The Synergetic Killing Effect Of Cispiatin And Radiation On Lung Cancer Cells: Via Both Autophagy And Apoptosis

Although great progress has been made in the diagnosis and treatment of cancer, lungcancer is still the leading cause of death worldwide. The overall5-year survival rate of lungcaner is only15.9%.At diagnosis,15%case is at the local stage. In22%case, the tumor hasmetastasized to the regional lymph nodes or invaded to the nearby structures directly while in56%case distant metastasis has taken place, demonstrating that only a few patients has theopportunity of curative surgery while most patients only can rely on radiotherapy andchemotherapy.During the process of radiotherapy and chemotherapy, multiple changes about growtharrest and death pathway can take place. Traditionally, irradiation can induce cell deathmainly through apoptosis. However, recent results show that autophagy play an important rolein irradiation induced cell death which can help to restore and improve radiosensitivity.Autophagy is a highly conserved cellular process with the degradation of intracellularorganelles and long-lived protein to remain homeostasis.Autophagy has been appreciated toact as a double-edged sword in the cancer initiation, development and metastasis. On onehand, autophagy plays an anti-tumor role. On the other hand, autophagy, an adaptive response,protects tumor cells to survive against stress, once autophagy is inhibited, the therapeuticeffect is supposed to be enhanced remarkably. Cisplatin, a cell cycle nonspecificantineoplastic agent, is usually viewed as a radiosensizer during chemoradiotherapy becauseCisplatin can effectively inhibit the repair of sublethal damage resulted from irradiation.Our research aim to explore the effect and mechanism of synergetic killing effect ofinduced by different fractionation protocols and the combination of Cisplatin in vitro and vivo,find out the optimum of fraction and combination and offer promising option for the clinicalapplication. Objective: Cisplatin is a common used drug for chemotherapy, but if it could be usedsynergetically with radiotherapy is still unclear. In this study, the synergetic kills effects ofCisplatin and radiation on lung cancer and the underlying mechanisms were explored both invitro and tumor xenograft in vivo.Methods: A549cells were used for in vitro experiments and divided into four groups:(A) Sham-irradiation;(B) CRT (conventional radiotherapy);(C) HRT (hyper fractionationradiotherapy);(D) CRT+cisplatin (conventional radiotherapy combined with cisplatin).Xenograft of Tumor-bearing C57BL/6model was established and the above-mentionedtreatments were introduced, MTT and colony formation assay were used to detect cellviability; Western blot was used to detect protein expression; monodansylcadaverine (MDC)staining and immunofluorescence technique was used to analyze autophagy rate, flowcytometry and TUNEL assay were used to analyze apoptosis rate, immunohistochemisty wereused to detect expression of genes related to apoptosis and autophagy.Results:1-Cisplatin increase the killing effects induced by radiation.A549cells were seeded into a96-well plate after70%-80%confluence cells were treatedwith various doses of cisplatin for48h, the viability of the cells was analyzed with MTT assay.Cisplatin showed dose-dependent cytotoxicity on A549cells (p<0.05). Using colonyformation assay, the responses of A549cells to radiation were analyzed with or without5μM of Cisplatin. Cisplatin enhanced the radiation-induced cytotoxicity on A459cells ascompared with cells treated with IR alone. We used colony formation and IHC studies to finda similar trend when treated with fractionated IR in the absence or presence of Cisplatin.These findings indicate that Cisplatin potently suppresses A549tumor cells growth andsynergizes with radiation to promote the cell-killing effect of radiation.2-Combination of Cisplatin with radiation yields better killing effects in vivo.To verify the effects of Cisplatin and radiation on tumor growth in vivo, Lewis cells wereimplanted to establish the exograft model.When the exograft growed to same size, mice wererandomly grouped and treated as described in material and methords. Compared with controlgroups, the tumor size with different treatments, especial HRT+Cisplatin treatment wassmaller. These data provide additional evidence that the combination of Cisplatin with IR ledto promotion of the radiotherapy outcome. 3-Cisplatin enhances the radiation-induced autophagyNext, we sought to delineate the underlying mechanism that stands behind the synergisticeffect of Cisplatin and radiation. Autophagy has been shown to play paradoxical role duringcancer radio-therapy under a certain circumstance, either confer to radioresistance, or enhancethe radiation-induced cytotoxicity. Exposure of A549cells to fractionated IR resulted insignificant elevation of autophagy rates as indicated by MDC staining. Cisplatin promoted theIR-induced autophagy. During the process of autophagy, cytoplasmic MAPLC3-I protein(ATG-8homologue) is converted to lapidated form, MAPLC3-II which tightly bind toautophagosome membrane, also Beclin-1was up regulated upon exposure of A459cells tofractionated IR.4-Cisplatin enhances the IR-induced apoptosisApoptosis has been well appreciated to be the main killing mechanism after thetreatment of radiation. Fractionated IR up-regulated apoptosis significantly in A459cells, wefound that Cisplatin markedly promoted the fractionated IR-induced apoptosis. To elucidatethe underlying mechanism, the cleaved caspase-3in the irradiated cells evaluated at48h forboth fractionated IR and fractionated IR with Cisplatin. The results demonstrated that thecaspase-3protein procession is involved in fractionated IR induced apoptosis. The productionof the active cleaved fragment of caspase-3after fractionated IR treatment supported theinvolvement of this protease in apoptosis. Caspase-3processing was more enhanced aftercombining Cisplatin with radiation as compared with radiation alone.In A549we found that fractionated IR resulted in dramatic increase in p21expression,which was further enhanced when we added Cisplatin to radiation. Such findings suggest thatCisplatin promotes the radiation-induced apoptosis, through activation of caspase-3procession and p21expression.5-The combination effect of Cisplatin and IR on autophagy regulatory genesAfter we confirmed the association between combination therapy and autophagy, weinvestigated the impact of radiation alone and radiation plus Cisplatin on diverse genes thatare well appreciated to play crucial roles in autophagy signaling pathways. Using IHCanalysis, we found that combining Cisplatin with fractionated IR resulted in more significantelevation of PI3KIII and Beclin-1expression (p<0.05) than exposure to fractionated IR alone(p<0.05). Class I PI3Ks activates Akt/PKB by phosphorylation, which in turn inhibitsautophagy, the level of phosphorylated Akt (p-Akt) was declined after exposure tofractionated IR in the presence or absence of Cisplatin. DRAM1gene has been reported topromote the autophagy process. We found that DRAM1showed marked up-regulation upon exposure of A459cells to the combined treatment as compared with radiation alone.MAPLC3-II protein (which indicates autophagosome formation) was up-regulatedsignificantly after exposure to radiation with or without Cisplatin (p<0.05). These findingsindicate that Cisplatin, synergistically with radiation, triggers autophagy signaling pathways.6-The impact of combination therapy on apoptosis related genesBcl-2and Bax constitute crucial part in the apoptotic machinery. As shown in UsingTUNEL assay and IHC analysis, the combined Cisplatin and fractionated IR therapy resultedin more significant decrease of Bcl-2expression (p<0.05) than in fractionated IR alone(p<0.05). On the other hand, Bax expression was significantly elevated after differenttreatment. Conversely, P21expression was markedly up-regulated after combined therapy.These results indicate that combination of Cisplatin with radiation affects apoptosis signalinggenes more potently than radiation alone does.Conclusion1. Cisplatin can increase the killing effects induced by radiation both in vitro and vivo.2. Cisplatin and radiation can execute synergetic killing effect on lung cancer cells viaboth autophagy and apoptosis.3. Cisplatin enhances the radiation-induced autophagy via inhibition of PI3KI/Aktpathway and up-regulation of DRAM1and Beclin-1gene.4. Cisplatin enhances the IR-induced apoptosis via inhibition of Bcl-2and up-regulationof P21and Bax gene.Summarily, our research offered strong evidence to adding Cisplatin to radiation tostrengthen the killing effect of radiation by means of both pro-apoptosis and pro-autophagycell death.