Seiji Yoshitomi

Proteasome inhibitors can alter the signaling pathways and attenuate the P‐glycoprotein‐mediated multidrug resistance

Numerous signaling pathways were reported to be involved in the resistance for conventional cytotoxic drugs, although one of the main reasons is the overexpression of P‐glycoprotein (P‐gp) in multidrug resistant cancer cells. The overexpression of P‐gp has been associated with the resistance to a wide range of anticancer drugs. Doxorubicin and paclitaxel are substrates of this transporter system and have an important role for the various human malignancies. In the present study, drug‐sensitive MCF7 and multidrug resistant MCF7/ADR (characterized by overexpression of P‐gp) human breast cancer cell lines were used as an experimental model. We have found that PS341 and MG132, proteasome inhibitors, reduced the degree of the multidrug resistance (MDR) in MCF7/ADR cells. This phenomenon was accompanied by a decrease in the IC50 value of doxorubicin and paclitaxel from 55.9 ± 3.46 to 0.60 ± 0.08 μM, and from 17.61 ± 1.77 to 0.59 ± 0.12 μM, respectively. The IC50 values of sensitive cells for doxorubicin and paclitaxel were about 0.42 and 0.83 μM, respectively. The effect of PS341 and MG132 on MCF7/ADR cells was associated with a significant decrease in both protein and gene levels of P‐gp expression. Moreover, with regard to the expression of possible signal transduction pathways of mitogen‐activated protein kinase (MAPK) related to the activation of mdr1, proteasome inhibitors did significantly influence the activation of these proteins. Western blot analysis revealed that 24 hr exposure of multidrug resistant MCF7/ADR cells with proteasome inhibitors did change the levels of DNA binding activity of nuclear factor‐kappaB (NF‐kappaB), pERK1/2, c‐Jun, and p‐c‐Jun. In conclusion, we could remark that proteasome inhibitors (especially PS341) attenuate the resistance of MCF7/ADR cells for P‐gp substrate drugs of doxorubicin and paclitaxel. Several proteins are supposed to be associated with the resensitization of the cells to conventional cytotoxic drugs, although decreased activity of P‐gp is at least involved in the proteasome inhibitor‐related resensitization. And influence with MAPK pathways, which have been reported to be associated with the regulation of P‐gp, might be contributed to the resensitization brought by proteasome inhibitors.

Tumor inhibitory effect of gefitinib (ZD1839, Iressa) and taxane combination therapy in EGFR-overexpressing breast cancer cell lines (MCF7/ADR, MDA-MB-231)

Some kinds of breast cancer cell lines, similar to several types of solid tumors, express epidermal growth factor receptor (EGFR). However, gefitinib, an EGFR tyrosine kinase inhibitor, is not effective for all these cell lines. Similarly, taxane is effective for many of the cell lines, although some, such as the multidrug‐resistant MCF7/ADR cell line, show taxane‐resistance. Here, we examined the growth inhibitory effect of combination treatment with gefitinib and taxane on the breast cancer cell lines MDA‐MB‐231 (EGFR‐positive) and MCF7/ADR (EGFR‐ and HER2‐positive). To estimate the combined effect, a Combination Index was calculated for each cell line. The combination of gefitinib and taxane showed a strong synergistic effect on MCF7/ADR cells, but an invitro additive‐antagonistic effect on MDA‐MB‐231 cells. Similarly, the combination treatment showed a significantly increased tumor inhibitory effect on MCF7/ADR xenografts, but not on MDA‐MB‐231 xenografts. Regarding the mechanism of the synergistic effect, Western blotting analysis revealed that taxane activated the EGFR‐Akt pathway in MCF7/ADR cells but not in MDA‐MB‐231. To determine the optimal sequential administration of gefitinib and taxane for MCF7/ADR cells, we used flow cytometry to analyze the cell cycle and apoptosis; finding that taxane treatment followed by gefitinib produced a higher rate of G2 arrest and apoptosis than gefitinib treatment followed by taxane. These results suggest gefitinib overcomes the drug‐resistance of these cells, thereby increasing the effects of taxane on MCF7/ADR cells. Further, activation of the EGFR‐Akt pathway by taxane is related to this synergistic effect.

PLC and PI3K Pathways are Important in the Inhibition of EGF-Induced Cell Migration by Gefitinib ('Iressa', ZD1839)

BackgroundExpression of epidermal growth factor receptor (EGFR) by human breast cancer tissues is associated with poor clinical response. The EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib (‘Iressa’, ZD1839), is a leading example of a molecular targeted agent, and has an anti-proliferative effect on various cancer cells. But the details of the anti-cancer effect and mechanism have not been elucidated. We studied the anti-cancer effect of gefitinib in breast cancer cell lines and the intracellular pathway downstream of EGFR associated with cell migration.MethodsIn this study, we analysed the anti-proliferative and anti-migratory effect of gefitinib in EGFR (+) breast cancer cell lines by WST-1 analysis and chemotaxis chamber analysis. We analyzed several intracellular phosphorylated pathways which are activated by mitogen activated kinases (extracellular signal-regulated protein kinase 1 and 2: MEK), phosphatidylinositol 3’-kinase (PI3K) and phpspholipase C (PLC), by blocking those pathways using inhibitors of each kinase, and also investigated the effects on the phosphorylation of myosin light chain (MLC).ResultsGefitinib inhibited proliferation in most of these cell lines. MDA-MB231 was shown to be resistant. Furthermore, proliferation of MDA-MB231 cells was not affected by EGF stimulation, but migration of MDA-MB231 cells was significantly inhibited. PI3K and PLC inhibitors blocked EGF-stimulated cell migration and MLC phosphorylation, but the MEK inhibitor did not influence cell migration.ConclusionsGefitinib has an anti-migratory effect on MDA-MB231 that results in an anti-proliferative effect. PI3K and PLC are important for the migration of MDA-MB231 cells, and gefitinib may inhibit migration by blocking these signalling pathways.

Clinical Significance of Preoperative Lymphoscintigraphy for Sentinel Lymph Node Biopsy in Breast Cancer

BACKGROUND Lymphoscintigraphy (LSG) has been widely used as an additional modality to sentinel lymph node biopsy (SLNB) using isotope. However, the significance of the number of LSG-visualized axillary nodes has not been fully understood. We analyzed this and discussed its potential as a modality to complement SLNB. METHODS Ninety-one breasts and axillary lymph nodal status were evaluated retrospectively. All patients were examined by LSG using isotope and subsequently by SLNB. RESULTS Nine patients (9.9%) had no LSG-visualized axillary node, while 61 patients (67.0%) had only 1 node, and 21 patients (23.1%) had multiple nodes. Overall, sentinel lymph node (SLN) identification rate was 96.7%, and the mean number of removed SLNs was 1.5 nodes per patient. In patients with nonvisualized nodes, 66.7% of SLNs were successfully identified, while 100% of SLNs were identified in those with LSG-visualized nodes. Compared with patients with less than one visualized node, significantly more SLNs were removed in patients with multiple visualized nodes. The number of LSG-visualized nodes correlated with that of metastatic nodes. CONCLUSIONS Preoperative LSG is effective in evaluating SLN status, and the LSG status could be associated with the number of dissected SLN. Moreover, the results of LSG potentially reflect the histological nodal status.

Less invasive surgery for primary hyperparathyroidism based on preoperative 99mTc-hexakis-2-methoxyisobutylisonitrile imaging findings.

PurposeThe clinical benefits acquired by introducing hexakis-2-methoxyisobutylisonitrile (MIBI), the reliability of MIBI, and the adequacy of applying less invasive surgeries based on MIBI imaging were retrospectively investigated.MethodsThe surgical results before introducing 99mTc-MIBI scintigraphy (MIBI) in 10 patients with primary hyperparathyroidism were compared with those after introducing MIBI in 22 patients.ResultsSurgical failure occurred in two patients (20.0%) before introducing MIBI, and in one (4.5%) after introducing MIBI, thus suggesting that the surgical results improved after introducing MIBI. This was because the diagnosis of ectopic adenoma improved with MIBI. Less invasive surgeries were performed in two patients before introducing MIBI, and in 13 patients after introducing MIBI. Consequently, the operation time decreased significantly. The sensitivity of MIBI, ultrasonography, and computed tomography to adenoma was 100%, 72.2%, and 57.1%, respectively, thus indicating MIBI to have the highest sensitivity. The positive predictive value of MIBI was 85.7%, and all false-positive results were caused by nodular lesions of the thyroid gland. However, the preoperative differentiation of false-positive results could be successfully performed by combining MIBI with neck ultrasonography.ConclusionThe sensitivity of MIBI to adenoma was thus found to be highly satisfactory. As a result, it might be possible to perform less invasive surgeries based on MIBI imaging without any decrease in the surgical results.

Safety and efficacy of gemcitabine and trastuzumab in HER2-directed therapy pretreated patients with HER2-positive metastatic breast cancer: SBP-01 study.

142 Background: Prognosis of HER2-positive metastatic breast cancer (MBC) has been dramatically improved by trastuzumab (Tmab). More recently, newer anti-HER2 agents such as lapatinib, pertuzumab and T-DM1 have prolonged survival. Despite the efficacy of these drugs, most patients develop progressive disease during or after treatment, and alternative anti-HER2 agents plus chemotherapies are required in subsequent lines of treatment. However, there are few evidence on efficacy of Tmab-containing regimens after disease progression. Gemcitabine (GEM) is non-cross resistant to anthracycline and taxane. Preclinical studies have shown that the combination of Tmab and GEM has synergistic effect against HER2-positive breast cancer cell lines. SBP-01 study assessed the efficacy and safety of the combination of Tamb and GEM in patients with HER2-positive MBC previously treated with anti-HER2 therapy. METHODS SBP-01 study included patients treated with one or more anti-HER2 directed regimens for MBC. Patients were administered with GEM 1250 mg/m2 on days 1 and 8 of each 21-day cycle and Tmab 4mg/kg loading dose and then 2mg/kg weekly. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression free survival (PFS), overall survival, and safety. RESULTS Between June 2011 and June 2014, 35 patients were enrolled. Patients had ER positive tumor (37.1%), a median of 2 metastatic organ sites, visceral metastasis (80.0%), prior (neo) adjuvant Tmab (22.9%) and a median of 2 prior chemotherapy regimens for MBC. Previous HER2-directed drugs included Tmab (94.3%), lapatinib (37.1%), T-DM1 (8.6%) and pertuzumab (2.9%). ORR was 22.9% (95% CI, 8.6%-36.8%). Median PFS was 146 days. Patients with stable disease response received a median of 7 cycles (6-28 cycles) of treatment. Grade3/4 leukopenia (20.0%) and neutropenia (48.6%) were observed. All non-hematological toxicities were less than grade3. CONCLUSIONS The Combination Tmab and GEM is effective and well-tolerated regimen for patients previously treated with HER2-directed therapy, and appears to make disease stable for long time period. CLINICAL TRIAL INFORMATION UMIN000005881.

Attenuating effect of PS341 for MDR1 in multidrug resistant breast cancer

2087 Background: Over-expression of P-gp has been associated with the resistance to a wide range of anti-cancer drugs. Doxorubicin and paclitaxel are substrate of this transporter system and have important role for the various human malignancies, especially in the treatment of breast cancer. Here we demonstrate that proteasome inhibitors could remarkably enhance drug sensitivities for MDR1 overexpressed multidrug resistant breast cancer and analyzed the estimated pathways involved in it Methods: Drug sensitive MCF7 and multidrug resistant MCF7/ADR (characterized by overexpression of P-gp) human breast cancer cell lines were used as an experimental model. And two proteasome inhibitors;PS341 and MG132 were used as treatment drugs. Results: PS341 and MG132, proteasome inhibitors, reduced the degree of the multidrug resistance in MCF7/ADR cells. This phenomenon was accompanied by a decrease in the IC50 value of doxorubicin and paclitaxel from 55.9±3.46 to 0.60±0.08 uM, and from 17.61±1.77 to 0.59±0.12 uM. The...