Lethal Effects And Mechanism Of Nicotinamide For None-Focused Ultrasound On Different Pulmonary Adenocarcinoma Cell Lines In Suspension

BackgroundMalignant pleural effusion is a common complication of patients with cancer, especially lung cancer.In most cases,pleural effusion is the rusults of disease progression and recurrence and indicates unfavorable prognosis.Lung cancer (about 35%) is the most common reason,predominantly lung adenocarcinoma. Treatment aimed directly at hydrothorax can abate or eliminate symptoms and raise or improve survival quality of cancer patients.The commonly used method,local injection of chemotherapeutics after chemotherapy all over the body coupled with thoracentesis,is not effective to malignant hydrothorax caused by lung adenocarcinoma.This method may cause sever injuries to normal cells unselectively while killing cancer cells,which may jeopardize cancer patients. Repeated application of chemotherapeutics results in the resistance of tumor cells to drugs,which is an important factor for effectiveness of chemotherapy.Multidrug resistance is one of the main reasons of chemotherapy failure.As a result,novel treatment with good selectivity and minor side effects has become the hot spot of research.Ultrasound treatment is a novel developed technique for tumor treatment after physical treatments like tumor thermotherapy,freezing and microwave.Searches on tumor treatment by high intensity focused ultrasound(HIFU) have been widely reported.However,when tumor cells are within the hydrothorax,hydroperitonia, or diffused in tissues,it is not likely to kill these cells by HIFU.Previous researches conducted in our laboratory indicate that using NFU to kill tumor cells in suspension is an effective method.During the spread of ultrasound in tissues,its energy can decrease exponentially with the increase of the spreading distance, which causes decreased energy in target areas and decreased therapeutic effects.Nicotinamide(NA),an acid amide derivative of nicotinic acid,and moity of coenzymeâ… and coenzymeâ…¡,plays a role in hydrogen transmission during biological oxidation,and promotes biological oxidation and metabolism.It has a fairly wide rage of safety.Only large dosage can lead to vitaminosis.In recent years,it has drawn special attention used as a kind of radiosensitizer.Can we enhance the lethal effects of NFU to tumor cells in suspension using NA?What is the killing mechanism of the unification? Are the lethal effects in conformity? Dose it cause injury to normal cells while killing cancer cells? What is the effectivenese compared with chemotherapeutics? In order to elucidate these questions,we design this study answer these questions.Objective and SignificanceExplore whether NA can enhance the lethal effects of NFU to tumor cells and the killing mechanism,and identify the specificity.Compare the effectiveness of NFU sensitized with NA and four chemotherapeutics for lung cancer.Provide experimental bases for the clinical treatment of malignant pleural effusion with NFU sensitized with NA.Methods1.Lethal effects of NFU sensitized with NA on human pulmonary carcinoma cell line A549 in suspension(1)Material and groupsHuman pulmonary adenocarcinoma cell line A549 Blank control group;NA group;NFU group;NA sensitization NFU group(2)Determination of experimental doses through measuring the inhibition ratio and sensitization ratioWe observe cytostasis ratio with MTT colorimetric method.The concentrations of NA were 0,1,2,3,4 and 5mg/ml respectively.The dosage of ultrasound wave irradiation was increased from 0W/cm²Ã—5s to 70W/cm²Ã—5s. SPSS13.0 was used to obtain the Inhibitory concentration 50(IC₅₀) and sensitization ratio(SER).(3)MethodsWe observed the morphologic changes of A549 both directly and after HE stain with inverted microscope.Instant and 24 hours' survival rates of A549 cells treated by different methods were determined based on the trypan blue exclusive assay.Growth rates were detected by MTT after 24 hours.The percentage changes of apoptosis and the distributional percentage of cell cycle were analyzed with flow cytometry.The capability of cell colony formation was detected by clone formation test.2.Lethal Mechanisms of NFU sensitized with NA on human pulmonary carcinoma cell line A549 in suspension(1)Material and groupsHuman pulmonary adenocarcinoma cell line A549Blank control group;NA group;NFU group;NA sensitization NFU group(2)MethodsNucleolar organi-zer regions staining were used to observe the argyrophilic nucleolar organi-zer regions(AgNOR)grains.The apoptotic changes of four groups were analyzed with Hoechst33342 fluorescence staining,AO/EB double fluorescence staining and transmission electron microscope.Apoptosis DNA Ladder was studied by Agarose Gel Electrophoresis.Changes of cell:count was obtained by observing cell culture fluid color and counting chamber.Immunocytochemistry was used to detect expressions of p53,Bcl-2 and Ki-67.Protein changes in cell culture fluid and with in cells were detected by BCA method and protein gel electrophoresis. 3.Lethal Effect Comparisons of None Focused Ultrasound sensitized with nicotinamide on different human pulmonary adenocarcinoma cell Lines and immortalized bronchus epithelial cells(1)Cell LinesFive cell lines were chosen.Human pulmonary adenocarcinoma cell line A549, GLC-82,GLC-82/CDDP(resistant to cisplatin),mouse Lewis pulmonary adenocarcinoma cell line LLC,and human immortalized bronchus epithelium HBE.(2)GroupsBlank control group;NA group;NFU group;NA sensitization NFU group(3)MethodsShock and intermitant method of large dose were used to induce GLC-82/CDDP cells.MTT was used to measure the inhibition ratio of GLC-82 and GLC-82/CDDP in response to different concentration cisplatin was calculated IC₅₀ value and Resistance Index(RI).Instant and 24 hours'survival rates treated by different methods were determined based on the trypan blue exclusive assay. Growth rates were detected by MTT after 24 hours.The percentage changes of apoptosis were analyzed with flow cytometry.Survival rates,growth rates and apoptosis rates of five cells were compared after different procedures.4.Comparisons of lethal effect between None Focused Ultrasound sensitized with nicotinamide and four chemotherapeutics(1)Cell LinesHuman pulmonary adenocarcinoma cell line A549,GLC-82,GLC-82/CDDP and human immortalized bronchus epithelium HBE.(2)Chemotherapeutics and concentrationCDDP:concentration in vitro(plasma drug concentration)13.6μg/mlPTX:concentration in vitro(plasma drug concentration)119μg/mlGEM:concentration in vitro(plasma drug concentration)850μg/mlBLM:concentration in vitro(plasma drug concentration)3μg/ml(3)GroupsSix groups were divided.NA-NFU 10s group;NA-NFU 5s group;DDP group; PTX group;GEM group;BLM group(4)MethodsInhibition ratios were detected by MTT.Lethal effect were according to the inhibition ratios.Results1.Lethal effects of NFU sensitized with NA on human pulmonary carcinoma cell line A549 in suspension(1) We found microscopically that large amount of cell debris and charring-like materials were produced after NA-NFU treatment.Cells became shrinked,round,low refractive or floated.Obsered after HE staining, morphological changes of A549 were very obrious.The cells become slender with scarce cytoplasm.The number of pyknosis and karyolysis was increased.The pathological mitosis was decreased dramatically.(2) The lethal effect of NFU was improved significantly when NA concentration was≥3mg/ml.The sensitization and dosage were positively correlated in a certain range(2-5mg/ml).Under the circumstances of 30W/cm²Ã—5s NFU sensitized with 3mg/ml NA,IC₅₀ was near 50%with an inhibition ratio of 51.48%,which was settled as experimental dose.(3) We found that there was no significance among the instant survival rate,24 hours'survival rate,growth rate;clone formation capability,apoptotic rate,cell cycle changes and the negative control group when NA(3mg/ml) was used solely (P＞0.05).(4) The instant survival rate,24 hours' survival rate,24 hour growth rate and clone formation capability were significantly different among four groups(P＜0.05). The instant survival rate,24 hours' survival rate,24 hour growth rate and clone formation capability were decreased significantly in groups treated with NA combined with NFU compared with the other three groups.Twenty-four hours' apoptotic rate was increased dramatically(P＜0.05).2.The lethal Mechanism of NFU sensitized with NA on human pulmonary carcinoma cell line A549 in suspension(1) Endonuclear AgNOR particles were changed markedly both in quantity and distribution after NA-NFU treatment.Quantity was reduced or absent. There were statistical significances compared with control group,NA group and NFU group(P＜0.05).Growth rate was descended(P＜0.05).Growth curves were flat.NA was interacted with NFU.NA could enhance the inhibition of NFU to A549 cells.(2) Typical apoptosis changes could be seen under fluorescence microscope with Hoechst33342 fluorescence staining,AO/EB double fluorescence staining and transmission electron microscope after 24h of NA-NFU treatment.Typical DNA Ladder strip was obtained from Agarose Gel electrophotogram.(3) There were more cells in NFU and NA-NFU before treatment than those after treatment(P＜0.05).There was no signiticance between the control group and NA group(P＞0.05).(4) Immunohistochemisty revealed that:p53 was expressed in small amount of cells in NFU group and NA-NFU group.The expressions in control and NA were negative.Bcl-2 was negative in four groups.Ki-67 was positive in four groups: NA-NFU＞NFU＞Control＞NA.This was different from MTT.We considered this as false positive results due to the presence of antigens after cell injuries.(5) Protein concentration was raised in cell culture fluid but descended intra-cellularly.This was more obvious in NA-NFU group.This showed that NFU could destroy cell membrane or increase permeability.3.Lethal effect comparisons of None Focused Ultrasound sensitized with uicotinamide on different human pulmonary adenocarcinoma cell line and immortalization bronchus epithelium(1) Differences of inhibition ratio between GLC-82 and GLC-82/CDDP were significant(P＜0.001).IC₅₀ of GLC-82 was 0.190μg/ml CDDP.IC₅₀ of GLC-82/CDDP was 1.444μg/ml CDDP.RI was 7.6.(2) Survival rates,growth rates and apoptosis rates of five cells were not significant after NFU only.After sensitized with nicotinamide survival rates and growth rates were significantly different(P＜0.05).Survival rates of human immortalization bronchus epithelium HBE was higher than that of human pulmonary adenocarcinoma cell line A549,GLC-82,and GLC-82/CDDP(P＜0.05).Survival rates of mouse Lewis pulmonary adenocarcinoma cell line LLC was lower than those four cells above(P＜0.05).4.Comparisons of lethal effect between None Focused Ultrasound sensitized with nicotinamide and four chemotherapeutics(1) The inhibition ratio in NA combined with NFU 10s group was higher than NA combined NFU 5s group.The inhibition ratios with CDDP and PTX were higher than NA combined with NFU.GEM and BLM were lower.(2) The inhibition ratio in HBE as a control was a little lower than pulmonary adenocarcinoma cell line A549,GLC-82,and GLC-82/CDDP after NFU treatment. The inhibition ratio in GLC-82/CDDP was lower than other three cells obviously. HBE was lower than A549 and GLC-82 only after PTX treatment,while the difference was not significance with other three chemotherapeutics.(3) The highest inhibition ratio was exhibited on the first day,then lower on the second and third day.The opposite situation was shown in chemotherapeutics.Conclusions1.NA-NFU has lethal effects on some pulmonary carcinoma cell lines,but it is not better than chemotherapeutics.NA-NFU shows advantages over resistant cells. It also causes injury to normal cells while killing cancer cells,which are slight, compered to chemotherapeutics.2.NA(3mg/ml) has little lethal effects on cells.NFU can reduce survival ratio, suppress cell multiplication,increase permeability of cell membrane and induce apoptosis.Some cells are made into fragmentation directly.This is related to mechanical effect,cavatition erosion and thermal effect of ultrasound.NA plays sensitizing effects mainly through inhibiting injury and repair abilities of cells.3.The lethal effect of NFU is improved significantly when NA concentration is≥3mg/ml.