Combination of standard cytotoxic agents with polyamine analogues in the treatment of breast cancer cell lines.

Polyamines are essential for cell growth and differentiation. Structural polyamine analogues have been shown to have antitumor activity in experimental models including breast cancer. The ability of polyamine analogues to alter activity of cytotoxic chemotherapeutic agents in breast cancer models has not been evaluated. This study evaluates the ability of two polyamine analogues, N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane (CPENSpm) and N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm) to synergize with cytotoxics in five human breast cancer cell lines. Antagonism, additivity, or synergy of the combinations was determined using the median effect/combination index model. The chemotherapeutic agents chosen, cis-diaminechloroplatinum(II), doxorubicin, 5-fluorouracil, fluorodeoxyuridine, 4-hydroperoxycyclophosphamide, paclitaxel, docetaxel, and vinorelbine, all have antitumor activity in breast cancer and represent a spectrum of mechanisms. Three treatment schedules of polyamine analogue and cytotoxic were tested in MCF-7 and MDA-MB-468 lines, demonstrating a schedule-dependence of synergistic growth inhibition. Cytotoxic agent alone for 24 h followed by polyamine analogue alone for 96 h resulted in the most synergistic combinations and the greatest synergy. This schedule was then tested in three additional breast cancer lines, and several synergistic combinations were again identified. Two cytotoxics, vinorelbine and the fluoropyrimidines, showed the most promise in combination with the polyamine analogues. They were able to synergize with one or both polyamine analogues in most of the breast cancer cell lines. CPENSpm was also able to synergize with virtually all of the cytotoxics in the estrogen receptor alpha-positive MCF-7 and T-47D lines. These preclinical data demonstrate a treatment schedule and combinations of polyamine analogues and cytotoxics that will be important to study mechanistically and clinically for breast cancer.

Novobiocin in combination with high-dose chemotherapy for the treatment of advanced breast cancer: a phase 2 study.

We conducted the first phase 2 and pharmacologic study to evaluate the combination of novobiocin (a coumeromycin antibiotic that has been shown to augment alkylating agent cytotoxicity in experimental models) and high-dose cyclophosphamide and thiotepa followed by autologous marrow support in women with chemosensitive advanced breast cancer. Its aims were (1) to determine progression-free survival (PFS) and overall survival (OS), (2) to evaluate the pharmacokinetics of cyclophosphamide and thiotepa, and (3) to measure the ability of novobiocin to reverse alkylator drug resistance in vitro. Forty-one women with chemotherapy-responsive advanced breast cancer received cyclophosphamide (4 g/m2) for peripheral blood stem cell mobilization (treatment 1) followed by high-dose cyclophosphamide (1.5 g/m2 per day for 4 days), thiotepa (200 mg/m2 per day for 4 days), and novobiocin (4 g/day orally for 7 days) (treatment 2) and autologous marrow support. The median PFS was 10 months (range, 0.2-70.6 months) and OS, 21.5 months (range, 0.2-70.6 months). There was no statistically significant relationship between PFS or OS and area-under-the-curve values of cyclophosphamide, thiotepa, or 4-hydroxycyclophosphamide. Patient plasma samples (n = 12) obtained during novobiocin therapy were able to reverse alkylator drug resistance in an in vitro colony-forming assay. Correlative laboratory studies in an in vitro model system demonstrated that patient plasma after novobiocin treatment resulted in the magnitude of resistance reversal that had been predicted by prior preclinical experiments. Clinically, however, this activity of novobiocin did not translate into a substantial increase in PFS or OS compared with historical controls treated with high-dose alkylator therapy alone.

Delayed micromolar elevation in intracellular calcium precedes induction of apoptosis in thapsigargin-treated breast cancer cells.

Thapsigargin (TG), a highly specific inhibitor of the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-ATPase pump, can induce apoptosis in a variety of epithelial and lymphoid cell types. In prostate cancer cell lines, TG induces an initial 5- to 10-fold elevation of intracellular calcium ([Ca2+]i) within a few minutes of exposure. With prolonged exposure times (i.e., 12-36 h) a second elevation of [Ca2+]i to >10 microM is observed. In this study, the human breast carcinoma cell lines MCF-7 and MDA MB 468 cells were used to determine the temporal relationship between TG-induced elevation of [Ca2+]i and activation of programmed cell death. Using a microinjection method that allows for long-term analysis of [Ca2+]i changes, we found that after TG exposure, calcium measurements in these cells demonstrated an initial rise (>4-fold) in [Ca2+]i that occurred within minutes and returned to baseline within a few hours. With prolonged TG exposure, the cells underwent a second elevation (>5 microM) of [Ca2+]i occurring stochastically between 12 and 36 h after the initial exposure to TG. Both of the cell lines were growth-inhibited by 100 nM TG after only 1 h of exposure, but clonogenic ability in the MCF-7 cells was significantly reduced only after 48 h of exposure. The induction of apoptosis by TG was demonstrated by morphological changes typical for programmed cell death and DNA fragmentation (both high molecular weight and oligonucleosomal-sized fragments were detected) after 48 h of treatment. TG induction of apoptosis in these breast cancer cells occurred subsequent to the secondary rise in [Ca2+]i, which confirmed that this secondary rise in [Ca2+]i is not prostate cancer-specific. The secondary rise in [Ca2+]i to micromolar levels may directly activate the endonucleases responsible for DNA fragmentation that occurs as part of the apoptotic process. These studies indicate that TG is an active agent in vitro against breast cancer cells. Inactive prodrug analogues of TG are currently being developed that can be activated by tissue-specific proteases, and further pursuit of this strategy as a potential treatment for breast cancer is warranted.

Clinical aspects of cell death in breast cancer: the polyamine pathway as a new target for treatment.

Because intracellular polyamines have a critical role in cell proliferation and death pathways, the polyamine metabolic pathway represents a potential target for intervention in cancers. A number of polyamine analogues have been identified that downregulate polyamine synthesis and enhance polyamine catabolism, thereby depleting intracellular polyamines. Treatment of human breast cancer cell lines in culture with these analogues has been shown to decrease cell proliferation and induce programmed cell death. Phase I studies with one analogue are now complete, setting the stage for phase II trials to determine efficacy, in addition to preclinical studies to examine combinations of polyamine analogues and conventional cytotoxics.

Induction of jun gene family members by transforming growth factor alpha but not 17 beta-estradiol in human breast cancer cells.

To investigate whether estrogen treatment of hormone-responsive human breast cancer cells was associated with activation of members of the jun family of immediate early response genes, the expression of these oncogenes in human breast cancer cells was examined. 17 beta-Estradiol had little effect on expression of c-jun, jun B, jun D, or c-fos mRNA by MCF-7 cells over 12 h, although it stimulated c-myc expression 4-fold within 30 min. In contrast, several peptide growth factors, including transforming growth factor-alpha (TGF-alpha), rapidly and transiently induced expression of c-jun, jun B, and c-fos mRNA 4- to 10-fold over control. A similar pattern of expression was seen in two other estrogen-responsive human breast cancer cell lines, ZR-75-1 and T47D. Inhibition of protein synthesis by cycloheximide did not abrogate induction of c-jun or jun B mRNA by TGF-alpha in MCF-7 cells, suggesting that new protein synthesis was not required. In addition, nuclear runoff transcription analysis demonstrated that increased expression of c-jun and jun B mRNA after TGF-alpha treatment of MCF-7 cells was regulated at least in part at the transcriptional level. Chronic exposure of MCF-7 cells to 17 beta-estradiol for 24-48 h was associated with decreased expression of jun B mRNA only, while similar treatment with TGF-alpha did not change mRNA expression of any jun family member. Thus, expression of jun family members is induced by peptide growth factors like TGF-alpha but not 17 beta-estradiol in human breast cancer cells. These results suggest that these nuclear protooncogenes play different roles in modulating gene expression by MCF-7 cells after exposure to TGF-alpha or 17 beta-estradiol.

Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane.

We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors.

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mammary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 micrograms/ml insulin, 1 microgram/ml prolactin, 1 microgram/ml progesterone, 1 microgram/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 micrograms/ml transferrin, and 5 microM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two- and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an important tool to study the hormonal regulation of the growth and development of rat mammary cells.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions.

A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, beta-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated.