Tripeptide mimetics inhibit the 20 S proteasome by covalent bonding to the active threonines.

Proteasomes play an important role in protein turnover in living cells. The inhibition of proteasomes affects cell cycle processes and induces apoptosis. Thus, 20 S proteasomal inhibitors are potential tools for the modulation of neoplastic growth. Based on MG132, a potent but nonspecific 20 S proteasome inhibitor, we designed and synthesized 22 compounds and evaluated them for the inhibition of proteasomes. The majority of the synthesized compounds reduced the hydrolysis of LLVY-7-aminomethylcoumarin peptide substrate in cell lysates, some of them drastically. Several compounds displayed inhibitory effects when tested in vitro on isolated 20 S proteasomes, with lowest IC(50) values of 58 nm (chymotrypsin-like activity), 53 nm (trypsin-like activity), and 100 nm (caspase-like activity). Compounds 16, 21, 22, and 28 affected the chymotrypsin-like activity of the beta5 subunit exclusively, whereas compounds 7 and 8 inhibited the beta2 trypsin-like active site selectively. Compounds 13 and 15 inhibited all three proteolytic activities. Compound 15 was shown to interact with the active site by x-ray crystallography. The potential of these novel inhibitors was assessed by cellular tolerance and biological response. HeLa cells tolerated up to 1 microm concentrations of all substances. Intracellular reduction of proteasomal activity and accumulation of polyubiquitinated proteins were observed for compounds 7, 13, 15, 22, 25, 26, 27, and 28 on HeLa cells. Four of these compounds (7, 15, 26, and 28) induced apoptosis in HeLa cells and thus are considered as promising leads for anti-tumor drug development.

Stimulation of TNF-alpha production by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) - a novel immunomodulator with potential application in tumour immunotherapy.

The immunomodulatory effects of a recently synthesized adamantane derivative of aminopyridine - 2-(1-adamantylamino)-6-methylpyridine (AdAMP) - were tested on normal and neoplastic cells in vitro. When incubated with TNF-alpha gene-transduced mouse melanoma cells (B78/TNF), AdAMP significantly enhanced basal production of TNF-alpha by these cells, both by "high" and "moderate" TNF-alpha-producer cells. A similar TNF-alpha production-enhancing effect was observed in cultures of human ovarian carcinoma cells (CAOV1) which spontaneously produce TNF-alpha but not in cultures of tumour cells incapable of TNF-alpha secretion. RT-PCR analysis showed that the enhancement of TNF-alpha production by AdAMP was associated with an increase in TNF-alpha mRNA expression in the treated cells. The results of an electrophoretic mobility shift assay (EMSA) showed that AdAMP significantly activated nuclear factor kappaB (NF-kappaB) in both CAOV1 and B78/TNF cells. The role of NF-kappaB in enhancement of TNF-alpha production was confirmed in experiments in which MG132, an inhibitor of NF-kappaB activation, reversed the effect of AdAMP. Unexpectedly, dexamethasone, a potent antiinflammatory agent and a strong inhibitor of TNF-alpha production in vivo, increased both spontaneous and AdAMP-augmented production of TNF-alpha in in vitro cultures of ovarian carcinoma cells and B78/TNF cells. AdAMP also enhanced TNF-alpha secretion by LPS-induced monocytes. AdAMP-induced augmentation of TNF-alpha production by B78/TNF cells was accompanied by morphological changes in the treated cells and a decrease in their adherence to fibrinogen and collagen IV. In view of these properties, AdAMP seems to be a therapeutically promising compound with potential application as an adjuvant augmenting the efficacy of cancer vaccine-based therapies or in the local treatment of certain tumours.

Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism.

Lovastatin, a drug successfully used in the clinic to prevent and to treat coronary heart disease, has recently been reported to decrease the incidence of melanoma in lovastatin-treated patients. Lovastatin has also been proved to potentiate antitumor effects of both cisplatin and TNF-alpha in murine melanoma models. Recently, an augmented therapeutic effect of lovastatin and doxorubicin has been reported in 3 tumor models in mice. In our preliminary study lovastatin caused retardation of melanoma growth in mice treated with doxorubicin (Feleszko et al. J Natl Cancer Inst 1998;90:247-8). In the present report, we supplement our preliminary observations and demonstrate in 2 murine and 2 human melanoma cell lines that lovastatin effectively potentiates the cytostatic/cytotoxic activity of doxorubicin in vitro via an augmentation of apoptosis (estimated with PARP-cleavage assay, annexin V assay and TUNEL). The combined antiproliferative activity of lovastatin and doxorubicin was evaluated using the combination index (CI) method of Chou and Talalay, revealing synergistic interactions in melanoma cells exposed to lovastatin and doxorubicin. In B16F10 murine melanoma model in vivo, we have demonstrated significantly increased sensitivity to the combined treatment with both lovastatin (5 mg/kg for 14 days) and doxorubicin (4 x 1 mg/kg) as compared with either agent acting alone. Lovastatin treatment resulted also in significant reduction of the number of experimental metastasis in doxorubicin-treated mice. The results of our studies suggest that lovastatin may enhance the effectiveness of chemotherapeutic agents in the treatment of malignant melanomas.

Synergistic interaction between highly specific cyclooxygenase-2 inhibitor, MF-tricyclic and lovastatin in murine colorectal cancer cell lines.

Statins, anti-hypercholesterolemic agents, have previously been reported to induce apoptosis and exert antitumor activity when combined with other antitumor agents. The potential of lovastatin in combination with highly specific COX-2 inhibitor (MF-tricyclic) to induce anti-proliferative activity against tumour cells was evaluated using the combination index (CI) method. Murine colorectal cancer (colon-26, CMT-93), melanoma (B16F10) and human bladder carcinoma cells (T24) were tested. Exposure of colon-26 and CMT-93 cells resulted in synergistic interactions in both cell lines with CI<1 for 20-80% inhibition of cell growth in both cell lines. This synergy was not observed in the B16F10 melanoma and T24 bladder carcinoma cells. MF-tricyclic (40 microg/ml), augmented lovastatin-induced apoptosis up to 2.5-fold in colon-26 cancer cells. Combination of a specific COX-2 inhibitor, MF-tricyclic, may increase antiproliferative effects of lovastatin in colon cancer cells and this effect was due to an augmented apoptosis.