HCT-116, DLD-1 and NCM460 cell lines were obtained from Professor Miyagi Yohei (Clinical Research Institute, Kanagawa Cancer Center, Yokohama, Japan). The cell lines were cultured as monolayers in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) with 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. All cells were harvested by centrifugation (1,500 × g for 10 min at 4°C) and rinsed with phosphate-buffered saline (PBS). Anti-LAMP2A (cat. no. ab18528) and anti-PLD2 (cat. no. ab78907) antibodies were purchased from Abcam. NF-κB Pathway Sampler kit (cat. no. 9936T) was purchased from Cell Signaling Technology, Inc. Anti-cluster of differentiation (CD)147/extracellular matrix metalloproteinase inducer (EMMPRIN; cat. no. SC-21746) antibody was purchased from Santa Cruz Biotechnology, Inc. Anti-lung resistance-related protein 1 (LRP1; cat. no. ab92544), anti-P-glycoprotein 1 (PGP)/multidrug resistance (MDR; cat. no. ab242104), anti-glutathione S-transferase π (GST-π; cat. no. ab138491), anti-MutL homologue 1 (MLH1; cat. no. ab92312), Anti-F-box and WD40 domain protein 7 (FBXW7) antibody (cat. no. ab12292) and anti-multidrug resistance-associated protein 1 (MRP1; cat. no. ab233383) antibodies were purchased from Abcam. Anti-Bcl-2 (cat. no. 15071) and anti-BAX (cat. no. 14796) antibodies were purchased from Cell Signaling Technology, Inc. LysoTracker-LysoGreen was purchased from Nanjing KeyGen Biotech Co., Ltd. (cat. no. KGMP006-2). Pyrrolidine dithiocarbamate (PDTC; cat. no. P8765.) and chloroquine (CQ; cat. no. C6628.) were purchased from Sigma-Aldrich (Merck KGaA) and 5-fluorouracil (5-FU) was purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. Oxaliplatin (OXA) was purchased from Nanjing Pharmaceutical Factory, Co. Ltd.

Cell viability was measured using the MTT assay. CRC and 5-FU-resistant CRC cells were seeded in triplicate in 96-well plates at 6,000 cells/well and incubated at 37°C for 24 h in RPMI-1640 medium supplemented with 10% FBS. Subsequently, the cells were treated with the indicated doses of 5-FU (10 mM), OXA (10 mM), or FOX (5-FU: 10 mM + OXA: 10 mM) for 96 h at 37°C. The same volume of dimethyl sulfoxide (DMSO) was used as the negative control. Following this, 25 µl MTT solution (5 mg/ml) was added to each well for 4 h at 37°C, then the cell culture supernatants were carefully removed and 200 µl DMSO was added to dissolve the purple formazan. Finally, the optical density was measured at a wavelength of 570 nm using a microplate reader (Model 550; Bio-Rad Laboratories, Inc.).

For the migration assay, 2×10⁵ cells were resuspended in serum-free RPMI-1640 and seeded in the Matrigel-coated insert (BD Biosciences) insert on the top portion of the chamber (BD Biosciences). The lower compartment of the chamber contained 10% FBS as a chemoattractant. After incubation for 24 h, cells on the membrane were scrubbed, washed with PBS, fixed in 100% methanol for 10 min at room temperature and stained with 10% Giemsa dye for 10–15 min at room temperature. For the invasive assay, the procedures were the same as above with a control-membrane insert (BD Biosciences). A total of five random fields were selected to count and images were captured under an inverted fluorescence microscope (×10 magnification; Nikon Corporation).

Flow cytometry was performed with 7-aminoactinomycin and phycoerythrin labeled Annexin V (BD Pharmingen; BD Biosciences) to detect phosphatidylserine externalization as an endpoint indicator of apoptosis. Cell apoptosis was detected by staining with Annexin V-PE/7AAD (KeyGen Biotech Co., Ltd.; cat. no. KGA214) according to the manufacturer's instructions. Apoptotic rate was calculated by the percentage of early + late apoptotic cells. After washing with PBS and centrifuging twice (both 1,000 × g, 5 min), cells were resuspended in Annexin-V binding buffer. They were stained with Annexin V-PE/7AAD and incubated for another 15 min at room temperature in a dark room. Cell samples were analyzed by flow cytometry (FACScan; BD Biosciences) to acquire the apoptotic fractions. Apoptotic fractions were investigated by Cell Quest 3.0 software (BD Biosciences).

The short hairpin (sh)RNA and LAMP2A expression plasmids were designed, constructed and purified by Shanghai GenePharma Co., Ltd. The shRNA and LAMP2A expression plasmids were transfected using Attractene Transfection Reagent (Qiagen GmbH) in accordance with the manufacturer's instructions. Cells cultured in 6-well plates were transfected with 1.2 ug/well shRNA or LAMP2A expression plasmids. At 24 h after transfection, the cells were collected to extract RNA and total protein for subsequent experimentation.

Total RNA was extracted from cultivated cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocols. RNA purification was performed using the RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's instructions.

Total RNA was extracted from CRC cells (1×10⁶) using the Qiagen RNeasy Mini kit (Qiagen GmbH), according to the manufacturer's protocols. Total RNA was subjected to cDNA synthesis using avian myeloblastosis virus reverse transcriptase and random primer (Takara Bio, Inc.). General- and RT-qPCR amplification was performed using TaKaRa Hot Start Taq Polymerase (Takara Bio, Inc.) and SYBR Premix Ex Taq TM II kit (Takara Bio, Inc.), respectively, all according to the manufacturer's protocols. All experiments were performed three times. mRNA expression analysis was performed using SYBR Green Master Mix (Life Technologies; Thermo Fisher Scientific, Inc.) on a LightCycler 96 Detection system (Roche Diagnostics) using β-actin for normalization. The cycling parameters were: 95°C for 5 min, followed by 59 cycles at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. Expression levels were normalized to endogenous controls and relative quantification (2^−ΔΔCq) was used for fold-change calculation (24). Using GenBank, oligonucleotide primers were designed as follows: LAMP2A: Forward primer: 5′-GCCGTTCTCACACTGCTCTA-3′; reverse primer: 5′-CCGCTATGGGCACAAGGAA-3′. PLD2: Forward primer: 5′-CCACAAACAGGGGTGGTGTT-3′; reverse primer: 5′-GAATGGCCTGGATGGAGTTG-3′; β-actin: Forward primer: 5′-GGTGGCTTTTAGGATGGCAAG-3′; reverse primer: 5′-ACTGGAACGGTGAAGGTGACAG-3′.

Cells were lysed in SDS cell lysis buffer containing 50 mM Tris (pH, 8.1) and 1% SDS, supplemented with protease inhibitors (Roche Diagnostics) for 30 min on ice. The homogenates were centrifuged at 18,000 × g for 10 min at 4°C. Supernatants were collected, and the protein concentration was determined by Bradford's assay (Bio-Rad Laboratories, Inc.); 30 µg protein was loaded for analysis. The proteins were separated via 12% SDS-PAGE and electrotransferred onto a PVDF (polyvinylidene fluoride) membrane, which was then blocked overnight in 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST, 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) at 4°C. For immunoblotting, the membrane was incubated with primary antibody (all 1:1,000) for 2 h at room temperature (20°C) as follows: LAMP2A, PLD2, NF-κB, Bcl-2, BAX, MRP1, PGP, CD147, FBXW7, MLH1, LRP1 and GSTπ. Then, it was incubated with anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (Dako; Agilent Technologies, Inc.) at a dilution of 1:5,000 for 2 h at room temperature. The bands were visualized using ECL-Plus detection reagents (Santa Cruz Biotechnology, Inc.). The densitometric quantification was performed with β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-47778) control using - ImageJ software (ImageJ 1.52a; National Institutes of Health).

CRC cells (1×10⁷) were seeded onto 100 mm plates and allowed to attach overnight at 37°C. The cells were then washed twice with PBS and lysed in lysis buffer (1% Triton X-100, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride and 2 µg/ml aprotinin) on ice. Co-immunoprecipitation was performed using 200 µg lysates with 4 µl of rabbit anti- NF-κB or control IgG mixed with protein G agarose beads (GE Healthcare Bio-Sciences). The final mixture was gently agitated overnight at 4°C. Following this, the beads were centrifuged for 1 min at 13,000 × g at 4°C and washed four times with lysis buffer for 1 min. Finally, 40 µl sampling buffer (Beyotime Institute of Biotechnology) was added, boiled at 95°C for 5 min and subjected to western blot analysis. A total of 30 µg protein was loaded for analysis. The proteins were separated via 10% SDS-PAGE and transferred onto a PVDF membrane, which was then blocked overnight in 5% skimmed milk in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) at 4°C. For immunoblotting, the membrane was incubated with primary antibodies (all 1:1,000) for 2 h at room temperature (20°C) as follows: LAMP2A, PLD2, NF-κB, MRP1, PGP, CD147, FBXW7, MLH1, LRP1 and GSTπ. Then, it was incubated with anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (Dako; Agilent Technologies, Inc.) at a dilution of 1:5,000 for 2 h at room temperature. The bands were visualized using ECL-Plus detection reagents (Santa Cruz Biotechnology, Inc.). The densitometric quantification was performed with β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-47778) control using ImageJ software (version 1.52a; National Institutes of Health).

All statistical analyses were performed by SPSS v24.0 (IBM Corp.) with the Student's t-test (two tailed) and by one-way analysis of variance with Tukey's post hoc test for multiple groups. The results are representative of three different experiments and data are expressed as mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To obtain 5-FU-resistant cells, CRC cells were first selected using increasing concentrations of 5-FU on parental HCT-116/DLD-1 cells. They were then intermittently treated with large doses of 5-FU. Cells that acquired 5-FU resistance were identified by cell survival assay and showed increased expression of drug resistance proteins, CD147/EMMPRIN (25); LRP1 (26); PGP/MDR (26); GST-π (27); MLH1 (28,29); MRP1 (26); FBXW7 (30), as verified by western blotting. They were labeled as HCT116-R or DLD-1-R cells (Fig. 1A-D). HCT116-R or DLD-1-R cells increased in size and the nuclei were irregular. Some cells had multiple, enlarged lysosomes. The proliferation rate of HCT116-R or DLD-1-R cells was similar to the parental cells, but the doubling time was prolonged in vitro (Fig. 1A and B). Co-resistance to other chemotherapy drugs such as OXA were also observed in HCT116-R or DLD-1-R cells (Fig. 1E).

Compared to parental cells, a significant elevation of LAMP2A was found in HCT116-R/DLD-1-R cells. However, the expression of LAMP2A was minimal in their corresponding normal colonic epithelial cells, NCM460 (Fig. 2A). In addition, HCT116-R/DLD-1-R cells had multiple, enlarged lysosomes when examined by LysoTracker staining (Fig. 2B). Meanwhile, enhanced PLD2 and Bcl-2 protein expression was found in HCT116-R/DLD-1-R cells (Fig. 2C). The same results were observed in the mRNA levels of LAMP2A and PLD2 by a RT-qPCR (Fig. 2D).

Excessive LAMP2A expression is one of the major features of HCT116-R/DLD-1-R cells. It was hypothesized that the silence or overexpression LAMP2A expression in HCT116-R/DLD-1-R cells would affect the sensitivity to 5-FU. LAMP2A expression was decreased in HCT116-R/DLD-1-R cells by transfection with shLAMP2A plasmid, which resulted in decreased cell growth rate and increased 5-FU-mediated cell death (Figs. 3A and B; 4A and B). Conversely, when LAMP2A expression was increased in HCT116/DLD-1 cells by transfection with the LAMP2A overexpression plasmid, markedly increased cell growth rate and decreased 5-FU-mediated cell death was observed (Figs. 3A and B and 4A and B). The same results were observed following treatment with OXA and FOX (Figs. 3A and 4A). In addition, the knockdown of LAMP2A in HCT116-R/DLD-1-R cells or overexpression of LAMP2A in HCT116/DLD-1 cells was associated with decreased or increased expression of Bcl-2. The expression of Bax was opposite (Figs. 3B and 4B). Migration and invasion were lower in cells with knockdown of LAMP2A, but apoptosis was higher. Adverse results were found in cells overexpression of LAMP2A (Figs. 3C and D and 4C and D). Therefore, it was confirmed that HCT116-R/DLD-1-R cells can regain sensitivity to 5-FU if LAMP2A expression is reduced.

Knockdown of LAMP2A in HCT116-R/DLD-1-R cells or overexpression of LAMP2A in HCT116/DLD-1 cells was associated with decreased or increased expression of PLD2 and NF-κB, respectively. It was hypothesized whether high expression of LAMP2A enhanced PLD2 expression through activation of NF-κB pathway. Therefore, HCT116-R/DLD-1-R cells were treated with CQ or PDTC to inhibit the lysosomal or NF-κB pathways. When HCT116/DLD-1 and HCT116-R/DLD-1-R cells were treated with CQ (50 mM) and harvested at specific time points (0, 2, 4, 6, 8 and 10 h), it was identified that PLD2 first decreased and then increased (Fig. 5A). The lowest level was observed at 4 h. Next, HCT116/DLD-1 and HCT116-R/DLD-1-R cells were treated with CQ (50 mM) for 4 h and a clear and significant reduction of PLD2 was observed compared with non-treated cells in the western blot analysis (Fig. 5B and C). Notably, similar results were obtained with PDTC (10 mM, 2 h; Fig. 5B and C). Co-immunoprecipitation experiments further confirmed the interaction of NF-κB with PLD2 (Fig. 5D). In addition, some drug-resistance proteins (CD147, GST3 and MLH1) were observed to be associated with NF-κB by co-immunoprecipitation experiments (Fig. 5D).