Sensitization Of Nicotinamide For Non-Focused Ultrasound On Human Pulmonary Carcinoma Cell Line GLC-82

BACKGROUD: High intensity focused ultrasound (HIFU) has the potential toprovide the clinician with a truly noninvasive, targeted treatment option in thetreatment of cancers. Studies have indicated that HIFU caused tissue and cell damagethrough three predominant mechanisms. The first is through mechanical effect, thesecond is by the conversion of mechanical energy into heat, and the last one isthrough cavitation. However, the use of HIFU is limited to the treatment of sharplydefined solid tumors; However, when tumor cells are situated in hydrothroax andhydroperitonia, or the malignant diffusely infiltrate into tissues, it is difficult forHIFU to kill them. Thus far, there has been no research report about this question. Inthis study we aim to solve this problem with non-focused ultrasound (NFU). Theeffects of cavatition erosion and caloric effect of ultrasound can cause change in thebimolecular lipid leaflet structure of the cytomembrane, increase fluidity andpermeability, induce membranolysis. And raise cytotoxicity of chemotherapeutics.,damage DNA, prevent cell proliferation and promote cell apoptosis. However, theintensity of NFU is a limitation, it will cause widespread damage to viable tissues ifthe dosage of NFU is increased.Nicotinamide (NA) sensitizes the radiation breakdown of tumor cells through two main mechanisms. One is by increasing tumor cell sensitivity to radiation, andthe second is through preventing DNA damage repairing. Thus, NA can also be asensitizer for NFU, can not only increase cell sensitivity to NFU, but also can preventcell DNA repair after damage by NFU. So, NA has synergistic effect with NFU, aswell as plays as a sensitizer in enhancing the effect of NFU. The sensitization of NAcan increase the damage of NFU to tumor cells, as a result of which the feasibility oflow dose-intensity ultrasound in tumor therapy with low damages to normal adjacenttissues become true. Therefor, whether NA can be used as a sensitizer to enhance theeffect of NFU in tumor therapy, there were no reports about this in previous literature.OBJECTIVE AND SIGNIFICANCE: The objective of this study is to investigatewhether NA can increase the effects of NFU when tumor cells are situated inhydrothroax and hydroperitonia, or malignant cells infiltrating into tissue, throughincreasing cells sensitivity to NFU. Providing basis for lung cancer therapy in clinical.METERIAILS AND METHODS:一,The Effects of Non-Focused Ultrasound on GLC-82 Cell live in SuspensionExperimental groups:1,Groups dependent on dosage: Cells were divided into 5 groups, the intensities ofNFU were 0W/cm²,20W/cm²,30W/cm²,40W/cm²,50W/cm²,time was 5s.2,Groups dependent on time: Cells were divided into 5 groups, the times of NFUwere 0s,5s,10s,15s,20s, the intensities was 20 W/cm².Methods:Survival rates of GLC-82 cells irradiated by NFU with different intensitiesand times were determined based on the Trypan blue exclusive assay; MTTassay was used to evaluate survival rates 24 hours after irradiated by NFU; the percentage changes of apoptosis and the distributional percentage of cell cyclewere analyzed with flow cytometry.二,Study on the Influence of Nicotinamide on GLC-82 Cells in vitroExperimental groups:Cells were divided into 6 groups, the concentration of NA were 0mg/ml,1mg/ml,2mg/ml,3mg/ml,4mg/ml and 5 mg/ml.Methods:Morphologic change of cells were observed with fluorescence microscope;MTT assay was used to evaluate survival rates Survival rates of GLC-82 cellsThe influence of NA to cell migration was analyzed by scarification test;formation of clone cell colony; the percentage changes of apoptosis and thedistributional percentage of cell cycle were analyzed with flow cytometry.三,Sensitization of Nicotinamide for Non-Focused Ultrasound on GLC-82 CellsExperimental groups:Cells were divided into Control,NA,NFU and NFU-NA four groups, GroupControl (Blank group): Normal GLC-82 Cells; Group NA (pure NA group): theconcentration of NA was 3mg/ml; Group NFU (pure NFU group): Without NA, NFUwas 30 W/cm²Ã—5 S; Group NFU-NA: Given NA(concentration was 3 mg/ml) 30 minpre-irradiation with NFU (30 W/cm²Ã—5 S).Methods:MTT assay was used to evaluate survival rates 24 hours after irradiation by NFUand culture in the presence of Nicotinamide at different concentrations; Morphologicchange of cells were observed with light microscope, fluorescence microscope andscanning electron microscope; Formation of clone cell colony; Nucleolus-organizingregion was tested by silver staining; the percentage changes of apoptosis and thedistributional percentage of cell cycle were analyzed with flow cytometry. RESULTS AND DISCUSSION:一,The Effects of None Focused Ultrasound on GLC-82 Cell live in Suspension1,The instant survival rates of GLC-82 cells went down obviously as theintensities and times of NFU exposure was raised;2,Cell debris and dead cells increased with increasing intensities and times ofNFU even 24 hours after exposure;3,24 hours after irradiation, the suvival rates of GLC-82 cells among groups ofdifferent intensities and groups of different times were compared, all of thesedifferences were statistically significant (P＜0.05);4,The percentage of apoptotic cells in irradiated groups were higher thanunirradiated groups, among all, instant and 24th hr apoptosis rates of group 30 W/cm²were also high than other groups.二,Study on the Influence of Nicotinamide on GLC-82 Cells in vitro1,Nicotinamide at 1-5mg/ml induced apoptosis of GLC-82 cells in adose-dependent manner, and group 4 mg/ml and group 5 mg/ml showed increasedapoptosis than other groups. Distinct morphologic changes of cell apoptosis such askaryopyknosis and conglomeration were observed;2,Nicotinamide also had evident dose-dependent inhibitory function to GLC-82cells, and IC₅₀ of 24 hour higher than other times;3,Cell migration became slower as the dosage of Nicotinamide increased, 80h,the scratching distance of negative control group almost disappeared, and groups 1-3mg/ml became definately narrow, but groups 4-5 mg/ml were wider than at 0 hr;Accompanying parts of cells flowing in the culture fluid and intercellular spacebecome obviously less;4,Cells produced an arrested at G₁ phase, accompanied by a reduction of cellstransiting through S phase, respectively at 3 mg/ml-5 mg/ml, and the percentages of apoptosis in groups 4 mg/ml and 5 mg/ml were higher than other groups, they were20.1% and 27.2 %, respectively.三,Sensitization of Nicotinamide for None Focused Ultrasound on GLC-82Cells1,The cytotoxicity of NA increased in a dose-dependent manner following24-hour treatment, with the optimal dose range of 1 mg/ml-5 mg/ml. A sub-toxic doseof NA at 3 mg/ml was used in the subsequent experiments;2,NA significantly reduced the growth rates and enhanced the sensitization forNFU on GLC-82 cells with SER of 1.765, 2.108 and 2.352 at the doses of 3 mg/ml,4mg/ml and 5 mg/ml, repectively; After treatment with NFU-NA, microvilli andfilipodium of GLC-82 cells reduced, shortened and shrunk into corpuscule-shape;3,The cloning efficiency of NFU-NA was exposed cells 2.2%, less than theothers; Treated with NFU-NA the pathological karyokinesis were fewer, butkaryopyknosis and karyorrhexis was more than other groups;4,The level of AgNORs decreased obviously after the treatment of NFU-NA,the number of AgNORs was went down5,The cell cycle of GLC-82 cells was arrested at G2-phase, and the percentagesof GLC-82 cells in S and G2 phase were19.0%,24.5% and 43.6% after exposure to 30W/cm² NFU, NA 3 mg/ml, and NFU-NA at 24h, respectively.CONCLUSION:1,Non-focused ultrasound can inhibit the growth of GLC-82 cells in suspension,and low intensities of NFU mainly induce apoptosis of GLC-82 cells while highintensities kill these cells. NFU has tardive effect to the distributional percentages ofcell cycle.2,As the dosage of Nicotinamide increasing, the survival rates of GLC-82 Cellsincreased, while apoptosis rate went down, and the cell cycle of GLC-82 cells was arrested at G1-phase.3,Nicotinamide can enhance the effect of NFU on GLC-82 cells in suspension.