Evaluate The Effect Of LB-100 On The Radiosensitivity Of Cervical Cancer Cell Ca Ski And Intestine

Background and objectThe Serious environmental pollution and aging population lead the incidence of tumours rising,and made it become the leading causes of death around the word.Cervical cancer is one of the major threats to the health of women,especially in Asian countries including China,where the HPV widely spread.Radiotherapy or with operation is the major therapy for cervical cancer in early stage,and have a good effect.But the damage effect of radiation is not selective,and the cervix locate in the pelvic cavity,its neighboring—intestine is very sensitive to radiation.Therefore,the patients accepted radiotherapy always inevitably accept intestinal irradiation injury.They suffer from abdominal pain,diarrhea,hematochezia and even intestinal obstruction.Those increase the patients’ psychological and economic burden,and greatly increase the clinical nursing work at the same time.However,if reducing the radiation dose to prevent intestinal complications,will lead the cervical cancer to eliminate incompletely,metastasis and recurrence.Therefore,It is urgent to find a new drug that can enhance the radiosensitivity of cervical cancer cells under radiotherapy.So it can reduce the radiation dose and aviod the intestinal injury as much as possible,and reduce the burden of patients and nurses,but not reduce the antitumor effect.Protein phosphatase 2A(PP2A)is a regulatory protein involved in cell cycle and DNA damage response.The research showed that,exogenous inhibitors such as cantharidin and okadaic acid can enhance the sensitivity of chemoradiotherapy on tumor cells by inhibiting PP2 A activity,and then by changing the cell cycle and DNA damage response of tumor cells.LB-100 is a water-soluble inhibitor of PP2 A.It has been proved that LB-100 enhanced the sensitivity of chemoradiotherapy on tumor cells,such as osteosarcoma,ovarian cancer and so on.And it has been in phase I clinical research.However,there is no report about the effect of LB-100 on cervical cancer cell lines.In cervical cancer patients who received radiotherpy would suffer radioactive intestinal injury inevitably.This made patients suffer pains in the physical and mental,and made it to be a big trouble for clinical treatment and nurse care.so when we are studying a new drug as a radiosensitizer for abdominopelvic cancer radiotherapy in the lab,we should pay attention to its effect on bowel.In this study,we used the cell Cas ki and CCD 841 Co N and HIEC,to explore the effects of LB-100 on the radiation-sensitivity of those cells.Then we established a radiation enteritis model on mouse to observe the effect of LB-100 on normal intestine and damaged intestine after radiation.Our purpose is to provide some guidance for clinical radiotherapy and nursing of patients with cervical cancer.Methods:1.LB-100 concentration screening:Treated on Ca ski,CCD 841 Co N and HIEC with different concentrations of LB-100 for 24 hours respectively,then detected the cell activity by CCK-8.And regard the concentration that had no inhibition effect on the two kinds of normal intestinal epithelial cells as safe drug concentrarion.2.48 hr cell activity detection(Ca ski,CCD 841 CoN and HIEC):Cells were divided into 4 groups respectively including control、LB-100、R、LB100+R.2.5μM LB100 and(or)irradiation(6Gy)were treated on them respectively.And then detected the activity of these cells 48 hours after irradiation.3.Tablet clone forming experiments(Ca ski,CCD 841 Co N and HIEC):The grouping method was same as 2,2.5μM LB100 treated on Ca ski,CCD 841 Co N and HIEC 3 hours before irradiation(0Gy、2Gy、4Gy、6Gy).Removed LB-100 at 24 hours after radiation,then incubated cells with conventional medium until it formated the clone,then dying and counting.4.Flow cytometry apoptosis detection(Ca ski):The grouping method was same as 2,2.5μM LB100 and(or)radiation(6Gy)were treated on Ca ski,and then detected the apopotosis by FCM 24 hours and 48 hours after radiation.5.Flow cytometry cell cycle detection(Ca ski):Ca ski cell grouping method and teatment were same as 4.Cell-cycle was detected by flow cytometry at 24 huors after radiation.6.immunofluorescence staining(Ca ski):Ca ski cell was divided into control and LB-100 only group(LB).LB-100(2.5μM)treated on Ca ski for 48 hours,then give it Immunofluorescence staining(DAPI and Tublin).7.In vivo experiment:BALB/c mice were divided into 4 groups:control,LB-100 only(LB),radiation only(R)and LB-100 plus radiation(LB+R).Give them intraperitoneal injection LB-100(2.5mg/kg)or saline,then give them whole body radiation 11 Gy or 0Gy 3 hours latter.And then give them LB-100(2.5mg/kg)or saline intraperitoneal injection at 24 huors,48 hours and 72 hours after radiation once respectively,and watch their general condition at the same time.Then killed them by cervical dislocate and obtained their gut tissue,measured the small intestine,and stained the intestinal tissue by HE and Brdu.Results:1.The results of LB-100 concentration screening showed: When the concentration was less than 5μM,LB-100 had no cytotoxicity on the three kinds of cells above,but significantly inhibited those cells in a dose-dependent manner when the concentration was more than 5μM.2.The results of 48 hr cell activity detection showed:When compared with control,the cell viability of Ca ski were lower in LB group.But that of CCD 841 CoN and HIEC had no difference between control and LB group.LB+R group showed lower cell viability than R group in Cas ki,but the cell viability of CCD 841 CoN and HIEC in LB+R group were same with R group.3.The results of tablet clone forming experiments showed: The clone number of Ca ski and CCD 841 CoN in LB group were less than that in control.But there is no difference in HIEC clone number between control and LB group.And the clone number of three kinds of cells above in R group were all less than that in control.The clone number of Ca ski in LB+R were less than that in R group and LB group.The clone number of CD 841 CoN and HIEC in LB+R were no difference to that in R group.4.The results of flow cytometry apoptosis detection showed : The apoptosis rate of Ca ski in LB group and R group are more than that in control.And the apoptosis in LB+R was more than that in LB group and R group.5.The results of flow cytometry cell cycle detection showed: The rate of Ca ski cells in G0/G1 phase,LB group and R group was lower than control,but LB+R group was lower than LB group and R group.6.The results of immunofluorescence staining showed:The nucleus morphometry and the microtuble structure are normal in control.But there appeared a large number of abnormal and huge nuclear cells,and the microtuble structure was disappeared in LB group.7.The results of in vivo experiment showed: LB group compared with control,and LB+R group compared with R group,there is no difference in the aspects of general condition,body weight,small intestine length and histopathology.Conclusions:1.When the concentration of LB100 was less than 5μM,there was little cytotoxicity on Ca ski.So we can regard it as safe drug concentration,therefore,we chose lower concentration,2.5μM to conduct the latter experment.2.5μM LB-100 can enhance the radiation-sensitivity of Ca ski.The mechanism of LB-100 enhancing the radiationsensitivity of Ca ski might be it that LB-100 induced the apoptosis,promoted the process of cell cycle,damaged the microtuble structure,leaded to polyloid and abnormal nuclear,and leaded Ca ski to mitotic death at last.2.When the concentration of LB100 was less than 5μM,there was little cytotoxicity on CCD 841 Co N and HIEC,so we can regard it as safe drug concentration.2.5μM LB-100 had no effect on radiation-sensitivity of CCD 841 CoN and HIEC.And there was no side-effect about LB-100 on normal intestinal tissue of mouse.And LB-100 didn’t make the intestinal irradiation injury of mouse worse,however,it had no protective effect on it as well.