Study On The Pharmacodynamics And Mechanism Of Curcumol Against Human Lung Cancer H460 Cells

The problem at present is still accepted the treatment of cancer,the incidence and mortality of lung cancer in our country is expected to continue to high.The selectivity of traditional chemotherapeutic drugs had low efficiency.While killing cancer cells,it also causes some damage to some normal cells in the body with serious impact on quality of life and even life.So it is extremely important to study the targeted therapy of lung cancer and to find new anti-lung cancer drugs with high toxicity and low toxicity.The traditional Chinese medicine with a long history had great potential of patent medicine in the field of unknown treatment.The application of modern molecular pharmacology to the evaluation of drug efficiency and the clarifying of its mechanism of action can provide a novel idea for the modernization of Chinese medicine.Therefore,it is of great significance to explore the anti-cancer effect of curcumol and to clarify the target of its action.This article explores the efficacy of curcumol in the following aspects:The first section:Study of the effect of curcumol on the growth of lung cancer cells in vitro:?1?The effective concentration and time of curcumol to inhibit the growth of H460 lung cancer cells was screened by MTT method.The MRC-5 normal lung fibroblast cells were used to measure the toxicity of curcumol.The results showed that the inhibition of the growth of human lung cancer H460 cells was more obvious with the increase of curcumol concentration and the prolongation of time.The IC₅₀0 values are 248.2?mol/L?24 h?,165.3?mol/L?48 h?and 105.3?mol/L?72 h?.There was no observable toxic effect on human normal lung MRC-5 cells.?2?Trypan blue staining experiment found that the IC₅₀0 values of curcumol against H460 were 307.8?mol/L?24h?,175.9?mol/L?48 h?and 112.4?mol/L?72 h?.It was utilized to verify the above conclusions.?3?By cell cloning experiments proved that curcumol inhibited the proliferation of H460 cells.The cell clone formation rates of curcumol were 59.63%?20?mol/L?,20.42%?40?mol/L?,0.52%?80?mol/L?and 0%?160?mol/L?,respectively.The second section:Study on the anti-tumor effect of curcumol on H460 tumor bearing nude mice:?1?The acute toxicity test of mice was designed.And the median lethal dose(LD₅₀)of curcumol was calculated.According to the dose of LD50,the dosage of curcumol in nude mice was measured?70?mol/L.That is 165.4 mg/kg?.?2?The H460 tumor bearing nude mice models was established,including control group,gastric lavage group,intravenous injection group,curcumol gavage group,curcumol intravenous injection group and positive?-elemene group.The results showed that the tumor volume inhibition rate and tumor weight inhibition rate of curcumol?at 165.4mg/kg doses?were 37.61%and 37.45%.There were no significant difference in the organ coefficient and body weight growth curve of curcumol group compared with the model group,indicating that curcumol had no significant side effects on mice under the treatment dose.The third section:After confirming the anti-tumor pharmacodynamics of curcumol in vivo and in vitro,we systematically studied its anti-tumor mechanism.The main contents of this section were as follows:?1?Flow cytometry was used to detect the H460 cells cyclical changes of curcumol incubated.The results showed that the role of curcumol in anti-cancer H460 cells were to block the cell proliferation in the G₀/G₁phase,at the same time,the apoptotic peak appeared at high concentration.?2?Qualitative observation of morphologic changes in H460 cells after curcumol incubated by Hoechst33258 staining.The results of cell nuclear staining suggest that curcumol?100?mol/L?can induce apoptosis.?3?Detection of apoptosis rate of H460 cells treated by curcumol by Annexin V/PI double staining.The results showed that the apoptosis rate of curcumol?100?mol/L?was 86.75%.?4?The changes in mitochondrial membrane potential of H460 cells were monitored by flow cytometry to reflect the cell apoptosis.The results suggest that curcumol can lead to a significant decrease in mitochondrial membrane potential?p<0.05?.?5?The activity of Caspase3 in H460 cells was determined by spectrophotometer.The results showed that Caspase3activity in H460 cells was significantly increased compared with that in control group after curcumol treatment.The fourth section:On the basis of the previous work,we further study the resistance of curcumol to H460 lung cancer cells from molecular mechanism.?1?Used Western Blot test to screen the expression changes of protein and signal pathway such as AKT-mTOR,JAK2-STAT3,BAX-BCL-2,Caspase3 and PTEN to determine the signal pathway regulated by curcumol in inducing H460 cells apoptosis.It was found that curcumol can play an important role by down regulating the protein expression level of JAK2 and STAT3 and increasing the expression level of Cleaved-Caspase3protein in H460 cells.?2?The qualitative analysis of the target protein or related signaling pathway protein by immunofluorescence?IF?method.The results showed that the JAK2 and Caspase3 proteins were mainly distributed in the cytoplasm,and the STAT3 protein was distributed in the cytoplasm and nucleus.?3?Further,detection by IF method,Western Blot method and qRT-PCR method respectively from the qualitative to quantitative analysis in protein level and mRNA level after curcumol treatment in H460 cells,which the expression of JAK2,STAT3,p-JAK2?Tyr-1007/1008?,p-STAT3?Tyr-705?and Caspase3 proteins were down-regulation,simultaneously,the expression of Cleaved-Caspase3 protein was up-regulation.It elucidated that the JAK2-STAT3 signaling pathway is an important target for the anti-tumor effect of curcumol.?4?HE staining,Western Blot,immunohistochemistry?IHC?and qRT-PCR were used to detect the expression of JAK2-STAT3 signaling pathway in tumor tissues from qualitative to quantitative in protein level and mRNA level respectively to elucidate the mechanism and target of curcumol.It was suggested that the expression of JAK2 and STAT3 protein in the tumor tissue specimens will provide a basis for the accurate application of curcumol.In summary,Chinese medicine monomer of curcumol has a good application prospect in anti-lung cancer.This article will be thoroughly studied and fully elucidated to provide a precise treatment for the clinical use of curcumol.