GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (?Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

PIWIL4在人卵巢癌中的表达及其功能研究

采用半定量RT-PCR方法检测人卵巢透明细胞癌ES-2细胞中PIWIL1、PIWIL2、PIWIL3、PIWIL4 mRNA表达水平,以及PIWIL4在卵巢癌组织和癌旁正常组织中的表达情况．设计并化学合成针对PIWIL4的siRNA,用脂质体转染法将其转入ES-2细胞内,通过MTT和克隆形成实验,观察PIWIL4-siRNA对ES-2细胞生长活性和增殖能力的影响．半定量RT-PCR实验结果发现,ES-2细胞中,PIWIL4相对PIWIL1、PIWIL2、PIWIL3,其表达水平最高(P < 0.05),而且PIWIL4在卵巢癌组织中的表达明显高于癌旁正常组织(P < 0.01)．MTT实验和克隆形成实验结果显示,转染PIWIL4-siRNA的ES-2细胞生长活性和克隆形成率明显低于对照组(P < 0.05)．由此得出结论：PIWIL4在卵巢癌细胞系ES-2细胞表达较高,且它在卵巢癌组织中的表达明显上调,同时PIWIL4-siRNA可有效抑制ES-2细胞的生长活性和增殖能力,提示PIWIL4可能与卵巢癌发生、发展相关．     

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.     

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.     

Antagonistic analogs of growth hormone releasing hormone (GHRH) inhibit cyclic AMP production of human cancer cell lines in vitro.

Antagonistic analogs of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers both in vivo and in vitro. GHRH, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide stimulate cyclic AMP (cAMP) release from various human cancer cell lines in vitro. Thus, in the present study, we investigated the effects of antagonistic analogs of GHRH on the GHRH- and VIP-induced cAMP release from cultured human cancer cells in a superfusion system. Various human cancer cell lines were exposed to human GHRH(1-29)NH2 (2-20 nM) or VIP (0.1-5 nM) repeatedly for 12 min or continuously for 96 min. GHRH antagonist MZ-5-156 at 100 to 200 nM concentration inhibited the GHRH- or VIP-induced cAMP release from mammary (MDA-MB-468), prostatic (PC-3), and pancreatic (SW-1990 and CAPAN-2) cancer cells. These results show that antagonistic analogs of GHRH suppress the stimulatory effects of GHRH and VIP on the cAMP production of various cancer cells. Because cAMP is a potent second messenger controlling many intracellular functions, including the stimulation of cell growth, an inhibition of autocrine/paracrine action of GHRH by the GHRH antagonists may provide the basis for the development of new methods for cancer treatment.     

Potentiating effects of GHRH analogs on the response to chemotherapy

Growth hormone releasing hormone (GHRH) from hypothalamus nominatively stimulates growth hormone release from adenohypophysis. GHRH is also produced by cancers, acting as an autocrine/paracrine growth factor. This growth factor function is seen in lymphoma, melanoma, colorectal, liver, lung, breast, prostate, kidney, bladder cancers. Pituitary type GHRH receptors and their splice variants are also expressed in these malignancies. Synthetic antagonists of the GHRH receptor inhibit proliferation of cancers. Besides direct inhibitory effects on tumors, GHRH antagonists also enhance cytotoxic chemotherapy. GHRH antagonists potentiate docetaxel effects on growth of H460 non-small cell lung cancer (NSCLC) and MX-1 breast cancer plus suppressive action of doxorubicin on MX-1 and HCC1806 breast cancer. We investigated mechanisms of antagonists on tumor growth, inflammatory signaling, doxorubicin response, expression of drug resistance genes, and efflux pump function. Triple negative breast cancer cell xenografted into nude mice were treated with GHRH antagonist, doxorubicin, or their combination. The combination reduced tumor growth, inflammatory gene expression, drug-resistance gene expression, cancer stem-cell marker expression, and efflux-pump function. Thus, antagonists increased the efficacy of doxorubicin in HCC1806 and MX-1 tumors. Growth inhibition of H460 NSCLC by GHRH antagonists induced marked downregulation in expression of prosurvival proteins K-Ras, COX-2, and pAKT. In HT-29, HCT-116 and HCT-15 colorectal cancer lines, GHRH antagonist treatment caused cellular arrest in S-phase of cell cycle, potentiated inhibition of in vitro proliferation and in vivo growth produced by S-phase specific cytotoxic agents, 5-FU, irinotecan and cisplatin. This enhancement of cytotoxic therapy by GHRH antagonists should have clinical applications.     

High expression of LASS2 is associated with unfavorable prognosis in patients with ovarian cancer

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), is a gene isolated from a human liver complementary DNA library. In this study, we found that LASS2 protein level was positively related to International Federation of Gynecology and Obstetrics (FIGO) stage and LASS2-negative tumors showed significant association with longer disease-free survival (DFS) and overall survival (OS) in ovarian cancer patients. The heterogeneous expression of LASS2 had been exhibited in diverse ovarian cancer cells. A significantly lower messenger RNA (mRNA) and protein level of LASS2 was seen in 3AO cell compared with those in other types of ovarian cancer cells. Meanwhile, the mRNA and protein levels of LASS2 in ES-2 and NIH:OVCAR-3 cells were obviously higher. LASS2 overexpression in 3AO cell could promote migration, invasion, and metastasis abilities in vitro and in vivo, while LASS2 knockdown in ES-2 and NIH:OVCAR-3 cells had the opposite effects. The oncogenic capacity of LASS2 in ovarian cancer may be mediated by increased expression of YAP/TAZ. It is indicated that lowering the expression of LASS2 is likely to serve as an unprecedented approach for the treatment of ovarian cancer.     

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.     

TRPC3对上皮性卵巢癌细胞株迁移和侵袭的影响

目的:探讨瞬时受体电位通道C3(TRPC3)对人卵巢癌细胞迁移、侵袭能力的影响。方法:采用蛋白免疫印迹法和实时荧光定量PCR法分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30中TRPC3蛋白和m RNA的表达水平。通过Transwell迁移实验(不含Matrigel胶的Transwell小室)和Transwell侵袭实验分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30的迁移、侵袭能力。结果:在SKOV3、ES-2和HEY-T30三种卵巢癌细胞株中,ES-2中TRPC3的蛋白和m RNA表达均显著高于其他两株(P0.05)。Transwell迁移实验和Transwell侵袭实验显示卵巢癌细胞株ES-2的迁移、侵袭能力均显著高于其他两种细胞株(P0.05)。结论:瞬时受体电位通道C3(TRPC3)在ES-2人卵巢癌细胞中高表达,并可能促进人卵巢癌细胞的迁移、侵袭。     

Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells

Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

GHRH antagonist MZ-5-156 increases the expression of AMPK in A549 lung cancer cells

AMP-activated protein kinase (AMPK) regulates cellular proliferation, growth and metabolism. Targeted activation of AMPK is considered an important therapeutic strategy for cancer treatment. To evaluate the effect of growth hormone-releasing hormone (GHRH) and its antagonist MZ-5-156 on the phosphorylation of AMPK and other related regulatory intracellular proteins we employed human non-small cell lung cancer cell line A549, which expresses GHRH receptors. Treatment of A549 cells with GHRH antagonist decreased cell proliferation and activated AMPK as well as glycogen synthase kinase (GSK)3β. Furthermore, MZ-5-156 inhibited Akt, the mammalian target of rapamycin (mTOR) and its downstream target eIF4E which controls protein synthesis and cell growth. GHRH(1-29)NH2 counteracted all these effects. HeLa human endometrial cancer cells which do not express any GHRH receptors were used as a negative control and GHRH did not induce the AMPK activation in these cells. Our results demonstrate for the first time that GHRH antagonists can regulate the AMPK metabolic pathway, which is crucial for the growth of non-small cell lung cancer and other major cancers.     

Antiproliferative actions of growth hormone-releasing hormone antagonists on MiaPaCa-2 human pancreatic cancer cells involve cAMP independent pathways

We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10-30 microM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [(125)I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.     

Experimental therapy of ovarian cancer with synthetic makaluvamine analog: in vitro and in vivo anticancer activity and molecular mechanisms of action

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer.