IL-8-NF-κB-ALDH1A1 loop promotes the progression of intrahepatic cholangiocarcinoma

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is a poor prognosis of malignant cancer with high lymph node metastasis and resistance to systemic therapies. Recent studies suggested that the involvement of IL-8 could promote ICC metastasis through epithelial-mesenchymal transition while the ICC-ALDH1A1high subtype is clarified by multi-omics study. The correlation between ALDH1A1 and IL-8 in ICC remains elusive. This study aims to further explore the roles and regulatory mechanisms of ALDH1A1 and IL-8 in ICC.

Methods: We analyzed IL-8 and ALDH1A1 expression in ICC patients and cells. CXCR2 inhibitor (SB225002) was applied to inhibit the function of IL-8, and JSH-23 was applied to inhibit the NF-κB signaling pathway. We examined the effects of IL-8 inhibition on NF-κB, ALDH1A1 expression, and cell growth, migration, invasion, and stemness. Moreover, we examined the effects of ALDH1A1 on NF-κB, IL-8 expression, and cell growth, migration, invasion, and stemness. The effects of IL-8 and ALDH1A1 on tumor growth and NF-κB expression were validated using subcutaneous tumors in nude mice.

Results: IL-8-derived tumor cells could promote ICC progression. The high expression of IL-8 in serum was associated with lymph node metastasis. IL-8 could upregulate ALDH1A1 expression by activating the NF-κB signaling pathway, promoting tumor progression. Upregulation of ALDH1A1 could activate NF-κB to promote IL-8 secretion, forming a positive feedback loop to promote tumor invasiveness and cell stemness in ICC.

Conclusions: IL-8-derived tumor cells could upregulate ALDH1A1 expression by activating the NF-κB signaling pathway, promoting tumor progression. Upregulation of ALDH1A1 could activate NF-κB to promote IL-8 secretion, forming a positive feedback loop to promote tumor invasiveness and cell stemness in ICC.

FIGURE 1

IL-8 promotes ICC progression. (A) The level of IL-8 concentration in 30 patients with ICC. (B) The lymph node metastasis of different IL-8 expression. (C) The proliferation ability of HuCCT1 cells with IL-8 treatment. (D) The clone formation ability of HuCCT1 cells with 10 ng/mL IL-8 treatment. (E) The apoptosis ratio of HuCCT1 cells with 10 ng/mL IL-8 treatment. (F) The migration ability of HuCCT1 cells with 10 ng/mL IL-8 treatment (scale bar=200 μm). (G) The invasion ability of HuCCT1 cells with 10 ng/mL IL-8 treatment (scale bar=200 μm). *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: FITC, fluorescein isothiocyanate; ICC, intrahepatic cholangiocarcinoma; PE, phycoerythrin.

FIGURE 2

IL-8/CXCR2 promotes intrahepatic cholangiocarcinoma by activating NF-κB. (A) The proliferation ability of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment. (B) The clone formation ability of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment. (C) The apoptosis ratio of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment. (D) The migration ability of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment (scale bar=200 μm). (E) The invasion ability of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment (scale bar=200 μm). (F) The P-P65 fluorescence intensity of HuCCT1 cells with IL-8 treatment or SB225002 combined IL-8 treatment (scale bar=50 μm). (G) The expression of P-P65 protein with IL-8 treatment or SB225002 combined IL-8 treatment. Compared to the control group, *p<0.05, **p<0.01, and ***p<0.001. Compared to the IL-8 treatment group, #p<0.05, ##p<0.01, and ###p<0.001. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 3

ALDH1A1 is highly expressed in ICC, and ALDH1A1 promotes ICC by activating NF-κB. (A) The level of ALDH1A1 concentration in 30 patients with ICC. (B) The expression of ALDH1A1 protein in ICC adjacent and tumor tissues. (C) The immunohistochemistry staining of ALDH1A1 in ICC adjacent and tumor tissues (scale bar=100 μm). (D) The proliferation ability of ICC cells with different ALDH1A1 expression. (E) The clone formation ability of ICC cells with different ALDH1A1 expression. (F) The apoptosis ratio of ICC cells with different ALDH1A1 expression. (G) The migration ability of ICC cells with different ALDH1A1 expression (scale bar=200 μm). (H) The invasion ability of ICC cells with different ALDH1A1 expression (scale bar=200 μm). (I) The P-P65 fluorescence intensity of ICC cells with different ALDH1A1 expression (scale bar=50 μm). (J) The expression of P-P65 protein of ICC cells with different ALDH1A1 expression. *p<0.05, ** p<0.01, and ***p<0.001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICC, intrahepatic cholangiocarcinoma.

FIGURE 4

IL-8 participates in promoting tumor progression by upregulating the expression of ALDH1A1 via activating NF-κB. (A) The relationship of IL-8 and ALDH1A1 expression in 30 patients with intrahepatic cholangiocarcinoma. (B) The expression of ALDH1A1 mRNA by quantitative reverse transcription polymerase chain reaction. (C) The expression of ALDH1A1 protein by western blot. (D) The expression of P-P65 protein. (E) The expression of N-cadherin protein. (F) The expression of stem cell markers (CD133, OCT-4, CD44). (G) The tumorsphere formation ability of intrahepatic cholangiocarcinoma cells (scale bar=200 μm). (H) The expression of CD133 by flow cytometry. (I) The CD133 fluorescence intensity of intrahepatic cholangiocarcinoma cells (scale bar=20 μm). *p<0.05, ** p<0.01, and ***p<0.001. Abbreviations: GADPH, glyceraldehyde-3-phosphate dehydrogenase; N-cad, N-cadherin.

FIGURE 5

ALDH1A1 promotes tumor progression by regulating IL-8 expression through activating NF-κB. (A) The expression of IL-8 mRNA in ICC cells with different ALDH1A1 expression. (B) The expression of IL-8 concentration in ICC cells with different ALDH1A1 expression. (C) The expression of IL-8 mRNA in ICC cells with JSH-23 treatment. (D) The expression of IL-8 concentration in ICC cells with JSH-23 treatment. (E) The proliferation ability of si-ALDH1A1 cells with IL-8 treatment. (F) The proliferation ability of si-ALDH1A1 cells with SB225002 treatment. (G, H) The effect of IL-8 treatment or SB225002 treatment on clone formation ability in different ALDH1A1 expression ICC cells. (I, J) The effect of IL-8 treatment or SB225002 treatment on apoptosis ratio in different ALDH1A1 expression ICC cells. (K, L) The effect of IL-8 treatment or SB225002 treatment on migration ability in different ALDH1A1 expression ICC cells (scale bar=200 μm). (M, N) The effect of IL-8 treatment or SB225002 treatment on invasion ability in different ALDH1A1 expression ICC cells (scale bar=200 μm). (O) The effect of IL-8 treatment or SB225002 treatment on expression of N-cadherin and P-P65 protein in different ALDH1A1 expression ICC cells. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: GADPH, glyceraldehyde-3-phosphate dehydrogenase; N-cad, N-cadherin.

FIGURE 5

ALDH1A1 promotes tumor progression by regulating IL-8 expression through activating NF-κB. (A) The expression of IL-8 mRNA in ICC cells with different ALDH1A1 expression. (B) The expression of IL-8 concentration in ICC cells with different ALDH1A1 expression. (C) The expression of IL-8 mRNA in ICC cells with JSH-23 treatment. (D) The expression of IL-8 concentration in ICC cells with JSH-23 treatment. (E) The proliferation ability of si-ALDH1A1 cells with IL-8 treatment. (F) The proliferation ability of si-ALDH1A1 cells with SB225002 treatment. (G, H) The effect of IL-8 treatment or SB225002 treatment on clone formation ability in different ALDH1A1 expression ICC cells. (I, J) The effect of IL-8 treatment or SB225002 treatment on apoptosis ratio in different ALDH1A1 expression ICC cells. (K, L) The effect of IL-8 treatment or SB225002 treatment on migration ability in different ALDH1A1 expression ICC cells (scale bar=200 μm). (M, N) The effect of IL-8 treatment or SB225002 treatment on invasion ability in different ALDH1A1 expression ICC cells (scale bar=200 μm). (O) The effect of IL-8 treatment or SB225002 treatment on expression of N-cadherin and P-P65 protein in different ALDH1A1 expression ICC cells. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: GADPH, glyceraldehyde-3-phosphate dehydrogenase; N-cad, N-cadherin.

FIGURE 6

Verification of the effect of IL-8/CXCR2–NF-κB–ALDH1A1 loop promotion of the progression of ICC in vivo. (A) The effect of IL-8 treatment or SB225002 treatment on the tumor size in mice. (B) The effect of IL-8 treatment or SB225002 treatment on tumor weight in mice. (C) The effect of IL-8 treatment or SB225002 treatment on tumor volume in mice. (D) The hematoxylin and eosin staining of tumors (scale bar=100 μm). (E) The immunohistochemistry staining of P-P65 and PCNA in tumors (scale bar=100 μm). (F) Summary of IL-8/CXCR2–NF-κB–ALDH1A1 loop promotion the progression of ICC. *p<0.05, ** p<0.01, and ***p<0.001. Abbreviation: PCNA, proliferating cell nuclear antigen.