Ishikawa Endometrial Adenocarcinoma Research Articles

Antiproliferative and apoptotic activities of tosylcyclonovobiocic acids as potent heat shock protein 90 inhibitors in human cancer cells

We evaluated whether inhibition of heat shock protein 90 (hsp90) function by novobiocin derivatives could induce the degradation of signal transducers that drive cancer cell growth and thereby promote apoptosis. Removal of the noviose moiety in novobiocin and introduction of a tosyl substituent at C-4 or C-7 coumarin nucleus provided derivatives 4TCNA and 7TCNA which compared favourably with novobiocin in MCF-7 breast cancer cells. Here we extend the antiproliferative and apoptotic properties of these analogues to a panel of cancer cell lines. Destabilization of hsp90 client proteins Raf-1, HER2, and cdk4 suggests inhibition of hsp90 chaperoning function. In HT29 colon and IGROV1 ovarian cancer cells, the growth inhibiting effect of 4TCNA and 7TCNA was consistent with the stimulation of cell death as assessed by the processing and activation of caspase 9, 8, 7 and 3 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP). In Ishikawa endometrial adenocarcinoma cells, 4TCNA also promoted apoptosis and the processing of PARP. These derivatives impacting multiple pathways involved in the neoplastic process may represent promising drugs for cancer therapy.

Epigenetic up-regulation of CXCR4 and CXCL12 expression by 17 β-estradiol and tamoxifen is associated with formation of DNA methyltransferase 3B4 splice variant in Ishikawa endometrial adenocarcinoma cells

Increased risk for the development of endometrial cancer has been associated with unopposed oestrogen exposure, hyperoestrogenic factors, and a history of breast cancer treated long-term with tamoxifen (Tam). Stromal cell-derived factor-1, currently named as CXCL12, is a chemokine that, via binding to CXCR4 receptor, activates several downstream effectors and signalling pathways responsible for proliferation, survival, and migration of cancer cells.We observed that 17β-estradiol (E2) and tamoxifen (Tam) increase the expression of CXCR4 and CXCL12 transcripts and proteins in oestrogen receptor positive (ER+) but not in negative (ER−02) Ishikawa endometrial adenocarcinoma (ISH) cell lines. However, the demethylating agent 5-Aza-2′-deoxycytidine profoundly elevated CXCR4 and CXCL12 expression in both ER+ and ER−02 ISH cells. Bisulfite sequencing revealed that E2 and Tam up-regulate expression via demethylation of cytosine in the cytosine–guanosine dinucleotide island of CXCR4 and CXCL12 promoters. We also found that E2 and Tam significantly increased, for several hours, the expression of DNA methyltransferase 3B4 enzymatically inactive splice variant in ER+ but not in ER−02 ISH cells.Our results suggest that E2 and Tam, through their ability for gene-transcription regulation, change the cellular milieu that maintains the hypermethylated stage of CpG islands of CXCR4 and CXCL12 promoters.

Regulation of SULT1E1 expression in Ishikawa adenocarcinoma cells by tibolone

Tibolone is used therapeutically as a hormone replacement agent and has beneficial effects on osteoporosis and hot flushes as well as libido in post-menopausal women without stimulatory effects in the breast and endometrium. The lack of effect in the endometrium is due in part to the tissue specific sulfation of tibolone and its active metabolites in endometrial tissues. Tibolone is metabolized into 3α-OH and 3β-OH tibolone as well as the Δ4-isomer. Tibolone and the Δ4-isomer bind and activate progesterone and androgen receptors whereas 3α-OH and 3β-OH tibolone activate the estrogen receptors. Human endometrium and Ishikawa endometrial adenocarcinoma cells express SULT1E1 that efficiently sulfates both 3-OH tibolone metabolites and has trace activity with tibolone but no activity with the Δ4-isomer. Treatment of Ishikawa cells with all four tibolone compounds resulted in the induction of SULT1E1 activity similar to the induction by progesterone. The induction of SULT1E1 was inhibited by RU486 indicating a role for the progesterone receptor. Sulfation of the tibolone compounds by Ishikawa cells and Ishikawa cells expressing physiological levels of SULT1E1 activity resulted in the sulfation of tibolone and the 3-OH metabolites but not Δ4-tibolone. These results indicate that the lack of endometrial stimulation involves induction of SULT1E1 and the selective sulfation and inactivation of the estrogenic 3-OH tibolones and interconversion of the tibolone metabolites to generate the progestagenic non-sulfated Δ4-isomer.

P38 Mitogen-Activated Protein Kinase Stimulates Estrogen-Mediated Transcription and Proliferation through the Phosphorylation and Potentiation of the p160 Coactivator Glucocorticoid Receptor-Interacting Protein 1

Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.

Effect of mifepristone on proliferation and apoptosis of Ishikawa endometrial adenocarcinoma cells

To determine the mechanism by which mifepristone improves breakthrough bleeding, the effects of mifepristone on proliferation and apoptosis of Ishikawa endometrial carcinoma cells were evaluated in the presence and absence of progestin. Prospective basic research study. Research laboratories for reproductive health at a university medical school. None. Ishikawa cells were cultured in vitro. Mifepristone and/or medroxyprogesterone acetate at various concentrations were added to the cells. Determination of cell proliferation and apoptosis. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine (BrdU) labeling analysis demonstrated that mifepristone inhibited the growth of Ishikawa cells and percentage of BrdU-labeled cells in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that mifepristone at 100 micromol/L, which completely inhibited cell proliferation, increased the proportion of cells in the S phase and diminished the cells in the G2/M phase. Apoptosis was identified by annexin-V-fluorescein isothiocyanate binding and caspase-3 activation. Immunofluorescent double labeling of Ishikawa cells in the absence or presence of mifepristone revealed that BAX protein expression increased and translocated from cytosol to mitochondria. Mifepristone inhibited cell growth by arresting cell cycle progression at S phase, induced apoptosis through caspase-3 activation, and modulated apoptosis regulatory genes BCL2/BAX and FAS/FASLG in Ishikawa cells. Together, these data imply that the improvement in breakthrough bleeding observed with mifepristone might be due to diminished volume of endometrial tissue similar to that seen with endometrial atrophy.

Prostaglandin E2Mediates Phosphorylation and Down-Regulation of the Tuberous Sclerosis-2 Tumor Suppressor (Tuberin) in Human Endometrial Adenocarcinoma Cells via the Akt Signaling Pathway

Prostaglandin (PG) E2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted down- regulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nm PGE2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE2-induced phosphorylation of Akt and tuberin. These data suggest that PGE2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.

The Src Kinase Pathway Promotes Tamoxifen Agonist Action in Ishikawa Endometrial Cells through Phosphorylation-Dependent Stabilization of Estrogen Receptor α Promoter Interaction and Elevated Steroid Receptor Coactivator 1 Activity

Tamoxifen is the most widely used selective estrogen receptor modulator for breast cancer in clinical use today. However, tamoxifen agonist action in endometrium remains a major hurdle for tamoxifen therapy. Activation of the nonreceptor tyrosine kinase src promotes tamoxifen agonist action, although the mechanisms remain unclear. To examine these mechanisms, the effect of src kinase on estrogen and tamoxifen signaling in tamoxifen-resistant Ishikawa endometrial adenocarcinoma cells was assessed. A novel connection was identified between src kinase and serine 167 phosphorylation in estrogen receptor (ER)-alpha via activation of AKT kinase. Serine 167 phosphorylation stabilized ER interaction with endogenous ER-dependent promoters. Src kinase exhibited the additional function of potentiating the transcriptional activity of Gal-steroid receptor coactivator 1 (SRC-1) and Gal-cAMP response element binding protein-binding protein in endometrial cancer cells while having no effect on Gal-p300-associated factor and Gal fusions of the other p160 coactivators glucocorticoid-interacting protein 1 (transcriptional intermediary factor 2/nuclear coactivator-2/SRC-2) and amplified in breast cancer 1 (receptor-associated coactivator 3/activator of transcription of nuclear receptor/SRC-3). Src effects on ER phosphorylation and SRC-1 activity both contributed to tamoxifen agonist action on ER-dependent gene expression in Ishikawa cells. Taken together, these data demonstrate that src kinase potentiates tamoxifen agonist action through serine 167-dependent stabilization of ER promoter interaction and through elevation of SRC-1 and cAMP response element binding protein-binding protein coactivation of ER.

Effects of raloxifene hydrochloride on endometrial cancer cells in vitro

Objective. Determine effects of raloxifene hydrochloride, a selective estrogen receptor modulator (SERM), on growth and proliferation of an estrogen-responsive endometrial cancer cell line in vitro. Materials and methods. Studies were performed with Ishikawa endometrial adenocarcinoma cells, a well-differentiated cancer that expresses estrogen receptors and progesterone receptors. Raloxifene was purified as the hydrochloride salt. The four arms of the study were cells grown (1) without any further addition (control), (2) with estradiol only, (3) with raloxifene only, or (4) with estradiol and raloxifene. Three concentrations of estradiol (10, 100, 1000 pg/ml) and raloxifene (1, 10, 100 ng/ml) were used. After 1 week of culturing, the number of living cells for each experimental group was determined and expressed as a percentage of the control group. Results. Cells treated with raloxifene 10 or 100 ng/ml alone grew significantly faster than control cells: 10 ng/ml [115.25%; SD, 11.05; 95% confidence interval (CI), 107.35–123.16; P = 0.002] and 100 ng/ml (111.14%; SD, 14.19; 95% CI, 100.98–121.29; P = 0.03). Estradiol 10 or 100 pg/ml did not stimulate cell growth, whereas cells treated with 1000 pg/ml grew significantly faster than control cells (114.69%; SD, 16.84; 95% CI, 102.65–126.74; P = 0.02). Raloxifene and estradiol together in any concentration did not affect cell growth. Conclusions. Raloxifene did not inhibit the growth of endometrial cancer cells in vitro. High concentrations even promoted cell growth. Estradiol in physiologic concentrations did not stimulate the growth of endometrial cancer cells in vitro.

Selective estrogen receptor modulator regulated proteins in endometrial cancer cells

Tamoxifen is the primary hormonal therapy for breast cancer and is also used as a breast cancer chemopreventative agent. A major problem with tamoxifen therapy is undesirable endometrial proliferation. To identify proteins associated with the growth stimulatory effects of tamoxifen in an ER-positive model, the present study profiled total cellular and secreted proteins regulated by estradiol and selective estrogen receptor modifiers (SERMs) in the Ishikawa endometrial adenocarcinoma cell line using two-dimensional gel electrophoresis. Following 24 h incubation with 10 −8 M estradiol, 10 −7 M 4-hydroxytamoxifen, or 10 −7 M EM-652 (Acolbifene), nine proteins exhibited significant increase in expression. The proteins identified were heat shock protein 90-α, and -β, heterogeneous nuclear ribonucleoprotein F, RNA polymerase II-mediating protein, cytoskeletal keratin 8, cytoskeletal keratin 18, ubiquitin-conjugating enzyme E2-18 kDa and nucleoside diphosphate kinase B. These protein profiles may serve as novel indices of SERM response and may also provide insight into novel mechanisms of SERM-mediated growth.

Effects of a combination of exemestane and paclitaxel on human tumor cells in vitro.

Exemestane, a non-steroidal aromatase inhibitor that shuts down estrogen synthesis, and paclitaxel, an antineoplastic drug, inhibiting microtubule formation and interfering with the cells potential to proliferate, are well established treatments for metastatic breast cancer. Given that exemestane is a treatment for hormone-sensitive tumors in postmenopausal women with more favorable prognosis, while paclitaxel is normally used for women suffering from hormone-insensitive breast cancers with less favorable prognoses, there is currently no experience with the combination of the two drugs. In order to find out to what extent exemestane and paclitaxel add to each other's effects when given concomitantly, the effect of the two drugs alone and in combination on the growth of various gynecological tumor cell lines was assessed. Tumor cell growth was measured according to the cell titer cell proliferation technique, also referred to as the MTS assay, by measurement of relative cell numbers. In gynecological cancer cells expressing aromatase, the effect of a treatment with paclitaxel (10 nM) on cell growth was enhanced by co-treatment with exemestane. This additive effect was independent of ERalpha expression, but dependent on the presence of androstenedione. It was observed in HEC-1A and Ishikawa endometrial adenocarcinoma cells as well as in SK-OV-3 ovarian cancer and in MDA-MB-231 breast cancer cells. Our findings suggest that a combination of paclitaxel with exemestane might be beneficial for the treatment of aromatase-positive gynecological cancer, because it may allow us to reduce the paclitaxel dosage and therefore the toxicity of the treatment.

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100 nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of “tissue specificity” and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.

Effekte von Kinase-Inhibitoren auf die Östradiolwirkung in der endometrialen Adenokarzinomzelllinie Ishikawa

Purpose: Interactions between signal transduction cascades involving tyrosine kinase and its receptor and the cellular response to stimulation with estradiol have received a great deal of study. We studied the effect of inhibitors of receptor tyrosine kinases and cytoplasmatic kinases on the cellular response to estradiol in an endometrial adenocarcinoma cell line that expresses estrogen receptors-alpha. Material and Methods: Estrogen effects were measured with a transient reporter gene assay. Ishikawa endometrial adenocarcinoma cells were cultured without serum and transfected with the vector pERE-TA-SEAP via lipofectamines. The vector contains the secreted alkaline phosphatase (SEAP) reporter gene under the control of an estrogen response element (ERE). After transfection the cells were treated with estradiol, the EGFR inhibitor AG1478, and the MEK inhibitors PD98059 and U0126 for 48 hours. The activity of the EREs was quantified with luminometric measurement of the SEAP concentration in the culture supernatant. Cellular proliferation was measured with an ELISA that quantified the uptake of BrDU into replicating DNA. Results: Physiologic estradiol stimuli caused activation of the ERE. Simultaneous treatment with the EGFR inhibitor AG1478 significantly increased the estradiol-induced activation of ERE. In contrast, both MEK inhibitors reduced both ERE activation and estradiol-induced cellular proliferation. Conclusion: Treatment with the EGFR inhibitor AG1478 increased the response of endometrial adenocarcinoma cells to estrogen stimuli. MEK inhibitors can block MAP kinase signal transduction and inhibit cellular estradiol effects. This suggests that inhibitors of cytoplasmatic kinases may be more useful than EGFR inhibitors in the treatment of endometrial cancers that express estrogen receptors.

Regulation of estrogen activity by sulfation in human Ishikawa endometrial adenocarcinoma cells

Sulfation is an important conjugation reaction in the metabolism of steroids. Steroids sulfates do not interact with the appropriate hormone receptors; additionally, the presence of the charged sulfate moiety increases the aqueous solubility and excretion of most steroids. Estrogen sulfotransferase (EST) is the major form of human cytosolic ST involved in the conjugation of estrogens. EST is important in the inactivation of β-estradiol (E2) during the luteal phase of the menstrual cycle. EST has a significantly higher affinity for the sulfation of E2 and 17α-ethinylestradiol (EE2) than for other potent estrogens such as diethylstilbestrol (DES) and equine estrogens. The ability of EST to sulfate these estrogenic compounds at physiologic concentrations is important in regulating their activation of the ER in estrogen responsive cells. Human Ishikawa endometrial adenocarcinoma (ISH) cells possess an estrogen receptor (ER)-regulated alkaline phosphatase (AlkPhos) which is used to assay ER activation. To study the effects of EST activity on the ER activation of different estrogenic compounds, ISH cells were stably transformed with an EST expression vector. Dose–response curves for the induction of AlkPhos activity by the different estrogenic compounds were generated with EST/ISH and control pcDNA/ISH cells. EST/ISH cells were 200-fold less sensitive to E2 and EE2 than were control cells. No differences were observed in the dose–response curves for DES between EST/ISH and pcDNA/ISH cells. EST/ISH cells were approximately 3–10-fold less sensitive to the equine estrogens equilin and 17-equilin as compared to control cells. The ability of EST to decrease the ER activation of an estrogen correlates with the sulfation of these compounds at nanomolar concentrations by EST/ISH and pcDNA/ISH ISH cells. These results indicate that EST is capable of efficiently inactivating E2 and EE2 but is significantly less effective in inhibiting the ER binding of other potent estrogenic compounds.

Steroid sulphotransferase and 17beta-hydroxysteroid dehydrogenase activities in Ishikawa human endometrial adenocarcinoma cells.

The present studies concern sulphotransferase activities for estrogens and other steroids, and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities for estrogens in Ishikawa endometrial adenocarcinoma cells. When physiological concentrations of various estrogens (estrone, estradiol, estriol) are incubated, most of the transformation product is the respective sulphate. The sulphotransferase activity is very rapid, and 2 h after incubation 70-95% are converted to the sulphated form. Sulphates are found exclusively in the culture medium, which suggests that as soon as the sulphate is biosynthesized it is secreted to the medium. Comparative data using neutral steroids (dehydroepiandrosterone, testosterone, and pregnenolone) show that sulphotransferase activity for these compounds is very limited. In another series of studies, 17beta-HSD activity was explored for the interconversion estrone estradiol. At low concentrations (5 x 10(-9)-5 x 10(-8) M), when estradiol (E2) is incubated, most of the unconjugated material remains as E2 in the cellular compartment, but at high concentrations (5 x 10(-7)-5 x 10(-6) M) a great proportion (70-80%) of the E2 is converted to estrone (E1). On the other hand, after incubation of E1 at all concentrations most remained as unchanged E1. It is suggested that, in Ishikawa cells, at very low concentrations of E1 or E2, sulphotransferases are predominant, but when this enzyme is saturated 17beta-HSD activity is orientated to the oxidative form.

Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone.

During the secretory phase of the human menstrual cycle, the endometrium is minimally responsive to the estrogens secreted from the ovaries. Conjugation of beta-estradiol (E2) with sulfate is thought to be an important mechanism in the regulation of the levels of active E2 in endometrial tissue. Estrogen sulfation is reportedly increased during the secretory phase in response to the high levels of progesterone secreted by the ovaries. Estrogen sulfotransferase (hEST), a distinct form of human cytosolic sulfotransferase (ST) with an affinity for E2 and estrone at low nanomolar concentrations, has recently been cloned and expressed in mammalian cells and in bacteria (J Steroid Biochem Mol Biol 52:529, 1995). At least two other forms of human cytosolic ST, dehydroepiandrosterone ST (hDHEA-ST) and the phenol-sulfating form of phenol-ST (hP-PST), also conjugate estrogens but at micromolar concentrations. This report describes the specific induction of hEST in human Ishikawa endometrial adenocarcinoma cells by progesterone as a model for the increases in estrogen sulfation observed in women during the secretory phase of the menstrual cycle. Treatment of Ishikawa cells with 10 microns progesterone for 48 h resulted in a 7-fold increase in the sulfation of 20 nM E2. The sulfation of selective substrates for human dehydroepiandrosterone sulfotransferase (hDHEA-ST) and the two forms of phenol sulfotransferase (hP-PST, hM-PST) were not affected by treatment with progesterone. The levels of immunoreactive hEST and hEST mRNA in the Ishikawa cells were both increased by progesterone, whereas the levels of immunoreactive hDHEA-ST, hP-PST, and hM-PST were not altered. hEST activity was not induced by treatment of Ishikawa cells with varying concentrations of E2, testosterone, or cortisol. The induction of hEST by progesterone was inhibited by RU-486, indicating that progesterone is acting via the progesterone receptor. These results indicate that progesterone is capable of specifically inducing hEST and estrogen sulfation in human Ishikawa adenocarcinoma cells and suggest a mechanism for increasing estrogen sulfation in the endometrium during the secretory phase of the menstrual cycle.

Hormonal control of proliferation in the Ishikawa endometrial adenocarcinoma cell line

Steroid hormone regulation of proliferation of the Ishikawa human endometrial adenocarcinoma cell line was investigated in defined tissue culture medium. Oestradiol increased cell number following treatment for greater than 8 days; 4-OH tamoxifen, used alone, induced growth in a similar manner to oestradiol and was not antagonistic when used in combination with oestradiol. Progesterone decreased cell number 4 days after treatment but thereafter the effect was lost; the effect of progesterone was abolished in the presence of Phenol Red, consistent with the oestrogenic properties of this indicator. Oestradiol together with progesterone for greater than 8 days resulted in maximal growth and was preceded by an apparent increase in synthesis of a protein of molecular weight 36 kDa pI 8.