Ability of breast cancer cell lines to stimulate bone resorbing activity of mature osteoclasts correlates with an anti-apoptotic effect mediated by macrophage colony stimulating factor

We compared the effect of conditioned medium (CM) from several human breast carcinoma cell lines on osteoclast bone resorbing activity and osteoclast apoptosis. Our findings indicate that ability of cancer cell line to increase the in vitro bone resorbing activity is linked to their potential to inhibit osteoclast apoptosis. Cancer cells producing the higher level of M-CSF have the higher osteolytic activity, suggesting that M-CSF originating from cancer cells may contribute, at least in part, to the osteoclast activity at the metastatic site by enhancing their survival. Given that M-CSF plays an important role in the anti-apoptotic effect, we speculated that blocking M-CSF pathway would prevent the CM effects. Small interfering RNA (siRNA) targeting M-CSF and imatinib, a protein tyrosine kinase inhibitor targeting M-CSF receptor, almost completely reversed the CM effect on both osteoclast apoptosis and bone resorption. Blockade of M-CSF pathway could be thus of clinical value in the treatment of breast cancer related bone destruction.     

小鼠鳞癌细胞培养液中溶骨因子的分离纯化

以7、12,二甲基(α)苯基蒽诱导建立了小鼠鳞癌细胞株,接种至裸鼠中可致高血钙。其细胞培养液经超滤浓缩100倍后,通过DEAE层析、Ultrogel AcA54层析、麦胚凝集素琼脂糖层析及HPLC(C_(18))。初步分离纯化了溶骨因子。这一溶骨因子在体外可显著刺激小鼠颅骨片中~(45)Ca的释出,伴有PGE2的生成及腺苷酸环化酶活性的增高,其分子量约15,000道尔顿。     

Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation

Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar.     

Bone resorption and circulating PTH-like bioactivity in an animal model of hypercalcaemia of malignancy

An in vivo model of humoral hypercalcaemia of malignancy has been used to examine the role of circulating PTH-like bioactivity in the development of bone resorption and hypercalcaemia. After inoculation of cells from a renal carcinoma cell line into nude mice, circulating PTH-like bioactivity as measured by the sensitive renal and metatarsal cytochemical bioassays for PTH was elevated in only 18% and 53% of the mice respectively. Bone resorption was elevated in all the mice investigated irrespective of the level of PTH-like bioactivity. Thus, in this model, while the circulating PTH-like moiety is more potent when acting on bone, it did not correlate with the degree of bone resorption suggesting that it may not be the sole cause of the hypercalcaemia.     

Human pancreatic adenocarcinoma: In vitro and in vivo morphology of a new tumor line established from ascites

Summary A human pancreatic tumor cell line has been established from the ascites of a patient with histopathologically confirmed adenocarcinoma of the head of the pancreas and maintained for more than 12 months in the laboratory. Epitheloid tumor cell colonies, which resulted from primary tissue cultures of the ascitic cell component, were mechanically isolated by needle micromanipulation. Tumorigenicity was proven in athymic nude mice. Morphologically the pancreatic tumor epithelial cells grew to confluency with moderately tight adhesion to the culture plastic surface and with free-floating cells in the medium. Upon re-establishment of the tumoral xenograft in tissue culture, the epithelial cells retained their original morphology. Histologically the tumor grown in nude mice exhibited prototypic characteristics of the primary adenocarcinoma in the patient, producing abundant mucin and displaying a broad spectrum of glandular differentiation, which ranged from well to poorly differentiated adenocarcinomas with occasionally localized lymphocytic infiltrations. Furthermore, the tumor expressed carcinoembryonic antigen and human pancreas cancer associated antigen. This tumor line, designated AsPC-1, has been cultured for at least 10 passages in vitro and 3 in vivo. It represents a new model for human pancreatic cancer. This work was supported in part by Research Grant CA-18410 awarded by the National Cancer Institute through the National Pancreatic Cancer Project.     

The effects of a constitutive expression of transforming growth factor-alpha on the growth of MCF-7 human breast cancer cells in vitro and in vivo

It has been suggested that transforming growth factor-alpha (TGF-alpha) is a mitogenic autocrine growth factor for human breast cancer cells, responsible for mediating the mitogenic effects of 17 beta-estradiol (E2) in responsive cells. To test this hypothesis we have introduced eukaryotic expression vectors directing the expression of TGF-alpha mRNA into E2-responsive MCF-7 human breast cancer cells. Transfected cells produce levels of TGF-alpha equivalent to or greater than those produced by both E2-stimulated MCF-7 cells and hormone-independent MDA-MB-231 cells. One transfected clone (H8) secretes sufficient TGF-alpha to fully down-regulate EGF-R expression. However, both of the transfected clones that constitutively secrete elevated levels of TGF-alpha (A8 and H8) respond to E2 stimulation in vitro by increasing the rate of cellular proliferation and inducing PGR synthesis. The basal proliferative capacity of H8 and A8 cells is equivalent to that of the parental cells and to cells transfected only with the G418 (neomycin) resistance gene. Furthermore, the TGF-alpha cDNA-transfected clones do not form tumors in ovariectomized athymic nude mice without E2 supplementation. Thus, the precise role of TGF-alpha in mediating either the in vivo or the in vitro mitogenic effects of E2 in MCF-7 human breast cancer cells remains unclear. While TGF-alpha expression may be essential, it is not sufficient alone to induce the fully E2-independent phenotype. Thus, TGF-alpha may function in combination with other E2-induced growth factors to control breast cancer proliferation and tumorigenesis.     

Establishment of a CSF-producing cell line from a human gastric carcinoma and characteristics of the CSF produced by that cell line

A human cell line producing colony-stimulating factor has been established in vitro from a human gastric carcinoma. The cell line was transplantable into nude mice which developed a marked neutrophilia. The cell line has been maintained for three years. The cells grew in a monolayered sheet and produced colony-stimulating factors that enhanced the formation of granulocyte and monocyte colonies in vitro with mouse bone marrow cells as the target and granulocyte colonies with human bone marrow cells as the target.     

胰腺癌原位种植瘤中VEGF-C的器官差异表达和作用

目的 研究胰腺癌裸鼠原位种植瘤自发性淋巴结转移模型中VEGF-C表达的器官差异,以及VEGF-C反义寡核苷酸对不同生长部位胰腺癌细胞生长、凋亡能力的影响。方法 建立人胰腺癌细胞株PANC-1裸鼠原位种植瘤自发性淋巴结转移模型,分离、原代培养原发灶和自发性淋巴结转移灶中的胰腺癌细胞,并应用荧光定量PCR、MTT、流式细胞术检测VEGF-C反义寡核苷酸转染对原发胰腺癌细胞和淋巴结转移的胰腺癌细胞各自生长特性、凋亡能力的影响。结果 淋巴结转移胰腺癌细胞VEGF-C的mRNA表达水平显著高于原发灶胰腺癌细胞（P〈0.05）。VEGF-C反义核苷酸抑制胰腺癌细胞VEGF-C的表达后,淋巴结转移灶中胰腺癌细胞生长抑制率、凋亡率均显著提高（P〈0.01）,而原发灶中胰腺癌细胞无明显影响（P〉0.05）。结论 VEGF-C反义寡核苷酸能显著抑制淋巴结转移灶中胰腺癌细胞生长、促进凋亡,但对原发灶胰腺癌细胞无影响;VEGF-C基因的表达和作用存在器官差异性。     

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.     

Purification of a lipoprotein lipase-inhibiting protein produced by a melanoma cell line associated with cancer cachexia

A human melanoma cell line, SEKI, induces severe cachexia in tumor-bearing nude mice. A factor with the ability to inhibit lipoprotein lipase (LPL) was isolated from the conditioned medium of this cell line. This factor was 40-K-dalton protein, and designated temporarily as melanoma-derived LPL inhibitor (MLPLI). Amino acid sequencing revealed that the amino-terminal portion consists of SPLPITPV-AT--IR-P. Unexpectedly, the sequence, as far as determined, was identical to those of leukemia inhibitory factor (LIF), suggesting that MLPLI is a protein closely related to LIF. The findings that MLPLI inhibits LPL activity and that MLPLI is produced by human cancer cells inducing cancer cachexia also suggest that this protein is a candidate for the factor responsible for cancer cachexia.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

MT1-MMP cooperates with Kras(G12D) to promote pancreatic fibrosis through increased TGF-β signaling

Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14), which is required for growth and invasion in the collagen-rich microenvironment. In this article, we show that compared with control mice (Kras(+)/MT1-MMP(-)) that express an activating Kras(G12D) mutation necessary for pancreatic cancer development, littermate mice that express both MT1-MMP and Kras(G12D) (Kras(+)/MT1-MMP(+)) developed a greater number of large, dysplastic mucin-containing papillary lesions. These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin (+) cells in the stroma, indicative of activated myofibroblasts, and increased Smad2 phosphorylation. To further understand how MT1-MMP promotes fibrosis, we established an in vitro model to examine the effect of expressing MT1-MMP in pancreatic ductal adenocarcinoma (PDAC) cells on stellate cell collagen deposition. Conditioned media from MT1-MMP-expressing PDAC cells grown in three-dimensional collagen enhanced Smad2 nuclear translocation, promoted Smad2 phosphorylation, and increased collagen production by stellate cells. Inhibiting the activity or expression of the TGF-β type I receptor in stellate cells attenuated MT1-MMP conditioned medium-induced collagen expression by stellate cells. In addition, a function-blocking anti-TGF-β antibody also inhibited MT1-MMP conditioned medium-induced collagen expression in stellate cells. Overall, we show that the bona fide collagenase MT1-MMP paradoxically contributes to fibrosis by increasing TGF-β signaling and that targeting MT1-MMP may thus help to mitigate fibrosis.     

Differences between cell lines of uterine cervical glassy cell carcinoma and large cell nonkeratinizing squamous cell carcinoma.

Cell lines were established from two uterine cervical cancers, a glassy cell carcinoma (GCC) and a large cell nonkeratinizing squamous cell carcinoma (LCSC), and studied by a variety of techniques, including histology, chromosome analysis, heterotransplantation and tumor marker analyses. There were radical differences in the morphology, heterotransplantability, production of tumor markers, etc., between the cultures of these morphologically similar cancers. The LCSC line (HKMUS) consisted of polygonal and round cells containing tonofilaments; these cells discharged tumor antigen-4 (TA-4) into the conditioned media. HKMUS was heterotransplantable into the subcutis of nude mice to form LCSC. On the other hand, the GCC line (HOKUG) consisted of round or spindle-shaped cells. HOKUG was easily transplanted into the subcutis or intraabdominal cavity of nude mice and metastasized easily. It discharged TA-4, carbohydrate antigen 125 (CA125) and neuron-specific enolase (NSE) into the conditioned media. The histologic picture of GCC revealed numerous blood vessels and a rapid proliferation of the cells. GCC, which is considered to be a mixed carcinoma having the characteristics of both squamous carcinoma and adenocarcinoma, seems to be a cancer of unpredictable prognosis as compared to LCSC, possibly due to its rapid proliferation and easy metastasis, leading to peritonitis carcinomatosa.     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

Embelin Suppresses Growth of Human Pancreatic Cancer Xenografts,and Pancreatic Cancer Cells Isolated from KrasG12D Mice by Inhibiting Akt and Sonic Hedgehog Pathways

Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from Kras^G12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh) and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old Kras^G12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2) and cell cycle (cyclin D1, CDK2, and CDK6), and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax). In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9) in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from Kras^G12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and Shh pathways, and can be developed for the treatment and/or prevention of pancreatic cancer.     

Establishment and characterization of human pancreatic adenocarcinoma cell line in tissue culture and the nude mouse

We established a human pancreatic carcinoma cell line, designated SPH, from cancerous ascites of a 57-year-old male patient with ductal adenocarcinoma of the pancreas. The cells have been cultured for 32 months with RPMI-1640 medium supplemental with 10% fetal calf serum. The population doubling time of this cell line was about 35 h, and the modal number of chromosomes was 85 at passage 20. The cells produced CA19-9, SPan-1, and DUPAN-2 in the conditioned medium and formed tumors in nude mice, the histology of which was similar to that of the primary tumor. Based on these findings, this cell line is considered to be a very useful model for studying many aspects of primary and metastatic pancreatic cancer cell biology.     

Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice

Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-alpha production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with beta-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-alpha is added or overexpressed. Thus, (c) the enhanced TGF-alpha production when cells are not in three- dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-alpha. The data suggest that ECM upregulates WAP by downregulating TGF-alpha production. We also propose that changes in TGF-alpha activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.     

Calcitonin-suppressed expression of parathyroid hormone-related protein in breast cancer cells.

Parathyroid hormone-related protein (PTHrP) is a key factor behind humoral hypercalcemia of malignancy (HHM). It is produced in most breast tumors and may be an important local mediator of skeletal metastases due to breast cancer. PTHrP may mediate local bone destruction in the absence of increased circulating PTHrP. Calcitonin (CT) is used for treatment of HHM, but there are data showing that CT can increase PTHrP expression and secretion in vitro. We have therefore studied the effect of CT on PTHrP gene expression and secretion in MCF-7 breast cancer cells. PTHrP mRNA decreased significantly after 4, 8, and 16 h incubation with 10 nM salmon calcitonin (sCT) when compared with the respective controls. PTHrP mRNA also decreased significantly and dose-dependently after incubation with sCT at 0.1 to 10 nM for 16 h. The PTHrP levels in the conditioned medium also decreased in a similar dose-dependent manner. The adenylate cyclase agonist forskolin lowered the PTHrP mRNA dose-dependently. In cells exposed to varying concentrations of sCT for 15 min, the cAMP levels increased dose-dependently. In conclusion, sCT can suppress PTHrP gene expression in MCF-7 breast cancer cells. The suppressive effect is probably exerted mainly via the cAMP-protein kinase A pathways.