The biological effects of delphinidin in breast cancer cell lines included inhibition of proliferation, as measured by MTS assay, and increased apoptosis, as measured by DNA fragmentation and reduced survivin levels. Delphinidin did not promote cell death in non-transformed MCF10A breast epithelial cells. However, MCF10A cells that were chemically transformed by chronic exposure to the PhIP carcinogen were sensitive to the growth inhibitory action of delphinidin. The most pronounced biological effects of delphinidin included inhibition of anchorage-independent growth, induction of apoptosis, and prevention of PhIP-mediated transformation. These results provide compelling evidence that delphinidin specifically targets breast cancer cells without non-specific toxicity to non-cancerous breast epithelial cells.
Our data suggested that delphinidin reduced proliferation of triple negative, ER-positive, and HER2-overexpressing breast cancer molecular subtypes. Consistent with our results, delphinidin has been shown to inhibit proliferation of the ER-positive MCF7 breast cancer cell line.17
Growth inhibition of ER-positive breast cancer may occur independent of ER-targeted effects, however, as published data suggested that delphinidin does not reduce ER alpha or ER beta expression levels.17
In fact, delphinidin-mediated inhibition of MAPK signaling may be a critical molecular mechanism of growth inhibition. We observed reduced Erk1/2 phosphorylation after delphinidin exposure in triple negative HCC1806 and MDA468 cells and in HER2-overexpressing SKBR3 cells. Reduced Erk1/2 signaling has been reported in delphinidin-treated A431 cells downstream of EGFR and HER2 inhibition,14
consistent with our data in SKBR3 cells. We found that delphinidin did not reduce phosphorylation of other MAPK proteins (p38 and JNK) in HCC1806 cells. However, delphinidin has previously been shown to inhibit UV-induced Erk1/2, p38, and JNK MAPK phosphorylation in mouse epidermal cells.13,18
In addition, delphinidin did not significantly block Akt phosphorylation in MDA468 or HCC1806 triple negative cells. In contrast, UV-induced Akt phosphorylation was reduced by delphinidin in epidermal cells.18
Collectively, these results indicate that delphinidin targets the MAPK signaling pathway, but also suggest that delphinidin has potential cell type-specific or context-specific molecular effects.
Cellular transformation by carcinogens that induce MAPK signaling appears to be inhibited by delphinidin. Kang et al13
showed that 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced neoplastic transformation of mouse epidermal cells was blocked by delphinidin. The authors found that delphinidin directly blocked TPA-induced phosphorylation of MEK-Erk1/2. Similarly, we found that the carcinogen PhIP activated Erk1/2 phosphorylation, consistent with past findings.19
Delphinidin blocked PhIP-mediated Erk1/2 activation, and inhibited anchorage-independent growth of PhIP-transformed MCF10A cells. PhIP is one of the most abundant heterocyclic amines consumed by humans in cooked meat and fish, and has been shown to have carcinogenic activity in the mammary gland, colon, and prostate of rats.15,19–22
Natural antioxidant compounds such as those derived from green tea have been shown to block PhIP-mediated carcinogenesis.24
Our data further support the role of polyphenolic compounds such as delphinidin in blocking cellular transformation by environmental or diet-derived carcinogens such as PhIP.
Delphinidin-mediated apoptosis was induced at the highest levels in HER2-overexpressing cell lines. Use of delphinidin in HER2-positive disease would possibly be limited to single agent treatment, however, since combining delphinidin with Herceptin or lapatinib appeared to reduce efficacy of these currently approved HER2-targeted therapies. Our molecular data indicated that delphinidin inhibits HER2 signaling. Similarly, delphinidin has been reported to be a potent inhibitor of EGFR and HER2 phosphorylation in the human vulva carcinoma cell line A431.14,23,24
Inhibition of HER2 signaling may be an important growth inhibitory mechanism of delphinidin in HER2-overexpressing breast cancer since these cancers have oncogenic addiction to HER2, and since the combination of delphinidin with HER2-targeted drugs did not show synergy. If delphinidin affected multiple signaling pathways independently of HER2, we might expect synergy between HER2 inhibitors and delphinidin since multiple molecular pathways would be blocked. Instead, the combination did not produce additive or synergistic effects, but yielded slightly antagonistic effects. Thus, delphinidin-mediated HER2 inhibition is likely to be an important molecular effect of delphinidin in this breast cancer subtype.
Based on our data, delphinidin may serve as an important lead compound for developing novel therapies for breast cancer. The concentrations of delphinidin that we used in this study were in the μg/mL range. These are likely to be physiologically tolerable doses as humans are estimated to consume between 80–215 mg of anthocyanidins daily,25,26
with delphinidin being a major source of anthocyanidins. Delphinidin has demonstrated anti-inflammatory and anti-angiogenic activities in prostate cancer models,7
and appears to be a broad-spectrum inhibitor of multiple kinase signaling pathways including HER2 and downstream MAPK in multiple cell types.13,23,24
Our own data indicates that delphinidin is an inhibitor of HER2 and Erk1/2 signaling, with growth inhibition and apoptosis observed in multiple molecular subtypes of breast cancer. Thus, delphinidin could represent a novel lead agent for development of future breast cancer chemotherapeutic drugs.