A series of calothrixin B (2) analogues bearing substituents at the 'E' ring and their corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The cytotoxicities of calothrixins 1, 2, and 15b-p and quinocarbazole analogues were investigated against nine cancer cell lines. The quinocarbazoles 21a and 25a inhibited the catalytic activity of human topoisomerase II. The plasmid DNA cleavage abilities of calothrixins 1, 2, and 15b-p identified compound 15h causing DNA cleavage comparable to that of calothrixin A (1). Calothrixin A (1), 3-fluorocalothrixin 15h and 4-fluoroquinocarbazole 21b induced extensive DNA damage followed by apoptotic cell death. Spectral and plasmid unwinding studies demonstrated an intercalative mode of binding for quinocarbazoles. We identified two promising drug candidates, the 3-fluorocalothrixin B 15h with low toxicity in animal model and its deoxygenated derivative 4-fluoroquinocarbazole 21b as having potent cytotoxicity against NCI-H460 cell line with a GI of 1 nM.

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The broad-range cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) is known to induce both a G1 cell cycle arrest and apoptosis. The mechanism of UCN-01-induced apoptosis is largely unknown. We analysed the mechanism of cytotoxicity of UCN-01 in four established colon carcinoma cell lines. The cell lines SW48 and LS513 responded to UCN-01 treatment by undergoing apoptosis in a concentration-dependent manner while the cell lines HT-29 and WiDr were completely resistant. Apoptosis in LS513 and SW48 cell lines was concomitant with the suppression of Bcl-x L on mRNA and protein level. In contrast, in the apoptosis-resistant cell lines, Bcl-x L expression was not affected by UCN-01. Stable overexpression of the Bcl-x L protein abrogated UCN-01-triggered apoptosis, but only partially restored growth, indicating that both cell cycle arrest and apoptosis exert the anticancer effect in a coordinated manner. The inhibition of Akt phosphorylation did not correlate with the apoptotic phenotype. UCN-01 inhibited the activating STAT3 phosphorylations on Ser727 and, notably, on Tyr705, but STAT3 did not contribute to Bcl-x L expression in colon carcinoma cells. Moreover, we show for the first time that UCN-01 induces apoptosis by suppression of Bcl-x L expression. The inhibition of this pathway is a new aspect of cytotoxic and modulatory potential of UCN-01.

Here we used five p53 WT and five p53 MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53 MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53 MUT cells than in the cell cycle-arrested p53 WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53 MUT cells after SN-38 treatment contributes to DNA damageinduced apoptosis, whereas their suppression in p53 WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53 MUT cells activates the checkpoint and contributes to apoptosis.The function of p53 protein as a guardian of the genome is to prevent the organism from propagation of potentially harmful DNA lesions. The p53 WT 2 cells react to a mutagenic offense by a cell cycle arrest, allowing for the proper repair of the damaged DNA, or in the case of overwhelming damage by apoptosis triggered by the p53-mediated activation of proapoptotic genes such as BAX, PUMA, and NOXA (3).The majority of colorectal carcinomas have a mutated p53 gene that in most of the cases is associated with a loss of tumor suppressor function. These cells are unable to execute a proper cell cycle arrest after DNA damage and are also frequently resistant to apoptosis, which results in propagation of genetic lesions and cell survival.We showed previously that despite this general concept, colon carcinoma cells may respond differently to the chemotherapeutic irinotecan (CPT-11); the p53 WT cells execute a long term cell cycle arrest after irinotecan treatment, whereas the p53-deficient cells perform a transient cell cycle arrest followed by apoptosis (1).Because of the frequent p53 mutations in colon carcinoma cells, the p53-independent apoptosis is of particular interest. The p53 MUT cells usually do not maintain the G 2 /M arrest after DNA damage, but initiate a premature mitosis that leads to mitotic catastrophe, accompanied by the emergence of aberrant mitoses and distinct nuclear forms of micronucleation and apoptotic nuclear condensation (4 -6).Mitotic catastrophe can be also induced in p53 WT cells by DNA damage and is concomitant with elimination of genes responsible for triggering of G 2 /M arrest, like Chk1 or ATR (7, 8), or those responsible for its maintenance, like p21 WAF1/Cip1 (9) or 14-3-3 (10), o...