Differences in protein kinase C and estrogen receptor alpha, beta expression and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants

Widespread use of MCF-7 human breast cancer cells as a model system for breast cancer has lead to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, differences in sensitivity to apoptosis have just begun to be described. Based on the possible differences in apoptotic sensitivity that may arise due to the existence of MCF-7 cell variants, we determined the relative sensitivity of MCF-7 cell variants from three established laboratories (designated M, L and N) to known inducers of apoptosis. Consistent with our previous studies we demonstrate that differences exist among these variants in regards to tumor necrosis factor alpha (TNF)-induced cell death and inhibition of proliferation in a dose-dependent manner. To establish if the difference in apoptotic susceptibility was specific to TNF, the three MCF-7 cell variants were tested for their response to other known inducers of apoptosis: okadaic acid, staurosporine and 4-hydroxy-tamoxifen. Viability and DNA fragmentation analysis revealed a similar pattern of resistance to apoptosis by all agents in the MCF-7 M variant. The MCF-7 L variant was resistant to okadaic acid and 4-hydroxy-tamoxifen but not staurosporine. In contrast, MCF-7 N cells were sensitive to induction of apoptosis by all agents. The role of both protein kinase C (PKC) and estrogen signaling in the regulation of cell survival prompted investigation of these pathways as a mechanism for differential sensitivity of MCF-7 cell variants to apoptosis. While both estrogen receptor alpha (ERalpha) and ERbeta were expressed in MCF-7 M and N cells, the absence of ERbeta in MCF-7 L cells correlated with decreased estrogen responsiveness of the L variant. Variations in estrogenic responsiveness and PKC isoform expression may account for the enhanced susceptibility of both the L and N variants to staurosporine.     

Regulation of MCF-7 breast cancer cell growth by beta-estradiol sulfation

Estrogen stimulation is an important factor in human breast cancer cell growth and development. Metabolism of -estradiol (E₂), the major endogenous human estrogen, is important in regulating both the level and activity of the hormone in breast tissues. Conjugation of E₂ with a sulfonate moiety is an inactivation process since the sulfate ester formed by this reaction can not bind and activate the estrogen receptor. In human tissues including the breast, estrogen sulfotransferase (EST, SULT1E1) is responsible for high affinity E₂ sulfation activity. EST is expressed in human mammary epithelial (HME) cells but not in most cultured breast cancer cell lines, including estrogen responsive MCF-7 cells. Stable expression of EST in MCF-7 cells at levels similar to those detected in HME cells significantly inhibits cell growth at physiologically relevant E₂ concentrations. The mechanism of cell growth inhibition involves the abrogation of responses observed in growth factor expression in MCF-7 cells following E₂ stimulation. MCF-7 cells expressing EST activity did not show a decrease in estrogen receptor- levels, nor a characteristic increase in progesterone receptor or decrease in transforming growth factor- expression upon exposure to 100 pM or 1 nM E₂. The lack of response in these MCF-7 cells is apparently due to the rapid sulfation and inactivation of free E₂ by EST. These results suggest that loss of EST expression in the transformation of normal breast tissues to breast cancer may be an important factor in increasing the growth responsiveness of preneoplastic or tumor cells to estrogen stimulation.     

内质网应激对乳腺癌MCF-7细胞CCL5表达的影响

目的探讨内质网应激水平与人乳腺癌MCF-7细胞CCL5表达之间的关系,明确CCL5与人乳腺癌MCF-7细胞增殖侵袭转移能力之间的关系。方法使用内质网应激诱导剂(Tuniamycin）和内质网应激抑制剂(4-PBA）分别处理人乳腺癌MCF-7细胞,24小时后提取细胞总蛋白。Western blot检测MCF-7细胞内质网应激水平及CCL5表达情况。MTT比色法,Transwell小室法检测细胞增殖和侵袭转移能力。结果内质网应激诱导剂处理组细胞内质网应激处于高水平,同时CCL5表达量增高;抑制剂组细胞内质网应激水平低,CCL5表达量也随之降低。并且诱导剂组肿瘤细胞增殖较抑制剂组活跃。升高的CCL5部分分泌到培养液中,影响细胞侵袭转移能力。结论内质网应激能够诱导人乳腺癌MCF-7细胞CCL5的表达;内源性CCL5能促进MCF-7细胞的增殖;外源性CCL5能促进MCF-7细胞侵袭转移。     

VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors

The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of breast cancer. In several human cancers including those of the breast and prostate, the expression of vascular endothelial growth factor C (VEGF-C) is associated with lymph node metastasis. Our study was undertaken to evaluate the effect of VEGF-C on metastasis of poorly invasive, estrogen dependent human MCF-7 breast cancer cells. MCF-7 breast cancer cells transfected with VEGF-C (MCF-7-VEGF-C) were grown as tumors in the mammary fat pads of nude mice implanted with subcutaneous estrogen pellets. Tumor lymphangiogenesis and lymph node metastasis were studied immunohistochemically using antibodies against lymphatic vessel hyaluronan receptor -1 (LYVE-1), VEGF receptor-3 (VEGFR-3), PECAM-1, pan-cytokeratin and estrogen dependent pS2 protein. Overexpression of VEGF-C in transfected MCF-7 cells stimulated in vivo tumor growth in xenotransplanted mice without affecting estrogen responsiveness. The resulting tumors metastasized to the regional lymph nodes in 75% (in 6 mice out of 8, Experiment I) and in 62% (in 5 mice out of 8, Experiment II) of mice bearing orthotopic tumors formed by MCF-7-VEGF-C cells whereas no metastases were observed in mice bearing tumors of control vector-transfected MCF-7 cells (MCF-7-Mock). The density of intratumoral and peritumoral lymphatic vessels was increased in tumors derived from MCF-7-VEGF-C cells but not MCF-7-Mock cells. Taken together, our results show that VEGF-C overexpression stimulates tumor lymphangiogenesis and induces normally poorly metastatic estrogen-dependent MCF-7 tumors to disseminate to local lymph nodes. These data suggest that VEGF-C has an important role in lymph node metastasis of breast cancer even at its hormone-dependent early stage.     

The role of growth factor receptor pathways in human breast cancer cells adapted to long-term estrogen deprivation

To study the long-term effects of estrogen deprivation on breast cancer, MCF-7Ca human estrogen receptor-positive breast cancer cells stably transfected with human aromatase gene were cultured in the steroid-depleted medium for 6 to 8 months until they had acquired the ability to grow. Proliferation of these cells (UMB-1Ca) was accompanied by increased expression of human epidermal growth factor receptor 2, increased activation of AKT through phosphorylation at Ser473 and Thr308, and increased invasion compared with parental MCF-7Ca cells. Estrogen receptor expression was also increased 5-fold. Although growth was inhibited by the antiestrogen fulvestrant, the IC50 was 100-fold higher than for parental MCF-7Ca cells. Aromatase inhibitor letrozole also inhibited growth at 10,000-fold higher concentration than required for MCF-7Ca cells, whereas anastrozole, exemestane, formestane, and tamoxifen were ineffective at 100 nmol/L. Growth of UMB-1Ca cells was inhibited by phosphatidylinositol 3-kinase inhibitor wortmannin (IC50 approximately 25 nmol/L) and epidermal growth factor receptor kinase inhibitor gefitinib (ZD 1839; IC50 approximately 10 micromol/L) whereas parental MCF-7Ca cells were insensitive to these agents. Concomitant treatment of UMB-1Ca cells with the signal transduction inhibitors and anastrozole and tamoxifen restored their growth inhibitory effects. These studies show that estrogen deprivation results in up-regulation of growth factor signaling pathways, which leads to a more aggressive and hormone refractory phenotype. Cross-talk between ER and growth factor signaling was evident as inhibition of these pathways could restore estrogen responsiveness to these cells.     

Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.     

The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.

We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.     

Estrogen up-regulates neuropeptide Y Y1 receptor expression in a human breast cancer cell line

Normal breast tissue mainly expresses the neuropeptide Y (NPY) Y2 receptor whereas primary human breast carcinomas express the Y1 receptor (Y1R) subtype. We hypothesized that activation of estrogen signaling systems plays a role in the induction of Y1R. To investigate this possibility, we used estrogen receptor-positive (ER+) human breast carcinoma cell line, MCF-7, and examined the effect of estrogen on Y1R gene expression and its signaling pathways. Saturation binding studies revealed that MCF-7 cells express high-affinity NPY receptor. NPY inhibited forskolin-stimulated adenosine 3'5'-cyclic monophosphate (cAMP) accumulation and mobilized intracellular Ca(2+) in MCF-7 cells. Chronic estrogen treatment enhanced NPY-mediated inhibition of cAMP accumulation by 4-fold and caused a significant increase in Y1R mRNA expression through ERalpha. Similarly, estrogen increased Y1R mRNA expression in T-47D (ER+) but not in MDA-MB231 or MDA-MB468 (ER-) cell lines. Cycloheximide decreased basal Y1R mRNA expression; however, it did not affect its increase by estrogen. Moreover, estrogen treatment of MCF-7 cells did not increase Y1R mRNA stability. The up-regulation of Y1R expression by estrogen is prevented by hydroxyurea but not by nocodazole or IB-MECA (cell cycle inhibitors). Lastly, NPY inhibited estrogen-induced cell proliferation through Y1R. In conclusion, MCF-7 cells express a functional Y1R coupled to both Ca(2+) and cAMP pathways. Estrogen up-regulates Y1R expression through ERalpha. This effect is independent of increased Y1R mRNA stability or new protein synthesis, and likely occurs during S phase completion of the cell cycle. Estrogen plays an important role in the up-regulation of Y1R, which in turn regulates estrogen-induced cell proliferation in breast cancer cells.     

Trans-resveratrol boronic acid exhibits enhanced anti-proliferative activity on estrogen-dependent MCF-7 breast cancer cells

Resveratrol (RSV), a natural compound present in the skin and seeds of red grapes, is considered a phytoestrogen and has structural similarity to the synthetic estrogen diethylstilbestrol. RSV inhibits tumor cell growth in estrogen receptor-positive (ER+) and negative (ER-) breast cancer cell lines resulting in cell specific regulation of the G 1/S and G 2/M stages of the cell cycle. However apoptotic cell death was only observed in ER+ MCF-7 cells. In this study, we designed and synthesized boronic acid derivative of RSV and evaluated their biological effects on ER+ MCF-7 breast cancer cells. The trans-4 analog inhibited the growth of MCF-7 cells and is not a substrate for p-glycoprotein. The trans-4 analog induces G 1 cell cycle arrest, which coincides with marked inhibition of G 1 cell cycle proteins and a greater pro-apoptotic effect. Finally, the trans-4 analog had no effect on the estrogen-stimulated growth of MCF-7 cells. Our results demonstrate that the trans-4 analog inhibits MCF-7 breast cancer cells by a different mechanism of action than that of RSV (S-phase arrest), and provides a new class of novel boronic acids of RSV that inhibit breast cancer cell growth.     

Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50 mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24 h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.     

Estrogen receptor and hormone responsiveness of medullary thyroid carcinoma cells in continuous culture

We have examined the estrogen responsiveness and estrogen receptor in medullary thyroid carcinoma using a model of an established human cell line, TT. TT cells bind [3H]estradiol with high affinity. Scatchard analysis reveals a single class of binding site with a concentration of 173 fmol/10(6) cells and a dissociation constant of 2.1 x 10(-9) M, values which are comparable to those of a well established model cell line for estrogen responsiveness, MCF-7 human breast cancer cell line. Estradiol in physiological concentrations moderately stimulated TT cell proliferation, whereas in pharmacological concentrations it markedly inhibited cell growth. [3H]Thymidine incorporation into acid-insoluble material was also stimulated following a 5-day treatment with 5 x 10(-9) M estradiol. Tamoxifen at a concentration of 1 microM reduced cell proliferation by 43-48% after 5-7 days of treatment. The growth suppression induced by tamoxifen was reversed by addition of 10 nM estradiol. This is the first report of estrogen growth stimulation and tamoxifen growth inhibition of a tumor cell line derived from human medullary thyroid carcinoma.     

Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture

MCF-7 human breast cancer cells, grown in long-term tissue culture, were found to be highly responsive to prolactin in terms of growth even in the presence of serum. Human prolactin, placental lactogen, and growth hormone (50-250 ng/ml) stimulated MCF-7 cells to grow when added to culture medium of cells in the presence of charcoal-stripped serum. Within 3 days of the hormone addition, a 4.4-fold increase in cell number was achieved with human prolactin at 100 ng/ml in the presence of 10% charcoal-stripped serum. Under these same conditions, estradiol-17 beta at 10(-8) M achieved only a 2-fold increase. After 6 days of culture, both estradiol-17 beta and prolactin gave a total 5-fold increase in cell number. No prolactin effect was achieved in the presence of 10% fetal bovine serum. Stripping fetal bovine serum with dextran-coated charcoal removes as much as 85% of the endogenous lactogens. Removal of these hormones is essential for demonstration of subsequent prolactin-induced growth response in MCF-7 cells, since bovine prolactin binds effectively to lactogen receptors on the surface of the cells but does not transmit a growth signal. When added simultaneously with human prolactin, bovine prolactin blocks the growth response to the former hormone. These results clearly demonstrate that, under the proper conditions of culture, the human breast cancer cell line MCF-7 is highly responsive to growth stimulation by homologous lactogenic hormones. This then affords us an excellent model for further studies on the possible role of prolactin in growth and maintenance of human breast cancer.     

Inhibition of colon and breast carcinoma cell growth by interleukin-4.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.     

The biology of breast tumor progression. Acquisition of hormone independence and resistance to cytotoxic drugs.

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCC1 cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.     

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Regulation of human estrogen receptor gene, epidermal growth factor receptor gene, and oncogenes by estrogen and antiestrogen in MCF-7 breast cancer cells

It is generally believed that estrogen may act either as an initiator or as a promoter in carcinogenesis of human breast cancer. This estrogenic action is generally dependent on the estrogen receptor. In the human estrogen receptor, cDNA has a homology to V-erb-A oncogene. Experiments using MCF-7 human breast cancer cells were carried out to study the regulatory effect of estrogen and antiestrogen on RNA activities of oncogenes, estrogen receptor gene, and epidermal growth factor (EGF) receptor gene. The effect of estradiol on activation of estrogen and EGF receptor genes and myc, ras, and fos oncogenes was positive in relation to the concentrations of supplemented estradiol. In addition, the effects of antiestrogen (tamoxifen) were investigated. Tamoxifen suppressed MCF-7 cell growth, and spot hybridization of the RNA of MCF-7 cells revealed that RNA activities of estrogen and EGF receptor genes and myc, ras, and fos oncogenes were suppressed by tamoxifen. These results suggest that the three oncogenes and two receptor genes are partly regulated by estrogen and antiestrogen (tamoxifen) in MCF-7 human breast cancer cells. This regulatory system may have a role in carcinogenesis and in the treatment of human breast cancer.     

The process of malignant progression in human breast cancer

Malignant progression in breast cancer represents the processes through which localized, hormone-dependent tumor cells become resistant to endocrine manipulations and metastasize to sites distant from the primary tumor. By selection in ovariectomized athymic nude mice, we have isolated a variant (MIII) of the hormone-dependent, poorly invasive, human breast cancer cell line MCF-7. MIII cells have lost their absolute requirement for estrogen to form proliferating tumors in nude mice. Furthermore, these tumors are significantly more invasive than the parental MCF-7 cell line. MIII cells retain some responsivity to estrogens and antiestrogens, indicating that they have progressed to a hormone-independent but hormone-esponsive phenotype. In an attempt to determine the nature of this process, we have compared the phenotype of MIII cells with that of other MCF-7 variants. These comparisons strongly suggest that the factors contributing to perturbations in antiestrogen sensitivity, hormone-dependent growth, metastatic potential and tumorigenicity are essentially independent of each other and acquired in a random manner. Loss of estrogen receptor expression and overexpression of EGF receptors tend to occur later in the process of malignant progression.     

Effects of the pineal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCF-7) in culture

Since melatonin, the major hormone of the pineal gland, has been shown to inhibit the growth of mammary tumors in animal models of human breast cancer, we examined the hypothesis that this indoleamine has the potential to inhibit breast cancer growth by directly inhibiting cell proliferation as exemplified by the growth of the estrogen-responsive human breast cancer cell line MCF-7 in culture. Concentrations of melatonin (10(-9) M; 10(-11) M), corresponding to the physiological levels present in human blood during the evening hours, significantly inhibited (P less than 0.001) cell proliferation by as much as 60% to 78% as measured by either DNA content or hemocytometer cell counts. Melatonin's inhibitory effect was reversible since the logarithmic growth of MCF-7 cells was restored after melatonin-containing medium was replaced with fresh medium lacking melatonin. Not only was the inhibitory effect of melatonin absent at either pharmacological (10(-7) M; 10(-5) M) or subphysiological (10(-15) M; 10(-13) M) concentrations, but melatonin also failed to inhibit the proliferation of either human foreskin fibroblasts or the estrogen receptor-positive human endometrial cancer cell line RL95-2. Both transmission and scanning electron microscopy revealed several morphological changes that correlated with melatonin's inhibition of cell growth. After just 4 days of exposure to melatonin, MCF-7 cells exhibited reduced numbers of surface microvilli, nuclear swelling, cytoplasmic and ribosomal shedding, disruption of mitochondrial cristae, vesiculation of the smooth endoplasmic reticulum, and an increase in the numbers of autophagic vacuoles. These results support the hypothesis that melatonin, at physiological concentrations, exerts a direct but reversible, antiproliferative effect on MCF-7 cell growth in culture. This antiproliferative effect is associated with striking changes in the ultrastructural features of these cells suggestive of a sublethal but reversible cellular injury.