Plumbagin Induces Apoptotic And Autophagic Cell Death Through Inhibition Of The PI3K/Akt/mTOR Pathway In Human Non-small Cell Lung Cancer Cells

Background and Objecitive:Lung cancer is the leading cause for cancer-related mortality in humans comprising of two major types:small-cell lung cancer （SCLC） and non-small-cell lung cancer （NSCLC）. NSCLC accounts for85%of all cases of lung cancers. There is an increasing trend of incidence of cancer with an estimation of228,190new cases of lung cancer in America, which accounting for14%of cancer diagnosis. Although current therapeutic strategies for the treatment of NSCLC have made advancement by the platinum-based standard initial treatment with other agents, the treatment options for NSCLC remain insufficient. In addition, the average5-year survival rate is about17%which has not been significantly improved over the last40years. It highlights the need for new therapeutics either to provide a cure or even an improved survival rate. Identifying new agents and characterizing the underlying mechanism will provide more effective therapeutics with fewer side effects for NSCLC treatmen. Plumbagin （5-hydroxy-2-methyl-1,4-naphthoquinone）, a second metabolite naturally occurs in Plumbaginaceae, Droseraceae and Ebenceae with various pharmacological activities, including antioxidant, antiinflammatory, anticancer, antibacterial and antifungal activities in vitro and in vivo. Of note, it has been reported that plumbagin exhibited anti-proliferative effect various cancer cell lines via different actions. These bioactive activities of plumbagin are mainly attributed to its capability of inducing oxidative stress, apoptosis and cell cycle arrest. The anticancer effect of plumbagin in NSCLC has been reported as well. It has been showed that plumbagin induced apoptosis in A549by inhibiting the activation of NF-kB or p53accumulation via c-JNK phosphorylation. However, the anticancer mechanisms of plumbagin remain inconclusive.Therefore, in order to elucidate the underlying mechanism（s） of plumbagin in NSCLC, two NSCLC cell lines A549, H23will be used in this study. Previous research indicated that the anti-cancer effect related to the pro-apoptosis and pro-autophagy ability of plumbagin. We first time researched both the mechanisms of apoptosis and autophagy, and their relationship.Which gave a new way to cure NSCLC.Methods:1. Cell lines and cell cultureThe NSCLC cell lines A549and H23were obtained from the American Type Culture Collection （Manassas, VA） and cultured in RPMI-1640medium containing L-glutamine, phenol red, L-cystine and L-methionine, sodium bicarbonate and sup-plemented with10%heat-inactivated fetal bovine serum. The cells were maintained at37℃in a5%CO2/95%air humidified incubator. PLB was dissolved in DMSO, and was freshly diluted to the desired concentration with culture medium. The final concentration of DMSO was at0.05%（v/v）. The control cells received the vehicle only.2. Cell viability assay （WST-1assay）A549and H23cells were plated in96-well culture plates （1X104 cells/well）. After24h incubation, the cells were treated with vehicle alone （0.1%DMSO） and plumbagin （0,1,5,10,25,50μM） for24h. General viability of cultured cells was determined by reduction of a water-soluble tetrazolium salt （WST-1） as described else where.3. Quantification of apoptosisAfter treated with plumbagin （0,0.5,3,6,10μM） for24h, Apoptotic cells were quantitated using AnnexinV:PE apoptosis detection kit （BD Pharmingen Biosciences, USA）. Cells were trypsinized, washed twice with cold PBS and resuspended in1×binding buffer at a concentration of1×105/ml cells in a total volume of100μl To this,5μl of PE Annexin V-FITC and5μl of7-AAD were added. The tubes were gently mixed and incubated in the dark for15mins at room temperature. Four hundred microliters of1×binding buffer was then added to each tube and the number of apoptotic cells was analyzed by flow cytometry within1h. DMSO-treated control cells were used to serve as PE Annexin V-or7-AAD-only control.4. Cell cycle analysis using flow cytometeryTo determine cell cycle distribution, A549and H23cells treated with0,1,3,6μM plumbagin for24h, stained by propidium iodide （PI）. Analysis was performed with flow cytometry according to a previously published procedure.5. Measurement of intracellular reacitve oxygen species （ROS） levelsIntracellular ROS levels were measured by a fluorometer using CM-H2DCFDA （Invitrogen, Carlsbad, CA, USA）, a chloromethyl derivative of H2DCFDA. CM-H2DCFDA is a fluorescent dye which diffuses through cell membranes and is hydrolyzed by intracellular esterases to DCFH. In the presence of ROS, DCFH is oxidized to DCF, which is highly fluorescent and can be detected by flow cytometry. Briefly, cells were cultured on96-well microplate at density of1×104cells/well. After treatment for24hr, the cells were incubated with5μM CM-H2DCFDA in Dulbecco’s phosphate buffered saline （PBS） for30min. Fluorescence intensity was analyzed at an excitation wavelength of485nm and emission wavelength of530nm.6. Western blot analysisMonoclonal or polyclonal antibodies specific for p53, p21, cyclin B, cdc2, Puma, Bcl-2, cytochrome C, caspase9, caspase3were purchased from Cell Signaling Technology （Danvers, MA, USA）, and for actin from Santa Cruz Biotechnology （Santa Cruz, CA, USA）. A549and H23cells were untreated or treated with plumbagin （0,0.5,1,2.5μM） for24h, after which cells were collected and lysed, then protein was extracted. After sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were transferred onto immobilon nitrocellulose membrane （Millipore） at200mA for3hr at4℃. Blots were probed with indicated primary antibody. Immune complexes were detected using anti-mouse or anti-rabbit peroxidase-conjugated secondary immunoglobulin G antibody （Boehringer Mannheim, Mannheim, Germany） and visualized by electro-chemiluminescence Western Blotting Detection Reagents （Amersham, Piscataway, NJ）.7. Analysis of the uptake and subcellular distribution of PLB by confocal microscopyThe drug uptake process was observed using confocal microscopy. Cells were plated onto8wells chamber slide, and incubated over night, then were treated with plumbagin for24h,12h,6h,4h,2h, Oh. After being washed with PBS for3times, the cells were fixed by Para formaldehyde and dried, then were stained with DAPI which can stain nucleus, covered with the cover glass and sealed by polish oil. Samples were observed by Leica TCS SP2laser scanning confocal microscope using405nm （DAPI） and488nm （plumbagin） wavelength. 8. Determination of autophagyA549and H23cells were seeded at an intensity of5₁04cells/well in a24-well plate with glass black bottom （In Vitro Scientific Inc, Sunnyvale, CA）. After24h incubation, the cells were treated with fresh medium alone, control vehicle alone （0.05%DMSO, v/v）, or PLB（0.5,2.5, and101M） for24h at37_C.To check the effect, of autophagy inducers on PLB-induced autophagy, cells were treated with2.51M, PLB plus the autophagy inducer SB202190（201M） or SB203580（201M） for24h. To examine the effect of autophagy inhibitors on PLB-induced autophagy, cells were pre-treated with chloroquine （101M）, bafilomycin （0.11M）, or wortmannnin （101M） for1h, then treated with2.51M PLB. All autophagy inducers and inhibi-tors were dissolved in DMSO at a final concentration of0.05%（v/v）. In the mean-time,1511of CellLight_Lysosomes-RFP （a modified baculovirus expressing a fusion construct of a lysosomal marker and red fluorescent protein, from Invitrogen Inc. Carlsbad, CA） was added to each well. After incubation for24h, cells were examined for presence of autophagic puncta using a Leica TCS SP2laser scanning confocal microscopy （Wetzlar, Germany） at wavelengths of555/584nm. The fluorescence was read using a Synergy H4Hybrid microplate reader （BioTek Inc, Winooski, VT） at wavelengths of555/584nm. The WST-1assay was performed to monitor cell viability. In addition, the expression levels of autophagy-associated proteins including p38, p-p38, Akt, p-Akt, p-mTOR, mTOR, beclinl, and LC3I/D in both A549and H23cells treated with PLB with or without the presence of an autophagy inducer or inhibitor were determined using Western blotting analysis.9. Simultaneous determination of apoptosis and autophagy using flow cytometry To explore the potential crosstalk between PLB-induced apoptosis and autophagy, we further determined the two modes of programmed cell death simultaneously. A549 and H23cells were plated in6-well cell culture plates （Corning, NY） at an intensity1.5X105cells per well. After overnight incubation, the cells were treated with fresh medium, vehicle alone （0.05%DMSO, v/v）, or PLB at0.5,2.5, and10μM for24h. For comparison purpose, the autophagy inducer SB202190（20μM）, autopahagy inhibitor wortmannnin （10μM）, or ROS inhibitor GSH （1mM） were used alone or in combination with2.5μM PLB to treat the cells for24h. At the end of the treatment, the cells were trypsinized and centrifuged at100g for5min to pellet the cells. The cells were divided into two equal volume279samples, and washed with PBS. For apoptosis detection using annexin V, each sample of cells was resuspended in1ml binding buffer （10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaC12, fitered through0.2μm filter）. An quota of cell suspension （19511） was mixed with511Annexin-ATTO647N （Enzo life science, CA, No.ALX-209-259-T100）, and incubated for10min in dark. Cells were washed once using PBS and resuspended in19011binding buffer,1011of20lg/ml propidium iodide （PI, Sigma-Aldrich Inc, MO, No. P4170） was added, and the cells were subject to apoptotic analysis using flow cytometry （Becton Dickinson Immunocytometry Systems, San Jose, CA）. For autophagy detection, each sample was washed by resuspending the cell pellet in1_assay buffer （Enzo Life Sciences Inc, Farmingdale, NY, No. ENZ-51031-K200） and collected by centrifugation. Cells were resuspended in25011of phenol red-free culture medium （Invitrogen, Carlsbad, CA, No.1294895） containing5%FBS, and25011of the diluted Cyto-ID_Green stain solution （Enzo Life Sciences Inc, Farmingdale, NY, No. ENZ-51031-K200） was added to each sample and mixed well. Cells were incubated for30min at37℃in dark, collected by centrifugation, washed with1_assay buffer, and resuspended in500μl fresh assay buffer. Cells were analyzed using the green （FL1） channel of a flow cytometer（Becton Dickinson Immunocytometry Systems, San Jose, CA）. Results:The IC50value for PLB was10.87μM in A549cells and7.80μM in H23cells, a concentration-dependent increase in the cell number in G2/M phase in both325.A549and H23cells after PLB treatment. PLB induced remarkable cell cycle arrest and down-regulation of Cdc2and cyclin B1in A549and H23cells. PLB up-regulates p53and p21expression in A549, but not in H23cells.PLB induces the apoptosis of A549and H23cells, treatment of A549and H23cells with PLB at0.5-10μM significantly increased the expression level of Bax, but significantly decreased that of Bcl-2in a concentration-dependent manner, resulting in significantly increased Bax/Bcl-2ratio values. Treatment of with PLB at0.5-2.51M significantlyincreased the expression level of PUMA in a concentration-dependent manner in A549cells, but not increased in H23cells. Treatment of A549and H23cells with PLB significantly increased the release of cytochrome c from mitochondria in aconcentration-dependent manner.Cleaved caspase-3（Asp175） and caspase-9（Asp315） were activated by PLB concentration-dependently in both A549and H23cells. Incubation of PLB induces the generation of intracellular ROS in A549and H23cells. Antioxidants attenuates PLB-induced ROS production and growth inhibition and in A549and H23cells.PLB induces autophagy in A549and H23cells. PLB treatment significantly decreased the phosphorylation of PI3K （p55） at Tyrl99in both A549and H23cells in a concentration-dependent mannerthe, but did not significantly affected PI3K, the ratio of p-PI3K level over that of PI3K was significantly increased in both A549and H23cells. With increasing concentrations of PLB, an increasing amount of phosphorylated p38at Thr180/Tyr182was observed, but the amount of p38remained almost the same, the ratio of p-p38/p38was increased. PLB treatment significantly decreased the phosphorylation level of Akt, while the level of Akt was not affected significantly, and decreased the ratio of p-Akt/Akt significantly. PLB significantly decreased the phosphorylation of mTOR at Ser2448in both A549and H23cells in a concentration-dependent manner, but not affected the level of mTOR significantly, and increased the ratio of p-mTOR/mTOR. PLB significantly induced the expression of beclin1in both A549and H23cells in a concentration-dependent manner. With increasing concentrations of PLB, an increased amount of LC3-I and II was observed, and the ratio of LC3-II/LC3-I increased. Treatment of Chloroquine, bafilomycin, and wortmannin inhibits PBL-induced autophagy in both A549and H23cells. Both SB202190and SB203580increased the ratio of p-p38over p38in A549and H23cells. In both A549and H23, SB202190and SB203580significantly enhanced PLB-induced mTOR phosphorylation inhibition in A549and H23cells.After0.5h treatment with PLB, the green fluorescence of PLB was detected in the cytosol. The green fluorescence was observed in nucleus after12h. After24h, there was only extracellular green fluorescence detected.Conclusions:1、PLB induced concentration-dependent inhibitory effect on the growth of A549and H23cells.2、PLB induced remarkable cell cycle arrest in A549and H23cells. This effect could contribute to the grow inhibition by PLB. The cell cycle arrest could be partially ascribed to down-regulation of Cdc2and cyclin B.3、PLB may increase the level of p21via up-regulating p53in A549cells, but not in H23cells. This may be related to the mutation of p53gene （M246I） that partially inactivates this protein in H23cells.4、PLB induced remarkable activation of caspase-3and caspase-9and eventually led to apoptotic death in both A549and H23cells.5、PLB significantly increased the intracellular level of ROS in both A549and H23cells. While The intracellular levels of ROS were significantly decreased after cells were pretreated with ROS scavengers.6、PLB induces autophagy in A549and H23cells via inhibition PI3K/Akt/mTOR pathway.7、Chloroquine, bafilomycin, and wortmannin inhibits PBL-induced autophagy.8、SB202190and SB203580enhances PLB-induced autophagy through increased inhibition of PI3K/Akt/mTOR pathway.9、There is crosstalk between PLB-induced apoptosis and autophagy in A549and H23cells. PLB was readily and rapidly taken up by the cells and then distributed to the cytosol and nucleus.