Felicia Y.-H. Wu

Comparison of antitumor activity of vitamins K1, K2 and K3 on human tumor cells by two (MTT and SRB) cell viability assays

Vitamin K (VK) congeners (VK1, VK2, and VK3) have been used as antihemorrhagic agents, while VK3 has also been found to inhibit growth in various rodent and human tumor cells. We have compared the antitumor activities of vitamin K1, K2, and K3 against a panel of human cancer cell lines. For each test agent, a dose-response profile was generated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and an SRB (sulforhodamine B) assay. Both assays yielded similar results. The respective ID50 values of VK3 in five hepatoma cell lines, HA59T, HA22T, PLC, HepG2, and Hep3B, of increasing differentiation state, were 42, 36, 28, 27, and 20 microM. For nasopharyngeal carcinoma (CG1), leukemia (U937), oral epidermoid carcinoma (KB), and breast carcinoma (BC-M1) cells, the ID50 values of VK3 were 26, 15, 25, and 33 microM, respectively. For all the above cells, the ID50 values of VK1 ranged from 6 to 9 mM, and the ID50 values of VK2 ranged from 1 to 2 mM. Thus, the relative potencies of antitumor activity of VK3 compared to VK2 and to VK1 are about 60- and 300-fold, respectively. These results support the preference for use of VK3 over VK1 and VK2 in cancer therapy.

Determination of anticancer drug vitamin K3 in plasma by high-performance liquid chromatography

Synthetic vitamin K3 (VK3, 2-methyl-1,4-naphthoquinone, or menadione) has been found to exhibit antitumor activity against various human cancer cells at relative high dose. Parallel to our study on the mechanism of VK3 action and for future clinical trials in Taiwan, we developed a simple, sensitive and accurate high-performance liquid chromatographic method for the determination of VK3 in biological fluids. VK3 was extracted from the plasma samples with n-hexane. The chromatographic separation employed an ODS analytical column (5 microns, 250 x 4.6 mm I.D.) with a mobile phase of methanol-water (70:30, v/v) and UV detection at 265 nm. On completely drying of the extraction solution, n-hexane, by a stream of nitrogen, menadione was lost to a great extent. Methanol (70%, 200 microliters) was added to the extraction solvent after extraction and centrifugation to prevent the loss of menadione. The absolute recovery was 82.4 +/- 7.69% (n = 7). The within-day and between-day calibration curves of VK3 in plasma in the ranges of interest (0.01-10.00 micrograms/ml; 0.01-5.00 micrograms/ml) showed good linearity (r > 0.999) and acceptable precision. The limit of quantitation of VK3 was 10 ng/ml in plasma. This method has been successfully applied to a pilot pharmacokinetic study of VK3 in rabbits receiving an intravenous high-dose bolus injection of 75 mg menadiol sodium diphosphate (Synkayvite). The pharmacokinetic properties of menadione could be described adequately by an open two-compartment model. The mean half-life of menadiol (transformation to menadione) was 2.60 +/- 0.12 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of two growth inhibitory K vitamin analogs on cell cycle regulating proteins in human hepatoma cells.

A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.

Tissue-specific cancer-related serpin gene cluster at human chromosome band 3q26

Approximately one quarter of the identified human serpin genes are cancer‐related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas‐specific protease inhibitor, pancpin (PI14), and the brain‐associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov‐serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor‐1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33–35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down‐regulated in tumor brain tissues and in two brain cancer cell lines: U‐87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI‐10077 (telomere) is located downstream of the 3′‐flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is ∼100 kb, and hence we speculate that other tissue‐specific cancer‐related serpin genes are likely to reside within this 3q26.1 cluster region.

A pharmacokinetic study with the high-dose anticancer agent menadione in rabbits.

The aim of this investigation was to assess the pharmacokinetic properties of high-dose menadione (VK3), as an anticancer agent, in plasma and red blood cells (RBCs) in rabbits. An extremely high dose of 75 mg menadiol sodium diphosphate (Synkayvite) was intravenously injected. HPLC analysis was applied to measure the major metabolite, menadione, VK3. The kinetic properties of VK3 in both plasma and red blood cells showed a short elimination half-life, high clearance, and large volume of distribution in plasma and RBCs. The mean elimination t1/2 values of menadione in plasma and in RBCs were 27.17 +/- 10.49 min and 35.22 +/- 11.82 min, respectively. The plasma clearance (CL/F) of VK3 was 0.822 +/- 0.254 L min-1. The systemic clearance in RBCs was 0.407 +/- 0.152 L min-1. The apparent volume of distribution (Vd/F) in plasma was 30.833 +/- 12.835 L and that in RBCs 20.488 +/- 9.401 L. The plasma AUC was 32.453 +/- 9.785 micrograms min mL-1 and that of RBCs 67.219 +/- 24.449 micrograms min mL-1. Menadiol was rapidly biotransformed to menadione in blood. The formation rate constant (kf) of menadione in plasma was 0.589 +/- 0.246 min-1, and that of RBCs 1.520 +/- 1.345 min-1. Through this study the estimated menadione dosage needed to maintain a plasma level of 1 microgram mL-1 for anticancer purposes was 19.7 mg kg-1 every hour.

Cloning and characterization of the endogenous retroviral-tRNAGlu multigene family from human genomes of different racial backgrounds

An 8.3-kb human endogenous retroviral-tRNA(Glu) (HERV-E)-encoding cDNA clone and a 1.5-kb genomic clone were isolated from a Chinese-derived cervical cancer cell line, CC7T, and their sequences determined. The former is a full-length endogenous retroviral cDNA containing corresponding u5-gag-pol-env-u3-r regions. The latter is a partial retroviral DNA segment, covering the gag and pol genes. Analysis of normal human DNA by Southern blot hybridization with three specific HERV-E molecular DNA probes revealed complex restriction-fragment length polymorphisms (RFLP), implying that the human genome contains diverse proviral structures and dispersed integration sites. The complex patterns were virtually identical between DNAs from African-Americans, Asians and Caucasians, with only a few minor variations. The data suggest that these proviral sequences were mostly incorporated into the human genome before racial divergence and, hence, may serve as markers for distinct chromosomal sites.