Expression of Somatostatin Receptor Subtypes (SSTR-1–SSTR-5) in Pediatric Hematological and Oncological Disorders

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1–5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.     

Cyclooxygenase-2 parallels invasive depth and increased MVD in transitional cell carcinoma

To investigate the relationship between the expression of cyclooxygenase-2 (COX-2), and invasive depth and angiogenesis in human transitional cell carcinoma (TCC), the expression of COX-2 and von Willebrand factor (vWF) was detected in paraffin-embedded transitional cell carcinoma specimens from 50 patients with histologically confirmed bladder transitional cell carcinoma. Microvessels were counted and microvessel density (MVD) was calculated. The correlation between COX-2 and invasive depth, MVD and invasive depth, COX-2 and MVD were analyzed. COX-2 expression was significantly related to the invasive depth of tumors; it solely expressed in invasive TCC. The ratio of COX-2 positively stained samples in each stage correlated to the invasive stages; so did the proportion of COX-2 (+) cells in whole tumor cells. Also, MVD increased with the invasive stages of tumors and positively correlated to the invasive depth. The COX-2-positive TCC samples were highly vascular, their microvessel density was significantly higher than that of the COX-2-negative TCC samples. There was a positive correlation between MVD and COX-2 (+) cells proportion. Our findings suggested that COX-2 was positively correlated with the invasive depth and involved in the process of angiogenesis in human transitional cell carcinoma.     

Diverse effects of β-carotene on secretion and expression of VEGF in human hepatocarcinoma and prostate tumor cells

Oral administration of β-carotene (BC) was found to exert opposite effects on plasma levels of vascular endothelial growth factor (VEGF) in two animal models. One study in nude mice injected via tail vein with hepatocarcinoma SK-Hep-1 cells showed that BC decreases the plasma VEGF level, whereas the other study in nude mice injected subcutaneously with prostate tumor PC-3 cells showed that BC increases the plasma VEGF level. Herein we investigated whether BC (0.5-20 μM) possesses diverse effects on VEGF secretion in SK-Hep-1, PC-3 and melanoma B16F10 cells. We found that incubation of SK-Hep-1 cells with BC (1-20 μM) for 6 h significantly decreased VEGF secretion, whereas BC (1-10 μM) significantly increased the VEGF secretion in PC-3 cells. However, these effects disappeared at 12 h of incubation. Similar effects occurred in VEGF mRNA and protein expression after treatment of SK-Hep-1 and PC-3 cells with BC for 6 h. In contrast, BC (0.5-20 μM) did not affect mRNA and protein expression and secretion of VEGF in B16F10 cells. We also found that the proliferation of SK-Hep-1 and B16F10 cells was significantly inhibited by 20 μM BC at 6 and 12 h of incubation, whereas the proliferation of PC-3 cells was significantly inhibited by 20 μM BC at 12 h of incubation. In summary, the present study demonstrated the tumor-specific effect of BC on VEGF secretion in different cancer cell lines.     

Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine. Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-inducible protein gamma3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniques that included microsequencing, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/cgi-bin/celis). Proteome profile analysis of primary cultures from a low-grade lesion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN-gamma showed that all of the proteins disregulated in vivo were also affected in the cultures. The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first glance at the effect of IFN-gamma on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.     

Mutation and expression of the p27KIP1 and p57KIP2 genes in human gastric cancer

Cyclin-dependent kinase inhibitors (CDKI) are negative regulators of cell cycle progression by binding the cyclin-CDK complex and inhibiting the CDK activity. Genetic alteration in the CDKI genes has been implicated for carcinogenesis. To test the genetic alteration in the p27 and p57 genes, KIP family CDKI genes, 30 gastric tumor-normal pairs and 8 gastric cancer cell lines were analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). No mutation was detected in these genes although length polymorphisms in the proline-alanine repeat of the p57 gene were detected. When the p27 and p57 mRNAs were analyzed in gastric cancer cell lines by RT-PCR, the p27 mRNA was expressed considerably high in tumor cells but expression of the p57 mRNA was much low in gastric cancer cell lines compared to that of normal cells. The result suggests that inactivation of gene expression rather than mutations in the p57 gene accounts possibly for the involvement of this gene in tumorigenesis of gastric cancer. However, expression of the p27 gene seems to be essential for cell survival.     

Transthyretin (prealbumin) gene in human primary hepatic cancer.

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC) a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a hereditary disorder, familial amyloidosis (FAP), and related to thyroxine transport and binding to retinol-RBP (the retinol binding protein) complex. This is the first report of a study on TTR in human primary hepatic cancer. Since TTR gene was strikingly suppressed in mRNA expression and possibly defective in its gene structure, it was strongly implicated that TTR might be an important gene marker or a candidate of anti-oncogene for human PHC. The biological activity of TTR gene is under study.     

TRANSTHYRETIN (PREALBUMIN) GENE IN HUMAN PRIMARY HEPATIC CANCER

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC), a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a     

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Primary Human Endometrial Carcinomas: Novel Therapeutic Approaches

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.     

d-Poly(lactide) and LHRH decapeptide stereointeractions investigated by vibrational spectroscopy

The interactions between poly(d-lactic acid) (PDLA) and l-configured leuprolide (LHRH) to form heterocomplexes were investigated by a combination of infrared (IR), Raman and near infrared spectroscopy (NIR). It was found that an α crystal with 10₃ helical conformation of PDLA is formed in the PDLA/LHRH heterocomplexes with various LHRH loadings, whereas the secondary structures of LHRH in these heterocompelexes are greatly affected by the blend ratio of LHRH and PDLA. Based on the analysis of IR, Raman and NIR spectra of the various heterocomplexes, it is suggested that stereoselective Van der Waals interactions, consisting of interwined α helices of PDLA and LHRH, is responsible for the driving force of PDLA and LHRH stereocomplexation. To clarify the solid structure and the potential interaction in these heterocomplexes with various LHRH loadings, temperature-dependent IR spectral measurements were also employed. On the basis of the results presented, a model for the PDLA and LHRH stereointeraction process was proposed.     

Ureido-modification of the resin-bound LHRH analogue with N,N''-carbonyldiimidazole

The ureido-modification of the resin-bound a luteinizing hormone releasing hormone (LHRH) analogue was investigated by CDI-activating method. The amino group at the side chain of LHRH analogue could be transformed into various substituted urea moieties in high yields. However, its terminal amino group was partially converted to a hydantoin structure due to the attack of the N atom of the adjacent amide bond.     

Effects of changes in pattern of LHRH pulse on LH secretion by perfused anterior pituitary cells of male rats

In the present study, a perfusion system of dispersed cells was used to investigate the effects of LHRH pulse amplitude and frequency, and LHRH continuous stimulation on LH secretion by anterior pituitary cells of adult male rats. The results have shown that, in the range of LHRH concentrations from 1 X 10(-10) to 1 X 10(-6) mol/L, the dose-response curve of LH secretion was linear. LHRH pulse frequency generated a biphasic LH response: increasing LHRH pulse frequency increased the basal LH secretion and decreased LH/pulse. When 1 X 10(-9) mol/L or greater LHRH was given at frequencies of 3 pulses/h or higher, it was observed that a maximal LH peak was induced and then the LH release declined progressively to its LH basal level, i.e. LHRH self-priming effect and LH desensitization occurred. Enhancement of amplitude of LHRH pulses could reduce pulse frequency required for priming. Increases in frequency of LHRH pulses with high amplitude would provoke the priming effect more quickly. In addition, continuous perfusion of LHRH with different concentrations could also elicit the LHRH self-priming effect and lH desensitization. LHRH with low concentration (1 X 10(-10) mol/L) would take much longer to evoke a self-priming effect. These results indicate that the LH secretion pattern is dependent on LHRH pulsatile amplitude and frequency, and will help to clarify the kinetics mechanisms by which LH pulses fluctuate in vivo.     

PAK1 as a Potential Therapeutic Target in Male Smokers with EGFR-Mutant Non-Small Cell Lung Cancer

P21-activated kinases (PAKs) are serine/threonine protein kinases that contribute to several cellular processes. Here, we aimed to determine the prognostic value of PAK1 and its correlation with the clinicopathological characteristics and five-year survival rates in patients with non-small cell lung cancer (NSCLC). We evaluated PAK1 mRNA and protein expression in NSCLC cells and resected tumor specimens, as well as in healthy human bronchial epithelial cells and adjacent healthy lung tissues, respectively, for effective comparison. Immunohistochemical tissue microarray analysis of 201 NSCLC specimens showed the correlation of PAK1 expression with clinicopathological characteristics. The mRNA and protein expression of PAK1 were 2.9- and 4.3-fold higher in six of seven NSCLC cell types and human tumors (both, p < 0.001) than in healthy human bronchial epithelial BEAS-2B cells and adjacent healthy lung tissues, respectively. Decreased survival was significantly associated with PAK1 overexpression in the entire cohort (χ² = 8.48, p = 0.0036), men (χ² = 17.1, p < 0.0001), and current and former smokers (χ² = 19.2, p < 0.0001). Notably, epidermal growth factor receptor (EGFR) mutation-positive lung cancer patients with high PAK1 expression showed higher mortality rates than those with low PAK1 expression (91.3% vs. 62.5%, p = 0.02). Therefore, PAK1 overexpression could serve as a molecular target for the treatment of EGFR mutation-positive lung cancer, especially among male patients and current/former smokers.     

Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

Introduction Malignantdiseasesarecharacterizedbytheunreg ulatedgrowthoftransformedcells.Inrecentyears,dramaticinsightsintothemolecularmechanismsofthis phenomenonhavebeenachievedfrombasiccancerre search.Manycellularfunctionsareregulatedbychan gesingeneexpr…     

LHRH/TAT dual peptides-conjugated polymeric vesicles for PTT enhanced chemotherapy to overcome hepatocellular carcinoma

Combination therapy such as photothermal therapy (PTT) enhanced chemotherapy is regarded as a promising strategy for cancer treatment. Herein, we developed redox-responsive polymeric vesicles based on the amphiphilic triblock copolymer PCL-ss-PEG-ss-PCL. To avoid the limited therapeutic effect of chemotherapeutic drugs caused by systemic exposures and drug resistance, the redox-sensitive polymeric vesicles were cargoed with two chemotherapeutics: doxorubicin (DOX) and paclitaxel (PTX). Besides, indocyanine green (ICG) was encapsulated, and cell-penetrating peptides and LHRH targeting molecule were modified on the surface of polymeric vesicles. The results indicated that the polymeric vesicles can load different kinds of drugs with high drug loading content, trigger drug release in responsive to the reductive environment, realize high cellular uptake via dual peptides and laser irradiation, and achieve higher cytotoxicity via chemo-photothermal combination therapy. Hence, the redox-responsive LHRH/TAT dual peptides-conjugated PTX/DOX/ICG co-loaded polymeric micelles exhibited great potential in tumor-targeting and chemo-photothermal therapy.     

Synthesis,crystal structure of a novel tetranuclear Cu (Ⅱ) complex and its application in GSH-triggered generation of reactive oxygen species for chemodynamic therapy

A novel tetranuclear copper complex (TCC) has been successfully synthesized and characterized using high resolution mass spectrometry (HRMS), Fourier transform infrared spectroscopy (FTIR) and X-ray single crystal diffraction technique. TCC was capable of triggering by Glutathione (GSH) to produce Cu (Ⅰ), then the resulted Cu (Ⅰ) further converted the overexpression of endogenous H₂O₂ into •OH, a highly cytotoxic reactive oxygen species (ROS), thereby killing the cancer cells. TCC showed high cytotoxicity to 4T1, MCF7, HepG2 cancer cells. Interestingly, the cytotoxicity of TCC to non-cancerous cells is much lower than that of cancer cells. Cell cycle experiments demonstrated that TCC was capable of arresting the cancer cell cycle in the G2/M phase. The apoptosis experiments shown that TCC could induce apoptosis rather than necrosis.     

长效LHRH拮抗剂的设计、合成和生物活性

根据抗蛋白酶降解的长效肽设计思想, 合成了一系列新型结构的LHRH拮抗剂类似物. 体内生物活性评价结果表明, 所设计的多肽具有比母体肽和阳性对照更长的体内抑制睾酮作用时间和较低的最低有效剂量, 证实了该设计思想的可行性, 并为开发长效LHRH拮抗剂药物提供了新的候选化合物.