The Correlation Of Radiosensitizing Effect Of Elemene To Anoxia Lung Cancer Cells With MTOR And HIF-1α/Survivin Signal Pathway

Lung cancer is a malignant tumor threaten to human life worldwide with themortality is1.3million and the non-small-cell lung carcinoma is about80%. Moreadvanced stage patients when diagnosed received modality therapy of radiation andchemotherapy remain the main treatment for them. Researches found that more anoxictumor cells were presented in tumor tissue and the resistances of anoxic cells to radialline caused failure of tumor radiotherapy. To overcome the anoxia condition, peoplesimmediately or indirectly increased oxygen content in tumor tissue but the effectivenesswas also poor. So develop and study tumor anoxic cell radiosensitizer to increase theradiosensitivity become a hot topic in the radiotherapy studies.Elemene has antitumor effects and also augments the sensitivity to radiotherapyand studies have already confirmed that. Our group discovered that the radiosensitizingeffects of elemene to lung adenocarcinoma A549cell were related with G2/M blocking;up-regulating Bcl-2and down-regulating p53expression resulted to cell apoptosis. Theeffction also related with inhibiting the telomerase activation, inducing DNA damageand inhibiting the repair of DNA damage. Also in vitro study confirmed that elemene incombination with irradiation could improve the tumor anoxic condition to playradiosensitizing effect. Index documents and found that there were few reports about thesignal pass which related with theradiosesiting effects of elemene.Mammalian target of rapamycin (mTOR), Hypoxia inducible factor1alpha (HIF-α)and Survivin have overexpression in tumors and have close association in rumorigenesisand growth. Also, mTOR and HIF-α are in touch with proliferation, invasion andmigration of tumor cells. If the radiosensitizing effect of elemene has relationship withmTOR and HIF-α/Survivin signal pass, there are few reports about it. This is why to engineer the discussion. The in vivo study of our experimental group detected thatelemene in combination with irradiation could decrease HIF-α and Survivin expressioninduced by irradiation and the inhibition to mTOR was higher than elemene alone, thusto increase the cell apoptosis in tumor. Prompted that mTOR, HIF-α and Survivinmaybe the target points of radiosensitizing effect of elemene. But if it had role mTOR toaffect HIF-α and Survivin was not clearly. The in vitro study of radiosensitizing effectof elemene was under normoxia condition in the past. So in this study, we will detectthe the radiosensitizing effect of elemene to lung adenocarcinoma A549cell andsquamous cell carcinoma H520cell. Also, we probe the effection to mTOR, HIF-1α andSurvivin expression when combine elemene and irradiation to anoxia lung cancer cells.Also, the accommodation of mTOR and HIF-1α/Survivin to cytoactive and the relationto radiosensitizing effect of elemene are under detected.Objective:1. Using the light concentration of elemene on anoxia A549and H520cells toapproach the radiosensitizing effect of elemene, and study the influnce of elemene withirradiation to mTOR, HIF-1α and Survivin in anoxic lung cancer A549and H520cells.2. To validate the regulate relation of mTOR and HIF-1α/Survivin signal pass andobserve the inflrence of the two pathways to A549and H520cell proliferation, invasionand migratory.3. To investigate the correlation of radiosensitizing effect of elemene to lungcancer cells with mTOR and HIF-1α/Survivin signal pathway.Methods:Part one: The influence to mTOR, HIF-1α and Survivin expression ofradiosensitizing effect of elemene to anoxia A549and H520cells.1. MTT method to detect the lethal effect with different concentration of elemeneto anoxia A549and H520cells and to vent the IC50of A549and H520cells whenelemene effect for24h.2. Clone formation to observe the radiosensitizing effect of light concentration ofelemene to anoxia A549and H520cells.3. Grouped A549and H520cells into normoxia control, anoxia control, anoxiaelemene, anoxia irradiation and anoxia combination groups. After20μg/ml β-elemenefor24h, puted the anoxia group cells into anoxia box for30min then cultured innormoxia incubator.Taked out the irradiation and combination groups to receive X-rayirradiation2h later and culture for24h. To detect the expression of mTOR, HIF-1α and Survivin in lung cancer cells using RT-PCR and WseternBlot methods.4. Gived the same groups and treatments above-mentioned. To detect cellapoptosis of A549and H520by flow cytometer.Part two: The influence of mTOR and HIF-1α/Survivin signal pathway to anoxiaA549and H520cell proliferation, invasion and migration.1. Divided A549cells into un-transfect, negative transfect and mTOR-siRNA orHIF-1α-siRNA transfect groups. To detecte the mTOR and HIF-1α expression in A549cell to make sure the silence efficiency by RT-PCR. Then to detecte the HIF-1α andSurvivin expression after silencing mTOR and Survivin expression after silencingHIF-1α using RT-PCR and WesternBlot methods.2. Divided A549cells into the same groups above-mentioned. MTT method usedto detect the absorbance of lung cancer cells to analysis fell proliferation. Transwellused to detect the ability of invasion and migration of lung caner cells. Flow cytometryused to detect cell apoptosis.Part three: The correlation of radiosensitizing effect of elemene to A549andH520cell with mTOR and HIF-1α/Survivin signal pathway.1. Divided A549cells into un-transfect and transfect groups, then divided intothree groups each: elemene, irradiaton and combination groups. The transfect groupsfirst transfect the siRNA into lung cancer cells for6h. After giving the drug with24h, todeal all groups with anoxia and continue to incubation for24h. The cells that needed toirradiate were gived after anoxia. Cells were incubated for24h after theabove-mentioned process, detecting the express of HIF-1α and Survivin after silencingmTOR and the Survivin expression after silencing HIF-1α by RT-PCR andWesternBlot.2. Detected the cell apoptosis in each group by flow cytometer.Results:Part one:1. The different concentration of elemene had the inhibition effect toanoxia A549and H520cells with increasing by the concentration raising. At the sameconcentration, the inhibition to H520cell was higher than A549cell. The IC50of A549cell was112μg/ml and H520cell was93μg/ml.2. Clone formation to decte the sensetive effect of elemene to lung cancer cells: Atthe same irradiation dose, the cell survival of A549and H520cells were reducing alongwith the dose increasing. The light concentration of elemene with irradiation causedlower cell survival than irradiation alone. 3. The expression of mTOR, HIF-1α and Survivin in A549and H520cells: inA549cells, compared to the normoxia group, the expression of mTOR and HIF-1α wereincreased in different levels and had statistical significance (mTOR: p<0.05; HIF-1α:p<0.01). Survivin mRNA and protein expression were increased in different levels(protein: p<0.01; mRNA: p>0.05). In H520cells, the mTOR, HIF-1α and SurvivinmRNA expression were increased compaired to the normoxia group (p<0.01).4. The mTOR expression in anoxia A549and H520cells: in A549cell, the mTORmRNA and protein expression in the elemene group and irradiation group wereincreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to anoxiacontrol group. The expression of mTOR mRNA and protein in combination group weredecreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to elemenegroup. The mTOR mRNA and protein expression in the joint group were also decreasedin different levels (mRNA: p<0.01; protein: p>0.05) compaired to irradiation group. InH520cell, the mTOR mRNA expression in anoxia elemene, irradiation groups werelower than the anoxia control group (p<0.05) and in combination group was decreasedcompaired to elemene and irradiation group (p<0.01).5. The expression of HIF-1α in anoxia A549and H520cells: in A549cell, theHIF-1α mRNA and protein expression in the elemene group were decreasedsignificantly contrasted to the anoxia group (p<0.01), but the HIF-1α mRNA andprotein expression in irradiation group were increased in defferent levels (mRNA:p<0.01; protein: p>0.05). The HIF-1α mRNA and protein expression in thecombination group were obviously decreased contrasted to elemene and irradiationgroups (p<0.01). In H520cell, compaired to the anoxia control, the HIF-1α mRNAexpression in the anoxia elemene group was decreased (p<0.01) and in anoxiairradiation group was increased (p>0.05). The HIF-1α mRNA expression incombinition group was lower than the elemene and irradiation groups (p<0.01).6. The expression of Survivin in anoxia A549and H520cells: Contrast to theanoxia control, in A549cell, Survivin mRNA and protein expression in elemene groupand mRNA expression in irradiation were decreased (p<0.01), but the proteinexpression in irradiation group were increased (protein: p>0.05). Survivin mRNA andprotein expression in the combination group were decreased significantly contrasted toelemene and irradiation groups (p<0.01). In H520cell, Survivin mRNA and proteinexpression in elemene and irradiation groups were decreased compaired to anoxiacontrol group (p<0.01) and Survivin mRNA in the combination group was decreased contrasted to elemene and irradiation groups (p<0.01).7. The cell apoptosis in lung cancer cells: in A549cell, apoptosis in control groupwas higher than normoxia group (p>0.05) and lower than the rest and has statisticalsignificance (p<0.01). The cell apoptosis in combination group was higher thanelemene group and irradiation group (p<0.01). The same tendency was observed inH520cell.Part two:1. Screening of mTOR and HIF-1α-siRNA: Transfected mTOR siRNA and HIF-1αsiRNA into A549and H520cells to make sure the gene silence effectivencess. ThemTOR and HIF-1α mRNA expression were both ihibited after transfecting (p<0.01).2. The expression of HIF-1α and Survivin after transfecting with mTOR siRNA inA549and H520cells: in A549cell, contrasted to control and negative groups, HIF-1αand Survivin mRNA and protein expression were decreased when the mTOR expressionwas inhibited (p<0.01). In H520cell, both HIF-1α and Survivin mRNA expressionwere decreased (p<0.01).3. The expression of Survivin after transfecting with HIF-1α siRNA in A549andH520cells: in A549cell, compaired to control and negative groups, Survivin mRNAand protein expression were decreased when the HIF-1α expression was inhibited(p<0.01). In H520cell, Survivin mRNA expression had the same tendency compared tocontrol and negative groups (p<0.01).4. The cell apoptosis of A549and H520cells after transfect: contrasted to controlgroups, the apoptosis were increased in the mTOR or HIF-1α siRNA transfect cells andhad statistical significance (p<0.01).5. Cell proliferation: Absorbances of A549and H520cells after transfect withmTOR or HIF-1α siRNA were decreased contrasted to control groups (p<0.01).6. Cell invasion and migeration: The numbers of A549and H520cells passedMatrigel and microporous membrane of Transwell were decreased compared to controlgroups (p<0.01).Part three:1. The expression of mTOR and HIF-1α after transfecting with mTOR or HIF-1αsiRNA in lung cancer cells: in A549cell, mTOR and HIF-1α mRNA and proteinexpression were decreased obviously in the transfect groups contrasted to theun-transfect groups (p<0.01). In H520cell, mTOR and HIF-1α mRNA expression werealso decreased compaired to corresp un-transfect groups (p<0.01). 2. The expression of HIF-1α and Survivin after transfecting with mTOR-siRNA inlung cancer cell: in A549cell, the HIF-1α and Survivin mRNA in elemene, irradiationand combination groups were decreased a little in every group (p>0.05) whendown-regulated mTOR, but the HIF-1α protein expression were decreased obviouslyand had statistical significance (p<0.01) compared to corresp un-transfect groups.Survivin mRNA and protein expression in A549cell were decreased compared toun-transfect groups (p<0.01). In H520cell, HIF-1α mRNA expression in elemene andcombination groups were decreased a little (p>0.05) but in irradiation group wasdecreased obviously (p<0.01) contrasted to un-transfect groups. Survivin mRNAexpression in transfect groups of H520cell were down-regulated after mTOR wasdecreased (p<0.01).3. The expression of Survivin after transfecting with HIF-1α-siRNA in lung cancercells: in A549cell, Survivin mRNA and protein expression in elemene, irradiation andcombination groups were increased (p<0.01) compared to corresp un-transfect groups.In H520cell, Survivin mRNA expression were also increased obviously in everytransfect groups contrasted to corresp un-transfect groups (p<0.01).4. The cell apoptosis of lung cancer cells: The apoptosis in transfect groups ofA549and H520cells were increased contrast to the corresp un-transfect groups and hadstatistical significance (p<0.01).Conclusion:Part one:1. The light concentration of elemene had radiosensitizing effect to anoxia A549and H520cells and rose up along with the concentration increasing.2. Hypoxia could increase mTOR, HIF-1α and Survivin expression in differentlevels in A549and H520cells and cell apoptosis were decreased. So improved thatmTOR, HIF-1α and Survivin were correlated with tumor cell survival in anoxiccondiation.3. Elemene in combination of irradiation could decrease the expression of HIF-1αand Survivin in anoxia A549and H520cells. Meanwhile the inhibition to mTOR wasobviously contrasted to elemene alone. All illustrate that mTOR, HIF-1α and Survivinwere the targets of radiosensitizing effect of elemene.4. Elemene in combination of irradiation could increase apoptosis of anoxia A549and H520cells. Imply that elemene possible promoted the tumor cell apoptosis througheffecting to mTOR, HIF-1α and Survivin. Part two:1. After down-regulating the mTOR expression, the expression of HIF-1α andSurvivin in anoxia A549and H520cells were decreased and cell apoptosis wereincreased. The Survivin expression is decreased when HIF-1α is down-regulated andthe lung cancer cell apoptosis were increased. Prompt that mTOR could influence theactivity of HIF-1α to regulate Survivin expression to induce cell apoptosis.2. Down-regulation of mTOR could decrease HIF-1α and Survivin expression andSurvivin expression was down-regulated after HIF-1α was inhibated. Both mTOR andHIF-1α was inhibated could reduce the A549and H520cell proliferation, invasion andmigeration. Confirm that mTOR and HIF-1α/Survivin signal pass possibly related withthe regulation of cytoactive of A549and H520cells.Part three:1. Elemene and irradiation decreased HIF-1α and Suvivin expression in A549andH520cells after silencing mTOR and cell apoptosis also decreased. Imply that theradiosensitizing effect of elemene possible not through regulating HIF-1α/Suvivinexpression by acting to mTOR. Maybe there were other pathways to regulateHIF-1α/Suvivin expression to induce apoptosis.2. Survivin levels were increased when Elemene and irradiation effected A549andH520cells after silencing HIF-1α but cell apoptosis were increased. Imply that theradiosensitizing effect of elemene possible correlated with the regulation ofHIF-1α/Survivin.