Gonadotropin-releasing hormone retards doxorubicin-induced apoptosis and serine/threonine phosphatase inhibition in ovarian cancer cells

We recently demonstrated serine/threonine phosphatase (protein phosphatase 2A, PP2A), a crucial enzyme in apoptosis control, within the plasma membrane as well as soluble fraction. This study aimed to determine whether gonadotropin-releasing hormone (GnRH) affects the membrane PP2A-associated apoptosis and the enzyme activity in ovarian cancer cells. PP2A activity was assessed by measuring the dephosphorylation of phosphopeptide highly selective for the PP2A in plasma membranes isolated from ovarian cancer SK-Ov-3 and Caov-3 cells. Apoptosis was quantified by nuclear morphology after staining with Hoechst 33342 and by loss of plasma membrane phospholipid asymmetry using Cy3-conjugated annexin-V. Treatment with doxorubicin (1 microM) for 48 h caused parallel increases in the percentage of cells undergoing apoptosis (71.3+/-5.5% vs. 2.2+/-0.8% for control, P<0.01) and decrease in the membrane-associated PP2A activity (to 38.5+/-8.2% of control, P<0.01). In cells simultaneously incubated with GnRH agonist leuprolide (1 microM) for 48 h, the maximum doxorubicine-induced apoptosis and membrane PP2A inhibition were reduced to 38.4+/-6.2% (P<0.01) and to 82.1+/-7.3% of control (P<0.01), respectively. The GnRH agonist alone caused apoptotic cell death accounting for only 6.8+/-2.1% (P<0.01) and had no effect on membrane PP2A activity. These findings demonstrate that PP2A inhibition is closely coupled to the onset of apoptosis in ovarian cancer cells exposed to doxorubicin. GnRH agonist may protect against the inhibition of membrane-associated PP2A activity and thus retard doxorubicin-induced apoptosis, suggesting an additional activity of GnRH to protect ovarian cancer cells from stimulated apoptotic cell death.     

The extending range of the tumor is a more suitable predictive risk factor for lymph node metastases than the location of the deepest tumor invasion in distal thoracic esophageal and cardiac cancer

In the regional lymph node grouping of esophageal cancer by the Japanese Guide Line (the 9th edition), the location of the deepest tumor invasion has precedence in determination. In this study, we investigated the extending range of the tumor and the location of the deepest tumor invasion to ascertain which was the more important factor in predicting lymph node metastases. We examined 52 patients with distal thoracic esophageal and cardiac cancer who had undergone esophagectomy with three-field lymph node dis-section. Among the 52 patients, 16 were found to be positive and 36 negative, for middle-upper mediastinum and cervical lymph node metastases, and the two groups were compared in terms of detailed parameters of the tumor's location. In the result, the distance from the esophagogastric junction (EGJ) to the proximal margin of the tumor was significantly longer in the metastatic group (p=0.005). In univariate logistic regression with this parameter as the independent variable, we obtained a statistically significant result (p=0.0115, odds ratio=1.041, 95% confidence interval=1.009-1.073, R2=0.1169). On the other hand, when the distance from the EGJ to the deepest portion of the tumor was used as the independent variable, p=0.0742, odds ratio=1.045, 95% confidence interval=0.996-1.096 and R2=0.0577. Multiple logistic regression was performed with these two parameters, and the distance from the EGJ to the proximal margin of the tumor was a more important factor than the distance from the EGJ to the deepest portion of the tumor (p=0.0553 vs. 0.9161). We concluded that the extending range of the tumor was a more suitable predictive risk factor of lymph node metastases than the location of the deepest tumor invasion in distal thoracic esophageal and cardiac cancer. We should seriously consider the extending range of the tumor in the regional lymph node grouping of the Japanese Guide Line.     

The modifier subunit of glutamate cysteine ligase relates to cisplatin resistance in human small cell lung cancer xenografts in vivo

Glutamate cysteine ligase (GCL) plays an important role in the intracellular detoxification of cisplatin (CDDP). GCL is composed of a modifier or light chain subunit (GCLM) and a catalytic or heavy chain subunit (GCLC). Previously, we showed that the GCL subunits enhanced CDDP-resistance in non-small cell lung cancer (NSCLC) xenografts. In small cell lung cancer (SCLC), it is unclear whether the GCL subunits are essential to CDDP-resistance. We examined the gene expression of GCLM and GCLC in four human SCLC xenografts with the real-time polymerase chain reaction (PCR). An in vivo drug sensitivity test with CDDP was performed on the SCLC xenografts. CDDP-resistance was examined as the growth ratio of the relative volume of the treated xenografts to the controls (T/C%). The expression level of GCLM gene in SCLC was significantly lower than that in NSCLC (p=0.0026, Welch's t-test). One of four SCLC xenografts showed 62% of T/C and this was evaluated as CDDP-resistance, while the other three xenografts were sensitive to CDDP in vivo (Mann-Whitney U-test, p<0.01, one-sided). The expression level of the GCLM gene was significantly correlated to T/C% (Fisher's test, p=0.0289, correlations = 0.975), while the GCLC gene expression level was not associated with T/C%. These results suggest that the overexpression of GCLM is correlated with CDDP-resistance in SCLC xenografts in vivo.     

Efficacy of lamivudine therapy for decompensated liver cirrhosis due to hepatitis B virus with or without hepatocellular carcinoma

The prognosis for patients with decompensated hepatitis B virus (HBV) related liver cirrhosis (LC-B), especially for those with LC-B complicated with hepatocellular carcinoma (HCC), is poor. We investigated the effects of lamivudine in patients with decompensated LC-B, with and without HCC. Decompensated LC-B patients (n=55) with Child-Pugh classification scores (CPS) >7 points were enrolled. All were admitted to the hospitals of the authors between January 1997 and December 2004. Decompensated cases due to a severe exacerbation of hepatitis with CH-B and patients with HCC showing an extra hepatic metastasis or portal vein tumor thrombus were excluded. Some 19 cases (including 5 cases complicated with HCC at the start of therapy) were treated with lamivudine at 100 mg/day (L group), and 36 (including 7 cases with HCC at time of admittance) were treated without lamivudine (non-L group). The median of CPS points in the L group was higher than that of non-L group (11 points versus 9 points, p<0.02). Prothrombin time (%), albumin, ascites, CPS, and HBV-DNA quantity were each significantly improved after 6 months in the L group (p<0.05). A mutation in the YMDD motif was observed in 5 patients in the L group, however liver function did not deteriorate. Further, the survival rate was significantly higher in the L group (p<0.05). HCC was found in 3 L group and 4 non-L group patients during the study. In the L group, all patients complicated with HCC were treated repeatedly or until cured, whereas 91% of those in the non-L group could not be treated (p<0.01). Our results suggest that lamivudine is a useful and important therapy for patients with decompensated LC-B with and without HCC, as well as those who are restricted from having liver transplantation.     

Multiple endocrine neoplasia type 1 gene mutations in sporadic gastrinomas in Japan

The molecular mechanisms responsible for the development and/or progression of gastrinomas are largely unknown. Studies involving sporadic enteropancreatic neuro-endocrine tumors suggest that mutations in the MEN1 gene occur in some tumors and probably play an important role in their pathogenesis. In this study, we examined whether somatic mutations in the MEN1 gene are also responsible for sporadic gastrinomas and correlate with clinical manifestations of gastrinomas in Japanese patients. Genomic DNA was extracted from paraffin-embedded gastrinoma tissues from 12 patients. Nucleotide sequences in the MEN1 genes were determined by direct sequencing. We identified 6 mutations in 7 out of 12 examined gastrinomas (58%). The identified mutations were 1 non-sense, 2 missense, 1 deletion leading to frame shifts, 1 insertion and 1 splicing mutation. Identical mutations were found in three gastrinoma tissues. The age at surgery, the rate of hepatic metastasis, and tumor status were not significantly different in the 2 groups. This study demonstrates that alterations in the MEN1 gene are involved in about half of all sporadic gastrinomas, although no correlation between the presence of mutations and location and clinical phenotype or severity of disease has been found.     

Immunohistochemical analysis of p53 and MIB-1 in tissue specimens obtained from endoscopic ultrasonography-guided fine needle aspiration biopsy for the diagnosis of solid pancreatic masses

Endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) has been shown to be a highly accurate technique for distinguishing benign from malignant pancreatic masses. In this study, we examined p53 immunohistochemical analysis in FNAB specimens obtained from solid pancreatic diseases, and prospectively evaluated clinical applications for the diagnosis of malignancy in combination routine histological examination. Tissue specimens obtained from 62 pancreatic masses (51 pancreatic cancers and 11 chronic pancreatitis) by EUS-FNAB were evaluated by routine histological examination and p53 immunostaining. The conventional EUS-FNA diagnostic test statistics for the pancreatic masses were as follows: 76% sensitivity, 91% specificity and 79% accuracy. p53 protein overexpression was observed in 67% patients with pancreatic cancer, but not in patients with chronic pancreatitis. If the diagnosis of malignancy was made using the combination of p53 protein overexpression and conventional histological examination, the diagnostic test statistics changed as follows: 90% sensitivity, 91% specificity and 92% accuracy. p53 immunostaining in combination with routine histological examination of EUS-FNAB may improve the diagnostic accuracy for pancreatic cancer.     

Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE.

PURPOSE: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE. EXPERIMENTAL DESIGN: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8(+) T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8(+) T cells sensitized with autologous CD4(+) PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. RESULTS: After DNA vaccination, murine CD8(+) T cells recognizing MAGE-A4(143-151) or SAGE(715-723) in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8(+) T cells with autologous CD4(+) PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE(715-723)-specific precursors was observed in HLA-A2402-positive healthy individuals. CONCLUSIONS: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4(143-151) and SAGE(715-723), were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.     

Important prognostic histological parameters for patients with invasive ductal carcinoma of the pancreas

For clinicians, the orthodox histological investigation of patients with invasive ductal carcinoma (IDC) remains important for predicting prognoses. The purpose of the present study was to determine the most important of the known prognostic histological parameters (including fibrotic focus and tumor necrosis), enabling the outcomes of 101 patients with IDC of the pancreas to be predicted accurately. Furthermore, we established a scoring classification consisting of important prognostic histological parameters examined in this study. Multivariate survival analyses showed that invasive tumor size of more than 3 cm, the presence of tumor necrosis, the presence of nerve plexus invasion and lymph vessel invasion scores of 2 or 3 were important prognostic factors. Our scoring classification, consisting of the above four parameters, accurately classified the outcome of patients independent of the invasive tumor size of IDC. We concluded that invasive tumor size of 3 cm or more, the presence of tumor necrosis, the presence of nerve plexus invasion and lymph vessel invasion scores of 2 or 3 are important histological prognostic parameters for patients with IDC of the pancreas. Furthermore, the scoring system consisting of the above four histological parameters is probably a very useful prognostic histological classification for patients with IDC of the pancreas.     

Japanese multicenter phase II study of CHOP followed by radiotherapy in stage I-II, diffuse large B-cell lymphoma of the stomach

CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) followed by radiotherapy is regarded as standard care for localized aggressive lymphoma; however, prospective confirmation of its applicability to localized primary gastric lymphoma is inadequate, and most patients in Japan have been initially treated with gastrectomy. We conducted a multicenter phase II study to evaluate the feasibility and efficacy of the non-surgical treatment. Eligibility criteria required primary gastric diffuse large B-cell lymphoma, stage I-II(1), age 20-75, performance status 0-1 and adequate organ function. Treatment consisted of three cycles of CHOP followed by radiotherapy 40.5 Gy. Fifty-five patients were enrolled between December 1999 and February 2003, and 52 eligible patients were analyzed. Patient characteristics were as follows: median age, 61 years; 28 men, 24 women; 36 with stage I, 16 with stage II(1); 47 with a low International Prognostic Index (IPI) and five with a low-intermediate IPI. All but one patient completed planned treatment. No serious complications including massive hemorrhage or perforation were observed. A complete response was achieved in 48 of the 52 patients (92%, 95% confidence interval: 82-98%) and progressive disease in three. Two patients underwent salvage gastrectomy due to disease persistence or recurrence. With a median follow-up period of 28 months, 2-year progression-free and overall survivals were 88 and 94%, respectively. CHOP followed by radiotherapy is safe and highly effective in localized gastric diffuse large B-cell lymphoma. This organ-preserving treatment should be considered as a very reasonable therapeutic option.