Pyrrolotetrazinones deazaanalogues of temozolomide induce apoptosis in Jurkat cell line: involvement of tubulin polymerization inhibition.

PURPOSE: Pyrrolotetrazinones are a new class of azolotetrazinones endowed with a high, remarkable antiproliferative activity in human tumor cultured cells. They hold the deaza skeleton of the antitumor drug temozolomide, although preliminary investigations indicated a different mechanism of action. To understand their mechanism(s) of action along with their target at molecular level, four derivatives were selected on the basis of their activity on a panel of human tumor cell lines and they were investigated in depth in a T leukemia cell line (Jurkat). METHODS AND RESULTS: Flow cytometric analysis of cell cycle after treatment with pyrrolotetrazinones has demonstrated that they were able to induce an arrest of the cell cycle in G2/M phase. This effect was accompanied by apoptosis of the treated cells which is further characterized by exposure of phosphatidylserine on the external surface of the cell membranes. Mitochondria were strongly involved in the apoptotic pathway as demonstrated by the induced mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3. Western blot analysis showed that Bcl-2 expression was down regulated whereas the proapototic protein Bax was upregulated in a time dependent manner. Moreover, these compounds induced a clear increase in the mitotic index, and inhibited microtubule assembly in vitro indicating that pyrrolotetrazinones, at variance with temozolomide, involved an efficacious inhibition of tubulin polymerization in their mechanism of action. Interestingly compound 3 at the concentration of 50 mg/kg body weight significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice. CONCLUSION: These results suggest that pyrrolotetrazinones inhibit microtubule polymerization, induce G2/M arrest of cell cycle and cause apoptosis through the mitochondrial pathway identifying them as novel effective antimitotic agents with potential for clinical development.

Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544.

Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21(Waf1/Cip1), a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21(Waf1/Cip1) paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21(Waf1/Cip1) induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

Photophysical properties and photobiological behavior of amodiaquine, primaquine and chloroquine.

This article describes the results of a coupled photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs amodiaquine (AQ), primaquine (PQ) and chloroquine (CQ). Photophysical experiments were carried out in aqueous solutions by steady-state and time-resolved spectrometric techniques to obtain information on the different decay pathways of the excited states of the drugs and on the transient species formed upon laser irradiation. The results showed that all three drugs possess very low fluorescence quantum yields (10(-2)-10(-4)). Laser flash photolysis experiments proved the occurrence of photoionization processes leading to the formation of a radical cation in all three systems. In the case of AQ the lowest triplet state was also detected. Together with the photophysical properties the photobiological properties of the antimalarial drugs were investigated under UV irradiation, on various biological targets through a series of in vitro assays. Phototoxicity on mouse 3T3 fibroblast and human keratinocyte cell lines NCTC-2544 was detected for PQ and CQ but not for AQ. In particular, PQ- and CQ-induced apoptosis was revealed by the externalization of phosphatidylserine. Furthermore, upon UV irradiation, the drugs caused significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry. The photodamages produced by the drugs were also evaluated on proteins, lipids and DNA. The combined approaches were useful in understanding the mechanism of phototoxicity induced by these antimalarial drugs.