Uterine fibroids are the most common benign tumor of the female genital tract. Recent longitudinal studies have estimated that the lifetime risk of fibroids in a woman over the age of 45 years is more than 60% (24
). There are currently no effective, long-term orally administered drug therapies for leiomyomas (2
A number of recent studies have found that EGCG could inhibit proliferation of several cancer cells (12
). In the present study, HuLM cell line is used to evaluate the antitumor effects of EGCG. We have shown that antiproliferative effects of EGCG were both time- and dose-dependent. EGCG at 50µM and above significantly inhibited the growth of HuLM cells as early as 72h of incubation, while there was no significant difference in either the morphology or proliferation at 1µM and lower concentration at same time points. The inhibitory effect of 10 µM EGCG on HLM cells was not significant until 5 days post-treatment. However these levels are within physiologically achievable range (26
The signal transduction pathway by which EGCG exerts cell cycle arrest and induction of apoptosis remains to be clarified. Several mechanisms of cell-cycle arrest by EGCG have been postulated (8
). The d
-type cyclins, through the interaction with CDKs-forming cyclin d1-CDK4/6 complexes, are mainly responsible for driving the cell cycle from G1 to S phase (12
). In the present study, we demonstrate a significant decrease in the expression of CDK4 and PCNA in EGCG treated HuLM cells.
The induction of apoptosis by 100 µM EGCG treatment on HuLM cells was revealed by TUNEL assay. It is consistent with the changes in BCL2 at RNA and protein levels. The BCL family includes proapoptotic members such as BAX and antiapoptotic members such BCL-2. Antiapoptotic BCL-2 members act as repressors of apoptosis by blocking the release of cytochrome-c, whereas proapoptotic members act as promoters. BCL2 is over-expressed in many tumors, including leiomyoma, that cause resistance to chemotherapeutic drugs and radiation therapy, while decreasing BCL2 expression may promote apoptotic responses to anticancer drugs (27
). These effects are more dependent on the balance between BCL2 and BAX than on BCL2 quantity alone (28
). We found that the level of BCL2 protein was significantly down-regulated while BAX was up-regulated in a dose-dependent manner, when HuLM cells were incubated with ascending concentration of EGCG. Quantitative RT-PCR results showed that BCL2 and BCL2A1 mRNA was significantly down regulated after EGCG treatment. These changes contributed synchronously to the EGCG-induced apoptosis in HuLM cells. Additionally, EGCG-treated HuLM cells exhibited increased expression of several genes that represent p53 pathway such as BAX, p21, transformed 3T3 cell double minute 2 (MDM2) and TP53I3. As a transcription factor, p53 regulates downstream genes important in cell cycle arrest, DNA repair, and apoptosis. Loss of p53 in many cancers leads to genomic instability, impaired cell cycle regulation, and inhibition of apoptosis. (29
Studies have shown that EGCG induces apoptosis and growth inhibition of cancer cells, but not normal cells (31
). EGCG was shown to reduce NFκB levels in human epidermoid carcinoma; cells at a much lower dose than an equivalent inhibition in normal human epidermal keratinocytes (32
). The mechanism of these different effects underscores a therapeutic window demonstrated by EGCG, where the inhibitory effects were only observed in tumor cells. Our results also showed that the expression of bcl2A1, a key factor in NFκB pathway, was reduced 11.8-fold in 100µM EGCG treated huLM cells, compared to untreated control.
Of 18 pathways, we found the expression of 15 genes that represent 10 pathways changed significantly (), while remaining 8 pathways did not show significant changes in this study. Impressively, the expression of BMP2 gene in EGCG-treated HuLM cells in our study was 14 fold more than untreated control. BMPs are members of the transforming growth factor-beta superfamily which regulate cell differentiation, proliferation and apoptosis. The BMP-signaling network plays a pivotal role during embryogenesis and tumorigenesis. Prior studies found that BMP2 acts as a tumor suppressor, promoting apoptosis in human primary colonic epithelial cells (33
). Another report demonstrated that the effect of BMP signaling and growth suppression in cancer cells was facilitated by CDKN1A. Additionally, loss of BMP2 is related to progression towards aggressive phenotype of prostate cancer (34
HSF1, which regulates transcription of heat-shock genes induced by stress, was 16 fold higher in EGCG-treated HuLM cells than the untreated control. HSF1 is increasingly implicated in cancer, and earlier studies have found that over-expressing a constitutively activated form of HSF1 could sensitize HeLa cells to Fas-mediated killing (35
). The implication is that up-regulation of the expression of HSF1 in tumor cells, either through pharmacologic or gene-therapy approaches, could sensitize tumors to the killing effects of cancer therapies operating through the Fas receptor. Interestingly, HSF1 binds to the corepressor metastasis-associated protein 1 (MTA1) and participates in the repression of estrogen-dependent transcription in breast carcinoma cells (36
). This repression effect of HSF1 may conceivably contribute to the role of EGCG in the treatment of leiomyoma, which is another example of estrogen-dependent tumors (2
Although higher concentration of EGCG displayed more profound antiproliferation effects on HuLM cells, it is impractical to expect such high plasma levels of EGCG to be achieved in vivo
. The fact that a significant inhibitory effect on HuLM cells was detected at doses of EGCG as low as 10µM is encouraging. Such levels are achievable in plasma and tissue by regular consumption of brewed green tea or EGCG supplement (9
). Theoretically, the maximum blood concentration of EGCG may reach 60 µM after drinking a cup of tea (9
). A previous study, however, reported that peak plasma EGCG levels of 200–400 ng/ml (0.4–0.8 µM) can be achieved after the oral administration of 800 mg EGCG (equivalent to 3–4 cups of green tea) daily in healthy individuals (37
). The reason could be due to the instability of EGCG and metabolic transformation such as methylation (38
). A wide range of EGCG doses (0.5–10mM) have shown antitumor effects in animal studies (39
). Besides inhibiting tumor cells directly, EGCG at lower concentration may trigger other multiple pathways to suppress tumor growth indirectly. We have recently confirmed such observation in a nude mouse model of uterine fibroids treated daily with EGCG added to drinking water (unpublished data).
In this report we demonstrate that the antiproliferative effect of 10 µM EGCG on HuLM cells occurred after 5 days which suggests that such intervention in humans might be more suited for regular use over a prolonged period of time. Thus, such intervention would be an option more appropriate for slowly growing benign tumors such as uterine fibroids, where a decrease in tumor burden but not necessarily complete eradication is sufficient to produce significant clinical improvement.
Based on the down-regulation of BCL2 and up-regulation of BAX at both protein and mRNA levels, we suggest that the altered expression of BCL2 family members, could be key in the EGCG-mediated induction of apoptosis HuLM cells. Down-regulation of CDK4 protein may also contribute to the cell cycle arrest effects of EGCG, because down-regulation of both cyclin D1 mRNA and cdk4 protein could lower the kinase activities associated with cyclin Dl-CDK4 complexes, leading to cell-cycle arrest. This was evident by the significant reduction of cyclin-dependent kinase 2 (CDK2), and increase in cyclin-dependent kinase inhibitor 1A (CDKN1A, p21) at mRNA levels after EGCG treatment.
In conclusion, this current study demonstrate for the first time a growth inhibition and apoptosis induction effect of EGCG in HuLM cells, and suggests that such effects are mediated through the modulation of multiple cellular signaling pathways. Pending further in vivo evaluation, this nutritional supplement can potentially be used as a simplified oral alternative for the prevention or treatment for uterine fibroids.