Doxorubicin was purchased from Selleck Chemicals (Houston, TX, USA). RPMI-1640, radioimmunoprecipitation assay (RIPA) buffer, Hanks buffer and MTT reagent were obtained from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany). Tocilizumab (an anti-IL6R antibody) was purchased from Roche Diagnostics, Basel, Switzerland. Primers, probes and cDNA kits for mRNA quantification were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and foetal bovine serum (FBS), Lipofactamine 2000 and Antibiotic-Antimycotic were procured from Gibco (Thermo Fisher Scientific, Inc). Lentiviral particles for knockdown of PTEN were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All antibodies were obtained from CST (Cell Signaling Technology, Inc., Danvers, MA, USA).

The LS180 cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 supplemented with 1% antibiotics Antibiotic-Antimycotic and 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂. For the generation of doxorubicin-resistant cells, LS180 and LS180 short hairpin (sh)PTEN (shPTEN-treated LS180) cells were treated with 1 µM of doxorubicin for a period of nine months. However, resistant cells were grown in drug-free media for 48 h prior to experimentation.

shRNA PTEN lentiviral particles from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) were used for PTEN knockdown. Briefly, LS180 and doxorubicin-resistant LS180 cells were grown in 6 well plates in an incubator at 37°C with 5% CO₂ and 95% humidity for 24 h. shRNA PTEN Lentiviral particles Santa Cruz Biotechnology Inc. at a concentration of 1 µg/ml with polybrene [Sigma-Aldrich (5 µg/ml)] were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to the cells for 24 h. Following this 24 h incubation, media was replaced with complete DMEM. Puromycin (4 µg/ml) was used as a selection marker and three rounds of selection were performed for 48 h each. Only the cells resistant to puromycin were cultured for subsequent experiments.

The LS180 cell line and its LS180 PTEN knockdown model, as well as doxorubicin-resistant cells were seeded in 96-well plates at a density of 1.5×10⁴ cells/well and allowed to grow for 24 h at 37°C. At 24 h, parental and resistant cells were treated with doxorubicin at a concentration dependent manner (0.1, 0.2, 0.4, 0.8, 1, 2, 5, 10, 15 and 20 µM) for 48 h. MTT solution was added into each well at a concentration of 2.5 mg/ml and cells were incubated for 4 h at 37°C. Finally, a total of 150 µl dimethylsulfoxide (DMSO) was added to each well to dissolve the formazen crystals and absorbance was detected at 570 nm by a synergy MX plate reader (BioTek Instruments, Inc., Winooski, VT, USA).

LS180, LS180 shPTEN, doxorubicin LS180 and doxorubicin LS180 shPTEN cells were seeded at a density of 1×10⁶ in 60 mm dishes for 24 h. Cells were treated with doxorubicin at a concentration of 1 µM for 24 h and were lysed using RIPA buffer. Protein determination was performed by the Bradford method, and proteins (70 µg) were separated on 10% SDS-PAGE and then transferred onto a nitrocellulose membrane at 100 V for 2 h. To avoid non-specific binding, membranes were blocked in 5% fat-free milk for 1 h at room temperature and primary antibodies p-AKT ser473 (10% SDS-PAGE; cat. no. 4060; dilution 1:1,000), AKT (10% SDS-PAGE; cat. no. 4691; dilution 1:1,000), NF-κB p65 (10% SDS-PAGE; cat. no. 8242; dilution 1:1,000), EpCAM (10% SDS-PAGE; cat. no. 93790; dilution 1:1,000), claudin-3; 15% SDS-PAGE; cat. no. 83609; dilution 1:1,000), PTEN; 10% SDS-PAGE; cat. no. 9188; dilution 1:1,000), TGFR2 (10% SDS-PAGE; cat. no. 79424; dilution 1:1,000), vimentin (10% SDS-PAGE; cat. no. 5741; dilution 1:1,000), β-actin (cat. no. 4970; dilution 1:2,000), TWIST (15% SDS-PAGE; cat. no. 14472; dilution 1:1,000), E-cadherin (6% SDS-PAGE; cat. no. 14472; dilution 1:1,000), HRP conjugated mouse anti-rabbit secondary antibody (cat. no. 93702; dilution 1:2,500), anti-mouse secondary antibody (cat. no. 14709; dilution 1:2,500) were obtained from Cell Signaling Technology Inc., (Danvers, MA, USA) incubated overnight at 4°C. Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST) two times for 5 min. The horseradish peroxidase (HRP)-labeled antibody was added at room temperature for 1 h, after which the protein blots were again washed twice with TBST at room temperature for 5 min each. Finally, protein bands were visualized using enhanced chemiluminescence (ECL; GE Healthcare, Chicago, IL, USA) and an X-ray film.

This assay was performed to detect the cancer stem cell fraction within the colon cancer cell line LS180. Cells (LS180, LS180 shPTEN, doxoLS180 and doxoLS180 shPTEN) were incubated with anti-CD44-PE (cat. no. ab46793; dilution 1:200) and anti-CD24-FITC (cat. no. ab30350; dilution 1:200) or stained with their isotype controls IgG (cat. no. ab172730; dilution 1:200; all Abcam) for 30 min on ice. Following this incubation, cells were washed in Hanks' balanced salt solution (HBSS) supplemented with 2% FCS and analysed by flow cytometry using FACSDiVa 6.2 software with a (BD Accuri^™ C6 Flow Cytometer (both BD Bioscience).

IL6, IL2 and IL8 levels were detected using ELISA. Briefly, cells (LS180, LS180 shPTEN, doxoLS180 and doxoLS180 shPTEN) were seeded at a density of 0.25×10⁶ in a 24-well plate and allowed to grow in an incubator at 37°C with 5% CO₂ and 95% humidity for 3 days. The media from the cultured cells was then removed and was analysed for the IL6, IL2 and IL8 levels using an antibody array 5 raybio human cytokine kit according to the manufacturer's protocol.

Using mammocult medium (Stem Cell Technologies, Inc., Vancouver, BC, Canada), cells (LS180, LS180 shPTEN, doxoLS180 and doxoLS180 shPTEN) were seeded in ultra-low attachment plates at a density of 1×10⁵ cells/well and allowed to grow for 7 days and were then treated with tocilizumab at a concentration of 1 mg/l for 48 h. Following treatment, the primary spheres were dissociated by pippeting and single cells were reseeded in ultra-low attachment 6-well plates at a density of 5×10⁴ cells/well in mammary epithelial growth medium (MEGM, Lonza), supplemented with B27 (Invitrogen; Thermo Fisher Scientific, Inc.), 20 ng/ml bFGF (Sigma-Alrich; Merck KGaA) and 30 ng/ml EGF were incubator at 37°C with 5% CO₂ and 95% humidity for 21 days of incubation. A total of 20 fields of view were randomly selected, observed and secondary spheres were counted using a light microscope at a magnification of ×30.

Primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc.) vimentin forward, 5′-GGCTCAGATTCAGGGGAACAGC-3′ and reverse, 5′-CAGGTTGTGCAGGTTGTTCTA-3′; TWIST forward, 5′-TGTAAAACGACGGCCAGT-3′ and reverse, 5′-CAGGAAACAGCTATGACC-3′; N-cadherin forward, 5′-CACTGCTCAGGACCCAGAT-3′ and reverse, 5′-TAAGCCGAGTGATGGTCC-3′; TGFR forward, 5′-TGTAAAACGACGGCCAGT-3′ and reverse, 5′-CAGGAAACAGCTATGACC-3′; E-cadherin forward, 5′-GGGGTACCTGTCTCTCTACAAAAAGGCA-3′ and reverse, 5′-GGAAGATCTGGGCTGGAGCGGGCTGGAGT-3′; CLDN3 forward, 5′-CTGCTCTGCTGCTCGTGTCC-3′ and reverse, 5′-TTAGACGTAGTCCTTGCGGTCGTAG-3′; EpCAM forward, 5′-CGCAGCTCAGGAAGAATGTG-3′ and reverse, 5′-TGAAGTACACTGGCATTGACG-3′; and GAPDH forward, 5′-GGTGTGAACGGATTTGGCCGTATTG-3′; and reverse, 5′-CCGTTGAATTTGCCGTGAGTGGAGT-3′ Total RNA was isolated from parental and doxorubicin-resistant cells using the TRIzol reagent (Sigma-Aldrich; Merck KGaA). RNA was purified by using RNeasy mini kit (Qiagen GmbH, Hilden, Germany). RNA was reverse-transcribed into cDNA using M-MLV RT kit (Promega Corporation, Madison, WI, USA). RT-qPCR was performed using a TaqMan universal PCR master mix from Roche (Roche Diagnostics, Basel, Switzerland) with reverse transcription involving denaturation at 94°C for 30 sec and annealing and elongation at 72°C for 1 min followed by aforementioned primers on an ABI PRISM sequencing detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) The relative fold change of differential inducible expression of the genes vs. control group was quantified by using the 2^−∆∆Cq method (27).

For statistical analysis graphPad Instat3 software (GraphPad Software Inc., La Jolla, CA, USA) was used. All the experiments were performed three times. The relevant data are expressed as the mean ± standard deviation (SD). One-way analysis of variance by post-hoc analysis with Tukey's multiple-comparisons test was performed was used to examine differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

LS180, a colon cancer cell line was selected, and a stable PTEN knockdown model of LS180 was generated. LS180 and LS180 shPTEN cells were treated with increasing concentrations of doxorubicin for a period of over nine months. Resistance developed by doxorubicin was calculated by cell viability. The IC₅₀ of doxorubicin had increased in the resistant cells as compared with their parental cell lines. Furthermore, the knockdown of PTEN also decreased the response of LS180 cells towards doxorubicin (Fig. 1).

The IL6 signalling pathway is a crucial cellular pathway and its deregulation has been reported in various types of cancer. Doxorubicin resistance in LS180 cells led to increased level of IL6 which was further elevated in response to shPTEN treatment (Fig. 2A). It was also observed that shPTEN in doxorubicin resistance LS180 leads to an increase in the level of IL2 and IL8 (Fig. 2B). Furthermore, tocilizzumab (an IL6 inhibitor) led to almost complete inhibition of IL6 production in the parental cells as well as doxorubicin-resistant and PTEN knockdown models and this effect was also observed in IL2 and IL8 (Fig. 2A and B). Furthermore, the expression of p-AKT 473 has been increased in doxorubicin resistance LS180 cells that leads to the upregulation of NF-kB as compared with LS180 cells. However, the effect was further elevated in response to shPTEN treatment (Fig. 2C and D).

To investigate the stem properties of different subtype, the present study compared the expression of CD44, CD24 in LS180, LS180 shPTEN, doxoLS180 and doxoLS180 shPTEN cell lines using flow cytometry analysis. As expected doxorubicin resistance led to an increase in the population of these cancer-like stem cells whose numbers were further elevated by knockdown of PTEN by analysing the markers of stem cells such as CD44⁺/CD24⁻ (Fig. 3A). Doxorubicin-resistant cells exhibited an increased ability to form mammospheres. PTEN knockdown further increased the formation of mammospheres. Notably, anti-IL6R has decreased the formation of mammospheres in PTEN knockdown doxorubicin resistant LS180 cells (Fig. 3B).

Cancer cells undergo reprogramming and change morphologically when treated with chemoresistant drugs over a period of time (28). The results of the present study demonstrated that RT-qPCR analysis of the epithelial to mesenchymal responsive genes including neural (N)-cadherin, vimentin, TWIST and TGFR2 was markedly increased in doxorubicin-resistant cells which was further enhanced by shPTEN doxorubicin-resistant cells as compared with LS180 (Fig. 4A). Furthermore, the expression of E-cadherin, epithelial cell adhesion molecule (EpCAM) and claudin-3 was decreased in both doxorubicin-resistant and shPTEN doxorubicin-resistant cells compared with LS180 cells (Fig. 4A). It was further confirmed by western blotting that there was a increase in the expression of N-cadherin, vimentin, TWIST and TGFR2 in doxorubicin-resistant cells as compared to parental cells (Fig. 4C) whereas an increase in the expression of mesenchymal proteins, including E-cadherin, epithelial cell adhesion molecule (EpCAM) and claudin-3 in the doxorubicin-resistant cells compared with parental cells (Fig. 4D. Taken together, these results demonstrate that doxorubicin leads to resistance in colon cancer and induces EMT-like phenotype which is further elevated by the knockdown of PTEN.