NF-kappaB activation contributes to anthracycline resistance pathway in human ovarian carcinoma cell line A2780.

The development of chemoresistance is a major obstacle for successful anticancer therapy. Understanding the molecular mechanisms leading to chemoresistance is a rational step to improve the therapeutic efficacy of cytotoxic drugs. Since anthracyclines play an important role in cancer chemotherapy, we have generated a human ovarian tumor cell line resistant to sabarubicin (MEN 10755), the newest anthracycline molecule in clinical development. Expression of the transporter protein MRP that affected sabarubicin uptake, and a reduced DNA topoisomerase II content in A2780/saba cells was observed. Since the poisoning of DNA topoisomerase II results in DNA damage, which is a critical signal for NF-kappaB activation, we explored if this transcription factor has a role in the chemoresistance to anthracyclines. We showed a reduced NF-kappaB activation in the resistant cell line. Moreover, qualitative changes in NF-kappaB dimer formation between the two cell lines were observed. In agreement with the hypothesis of a role of NF-kappaB in mediating drug resistance, we showed that the pharmacological inhibition of NF-kappaB activation attenuated drug resistance in A2780/saba cells whereas it had no effect in A2780 cells. Altogether, these findings show that anthracycline resistance in A2780 cell lines is due to the coexpression of several molecular mechanisms.

Involvement of nitric oxide in both central and peripheral haemodynamic effect of D/L-nebivolol and its enantiomers in rats.

The cardiovascular profile of the racemate D/L-nebivolol and its enantiomers administered by intravenous (i.v.) or by intracerebroventricular (i.c.v.) route was investigated in anaesthetized normotensive rats. D/L-Nebivolol (0.1-0.5 mg/kg) induced a dose-related reduction in blood pressure when administered by i.c.v. route. These hypotensive effects were more marked as compared to those achieved by peripheral administration of D/L-nebivolol (0.1-1 mg/kg i.v.). Both enantiomers contributed to the hypotensive effect of D/L-nebivolol by i.c.v. route, while the effects of the drug on blood pressure by i.v. route were due to the d-enantiomer. The bradycardic effect of the racemic form given i.v. was dose-related and, at the highest dose (1 mg/kg), was more pronounced as compared to i.c.v. route. D-Nebivolol was responsible for chronotropic effects by both the i.v. and i.c.v. route, although by i.c.v. route L-nebivolol also induced a reduction in heart rate. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) administered at 5 mg/kg i.v. bolus + 0.1 mg/kg/min infusion or at 2.5 mg/kg i.c.v. counteracted the effects of D/L-nebivolol (either 1 mg/kg i.v. or 0.5 mg/kg i.c.v.) on blood pressure, while it did not inhibit the cardiovascular changes induced by isoprenaline (300 ng/kg i.v.) or calcitonin gene-related peptide (CGRP; 400 ng/kg i.v.). In addition, i.c.v. effects of D/L-nebivolol on blood pressure and heart rate were not affected by pre-treatment with atropine (2 mg/kg i.v.). The present findings demonstrate that D/L-nebivolol produced haemodynamic changes following both peripheral and central administration; these latter findings are mainly due to its L-enantiomer and these effects involve the L-arginine/nitric oxide pathway.

Sabarubicin- (MEN 10755) and paclitaxel show different kinetics in nuclear factor-kappaB (NF-kB) activation: effect of parthenolide on their cytotoxicity.

BACKGROUND: Several antitumor drugs have been described to induce nuclear factor kappaB (NF-kappaB), but results about its role in regulating apoptotic cell death are quite controversial. In this paper, we studied NF-kappaB induced by the two anticancer agents Sabarubicin (MEN 10755) and paclitaxel (Taxol) and the effects of its pharmacological inhibition. MATERIALS AND METHODS: In the human colon cancer cell line HCT-116, we investigated NF-kappaB activation induced by the two anticancer agents using electrophoretic mobility shift assay (EMSA), while drug-induced cytotoxicity was measured by trypan blue staining. Apoptosis was analyzed using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit, flow cytometry and caspase-3 activation assay. RESULTS: The combination with the NF-kappaB inhibitorparthenolide increased Sabarubicin- but not paclitaxel-induced cell death. EMSA experiments demonstrated that the two antitumor drugs induced NF-kappaB complexes with different kinetics but similar subunit composition. Moreover, Sabarubicin elicited NF-kappaB activation definitely earlier than DNA fragmentation, whereas with paclitaxel the kinetics of the two phenomena were similar.

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity.

(1) The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues--like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755--which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. (2) DOX induced electrocardiographic and haemodynamic alterations, like elongation of QalphaT or SalphaT intervals and suppression of isoprenaline-induced dP/dt increases, which developed in a time-dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. (3) DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed approximately 60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol approximately 40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). (4) DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe-S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe-S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. (5) These results show that (i) secondary alcohol metabolites are important determinants of anthracycline-induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

Expression and characterization of biologically active human Fas ligand produced in CHO cells.

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh- FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 microg/L x 10(6) cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays.

Maintenance therapy in ovarian cancer: Molecular basis and therapeutic approach.

Ovarian cancer has the highest mortality rate among gynaecological tumours despite the fact that the majority of patients with advanced disease achieve complete remission after first-line surgery and chemotherapy. Unfortunately, disease recurrence occurs in the majority of patients and second-line treatments are not curative. Clearly, the persistence of dormant and drug-resistant cells after front-line treatments results in the inability to cure the disease. The identification of cancer-initiating cells or cancer stem cells as key players in the development of recurrence has opened up a novel field of research aimed at identifying additional innovative therapeutic approaches. Strategies of maintenance therapy to extend the survival of patients have been studied, but to date no overall survival benefit has been detected. Currently, numerous clinical trials have just been completed or are ongoing involving patients achieving a complete clinical response after first-line chemotherapy in order to evaluate the efficacy of different therapeutic approaches in terms of disease-free survival and overall survival. At the 2010 ASCO meeting, the first positive results of a phase III clinical trial in this setting were presented: bevacizumab (15 mg/kg i.v. every 21 days) added to first-line chemotherapy and continued for an additional 15 cycles was found to prolong progression-free survival of 3.8 months in comparison to 6 cycles of chemotherapy alone or only 6 cycles of chemotherapy plus bevacizumab. In addition, positive results were announced for a second phase III trial testing bevacizumab in the same setting, but at half dose. The final assessment of the overall clinical benefit and the approval of bevacizumab in maintenance therapy by regulatory agencies is expected to be positive, as are the final results of abagovomab phase III trial MIMOSA, another antibody-based therapy tested as a maintenance treatment for advanced ovarian cancer patients. Encouraging preliminary results confirming the safety profile and the immunogenic activity of abagovomab were presented at the last ASCO meeting. The final results are expected to be released in the first half of 2011.

Forces determining the adsorption of a monoclonal antibody onto an aluminium hydroxide adjuvant: influence of interstitial fluid components.

This study evaluates the forces involved in the adsorption of abagovomab onto an aluminium hydroxide adjuvant in interstitial fluid and the influences of interstitial fluid and its components on this process. Abagovomab is a monoclonal, anti-idiotypic antibody developed as an immunovaccine for the treatment of ovarian cancer. Partial elution of abagovomab by a change in ionic strength indicates that electrostatic interactions influenced adsorption. Studies on the role of phosphate and simulated interstitial fluid on the adsorption demonstrated that ligand exchange is the main force of adsorption. A comparison of the adsorption capacity of abagovomab in the formulation with that in an environment imitating the in vivo environment using simulated interstitial fluid showed the adsorption capacity to decrease, the more the conditions resemble the in vivo environment after subcutaneous or intramuscular administration.

Human and murine macrophages mediate activation of MEN 4901/T-0128: a new promising camptothecin analogue-polysaccharide conjugate.

MEN 4901/T-0128 is a new cytotoxic prodrug constituted by the camptothecin analogue T-2513 bound to carboxymethyl dextran through a triglycine linker. MEN 4901/T-0128 was designed to target the active camptothecin at the tumour site. MEN 4901/T-0128 is weakly cytotoxic in vitro and thus T-2513 must be released from the conjugate to become active. Here, we demonstrated that human purified cathepsin B releases T-2513 from MEN 4901/T-0128 at pH values ranging from 3 to 5. pH dependency of this reaction suggests that cleavage of the linker should mainly occur in the lysosomes. As elevated cathepsin B activity has been described in macrophages, human tumour monocytic THP-1 cells differentiated into macrophage-like cells were used to study the cellular mechanisms responsible for MEN 4901/T-0128 antitumour activity. Here, we show that differentiated THP-1 internalizes MEN 4901/T-0128 efficiently in a time-dependent and concentration-dependent manner. After phagocytosis, THP-1 cells can cleave the prodrug and release T-2513 in the media. On the contrary, undifferentiated THP-1 cells or pancreatic ASPC-1 tumour cells, although expressing high levels of cathepsin B, are much less efficient in the release of cytotoxic moieties in the culture media. Moreover, normal murine macrophages, recovered from the peritoneal cavity or from the spleen, when activated (in vitro by 100 ng/ml phorbol 12-myristate-13-acetate and in vivo by 300 microl of 3% w/v thioglycollate solution), were able to release (after incubation with 10 microg/ml MEN 4901/T-0128) cytotoxic moieties in the culture supernatant, in an amount sufficient to kill human carcinoma A2780 cells. Thus, we suggest that tumour-associated macrophages may play a key role in the uptake of MEN 4901/T-0128, cleavage and local release of active moiety T-2513. This mechanism should support a tumour targeting of the cytotoxic moieties, allowing an improved antitumour efficacy/safety ratio for MEN 4901/T-0128.

Role of NK-1 and NK-2 tachykinin receptor antagonism on the growth of human breast carcinoma cell line MDA-MB-231.

We demonstrate that neurokinin A (NKA) and substance P (SP) play a role in the proliferation of the estrogen receptor-negative (ER-) cell line MDA-MB-231, a human breast carcinoma expressing both NK-1 and NK-2 receptors. In vitro experiments showed that the specific receptor antagonists MEN 11,467 (NK-1) and nepadutant (MEN 11,420; NK-2) inhibited tumor cell proliferation, and blocked the stimulatory effect of SP and NKA. Anti-tumoral activity of NK-1 and NK-2 receptor antagonists was demonstrated in nude mice, measuring growth inhibition of MDA-MB-231 tumor cells xenografted s.c. and by using the hollow-fiber assay. In both systems a significant inhibition was found when compounds were administered at 5 mg/kg i.v. every day for 2 weeks. Results obtained from both these models suggest that the in vivo activity of NK-1 and NK-2 antagonists may be a result of a cytostatic effect rather than a cytotoxic effect. Our results suggest that the control of breast carcinoma (ER-) growth by tachykinin receptor antagonists may become a new form of targeted therapy for these human tumors.

Nuclear factor-kappaB, induced in human carcinoma cell line A2780 by the new anthracycline men 10755, is devoid of transcriptional activity.

The new disaccharide anthracycline MEN 10755 induces activation of both NF-kappaB and p53 transcription factors in A2780 cells. Nevertheless, pharmacologic inhibition of NF-kappaB activation does not modify the sensitivity of A2780 cells to MEN 10755 treatment. To better characterize the role of NF-kappaB in MEN 10755-induced cytotoxicity, we analyzed the expression of a number of genes that are known to be regulated by NF-kappaB. None of these genes is modified by MEN 10755 treatment. On the contrary, our results suggest that the p53 DNA damage-responsive pathway is fully activated in A2780 cells, several genes controlled by p53 being up- or downregulated according to the described action of p53 on their promoters. Thus, in the A2780 cell line, the role of p53 in transducing the DNA-damage signal appears to be relevant, whereas NF-kappaB, although activated, appears to be nonfunctional. Other human carcinoma cell lines besides A2780 activate NF-kappaB DNA binding in response to MEN 10755 treatment, but again, this binding does not always lead to target gene activation. These results suggest that other factors, tumor type-specific and different from mere activation, could influence NF-kappaB transcriptional activity. Therefore, care should be taken when considering the pharmacologic inhibition of NF-kappaB as a means to improve anticancer therapy efficacy.

MEN15658: a new promising anti-tumoral drug active on resistant tumor cells.

We describe the identification of MEN15658, a molecule characterized by a promising cytotoxic effect against human tumor cell lines, including platinum- and anthracycline-resistant ovarian carcinoma. MEN15658 induces p53 accumulation, and activation of gadd-45, p21, c-fos and bcl-2 family genes in human ovarian carcinoma A2780 cell line. The compound causes a block in S phase of the cell cycle, inducing apoptotic cell death, thus suggesting an involvement of DNA damage in the MEN15658 effect on tumor cells. The anti-tumoral activity observed against the human ovarian carcinoma A2780 cell line xenotransplanted in nude mice makes this compound a new promising anti-tumoral drug.

CD137 and CD137 ligand constitutively coexpressed on human T and B leukemia cells signal proliferation and survival.

CD137, a member of the tumor necrosis factor receptor family, provides expansion and survival signal to T cells. Its ligand, CD137L, in addition to its ability to costimulate T cells, signals back into antigen presenting cells promoting their activation and differentiation. Recently, CD137 has been proposed as a therapeutic target to improve and sustain anticancer immune response. Several activated T leukemia and B lymphoma cell lines expressed CD137 or CD137L, respectively, and soluble CD137L has been found in sera of leukemia patients. However, the functionality and role of these costimulatory molecules in hematologic malignancies are until now unknown. Interestingly, we observed constitutive CD137 and CD137L coexpression on both human T and B leukemia cell lines. The constitutive CD137 expression on unstimulated T or B leukemia cells presents some differences compared to CD137 expressed on PMA/ionomycin-activated T leukemia cells. Surprisingly, in spite of the low expression level, both tumor CD137 and CD137L molecules signaled in T and B leukemia cells inducing proliferation and prolonging survival. In addition, CD137/CD137L system ligation opposed the anticancer drug cytotoxic effects, reducing the apoptotic DNA fragmentation and stimulating proliferation of doxorubicin-escaped leukemia cells. Although the role of leukemia CD137/CD137L system in vivo is unknown, these data suggest that these costimulatory molecules might confer an advantage to hematologic tumors promoting survival, sustaining cellular growth and contributing to drug resistance.