NMDA receptors are important regulators of pancreatic cancer and are potential targets for treatment.

Pancreatic cancer, particularly adenocarcinoma of the pancreas, is a common disease with a poor prognosis. In this study, the importance of N-methyl-D-aspartate (NMDA) receptors for the growth and survival of pancreatic cancer was investigated. Immunohistochemistry performed with antibodies against GluN1 and GluN2B revealed that all invasive adenocarcinoma and neuroendocrine pancreatic tumors likely express these two NMDA receptor proteins. These proteins were found to be membrane components of pancreatic cancer cell lines, and both channel-blocker antagonist and GluN2B antagonist significantly reduced cell viability in vitro. Both types of antagonists caused an internalization of the receptors. Dizocilpine maleate (MK-801) and ifenprodil hemitartrate both significantly inhibited the growth of pancreatic tumor xenografts in nu/nu mice. These findings predict that, as for other solid tumors investigated by us, pancreatic cancer could be successfully treated, alone or in combination, with NMDA receptor antagonists or other receptor-inhibiting blocking agents.

NMDA receptors are expressed in human ovarian cancer tissues and human ovarian cancer cell lines.

We have earlier demonstrated that breast cancer and small-cell lung cancer express functional NMDA receptors that can be targeted to promote cancer cell death. Human ovarian cancer tissues and human ovarian cancer cell lines (SKOV3, A2008, and A2780) have now been shown to also express NMDA-receptor subunit 1 (GluN1) and subunit 2B (GluN2B). Seventeen ovarian cancers in two arrays were screened by immunohistochemistry using polyclonal antibodies that recognize an extracellular moiety on GluN1 and on GluN2B. These specimens comprised malignant tissue with pathology diagnoses of serous papillary cystadenocarcinoma, endometrioid adenocarcinoma, and clear-cell carcinoma. Additionally, archival tissues defined as ovarian adenocarcinoma from ten patients treated at this institute were also evaluated. All of the cancerous tissues demonstrated positive staining patterns with the NMDA-receptor antibodies, while no staining was found for tumor-adjacent normal tissues or sections of normal ovarian tissue. Human ovarian adenocarcinoma cell lines (A2008, A2780, SKOV3) were demonstrated to express GluN1 by Western blotting, but displayed different levels of expression. Through immunocytochemistry utilizing GluN1 antibodies and imaging using a confocal microscope, we were able to demonstrate that GluN1 protein is expressed on the surface of these cells. In addition to these findings, GluN2B protein was demonstrated to be expressed using polyclonal antibodies against this protein. Treatment of all ovarian cell lines with antibodies against GluN1 was found to result in decreased cell viability (P<0.001), with decreases to 10%-25% that of untreated cells. Treatment of control HEK293 cells with various dilutions of GluN1 antibodies had no effect on cell viability. The GluN1 antagonist MK-801 (dizocilpine maleate) and the GluN2B antagonist ifenprodil, like antibodies, dramatically decreased the viability of A2780 ovarian tumor cells (P<0.01). Treatment of A2780 tumor xenografts with ifenprodil (2.5 mg/kg body weight/day) significantly reduced tumor growth in nu/nu mice. Our findings suggest that both GluN1 and GluN2B proteins as membrane components could be readily available targets for the treatment of most ovarian cancers.

Growth Impairment of Small-Cell Cancer by Targeting Pro-Vasopressin with MAG-1 Antibody.

Previously we demonstrated that human small-cell lung cancer (SCLC) seems to universally express the vasopressin gene, and this leads to the presence of a cell surface marker representing the entire pro-hormone precursor. In this study, we show this marker can be targeted with MAG-1, a mouse monoclonal antibody against a C-terminal moiety on pro-vasopressin. In vitro targeting of cell lines derived from primary and recurrent disease demonstrates attachment of antibody to the cell surface followed by internalization. In vivo targeting with (99)Tc-labeled Fab fragments of MAG-1 shows selective attachment to xenografts. In vivo treatment of tumors from classical cell line, NCI H345, with either ~1.65 mCi (~1.65 mg)/kg body weight (BW) of (90)Yttrium-labeled MAG-1, or ~1.65 mg/kg BW native MAG-1, delivered every second day for 6 days produced similar reductions in the growth rate to ~50% (p < 0.03). When dosing with native MAG-1 was escalated to daily amounts of ~3.3 mg/kg BW over 16 days, tumor growth rates fell to ~33% of saline controls (p < 0.005). Examination of tumors treated with this higher dosing demonstrated the presence in several of extensive apoptosis. Normal tissues seemed to be unaffected. A larger dosage of MAG-1 (~6.6 mg/kg BW) given daily for 14 days was used to treat xenografts of the variant cell line NCI H82 representing recurrent disease. This treatment decreased the rate of increase in tumor size by half, and doubling time ~3-fold. Increases in cleaved PARP supported increased apoptosis with antibody treatment. We believe these data provide evidence that the growth rate of SCLC tumors can be extensively reduced by treatment with MAG-1 antibody, and that a humanized form of this antibody could, in future, be potentially used for targeting therapy onto recurrent SCLC in patients.

Provasopressin expression by breast cancer cells: implications for growth and novel treatment strategies.

The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer.