Teruhiko Fujii

Protein Kinase C Promotes Apoptosis in LNCaP Prostate Cancer Cells through Activation of p38 MAPK and Inhibition of the Akt Survival Pathway

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKCα and the novel PKCδ isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574–7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645–655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKCα in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Pattern of Recurrence after Extended Radical Esophagectomy with Three-Field Lymph Node Dissection for Squamous Cell Carcinoma in the Thoracic Esophagus

Abstract. Factors responsible for recurrence of esophageal cancer were investigated in 90 patients who underwent extended radical esophagectomy with three-field dissection for a squamous cell carcinoma in the thoracic esophagus. The initial tumor recurrence was grouped as either locoregional (site of the primary tumor, anastomotic site, or lymph nodes) or as distant (distant organs, pleura, or peritoneum). Nineteen patients (21%) developed a locoregional recurrence, and 19 (21%) developed a distant recurrence. One (1%) developed both recurrences simultaneously and was classified as a distant recurrence. The locoregional recurrence was correlated with the stage factors, particularly the number of metastasis-positive nodes. For the distant recurrence, vascular invasion was found to have been the most important prognostic factor. Our findings suggested that locoregional recurrence was due to tumor progress related to the extent of lymph node metastasis, whereas distant recurrence was due to the oncologic behavior of the tumor. Locoregional recurrence in patients with limited disease may be reduced by extended radical esophagectomy with three-field dissection. Distant recurrence cannot be controlled by surgery. Adopted postoperative adjuvant therapies showed no effect on recurrence.

Optimal Lymphadenectomy for Squamous Cell Carcinoma in the Thoracic Esophagus: Comparing the Short- and Long-term Outcome among the Four Types of Lymphadenectomy

Controversy continues over the optimal extent of lymphadenectomy (regional versus three-field) for a potentially resectable squamous cell carcinoma in the thoracic esophagus. In the Consensus Conference of the International Society for Diseases of the Esophagus (ISDE), held in Munich in 1994, the types of lymphadenectomy were classified as standard, extended, total, or three-field lymphadenectomy. The objective of the present study was to determine the optimal procedure among these four types of lymphadenectomy. The mortality and morbidity rates, postoperative course, and survival rates were compared among 302 patients who underwent curative (R0) transthoracic esophagectomy with one of these four types of lymphadenectomy at Kurume University Hospital, Fukuoka, Japan, from 1986 to 1998. Three-field lymphadenectomy resulted in better survival than any other type of lymphadenectomy for patients with positive lymph node metastasis from a cancer in the upper or middle thoracic esophagus. A postoperative complication, such as recurrent laryngeal nerve paralysis, anastomotic leakage, and tracheal ischemic lesion, was significantly more common after three-field lymphadenectomy. However, the mortality rate was the same among the four procedures. Three-field lymphadenectomy was optimal for an upper or middle thoracic esophageal cancer with metastasis in the lymph node(s) based on improved long-term survival, whereas there was not a large difference in short-term and long-term outcomes after the four types of lymphadenectomy for a lower thoracic esophageal cancer.

Expression of HER2 and Estrogen Receptor α Depends upon Nuclear Localization of Y-Box Binding Protein-1 in Human Breast Cancers

In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER)alpha, ER beta, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ER alpha expression in ER alpha-positive, but not ER alpha-negative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ER alpha (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt-dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ER alpha.

Impact on Outcome of Additional Microvascular Anastomosis—Supercharge—on Colon Interposition for Esophageal Replacement: Comparative and Multivariate Analysis

Abstract. The impact on the outcome of an additional microvascular anastomosis—supercharge—on colon interposition for esophageal replacement was retrospectively evaluated by comparing it with colon interposition without supercharge. A series of 53 patients had undergone colon interposition for esophageal replacement at Kurume University Hospital from 1981 to 1996. The postoperative courses and the morbidity and mortality rates were compared between the 24 patients who underwent colon interposition without supercharge from 1981 to 1988 and the other 29 patients who underwent colon interposition with supercharge from 1989 to 1996. Risk factors for leakage of the esophagocolostomy and for hospital mortality after colon interposition were evaluated by multivariate analysis. Colon interposition with supercharge required a longer operation time but resulted in a lower incidence of necrosis in the colon graft and leakage in the esophagocolostomy (Odds ratio = 34), a shorter duration until peroral intake, and a shorter hospital stay compared to colonic interposition without supercharge. The addition of supercharge to colon interposition for esophageal replacement has been an effective option that has prevented serious complications caused by graft ischemia.

Bortezomib sensitizes human esophageal squamous cell carcinoma cells to TRAIL-mediated apoptosis via activation of both extrinsic and intrinsic apoptosis pathways

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive human cancers, and novel treatment modalities are required. We investigated the therapeutic potential of the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) in combination with the proteasome inhibitor bortezomib (Velcade) on human ESCC cell lines. Bortezomib enhanced the susceptibility to TRAIL in 12 of the 15 ESCC cell lines tested, although most showed low sensitivity to TRAIL as a single agent. The enhancement of TRAIL-induced apoptosis by bortezomib was caspase dependent. Increased processing of caspase-8 often accompanied enhancement of TRAIL-induced apoptosis by bortezomib. However, the increased cell surface expression of death receptors observed on bortezomib treatment did not seem to be crucial for this effect. For some ESCC, bortezomib treatment resulted in a more efficient recruitment of caspase-8 and the Fas-associated death domain to the death-inducing signaling complex. Additional downregulation of the cellular FLICE-inhibitory protein long isoform [c-FLIP(L)] could cooperate in the activation of the extrinsic pathway in some cases. For other ESCC, the crucial effect of bortezomib treatment seemed to be increased signaling via the intrinsic apoptotic pathway on subsequent exposure to TRAIL. Thus, bortezomib could sensitize ESCC to TRAIL apoptosis by multiple molecular mechanisms of action. Therefore, the combination of bortezomib and TRAIL might be a novel therapeutic strategy for ESCC patients who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway. Mol Cancer Ther; 9(6); 1842–51. ©2010 AACR.

FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis

The forkhead box protein 3 (FOXP3) transcription factor is highly expressed in tumor cells as well as in regulatory T cells (Tregs). It plays a tumor-enhancing role in Tregs and suppresses carcinogenesis as a potent repressor of several oncogenes. The clinical prognostic value of FOXP3 expression has not yet been elucidated. In this study, immunohistochemistry was used to investigate the prognostic significance of FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in breast cancer patients. Of the 100 tumor specimens obtained from primary invasive breast carcinoma, 63 and 57% were evaluated as FOXP3+ tumor cells and as being highly infiltrated by FOXP3+ lymphocytes, respectively. Although FOXP3 expression in tumor cells was of no prognostic significance, FOXP3+ lymphocytes were significantly associated with poor overall survival (OS) (n=98, log-rank test P=0.008). FOXP3 exhibited a heterogeneous subcellular localization in tumor cells (cytoplasm, 31%; nucleus, 26%; both, 6%) and, although cytoplasmic FOXP3 was associated with poor OS (P= 0.058), nuclear FOXP3 demonstrated a significant association with improved OS (P=0.016). Furthermore, when patients were grouped according to their expression of tumor cytoplasmic FOXP3 and lymphocyte FOXP3, there were notable differences in the Kaplan-Meier curves for OS (P<0.001), with a high infiltration of FOXP3+ lymphocytes accompanied by a cytoplasmic FOXP3+ tumor being the most detrimental phenotype. These findings indicated that FOXP3 expression in lymphocytes as well as in tumor cells may be a prognostic marker for breast cancer. FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its localization, which may help establish appropriate cancer treatments.

Cell growth inhibition by all‐trans retinoic acid in SKBR‐3 breast cancer cells: Involvement of protein kinase Cα and extracellular signal‐regulated kinase mitogen‐activated protein kinase

All‐trans retinoic acid (ATRA), a synthetic derivative of vitamin A, inhibits the growth of breast cancer cells. To elucidate the mechanism by which ATRA causes cell growth inhibition, we examined changes in cell cycle and intracellular signaling pathways, focusing on protein kinase C (PKC) and mitogen‐activated protein kinase (MAPK). Using the estrogen receptor‐negative, retinoid receptor–positive breast cancer cell line SKRB‐3, we found that treatment with ATRA significantly decreased the expression of PKCα, as well as reducing ERK MAPK phosphorylation. ATRA treatment leads to dephosphorylation of Rb, and consequently to G1 arrest. Marked changes in the expression of cyclins (particularly cyclins A and E) were observed in SKBR‐3 cells treated with ATRA. Using a series of pharmacological and molecular approaches, we found evidence that ATRA‐induced SKBR‐3 cell growth inhibition involves the deregulation of the PKCα‐MAPK pathway. These data suggest that retinoids interfered with signal transduction pathways that are crucial for cell cycle progression, and highlight the complexities of the biological effects of retinoid derivatives.

Apparent Diffusion Coefficient in Invasive Ductal Breast Carcinoma: Correlation with Detailed Histologic Features and the Enhancement Ratio on Dynamic Contrast-Enhanced MR Images.

Purpose. To investigate the correlation of Apperent Diffusion Coefficient (ADC) values in invasive ductal breast carcinomas with detailed histologic features and enhancement ratios on dynamic contrast-enhanced MRI. Methods and Materials. Dynamic MR images and diffusion-weighted images (DWIs) of invasive ductal breast carcinomas were reviewed in 25 (26 lesions) women. In each patient, DWI, T2WI, T1WI, and dynamic images were obtained. The ADC values of the 26 carcinomas were calculated with b-factors of 0 and 1000 s/mm2 using echoplanar DWI. Correlations of the ADC values were examined on dynamic MRI with enhancement ratios (early to delayed phase: E/D ratio) and detailed histologic findings for each lesion, including cellular density, the size of cancer nests, and architectural features of the stroma (broad, narrow, and delicate) between cancer nests. Results. The mean ADC was 0.915 ± 0.151 × 10−3 mm2/sec. Cellular density was significantly correlated with ADC values (P = .0184) and E/D ratios (P = .0315). The ADC values were also significantly correlated to features of the stroma (broad to narrow, P = .0366). Conclusion. The findings suggest that DWIs reflect the growth patterns of carcinomas, including cellular density and architectural features of the stroma, and E/D ratios may also be closely correlated to cellular density.

Primary amebic meningoencephalitis due to Naegleria fowleri: An autopsy case in Japan

Free‐living amebas represented by Naegleria fowleri, Acanthamoeba and Balamutia have been known to cause fatal meningoencephalitis since Fowler and Carter (1965) reported the first four human cases. An autopsy case of a 25‐year‐old female with primary amebic meningoencephalitis (PAM) due to Naegleria fowleri is described. Headache, lethargy and coma developed in this patient, and her condition progressed to death 8 days after the onset of clinical symptoms. Cerebral spinal fluid examination confirmed clusters of amebas, which were grown in culture and identified as Naegleria fowleri. At autopsy, lesions were seen in the central nervous system (CNS) and the ethmoid sinus. The CNS had severe, suppurative meningoencephalitis with amebic trophozoites mingled with macrophages. This case is the first report of PAM due to Naegleria fowleri in Japan.