Masakazu Souda

Aldehyde dehydrogenase 1 expression predicts poor prognosis in triple-negative breast cancer

Ohi Y, Umekita Y, Yoshioka T, Souda M, Rai Y, Sagara Y, Sagara Y, Sagara Y & Tanimoto A
(2011) Histopathology59, 776–780

High mobility group box 1 is upregulated after spinal cord injury and is associated with neuronal cell apoptosis.

Study Design. Cerebrocortical culture and rat spinal cord injury (SCI) model were used to examine the expression of high mobility group box 1 (HMGB1), TNF-&agr;, and Rage by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical examination. In addition, relationship between upregulation of HMGB1 and neural cells apoptosis was evaluated after SCI. Objective. To evaluate the upregulation of HMGB1, TNF-&agr;, and Rage after SCI. Summary of Background Data. It is known that the mode of delayed neuronal cell death after SCI is apoptosis. Apoptotic cell death is influenced by several injury-promoting factors which include pro-inflammatory cytokines. Inhibition of apoptosis promotes neurologic improvement following SCI. However, the factors which transmit inflammatory signaling following SCI have not yet been clarified in detail. HMGB1 was reported as an important mediator of inflammation. We examined the expression of HMGB1, TNF-&agr; and Rage following acute SCI. Methods. Expression of HMGB1, TNF-&agr; and Rage was examined by RT-PCR and immunohistochemical examination. Apoptotic cell death was evaluated by TUNEL methods. Results. HMGB1 was exported from nuclei to cytoplasm in active caspase-3 positive apoptotic cell in vitro. In addition, HMGB1, TNF-&agr;, and Rage was expressed in same cell after NMDA treatment. RT-PCR revealed that expression of HMGB1 and TNF-&agr; was upregulated following SCI. Immunohistochemical examination revealed that the numbers of HMGB1-, TNF-&agr;-, and Rage-positive cells were increased following SCI. The number of TUNEL-positive cells was significantly increased at 12 hours after injury, and was maximal at 72 hours after injury. However, HMGB1- and TNF-&agr;-positive cells were maximal in number 48 hours after injury, while Rage-positive cells were maximal in number at 24 hours after injury. These data suggest that HMGB1, TNF-&agr;, and Rage were upregulated following SCI but preceding the apoptotic cell death. Conclusion. Our findings suggest that HMGB1 play a role in the induction of apoptosis via inflammatory reaction.

Aldehyde dehydrogenase 1 expression is a predictor of poor prognosis in node-positive breast cancers: a long-term follow-up study

Yoshioka T, Umekita Y, Ohi Y, Souda M, Sagara Y, Sagara Y, Sagara Y, Rai Y & Tanimoto A
(2011) Histopathology58, 608–616
Aldehyde dehydrogenase 1 expression is a predictor of poor prognosis in node‐positive breast cancers: a long‐term follow‐up study

Expression of wild-type estrogen receptor β protein in human breast cancer: Specific correlation with HER2/neu overexpression

Expression of estrogen receptor β (ERβ) protein in human breast cancer and correlation with clinicopathological factors have been reported by many investigators, but many of them used ERβ antibodies that react with both wild‐type ERβ (ERβwt) and splicing variant isoform. Therefore, the frequency and correlation with clinicopathological factors of ERβwt expression remain to be established. In the present study a monoclonal antibody EMR02, specific for ERβwt, was used in formalin‐fixed paraffin‐embedded sections from 225 female primary breast cancer patients diagnosed as having invasive ductal carcinoma. Expression of ERα, progesterone receptor (PgR) and HER2/neu were also investigated by immunohistochemistry. For ERβwt, ERα and PgR, positivity was defined as nuclear staining in >10% of the cancer cells. HER2/neu overexpression was defined as a Hercep test score 3+. Positivity for ERβwt, ERα, PgR and HER2/neu overexpression was 55%, 74%, 61% and 25%, respectively. The expression of ERβwt had a positive correlation with ERα (P = 0.018) and PgR (P = 0.02). There was significant positive correlation between ERβwt expression and HER2/neu overexpression (P < 0.0001). According to multivariate logistic regression analysis the most significant association was between ERβwt expression and HER2/neu overexpression (P < 0.0001). These results suggest that clinical significances of ERβwt expression in human breast cancer patients may be more complex.

Effects of Neonatally Administered Diethylstilbestrol on Induction of Mammary Carcinomas Induced by 7, 12-Dimethylbenz[A]Anthracene in Female Rats

Various doses of diethylstilbestrol (DES) were administered to rats once at birth. Thereafter, at 50 days after birth, the rats in all groups were given 10 mg 7, 12-dimethylbenz[a]anthracene (DMBA) and undergone necropsy at 300 days after birth. The incidence of mammary carcinomas (MCs) were 50, 54, 91, 39, 19% at 175 days after birth, and 77, 87, 100, 85, 75% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. The incidence of rats without corpus luteum were 0, 0, 0, 30, 100% at 50 days after birth, and 0, 40, 53, 93, 100% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. Observation of the whole mount specimens showed a higher number of terminal end buds (TEBs) in the 1 μg group and a lower number in the 100 μg group compared with the control at 50 days after birth. It suggested that the administration of a relatively low dose (1 μg) of DES during neonatal period may increase TEBs, thus resulting in a stimulatory effect on the initiation of MCs.

Maspin expression is frequent and correlates with basal markers in triple-negative breast cancer.

BackgroundMaspin is a unique member of the serine protease inhibitor superfamily and its expression is found in myoepithelial cells of normal mammary glands; therefore, it has been considered to be a myoepithelial marker. We previously reported that maspin was frequently expressed in biologically aggressive breast cancers. In turn, triple-negative (TN) breast cancer is a subtype of tumor with aggressive clinical behavior and shows frequent expression of basal markers. We hypothesized that maspin expression may be frequent and correlate with basal rather than myoepithelial markers in TN breast cancer.MethodsParaffin-embedded 135 TN invasive ductal carcinoma tissue samples were immunohistochemically investigated using the Dako Envision+ kit and primary antibodies for maspin, basal (CK5/6, EGFR, CK14) and myoepithelial markers (p63, CD10). The correlation between maspin expression and relapse-free survival (RFS) was investigated by the log-rank test.ResultsThe positive rate for maspin was 85.9% and significantly correlated with younger age (P = 0.0015), higher histological grade (P = 0.0013), CK5/6 positivity (P < 0.0001), CK14 positivity (P = 0.0034) and the basal-like subtype defined by CK5/6, EGFR and CK14 positivity (P = 0.013). The positive rates for CK5/6, EGFR, CK14, CD10 and p63 were 59.2%, 48.9%, 34.1%, 17.8% and 12.6%, respectively. There was no significant correlation between maspin expression and RFS.ConclusionsThe positive rate for maspin is the highest among known basal and myoepithelial markers, and strongly correlates with basal markers in TN breast cancer. These results suggested that maspin could be a candidate for a therapeutic target for TN breast cancer.

Gene expression profile of terminal end buds in rat mammary glands exposed to diethylstilbestrol in neonatal period

Diethlstilbestrol (DES) is a synthetic estrogen prescribed to several millions of pregnant women worldwide. The risk for breast cancer after age 40 in women prenatally exposed to DES has been reported; however, the precise mechanism of susceptibility to breast cancer remains to be resolved. We investigated the global gene expression profile of terminal end buds (TEBs), the target of carcinogen, in rat mammary glands neonatally exposed to a low- or high-dose DES at postnatal days (PND) 35 and 45, equivalent to the peripubertal stage in humans. In all groups, the number of TEBs gradually increased, peaked at PND35 and decreased at PND49. At PND35 and 49, the low-dose DES-treated group (low-DES group) showed the highest number of TEBs. In the low-DES group at PND35, β-casein, γ-casein and whey acidic protein (WAP) mRNA expression increased 8.2-fold, 26.1-fold and 13.3-fold, respectively, whereas γ-casein and WAP mRNA decreased 17.6-fold and 27.7-fold, respectively, at PND49. The most significant network revealed by Ingenuity Pathway Analysis (IPA) software showed the relevance of NF-κB in low-DES group. The present findings suggest that the deregulation of mammary gland differentiation and development-related genes could be induced and cause the increased number of terminal duct lobular units (TDLUs) in human mammary glands of DES daughters in a critical period of mammary gland development.

Induction of Sarcomas by a Single Subcutaneous Injection of 7,12-Dimethylbenz[a]anthracene into Neonatal Male Sprague–Dawley Rats: Histopathological and Immunohistochemical Analyses

Animal experiments have shown that carcinogenicity of chemicals is higher in fetal or neonatal periods than adult. We investigated sensitivities to a carcinogen in peri-neonatal rats with a model of sarcomas-induction by a subcutaneous injection of chemo-carcinogen that has rarely done in neonatal rats. Neonatal male SD rats were injected with 7,12-DMBA 10, 100, and 500 μg, which resulted in sarcomas-induction in 0, 62, and 94% of rats. Male SD rats were injected with DMBA 500 μg at 0, 3, 7, 14, and 21 days, which resulted in sarcomas-induction in 94, 70, 64, 50, and 44% of rats. Although the induced sarcomas were occasionally in mixed morphological feature as previous reports for sarcomas of rat, each was immunohistochemically in almost monotonous pattern, and classification was feasible. The incidence of rhabdomyosarcomas was higher in rats neonatally injected with a higher dose of DMBA than a lower dose, and in rats injected at peri-neonatal periods than later periods. In histological observations for the site of injection before overt sarcomas develop, clusters of atypical mesenchymal cells emerged as previous studies, but also those were immunohistochemically differentiated into rhabdomyocytes and other mesenchymal cells. We consider these findings may contribute a little to elucidation of process of sarcomas-induction in rats.

Gene expression profiling during rat mammary carcinogenesis induced by 7,12‐dimethylbenz[a]anthracene

7,12‐Dimethylbenz[a]anthracene (DMBA)‐induced rat mammary carcinoma is a well‐recognized model; however, the genetic alterations during its carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate cells from terminal end buds (TEBs), the origin of carcinoma, at 2 weeks after sesame oil treatment (control) or DMBA treatment (DMBA‐TEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). Using an oligonucleotide microarray representing 20,600 rat probe sequences, we analyzed gene expression profiles and validated mRNA and protein levels of genes of interest byreal‐time quantitative PCR and immunohistochemistry. The number of differentially expressed genes dramatically increased from DMBA‐TEBs (63) to DCIS (798) and MC (981). Only the expression of PEP‐19, an anti‐apoptotic gene, showed significant increases in DMBA‐TEBs (4‐fold), DCIS (10‐fold) and MC (16‐fold). MMP‐13 expression was increased markedly in DCIS (19‐fold) and MC (61‐fold) while OPN expression was increased 6‐fold in DCIS and 8‐fold in MC. MMP‐7 expression was increased 4‐fold in MC. Nidogen‐1; a participant in the assembly of basement membranes, TSP‐2; an inhibitor of angiogenesis and COUP‐TFI; a transcription repressor showed significant decreases in DCIS (4‐, 9‐ and 17‐fold, respectively) and MC (10‐, 37‐ and 100‐fold). Network analyses with IPA software revealed that the most significant network included Akt groups in DCIS and ERK groups in MC. The present findings provide us with a better understanding of the molecular alteration that occur during mammary carcinogenesis and suggest the importance of PEP‐19 overexpression in the very early stage of mammary carcinogenesis.

Ghrelin improves intestinal mucosal atrophy during parenteral nutrition: An experimental study

BACKGROUND/PURPOSE Total parenteral nutrition (TPN) has been reported to be associated with mucosal atrophy of the small intestine. Ghrelin has hormonal, orexigenic, and metabolic activities. We investigated whether ghrelin improved intestinal mucosal atrophy using a TPN-supported rat model. METHODS Rats underwent jugular vein catheterization and were divided into four groups: TPN alone (TPN), TPN plus low-dose ghrelin (TPNLG), TPN plus high-dose ghrelin (TPNHG), and oral feeding with normal chow (OF). Ghrelin was administered continuously at dosages of 10 or 50 μg/kg/day. On day 6 rats were euthanized, and the small intestine was harvested and divided into the jejunum and ileum. Then the villus height (VH) and crypt depth (CD) were evaluated. RESULTS The jejunal and ileal VH and CD in the TPN group were significantly decreased compared with those in the OF group. TPNHG improved only VH of the jejunum. TPNLG improved VH and CD of the jejunum and CD of the ileum. The improvement of TPNLG was significantly stronger than that in CD of the jejunum and ileum. CONCLUSIONS TPN was more strongly associated with mucosal atrophy in the jejunum than in the ileum. Low-dose intravenous administration of ghrelin improved TPN-associated intestinal mucosal atrophy more effectively than high-dose administration.