The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells

Kowalczyk, Aleksandra; Gil, Malgorzata; Horwacik, Irena; Odrowaz, Zaneta; Kozbor, Danuta; Rokita, Hanna

doi:10.1016/j.canlet.2009.02.040

Cited by 57 publications

(63 citation statements)

References 46 publications

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“…Phosphorylation of P53 at Ser46 is strongly associated with its proapoptotic activity, therefore decrease in phosphorylation at the amino acid residue could prevent apoptosis. This finding is in agreement with our earlier results, showing that the mAb-induced cell death is only partially caused by apoptosis (8). P53 can be also phosphorylated at Ser15 by different kinases and thus impairs the ability of MDM2 to bind P53, promoting both the accumulation and activation of P53 in response to DNA damage (62).…”

Section: Discussionsupporting

confidence: 93%

“…Moreover, the anti-GD2 ganglioside mAbs were very efficient in combination with anticancer drugs to exert enhanced cytotoxicity against the afore-mentioned cells (7). Our studies showed that the anti-GD2 14G2a mAb decreases survival of IMR-32 human neuroblastoma cells in a dose-dependent manner through induction of apoptosis and also exerts a synergistic effect with doxorubicin and topotecan in killing IMR-32 cells in culture (8). It was also recently shown by us that the same mAb inhibits IMR-32 and LA-N-1 human neuroblastoma cells survival in vitro, through significant decrease in expression of all three Aurora kinases and phosphorylation (9).…”

Section: Introductionmentioning

confidence: 67%

“…Several studies have evidenced that GD2-specific antibodies inhibit tumor growth without involvement of the immune system (6)(7)(8). The mechanisms of the anti-proliferative effects of the antibodies are only partially elucidated.…”

Section: Introductionmentioning

confidence: 99%

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GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Durbas

Horwacik

Boratyn

et al. 2015

International Journal of Oncology

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Abstract. Mechanisms leading to inhibitory effects of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) and PI3K/Akt/mTOR pathway inhibitors on human neuroblastoma cell survival were studied in vitro. We have recently shown on IMR-32, CHP-134, and LA-N-1 neuroblastoma cells that targeting GD2 with the mAb decreases cell viability of the cell lines. In this study we used cytotoxicity assays, proteomic arrays and immunoblotting to evaluate the response of the three cell lines to the anti-GD2 14G2a mAb and specific PI3K/Akt/mTOR pathway inhibitors. We show here that the mAb modulates intracellular signal transduction through changes in several kinases and their substrates phosphorylation. More detailed analysis of the PI3K/Akt/ mTOR pathway showed significant decrease in activity of Akt, mTOR, p70 S6 and 4E-BP1 proteins and transient increase in PTEN (a suppressor of the pathway), leading to inhibition of the signaling network responsible for stimulation of translation and proliferation. Additionally, combining the GD2-specific 14G2a mAb with an Akt inhibitor (perifosine), dual mTOR/PI3K inhibitors (BEZ-235 and SAR245409), and a pan-PI3K inhibitor (LY294002) was shown to enhance cytotoxic effects against IMR-32, CHP-134 and LA-N-1 cells. Our study extends knowledge on mechanisms of action of the 14G2a mAb on the neuroblastoma cells. Also, it stresses the need for further delineation of molecular signal orchestration aimed at more reasonable selection of drugs to target key cellular pathways in quest for better cure for neuroblastoma patients.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 67%

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GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Durbas

Horwacik

Boratyn

et al. 2015

International Journal of Oncology

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“…It will be interesting to elucidate if antibodies and/or the agglutination are responsible of the apoptosis in T. cruzi. Recently, it has been shown that antibodies can induce cell death with exhibition of several characteristics that are typical for apoptosis such as increased number of annexin V and propidium-iodide-positive cells, cleavage of caspase 3, and prominent rise in caspase activity (Kowalczyk et al 2009). Yet it remains to be established how antibodies can lead to apoptosis, both in cells and possibly in T. cruzi epimastigotes.…”

Section: Discussionmentioning

confidence: 98%

Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes

et al. 2010

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The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.

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“…The tumor-targeting ability of these antibodies and the immune response they elicit have been extensively studied (19,(29)(30)(31). However, there has been little investigation of their retention in other organs (possibly related to the observed toxicity) or of the role of GD2 expression levels in patient response.…”

Section: Discussionmentioning

confidence: 99%

⁶⁴Cu-p-NH₂-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma

et al. 2012

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The hu14.18K322A variant of the GD2-targeting antibody hu14.18 has been shown to elicit a level of antibody-dependent cellmediated cytotoxicity toward human neuroblastoma cells similar to that of the parent antibody. However, hu14.18K322A exhibited a decreased complement activation and associated pain, the dose-limiting toxicity in neuroblastoma immunotherapy. PET with a radiolabeled analog of the same antibody used in treatment will provide insight into the ability of hu14.18K322A to reach its target, as well as nontarget uptake that may cause side effects. Such antibody radiotracers might also provide a method for measuring GD2 expression in tumors, thus enabling the prediction of response to anti-GD2 therapy for individual patients. Methods: The conjugation of hu14.18K322A with p-NH 2 -Bn-DOTA was accomplished using N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide with subsequent 64 Cu radiolabeling at 37°C for 30 min. Immunoreactivity of the conjugate was assessed by a dose-escalation blocking experiment measuring binding to purified GD2 versus GD1b as a negative control. Cell uptake and biodistribution studies in M21 (GD2-positive) and PC-3 (GD2-negative) tumor models were performed, as was small-animal PET/CT of M21 and PC-3 tumor-bearing mice. Results: The labeling of 64 Cu-p-NH 2 -Bn-DOTA-hu14.18K322A was achieved at more than 95% radiochemical purity and a specific activity of 127-370 MBq/mg (3.4-10 mCi/mg) after chromatographic purification. Preliminary in vitro data demonstrated a greater than 6-fold selectivity of binding to GD2 versus GD1b and dose-dependent inhibition of binding by unmodified hu14.8K322A. In vivo data, including small-animal PET/CT, showed significant GD2-positive tumor-targeting ability, with a persistent 2-fold-higher uptake of radiotracer than in GD2-negative tumors. Conclusion: 64 Cu-p-NH 2 -Bn-DOTAhu14.18K322A represents a novel PET radiotracer to facilitate clinical investigations of anti-GD2 immunotherapies and to complement other imaging modalities in the staging and treatment of neuroblastoma.

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The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells

Cited by 57 publications

References 46 publications

GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes

⁶⁴Cu-p-NH₂-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma

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The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells

Cited by 57 publications

References 46 publications

GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes

64Cu-p-NH2-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma

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⁶⁴Cu-p-NH₂-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma