Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines

In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIP(S) and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

Dehydroepiandrosterone stimulates inflammation and impairs ovarian functions of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in child-bearing-age women. It is characterized by ovulation dysfunction, polycystic ovaries, and hyperandrogenism. Inflammation is likely to be a crucial contributor to the pathogenesis of PCOS. However, the association between the inflammatory cytokines and the development of PCOS has not been reported. To explore the relationship between the inflammatory cytokines and PCOS, alterations of serum proteins in dehydroepiandrosterone (DHEA)-induced PCOS rats were screened by protein array, and the concentration of IFN-γ was further measured by using enzyme-linked immunosorbent assay (ELISA). DHEA-induced PCOS rats had a decreased level of IFN-γ compared with the control rats, which was restored partly in flutamide (an androgen receptor antagonist)-treated rats. Moreover, the level of IFN-γ in serum of patients with PCOS was also lower than that in healthy women. Using the ovarian granulosa cells (KGN), we demonstrated that DHEA downregulated the expression and secretion of IFN-γ in dose- and time-dependent manners, which could be restored to some extent by treating with flutamide. Furthermore, flutamide ameliorated the inhibitory effect on cell proliferation and promotive effect on cell apoptosis by DHEA. The results also revealed that IFN-γ promoted the proliferation but inhibited the apoptosis of KGN cells, which was suppressed by DHEA via activating the downstream PI3K/AKT signaling pathway. Taken together, these results showed that DHEA inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells through downregulating the expression of IFN-γ which could be restored by flutamide, and IFN-γ may serve as a potential inflammatory biomarker for PCOS detection.     

Green tea extract affects porcine ovarian cell apoptosis

Green tea is a commonly used beverage and green tea extract is a common dietary herbal supplement manufactured into different over-the-counter products. The aim of this in vitro study was to examine the steroid hormone secretion (progesterone and 17-β estradiol), proliferation and apoptosis of porcine ovarian granulosa cells after addition of green tea extract. Granulosa cells were incubated with green tea extract at five doses (0.1, 1, 10, 100 and 200?μg/ml) and the release of hormones by granulosa cells was assessed by EIA after 24?h exposure. The presence of proliferation and apoptotic markers was assessed by immunocytochemistry. Secretion of steroid hormones was not affected by green tea extract at all the doses in comparison to control. Also, markers of proliferation (PCNA and cyclin B1) were not affected by green tea extract. However, the highest dose (200?μg/ml) of green tea extract used in this study increased the accumulation of apoptotic markers caspase-3 and p53 in granulosa cells. In conclusion, our results indicate the impact of green tea extract at the highest dose used in this study on ovarian apoptosis through pathway that includes activation of caspase-3 and p53. Potential stimulation of these intracellular regulators could induce the process of apoptosis in ovarian cells.     

Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells

We investigated the impact of bisphenol A (BPA) on murine ovarian granulosa cells. Ovarian granulosa cells were cultured with 100 fM to 100 microM BPA for 24 h to 72 h. BPA decreased granulosa cell viability in a dose- and time-dependent manner. The lowest concentration that induced a significant decrease was 100 pM (89.2 +/- 4.0% of the control). TUNEL analysis demonstrated that treatment with BPA increased apoptosis of granulosa cells in a dose- and time-dependent manner. In addition, flow cytometry analyses revealed that treatment with BPA resulted in G2-to-M arrest, which was most prominent at 48 h. BPA increased the expression of Bax and concomitantly decreased the expression of Bcl2 at both protein and mRNA levels of granulosa cells. These findings suggest that low, presumably environmentally relevant doses of BPA, decrease the viability of granulosa cells by inducing apoptosis and G2-to-M arrest. Up-regulation of Bax and down-regulation of Bcl2 were suggested to be involved in this apoptotic effect.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

In vitro assessment of iron effect on porcine ovarian granulosa cells: secretory activity, markers of proliferation and apoptosis

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.     

Impact of vitrification on granulosa cell survival and gene expression

IntroductionCryopreservation of ovarian tissue is an essential step in Ovarian Tissue Banking. In order to prevent the formation of ice crystals, typically the tissue is slowly frozen using a cryoprotectant. As an alternative the method of ultra-fast freezing by vitrification becomes more attention for freezing ovarian tissue because it has successfully been used for oocytes, embryos and sperm. However the impact of vitrification on granulosa cells, which are an essential part of ovarian tissue is uncertain.AimIn this study, we have therefore analysed the influence of vitrification on the survival rates of granulosa cells, the impact of DMSO or ethylenglycol containing vitrification protocols and investigated to what extent the gene expression of apoptosis- and temperature-sensitive genes changes.Material and methodsWe used the human granulosa cell line KGN as a model for human granulosa cells and determined the survival rate and cell cycle stages by FACS analyses. The change in gene expression was determined by quantitative PCR analyses.ResultsOur results show that vitrification is possible in granulosa cells but it reduces cell viability and leads to fluctuations in the cell cycle. The DMSO containing protocol results in a lower amount of dead cells than the ethylenglycol containing protocol. Gene expression analysis reveals that TNF-alpha expression is strongly increased after vitrification, while other apoptosis or temperature-related genes seem to stay unaffected.ConclusionWe conclude that vitrification influences the viability of human granulosa cells. Furthermore, our results suggest that this could be mediated by a change in TNF-alpha gene expression.     

Plumbagin inhibits proliferation and promotes apoptosis of ovarian granulosa cells in polycystic ovary syndrome by inactivating PI3K/Akt/mTOR pathway

ABSTRACT

Polycystic ovary syndrome (PCOS) is recognized as a general endocrine disease and reproductive disorder. Although evidence indicates that PCOS has a complex etiology and genetic basis, the pathogenic mechanisms and signal pathway in PCOS remain unclear. In this study, the normal structure of follicle and corpus luteum were observed, and no cyst nor hyperemia was observed under the light microscopic study with hematoxylin and eosin (H&E) staining. Eestosterone and progesterone were evaluated by radioimmunoassay in rat serum. The alterations of proliferative ability and cell cycle distribution of each group were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry. The protein expression of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, and GAPDH were analyzed by western blotting. Both doses of PLB could benefit the ovarian morphology and polycystic property. PLBinduced a suppress effect on the proliferation of rat ovarian granulosa cells. In addition, PLB also induced concentration-dependent apoptosis in rat ovarian granulosa cells. The rat ovarian granulosa cells treated with PLB that the expression levels of p-AKT, p-mTOR, and p-PI3K were significantly decreased in a concentration-dependent manner. PLB not only plays a critical role in attenuating the pathology and polycystic property changes in the ovary but can also induce rat ovarian granulosa cell apoptosis through the PI3K/Akt/mTOR signal pathway. This study showed the innovative role of PLB in the pathogenesis of PCOS and provides a new therapeutic modality for the treatment of PCOS.     

Insulin mitigates apoptosis of porcine follicular granulosa cells by downregulating BimEL

Follicular growth or atresia is governed by the survival and apoptosis of granulosa cells. Increasing evidence shows that follicle growth is influenced by energy intake, which is positively related to insulin levels. However, the function of insulin in granulosa cell survival is poorly understood. This study focused on the effects of insulin on porcine medium follicle granulosa cell survival. In the present study, we showed that insulin markedly mitigated the apoptosis of porcine granulosa cells following serum starvation. Moreover, insulin activated the PI3K/Akt pathway to downregulate bim mRNA expression and simultaneously promoted the phosphorylation of BimEL through activating ERK 1/2, both of which reduced the level of BimEL. The results demonstrate that insulin protected the granulosa cells against apoptosis by reducing levels of the pro-apoptotic protein BimEL. However, the concentration of insulin (1 μg/ml) was relatively high. High levels of insulin partly combined with the IGF-1 receptor to play its roles in granulosa cells. This experiment provides new insight into the role of insulin in granulosa cells and sheds light on nutrition-reproduction interactions.     

Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro

The small ubiquitin-related modifier-1 (SUMO-1) with broad cellular expression has been implicated in a range of cellular processes, such as cell proliferation, differentiation, and apoptosis. As shown recently, SUMO-1 is expressed and regulated by gonadotropins, in particular an ovulatory hCG stimulus in mouse granulosa cells in vivo. To test the hypothesis that modulation of granulosa cell apoptosis changes SUMO-1 expression during granulosa cell differentiation in the mouse ovary, we demonstrate that progesterone receptor (PR) proteins are absent in pre-ovulatory granulosa cell nuclei, whereas they are expressed in periovulatory granulosa cell nuclei in parallel with decreases in SUMO-1 expression, caspase-3 activation, and DNA fragmentation in vivo. Second, treatment with either PR antagonists or a cell permeable ceramide analog consistently increases SUMO-1 expression in parallel with an increase in apoptosis as well as a decrease in cell proliferation in periovulatory granulosa cells in vitro. However, we do not observe an increase in SUMO-1 expression in pre-ovulatory granulosa cells that have undergone the same treatment. Third, we have also demonstrated, in pre-ovulatory granulosa cells in vitro, neither induction of spontaneous apoptosis nor the protective effect of EGF against spontaneous apoptosis changes SUMO-1 protein expression. Fourth, we show that induction of apoptosis enhances SUMO-1 conjugation in periovulatory granulosa cells in vitro, pointing to the pivotal link between the SUMO-1 conjugation and cell death. Taken together, our observations suggest that SUMO-1 via sumoylation has an important role in the regulation of granulosa cell apoptosis during granulosa cell differentiation in the mouse ovary.     

Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles

Previously, we prepared an IgM monoclonal antibody(PFG-1) which specifically recognized a cell-membraneglycoprotein (PFG-1 antigen; 55 kD, pI 5.9),immunohistochemically reacted with granulosa cells ofhealthy follicles but not of atretic follicles, andinduced granulosa cell apoptosis. In the presentstudy, an IgM monoclonal antibody (PFG-3) capable ofinducing granulosa cell apoptosis and an IgGmonoclonal antibody (PFG-4) not capable of inducingapoptosis were produced against granulosa cellsprepared from healthy antral follicles of porcineovaries. Two-dimensional Western blotting analysisrevealed that PFG-3 specifically recognized twocell-membrane proteins (named PFG-3-1 andPFG-3-2/PFG-1 antigens; 42 kD, pI 5.2 and 55 kD, pI5.9, respectively) of healthy granulosa cells, andthat PFG-4 recognized the same two cell-membraneproteins. In atretic granulosa cells, PFG-3-2/PFG-1antigen disappeared. Immunochemical reactions of theseantibodies were only detected in follicular granulosacells but not any other ovarian tissues or organs.PFG-3 and PFG-4 immunohistochemically reacted withgranulosa cells of healthy and atretic follicles. Whenthe isolated granulosa cells prepared from healthyfollicles were cultured in medium containing PFG-3,the cells underwent apoptosis, and co-incubation withPFG-4 inhibited PFG-3-inducible apoptosis. Theseobservations suggested that PFG-3-2/PFG-1 antigen isa novel cell death receptor which is different fromthe apoptosis-mediating receptors (Fas/Apo-1/CD95 orTNF receptor), and that PFG-3-1 antigen may act as adecoy receptor and inhibit apoptotic signal transmission.     

人卵巢颗粒细胞的体外培养方法探讨

目的建立人卵巢颗粒细胞分离纯化、体外培养的有效方法。方法收集体外受精—胚胎移植(IVF-ET)穿卵时的卵泡液,用胰蛋白酶消化法及密度梯度离心法分离纯化颗粒细胞并用不同培养基进行培养。结果用体积分数为50%的Percoll细胞分离液分离,DMEM/F12或McCoy’5a液体培养基进行培养,细胞纯度高,存活率高,后续生长良好。结论建立了人卵巢颗粒细胞体外培养的稳定模型,为颗粒细胞的体外研究奠定良好的基础。     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

Cell-to-cell communication competence in simian virus 40-transfected rat ovarian cells is reduced following tumor selection

Summary A pSV3neo-transfected rat ovarian cell line (SV-GC) was developed from a primary granulosa culture (GC) to study gap junctional intercellular communication (GJIC) during Simian virus 40 (SV40) transformation. SV-GC expressed SV40 large T-antigen (T-ag), grew indefinitely in culture without luteinization, was anchorage independent, and formed tumors in nude mice. Ultrastructural analysis identified abundant gap junctional membrane and suggested that SV-GC was arrested at an early stage of differentiation. Functional GJIC, measured by a dye transfer technique (gap FRAP), was comparable to that observed in normal granulosa cells, suggesting that the expression of T-ag alone was insufficient to reduce GJIC. However, there was approximately a 50% loss in the rate of GJIC in the nude mouse SV-GC-tumor derived and G418 selected cell line (T-SV-GC). SV-GC→T-SV-GC also resulted in a transition from migration of cells as an epithelial sheet to the dissociation of individual fibroblastoid cells. Tumor cell detachment was also seen in migrating malignant human (A2780 and 547) and rat (DC3) ovarian cell lines. Co-culture combinations of normal (GC)→transformed (SV-GC) → tumor-derived (T-SV-GC) cells indicated that the rate of heterologous GJIC was characteristic of the least communicating partner. Taken together, these data suggested that SV-GC → T-SV-GC represented progression toward metastasis with concomitant reduction of GJIC and adhesiveness. These sequentially derived cell lines may be a useful in vitro model system for studies focusing on the mechanisms involved in the detachment of cells during the progression of ovarian cancer.     

FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation,induction of apoptosis via the ERK and JNK-dependent mechanism,and suppression of sIL-6R and VEGF production

BackgroundXanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue.MethodsAnti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays.ResultsXN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells.ConclusionsERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells.General Significance: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.