| Pancreatic cancer is a common and lethal malignancy resulting in more than250,000deaths per year worldwide. With the improvement of diagnosis and changes of lifestyle, the incidence of pancreatic cancer has increased every year, which was nearly five times from the1950s to the1990s in China. It is difficult to detect in early stage for lack of typical clinical manifestations. Most cases were in advanced stages and lost the opportunity of surgical resection when they were diagnosed. The administration of cytotoxic agents for the treatment of advanced pancreatic cancer has had disappointing results.The1-year survival rate in advanced or metastatic pancreatic cancer was low (less than20%) after gemcitabine monotherapy.There are few effective therapies available for the control of locally advanced or metastatic pancreatic cancer.Endoscopic ultrasonography (EUS) was introduced to clinical medicine for more than30years ago and has contributed to the diagnosis and management of pancreatic tumors. With the advent of linear-array echoendoscope, it is possible to trace needle tract under ultrasound and to do EUS-guided fine needle aspiration (EUS-FNA). More recently,fine needle injection (FNI) has followed.With the development of interventional endoscopic ultrasonography (EUS), multiple agents can be delivered by EUS for the treatment of pancreatic cancer such as fine needle injection (FNI), photodynamic therapy, gene therapy and radioactive seed implantation. Recently EUS-guided ethanol ablation was reported for therapy of pancreatic tumors.The mechanism of high-concentration ethanol ablation mainly involves cell death by causing cell membrane lysis, protein denaturation, and vascular occlusion. Percutaneous ethanol injection has been used in the ablation of renal cysts, hepatic cysts, and solid tumors, such as liver or adrenal tumors. EUS-guided ethanol injection is superior to percutaneous application because it offers real-time imaging monitoring of the lesions deeply located in the pancreas. EUS can provide precise measurement of lesions and identification of surrounding structures, and it readily delivers therapeutic agents to a target site and minimizes damage to nontumor tissue. Some EUS-guided ethanol ablation procedures for pancreatic tissue have been shown to be safe and feasible in animal models. Recently, some studies have shown that EUS-guided ethanol injection into pancreatic cysts is safe and feasible, slows or inhibits growth, and avoids surgical resection. EUS-guided ablation therapy using ethanol has been reported for the treatment of pancreatic neuroendocrine tumors in some case reports.The high-concentration ethanol is spreaded from the injection site to the surrounding tumor tissue after it is injected into tumor. Tumor cells occur dehydrated and denatured in places with high concentration of ethanol, resulting in tumor necrosis. However, the low concentration of ethanol is insufficient to cause tumor cell necrosis in the surrounding tumor tissue. It is unclear how low concentration of ethanol acts on the tumor cells.In recent years, some studies showed that low concentration of ethanol can induce apoptosis in intestinal epithelial cells, neural cells,corneal epithelial cells and hepatocellular carcinoma cells. Some researchers used volume percentage concentration of ethanol to treat cells. Hepatocellular carcinoma (HepG2) cells were treated with ethanol in vitro. The results showed that apoptosis induced by low concentration of ethanol in human HepG2cells was associated with Fas-receptor activation and subsequent caspase-8activation.Based on these studies, we performed experimental study of ethanol injection in treatment of pancreatic cancer.First, different concentrations of ethanol were injected into subcutaneous xenograft tumor of nude mice and an appropriate concentration was screened to treat pancreatic cancer. Second, orthotopic human pancreatic cancer models were etablished in nude mice. Ethanol is injected into tumor by high-frequency endoscopy ultrasound prob. The efficacy and safety of ethanol injection in treatment of pancreatic cancer are evaluated. In addition, whether the low ethanol concentration can induce tumor cell apoptosis was studied. These studies will provide a theoretical basis for treatment unresectable pancreatic carcinomas of ethanol ablation. The current study mainly includes the following three parts:1. Experimental study on intratumoral injection of different concentrations ethanol in treatment of pancreatic cancer xenograftsObjective To study the efficacy of intratumoral injection of different concentrations of ethanol for Pancreatic Cancer Xenografts and choose appropriate concentration of ethanol for treatment of pancreatic cancerMethods To etablish human pancreatic cancer xenografts in nude mice,48nude mice with similar tumor size were randomly divided into6groups(8mice per group):A0%normal saline;B20%ethanol;C40%ethanol; D60%ethanol; E80%ethanol; F95%ethanol. The longest and the shortest diameters of tumor of nude mice were respectively measured. Tumor volume, relative tumor volume (RTV) and the relative rate of tumor proliferation (T/C) were calculated. One week after the injection the nude mice were killed. The tumor tissue was sent for pathologic histology.Results RTV in20%ethanol group was similar that of normal saline (NS) group (P=0.212). RTV in40%、60%、80%and95%ethanol groups were lower than that of NS group (P<0.01). T/C was less than30%in60%,80%and95%ethanol groups. RTV in80%and95%ethanol groups were lower than that of60%ethanol group (P=0.003and P=0.009). RTV was similar in two groups of80%and95%(P=0.819). The severe coagulation necrosis was seen through optical microscope in60%、80%and95%ethanol groups.Conclusion There are similar efficacy for nude mice subcutaneously transplanted pancreatic cancer in80%ethanol group and95%ethanol group.2. Experimental study on endoscopy ultrasound probe-guided intra-tumoral injection of ethanol in treatment of orthotopic human pancreatic cancer model in miceObjective To study the feasibility of intratumonal injection of80%ethanol for orthotopic human pancreatic cancer in mice with intervenfional endoscopy ultrasonography.Methods To etablish orthotopic human pancreatic cancer model in mice,20mice with similar tumor size were randomly divided into2group(10mice per group):80%ethanol group and normal saline (NS) as the control group.Agents were injected intratumor by high-frequency endoscopy ultrasound probe. Tumor size was measured by high-frequency endoscopy ultrasound probe before and the seventh day after treatment.Tumor volume, relative tumor volume (RTV) and the relative rate of tumor proliferation (T/C%) were calculated. The serum amylase was detected before and twenty-four hours after treatment; One week after the injection the nude mice were killed. The tumor tissue was sent for pathologic histology.Results RTV in experimental group was significantly lower than that of NS group(0.47±0.09v1.24±0.04, P<0.01). T/C%was38%in experimental group. Twenty-four hours after treatment, the serum amylase slightly increased in experimental group,but no difference was seen before and after treatment (P=0.059). There was also no significant difference in the experimental group and the control group (P=0.13).The severe coagulation necrosis was seen through optical microscope in experimental group compared with control group.Conclusion Intratumoral injection of80%ethanol guided by high-frequency endoscopy ultrasound probe is feasible therapy method for orthotopic human pancreatic cancer model in nude mice.3. Low concentrations of ethanol induce apoptosis in human pancreatic cancer cell linesObjective:The present study was designed to determine the effect of low concentrations of ethanol on apoptosis of cells in a human pancreatic cancer cell lines.Methods:Pancreatic cancer cells SW1990were incubated with low concentrations of ethanol (2%,4%and6%) or without ethanol (control group,0%) for6h and analyzed immediately. Cell proliferation rate was measured by means of CCK8. Apoptosis was assessed by means of flow cytometry. Caspase-8and caspase-9were analyzed by real-time quantitative PCR and caspase-3protein was analyzed by westernblot.Results:Six hours exposure to2%,4%and6%ethanol inhibited cell proliferation in SW1990cells compared with control group (P<0.05). As the concentration increased, the rate of cell proliferation decreased significantly. Flow cytometry showed that2%,4%and6%ethanol group can induce apoptosis of SW1990cells.There was statistically significance compared with to the control group (0%) groups (P<0.05). Expression of caspase8mRNA didn’t increased significantly after6h incubation of SW1990cells with2%,4%and6%ethanol compared with to the control group (0%) groups (P>0.05). Caspase9mRNA expression increased markedly in2%,4%and6%ethanol compared with to the control group (0%) groups (P<0.05). Caspase-3protein expression increased significantly after ethanol treatment in SW1990cells, whereas wasn’t observed in control cells.Conclusion:Low ethanol concentrations inhibit cell proliferation and increase apoptosis in SW1990cells. These findings show that apoptosis induced by low concentrations of ethanol in human pancreatic cancer cells SW1990may be associated with mitochondria path activation and subsequent caspase-9activation. |
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