Yonsei Med J.  2014 Sep;55(5):1196-1205. 10.3349/ymj.2014.55.5.1196.

LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells

• Guo Z
• Chen Q
• Liu B
• Tian D
• Zhang S
• Li M

• KMID: 1799481
• DOI: http://doi.org/10.3349/ymj.2014.55.5.1196

Abstract

PURPOSE
Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) are an inhibitor of receptor tyrosine kinases (RTKs) that was discovered in recent years, and many studies showed that LRIG1 is a tumor suppressor gene and may be related to tumor drug resistance. In this study, we explored whether LRIG1 protein expression can improve the chemosensitivity of glioma cells and what was its mechanism.
MATERIALS AND METHODS
We collected 93 cases of glioma tissues and detected the expression of LRIG1 and BCL-2. We constructed a multidrug resistance cell line U251/multidrug resistance (MDR) and examined the change of LRIG1 and BCL-2 at mRNA and protein expression levels. LRIG1 expression was upregulated in U251/MDR cells and we detected the change of multidrug resistance. Meanwhile, we changed the expression of LRIG1 and BCL-2 and explored the relationship between LRIG1 and BCL-2. Finally, we also explored the relationship between LRIG1 and RTKs.
RESULTS
LRIG1 was negatively correlated with BCL-2 expression in glioma tissue and U251/MDR cells, and upregulation of LRIG1 can enhance chemosensitivity and inhibit BCL-2 expression. Furthermore, LRIG1 was negatively correlated with RTKs in U251/MDR cells.
CONCLUSION
These results demonstrated that LRIG1 can improve chemosensitivity by modulating BCL-2 expression and RTK signaling in glioma cells.

Keyword

Glioma; LRIG1; chemosensitivity; BCL-2; RTKs

MeSH Terms

Astrocytoma/drug therapy/genetics/metabolism
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics/*physiology
Gene Expression Regulation, Neoplastic
Gene Knockdown Techniques
Glioma/drug therapy/*metabolism
Humans
Membrane Glycoproteins/metabolism/*physiology
Proto-Oncogene Proteins c-bcl-2/*metabolism
RNA, Messenger/metabolism
Receptor Protein-Tyrosine Kinases/metabolism
Membrane Glycoproteins
Proto-Oncogene Proteins c-bcl-2
RNA, Messenger
Receptor Protein-Tyrosine Kinases

Figure

• 

  Fig. 1 Immunohistochemical analysis of LRIG1 and BCL-2 protein expression in human astrocytomas (light microscope). LRIG1 was localized in the cytoplasm, nucleus and perinuclear area and BCL-2 was mainly localized in the cytoplasm in grades II and IV of astrocytoma. Arrows indicate LRIG1 protein expression. Moreover, LRIG1 expression was negatively correlated with BCL-2 expression (r=-0.713, p<0.05). LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; BCL-2, B cell lymphoma/lewkmia-2.

• 

  Fig. 2 Establishment of an MDR cell line and the expression of LRIG1 and BCL-2. A multidrug resistance cell line U251/MDR was isolated by stepwise exposure to increasing temozolomide (TMZ) concentrations for approximately 6 months. The initial concentration was 0.25 mg/mL, which was doubled after the cells had been cultured for 15 days; the final concentration was 16 mg/mL. The U251/MDR cells were seeded in 96-well plates, and chemotherapy drugs were added to each well 12 h later [VP-16: 10 mg/mL, TMZ: 16 mg/mL, vincristine sulfate (VCR): 10 mg/mL]. The cell proliferation was detected using the CCK-8 assay after 48 h (A), and the results showed that the U251/MDR cells were more resistant to TMZ, etoposide (VP-16), and VCR than the U251 cells. The results are represented as the survival rate. We also detected the mRNA and protein expression of LRIG1 and BCL-2 in the U251/MDR and U251 cells (B and C), and the results showed that LRIG1 expression was reduced and that BCL-2 expression was increased in the U251/MDR cells, compared to the U251 cells. The results are represented as the ratio of absorbance. *p<0.05 vs. U251 cells. MDR, multidrug resistance; LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; BCL-2, B cell lymphoma/lewkmia-2; VP-16, etoposide; CCK, Cell Counting Kit.

• 

  Fig. 3 The effect of LRIG1 in MDR. U251/MDR cells at 50-80% confluence were transfected with the LRIG1 plasmid (2 mg/well, 6-well plate) using the FUGENE HD transfection reagent (Roche) according to the manufacturer's instructions. After 48 h, the BCL-2 expression was detected (A). At the same time, the LRIG1-U251/MDR cells were seeded in 96-well plates and then treated with VP-16 (10 mg/mL), TMZ (16 mg/mL), and VCR (10 mg/mL) for 48 h. Following incubation, we detected the inhibition of VP-16, TMZ, and VCR on the U251/MDR cells using the CCK-8 assay (B). The results are represented as the survival rate, *p<0.05 vs. control group. MDR, multidrug resistance; LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; TMZ, temozolomide; VCR, vincristine sulfate; VP-16, etoposide; BCL-2, B cell lymphoma/lewkmia-2; CCK, Cell Counting Kit.

• 

  Fig. 4 BCL-2 knockdown reverses MDR in U251/MDR cells. U251/MDR cells at 50% confluence were transfected with siBCL-2 (2 mg/well, 6-well plate) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, the cells were seeded in 96-well plates and then treated with VP-16 (10 mg/mL), TMZ (16 mg/mL), and VCR (10 mg/mL) for 48 h. The inhibition of cell proliferation was detected using the CCK-8 assay. The results are represented as the survival rate. *p<0.05 vs. control group. MDR, multidrug resistance; TMZ, temozolomide; VCR, vincristine sulfate; BCL-2, B cell lymphoma/lewkmia-2; VP-16, etoposide; CCK-8, Cell Counting Kit.

• 

  Fig. 5 siLRIG1 can reduce chemosensitivity by regulating BCL-2 gene expression. First, we seeded U251/MDR cells in 6-well plates when they reached 50% confluence. siBCL-2 was transfected into U251 cells using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions (A). Next, siLRIG1 was transfected into the siBCL-2 cells using the same method. We then detected the inhibition of cell proliferation of VP-16 (10 mg/mL), TMZ (16 mg/mL), and VCR (10 mg/mL) according to the method described previously (B). The results are represented as the survival rate. *p<0.05 vs. siBCL-2 alone. MDR, multidrug resistance; LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; TMZ, temozolomide; VCR, vincristine sulfate, BCL-2, B cell lymphoma/lewkmia-2; VP-16, etoposide.

• 

  Fig. 6 The expression of EGFR and c-Met in U251 and U251/MDR cells. We seeded U251 and U251/MDR cells in 6-well plates. After 48 h, total protein was extracted, and the expression levels of EGFR, c-Met, and LRIG1 were detected using immunoblot analysis. Compared to U251, LRIG1 expression was reduced, and the expression levels of EGFR and c-Met were increased in the U251/MDR cells. *p<0.05 vs. U251 cells. MDR, multidrug resistance; LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; EGFR, epidermal growth factor receptor.

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