Human CRC cell lines HCT116 and LoVo were obtained from ATCC. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 23.05 mmol/L glucose (Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO₂.

To mimic the GD condition of tumor microenvironment, HCT116 and LoVo cells were incubated for 48 h in DMEM containing 1.5 mmol/L glucose and 10% FBS containing about 0.5 mmol/L glucose at 37 °C in a humidified atmosphere containing 5% CO₂.

For the cell proliferation assay, 1000 CRC cells were plated in 96-well plates and incubated for different time periods (24, 48, 72, and 96 h), and then the cell growth was detected with the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. For the cell chemotherapy sensitivity assay, 2000 LoVo or HCT116 cells were plated in 96-well plates and treated with oxaliplatin (LOHP; range, 0-16 μg/mL) and 5-fluorouracil (5-FU; range, 0-1.6 μg/mL) for 48 h, and cell inhibition was then assessed by the CCK-8 assay.

Hoechst Staining was performed according to the manufacturer’s protocol (Beyotime, China). Cells were visualized with a DP70 inverted immunofluorescence microscope (Olympus). Cells with condensed and fragmented nuclei were judged to be apoptotic.

Total RNA was prepared from cultures on day 10, using the RNAiso reagent (TaKaRa, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the HiFiScript cDNA Kit (CWBIO, China). Quantitative real-time RT-PCR analysis was performed to detect mRNA expression using UltraSYBR Mixture (CWBIO), with β-actin as an internal control. The sequences of primers used in this study were as follows: 5’-CGTGGTCTTTGCTTGGG TG-3’ and 5’-TGCGGTGCTTTGCTGGAAT-3’ for ATF4; 5’-GACCTATTGGGGTGTTTCG-3’ and 5’-CCTCAGCGGTTTCTTTCAT-3’ for Grp78; 5’-ATAGTGATAAAGGTTTCGGTT-3’ and 5’-ACAGGAGTTCTGGAAGGAG-3’ for PERK; 5’-AGTGTGACGTGGACATCCGCAAAG-3’ and 5’-ATCCACATCTGCTGGAAGGTGG AC-3’ for β-actin.

Cells were lysed with RIPA buffer and incubated on ice for 30 min. After centrifugation, protein concentration was measured with BCA Protein Assay Reagent (CWBIO). Cell lysates dissolved in sample buffer were separated using SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking with Tris-buffered saline containing Tween-20 containing 5% milk, the membrane was immunoblotted with appropriate primary antibodies, including anti-Grp78 (Santa Cruz, United States), anti-PERK (Cell Signaling, United States), anti-ATF4 (Santa Cruz), anti-MDR1 (Santa Cruz) and anti-β-actin (Abcam, United States), followed by incubation with goat anti-mouse immunoglobulin (Ig) or anti-rabbit Ig conjugated with horseradish peroxidase. After washing, the membrane was developed using Chemiluminescent Substrate (CWBIO).

Green fluorescent protein-expressing lentiviral plasmids expressing short hairpin RNA (shRNA) against human ATF4 (Vehicle-shATF4) were obtained from Open Biosystems (Carlsbad, CA), and ATF4 lentiviral plasmid (Vehicle-ATF4) was constructed as described in our previous work[8]. The Vehicle, Vehicle-shATF4, Vehicle and Vehicle-ATF4 plasmids were cotransfected into HEK-293T cells along with the packaging plasmid ps-PAX2 and the envelope plasmid pMD2G using Lipofectamine 2000 (Invitrogen). Virus particles were harvested 48 h after cotransfection. Then, the particles were individually used to infect HCT116 and LoVo cells. The cells were then harvested 3 d after infection for Western blot and qRT-PCR validation.

LoVo or HCT116 cells were plated in 6-well plates and treated with LOHP (0.1 μg/mL) and 5-FU (0.05 μg/mL) for 48 h. The cells were then harvested and subjected to apoptosis analysis using an Annexin V/7-AAD Apoptosis Detection Kit (CWBIO).

Each experiment was repeated at least three times. The data are presented as the mean ± SD. Differences between groups were analyzed with Student’s t test. All statistical analyses were performed using GraphPad Prism 5 software. The significance level was set at 0.05.

To investigate whether the surviving CRC cells under GD could acquire drug resistance, we assessed the potential effect of GD on the sensitivity of CRC cells to LOHP and 5-FU, two of the most commonly used drugs for CRC treatment[15]. The results revealed that the IC₅₀ values of GD-treated HCT116/LoVo cells were significantly higher than those of their corresponding control cells (Figure 1A and Figure 2), suggesting that GD strongly decreases the sensitivity of CRC cells to LOHP and 5-FU. These data indicate that GD induces a MDR phenotype in CRC cells. Next, to determine whether GD inhibits chemotherapy-induced apoptosis in CRC cells, we used Hoechst staining to investigate the apoptotic rates. After incubation under GD condition for 24 h, CRC cells were treated with LOHP or 5-FU for subsequent 48 h under normal culture conditions. These cells were then subjected to Hoechst staining. The results revealed that the apoptotic rates were much lower in the GD-treated CRC cells than in the control cells (Figure 1B). To confirm the MDR phenotype of the GD-treated CRC cells, we examined the expression levels of multidrug resistance gene 1 (MDR1), a major marker of MDR. As shown in Figure 1C and D, both the mRNA and protein expression levels of MDR1 were increased in the GD-treated CRC cells as compared to the control cells. Taken together, these observations suggest that GD, through inhibiting apoptosis, significantly decreases the sensitivity of CRC cells to chemotherapy.

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Figure 1 Glucose deprivation promotes drug resistance of colorectal cancer cells. A: GD decreased drug susceptibility to CRC cells. LoVo cells were treated with the indicated doses of the different drugs for 48 h under GD or normal culture condition. The in vitro drug sensitivity was tested by the CCK-8 assay; B: GD inhibited LOHP- and 5-FU-induced apoptosis. LoVo cells were treated with 0.1 μg/mL LOHP or 0.05 μg/mL 5-FU for 48 h. Hoechst 33258 nuclear staining and Annexin V/7-AAD staining assays were performed to detect apoptosis. C and D: GD promoted the expression of resistance gene MDR1. The mRNA and protein levels of MDR1 were examined by qRT-PCR and Western blot, respectively. ^aP < 0.05, ^bP < 0.01,^cP < 0.001, control vs GD. GD: Glucose deprivation; CRC: Colorectal cancer.

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Figure 2 Glucose deprivation promotes drug resistance of HCT116 cells to LOHP and 5-FU. HCT116 cells were treated with the indicated doses of the different drugs for 48 h under GD or the normal condition. The in vitro drug sensitivity was tested by the CCK-8 assay. ^cP < 0.001, control vs GD. GD: Glucose deprivation.

Our previous work showed that GD induces tumor growth and angiogenesis by activating PERK/ATF4 arm of UPR signaling. To investigate the role of PERK/ATF4 pathway in GD-induced MDR in CRC cells, we examined the mRNA and protein expression of UPR markers (Grp78, PERK and ATF4), which are well-known to be induced by stressful microenvironments such as GD and hypoxia[8,16]. As expected, the mRNA levels of Grp78 and ATF4 were significantly increased in GD-treated CRC cells. Although the mRNA and protein expression of PERK was not significantly increased as that of Grp78 and ATF4, the phosphorylation (activation) of PERK (upward shift in the bands) was clearly observed in GD-treated CRC cells (Figure 3A and B). These data suggest the activation of UPR upon GD treatment and the potential key role of Grp78/PERK/ATF4 pathway in GD-induced MDR phenotype in CRC cells.

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Figure 3 Grp78/PERK/ATF4 pathway is activated in glucose deprivation. A and B: GD promoted the expression of genes involved in UPR. The mRNA and protein expression were examined by qRT-PCR and Western blot, respectively, and β-actin was used as an internal control. The phosphorylation of PERK (upward shift in the bands) indicated its activation in GD-treated CRC cells. ^aP < 0.05, ^cP < 0.001, control vs GD. GD: Glucose deprivation; CRC: Colorectal cancer.

To explore whether the acquisition of anti-apoptotic property in glucose-depleted CRC cells was due to the activation of ATF4, we silenced the expression of ATF4 using shATF4 in the GD-treated LoVo and HCT116 cells (Figure 4A). The results showed that silencing ATF4 expression counteracted GD-induced drug resistance of CRC cells to both drugs (LOHP and 5-FU) compared with the control cells (Figure 4B and Figure 5A). Moreover, both Hoechst nuclear staining (Figure 5B) and Annexin V/7-AAD staining assays (Figure 5C) showed that ATF4 knockdown significantly increased apoptotic rates of GD-treated CRC cells compared with the control cells. These results suggest that GD inhibits apoptotic activity in CRC cells by activating ATF4 expression. In addition, down-regulation of MDR1 was observed in the ATF4-depleted CRC cells treated with LOHP compared with the control cells, suggesting that ATF4 may mediate GD-induced MDR effect in CRC cells by up-regulating MDR1 expression (Figure 5D). Collectively, these results suggest that the activation of ATF4 plays a crucial role in the GD-induced MDR phenotype in CRC cells.

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Figure 4 Down-regulation of activating transcription factor 4 significantly reverses the glucose deprivation-induced resistance of HCT116 cells to chemotherapy. A: Silencing ATF4 expression using shATF4 in GD-treated LoVo and HCT116 cells; B: Depletion of ATF4 enhanced the sensitivity of HCT116 cells to LOHP and 5-FU. The in vitro drug sensitivity was tested using the CCK-8 assay. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, control vs GD. GD: Glucose deprivation.

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Figure 5 Down-regulation of activating transcription factor 4 significantly reverses the glucose deprivation-induced resistance of colorectal cancer cells to chemotherapy. A: Depletion of ATF4 enhanced the sensitivity of CRC cells to chemical drugs. Vector and shATF4 stably transfected LoVo cells were treated with the indicated doses of the different drugs for 48 h. In vitro drug sensitivity was tested using the CCK-8 assay; B and C: The apoptotic rates were much higher in the ATF4-depleted cells than in the control cells. Hoechst 33258 nuclear staining and Annexin V/7-AAD staining assays were performed to detect apoptosis; D: Depletion of ATF4 by shRNA in CRC cells led to significantly reduced expression of MDR1. The mRNA and protein levels of MDR1 were detected by qRT-PCR and Western blot, respectively, and β-actin was used as an internal controls. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vehicle GD vs shATF4-GD. GD: Glucose deprivation; CRC: Colorectal cancer.

To further investigate the role of ATF4 in the drug resistance of CRC cells, forced expression of ATF4 was induced in LoVo and HCT116 cells (LoVo-ATF4 and HCT116-ATF4) using lentivirus transduction. ATF4-overexpressing CRC cells were co-treated with shATF4 and therapeutic drugs, and the results demonstrated that inhibition of ATF4 increased the sensitivity of LoVo-ATF4 and HCT116-ATF4 cells to chemotherapy (Figure 6A and Figure 7A). Moreover, we detected the apoptosis in ATF4-overexpressing cells treated with shATF4, and revealed that the apoptotic rates were much higher compared with the control cells (Figure 6B and Figure 7B). Meanwhile, qRT-PCR and Western blot results also showed the decreased expression of MDR1 (Figure 6C and D). These findings further demonstrate that ATF4 contributes to the induction of chemoresistance in CRC cells.

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Figure 6 Inhibition of activating transcription factor 4 activity reintroduces drug sensitivity in activating transcription factor 4-overexpressing colorectal cancer cells. A: After transfection with shATF4 or vector, ATF4-overexpressing cells were exposed to the indicated doses of LOHP or 5-FU for 48 h. Cell viabilities were determined by the CCK-8 assay; B: The apoptotic rates were much higher in the shATF4-transfected cells than in the control cells. Annexin V/7-AAD staining assay was performed to detect apoptosis; C and D: Inhibition of ATF4 decreased the MDR1 expression. The mRNA and protein levels of MDR1 in the ATF4-depleted cells were examined by qRT-PCR and Western blot, respectively, and β-actin was used as an internal controls. ^aP < 0.05, ^bP < 0.01, vehicle vs shATF4. GD: Glucose deprivation; CRC: Colorectal cancer.

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Figure 7 Inhibition of activating transcription factor 4 activity reintroduces drug sensitivity in activating transcription factor 4-overexpressing HCT116 cells. A: ATF4 knockdown increased sensitivity of HCT116-ATF4 cell to chemotherapy. The in vitro drug sensitivity was tested using the CCK-8 assay; B: ATF4 knockdown counteracted drug-induced apoptosis. The apoptotic rates were much higher in the shATF4-treated HCT116-ATF4 cells than in the control cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vehicle vs shATF4.

Previous studies have proved the role of ATF4 in tumor proliferation. To investigate the proliferation-prompting function of ATF4 in CRC, we overexpressed ATF4 in LoVo and HCT116 cells and then inhibited ATF4 in these cells or their control cells, respectively (As shown in Figure 8A). ATF4 overexpression significantly increased the growth rates of HCT116 and LoVo cells compared to the vector control (Figure 8B). In contrast, inhibition of ATF4 in the ATF4-overexpressing CRC cells significantly decreased the growth rates compared to the control cells (Figure 8C). The results suggest that ATF4 may play multiple roles in CRC progression.

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Figure 8 Activating transcription factor 4 promotes the proliferation of colorectal cancer cells. A: We overexpressed ATF4 in LoVo and HCT116 cells and then inhibited ATF4 in these cells or their control cells, respectively; B: ATF4 overexpression enhanced the cell growth of LoVo and HCT116 cells; C: Silencing ATF4 expression inhibited the proliferation of ATF4-overexpressing cells in a dose- and time-dependent manner in LoVo and HCT116 cells. The CCK-8 assay was used to determine the cell growth rate. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, control vs ATF4. GD: Glucose deprivation; CRC: Colorectal cancer.

Chemoresistance is an important reason for clinical chemotherapy failure. Recent studies suggest that that tumor microenvironment is an important determinant of malignant progression and chemoresistance. Changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can prompt tumor progression and drug resistance. However, the role and mechanism of GD in colorectal cancer (CRC) drug resistance are unknown.

GD has been found to be involved in the regulation of multiple pathological processes that contribute to tumorigenesis and metastasis, such as tumor cell proliferation, multidrug resistance, and autophagy. Activating transcription factor 4 (ATF4) is a key player in UPR signaling. Many studies recently revealed that ATF4 participates in drug resistance of cancer. However, the role of ATF4 in GD-induced CRC drug resistance remains unclear and needs further exploration.

The authors clearly identified that GD induces the MDR phenotype of CRC cells by activating PEKR/ATF4 signaling and targeting the ATF4 pathway may provide a clinical perspective for treating drug resistant of CRC to conventional therapy.

ATF4 may be a therapeutic target to combat therapeutic resistance in CRC cells.

GD: In the tumor microenvironment, the abnormal development of vasculature results in insufficient blood supply, such as hypoxia and GD. Changes in the tumor microenvironment can promote tumor progression and drug resistance.

This is a very interesting study which explored the role and mechanism of GD in the chemoresistance of CRC. The results suggest that ATF4 may be a new target for overcoming CRC resistance to conventional chemotherapy.