Luisa Dusonchet

Resistance to diverse apoptotic triggers in multidrug resistant HL60 cells and its possible relationship to the expression of P-glycoprotein, Fas and of the novel anti-apoptosis factors IAP (inhibitory of apoptosis proteins)

We studied the human HL60 leukemia cell line and its multidrug resistant (MDR) variant HL60R. In contrast to the HL60, HL60R showed an inability to undergo apoptosis from doxorubicin (Dox) or other different stimuli, including cisplatin, Fas ligation and serum withdrawal. HL60R cells lost surface Fas expression, but we found no evidence that Fas/FasL mediates the apoptotic effects of Dox in HL60. P-glycoprotein (P-gp) did not seem to play a major role as a specific inhibitor of apoptosis. In fact, the P-gp inhibitor verapamil reversed only partially the resistance to Dox-induced apoptosis of the MDR cells. In addition, it did not modify the rate of apoptosis induced from the other stimuli in the same cells. The expression of p53 or Bcl-2 was not different between HL60 and HL60R. However, in HL60R there was an increase in the mRNAs of inhibitory of apoptosis proteins (IAPs) like neuronal apoptosis inhibitory protein (NAIP), c-IAP-2 and survivin. Treatment with Dox or serum starvation strongly down-regulated X-linked IAP and survivin mRNAs in HL60. Cisplatin decreased NAIP and survivin mRNAs in the same cells. However, in HL60R the levels of these IAP mRNAs were much less affected by the treatments. These results support that IAPs may be involved in tumor resistance to chemotherapeutic drugs or other apoptotic agents.

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 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001–1 μg/ml, and VP16, at concentrations ranging from 0.05–1 μg/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 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) 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 24 h but not at 48 h. 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.

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.

Influence of Pharmacokinetic Variations on the Pharmacological Properties of Adriamycin

Whenever it appears impossible to modify the chemical structure of drugs with a high and established therapeutic activity but a low chemotherapeutic index, pharmacological research has to find other ways of improving the chemotherapeutic index. This problem is particularly important in the case of antitumor drugs, thus justifying research into the most suitable choice of dosage and routes of administration, as well as into the pharmacological associations which enable tumor cells to be hit at various stages of the reproductive cycle. Alternatively, the therapeutic index could be improved by the use of antagonistic compounds (like, for example, methotrexate and folinic acid) which act upon the same organic functions.

Spectrophotofluorometric characterization of adriamycin a new antitumour drug

Summary Adriamycin, a new antitumour drug structurally similar to daunomycin, shows evident spectrophotofluorometric characteristics. A technique previously employed by Kohn for tetracyclines is proposed by the present workers for the identification and estimation of adriamycin in tissues. Furthermore, a transformation of the molecule was revealed in some organs, using the same technique.

Does catalase play a role in Adriamycin induced cardiotoxicity

Summary Adriamycin causes an increase of lipid peroxidation in mouse cardiac homogenates that is dependent on the concentration of the antiblastic. The same phenomenon is not observed in the hearts of mice treated with an elevated dose of Adriamycin in which, conversely, an increase of the antioxidizing enzyme catalase was noticed. The significance of these findings is discussed with relationship to the hypothesis of an enhanced free radicals formation at the basis of Adriamycin induced cardiotoxicity.

NAIP-ΔEx10-11: a novel splice variant of the apoptosis inhibitor NAIP differently expressed in drug-sensitive and multidrug-resistant HL60 leukemia cells

Alterations of neuronal apoptosis inhibitory protein (NAIP), a member of the inhibitory of apoptosis protein (IAP) family of inhibitors of apoptosis, have been previously associated with different neurodegenerative disorders. This study indicated the existence of a novel NAIP splice variant. This isoform, NAIP-deltaEx10-11, was found in tumor cell lines of different origin and in normal adult brain. Analysis of the putative protein predicted that the NAIP variant lacks part of the third BIR domain as well as the COOH-terminal tail of regular NAIP. This might suggest that it is endowed with a reduced antiapoptotic activity. This view is supported by the fact that NAIP-deltaEx10-11 mRNA and protein were much less expressed in the multidrug- and apoptosis-resistant HL60R leukemia than in its parental counterpart HL60. The opposite occurred for regular NAIP. Overall, the NAIP transcripts might be involved in tumor resistance to chemotherapeutic agents.