A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

Abstract

Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.

Figure 1

Expression of galectin-1 in HeLa and C33A cells. (a) Cells transfected with GFP only, GFP plus galectin-1, or shRNA were compared with cells without a vector. Lane 1: wild type; lane 2: empty vector; lane 3: shRNA; lane 4: GFP; lane 5: GFP+galectin-1; lane 6: wild type; lane 7: GFP; and lane 8: GFP+galectin-1. (b) Interaction between endogenous galectin-1 and GFP-galectin-1 fusion protein in C33A and HeLa cells. IP was performed for GFP or galectin-1, and then immunoblotting was performed for galectin-1 in both cells transfected with fusion protein. A band (43 kDa) on each lane indicates fusion protein expression formation. A band (14 kDa) on Lane 1 indicates heterodimer formation of endogenous galectin-1 and fusion protein. (c) Distribution of galectin-1 (red color) and GFP-galectin-1 fusion protein (green color) in HeLa cells transfected with GFP-galectin-1 fusion protein

Figure 2

Galectin-1 inhibits radiation-induced cell death. Clonogenic survival curves were compared between (a) galectin-1 knockdown and scrambled RNA in HeLa cells 48 h after transfection (b) galectin-1 cDNA and GFP vectors in HeLa cells (c) galectin-1 knockdown and scrambled RNA in C33A cells 48 h after transfection (d) galectin-1 cDNA and GFP vectors in C33A cells. Cells were irradiated and clonogenic assay performed 12 to 14 days following irradiation. Clonogenic assays were performed during three independent experiments at each paired condition (with and without galectin-1 modulation). The error bar represents the standard error of mean: ^*P<0.05, ^**P<0.01, and ^***P<0.001

Figure 3

Galectin-1-mediated radioresistance was dependent on H-Ras but not on K-Ras. The knock down of HeLa cells by the siRNA of galectin-1 and H-Ras was confirmed by reverse transcription-polymerase chain reaction analysis and western-blot analysis in cells transfected with siRNA. Control cells were treated with scrambled RNA. The clonogenic survival curves were compared among scrambled RNA, H-Ras knockdown/scrambled RNA, and H-Ras knockdown/galectin-1 knockdown. After 48 h following transfection, cells were irradiated, and a clonogenic assay was performed 12 to 14 days after irradiation. Galectin-1 (a) knockdown or (b) overexpression did not affect the radiosensitivity of cervical cancer cells with H-Ras knockdown. However, galectin-1 (c) knockdown or (d) overexpression altered the radiosensitivity of cervical cancer cells with K-Ras knockdown. (e) Galectin-1 knockdown decreased clonogenic survival in HeLa cells transfected with mutated H-Ras at high doses but not at low doses. Clonogenic assays were performed during three independent experiments at each paired condition (with and without galectin-1 modulation). The error bar represents the standard error of mean: ^*P<0.05

Figure 4

The interaction between H-Ras and galectin-1 is more intense than that between K-Ras and galectin-1 in C33A and HeLa cells. An in situ proximity ligation assay (PLA) and IP were performed for the interaction between galectin-1 and Ras. (a) PLA for galectin-1 and H-Ras in C33A cells with or without galectin-1 overexpression. (b) PLA for galectin-1 and K-Ras in C33A cells with or without galectin-1 overexpression. (c) PLA for galectin-1 and H-Ras in HeLa cells with or without galectin-1 knockdown. (d) PLA for galectin-1 and K-Ras in HeLa cells with or without galectin-1 knockdown. (e) PLA for galectin-1 and H-Ras in HeLa cells with or without the galectin-1 inhibitor anginex (10 μM), which was administered for 24 h before cell fixation. (f) IP for galectin-1 and Ras in C33A and HeLa cells transfected with GFP plus galectin-1. IP was performed for GFP, H-Ras, and galectin-1 before immunoblotting for galectin-1, H-Ras, and K-Ras in both cells

Figure 5

Effects of galectin-1 on the Ras-transmitted downstream signals. Scrambled RNA or galectin-1 shRNA were transfected in HeLa cells. GFP or galectin-1 cDNA were transfected in C33A cells. The cells were irradiated with 6 Gy and harvested 5 or 10 min later. Expressions of activated H-Ras, p-Raf-1, and p-ERK using western blots in HeLa and C33A cells were compared with or without galectin-1 modulation

Figure 6

Galectin-1 enhanced the repair of DNA damage. GFP or galectin-1 plus GFP were transfected into C33A cells. Stable clones of scrambled RNA or galectin-1 shRNA were transfected into HeLa cells. Cells were harvested immediately, 4 or 24 h following 6-Gy irradiation. Comet assay was performed. (a) A longer tail was noted in galectin-1 shRNA compared with scrambled RNA HeLa cells 24 h following irradiation. (b) A shorter tail was noted in galectin-1-overexpressed cells compared with GFP-transfected C33A cells 24 h following irradiation. (c) Western blots were performed after harvesting the cells. Greater γ-H2AX expression was noted in galectin-1-knocked down HeLa cells and GFP-transfected C33A cells 4 h and 24 h following irradiation, respectively

Figure 7

A proposed model of cooperation between H-Ras and galectin-1 for radioresistance. Galectin-1 enhances activation (GTP form) of H-Ras following irradiation. Galectin-1 potentiates downstream signals of H-Ras, such as Raf-1 and ERK, that may mediate DNA damage repair and radioresistance.^, Galectin-1 may regulate p21 expression through Raf-1. Hence, Raf-1 may have a significant role in galectin-1-mediated radioresistance