CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk

Author’s Accepted Manuscript CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk Pinjia Chen, Xiaoyong Luo, Peipei Nie, Baoyan Wu, Wei Xu, Xinpeng Shi, Haocai Chang, Bing Li, Xiurong Yu, Zhengzhi Zou

PII: DOI: Reference:

www.elsevier.com

S0891-5849(17)30042-4 http://dx.doi.org/10.1016/j.freeradbiomed.2017.01.033 FRB13189

To appear in: Free Radical Biology and Medicine Received date: 28 September 2016 Revised date: 16 January 2017 Accepted date: 24 January 2017 Cite this article as: Pinjia Chen, Xiaoyong Luo, Peipei Nie, Baoyan Wu, Wei Xu, Xinpeng Shi, Haocai Chang, Bing Li, Xiurong Yu and Zhengzhi Zou, CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2017.01.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk Pinjia Chen1, 1 , Xiaoyong Luo1,†, Peipei Nie2,†, Baoyan Wu3, Wei Xu2, Xinpeng Shi1, Haocai Chang3, Bing Li1, Xiurong Yu1, Zhengzhi Zou3,2* 1

Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou

University, Luoyang, China 2

KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou

Medical University, Guangzhou, China 3

MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Joint

Laboratory of Laser Oncology with Cancer Center of Sun Yat-sen University, College of Biophotonics, South China Normal University, Guangzhou, China *

Correspondence to: Zhengzhi Zou, E-mail: [email protected]

ABSTRACT Autophagy

plays

a

key

role

in

supporting

cell

survival

against

chemotherapy-induced apoptosis. In this study, we found the chemotherapy agent SN-38 induced autophagy in colorectal cancer (CRC) cells. However, inhibition of autophagy using a small molecular inhibitor 3-methyladenine (3-MA) and ATG5 siRNA did not increase SN-38-induced cytotoxicity in CRC cells. Notably, another autophagy inhibitor chloroquine (CQ) synergistically enhanced the anti-tumor activity of SN-38 in CRC cells with wild type (WT) p53. Subsequently, we identified a potential mechanism of this 1

These authors contributed equally to this work. Requests for reprints: Zhengzhi Zou, MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, No. 55 Zhongshan Road West, Guangzhou, 510631, PR China. Phone: +86-20-8521-1436; Fax: +86-20-85216052; E-mail: [email protected] 2

cooperative interaction by showing that CQ and SN-38 acted together to trigger reactive oxygen species (ROS) burst, upregulate p53 expression, elicit the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). In addition, ROS induced by CQ plus SN-38 upregulated p53 levels by activating p38, conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death. Moreover, we showed induction of ROS and p53 by the two agents provoked the loss of LMP and ∆ψm. Altogether, all results suggested that CQ synergistically sensitized human CRC cells with WT p53 to SN-38 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 (a prodrug of SN-38) in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.

Abbreviations: CRC, Colorectal cancer; CQ, chloroquine; 3-MA, 3-methyladenine; ROS, reactive oxygen species; LMP, lysosomal membrane potential; ∆ψm, mitochondrial membrane potential; NAC, N-acetyl-L-cysteine; Vit-C, vitamin C; DMSO, dimethyl sulfoxide; Pep-A, pepstatin A; CA, CA-074-Me; siRNA, small interfering RNA; combination index (CI); AO, acridine orange; Cat-B, cathepsin B; Cat-D, cathepsin D. Keywords: chloroquine, SN-38, p53, ROS, apoptosis, colorectal cancer

1. Introduction Colorectal cancer (CRC) is one of the most common cancers. Chemotherapy is

considered be one of the best option treatments against CRC. CPT-11 (Irinotecan), a DNA topoisomerase I inhibitor, is an antitumor prodrug and has been demonstrated to be active against many types of solid tumors including lung, uterine, ovarian, gastric carcinomas and CRC [1-4]. CPT-11 is converted by carboxylesterase in the liver to produce the active metabolite SN-38, which has at least 1000-times more effective anticancer activity than CPT-11 [5]. Although CPT-11 showed remarkable anticancer activity in preliminary clinical trials, unfortunately chemotherapy with CPT-11 appears to be contraindicated in advanced CRC [6]. Recently, there have been many efforts to administer combination chemotherapy with CPT-11 and other drugs in CRC patients. For example, CPT-11 combined with proteasome inhibitors displays synergistic anti-cancer effects in CRC [7]. Moreover, CPT-11, leucovorin and 5-FU in combination chemotherapy has been approved as standard therapeutic regimen by the US FDA for treating metastatic CRC [8].

Lysosomotropic agent chloroquine (CQ) is a classical anti-malarial and -inflammatory drug [9]. CQ has been shown to accumulate in the lysosomal lumen and inhibit lysosomal acidification [10]. In addition, CQ is capable to prevent autophagy by blocking autophagosome-lysosomal fusion events and inhibiting lysosomal proteases [11]. Hence, CQ is the most widely used autophagy inhibitor in vitro [11, 12]. Autophagy, a regulated degradative process, is involved in the turnover of cytoplasmic macromolecules and damaged organelles [13]. Autophagy is thought to act as a cell-survival pathway in eucaryote cells under stress circumstance [14]. Several reports have shown that autophagy ameliorates cancer cell apoptosis induced by chemotherapy drugs [15]. Our

recent study indicated that Aurora-A inhibition-stimulated autophagy increases drug resistance in breast cancer cells [12]. Moreover, autophagy inhibitors including CQ have been used in combination with diverse chemotherapeutic drugs and have been shown to enhance tumor cell killing [16]. Besides, single-drug therapy with CQ exerts high anti-cancer effects in myc-induced model of lymphoma [17]. Additionally, CQ increases survival of breast cancer xenografts nude mice when used as a single agent [18].

In this study, we found SN-38 induced autophagy in CRC cells. To examine whether inhibition of autophagy increased the cytotoxicity of SN-38 in CRC cells, we determined the effects of SN-38 on the CRC cell death under attenuation of autophagy condition by using siRNA and autophagy inhibitors. Notably, we found the cytotoxicity of SN-38 was not obviously aggravated by 3-methyladenine (3-MA, a widely used autophagy inhibitor) and siRNA against ATG5 (a gene required for the formation of autophagosomes) in CRC cells. However, CQ significantly enhanced the anti-tumor activity of SN-38/CPT-11. Furthermore, we showed that combination of CQ and SN-38 could synergistically inhibit cell viability and induce cell apoptosis in CRC cell lines. In addition, we identified a potential mechanism of this cooperative interaction by showing that the two agents acted together to trigger reactive oxygen species (ROS) burst and up-regulate p53 expression. And then ROS and p53 elicited the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). Moreover, ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death.

2. Materials and methods 2.1. Cell culture The human colon cancer-derived cell lines SW1116 (WT (wild type) p53), HCT116 (WT p53), LOVO (WT p53), HT-29 (p53 R273H mutation), SW480 (p53 R273H/P309S mutation), and a normal human colon mucosal epithelial cell line NCM460 (WT p53) were obtained from the American Type Culture Collection (ATCC) [19; 20; 21]. All cell lines have a stringent quality control in cell authenticity and have incorporated short-tandem repeat (STR) profiling for cell line validation. All cells were grown in DMEM medium (Gibco; Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco; Life Technologies, Carlsbad, CA) at 37°C in 5% CO2 incubator. Cells were grown in monolayer and passaged routinely 2-3 times a week [22].

2.2. Reagents and drugs treatment SN-38, SB202190, SB203580 and CPT-11 were purchased from Selleck Chemicals LLC (Houston TX, USA). N-acetyl-L-cysteine (NAC), vitamin C (Vit-C), dimethyl sulfoxide

(DMSO),

PFT-,

pepstatin

A

(Pep-A),

CA-074-Me

(CA),

E64d,

3-Methyladenine (3-MA) and chloroquine (CQ) were purchased from Sigma (St. Louis, MO, USA). Vit-C was dissolved in water. Other reagents were dissolved in DMSO and stored at -80 °C. For drugs treatment, stock solutions were diluted to the indicated final doses with growth medium just before use. The final concentration of DMSO in medium was 0.02% in all cases. Prior to drugs treatment, cells were incubated for at least 8 h and thereafter replaced with fresh media containing drugs; Control cells received an equivalent amount of DMSO.

2.3. Cell viability and apoptosis assays CCK8 assay was conducted to assess the cell viability. Briefly, cells were plated into 96-well plates at a density of 0.5-1×104 cells per well and incubated for at least 8 h in a 5% CO2 atmosphere at 37 °C before exposure to drugs. The media were then removed, and cells were treated with drugs. After the cells were incubated for indicated time, CCK8 reagent were added to each well and the plate was incubated for another 2 h at 37 °C. Absorbance of the media was then measured using a Micro-plate Reader (Bio-Rad, Hercules, CA) at 450 nm. This assay was conducted in triplicate.

Cells apoptosis were measured by Annexin V-FITC (fluorescein isothiocyanate)/PI analysis as described previously [23]. Briefly, cells were plated and treated with the reagents. Subsequently, the cells were washed twice with PBS and resuspended in 1 mL of 1×binding buffer. Cells undergoing apoptosis were analyzed by counting the early-stage apoptotic cells that stained positive for Annexin V-FITC and negative for PI, and late-stage apoptotic cells as Annexin V-FITC and PI positive using FACS CaliburTM flow cytometer. Experiments of cell cycle and apoptosis were performed in triplicate independently.

2.4. Quantification of EGFP-LC3 puncta assay. Cells were transfected with EGFP-LC3 constructs (Addgene, 11546)56 in Opti-MEM (Invitrogen, 11058) using Lipofectamine 2000 (Invitrogen, 11668). Afterward, cells were fixed in 4% paraformaldehyde at RT for 20 min, and washed twice with PBS. EGFP-LC3 distribution was subsequently monitored using an Olympus FV-1000

confocal microscope. Autophagy was quantified. Simply, the percentage of cells showing accumulation of EGFP-LC3 in dots or vacuoles (EGFP-LC3vac, of a minimum of 500 cells per preparation in three independent experiments) was counted. Cells representing several intense punctate EGFP-LC3 aggregates with no nuclear EGFP-LC3 were classified as autophagic. On the contrary, cells presenting a mostly diffuse distribution of EGFP-LC3 in the cytoplasm and nucleus were considered as nonautophagic.

2.5. Combination index For combination treatment of CQ and/or SN-38, CCK8 assay data were converted to fraction of growth affected by the individual drug or the combination treated cells compared with untreated cells and analysed using CalcuSyn software (Biosoft, Ferguson, MO, USA) to determine whether the combination was synergistic. This program is based upon the Chou-Talalay equation [24], which calculates a combination index (CI). The general equation for the classic isobologram is given by: CI = (D)1/(Dx)1 + (D)2/(Dx)2. Where Dx indicates the dose of one compound alone required to produce an effect, (D)1 and (D)2 are the doses of compounds 1 and 2, respectively, necessary to produce the same effect in combination. From this analysis, the combined effects of the two compounds can be summarized as follows: CI < 1, CI = 1, CI > 1 indicate synergistic, additive and antagonistic effects, respectively.

2.6. RNA interference SiRNAs for down-regulating ATG5 and TP53 gene expression were done by transfection of RNA oligonucleotides with lipofectamine 2000 (Invitrogen, USA)

according to the manufacturer’s instructions. One day before transfection, cells were plated on a 6-well (35 mm) tissue culture plate in DMEM complete medium. After cells reached 30% confluence, they were transfected with siRNAs as previously described [19]. Briefly, cells were placed in 1 mL of siRNA mixture with 50 nM siRNA and 3 μL lipofectamine 2000. After 8 h of transfection, the medium with siRNA and lipofectamine 2000 were replaced with 2 mL of DMEM complete medium, and experiments were conducted 48 h after transfection. Protein levels were analyzed by Western blot. The negative control (NC) siRNA and siRNAs against ATG5 and TP53 were synthesized by Shanghai GenePharma Co.. For ATG5 and TP53: 5'-TGATATAGCGTGAAACAAG-3'; and 5’-CUACUUCCUGAAAACAAC-3’, respectively.

2.7. Measurement of lysosomal membrane potential (LMP) and mitochondrial membrane potential (MMP) LMP and MMP were assessed by acridine orange (AO) and JC-1 staining respectively. To determine the assessment of LMP, cells were exposed to 1mM of the lysosomotropic metachromatic fluorophore AO for 20 min at 37°C. And then cells were washed and resuspended in PBS. Changes in LMP were monitored with the FL3 channel in a FACS CaliburTM flow cytometer. For the assessment of MMP, cells were incubated with 10 μg/mL JC-1 in growth culture medium for 15 min at 37 °C, washed twice, and resuspended in PBS and followed by determination of the JC-1 fluorescence with the FL1 and FL2 channels by FACS CaliburTM flow cytometry (BD Biosciences, San Jose, CA, USA).

2.8. Western blot analysis Total proteins were isolated from cells with lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP40; 2.5 mM sodium pyrophosphate; 0.02% sodium azide; 1 mM EGTA, 1 mM EDTA; 1 mM β-glycerophosphate; 1 mM Na3VO4; 1 mM PMSF; 1 μg/mL leupeptin). The lysates were centrifuged at 12,000 rpm for 30 min at 4 °C. The protein concentration was determined by Bradford dye method. Equal amounts (20 to 50 μg) of cell

extract

were

subjected

to

electrophoresis

in

6-15%

sodium

dodecyl

sulfate-polyacrylamide (SDS-PAGE) and transferred to PVDF or nitrocellulose membranes (Millipore, Darmstadt, Germany) for antibody blotting. The membranes were blocked and then incubated with ATG5, cleaved-PARP, cleaved-caspase3, p53, p-p53, p38 MAPK, p-p38 MAPK, Cathepsin B, Cathepsin D, COX IV, Cytochrome c and Actin antibodies (all from Cell Signaling Technologies, Massachusetts, USA), γ-tubulin and LAMP2 (Abcam), p62 and Actin (Santa Cruz Biotechnology, Santa Cruz, CA), LC3 (Novus Biologicals, Inc., Littleton, CO) overnight at 4 °C. Subsequently, the membranes were incubated with a HRP-conjugated anti-mouse or -rabbit secondary antibody (Protein Tech Group, Chicago, IL) at RT for 1 h. The protein bands were visualized using an enhanced chemiluminescence reagent (ECL) kit (GE Healthcare; Munich, Germany), according to the manufacturer’s instructions [25].

2.9. Measurement of intracellular reactive oxygen species (ROS) generation Dihydroethidium (DHE) assay Dihydroethidium (DHE, Sigma, St. Louis, MO, USA) was dissolved at 10 mM in anhydrous DMSO and diluted to 100 μM with PBS just before starting the experiment.

Once DHE solution was ready, all medium was removed from the plates and 1 ml of serum free DMEM was added to each dish. Then 10 μM DHE were added to each dish and incubated for 30 min. Finally, the cells were washed three times with PBS, scraped into 500 μl of PBS and fluorescence was measured in a 96-well black microplate in a Micro-plate Reader (Bio-Rad, Hercules, CA) using excitation and emission filters of 540 and 590 nm, respectively.

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay ROS generation inside living cells was detected using flow cytometer with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Wako Ltd, Osaka, Japan), an oxidation-sensitive probe, which is cleaved by intracellular nonspecific esterases and turns into highly fluorescent molecule DCF upon oxidation by ROS. Briefly, cells were plated and treated with the reagents. Subsequently, the cells were washed twice with PBS and suspended dead cells were removed. Cells were trypsinized and loaded with 10 μM DCFH-DA for 30 min at 37 °C in the dark. Subsequently cells were assayed with the FL1 channel by FACS CaliburTM flow cytometer as described in detail previously [26].

2.10. Hydrogen peroxide (H2O2) assay The contents of H2O2 in treated cells were analyzed with Amplex Red Peroxide Assay kit (Life Technologies, Carlsbad, CA) according to the manufacturer protocol. In brief, confluent cells on 12-well plates were washed using serum free DMEM medium and harvested, lysised, centrifuged at 12,000 g for 5 min. Then, test tubes containing 50 μl of supernatants and 100 μl of test solution were placed at room temperature for 30 min, and

were then measured immediately using a Micro-plate Reader (Bio-Rad, Hercules, CA) at a wavelength of 560 nm. Absorbance values were calibrated to a standard concentration curve to calculate the concentration of H2O2. This assay was conducted in triplicate.

2.11. Caspase activity assay Assays of caspase-9 and caspase-3 activity were carried out by using caspase-9 assay kit (abcam, ab65607) and caspase-3 assay kit (abcam, ab39383) according to the manufacturer’s protocol. Briefly, cells were lysed in pH 7.2 lysis buffer (10 mM HEPES, 5 mM MgCl2, 142 mM KCl, 1 mM EDTA, 0.2% NP-40) with 10 mM DTT. Following incubation for 0.5 h on ice, cell lysate was centrifuged at 12,000 rpm for 30 min at 4 °C and the protein concentration in supernatants was measured by Bradford dye method. Aliquots of 10 mg/100 mL assay volume were treated with 140 mM site-specific tetrapeptide substrates Ac-DEVD-AFC for caspase-3 and LEHD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin) for caspase-9 in a caspase assay buffer (20 mM HEPES, 100 mM NaCl, 0.01% (w/v) CHAPS, 1 mM EDTA, 10% (w/v) sucrose, pH = 7.2) at 37 °C with 10 mM DTT for 0.5 h. The release of the fluorogenic group AFC was determined in a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) with excitation at 400 nm and emission at 505 nm respectively. The relative fluorescent units (RFU) were normalized with protein concentrations [27].

2.12. Cytosolic cathepsin activity assay Assays of cathepsin B (Cat-B) and cathepsin D (Cat-D) activity were carried out by using cathepsin B assay kit (abcam, ab65300) and cathepsin D assay kit (abcam, ab65302)

according to the manufacturer’s protocol. Briefly, 50 g of cytosolic fraction protein prepared from control or stimulated cells was added up to the final reactive to 200 L per well in a 96-well plate. Aliquots of assay volume were treated with 140 mM site-specific substrates in assay buffer at 37 °C with 10 mM DTT for 0.5 h. Cleavage of the preferred Cat-B substrate [sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin)] and Cat-D substrate [sequence GKPILFFRLK(Dnp)-D-R-NH2, labeled with MCA] by Cat-B and Cat-D release AFC and MCA respectively. The AFC fluorescence is measured at 400 nm excitation and 505 nm emission, and the MCA fluorescence is measured at 328 nm excitation 460 nm emission with a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) respectively. The relative fluorescent units (RFU) were normalized with protein concentrations.

2.13. Preparation of cytosolic and mitochondrial fractions Cytoplasmic and mitochondrial fractions were prepared using a ProteoExtractSubcellular Proteome Extraction Kit (Calbiochem) according to the manufacturer’s instructions. The purity of each fraction was analyzed by Western blot using antibodies against γ-tubulin (cytoplasmic fraction) and COX IV (mitochondrial fraction).

2.14. Xenografted colon cancer cells in nude mice Athymic BALB/c nude mice (4-6 weeks old) were obtained from Si-Lai-Ke-Jing-Da Experimental Animal Co. Ltd (Changsha, China). All of the procedures of animal experiments were performed according to approved protocols and in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals (Institute of

Laboratory Animal Resources, Commission on Life Sciences, National Research Council). It was approved by the Institutional Animal Care and Use Committee of our university (Zhengzhou

University,

Zhengzhou, China).

Mice were implanted

subcutaneously into the right flanks with 4×106 SW1116 and LOVO (6 mice in each group). After 10 days, the tumors became palpable. And then all mice were divided into four groups (6 mice per group) at random. For drugs treatment, 60 mg/kg CQ, 20 mg/kg CPT-11, or combination of them dissolved in DMSO at a volume of 50 μL was administered to nude mice by intraperitoneal injection (for CQ: once per two days for 6 times; for CPT-11: once per four days for three times). Tumor volume was measured two times a week by using calipers (as indicated at each time point) for 25 days. The tumor volume was estimated by the following formula: length × (width)2 × 3.14/6. The mice whole body weight was measured two times a week as indicated at each time point. All mice were euthanized by intraperitoneal injection of 200 mg/kg pentobarbital at the end of the experiment.

2.15. Statistics All experiments were repeated three times and were expressed as mean ± SD. P values were calculated using student’s t test and P value < 0.05 was considered significant. Statistical analysis was analyzed using the Statistical Package for Social Sciences (SPSS) software (version 16.0).

3. Results 3.1. Inhibition of autophagy fails to enhance SN-38-induced cytotoxicity in human colorectal cancer cells To determine if SN-38 induced autophagy, we examined the protein expression of LC3-II, an LC3-phosphatidylethanolamine conjugate molecule, which is located on the autophagosomal membrane. We found a significant increase of LC3-II levels after 48 h of treatment with SN-38 in LOVO and HCT116 cells (Fig. 1A). In addition, the reduction of p62, a marker for autophagy, was found in the two SN-38 treated CRC cells (Fig. 1A). As shown in Fig. 1B, we showed that SN-38 decreased p62 expression in time- and dose-dependent manner in HCT116 cells. Moreover, the numbers of cells with EGFP-LC3 puncta increased to approximately 50% in HCT116 cells treated with 4 nM of SN-38 for 48 h (Fig. 1C). All above results suggested SN-38 induced autophagy in CRC cells.

To assess the role of SN-38-induced autophagy in CRC cells, we attempted to inhibit the autophagic process using autophagy inhibitor 3-MA. As shown in Fig. 1D, the p62 levels were upregulated following treatment with 3-MA in CRC LOVO and HCT116 cells. These results indicated 3-MA blocked the autophagy induced by SN-38. However, 3-MA did not remarkably enhance the cell apoptosis by SN-38 in three CRC cell lines (Fig. 1E). Furthermore, we used siRNA against ATG5 to inhibit autophagosomal formation. Similarly, suppression of ATG5 also did not enhance the cytotoxicity induced by SN-38 in LOVO and HCT116 cells (Fig. 1F and G).

3.2. Synergistic antineoplastic effects induced by CQ and SN-38 in colorectal cancer cells

Of note, we found another autophagy inhibitor CQ apparently amplified the toxicity of SN-38 in SW1116, LOVO and HCT116 cells with WT p53 status (Fig. 2A). To confirm this synergism, all three CRC cell lines were treated with CQ and SN-38 in combination in a constant ratio to one another, and combination index (CI) was calculated using Calcusyn software following Chou and Talalay’s method. As shown in Fig. 2B, when CRC cells were treated with high doses of SN-38 and CQ, the CI value were less than 1. These results indicated the two agents exerted significantly synergistic cytotoxic effects in the three CRC cells with WT p53. In addition, we also assessed the effects of the two drugs in combination against CRC cell lines SW480 and HT-29, which overexpressed mutant p53. As shown in Fig. S1A and B, compared with CRC cells expressing WT p53, both SW480 and HT-29 cells were more resistant to SN-38 with IC50 values of > 8 nM and 24 nM respectively. Interestingly, SW480 and HT-29 appeared to be insensitive to SN-38 and CQ in combination. By calculating CI, we found the combination of the two agents resulted in antagonistic effect (CI > 1) in SW480 cells, and antagonistic action or slightly synergy (0.6 < CI < 1) in HT-29 cells (Fig. S1C and D).

By annexin-V/PI assays, we also found CQ obviously augmented the apoptotic effects induced by SN-38 in all three CRC cell lines with WT p53 status (Fig. 2C). In addition, the apoptotic marker cleaved-PARP and -caspase3 were studied in HCT116 cells after treatment with CQ, SN-38 or their combination by Western blot analysis. As shown in Fig. 2D, CQ combined with SN-38 notably increased the expression of cleaved-PARP and -caspase3 in HCT116 cells. However, compared with CQ or SN-38

alone, the combination of these two drugs only led to a modest increased number of apoptotic cells in both SW480 and HT-29 cells with mutant p53 status (Fig. S1E and F). Notably, we assessed the effects of the two drugs in NCM460 cells, a normal human colorectal mucosal epithelial cell line. As shown in Fig. S2, no synergistic killing was observed in NCM460 cells.

3.3. CQ and SN-38 in combination treatment induces cell apoptosis involved in ROS production in colorectal cancer cells Above results suggested that CQ-inhibited autophagy was dispensable for the synergistic induction of cell death by SN-38 plus CQ. Therefore, we focused on this combination to clarify the potential molecular mechanisms of this synergism in the subsequent studies. To examine whether cell death by CQ and SN-38 was involved in ROS production in CRC cells, LOVO and HCT116 cells were treated for 48 h with the two compounds alone or in combination, and then the levels of ROS were evaluated by DCFH-DA staining and flow cytometric assays. As shown in Fig. 3A and B, CQ combined with SN-38 resulted in a burst of ROS in both CRC cells. The content of ROS increased to more than 7 times and 9 times in LOVO and HCT116 cells respectively (Fig. 3A and B). Next, we monitored the kinetics of ROS production upon treatment with SN-38 and CQ in combination. LOVO cells were treated with 80 μM of CQ in combination with 4 nM SN-38 for different time (1-48 h). We showed the levels of ROS were gradually enlarged in a time-dependent manner during 0-24 h (Fig. 3C). And then the intracellular ROS concentrations were slightly decreased after 24 h time points (Fig. 3C). Additionally, SW480 and HT-29 cells with mutant p53 gene were also treated with CQ and SN-38 in combination. Results indicated intracellular ROS levels induced by the

two drugs increased to no more than 4 times in both cells (Fig. S3A and B).

Additionally, the levels of ROS in CRC cells treated by CQ and SN-38 were also evaluated by dihydroethidium assay. As shown in Fig. 3D and E, CQ combined with SN-38 significantly increased ROS levels in both LOVO and HCT116 cells. By dihydroethidium assay, we also showed the levels of ROS were gradually increased in a time-dependent manner during 0-24 h. And then the intracellular ROS concentrations were slightly attenuated after 24 h time points (Fig. 3H). Additionally, we evaluated the levels of ROS by dihydroethidium assay in HT-29 cells with mutant p53 gene. Results indicated intracellular ROS levels induced by the two drugs increased to only 2 times (Fig. S3D). To examine whether specific oxidant H2O2 was induced by CQ and SN-38 in CRC cells, the contents of H2O2 were analyzed with Amplex Red Peroxide Assay. As shown in Fig. 3F and G, CQ combined with SN-38 slightly increased H2O2 levels in both LOVO and HCT116 cells. In addition, we showed the intracellular ROS concentrations were slightly increased after 8 h time points (Fig. 3I). However, CQ and SN-38 alone or in combination failed to increase the H2O2 levels in HT-29 cells (Fig. S3E). Above results suggested the induction of ROS by CQ plus SN-38 was lower in SW480 and HT-29 cells than that in CRC cells with wide p53. Moreover, as depicted in Fig. S3C, CQ plus SN-38 also led to a dramatic induction of ROS levels in normal colonic epithelial cell line NCM460 with wild type p53.

To further examine whether CQ plus SN-38 induced cell apoptosis involved in ROS production, we pretreated LOVO and HCT116 cells with ROS scavengers N-acetyl-L-cysteine (NAC) and vitamin C (Vit-C) prior to the two drugs treatment. We

showed the inductions of ROS by CQ and SN-38 were completely blocked by pretreatment with 8 mM NAC or 10 mM Vit-c in CRC cells (Fig. S4). Moreover, Fig. 3J and K indicated that both NAC and Vit-C significantly decreased cells apoptosis induced by the two drugs. Our results suggested that CQ enhanced the apoptotic effects of SN-38 were associated with ROS production. However, abolishment of ROS burst by NAC or Vit-C failed to completely inhibit the induction of apoptosis by CQ plus SN-38. These data hinted cell apoptosis evoked by CQ and SN-38 in combination was involved in some other apoptotic factors besides ROS.

3.4. CQ and SN-38 cooperate to trigger production of p53 involved in ROS-p38 pathway Several reports show that p53 is potential mediator of induction of tumor cells apoptosis by CQ [17]. In addition, SN-38 also activates the p53 pathway to trigger cell death in human tumor cells [28]. These studies led us to hypothesize that anti-apoptotic effect of CQ plus SN-38 in this study might be associated with p53. Thus, we detected the levels of p53 in SW1116, LOVO and HCT116 cells treated with CQ and SN-38. As shown in Fig. 4A, CQ combined with SN-38 apparently increased p53 expression. Subsequently, LOVO cells were treated with CQ (80 μM) in combination with SN-38 (2 nM) for different time (4, 8, 16, 32 and 48 h). Fig. 4B showed the levels of p53 were increased in a time-dependent manner.

Interestingly, we found that abolishment of ROS burst using NAC or Vit-C significantly decreased the p53 levels by SN-38 and CQ in combination in LOVO and HCT116 cells (Fig. 4C). Since recent reports suggest ROS induces p53 by activating p38 MAPK (p38) [29], we next verified if CQ plus SN-38-induced ROS activated p38. As

shown in Fig. 4D, CQ combined with SN-38 induced p38-phosphorylation and the effect was abrogated by both NAC and Vit-C. Moreover, p38 inhibitors SB203580 and SB202190 inhibited the increase of p53 by CQ plus SN-38. Above results implied that CQ and SN-38 cooperated to trigger production of p53 involved in ROS-p38 pathway

3.5. CQ and SN-38 cooperate to trigger production of ROS involved in p53 Prior studies have demonstrated that p53 activation plays a crucial role in chemotherapy drugs induced ROS generation [29]. We next explored the involvement of p53 in induction of ROS by CQ and SN-38 in combination. As shown in Fig. 5A and B, p53-silencing using siRNAs in LOVO and HCT116 cells prior to the two compounds treatment significantly reduced burst of ROS. Moreover, depletion of p53 apparently abrogated cell apoptosis stimulated by CQ plus SN-38 in both CRC cell lines (Fig. 5C and D). Additionally, inhibition of p53 by use of pharmacological inhibitors pifithrin-α (PFT-α, a small molecule inhibitor of p53 transcriptional activity) (50 μM) also resulted in decrease of ROS levels (Fig. 5E and F). Similarly, we showed PFT-α prevented the cell apoptosis induced by CQ plus SN-38 in LOVO and HCT116 cells (Fig. 5G and H). Notably, in SW480 and HT-29 cells with mutant p53, PFT-α did not prevent ROS production by CQ and SN-38 in combination (Fig. S5A and B). Together, our data showed that p53 contributed production of ROS triggered by CQ in combination with SN-38 in CRC cells with wild type TP53 gene.

3.6. CQ and SN-38 cooperate to trigger loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆Ψm) Since high concentration of ROS can damage lysosomal and mitochondrial

membrane, we next measured the LMP and ∆Ψm in CQ and SN-38 treated CRC cells. As shown in Fig. 6A and B, we found treatment with CQ and SN-38 in combination markedly increased loss of LMP and ∆Ψm. Since loss of LMP brings about lysosomal proteases cathepsins are released from lysosome into cytoplasm [30], the expression of cathepsin B and D in cytoplasm were measured by Western blot analysis of cytosolic and mitochondrial fractions. As shown in Fig. 6C, CQ combined with SN-38 significantly increased the amounts of cathepsin B and D in cytoplasm, conversely, the lysosomal cathepsin B and D levels were sharply attenuated by the two agents in combination. Mitochondrial dysfunction results in cytochrome c were released from mitochondria into cytoplasm. Therefore, we measured the levels of cytosolic and mitochondrial cytochrome c in LOVO cells treated with CQ plus SN-38. As shown in Fig. 6C, the amounts of cytochrome c in cytoplasm were significantly increased by CQ plus SN-38, whereas the mitochondrial cytochrome c levels were markedly reduced.

3.7. CQ and SN-38 in combination treatment induces cell apoptosis involved in caspases and cathepsins The results shown above showed the CQ in combination with SN-38 caused loss of LMP and ∆Ψm. To further determine the loss of LMP and ∆Ψm induced by CQ combined with SN-38, activities of caspase-3, -9 and cathepsin B, D in cytoplasm were measured by fluorogenic substrate cleavage. As shown in Fig. 7A-D, activities of caspase-3, -9 and cathepsin B, D were obviously increased in CRC cells treated with the two drugs in combination. Increased lysosomal cathepsin activities in cytoplasm are associated with cell apoptosis [30]. We next used several different inhibitors of cathepsins to pretreat CRC cells prior to treatment with CQ and SN-38. We found that

E64d (cathepsin B/L inhibitor), CA-074-Me (CA, a selective irreversible inhibitor of cathepsin B) and pepstatin A (Pep-A, inhibitor of cathepsin D/E) all evidently reduced cells apoptosis induced by the two drugs in combination (Fig. 7E and F).

3.8. CQ plus SN-38 induced loss of LMP and ∆Ψm is involved in p53 and ROS The data shown above indicated induction of p53 and ROS by the combination treatment with CQ and SN-38 induced CRC cells apoptosis. Next, we investigated whether loss of LMP and ∆Ψm by the two drugs was linked in p53 and ROS. To explore p53 contributed to loss of LMP and ∆Ψm, p53 was depleted by siRNA in LOVO and HCT116 cells. And then CRC cells were treated with the two compounds alone or in combination. As shown in Fig. 8A-D, p53 siRNA effectively blocked the loss of LMP and ∆Ψm. Additionally, we also explored the effects of ROS in the loss of LMP and ∆Ψm. LOVO and HCT116 cells were pretreated with NAC prior to CQ and SN-38 treatment. After 48 h treatment with the two agents, LMP and ∆Ψm were detected. As shown in Fig. 8E-H, NAC significantly reduced loss of LMP and ∆Ψm in both cells after CQ in combination with SN-38 treatment.

3.9. CQ and SN-38 synergistically inhibit tumor growth in xenograft tumor models In order to determine whether the combination of CQ and SN-38 also synergistic against CRC in vivo, the growth inhibitory effect of the combination was evaluated against the CRC xenografts in nude mice. As shown in Fig. 9A, BALB/c-nude mice bearing xenograft tumors were administered CQ (60 mg/kg, intraperitoneal injection once per two days for 10 days) and CPT-11 (20 mg/kg intravenous injection once per four days

for 8 days) alone or combination. Volumes of tumors as well as body weights of mice were measured twice weekly during the whole experimental period. As shown in Fig. 8B and D, CQ and SN-38 in combination exerted greater antitumor effects in both SW1116 and HCT-116 xenograft tumor models compared to these drugs alone (P < 0.01). In addition, we found no significant differences of body weight between DMSO-treated control mice and CQ-treated mice (Fig. 9C and E). However, CPT-11 alone or CPT-11 combined with CQ decreased body weight of mice (about 15% and 9% at day 28 in SW1116 and LOVO xenograft respectively) compared with the vehicle controls (Fig. 9C and E). Notably, no significant differences of body weight were found between those mice treated with CPT-11 and CQ in combination with that treated with CPT-11 alone (Fig. 9C and E). Importantly, we observed the body weight partly recovered at day 35 in SW1116 xenografts treated with CPT-11 alone or combined with CQ (Fig. 9E). No deaths were found during the whole treatment period. All these results suggested CQ enhanced the CRC tumor sensitivity to CPT-11, resulting in the synergistic effect of CQ and CPT-11 in vivo. More importantly, toxic side effects by CPT-11 were not strengthened by CQ.

Since CQ and SN-38 cooperated to trigger cell apoptosis involved in p53 in vitro, we also observed the effects of CQ plus CPT-11 on the expression of p53 in vivo. As shown in Fig. 9F and G, by Western blot analysis, a significantly greater expression of p53 was observed in those tumors treated by CQ in combination with CPT-11 than those tumors treated by the two drugs alone.

4. Disscussion In this study, we showed that combination of CQ and SN-38 could synergistically inhibit cell viability and induce cell apoptosis in CRC cell lines. In addition, as shown in Fig. 10, we identified a new potential mechanism of this cooperative interaction by showing that CQ and SN-38 acted together to trigger ROS burst and p53 expression, subsequently resulted in loss of LMP and ∆Ψm. More importantly, the production of ROS increased p53 levels; conversely p53 stimulated ROS generation. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.

SN-38 and its prodrug CPT-11 are both topoisomerase I inhibitors. This drug is used as anti-cancer drugs from the effectiveness in inhibiting cells growth by blocking DNA replication, leading to cells death. Moreover, previous study suggests CPT-11 induces autophagic cell death in p53-null colon cancer cell [31]. Here we found that SN-38 induced cell autophagy in CRC cells. Induction of autophagy has been demonstrated as a mechanism of tumor resistance to chemotherapeutic drugs [32, 33]. We showed inhibition of autophagy using 3-MA and ATG5 siRNA failed to enhance SN-38-induced cytotoxicity in human CRC cells. These results suggested SN-38 induced autophagy was dispensable for cell death. Notably, CRC cell death by SN-38 was observably enlarged by another autophagy inhibitor CQ. These results implied CQ increased SN-38-induced cell death independent of its function in autophagy inhibition. Indeed, the majority of studies have showed CQ exerts anti-cancer effects beyond autophagy inhibitor. For example, CQ

sensitizes mTOR and PI3K inhibitors in an autophagy-independent manner in breast cancer cells [18]. However, the mechanisms have not been clarified. Here, we found that CQ synergistically enhanced SN-38-induced cytotoxicity was associated with loss of LMP and ∆Ψm triggered by ROS and p53.

It has been reported that SN-38 is capable of generating ROS in gastric cancer cells [34]. Consistent with previous study, we also observed ROS generation in CRC cells after treatment with SN-38. Moreover, SN-38-triggered ROS was sharply increased by CQ. Notably, we found that DCF fluorescence values were greater than DHE fluorescence values in CRC cells treated with CQ and SN-38 in combination. Previous studies reported cytochrome c released from mitochondria to the cytosol during apoptosis, is capable of oxidizing DCFH directly or indirectly via a peroxidase-type mechanism to form DCF fluorescence [35]. In addition, redox-active metals are also capable of promoting DCFH oxidation [35]. All these studies suggested artifactual amplification of the DCF fluorescence intensity in CRC cells treated with the two drugs. It hinted DHE was more suitable for detection of intracellular ROS. The contents of specific oxidant H2O2 were analyzed with Amplex Red Peroxide Assay. Our results showed CQ combined with SN-38 only slightly increased H2O2 levels in CRC cells. These results suggested other oxidants besides H2O2 were also induced by CQ plus SN-38 in CRC cells.

ROS has been demonstrated to disrupt lysosomal and mitochondrial membrane integrity and cause loss of LMP and ∆Ψm [36, 37]. Therefore, ROS-inducing agents have been used to kill tumor cells in cancer therapy [38-40]. Consistent with this notion, we

showed that ROS production induced by CQ plus SN-38 markedly increased CRC cell death. Interestingly, we found that cells death induced by SN-38 was not obviously enhanced by CQ in the normal human colonic epithelial cells NCM460. It has been proposed that normal cells are not sensitive to ROS-causing agents [41]. Therefore, a possible explanation for such findings might reflect NCM460 cells were more resistant to ROS induced by CQ plus SN-38 than CRC cells.

Our present work provided evidences that CQ enhanced SN-38-induced CRC cell apoptosis by increasing ROS and p53 levels. Moreover, we showed intracellular ROS and p53 levels induced by the two drugs were mutually regulated. Previous studies indicate ROS stimulates p53 expression [29]. Similar results were found in this study. To decipher the mechanisms of ROS-increased p53 expression, we showed ROS-induced phosphorylation of p53 in Ser15 residue was mediated by p38 MAPK in these CRC cells. This signaling cascade was consistent with previous findings that ROS enhances the activation of p38 MAPK, which in turn phosphorylates p53 in Ser15 [42]. Ser15 phosphorylation of p53 impairs MDM2-mediated degradation of p53 by attenuating the p53-MDM2 interaction, leading to stabilization of p53 [43]. p53 has been understood to perform the function of inducing apoptosis by transcriptional induction of mitochondrial proapoptotic Bcl2 family proteins like BAX, PUMA and NOXA [44].

Interestingly, our study showed that p53 could induce CRC cell apoptosis by an additional way involved in increasing ROS production. Moreover, we found ROS levels stimulated by CQ plus SN-38 were observably attenuated by inhibition of p53

transcriptional activity using PFT-α in CRC cells with wild type p53. However, PFT-α failed to prevent ROS production by the two drugs in CRC cells with mutant p53 (p53-R273H) lacking transcriptional activity. These results suggested some genes regulated by p53 played a crucial role in CQ plus SN-38 induced ROS generation. However, the mechanisms behind the induction of ROS by p53 in CRC cells remained to be identified. Several previous studies have indicated p53 promotes ROS by transcriptional downregulation of anti-oxidant enzymes and upregulation of pro-oxidant enzymes [45]. In addition, p53 can induce uncoupling of mitochondria by transactivating BAX and PUMA, resulting in ROS being produced from a less efficient electron transport chain [46, 47]. Notably, we observed that CQ and SN-38 in combination produced significantly lower ROS levels in both p53-null cancer cells Hep3B and Saso2 relative to CRC cells expressing p53 (data not shown). Moreover, Hep3B and Saso2 cells were relatively insensitive to CQ combined with SN-38 treatment (data not shown). In conclusion, above findings thus confirmed the existence of ROS-p53-ROS feed back loop that induced CRC cells death.

In summary, we reported that synergistic induction of SN-38-mediated apoptosis by CQ in human CRC cells was involved in loss of LMP and ∆Ψm by stimulating ROS burst and p53 expression. In addition, CQ and SN-38 synergistically inhibited tumor growth in vivo colorectal cancer mice xenograft studies. Of note, the two agents in combination did not cause more toxicity than the drugs alone in mice with the concentrations and schedules used in these studies. On the basis of these findings, we hypothesized that CQ and SN-38 in combination might be an attractive therapeutic regimen for the treatment of

CRC.

Conflict of Interest The authors declare no conflict of interest.

Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (No. 81402187 and No. 81371646), the Ph.D Start-up Fund of Natural Science Foundation of Guangdong Province (No. 2014A030310505 to Z. Zou), the Foundation for Distinguished Young Talents in Higher Education of Guangdong (No. C1085229 to Z. Zou). Supplementary figure legends Fig. S1. Antineoplastic effects induced by CQ and SN-38 in colorectal cancer cells with mutant p53. (A and B) SW480 and HT-29 cells were cultured in control conditions (DMSO) or in the presence of the indicated concentrations of CQ and SN-38 alone or in combination for 48 h. Cell viability was assessed for viability by CCK8 assay. Mean ± SD of three independent experiments performed in triplicate were shown. (C and D) Cells were treated and processed as in A. The combination index (CI) values for SN-38 and CQ were calculated according to the Chou-Talalay’s method at the 48 h time point, with the biological response being expressed as the fraction of affected (Effect) cells. CI < 1, CI = 1, CI > 1 indicated synergistic, additive and antagonistic effects, respectively. The effect ranges from 0 (no inhibition) to 1 (complete inhibition). The data were representative of three independent experiments. (E and F) Cells were treated with indicated

concentrations of CQ and SN-38 for 48 h. Cell apoptosis was assessed by Annexin V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, SD. **, P < 0.01 ***, P < 0.001.

Fig. S2. Antineoplastic effects induced by CQ and SN-38 in NCM460 cells. (A) NCM460 cells were cultured in control conditions (DMSO) or in the presence of the indicated concentrations of CQ and SN-38 alone or in combination for 48 h. Cell viability was assessed for viability by CCK8 assay. Mean ± SD of three independent experiments performed in triplicate were shown. (B) Cells were treated and processed as in A. The combination index (CI) values for SN-38 and CQ were calculated according to the Chou-Talalay’s method at the 48 h time point, with the biological response being expressed as the fraction of affected (Effect) cells. CI < 1, CI = 1, CI > 1 indicated synergistic, additive and antagonistic effects, respectively. The effect ranges from 0 (no inhibition) to 1 (complete inhibition). The data were representative of three independent experiments.

Fig. S3. CQ and SN-38 in combination treatment induces modest ROS production in SW480, HT-29 and NCM460 cells. (A-C) The levels of ROS were measured with DCFH-DA staining by flow cytometric analyses at 48 h after CQ and SN-38 treatment in SW480, HT-29 and NCM460 cells. (D and E) The levels of ROS and H2O2 were measured with DHE fluorescence assay and Amplex Red fluorescence assay in HT-29 cells treated by CQ and SN-38. Columns, means of three determinations; bars, SD. Results shown were representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. S4. N-acetyl-L-cysteine and Vitamin C attenuates the ROS production induced by CQ plus SN-38 in colon cancer cells. (A and B) LOVO and HCT116 cells were pretreated with indicated concentration of N-acetyl-L-cysteine (NAC) and vitamin C (Vit-C) for 30 min before SN-38 and CQ treatment. And then LOVO and HCT116 cells were treated with CQ (80 μM) plus SN-38 (2 nM) and CQ (80 μM) plus SN-38 (4 nM) for additional 48 h respectively. The levels of ROS were measured with DCFH-DA staining by flow cytometric analyses. Columns, means of three determinations; bars, SD. Results shown were representative of three independent experiments. ***, P < 0.001.

Fig. S5. PFT-α did not attenuate the ROS production induced by CQ plus SN-38 in SW480 and HT-29 cells. (A and B) SW480 and HT-29 cells were pretreated with 50 μM of PFT-α for 30 min before SN-38 and CQ treatment. And then cells were treated with CQ (80 μM) plus SN-38 (4 nM) and CQ (80 μM) plus SN-38 (16 nM) for additional 48 h respectively. The levels of ROS were measured with DCFH-DA staining by flow cytometric analyses. Columns, means of three determinations; bars, SD. Results shown were representative of three independent experiments.

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Fig. 1. Inhibition of autophagy fails to enhance SN-38-induced cytotoxicity in human colorectal cancer cells. (A) LOVO and HCT116 cells were treated with 2 nM and 4 nM of SN-38 for 48 h respectively. (B) HCT116 cells were treated with increasing doses of SN-38 and treated with 4 nM SN-38 for different time. Cell extracts were prepared and analyzed by Western blotting with indicated antibody. These experiments were repeated thrice. (C) HCT116 cells were transfected with 2 μg of EGFP-LC3 construct. At 8 h post-transfection, cells were treated with 4 nM of SN-38 for 48 h. And then cells were examined by confocal microscopy (magnification × 400). The percentage of cells showing accumulation of EGFP-LC3 in puncta (EGFP-LC3vac) was quantified. (D) LOVO and HCT116 cells were treated with 2 nM and 4 nM of SN-38 combined with 10 mM of 3-Methyladenine (3-MA) for 48 h respectively. Cell extracts were prepared and analyzed by Western blotting with indicated antibody. These experiments were repeated thrice. (E) Cells were treated with indicated concentrations of 3-MA and SN-38 for 48 h. Cell apoptosis was assessed by Annexin V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, SD. (F) and (G) LOVO and HCT116 cells were transfected with 50 nM of NC siRNA, ATG5 siRNA respectively, and then were treated with increasing doses of SN-38 for 48 h, the knockdown effects on ATG5 were confirmed by Western blot analysis (upper panel). Cell viability was measured using CCK8 assay. Columns, means of three determinations; bars, SD.

Fig. 2. Synergistic antineoplastic effects induced by CQ and SN-38 in colorectal cancer cells with wild-type p53. (A) SW1116, LOVO and HCT116 cells were cultured in control

conditions (DMSO) or in the presence of the indicated concentrations of CQ and SN-38 alone or in combination for 48 h. Cell viability was assessed for viability by CCK8 assay. Mean ± SD of three independent experiments performed in triplicate were shown. (B) Cells were treated and processed as in A. The combination index (CI) values for SN-38 and CQ were calculated according to the Chou-Talalay’s method at the 48 h time point, with the biological response being expressed as the fraction of affected (Effect) cells. CI < 1, CI = 1, CI > 1 indicated synergistic, additive and antagonistic effects, respectively. The effect ranges from 0 (no inhibition) to 1 (complete inhibition). The data were representative of three independent experiments. (C) Cells were treated with indicated concentrations of CQ and SN-38 for 48 h. Cell apoptosis was assessed by Annexin V-FITC/PI staining assay by flow cytometry. Columns, means of three determinations; bars, SD. **, P < 0.01 ***, P < 0.001. (D) HCT116 cells were treated with indicated concentrations of CQ and SN-38 alone or in combination for 48 h. Cell extracts were prepared and analyzed by Western blotting with indicated antibody. These experiments were repeated thrice.

Fig. 3. CQ and SN-38 in combination treatment induces cell apoptosis involved in ROS production in colorectal cancer cells. (A) and (B) (a) The levels of ROS were measured with DCFH-DA staining by flow cytometric analyses at 48 h after CQ and SN-38 treatment in SW1116 and HCT116 cells. (b) The levels of ROS were presented as fold change compared to the levels in control cells. (C) (a) The levels of ROS were measured at indicated time point after treatment of CQ and SN-38 in combination in LOVO cells. (b) The levels of ROS were presented as fold change compared to the levels in control

cells. (D-I) The levels of ROS and H2O2 were measured with DHE fluorescence or Amplex Red Peroxide assay at 48 h after CQ and SN-38 treatment in LOVO (D and F) and

HCT116 cells (E and G). The levels of ROS were measured with DHE fluorescence or Amplex Red Peroxide assay at indicated time point after treatment of CQ and SN-38 in

combination in LOVO cells (H and I). The levels of ROS and H2O2 were presented as fold change compared to the levels in control cells. (J and K) LOVO and HCT116 cells were pretreated with different concentration of N-acetyl-L-cysteine (NAC) and vitamin C (Vit-C, dehydroascorbic acid) for 30 min before CQ and SN-38 treatment. And then cells were treated with indicated concentrations of CQ and SN-38 alone and in combination for additional 48 h. Cells apoptosis was measured using an Annexin V-FITC/PI staining assay. Columns, means of three determinations; bars, SD. Results shown were representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with DMSO-treated cells.

Fig. 4. CQ and SN-38 cooperate to trigger production of p53 involved in ROS-p38 pathway. (A) SW1116, LOVO and HCT116 cells were treated with SN-38 and CQ alone or in combination for 48 h respectively. (B) LOVO cells were treated with SN-38 and CQ in combination for indicated time. (C and D) LOVO and HCT116 cells were pretreated with indicated concentration of N-acetyl-L-cysteine (NAC) and vitamin C (Vit-C) for 30 min before SN-38 and CQ treatment. And then LOVO and HCT116 cells were treated with CQ (80 μM) plus SN-38 (2 nM) and CQ (80 μM) plus SN-38 (4 nM) for additional 48 h respectively. (E) LOVO and HCT116 cells were pretreated with SB202190 (1 μM) or SB203580 (10 μM) for 30 min before SN-38 and CQ treatment. And then cells were

treated with CQ (80 μM) plus SN-38 (2 nM) and CQ (80 μM) plus SN-38 (4 nM) for additional 48 h respectively. Cell extracts were prepared and analyzed by Western blotting with indicated antibody. This experiment was repeated thrice.

Fig. 5. CQ and SN-38 cooperate to trigger production of ROS involved in p53. (A) and (B) LOVO and HCT116 cells were transfected with 50 nM NC siRNA, p53 siRNA (si p53-a and si p53-b) respectively, and then were treated with indicated dose of CQ and SN-38 alone or in combination for 48 h, the levels of ROS were measured with DCFH-DA staining by flow cytometric analyses. The knockdown effects on p53 were confirmed by Western blot analysis (upper panel). (C) and (D) Cells were treated and processed as in A. Cells apoptosis was measured using an Annexin V-FITC/PI staining assay. (E) and (F) LOVO and HCT116 cells were pretreated with PFT-α for 30 min before SN-38 and CQ treatment. And then cells were treated with CQ plus SN-38 for additional 48 h respectively. The levels of ROS were measured with DCFH-DA staining by flow cytometric analyses. (G) and (H) Cells were treated and processed as in E and F. Cells apoptosis was measured using an Annexin V-FITC/PI staining assay. Columns, means of three determinations; bars, SD. Results shown are representative of three independent experiments. **, P < 0.01 vs. control; ***, P < 0.001 vs. control.

Fig. 6. CQ and SN-38 cooperate to trigger loss of lysosomal membrane potential and mitochondrial membrane potential. (A) (a) The levels of lysosomal membrane potential (LMP) were measured with Acridine Orange (AO) staining by flow cytometric analyses at 48 h after CQ and SN-38 treatment. (b and c) The percentage of cells with loss of LMP

was shown. (B) (a) The levels of mitochondrial membrane potential (∆ψm) were measured with JC-1 staining by flow cytometric analyses at 48 h after CQ and SN-38 treatment. (b and c) The percentage of cells with loss of ∆ψm was shown. This experiment was repeated thrice. Columns, means; bars, SD. **, P < 0.01 vs. control; ***, P < 0.001 vs. control. (C) LOVO cells were treated for 48 h with the indicated concentrations of CQ and SN-38 either alone or in combination, fractionated into cytosol and mitochondria, and analyzed for the distribution of cytochrome c, cathepsin B (Cat-B) and cathepsin D (Cat-D), by Western blot analysis. The fractionation quality was verified by the distribution of specific subcellular markers: COX IV for mitochondria, LAMP2 for lysosome and tublin for cytosol. This experiment was repeated thrice.

Fig. 7. CQ and SN-38 in combination treatment induces cell apoptosis involved in caspases and cathepsins. (A-D) CQ and SN-38 combination therapy increased the caspase-3, -9 and cathepsin B, D activity in colon cancer cells. LOVO and HCT116 cells were plated, treated for 48 h with the indicated concentrations of CQ and SN-38 either alone or in combination. The caspase and cathepsin activities were quantified as described under Methods. This experiment was repeated thrice. (E) and (F) LOVO and HCT116 cells were pretreated with indicated concentration of E64d, CA-074-Me (CA) and pepstatin A (Pep-A) for 1 h, and then cells were treated with indicated concentrations of CQ and SN-38 alone or in combination for additional 48 h. Cells apoptosis was measured using an Annexin V-FITC/PI staining assay. This experiment was repeated thrice. Columns, means; bars, SD. *, P < 0.05 vs. control; **, P < 0.01 vs. control; ***, P < 0.001 vs. control.

Fig. 8. CQ plus SN-38 induces loss of lysosomal membrane potential and mitochondrial membrane potential involved in p53 and ROS. (A-D) LOVO and HCT116 cells were transfected with 50 nM NC and p53 siRNA respectively, and then were treated with CQ and SN-38 alone or in combination for 48 h. The loss levels of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm) were measured with Acridine Orange (AO) and JC-1 staining by flow cytometric analyses respectively. The knockdown effects on p53 were confirmed by Western blot analysis (upper panel). (E-H) LOVO and HCT116 cells were pretreated with indicated concentration of NAC for 1 h, and then were treated with indicated concentrations of CQ and SN-38 alone or in combination for additional 48 h. The loss of LMP and ∆ψm were measured with AO and JC-1 staining by flow cytometric analyses respectively. This experiment was repeated thrice. Columns, means; bars, SD. **, P < 0.01 vs. control; ***, P < 0.001 vs. control.

Fig. 9. CQ and SN-38 synergistically inhibit tumor growth in xenograft tumor models. (A) Treatment schedule. (B and D) Combination of suboptimal concentrations of CQ and SN-38 displays significantly greater efficacy compared with either drug alone in xenograft tumor models. Nude mice bearing LOVO and SW1116 xenografts were administered with the indicated treatments. (CQ: 60 mg/kg, intraperitoneal injection; CPT-11: 20 mg/kg, intravenous injection). Tumor volume was measured two times a week by using calipers (as indicated at each time point) for 25 days. Data were shown as mean ± SD. (n = 6 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control. (C and E) Average body weight changes were measured over the course of the study. (F and

G) Tumor tissue were harvested from SW1116 and LOVO xenograft mice and homogenized in lysis buffer. Lysates were subjected to Western blotting with anti-p53 and β-actin.

Fig. 10. CQ plus SN-38 induced cell death of CRC cell with WT p53 via ROS/p38/p53 loop of amplification. CQ and SN-38 acted together to enhance production of reactive oxygen species (ROS) and p53. And then ROS and p53 elicited loss of mitochondrial membrane potential (MMP) and lysosomal membrane potential (LMP) respectively. Moreover, ROS induced by CQ and SN-38 increased levels of p53 through p38 pathway. Conversely, p53 stimulated ROS. Additionally, cytoplasmic cathepsins induced by CQ and SN-38 contributed to the caspase-independent cell death. CQ plus SN-38 enhanced cyto-c (cytochrome c) translocation into the cytosol and induced subsequently caspase-3 dependent cell apoptosis.

Highlights Chloroquine synergistically enhances the anti-tumor activity of SN-38 in colorectal cancer cells with wild type p53. Chloroquine and SN-38 act together to trigger ROS burst, upregulate p53 expression. Chloroquine

synergistically SN-38

induces

apoptosis

through

lysosomal

and

mitochondrial apoptotic pathway via p53-ROS cross-talk. Chloroquine enhances colorectal cancer cells response to CPT-11 in xenograft models.