The study of estrogen metabolic and conjugation enzymes have focused on the relationship between enzyme activity, polymorphism, cancer susceptibility and response to chemotherapy (8
). Similar to other enzymes, there have been a number of polymorphism studies on UGT2B17 (15
), and functional studies of UGT2B17 have been focused on the glucuronidation of endogenous and exogenous compounds including steroid hormones (17
). UGT2B17 is expressed in the liver and various extrahepatic tissues including testis, uterus, prostate and lung (19
). Recently Nakamura et al.
) investigated the expression of the UGT2B family in various normal tissues and observed UGT2B17 mRNA expression in normal endometrial tissues. However, there have been no reports comparing the expression level of UGT2B17 in EC tissues and adjacent normal tissues. Therefore, we looked at UGT2B17 mRNA levels in both EC tissues and matched normal endometrial tissues using real-time RT–PCR. Unexpectedly, UGT2B17 mRNA expression was significantly higher in EC tissues. Next, we also observed UGT2B17 mRNA expression in EC Ishikawa cells as previously reported (11
) and used these cells for functional analysis of UGT2B17.
The UGT family is involved in the glucuronidation of endogenous and exogenous steroid hormones and detoxification of environmental carcinogens, and hence, low UGT expression or activity may increase the risk of various cancers including EC (20
). However, we observed that UGT2B17 mRNA expression was higher in EC tissues than in adjacent normal tissues in this study. Given that UGT2B17 mRNA was not regulated by estrogen level in breast cancer MCF-7 cells (12
), we hypothesized that UGT2B17 might have other functions independent of estrogen, which promote EC. To test this hypothesis, we knocked down UGT2B17
mRNA in Ishikawa cells with an siRNA and observed its effects on cell growth, invasion, apoptosis and the cell cycle. In si-UGT2B17
-transfected Ishikawa cells, the number of viable cells was significantly decreased compared with si-negative control-transfected cells. In addition, the number of cells in the apoptotic and G0
phase was significantly increased after si-UGT2B17
transfection. The expression level of cleaved PARP was also significantly increased in si-UGT2B17
-transfected Ishikawa cells compared with control cells. However, there was no significant change in the number of invasive cells after transfection of si-UGT2B17
. Therefore, these data suggest that UGT2B17 may play a role in apoptosis and the cell cycle but not invasion ability.
In order to further investigate the molecular mechanisms of UGT2B17 function, we used microarrays to screen for apoptosis-related genes and found altered expression of several genes in si-UGT2B17
transfectants and control cells. In the microarray data, five genes (BAX, LTBR, TNF, TNFRSF10A, CD27
) were upregulated in si-UGT2B17
-transfected cells, whereas only Mcl-1 was downregulated. To confirm these data, we performed real-time RT–PCR to validate the microarray data. We also confirmed increased Mcl-1 protein levels by western analysis. Mcl-1 is an anti-apoptotic member of the Bcl-2 family and functions to suppress apoptosis (21
). The function and sequence of Mcl-1 is very similar to Bcl-2 and both anti-apoptotic proteins share the ability to promote cell survival (22
). In addition, Mcl-1 plays an important role in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-resistant cancers (21
). Usually TRAIL death receptor signaling leads to increased apoptosis and is useful in cancer therapy (26
). However, the therapeutical potential is limited because TRAIL resistance can occur through upregulation of FLIP or through an increase in anti-apoptotic proteins of the Bcl-2 family including Mcl-1 (28
). In TRAIL-resistant cells, Mcl-1 inhibits BH3-only proteins (Bim, Puma and Bak) and inhibits apoptosis (21
). The combination of TRAIL with a multikinase inhibitor (sorafenib) has been used for the treatment of advanced renal cancer and liver cancer as the next step to TRAIL alone treatment (30
). This combination therapy has been successfully used in other cancers (32
). The Raf–MAP kinase kinase–extracellular-signal-related kinase pathway is a target of sorafenib (mitogen activated protein kinase pathway). One of the mechanisms of apoptosis induction by sorafenib is Mcl-1 downregulation through reduced translation (21
). Llobet et al.
) found that EC cell death triggered by sorafenib was mediated by downregulation of Mcl-1. In this study, Mcl-1 expression was downregulated in UGT2B17-depleted cells. Therefore, UGT2B17-depleted cells are similar to those treated with sorafenib, with alterations of the target genes of Mcl-1. As expected, the expression of Puma was increased in UGT2B17-knockeddown cells. As Akt and Mcl-1 have been shown to play an important role in TRAIL-induced apoptosis, we looked at Akt, phosphorylated-Akt and Mcl-1 expression (35
). However, no relationship was found in western blot results suggesting that UGT2B17 itself may not affect the Mcl-1–Akt pathway.
We also examined Mcl-1 mRNA expression levels in EC tissues and matched adjacent normal endometrial tissues to see the relationship between UGT2B17 mRNA and Mcl-1 mRNA expression. Mcl-1 expression was increased in four of nine EC tissue samples. Among cancer tissue samples (n
8) with increased UGT2B17 mRNA expression, Mcl-1 expression was also increased in four samples (50%) but not changed in two others. A tissue sample with decreased UGT2B17 expression in cancer similarly had decreased expression of Mcl-1. Thus, there may be a positive correlation between UGT2B17 and Mcl-1 mRNA expression. We also examined the relationship between UGT2B17 expression and clinical parameters such as stage and grade. However, we did not find a significant relationship. Since our sample number is relatively small, a larger study will be needed to look at the correlation between UGT2B17 expression and clinical parameters.
In conclusion, this is the first report to show that UGT2B17 and Mcl-1 expression is upregulated in EC tissues and that UGT2B17 knockdown inhibited cell growth and increased apoptosis in EC cells through Mcl-1 downregulation. However, additional studies will be required to reveal the exact molecular mechanisms involved in the regulation of Mcl-1 expression by UGT2B17. These findings also suggest that the expression level of UGT2B17 may be an endometrial tumor marker and silencing UGT2B17 may be beneficial as a therapeutic tool for EC. Since there are many UGT families, similar functional studies of these families have not been carried out. Therefore, the expression pattern of all UGT families in EC and adjacent normal tissues may be useful to predict them oncogenic or tumor suppressive effects.