Synergistic interactions between aminoflavone, paclitaxel and camptothecin in human breast cancer cells

Purpose

Aminoflavone is a unique DNA damaging agent currently undergoing phase I evaluation in a prodrug form (AFP464). In anticipation of combination regimens, interactions between aminoflavone and several anticancer drugs were investigated in MCF-7 breast cancer cells to determine whether synergistic cancer cell killing effects were observed.

Methods

Colony formation assays were performed to assess the effect of combining aminoflavone with a variety of anticancer drugs. Changes in initial uptake, retention or efflux of aminoflavone and the second agent were compared to the behavior of drugs alone. Key features required for aminoflavone activity in cell culture models were also explored, focusing on the obligatory induction of CYP1A1/1A2 and binding of reactive aminoflavone metabolites to tumor cell total macromolecules and DNA.

Results

Aminoflavone was synergistic when co-incubated with paclitaxel, camptothecin or SN38. Uptake of neither aminoflavone nor any of the other three compounds was altered in combination incubations. Paclitaxel did not inhibit DNA binding of aminoflavone metabolites, while camptothecin did. Aminoflavone-induced CYP1A1 induction was observed in the presence of camptothecin or paclitaxel.

Conclusions

Aminoflavone is a promising therapeutic agent for breast cancer due to its unique mechanism of action compared to commonly used drugs. Combined treatments utilizing aminoflavone in conjunction with paclitaxel or camptothecin may provide an even greater cytotoxic effect than achieved with aminoflavone alone.     

Aromatase activity in primary and metastatic human breast cancer

Aromatase activity was measured in 104 primary and 24 metastatic breast cancer patients. The assay employed quantitates the production of 3H water release from 1 beta-[3H]androstenedione after aromatization. Of 104 human primary breast tumors studied, 64 contained measurable aromatase activity, ranging from 5-70.5 pmol estrone formed/g protein/hour. In primary breast cancers there was no difference in levels of aromatase activity when analyzed by menstrual status or age by decade. Aromatase activity was similar in small and large primary tumors. The median aromatase activity of primary breast tumors (8.6 pmol/g/h) was similar to that found in metastatic breast cancer deposits (12.0 pmol/g/h). Aromatase activity did not correlate with either estrogen (ER) or progesterone (PR) receptor concentration in the tissues assayed. In this regard there were 33 ER- PR- tumor biopsies. Twelve of these 33 tumors contained aromatase activity greater than 10 pmol/g/hour.     

Aromatase and breast cancer

Aromatase activity may be detected using in vitro and in vivo techniques in most breast cancers and mammary adipose tissue. This activity makes a variable contribution to endogenous estrogens within the breast and in many cases represents the major source of these hormones. Such local biosynthesis may maintain the growth of some hormone-dependent tumors. The factors which regulate aromatase activity within the breast are not defined but are likely to include growth factors and cytokines which may be produced by breast tissues so that autocrine and paracrine loops may exist. Estrogen biosynthesis within the breast, like other peripheral systems, appears sensitive to classical aromatase inhibitors and the new generation of drugs are capable of profoundly blocking the activity and markedly reducing endogenous estrogen levels; in turn these endocrine effects are translated in dramatic anti-tumor influences in hormone-dependent breast cancer.     

Metabolic interaction between cancer cells and stromal cells according to breast cancer molecular subtype

Introduction

The aim of this study was to investigate the differential expression of markers related to metabolic, mitochondrial and autophagy status in different molecular subtypes of breast cancer.

Methods

Using tissue microarray sections generated from 740 cases of breast cancer, we performed immunohistochemical staining for Glut-1, CAIX, MCT4, ATP synthase, glutaminase, BNIP3, Beclin-1, LC3A, LC3B and p62. Based on the immunohistochemical expression of estrogen receptor (ER), progesterone (PR), HER2, and Ki-67 labeling index, the cases were classified into luminal A, luminal B, HER2 and triple-negative breast cancer (TNBC). We further classified metabolic phenotypes of tumors according to glycolytic status by assessing Glut-1 and CAIX expression as follows: Warburg type: tumor (glycolysis type), stroma (nonglycolysis type); reverse Warburg type: tumor (nonglycolysis type), stroma (glycolysis type); mixed type: tumor (glycolysis type), stroma (glycolysis type); and null type: tumor (nonglycolysis type), stroma (nonglycolysis type).

Results

Expression of Glut-1, MCT4 and LC3A was highest in TNBC and lowest in luminal A type (P < 0.001). Tumors were classified into 298 Warburg type (40.3%), 54 reverse Warburg type (7.3%), 62 mixed type (8.4%) and 326 null type (44.0%). The mixed type had a higher histologic grade, ER negativity, PR negativity and Ki-67 index, whereas the null type showed lower histologic grade, ER positivity, PR positivity and Ki-67 index (P < 0.001). TNBC constituted the major portion of Warburg and mixed types, and luminal A consisted mainly of reverse Warburg and null types (P < 0.001).

Conclusion

Breast cancer is heterogeneous in its metabolic status, and therefore it can be classified into various metabolic phenotypes. Specifically, the Warburg and mixed types had strong associations with TNBC, whereas reverse the Warburg and null types had associations with the luminal type, suggesting a correlation between metabolic phenotype and the biology of breast cancer.     

Secretome mediated interactions between sensory neurons and breast cancer cells

The role of the nervous system in aiding cancer progression and metastasis is an important aspect of cancer pathogenesis. Interaction between cancer cells and neurons in an in vitro platform is a simple and robust method to further understand this phenomenon. In our study, we aimed to examine in vitro reciprocal effect between breast cancer cells and cancer-sensitized peripheral primary sensory neurons. Secretome obtained from either cultured DRG neurons from tumor-burdened rats, or MRMT1 breast cancer cells were used to study neuronal and cancer cell reciprocity. We utilized neurite analysis, modified cell migration assay and cell signaling pathway inhibitors to determine neurite growth patterns and cell migration in PC12/DRG neurons and MRMT1 cells, respectively. MRMT1 secretome was found to induce significant neurite outgrowth in PC12 and primary sensory neurons. Secretome-induced neurite growth in PC12 cells was partly mediated by PI3K and ERK pathways, but not by adenylyl cyclase. Conversely, secretome from tumor-sensitized sensory neuron cultures induced increased rate of migration in cultured MRMT1 cells. Results from our study provide additional support to the hypothesis that both breast cancer cells and nerve terminals secrete signaling messengers that have a reciprocal effect on each other.     

Aromatase inhibitors in breast cancer: an overview

We examined published reports on the use of aromatase inhibitors in postmenopausal patients with hormone receptor-positive breast cancer. Our data were obtained through a MEDLINE search of literature published in English. Current data indicate that aromatase inhibitors are equivalent or superior to tamoxifen as first-line therapy for metastatic breast cancer and as neoadjuvant treatment for primary breast cancer. In addition, randomized studies have shown that aromatase inhibitors can be administered instead of tamoxifen as a single agent for 5 years or sequentially with tamoxifen for 5 or 10 years. These choices should be discussed with the patient, considering the estimated risk for recurrence and other associated comorbid conditions such as osteoporosis and thromboembolism.     

Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues

A mouse monoclonal antibody (MAb) recognizing alpha-smooth-muscle actin has been used to study smooth-muscle differentiation features in the stromal cells of desmoplastic reactions accompanying mammary tumors. We have studied, by the same immunohistochemical technique, a series of malignant and non-malignant human breast tissues. Cells composing the desmoplastic reaction were found to express alpha-smooth-muscle actin in all the 11 breast carcinomas examined, whereas no immunostain was demonstrated in the stromal cells of 7 breast tissue samples histologically defined as normal. Three of 9 cases of fibrocystic disease showed a minority of positively stained stromal cells, generally in association with epithelial hyperplasia. All the 7 cases of sclerosing adenosis, 3 of 4 cases of diffuse papillomatosis and all 3 intraductal papillomas exhibited a majority of immunoreactive stromal cells. Numerous stromal cells in 3 of 11 circumscribed fibroadenomas analyzed expressed low amounts of alpha-smooth-muscle actin. The factor(s) responsible for smooth-muscle differentiation in stromal cells are presently unknown, but the detection of this previously unsuspected stromal cell phenotype in nonmalignant mammary tissues might help in characterizing the variant morphological aspects designated under the label "fibrocystic disease" and in understanding the biology of premalignant or early malignant lesions of the breast.     

Aromatase inhibitors in breast cancer therapy

Estrogens are involved in numerous physiological processes and have crucial roles in certain disease states, such as mammary carcinomas. Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is found in breast tissue and the importance of intratumoral aromatase and local estrogen production is being unraveled. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy for the treatment of breast cancer. The initial nonselective nature of nonsteroidal inhibitors, such as aminoglutethimide, has been greatly reduced in the later generations of inhibitors, anastrozole and letrozole. Mechanism-based steroidal inhibitors, such as 4-hydroxyandrostenedione and exemestane produce potent aromatase inhibition in patients. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole and exemestane, are approved for clinical use as first-line endocrine therapy in postmenopausal women with metastatic hormone-dependent breast cancer and as second-line endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies.     

Aromatase inhibitors and male breast cancer

Abstract The majority of breast cancers in male patients are hormone receptor positive. Tamoxifen has proven to be successful in both adjuvant and metastatic settings and remains the standard of care. Given the improved outcomes in female patients with aromatase inhibitors (AI), these drugs have become a potential therapeutic tool for male patients. Preliminary data show effective suppression of oestradiol levels in males treated with AI and some reports have demonstrated objective responses. Here we report a case of a male patient with metastatic breast cancer treated with letrozole who achieved clinical response associated with a decrease in blood oestradiol levels.     

Aromatase inhibitors in breast cancer therapy

Recent advances have been made in the hormonal treatment of breast cancer with the advent of third-generation aromatase inhibitors (anastrozole, letrozole, and exemestane). These newer agents have substantial antitumor activity and appear to be as effective as tamoxifen, with fewer adverse effects. Recent reports indicate that anastrozole is more effective than tamoxifen as adjuvant endocrine therapy in postmenopausal women with breast cancer. This report provides an overview of the clinical trials conducted to date with the aromatase inhibitors as first- and second-line therapies, with an emphasis on recently updated analyses comparing anastrozole with tamoxifen in the adjuvant setting.     

Aromatase inhibitors in breast cancer therapy

Estrogens are involved in numerous physiological processes and have crucial roles in certain disease states, such as mammary carcinomas. Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is found in breast tissue and the importance of intratumoral aromatase and local estrogen production is being unraveled. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy for the treatment of breast cancer. The initial nonselective nature of nonsteroidal inhibitors, such as aminoglutethimide, has been greatly reduced in the later generations of inhibitors, anastrozole and letrozole. Mechanism-based steroidal inhibitors, such as 4-hydroxyandrostenedione and exemestane produce potent aromatase inhibition in patients. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole and exemestane, are approved for clinical use as first-line endocrine therapy in postmenopausal women with metastatic hormone-dependent breast cancer and as second-line endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies.     

CCL2 mediates cross-talk between cancer cells and stromal fibroblasts that regulates breast cancer stem cells

Cancer stem cells (CSC) play critical roles in cancer initiation, progression, and therapeutic refractoriness. Although many studies have focused on the genes and pathways involved in stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer stem cells. We found that compared with normal fibroblasts, primary cancer-associated fibroblasts (CAF) and fibroblasts activated by cocultured breast cancer cells produce higher levels of chemokine (C-C motif) ligand 2 (CCL2), which stimulates the stem cell-specific, sphere-forming phenotype in breast cancer cells and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3 activation by diverse breast cancer-secreted cytokines, and in turn, induced NOTCH1 expression and the CSC features in breast cancer cells, constituting a cancer-stroma-cancer signaling circuit. In a xenograft model of paired fibroblasts and breast cancer tumor cells, loss of CCL2 significantly inhibited tumorigenesis and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated with poor differentiation in primary breast cancers, further supporting the observation that NOTCH1 is regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic target that can block the cancer-host communication that prompts CSC-mediated disease progression.     

Aromatase expression in the human breast

The plasma levels of free estradiol are very low in postmenopausal women. However, concentrations of estrogens within breast tissue have been reported to be higher than in plasma and similar to plasma concentrations in premenopausal women. One mechanism by which this may occur is for breast cells to synthesize estrogens themselves and produce high concentrations locally. Thus, tumor aromatase may be a significant source of estrogen which stimulates tumor growth. To address the question of the importance of this pathway, we have investigated the expression of aromatase within the normal breast and breast cancers. Because conventional biochemical assays for measuring aromatase activity require relatively large amounts of tissue, we developed an immunocytochemical method using a monoclonal antibody to determine the expression of aromatase. The method can be applied to sections of tumors embedded in paraffin blocks as routinely prepared for pathology. Since we have previously shown that mRNA for aromatase (P450 arom) and the protein are expressed in the same cells of the human placenta, we used in situ hybridization of sequence specific probes to P450 arom mRNA in breast tissue as one method to verify the specificity of the immunocytochemical detection of the enzyme. Both immunocytochemistry and in situ hybridization identified aromatase enzyme and mRNA expression in the epithelial cells of the terminal ductal lobula units (TDLU) and surrounding stromal cells of the normal human breast, and in the tumor epithelial cells and stromal cells of breast cancers. In addition, evidence for the functional significance of tumor aromatase was indicated by a correlation between aromatase activity and expression of proliferating cell nuclear antigen (PCNA) in the tumor, and by increased thymidine incorporation into DNA in response to testosterone in tumors in histoculture which had high aromatase activity but not in those with low activity. The findings suggests that estrogen produced locally is important in enhancing proliferation of the tumor.     

Synergistic and antagonistic interactions between docetaxel and interferon-beta on growth of human breast cancer cells in vitro

Docetaxel and interferon-beta (IFN-beta) were tested alone and in combination for their antiproliferative activity against the estradiol receptor (ER) positive MCF-7 and the ER negative MDA-MB231 breast cancer cell lines. Cell growth inhibition was determined after a 3 day incubation by the sulforhodamine B assay. The antiproliferative effects of the drug combinations were analysed using Berenbaum's hyperplane theorem to determine additive, synergistic and antagonistic effects. Docetaxel was found to be equally effective in inhibiting cell growth of both cell lines. On the other hand MCF-7 cells were more sensitive to the antiproliferative activity of IFN-beta than MDA-MB231 cells. At low docetaxel:IFN-beta molar concentration ratios a synergistic interaction was observed for MCF-7 cells, whereas an additive interaction was found for MDA-TMB231 cells. Higher molar ratios resulted in additive interactions on MCF-7 and antagonistic interactions on MDA-MB231 cells. MCF-7 cells seem to be more sensitive to treatment by the combination of docetaxel and IFN-beta than the MDA-MB231 cells. Therefore an ER positive breast carcinoma may possibly profit by the combination of docetaxel and IFN-beta, but further studies are necessary to clarify the therapeutic usefulness and optimal scheduling of the drug combination.     

Gene expression profiling of tumor-stromal interactions between pancreatic cancer cells and stromal fibroblasts

The interactions between cancer cells and surrounding stroma play a critical role in tumor progression, but their molecular basis is largely unknown. Global gene expression profiling was performed using oligonucleotide microarrays to determine changes in the gene expression of pancreatic cancer cells (CFPAC1) and stromal fibroblasts induced by coculture. This analysis identified multiple genes as differentially expressed in pancreatic cancer cells and in fibroblasts as a consequence of their mutual interactions, including those that encode for proteins associated with tumor invasion, metastasis, and angiogenesis. Among the genes identified, the cyclooxygenase-2 (COX-2)/PTGS2 gene was of particular interest because COX-2 expression was markedly augmented in both cell types (cancer cells and fibroblasts) in response to coculture. Coculture with fibroblasts also induced COX-2 expression in additional pancreatic cancer cells with an unmethylated COX-2 promoter, but not in those with a methylated COX-2 promoter. Using an in vitro invasion assay, we found an increase in the invasive potential of CFPAC1 cells when they were cocultured with fibroblasts, an effect blocked partially by the addition of a selective COX-2 inhibitor, NS-398, or by COX-2 knockdown with small interfering RNA. Thus, COX-2 inhibitors can decrease the invasive properties of pancreatic cancer cells acquired through tumor-stromal interactions.