Anti-inflammatory bicyclic polyprenylated acylphloroglucinols with diverse architectures including an unprecedented 6/6/6 tricyclic core from Garcinia yunnanensis.

Garciyunnanol A (1), an unprecedented 1,2-seco-bicyclic polyprenylated acylphloroglucinol (BPAP) possessing a unique 6/6/6 tricyclic core, was characterized from Garcinia yunnanensis together with 16 BPAPs, including eight new compounds (garciyunnanols B-I, 2-9). Biogenetically, the bicyclo[3.3.1]nonane-2,4,9-trione moiety of 12 reconstructed the bicyclic δ-lactone core of 2 through Norrish type Ⅰ cleavage and cyclization, followed by a cyclization of two side chains to form an intriguing 6/6/6 tricyclic core of 1. Their structures were elucidated through analysis of spectroscopic data, calculation and comparison of ECD spectra. Bioactivity evaluation manifested that compounds 1, 2, 5, 6 and 14 demonstrated superior inhibition of NO production compared to the positive control dexamethasone. Notably, compound 5 exhibited a dose-dependent inhibitory effect on NO production, with an IC50 value of 0.25 ± 0.87 µM. Furthermore, experiments involving ELISA, Western blotting, and immunofluorescence staining revealed that 5 effectively reduced the secretion of interleukin-1ß in LPS plus nigericin-stimulated THP-1 macrophages by inhibiting the activation of the NLRP3 inflammasome.

Analysis of the expression and prognostic significance of DDK complex in Hepatocarcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide. Although DBF4-dependent kinase (DDK) complex composed of CDC7 kinase and its regulatory subunit DBF4 has been shown to be overexpressed in primary tumors and promotes tumor development, while its role and prognostic value in HCC remain largely unknown. In the present study, the expression of DBF4 and CDC7 and their relationship with clinical characteristics were comprehensively analyzed. METHODS: The mRNA expression profiles of HCC and the corresponding clinical data of HCC patients were downloaded from TCGA and GEO databases, respectively. The differences in DBF4 and CDC7 expression in tumor tissues and adjacent normal tissues were analyzed. HCC-derived tissue microarray (TMA) was used to evaluate and score the expression of CDC7 by immunohistochemistry (IHC) staining. The Kaplan-Meier method and the Cox regression method were used to analyze the relationship between overall survival and clinical characteristics of the patients. Gene set enrichment analysis (GSEA) was used to analyze the pathway enrichment of DBF4 and CDC7. RESULTS: DBF4 and CDC7 had similar expression patterns in HCC patients. Detailly, compared with adjacent tissues, both mRNA and protein of DBF4 and CDC7 were significantly higher in HCC, and their expression was positively correlated with AJCC_T stage, clinical stage and G stage (grade) of liver cancer patients, and higher DBF4 or CDC7 expression predicted a worse prognosis in HCC patients with shorter overall survival (OS), recurrence-free survival (RFS), disease-specific survival (DSS) and progress-free survival (PFS). Cox regression analysis suggested that both DBF4 and CDC7 were independent risk factors for the prognosis of HCC patients in TCGA dataset. GSEA suggested that both DBF4 and CDC7 were positively correlated with cell cycle and DNA replication. Finally, the prognostic value of CDC7 was furtherly confirmed by TMA-based IHC staining results. CONCLUSIONS: Our study showed that DDK complex was significantly increased in HCC. Both DBF4 and CDC7 may be potential diagnostic and prognostic markers for HCC, and high expression of DDK members predicts a worse prognosis in patients with HCC, which may be associated with high tumor cell proliferation rate.

Modification effects of T2DM-susceptible SNPs on the reduction of blood glucose in response to lifestyle interventions.

In recent years, it has been demonstrated that some susceptible gene loci of type 2 diabetes mellitus (T2DM) are not only associated with the susceptibility risk of T2DM, but also the modifying effects of lifestyle interventions. To further explore the modifying effects of the single nucleotide polymorphism (SNP) on the onset of T2DM and the reduction of blood glucose in response to lifestyle interventions among the high-risk population, we performed a lifestyle intervention study in two Deqing rural communities during the period from June to December in 2017. The intensive lifestyle interventions were conducted among the study subjects of the intervention group while those in the control group only received conventional and general health education. All participants were genotyped by the MassARRY system. This study showed that for SNP rs9502570, fasting blood glucose showed a significantly greater reduction for individuals with CC + CT genotype than those with TT genotype (P=0.031). In the intervention group, the glycated hemoglobin A1C (HbA1C) decreased by 0.03% for those with CC+CT genotype, while HbA1C increased by 0.27% for those with TT genotype (P=0.012). The difference in the reduction of fasting blood glucose and HbA1c between the intervention and control groups was also statistically significant between individuals with TT and those with CC+CT genotype. For SNP rs10811661, the reduction of fasting blood glucose was significantly higher in people with TT genotype than those with CC + CT genotype (0.44 mmol/L vs 0.12 mmol/L, P=0.021). The difference in reduction of fasting blood glucose between the intervention group and control group was also statistically significant between TT and CC+CT genotype (P<0.001). In summary, the SNP genotypes of both rs9502570 and rs10811661 could modify the effects of lifestyle interventions on reducing fasting blood glucose and HbA1C among the high risk rural population for T2DM. The present study has provided supporting evidence for future development of individualized intervention measures for high-risk population of T2DM.

Exonuclease 1 expression is associated with clinical progression, metastasis, and survival prognosis of prostate cancer.

Prostate cancer (PCa) is the most prevalent malignancy and the second leading cause of cancer-related deaths in the male population in western countries, and we explored the association between exonuclease 1 (EXO1) expression and clinical progression, metastasis (Met), and survival prognosis of PCa. EXO1 expression of high/low-metastatic patient-derived xenografts model was investigated and clinical correlation and prognosis outcomes were validated. EXO1 in high-metastatic models was significantly increased compared with low-metastatic lines. In memorial sloan-kettering cancer center (MSKCC) cohort, EXO1 expression positively correlated with PCa Met, and patients with high EXO1 had poor biochemical recurrence-free survival in primary PCa cohort. Validation in The Cancer Genome Atlas primary cohort indicated EXO1 expression was significantly associated with lymph node Met and disease-free survival. The overexpression of EXO1 is significantly associated with PCa poor survival outcome, and is a promising biomarker for PCa, especially for primary PCa. A prospective study is clearly needed to validate these findings.

Association Between Sleep Duration and Stroke in Different Status of Metabolic Syndrome: A Cross-Sectional Study in Shanghai Adult Residents.

Purpose: This study aimed to investigate the relationship between sleep duration (SD) and stroke, and examine the effects of SD on stroke with or without metabolic syndrome (Mets) and its components among the adult residents in Shanghai, China. Participants and Methods: A total of 20,245 participants (51.72% male, mean age 44.66 years) were included from Shanghai Chronic Disease and Risk Factors Surveillance (SCDRFS) in 2017. The weighted logistic regressions were performed to examine the associations between SD and stroke in different status of Mets and its components. Results: The mean SD was 7.51±0.03 h/d. After adjusting for all the potential factors, SD<6 h/d (OR=1.73, 95% CI: 1.35-2.20) or ≥10 h/d (OR=1.66, 95% CI: 1.08-2.57) was significantly positively associated with stoke in the total participants; moreover, in the non-Mets group, only SD<6 h/d (OR=1.77, 95% CI: 1.19, 2.64) significantly increased the risk of stroke; while, in the Mets group, SD<6 h/d (OR=1.80, 95% CI:1.17-2.76) and ≥10 h/d (OR=1.97, 95% CI: 1.00-3.88) both had a positive significantly association with stoke. In addition, the effects of SD<6 h/d on stroke were more pronounced among those with high WC (OR=2.24, 95% CI: 1.40-3.58) and high TG (OR=2.60, 95% CI: 1.86-3.62), and the effects of SD≥10 h/d on stroke were more evident among those with high TG (OR=2.28, 95% CI: 1.02-5.08) and high FBG (OR=2.58, 95% CI: 1.30-5.10). Conclusion: Both short and long SD were significantly positively associated with stroke in the total participants, and the associations were stronger in the Mets group; conversely, in the non-Mets group, only short SD was significantly positively associated with stroke, and no significant association was observed between long SD and stroke. Therefore, more precise sleep measures may be needed to prevent stroke according to the different status of Mets.

Mechanistic study on the side arm effect in a palladium/Xu-Phos-catalyzed enantioselective alkoxyalkenylation of γ-hydroxyalkenes.

Recently, the asymmetric bifunctionalization of alkenes has received much attention. However, the development of enantioselective alkoxyalkenylation has posed a considerable challenge and has lagged largely behind. Herein, we report a new palladium-catalyzed enantioselective alkoxyalkenylation reaction, using a range of primary, secondary, and tertiary γ-hydroxy-alkenes with alkenyl halides. By employing newly identified Xu-Phos (Xu8 and Xu9) with a suitable side-arm adjacent to the PCy2 motif, a series of allyl-substituted tetrahydrofurans were obtained in good yields with up to 95% ee. Besides (E)-alkenyl halides, (Z)-alkenyl halide was also examined and provided the corresponding (Z)-product as a single diastereomer, supporting a stereospecific oxidative addition and reductive elimination step. Moreover, deuterium labeling and VCD experiments were employed to determine a cis-oxypalladation mechanism. DFT calculations helped us gain deeper insight into the side-arm effect on the chiral ligand. Finally, the practicability of this method is further demonstrated through a gram-scale synthesis and versatile transformations of the products.

[Improvement of carbon tetrachloride drug-induced liver injury model in vitro].

OBJECTIVE: To establish an ideal CCl4 drug-induced liver injury model in vitro. METHOD: Traditional method and improved method were adopted for preparing CCl4 injury liquid and drug-induced human liver HepG2 cell injury. Cell morphological change was observed under a bright-field microscope. The level of alanine aminotransferase (ALT) in supernatant was detected by biochemical method. 4-Methyl-tetrazolium (MTT) chromatometry was adopted for determining cell activity. RESULT: The improved method showed better CCl4-induced injury effect than the traditional method. With the increase in the concentration of CCl4 injury liquid, the ALT level significantly increased, whereas the cell activity notably decreased. Particularly, 70% CCl4 injury liquid use for 4 hours could achieve the best injury effect. CONCLUSION: The improved method could be used to establish an ideal CCl4 drug-induced liver injury model in vitro, which can lay foundation for further in vitro studies.

Stature estimation based on the length of tibia and fibula measured by digital X-ray in Chinese Han teenagers.

OBJECTIVE: To derive regression formulae for stature estimation using forensic radiography from the tibia and fibula of Chinese Han teenagers in Sichuan Province. METHODS: To construct equations, measurements were conducted on the training sample (412 adults, 201 males and 211 females). The whole length of the fibula and four measurements of tibia were determined using CR radiography, rectified through theoretical magnification. The regression formulae were relatively constructed to the real stature measured in an erect position. Through using the testing sample (40 adults) for the regression formulae, the reliability of the regression formulae was assessed. RESULTS: The range of correlation coefficients of four measurements for tibia was 0.880-0.895 in the sex-unknown group, 0.869-0.893 in the male, and 0.845-0.855 in the female. The five measurements were found to be better correlated with stature in the male than in the female. CONCLUSION: The digital X-ray of the tibia and fibula for stature estimation is proved to be effective in forensic individual identification; therefore, these equations can be of great assistance to the stature estimation of the contemporary Chinese Han teenagers.

[Stature estimation of teenagers by limb long bones with computerized radiography].

OBJECTIVE: Relative parameters of upper limb bones, tibia and fibula were measured with computed radiography and used to establish the mathematical models for stature estimation of teenagers (from 14 to 18 years old) of Han population in Sichuan Province. METHODS: The upper limb bones, tibia and fibula of 194 subjects were taken computerized radiography on normal position and were measured the lengths between relative landmarks. The body height of each subject was recorded. Linear regression equations for stature estimation between body height and the lengths of upper limb bones, tibia and fibula were established. RESULTS: Forty-two single linear regression equations and 4 multiple regression equations were obtained. The coefficients of correlation(r) were 0.689-0.917 and the standard errors of estimate(SE) were between 3.075 and 5.485 cm. All of the equations were statistically tested and diagnosed with good applicability. CONCLUSION: These equations could be used to estimate the body height of Sichuan Han population aged from 14 to 18. The lengths of the upper limb bones, tibia and fibula measured on the CR films could be useful to stature estimation of the adolescence and the forensic personal identification.

Investigation of the Association between 45 Tag SNPs and Type 2 Diabetes Mellitus in Han Chinese Adults: A Prospective Cohort Study.

INTRODUCTION: The objective of this study was to examine the association between type 2 diabetes mellitus (T2DM) and genes identified in previous genome-wide association studies (GWASs) in rural Han Chinese adults. METHODS: This prospective study included 1,832 adults aged ≥18 years in Deqing without diabetes at baseline. The subjects were followed up for 8.7 years on average. We selected 45 susceptible tag single-nucleotide polymorphisms (SNPs) for T2DM that have been identified in GWASs and genotyped. A Cox model was constructed to calculate the adjusted hazard ratios (aHRs) for the association between SNPs and incident T2DM. RESULTS: The incidence rate of T2DM was 12.0 per 1,000 person-years. After adjustment for covariates and a Bonferroni correction, rs17584499 of protein tyrosine phosphatase, receptor-type D (PTPRD), rs11257655 and rs10906115 of cell division cycle 123 gene (CDC123), and rs12970134 of melanocortin-4 receptor (MC4R) were significantly associated with incident T2DM. The aHRs for incident T2DM were 1.75 (95% confidence interval [CI]: 1.28-2.40) and 1.61 (95% CI: 1.27-2.04) in association with an increasing number of T alleles in rs17584499 and rs11257655 under an additive genetic model, and the aHR was 1.72 (95% CI: 1.33-2.22) with an increasing number of A alleles in rs10906115. The aHRs under the dominant model were 1.82 (95% CI: 1.25-2.66) for TT + CT versus CC of rs17584499 and 2.04 (95% CI: 1.47-2.86) for AA + AG versus GG of rs10966115. The aHRs under the recessive model were 2.99 (95% CI: 1.30-6.89) for TT versus CT + CC of rs17584499, 1.92 (95% CI: 1.39-2.70) for TT versus CT + CC of rs11257655, and 2.54 (95% CI:1.22-5.29) for AA versus AG + GG of rs12970134. In addition, an increased incidence of T2DM was significantly associated with the TA haplotype of rs11257655 and rs10906115 (aHR = 1.81, 95% CI: 1.12-2.92), while a decreased incidence was associated with the CG haplotype (aHR = 0.49, 95% CI: 0.35-0.68) and the CT haplotype of rs1111875 and rs5015480 (aHR = 0.61, 95% CI: 0.37-0.98). CONCLUSION: Variants of the PTPRD, CDC123, and MC4R genes were associated with the T2DM incidence in a rural Han Chinese population.

The x(c)- cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases.

The x(c) (-) cystine/glutamate antiporter is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. Its main functions in the body are mediation of cellular cystine uptake for synthesis of glutathione essential for cellular protection from oxidative stress and maintenance of a cystine:cysteine redox balance in the extracellular compartment. In the past decade it has become evident that the x(c) (-) transporter plays an important role in various aspects of cancer, including: (i) growth and progression of cancers that have a critical growth requirement for extracellular cystine/cysteine, (ii) glutathione-based drug resistance, (iii) excitotoxicity due to excessive release of glutamate, and (iv) uptake of herpesvirus 8, a causative agent of Kaposi's sarcoma. The x(c) (-) transporter also plays a role in certain CNS and eye diseases. This review focuses on the expression and function of the x(c) (-) transporter in cells and tissues with particular emphasis on its role in disease pathogenesis. The potential use of x(c) (-) inhibitors (e.g., sulfasalazine) for arresting tumor growth and/or sensitizing cancers is discussed.

Establishment in severe combined immunodeficiency mice of subrenal capsule xenografts and transplantable tumor lines from a variety of primary human lung cancers: potential models for studying tumor progression-related changes.

PURPOSE: Lung cancer is a biologically diverse disease and relevant models reflecting its diversity would facilitate the improvement of existing therapies. With a view to establishing such models, we developed and evaluated xenografts of a variety of human lung cancers. EXPERIMENTAL DESIGN: Using nonobese diabetic/severe combined immunodeficiency mice, subrenal capsule xenografts were generated from primary lung cancer tissue, including moderately and poorly differentiated squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, and carcinosarcoma. After 4 to 12 weeks, xenografts were harvested for serial transplantation and comparison with the original tissue via histologic, chromosomal, and cytogenetic analyses. RESULTS: Xenografts were successfully established. H&E staining showed that xenografts retained major histologic features of the original cancers. Immunohistochemistry and fluorescence in situ hybridization confirmed the human origin of the tumor cells and development in xenografts of murine supportive stroma. Four transplantable lines were developed from rapidly growing tumors (>5 generations), i.e., a small cell lung carcinoma, large cell undifferentiated carcinoma, pulmonary carcinosarcoma, and squamous cell carcinoma. Analyses including spectral karyotyping, comparative genomic hybridization, and fluorescence in situ hybridization, revealed that the xenografts were genetically similar to the original tumors, showing chromosomal abnormalities consistent with karyotypic changes reported for lung cancer. CONCLUSIONS: The subrenal capsule xenograft approach essentially provides a living tumor bank derived from patient material and a means for isolating and expanding specific cell populations. The transplantable tumor lines seem to provide good models for studying various aspects of tumor progression and a platform for developing novel therapeutic regimens, with the possibility of patient-tailored therapies.

Preparation, chromatographic evaluation and application of adenosine 5'-monophosphate modified ZrO2/SiO2 stationary phase in hydrophilic interaction chromatography.

The zirconia-coated silica (ZrO2/SiO2) material was obtained by coupling layer-by-layer (LbL) self-assembly method and sol-gel technology, to take dual advantages of the suitable porous structure of SiO2 and basic resistance of ZrO2. Adenosine 5'-monophosphate (5'-AMP) was then self-assembled onto ZrO2/SiO2 via Lewis acid-base interaction, generating 5'-AMP-ZrO2/SiO2. The chromatographic properties of 5'-AMP-ZrO2/SiO2 were systemically studied by evaluating the effect of acetonitrile content, pH and buffer concentration in the mobile phase. The results demonstrated that the 5'-AMP-ZrO2/SiO2 possessed hydrophilic interaction chromatographic (HILIC) property comprising hydrophilic, hydrogen-bonding, electrostatic and ion-exchange interactions. For basic analytes, the column efficiency of ZrO2/SiO2 and 5'-AMP-ZrO2/SiO2 was superior to the bare ZrO2, and different selectivity was obtained after the introduction of 5'-AMP. For acidic analytes, good resolution was obtained on 5'-AMP-ZrO2/SiO2 while the analysis failed on the bare ZrO2 column owing to strong adsorption. Hence, the proposed 5'-AMP-ZrO2/SiO2 had great potential in analyzing acidic compounds in HILIC mode. It was an extended application of ZrO2 based SP.

Identification of novel therapeutic targets in microdissected clear cell ovarian cancers.

Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients.

Induction of neuronal apoptosis inhibitory protein expression in response to androgen deprivation in prostate cancer.

A mechanism for survival of prostate cancer cells in an androgen-deprived environment remains elusive. Here, we find that expression of neuronal apoptosis inhibitory protein (NAIP) was significantly increased in vivo and in vitro in response to androgen deprivation therapy (ADT). Increased expression of NAIP corresponded to increased DNA-binding activity of NF-kappaB that physically associated to previously uncharacterized kappaB-like sites in the NAIP locus. Importantly, expression of NAIP was significantly increased (p=0.04) in clinical samples of prostate cancer from patients receiving ADT. Expression of NAIP may be associated with enhanced survival of prostate cancer in response to castration.

The xc- cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine.

PURPOSE: To determine whether the xc- cystine transporter could be a useful therapeutic target for small-cell lung cancer (SCLC). METHODS: Human SCLC cell cultures were examined for growth dependence on extracellular cystine, xc- expression, glutathione levels and response to highly specific xc- inhibitors, i.e., monosodium glutamate (MSG) and the anti-inflammatory drug, sulfasalazine (SASP). In studying tumor growth inhibition by SASP, use was also made of a novel SCLC tissue xenograft model, LU6-SCLC, derived from a chemoresistant patient's SCLC specimen. RESULTS: Growth of NCI-H69 and NCI-H82 SCLC cells greatly depended on xc- -mediated uptake of cystine. SASP substantially reduced their glutathione levels (>70%; 0.3 mM SASP; 24 h) and growth (72 h) with IC(50)s of 0.21 and 0.13 mM, respectively; MSG also inhibited growth markedly. Both SASP- and MSG-induced growth arrests were largely prevented by cystine uptake-enhancing 2-mercaptoethanol (66 approximately microM) indicating they were primarily due to cystine starvation. Without major side-effects, SASP (i.p.) restrained growth of NCI-H69 cell xenografts (approximately 50%) and, importantly, substantially inhibited growth of the clinically more relevant LU6-SCLC tissue xenografts (approximately 70% by stereological analysis), reducing tumor glutathione contents. CONCLUSIONS: The xc- cystine/glutamate antiporter is potentially useful as a target for therapy of SCLC based on glutathione depletion. Sulfasalazine may be readily used for this approach, especially in combination chemotherapy.

ASAP1, a gene at 8q24, is associated with prostate cancer metastasis.

Metastatic prostate cancer is a terminal disease, and the development of reliable prognostic tools and more effective therapy is critically important for improved disease survival and management. This study was aimed at identifying genes that are differentially expressed in metastatic and nonmetastatic prostate cancer cells and, as such, could be critical in the development of metastasis. Long-SAGE analysis was used to compare a transplantable human metastatic prostate cancer subline, PCa1-met, with a nonmetastatic counterpart, PCa2. Both sublines were developed from a patient's prostate cancer specimen via subrenal capsule grafting and subsequent orthotopic implantation into SCID mice. Among various differentially expressed genes identified, ASAP1, an 8q24 gene encoding an ADP-ribosylation factor GTPase-activating protein not previously associated with prostate cancer, was up-regulated in the metastatic subline as confirmed by quantitative real-time PCR. Immunohistochemistry of xenograft sections showed that cytoplasmic ASAP1 protein staining was absent or weak in benign tissue, significantly stronger in nonmetastatic PCa2 tissue, and strongest in PCa1-met tissue. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared with benign prostate tissue. Moreover, additional ASAP1 gene copies were detected in 58% of the primary prostate cancer specimens. Small interfering RNA-induced reduction of ASAP1 protein expression markedly suppressed in vitro PC-3 cell migration (approximately 50%) and Matrigel invasion (approximately 67%). This study suggests that the ASAP1 gene plays a role in prostate cancer metastasis and may represent a therapeutic target and/or biomarker for metastatic disease.

Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy.

BACKGROUND: Certain cancers depend for growth on uptake of cystine/cysteine from their environment. Here we examined advanced human prostate cancer cell lines, DU-145 and PC-3, for dependence on extracellular cystine and sensitivity to sulfasalazine (SASP), a potent inhibitor of the x(c)(-) cystine transporter. METHODS: Cultures were evaluated for growth dependence on exogenous cystine, x(c)(-) transporter expression, response to SASP (growth and glutathione content). In vivo, effect of SASP was determined on subrenal capsule xenograft growth. RESULTS: Cystine omission from culture medium arrested DU-145 and PC-3 cell proliferation; both cell lines expressed the x(c)(-) transporter and were growth inhibited by SASP (IC(50)s: 0.20 and 0.28 mM, respectively). SASP-induced growth inhibition was associated with vast reductions in cellular glutathione content - both effects based on cystine starvation. SASP (i.p.) markedly inhibited growth of DU-145 and PC-3 xenografts without major toxicity to hosts. CONCLUSIONS: SASP-induced cystine/cysteine starvation leading to glutathione depletion may be useful for therapy of prostate cancers dependent on extracellular cystine.

An orthotopic metastatic prostate cancer model in SCID mice via grafting of a transplantable human prostate tumor line.

Metastasis is the major cause of prostate cancer deaths and there is a need for clinically relevant in vivo models allowing elucidation of molecular and cellular mechanisms underlying metastatic behavior. Here we describe the development of a new in vivo model system for metastatic prostate cancer. Pieces of prostate cancer tissue from a patient were grafted in testosterone-supplemented male NOD-SCID mice at the subrenal capsule graft site permitting high tumor take rates. After five serial transplantations, the tumor tissues were grafted into mouse prostates. Resulting tumors and suspected metastatic lesions were subjected to histopathological and immunohistochemical analysis. Samples of metastatic tissue were regrafted in mouse anterior prostates and their growth and spread examined, leading to isolation from lymph nodes of a metastatic subline, PCa1-met. Orthotopic grafting of PCa1-met tissue in 47 hosts led in all cases to metastases to multiple organs (lymph nodes, lung, liver, kidney, spleen and, notably, bone). Histopathological analysis showed strong similarity between orthotopic grafts and their metastases. The latter were of human origin as indicated by immunostaining using antibodies against human mitochondria, androgen receptor, prostate-specific antigen and Ki-67. Spectral karyotyping showed few chromosomal alterations in the PCa1-met subline. This study indicates that transplantable subrenal capsule xenografts of human prostate cancer tissue in NOD-SCID mice can, as distinct from primary cancer tissue, be successfully grown in the orthotopic site. Orthotopic xenografts of the transplantable tumor lines and metastatic sublines can be used for studying various aspects of metastatic prostate cancer, including metastasis to bone.