Mullerian Inhibiting Substance induces NFkB signaling in breast and prostate cancer cells

The MIS type II receptor is expressed at high levels in the Mullerian duct and in Sertoli cells and granulosa cells of the embryonic and adult gonads. The presence of MIS type II and type I receptors in tissues and cell lines derived from breast and prostate suggests that the prostate and mammary glands may be additional targets for MIS action. In both breast and prostate cancer cells, MIS activated NFkB DNA binding activity and induced IEX-1, an immediate early gene which regulates cell growth and apoptosis. Exposure of cells to MIS inhibited growth by increasing the fraction of cells in the G1 phase of the cell cycle and by inducing apoptosis. These results suggest that MIS may be a putative mediator of growth regulatory signals in the breast and prostate.     

The stimulation of mitogenic signaling pathways by N-POMC peptides

The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC_1–28 stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC_1–28 and N-POMC_1–49 in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5 min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC_1–49 stimulated the phosphorylation of Akt more robustly than N-POMC_1–28.We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal.     

Efavirenz directly modulates the oestrogen receptor and induces breast cancer cell growth

Objectives

Efavirenz‐based HIV therapy is associated with breast hypertrophy and gynaecomastia. Here, we tested the hypothesis that efavirenz induces gynaecomastia through direct binding and modulation of the oestrogen receptor (ER).

Methods

To determine the effect of efavirenz on growth, the oestrogen‐dependent, ER‐positive breast cancer cell lines MCF‐7, T47D and ZR‐75‐1 were treated with efavirenz under oestrogen‐free conditions in the presence or absence of the anti‐oestrogen ICI 182,780. Cells treated with 17β‐oestradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure the ER binding affinity of efavirenz.

Results

Efavirenz induced growth in MCF‐7 cells with an estimated effective concentration for half‐maximal growth (EC₅₀) of 15.7 μM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to the ER [inhibitory concentration for half maximal binding (IC₅₀) of ～52 μM] at a roughly 1000‐fold higher concentration than observed with 17β‐oestradiol.

Conclusions

Our data suggest that efavirenz‐induced gynaecomastia may be caused, at least in part, by drug‐induced ER activation in breast tissues.     

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.     

Piperlongumine induces cell death through ROS-mediated CHOP activation and potentiates TRAIL-induced cell death in breast cancer cells

Purpose

Piperlongumine (PL) has been shown to selectively induce apoptotic cell death in cancer cells via reactive oxygen species (ROS) accumulation. In this study, we characterized a molecular mechanism for PL-induced cell death.

Methods

Cell viability and cell death were assessed by MTT assay and Annexin V-FITC/PI staining, respectively. ROS generation was measured using the H₂DCFDA. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein expression were analyzed by RT-PCR and Western blot analysis, respectively.

Results

We found that PL promotes C/EBP homologous protein (CHOP) induction, which leads to the up-regulation of its targets Bim and DR5. Pretreatment with the ROS scavenger N-acetyl-cysteine abolishes the PL-induced up-regulation of CHOP and its target genes, suggesting an essential role for ROS in PL-induced CHOP activation. The down-regulation of CHOP or Bim with siRNA efficiently attenuates PL-induced cell death, suggesting a critical role for CHOP in this cell death. Furthermore, PL potentiates TRAIL-induced cytotoxicity in breast cancer cells by upregulating DR5, as DR5 knockdown abolished the sensitizing effect of PL on TRAIL responses.

Conclusions

Overall, our data suggest a new mechanism for the PL-induced cell death in which ROS mediates CHOP activation, and combination treatment with PL and TRAIL could be a potential strategy for breast cancer therapy.     

A novel mechanism for mitogenic signaling via pro-transforming growth factor alpha within hepatocyte nuclei

Transforming growth factor (TGF) alpha, an important mediator of growth stimulation, is known to act via epidermal growth factor receptor (EGF-R) binding in the cell membrane. Here we show by immunohistology, 2-dimensional immunoblotting, and mass spectrometry of nuclear fractions that the pro-protein of wild-type TGF-alpha occurs in hepatocyte nuclei of human, rat, and mouse liver. Several findings show a close association between nuclear pro-TGF-alpha and DNA synthesis. (1) The number of pro-TGF-alpha+ nuclei was low in resting liver and increased dramatically after partial hepatectomy and after application of hepatotoxic chemicals or the primary mitogen cyproterone acetate (CPA); in any case, S phase occurred almost exclusively in pro-TGF-alpha+ nuclei. The same was found in human cirrhotic liver. (2) In primary culture, 7% of hepatocytes synthesized pro-TGF-alpha, which then translocated to the nucleus; 70% of these nuclei subsequently entered DNA replication, whereas only 2% of pro-TGF-alpha- hepatocytes were in S phase. (3) The frequency of hepatocytes coexpressing pro-TGF-alpha and DNA synthesis was increased by the hepatomitogens CPA or prostaglandin E(2) and was decreased by the growth inhibitor TGF-beta1. (4) Treatment with mature TGF-alpha increased DNA synthesis exclusively in pro-TGF-alpha- hepatocytes, which was abrogated by the EGF-R tyrosine kinase inhibitor tyrphostin A25. In conclusion, TGF-alpha gene products may exert mitogenic effects in hepatocytes via 2 different signaling mechanisms: (1) the "classic" pathway of mature TGF-alpha via EGF-R in the membrane and (2) a novel pathway involving the presence of pro-TGF-alpha in the nucleus.     

吉西他滨对人胰腺癌SW1990和BxPC3细胞株Notch信号通路的诱导作用

目的 观察吉西他滨对人胰腺癌细胞(SW1990和BxPC3)Notch信号通路活性的影响,探讨其与胰腺癌对吉西他滨化疗耐药的关系.方法 不同浓度吉西他滨处理人胰腺癌SW1990和BxPC3细胞株48 h,实时定量PCR检测Notch信号通路受体Notch1、2、3、4,配体Jagged1、2和下游靶基因Hes1 mRNA的表达,Western blotting测定细胞Hes1蛋白表达.结果 2μmol/L吉西他滨作用胰腺癌细胞株48 h,SW1990细胞的Notch1、2、3、Jagged1、2和Hes1 mRNA表达量分别为8.26±0.48、39.12±4.87、0.84±0.06、105.8±17.92、6.59±0.32和17.30±2.96,均较未处理细胞的1.02±0.15、15.25±1.28、0.12±0.02、32.66±1.98、1.88±0.29和5.02±0.64明显升高(P<0.05或P<0.01);BxPC3细胞上述各项表达量分别为7.87±0.59、109.4±10.98、0.74±0.19、62.73±13.50、2.09±0.16和15.38±1.06,也均较未处理细胞的1.14±0.43、58.96±2.63、0.10±0.02、16.95±3.79、0.98±0.02和2.04±0.16,明显升高(P<0.05或P<0.01).1、2 μmol/L吉西他滨作用胰腺癌细胞株48 h,SW1990细胞Hes1蛋白表达量分别为0.30±0.03、0.42±0.03;BxPC3细胞分别为0.33±0.02、0.45±0.03,均较未处理细胞显著增高(0.13±0.01、F=33.71;0.09±0.02、F=38.54,P值均<0.01).结论 吉西他滨可明显激活SW1990和BxPC3细胞的Notch信号通路,这可能是胰腺癌细胞获得化疗耐受性的机制之一.     

Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor G(i alpha) inhibitory to G(beta gamma) stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of G(beta gamma)-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655-662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949--953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, beta-arrestin, and G(beta), in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or G(beta) antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with G(beta) antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, beta-arrestin, and G(beta) has varying consequences on their ability to associate. As a result, increased availability of and signaling via G(beta gamma) could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing G(beta gamma) signaling and opioid tolerance formation.     

Bradykinin stimulates cell proliferation through an extracellular-regulated kinase 1 and 2-dependent mechanism in breast cancer cells in primary culture

We have previously reported that bradykinin (BK) represents an influential mitogenic agent in normal breast glandular tissue. We here investigated the mitogenic effects and the signalling pathways of BK in primary cultured human epithelial breast cells obtained from a tumour and from the histologically proven non-malignant tissue adjacent to the tumour. BK provoked cell proliferation, increase in cytosolic calcium, activation of protein kinase C (PKC)-alpha, -beta, -delta, -epsilon and -eta and phosphorylation of the extracellular-regulated kinases 1 and 2 (ERK1/2). The following compounds blocked the proliferative effects of BK: Hyp3-BK, a B2 receptor subtype inhibitor; U73122, a phospholipase C-beta inhibitor; GF109203X, a protein kinase C (PKC) inhibitor; and PD98059, a mitogen-activated protein kinase kinase inhibitor. G?6976, a Ca(2+)-dependent PKC inhibitor, did not have any effect. In conclusion, the mitogenic effects of BK are retained in peritumour and tumour cells; hence, it is likely that BK has an important role in cancer endorsement and progression.     

Endotoxin signaling in human macrophages: signaling via an alternate mechanism

Lipopolysaccharide (LPS) signals through Toll-like receptors (TLRs) in the course of sepsis, resulting in the release of inflammatory factors. In cell lines and murine models, parts of the signaling pathways involved have been elucidated with MyD88, Mal/TIRAP and IKK2 playing an important role in the induction of NF-kappaB. By focusing on primary human cells, we have shown that there are fundamental signaling differences between human and murine macrophages and between cells of myeloid and non-myeloid origins. In primary human cells, there are no available knockouts so we employed the use of dominant negatives to investigate the signaling cascades. We show that in primary human macrophages MyD88, Mal/TIRAP and IKK2-independent alternative pathways activate NF-kappaB and induce the expression of inflammatory cytokines, whereas in non-myeloid synovial fibroblasts MyD88 and/or Mal/TIRAP are essential adaptors for LPS signaling.     

Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines

Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-alpha. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation of AP-1/c-Jun in MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-alpha. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a role in vivo.     

Expression of Indian Hedgehog signaling molecules in breast cancer

Purpose  To investigate the clinicopathological significance and expression pattern of Hedgehog (Hh) signaling molecules in breast normal glands and invasive ductal carcinoma. Materials and methods  A total of 142 cases, including 21 of normal breast and 121 of invasive ductal carcinoma of the breast, were immunohistochemically analyzed for Ihh, Ptch, Smo, Gli-1, Gli-2, and Gli-3 protein expression. Results  All of Hh signaling molecules were greatly enhanced in invasive ductal carcinoma compared with the normal breast epithelia. The expressions of Ihh, Smo, and Gli-2 were increased in PR negative cases, and the expressions of Ihh, Ptch, and Gli-1/2/3 were statistically correlated with increased proliferating index of Ki-67 in invasive ductal carcinoma. Ihh and Gli-1/2/3 expressions were correlated with node metastasis. Additionally, the protein expressions of Ihh, Ptch, and Gli-2 were correlated with the clinical stage of breast cancer. Conclusions  Hedgehog signaling molecules play an important role in the progression of invasive ductal carcinoma of breast.     

Resveratrol induces apoptosis in thyroid cancer cell lines via a MAPK- and p53-dependent mechanism

Two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines treated with resveratrol (RV), 1-10 microM, showed activation and nuclear translocation of MAPK (extracellular signal-regulated kinase 1/2). Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by RV. Inhibition of the MAPK pathway by either H-ras antisense transfection or PD 98059, an MAPK kinase inhibitor, blocked these RV-induced effects. Addition of pifithrin-alpha, a specific inhibitor of p53, or transfection of p53 antisense oligonucleotides caused decreased RV-induced p53 and p21 expression in PTC and FTC cells. Studies of nucleosome levels estimated by ELISA and of DNA fragmentation showed that RV induced apoptosis in both papillary and follicular thyroid cancer cell lines; these effects were inhibited by pifithrin-alpha and by p53 antisense oligonucleotide transfection. PD 98059 and H-ras antisense transfection also blocked induction of apoptosis by RV. Thus, RV acts via a Ras-MAPK kinase-MAPK signal transduction pathway to increase p53 expression, serine phosphorylation of p53, and p53-dependent apoptosis in PTC and FTC cell lines.     

A study on Notch signaling in human breast cancer

Breast cancer is one of the leading causes of cancer death in women. The Notch family of proteins plays crucial roles in determining cell fates such as proliferation, differentiation and apoptosis. A role for Notch signaling in human breast cancer has been suggested by the development of adenocarcinomas in the murine mammary gland. However, it is not clear currently whether Notch signaling is frequently expressed and activated in breast cancers. Here we show that Notch signaling is overexpressed and highly activated in breast cancers. More significantly, the attenuation of Notch signaling by gamma-secretase inhibitor can inhibit the proliferation of breast cancer cells by both causing cell cycle arrest and apoptosis. Thus, targeting Notch signaling may be of therapeutic value in breast cancers.     

Enhanced insulin signaling via Shc in human breast cancer

Insulin is a mild mitogen and has been shown to potentiate mitogenic influence of other growth factors. Because hyperinsulinemia and/or overexpression of insulin receptors have been linked to development, progression, and outcome of breast cancer, we attempted to evaluate the mechanism of these associations. We have compared the expression of insulin receptors and the magnitude of insulin signaling in breast tumors and adjacent normal mammary tissue samples obtained from 20 patients. We observed that insulin binding more than doubled in the tumors as compared with the normal tissue (P <.01 by paired t test). Insulin signaling to Shc, judged by the magnitude of its phosphorylation, was also significantly enhanced in the tumors. In contrast, the phosphorylation of the insulin-receptor substrate-1 (IRS-1), Akt, and mitogen-activated protein (MAP) kinase were identical in the tumorous and normal mammary tissues. Finally, tumors displayed significantly increased amounts of farnesylated p21 Ras and geranylgeranylated Rho-A (P <.01), consistent with Shc-dependent activation of farnesyl (FTase) and geranylgeranyl transferases (GGTase) in the tumor tissue. We conclude that the mechanism of the mitogenic influence of insulin in breast cancer may include increased expression of insulin receptors, preferential hyperphosphorylation of Shc, and increased amounts of prenylated p21 Ras and Rho-A in tumor tissue as compared with adjacent normal mammary tissue.     

Differential mitogenic signaling in insulin receptor-deficient fetal pancreatic beta-cells

Insulin receptor (IR) may play an essential role in the development of beta-cell mass in the mouse pancreas. To further define the function of this signaling system in beta-cell development, we generated IR-deficient beta-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing beta-cell lines (beta-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (beta-IR(-/-)). beta-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In beta-IRLoxP beta-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S(6)K phosphorylation and beta-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S(6)K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S(6)K signaling and mitogenesis. Interestingly, in beta-IR(-/-) beta-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S(6)K phosphorylation and beta-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S(6)K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of beta-IR(-/-) cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic beta-cell lines. However, in the absence of the insulin receptor, glucose induces beta-cell mitogenesis.