Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression.

Programmed cell death ligand 1 (PD-L1) on tumor cells suppresses anti-tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD-L1 disruption in ovarian cancer. PD-L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. PD-L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD-L1-KO ID8-inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD-L1-KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4+ T cells, CD8+ T cells, NK cells and CD11c+ M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD-L1-KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon-γ, tumor-necrosis factor-α, interleukin (IL)-2, IL-12a, CXCL9 and CXCL10 was significantly stronger, while that of IL-10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD-L1-KO ID8 groups. These results indicate that CRISPR/Cas9-mediated PD-L1 disruption on tumor cells promotes anti-tumor immunity by increasing tumor-infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD-L1-targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer.

Levels of serum-circulating angiogenic factors within 1 week prior to delivery are closely related to conditions of pregnant women with pre-eclampsia, gestational hypertension, and/or fetal growth restriction.

AIM: We aimed to investigate maternal serum angiogenic marker profiles within 1 week prior to delivery in cases of gestational hypertension (GH), pre-eclampsia (PE), and/or fetal growth restriction (FGR) with different clinical conditions. METHODS: We enrolled 165 women with singleton pregnancy. The participants were classified based on three characteristics: (i) proteinuria (GH and PE); (ii) FGR (PE with FGR [PE + FGR], PE alone, and FGR alone); and (iii) onset (early onset PE [EO PE] and late-onset PE [LO PE]). All sera were obtained within 1 week prior to delivery, and soluble fms-like tyrosine kinase 1 (sFlt-1), soluble endoglin (sEng), and placental growth factor (PlGF) were measured with enzyme-linked immunosorbent assay. RESULTS: (i) In PE, a significantly increased sFlt-1, sEng, and sFlt-1 to PlGF ratio (sFlt-1/PlGF) and significantly decreased PlGF were observed compared with GH and Term control, whereas in GH, only sFlt-1/PlGF was significantly higher than Term control. (ii) In PE + FGR, similar changes were more markedly shown compared with PE alone. The FGR alone group exhibited similar tendencies as PE, although significant differences were found in PlGF and sEng levels. (iii) In EO PE, significant changes were observed in all factors compared with LO PE or Term control, while no significant change in PlGF levels was observed between LO PE and Term control. CONCLUSION: We demonstrated that the levels of circulating angiogenic factors just before delivery are correlated with the severity of hypertensive disorders of pregnancy and FGR. Profiling these specific markers may contribute to better understanding of the clinical conditions in individual patients and their pathogenesis.

Indoleamine 2,3-dioxygenase promotes peritoneal metastasis of ovarian cancer by inducing an immunosuppressive environment.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that has immunoregulatory functions. Our prior study showed that tumoral IDO overexpression is involved in disease progression and impaired patient survival in human ovarian cancer, although its mechanism remains unclear. The purpose of the present study is to clarify the role of IDO during the process of peritoneal dissemination of ovarian cancer. Indoleamine 2,3-dioxygenase cDNA was transfected into the murine ovarian carcinoma cell line OV2944-HM-1, establishing stable clones of IDO-overexpressing cells (HM-1-IDO). Then HM-1-IDO or control vector-transfected cells (HM-1-mock) were i.p. transplanted into syngeneic immunocompetent mice. The HM-1-IDO-transplanted mice showed significantly shortened survival compared with HM-1-mock-transplanted (control) mice. On days 11 and 14 following transplantation, the tumor weight of peritoneal dissemination and ascites volume were significantly increased in HM-1-IDO-transplanted mice compared with those of control mice. This tumor-progressive effect was coincident with significantly reduced numbers of CD8(+) T cells and natural killer cells within tumors as well as increased levels of transforming growth factor-ß and interleukin-10 in ascites. Finally, treatment with the IDO inhibitor 1-methyl-tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor-ß secretion. These findings showed that tumor-derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor-infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity. Therefore, IDO may be a promising molecular target for the therapeutic strategy of ovarian cancer.

Comprehensive Gene Mutation Profiling of Circulating Tumor DNA in Ovarian Cancer: Its Pathological and Prognostic Impact.

Liquid biopsies from circulating tumor DNA (ctDNA) have been employed recently as a non-invasive diagnostic tool for detecting cancer-specific gene mutations. Here, we show the comprehensive gene mutation profiles of ctDNA in 51 patients with different histological subtypes of stage I-IV ovarian cancer, and their association with clinical outcomes. The ctDNA extracted from pre-treatment patients' plasma were analyzed using Cancer Personalized Profiling by Deep Sequencing targeting 197 genes. Of 51 patients, 48 (94%) showed one or more non-synonymous somatic mutations, including TP53 (37.3%), APC (17.6%), KRAS (15.7%), EGFR (13.7%), MET (11.8%), PIK3CA (11.8%), NPAP1 (11.8%), and ALK (9.8%). The most frequently mutated genes were as follows: TP53 in high-grade serous carcinoma (66.7%), APC in clear cell carcinoma (30.8%), PIK3CA in endometrioid carcinoma (40%), and KRAS in mucinous carcinoma (66.7%). Higher cell-free (cf)DNA concentration significantly correlated with worse progression-free survival (PFS) in all patients as well as stage III-IV patients (p = 0.01 and 0.005, respectively). Further, patients with any pathogenic mutations showed significantly worse PFS (p = 0.048). Blood tumor mutational burden detected from ctDNA did not significantly correlate with the histological subtypes or survival. Collectively, clinico-genomic profiles of individual ovarian cancer patients could be identified using ctDNA and may serve as a useful prognostic indicator. These findings suggest that ctDNA-based gene profiling might help in establishing personalized therapeutic strategies.

Changes in the gene mutation profiles of circulating tumor DNA detected using CAPP-Seq in neoadjuvant chemotherapy-treated advanced ovarian cancer.

Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) is a novel ultrasensitive next-generation sequencing-based approach that is used to detect circulating tumor DNA (ctDNA). The aim of the present study was to compare the gene mutation profiles and blood tumor mutation burden (bTMB) measured between pre- and post-neoadjuvant chemotherapy (NAC), utilizing CAPP-seq for plasma ctDNA in patients with advanced ovarian cancer. The current study included 10 patients (6 NAC-sensitive and 4 NAC-resistant) clinically diagnosed as having stage III or IV ovarian cancer and were administered NAC between May 2017 and February 2019. The plasma ctDNA samples were collected at pre- and post-NAC, and comprehensive gene mutation analysis was performed using CAPP-seq. In 5 out of 6 NAC-sensitive cases, the variant allele frequency (VAF) of non-synonymous somatic mutations decreased following NAC. In 2 out of the 4 NAC-resistant cases, the VAF of non-synonymous somatic mutations increased, and new somatic mutations emerged following NAC. In regard to TP53 mutation, the rate of TP53 mutation in the NAC-resistant cases was significantly higher compared with NAC-sensitive cases. Finally, the bTMB decreased significantly after NAC treatment in the NAC-sensitive cases, even though there were no significant differences in the pretreatment bTMB levels between the NAC-sensitive and NAC-resistant cases. These results indicated that gene mutation can be profiled and monitored using liquid biopsy-based CAPP-Seq in patients with advanced ovarian cancer with NAC treatment, and TP53 mutation in the ctDNA and bTMB may be novel biomarkers that can be used for patient monitoring during NAC treatment.

Calreticulin Regulates Syncytialization Through Control of the Synthesis and Transportation of E-Cadherin in BeWo Cells.

During placental development, mononuclear cytotrophoblasts differentiate and fuse to syncytiotrophoblasts (STBs) to form syncytia, which secrete human chorionic gonadotropin (hCG). Decreased maternal perfusion of the placenta, which leads to placental dysfunction, induces changes in trophoblast syncytialization. Our previous study showed that calreticulin (CRT), a Ca2+-binding molecular chaperone found in the endoplasmic reticulum, is expressed in the human placenta and is involved in regulating extravillous trophoblast invasion, although its role in villous trophoblasts remains unclear. In this study, we investigated the functional role of CRT in trophoblast differentiation using the human trophoblast-like cell line BeWo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in human third-trimester placentas and mainly localized in STBs. The fetal growth restriction group exhibited significantly lower CRT expression levels than did the control group. In BeWo cells, CRT knockdown markedly suppressed forskolin-induced cell fusion and ß-hCG secretion. As for the mechanism responsible for these effects, the cell surface expression of E-cadherin, a key adhesion molecule related to syncytialization, was decreased, and E-cadherin accumulated adjacent to the Golgi apparatus in the CRT-knockdown cells, which led to dysfunctional cell-to-cell adhesion. Additionally, metabolic labeling and a pulse-chase study revealed that the protein expression of E-cadherin was suppressed at the translational level in the CRT-knockdown cells. Collectively, these results demonstrate that CRT regulates syncytialization by ensuring appropriate control of both the synthesis and transportation of E-cadherin, suggesting that CRT expression is important for placental development during pregnancy.

Liquid biopsy-based comprehensive gene mutation profiling for gynecological cancer using CAncer Personalized Profiling by deep Sequencing.

Liquid biopsies of circulating tumor DNA (ctDNA) have recently been used as a non-invasive diagnostic tool for detecting tumor-specific mutations. We present a study of ctDNA liquid biopsies in gynecological cancer using an ultrasensitive next-generation sequencing-based method for ctDNA detection named CAncer Personalized Profiling by deep Sequencing (CAPP-Seq). We performed CAPP-Seq with plasma-ctDNA obtained from 16 patients with gynecological cancer. In all cases, at least one non-synonymous somatic mutation was detected in the ctDNA. In the pre-treatment ctDNA, 4 of 16, 4/16, 5/16, 2/16, 2/16, and 2/16 patients had TP53, KRAS, APC, PIK3CA, BRCA1, and EGFR mutations, respectively. MET gene copy-number gains were detected in the ctDNA of 2 of 16 patients, and FISH analysis of the paired tumor samples confirmed these results. In 2 neoadjuvant chemotherapy-treated ovarian cancer patients, the changes in gene mutation patterns were associated with the treatment response. These findings suggest that CAPP-Seq-based liquid biopsies can be used for the genetic characterization of independent gynecological cancers with high frequency, and might be clinically useful for non-invasive tumor genotyping and therapeutic response monitoring.

Experimental characterization of recurrent ovarian immature teratoma cells after optimal surgery.

Minimal optimal surgery without chemotherapy is often performed for patients with ovarian immature teratoma, which frequently occurs in young women who hope for future pregnancies. If tumors recur after the operation, anticancer drug chemotherapy is often administered, although few studies have highlighted differences between the recurrent and the primary tumor cells. Therefore, we have established experimental animal models of recurrent ovarian immature teratoma cells after optimal surgery and characterized the anticancer drug sensitivity and antigenicity of the recurrent tumors. Surgically-excised tumor cells of a grade II ovarian immature teratoma were cultured in vitro and transplanted into nude mice to establish stable cell lines. Differential drug sensitivity and antigenicity of the tumor cells were compared between the primary and the nude mouse tumors. Nude mouse tumor cells showed a normal 46XX karyotype. Cultured primary cells showed a remarkably high sensitivity to paclitaxel, docetaxel, adriamycin and pirarubicin, compared to peritoneal cancer cells obtained from a patient with ovarian adenocarcinomatous peritonitis. The drug sensitivity of teratoma cells to 5-fluorouracil, bleomycin or peplomycin was also significantly higher. However, there was no significant difference in sensitivity to platinum drugs between the primary teratoma and the peritoneal adenocarcinoma cells. As for nude mouse tumor cells, sensitivity to 12 anticancer drugs was significantly lower than that of the primary tumor cells, while there was little difference in sensitivity to carboplatin or peplomycin between the primary and nude mouse tumor cells. Flow cytometry showed that the expression of smooth muscle actin (SMA) significantly decreased in nude mouse tumor cells when compared to cultured primary cells. In conclusion, ovarian immature teratomas with normal karyotypes have a malignant potential to recur after minimal surgery. During nude mouse transplantation, SMA-overexpressing cells appeared to be selectively excluded and nude mouse tumor cells were less sensitive to the majority of anticancer drugs than the primary tumor cells. These results indicate that after optimal surgery for ovarian immature teratoma, recurrent cells can be more resistant to anticancer drugs than the primary tumors. Therefore, it is likely that adjuvant chemotherapy lowers the risk of ovarian immature teratomas recurring after optimal surgery. BEP and PBV regimens are frequently given to teratoma patients. However, paclitaxel/carboplatin or docetaxel/carboplatin, which are the most effective chemotherapy treatments for epithelial ovarian cancer patients, are considered to be an alternative regimen, especially in the prevention of reproductive toxicity.

A comprehensive gene mutation analysis of liquid biopsy samples from patients with metastatic colorectal cancer to the ovary: A case report.

Liquid biopsies of circulating tumor DNA (ctDNA) can detect molecular alterations, including tumor-specific mutations, and have recently been used as a non-invasive diagnostic, prognostic, and predictive tool. However, this technique is not commonly used in the gynecological field. Gene mutation profiling of liquid biopsy samples was performed using CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), a novel next-generation sequencing-based approach to ultrasensitive ctDNA detection, in order to make it possible to molecularly diagnose metastatic colorectal cancer to the ovary. Liquid biopsy (plasma) samples and formalin-fixed paraffin-embedded tumor samples were obtained from two patients with ovarian tumors, who had a history of surgery for colorectal cancer, and comprehensive gene mutation profiling was conducted using CAPP-Seq. In patient 1, mutations were identified in the same three regions in both the ovarian tumor and preoperative plasma sample (in the KRAS G13D, APC E1306*, and TP53 H193Y genes). In patient 2, mutation was identified in the same one region in all the primary colorectal tumor, the ovarian tumor, and preoperative plasma sample (in APC R216* gene). These mutations are well-known genetic signatures of colorectal cancer, suggesting that the ovarian tumor was metastatic. Tthe gene mutation patterns of colorectal cancer were examined by subjecting liquid biopsy samples from patients with suspected metastatic ovarian tumors to CAPP-Seq. Gene mutation profiling of liquid biopsy samples can contribute to the preoperative differential diagnosis of metastatic ovarian cancer and its subsequent personalized treatment.

Prevention of lipopolysaccharide-induced preterm labor by the lack of CX3CL1-CX3CR1 interaction in mice.

Preterm labor (PTL) is the most common cause of neonatal death and long-term adverse outcome. The pharmacological agents for PTL prevention are palliative and frequently fail to prevent PTL and improve neonatal outcome. It is essential to fully understand the molecular mechanisms of PTL in order to develop novel therapeutic methods against PTL. Several lines of evidence indicate some chemokines are expressed in gestational tissues during labor or PTL. To reveal the pathophysiological roles of the CX3CL1-CX3CR1 axis in PTL, we performed present study using LPS-induced PTL mice model in CX3CR1-deficient (Cx3cr1-/-) mice. We indicated that PTL was suppressed in Cx3cr1-/- mice and immunoneutralization of CX3CL1 in WT mice. From immunohistochemical and the gene expression analyses, the CX3CL1-CX3CR1 axis has detrimental roles in PTL through intrauterine recruitment of macrophages and the enhancement of macrophage-derived inflammatory mediators. Thus, the CX3CL1-CX3CR1 axis may be a good molecular target for preventing PTL.

Downregulation of indoleamine 2, 3-dioxygenase expression in the villous stromal endothelial cells of placentas with preeclampsia.

INTRODUCTION: Previous studies have shown that indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that converts tryptophan to kynurenine, is expressed in the placenta and might play a role in the maintenance of pregnancy, although its associations with the pathogeneses of preeclampsia (PE) and fetal growth restriction (FGR) remain unclear. The objective of this study was to investigate the differences in IDO expression among normal, PE, and FGR placentas, and the associations between IDO expression and clinical symptoms, or the expression of fms-like tyrosine kinase receptor-1 (Flt-1). METHODS: Immunohistochemical studies of IDO and Flt-1 expression were performed in human placentas that were complicated with FGR alone (n=19), PE alone (n=20), or both PE and FGR (n=39), and gestational age-matched controls (n=23). RESULTS: It was found that IDO was expressed on endothelial cells in the villous stroma, while Flt-1 was located on trophoblast cells. The IDO expression level of the PE alone group was significantly lower than those of the FGR alone and control groups. The IDO expression of the PE+FGR group was significantly lower than that of the FGR alone group. Lower IDO expression was significantly correlated with more severe maternal hypertension or proteinuria in PE patients, who exhibited higher Flt-1 expression. The late onset PE patients exhibited significantly lower IDO expression than the early onset PE patients. CONCLUSION: This study demonstrated that the downregulation of IDO expression on the endothelial cells of the villous stroma was associated with PE, but not FGR, suggesting that IDO might be involved in the pathophysiology of PE.

Calreticulin Is Involved in Invasion of Human Extravillous Trophoblasts Through Functional Regulation of Integrin ß1.

Calreticulin (CRT), a molecular chaperone in the endoplasmic reticulum (ER), plays a variety of roles in cell growth, differentiation, apoptosis, immunity, and cancer biology. It has been reported that CRT is expressed in the human placenta, although its function in placental development is poorly understood. Appropriate invasion of extravillous trophoblasts (EVTs) into the maternal decidua is necessary for successful pregnancy. The objective of the present study was to investigate the expression and functional role of CRT in EVTs using the human EVT cell line HTR8/SVneo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in the human placenta in the early stage of pregnancy and localized to the EVTs. CRT knockdown markedly suppressed the invasion ability of HTR8/SVneo cells. Furthermore, the adhesion to fibronectin was suppressed in the CRT-knockdown cells via the dysfunction of integrin α5ß1. In the CRT-knockdown cells, terminal sialylation and fucosylation were decreased, and the core galactose-containing structure was increased in the N-glycans of integrin ß1. In addition, the expression levels of several critical glycosyltransferases were changed in the CRT-knockdown cells, consistent with the changes in the N-glycans. These results showed that CRT regulates the function of integrin ß1 by affecting the synthesis of N-glycans in HTR8/SVneo cells. Collectively, the results of the present study demonstrate that the ER chaperone CRT plays a regulatory role in the invasion of EVTs, suggesting the importance of CRT expression in placental development during early pregnancy.

Cell proliferation of bulky cervical squamous cell carcinoma is strongly associated with a paracrine tissue-specific growth factor(s).

To examine the possibility of a cervical squamous cell carcinoma-specific growth factor(s), previously suggested by clinical findings and in vitro culture experiments, we examined in vitro cultures and nude mouse transplantations of cervical squamous cell carcinoma cells obtained from a patient with a bulky cervical tumor without detectable distant metastases. The cancer cells did not proliferate without additional growth factors but remained viable in vitro. Fourteen weeks after transplantation of the tumor cells on their backs, 1 of the 3 nude mice developed large metastatic pelvic tumors without macroscopic metastatic lesions in their lungs or liver. When these pelvic tumor fragments were trans-planted onto the backs of other nude mice, ulcerated back tumors and larger metastatic pelvic tumors, but no macroscopic metastatic lesions in the lungs or liver, were observed. Histopathological examination of these pelvic tumors showed that all were the same squamous cell carcinoma as the primary tumor. These results indicate that cell proliferation of bulky cervical squamous cell carcinoma is strongly associated with a squamous cell carcinoma-specific growth factor(s) in a paracrine manner.

AG490, a Jak2 inhibitor, suppressed the progression of murine ovarian cancer.

Ovarian cancer is the major cause of cancer death among female genital malignancies, and requires developing novel therapeutic measures. Immune escape and acquisition of tolerance by tumor cells are essential for cancer growth and progression. An immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) overexpression in tumors is essential for host immune tolerance. Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) pathway is involved in various kinds of tumor biology. Thus, we examined the effects of STAT1 inhibition by AG490 (a JAK2 inhibitor) on ovarian cancer progression in mice. In vitro study, IFN-γ treatment up-regulated Ido mRNA expression with STAT1 activation in OV2944-HM-1 cells, whereas AG490 treatment significantly inhibited this effect with the suppression of STAT1 phosphorylation. In vivo model, OV2944-HM-1 cells were intraperitoneally/subcutaneously transplanted into syngeneic immunocompetent female mice. AG490 treatment significantly suppressed subcutaneous tumor growth, compared with control. Consistently, in mice intraperitoneally inoculated HM-1 cells, the same treatment significantly improved survival rate with the reduced number of intraperitoneal tumors. Actually, intratumoral IDO expression was significantly suppressed with the reduction of STAT1 activation in AG490-treated mice. Moreover, in tumor microenvironment of mice treated with AG490, the accumulation of anti-tumor leukocytes such as CD8(+) T-cells, M1 macrophages, and NK cells was apparently exaggerated with the reciprocal reduction of regulatory T cells. Furthermore, intratumoral expression of anti-tumor cytokines such as IL-1α, IL-1ß and IL-12 expression was significantly enhanced in mice treated with AG490. Collectively, JAK/STAT signal pathways may be good molecular target for immunotherapy of ovarian cancer.

Expression and function of activin receptors in human endometrial adenocarcinoma cells.

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

Activin A inhibits growth-inhibitory signals by TGF-beta1 in differentiated human endometrial adenocarcinoma cells.

Although human endometrial adenocarcinoma tissues express high levels of activin A, the pathophysiological functions of endometrial activin A remain unclear. Human endometrial cancer cells express both activin receptor and TGF-beta receptor, and TGF-beta1 utilizes the same intracellular signaling molecules as activin A. Using three differentiated human endometrial adenocarcinoma cell lines, we investigated whether there are interactions between TGF-beta1- and activin A-mediated signaling. Flow cytometric analysis revealed cell surface expression of two types of TGF-beta receptor subunits and four types of activin receptor subunits. TGF-beta1 inhibited cell proliferation in three endometrial adenocarcinoma cell lines. Activin A did not affect the growth of the three endometrial cell lines, and pre-incubation with activin A dose-dependently reduced TGF-beta1-mediated inhibition of cell growth. These results suggest that in endometrial adenocarcinoma cells, the intracellular signals underlying TGF-beta1-mediated inhibition of growth can themselves be inhibited by activin A. Therefore, the increased expression of activin A may be involved in carcinogenesis by reducing TGF-beta-mediated signals inhibiting cell growth in human endometrial adenocarcinoma tissues.

Growth-inhibitory signals by activin A do not affect anticancer drug-sensitivity and acquired multi-drug-resistance in human ovarian endometrioid adenocarcinoma OVK-18 cells.

Using the human ovarian adenocarcinoma cell line, OVK-18, which is sensitive to activin A-mediated inhibition of growth and various anticancer drugs, we determined whether activin A altered the sensitivity of these cells to seven anticancer drugs. The relationship between the sensitivity to activin and the resistance to anticancer drugs was also investigated in OVK-18 parent cells and OVK-18-derived CDDP-resistant cells. Activin A inhibited proliferation of OVK-18 parent cells in a dose-dependent manner, although it did not affect the sensitivity of OVK-18 parent cells to the seven anticancer drugs, CDDP, CBDCA, adriamycin, paclitaxel, SN38, terarubicin and etoposide (VP16). Both the sensitivity to activin A-mediated inhibition of growth and the sensitivity to anticancer drug-induced apoptosis were reduced in CDDP-resistant cells, while their sensitivity to the seven anticancer drugs was not affected by activin A. Flow cytometric analysis revealed a significant reduction in type IIA activin receptor expression on the surface of CDDP-resistant cells. These results indicate that the activin A-induced intracellular signals inhibiting cell growth are independent of the inhibition caused by the seven anticancer drugs, and suggest that the reduced sensitivity of CDDP-resistant cells to activin A is derived in part from reduced activin receptor expression and not acquired drug-resistance.

Differential sensitivity to paclitaxel-induced apoptosis and growth suppression in paclitaxel-resistant cell lines established from HEC-1 human endometrial adenocarcinoma cells.

To investigate acquired paclitaxel (PTX) resistance in cancer cells, we established five monoclonal PTX-resistant cell lines from HEC-1 human endometrial adenocarcinoma cells by means of long-term PTX-exposed cultures and limiting dilution cultures. The established PTX-resistant subclones showed apparent resistance to PTX-induced DNA fragmentation but not to PTX-induced growth suppression. None of the five PTX-resistant subclones showed apparent resistance to other anticancer drugs such as cisplatin, etoposide, 5-fluorouracil, pirarubicin-HCl, 4-hydroxy-cyclophosphamide or mitomycin C. Semiquantitative flow cytometric analysis revealed no apparent differential expression of 17 molecules that were previously reported to regulate apoptosis or drug resistance, between the five PTX-resistant subclones and the parent cells. Karyotyping analysis revealed common changes in chromosomes 4 and 18 in the five PTX-resistant subclones but not in the HEC-1 parent cells. These results indicate that PTX-induced growth suppression is regulated by different mechanisms from those involved in PTX-induced apoptosis. It was concluded that these established PTX-resistant subclones can be useful models in studies related to the prevention or treatment of recurrent cancers after PTX chemotherapy.

Impaired death-associated protein kinase-mediated survival signals in 5-fluorouracil-resistant human endometrial adenocarcinoma cells.

A recent study showed that both 5-fluorouracil (5FU)-stimulated apoptosis and Fas-mediated apoptosis in human endometrial adenocarcinoma cells are enhanced by targeted knockdown of endogenous death-associated protein kinase (DAPK) with DAPK small-interfering RNAs. Therefore, we investigated the DAPK survival signals in three 5FU-resistant subclones. DAPK knockdown did not enhance 5FU-stimulated or Fas-mediated apoptosis in any of the three 5FU-resistant subclones, but the subclones acquired resistance to VP16-stimulated cell death that was DAPK-independent. Semi-quantitative flow cytometric analyses showed that there was no differential expression in nine cell surface antigens, including Fas, and six intracellular molecules, including DAPK, that may regulate cell death or survival between the parent cells and 5FU-resistant cells. DAPK mRNA and protein were expressed in the 5FU-resistant subclones at similar levels to the parent cells. These results indicate that acquisition of 5FU-resistance may be accompanied by impairment of common apoptotic signals regulating both DAPK-dependent and DAPK-independent pathways.

Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells.

Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2'-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers.