Bax/Bcl-2 expression levels of 2-methoxyestradiol-exposed esophageal cancer cells

Abstract

ABSTRACT2-Methoxyestradiol (2ME), an endogenous metabolite of 17β-estradiol, has been reported to play an active role in the induction of apoptosis in both proliferating endothelial and cancer cells. Since it has been indicated that an increased ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2 protein expression can be associated with apoptosis, and since the exact action mechanism of 2ME is still not clearly defined and appears to vary according to cell type, the influence of 1 μM 2ME was investigated on Bax and Bcl-2 expression levels in squamous esophageal carcinoma cells. 2ME exposure led to statistically significant decreases (0.69 over DMSO controls) in Bcl-2 expression levels. In contrast, no statistically significant effects were observed on Bax expression levels after exposure to 2ME. The Bax/Bcl-2 ratio for 2ME-exposed cells was 1.45, normalised against Bcl-2 levels. Although the exact mechanisms of apoptosis induction in squamous esophageal cancer cells require further investigation, the present study suggests that this altered ratio in favor of Bax could lead to the induction of apoptosis in these cells.2-Methoxyestradiol (2ME), once considered an inactive end-metabolite of 17β-estradiol, has been reported to contain antiproliferative effects on various tumor cell lines regardless of the cell's hormone receptor status (10). Early research on 2ME's biological activity stated its effect on mitotic spindles and cell cycle progression. 2ME caused mitotic accumulation and abnormal mitotic spindle formation in both estrogen receptor (ER) positive-and ER negative cells (12). Furthermore, 2ME targeted proliferating endothelial cells as well as tumor cells, culminating into the initiation of apoptosis. As a consequence, this endogenous estradiol metabolite has emerged as a promising anticancer agent (9).New evidence with regard to 2ME's action mechanism implicates the involvement of both the extrinsic and intrinsic pathways of apoptosis induction. However, the exact mechanism of 2ME is still not clearly defined and appears to vary according to cell type (11). In addition, we have previously shown that degree of differentiation of esophageal carcinoma cells influences the susceptibility of tumor cells to the anti-mitogenic effects of prostaglandinA 2 , another endogenous metabolite playing an active role in the induction of apoptosis. More pronounced effects were observed in less differentiated squamous esophageal cancer cell lines, while more differentiated and normal cells appeared to be less affected (5).Dose-dependent studies revealed that 10 −6 M 2ME was most inhibitory to WHCO3 cell proliferation and decreased cell numbers by almost 60% (13). Light microscopy as well as electron microscopy demonstrated hallmarks of apoptosis in WHCO3 cells. These hallmarks included cell shrinkage,

Influence of 2-methoxyestradiol on cell morphology and Cdc2 Kinase activity in WHCO3 esophageal carcinoma cells

“…In addition, levels of p53, Bax and p21 were increased, while that of the anti-apoptotic Bcl-2 protein was undetectable in cells treated with 2ME (10). As already mentioned, we have previously demonstrated that the pro-apoptotic Bax/anti-apoptotic Bcl-2 ratio for 2ME-treated WHCO3 cells was 1.45 normalized against Bcl-2 levels, thus favouring induction of apoptosis (14). The increased Cdc2 activity observed in 2ME-treated WHCO3 cells is consistent with the morphological hallmarks of mitotic arrest observed.…”

“…JNK plays a key role in the phosphorylation and inactivation of the pro-apoptotic protein Bcl-2, thus contributing to the induction of apoptosis (3,5). Furthermore, demonstrated that the Bax/Bcl-2 ratio for 2ME-treated WHCO3 cells was 1.45 normalized against Bcl-2 levels (14). Exposure of human cervical carcinoma cells (HeLa) to 2ME revealed a Bax/Bcl-2 ratio of 1.87 normalized against Bcl-2 levels in these cells, also suggesting that this altered ratio in favor of Bax could lead to the induction of apoptosis in these cells (13).…”

In vitro effects of 2‐methoxyestradiol on cell numbers, morphology, cell cycle progression, and apoptosis induction in oesophageal carcinoma cells

“…5 Current evidence has suggested that 2-ME is the causative agent leading to an increase in Cdc2 kinase activity, the activation of c-Jun NH 2 -terminal kinase signaling, generation of reactive oxygen species and an altered ratio of Bax/ Bcl-2 in favor of Bax, ultimately culminating into apoptosis. [8][9][10][11] Cell division cycle (Cdc) 2 kinase activity is a cell cycle regulatory component essential for commencement of mitosis, whereas Cdc2 inactivation is needed for mitotic exit. Prolonged Cdc2 activity can sustain the cell in mitosis for an indefinite period until particular conditions are met for mitotic exit.…”

“…12 JNK is involved in the phosphorylation and inactivation of Bcl-2, a pro-apoptotic protein, thus contributing to apoptotic induction. 8,9 Nevertheless, apoptotic induction via the extrinsic and intrinsic pathways appears to be dependent on cell type. [13][14][15] It is also known that 2-ME displays a dose-dependent biphasic pattern on cell proliferation at concentrations ranging from 10 À8 to 10 À5 M. Stimulatory effects have been demonstrated at low concentrations of 2-ME and inhibitory effects were observed at high concentrations.…”

In vitro effects of 2‐methoxyestradiol on cell morphology and Cdc2 Kinase activity in SNO oesophageal carcinoma cells

“…1,5,6,9,15,16 We have previously confirmed antiproliferative effects and morphological hallmarks of apoptosis in 2 ME-exposed HeLa, as well as WHCO3 squamous oesophageal carcinoma cells and revealed that 2 ME was responsible for an altered ratio of Bax/ Bcl-2 in favour of Bax suggesting that this could lead to the induction of apoptosis in both these cells. 17,18 However, the exact action mechanism of 2 ME is complicated, still not clearly defined and appears to vary according to cell type.…”