Prodigiosin induces autop- hagic alterations in human colon cancer cells

 
C. Zhao, et al. Cancer Letters 481 (2020) 15–23 Fig. 1. Prodigiosin induces autop- hagic alterations in human colon cancer cells. (A) The chemical structures of prodi- giosin (PG). (B and C) Prodigiosin pro- motes LC3B-II accumulation. The cells were exposed to prodigiosin at the dosesindicatedfor12h.(B),ortreated with prodigiosin (0.2 μM) for the time periods indicated. (C), and cell lysates were analyzed using western blotting along with an anti-LC3B antibody. (D) Prodigiosin induces LC3B-II accumula- tion in multiple colon cancer cell lines. Three different lines of colon cancer cells were treated with prodigiosin (0.2 μM) for 12 h and the cells lysates were analyzed using western blotting. (E and F) Effect of prodigiosin on GFP- LC3B punctation. SW480 cells stably expressingEGFP-LC3weretreatedwith various concentrations of prodigiosin for 12 h. (E) or were treated with 0.2 μM prodigiosin for the different durations indicated (F), and the EGFP- LC3 puncta were observed using con- focal microscopy. Scale bar = 10 μm. Quantification of average EGFP puncta per cell obtained from 3 independent experiments. The results are presented as mean ± SD; *, P < 0.05 versus controlgroup;50cellsundergoingeach treatment condition were analyzed. autophagicflux in CRC cells but also markedly decreased viability and 2.2. Reagents and antibodies increased caspase-dependent apoptosis in CRC cells treated with 5-Fu. The synergy between prodigiosin and 5-Fu was also confirmed in a Prodigiosin (AG-CN2-0105) was purchased from AdipoGen. mouse xenograft model. 

 

Thus, prodigiosin is a novel autophagy in- Chloroquine diphosphate salt, rapamycin, bafilomycin A1and Z-VAD- hibitor that can augment the effect of chemotheAMG 925</a>.youtube.com/embed/XDs7FlC8M6M\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen>rapeutic drugs, and FMK were obtained from MedChemExpress. LysoTraker Red and acri- potentially sensitize recalcitrant patients. dine orange were purchased from Sigma-Aldrich, and LysoTracker Red DND-99 (L7528) from Invitrogen. Antibodies against LC3B, cleaved caspase-3, cleaved caspase−9, PARP, GAPDH, mouse IgG, and rabbit 2. Materials and methods IgG horseradish peroxidase (HRP)-linked were purchased from Cell Signaling Technology Incorporation, and those targeting 2.1. Cell lines and cell culture SQSTM1, CTSB/cathepsin B, CTSD/cathepsin D, GFP, RFP and Alexa Fluor 555 anti-mouse IgG from Abcam PLC. Enhanced chemilumines- HCT116(CCL-247),SW480(CCL-228),HT-29(HTB-38),N87(CRL- cence (ECL) reagents were purchased from Santa Cruz Biotechnology 5822),AGS(CRL-1379)andLoVo(CCL-229)cellswerepurchasedfrom Incorporation. American Type Culture Collection (ATCC) and cultured in RMPI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin Tunicamycin. Caco-2 (ATCC, HTB-37) cells were cul- 2.3. Cell viability assay tured in Dulbecco\'s modified Eagle\'s medium (DMEM) with 10% FBS, 4 while SW480 cells stably expressing EGFP-LC3 were maintained in Cells were seeded in 96-well plates at the density of 1 × 10 cells/ RMPI 1640supplemented with10%FBS and200mg/ml G418.SW480 100μl/well,andculturedwiththevehicleorprodigiosinfor48h.Three cells stably expressing GFP-LC3B-RFP-LC3BΔG were maintained in hours before harvesting, 20 μl MTS reagent (CellTiter 96 Aqueous One DMEM supplemented with 10% FBS and 15 mg/ml puromycin (Sigma- Solutionreagent,Promega)wasaddedtoeachwell,andtheabsorbance Aldrich). density at 490 nm was read on a 96-well plate reader, and IC50 values 16
r> C. Zhao, et al. Cancer Letters 481 (2020) 15–23 Fig. 2. Prodigiosin inhibits autop- hagic flux and autophagic degrada- tion. (A-C) Prodigiosin increases SQSTM1 accumulation. HCT116 and SW480cellsweretreatedwithdifferent concentrations of prodigiosin for 12 h. (A), or were treated with prodigiosin (0.2 μM) for the durations indicated (B), and SQSTM1 protein level was analyzed using immunoblotting. Relative SQSTM1 mRNA expression le- vels (compared with 18s ribosomal RNA) were analyzed using quantitative real-time PCR (C). The results (mean ± SD) are representative of 3 independent experiments. N.S, Not significant. (D) Combined treatment withprodigiosinandCQdoesnotshow any significant changes in the accu- mulationofSQSTM1.SW480cellswere treated with prodigiosin (0.1 μM) for 12 h in the presence or absence of CQ, as indicated. CQ, chloroquine. (E–G) The degradation of GFP-LC3B, but not RFP-LC3BΔG, was inhibited by prodi- giosin. Wild-type or stably GFP-LC3B- RFP-LC3BΔG-expressing SW480 cells were treated with 0.1 μM prodigiosin, 100 μM CQ Cell Counting Kit-8 solubility, or 200 nM rapamycin for 12h,followedbyimmunoblottingwith anti-LC3 to detect LC3-fusion proteins, including RFP-LC3BΔG, GFP-LC3B-I and GFP-LC3B-II (E). Stably GFP-LC3B- RFP-LC3BΔG-expressing SW480 cells weretreatedasindicatedinE,followed by immunoblotting with anti-GFP or anti-RFP to detect LC3-fusion proteins (F) andflow cytometry (G). The results (mean ± SD) are representative of 3 independent experiments; *, P < 0.05 versus control group. Rapa, rapamycin. (H) Confocal microscopy images dis- playing the colocalization between SQSTM1 and LC3B. SW480 stably expressing EGFP-LC3, were treated with 0.1 μM prodigiosin or 100 μMCQ for 12 h, fixed and then stained with anti-SQSTM1(red).Scalebar=10μm. 

 

(For interpretation of the references to colorinthisfigurelegend,thereaderis referred to the Web version of this ar- ticle). with its cargo within these autophagosomes, the steady-state level of throughout the autophagic flux. SW480 cells stably expressing GFP- SQSTM1isamarkerofautophagicstatus[25].AsshowninFig.2Aand LC3-RFP-LC3ΔG were treated with the different drugs, and the lysates B, prodigiosin significantly upregulated SQSTM1 protein levels in a were analyzed for the levels of cleaved GFP-LC3B and RFP-LC3BΔG. In dose and time-dependent manner. However, SQSTM1 mRNA levels thecellstreatedwithCQorprodigiosin,GFP-LC3B-Iwasconvertedinto were unaffected by prodigiosin, indicating that SQSTM1 accumulation GFP-LC3-BII, and RFP-LC3BΔG was detected onlyin itsLC3B-I form.In is not related to its transcriptional activation (Fig. 2C). Finally, the contrast, treatment with the autophagy promoter rapamycin degraded cationic autophagy inhibitor chloroquine (CQ) failed to augment the GFP-LC3B-II while RFP-LC3BΔG was still detectable (Fig. 2E and F). prodigiosin-induced accumulation of SQSTM1 and LC3B-II (Fig. 2D), Furthermore, CQ and prodigiosin significantly increased the GFP/RFP indicating that prodigiosin inhibits the autophagicflux. To further va- ratio by preventing lysosomal degradation of GFP-LC3B (Fig. 2G), and lidate this hypothesis, we used a fluorescence autophagy probe based resulted in co-localization of SQSTM1 with GFP, indicating that au- on the GFP-LC3B-RFP-LC3BΔG fusion protein (Supplementary Fig. 2) tophagy was arrested at the autophagosome level (Fig. 2H). Taken to- [26]. The latter is cleaved by ATG4 proteases into an equimolar mix of gether, prodigiosin blocks the autophagy flux, and the likely me- the GFP-LC3B and RFP-LC3BΔG fragments. Upon autophagy induction, chanism is inhibition of autophagosome-lysosome fusion. GFP-LC3Bislocalizedtotheautophagosomesandispartiallydegraded, whileRFP-LC3BΔGisnotinternalizedintotheautophagosomesduetoa C-terminal glycine deletion and remains in the cytosol. Thus, RFP- LC3BΔG acts as an internal control for GFP-LC3B degradation 18