Ikuya Fujiwara

The molecular mechanism of apoptosis induced by xenogeneic cytotoxicity

Abstract: In order to clarify the role of natural killer (NK) cells in delayed xenograft rejection (DXR) of discordant xenotransplantation, we used in vitro xenogeneic combination of human NK cells and pig kidney target cells (PK13, and investigated the mechanism of xenogeneic cytotoxicity caused by human NK cells. In the presence of decomplemented human serum or human IgG, freshly isolated human peripheral blood lymphocytes (PBLs) caused both membrane (51Cr release) and DNA (3H release) damage on PK15. In contrast, only membrane damage was detected in the presence of normal human serum. To clarify the participation of perforin/granzymes‐cell mediated cytotoxicity (P/G‐CMC), when EGTA or concanamycin B (CMB) was added to the cytotoxicity assays, both cytotoxicities were completely inhibited by these drugs in a dose‐dependent manner. In terms of the involvement of Fas/FasL‐based cytotoxicity (F‐CMC), while the cytotoxicity assays were performed in the presence of antagonistic anti‐human FasL mAb, this antibody was not able to block the cytotoxicity. From these results, it is concluded that xenogeneic cytotoxicity is due to NK cell dependent ADCC (antibody‐dependent cell‐mediated cytotoxicity), and their effector mechanism can cause apoptosis on target cells via P/G‐CMC.

Video-assisted skin-sparing breast-conserving surgery for breast cancer and immediate reconstruction with autologous tissue.

Objective:To analyze therapeutic results of video-assisted breast-conserving surgery (VA-BCS) for early stage breast cancer. Background:VA-BCS for breast cancer has been developed in Japan, and is indicated for breast cancer unaccompanied by skin involvement. The surgical incision is made at an inconspicuous site, followed by skin-sparing partial mastectomy (SSPM) and immediate reconstruction of the breast. This technique affords good cosmetic results. The long-term results are reported herein. Methods:VA-BCS was performed on 551 patients. The skin incision was made as a peri-areolar incision or at the midaxillary line. Skin-sparing partial mastectomy was performed using an endoscope and the lifting and tunneling method. Morbidity, curability, and degree of satisfaction with regard to cosmesis were analyzed. Results:Skin necrosis in 22 patients (4.0%) and necrosis of fatty tissue-muscle flap in 17 patients (3.1%) were recorded as postoperative complications. No other serious complications were encountered. Local recurrence occurred in 23 patients (4.2%) after a mean follow-up of 38.4 months. Distant-metastasis-free survival rate at 66 months was 100% for Tis, 95.5% for T1, and 90.7% for T2. Overall survival rate was 100% for Tis, 97.3% for T1, and 95.7% for T2. Degree of satisfaction with surgery as investigated by questionnaire was “good” for 76.1% of patients. Conclusion:VA-BCS for early stage breast cancer showed no association with increases in local or distant organ recurrence. The technique yielded improved cosmesis and a high degree of patient satisfaction. Follow-up observation of patients for a longer period is necessary, but VA-BCS seems useful for local treatment of breast cancer.

Aberrant Expression of Fra-1 in Estrogen Receptor-negative Breast Cancers and Suppression of their Propagation In Vivo by Ascochlorin, an Antibiotic that Inhibits Cellular Activator Protein-1 Activity

Estrogen receptor-negative breast cancers generally are highly malignant, resistant to chemotherapy and poorly prognostic. Here we demonstrate that estrogen receptor-negative human breast cancer cell lines highly express Fra-1, c-Fos and c-Jun, components of the transcription factor, activator protein-1 (AP-1). Retrospective observation of breast cancer tissues obtained by core needle biopsy before surgery from stages II and III patients demonstrates that Fra-1 expression is high in estrogen receptor-negative human breast cancers, and negatively correlated to paclitaxel sensitivity. Ascochlorin, which suppresses cellular AP-1 activity, selectively kills estrogen receptor-negative human and mouse breast cancer cell lines, and prolongs the survival time of mice implanted with an estrogen receptor-negative mammary carcinoma. These results suggest that chemotherapy targeting AP-1 activity is a potent strategy for estrogen receptor-negative human breast cancers.

Development of HER2-antagonistic peptides as novel anti-breast cancer drugs by in silico methods

An antagonistic peptide called HRAP that binds to the human HER2 molecule was designed by our computational method. In silico docking study demonstrated the specific interaction of HRAP with the dimerization domain in the HER2 molecule. Interestingly, HRAP inhibited proliferation of HER2-overexpressed human breast cancer cell lines. However, it had little cellular cytotoxicity (apoptosis inducibility). The cell proliferation inhibition was associated with the suppression of phosphorylation of PTEN and Akt. Thus, HRAP is the first HER2-binding small peptide antagonist rationally designed by a computer-aided SBDD method and is useful for the development of peptide mimetics to generate novel anti-breast cancer drugs.

Caspase-dependent apoptosis by ectopic expression of E2F-4

E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

Enhancement of paclitaxel-induced apoptosis in HER2-overexpressing human breast cancer cells by a pertuzumab mimetic peptide, HRAP

HRAP, a pentapeptide designed to bind with the pertuzumab interacting site in an extracellular domain of the HER2 molecule, enhanced the cytotoxicity of paclitaxel in HER2-overexpressing human breast cancer cell lines, BT474 and SKBR-3, but not in MDA-231 cells, which express lower levels of HER2. HRAP enhanced mitochondria-dependent apoptosis induced by paclitaxel in SKBR-3 and BT-474, but not in MDA-231. HRAP enhanced the inhibition of phosphorylation of serine 473 in Akt and Ser380/Thy382/The383 in PTEN. These results suggest that HRAP enhances paclitaxel-induced apoptosis in a manner dependent on the PTEN/Akt signal transduction pathway.

Phase II Clinical Trial of First-line Eribulin Plus Trastuzumab for Advanced or Recurrent HER2-positive Breast Cancer

Background/Aim: Eribulin mesylate has been approved for advanced or metastatic breast cancers subjected to at least two previous chemotherapy regimens. The present multicenter, phase II, single-arm study assessed the efficacy and safety of a first-line regimen of eribulin plus trastuzumab for untreated advanced or metastatic HER2-positive breast cancer. Patients and Methods: Enrolled patients received eribulin (1.4 mg/m2 intravenously; I.V.) on days 1 and 8 of each 21-day cycle, an initial trastuzumab dose (8 mg/kg I.V.) on day 1, and 6 mg/kg of trastuzumab on day 1 of each subsequent cycle. The primary endpoint was the response rate (RR). The secondary endpoints were progression-free survival (PFS), overall survival (OS), duration of response (DOR), and safety. Twenty-eight patients (median age: 62.5 years) received a median of 12 (range: 2–53) cycles of eribulin plus trastuzumab. Results: The RR was 53.6% [complete response (CR), 4; partial response (PR), 11] with a median PFS of 344 days. The clinical benefit rate was 64.0%. Grade 3/4 adverse events were observed in 12 (42.9%) patients. For details, neutropenia in 8 (28.6%) patients, peripheral neuropathy in 2 (7.1%) patients, interstitial pneumonia in 1 (3.6%) patient, ALT elevation in 1 (3.6%) patient, osteonecrosis of the jaw in 1 (3.6%) patient, and fatigue in 1 (3.6%) patient. The patient with osteonecrosis received denosumab, too. No symptomatic congestive heart failure was observed. Conclusion: Combination therapy of eribulin plus trastuzumab is acceptable in efficacy and safety, and a capable option for first-line advanced or recurrent HER2-positive breast cancer.

Prognosis of Japanese breast cancer based on hormone receptor and HER2 expression determined by immunohistochemical staining

1122 Background: We classified Japanese breast cancer patients based on estrogen receptor (ER), progesterone receptor (PR) and HER2 protein expression and compared their prognoses. Methods: Background and prognostic factors were compared among 600 patients with breast cancer who were assigned to groups as follows: luminal A (ER+ and/or PR+ and HER2-; n = 431; 71.8%), luminal B (ER+ and/or PR+ and HER2+; n =27; 4.5%), HER2 subtype (ER-, PR- and HER2+; n = 39; 6.5%) and basal-like breast cancer (BBC) (ER-, PR- and HER2-; n = 103; 17.2%). Results: There were no differences among the groups regarding to age, menopausal state, size of tumors, and axillary metastasis. Disease-free survival rates were significantly lower for the luminal B, HER2 subtype and BBC, than for the luminal A subtype. Cancer tended to recur earlier and overall survival was significantly lower for the BBC, than that for the luminal A and HER2 subtype. Luminal B and HER2 subtype revealed same overall survival rate as luminal A. Conclusions...