Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models.

Polyoxomolybdates (PMs) as discrete molybdenum-oxide cluster anions have been investigated in the course of study of their medical applications. Here, we show the significant antitumour potency of the polyoxomolybdate [Me(3)NH](6)[H(2)Mo(V)(12)O(28)(OH)(12)(Mo(VI)O(3))(4)].2H(2)O (PM-17), which is a photo-reduced compound of [NH(3)Pr(i)](6)[Mo(7)O(24)].3H(2)O. The effect of PM-17 on the growth of cancer cell lines and xenografts was assessed by a cell viability test and analysis of tumour expansion rate. Morphological analysis was carried out by Hoechst staining, flow-cytometric analysis of Annexin V staining, terminal deoxynucleotidyl transferase-mediated 'nick-end' labelling staining, and electron-microscopic analysis. Activation of autophagy was detected by western blotting and fluorescence-microscopic analysis of the localisation of GFP-LC3 in transfected tumour cells. PM-17 inhibited the growth of human pancreatic cancer (AsPC-1) xenografts in a nude mice model, and induced morphological alterations in tumour cells. Correspondingly, PM-17 repressed the proliferation of AsPC-1 cells and human gastric cancer cells (MKN45) depending on the dose in vitro. We observed apoptotic patterns as the formation of apoptotic small bodies and translocation of phosphatidylserine by Hoechst staining and flow-cytometric analysis following Annexin V staining, and in parallel, autophagic conformation by the formulation of autophagosomes and localisation of GFP-LC3 by electron- and fluorescence-microscopic analysis.

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.

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.

Synergistic effect of polyoxometalates in combination with oxacillin against methicillin-resistant and vancomycin-resistant Staphylococcus aureus: a high initial inoculum of 1 x 108 cfu/ml for in vivo test.

Synergistic effect of polyoxometalates, K(6)[P(2)W(18)O(62)].14H(2)O (P(2)W(18)), K(4)[SiMo(12)O(40)].3H(2)O (SiMo(12)), K(7)[PTi(2)W(10)O(40)].6H(2)O (PTi(2)W(10)), and K(9)H(5)[alpha-Ge(2)Ti(6)W(18)O(77)].16H(2)O (Ge(2)Ti(6)W(18)), in combination with a beta-lactam oxacillin against methicillin-resistant and vancomycin-resistant Staphylococcus aureus (characterized by possessing the penicillin-binding protein 2' (PBP2') as a cell-wall synthesis enzyme) with a high initial inoculum of 1 x 10(8) cfu/ml (for in vivo test) was investigated with a help of the growth curve and the reverse transcription polymerase chain reaction (RT-PCR) analyses. The growth curves showing the suppression of cell proliferation of the strains based on the synergistic effect of the polyoxometalates in combination with oxacillin indicated a recovery of the cell proliferation during continuous cultivation. The duration of the suppression of the cell proliferation increased with increasing the concentration of the polyoxometalates, depending on the amounts of the initial inoculum of the strain. The RT-PCR results for P(2)W(18), SiMo(12), and PTi(2)W(10) indicated the suppression of expression of the PBP2'-encoding mecA gene in contrast to the ones for Ge(2)Ti(6)W(18). The difference in the RT-PCR results among the polyoxometalates suggests that there remain other factors for the inhibition of PBP2' production such as post-transcription process.

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.

A three-dimensional inorganic/organic hybrid vanadium oxide complex with pentacoordinate CoII, [CoV2O6(4,4'-bipy)].

The title compound, poly[[cobalt(II)-mu-(hexaoxodivanadium-O:O')]-mu-bipyridine-N:N'], [CoV2O6(C10H8N2)], has been prepared hydrothermally and characterized by elemental analyses, IR spectroscopy and single-crystal X-ray diffraction. The structure consists of bimetallic oxide layers, [Co2V4O12], linked through 4,4'-bipyridine ligands into a three-dimensional network.

A three-dimensional inorganic/organic hybrid material, [Ni(4,4'-bipy)3-(H2O)2V4O12]*2.5H2O.

The title compound, poly[[[diaqua(mu-4,4'-bipyridyl)dinickel(II)]-bis(mu-4,4'-bipyridyl)-di-mu-hexaoxodivanadate(2-)] 2.5-hydrate], [Ni2(V2O6)2(C10H8N2)3(H2O)2]*2.5H2O, has been prepared hydrothermally and characterized by elemental analyses, IR spectroscopy and single-crystal X-ray diffraction. The structure consists of [V2O6], [Ni(4,4'-bipy)4O2] and [Ni(H2O)2(4,4'-bipy)2O2] polyhedra, and water of crystallization. The Ni atoms and one bipyridyl group lie on centres of symmetry.

Inhibitory effect of polyoxotungstates on the production of penicillin-binding proteins and beta-lactamase against methicillin-resistant Staphylococcus aureus.

In our continuous work on the enhancement of the antibacterial activity of beta-lactam antibiotics against the cells of methicillin-resistant Staphylococcus aureus (MRSA) strains by Keggin-structural polyoxotungstates and their lacunary species, Wells-Dawson, double-Keggin, and Keggin-sandwich polyoxotungstates are also found to be synergistic but highly cytotoxic. The coexistence of polylysine or protamine sulphate decreased the synergistic potency of the polyoxotungstates, due to their electrostatic interaction with negatively charged polyoxotungstates. Inductively coupled plasma atomic emission spectrometry (ICP) analysis of the polyoxotungstate-treated cells indicated that the polyoxotungstates uptaken in the cell are preferentially located at the membrane fraction with intact composition. The polyoxotungstates depressed not only the production of PBP2', but also the production of beta-lactamase which hydrolyzes beta-lactam antibiotics on the membrane. This leads to the synergistic effect of polyoxotungstates against the MRSA cells in the coexistence of beta-lactam antibiotics which have high affinities to PBPs 1-4. MRSA cells which were modified to be susceptible to beta-lactam antibiotics during incubation in the presence of polyoxotungstates recovered their resistance to beta-lactam antibiotics when they were subcultured in the absence of the polyoxotungstate.

Quantitation of herpes simplex viral DNA in Vero cells for evaluation of an antiviral agent using the polymerase chain reaction.

A method for quantitation of the DNA of Herpes simplex virus type 2 (HSV-2)-infected Vero cells by the polymerase chain reaction (PCR) was developed. This method allowed recognition of several molecules of viral DNA among the total DNA extracted from cells. The method could be applied to a very large range (10(-0)-10(-7)) of initial amounts of template. Products of PCR were collected after each cycle for kinetic analysis. Products were subjected to electrophoresis and amplified bands were stained with ethidium bromide. The intensity of fluorescence of each band was measured with a charge-coupled device (CCD) image analyzer. The time course of increases in the relative yield of viral DNA was determined. Two-fold amplification of viral DNA occurred each 6-h cycle from 7 h after infection. Using this method, the yields of viral DNA after treatment with the drug acyclovir (ACV) at 0.1 and 2 microg/ml were about 1/10 and 1/80 of those from nontreated infected cells, respectively. These results indicate that this method makes clear the inhibitory effect of ACV on the synthesis of viral DNA.

In vitro antibacterial activity of vanadate and vanadyl compounds against Streptococcus pneumoniae.

All of the vanadate and vanadyl compounds tested in this study showed potent antibacterial activity against Streptococcus pneumoniae: the MIC (minimum inhibitory concentration) values of the vanadium compounds were 4-32 micrograms/ml, while the MIC values of tungstates and molybdates were 128-->8000 micrograms/ml. Scanning electron microscopy showed the elongation of S. pneumoniae strains under low concentrations (< MIC) of vanadium compounds such as (tert-BuNH3)4[V4O12] (1), (tert-BuNH3)6[V10O28] (2), Na3VO4 (7) and VOSO4 (8). The vanadium compounds non-selectively inhibited the incorporation of thymidine, uridine, leucine and glucose into the cells of S. pneumoniae and led to an efflux of potassium ions from the cells. It implies that the vanadium compounds interfere in or on the cell membrane with the transport of substrates and ions through the cell membrane, resulting in antibacterial activity against S. pneumoniae cells.

Synergistic anti-influenza virus A (H1N1) activities of PM-523 (polyoxometalate) and ribavirin in vitro and in vivo.

A Kegin-type polyoxometalate, PM-523, in combination with ribavirin, was tested for its therapeutic effectiveness against influenza virus (FluV) A (H1N1) infection in tissue culture and in mice. PM-523 [(PriNH3)6H [PTi2W10O38(O2)2] x H2O, where Pri is isopropanol] and ribavirin individually inhibited FluV A-induced cytopathic effects in Madin-Darby canine kidney (MDCK) cells at median effective concentrations (EC50s) of 30 and 34 microM, respectively, and at 70% effective concentrations (EC70s) of 48 and 72 microM, respectively. On the other hand, a combination of PM-523 and ribavirin at a ratio of 1:16 exhibited lower EC50s and EC70s than each compound used singly, and combination indices were less than 1. A wide range of combinations of PM-523 and ribavirin at ratios of from 1:128 to 1:1 exhibited additive or synergistic anti-FluV effects in MDCK cells. When these compounds were tested for their anti-FluV A activities in vivo by aerosol exposure of mice which had been infected with a lethal dose of FluV A by an intranasal route, a 1:16 combination of PM-523 and ribavirin was found to have a significantly better therapeutic effect than a single dose of either compound used singly with respect to both the survival rate of the mice and the virus titer in the lungs of the infected mice. PM-523 was effective for the treatment of experimental FluV infection, and in combination with ribavirin, PM-523 exhibited enhanced anti-FluV effects in vitro and in vivo compared with the effect of PM-523 alone.

Synergistic effect of polyoxotungstates in combination with beta-lactam antibiotics on antibacterial activity against methicillin-resistant Staphylococcus aureus.

The in vitro antibacterial effect of the combination of various polyoxometalates with beta-lactam antibiotics on methicillin-resistant Staphylococcus aureus (MRSA) strains is investigated by the use of both the National Committee for Clinical Laboratory Standards (NCCLS) disk method and the agar dilution method. Keggin-structural polyoxotungstates such as K7[PTi2W10O40].6H2O (5) and K7[BVW11O40].7H2O (8) and their lacunary species formulated by [XW11O39]n- and[XW9O34]n- potentiated the antibacterial activity of beta-lactam antibiotics such as oxacillin, piperacillin and cefazolin on MRSA with high selectivity. The depression of bacterial growth with the coexistence of polyoxotungstates and oxacillin was confirmed by the measurement of the bacterial turbidity at 660nm. Polyoxomolybdates and polyoxovanadates, on the other hand, exhibited hardly any synergistic effect in combination with oxacillin. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins separated from MRSA revealed that polyoxotungstates depressed the formation of penicillin-binding protein 2'(PBP2'), an enzyme which is essential for cell wall construction in the MRSA growth. It is concluded that polyoxotungstates make the MRSA strains susceptible to beta-lactam antibiotics.

Structure-activity correlationship and strain specificity of polyoxometalates in anti-human immunodeficiency virus activity.

The anti-human immunodeficiency virus (HIV) activity of polyoxometalates of representative structural families, such as Keggin, lacunary Keggin, trivacant Keggin, Keggin sandwich, Wells-Dawson and Wells-Dawson sandwich, was determined using two strains of HIV type 1 (HIV-1HTLV-IIIB and HIV-1SF-2H). The compounds were preferably selected to cover both polyoxotungstates and polyoxomolybdates in each structural family. In general, polyoxotungstates of Keggin, lacunary Keggin, trivacant Keggin, Keggin sandwich, Wells-Dawson and Wells-Dawson sandwich structures showed anti-HIV-1HTLVIIIB activity, whereas most compounds not included in these structural categories were inactive. Among the compounds with a potent anti-HIV-1HTLV-IIIB activity, those of Keggin and its closely related structural families (lacunary Keggin, trivacant Keggin and Keggin sandwich) inhibited the cytopathogenicity and syncytium formation caused by HIV-1SF-2 to a much higher extent compared with HIV-1HTLV-IIIB-related ones. The difference between the spectra of anti-HIV-1HTLV-IIIB activity and the specificity for HIV-1SF-2H might result from differential structural requirements in these functions.

Activity of the Keggin polyoxotungstate PM-19 against herpes simplex virus type 2 infection in immunosuppressed mice: role of peritoneal macrophage activation.

The in vivo antiviral activity of the Keggin polyoxotungstate PM-19 [K7(PTi2W10O40).6H2O] against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). When PM-19 was administered intraperitoneally to immunosuppressed mice for 3 days (once daily) starting at the time of infection, it prevented death due to HSV-2 encephalitis in a dose-dependent manner (10-25 mg/kg). The in vivo anti-HSV-2 activity of PM-19 was superior to that of acyclovir. Intraperitoneal administration of PM-19 to the immunosuppressed mice significantly increased the number of peritoneal cells, especially macrophages. PM-19 did not stimulate interferon-inducing activity or natural killer cell activity, but markedly enhanced peritoneal macrophage functions: (1) phagocytic activity as assessed by measuring the amount of 51Cr-labeled sheep red blood cells taken into the macrophages, and (2) extrinsic antiviral activity as monitored by reduction in the numbers of plaque formed upon cocultivation of HSV-2-infected HEL cells with the macrophages. These results point to the role of peritoneal macrophage activation in the activity of PM-19 against HSV-2 infection in immunosuppressed mice.

In vitro antiviral activity of polyoxomolybdates. Mechanism of inhibitory effect of PM-104 (NH4)12H2(Eu4(MoO4)(H2O)16(Mo7O24)4).13H2O on human immunodeficiency virus type 1.

A screening for inhibitors of human immunodeficiency virus type 1 (HIV-1) among various types of isopolyoxomolybdates and heteropolyoxomolybdates was carried out by using an in vitro assay system measuring the cytopathogenicity of HIV-1 in CD4+ human MT-4 cells. A novel heteropolyoxomolybdate named PM-104 with the chemical formula (NH4)12H2(Eu4(MoO4)(H2O)16(Mo7O24)4).13H2O was found to be associated with potent anti-HIV-1 activity. PM-104 interferes with virus infection at a very early step such as adsorption and/or penetration into the cells. In addition to the cytopathic effect of HIV-1 on MT-4 cells, syncytium formation between mock-infected MOLT-4 cells and MOLT-4 cells chronically infected with either HIV-1 or HIV-2 is suppressed by PM-104. PM-104 also blocks the replication of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The antiviral properties of PM-104 could be attributed to the combined effect of europium atoms and its peculiar three-dimensional anion structure.

Antitumor activity of new antitumor substance, polyoxomolybdate, against several human cancers in athymic nude mice.

Antitumor polyoxomolybdates have been recognized in the course of study on the medical utilization of polyoxometalates, inorganic polymers of metal oxide. [NH3Pri]6[Mo7O24].3H2O (PM-8) was found as a representative of antitumor polyoxomolybdates. The growth suppressions of PM-8 against Co-4 human colon cancer xenografted under the subrenal capsule in cd-1 mice were equal or superior to that of 5-FU, MMC, ACNU, ADM and CDDP. Potent antitumor activity of PM-8 is also established against MX-1 human breast and OAT human lung cancer xenografted in athymic nude mice. Polyoxomolybdate is a new type of antitumor substance.

Suppressive effect of polyoxometalates on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and their inhibitory activity against HIV-1 reverse transcriptase.

One isopolyoxometalate and 42 heteropolyoxometalates consisting of 3 compounds with the trivacant Keggin structure, 2 with the lacunary Keggin structure, 30 with the Keggin structure, one with the Wells-Dawson structure and 6 with miscellaneous structures were tested for their suppressive effect on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and inhibitory activity against HIV-1 reverse transcriptase. In contrast to the leading interpretations which attribute the suppressive effect of polyoxometalates on the cytopathogenicity of HIV-1 to the inhibition of HIV-1 reverse transcriptase by these compounds, there was no distinct correlation observed between these two functions of polyoxometalates.

In vitro antiviral activity of polyoxotungstate (PM-19) and other polyoxometalates against herpes simplex virus.

Polyoxotungstates with Keggin-type structure were found to demonstrate marked antiherpetic activity. K7[TiW10PO40].6H2O (PM-19) caused a decrease in plaque formation by several strains of herpes simplex virus (HSV) type 1, including acyclovir-resistant (thymidine kinase-negative) strains, at concentrations which were not toxic to the host cells. The 50% plaque-inhibiting concentration (EC50) for the different strains was between 20 and 50 micrograms/ml. Single-cycle HSV growth was also inhibited by PM-19. PM-19 inhibited viral DNA synthesis in HSV-infected cells at a concentration of 5 micrograms/ml but did not exhibit a virucidal effect, and pretreatment of the host cells with PM-19 did not provide resistance to herpes infection. Yet, virus adsorption to the cells was markedly affected at PM-19 concentrations higher than 25 micrograms/ml. PM-19 was also effective against human cytomegalovirus, but not against adenoviruses and varicella-zoster virus, although it did delay the development of the cytopathic effect of these viruses.

Antiviral activity of polyoxomolybdoeuropate PM-104 against human immunodeficiency virus type 1.

A polyoxomolybdoeuropate PM-104 (NH4)12H2[Eu4(MoO4)(H2O)16(Mo7O24)4].13H2O was found to be a potent inhibitor of the growth of human immunodeficiency virus type 1 (HIV-1), a causative agent of acquired immunodeficiency syndrome (AIDS). On the basis of TI50 [median cytotoxic concentration (CC50)/median effective concentration (EC50)], the in vitro anti-HIV-1 activity of PM-104 is favorably comparable to that of a heteropolyoxotungstate PM-19 K7[PTi2W10O40].6H2O, which is one of the most potent HIV-1 inhibitors among the polyoxometalates so far tested. The heteropolyoxomolybdate with a potent anti-HIV-1 activity is introduced for the first time in this communication.