Retention of tocilizumab and anti-tumour necrosis factor drugs in the treatment of rheumatoid arthritis.

OBJECTIVES: The retention of the anti-rheumatic agent tocilizumab (TCZ) has not been well documented in patients with rheumatoid arthritis (RA). We conducted an observational study to compare the retention of TCZ and anti-tumour necrosis factor (TNF) drugs in the treatment of patients with RA. METHOD: We reviewed continuation rates and causes of discontinuation of biological agents (biologics) by assessing medical records of patients with RA who were administered biologics at our institute from September 1999 to April 2012, using the Osaka University Biologics for Rheumatic Diseases (BiRD) registry. RESULTS: A total of 401 patients were included. TCZ, infliximab (IFX), etanercept (ETN), and adalimumab (ADA) were administered to 97, 103, 143, and 58 patients, respectively. There were some differences between the baseline characteristics of the groups. The median duration (range) of TCZ, IFX, ETN, and ADA administration was 2.5 (0.1-12.6), 1.9 (0.0-7.7), 2.9 (0.0-11.3), and 1.3 (0.0-3.4) years, respectively. Continuation rates for TCZ and ETN were significantly higher than those for IFX and ADA. Multivariate analyses showed that discontinuation due to lack or loss of efficacy was significantly less common in the TCZ group than in the other groups. Discontinuation due to overall adverse events was not significantly different between treatment groups. CONCLUSION: TCZ and ETN show better retention than IFX or ADA in the treatment of RA.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Effect of GDF-5 and BMP-2 on the expression of tendo/ligamentogenesis-related markers in human PDL-derived cells.

OBJECTIVES: The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers. MATERIALS AND METHODS: Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control. RESULTS: Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal ligament-derived cells. CONCLUSION: Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament-derived cells. The expression of scleraxis was higher in STRO-1-negative fraction, suggesting more differentiated state of the cells.

Oncostatin M induces association of Grb2 with Janus kinase JAK2 in multiple myeloma cells.

Oncostatin M (OSM) is a 28-kD glycoprotein recently identified as a growth factor for human multiple myeloma cells. It belongs to a family of distantly related cytokines that includes interleukin 6, ciliary neurotrophic factor, leukemia-inhibitory factor, and interleukin 11. These cytokines initiate signaling by inducing either homodimerization of gp130 or heterodimerization of gp130 with leukemia-inhibitory factor receptor beta components. Such dimerization in turn activates receptor-associated tyrosine kinases. In the present study using U266B1 human multiple myeloma cells, we show that OSM induces tyrosine phosphorylation and activation of JAK2, but not JAK1 or Tyk2, kinases. The results also demonstrate that OSM induces direct interaction of JAK2 kinase with Grb2, an SH2/SH3 domain containing adaptor protein. The SH2 domain of Grb2 is directly associated with tyrosine-phosphorylated JAK2. Furthermore, the presence of Sos in the JAK2-Grb2 complex suggests a role for Ras in OSM-transduced signaling.

Oncostatin M, leukemia inhibitory factor, and interleukin 6 induce the proliferation of human plasmacytoma cells via the common signal transducer, gp130.

We analyzed the stimulatory effect of oncostatin M (OSM), leukemia inhibitory factor (LIF), interleukin 6 (IL-6), IL-11, and the inhibitory effect of anti-IL-6 antibody (Ab), anti-IL-6 receptor monoclonal antibody (mAb), and anti-gp130 mAb on the growth of human plasmacytoma cells freshly isolated from a patient with multiple myeloma. The purified cells showed a plasmacytoid morphology and expressed CD38, CD54, and CD56 antigens but no CD3, CD5, CD10, CD19, CD20, or very late antigen 5. IL-6 receptor (IL-6R) and its signal transducer, gp130, were expressed on their cell surface at a low level. Dose-dependent proliferation of the cells in response to OSM, LIF, and IL-6, but not to IL-11, was observed using [3H]TdR incorporation in vitro. Both anti-IL-6 Ab and anti-IL-6R mAb inhibited the growth of the cells in the presence or absence of exogenous IL-6. These cells release IL-6 but not OSM or LIF into the culture supernatant during short-term culture. Therefore, an autocrine growth mechanism mediated by IL-6, but not by OSM or LIF, was confirmed. Furthermore, anti-gp130 mAb completely inhibited the proliferation of the cells induced by OSM, LIF, as well as IL-6. These data indicate that OSM, LIF, and IL-6 can act as growth factors of human plasmacytoma cells through a common signal transducer, gp130, on their cell surface, and also suggest the potential therapeutic application of anti-gp130 mAb, as well as anti-IL-6R mAb against myeloma/plasmacytomas.

Serum levels of soluble vascular endothelial growth factor receptor-2 in patients with systemic sclerosis.

BACKGROUND: Although vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (KDR) signalling may play a major role in the microangiopathy of systemic sclerosis (SSc), serum levels of soluble KDR (sKDR) in this disease have not yet been determined. OBJECTIVES: To evaluate the possibility that serum sKDR levels can be a specific disease marker of SSc. METHODS: Serum sKDR levels of 42 patients with SSc, 10 patients with Raynaud's phenomenon (RP) and 22 healthy controls were measured with specific enzyme-linked immunosorbent assays. Quantitative real-time polymerase chain reaction (PCR) was performed to determine KDR mRNA levels. RESULTS: In females, the serum sKDR levels were significantly higher in patients with SSc, especially limited cutaneous SSc, than in patients with RP or healthy controls. Quantitative real-time PCR with RNA from skin sections revealed that KDR mRNA levels were also increased in the skin of patients with SSc with elevated serum sKDR levels. A significantly lower prevalence of pulmonary fibrosis, higher percentage vital capacity, and a higher incidence of telangiectasia were seen in female patients with SSc with elevated serum sKDR levels than those with normal levels. CONCLUSIONS: These results suggest that the skin can be one of the sources of elevated serum sKDR levels, and that serum sKDR levels are useful for diagnosis and may be a marker of microangiopathy in patients with SSc, especially females. The VEGF-A/KDR signalling system may be involved in the pathogenesis of the disease.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Quantification of hardness, elasticity and viscosity of the skin of patients with systemic sclerosis using a novel sensing device (Vesmeter): a proposal for a new outcome measurement procedure.

OBJECTIVES: No objective method to measure skin involvement in SSc has been established. We developed a novel method using a computer-linked device to simultaneously quantify physical properties of the skin such as hardness, elasticity and viscosity. METHODS: Skin hardness was calculated by measuring the depth of an indenter pressed onto the skin. The Voigt model was used to calculate skin elasticity, viscosity, visco-elastic ratio and relaxation time by analysing the waveform of skin surface behaviour. The results were compared with the modified Rodnan skin score (mRSS) obtained at 17 sites on the bodies of 20 SSc patients and 20 healthy controls. A functional assessment questionnaire was administered to determine how skin hardness represents a patient's disability. We also examined intra- and inter-observer variability to determine the reliability of this method. RESULTS: The crude hardness obtained with this device correlated well with the standard hardness specified by the American Society for Testing and Materials (ASTM, r = 0.957). A close relationship between hardness and total mRSS was also observed (r = 0.832). Skin elasticity correlated positively, and relaxation time negatively with mRSS. Functional disability correlated more closely with skin hardness (r = 0.643) than with mRSS (r = 0.517). Intra- and inter-observer variabilities were 7.63 and 19.76%, respectively, which were lower than those reported for mRSS. CONCLUSIONS: Increases in hardness and elasticity as well as shortening of relaxation time constitute objective characteristics of skin involvement in SSc. The system devised by us proved to be able to assess skin abnormalities of SSc with high reliability.

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells.

Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18. In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells. Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen. We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane. Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells. These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule. They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.

Particle size specific distribution of perfluoro alkyl substances in atmospheric particulate matter in Asian cities.

Seasonal and local characteristics of perfluorinated alkylated substances (PFASs) were examined using size-segregated particles including an ultrafine range. The examination included sampling and analysis of ambient particles collected at four sites located in different environments in three different countries, Japan (Kanazawa and Okinawa), Hong Kong and India. To minimize the evaporation artefacts derived from PFASs during the sampling, an air sampler that permitted particles smaller than 0.1 µm (PM0.1) to be separated at a moderate pressure drop (<5-15 kPa), was used for all of the air sampling procedures. In the case of Kanazawa, a local city in Japan, the concentration of PFASs was found to be dominated by carboxylates, especially PFOA, PFNA and PFDA regardless of the particle size and sampling period. Ultrafine particles were found to be the largest contributor to the mass fraction of PFCAs, while the maximum PFOS mass fractions were determined to be in the coarse-sized fractions. The seasonal difference in the total PFAS concentration can be largely attributed to precipitation. The results were basically similar for all sites that were examined. The type of land use may be a more influencing factor on the mass fraction of the PFASs than the country of origin. The dependency of PFAS mass fraction on the specific surface of the particle suggests that ultrafine PFAS particles are segregated, not only by gas deposition but could also be segregated by a mechanism involving compositional dependence or the primary source of the particles. Other possible sources of PFASs, other than from traffic are also possible.

Isatin, an endogenous MAO inhibitor, and a rat model of Parkinson's disease induced by the Japanese encephalitis virus.

A single dose of isatin (indole-2,3-dione)(i.p.), an endogenous MAO inhibitor, significantly increased norepinephrine and 5-hydroxytryptamine concentrations in the rat brain and also significantly increased acetylcholine and dopamine (DA) levels in the rat striatum. Urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. We have developed a rat model of Parkinson's disease induced by the Japanese encephalitis virus (JEV). The distribution of the pathological lesions of JEV-rats resemble those found in Parkinson's disease. Significant behavioral improvement was observed in JEV-rats after isatin, L-DOPA and selegiline administration using a pole test. Both isatin and selegiline prevented the decrease in striatum DA levels of JEV-rats. The increased turnover of DA (DOPAC/DA) induced by JEV was significantly inhibited by isatin, but not selegiline. These findings suggest that JEV-infected rats may serve as a model of Parkinson's disease and that exogenously administered isatin and selegiline can improve JEV-induced parkinsonism by increasing DA concentrations in the striatum.

Anticancer activity of polyoxomolybdate.

Anticancer polyoxomolybdates have been investigated for medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH3Pri]6[Mo7O24].3H2O (PM-8) has been recognized as one of significant antitumoral polyoxomolybdates. PM-8 had shown the growth suppression against several tumors, for examples, Co-4, human colon cancer, MX-1, human breast cancer, and OAT, human lung cancer. PM-8 showed the tumor growth suppression for MKN-45 human gastric cancer in tumor bearing mice. PM-8 inhibited the cell growth of AsPC-1 which depended on the dose with showing DNA ladder formation and DNA fragmentation, and positive Hoechst staining indicating apoptosis. The ratio of apoptotic cells on flow cytometry analysis were 35%, and 57% with treatment of PM-8 after 48, and 72 h, respectively. One of the anti-tumor activity of PM-8 result from the activation of apoptotic pathway. It is thought that polyoxomolybdates will be applied as a novel anti-tumor agent especially against cancers which are difficult to be treated clinically.

Antitumor activity of polyoxomolybdate, [NH3Pri]6[Mo7O24].3H2O, against, human gastric cancer model.

Polyoxometalates are negatively charged inorganic compounds which contain metal ions such as tungsten, molybdenum, vanadium etc. and which make clusters with the surrounding oxygen atoms. [NH3Pri]6[Mo7O24].3H2O (PM-8) was found to be a significant antitumor polyoxomolybdates. It had already been reported that the PM-8 suppressed the growth of Co-4 human colon cancer, MX-1 human breast cancer and OAT human lung cancer xenografted in nude mice. However, the mechanism of the antitumor activity has not been clarified. In this study, the antitumor activity of one of the metal oxide clusters (polyoxometalates), hexabis(isopropylammonium) heptamolybdate trihydrate, [NH3Pri]6[Mo7O24].3H2O (PM-8) were shown in an MTS assay. DNA ladder formation and detection of apoptotic bodies in nuclei were revealed that antitumor activity of PM-8 in MKN45 cells was due to apoptosis. It is concluded that the observation of significant tumor growth suppression of PM-8 in MKN45-bearing mice results from the induction of apoptosis. PM-8 shows promise as a novel anti-cancer agent.

Effects of tetrabromobisphenol A, brominated flame retardant, in ICR mice after prenatal and postnatal exposure.

Tetrabromobisphenol A (TBBPA), brominated flame retardant, is produced in the largest amounts globally for use in plastics or building materials. TBBPA has been detected in sediment, air at the dismantling plant or human serum samples. In the present study, we examined the effects of prenatal and postnatal exposure to TBBPA in mice. TBBPA (99.1% pure) in diet was administered to pregnant ICR mice at doses of 0% (control), 0.01%, 0.1% or 1.0% from gestational day 0 to weaning at postnatal day 27. The average daily food intake and body weight of dams showed no significant differences between the control and treated groups. There were no dose-related effects on reproductive data. Serum concentrations of total-cholesterol and liver weights of treated dams and offspring were higher than those of the control mice. Histological findings in treated dams or offspring showed the increase of focal necrosis of hepatocytes and inflammatory cell infiltration in the liver, and increase of dilation or atrophy of renal tubules and cyst in the kidney. TBBPA was developed as a new, safe class of flame retardant and was not highly toxic. However, the present data suggested that TBBPA caused a lipid metabolic disorder and hepatic or kidney lesion, under these conditions.

Rat maf related genes: specific expression in chondrocytes, lens and spinal cord.

maf is a family of oncogenes originally identified from avian oncogenic retrovirus, AS42, encoding a nuclear bZip transcription factor. We have isolated two maf related cDNA clones, maf-1 and maf-2, from a rat liver cDNA library. Comparison of the sequence homologies of the proteins encoded by maf-1 and maf-2 with those of c-maf and chicken mafB indicated that maf-1 and maf-2 are the rat homologues of mafB and c-maf, respectively. Both genes are expressed at low levels in a wide variety of rat tissues, including spleen, kidney, muscle and liver. Immunohistochemical studies and in situ hybridization analyses show that maf-1 and maf-2 are strongly expressed in the late stages of chondrocyte development in the femur epiphysis and the rib and limb cartilage of 15 day old (E15) embryo in rat. Cartilage cells, induced by subcutaneous implantation of bone morphogenic protein, also expressed maf-1 and maf-2. In situ hybridization analyses of E15 embryos show that both genes are expressed in the eye lens and the spinal cord as well as the cartilage. However, the expression patterns of maf-1 and maf-2 in lens and spinal cord are different.

Dexamethasone induces apoptosis of multiple myeloma cells in a JNK/SAP kinase independent mechanism.

The stress-activated protein kinases (SAPKs), also known as c-Jun amino-terminal kinases (JNKs), are activated in response to diverse stimuli including DNA damage, heat shock, interleukin-1, tumor necrosis factor-alpha and Fas. Although all these inducers cause apoptosis, whether SAPK/JNK activation is required for apoptosis is controversial. In this study, we demonstrate that ionizing radiation (IR) and dexamethasone (Dex) induce apoptosis in multiple myeloma (MM) derived cell lines, as well as in patient cells. IR-induced apoptosis is associated with activation of SAPK/JNK and p38 kinase, in contrast to Dex-induced apoptosis, which is not associated with activation of stress kinases. Moreover, Dex-induced apoptosis is associated with a significant decrease in the activities of mitogen activated protein kinase (MAPK) and p70S6K, whereas IR-treatment does not alter the activity of these kinases. Both IR and Dex induce poly (ADP ribose) polymerase (PARP) cleavage, a signature event of apoptosis. Finally, interleukin-6 (IL-6) inhibits Dex-induced apoptosis, downregulation of MAP and p70S6K growth kinases and PARP cleavage; in contrast, IL-6 does not inhibit IR-induced apoptosis, activation of SAPK/JNK, and PARP cleavage. Taken together, our findings suggest that SAPK/JNK activation is not required for apoptosis in MM cells, and that there are at least two distinct apoptotic signaling pathways: (i) SAPK/JNK-associated, which is induced by IR and unaffected by IL-6; and (ii) SAPK/JNK-independent, which is induced by Dex, associated with downregulation of MAPK and p70S6K and inhibited by IL-6.

Japanese encephalitis virus up-regulates expression of macrophage migration inhibitory factor (MIF) mRNA in the mouse brain.

Macrophage migration inhibitory factor (MIF) is known as a proinflammatory cytokine, glucocorticoid-induced immunomodulator, and pituitary hormone, and contributes to broad-spectrum immune and inflammatory response. To investigate the expression of MIF in the central nervous system in an event of viral infection, we evaluated MIF mRNA expression in the mouse brain infected with Japanese encephalitis virus (JEV). In situ hybridization revealed that MIF mRNA expression was significantly up-regulated in the whole brain by intracranial JEV inoculation at 2 days post-inoculation (d.p.i.). Neurons as well as glial cells expressed MIF transcripts in which some of these cells were co-labeled by double staining for JEV antigens and MIF mRNA. At 4 d.p.i., when typical symptoms of encephalitis were observed, JEV antigen-positive cells were much increased in parallel with enhanced MIF mRNA, consistent with the results of Northern blot analysis. Reverse transcription-polymerase chain reaction showed that MIF mRNA was minimally changed at 1 d.p.i. in comparison with that at 0 d.p.i., but markedly up-regulated after 2 d.p.i. and sustained up to 4 d.p.i. On the other hand, a significant increase of tumor necrosis factor (TNF)-alpha mRNA was observed after only 3 d.p.i. These data suggest the possibility that MIF is involved in virus-induced encephalitis with regard to not only immune responses in the early stage, but also the exacerbation of inflammation in concert with TNF-alpha in the late stages. This is the first evidence demonstrating that MIF is up-regulated in the case of virus-induced encephalitis, which should contribute to the further understanding of the pathological mechanism of JEV-induced encephalitis.

A novel anti-tumor agent, polyoxomolybdate induces apoptotic cell death in AsPC-1 human pancreatic cancer cells.

Anti-tumoral polyoxomolybdates have been investigated in the course of study of the medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH(3)Pr(i)](6)[Mo(7)O(24)].3H(2)O (PM-8) has been recognized as one of significantly anti-tumoral polyoxomolybdates. PM-8 inhibited the cell growth of human pancreatic cells (AsPC-1) depending on the dose. DNA ladder formation and DNA fragmentation were observed by Hoechst and TUNEL staining and flowcytometry analysis. The ratio of apoptotic cells were 29%, 35%, and 57% with treatment of PM-8 after 24, 48, and 72 h, respectively, which suggested that the anti-tumor activity of PM-8 results from the activation of the apoptotic pathway. Polyoxomolybdates provide promising, novel anti-tumor agent, especially for the treatment of cancers that are difficult to treat.