Maria Meli

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4u2009h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001–1u2009μg/ml, and VP16, at concentrations ranging from 0.05–1u2009μg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72u2009h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24u2009h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24u2009h but not at 48u2009h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

Multidrug resistance reverting activity and antitumor profile of new phenothiazine derivatives

A series of easily affordable phenothiazine derivatives bearing a rigid but-2-ynyl amino side chain were synthesized and tested to evaluate the MDR reverting activity and full antitumor profile. Some compounds endowed with remarkable MDR reverting effect were identified, and the most active one (6c) was shown to increase doxorubicin retention in multidrug resistant cells, suggesting a direct interaction with P-glycoprotein. Furthermore, a broad range of cellular activities were observed for different compounds. In particular, the ability of some derivatives to induce antiproliferative effects on resistant cell lines and to interfere with the G(1) phase of the cell cycle, a phase usually not affected by classical antitumor agents, was noted. Moreover, the most cytotoxic compounds of the series were able to induce apoptosis in resistant cell lines, via an atypical pathway of caspase cascade activation, and a synergistic effect in combination with doxorubicin was also found.

Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells

Resistance to imatinib mesylate is an emergent problem in the treatment of Bcr-Abl expressing myelogenous leukemias and additional therapeutic strategies are required. We observed that galangin, a non-toxic, naturally occurring flavonoid was effective as anti-proliferative, and apoptotic agent in Bcr-Abl expressing K562 and KCL22 cells and in imatinib mesylate resistant K562-R and KCL22-R cells. Galangin induced an arrest of cells in G0-G1phase of cell cycle and a decrease in pRb, cdk4, cdk1, cycline B levels; moreover, it was able to induce a monocytic differentiation of leukemic Bcr-Abl+ cells. Of note, galangin caused a decrease in Bcl-2 levels and markedly increased the apoptotic activity of imatinib both in sensitive or imatinib-resistant Bcr-Abl+ cell lines. In contrast, flavonoids unable to modify the Bcl-2 intracellular levels, such as fisetin and chrysin, did not increase the apoptotic effect of imatinib. These data suggest that galangin is an interesting candidate for a combination therapy in the treatment of imatinib-resistant leukemias.

Expression of IAPs and Alternative Splice Variants in Hepatocellular Carcinoma Tissues and Cells

Abstract: IAPs (inhibitors of apoptosis proteins) might have a major role in the apoptotic resistance that marks many cancers. The studies on IAPs in human HCC have focused on survivin or XIAP, indicating that their new or increased expression in this tumor is associated with a more unfavorable prognosis. The present results corroborate these findings, emphasizing the role that the coordinated expression of different IAPs and alternative splice variants might play in the adverse biology of hepatocellular carcinoma.

α‐Glycerophosphocholine in the Mental Recovery of Cerebral Ischemic Attacks

The clinical efficacy and the tolerability of alpha-glycerophosphocholine (alpha-GPC), a drug able to provide high levels of choline for the nervous cells of the brain and to protect their cell walls, have been tested in a clinical open multicenter trial on 2044 patients suffering from recent stroke or transient ischemic attacks. alpha-GPC was administered after the attack at the daily dose of 1000 mg im for 28 days and orally at the dose of 400 mg tid during the following 5 months after the first phase. The evaluation of the efficacy on the psychic recovery was done by the Mathew Scale (MS) during the period of im drug administration, and using the Mini Mental State Test (MMST), the Crichton Rating Scale (CRS), and the Global Deterioration Scale (GDS) during the following period of oral administration. The MS mean increased 15.9 points in 28 days in a statistically significant way (p < 0.001) from 58.7 to 74.6. At the end of the 5 month oral administration, the CRS mean significantly decreased 4.3 points, from 20.2 to 15.9 (p < 0.001); the MMST mean significantly increased (p < 0.001) from 21 to 24.3 at the end of the trial, reaching the normality score at the 3rd month assessment. The GDS score at the end of the trial corresponded to no cognitive decline or forgetfulness in 71% of the patients. Adverse events were complained of by 44 patients (2.14%); in 14 (0.7%) the investigator preferred to discontinue therapy. The most frequent complaints were heartburn (0.7%), nausea-vomit (0.5%), insomnia-excitation (0.4%), and headache (0.2%). The trial confirms the therapeutic role of alpha-GPC on the cognitive recovery of patients with acute stroke or TIA, and the low percentage of adverse events confirms its excellent tolerability.

NF‐κB Inhibition Restores Sensitivity to Fas‐Mediated Apoptosis in Lymphoma Cell Lines

Abstract: Failure to perform the Fas‐related apoptosis pathway can account for tumor resistance both to chemotherapeutic agents and to immunological effectors. We studied the role of NK‐κB in Fas‐resistance, employing the Fas‐sensitive human T‐lymphoma HuT78 cell line and its Fas‐resistant variants HuT78B1 and HuT78G9. All these cell lines expressed high levels of constitutively activated NF‐κB. Pretreatment of cells with NF‐κB inhibitors (PDTC, MG132, or SN50) strongly enhanced CH11‐induced apoptosis in HuT78 and Hut78G9 cells, while only MG132 showed a similar potentiating effect in HuT78B1. The described synergism was significantly inhibited by pretreatment with the anti‐Fas‐blocking antibody ZB4 or with the pancapsase inhibitor Z‐VAD‐FMK, but not by capsase‐8 or ‐9 inhibitors. Overall, these data suggest that NF‐κB inhibition may restore the Fas‐pathway in Fas‐resistant NF‐κB‐overexpressing tumors.

Inhibition of activated STAT5 in Bcr/Abl expressing leukemia cells with new pimozide derivatives.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkins lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

4,5,6,7-Tetrahydro-isoxazolo-(4,5-c)-pyridines as a new class of cytotoxic Hsp90 inhibitors

Hsp90 is considered an interesting therapeutic target for anticancer drug development. Here we describe a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine compounds. A small library of derivatives has been synthesized and investigated. Some reported compounds show interesting properties combining both notable binding to Hsp90 and potent cell growth inhibitory activity. N-5 substitution with a 2,4 resorcinol carboxamide appears crucial for activity. Moreover, a derivative bearing a hydroxamic acid residue bound to C-3 amide portion was found to inhibit both Hsp90 and HDAC6.

Programmed cell death (PCD) associated with the stilbene motif of arotinoids : Discovery of novel apoptosis inducer agents possessing activity on multidrug resistant tumor cells

Considering that the stereochemistry of the C9-C10 alkenyl portion of natural 9-cis-RA, as the one of the olefinic moiety of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel class of TTNPB analogues bearing both the cis or trans configuration of the alkenyl portion. The compounds were evaluated in vitro for their cytotoxic and apoptotic activities. We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with that of the retinoid 4. Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells. Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines. Therefore, to the best of our knowledge, 16c may represent the first known aminoarotinoid endowed with potent apoptotic activity in MDR cells. Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important feature in conferring cytotoxic and apoptotic activity to this class of compounds.

The new iodoacetamidobenzofuran derivative TR120 decreases STAT5 expression and induces antitumor effects in imatinib-sensitive and imatinib-resistant BCR-ABL-expressing leukemia cells.

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR–ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR–ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR–ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR–ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0–G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR–ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR–ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.