Immuno blottingforLC3and SQSTM1 were used to observe autophagy

 C. Zhao, et al. Cancer Letters 481 (2020) 15–23 Fig.4. Prodigiosinsensitizescolorectalcancercellsto5-Fu-inducedcelldeathpartlythroughacaspase-dependentprocess.(A)ImmunoblottingforLC3and SQSTM1 were used to observe autophagy. SW480 were pretreated with prodigiosin (0.1 μM) for 1 h, followed by 5-Fu (50 μM) for another 12 h LC3B-II and SQSTM1 proteinlevelswereanalyzedusingwesternblotting.GAPDHwasusedastheloadingcontrol.5-Fu,5-fluorouracil. (BandC)Prodigiosin sensitizesHCT116 andSW480cellsto5-Fu-inducedcelldeath.Thecellsweretreatedwith5-Fu(50μM)inthepresenceorabsenceof0.1μMprodigiosinfor48h,andMTSassayswere performedtoassesscellviability.Theresults(mean ± SD)arerepresentativeof3independentexperiments;**,P < 0.01.(D)Apoptosisinductionbycotreatment withprodigiosinand5-Fu.TheHCT116cellswerecotreatedwith0.1μMprodigiosinand50μM5-Fufor48h,andapoptoticcellsweredetectedusingAnnexinV/PI staining followed by fluorescence microscopy imaging (left) and flow cytometry (right). Representative images are shown. Scale bar = 50 μm. (E) Prodigiosin promotes 5-Fu-induced apoptosis. The HCT116 cells were pretreated with prodigiosin for 1 h, followed by 5-Fu (50 μM) for another 24 h. The cell lysates were collectedandsubjectedtowesternblottingusingantibodiesagainstPARP,cleaved-caspase-3,cleaved-cleaved-caspase-3.GAPDHwasusedastheloadingcontrol.(F) HCT116 cells were treated with prodigiosin (0.1 μM), 5-Fu (50 μM) or prodigiosin + 5-Fu with or without Z-VAD-FMK (25 μM) for 24 h and cell viability was analyzed using MTS assay. The results (mean ± SD) are representative of 3 independent experiments; **, P < 0.01. C). Similar results were obtained with HCT116 cells treated with Prodigiosin inhibited the autophagic flux in CRC cells by blocking 100μM CQ and 5-Fu (Supplementary Fig. 4C) Z-VAD-FMK, indicating that blocking autophagosome-lysosomefusionandautophagicdegradation,aswellas the autophagic flux significantly augments the toxic effects of 5-Fu. attenuatingtheproteolytic activityofseverallysosomalhydrolases.CQ Consistent with this, the combination treatment markedly increased andbafilomycinA1exertasimilarinhibitoryeffectonautophagosome- apoptosis intheCRCcells(Fig.4D),andincreased thelevelsofcleaved lysosome fusion by alkalinizing the lysosomes and impairing the func- caspase-3, caspase-9 and PARP (Fig. 4E) compared to prodigiosin or 5- tion of lysosomal hydrolases [35,36]. However, prodigiosin had no ef- Fu monotherapy. In addition, the pan-caspase inhibitor Z-VAD-FMK fect on lysosomal pH, but instead prevented cathepsin maturation. partly rescued prodigiosin/5-Fu-induced apoptosis (Fig. 4F), and in- Enhanced LC3B-II formation levels have been observed in multiple hibitedPARPcleavageandcaspase-3activation(SupplementaryFig.5). prodigiosin-treated cancer cells [20,21,37], which unlike our observa- Taken together, prodigiosin blocks 5-Fu-induced autophagy and sensi- tions,ledtoautophagiccelldeath[21,37].Itispossiblethatprodigiosin tizes the CRC cells to apoptosis by promoting caspase activation cck-8 chemicals. acts as an autophagy inducer at high concentrations (up to 5 μM) used in the previous studies [21]. The significantly lower dose of 0.1 μMon the other hand induced accumulation of LC3B-II and SQSTM1 without 3.5. Prodigiosin augments 5-Fu efficacy in CRC xenografts in vivo detectable cell death Cytochalasin D, which could not be augmented by CQ. The ma- turation of precursor cathepsins to their mature form is a marker of The synergistic effect of prodigiosin and 5-Fu seen in human CRC lysosomal activity [27]. CTSD and CTSB in particular are effectors of cells was validated in a xenograft model. As shown in Fig. 5A and B, 5- autophagosome cargo degradation and autophagic flux [38,39]. We Fuorprodigiosinaloneminimallyinhibitedtumorgrowthcomparedto found that prodigiosin blocked the lysosomal phase of autophagy by that in the untreated tumor-bearing mice. In contrast, both tumor increasingaccumulationoftheprecursorcathepsins.Furtherstudiesare weight and volume decreased significantly in the combination tr###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-4.png####eat- neededtoelucidatethemechanismsofprodigiosin-mediated inhibition ment compared to that in either single-agent group. Furthermore, no of autophagosome-lysosome fusion and cathepsin maturation. significant changes were seen in the body weight of any of the treated Recent studies have implicated autophagy as a mechanism of 5-Fu mice (Fig. 5C). The safety profile of prodigiosin was also assessed in resistanceincancercells[40,41].Furthermore,inhibitionofautophagy terms of the hematological and histological parameters. The serum le- by3-methyladenine(3-MA,anautophagyinhibitor)sensitizesCRCcells vels of creatinine, blood urea nitrogen (BUN), AST and ALT were not towards 5-Fu-induced apoptosis involving cytochrome c release and significantly affected by prodigiosin (Supplementary Fig. 6A), and no activation of caspases [30]. Consistent with these findings, we found changesweredetectedinthemajororganslikeheart,liver,spleen,lung that 5-Fu induced apoptosis and autophagy in CRC cells, and co-treat- and kidney of the prodigiosin-treated mice compared to the controls ment with prodigiosin and 5-Fu activated caspase-3, caspase-9 and (SupplementaryFig.6B).

Consistentwiththeinvitroresults,prodigiosin PARP. Although studies increasingly show that suppression of autop- and 5-Fu synergistically increased LC3B-II levels compared to either hagyinCRCcellsbyCQcanabrogatetheprotectiveeffectofautophagy drug alone (Fig. 5D). Furthermore, the in situ levels of SQSTM1 were and augment chemosensitivity [42–44], it is unclear whether the anti- also markedly increased in the prodigiosin and combination-treated cancer effects of CQ are the result of autophagy block since there is tumors, and decreased in the 5-Fu-treated tumors compared to the evidence indicating that the synergism between CQ and chemother- control group (Fig. 5D and E, bottom panels). The combination treat- apeuticagentsisindependentofautophagy[45,46].Nevertheless,some mentalsoenhancedtheinfiltrationofinflammatorycellsinthetumors, phase I clinical trials investigating the efficacy of CQ as a primary or decreased tumor cell density (Fig. 5E, top panels and Supplementary adjuvant therapeutic agent have shown promising results [47,48], al- Fig. 7), andincreased the proportion of apoptotic (Fig. 5E,middle though it is not particularly effective in hypoxic/acidic tumor tissues panels). Furthermore, 20 mg/kg CQ and 30 mg/kg 5-Fu remarkably since it can be neutralized at low pH [49]. 

Our findings provide new suppressed tumor growth compared to CQ alone, and a similar sy- insights into the therapeutic potential of blocking autophagy in cancer nergistic effect was seen with 1 mg/kg prodigiosin and 30 mg/kg 5-Fu cells, and present prodigiosin as a novel autophagy inhibitor that sy- (Supplementary Fig.8A-C).Takentogether,prodigiosin sensitizesCRC nergizes with chemotherapeutic agents to eliminate cancer cells. cells to 5-Fu-induced apoptosis in vivo by suppressing autophagy, re- sulting in enhanced tumor clearance without any adverse side-effects. Funding 4. Discussion This work was supported by the National Natural Science Foundation of China (No. 81871905, No. 81700486), the Guangzhou Autophagy is activated in some cancer cells as a pro-survival me- Science,TechnologyandInnovationCommission(201906010052),and chanismtoenhancechemoresistance.Thus,inhibitionofautophagyisa the Fundamental Research Funds for the Central Universities promising strategy to augment the cytotoxicity of chemotherapeutic (2017Q113). agentsintumorcells[31].SeveralphaseI/IIclinicaltrialsarecurrently analyzing the therapeutic effect of autophagy suppression using CQ or its derivative hydroxychloroquine in combination with other drugs on Authors contributions multiplecancers,includingCRC[32–34].Tothebestofourknowledge, this is the first study to explore the efficacy of autophagy inhibition Conception and design: C. Zhao, J. He, Y. Nie. using prodigiosin in combination with 5-Fu in C RC cells. Development of methodology: C. Zhao, S. Qiu, J. He. 21