Study On The Relationship Between Choline Uptake Of Prostate Cancer And Its Biological Behavior

Background and objectiveCholine is a substrate for the synthesis of phosphatidylcholine, which is a essential component of all cell membranes. As carcinogenesis is characterized by enhanced cell proliferation that may lead to increased membrane or fatty acid demands, theoretically the uptake of choline could reflect proliferative activity and then the biological behavior of malignant tumors. And11C-labeled choline (11C-choline) has been introduced as an oncologic tracer for molecular imaing with positron emission tomography (PET). However, its use for primary prostate cancer remains controversial. The results of different studies varied considerably. Some authors reported a significant overlap of choline standard uptake value (SUV) between prostate cancer and benign prostate hyperplasia (BPH) tissue. In this study, the maximal choline standard uptake value (SUVmax) of the prostate lesions and the pelvic muscles were measured and their ratios (SUVmax-P/M ratio) were calculated in the PET/CT. Then we evaluated whether tracer11C-Choline uptake, quantified as SUVmax and SUVmax-P/M ratio, correlated with tumor stage, Gleason score, and expression levels of several biomarkers of aggressiveness.Methods1.Patients Twenty-six patients with initial diagnosis of prostate cancer between April2007and March2010at our center were included in this study. Patients were excluded from this study if they had received antiandrogen therapy before PET/CT. All patients underwent ultrasound-guided transrectal biopsy of their tumor and histologically proved carcinomas. To avoid false positive accumulation of choline caused by existence of inflammatory cells, scanning was performed before histological intervention. And the results of PET/CT scanning were taken as reference for biopsy. The median age of the patients was68.1years (range,56-81years). There were10patients with stage Ⅱ,5patients with stage Ⅲ, and11patients with stage IV.14patients had a Gleason score≥7(4+3).2.11C-choline PET/CT scan11C-Choline was synthesized according to the solid-phase method in a modified commercial synthesis module. Five minutes after injection of7.4MBq/kg of11C-Choline, PET images were acquired in the supine position over2bed positions from the upper pelvis through the mid thigh. For patients with a serum prostate specific antigen (PSA)>10ng/ml, whole body PET/CT imaging was performed (6bed positions). CT and PET images were matched and fused into transaxial, sagittal, and coronal images. The11C-Choline SUVmax was determined using a circular (1cm diameter) region of interest. The SUVmax of the prostate lesions (target) and the pelvic muscles (nontarget) were measured and their ratios (P/M) were calculated.3.Immunohistochemistry For immunohistochemical staining, paraffin sections (5-μm thickness) on glass slides coated with poly-L-lysine were deparaffinized in turpentine, hydrated, and then placed in phosphate-buffered saline (PH7.6). Antigen retrieval was performed by boiling for15minutes in0.01mol/L citrate buffer (PH6.0). Using Envision immunohistochemistry, Ki-67, androgen receptor, CD31, Her-2/neu, Bcl-2and PTEN expressions were examined.4.Statistical analysis Statistical analysis on the relationship between uptake of the prostae cancer and Gleason score, tumor stage, expression levels of these biomarkers were performed using SPSS for Windows17.0software.ResultsThe mean value of SUVmax of the primary tumors in26patients was9.42±6.70(ranged from1.69to24.65), whereas the mean SUVmax-P/M ratios was4.33±1.41(ranged from1.18to7.44). Both SUVmax-P/M ratio and SUVmax showed no significant difference between patients with tumor stage Ⅱ and Ⅲ, but significantly elevated in patients with tumor stage Ⅳ. Furthermore, SUVmax-P/M was also significantly higher in lesions with Gleason score of4+3or higher versus less than or equal to3+4.The mean value of Ki-67labeling index and androgen receptor index was10.4±13.1(ranged from0to48) and86.5±61.8(ranged from0to180). The mean rate of MVD as assessed by CD31was26.4±11.8(ranged from7to48). Positive expression of Her-2/neu, Bcl-2and PTEN was recognized in6,13, and11patients, respectively. The correlation coefficients for SUVmax-P/M ratio with Ki-67and MVD were0.503and0.545, and the P values for both of these two biomarkers correlations were <0.05. SUVmax-P/M raito was higher in Her-2/neu positive subgroup than negative subgroup. In contrast, neither Gleason score nor expression levels of the biomarkers involved in the study associated with SUVmax.ConclusionThis pilot study indicate that the11C-Choline uptake in primary prostate cancer normalized by pelvic muscle does correlate with Gleason score, tumor stage and expression of several tumoral biomarkers. These findings confirm that PET/CT could be used as a non-invasive diagnostic modality that can provide information currently available only through pathological examination of tumor tissues. We might be able to pretherapeutically evaluate the biological aggressiveness of primary prostate cancer by means of11C-Choline PET/CT scan, and monitoring the SUVmax-P/M ratio would be informative for work-up and management of primary prostate cancer. Background and objectiveProstate cancer is one of the most common malignant tumor in western countries, which is the second leading cause of cancer deaths in American men. In recent years, the morbidity of prostate cancer increases obviously in China, and now it is reported the third place of malignancies of genitourinary system. However, unlike in western countries, over60%of the patients with prostate cancer in China had already developed to the advanced stage when they came to the clinics. For these patients, androgen blocking therapy would be the only choice because they had lost the opportunity of radical operation. Androgen ablation could trigger cell death and cell cycle arrest of prostate cancer cells and remains the primary choice of treatment for patients with metastatic disease. Unfortunately, it is not a curative method. Generally, recurrent tumors arise within a median of2-3years, and the tumor turns to be androgen-independent. At present, few therapeutic methods have been proven to be effective in treating castrate resistant prostate cancers, and it is considered the incurable stage of prostate cancer.Calmodulin is a small, highly conserved and the most abundant calcium-binding protein. It plays a critical role in regulateing basic cellular processes including cell proliferation, growth and movement and calmodulin levels are increased in human malignant tumor cells. Some researchers observed a structural and functional interaction of androgen receptor with calmodulin in prostate cancer cells. Furthermore, calmodulin agonist could block androgen receptor transcriptional activity in androgen-sensitive prostate cancer cell lines as effectively as antiandrogen drugs like bicalutamide in vitro. However, clinical application of current calmodulin agonists like W7and trifluoperazine is of limited use in clinical practice because of great side effects.Berbamine is a kind of bis-benzylisoquinoline alkaloids extracted from Chinese berberis plants, which has been used for the treatment of leucopenia led by cancer chemotherapy for many years. It has been proven to be a calmodulin antagonist which could inhibit the proliferation of different kinds of malignant cell lines. Our previous studies showed that berbamine could inhibit the growth of prostate cancer cell lines in vitro and induce apoptosis by down regulating the expression of surviving and up regulating the expression of bax. On the basis of our previous research, in this study we tried to elucidate the anti prostate cancer effects of berbamine in vitro and in vivo, and to explore its possible molecular mechanisms.MethodsIn vitro Human prostate cancer cell lines LNCAP (androgen-sensitive), CWR22Rvl (androgen-responsive but androgen-independent) and PC3(androgen-independent) were involved in the present study. Various working concentrations of berbamine were used. The growth of these3cell lines were examined using MTT assay and the IC50was calculated. Cell cycle distribution was analyzed on flow cytometer. Caspase-3/8/9activities were determined by a colorimetric assay kit and the caspase activities were expressed as percentage of enzyme activity compared to the control. Expression levels of Fas protein and androgen receptor were exmined using western-blotting analysis.In vivo24balb-c/nu-nu mice (4-6weeks old) were bred and maintained in a specific-pathogen-free (SPF) environment.10'CWR22RV1cells were injected subcutaneously in the mid line dorsal region of the mice. When tumor xenograft volumes reached100mm3, nude mice were randomly assigned to treated group and control group. In the treated group, berbamine was injected intraperitoneally at a dose of60mg per kg a day for4weeks, while equal volumes of isotonic saline was administered in the control group. Tumor volumes were calculated weekly using caliper and their volumes calculated. A growth-inhibition effect of berbamine was generally observed, as shown by the in vivo growth curve. At the conclusion of the study, mice were euthanized and the tumors were harvested and embedded in paraffin. Apoptosis was evaluated by using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the apoptosis index (AI) was calculated. Expression levels of PCNA, a biomarker of proliferation, were examined by immnunohistochemistry.ResultsBerbamine demonstrated a dose-dependent growth inhibition in all of the3cell lines. LNCAP was the most sensitive to berbamine, whereas PC3was the least sensitive, with the48hours'IC50values ranging from15.06ug/ml (LNCAP) to37.04μg/ml (PC3). To further probe its effects on cell growth, cell cycle analysis revealed that berbamine significantly impeded G1to S phase progression in LNCAP cells, while S to G2phase progression in CWR22RV1cells and PC3cells. A stronger increase in Caspase-8activities was observed in LNCAP cells treated by berbamine than in PC3cells. Mechanistically, berbamine treatment led to a increased expression level of Fas protein in LNCAP cells while no significant increase in PC3cell line, which might result in a activation of Caspase-8. Western-blotting analysis also showed that40μg/ml berbamine treatment for48hours could reduce the expression level of androgen receptor in LNCAP cells.To investigate whether the effects of berbamine corresponded to anti-tumor effects in vivo, we examined the effect of berbamine on CWR22RV1tumor growth in a human tumor xenograft model. We found that a daily regimen of berbamine injection significantly suppressed the growth of CWR22RV1tumors. The inhibitory rate of berbamine administration was45.1%. The average apoptosis index determined by TUNEL was21.2%in the treated group and4.7%in the control group. Average PCNA expression levels was40.9%in the treated group and78.8%in the control groupConclusionBerbamine could inhibit the cell proliferation in vitro in three kinds of prostate cancer cell lines, including LNCAP, CWR22RV1and PC3. In androgen independent prostate cancer cells, berbamine significantly impeded S to G2phase progression in cell cycle and induced apoptosis through endogenous pathway. In contrast, it could impeded G1to S phase in androgen sensitive cells and induce apoptosis through both endogenous and exogenous pathways. This might result from the up regulation of expression of Fas protein and its effects on androgen receptor in the androgen sensitive prostate cancer cells. In vivo, berbamine also displayed a anti prostate cancer effect through inducing apoptosis and inhibiting proliferation of CWR22RV1xenograft model. It could be concluded that berbamine might be a promising candidate of new drug for the treatment of prostate cancer.