Folate Receptor Targeted Diagnosis And Therapy Of Nonfunctional Pituitary Adenoma And Its Involved Mechanisms

Pituitary adenomas (PAs) are the second most common intracranial neoplasms, accounting for approximately20%of primary brain tumors, with an increaing morbidity. Autopsy studies demonstrate a higher incidence of PAs at25%in population. PAs are associated with significant morbidity and mortality due to hormone hypersecretion and mass effects. Nonfunctional pituitary adenomas (NFPAs) account for approximately30%of PAs and are the most common type. Because of their hormonal inactivity, NFPAs are often difficult to diagnose until they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects, or headaches. The first-line treatment is surgery, but more than40%of NFPAs are locally invasive, and infiltrate the dura, bones, and sinuses. Consequently, total resection may not be possible which leads to a high recurrence rate with a poor clinical outcome. Although postoperative radiotherapy may be effective in preventing tumor regrowth, it is associated with severe complications and has been frequently withheld by some clinicians in the past decade. Furthermore, present chemotherapy for treating NFPAs is mainly based on dopamine agonists (DA) and somatostatin (SRIF) analogs. Unfortunately, these strategies all harbor clinical limitations. As a result, there is an urgent need to determine new diagnositic and therapeutic biomarkers and construct novel antitumor agents to combat these refractory NFPAs.Recent research into folate receptors (FRs) has opened up new perspectives on the targeted imaging, diagnosis and therapy of NFPAs. Membrane FRs, including FRa and FRβ,are glycosylphosphatidylinositol (GPI)-anchored glycoproteins that recognize and internalize folate via endocytosis. FRa expression is amplified in over90%of ovarian carcinomas and at varying frequencies in other epithelial cancers. Meanwhile, FRβ is expressed in a non-functional form in neutrophils, and in a functional form in activated macrophages and myeloid leukemias. In contrast, most normal tissues do not express either FR isoform. Folic acid (folate) is a high affinity ligand for the FRs and retains high FR affinity after derivatization via one of its carboxyl groups. Because of its small size and bioavailability, folate has become one of the most investigated targeting ligands for tumor-specific drug delivery. Since targeted delivery via selective cellular markers can potentially increase the efficacy and reduce the toxicity of therapeutic agents, a variety of molecules and drug carriers, including chemotherapeutic agents, imaging agents, oligonucleotides, proteins, haptens, gene transfer vectors, nanoparticles and liposomes, have been conjugated to folate and evaluated for FRα-targeted diagnosis and therapy. These FRα-targeted agents have shown promising efficacy in preclinical models and have significant potential for future clinical application in a wide range of cancers, such as ovarian carcinomas and non-small cell lung cancer.Previous studies have demonstrated that FRα was uniquely overexpressed in NFPAs but not in functional pituitary adenomas (FPAs) or normal pituitary glands. However, whether the expression of FRα in NFPAs was positively correlated with tumor invasiveness and proliferation, whether FRα plays a important role in the tumorigenesis and progression of NFPA and whether FRα could be used as the molecular target for targeted diagnosis and therapy are unclear. In this study, we evaluated differential expression of folate receptor alpha in pituitary adenomas and its relationship to tumor behavior of NFPA. Then we assessed the targeting ability and cytotoxic efficacy of a novel lipophilic folate derivative containing liposomes loaded with doxorubicin (F-L-DOX); We further evaluated the mechanisms involved in the anti-tumor effect of F-L-DOX on the treatment of NFPAs. At last, we studied the effect and involved mechanisms of F-L-DOX on human cervical cancer xenografts in nude mice.Section One Differential Expression of Folate Receptor Alpha in Pituitary Adenomas and Its Relationship to Tumor BehaviorObjective:To identify a possible biomarker for the diagnosis of nonfunctional pituitary adenomas (NFPAs) that could also be used to assess tumor behavior.Methods:Sporadic pituitary tumor specimens (n=76) and normal pituitary glands (n=7) were examined. FRa protein and mRNA expression were quantified by immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction, respectively. We verified the differential expression of FRa in pituitary adenomas and evaluated the associations of FRa expression with Ki-67labeling index (LI) and clinicopathologic characteristics of NFPAs. Statistical significance was determined by using the Student t test or one-way analysis of variance.Results:FRa mRNA and protein was uniquely overexpressed in NFPAs but not in functional adenomas (adrenocorticotropic hormone, growth hormone, and prolactin) or normal adenohypophysial tissues (P<.001). The expression of FRa was positively correlated with tumor invasiveness, size and Ki-67LI in NFPAs.Conclusion:FRa may play an important role in the development and progression of NFPAs. Therefore, FRa may be useful as a molecular biomarker for the diagnosis of NFPAs and assessment of tumor invasiveness. Section two Effects of a novel lipophilic folate derivative containing liposomes loaded with doxorubicin on cell viability in nonfunctional pituitary adenomasObjective:To determine whether the novel lipophilic folate derivative containing liposomes loaded with doxorubicin (F-L-DOX) could be a useful therapy for nonfunctional pituitary adenomas(NFPAs).Methods:the targeting ability, cytotoxic effect and the anti-invasive ability of F-L-DOX which targeted the overexpressed FRa on the cell membrane of primary human NFPA cells were evaluated in25cases of pituitary adenomas.Results:FRa-targeted liposomes could effectively target the NFPA cells through the FRa and the endocytosis was blocked by1mM free folic acid. F-L-DOX more effectively inhibited tumor growth and promoted the apoptosis of NFPA cells through activating caspase-8, caspase-9and caspase-3/7, compared to non-targeted liposomal doxorubicin (L-DOX). Furthermore, F-L-DOX also exerted greater anti-invasive ability by suppressing the secretion of MMP-2and MMP-9from NFPA cells compared to L-DOX. In contrast, when1mM free folic acid was added, the pleotropic effects of F-L-DOX on NFPA cells were significantly blunted.Conclusion:Our findings suggest that FRa may play a critical role in mediating the antitumor effect of F-L-DOX which could be an excellent alternative therapeutic strategy to treat NFPAs. Section Three Effect of F-L-DOX on human cervical cancer xenografts in nude miceObjective:To study the effect of F-L-DOX on human cervical cancer xenografts in nude mice.Methods:Human cervical cancer cell line Hela was implanted into36nude mice. Different kinds of Doxorubicin formulations were injected around the tumor bearing nude mice divided into4groups. The tumor growth, morphological changes of tumor tissues, apoptosis rate of tumor cells were determined and compared.Results:The growth speed of tumor in the group of F-L-DOX significantaly slowed down than other groups. The rate of tumor restrain in tumor weight and tumor apoptosis of F-L-DOX group were72.33%and71.80%, which were remarkably higher than those of the DOX (53.40%,51.40%) and L-DOX group(42.72%,42.30%)(all P<0.05). The body weight of nude mouse in DOX group failed to increase, while that in F-L-DOX group increased steadly.Conclusion:F-L-DOX as a new FRa targeted anti-tumor drug may have a favorable clinical prospect.