Research ArticleExperimental Studies
Open Access

Targeting Glypican-1 Reverses Resistance to 5-Fluorouracil in Esophageal Adenocarcinoma Cells

AKSHAY PRATAP, ANDREA QUALMAN, HEDLUND GARRETT, ERLINDA THE, ARGUDIT CHAUHAN, JUAN P. IDROVO, LINLING CHENG, SACHIN WANI, ROBERT ALEXANDER MEGUID and XIANZHONG MENG
Anticancer Research August 2023, 43 (8) 3411-3418; DOI: https://doi.org/10.21873/anticanres.16516

Abstract

Background/Aim: The primary mode of therapy for individuals with locally advanced esophageal adenocarcinoma (EAC) is neoadjuvant chemotherapy, commonly 5-Fluorouracil (5-FU). However, approximately 30% of these patients develop resistance to therapy. Glypican-1 (GPC-1) has been identified as one of the key drivers of chemoresistance in cancer; however, its role in EAC cells has not been explored. The objective of the present study was to evaluate the role of GPC-1 in chemoresistance to 5-FU in EAC cells. Materials and Methods: Cell viability to 5-FU was measured with CCK-8 assay, and GPC-1 expression was validated using western blot. 5-FU resistant cell lines were generated. The effect of lentivirus-mediated GPC-1 knockdown on FLO-1 cell viability, cell cycle, and apoptosis was evaluated. Results: 5-FU resistant EAC cells showed increased GPC-1 expression and knockdown of GPC-1 increased cell death and apoptosis. Importantly, the knockdown of GPC-1 enhanced the antitumor effects of 5-FU in vitro via down-regulating AKT/ERK/β-catenin signaling. Conclusion: Silencing GPC-1 has the potential to augment the efficacy of 5-FU chemotherapy in resistant EAC tumors.

Key Words:

Esophageal adenocarcinoma (EAC) is regarded as one of the most lethal malignant neoplasms worldwide, consequently ranking as the sixth primary cause of cancer-induced mortality (1). Although esophageal resection has been the mainstay of treatment, most patients are ineligible for curative surgery due to its delayed diagnosis, advanced stage, and progression of disease due to chemoresistance (2, 3). Despite significant treatment advances, the 5-year survival of EAC patients is only 23% (4, 5). The commonly approved primary treatment for EAC entails a combination of fluoropyrimidine and platinum-based chemotherapy. Nevertheless, the combination is known to have a median overall survival period of less than one year, which is attributed to progression of the disease on therapy due to multidrug resistance (6). Therefore, the current scenario necessitates the prompt exploration of novel therapeutic approaches that can effectively address the challenges of multidrug resistance in EAC. In a recent study, our group identified the critical role of Glypican-1 (GPC-1) in the progression of EAC (7). GPC-1 is a member of the family of glycoproteins known as membrane heparan sulfate proteoglycans (8). Glypicans serve as co-receptors to activate multiple intracellular signaling pathways, which drive tumorigenesis, chemoresistance, angiogenesis, and metastasis (9, 10). GPC-1 over-expression has also been linked to poor prognosis in esophageal cancer, pancreatic adenocarcinoma, and glioblastoma by promoting chemoresistance (11-13). However, the role of GPC-1 in EAC is still relatively unexplored. In this study, we investigated the role of GPC-1 in sensitizing resistant EAC cells to 5-Fluorouracil. We further analyzed the expression of Akt/Erk/β-catenin signaling in resistant EAC cell lines.

Materials and Methods

Chemical reagents. All chemicals and reagents were purchased from Millipore Sigma (St. Louis, MO, USA). 5-Flurouracil (5-FU) was purchased from Selleck Chem (Houston, TX, USA). Dimethyl sulfoxide was used to reconstitute 5-FU.

Cell lines culture and transfection. Cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). EAC cell lines, OE19 and FLO-1 were grown in Roswell Park Memorial Institute Medium (RPMI 1640; Gibco, Grand Island, NY, USA) and Dulbecco’s Modified Eagle Medium (DMEM, Gibco), respectively. Growth media were supplemented with 10% fetal bovine serum (FBS), 0.5% penicillin/streptomycin, and gentamicin/amphotericin (1:500 dilution). All cells were incubated in a humidified atmosphere with 95% air and 5% carbon dioxide at 37°C. Serum-reduced media for lentivirus transfection consisted of growth medium with 2-5% FBS, 0.5% penicillin/streptomycin, and gentamicin/amphotericin (1:500 dilution).

Transfection and establishment of stable cell line. Short hairpin RNA (shRNA) targeting GPC-1 was designed using a previously published protocol (7). The control scramble shRNA (SCR) was used as a negative control. Lentiviruses were prepared using a lentivirus packaging plasmid kit (Genecopeia, Rockville, MD, USA) according to the manufacturer’s protocol. HEK 293T cells were seeded in 6-well plates and transfected with packaging plasmid and constructs. Lentiviruses were collected 48 h later. Cells stably expressing shGPC-1 were grown in puromycin 2 μg/ml.

Western blotting. Cells were washed with ice cold phosphate-buffered saline (PBS) and then lysed with ice cold RIPA buffer (Sigma Aldrich, St Louis, MO, USA). Protein concentration was determined using BCA assay (DC assay, Bio-Rad, Hercules, CA, USA) and bovine serum albumin to generate a standard curve. Equal amounts of protein concentrations (25 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad 4%-20%) electrophoresis and transferred to 0.4 μm nitrocellulose membranes. Membranes were blocked in TBS-Tween 20 with 5% non-fat milk for 1 h and then incubated with primary antibodies (diluted in 5% BSA) at 4°C overnight. Secondary antibodies were diluted in TBS-Tween 20 with 5% non-fat milk. Protein quantification with densitometry analysis was performed using Image Lab Software (Bio-Rad).

Cell proliferation assay. Cell proliferation was determined by CCK-8 (GlpBio, Montclair, CA, USA). Cells were seeded in 96-well plates at 20,000 density and treated with CCK-8 at various time points. Plates were read on BioTek microplate reader at 450 nm.

Flow cytometry. For cell cycle analysis, cells were seeded in 12-well flat-bottom plates at a density of 200,000 cells per well for 24 h in antibiotic-free media. After 48 h of transfection, cells were harvested for cell cycle analysis using a Propidium Iodide Flow Cytometry Kit (ab139418; Abcam, Cambridge, UK). Samples were analyzed by flow cytometry using a BD FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA). For apoptosis analysis, cells were seeded into 24-well plates at 200,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with lentivirus particles at 48 h, and prepared for flow cytometry using Tonbo Bioscience PE Annexin V Apoptosis Kit (Tonbo Bioscience, San Diego, CA, USA). Samples were analyzed using a BD FACS Canto II. Data were analyzed using Flowjo v.10.8 software.

Statistical analysis. Experiments were performed three times. Data represent the mean±standard deviation (SD) and comparisons were made using Student’s t-test. Data with multiple comparisons were analyzed by ANOVA followed by Fisher’s least significant difference post hoc test. All statistical analysis was performed using Graph Pad prism software version 9.5.1(733). A p-value <0.05 was considered significant.

Results

Establishing 5-FU resistant cells. FLO-1 cells were treated with various concentrations of 5-FU to generate resistant cells. Cell viability was determined to 5-FU using CCK-8 assay, Figure 1. FLO-15-FU-R cells exhibited a significant increase in resistance to 5-FU, with IC50 of 89.21±2.2 μg/ml compared to parental FLO-1 cells (IC50=78.23±1.12 μg/ml) and OE-19 cells (IC50=60.33±3.10 μg/ml).

Figure 1.
Figure 1.

Establishing 5-FU resistant cells. Cell viability to 5-FU was calculated using CCK-8 assay in OE-19, parental FLO-1, and FLO-15-FU-R cells treated with given concentrations of 5-FU for 48 h.

Glypican-1 is up-regulated in 5-FU resistant EAC cells. Expression of GPC-1 was evaluated in 5-FU resistant FLO-1 cells (FLO-15-FU-R) using western blot. GPC-1 was markedly up-regulated in resistant cells (Figure 2A). To establish clear evidence of an induced GPC-1 expression by 5-FU, we treated wild-type (WT) EAC cells with different concentrations of 5-FU for 3 days. Western blot findings indicated a dose-dependent increase in the expression of GPC-1 (Figure 2B). These results suggest that GPC-1 is implicated in the mechanism that underlies resistance to 5-FU.

Figure 2.
Figure 2.

Glypican-1 is up-regulated in 5-FU resistant EAC cells. (A) Western blot analysis of GPC-1 expression in parental FLO-1 and FLO-15-FU-R cells. (B) Western blot analysis of response to 5-FU dose on GPC-1 expression in FLO-1 cells. β-actin was used as a loading control.

Knockdown of GPC-1 increases sensitivity to 5-FU. To further explore the role of GPC-1 in chemoresistance, we generated lentiviral mediated stably expressing GPC-1 short hairpin RNA (shRNA) FLO-15-FU-R cells (shGPC-1 FLO-15-FU-R). The efficiency of GPC-1 knockdown is shown in Figure 3A. To ascertain the potential sensitization of EAC cells towards 5-FU in response to GPC-1 silencing, we compared the viability of FLO-15-FU-R and shFLO-15-FU-R cells treated with varying concentrations of 5-FU. As shown in Figure 3B, CCK-8 assay showed that the efficacy of 5-FU treatment in FLO-15-FU-R was enhanced significantly as seen by a reduction in the IC50 from 85.3±3.3 to 42.1±2.5 μg/ml. These data suggest that GPC-1 over-expression mediates drug resistance, and its silencing may be a viable option for individuals with EAC who have become resistant to chemotherapeutic drugs.

Figure 3.
Figure 3.

Knockdown of GPC-1 increases sensitivity to 5-FU. (A) Western blot showing efficient knockdown of GPC-1 in FLO-15-FU-R cells. (B) Cell viability curve using CCK-8 assay in FLO-15-FU-R cells treated with either 5-FU or the combination with shGPC-1.

GPC-1 knockdown potentiates cell cycle arrest in 5-FU-resistant cells. To validate the impact of GPC-1 silencing on cell proliferation we analyzed the distribution of cells in the cell cycle and apoptosis using flow cytometry. As shown in Figure 4A, the percentage of cells in sub G1 phase increased with shGPC-1 and the combination of shGPC-1 and 5-FU significantly increased the number of cells in this cell cycle phase. In addition, there was an increased percentage of cells arrested in the G2/M phase with the combination of shGPC-1 and 5FU. Therefore, it appears that GPC-1 knockdown and 5-FU synergize to enhance apoptosis and G2/M arrest in FLO-1 cells. At the molecular level, the expression levels of cyclins and proteins that promote G2 phase entry to mitosis were significantly reduced by shGPC-1 alone or in combination with 5-FU, as shown in Figure 4B.

Figure 4.
Figure 4.

GPC-1 knockdown potentiates cell cycle arrest in 5-FU-resistant cells. (A) Cell cycle phase analysis using propidium Iodide staining in scramble FLO-15-FU-R, FLO-15-FU-R, and shGPC-1FLO-15-FU-R cells treated with 5-FU. (B) Western blot analysis of Cyclin B1, cdk1, and pHH-3 expression in FLO-15-FU-R cells silenced with shGPC-1 normalized to β-actin. ****p<0.0001.

GPC-1 knockdown potentiates apoptosis in 5-FU-resistant cells. To further evaluate the role of GPC-1 in apoptosis, Annexin V/FITC flow cytometric analysis was performed. As shown in Figure 5A, the percentage of apoptotic cells in FLO-15-FU-R were not significantly different compared to scramble control; however, GPC-1 knockdown alone caused an increase in the percentage of apoptotic cells and this effect was even more pronounced with the combination of GPC-1 and 5-FU. Protein expression of apoptosis markers cleaved PARP and Bax was significantly increased whereas that of anti-apoptosis protein Bcl2 was reduced with the combination of GPC-1 knockdown and 5-FU (Figure 5B).

Figure 5.
Figure 5.

GPC-1 knockdown induces apoptosis in 5-FU-resistant cells. (A) After 5-FU treatment for 48 h the GPC-1 silenced cells and scrambled control cells were collected and subjected to Annexin V/PI double staining. The Annexin V+/PI− cells (Q3 quadrant) and Annexin V−/PI− cells (Q4 quadrant) were then analyzed by flow cytometry. (B) Western blot analysis of key apoptosis protein marker expression after 5-FU treatment and GPC-1 silencing normalized to β-actin. *p<0.05, **p<0.01, ***p<0.001.

GPC-1 regulates chemoresistance via AKT/ERK/β-catenin signaling. Akt/Erk/β-catenin signaling is regarded as a key pro-survival and proliferation factor (14). To elucidate the regulation of Akt/Erk/β-catenin signaling by GPC-1 in 5-FU resistance, we treated FLO-15-FU-R either with 5-FU or its combination with shGPC-1 and analyzed the phosphorylation of Akt, Erk, and β-catenin using western blot. Silencing GPC-1 significantly attenuated the levels of active phospho-AkT, phospho-Erk, and phospho-β-catenin in FLO-15-FU-R cells (Figure 6). These results indicate that inhibition of GPC-1 dramatically improves the sensitivity of resistant cells to chemotherapy.

Figure 6.
Figure 6.

GPC-1 regulates chemoresistance via AKT/ERK/β-catenin signaling. After 5-FU treatment for 48 h, the GPC-1 silenced cells and scrambled control cells were collected and western blot analysis was performed for active and total Akt, Erk, and β-actin.

Discussion

The current recommended management of locally advanced or metastatic EAC is the administration of neoadjuvant chemotherapy (15). While the short-term efficacy of chemotherapy may be notable, its clinical effectiveness has been considerably restricted due to drug resistance development (16-18). Consequently, multiple research endeavors are being attempted to elucidate the molecular regulation and surmount the development of drug resistance in cancer therapy (19-22). In the present study, we show mechanistic insights into the regulation and specifically the contribution of GPC-1 in the development of chemoresistance in EAC. The significant results in this work indicate that: 1) GPC-1 is up-regulated in 5-FU resistant FLO-1 cells; 2) Silencing GPC-1 overcomes resistance to 5-FU; 3) Combination of GPC-1 knockdown and 5-FU produces a synergistic effect on cell cycle arrest/apoptosis; and 4) GPC-1 up-regulates AKT/ERK/β-catenin signaling to confer chemoresistance. GPC-1 is a newly identified oncogene that is linked to malignant transformation and can function as a survival factor via conferring drug resistance. However, the intricate control of GPC-1 expression and its underlying mechanisms regarding drug resistance in EAC cells has yet to be extensively examined. Our results indicate a noticeable up-regulation of GPC-1 in FLO-1 cells that were resistant to 5-FU, thus implying a direct correlation between the augmented GPC-1 expression and 5-FU resistance. Per our research, we noted that FLO-1 cells exhibited high GPC-1 expression and were more resistant to 5-FU-induced cell death compared to OE-19 cells that manifested low levels of GPC-1. Expression of GPC-1 in FLO-1 cells also correlated positively with exposure to increasing doses of 5-FU. In a comparable manner, reports have demonstrated the involvement of GPC-1 in the development of drug resistance in pancreatic cancer (10), glioblastoma (11), and esophageal cancer (8). Furthermore, it has been postulated that elevated GPC-1 expression levels in various solid tumors may serve as a prognosticator of reduced survival rates (12). In our previous report, we also showed that GPC-1 promotes the proliferation, migration, and invasion of EAC cells (13). In the present study, silencing GPC-1 with lentivirus shRNA made FLO-15-FU-R cells more susceptible to 5-FU. The results of this study indicate that the inhibition of GPC-1 could potentially serve as a vital factor in the control of cancer progression and growth, alongside the development of resistance to anti-cancer drugs in EAC cells. The evaluation of cellular responsiveness towards 5-FU using cell cycle analysis showed no significant difference in cell cycle phases in the scramble and FLO-15-FU-R cell groups. Silencing GPC-1 alone increased the percentage of cells in G2/M phase and reduced cells in G1/S phase. Combining 5-FU with GPC-1 knockdown in FLO-15-FU-R cells further increased the number of cells in G2/M phase, indicating the arrest of cells in G2/M phase. At the molecular level, the knockdown of GPC-1 reduced the expression of Cyclin B1, cdk1, and phospho-histone 3 (pHH-3), indicating that silencing GPC-1 reduced the expression of proteins involved in the progression of cells from the G2 phase to mitosis.

The balance between pro- and anti-apoptotic factors determines the fate of cancer cells and their sensitivity to chemotherapeutic agents. We, therefore, analyzed apoptosis in each of the groups using flow cytometry and western blot. Interestingly, combining 5-FU with GPC-1 knockdown overcame the repression of apoptosis in FLO-15-FU-R cells. Western blot results corroborated with flow cytometric findings showing increased cleavage of PARP and Bax and reduced expression of Bcl2 with combinatorial treatment with GPC-1 knockdown and 5-FU. Several studies suggest that GPC-1 plays a crucial role in cell division, migration, and apoptosis (23, 24). Studies have also shown that the knockdown of GPC-1 induces G2 phase arrest in various cell types (25). The Akt/Erk/β-catenin signaling pathway is widely recognized as a significant contributor to chemoresistance in cancer (26, 27). Studies have indicated that aberrant activation of the Akt/Erk pathway inhibits cell apoptosis and promotes cell survival (28). There is also strong evidence that there is crosstalk between Akt/Erk and Wnt/β-catenin pathways, which is believed to play a major role in tumor progression and resistance (29). Our study demonstrated that silencing GPC-1 reduced phosphorylation of Akt, Erk, and β-catenin and the effect was more pronounced when GPC-1 silencing and 5-FU were combined.

Conclusion

Collectively, the present study demonstrates that GPC-1 serves as a potential biomarker underlying the resistance of EAC cells to therapeutic intervention with 5-FU via its regulation of the Akt/Erk/β-catenin pathway. Our findings imply that a complementary approach involving the inhibition of GPC-1 and administration of 5-FU may represent a potent therapeutic tactic for individuals afflicted with EAC.

Acknowledgements

This work was supported by Intuitive Foundation Inc Robotic Fellowship grant, and ACS IRG#16-184-56 from American Cancer Society to the University of Colorado Cancer Center, Colorado, USA.

Footnotes

  • Authors’ Contributions

    A.P, A.Q, G.H, E.T and A.C designed and performed the experiments. A.C, J.P.I and L.C analyzed the data. S.W, R.A.M and X.M interpreted the data. A.P and X.M wrote and revised the manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received May 13, 2023.
  • Revision received June 11, 2023.
  • Accepted June 13, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Targeting Glypican-1 Reverses Resistance to 5-Fluorouracil in Esophageal Adenocarcinoma Cells
AKSHAY PRATAP, ANDREA QUALMAN, HEDLUND GARRETT, ERLINDA THE, ARGUDIT CHAUHAN, JUAN P. IDROVO, LINLING CHENG, SACHIN WANI, ROBERT ALEXANDER MEGUID, XIANZHONG MENG
Anticancer Research Aug 2023, 43 (8) 3411-3418; DOI: 10.21873/anticanres.16516
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