The Effect Of Proliferation And Apoptosis For Human Renal Clear Cell Adenocarcinoma Cell Through Inhibition Of PI3K/AKT Signaling Pathway By LY294002

Kidney cancer (takes cell carcinoma, RCC) is one of the most common malignant tumorin urinary system and kidney cancer onset hidden and difficult to be diagnosed early, highmortality, currently for T3previously dominated by surgical removal of the kidney, is worth apound of cure. Treatment of advanced metastatic kidney cancer mainly rely on biologicaltreatment, because of the lack of specific target, to inhibit and kill cancer cells is very limited.Therefore, desire for kidney cancer development and metastasis has a deep understanding,from genes or molecular level breach. PI3K-Akt signaling pathway is the study of a maturesignaling pathway and its abnormal activation state plays an important role in thetumorigenesis, including the occurrence of renal tumor, so PI3K-Akt signaling pathway isexpected to become the new target for malignant tumor treatment.LY294002is a kind of permeable cell specific PI3K inhibitors, its main function is tomake the PKB/Akt inactivation, thus inhibiting cell proliferation and induce cell apoptosis.LY294002effects on tumor cells, induce obvious nuclear pyknosis and reduce the volume ofthe cytoplasm. In colon cancer cells, making the phosphorylated Akt (Ser473) expressiondecreased, significantly inhibited the growth of tumor cells and induce apoptosis, LY294002role in cell apoptosis can be used to study the effect of PI3K activation pathway and therelationship between cancer cells apoptosis, however LY294002research in kidney is seldomreported. LY294002is applied in this article to act on human renal clear cell carcinoma cellline786-0, explore LY294002on renal clear cell, the influence of gland cancer cellproliferation and apoptosis and reveals LY294002role in kidney cancer cells, lay a foundation for the clinical treatment of renal cancer.Objective The purpose of this study is to apply high specific PI3K inhibitors2-(4-morpholine)-8-phenyl-4h-1-benzo pyran-4-ketone (LY294002) to act on humanrenal clear cell carcinoma cell line786-0, explore LY294002on the effects of renal clear cellcancer cell apoptosis and the mechanism, so as to lay a foundation to the research of theclinical treatment.Methods (1) Human renal clear cell carcinoma cell line786-0, cell model isestablished.(2) The logarithmic phase of renal clear cell cancer group training. With differentdrug concentrations LY294002(5μg/mL,10μg/mL,20μg/mL,40μg/mL) with renal clearcell cancer, determined by MTT method to detect the cancer cells in the role of different time(24h,48h,72h) under the OD value, calculate the proliferation inhibition rate, kidney cancercell concentration-inhibition rate curve drawing and time-inhibition rate curve. WithHoechst staining to observe the renal clear cell cancer cells, via LY294002(5μg/mL,10μg/mL,20μg/mLL,40μg/mL), the effect of different time (24h,48h,72h), the cellapoptosis.(3) Respectively by rt-pcr method to detect LY294002at different concentrationand time points of786-O cells after treatment of PI3K, mTOR, Akt mRNA expressionsituation, discuss the of PI3K, mTOR, Akt mRNA expression, the influence of and itsinfluence on786-O cell proliferation.Results (1) Through LY294002determined by MTT method in24,48,72h all showedproliferation inhibition, when the concentration of LY294002from1increased to40μg/mL,24h cells inhibition rate increased from6%to55%, action at48h cell inhibition rateincreased from12%to78%, at72h cells inhibition rate increased from14%to84%, at thesame time different concentrations of the cells compared with negative control group, theinhibition rate was statistically significant (P≤0.05), suggesting as LY294002concentrationincreases, the role of786-O cell proliferation inhibition increased gradually. With invertedmicroscope LY294002medication for786-O cell growth and form, the influence of different concentration LY294002action at48h cell morphological changes the most typical. Resultsshow that, compared with normal cells, LY294002can inhibit the cell growth by differentlevels of, express the change of the concentration dependence. Normal cell morphology, theedge is clear, cells arranged closely, and with the increase of drug concentration, the drugunder the action of cells floating cannot stick wall, normally it become pyknotic cells, celldensity decreased.(2) By Hoechst staining in fluorescence microscope the nuclei are uniformdispersion in light blue fluorescence; Cell apoptosis, cell nucleus or cytoplasm concentrateddye dense can be seen in the block light blue fluorescence; Relative to the786-0cells withoutmedication1μg/mL48h after drug treatment, cell morphology has not appeared obviouschange; Drug treatment is2.5μg/mL individual cells appear chromatin condensation, appearapoptotic body;5μg/mL, chromatin fluorescence enhancement drug treatment, a increase inthe number of cells of apoptosis body;10μg/mL drug treatment is further increased apoptosisof cells;20μg/mL medication visible break through the cell membrane of apoptosis bodybegins to release;40μg/mL small handle most visible apoptosis of drug release, complete theapoptosis.(3) In LY294002processing cells48h, using1,2.5,5,10,20,30,40u g/mLconcentration treatment, PI3K, and Akt, mTOR mRNA expression significantly suppressed,and presents with the increase of drug concentration changes occur inhibition degree isproportional relationship; Cell proliferation experiments show that with the extension of drugconcentration and its role in time, the role of tumor inhibition increased gradually.Conclusion Our results found that LY294002can significantly inhibit human renalclear cell gland cancer cells786-O cells in vitro proliferation, the proliferation inhibition wastime and dose dependence, in small doses of786-O cells had significant effect on themorphology and growth; At the same time LY294002can promote the apoptosis of786-Ocells. LY294002can restrain through inhibition of PI3K/Akt signal transduction pathway of786-O cell proliferation and induce its apoptosis. PI3K/Akt signal transduction pathway maybe involved in the occurrence of kidney development, inducing tumor cell apoptosis may be LY294002important give play to the role of anti-cancer mechanism. PI3K/Akt may become anew kidney cancer treatment targets, using LY294002may bring the Gospel of clinicaltreatment in patients with kidney cancer.