Yuzuru Yamazoe

Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil- containing protein kinase pathway

BackgroundMelanomas are highly malignant and have high metastatic potential; hence, there is a need for new therapeutic strategies to prevent cell metastasis. In the present study, we investigated whether statins inhibit tumor cell migration, invasion, adhesion, and metastasis in the B16BL6 mouse melanoma cell line.MethodsThe cytotoxicity of statins toward the B16BL6 cells were evaluated using a cell viability assay. As an experimental model, B16BL6 cells were intravenously injected into C57BL/6 mice. Cell migration and invasion were assessed using Boyden chamber assays. Cell adhesion analysis was performed using type I collagen-, type IV collagen-, fibronectin-, and laminin-coated plates. The mRNA levels, enzyme activities and protein levels of matrix metalloproteinases (MMPs) were determined using RT-PCR, activity assay kits, and Western blot analysis, respectively; the mRNA and protein levels of vary late antigens (VLAs) were also determined. The effects of statins on signal transduction molecules were determined by western blot analyses.ResultsWe found that statins significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not have cytotoxic effects on B16BL6 cells. Statins also inhibited the mRNA expressions and enzymatic activities of matrix metalloproteinases (MMPs). Moreover, they suppressed the mRNA and protein expressions of integrin α2, integrin α4, and integrin α5 and decreased the membrane localization of Rho, and phosphorylated LIM kinase (LIMK) and myosin light chain (MLC).ConclusionsThe results indicated that statins suppressed the Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion, and metastasis. Furthermore, they markedly inhibited clinically evident metastasis. Thus, these findings suggest that statins have potential clinical applications for the treatment of tumor cell metastasis.

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways.

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCalpha and PKCdelta phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.

Statin-induced apoptosis via the suppression of ERK1/2 and Akt activation by inhibition of the geranylgeranyl-pyrophosphate biosynthesis in glioblastoma

BackgroundStatins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. The inhibition of this key enzyme in the mevalonate pathway leads to suppression of cell proliferation and induction of apoptosis. However, the molecular mechanism of apoptosis induction by statins is not well understood in glioblastoma. In the present study, we attempted to elucidate the mechanism by which statins induce apoptosis in C6 glioma cells.MethodsThe cytotoxicity of statins toward the C6 glioma cells were evaluated using a cell viability assay. The enzyme activity of caspase-3 was determined using activity assay kits. The effects of statins on signal transduction molecules were determined by western blot analyses.ResultsWe found that statins inhibited cell proliferation and induced apoptosis in these cells. We also observed an increase in caspase-3 activity. The apoptosis induced by statins was not inhibited by the addition of farnesyl pyrophosphate, squalene, ubiquinone, and isopentenyladenine, but by geranylgeranyl-pyrophosphate (GGPP). Furthermore, statins decreased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt.ConclusionsThese results suggest that statins induce apoptosis when GGPP biosynthesis is inhibited and consequently decreases the level of phosphorylated ERK1/2 and Akt. The results of this study also indicate that statins could be used as anticancer agents in glioblastoma.

Risk Factors for Cisplatin-Induced Nephrotoxicity and Potential of Magnesium Supplementation for Renal Protection

Background Nephrotoxicity remains a problem for patients who receive cisplatin chemotherapy. We retrospectively evaluated potential risk factors for cisplatin-induced nephrotoxicity as well as the potential impact of intravenous magnesium supplementation on such toxicity. Patients and Methods We reviewed clinical data for 401 patients who underwent chemotherapy including a high dose (≥60 mg/m2) of cisplatin in the first-line setting. Nephrotoxicity was defined as an increase in the serum creatinine concentration of at least grade 2 during the first course of cisplatin chemotherapy, as assessed on the basis of National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. The severity of nephrotoxicity was evaluated on the basis of the mean change in the serum creatinine level. Magnesium was administered intravenously to 67 patients (17%). Results Cisplatin-induced nephrotoxicity was observed in 127 patients (32%). Multivariable analysis revealed that an Eastern Cooperative Oncology Group performance status of 2 (risk ratio, 1.876; P = 0.004) and the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) (risk ratio, 1.357; P = 0.047) were significantly associated with an increased risk for cisplatin nephrotoxicity, whereas intravenous magnesium supplementation was associated with a significantly reduced risk for such toxicity (risk ratio, 0.175; P = 0.0004). The development of hypomagnesemia during cisplatin treatment was significantly associated with a greater increase in serum creatinine level (P = 0.0025). Magnesium supplementation therapy was also associated with a significantly reduced severity of renal toxicity (P = 0.012). Conclusions A relatively poor performance status and the regular use of NSAIDs were significantly associated with cisplatin-induced nephrotoxicity, although the latter association was marginal. Our findings also suggest that the ability of magnesium supplementation to protect against the renal toxicity of cisplatin warrants further investigation in a prospective trial.

Mangiferin induces apoptosis by suppressing Bcl-xL and XIAP expressions and nuclear entry of NF-κB in HL-60 cells

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-d-glucoside (C-glucosylxanthone), is a xanthone derivative that is widely distributed in higher plants. Recently, mangiferin was found to exhibit potential antitumor effects. However, the molecular mechanisms of this effect have not been elucidated. In the present study, we attempt to clarify the mechanism of mangiferin-induced apoptosis in the human acute myeloid leukemia cell line HL-60; mangiferin was found to induce apoptosis. We also observed a concurrent increase in caspase-3 activity and DNA fragmentation. Furthermore, on examining the survival signals expressed during apoptotic induction, we observed that mangiferin caused a remarkable decrease in the nuclear entry of NF-κB p65. However, there were no changes in the expression of other survival signals, such as extracellular signal-regulated kinase 1/2, protein kinase B, and p38 mitogenactivated protein kinase. In addition, mangiferin suppressed the expressions of Bcl-xL and XIAP; however, we did not note any changes in the levels of Bcl-2, Bax, and Bim. These results indicate that mangiferin induces apoptosis by suppressing NF-κB activation and expressions of Bcl-xL and XAIP. These findings suggest that mangiferin may be useful as an anticancer agent and can be used in combination therapy with other anticancer drugs for the treatment of acute myeloid leukemia.

Blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathways by statins reduces the expression of bFGF, HGF, and TGF-β as angiogenic factors in mouse osteosarcoma.

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-β, and bFGF, HGF, and TGF-β secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-β mRNA expression, and bFGF, HGF, TGF-β secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore, statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-β expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma.

Dimethylfumarate inhibits tumor cell invasion and metastasis by suppressing the expression and activities of matrix metalloproteinases in melanoma cells

NF‐κB acts as a signal transducer during tumor progression, cell invasion, and metastasis. Dimethylfumarate (DMF) is reported to inhibit tumor necrosis factor‐α‐induced nuclear entry of NF‐κB/p65. However, only a few reports suggest that DMF inhibits tumor metastasis; also the molecular mechanisms underlying the inhibition of metastasis are poorly understood. We investigated the inhibition of tumor invasion and metastasis by DMF in a melanoma cell line, B16BL6. DMF inhibited B16BL6 cell invasion and metastasis by suppressing the expression and activities of MMPs. DMF also inhibited the nuclear entry of NF‐κB/p65, thus inhibiting B16BL6 cell invasion and metastasis. These results suggest that DMF is potentially useful as an anti‐metastatic agent for the treatment of malignant melanoma.

Overexpression of MDR1 and survivin, and decreased Bim expression mediate multidrug-resistance in multiple myeloma cells

Multidrug resistance represents a major obstacle for the chemotherapy of a wide variety of human tumors. To investigate the underlying mechanisms associated with resistance to anti-cancer drugs, we established anti-cancer drug-resistant multiple myeloma (MM) cell lines RPMI8226/ADM, RPMI8226/VCR, RPMI8226/DEX, and RPMI8226/L-PAM, the 50% inhibitory concentration values of which were 77-, 58-, 79-, and 30-fold higher than their parental cell lines, respectively. The resistant cell lines overexpressed MDR1 and survivin, or showed decreased Bim expression. These results indicated that regulating these factors with inhibitors might be a viable approach to increasing the susceptibility of quiescent MM cells to chemotherapy.

Nitrogen-containing bisphosphonate, YM529/ONO-5920, inhibits macrophage inflammatory protein 1α expression and secretion in mouse myeloma cells

Macrophage inflammatory protein 1α (MIP‐1α) is detected at high concentrations in patients with multiple myeloma, and it is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, we investigated whether or not YM529/ONO‐5920, a new bisphosphonate, inhibited MIP‐1α mRNA expression in, and MIP‐1α secretion from, mouse myeloma cells. When the cells were stimulated by lipopolysaccharide, increased MIP‐1α mRNA expression and MIP‐1α secretion were observed. YM529/ONO‐5920 inhibited MIP‐1α mRNA expression and MIP‐1α secretion in a concentration‐dependent manner. A transient increase in the phosphorylation of extracellular‐regulated kinase 1/2 (ERK1/2) and Akt was observed after lipopolysaccharide stimulation. After YM529/ONO‐5920 was given, there was no transient increase in the phosphorylation of ERK1/2 or Akt. These results indicated that YM529/ONO‐5920 inhibited the expression and secretion of MIP‐1α through blocking the signaling pathway of the Ras/mitogen‐activated protein kinase kinase/ERK and Ras/phosphatidylinositol‐3 kinase/Akt. Accordingly, YM529/ONO‐5920 appears to have promise for use in effective future therapy for osteolysis and myeloma cell growth that depends on MIP‐1α. (Cancer Sci 2008; 99: 152–158)

Fluorodeoxyglucose uptake in the bone marrow after granulocyte colony-stimulating factor administration in patients with non-Hodgkinʼs lymphoma

PurposeTo clarify the change in the fluorodeoxyglucose (FDG) uptake by the bone marrow over time after administration of granulocyte colony-stimulating factor (G-CSF), we evaluated the correlation between the interval from the last day of administration of G-CSF to positron emission tomography/computed tomography (PET/CT) study and spinal bone marrow accumulation in patients with non-Hodgkins lymphoma. MethodsA total of 127 patients with confirmed non-Hodgkins lymphoma who underwent FDG PET within 60 days from the last administration of G-CSF were retrospectively reviewed. Thirty age-matched and sex-matched healthy controls were also included to evaluate physiological FDG uptake. PET/CT examinations were retrospectively reviewed, and maximum standardized uptake value (SUVmax) was measured by placing volumetric regions of interest over each thoracic and lumbar vertebra on PET images referring to CT images. Bone marrow SUV was defined as the mean SUVmax of the vertebra. The correlation between the interval after G-CSF and the bone marrow SUV was plotted and analyzed with polynomial approximation. ResultsIn controls, physiological bone marrow SUV of the spine was determined. In patients with lymphoma, bone marrow SUV decreased over time and reached a plateau at about 14 days after G-CSF administration, and this was higher by 5% than the plateau at 10 days. SUV declined to the ‘physiological range’, that is, mean+1 standard deviation of patients, at about 7 days. ConclusionFor a PET/CT study, an interval of 10 days after G-CSF administration is recommended to minimize the influence of G-CSF on the bone marrow when evaluating treatment response in patients with non-Hodgkins lymphoma.