Delphinidin Inhibits HER2 and Erk1/2 Signaling and Suppresses Growth of HER2-Overexpressing and Triple Negative Breast Cancer Cell Lines.

Delphinidin is a polyphenolic compound found in many brightly colored fruits and vegetables. Delphinidin is also the major bioactive component found in many dietary supplements that are currently consumed as complementary cancer medicine including pomegranate extract. The purpose of the current study was to determine the in vitro biological effects of delphinidin on established breast cancer cell lines of varying molecular subtypes in comparison to non-transformed breast epithelial cells. We examined cell proliferation, apoptosis, and growth inhibition in response to delphinidin using a tetrazolium salt-based assay, DNA fragmentation assay, and anchorage-independent growth assay. In comparison to vehicle control, delphinidin inhibited proliferation (P < 0.05), blocked anchorage-independent growth (P < 0.05), and induced apoptosis (P < 0.05) of ER-positive, triple negative, and HER2-overexpressing breast cancer cell lines with limited toxicity to non-transformed breast epithelial cells. MAPK signaling was partially reduced in triple negative cells and ER-negative chemically transformed MCF10A cells after treatment with delphinidin. In addition, delphinidin induced a significant level of apoptosis in HER2-overexpressing cells in association with reduced HER2 and MAPK signaling. Since delphinidin is often consumed as a complementary cancer medicine, the effect of delphinidin on response to specific HER2-targeted breast cancer therapies was examined by proliferation assay. Results of these drug combination studies suggested potential antagonism between delphinidin and HER2-directed treatments. In summary, the data presented here suggest that single agent delphinidin exhibits growth inhibitory activity in breast cancer cells of various molecular subtypes, but raise concerns regarding potential drug antagonism when used in combination with existing targeted therapies in HER2-overexpressing breast cancer.

In vitro evaluation of pan-PI3-kinase inhibitor SF1126 in trastuzumab-sensitive and trastuzumab-resistant HER2-over-expressing breast cancer cells.

PURPOSE: The purpose of the current study is to determine the in vitro cytotoxic effects of the novel pan-PI3-kinase inhibitor SF1126 in HER2-over-expressing breast cancer cells. METHODS: Cell proliferation and cytotoxicity were examined by MTS colorimetric assay, FACS analysis, colony formation assay, and immunoblotting. Phosphoinositol-3-kinase signaling was assessed by immunoblotting for phosphorylated Akt. Combination effects of trastuzumab and SF1126 were examined in resistant cells by MTS and soft agar assay. RESULTS: SF1126 inhibited proliferation, and induced G1 arrest and apoptosis of SKBR3 and BT474 parental and trastuzumab-resistant HER2-over-expressing cells. Colony formation was inhibited by SF1126, caspase 3 and PARP proteins were cleaved, and survivin was down-regulated. Inhibition of PI3-kinase was confirmed by reduced phosphorylation of Akt. Finally, the combination of SF1126 and trastuzumab synergistically inhibited proliferation of resistant cells, with SF1126-treated cells showing reduced anchorage-independent growth. CONCLUSIONS: These results provide evidence that a clinically relevant pan-PI-3 kinase inhibitor can reverse trastuzumab resistance in breast cancer cells, and support further study of PI3-kinase inhibitor SF1126 in HER2-over-expressing breast cancer cells, including those that have progressed on trastuzumab.

Modulation of the BRCA1 Protein and Induction of Apoptosis in Triple Negative Breast Cancer Cell Lines by the Polyphenolic Compound Curcumin.

In the current study, we sought to examine the effects of curcumin in a specific type of breast cancer called triple negative breast cancer. These cancers lack expression of the estrogen and progesterone receptors and do not over-express HER2. Current treatment for triple negative breast cancers is limited to cytotoxic chemotherapy, and upon relapse, there are not any therapies currently available. We demonstrate here that the bioactive food compound curcumin induces DNA damage in triple negative breast cancer cells in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein. In addition, curcumin promotes apoptosis and prevents anchorage-independent growth and migration of triple negative breast cancer cells. Apoptosis and BRCA1 modulation were not observed in non-transformed mammary epithelial cells, suggesting curcumin may have limited non-specific toxicity. This study suggests that curcumin and potentially curcumin analogues should be tested further in the context of triple negative breast cancer. These results are novel, having never been previously reported, and suggest that curcumin could provide a novel, non-toxic therapy, which could lead to improved survival for patients with triple negative breast cancer. Curcumin should be studied further in this subset of breast cancer patients, for whom treatment options are severely limited.

Nordihydroguaiaretic acid, a cytotoxic insulin-like growth factor-I receptor/HER2 inhibitor in trastuzumab-resistant breast cancer.

The majority of patients with HER2-overexpressing metastatic breast cancer who initially respond to the HER2-targeted antibody trastuzumab show disease progression within 1 year. The identification of novel agents that effectively inhibit survival of cancer cells that have progressed on trastuzumab is critical for improving outcome for this patient population. In the current study, we show that the phenolic compound nordihydroguaiaretic acid (NDGA) promoted cell death of trastuzumab-naive and trastuzumab-refractory HER2-overexpressing breast cancer cells. NDGA induced DNA fragmentation, cleavage of poly(ADP-ribose) polymerase and caspase-3, and inhibition of colony formation. In addition, NDGA inhibited insulin-like growth factor-I and HER2 signaling in trastuzumab-refractory cells, with reduced downstream phosphatidylinositol-3 kinase/Akt signaling. Importantly, combination treatment with NDGA and trastuzumab suppressed proliferation and survival of trastuzumab-refractory cells to a greater degree than either agent alone, suggesting that NDGA increases the sensitivity of refractory cells to trastuzumab. Derivatives of NDGA are currently in clinical trial for other solid tumors. Our data strongly support further study of NDGA as a potential therapeutic against breast cancers that have progressed on trastuzumab.

A novel unidirectional cross-talk from the insulin-like growth factor-I receptor to leptin receptor in human breast cancer cells.

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells.

Cyclic adenosine 5'-monophosphate-dependent sphingosine-1-phosphate biosynthesis induces human CYP17 gene transcription by activating cleavage of sterol regulatory element binding protein 1.

In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the -520/-331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.

ACTH regulates steroidogenic gene expression and cortisol biosynthesis in the human adrenal cortex via sphingolipid metabolism.

Sphingolipids are a diverse family of phospholipids and glycolipids that mediate cell-cell interactions, participate in signal transduction pathways and modulate the activity of various cellular proteins and receptors. The objective of the present studies was to characterize the role of the sphingolipid biosynthetic pathway in adrenocorticotropin (ACTH)-dependent steroidogenic gene expression and cortisol production. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of sphingolipids was quantified by mass spectrometry. Treatment of H295R cells with ACTH and Bt2cAMP activated sphingolipid metabolism within five minutes. Decreases were found in the cellular levels of several sphingolipids, including sphingomyelin (SM) and glucosylceramide. ACTH/cAMP rapidly decreased levels of the signaling molecules ceramide, sphingosine and sphingosine-1-phosphate (S1P). The effect of these bioactive sphingolipids on steroidogenic gene expression was also examined. Both sphingosine and S1P were found to increase endogenous CYP17 mRNA and activate the transcriptional activity of CYP17-luciferase reporter constructs. Further, sphingosine and S1P rapidly increase cortisol biosynthesis in H295R cells. In summary, our studies establish a link between ACTH/cAMP-dependent steroidogenesis and sphingolipid metabolism in the human adrenal cortex. Finally, these findings suggest that sphingolipids may serve as signaling mediators in ACTH-stimulated cortisol biosynthesis.