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Clinical Cancer Research Vol. 6, 1546-1556, April 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Bis(4,7-dimethyl-1,10-phenanthroline) Sulfatooxovanadium(IV) as a Novel Apoptosis-inducing Anticancer Agent1

Rama Krishna Narla, Yanhong Dong, Osmond J. D’Cruz, Christopher Navara and Fatih M. Uckun2

Departments of Experimental Oncology [R. K. N., O. J. D., C. N., F. M. U.] and Chemistry [Y. D.] and Drug Discovery Program [R. K. N., Y. D., O. J. D., C. N., F. M. U.], Parker Hughes Institute, St. Paul, Minnesota 55113


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In a systematic effort to identify a potent anticancer agent, we synthesized 15 oxovanadium(IV) complexes and examined their cytotoxic activity against 14 different human cancer cell lines. The oxovanadium compounds included mono and bis ancillary ligands of 1,10-phenanthroline (phen) [VO(phen), VO(phen)2, VO(Me2-phen), VO (Me2-phen)2, VO(Cl-phen), VO(Cl-phen)2, VO(NO2-phen), VO(NO2-phen)2], 2,2'-bipyridyl (bipy) [VO(bipy), VO(bipy)2, VO(Me2-bipy), VO(Me2-bipy)2], and 2-2'-bipyrimidine(bipym) [VO(bipym) and VO(bipym)2], linked via nitrogen atoms, and 5'-bromo-2'-hydroxyacetophenone (acph) [VO(acph)2], linked via oxygen donor atom. The mono-chelated [VO(Me2-phen), compound 3] and bis-chelated-phen[VO(Me2-phen)2, compound 4] complexes were the most potent oxovanadium compounds and killed target cancer cells at low micromolar concentrations. Notably, the dimethyl substitution of the phenanthroline rings was essential for the anticancer activity of both compound 4 [VO(Me2-phen)2] and compound 3 [VO(Me2-phen)] because unsubstituted bis-chelated and mono-chelated phen oxovanadium(IV) complexes [VO(phen), compound 1, or VO(phen)2, compound 2] were less active. Addition of a chloro or nitro group to the phen complexes did not significantly improve the cytotoxic activity of the unsubstituted oxovanadium(IV) complexes. Irrespective of the ligands, bis-chelated phenanthroline containing compounds showed better activity than the mono-chelated phenanthroline containing complexes. The marked differences in the cytotoxic activity of oxovanadium(IV) complexes containing different heterocyclic ancillary ligands suggest that the cytotoxic activity of these compounds is determined by the identity of the five-member bidentate ligands, as well as the nature of the substitutents on the heterocyclic aromatic rings. Our results presented herein provide experimental evidence that oxovanadium compounds induce apoptosis in human cancer cells. Oxovanadium compounds, especially the lead compound VO(Me2-phen)2, may be useful in the treatment of cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Vanadium is a physiologically important trace element that is found in both anionic and cationic forms with oxidation states ranging from –1 to +5 (I–V; Refs. 1, 2, 3 ). The cationic form of vanadium complexes with oxidation state +4 (IV) have been shown to function as modulators of cellular redox potential, regulate cellular phosphorylation events, and exert pleiotropic effects in multiple biological systems (4, 5, 6, 7, 8, 9, 10) . Besides the ability of the vanadium metal to assume various oxidation states, its coordination chemistry also plays a key role in its interactions with various biomolecules. In particular, organometallic complexes of vanadium(IV) linked to bis(cycopentadienyl) moieties or vanadocenes exhibit antitumor properties both in vitro and in vivo (11, 12, 13, 14) .

In a systematic effort aimed at identifying new cytotoxic agents with potent activity against cancer cells, we synthesized 15 oxovanadium compounds and examined their cytotoxicity against a panel of 14 human cancer cell lines. The oxovanadium compounds included mono and bis ancillary ligands of phen3 and bipy substituted with dimethyl, chloro, and nitro groups and acph [VO(Br, OH-acph)2]. The mono-chelated [VO(Me2-phen), compound 3] and bis-chelated-1,10-phenantroline [VO(Me2-phen)2, compound 4] complexes were the most potent oxovanadium compounds and killed target cancer cells at low micromolar concentrations. Notably, the presence of two phenanthroline rings and their dimethyl substitution were essential for the anticancer activity of both compound 4 [VO(Me2-phen)2] and compound 3 [VO(Me2-phen)] because unsubstituted bis-chelated and mono-chelated phen oxovanadium(IV) complexes [VO(phen), compound 1, or VO(phen)2, compound 2] were less active. Addition of a chloro or nitro group to the phen complexes did not significantly improve the cytotoxic activity of the unsubstituted oxovanadium(IV) complexes. Irrespective of the ligands, bis-chelated phenanthroline containing compounds showed better activity than the mono-chelated phenanthroline containing complexes. The marked differences in the cytotoxic activity of oxovanadium(IV) complexes containing different heterocyclic ancillary ligands suggest that the cytotoxic activity of these compounds is determined by the identity of the five-member bidentate ligands, as well as the nature of the substitutents on the heterocyclic aromatic rings. Our results presented herein provide experimental evidence that oxovanadium compounds induce apoptosis in human cancer cells. Oxovanadium compounds, especially the lead compound VO(Me2-phen)2 may be useful in the treatment of cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Chemistry of Oxovanadium(IV) Complexes Containing Phen, Bipy, or 5'-Bromo-2'-Hydroxyacetophenone.
Reagents and solvents were purchased commercially and used as received. IR spectra were obtained as KBr pellets on a Nicolet Protege 460 spectrometer. Elemental analyses were performed by the Atlantic Microlab, Inc. (Norcross, Georgia). The oxovanadium(IV) complexes (compounds 1–14) were synthesized based on the previously published chemistry of VO(phen) and VO(phen)2 complexes (15) . Briefly, these complexes were synthesized by reacting an aqueous solution of vanadyl sulfate with an ethanol solution or a chloroform solution of the ligands. The solid products were purified from chloroform, ether, and/or water. Compound 15 was prepared from the reaction of VOSO4·3H2O with two equivalents of 5'-bromo-2'-hydroxyacetophenone and two equivalents of NaOH. An ethanol solution of 5'-bromo-2'-hydroxyacetophenone was added to an aqueous solution of VOSO4·3H2O, and then an aqueous solution of NaOH was added after 1 h. The reaction solution was stirred at room temperature for 2 h, and the yellow solid product was obtained by filtration, washing with water and ether, and drying in air. The purified complexes were characterized by Fourier transform IR spectroscopy (FT-Nicolet model Protege 460; Nicolet Instrument Corp., Madison, WI), UV-visible spectroscopy (DU 7400 spectophotometer; Beckman Instruments, Fullerton, CA), and elemental analysis (Atlantic Microlab, Inc., Norcross, GA). These oxovanadium(IV) complexes have an octahedral geometry except for compound 15 which has a square pyramidal geometry. The oxovanadium(IV) complexes are stabilized with bidentate ligands that form a five-member ring with the vanadium atom. The choice of these three organic ligands (phenanthroline, bipyridyl, and acetophenone) was based on the reported fact that the cationic oxovanadium(IV) complex of phenanthroline is superior to cisplatin [cis-diamminedichloroplatinum(II)] with respect to antitumor activity (15) , the structural similarity of bipyridyl ring to phenanthroline, and the neutral nature of acetophenone complex of oxovanadium(IV). Structural variations of the ligands included addition of bromo, chloro, or methyl groups on the phenanthroline, bipyridyl or acetophenone rings. The chemical structures of the 15 oxovanadium(IV) complexes, including 8 complexes with phen and 4 complexes with bipy, 2 complexes with bipym and 1 neutral complex, bis-5'-bromo-2'hydroxyacetophenone, are depicted in Table 1Citation . Among 15 compounds, 12 compounds (compounds 3–9, 11–15) are novel. Compounds 2, 4, 6, 8, 10, 12, and 14 are shown as having a cis-octahedral configuration based on the recently resolved crystal structures of the compounds 2 and 4 (16) .


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  		Table 1 Characterization of oxovanadium (IV) compounds

 
Cell Lines.
Human B-lineage ALL cell line NALM-6 (17) , T-lineage ALL cell line MOLT-3 (18) , acute myeloid leukemia cell line HL-60 (19) , and multiple myeloma cell lines ARH-77, U266BL, HS-SULTAN, brain tumor/glioblastoma cell line U373, breast cancer cell line BT-20, testicular cancer cell lines, 833K, 64cp, Tera-2 (embryonal carcinoma), and NT2D1 (pluripotent embryonal carcinoma), and the Hodgkin’s lymphoma cell line HS445 were obtained from American Type Culture Collection (Manassas, VA). Cell lines were propagated in RPMI 1640 (NALM-6, MOLT-3, HL-60, ARH-77, U266BL and HS-SULTAN), DMEM (U373 and BT-20), McCoy’s 5A medium (Tera-2 and NT2D1), or {alpha}-MEM (833K and 64cp). All media were supplemented with 10% FCS, 4 mM glutamine, 100 units/ml penicillin G, and 100 mg/ml streptomycin sulfate. All tissue culture reagents were obtained from Life Technologies Inc. (Life Technologies, Inc., Gaithersburg, MD). Cell lines were cultivated for a minimum of two passages after thawing prior to experimentation.

MTT and Clonogenic Assays.
The cytotoxicity of oxovanadium(IV) complexes were tested against human cancer cell lines using MTT assays (Roche Molecular Biochemicals, Indianapolis, IN), as described previously (20) . Briefly, exponentially growing tumor cells were seeded into a 96-well plate at a density of 4 x 104 cells/well and incubated in medium containing the oxovanadium(IV) compounds at concentrations ranging from 0.1 to 250 µM for 48 h at 37°C in a humidified 5% CO2 atmosphere. Triplicate wells were used for each treatment. To each well, 10 µl of MTT (final concentration, 0.5 mg/ml) was added, and the plates were incubated at 37°C for 4 h to allow MTT to form formazan crystals by reacting with metabolically active cells. The formazan crystals were solubilized overnight at 37°C in a solution containing 10% SDS and 0.01 M HCl. The absorbance of each well was measured in a microplate reader (Labsystems) at 540 nm and a reference wavelength of 690 nm. To translate the A540 values into the number of live cells in each well, the A540 values were compared to those on standard A540 versus cell number curves generated for each cell line. The percentage of survival was calculated using the formula: % survival = [Live cell number(test)/Live cell number(control)] x 100. The IC50 values were calculated by nonlinear regression analysis using the graphed Prism Software version 2.0 (GraphPad Software, Inc., San Diego, CA).

The anticancer activity of oxovanadium complexes against clonogenic NALM-6 cells was examined using a methylcellulose colony assay system, as described previously (21, 22, 23) . In brief, cells (105 cells/ml in RPMI-10% fetal bovine serum) were treated overnight at 37°C with oxovanadium complexes at varying concentrations. After treatment, cells were washed twice, plated at 104 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum, and 0.9% methylcellulose in 35-mm Petri dishes, and cultured for 7 days at 37°C in a humidified 5% CO2 incubator. Subsequently, leukemic cell colonies were enumerated using an inverted phase-contrast microscope, and the percentage of inhibition of colony formation was determined using the following formula: % Inhibition = [1 – (Number of colonies in compound-treated test cultures/Number of colonies in vehicle-treated control cultures)] x 100.

Apoptosis Assays.
The demonstration of apoptosis was performed as described earlier (20 , 24) by the in situ nick end labeling method using in situ cell death detection kit (Roche Molecular Biochemicals) according to the manufacturer’s recommendations. Exponentially growing cells were seeded in 6-well tissue culture plates and incubated with fresh medium containing compounds. After a 24-h incubation at 37°C in a humidified 5% CO2 incubator, the cells were collected into a 15-ml centrifuge tube, washed with PBS, and pelleted by centrifugation at 1000 rpm for 5 min. The cells were fixed in 2% paraformaldehyde, washed with PBS, and pelleted by centrifuging the tubes at 1000 rpm for 5 min. Cell pellets were resuspended in 50 µl of PBS, transferred to Superfrost Plus slides and allowed to attach for 15 min. The cells were permeabilized with 0.1% Triton X-100 in 0.1% citrate buffer and incubated for 1 h at 37°C with the reaction mixture containing TdT and FITC-conjugated dUTP. Cells were washed with PBS to remove unbound reagents, and the coverslips were mounted onto slides with Vectashield containing PI (Vector Laboratories, Burlingame, CA). Slides were viewed with a confocal laser scanning microscope (Bio-Rad MRC 1024) mounted on a Nikon Eclipse E800 series upright microscope, as reported previously (20 , 24 , 25) . Nonapoptotic cells do not incorporate significant amounts of dUTP due to lack of exposed 3'-hydroxyl ends, and consequently have much less fluorescence than apoptotic cells that have an abundance of exposed 3'-hydroxyl ends. In control reactions, the TdT enzyme was omitted from the reaction mixture.

A flow cytometric two-color TUNEL assay was used to detect apoptotic nuclei. Exponentially growing cells (106/ml) were incubated in DMSO alone (0.1%) or treated with one of the 15 oxovanadium compounds (each at 50 µM in 0.1% DMSO) for 24 h. Cells were washed in PBS and fixed in 4% paraformaldehyde in PBS for 15 min on ice. After two washings in PBS, they were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice and washed twice in PBS. Labeling of exposed 3'-hydroxyl (3'-OH) ends of fragmented nuclear DNA was performed using TdT and FITC-conjugated dUTP according to the manufacturer’s recommendations (Roche Molecular Biochemicals). Cells were counterstained with 5 µg/ml PI. Control samples included: (i) untreated cells; and (ii) cells incubated with the reaction mixture without the TdT enzyme. Cells were analyzed with a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). Relative DNA content (PI emission) was detected with band-pass filter 585/42, and dUTP incorporation (FITC emission) was detected with band-pass filter 530/30. Fluorescence was compensated for in the acquisition software using single-label control samples. Data were acquired in a listmode, gated to 10,000 events per sample, and analyzed using the CellQuest software program (Becton Dickinson). Nonapoptotic cells do not incorporate significant amounts of dUTP due to lack of exposed 3'-OH ends, and consequently have relatively little or no fluorescence compared to apoptotic cells that have an abundance of 3'-OH (M2 gates). Apoptosis was detected by an increase in the number of cells staining with FITC-dUTP. The M1 and M2 gates were used to demarcate nonapoptotic and apoptotic PI-counterstained cell populations, respectively.

Cell Cycle Analysis.
Exponentially growing cells were incubated with various concentrations ranging from 1 to 25 µM of oxovanadium compounds for 24 h at 37°C. Cells were harvested by trypsin release and resuspended in DNA staining solution (10 µg/ml RNase, 0.1% Triton X-100, 0.1 mM EDTA, 0.1% sodium citrate, 50 µg/ml PI, and 1 mM Tris-HCl) 1–2 h prior to flow cytometric analysis. The fluorescence of 10,000 cells was measured with a Becton Dickinson flow cytometer with excitation of 488 nm. The percentage of cells in G1, S, and G2-M was determined using the CellQuest software, version 3.1.

In some experiments, immunofluorescence was used to examine the morphological features of oxovanadium compound-treated cancer cells. At the end of the indicated treatment period, cells were washed twice with PBS and fixed in 2% paraformaldehyde. The cells were permeabilized, and nonspecific binding sites were blocked with 2.5% BSA in PBS containing 0.1% Triton X-100 for 30 min. Tubulin expression was examined by immunofluorescence using a monoclonal antibody against {alpha}-tubulin (Sigma Chemical Co.) at a dilution of 1:1000 and an antimouse IgG conjugated to FITC. Cells were washed in PBS and counterstained with TOTO-3 (Molecular Probes Inc., Eugene, OR) for 10 min at a dilution of 1:1000. Cells were washed again with PBS, and the coverslips were mounted with Vectashield (Vector Laboratories) and viewed with a confocal microscope.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Cytotoxicity of Oxovanadium Compounds against Human Cancer Cell Lines.
We have synthesized a series of 15 oxovanadium(IV) complexes (Table 1)Citation including 8 phen-linked [VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), VO(Cl-phen)2, VO(NO2-phen), and VO(NO2-phen)2], 4 bipy-linked [VO(bipy), VO(bipy)2, VO(Me2-bipy), VO(Me2-bipy)2], 2 bipym-linked [VO(N2-bipym) and VO(N2-bipym)2], and 1 acph-linked) [VO(Br,OH-acph)2], and tested their cytotoxic activity against 14 different human cancer cell lines, including the B-lineage ALL cell line NALM-6, T-lineage ALL cell line MOLT-3, acute myeloid leukemia cell line HL-60, multiple myeloma cell lines ARH-77, U266BL, and HS-SULTAN, Hodgkin’s lymphoma cell line HS445, and the testicular cancer cell lines 833K, 64cp, TERA-2, and NT2D1, prostate cancer cell line PC3, breast cancer cell line BT-20, and glioblastoma cell line U373 using MTT assays and/or confocal laser scanning microscopy. Compounds were tested side-by-side at eight different concentrations ranging from 0.1 to 250 µM.

Each of the 15 oxovanadium complexes exhibited significant cytotoxicity against several of the cancer cell lines in a concentration-dependent fashion (Tables 2Citation and 3Citation and Fig. 1Citation ). Fig. 1Citation shows the concentration-dependent MTT-based cytotoxicity curves of 12 representative oxovanadium(IV) compounds against NALM-6 leukemia cells. Table 3Citation shows the effects of oxovanadium compounds on breast cancer, prostate cancer, glioblastoma, and testicular cancer cell lines in MTT assays. The cytotoxic activity of the oxovanadium(IV) complexes was strongly dependent on the type of coordinated heteroligands. When compared to diaqua mono-chelated complexes, the octahedral structure oxovanadium complexes stabilized with five-member bis-chelated ligands of phenanthroline or bipyridyl showed superior cytotoxic activity against cancer cells. The mono-chelated [VO(Me2-phen), compound 3] and bis-chelated dimethyl-phen [VO(Me2-phen)2, compound 4] complexes were the most potent oxovanadium compounds and killed each of the cancer cell lines examined at low micromolar concentrations (Fig. 1Citation and Tables 2Citation and 3Citation ). Notably, the presence of two phenanthroline rings and their dimethyl substitution was essential for the anticancer activity of both compounds 4 [VO(Me2-phen)2 ] and 3 [VO(Me2-phen)] because unsubstituted bis-chelated and mono-chelated phen oxovanadium(IV) complexes [VO(phen), compound 1, or VO(phen)2, compound 2] were less active. Addition of a chloro or nitro group to the phen compounds did not significantly improve the cytotoxic activity of the unsubstituted oxovanadium(IV) complexes (Table 2)Citation . Irrespective of the ligands, bis-chelated phenanthroline containing compounds showed better activity than the mono-chelated phenanthroline containing compounds. The marked differences in the cytotoxic activity of oxovanadium(IV) complexes containing different heterocyclic ancillary ligands suggest that the cytotoxic activity of these compounds is determined by the identity of the five-member bidentate ligands, as well as by the nature of the substitutents on the heterocyclic aromatic rings. We also examined the ability of oxovanadium compounds to inhibit the in vitro clonogenic growth of NALM-6 leukemia cells (Table 4)Citation . All of the oxovanadium compounds inhibited in vitro colony formation of NALM-6 leukemic cells with IC50 values of 0.53 (compound 1), 0.31 (compound 2), 0.19 (compound 3), 0.03 (compound 4), 6.28 (compound 5), 3.31 (compound 6), 4.61 (compound 11), and 4.4 µM (compound 12). Similar to MTT assays, the data on clonogenic assays show an order of activity (i.e., compound 4 > 3 > 2 > 1) that indicates that the number of phenanthroline rings and dimethyl substitution on phenanthroline rings are important for the biological activity and potency of oxovanadium compounds.


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  		Table 2 Cytotoxic activity of oxovanadium (IV) compounds against leukemia (NALM-6, MOLT-3, and HL-60), Hodgkin’s lymphoma (HS445), and multiple myeloma (U266BL, ARH77, and HS-SULTAN) cells

Cells were treated with various concentrations ranging from 0.1 to 100 µM oxovanadium (IV) complexes for 48 h; cell survival was measured with MTT assays, and IC50 values were calculated with nonlinear regression analysis.

 

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  		Table 3 Cytotoxic activity of oxovanadium (IV) compounds against testicular cancer (833K, 64cp, TERA-2, and NT2D1), glioblastoma (U373), breast cancer (BT-20), and prostate cancer (PC3) cells

Cells were treated with various concentrations ranging from 0.1 to 100 µM oxovanadium (IV) complexes for 48 h; cell survival was measured with MTT assays, and IC50 values were calculated with nonlinear regression analysis.

 


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  		Fig. 1. Cytotoxic activity of oxovanadium(IV) complexes with mono- or bis-phen, mono- or bis-bipy, and mono- or bis-bipym against human leukemic NALM-6 cells. Cells were incubated with increasing concentrations (0.1–100 µM) of compounds 1–4 and 7–14 for 48 h in 96-well plates, and the cell survival was determined by MTT assay. The data points represent the mean (± SE) values from three independent experiments.

 

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  		Table 4 Cytotoxicity of oxovanadium (IV) compounds against clonogenic NALM-6 leukemia cells

 
Oxovanadium(IV) Compounds Induce Apoptosis in Human Cancer Cells.
To determine whether the cytotoxicity of the oxovanadium compounds is associated with apoptotic cell death, 833K testicular cancer cells were cultured with the oxovanadium compounds (50 µM) for 24 h and then subjected to flow cytometric analysis for dUTP incorporation by the TdT-mediated TUNEL assay. Fig. 2, A, C, E, and G,Citation depicts the two-color flow cytometric contour plots of cells from representative TUNEL assays. Control 833K cells were treated for 24 h at 37°C with 0.1% DMSO, whereas test cells were treated for 24 h at 37°C with an oxovanadium compound at a final concentration of 50 µM. The TdT-dependent incorporation of FITC-dUTP was dramatically increased in cells treated with the oxovanadium compounds as a result of abundance of free 3'-hydroxyl DNA ends created by apoptotic DNA fragmentation (Fig. 2, D, F, and H).Citation Apoptosis after treatment with oxovanadium compounds was also evident from the concentration-dependent emergence of a hypodiploid (<2 N) peak in the DNA histograms of PI-stained cells, which was accompanied by nonselective loss of G0/1, S, and G2-M cells (Fig. 3)Citation . Similar results were obtained with NALM-6 leukemia and HS-SULTAN multiple myeloma cells. The TUNEL assay-based IC50 values against NALM-6 cells were 4.1 µM for compound 1, 2.8 µM for compound 2, 1.8 µM for compound 3, 0.5 µM for compound 4, 6.7 µM for compound 5, 3.7 µM for compound 6, 12.6 µM for compound 11, and 8.7 µM for compound 12. As evidenced by the confocal laser scanning microscopy images depicted in Fig. 4Citation , VO(Me2-phen)2 (compound 4)-treated and VO(Me2-phen) (compound 3)-treated [but not VO(Cl-phen)2 (compound 6)-treated] leukemic NALM-6 and HS-SULTAN cells examined for FITC-conjugated dUTP incorporation (green fluorescence) and PI counterstaining (red fluorescence) showed an abundance of apoptotic yellow nuclei with superimposed green and red fluorescence at 48 h after treatment. We also used immunofluorescence staining with anti-{alpha}-tubulin antibody and the nuclear dye TOTO-3 in combination with confocal laser scanning microscopy to examine the morphological features of cancer cells treated with oxovanadium compounds. Fig. 5Citation depicts the two-color confocal laser scanning microscopy images of BT-20 breast cancer and U373 glioblastoma cells after treatment with oxovanadium compounds 4 and 6. Most of the oxovanadium compound-treated cells displayed the characteristic morphological features of apoptotic cell death, including an abnormal architecture with complete disruption of microtubules, marked shrinkage, chromatin condensation, nuclear fragmentation, the appearance of typical apoptotic bodies and inability of the cells to adhere to the substratum. Similar data were also obtained with prostate cancer PC3 cells (data not shown).



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  		Fig. 2. Apoptosis-inducing profile of oxovanadium compounds. Testicular cancer cells (833K) were incubated with compounds 2, 6, and 8 (50 µM) for 24 h, fixed, permeabilized, and processed for TUNEL assay as described in "Materials and Methods." Apoptotic nuclei were visualized with confocal laser scanning microscopy and quantitated by two-color flow cytometry. Two-color flow cytometric contour plots (left panels; A, C, E, and G) and two-color confocal laser scanning microscopy images (right panels; B, D, F, and H) of green/yellow (i.e., superimposed green plus red) apoptotic nuclei of 833K cells treated with compound 2 (C and D), 6 (E and F), or 8 (G and H) or vehicle (A and B). Red fluorescence represents nuclei stained with propidium iodide. Percentages in the left panels indicate cells with apoptotic nuclei.

 


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  		Fig. 3. Oxovanadium compounds affect DNA content/cell cycle distribution in human testicular cancer cells. 833K testicular cells were treated with vehicle or 25, 12.5, 6.3, or 3.1 µM of compounds 2, 4, and 8 for 24 h at 37°C, trypsinized, fixed, stained with propidium iodide, and analyzed by flow cytometry for DNA content. The percentages of cells in each cell cycle were determined using the CellQuest Software and are indicated in each of the histograms. An increase in G2-M cells and a selective loss of G0/1 cells preceded apoptosis. The apoptotic nuclei were evident as a hyperdiploid sub G1-peak (AP).

 


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  		Fig. 4. Oxovanadium(IV) complexes induce apoptosis in leukemia and multiple myeloma cells. Human leukemia NALM-6 (A.1–A.4) and multiple myeloma HS-SULTAN (B.1 and B.2) cells were incubated with 5 µM of compound 3, compound 4, or compound 6 for 24 h, fixed, permeabilized, and visualized for DNA degradation in a TUNEL assay using dUTP-labeling. Red fluorescence represents nuclei stained with propidium iodide. Green or yellow (i.e., superimposed red plus green) represents apoptotic nuclei containing fragmented DNA. A.1 and B.1, vehicle-treated control cells. A.2, cells treated with compound 3; A.3 and B.2, cells treated with compound 4; A.4, cells treated with compound 6.

 


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  		Fig. 5. Confocal images of apoptotic cancer cells treated with oxovanadium compounds. Cells were treated with 50 µM compound 4 or compound 6 for 24 h at 37°C. After treatment, cells were processed for immunofluorescence using a monoclonal antibody to {alpha}-tubulin (green fluorescence). Control cells (Con) were treated with vehicle only. Cells treated with compound 4 or 6 showed marked shrinkage with disruption of microtubules and lost their ability to adhere to the substratum. Blue fluorescence, nuclei stained with TOTO-3.

 
Several types of vanadium-containing compounds have been tested for antitumor activity, among which bis(cyclopentadienyl)vanadium(IV) and peroxovanadates(V) were most thoroughly investigated (26) . It was proposed that DNA is the target for bis(cyclopentadienyl)vanadium(IV) compounds. Peroxovanadates(V) were proposed to undergo one electron intramolecular transfer, producing Vanadium(IV) and superoxide, a process triggers the generation of other ROS, including hydroxyl radical, which ultimately causes the cell death (27 , 28) . Peroxovanadates(V) were also known to inhibit protein-tyrosine phosphatases, the function of which is essential for mitosis progression, thereby inhibiting the cell cycle (29) . All of the vanadium(IV)-containing compounds showed substantial in vitro anticancer activity, with compound 4 being the most active compound. In a previous study, these oxovanadium complexes showed potent spermicidal activity by rapidly and irreversibly immobilizing the human sperm and inducing apoptosis at micromolar concentrations (30) . This spermicidal activity was proposed to be mediated by the reactive oxygen intermediates inducing activity of oxovanadium compounds. Although we do not know the molecular basis for the in vitro anticancer properties of our oxovanadium(IV) compounds at the present time, it has been shown that this type of compounds can interact with DNA and causes DNA cleavage (31) . Our studies and others suggested that the generation of ROS and the cell cycle arrest by the compounds may also contribute to the antitumor activity (15 , 16) . The presence of two phenanthroline rings and dimethyl substitution on phenanthroline ligands of oxovandium(IV) complexes substantially improved the cytotoxic activity. This superior activity may be due to the electron-donating dimethyl groups, which may contribute to the generation of ROS (16) , to the cell permeable nature of the complex coupled with intrinsic metal chelating activity (32) , and to p53 transactivation activity of phen. The transactivation of p53 by phen was shown to induce p53 target genes such as Waf-1 and Mdm-2 (33) ; this induction results in cell cycle arrest at G1 and apoptosis (34 , 35) .

In conclusion, our results provide experimental evidence that oxovanadium(IV) complexes with phen, bipy, bipym, or 5'-bromo-2'-hydroxyacetophenone and that their derivatives, linked to vanadium(IV) via nitrogen or oxygen atoms, have potent in vitro anticancer activity against human cancer cells. Because of its potent apoptosis-inducing activity, further development of our lead compound VO (Me2-phen)2 (compound 4) may provide the basis for the design of potentially effective treatment programs for cancer patients. The previously reported cytotoxic effects of oxovanadium compounds on sperm and germ cells (30) indicates that cancer treatment programs using these compounds may be associated with adverse side effects on the male reproductive system and sterility.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a special grant from the Parker Hughes Trust. F. M. U. is a Stohlman Scholar of Leukemia Society of America. Back

2 To whom requests for reprints should be addressed, at Parker Hughes Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113. Phone: (651) 697-9228; Fax: (651) 697-1042. Back

3 The abbreviations used are: phen, 1,10-phenanthroline; ALL, acute lymphoblastic leukemia; acph, 5'-bromo-2'-hydroxyacetophenone; bipy, 2,2'-bipyridyl; dUTP, digoxigenin-UTP; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IR, infrared; PI, propidium iodide; ROS, reactive oxygen species; TdT, terminal dideoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick-end labeling; bipym, 2,2'-bipyrimidine; CL-phen, 5-chlorophen; NO2-phen, 5-nitrophen; Me2-bipy, 4,4'-dimethylbipy; Me2-phen, 4,7-dimethyl-phen. Back

Received 9/13/99; revised 1/ 4/00; accepted 1/ 4/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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