Chlorpromazine overcomes temozolomide resistance in glioblastoma by inhibiting Cx43 and essential DNA repair pathways.

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 γ-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.

Deciphering specific miRNAs in brain tumors: a 5-miRNA signature in glioblastoma.

MicroRNAs are endogenous non-coding RNAs with a marked impact on the development and progression of brain tumors. However, they commonly share different expression patterns in other types of tumors, thereby exhibiting lack of tissue specificity. Here, an integrative holistic analysis of microarray data is established for deciphering dysregulated miRNAs in glioblastoma, distinguishing them from eight other CNS tumors. The identification of dysregulated miRNAs was performed in a pool of 176 patients, 118 of which diagnosed with glioblastoma. Dysregulated miRNAs commonly expressed in glioblastoma were then discriminated from those co-expressed in other CNS tumors and further characterized. Overall, 21 miRNAs were found to be commonly dysregulated in glioblastoma. Notwithstanding, 16 miRNAs also exhibited a differential expression in at least one other CNS tumor. The remaining 5, specifically, hsa-miR-21-3p, hsa-miR-338-5p, hsa-miR-485-5p, hsa-miR-491-5p and hsa-miR-1290, were solely associated to glioblastoma. This signature is in-depth characterized, with the spotlight on tumor progression, invasion and patient survival. These five endogenous molecules, differentially expressed in glioblastoma, are thus suggested as potential therapeutic targets, modulating several genes involved in major signalling pathways, including MAPK/ERK, calcium, PI3K/AKT, mTOR and Wnt. In summary, these findings lay a foundation for further research on the expression and function of specific patterns of miRNAs expression in glioblastoma, providing reference for potential novel targets.

The small molecule SI113 hinders epithelial-to-mesenchymal transition and subverts cytoskeletal organization in human cancer cells.

The small molecule SI113 is an inhibitor of the kinase activity of SGK1, a key biological regulator acting on the PI3K/mTOR signal transduction pathway. Several studies demonstrate that this compound is able to strongly restrain cancer growth in vitro and in vivo, alone or in associative antineoplastic treatments, being able to elicit an autophagic response, either cytotoxic or cytoprotective. To elucidate more exhaustively the molecular mechanisms targeted by SI113, we performed activity-based protein profiling (ABPP) proteomic analysis using a kinase enrichment procedure. This technique allowed the identification via mass spectrometry of novel targets of this compound, most of them involved in functions concerning cell motility and cytoskeletal architecture. Using a glioblastoma multiforme, hepatocarcinoma and colorectal carcinoma cell line, we recognized an inhibitory effect of SI113 on cell migration, invading, and epithelial-to-mesenchymal transition. In addition, these cancer cells, when exposed to this compound, showed a remarkable subversion of the cytoskeletal architecture characterized by F-actin destabilization, phospho-FAK delocalization, and tubulin depolimerization. These results were definitely concordant in attributing to SI113 a key role in hindering cancer cell malignancy and, due to its negligible in vivo toxicity, can sustain performing a Phase I clinical trial to employ this drug in associative cancer therapy.

Detection of phosphorylated insulin receptor in colorectal adenoma and adenocarcinoma: implications for prognosis and clinical outcome.

Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well-known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio-pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper-insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P < 0.0001) and, within the adenocarcinoma cohort, it was more frequent in low-grade tumors (P = 0.005). In adenomas, staining was exclusively cytoplasmic, while in adenocarcinomas it was cytoplasmic and/or nuclear (P < 0.0001). Interestingly, disease-free survival in colorectal adenocarcinoma patients pointed out a significantly better prognosis for those bearing a positive staining for phosphorylated insulin receptor (P = 0.02). From these data, we can argue that activated insulin receptor plays a fundamental role at the early stages of tumorigenesis, where late stages could be characterized by a shift toward more active oncogenic drivers. Determining the amount of phosphorylated insulin receptor could thus represent a novel prognostic/predictive tool in colorectal adenocarcinoma patients.

SI113, a specific inhibitor of the Sgk1 kinase activity that counteracts cancer cell proliferation.

BACKGROUND/AIMS: Published observations on serum and glucocorticoid regulated kinase 1 (Sgk1) knockout murine models and Sgk1-specific RNA silencing in the RKO human colon carcinoma cell line point to this kinase as a central player in colon carcinogenesis and in resistance to taxanes. METHODS: By in vitro kinase activity inhibition assays, cell cycle and viability analysis in human cancer model systems, we describe the biologic effects of a recently identified kinase inhibitor, SI113, characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold, that shows specificity for Sgk1. RESULTS: SI113 was able to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. In RKO cells, this kinase inhibitor blocked insulin-dependent phosphorylation of the Sgk1 substrate Mdm2, the main regulator of p53 protein stability, and induced necrosis and apoptosis when used as a single agent. Finally, SI113 potentiated the effects of paclitaxel on cell viability. CONCLUSION: Since SI113 appears to be effective in inducing cell death in RKO cells, potentiating paclitaxel sensitivity, we believe that this new molecule could be efficiently employed, alone or in combination with paclitaxel, in colon cancer chemotherapy.

Identification of pivotal cellular factors involved in HPV-induced dysplastic and neoplastic cervical pathologies.

Cervical carcinoma represents the paradigm of virus-induced cancers, where virtually all cervical cancers come from previous "high-risk" HPV infection. The persistent expression of the HPV viral oncoproteins E6 and E7 is responsible for the reprogramming of fundamental cellular functions in the host cell, thus generating a noticeable, yet only partially explored, imbalance in protein molecular networks and cell signaling pathways. Eighty-eight cellular factors, identified as HPV direct or surrogate targets, were chosen and monitored in a retrospective analysis for their mRNA expression in HPV-induced cervical lesions, from dysplasia to cancer. Real-time quantitative PCR (qPCR) was performed by using formalin-fixed, paraffin embedded archival samples. Gene expression analysis identified 40 genes significantly modulated in LSIL, HSIL, and squamous cervical carcinoma. Interestingly, among these, the expression level of a panel of four genes, TOP2A, CTNNB1, PFKM, and GSN, was able to distinguish between normal tissues and cervical carcinomas. Immunohistochemistry was also done to assess protein expression of two genes among those up-regulated during the transition between dysplasia and carcinoma, namely E2F1 and CDC25A, and their correlation with clinical parameters. Besides the possibility of significantly enhancing the use of some of these factors in diagnostic or prognostic procedures, these data clearly outline specific pathways, and thus key biological processes, altered in cervical dysplasia and carcinoma. Deeper insight on how these molecular mechanisms work may help widen the spectrum of novel innovative approaches to these virus-induced cell pathologies.

The human papillomavirus-16 E7 oncoprotein exerts antiapoptotic effects via its physical interaction with the actin-binding protein gelsolin.

The oncoprotein E7 from human papillomavirus-16 (HPV-16 E7) plays a pivotal role in HPV postinfective carcinogenesis, and its physical interaction with host cell targets is essential to its activity. We identified a novel cellular partner for the viral oncoprotein: the actin-binding protein gelsolin (GSN), a key regulator of actin filament assembly and disassembly. In fact, biochemical analyses, generation of a 3D molecular interaction model and the use of specific HPV-16 E7 mutants provided clear cut evidence supporting the crucial role of HPV-16 E7 in affecting GSN integrity and function in human immortalized keratinocytes. Accordingly, functional analyses clearly suggested that stable HPV-16 E7 expression induced an imbalance between polymeric and monomeric actin in favor of the former. These events also lead to changes of cell cycle (increased S phase), to the inhibition of apoptosis and to the increase of cell survival. These results provide support to the hypotheses generated from the 3D molecular interaction model and encourage the design of small molecules hindering HPV-induced host cell reprogramming by specifically targeting HPV-16 E7-expressing cells.

Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery.

The retinoblastoma (RB) family consists of three genes, RB1, RBL1, and RBL2, that code for the pRb, p107, and pRb2/p130 proteins, respectively. All these factors have pivotal roles in controlling fundamental cellular mechanisms such as cell cycle, differentiation and apoptosis. The founder and the most investigated RB family protein is pRb, which is considered to be the paradigm of tumor suppressors. However, p107 and pRb2/p130 clearly display a high degree of structural and functional homology with pRb. Interestingly, these factors were first identified as physical targets of the Adenovirus E1A oncoprotein. Indeed, RB family proteins are the most important and widely investigated targets of small DNA virus oncoproteins, such as Adenovirus E1A, human papillomavirus E7 and Simian virus 40 large T antigen. By interacting with pRb and with other RB family members, these oncoproteins neutralize their growth suppressive properties, thus stimulating proliferation of the infected cells, de-differentiation, and resistance to apoptosis. All these acquired features strongly favor the rise and selection of immortalized and mutation-prone cells, leading to a higher propensity in undergoing transformation. Our present work aims to illustrate and delve into these protein-protein interactions. Considering that these viral oncoproteins are dispensable for normal cellular functions, they can create "oncogene addiction" in the infected/transformed cells. This makes the possibility to dismantle these interactions extremely attractive, thus promoting the development of highly specific smart molecules capable of targeting only the infected/transformed cells that express these viral factors.

Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2.

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.

Efficacy and safety of chlorpromazine as an adjuvant therapy for glioblastoma in patients with unmethylated MGMT gene promoter: RACTAC, a phase II multicenter trial.

Introduction: Drug repurposing is a promising strategy to develop new treatments for glioblastoma. In this phase II clinical trial, we evaluated the addition of chlorpromazine to temozolomide in the adjuvant phase of the standard first-line therapeutic protocol in patients with unmethylated MGMT gene promoter. Methods: This was a multicenter phase II single-arm clinical trial. The experimental procedure involved the combination of CPZ with standard treatment with TMZ in the adjuvant phase of the Stupp protocol in newly-diagnosed GBM patients carrying an unmethylated MGMT gene promoter. Progression-free survival was the primary endpoint. Secondary endpoints were overall survival and toxicity. Results: Forty-one patients were evaluated. Twenty patients (48.7%) completed 6 cycles of treatment with TMZ+CPZ. At 6 months, 27 patients (65.8%) were without progression, achieving the primary endpoint. Median PFS was 8.0 months (95% CI: 7.0-9.0). Median OS was 15.0 months (95% CI: 13.1-16.9). Adverse events led to reduction or interruption of CPZ dosage in 4 patients (9.7%). Discussion: The addition of CPZ to standard TMZ in the first-line treatment of GBM patients with unmethylated MGMT gene promoter was safe and led to a longer PFS than expected in this population of patients. These findings provide proof-of-concept for the potential of adding CPZ to standard TMZ treatment in GBM patients with unmethylated MGMT gene promoter. Clinical trial registration: https://clinicaltrials.gov/study/NCT04224441, identifier NCT04224441.

New insights in endometrial carcinogenesis.

Endometrial carcinoma is the most common cancer of the female genital tract in Europe and in the United States. Despite advances in defining the biology of endometrial carcinomas, there has been poor progress in determining markers that distinguish preinvasive endometrial proliferations. The aim of this review is to highlight the most recent studies regarding the molecular markers involved in endometrial adenocarcinoma pathogenesis and carcinogenesis. We focus on studies that describe markers with potential to progress from endometrial hyperplasia to invasive disease.

Effects of assessing the productivity of faculty in academic medical centres: a systematic review.

BACKGROUND: Many academic medical centres have introduced strategies to assess the productivity of faculty as part of compensation schemes. We conducted a systematic review of the effects of such strategies on faculty productivity. METHODS: We searched the MEDLINE, Healthstar, Embase and PsycInfo databases from their date of inception up to October 2011. We included studies that assessed academic productivity in clinical, research, teaching and administrative activities, as well as compensation, promotion processes and satisfaction. RESULTS: Of 531 full-text articles assessed for eligibility, we included 9 articles reporting on eight studies. The introduction of strategies for assessing academic productivity as part of compensation schemes resulted in increases in clinical productivity (in six of six studies) in terms of clinical revenue, the work component of relative-value units (these units are nonmonetary standard units of measure used to indicate the value of services provided), patient satisfaction and other departmentally used standards. Increases in research productivity were noted (in five of six studies) in terms of funding and publications. There was no change in teaching productivity (in two of five studies) in terms of educational output. Such strategies also resulted in increases in compensation at both individual and group levels (in three studies), with two studies reporting a change in distribution of compensation in favour of junior faculty. None of the studies assessed effects on administrative productivity or promotion processes. The overall quality of evidence was low. INTERPRETATION: Strategies introduced to assess productivity as part of a compensation scheme appeared to improve productivity in research activities and possibly improved clinical productivity, but they had no effect in the area of teaching. Compensation increased at both group and individual levels, particularly among junior faculty. Higher quality evidence about the benefits and harms of such assessment strategies is needed.

Molecular Biology in Glioblastoma Multiforme Treatment.

Glioblastoma (GBM, grade IV astrocytoma), the most frequently occurring primary brain tumor, presents unique challenges to therapy due to its location, aggressive biological behavior, and diffuse infiltrative growth, thus contributing to having disproportionately high morbidity and mortality [...].

Tackling the Behavior of Cancer Cells: Molecular Bases for Repurposing Antipsychotic Drugs in the Treatment of Glioblastoma.

Glioblastoma (GBM) is associated with a very dismal prognosis, and current therapeutic options still retain an overall unsatisfactorily efficacy in clinical practice. Therefore, novel therapeutic approaches and effective medications are highly needed. Since the development of new drugs is an extremely long, complex and expensive process, researchers and clinicians are increasingly considering drug repositioning/repurposing as a valid alternative to the standard research process. Drug repurposing is also under active investigation in GBM therapy, since a wide range of noncancer and cancer therapeutics have been proposed or investigated in clinical trials. Among these, a remarkable role is played by the antipsychotic drugs, thanks to some still partially unexplored, interesting features of these agents. Indeed, antipsychotic drugs have been described to interfere at variable incisiveness with most hallmarks of cancer. In this review, we analyze the effects of antipsychotics in oncology and how these drugs can interfere with the hallmarks of cancer in GBM. Overall, according to available evidence, mostly at the preclinical level, it is possible to speculate that repurposing of antipsychotics in GBM therapy might contribute to providing potentially effective and inexpensive therapies for patients with this disease.

Reducing the risk of overdiagnosis in lung cancer: a support from molecular biology.

Early detection and swift treatment, when achievable, may significantly affect prognosis in lung cancer patients. Therefore, individuals with a high risk for lung cancer are invited to participate into international screening programs, such as the International Early Lung Cancer Action Program (I-ELCAP). An undesirable consequence of such massive enterprises is the detection of pulmonary nodules also in subjects who are unlikely to ultimately die from lung cancer. Nevertheless, the individuals with pulmonary nodule undergo stringent diagnostic procedures to assess the nature of the lesion. This implies a noticeable (physical and emotional) stress for our patients and the likelihood of overdiagnosis and, potentially, consequent overtreatment. Molecular markers, more specifically, microRNAs, might significantly add value to the workup process aiming at the distinction between benign and malignant lesions and, among the malignant ones, those concretely threatening for the patients' survival. We are confident that such a multidisciplinary approach would better suit our patients' diagnostic and/or therapeutic, actual needs.

Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells.

BACKGROUND: "High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. METHODS: In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance. RESULTS: The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. CONCLUSIONS: Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.

Physical Interaction between HPV16E7 and the Actin-Binding Protein Gelsolin Regulates Epithelial-Mesenchymal Transition via HIPPO-YAP Axis.

Human papillomavirus 16 (HPV16) exhibits a strong oncogenic potential mainly in cervical, anogenital and oropharyngeal cancers. The E6 and E7 viral oncoproteins, acting via specific interactions with host cellular targets, are required for cell transformation and maintenance of the transformed phenotype as well. We previously demonstrated that HPV16E7 interacts with the actin-binding protein gelsolin, involved in cytoskeletal F-actin dynamics. Herein, we provide evidence that the E7/gelsolin interaction promotes the cytoskeleton rearrangement leading to epithelial-mesenchymal transition-linked morphological and transcriptional changes. E7-mediated cytoskeletal actin remodeling induces the HIPPO pathway by promoting the cytoplasmic retention of inactive P-YAP. These results suggest that YAP could play a role in the "de-differentiation" process underlying the acquisition of a more aggressive phenotype in HPV16-transformed cells. A deeper comprehension of the multifaceted mechanisms elicited by the HPV infection is vital for providing novel strategies to block the biological and clinical features of virus-related cancers.

Chlorpromazine induces cytotoxic autophagy in glioblastoma cells via endoplasmic reticulum stress and unfolded protein response.

BACKGROUND: Glioblastoma (GBM; grade IV glioma) is characterized by a very short overall survival time and extremely low 5-year survival rates. We intend to promote experimental and clinical research on rationale and scientifically driven drug repurposing. This may represent a safe and often inexpensive way to propose novel pharmacological approaches to GBM. Our precedent work describes the role of chlorpromazine (CPZ) in hindering malignant features of GBM. Here, we investigate in greater detail the molecular mechanisms at the basis of the effect of CPZ on GBM cells. METHODS: We employed proteomics platforms, i.e., activity-based protein profiling plus mass spectrometry, to identify potential cellular targets of the drug. Then, by means of established molecular and cellular biology techniques, we assessed the effects of this drug on GBM cell metabolic and survival pathways. RESULTS: The experimental output indicated as putative targets of CPZ several of factors implicated in endoplasmic reticulum (ER) stress, with consequent unfolded protein response (UPR). Such a perturbation culminated in a noticeable reactive oxygen species generation and intense autophagic response that resulted in cytotoxic and abortive effects for six GBM cell lines, three of which growing as neurospheres, while it appeared cytoprotective for the RPE-1 human non-cancer neuro-ectodermal cell line. CONCLUSIONS: This discrepancy could be central in explaining the lethal effects of the drug on GBM cells and the relatively scarce cytotoxicity toward normal tissues attributed to this compound. The data presented here offer support to the multicenter phase II clinical trial we have undertaken, which consists of the addition of CPZ to first-line treatment of GBM patients carrying a hypo- or un-methylated MGMT gene, i.e. those characterized by intrinsic resistance to temozolomide.

Anticancer Properties of the Antipsychotic Drug Chlorpromazine and Its Synergism With Temozolomide in Restraining Human Glioblastoma Proliferation In Vitro.

The extremely poor prognosis of patients affected by glioblastoma (GBM, grade IV glioma) prompts the search for new and more effective therapies. In this regard, drug repurposing or repositioning can represent a safe, swift, and inexpensive way to bring novel pharmacological approaches from bench to bedside. Chlorpromazine, a medication used since six decades for the therapy of psychiatric disorders, shows in vitro several features that make it eligible for repositioning in cancer therapy. Using six GBM cell lines, three of which growing as patient-derived neurospheres and displaying stem-like properties, we found that chlorpromazine was able to inhibit viability in an apoptosis-independent way, induce hyperdiploidy, reduce cloning efficiency as well as neurosphere formation and downregulate the expression of stemness genes in all these cell lines. Notably, chlorpromazine synergized with temozolomide, the first-line therapeutic in GBM patients, in hindering GBM cell viability, and both drugs strongly cooperated in reducing cloning efficiency and inducing cell death in vitro for all the GBM cell lines assayed. These results prompted us to start a Phase II clinical trial on GBM patients (EudraCT # 2019-001988-75; ClinicalTrials.gov Identifier: NCT04224441) by adding chlorpromazine to temozolomide in the adjuvant phase of the standard first-line therapeutic protocol.

Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study.

BACKGROUND: Cathepsins represent a group of proteases involved in determining the metastatic potential of cancer cells. Among these are cysteinyl- (e.g. cathepsin B and cathepsin L) and aspartyl-proteases (e.g. cathepsin D), normally present inside the lysosomes as inactive proenzymes. Once released in the extracellular space, cathepsins contribute to metastatic potential by facilitating cell migration and invasiveness. RESULTS: In the present work we first evaluated, by in vitro procedures, the role of cathepsins B, L and D, in the remodeling, spreading and invasiveness of eight different cell lines: four primary and four metastatic melanoma cell lines. Among these, we considered two cell lines derived from a primary cutaneous melanoma and from a supraclavicular lymph node metastasis of the same patient. To this purpose, the effects of specific chemical inhibitors of these proteases, i.e. CA-074 and CA-074Me for cathepsin B, Cathepsin inhibitor II for cathepsin L, and Pepstatin A for cathepsin D, were evaluated. In addition, we also analyzed the effects of the biological inhibitors of these cathepsins, i.e. specific antibodies, on cell invasiveness. We found that i) cathepsin B, but not cathepsins L and D, was highly expressed at the surface of metastatic but not of primary melanoma cell lines and that ii) CA-074, or specific antibodies to cathepsin B, hindered metastatic cell spreading and dissemination, whereas neither chemical nor biological inhibitors of cathepsins D and L had significant effects. Accordingly, in vivo studies, i.e. in murine xenografts, demonstrated that CA-074 significantly reduced human melanoma growth and the number of artificial lung metastases. CONCLUSIONS: These results suggest a reappraisal of the use of cathepsin B inhibitors (either chemical or biological) as innovative strategy in the management of metastatic melanoma disease.