Pirkko Härkönen

Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula Retz. fruit

A 70% methanol extract of Terminalia chebula fruit, was studied for its effects on growth in several malignant cell lines including a human (MCF-7) and mouse (S115) breast cancer cell line, a human osteosarcoma cell line (HOS-1), a human prostate cancer cell line (PC-3) and a non-tumorigenic, immortalized human prostate cell line (PNT1A) using assays for proliferation ([(3)H]-thymidine incorporation and coulter counting), cell viability (ATP determination) and cell death (flow cytometry and Hoechst DNA staining). In all cell lines studied, the extract decreased cell viability, inhibited cell proliferation, and induced cell death in a dose dependent manner. Flow cytometry and other analyses showed that some apoptosis was induced by the extract at lower concentrations, but at higher concentrations, necrosis was the major mechanism of cell death. ATP assay guided chromatographic fractionation of the extract yielded ellagic acid, 2,4-chebulyl-beta-D-glucopyranose (a new natural product), and chebulinic acid which were tested by ATP assay on HOS-1 cell line in comparison to three known antigrowth phenolics of Terminalia, gallic acid, ethyl gallate, luteolin, and tannic acid. Chebulinic acid (IC(50) = 53.2 microM +/- 0.16) > tannic acid (IC(50) = 59.0 microg/ml +/- 0.19) > and ellagic acid (IC(50) = 78.5 microM +/- 0.24), were the most growth inhibitory phenolics of T. chebula fruit in our study.

Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and an important prognostic factor in breast cancer. Hypoxia is an important inducer of VEGF expression but less is known of the role of hormones in VEGF regulation. We have studied the regulation of VEGF, VEGF-B, VEGF-C, and VEGF-D mRNAs in human MCF-7 and mouse S115 breast carcinoma cells stimulated by estrogens and androgens, respectively. VEGF, VEGF-B, and VEGF-C were expressed in both cell lines, whereas VEGF-D was expressed only in S115 cells. Addition of estradiol (E2) caused a biphasic increase of VEGF mRNA in MCF-7 cells and led to accumulation of the VEGF protein in the culture medium. The VEGF-B mRNA was not affected, while a decrease occurred in VEGF-C mRNA. Similarly, testosterone upregulated the expression of VEGF mRNA in the S115 cells. Experiments with actinomycin D and cycloheximide suggested that estrogen induction of VEGF mRNA is dependent on the synthesis of new mRNA and increased mRNA half-life. The antiestrogen ICI 182.780 inhibited E2 stimulation of VEGF, suggesting that the effect was mediated by the estrogen receptor. In contrast, the antiestrogens tamoxifen and toremifene which inhibit MCF-7 cell growth in vivo and in vitro did not inhibit estrogen effect but induced VEGF mRNA expression when used alone. The antiandrogen cyprosterone acetate inhibited T induction of VEGF mRNA in S115 cells, thus suggesting that activation of androgen receptor must be involved in the increase of VEGF mRNA. Our results suggest that both estrogen and androgen stimulate the expression of VEGF by increasing gene transcription and mRNA stability. In addition, the antiestrogens tamoxifen and toremifene also increased VEGF expression. Estrogen and androgen induction of VEGF expression and promotion of new vessel formation may be an important paracrine mechanism by which these hormones contribute to the early phase of tumor growth of hormonal cancer.

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.

Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17beta-estradiol (E2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J Bone Miner Res 9:321-327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstrated by the appearance of osteoblastic markers such as alpha1(I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OP), and transforming growth factor-beta1 (TGFbeta1), which were detected by using reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation, including deposition of collagen fibers and matrix mineralization, was also studied at several time points of the 3-week culture period. The effect of E2 on the appearance of osteoblastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E2, the rate of cell proliferation was increased in the early phase of cultures, but not after day 6. The addition of E2 in subcultures resulted in an increase of levels of mRNA for COL1, ALP, OCN, OP, and TGF-beta1. ALP activity was also increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E2. All E2 concentrations used (0.01-10 nmol/L) were effective but the maximum response was obtained with 0.1 nmol/L E2. Addition of the antiestrogen ICI 182,780 abolished the E2-induced stimulation of proliferation and later an increase in ALP activity. Addition of ICI 182,780 without the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E2 stimulates sequential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner.

Prolactin and prolactin receptors are expressed and functioning in human prostate.

Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.

Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis

Carbonic anhydrase III (CA III; EC 4.2.1.1) is a cytoplasmic enzyme that exhibits a relatively low carbon dioxide hydratase activity. It is expressed at a very high level in skeletal muscle, where physical exercise has been shown to increase free radical production. In this work we show the effect of overexpression of CA III on cellular response to oxidative stress. Rat CA III cDNA was transfected to NIH/3T3 cells, which have no endogenous CA III expression. The isolated clones expressed CA III mRNA and protein. The protein was localized to cytoplasm and nuclei. Compared to parental cells, transfected cells showed lower basal oxidized state as judged by measurement of intracellular reactive oxygen species (ROS) using fluorescent dye and an image analysis system. Addition of exogenous H2O2 to cells induced a rapid increase of ROS in control but not in CA III overexpressing cells. Association of this phenomenon with CA III expression was further confirmed by showing that overexpression of CA II could not prevent H2O2‐stimulated increase of ROS. In proliferation assays, CA III overexpressing cells grew faster and were more resistant to cytotoxic concentrations of H2O2 than control cells. After a 16 h exposure to oxidative stress, the number of apoptotic cells was also reduced in transfectants. Our results suggest that CA III functions as an oxyradical scavenger and thus protects cells from oxidative damage. A lower level of free radicals in CA III overexpressing cells may also affect growth signaling pathways.—Räisänen, S. R., Lehenkari, P., Tasanen, M., Rahkila, P., Härkönen, P. L., Väänänen, H. K. Carbonic anhydrase III protects cells from hydrogen peroxide‐induced apoptosis. FASEB J. 13, 513–522 (1999)

Cleavable ErbB4 Isoform in Estrogen Receptor–Regulated Growth of Breast Cancer Cells

ErbB1 and ErbB2 receptors are well-characterized targets for anticancer drugs, but the clinical relevance of the related ErbB4 receptor is unknown. Here, we have assessed the clinical significance of the proteolytically cleavable ErbB4 isoforms in breast cancer patients and investigated their functions in vitro. The expression of transcripts encoding the cleavable ErbB4 isoforms associated with estrogen receptor-alpha (ER) expression (P < 0.001) and a high histologic grade of differentiation (P </= 0.002) in real-time reverse transcription-PCR analysis of 62 breast cancer samples. Despite high ErbB4 mRNA expression levels in a subset of samples, ErbB4 gene amplification was not observed. High ErbB4 protein expression levels, as assessed by immunohistochemistry, associated with a favorable outcome in ER-positive cases from a series of 458 breast cancer patients (P = 0.01), whereas no association between ErbB4 expression and survival was found among women with ER-negative cancer (P = 0.86). However, nuclear ErbB4 immunoreactivity was associated with poor survival as compared with women whose cancer had membranous ErbB4 staining (P = 0.04). In vitro, overexpression of a cleavable ErbB4 isoform in ER-positive breast cancer cells resulted in translocation of a proteolytically released intracellular ErbB4 receptor fragment into the nucleus, as well as, enhanced proliferation, anchorage-independent growth, and estrogen response element-mediated transcriptional activity. These results suggest that the association of ErbB4 expression with clinical outcome is dependent on the subcellular localization of ErbB4 and that a proteinase-cleavable ErbB4 isoform promotes growth of ER-positive breast cancer and enhances ER-mediated gene transcription.

Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer

Abstract Cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) each have an important role in angiogenesis. The expression of these genes was investigated in human prostate cancer by immunohistochemistry, the expression of COX-1 and COX-2 being confirmed by mRNA analysis. Prostate cancer specimens from 12 patients were compared to control prostates from 13 patients operated on for bladder carcinoma. The intensity of COX-2 and NOS-2 immunostaining was significantly stronger in prostate cancer cells than in the non-malignant glandular epithelium of the control prostates. COX-2 and NOS-2 were clearly also expressed in the lesions of prostatic intraepithelial neoplasia (PIN) in control prostates. COX-2 was detected in the muscle fibres of the hyperplastic stroma of some control prostates. No significant difference was detected in COX-1 expression between control and cancer prostates. These results indicate that the expression of COX-2 and NOS-2 is elevated in prostatic adenocarcinoma and in PIN.

Estrogen and Testosterone Use Different Cellular Pathways to Inhibit Osteoclastogenesis and Bone Resorption

Using human peripheral blood CD14+ osteoclast precursors, we show that testosterone directly inhibits osteoclast formation and bone resorption at physiological concentrations. Instead, estrogen has no direct effects, whereas its action seems to be mediated through osteoblasts by producing osteoprotegerin. Both estrogen and testosterone acts through their cognate receptors.

Selective estrogenic effects of a novel triphenylethylene compound, FC1271a, on bone, cholesterol level, and reproductive tissues in intact and ovariectomized rats.

FC1271a is a novel triphenylethylene compound with a tissue-selective profile of estrogen agonistic and weak antagonistic effects. It specifically binds to the estrogen receptor alpha and beta with affinity closely similar to that of toremifene and tamoxifen. To study the in vivo effects of the compound, 4-month-old rats were sham operated (sham) or ovariectomized (OVX) and treated daily for 4 weeks with various doses of FC1271a or vehicle (orally). FC1271a was able to oppose OVX-induced bone loss by maintaining the trabecular bone volume of the distal femur. Accordingly, the OVX-induced loss of bone strength was prevented at doses of 1 and 10 mg/kg. FC1271a also prevented the OVX-induced increase in serum cholesterol in a dose-dependent manner. No significant changes in uterine wet weight or morphology were observed in the OVX-rats treated with 0.1 or 1 mg/kg FC1271a, but at a dose of 10 mg/kg it had a slightly estrogenic effect. In immature rats the effect of FC1271a on uterine wet weight was less stimulatory than that of toremifene or tamoxifen, but more stimulatory than that of raloxifene or droloxifene. The appearance of the dimethylbenzanthracene (DMBA)-induced mammary tumors was inhibited by treatment of DMBA-treated rats with FC1271a in a dose-dependent manner. In human MCF-7 breast cancer cell tumors raised in nude mice in the presence of estrogen, the growth and expression of pS2 marker gene could not be maintained after estrogen withdrawal by treatment with FC1271a. No formation of DNA adducts was observed in the liver of the FC1271a-treated rats. In conclusion, the bone-sparing, antitumor, and cholesterol-lowering effects of FC1271a combined with a low uterotropic activity and lack of liver toxicity indicate that FC1271a could be an important alternative in planning antiosteoporosis therapy for estrogen deficiency.