In vivo investigation of progressive alterations in rat mammary gland tumors by near-infrared spectroscopy

We have investigated mammary gland tissues of female rats treated with 7,12-dimethylbenz[a]anthracene in sesame oil by a near infrared (NIR) spectroscopy finding that the DNA and water contents in the cancerous tissues were larger than those in the normal tissues but that the lipid content in the former was less than that in the latter. With protein contents, however, little difference was observed between the two. Thus, we used a lipid band around 1725 nm (the first overtone of n-alkane) and a protein band around 2054 nm (a combination band of amide A and amide II of polypeptides) for a quantitative evaluation of malignant changes in the mammary gland tissues. The lipid/protein band intensity ratios were calculated from the spectra of the mammary glands in the control animals and those of the noncancerous and cancerous sites in the treated animals. The lipid/protein ratios in the control animals, in the noncancerous sites, and in the cancerous sites were 1.452 +/- 0.221 (n = 5), 0.728 +/- 0.069 (n = 5), and 0.362 +/- 0.060 (n = 5), respectively. These values were significantly different from each other (P < 0.001). The lipid changes observed by near-infrared (NIR) spectroscopy were confirmed by the results obtained from chemical methods for the evaluation of lipid levels in the same samples. Thus, our NIR spectroscopic method would be able not only to discriminate between cancerous and normal tissues but also to distinguish animals with cancers from normal animals. In addition, as the cancer grew, the lipid band intensity decreased, this band was shifted to higher wavelengths, and collagen peaks appeared in the tissues. These findings were supported by histological examinations of the cancerous and normal tissues. The present study indicates that NIR spectroscopy has high specificity and sensitivity in discriminating cancerous tissues from normal mammary glands in animals and it may offer potential for noninvasive, in vivo diagnosis of female breast cancer in the near future.     

Effect of sex and size on the protein composition in the tissues of the freshwater crab,Paratelphusa (Barytelphusa) guerini Milne Edwards

Soluble (enzymic) and insoluble (structural) proteins were estimated quantitatively in different tissues of the crab as a function of sex and size. The different protein fractions in the tissues of females decreased with weight. But the proteins in the tissues of males increased gradually with size up to 30–40 g and decreased later. In both sexes, the relative increase and decrease varied in different tissues.The insoluble protein content of the muscle was always greater than the soluble content in both sexes. However, the two fractions in other tissues showed different trends depending upon the sex. The soluble content was always greater than the insoluble content in gill, heart and hepatopancreas of males. The hepatopancreas of females had a higher soluble content at all weights, as in males. But the insoluble proteins of the gill and soluble proteins of the heart were more in smaller animals (below 25–30 g) and less in larger animals (above 25–30 g).The soluble proteins dominated in the males. This relation was maintained in the muscle throughout the weight range studed (10 to 75 g), while in other tissues this relation was seen for the the greater part of this weight range. The insoluble proteins in the different tissues tended to be more in smaller females (up to 25–30 g) and larger males (above 25–30 g).The total protein content of the muscle was always larger in males. The proteins of the gill and heart were larger in males at higher sizes (above 20–25 g). Hepatopancreatic proteins were larger only at the intermediate-sized males (between 25 and 60 g).     

gp78 is specifically expressed in human prostate cancer rather than normal prostate tissue

Elevated expression of gp78 has been observed in many types of cancers including lung, stomach, colon, liver and skin cancer. But there is no report about its expression in prostate cancers. In this study, using immunohistochemical staining we found gp78 is highly expressed in prostate cancers especially early stage tumors, but not in normal prostate tissues. gp78 protein expression is heterogeneous. In some tumors it was expressed in basal cells, while others in stromal cells. For gp78 is a ubiquitin E3 ligase, we then investigated the expression pattern of its cognate E2 (ubiquitin conjugating enzyme)-Ube2g2 in prostate cancers. We found it was expressed in both cancerous and normal tissues of prostate without significant differences in expression level. And unlike gp78, it exhibited a homogeneous expression pattern in different cell types in prostate tissues. In conclusion, our results indicate that gp78 is expressed specifically in human prostate cancer rather than normal prostate tissues, it could be a putative biomarker for prostate cancer diagnosis.     

Angular optimization for cancer identification with circularly polarized light

Depolarization of circularly polarized light scattered from biological tissues depends on structural changes in cell nuclei, which can provide valuable information for differentiating cancer tissues concealed in healthy tissues. In this study, we experimentally verified the possibility of cancer identification using scattering of circularly polarized light. We investigated the polarization of light scattered from a sliced biological tissue with various optical configurations. A significant difference between circular polarizations of light scattered from cancerous and healthy tissues is observed, which is sufficient to distinguish a cancerous region. The line-scanning experiments along a region incorporating healthy and cancerous parts indicate step-like behaviors in the degree of circular polarization corresponding to the state of tissues, whether cancerous or normal. An oblique and perpendicular incidence induces different resolutions for identifying cancerous tissues, which indicates that the optical arrangement can be selected according to the priority of resolution.     

Enhanced expression of a novel protein in human cancer cells: a potential aid to cancer diagnosis

Cap43 is a protein whose RNA is induced under conditions of severe hypoxia or prolonged elevations of intracellular calcium. Cap43 protein is expressed at low levels in normal tissues; however, in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers, Cap43 protein is overexpressed in cancer cells. The low level of expression of Cap43 in some normal tissues compared to their cancerous counterparts combined with the high stability of Cap43 protein and mRNA makes the Cap43 gene a new, important cancer marker. We hypothesize that the mechanism of Cap43 overexpression in cancer cells involves a state of hypoxia characteristic of cancer cells where the Cap43 protein becomes a signature for this hypoxic state. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression of the novel human gene,UBE2Q1, in breast tumors

The novel human gene, designated ubiquitin-conjugating enzyme E2Q family member 1 (UBE2Q1) maps to chromosome 1q21.3. The gene has an open reading frame corresponding to 422 amino acids and contains a RWD domain and an E2 ubiquitin conjugating enzyme domain. Here, we investigated the expression levels of both mRNA and protein of UBE2Q1 gene in cancerous versus normal parts of breast specimens from 26 patients. Real-time PCR data showed that the relative expression level of UBE2Q1 mRNA was significantly greater in cancers than in non-cancerous tissues of breast specimens (Mean ± SEM, 0.064 ± 0.015 for cancers and 0.026 ± 0.01 for noncancerous tissues, P < 0.05 Mann–Whitney test). A rabbit polyclonal antibody was generated against an amino acid sequence predicted from the DNA sequence of UBE2Q1 gene. This antibody was used to perform Western blotting on 21 cases in our cohort of breast specimens. Thus, 13 (61.904%) of the cases showed an increase in the UBE2Q1 immunoreactivity in their cancerous tissues as compared with the corresponding normal tissues. This result along with the real-time PCR data shows that the novel human gene, UBE2Q1, is expressed in human breast and may have implications for pathogenesis of breast cancer.     

Dipeptidyl peptidase IV in the human lung and spinocellular lung cancer.

The isoelectric point and proportions of soluble and membrane bound dipeptidyl peptidase IV (DPP-IV) in human lung and spinocellular lung cancer tissue were tested. It was found that soluble DPP-IV is relatively less frequent in the cancer than in normal lung tissue. We demonstrated multiple molecular forms of DPP-IV in normal and cancer lung tissues, differing probably not only in the degree of sialylation. DPP-IV from lung cancer tissue consists of more basic molecular forms than that from normal lung tissue. These results suggest that the molecular properties of DPP-IV in normal and cancerous lung tissues may be different.     

A structural analysis of glycosaminoglycans from lethal and nonlethal breast cancer tissues: toward a novel class of theragnostics for personalized medicine in oncology?

Cancer is one of the leading noncommunicable diseases that vastly impacts both developed and developing countries. Truly innovative diagnostics that inform disease susceptibility, prognosis, and/or response to treatment (theragnostics) are seriously needed for global public health and personalized medicine for patients with cancer. This study examined the structure and content of glycosaminoglycans (GAGs) in lethal and nonlethal breast cancer tissues from six patients. The glycosaminoglycan content isolated from tissue containing lethal cancer tumors was approximately twice that of other tissues. Molecular weight analysis showed that glycosaminoglycans from cancerous tissue had a longer weight average chain length by an average of five disaccharide units, an increase of approximately 15%. Dissacharide analysis found differences in sulfation patterns between cancerous and normal tissues, as well as sulfation differences in GAG chains isolated from patients with lethal and nonlethal cancer. Specifically, cancerous tissue showed an increase in sulfation at the "6S" position of CS chains and an increase in the levels of the HS disaccharide NSCS. Patients with lethal cancer showed a decrease in HS sulfation, with lower levels of "6S" and higher levels of the unsulfated "0S" disaccharide. Although these findings come from a limited sample size, they indicate that structural changes in GAGs exist between cancerous and noncancerous tissues and between tissues from patients with highly metastatic cancer and cancer that was successfully treated by chemotherapy. Based on these findings, we hypothesize that (1) there are putative changes in the body's construction of GAGs as tissue becomes cancerous; (2) there may be innate structural person-to-person variations in GAG composition that facilitate the metastasis of tumors in some patients when they develop cancer.     

The structural and compositional changes of glycosaminoglycans are closely associated with tissue type in human laryngeal cancer

Hyaluronan and sulfated glycosaminoglycans, as intrinsic components of proteoglycans, are playing important roles in cancer biology. In the present study, we investigated in detail the glycosaminoglycans on both fine chemical and structural levels in laryngeal cartilaginous and non-cartilaginous tissues at different stages of laryngeal cancer. The results indicated that in cartilaginous tissues the amounts of chondroitin sulfate, keratan sulfate, dermatan sulfate and hyaluronan presented a dramatic decrease in contrast to the non-cartilaginous tissues, which showed a significant increase of these glycosaminoglycans compared to their normal counterparts. On fine chemical structure, the molar ratios of 4-sulfated to 6-sulfated and non-sulfated to sulfated disaccharides from both cartilaginous and non-cartilaginous cancerous tissues showed a significant increase. On molecular-size level, in laryngeal cancer, the chromatographic behaviour of the sulfated glycosaminoglycan chains from both tissue-types revealed their lower M(r) with a more polydisperse and heterogeneous distribution compared to the normal ones. In addition, in both tissues, a significant decrease of high molecular-size hyaluronan was observed. Of particular interest was the great increase of hyaluronan of low molecular mass in the laryngeal non-cartilaginous tissues, which ranged from 330 to 890 kDa. The kind and the extent of these alterations, which presented an intense stage-related behaviour, depended on the tissue origin and could be associated with the malignant phenotype of human laryngeal cancer.     

NMR in cancer. VII. Sodium-23 magnetic resonance of normal and cancerous tissues.

Sodium-23 magnetic resonance was performed on four types of cancers and six types of normal tissues of rats and mice. The spin-lattice relaxation time of the tumors was generally longer than that of the normal tissues, with the most marked difference occurring between rat liver (T1 = 6.5 msec) and Novikoff hepatoma (T1 =23.7 msec). Estimation of tissue sodium from the signal intensity of the resonance indicated that all four types of tumors contained more sodium than any of the normal tissues.     

Protein tyrosine phosphorylation in normal rat tissues

• 1.1. Exogenous and endogenous tyrosine protein phosphorylation activities were examined in soluble and partieulate fractions from various normal tissues by using poly-[Glu-⁸⁰Na, Tyr²⁰] and a monoclonal antibody specific for phosphotyrosine.
• 2.2. Phosphorylation of the exogenous substrate by the partieulate forms of TPKs was 2- to 10-fold higher than by soluble forms. The activities of partieulate and soluble enzymes decreased in the following order: spleen > (thymus = kidney) > testes ⩾ (pancreas = liver = brain) > heart.
• 3.3. The level of endogenous phosphorylation in the tissues decreased respectively in the following order: thymus > brain ⩾ (pancreas = liver) > spleen > testes > kidney > heart for the partieulate fractions, and spleen > thymus > brain > pancreas ⩾ liver > testes > kidney > heart for the soluble fractions.
• 4.4. A large number of phosphotyrosine-containing proteins were detected. In addition, several phosphotyrosine-containing proteins of similar molecular weight were found in different tissues and fractions.

     

Gene expression and methylation status of 14-3-3sigma in human renal carcinoma tissues

Loss of 14-3-3sigma expression mainly by methylation-mediated silencing has been reported in several human cancers, but the methylation status of 14-3-3sigma in human renal carcinoma is rarely studied so far. In this report, 14-3-3sigma expression was first examined by RT-PCR and immunohistochemistry, and further we investigated the methylation status by methylation-specific PCR and the correlation between 14-3-3sigma expression and its methylation. We found 14-3-3sigma expression was lost in 27 of 31 renal tissues including 16 renal carcinoma tissues, eight para-cancerous kidney tissues and seven normal kidney tissues. Among 16 renal carcinoma tissues, 14 cases had complete hypermethylation of 14-3-3sigma. Eight para-cancerous kidney tissues were almost completely methylated except one case had both methylation and unmethylation. Among seven normal kidney tissues, five cases had partial methylation, and the other two cases were completely methylated. In addition, 14-3-3sigma mRNA had weak expression in OS-RC-2 cells, but it increased with gradual demethylation after treatment by a demethylation agent, 5-aza-2'-deoxycytidine. In general, 14-3-3sigma mRNA was mostly unexpressed, and its DNA frequently hypermethylated within 14-3-3sigma coding region was closely associated with the gene silencing in cancerous and para-cancerous kidney tissues. 14-3-3sigma was also frequently methylated and almost silencing in normal kidney tissues. However, the methylation frequency was gradually reinforced with the extent of malignancy from normal to para-cancerous and cancerous kidney tissues.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.     

Prioritization of Cancer Marker Candidates Based on the Immunohistochemistry Staining Images Deposited in the Human Protein Atlas

Cancer marker discovery is an emerging topic in high-throughput quantitative proteomics. However, the omics technology usually generates a long list of marker candidates that requires a labor-intensive filtering process in order to screen for potentially useful markers. Specifically, various parameters, such as the level of overexpression of the marker in the cancer type of interest, which is related to sensitivity, and the specificity of the marker among cancer groups, are the most critical considerations. Protein expression profiling on the basis of immunohistochemistry (IHC) staining images is a technique commonly used during such filtering procedures. To systematically investigate the protein expression in different cancer versus normal tissues and cell types, the Human Protein Atlas is a most comprehensive resource because it includes millions of high-resolution IHC images with expert-curated annotations. To facilitate the filtering of potential biomarker candidates from large-scale omics datasets, in this study we have proposed a scoring approach for quantifying IHC annotation of paired cancerous/normal tissues and cancerous/normal cell types. We have comprehensively calculated the scores of all the 17219 tested antibodies deposited in the Human Protein Atlas based on their accumulated IHC images and obtained 457110 scores covering 20 different types of cancers. Statistical tests demonstrate the ability of the proposed scoring approach to prioritize cancer-specific proteins. Top 100 potential marker candidates were prioritized for the 20 cancer types with statistical significance. In addition, a model study was carried out of 1482 membrane proteins identified from a quantitative comparison of paired cancerous and adjacent normal tissues from patients with colorectal cancer (CRC). The proposed scoring approach demonstrated successful prioritization and identified four CRC markers, including two of the most widely used, namely CEACAM5 and CEACAM6. These results demonstrate the potential of this scoring approach in terms of cancer marker discovery and development. All the calculated scores are available at http://bal.ym.edu.tw/hpa/.     

Soluble L1CAM promotes breast cancer cell adhesion and migration <Emphasis Type="Italic">in vitro</Emphasis>, but not invasion

Background  

Neural recognition molecule L1CAM, which is a key protein involved in early nervous system development, is known to be abnormally expressed and shed in several types of cancers where it participates in metastasis and progression. The distinction of L1CAM presence in cancerous vs. normal tissues has suggested it to be a new target for cancer treatment. Our current study focused on the potential role of soluble L1CAM in breast cancer cell adhesion to extracellular matrix proteins, migration, and invasion.     

Thermo-optic measurements and their inter-dependencies for delineating cancerous breast biopsy tissue from adjacent normal

The histopathological diagnosis of cancer is the current gold standard to differentiate normal from cancerous tissues. We propose a portable platform prototype to characterize the tissue's thermal and optical properties, and their inter-dependencies to potentially aid the pathologist in making an informed decision. The measurements were performed on 10 samples from five subjects, where the cancerous and adjacent normal were extracted from the same patient. It was observed that thermal conductivity (k) and reduced-scattering-coefficient (μ'_s) for both the cancerous and normal tissues reduced with the rise in tissue temperature. Comparing cancerous and adjacent normal tissue, the difference in k and μ'_s (at 940 nm) were statistically significant (p = 7.94e-3), while combining k and μ'_s achieved the highest statistical significance (6.74e-4). These preliminary results promise and support testing on a large number of samples for rapidly differentiating cancerous from adjacent normal tissues.     

Identification of a distinct 9S form of soluble clathrin in cultured cells and tissues

We have used a monoclonal antibody (CHC5.9) to identify clathrin (Mr 180,000; 'heavy chain') in coated vesicles, triskelion structures prepared in vitro and in high-speed supernatants (HSS) of cell homogenates from a variety of tissues and species (e.g., brain and liver from rat, cow and man; Xenopus ovaries). HSS proteins were subjected to sucrose density gradient centrifugation and gel filtration, and the fractions obtained were assayed for clathrin by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting. The native soluble clathrin identified in such fractions was indistinguishable from triskelions produced in vitro from purified bovine brain clathrin by several criteria, e.g. by its sedimentation coefficient (9S) and elution profile on gel filtration using Sephacryl S 300. No other major forms of soluble clathrin were detected. The results indicate that cells contain a soluble pool of clathrin and that the predominant molecular form of this soluble clathrin has properties similar to those of the triskelion obtained by dissociation studies in vitro. We hypothesize that this distinct 9S form represents a major oligomeric subunit involved in assembly and disassembly of clathrin polyhedron coats in the living cell.     

Sex-associated difference in estrogen receptor β expression in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric cancers in rats

Epidemiologic studies indicate that the incidence of gastric cancer is higher in males than in females. Although the mechanisms mediating this difference are unclear, a role for estrogens has been proposed. We used Western blotting to evaluate the role of estrogen receptor (ER) subtypes ERα and ERβ and proliferating cell nuclear antigen (PCNA) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats; ERα and ERβ mRNA levels also were analyzed by quantitative real-time RT-PCR analysis. The incidence of gastric cancer was significantly higher in male than female rats. In both sexes, ERα expression was similar in MNNG-treated cancerous and noncancerous tissues and normal gastric tissue. However, ERβ expression in MNNG-treated cancerous and noncancerous tissues was significantly lower in male rats and higher in female rats than that in normal gastric tissue; MNNG-induced cancerous tissue showed the highest ERβ expression. PCNA expression in MNNG-treated cancerous tissues was higher than that in noncancerous tissues, and was higher in male rats than female rats. Western blotting results were consistent with the mRNA changes determined by quantitative real-time RT-PCR. The present study provides evidence of a sex-associated difference in ERβ and PCNA expression in MNNG-induced gastric cancers in Wistar rats.     

Nickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteomic analysis of cancer tissues: shedding light on carcinogenesis and possible biomarkers

Lung, gastric, colorectal, pancreatic, and esophageal cancers, as well as hepatocellular carcinoma (HCC), were the six most common and highly fatal cancers for Japanese men in Japan in 2003, while for women uterine cervical cancer could also be added to this list. To identify diagnostic or therapeutic biomarkers for these cancers, investigators are nowadays performing proteomic analyses of cancer tissues and cells, and revealing a large number of molecules which are diagnostic, prognostic and informative of carcinogenesis. From reports of proteomic analyses of cancerous tissues and noncancerous tissues sampled from HCC, and pancreatic, esophageal, gastric, colorectal, lung and uterine cervical cancers, we classified the proteins into digestive enzymes, growth factors, cell adhesion molecules, calcium-binding proteins, proteases, protease inhibitors, transporter proteins, structural molecules, apoptosis inhibitor, molecular chaperone, as well as proteins related to cell growth, cell differentiation, cell transformation, tumor invasion, carcinogen metabolism, and others. The aim of this study was to understand carcinogenesis of major cancers from a proteomics perspective using samples from cancer patients, and to elucidate their tumor biomarkers.