Human Endometrial Cancer Cell Lines Research Articles

GnRH-II agonist [D-Lys6]GnRH-II inhibits the EGF-induced mitogenic signal transduction in human endometrial and ovarian cancer cells

The majority of human endometrial and ovarian cancers express receptors for GnRH type I (GnRH-I). Their proliferation is time- and dose-dependently reduced by GnRH-I and its analogs. GnRH-I analogs activate a phosphotyrosine-phosphatase (PTP) and inhibit EGF-induced mitogenic signal transduction. Recently we found that GnRH type II (GnRH-II) and its agonist [D-Lys6]GnRH-II also have antiproliferative effects on these tumor cells which are significantly greater than those of GnRH-I agonists. In a more recent study, we showed that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. The underlying signal transduction mechanisms of GnRH-II are still unknown. In this study we showed that the mitogenic effects of growth factors in endometrial and ovarian cancer cell lines were counteracted by GnRH-II agonist [D-Lys6]GnRH-II, indicating an interaction with the mitogenic signal transduction. We showed that [D-Lys6]GnRH-II reduces EGF-induced auto-tyrosine-phosphorylation of EGF-receptors via activation of a PTP and that EGF-induced activation of mitogen-activated protein kinase was blocked in cells treated with [D-Lys6]GnRH-II. Furthermore, EGF-induced expression of the immediate early gene c-fos was inhibited by treatment with [D-Lys6]GnRH-II. After knock-out of GnRH-I receptor expression, GnRH-II agonist [D-Lys6]GnRH-II still activated PTP and inhibited the EGF-induced mitogenic signal transduction. These data indicate, that the effects of GnRH-II are not due to a cross-reaction with the GnRH-I receptor. In conclusion these data suggest that the signaling of GnRH-II agonist [D-Lys6]GnRH-II is comparable to that of GnRH-I analogs.

Function of estrogen receptor isoforms α and β in endometrial carcinoma cells

The aim of this study was to explore how to modulate the expression of estrogen receptors (ER) alpha and beta and to verify the role of ERalpha and beta in relationship to estrogen and tamoxifen (TAM). A series of oligodeoxyribonucleotides (ODN) corresponding to regions of the ERalpha or beta was tested in human endometrial cancer cell lines (HEC-1B). The change in HEC-1B proliferation in response to 17beta-estradiol (E(2)) and TAM under the impact of antisense ODN was studied. The results of the study are as follows: 1) transfection with antisense ODN significantly inhibited ERalpha and ERbeta protein production, 2) the cells lost the ability to proliferate in response to E(2) after transfection with ERalpha antisense ODN especially at 24, 48, and 72 h. There was no obvious change in response to E(2) in HEC-1B cell lines that were transfected with ERbeta antisense ODN, and 3) after transfection with ERalpha antisense ODN, HEC-1B cells lost the ability to proliferate in response to TAM at 48 h. This inhibition was also observed after transfection with ERbeta antisense ODN at 24 h. ERalpha may be the primary receptor in the proliferation of HEC-1B cells in response to E(2). Both ERalpha and ERbeta are involved in the agonist impact of TAM on endometrial cancer cells.

Changes in gene expressions elicited by physiological concentrations of genistein on human endometrial cancer cells

The incidence of hormone-related diseases such as prostatic, breast, ovarian, and endometrial cancer is lower in Asian populations compared to Western countries. High consumption of soybean products that are rich in phytoestrogens, predominantly genistein, is postulated to be responsible for the lower incidence of hormone-related disease, although the mechanism through which this effect might be mediated is unclear. In this study, microarray analysis was used to identify the changes in gene expression elicited by treatment of the human endometrial cancer cell line, Ishikawa, with genistein at both physiologically achievable and supraphysiological concentrations. Genistein treatment at 5 microM concentration induced multiple changes in gene expression including some implicated in oncogenesis. In contrast, treatment with a supraphysiological concentration of genistein predominantly activated stress response genes and showed very limited overlap with the genes regulated at lower concentrations. Of the genes regulated by genistein, 9.3% were also regulated by 17beta-estradiol suggesting that genistein exerts its response via the estrogen pathway. These results indicate that at physiological concentrations, genistein is able to elicit pleiotropic effects on a variety of pathways believed to be involved in tumorigenesis. Supraphysiological concentrations of genistein, such as those used in many previous studies, elicit changes in gene expression that are unlikely to occur in vivo.

Estrogen-receptor-dependent regulation of telomerase activity in human endometrial cancer cell lines

Objectives. Given that prolonged exposure to unopposed estrogen is associated with endometrial cancer development and that the promoter region of the catalytic subunit of the telomerase enzyme, hTERT, contains putative estrogen response elements (EREs), we postulated that estrogen-receptor (ER)-mediated induction of telomerase activity may play an important role in endometrial carcinogenesis. Methods. ER-positive and ER-negative endometrial cancer cell lines were used. ERα expression was reconstituted in ER-negative cell lines by transient transfection. Telomerase activity was assayed using a PCR-based telomeric repeat amplification protocol (TRAP) after exposure to estradiol (E2). hTERT mRNA expression was assessed by real-time RT-PCR. Gel shift assays using oligonucleotide probes encoding each ERE and transient expression assays using luciferase reporter plasmids containing varying lengths of the 5′ promoter region of the hTERT gene were performed. Results. E2 induced both hTERT gene transcription and telomerase activity in the ER-positive cell lines, but not in the ER-negative cell lines. Transfection of ERα into ER-negative cell lines restored E2-induced hTERT gene transcription and telomerase activity. Gel shift assays revealed two EREs in the hTERT promoter that specifically bind to ERα. Luciferase assays demonstrated that at least the proximal ERE is responsible for transcriptional activation by ligand-stimulated ERα. Conclusions. Telomerase activity and hTERT mRNA were increased in response to estrogen in an ERα-dependent fashion in endometrial cancer cells. Binding of complexed estrogen with ERα to the EREs found within the hTERT promoter suggests a possible mechanism for telomerase induction that may facilitate the malignant transformation of hormone-dependent endometrial cells.

Determination of the mechanism of gemcitabine modulation of cisplatin drug resistance in panel of human endometrial cancer cell lines

Objectives. The primary objective of this study was to determine the mechanism(s) of cisplatin drug resistance in endometrial cancer cell lines. To evaluate the mechanism that gemcitabine modulates cisplatin drug resistance in endometrial cancer cell lines. Methods. Combination treatment was completed in panel of four human endometrial cancer cell lines. Growth inhibition assays were conducted in each cell line evaluating combinations of the Ic 25, Ic 50, and Ic 90 to determine optimal dosing for the combination of gemcitabine plus cisplatin. Evaluation of the correlative biological targets for modulation of platinum drug resistance was completed by the respective immunohistochemistry assays. Results. Downregulation of glutathione- S-transferase (GST) activity by 11% to 100% was observed with an associated 78.6% to 100% decrease in intracellular glutathione (GSH) concentrations. In the gemcitabine plus cisplatin treatment arm compared to either alone, there was also downregulation of MSH2, p53, and ERCC1 expression. No changes observed in the pro-apoptotic proteins, BAX or BAD, expression, AKT activation, or MDR1/PGP expression regardless of treatment with combination of gemcitabine plus cisplatin or either agent alone. Conclusions. There is likely more than one mechanism contributing to the increase synergistic in vitro platinum-resistant cell lines and increase clinical activity that has been observed in patients with platinum-resistant tumors. In this in vitro study, we determined the downregulation of intracellular GST activity and GSH concentration were the predominant mechanisms involved in the modulation of platinum resistance. Downregulation of MSH2, p53 and ERCC1 expression may also contribute to increase cytotoxic activity compared to cisplatin alone.

Expression of Monocyte Chemotactic Protein-1 in Human Endometrial Cancer Cells and the Effect of Treatment with Tamoxifen or Buserelin

Monocyte chemotactic protein-1 (MCP-1) is an important determinant of macrophage infiltration in tumours. This study investigated the effect of tamoxifen and the gonadotrophin-releasing hormone agonist buserelin on MCP-1 in the human endometrial cancer cell line EFE-184. Reverse transcription polymerase chain reaction and Western blot analysis were used to determine MCP-1 mRNA and protein expression, respectively. Immunoreactive MCP-1 in the cell culture media was quantified by enzyme-linked immunosorbent assay. Tamoxifen inhibited MCP-1 mRNA and protein expression in endometrial cancer cells and inhibited MCP-1 secretion in a time- and dose-dependent manner at concentrations of 10(-7) to 10(-5) M. Buserelin had no significant effect on MCP-1 mRNA and protein expression. These results suggest that tamoxifen directly inhibits the expression of MCP-1 in this cell line by blocking the MCP-1 signalling pathways. These findings may contribute to the understanding of the mechanisms underlying the different effects of tamoxifen and gonadotrophin-releasing hormone agonists in the treatment of endometrial cancer.

Function of Estrogen-Related Receptor α in Human Endometrial Cancer

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily that is closely related to estrogen receptor alpha (ERalpha). ERRalpha binds an estrogen response element (ERE), directly competes with ERalpha for binding ERE, and represses ERE-dependent transcription in MCF-7 cells, ER-positive breast cancer cells. We investigated whether ERRalpha modulate some ER-dependent activities in endometrial cancer. We investigated protein and mRNA expression of ERRalpha in endometrial cancer using immunohistochemistry and RT-PCR, respectively. After transient transfection using the ERRalpha expression vector (pCI-ERRalpha) or ERRalphaSi, which suppressed the expression of endogenous ERRalpha, Ishikawa cells were assayed for ERE-dependent luciferase activity. Cells stably overexpressing ERRalpha were generated and compared with estrogen-dependent and -independent cell growth. ERRalpha was detected in human endometrial cancer tissues by immunohistochemistry. An RT-PCR study showed that mRNA of ERRalpha was expressed in four endometrial cancer cell lines (Ishikawa, Hec1a, KLE, and SNGII) and 11 human endometrial tissues. Overexpression of ERRalpha repressed estrogen-induced ERE-dependent transcriptional activity in Ishikawa cells. After transfection with ERRalphaSi1, the expression of endogenous ERRalpha decreased to 0.5-fold, and estrogen-induced ERE luciferase activity increased to 1.5-fold. The cells stably overexpressing ERRalpha grew up more slowly than control cells in the presence of 10 nm estradiol. ERRalpha is expressed in human endometrial cancer tissues and cell lines and suppresses ERE-dependent transcriptional activity in the presence of estrogen. ERRalpha modulates estrogen-induced activity in estrogen-dependent endometrial cancer.

Growth regulation and transcriptional activities of estrogen and progesterone in human endometrial cancer cells

Estrogen-stimulated growth of the malignant human endometrium can be balanced by the differentiating properties of progesterone. To study the molecular basis behind this, gene expression profiling was performed using complementary DNA microarray analysis. In this study, the human endometrial cancer cell lines ECC-1 and PRAB-36 were used as models. The ECC-1 cell line, which expresses high levels of estrogen receptor alpha and is stimulated in growth by estrogens, was used to study estrogen regulation of gene expression. The Ishikawa sub-cell line PRAB-36, expressing both PRA and PRB, progesterone receptor isoforms, and inhibited in growth by progestagens, was used to study progesterone regulation of gene expression. Using these two well-differentiated human endometrial cancer cell lines, 148 estrogen- and 148 progesterone-regulated genes were identified. After functional classification, the estrogen- and progesterone-regulated genes could be categorized in different biologically relevant groups. Within the group of "cell growth and/or maintenance," 81 genes were clustered, from which a number of genes could be involved in arranging the cross talk that exists between estrogen and progesterone signaling. On the basis of analysis of the current findings, it is hypothesized that cross talk between estrogen and progestagen signaling does not occur by counterregulation of single genes, but rather at the level of differential regulation of different genes within the same functional families.

Growth regulation and transcriptional activities of estrogen and progesterone in human endometrial cancer cells

Estrogen-stimulated growth of the malignant human endometrium can be balanced by the differentiating properties of progesterone. To study the molecular basis behind this, gene expression profiling was performed using complementary DNA microarray analysis. In this study, the human endometrial cancer cell lines ECC-1 and PRAB-36 were used as models. The ECC-1 cell line, which expresses high levels of estrogen receptor α and is stimulated in growth by estrogens, was used to study estrogen regulation of gene expression. The Ishikawa sub–cell line PRAB-36, expressing both PRA and PRB, progesterone receptor isoforms, and inhibited in growth by progestagens, was used to study progesterone regulation of gene expression. Using these two well-differentiated human endometrial cancer cell lines, 148 estrogen- and 148 progesterone-regulated genes were identified. After functional classification, the estrogen- and progesterone-regulated genes could be categorized in different biologically relevant groups. Within the group of “cell growth and/or maintenance,” 81 genes were clustered, from which a number of genes could be involved in arranging the cross talk that exists between estrogen and progesterone signaling. On the basis of analysis of the current findings, it is hypothesized that cross talk between estrogen and progestagen signaling does not occur by counterregulation of single genes, but rather at the level of differential regulation of different genes within the same functional families.

Estrogen and Tamoxifen Induce Cytoskeletal Remodeling and Migration in Endometrial Cancer Cells

Much research effort has been directed toward understanding how estrogen [17beta-estradiol (E2)] regulates cell proliferation and motility through the rapid, direct activation of cytoplasmic signaling cascades (i.e. nongenomic signaling). Cell migration is critical to cancer cell invasion and metastasis and involves dynamic filamentous actin cytoskeletal remodeling and disassembly of focal adhesion sites. Although estrogen is recognized to induce cell migration in some model systems, very little information is available regarding the underlying pathways and potential influence of selective estrogen receptor modulators such as 4-hydroxytamoxifen on these processes. Using the human endometrial cancer cell lines Hec 1A and Hec 1B as model systems, we have investigated the effects of E2 and Tam on endometrial nongenomic signaling, cytoskeletal remodeling, and cell motility. Results indicate that both E2 and Tam triggered rapid activation of ERK1/2, c-Src, and focal adhesion kinase signaling pathways and filamentous actin cytoskeletal changes. These changes included dissolution of stress fibers, dynamic actin accumulation at the cell periphery, and formation of lamellipodia, filopodia, and membrane spikes. Longer treatments with either agent induced cell migration in wound healing and Boyden chamber assays. Agent-induced cytoskeletal remodeling and cell migration were blocked by a Src inhibitor. These findings define cytoskeletal remodeling and cell migration as processes regulated by E2 and 4-hydroxytamoxifen nongenomic signaling in endometrial cancer. This new information may serve as the foundation for the development of new clinical therapeutic strategies.

Gene regulation profiles by progesterone and dexamethasone in human endometrial cancer Ishikawa H cells

Objective.Progesterone and glucocorticoids such as dexamethasone mediate distinct biological functions, yet they bind to receptors that recognize the same consensus DNA response element. In breast cancer, progestins are associated with the incidence and progression of tumors, whereas glucocorticoids are growth-suppressive in mammary cancer cells; the differential effects of these two steroids are less well understood in the hormone-dependent disease cancer of the uterine endometrium. We set out to identify genes that are regulated by progesterone through progesterone receptors and dexamethasone through glucocorticoid receptors in a well-differentiated human endometrial cancer cell line. Methods.PR- and GR-positive Ishikawa H endometrial cancer cells were treated with vehicle, dexamethasone (100 nM) or progesterone (100 nM) for 2 h, 6 h, 12 h and 24 h, and RNA was isolated. Affymetrix microarrays were performed using the human HG-U133A chip, querying the expression of 22,000 genes. Expression of genes of particular interest was confirmed by real-time RT-PCR. Results.Expression analysis demonstrated that dexamethasone and progesterone regulate overlapping but distinct sets of genes and presumably exert many similar but also unique biological effects. Using real-time RT-PCR, we confirmed three particular genes of interest: the transcript for cysteine 1 (legumain), a gene associated with metastasis, that is strongly downregulated by progesterone, upstream c-fos relating transcription factor-2 (USF-2), an anti-proliferative factor that is induced by both progesterone and dexamethasone and N-cadherin, a cellular adhesion molecule downregulated by dexamethasone. Conclusion.These studies provide new insight into the effects of progesterone and dexamethasone in endometrial cancer cells and provide an extensive list of regulated pathways which can be assessed in the future as biomarkers and molecular targets for new therapies. Taken together, our findings indicate that progesterone and dexamethasone are primarily growth inhibitors in Ishikawa H endometrial cancer cells.

Lysophosphatidic Acid Acyltransferase-β Is a Prognostic Marker and Therapeutic Target in Gynecologic Malignancies

Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase beta (LPAAT-beta), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-beta converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-beta was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-beta using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-beta also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-beta was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-alpha. These findings identify LPAAT-beta as a potential prognostic and therapeutic target in ovarian and endometrial cancer.

GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs

We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.

GnRH-II receptor-like antigenicity in human endometrial, ovarian and breast cancer cells

The majority of human endometrial, ovarian and breast cancer cell lines express receptors for GnRH. Their proliferation is time- and dose-dependently reduced by GnRH-I, GnRH-II and its analogs. Recently we have demonstrated that the antiproliferative effects of GnRH-II are not mediated through the GnRH-I receptor. A functional receptor for the human GnRH-II receptor was not identified till now.

Significant anti-proliferation of human endometrial cancer cells by combined treatment with a selective COX-2 inhibitor NS398 and specific MEK inhibitor U0126

The extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a critical role in the anticancer action in vitro. ERK1/2 activation or phosphorylation is responsible for increased cyclooxygenase-2 (COX-2) protein expression in some cancer cells treated with selective COX-2 inhibitor NS398. We determined the effect of NS398 on ERK signaling and the synergistic effect of combined treatment with NS398 and a specific MEK inhibitor U0126 on three human endometrial cancer cell lines: Ishikawa, HEC-1A and AN3CA cells. Results showed that NS398 and U0126 individually, and especially the combination of both exhibited profound anti-proliferation of all three cell lines in a time- and concentration-dependent manner by [3-(4, 5)-dimethylthiazol-z-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. The phosphorylated ERK1/2 was up-regulated in HEC-1A and AN3CA cells, but the COX-2 protein expression was unchanged in the three cancer cell lines treated with NS398 alone. However, both phosphorylated ERK1/2 and COX-2 protein expression were concentration-dependently decreased in all three cell types by combined treatment with NS398 and U0126 assessed by western blot analysis. Simultaneously, the combination of NS398 and U0126 resulted in 2-fold increase in apoptosis of all three lines over that by the individual alone, and enhanced G0/G1 phase arrest of Ishikawa and HEC-1A cells induced by U0126 treatment determined by flow cytometry. The synergistic and complementary effects of combining NS398 and U0126 were found to be associated with activation of caspase-3, alterations of Bcl-2 family proteins and cell cycle regulatory proteins detected by western blot analysis. Taken together, these findings correlate with blocking MEK-ERK signaling cascade and down-regulating COX-2 protein expression in endometrial cancer cells with combination treatment of NS398 and U0126, suggesting that the combinatory use of NS398 and specific MEK inhibitors may be valuable for chemotherapy or chemoprevention of human endometrial cancer.

Endometrial cancer is a receptor-mediated target for Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS), a 140-kDa homodimer glycoprotein member of the TGF-beta superfamily of biological-response modifiers, causes regression of the Mullerian ducts in developing male embryos. MIS also can induce growth arrest and apoptosis in ovarian and cervical cancer cell lines. The embryonic progenitor of the ovarian and cervical epithelium is the coelomic epithelium, the same tissue that regresses under the direction of MIS in the male. The endometrium and uterus also arise from the coelomic epithelium and the Mullerian ducts. Here, we show that both normal human endometrium and endometrial cancers express the receptor for MIS and that MIS can inhibit the proliferation of a number of human endometrial cancer cell lines that express the MIS type II receptor. In the representative endometrial cancer cell line AN3CA, MIS affects the expression of key cell-cycle regulatory proteins. This work broadens the scope of tumors that MIS can potentially control and, by elucidating the MIS signaling pathway, identifies other potential avenues for intervention.

Importance of the central region of lamprey gonadotropin-releasing hormone III in the inhibition of breast cancer cell growth

Naturally occurring isoforms of the decapeptide gonadotropin-releasing hormone (GnRH) share residues 1–4 and 9–10. lGnRH-III, the third isoform isolated in the sea lamprey has no endocrine effect in mammals but shows a direct antiproliferative effect on human breast, prostate and endometrial cancer cell lines. To investigate these features, residues 5–8 of lGnRH-III were systematically replaced with Ala. The ability of the synthetic analogs to interact with receptors on MDA-MB 231 human breast cancer cells and their effect on the growth of the same cell line were investigated. [Ala 6]lGnRH-III and [Ala 7]lGnRH-III have neither receptor binding nor antiproliferative activity. Replacement of His 5 with Ala resulted in an analog that binds to the receptor but does not have antiproliferative activity. The results are in agreement with previous reports that modifications of Lys at position 8 are well tolerated.

HMMC-1

The purpose of this research was to generate a human monoclonal antibody specific to gynecological cancers and to evaluate such an antibody as therapy for gynecological cancers. Transchromosomal KM mice were immunized with the human uterine endometrial cancer cell line SNG-S. Hybridomas were constructed between spleen cells from KM mice and mouse myeloma cells. Reactivity of the antibody was evaluated by immunohistochemistry of pathological specimens of gynecological cancers. Cytotoxicity of HMMC-1 against SNG-S cells was tested by in vitro cytotoxicity assays. The epitope of HMMC-1 was determined by transfection with a panel of glycosyltransferase cDNAs and by inhibition assays with chemically synthesized oligosaccharides. HMMC-1 is a human IgM monoclonal antibody that reacts positively with mullerian duct-related carcinomas with positive rates of 54.6% against uterine endometrial adenocarcinoma, 76.9% against uterine cervical adenocarcinoma, and 75.0% against epithelial ovarian cancer. HMMC-1 does not react with normal endometrium at proliferative or secretory phases, normal uterine cervix, or normal and malignant tissue from other organs, whereas it reacts weakly with the epithelium of the gall bladder and the collecting duct of the kidney. HMMC-1 exhibits antigen-dependent and complement-mediated cytotoxicity. Upon cotransfection with cDNAs encoding two glycosyltransferases required for fucosylated extended core 1 O-glycan, mammalian cells express HMMC-1 antigen. Finally, binding of HMMC-1 to SNG-S cells is inhibited by synthetic Fucalpha1-->2Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->3GalNAcalpha1-octyl. These results indicate that HMMC-1 specifically recognizes a novel O-glycan structure. The unique specificity and cytotoxicity of HMMC-1 strongly suggest a therapeutic potential of this antibody.

Internalization of cytotoxic analog AN-152 of luteinizing hormone-releasing hormone induces apoptosis in human endometrial and ovarian cancer cell lines independent of multidrug resistance-1 (MDR-1) system

ObjectiveEighty percent of human ovarian and endometrial cancers express receptors for luteinizing hormone–releasing hormone (LHRH-R). These receptors can be used for targeted chemotherapy with agents such as AN-152, in which doxorubicin is linked to analog [D-Lys6]-LHRH. Direct receptor-mediated antiproliferative effects of AN-152 have been shown in vitro and in vivo. In LHRH-R positive cell lines, AN-152 was more effective than doxorubicin at equimolar concentrations. This study was designed to investigate the mechanism of action of AN-512 in ovarian and endometrial cancer cells in vitro. Study designThree ovarian (SKOV-3, NIH:OVCAR-3, EFO-21) and 2 endometrial carcinoma cell lines (Ishikawa, HEC-1A) were evaluated for doxorubicin- or AN-152–induced apoptosis. Internalization and cytoplasmic release of AN-152 was monitored by confocal laser scanning microscopy and inhibited by chloroquine. Cleavage of doxorubicin from AN-152 was inhibited by carboxylesterase inhibitor, diisopropyl fluorophosphate (DFP). The surface expression of multidrug resistance-1 (MDR-1) gene product P-glycoprotein (Pgp) was measured by flow cytometry. ResultsInduction of apoptosis by AN-152 in LHRH-R positive Ishikawa, HEC-1A, EFO-21, and NIH:OVCAR-3 cells was significantly higher than that induced by doxorubicin, whereas the percentage of apoptotic cells in LHRH-R negative SKOV-3 was higher after treatment with doxorubicin. In EFO-21 cells, apoptosis induced by AN-152 was inhibited by pretreatment with chloroquine. Pretreatment with DFP increased AN-152–induced apoptosis in LHRH-R positive cells and reduced apoptosis in LHRH-R negative SKOV-3. Both AN-152 and doxorubicin induced surface expression of MDR-1 gene product Pgp, but the effect of AN-152 was smaller than that of doxorubicin. Pgp surface expression induced by AN-152 was inhibited by pretreatment with DFP. ConclusionAN-152 is internalized through the LHRH-R and induces apoptosis in LHRH-R–positive human ovarian and endometrial cancer cell lines without activating the MDR-1 efflux pump system. The efficacy and specificity of AN-152 is inversely correlated with carboxylesterase activity.

Endometrial cancer expresses the Mullerian Inhibiting Substance (MIS) type II receptor and is growth inhibited by MIS

Introduction: In female embryos, the reproductive organs arise from the Mullerian ducts and associated epithelium; these structures regress under the influence of MIS produced by male embryos. The MIS type II receptor (MISIIR) can mediate MIS-induced growth inhibition of malignant ovarian and cervical cells. We propose that endometrial cancers will express the MISIIR and MIS will inhibit endometrial cancer growth. Methods: Human samples were obtained under IRB approval (2002-P-001324). Rat tissue was collected under institutional protocol 1999-N-00138-Rats. Cells lines were purchased from American Tissue and Cell Culture. Tissue and cell samples were analyzed by Western, PCR, growth inhibition assays, and FACS. Results: Western detected MISIIR protein in adult rat uterus; reverse transcriptase PCR detected MISIIR RNA in normal and malignant human endometrium. AN3CA and KLE, two human endometrial cancer cell lines, expressed MISIIR. MIS inhibited AN3CA and KLE cell growth 67.3% (p < 0.0001, n = 4) and 52.5% (p = 0.0041, n = 3), respectively. MIS-treated AN3CA cells demonstrated G1 arrest, increased Caspase 3 cleavage indicating apoptosis, increased levels of cell cycle proteins p107 and p130 and transcription factor E2F3, and decreased levels of transcription factor E2F1. Conclusions: MISIIR is expressed in normal endometrium and in human endometrial cancer. MIS inhibits the growth of two human endometrial cancer cell lines, induces cell cycle arrest and apoptosis in AN3CA cells, and affects proteins important to the control of cell cycle progression. In the future, treatment targets for MIS may broaden to include advanced endometrial cancer