Inhibition of human hepatocellular carcinoma by L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13) in vitro and in vivo.

A new anticancer tripeptide, L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13), was investigated for its activity and mechanism in human hepatocellular carcinoma (HCC) cell lines. MF13 showed antiproliferative activities in the panel of 7 human HCC cell lines with IC50 in the range of 0.08-2.32 microM. A significant blockade in the S-phase occurred in tumor cells 12 h after their exposure to MF13. The inactivated Rb (phosphorylated Rb, pRb), which is present in the S-phase, was increased within 6 h of treatment. Bcl-2 expression was without change in hepatocarcinoma cells treated with MF13; however, a significant increase of bax was observed, resulting in a decreased ratio of bcl-2/bax. Increased activity of caspase-9, -8 and -3 was detected in the MF13 treated cells, indicating an activated pathway of apoptosis by MF13. Morphological examination as well as DNA gel electrophoresis demonstrated a nuclear fragmentation and DNA degradation in the form of multiple-unit DNA ladder in MF13 treated tumor cells. MF13 alone at 10 mg/kg (i.p.) inhibited HepG2 tumor in nude mice by more than 94% in volume. Bel-7402 tumor originated from a Chinese patient with HCC exhibited a sensitivity to MF13 similar to HepG2 in vivo. Antitumor effect of MF13 in the nude mice bearing human hepatocarcinoma (Bel-7402 or HepG2) was stronger than mitomycin C as well as its precursor m-sarcolysin (p<0.01), and comparable with cyclophosphamide. We believe MF13 merits consideration for further investigation as an agent against human hepatocellular carcinoma.

Inhibition of microtubule polymerization by 3-bromopropionylamino benzoylurea (JIMB01), a new cancericidal tubulin ligand.

3-Bromopropionylamino benzoylurea (JIMB01) is a small molecular weight compound (MW 313) that has been synthesized in our laboratory. This compound showed antiproliferative activities in a panel of thirteen human tumor cell lines with IC(50) values in the range of 0.25 to 0.51 micro M for leukemia and lymphoma cell lines and 0.33 to 9.26 micro M for solid tumor cell lines. The primary action of JIMB01 is to inhibit microtubule polymerization but not depolymerization. A 4 micro M concentration of the compound caused a complete inhibition of microtubule assembly in a cell-free reaction. An increase in the number of human hepatocarcinoma cells blocked in the M-phase was detected 12hr after exposure to JIMB01. The kinase activity of cyclin B1, which is responsible for the G(2)/M transition, was increased accordingly. Bcl-2 phosphorylation became visible, in a western blot, within 6hr in hepatocarcinoma cells treated with JIMB01 at 0.8 micro M or higher. JIMB01-induced apoptosis in liver cancer cells was confirmed by morphological methods, flow cytometry, as well as DNA gel electrophoresis, which clearly demonstrated DNA degradation in the form of a multiple-unit DNA ladder. Furthermore, in vivo experiments using nude mice showed that intraperitoneal injection of JIMB01 at 15mg/kg (with seven injections at 4-day intervals) significantly inhibited the growth of a human hepatocarcinoma (BEL-7402) by 66% in tumor volume (P=0.01), at least compatible to the inhibition by vincristine (43% inhibition), indicating good bioavailability of the compound in the circulation. Side-effects of the compound were not observed, and the body weight of the treated mice remained stable during the 4-week treatment. Since JIMB01 is a small compound, targets a specific molecule in tumor cells, and has promising activity against human hepatocarcinoma in vivo, we believe JIMB01 merits consideration for further investigation.

Double blockade of cell cycle at g(1)-s transition and m phase by 3-iodoacetamido benzoyl ethyl ester, a new type of tubulin ligand.

3-Iodoacetamido benzoyl ethyl ester (3-IAABE) is a new compound synthesized in our laboratory. The primary action of 3-IAABE is to inhibit microtubule assembly by interacting with -SH groups on tubulin. In contrast to other known microtubule disrupters, 3-IAABE caused a double blockade in the cell cycle at G(1)-S transition and in M phase. The blockade was determined by cell cycle analysis and chromosome distribution. Kinase activities of cyclin E and cyclin-dependent kinase 2 responsible for the G(1)-S transition were increased, as were the activities of mitotic cyclin B and cdc2. 3-IAABE treatment also increased p53 expression and dephosphorylated (or activated) retinoblastoma protein. Investigation of the signal transduction pathway showed that 3-IAABE induced bcl-2 phosphorylation, followed by activation of caspase-9, -3, and -6, but not caspase-8. DNA fragmentation factor and poly(ADP-ribose) polymerase, the downstream substrates of caspase-3 and -6, were cleaved after 3 h of exposure to 3-IAABE, followed by DNA fragmentation. Pretreatment of the cells with inhibitors of caspase-9, -3, or -6, respectively, inhibited the cleavage of DNA fragmentation factor and poly(ADP-ribose) polymerase and thus inhibited the onset of apoptosis. 3-IAABE showed antitumor activities in the panel of 60 National Cancer Institute human tumor cell lines with total growth inhibition in the range of 0.22-4.3 micro M for solid tumor lines and 0.025-0.22 micro M for leukemia/lymphoma cell lines. The 3-IAABU total growth inhibition of phytohemagglutinin-stimulated healthy human lymphocytes was 450-fold greater than that of leukemic cells. 3-IAABE significantly inhibited the growth of human hepatocarcinoma (BEL-7402) in nude mice by 72% in tumor volume, more strongly than did vincristine (43 percent inhibition). Besides being a novel lead for the design of new anticancer tubulin ligands, the activity of 3-IAABE in the cell cycle may also help us to understand the molecular pharmacology of microtubule-active drugs.