| BackgroundCarcinoma of the lungs have long ranked first in terms of incidence and mortality nationwide and are mainly classified into non-small cell lung cancer（NSCLC）and small cell lung cancer（SCLC）according to the classification.At present,the clinical treatment is mainly based on chemotherapy combined with surgery,but in the process of clinical application,traditional drugs gradually appear toxic side effects and even drug resistance phenomenon,and the above multiple factors limit the effectiveness of clinical chemotherapy treatment with drugs.Thus,it is essential to investigate a novel,effective anti-NSCLC drug with little side effects.In recent years,Anticancer drugs extracted from natural biological compounds of plant and marine products have received wide interest.Algae are salty herbs with softening and dispersing properties and have been documented to be used in cancer treatment.Mougeotia nummuloides and Spirulina major belong to natural drugs,which have shown better antitumor,anti-inflammatory,and antioxidant effects in pharmacological studies.1-Monopalmitin（1-Mono）is a monomeric compound extracted from Mougeotia nummuloides and Spirulina major,and its effects in NSCLC have not been reported.Therefore,the purpose of this paper is to explore the effect of 1-Mono on lung cancer cells and its molecular mechanism and to make a pre-experimental exploration for 1-Mono to step into clinical research and treatment.MethodsWe purchased the 1-Mono and cultured NSCLC cells A549 and SPC-A1 in vitro,measured the cell growth activity by using the CCK8 technique,calculated the half-inhibitory concentration(IC50)based on the measured results and determined the appropriate drug concentration for subsequent experiments.Firstly,the effects of 1-Mono on cell biological behaviors such as proliferation,cycle and apoptosis of lung cancer cells were investigated by morphological observation assay,CCK8 assay,Ed U labeling assay,crystalline violet assay and scratching assay.Subsequently,the mechanisms related to the anti-tumor effects induced by 1-Mono were investigated and elucidated by experimental methods such as Western blot and FACS.In this study,NSCLC cell lines（A549 and SPC-A1）were studied to investigate the effects of 1-Mono on the biological functions of NSCLC and the specific molecular mechanisms of its effects,and to elucidate its importance in the treatment of NSCLC.Focusing on the effect of 1-Mono on the expression of autophagy and PI3K/Akt signaling pathway-related proteins in NSCLC cells,and the effect of inhibiting autophagy and PI3K/Akt expression on the anti-NSCLC effect of 1-Mono were studied.This experiment was conducted in six sections.Results1.1-Mono suppressed the growth of lung cancer cells in a dose-dependent way.Firstly,after treating A549 and SPC-A1 cells with different concentrations of 1-Mono for 48 h,cell activity measurement by CCK8 test,and it was concluded that 1-Mono could effectively reduce the proliferation of A549 and SPC-A1 cells in a dose-dependent manner.The semi-inhibitory concentration values were calculated to be 50.12μg/m L and 58.30μg/m L for A549 and SPC-A1 cells,respectively.1-Mono was insensitive to the normal cell line HBE（semi-inhibitory concentration=161.34μg/m L）,proved 1-Mono have a low cytotoxicity.Next,Ed U staining was used to demonstrate that 1-Mono could reduce cell proliferation activity by inhibiting cellular DNA synthesis.Crystalline violet staining further confirmed that 1-Mono could reduce the proliferation of NSCLC.In conclusion,we found that 1-Mono could inhibit the proliferation of NSCLC cells.2.1-Mono induced cell cycle arrest in NSCLC at G2/M phase.Propidium iodide staining was used to study the effect of 1-Mono on the cell cycle distribution of lung cancer cells.It was concluded that 1-Mono suppressed the division of A549 and SPC-A1 cells and blocked them at the G2/M phase.Subsequently,Western blot observed that the expression of Cyclin D1 was lowered and that of p21 was elevated after1-Mono treatment,confirming that 1-Mono induced G2/M block by regulating p21 and Cyclin D1.In conclusion,1-Mono evoked G2/M blockade in lung cancer cells by regulating Cyclin D1 and p21.3.1-Mono inhibits the migration of NSCLC cellsThe effect of 1-Mono on the migration of lung cancer cells was primarily accomplished by scratch assays.The results were photographed after treated A549 and SPC-A1 cells with different concentrations of 1-Mono（0μg/m L and 50μg/m L）for 0 h,24h,and 48 h,respectively.And showed that the scratch repair ability of the 1-Mono-treated group（50μg/m L）at 0 h,24 h,and 48 h was remarkably lower than that of the control group（0μg/m L）for A549 and SPC-A1.It is suggested that 1-Mono inhibits the migration in A549 and SPC-A1 cells.4.1-Mono killed lung cancer cells mainly through induction of apoptosis.The percentage of apoptosis occurrence induced by 1-Mono in a dose-dependent manner was verified by flow cytometry assay.1-Mono also dramatically enhanced Caspase-3 activation and its downstream substrate PARP cleavage（two markers commonly used to detect apoptosis）.In addition,Western blot results showed that the expression of IAPs（c-IAP1,c-IAP2,RIP1,RIP3,XIAP,Survivin）（major regulators of cell death）proteins was markedly inhibited after 1-Mono treatment.Finally,1-Mono markedly facilitated the accumulation of reactive oxygen species（ROS）levels,which play a key role in apoptosis.The Western blot analysis suggested that 1-Mono also induces the phosphorylation level of H2A.X,which is integral to DNA fragmentation during apoptosis.It is suggested that 1-Mono mediates cytotoxicity through the induction of apoptosis.5.1-Mono induces protective autophagy in NSCLC.1-Mono induced p62 degradation and accumulation of LC3-II,which was further verified by autophagic flux assay.Inhibition of autophagic activity with the autophagy inhibitor chloroquine enhanced 1-Mono-induced cytotoxicity significantly,suggesting that autophagy may be a cytoprotective mechanism.6.PI3K/Akt pathway mediates 1-Mono-induced antitumor effects.Western blot results showed that 1-Mono over-activated the phosphorylation of PI3K and Akt.Both inhibition of PI3K/Akt activity using LY294002 and Wortmannin,respectively,effectively reversed 1-Mono-induced cytotoxicity.Taken together,these results suggest that PI3K/Akt acts an essential role in 1-Mono-induced cytotoxicity.ConclusionIn conclusion,our data indicate that 1-Mono kills cancer cells mainly by inducing apoptosis.Our data also suggest that 1-Mono induced cytoprotective autophagy,as the autophagy inhibitor chloroquine significantly enhanced 1-Mono-induced cytotoxicity.Furthermore,we study the function of PI3K/Akt pathway in 1-Mono cytotoxicity,Further evidence showed 1-Mono stimulated the PI3K/Akt pathway,as inhibition of PI3K/Akt activity by LY294002 and Wortmannin partially attenuated 1-Mono-mediated anticancer activity,indicating that 1-Mono-induced antitumor effect was dependent on the PI3K/Akt pathway.In conclusion,the results of our studies indicate that 1-Mono kills lung cancer through the PI3K/Akt pathway,providing a new option for developing therapeutic pathways for the administration of drugs in lung cancer.Further experiments in vitro and vivo are still necessary to validate these genes and to analyze other potential molecular mechanisms. |
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