Caspase-3is up-regulated, Bcl-2is down regulated after treatment of IK combing IR. The induction of protein expression is more obvious in Huh7cells after treatment of IK and IR. Conclusions1.Isoegomaketone is an effective radio-sensitizer and it is very useful in inhibiting the cell proliferation of Huh7and Huh7-HBx cells.2.IK induces an GO/G1arrest and apoptosis of HCC cells.3.Effects of inhibiting of proliferation and inducting of apoptosis after treated with IK combing IR is better than that after treated with IR alone.4.There is an synergistic sensitizing effect of IK to radiotherapy on HCC cells.Innovative PointsThis study applied a new herb extraction-Isoegomaketone to perform the examination of the sensitization of HCC cells to radiotherapy. This compound plays a role in protecting liver. This study is very interesting but there is no report about it.We detected to cell lines with or without HBx to determine the radiosensitization effect of Isoegomaketone, and verifed that it inhibits the cell proliferation of both.We explore the possible mechanism of the inhibition in cell proliferation after treated with IK combing IR. Cyclins are down-regulated, Bax is induced. The effect of IK combining IR may provide some guidance in clinic trails.We confirmed the synergistic sensitizing effects of IK to IR. For HBx positive cells, IK also exhibits the effect. These researches are helpful in editing a individual radiotherapy regimen, and provide some theoretic evidence for clinic trails.