The Molecular Mechanisms Of Cellular Senescence Induced By Shikonin In NSCLC

ObjectiveNon-small-cell lung cancer(NSCLC),predominant subclassfication of lung cancer,leads high incidence and mortality annually worldwide,is the most common malignancy.Cancer therapeutics are primarily thought to work by inducing apoptosis in tumor cells.However,the importance of cellular senescence,which is a stress response that stably blocks proliferation,is increasingly being recognized.Senescence is prevalent in premalignant tumours,and progression to malignancy requires evading senescence.Malignant tumours,however,may still undergo senescence owing to interventions that restore tumour suppressor function or trigger senescent signals.Cellular senescence is regard as a critical physiological barrier against tumor progression.Senescent tumour cells can be cleared by immune cells,which may result in efficient tumour regression.Standard chemotherapy also has the potential to induce senescence,which may partly underlie its therapeutic activity.Although these concepts are well supported in animal models,translating them to clinical oncology remains a challenge.Lithospermum erythrorhizon has been used as medicine in China for thousands of years.Shikonin(SHK),derived from Lithospermum erythrorhizon,has showed detoxification,anti-inflammatory,antitumor,and antiviral effects in clinical treatments.Evidences indicated that SHK has showed great biological activities for inhibiting various cancers,including colon cancer,breast cancer,gastric cancer,lung cancer and so on.It is reported that SHK could induce apoptosis,necrosis,and premature senescence in A549 cancer cells.However,the underlying mechanisms of SHK-induced and senescence in lung cancer cells are still unclear.Based on above information,this study was aimed to investigate the effects of shikonin on non-small-cell lung cancer,and explore the related signal pathways involved in senescence.Findings of our study can provide deeper theoretical basis in the development of drug and clinical application of lung cancer.MethodsCell viability was measure by MTT assay.EdU assay was performed to detect the proliferation of A549 and H1299 cells.Flow cytometry was used to detect the cell cycle,ROS,apoptosis and autophagy.Cells and frozen sections were stained with SA-?-Gal to identify senescent cells.Westrn blot was used to detect the signaling pathway proteins.Mice were subjected to subcutaneously injection with A459 cells in each right flank.When the tumors reached a mean volume of 50 mm3,eligible mice were randomized into four groups as following:gavage control(sterilized coin oil),afatinib(5 mg/kg),SHK(5,10 mg/kg).Tumor tissues were fixed,embedded in paraffin,and then sliced up(4 ?m).Then slices were incubated with diluted anti-p-H2A.X and anti-p53 overnight at 4 ?.Following steps were performed using the immunostaining kit based on the manufacturer's instructions.Results1.SHK suppressed lung cancer cell proliferation via arresting cell cycle at G0/G1 phaseSHK dose-and time-dependently suppressed viable cell percentages in A549 and H1299 cells,and the respective IC50 were 2.28± 0.19 p M and 0.64±0.07?M at 72 hr.In contrast to the abundant EdU-positive cells observed in control(Ctrl,0.1%DMSO,same as follows),SHK at 1 and 2 ?M significantly suppressed A549 cell proliferation.Simultaneously,SHK at 2 ?M notably increased the percentage of A549 cells at G0/G1 phase from 45.79%± 2.27%in control treatment to 54.12%± 0.10%.Similarly,remarkable inhibition of EdU fluorescence(range,36.51%-67.44%)were also found in H1299 cells after treated with 0.3 and 0.6 ?M of SHK.And 0.3 and 0.6 ?M of SHK also markedly arrested H1299 cells at G0/G1 phase by 13.78%± 0.58%and 19.81%± 0.47%.Compared to control treatment,SHK at 2 and 0.6 ?M significantly decreased the expression of cyclin D1.2.SHK notably promoted cell senescence in A549 and H1299 cellsAfter treated with SHK,the chromosomes in control were markedly condensed into numerous SAHF focus.In addition,compare to H1299 cells,SAHF focus was abundantly condensed in A549 cells,suggesting A549 was more sensitive for SHK-induced senescence.Furthermore,both in A549 and H1299 cells,SHK remarkably altered cellular morphology from spindle and round to flat and enlarged,and the presence of vacuoles were also observed,especially in A549 cells.Meanwhile,the proportions of senescent cells,positively labeled by?-Gal,were also gradually increased by SHK in A549 and H1299 cells.3.SHK induced cellular senescence through stimulating ROS generation and subsequently triggering DNA damage-p53/p21waf axisRas and MEK-1 expressions were unexpectedly decreased by SHK,meanwhile,no significant differences were observed on Rb and p16 expressions.On the contrary,ROS production was notably increased by SHK.After SHK treatment,we found that the expression of Kdm2b was significantly decreased in A549 and H1299 cells.In contrast,the protein level of H3K36me2 was dramatically increased with remarkable inductions.In A549 cells,SHK effectively stimulated p53 transcription,and p21waf expression was subsequently induced.Meanwhile,the phosphorylated p53 in nucleus was significantly increased by SHK.In p53-deleted H1299 cells,we also observed that SHK at 0.3 and 0.6 ?M could markedly induced p21waf protein level by 1.76-and 2.00-fold,respectively.Compared to control treatment,SHK enhanced the expressions of p-H2A.X,sensor for DNA damage,respectively in A549 and H1299 cells.4.SHK-induced senescence was specifically dependent on ROS generationNAC pretreatment notably attenuated SHK-induced intracellular ROS generation by 89.55%± 0.99%and 75.52%± 1.35%in A549 and H1299 cells,respectively.In contrast to gradually suppressed cell viability in SHK treatments,pretreated with NAC successfully reversed the cytotoxicity of SHK in A549 and H1299 cells.At the same time,SA-?-Gal staining results also showed that NAC pretreatment markedly reduced the percentages of SHK-induced senescent cells by 82.64%± 5.68%and 73.47%± 6.63%in A549 and H1299 cells,respectively.And the morphologies of SHK-induced enlarged and flat senescent cells were also effectively reversed by NAC to spindle and round.Moreover,SHK-induced p53 activation was also completely reversed by pretreated with NAC.Additionally,immunofluorescence results also revealed that both in A549 and H1299 cells,SHK-induced p-H2A.X overexpressions were dramatically diminished by NAC pretreatment.5.SHK-induced apoptosis was dependent on ROS generationCompared to control treatment,SHK significantly increased early and late phases of apoptosis.The percentage of total apoptosis is extremely low(range,1.31%-6.11%).Moreover,western blot results also showed that compared with control treatment,SHK markedly decreased the expression of anti-apoptosis protein Bcl-2,while that of pro-apoptotic Bax and Cleaved Caspase-3 were significantly increased.Meanwhile,SHK simultaneously decreased Bcl-2/Bax ratio.Moreover,NAC pretreatment significantly diminished SHK-induced apoptosis from 8.87%± 1.17%to 2.70%± 0.17%in A549 cells.6.SHK suppressed lung cancer growth in A549 xenograft murine modelNo significant alterations on average body weight were observed among treatments.Meanwhile,compared to control treatment(Corn oil),SHK,either at 5 mg/kg or at 10 mg/kg,did not remarkably alter the tissue indexes including heart,liver,spleen,lung,and kidney,suggesting no systemic toxicity was observed after SHK treatments.Notably,SHK dose-dependently suppressed tumor growth and tumor size.Meanwhile,compared to control,SHK at 5 mg/kg and 10 mg/kg also significantly decreased the tumor weights by 28.57%and 55.84%,respectively.Compared to control treatment,SHK treatments dramatically increased the percentages of ?-Gal-positive senescent cells in tumor tissues.In addition,the expressions of p-H2A.X and p53 were also remarkably decreased by SHK in a dose-dependent manner.These results suggested SHK could suppress lung tumorigenesis through stimulating cellular senescence.ConclusionsIn conclusion,in vitro,SHK could induce cellular senescence through stimulating ROS generation and subsequently triggering DNA damage-p53/p21waf axis.Moreover,in A549-xenograft model,SHK has the same anti-cancer effects,which was scarcely reported in previous studies.SHK,a novel ROS-dependent senescence inducer,could serve as a promising agent for further lung cancer treatment.