Reverse Phase Protein Arrays in cancer stem cells

Signore, Michele; Manganelli, Valeria

doi:10.1016/bs.mcb.2022.04.004

Cited by 4 publications

(3 citation statements)

References 64 publications

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“…Reverse-Phase Protein microArrays (RPPA) analysis was performed based on established protocols. Anchorage-dependent GBM cells U-87 MG, U-251 MG, and TS#1 and TS#163 neurospheres were seeded onto 6-well microtiter plates (3.5 × 10 3 cells/well) and treated either with vehicle or CPZ at the cell line-specific IC30 and IC50 for 2 and 8 h and processed as described [ 41 – 43 ]. Cells from three different passages were used for biological replicates of individual experimental conditions.…”

Section: Methodsmentioning

confidence: 99%

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

Abbruzzese,

Matteoni,

Matarrese

et al. 2023

Cell Death Dis

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Glioblastoma (GBM) is the most frequent and lethal brain tumor, whose therapeutic outcome - only partially effective with current schemes - places this disease among the unmet medical needs, and effective therapeutic approaches are urgently required. In our attempts to identify repositionable drugs in glioblastoma therapy, we identified the neuroleptic drug chlorpromazine (CPZ) as a very promising compound. Here we aimed to further unveil the mode of action of this drug. We performed a supervised recognition of the signal transduction pathways potentially influenced by CPZ via Reverse-Phase Protein microArrays (RPPA) and carried out an Activity-Based Protein Profiling (ABPP) followed by Mass Spectrometry (MS) analysis to possibly identify cellular factors targeted by the drug. Indeed, the glycolytic enzyme PKM2 was identified as one of the major targets of CPZ. Furthermore, using the Seahorse platform, we analyzed the bioenergetics changes induced by the drug. Consistent with the ability of CPZ to target PKM2, we detected relevant changes in GBM energy metabolism, possibly attributable to the drug’s ability to inhibit the oncogenic properties of PKM2. RPE-1 non-cancer neuroepithelial cells appeared less responsive to the drug. PKM2 silencing reduced the effects of CPZ. 3D modeling showed that CPZ interacts with PKM2 tetramer in the same region involved in binding other known activators. The effect of CPZ can be epitomized as an inhibition of the Warburg effect and thus malignancy in GBM cells, while sparing RPE-1 cells. These preclinical data enforce the rationale that allowed us to investigate the role of CPZ in GBM treatment in a recent multicenter Phase II clinical trial.

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Section: Methodsmentioning

confidence: 99%

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

Abbruzzese,

Matteoni,

Matarrese

et al. 2023

Cell Death Dis

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“…Reverse-Phase Protein microArrays (RPPA) analysis was performed following established procedures (40,41). Brie y, protein extracts were resuspended in Laemmli sample buffer (42) at a nal concentration of 0.5 mg/mL with 2.5% TCEP reducing agent (Thermo-Fisher Scienti c) and boiled for 3' prior to printing with an Aushon 2470 (Quanterix) microarrayer.…”

Section: Rppa Analysismentioning

confidence: 99%

Chlorpromazine Overcomes Temozolomide Resistance in Glioblastoma by inhibiting Cx43 and Essential DNA Repair Pathways

Matarrese,

Signore,

Ascione

et al. 2024

Preprint

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Background In the fight against GBM, drug repurposing emerges as a viable and time-saving approach to explore new treatment options. Chlorpromazine, an old antipsychotic medication, has recently arisen as a promising candidate for repositioning in GBM therapy in addition to temozolomide, the first-line standard of care. We previously demonstrated the antitumor efficacy of chlorpromazine and its synergistic effects with temozolomide in suppressing GBM cell malignant features in vitro. This prompted us to accomplish a Phase II clinical trial to evaluate the efficacy and safety of adding chlorpromazine to temozolomide in GBM patients with unmethylated MGMT gene promoter. In this in vitro study, we investigate the potential role of chlorpromazine in overcoming temozolomide resistance. Methods In our experimental set, we analyzed Connexin-43 expression at both the transcriptional and protein levels in control- and chlorpromazine-treated GBM cells. DNA damage and subsequent repair were assessed by immunofluorescence of g-H2AX and Reverse-Phase Protein microArrays in chlorpromazine treated GBM cell lines. To elucidate the relationship between DNA repair systems and chemoresistance, we analyzed a signature of DNA repair genes in GBM cells after treatment with chlorpromazine, temozolomide and Connexin-43 downregulation. Results Chlorpromazine treatment significantly downregulated connexin-43 expression in GBM cells, consequently compromising connexin-dependent cellular resilience, and ultimately contributing to cell death. In line with this, we observed concordant post-translational modifications of molecular determinants involved in DNA damage and repair pathways. Our evaluation of DNA repair genes revealed that temozolomide elicited an increase, while chlorpromazine, as well as connexin-43 silencing, a decrease in DNA repair gene expression in GBM cells. Conclusions Chlorpromazine potentiates the cytotoxic effects of the alkylating agent temozolomide through a mechanism involving downregulation of Cx43 expression and disruption of the cell cycle arrest essential for DNA repair processes. This finding suggests that chlorpromazine may be a potential therapeutic strategy to overcome TMZ resistance in GBM cells by inhibiting their DNA repair mechanisms.

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“…Anchorage-dependent GBM cells U-251 MG, U-87 MG, and TS#1 and TS#163 neurospheres were seeded onto 6-well microtiter plates (3.5 x 10 3 cells/well) and treated either with vehicle or CPZ at the cell linespeci c IC30 and IC50 for 2 and 8 h and processed as described (42)(43)(44). Cells from three different passages were used for biological replicates of individual experimental conditions.…”

Section: Rppamentioning

confidence: 99%

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

Abbruzzese

Matteoni

Matarrese

et al. 2023

Preprint

View full text Add to dashboard Cite

Glioblastoma (GBM) is the most frequent and lethal brain tumor, whose therapeutic outcome - only partially effective with current schemes - places this disease among the unmet medical needs, and effective therapeutic approaches are urgently required. In our attempts to identify repositionable drugs in glioblastoma therapy, we identified chlorpromazine (CPZ) as a very promising compound. Here we aimed to further unveil the mode of action of this drug. We performed a supervised recognition of the signal transduction pathways potentially influenced by CPZ via Reverse-Phase Protein microArrays (RPPA) and carried out an Activity-Based Protein Profiling (ABPP) followed by Mass Spectrometry (MS) analysis to possibly identify cellular factors targeted by the drug. Indeed, the glycolytic enzyme PKM2 was identified as a major target of CPZ. Furthermore, using the Seahorse platform, we analyzed the bioenergetics changes induced by the drug. CPZ hindered GBM anabolic pathways and stimulated autophagy. Consistent with the ability of CPZ to target PKM2, we detected relevant changes in GBM energy metabolism, possibly attributable to the drug’s ability to inhibit the oncogenic properties of PKM2. RPE-1 non-cancer neuroepithelial cells appeared resistant to the drug. PKM2 silencing reduced the effects of CPZ. 3D modeling showed that CPZ interacts with PKM2 tetramer in the same region involved in binding other known activators. The effect of CPZ can be epitomized as an inhibition of the Warburg effect and, thus, malignancy in GBM cells while sparing RPE-1 cells. These preclinical data enforce the rationale that allowed us to investigate the role of CPZ in GBM treatment in an ongoing multicenter Phase II clinical trial.

show abstract

Reverse Phase Protein Arrays in cancer stem cells

Cited by 4 publications

References 64 publications

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

Chlorpromazine Overcomes Temozolomide Resistance in Glioblastoma by inhibiting Cx43 and Essential DNA Repair Pathways

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2

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