Alessia Muzi

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma

Poly(ADP‐ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisom‐erase I inhibitor irinotecan (CPT‐11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT‐11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10‐(4‐methyl‐piperazin‐1‐ylm‐ethyl)‐2H‐7‐oxa‐1,2‐diaza‐benzo[de]anthracen‐3‐one) increases the efficacy of CPT‐11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP‐1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose‐limiting intestinal toxicity of CPT‐11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN‐38 (the active metabolite of CPT‐11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/day×5 days per os) in combination with TMZ (10 mg/kg/day×5 days) + CPT‐11 (4 mg/kg/day×5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2 × 3 days) prevented intestinal injury and diarrhea induced by CPT‐11 (30 mg/kg/day × 3 days) reducing inflammation and PARP‐1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP‐ribose) antibody(Ab). Inconclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.—Tentori, L., Leonetti, C., Scarsella, M., Muzi, A., Mazzon, E., Vergati, M., Forini, O., Lapidus, R., Xu, W., Dorio, A. S., Zhang, J., Cuzzo‐crea, S., Graziani, G. Inhibition of poly(ADP‐ribose) polymerase prevents irinotecan‐induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma. FASEB J. 20, E1024–E1036 (2006)

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine-autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Stable depletion of poly (ADP-ribose) polymerase-1 reduces in vivo melanoma growth and increases chemosensitivity.

Poly(ADP-ribose) polymerase (PARP)-1, which plays a key role in DNA repair, inflammation and transcription, has recently been shown to be involved in angiogenesis. The aim of this study was to investigate PARP-1 role in melanoma aggressiveness and chemoresistance in vivo using clones stably silenced for PARP-1 expression. Whilst the growth characteristics of PARP-1-deficient melanoma cells were comparable to those of PARP-1-proficient cells in vitro, their tumourigenic potential in vivo was significantly compromised. In fact, mice challenged intra-muscle with PARP-1-deficient cells showed a delayed development of measurable tumour nodules, which were also significantly reduced in size with respect to those of mice inoculated with PARP-1-proficient cells. Moreover, animals challenged intra-cranially with PARP-1-deficient cells, a model that mimics CNS localisation of melanoma, showed an increased survival. Immunohistochemical analyses of PARP-1-depleted melanoma grafts indicated a reduced expression of the angiogenesis marker PECAM-1/CD31 and of the pro-inflammatory mediators TNF-alpha and GITR. Notably, PARP-1-silenced melanoma was extremely sensitive to temozolomide, an anticancer agent used for the treatment of metastatic melanoma. These results provide novel evidence for a direct role of PARP-1 in tumour aggressiveness and chemoresistance.

PARP-1 Modulates Amyloid Beta Peptide-Induced Neuronal Damage

Amyloid beta peptide (Aβ) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aβ25–35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aβ25–35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aβ25–35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aβ25–35 induces DNA damage which in turn activates PARP-1. Challenge with Aβ25–35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aβ25–35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aβ and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide

BackgroundChemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy.MethodsTen human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test.ResultsAll GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01).ConclusionsThe combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5′ OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) activity in PARP-1 silenced tumour cells increases chemosensitivity to temozolomide and to a N3-adenine selective methylating agent.

We recently demonstrated that poly(ADP-ribose) polymerase (PARP)-1 is involved in angiogenesis and tumour aggressiveness. In this study we have compared the influence of abrogation of PARP-1 expression by stable gene silencing to that of the pharmacological inhibition of cellular PARP activity using PARP-1/-2 inhibitors on the chemosensitivity of tumour cells to the wide spectrum methylating agent temozolomide (TMZ) and to the N3-adenine selective methylating agent {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane (Me-Lex). Silencing of PARP-1 in melanoma or cervical carcinoma lines enhanced in vitro sensitivity to TMZ and Me- Lex, and induced a higher level of cell accumulation at the G2/M phase of cell cycle with respect to controls. GPI 15427, which inhibits both PARP-1 and PARP-2, increased sensitivity to TMZ and Me-Lex both in PARP-1-proficient and - deficient cells. However, it induced different cell cycle modulations depending on PARP-1 expression, provoking a G2/M arrest only in PARP-1 silenced cells. Treatment of PARP-1 silenced cells with TMZ or Me-Lex resulted in a more extensive phosphorylation of Chk-1 and p53 as compared to PARP-1 proficient cells. The combination of the methylating agents with GPI 15427 increased Chk-1 and p53 phosphorylation both in PARP-1 proficient or deficient cells. When mice challenged with PARP-1 silenced melanoma cells were treated with the TMZ and PARP inhibitor combination there was an additional reduction in tumour growth with respect to treatment with TMZ alone. These results suggest the involvement of PARP-2 or other PARPs, in the repair of DNA damage provoked by methylating agents, highlighting the importance of targeting both PARP-1 and PARP-2 for cancer therapy.

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2(+/+) and p53(+/+). HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT+PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT+PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug

BackgroundMost DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ.MethodsAKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA.ResultsTMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects.ConclusionNF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.

The glutathione transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) increases temozolomide efficacy against malignant melanoma

First line treatment of metastatic melanoma includes the methylating agent dacarbazine or its analogue temozolomide (TMZ) with improved pharmacokinetics and tolerability. However, the prognosis of the metastatic disease is poor and several trials are evaluating TMZ in polychemotherapy protocols. The novel glutathione transferase P1-1 (GSTP1-1) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has recently shown activity against melanoma through c-Jun N-terminal kinase activation. In this study we have investigated the in vitro and in vivo efficacy of NBDHEX and TMZ combination against melanoma. The results indicated that NBDHEX and TMZ exerted in vitro synergistic anti-proliferative effects in murine B16 and human A375 melanoma cells. In B16 cells TMZ as single agent caused cell accumulation at the G(2)/M phase of cell cycle, whereas NBDHEX induced mainly apoptotic effects. NBDHEX provoked a higher level of p53 phosphorylation with respect to TMZ and the drug combination caused a more than additive increase of p53 activation. The in vivo efficacy of NBDHEX and TMZ has been investigated in an orthotopic B16 model. Treatment with NBDHEX provoked a reduction of tumour growth comparable to that obtained with TMZ, whereas the drug combination significantly increased tumour growth inhibition with respect to the single agents, without worsening TMZ myelotoxicity. Immunohistochemical analysis of tumour grafts revealed a profound reduction of Cyclin D1 and CD31 in all treatment groups; VEGF expression was, instead, markedly decreased only in NBDHEX or NBDHEX and TMZ treated samples. These findings indicate that NBDHEX represents a good candidate for combination therapies including TMZ, offering new perspectives for the treatment of melanoma.