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.     

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.     

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.     

Cathepsin B and L activities in gastric cancer tissue: correlation with histological findings

Cathepsin B and L activities in cancerous and noncancerous mucosal tissues from 29 patients with gastric cancer were determined with a small amount of tissue homogenate. Both enzyme activities were significantly higher in cancerous tissues than in noncancerous tissues. The cathepsin B activity was higher with decreasing differentiation of the cancerous tissues, and also with increasing depth of invasion and metastasis to regional lymph nodes. Significantly high cathepsin B activity was observed in specimens of poorly differentiated adenocarcinomas, as well as in specimens from patients with extensive metastasis to n2 or n3 lymph nodes. These results suggest that high cathepsin B activity is characteristic of gastric cancer which invades and metastasizes. Therefore, in cases of marked elevation of cathepsin B activity in cancerous tissues, relatively extensive resection may be necessary to obtain a cure.     

Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy

Raman spectroscopy has strong potential for providing noninvasive dermatological diagnosis of skin cancer. In this study, confocal Raman microscopy was applied to the dermatological diagnosis for one of the most common skin cancers, basal cell carcinoma (BCC). BCC tissues were obtained from 10 BCC patients using a routine biopsy and used for confocal Raman measurements. Autofluorescence signals from tissues, which interfere with the Raman signals, were greatly reduced using a confocal slit adjustment. Distinct Raman band differences between normal and BCC tissues for the amide I mode and the PO2- symmetric stretching mode showed that this technique has strong potential for use as a dermatological diagnostic tool without the need for statistical treatment of spectral data. It was also possible to precisely differentiate BCC tissue from surrounding noncancerous tissue using the confocal Raman depth profiling technique. We propose that confocal Raman microscopy provides a novel method for dermatological diagnosis since direct observations of spectral differences between normal and BCC tissues are possible.     

Antioxidant potential of cancerous human kidney tissues

Antioxidant potentials (AOP) of cancerous and noncancerous adjacent human kidney tissues from 12 patients were measured. AOP of the cancerous tissues was found to be significantly lower than that of noncancerous ones. However, tissue malondialdehyde (MDA) levels were significantly higher in the cancerous tissues compared with noncancerous ones. In the intra-correlation analysis, carried out between AOP and MDA levels, significant correlation was found in the cancerous tissues (r = 0.9) but no correlation observed in the noncancerous ones. In the inter-correlation analysis, negative correlation was found between AOP's of cancerous and noncancerous tissues (r = -0.49) and positive correlation between MDA levels (r = 0.51). Results suggest that antioxidant potential of cancerous kidney tissues is significantly reduced compared with noncancerous ones. Therefore, they expose to high oxidant stress and free radical-induced peroxidative attacks, the results of which are cellular deformations.     

Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase Ⅱ in Hepatocellular Carcinoma

Early detection and intervention are key strategies to reduce mortality, increase longterm survival, and improve the therapeutic effects of hepatocellular carcinoma(HCC) patients.Herein, the isobaric tag for relative and absolute quantitation(iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues,surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II(CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.     

Rate of 14CO2 production from variously labeled forms of [14C]glucose in human breast invasive ductal carcinoma tissues

To establish possible cancerous aggressiveness between the metabolism of variously labeled [14C]glucose in the human breast invasive ductal carcinoma (IDC) tissues, we measured the rates of 14CO2 production from those tissues by using radiorespirometry, expressing the results as initial velocity (V) in nanomoles of 14CO2 min-1 g-1 of fresh tissues. The Vc data were compared with results of the SBR system, which grades up from I to III. Vc,1 values measured with [1-14C]glucose increased from 1.99-2.82 for SBR I to 3.90-4.09 for SBR II, finally reaching 4.83-7.04 for SBR III, thus matching clearly the increase of IDC cancerous aggressiveness. Conversely, data obtained from [3,4-14C]glucose and [6-14C]glucose decreased with increasing cancer stage: i.e., with [3,4-14C]glucose, Vc,3,4 values were 5.79-9.34 for SBR I, 4.45-4.84 for SBR II, and 2.35-1.90 for SBR III; with [6-14C]glucose, the corresponding Vc,6 values were 1.34-1.90, 1.33-1.41, and 0.72-0.79. The Vn,1/Vn,6 ratios were close to unity for normal tissues and for noncancerous tissues surrounding SBRI tumors. For cancerous tissues, however, the Vc,1/Vc,6 ratios were 1.5, 2.9, and 6.1-9.8 in IDC tissues graded as SBR I, II, and III, respectively. The results suggest the possible use of radiorespirometry as a tool to assess IDC aggressiveness.     

Investigation of the permeability and optical clearing ability of different analytes in human normal and cancerous breast tissues by spectral domain OCT

The potential of OCT applied for early breast cancer detection attracted significant efforts. The permeability coefficients and the percentages of OCT signal enhancement for normal and cancerous breast tissues have been investigated from the experiments of 20% glucose, 40% glucose, and 20% mannitol in vitro. Obtained results indicate that the permeability coefficient in breast cancer tissue is prominently larger than that in normal breast tissue, while the optical clearing effect is just the reverse to that for each agent. The results suggest that OCT has the ability to distinguish cancer tissue from different aspect. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

T-RFLP技术检测结直肠癌患者肠道黏膜相关菌群改变

目的研究结直肠癌患者肠道黏膜相关菌群组成差异,探索肠道菌群在结直肠癌发生发展中的作用。方法用末端限制片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术分析50例结直肠癌患者癌组织、癌旁正常黏膜与健康对照组肠道黏膜相关细菌组成差异。结果与健康对照组相比,结直肠癌患者肠道黏膜相关细菌丰度显著增加(P0.05),多样性显著降低(P0.05)。结直肠癌患者癌组织与癌旁正常黏膜的黏膜相关细菌组成相近,但与健康对照组存在显著差异。MspI酶切的160bp、560bp的T-RF片段在结直肠癌患者癌组织及癌旁正常黏膜中为优势片段,而在健康对照组中缺失。相反,MspI酶切的66bp、74bp、141bp的T-RF片段在健康对照组为优势片段,但在结直肠癌癌组织及癌旁正常黏膜中缺失。结论肠道菌群失调与结直肠癌的发生发展密切相关。MspI酶切的66bp、74bp、141bp、160bp、560bp的T-RF片段所代表的细菌可能在结直肠癌的发生发展中起重要作用。     

Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer

Recent reports have suggested the involvement of gut microbiota in the progression of colorectal cancer (CRC). We utilized pyrosequencing based analysis of 16S rRNA genes to determine the overall structure of microbiota in patients with colorectal cancer and healthy controls; we investigated microbiota of the intestinal lumen, the cancerous tissue and matched noncancerous normal tissue. Moreover, we investigated the mucosa-adherent microbial composition using rectal swab samples because the structure of the tissue-adherent bacterial community is potentially altered following bowel cleansing. Our findings indicated that the microbial structure of the intestinal lumen and cancerous tissue differed significantly. Phylotypes that enhance energy harvest from diets or perform metabolic exchange with the host were more abundant in the lumen. There were more abundant Firmicutes and less abundant Bacteroidetes and Proteobacteria in lumen. The overall microbial structures of cancerous tissue and noncancerous tissue were similar; however the tumor microbiota exhibited lower diversity. The structures of the intestinal lumen microbiota and mucosa-adherent microbiota were different in CRC patients compared to matched microbiota in healthy individuals. Lactobacillales was enriched in cancerous tissue, whereas Faecalibacterium was reduced. In the mucosa-adherent microbiota, Bifidobacterium, Faecalibacterium, and Blautia were reduced in CRC patients, whereas Fusobacterium, Porphyromonas, Peptostreptococcus, and Mogibacterium were enriched. In the lumen, predominant phylotypes related to metabolic disorders or metabolic exchange with the host, Erysipelotrichaceae, Prevotellaceae, and Coriobacteriaceae were increased in cancer patients. Coupled with previous reports, these results suggest that the intestinal microbiota is associated with CRC risk and that intestinal lumen microflora potentially influence CRC risk via cometabolism or metabolic exchange with the host. However, mucosa-associated microbiota potentially affects CRC risk primarily through direct interaction with the host.     

Usefulness of CDK5RAP3, CCNB2, and RAGE genes for the diagnosis of lung adenocarcinoma

We used oligonucleotide microarrays with probe sets to 22,283 genes to analyze the gene expression profile of lung adenocarcinoma. Cancerous and noncancerous tissue samples were obtained from 23 patients with stage I or II lung cancer; 18 tissue pairs and 5 cancerous tissues. A list of 2065 genes that differentiate between cancerous and noncancerous tissues was generated using Winsorized paired t-tests. We analyzed CDK5RAP3 and CCNB2, which are involved in cell cycle progression, and RAGE. The first 2 of these 3 genes proved to be overexpressed in tumor tissue, whereas the RAGE gene was suppressed in tumor tissue. When CDK5RAP3 and CCNB2 were examined in individual patients we found that in cases where one of these genes was only slightly overexpressed the other was highly overexpressed. The combined expression of the 2 cell cycle genes was found to be statistically significant for differentiating between cancerous and noncancerous tissues. Inclusion of the data for the RAGE gene made the differentiation more powerful. The gene expression ratio gave a clear result: when CDK5RAP3 was expressed more than RAGE, the tissue was carcinomatous, and vice versa. We therefore conclude that these 3 genes may be used as a very reliable biomarker of lung adenocarcinoma.     

Estrone sulfate and sulfatase activity in human breast cancer and endometrial cancer

Estrone sulfate (E1-S) in the serum and tissues of patients with breast cancer or endometrial cancer was measured by a direct radioimmunoassay without hydrolysis. The concentration of E1-S in breast cancer tissue was 1.64 +/- 0.28 ng/g wet wt (+/- SE), lower than in surrounding normal breast tissue (4.46 +/- 1.23). Estradiol-17 beta(E2)/E1-S was higher in endometrial cancer tissue than normal endometrial tissue. Estrone sulfatase activity in breast cancer tissue was 0.81 +/- 0.23 nmol/h/mg protein, higher than in surrounding normal breast tissue (0.35 +/- 0.11). These results suggest that E1-S, which is abundant in the peripheral circulation, is hydrolyzed by sulfatase in breast cancer tissue or endometrial cancer tissue and liberates free estrogens, which may stimulate the growth of these malignant tumors.     

Discovery of EST-SSRs in lung cancer: tagged ESTs with SSRs lead to differential amino acid and protein expression patterns in cancerous tissues

Tandem repeats are found in both coding and non-coding sequences of higher organisms. These sequences can be used in cancer genetics and diagnosis to unravel the genetic basis of tumor formation and progression. In this study, a possible relationship between SSR distributions and lung cancer was studied by comparative analysis of EST-SSRs in normal and lung cancerous tissues. While the EST-SSR distribution was similar between tumorous tissues, this distribution was different between normal and tumorous tissues. Trinucleotides tandem repeats were highly different; the number of trinucleotides in ESTs of lung cancer was 3 times higher than normal tissue. Significant negative correlation between normal and cancerous tissue showed that cancerous tissue generates different types of trinucleotides. GGC and CGC were the more frequent expressed trinucleotides in cancerous tissue, but these SSRs were not expressed in normal tissue. Similar to the EST level, the expression pattern of EST-SSRs-derived amino acids was significantly different between normal and cancerous tissues. Arg, Pro, Ser, Gly, and Lys were the most abundant amino acids in cancerous tissues, and Leu, Cys, Phe, and His were significantly more abundant in normal tissues than in cancerous tissues. Next, the putative functions of triplet SSR-containing genes were analyzed. In cancerous tissue, EST-SSRs produce different types of proteins. Chromodomain helicase DNA binding proteins were one of the major protein products of EST-SSRs in the cancerous library, while these proteins were not produced from EST-SSRs in normal tissue. For the first time, the findings of this study confirmed that EST-SSRs in normal lung tissues are different than in unhealthy tissues, and tagged ESTs with SSRs cause remarkable differences in amino acid and protein expression patterns in cancerous tissue. We suggest that EST-SSRs and EST-SSRs differentially expressed in cancerous tissue may be suitable candidate markers for lung cancer diagnosis and prediction.     

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.     

Real-time diagnosis of chemically induced hepatocellular carcinoma using a novel mass spectrometry-based technique

Real-time analyses of hepatocellular carcinoma were performed in living mice to assess the applicability of probe electrospray ionization–mass spectrometry (PESI–MS) in medical diagnosis. The number of peaks and the abundance of ions corresponding to triacylglycerols (TAGs) were higher in cancerous tissues than in noncancerous tissues. Multiple sequential scans of the specimens were performed along a predetermined line extending over the noncancerous region to detect the boundary of the cancerous region. Our system successfully discriminated the noncancerous and cancerous tissues based on the intensities of the TAG ions. These results highlight the potential application of PESI–MS for clinical diagnosis in cancer.     

Carcinogenesis alters fatty acid profile in breast tissue

Cancerogenesis is associated with cell membrane changes. The aim of this study was to investigate whether breast tissues with different degrees of cancer involvement have different fatty acid profiles. Fourteen breast cancer patients with a mean age of 61 years were recruited. Morphological features of the tumoral specimens were characterized. Approximately 60 % of patients had invasive ductal carcinoma, and 80 % were ER positive; 65 % were PR positive; and 65 % were HER2 negative. The segments with confirmed cancer had significantly less amounts of total lipids as compared with the corresponding grossly normal or interface tissues. The fatty acid profile in cancer tissue was significantly different from that in other tissues. Fatty acid composition of five classes of phospholipids revealed the variations between cancer tissue and the other two segments. A transition of changes in fatty acid composition in these fractions of phospholipids was observed. The interface tissue had intermediate amounts of several fatty acids including palmitic acid, stearic acid, and arachidonic acid. Interestingly, we observed significantly higher amounts of the n-3 fatty acid DHA in cancer tissue as compared to the other two tissues. Data from this study will provide evidence that biochemical changes particularly phospholipid composition may take place well in advance prior to morphological changes. Should this theory be confirmed by larger studies, deviation of phospholipid composition from normal values can be used as markers of susceptibility of tissue to cancer development.     

Disease-specific gene repositioning in breast cancer

Genomes are nonrandomly organized within the three-dimensional space of the cell nucleus. Here, we have identified several genes whose nuclear positions are altered in human invasive breast cancer compared with normal breast tissue. The changes in positioning are gene specific and are not a reflection of genomic instability within the cancer tissue. Repositioning events are specific to cancer and do not generally occur in noncancerous breast disease. Moreover, we show that the spatial positions of genes are highly consistent between individuals. Our data indicate that cancer cells have disease-specific gene distributions. These interphase gene positioning patterns may be used to identify cancer tissues.     

Human Breast Cancer Tissues Contain Abundant Phosphatidylcholine(36∶1) with High Stearoyl-CoA Desaturase-1 Expression

Breast cancer is the leading cause of cancer and mortality in women worldwide. Recent studies have argued that there is a close relationship between lipid synthesis and cancer progression because some enzymes related to lipid synthesis are overexpressed in breast cancer tissues. However, lipid distribution in breast cancer tissues has not been investigated. We aimed to visualize phosphatidylcholines (PCs) and lysoPCs (LPCs) in human breast cancer tissues by performing matrix assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a novel technique that enables the visualization of molecules comprehensively. Twenty-nine breast tissue samples were obtained during surgery and subjected to MALDI-IMS analysis. We evaluated the heterogeneity of the distribution of PCs and LPCs on the tissues. Three species [PC(32∶1), PC(34∶1), and PC(36∶1)] of PCs with 1 mono-unsaturated fatty acid chain and 1 saturated fatty acid chain (MUFA-PCs) and one [PC(34∶0)] of PCs with 2 saturated fatty acid chains (SFA-PC) were relatively localized in cancerous areas rather than the rest of the sections (named reference area). In addition, the LPCs did not show any biased distribution. The relative amounts of PC(36∶1) compared to PC(36∶0) and that of PC(36∶1) to LPC(18∶0) were significantly higher in the cancerous areas. The protein expression of stearoyl-CoA desaturase-1 (SCD1), which is a synthetic enzyme of MUFA, showed accumulation in the cancerous areas as observed by the results of immunohistochemical staining. The ratios were further analyzed considering the differences in expressions of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki67. The ratios of the signal intensity of PC(36∶1) to that of PC(36∶0) was higher in the lesions with positive ER expression. The contribution of SCD1 and other enzymes to the formation of the observed phospholipid composition is discussed.