The electrical pulse application enhances intra-cellular localization and potentiates cytotoxicity of curcumin in breast cancer cells.

Breast cancer is the most common cancer of women, and fifth leading cause of mortality worldwide. Existing breast cancer regimens are costly and produce severe side effects. This highlights a need for the development of efficient novel therapies, which are cost effective and limit side effects. An electrical pulse (EP)-based chemo therapy, known as electrochemotherapy (ECT) using the natural compound curcumin could be an effective alternative. ECT is a non-surgical modality, which produces excellent anti-tumor efficacy at small drug concentrations due to increased uptake of drugs. In clinics, ECT is shown to be effective in treating advanced, recurrent, and metastatic breast cancers, which are refractory to multiple modalities. ECT with curcumin triggers apoptotic cell death in breast cancer cells and could be an effective alternative, due to curcumin's low cost and reduced side-effects. However, there is a lack of studies quantifying the uptake of curcumin in response to EP application. Towards this, we determined the uptake of different curcuminoids (curcumin, desmethoxycurcumin, and bisdemethoxycurcumin) upon EP application and their impact on cell cytotoxicity. Additionally, we studied the combined effect of calcium chloride (CaCl2) and a curcuminoids (Cur) mixture, based on initial studies suggesting calcium electroporation as a potential inexpensive anti-cancer treatment. Our results indicate EP with Cur increases cellular uptake, cell shrinkage, and cytotoxicity. The EP + Cur resulted in the highest uptake of the bisdemethoxycurcumin. Further, EP also potentiated the cytotoxicity of CaCl2 and of the Cur and CaCl2 combination against breast cancer cells and caused apoptosis. Our preliminary data pave the way to further studies on Cur and CaCl2 combination treating breast cancer.

An ex vivo co-culture model system to evaluate stromal-epithelial interactions in breast cancer.

Breast cancer is the most commonly diagnosed cancer among women worldwide. High breast cancer incidence and mortality rates, especially in obese patients, emphasize the need for a better biological understanding of this disease. Previous studies provide substantial evidence for a vital role of the local extracellular environment in multiple steps of tumor progression, including proliferation and invasion. Current evidence supports the role of adipocytes as an endocrine organ, which produces steroid hormones, pro-inflammatory cytokines and adipokines, such as leptin. To further define the role of the mammary microenvironment on tumorigenesis, we have developed an adipose-tumor epithelial cell co-culture system designed to reproduce the in vivo mammary environment. We validate this model through use of coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. CARS analysis demonstrates the sustained viability of the adipocytes, and that mammary cancer cell morphology parallels that of tumors in vivo. Also, characterized was the influence of mammary adipose tissue on tumor cell growth and migration. Adipose tissue co-cultured with mammary tumor epithelial cells, in the absence of any serum or supplemental growth factors, resulted in substantial increases in growth and migration of tumor cells. In conclusion, this novel co-culture system provides an ideal model to study epithelial-stromal interactions in the mammary gland. Understanding the relationship between adipose tissue, the most abundant and least studied component of the breast stroma and tumor epithelial cells is critical to clarifying the influence of obesity on the development, progression and prognosis of breast cancer.

Prolactin receptor expression in the epithelia and stroma of the rat mammary gland.

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

Differential expression of the growth hormone receptor and growth hormone-binding protein in epithelia and stroma of the mouse mammary gland at various physiological stages.

Increasing evidence suggests that GH is important in normal mammary gland development. To investigate this further, we studied the distribution and levels of growth hormone receptor (GHR) and GH-binding protein (GHBP) in the mouse mammary gland. At three weeks of age, the epithelial component of the right fourth inguinal mammary gland of female mice was removed. These animals were then either maintained as virgins until they were killed or they were mated. One group of the mated mice was killed on day 18 of pregnancy and the remaining mated animals were allowed to carry their pups until term and were killed on day 6 of lactation. At the time of death, both the intact left and the de-epithelialized right mammary glands were collected from all three groups. Some of the intact glands served as a source of epithelial cells, free of stroma. The mRNA levels for GHR and GHBP were measured in intact glands, epithelia-cleared fat pads, and isolated mammary epithelial cells. GHR and GHBP mRNAs were expressed in both the mammary epithelium and stroma. However, the levels of both GHR and GHBP mRNAs were significantly higher in the stroma as compared with the epithelium component. This increase for both mRNAs was from 3- to 12-fold at each physiological state examined. In the intact gland, both GHR and GHBP transcripts were highest in virgins, declined during late pregnancy, and the lowest levels were found in the lactating gland. GHBP and GHR protein concentrations were also assessed in intact glands and epithelia-free fat pads. Similar to the mRNAs, GHR and GHBP protein levels (means+/-s.e.m.) in intact glands were highest in virgin mice (0.891+/-0.15 pmoles/mg protein and 0.136+/-0.26 pmoles/mg protein respectively), declined during late pregnancy (0. 354+/-0.111 pmoles/mg protein and 0.178+/-0.039 pmoles/mg protein respectively), and were lowest during lactation (0.096+0.037 pmoles/mg protein and 0.017+0.006 pmoles/mg protein respectively). Immunocytochemistry utilizing specific antisera against mouse (m) GHR and mGHBP revealed that the two proteins are localized to both the stroma and parenchyma of mouse mammary glands, with similar patterns of immunostaining throughout the different physiological stages analyzed. GHR immunolocalized to the plasma membrane and cytosol of mammary epithelial cells and adipocytes, whereas the GHBP immunostaining was nuclear and cytosolic. In conclusion, we report here that GHR and GHBP mRNAs and proteins are expressed in both the epithelium and the stroma of mammary glands of virgin, pregnant, and lactating mice. In intact glands, GHR and GHBP proteins, as well as their transcripts are higher in abundance in virgin relative to lactating mice. At all physiological stages, GHR and GHBP mRNA levels are higher in the stroma compared with the parenchyma. These findings indicate that the actions of GH in the mammary gland are both direct through its binding to the epithelia, and indirect by binding to the stroma and stimulation of IGF-I production which, in turn, affects mammary epithelial development.

Changes in numbers of prolactin receptors during the cell cycle of Nb2 cells.

Lactogenic hormones including prolactin (PRL) have mitogenic effects on Nb2 cells, a pre-T lymphoma cell line. Previous studies have characterized the PRL stimulation of cellular processes such as RNA/DNA synthesis, signalling molecule activation, and the expression of specific genes. The data presented here explores the fluctuations in plasma membrane PRL receptor (PRLR) number that occur in the Nb2 cells during the course of a 24 h cell cycle. PRLR abundance was determined by measuring specific binding of [I125] oPRL to G1 arrested-intact Nb2 cells in which the cell cycle was initiated by addition of nonradioactive oPRL. Preliminary studies revealed that 1 ng/ml oPRL was the minimum PRL concentration that causes a maximal stimulation of mitogenesis, without interfering with [I125] oPRL binding measurements. Subsequent experiments revealed that upon cell cycle initiation of G1 arrested Nb2 cells with 1 ng/ml oPRL, PRLR number remained constant for the initial 6 h. After 8 h PRLR numbers decreased and at 12 h, the PRLR number was less than 25% of the initial value. After 12 hr, PRLR numbers increased and reach initial values by 18 hr. These studies show that the expression of cell surface PRL receptors is modulated in a sequential fashion during the cell cycle of Nb2 cells.

Development of a homologous radioimmunoassay for mouse growth hormone receptor.

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338-351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5-200 nM, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 +/- 0.045 pmol/mg protein (mean +/- SEM; n = 5) in the virgin animals to 1.015 +/- 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 +/- 0.201 pmol/mg protein (mean +/- SEM; n = 5) to 0.299 +/- 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 +/- 0.063 pmol/mg protein (mean +/- SEM; n = 3) and 0.371 +/- 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 +/- 0.612 pmol/mg protein (mean +/- SEM; n = 3) and 3.693 +/- 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 +/- 0.01 pmol/mg protein (mean +/- SEM; n = 3) in virgin mice to 0.364 +/- 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 +/- 0.003 pmol/mg protein (mean +/- SEM; n = 3) in virgin animals to 6.369 +/- 0.607 pmol/mg protein (n = 3) in pregnant mice.

Lovastatin inhibits prolactin-induced Nb2 cell mitogenesis and milk product synthesis in mouse mammary gland explants.

The p21ras protein has been shown to be active in many growth factor signaling systems, including that of prolactin (PRL). In our studies, the main objective was to examine further the involvement of ras in prolactin-stimulated mitogenic and metabolic processes. We used the farnesylation inhibitor lovastatin to block effectively ras-dependent signaling in Nb2 cells, a pre-T lymphoma cell line, and mammary gland explants derived from 12- to 14-day pregnant mice. Lovastatin completely inhibited PRL-induced Nb2 cell mitogenesis at 1 micron. In the mammary gland, lovastatin at 0.1 micron inhibited the PRL stimulation of lipid and lactose synthesis; at 2 microns, lovastatin abolished the PRL stimulation of casein production. When p21ras was immunoprecipitated from lovastatin (25 microns)-treated Nb2 cells and mammary gland explants, the unfarnesylated (inactive) form of ras was shown to be redistributed from the cell membrane to the cytosolic compartment of the cell. This suggests that the anchoring of ras to the cell membrane is essential for its action in prolactin signal transduction. Thus, in addition to the well-known active participation of ras in mitogenic growth factor signaling, the presence of functional ras also appears necessary for the PRL stimulation of specific metabolic processes involved in lactation.

Differential tyrosyl-phosphorylation of multiple mitogen-activated protein kinase isoforms in response to prolactin in Nb2 lymphoma cells.

Prolactin (PRL) stimulates mitogenesis and differentiative processes in a variety of cell types. Not all of the molecules involved in PRL signaling, which follows an initial PRL-receptor interaction, have been identified. In the present studies, PRL is shown to stimulate the differential tyrosyl phosphorylation of three isoforms (ERK-1, 2, and 4) of mitogen-activated protein kinases (MAP kinase) in a rat pre-T lymphoma cell line (Nb2). Evidence also suggests that PRL stimulates the tyrosyl phosphorylation of ERK-3, a MAP kinase isoform recently identified. When G1-arrested Nb2 cells are treated with 50 ng/ml oPRL, ERK-1 through 3 become tyrosyl phosphorylated within minutes (an indication of enzyme activation) and then become dephosphorylated within 30 min. Conversely, ERK-4 is rapidly tyrosyl phosphorylated by 5 min, and remains in this state for at least 1 hr.