The major new findings from this study include: 1) a relative high oral dose of EGCG significantly inhibits the progression of mouse breast cancer growth in female immunocompetent mice; 2) EGCG significantly suppresses breast tumor angiogenesis and VEGF expression in these mice; 3) EGCG treatment does not significantly affect angiogenesis and VEGF expression in the normal tissues such as the heart and skeletal muscles in the same experiment; 4) EGCG directly inhibits proliferation and migration of cultured mouse and human breast cancer cells; and 5) the down-regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1α and NFκB activation. These findings support the hypothesis that EGCG, a major green tea catechin directly targets both of tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Also, EGCG treatment has no significant effects on angiogenesis and VEGF expression in normal tissues such as the heart and skeletal muscle.
An important finding of this study is that a relative high oral dose of EGCG treatment at 50 to 100 mg/kg/day in drinking water significantly slows a growth curve of breast cancer in C57BL/6 female mice compared to the control group, which is characterized by 65% and 68% reduction in the tumor cross section area and tumor weight, respectively. Clearly, oral EGCG treatment is very effective in suppressing progression of breast cancer in a wild type immunocompetent mouse model. Ullmann et al. reported that peak plasma concentrations were greater than 3 μg/ml after oral dose of 1600 mg in healthy human subjects . We believe that oral dose of 50 to 100 mg/kg/day in human can reach the effective plasma concentrations of EGCG against breast cancer. Recent methods developed for the stereoselective total synthesis of EGCG, and structurally related catechins, could provide new sources of these compounds for biomedical use . Our next step is clinical trial for EGCG in breast cancer therapy.
Cancer cells are under greater hypoxia and oxidative stress than normal cells. 8-hydroxy-2’-deoxyguanosine, a major marker of constitutive oxidative stress is almost 10 times more prevalent in invasive ductal breast carcinoma cells than in normal control samples from the same patient . Tumor cells overproduce reactive oxygen species (ROS) by alterations to metabolic pathways in tumor cells , an inadequate tumor vascular network , and macrophage infiltration of the tumor . Breast carcinomas support their growth by stimulating angiogenesis. Blood flow within these new vessels is often chaotic, causing periods of hypoxia followed by reperfusion. The generation of ROS by reperfusion further causes oxidative stress within breast carcinomas. Also, a breast carcinoma rapidly outgrows its blood supply, leading to glucose deprivation and hypoxia. Glucose deprivation rapidly induces oxidative stress within breast carcinoma cells . Clearly, hypoxia and oxidative stress are found together within the breast carcinoma, in which VEGF production can be augmented by synergy between oxygen radicals and tumor hypoxia. Oxygen radicals and hypoxia co-operatively promote tumor angiogenesis . Hypoxia causes the activation of HIF-1, in which it stimulates VEGF expression. HIF-1 levels are also increased by oxygen radicals. In addition, oxygen radicals activate NFκB that also increases VEGF expression. Thus, the compound blocking HIF-1and NFκB pathways can significantly inhibit VEGF expression and angiogenesis in carcinomas including breast carcinomas.
In this study, we found that the significant inhibitions of tumor growth and tumor angiogenesis of breast cancer in female mice by EGCG were associated with suppressing the activation of HIF-1α and NFκB, and decreasing VEGF expression in breast carcinoma cells. VEGF is a key angiogenic factor that stimulates the growth of tumors including breast cancer, in which VEGF exerts paracrine (especially angiogenesis) and autocrine (proliferation and migration) effects to promote progression of breast cancer . VEGF overexpression and the activation of HIF-1α and NFκB pathways in breast cancer are strongly linked to rapid growth of tumors and worse prognosis [16, 29, 30]. Oxygen radicals and hypoxia co-operatively promote tumor angiogenesis, in which VEGF overexpression is stimulated by the activation of HIF-1α and NFκB pathways in breast cancer . The present findings indicate that EGCG significantly inhibits VEGF expression by suppressing the activation of HIF-1α and NFκB pathways, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer. Our results are supported by the previous findings as follows: 1) EGCG suppressed tumor growth by blocking the induction of VEGF in human colon carcinoma cells ; 2) EGCG inhibited VEGF/VEGFR axis by suppressing the expression of HIF-1α in human colorectal cancer cells ; and 3) EGCG inhibited cancer progression by decreasing NFκB activation . Progression stage is the final phase of cancer development, an uncontrolled growth of cancer cells occurs. In this stage cancer cells are under greater hypoxia and oxidative stress, in which many transcription factors, such as HIF-1α and NFκB, are activated leading to transmit aberrant signals resulting in abnormal functions such as tumor angiogenesis, cancer invasiveness and metastasis. Present findings illustrate that EGCG can inhibit multiple key cellular signals resulting in inhibiting tumor angiogenesis and breast cancer progression. Also, accumulating evidence shows that EGCG can target all stages of cancer development by blocking multiple cellular proteins involved in diverse cellular signal transduction pathways: proliferation, differentiation, apoptosis, angiogenesis or metastasis . In future study, we will investigate the therapeutic potentials of EGCG combined with VEGF receptor inhibitor, Notch inhibitor, HIF-1 inhibitor, or NFκB blocker in breast cancer therapy.
In present study, we demonstrated that EGCG treatment reduced plasma VEGF levels by 35% over the control mice, which was associated with more than 65% reduction of tumor weight in EGCG treated breast cancer mice, compared to untreated breast cancer mice. These findings are consistent with breast cancer patients that EGCG treatment reduced serum levels of VEGF . A study on 200 women showed that serum VEGF levels were significantly higher in breast cancer patients compared to control . Systemic VEGF levels were reduced significantly in the breast cancer patients following tumor excision . We believe that oral EGCG treatment could reduce the tumor-related blood VEGF levels.
Interestingly, the present study shows that EGCG treatment does not significantly affect angiogenesis and VEGF expression in the normal tissues such as the heart and skeletal muscles in the same experiment. The present study first time shows that oral EGCG treatment significantly inhibits angiogenesis, VEGF expression, and growth in breast tumor, but no such effects on the normal tissues such as the heart and limb muscles in the same mice. The different effects of EGCG in tumor and normal tissues can be explained by that cancer cells are under greater hypoxia and oxidative stress than normal cells. VEGF expression and angiogenesis are very stable in normal matured tissues in which they are regulated by metabolic balance within the tissue. However, angiogenesis is stimulated by significantly increased VEGF levels, activated HIF-1α and NFκB pathways in cancer. We also found that there was no significant difference in the body weight, heart weight, or kidney weight between EGCG-treated mice and the control mice. This is an exciting possibility, because EGCG is a drug of low toxicity.
Antiangiogenic therapy is an attractive approach for cancer treatment including breast cancer, in which these agents include monoclonal antibodies (mAbs) and the tyrosine kinase inhibitors (TKIs) of VEGF pathway. Implicated in many physiological processes, VEGF pathway inhibition can lead to on-target side effects, such as hypertension, proteinuria, thromboembolic events, or congestive heart failure [37–39]. The incidence of hypertension rate was up to 35% with bevacizumab, a monoclonal antibody against VEGF-A [40, 41]. Ultimately, considering the modest clinical benefit on the one hand, and the increase in toxicity on the other, the US Food and Drug Administration withdraw its approval of the breast cancer treatment for bevacizumab . As mentioned above, EGCG is a drug of low toxicity, and significantly inhibits angiogenesis in breast tumor (under greater oxidative stress), but not in the normal tissues (no oxidative stress) such as the heart and limb muscles in the same mice. Thus, EGCG may overcome the existing barriers - the mAbs and TKIs of VEGF pathway-induced on-target side effects. However, the further studies are needed.
In conclusion, our results indicate that oral administration of EGCG, a major green tea catechin, significantly inhibits tumor growth and tumor angiogenesis of breast cancer, but no effect on angiogenesis in the heart and limb muscles in an immunocompetent mouse model using mouse breast cancer (E0771) cells. EGCG directly suppresses the proliferation or migration of cultured mouse breast cancer cells as well as the proliferation of human breast cancer cells (MCF-7 and MDA-MB-231). These anticancer effects of EGCG seem to be mediated by blocking multiple intracellular signaling cascades such as HIF-1α and NFκB pathways. The mechanistic advance of EGCG on inhibiting tumor angiogenesis is very unique, in which EGCG does not target angiogenesis in normal tissue. Accumulating evidence indicates that EGCG displays a vast array of cellular effects involved in all stages of cancer development. The multiple targets on cancer and less side effects of EGCG will lead a successful targeted therapy for cancers including breast cancer. The potential therapeutic targets of EGCG in cancer therapy are needed to be further explored. Our next step is clinical trial for EGCG in breast cancer therapy. The combination of EGCG with other targeted compounds such as VEGF receptor inhibitor, Notch inhibitor or HIF-1 inhibitor could lead to a very effective specific targeted breast cancer therapy.