Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells migration through casein kinase 2α intronless gene and neutral endopeptidase

Background: We have previously reported that a new intronless gene for casein kinase 2α (CK2α), CSNK2A3, is expressed in human cells. The promoter of the well-known CK2α, CSNK2A1, displays characteristics of a housekeeping gene, whereas CSNK2A3 has a characteristic of a regulated promoter with two TATA boxes and a CAAT box. GPR68, a family of the G protein-coupled receptors, is also known as ovarian cancer G protein-coupled receptor 1 (OGR1). In the current study, we analyzed the roles of CK2α genes and neutral endopeptidase (NEP), a key enzyme that influences a variety of malignancies, in the OGR1-induced inhibition of A549 cell migration. Methods: We analyzed the transcript expressions of both the CK2α genes (CSNK2A1 and CSNK2A3) and NEP upon OGR1 overexpression. Protein expression of CK2α and NEP were also analyzed. We further elucidated the functional roles of both CK2α and NEP in the OGR1-induced inhibition of A549 cell migration in vitro using a wound-healing assay. We also analyzed the molecular mechanisms involved in the OGR1-induced inhibition of lung cancer cell migration. Results: The findings of this study showed that OGR1 upregulated the expression of CSNK2A3 but not CSNK2A1 in the A549 cells. The findings further suggested OGR1 also upregulates the expression of NEP. The OGR1-induced inhibition of A549 cell migration was abrogated completely by inhibition of CK2α activity, whereas partial abrogation (~ 30%) was observed in the presence of NEP inhibition. The results also revealed that OGR1 regulates CSNK2A3 via activation of Rac1/cdc42 and MAPKs pathways. CK2 is ubiquitously expressed, and in contrast, is believed to be a constitutively active enzyme, and its regulation appears to be independent of known second messengers. Conclusion: In the current study, we report for the first time the OGR1-induced regulation of CSNK2A3, CK2αP, and NEP in A549 cancer cells. Our study also decoded the downstream cellular proteins of OGR1 as well as the molecular mechanism involved in OGR1-induced inhibition of A549 cell migration. The findings of this research suggest the potential therapeutic targets to inhibit lung cancer progression

Abnormalities in Osteoclastogenesis and Decreased Tumorigenesis in Mice Deficient for Ovarian Cancer G Protein-Coupled Receptor 1

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases

Anticancer properties and enhancement of therapeutic potential of cisplatin by leaf extract of Zanthoxylum armatum DC

BACKGROUND: Clinical use of chemotherapeutic drug, cisplatin is limited by its toxicity and drug resistance. Therefore, efforts continue for the discovery of novel combination therapies with cisplatin, to increase efficacy and reduce its toxicity. Here, we screened 16 medicinal plant extracts from Northeast part of India and found that leaf extract of Zanthoxylum armatum DC. (ZALE) induced cytotoxicity as well as an effect on the increasing of the efficiency of chemotherapeutic drugs (cisplatin, mitomycin C and camptothecin). This work shows detail molecular mechanism of anti-cancer activity of ZALE and its potential for combined treatment regimens to enhance the apoptotic response of chemotherapeutic drugs. RESULTS: ZALE induced cytotoxicity, nuclear blebbing and DNA fragmentation in HeLA cells suggesting apoptosis induction in human cervical cell line. However, the apoptosis induced was independent of caspase 3 activation and poly ADP ribose polymerase (PARP) cleavage. Further, ZALE activated Mitogen-activated protein kinases (MAPK) pathway as revealed by increased phosphorylation of extracellular-signal-regulated kinases (ERK), p38 and c-Jun N-ter-minal kinase (JNK). Inhibition of ERK activation but not p38 or JNK completely blocked the ZALE induced apoptosis suggesting an ERK dependent apoptosis. Moreover, ZALE generated DNA double strand breaks as suggested by the induction γH2AX foci formation. Interestingly, pretreatment of certain cancer cell lines with ZALE, sensitized the cancer cells to cisplatin and other chemotherapeutic drugs. Enhanced caspase activation was observed in the synergistic interaction among chemotherapeutic drugs and ZALE. CONCLUSION: Purification and identification of the bio-active molecules from the ZALE or as a complementary treatment for a sequential treatment of ZALE with chemotherapeutic drugs might be a new challenger to open a new therapeutic window for the novel anti-cancer treatment

Bioassay-Guided Isolation of 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol from Oroxylum indicum and the Investigation of Its Molecular Mechanism Action of Apoptosis Induction

The leaf crude extract of Oroxylum indicum (L.) Kurz induces genomic DNA fragmentation, comet formation, and the inhibition of cell proliferation in the prostate cancer cell line PC3, as assessed by agarose gel electrophoresis, comet assay and MTT assay, respectively. The bioactive compound was purified through bioassay-guided fractionation using preparative HPLC and MTT assay. The light brown and water-soluble compound was characterized using 1H and 13C nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR), and electrospray ionization (ESI) mass spectrometry. The compound was identified as a glycosylated hydroquinone derivative, 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl) tetrahy-dro-2H-pyran-3,4,5-triol (molecular formula, C13H18N2O8; molecular mass = 330). The identified phytocompound has not been reported earlier elsewhere. Therefore, the common name of the novel anticancer phytocompound isolated from Oroxylum indicum in this current study is oroxyquinone. The half-maximal inhibitory concentration (IC50) of oroxyquinone on PC3 cells was 58.9 µM (95% CI = 54.5 to 63.7 µM). Treatment of PC3 cells with oroxyquinone induced genomic DNA fragmentation and chromatin condensation, increased in the annexin-V positive cells, arrested the cell cycle at S phases, and inhibited the cell migration; as assessed by comet assay, DAPI staining, flow cytometry and a wound healing assay, respectively. On the investigation of the molecular mechanism of the induction of apoptosis, the results indicated that oroxyquinone induced caspase-3 and PARP independent apoptosis but through the p38 pathway and the localization of AIF into the nucleus. The present study identifies a novel anticancer molecule and provides scientific evidence supporting the therapeutic potency of Oroxylum indicum for ethnomedicinal uses

Planispine A Sensitized Cancer Cells to Cisplatin by Inhibiting the Fanconi Anemia Pathway

The use of cisplatin as a chemotherapeutic drug is impeded by the development of drug resistance. Combination therapies of a chemosensitizer for cisplatin have been studied, but with little success, and the search for an effective combination therapy is continuing. Our earlier reports have shown that Zanthoxylum armatum DC. extract enhances the apoptotic effect of cisplatin in cancer cell lines. In this study, we purified and identified the bioactive phytocompound through bio-assay-guided purification, using column chromatography and HPLC. Chemical characterization using NMR and mass spectrometry revealed the compound as planispine A, with molecular structure C25H30O6 and molecular weight, 426.16 g/mol. Planispine A was found to inhibit cancer cell proliferation in a dose-dependent manner and to sensitize the cancer cells to cisplatin-augmented apoptotic cell death, in a caspase-dependent manner. A combination of planispine A and cisplatin induced S-phase cell cycle arrest, and reduced the expression of survival proteins such as cyclin D1. Interestingly, planispine A inhibits the Fanconi anemia pathway, as shown by reduced FANCD2 foci formation and FANCD2 monoubiquitination, which revealed the molecular mechanism of chemo-sensitization of cancer cells to cisplatin. Evaluation of this combination therapy in cisplatin-resistant tumors may lead to more efficient cisplatin treatment

Ovarian Cancer G Protein–Coupled Receptor 1, a New Metastasis Suppressor Gene in Prostate Cancer

Background Metastasis is a process by which tumors spread from primary organs to other sites in the body and is the major cause of death for cancer patients. The ovarian cancer G protein–coupled receptor 1 (OGR1) gene has been shown to be expressed at lower levels in metastatic compared with primary prostate cancer tissues. Methods We used an orthotopic mouse metastasis model, in which we injected PC3 metastatic human prostate cancer cells stably transfected with empty vector (vector-PC3) or OGR1-expressing vector (OGR1-PC3) into the prostate lobes of athymic or NOD/SCID mice (n = 3–8 mice per group). Migration of PC3 cells transiently transfected with vector control or with OGR1- or GPR4 (a G protein–coupled receptor with the highest homology to OGR1)-expressing vectors was measured in vitro by Boyden chamber assays. G protein alpha–inhibitory subunit 1 (Gα i1 ) expression after treatment with pertussis toxin (PTX) was measured using immunoblotting analysis. The inhibitory factor present in the conditioned medium was extracted using organic solvents and analyzed by mass spectrometry. Results In vivo, all 26 mice carrying tumors that were derived from vector-PC3 cells developed prostate cancer metastases (mean = 100%, 95% confidence interval [CI] = 83.97% to 100%) but few (4 of 32) mice carrying tumors derived from OGR1-expressing PC3 cells (mean = 12.50%, 95% CI = 4.08% to 29.93%) developed metastases. However, exogenous OGR1 overexpression had no effect on primary prostate tumor growth in vivo. In vitro, expression of OGR1, but not GPR4, inhibited cell migration (mean percentage of cells migrated, 30.2% versus 100%, difference = 69.8%, 95% CI = 63.0% to 75.9%; Pi1 and the secretion of a chloroform/methanol–extractable heat-insensitive factor into the conditioned medium through a PTX-sensitive pathway. Conclusion OGR1 is a novel metastasis suppressor gene for prostate cancer. OGR1\u27s constitutive activity via Gα i contributes to its inhibitory effect on cell migration in vitro