IT202000022636A1 - “COMBINATION THERAPY OF SORAFENIB AND/OR REGORAFENIB WITH THE RECOMBINANT HUMAN PROTEOGLYCAN-4 PROTEIN FOR THE TREATMENT OF HEPATOCARCINOMA” - Google Patents
“COMBINATION THERAPY OF SORAFENIB AND/OR REGORAFENIB WITH THE RECOMBINANT HUMAN PROTEOGLYCAN-4 PROTEIN FOR THE TREATMENT OF HEPATOCARCINOMA” Download PDFInfo
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- IT202000022636A1 IT202000022636A1 IT102020000022636A IT202000022636A IT202000022636A1 IT 202000022636 A1 IT202000022636 A1 IT 202000022636A1 IT 102020000022636 A IT102020000022636 A IT 102020000022636A IT 202000022636 A IT202000022636 A IT 202000022636A IT 202000022636 A1 IT202000022636 A1 IT 202000022636A1
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- sorafenib
- regorafenib
- rhprg4
- combination
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Classifications
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Description
Descrizione dell?invenzione industriale dal titolo: ?TERAPIA DI COMBINAZIONE DI SORAFENIB E/O REGORAFENIB CON LA PROTEINA RICOMBINANTE UMANA PROTEOGLICANO-4 PER IL TRATTAMENTO DELL'EPATOCARCINOMA?; Description of the industrial invention entitled: ?COMBINATION THERAPY OF SORAFENIB AND/OR REGORAFENIB WITH THE RECOMBINANT HUMAN PROTEOGLYCAN-4 PROTEIN FOR THE TREATMENT OF HEPATOCARCINOMA?;
DESCRIZIONE DESCRIPTION
Campo di applicazione Field of application
Nel suo aspetto pi? generale, la presente invenzione riguarda un metodo di trattamento antitumorale, e pi? precisamente riguarda una composizione e un metodo di trattamento dell'epatocarcinoma e delle metastasi epatiche basato sulla terapia di combinazione di sorafenib e regorafenib con la proteina proteoglicano 4 (PRG4), in particolare con la forma ricombinante della proteina proteoglicano 4 umana (rhPRG4). In its most In general, the present invention relates to an anticancer treatment method, and more? precisely it concerns a composition and a method of treatment of hepatocarcinoma and liver metastases based on the combination therapy of sorafenib and regorafenib with proteoglycan protein 4 (PRG4), in particular with the recombinant form of human proteoglycan protein 4 (rhPRG4).
Tecnica nota Known technique
Il carcinoma epatocellulare (HCC) ? tra le cause pi? frequenti di morte per cancro in tutto il mondo. Per le caratteristiche strutturali e funzionali dell'organo interessato da questa neoplasia, la maggior parte dei pazienti con carcinoma epatocellulare non pu? ricevere una terapia basata su un approccio chirurgico o ablazione con radiofrequenza, pertanto la terapia sistemica a base di farmaci antitumorali rimane il principale approccio terapeutico perseguito. L?accumulo progressivo di conoscenze sui meccanismi cellulari e molecolari che governano o contribuiscono all?evoluzione di questo tumore si ? tradotto negli anni nella concezione di nuove molecole farmacologiche, in forma di inibitori a basso peso molecolare, o di anticorpi monoclonali umanizzati. Hepatocellular carcinoma (HCC) ? among the causes incidence of cancer deaths worldwide. Due to the structural and functional characteristics of the organ affected by this neoplasm, most patients with hepatocellular carcinoma cannot receive therapy based on a surgical approach or radiofrequency ablation, therefore systemic therapy based on anticancer drugs remains the main therapeutic approach pursued. The progressive accumulation of knowledge on the cellular and molecular mechanisms that govern or contribute to the evolution of this tumor is translated over the years into the conception of new pharmacological molecules, in the form of low molecular weight inhibitors, or humanized monoclonal antibodies.
Tuttavia, la resa in termini prognostici di questi trattamenti ? ancora insoddisfacente. Ci? dipende da una comprensione comunque molto parziale della natura e del funzionamento dei suddetti meccanismi, nonch? dall?alta inter- e intra-eterogeneit? del tumore, a causa della spesso concomitante malattia epatica preesistente. Complessivamente, tali limiti di conoscenza e fattori di complessit? ostacolano significativamente lo sviluppo di un approccio di medicina di precisione. However, the prognostic performance of these treatments? still unsatisfactory. There? depends on a very partial understanding of the nature and functioning of these mechanisms, as well as? from? high inter- and intra-heterogeneity? of the tumour, due to the often concomitant pre-existing liver disease. Overall, such knowledge limitations and complexity factors significantly hinder the development of a precision medicine approach.
Un farmaco approvato per il trattamento del carcinoma epatocellulare (HCC) ? il sorafenib, 4-[4-[[4-cloro-3-(trifluorometil)fenil] carbamoilammino] fenossi]-N-metil-piridina-2-carbossammide, che per anni ha costituito l'unica forma di terapia sistemica per questo tipo di tumore. A drug approved for the treatment of hepatocellular carcinoma (HCC)? sorafenib, 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl] carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide, which for years was the only form of systemic therapy for this type of tumor.
Il meccanismo d'azione del sorafenib ? mediato dall'attivit? inibitoria esercitata sulle chinasi sovraespresse in una serie di vie molecolari coinvolte nella trasformazione di cellule normali in cellule tumorali, in particolare recettori con attivit? delle chinasi e sulle chinasi di Raf. Il farmaco ? disponibile in commercio in compresse somministrate per via orale come NEXAVAR<?>, che ? il sale tosilato del sorafenib. Sorafenib tosilato ? praticamente insolubile in acqua, leggermente solubile in etanolo e solubile in PEG 400; il farmaco ha una bassa biodisponibilit?. Inoltre, spesso alla somministrazione si associa una rilevante irritazione gastrointestinale Altri effetti collaterali come rash cutaneo, diarrea e ipertensione ne limitano fortemente l'applicazione clinica. The mechanism of action of sorafenib ? activity mediated? inhibitory activity exerted on kinases overexpressed in a number of molecular pathways involved in the transformation of normal cells into tumor cells, in particular receptors with activity of kinases and on Raf kinases. The drug? commercially available in orally administered tablets as NEXAVAR<?>, which is ? the tosylate salt of sorafenib. Sorafenib tosylate? practically insoluble in water, slightly soluble in ethanol and soluble in PEG 400; the drug has a low bioavailability. Furthermore, administration is often associated with significant gastrointestinal irritation. Other side effects such as skin rash, diarrhea and hypertension severely limit its clinical application.
Sorafenib tosilato ? descritto nei brevetti statunitensi US 7,235,576, US 7,897,623 e US 8,124,630. Sorafenib tosylate? described in US patents US 7,235,576 , US 7,897,623 and US 8,124,630 .
Il dosaggio di somministrazione del sorafenib approvato ? di 400 mg (2 compresse) per via orale due volte al giorno a digiuno. Tuttavia, l'interruzione del trattamento e/o una riduzione della dose possono essere necessarie per gestire sospette reazioni avverse al farmaco. In tali casi, la dose pu? essere ridotta a 400 mg una volta al giorno o a 400 mg a giorni alterni. The approved sorafenib administration dosage? of 400 mg (2 tablets) orally twice daily on an empty stomach. However, treatment interruption and/or dose reduction may be necessary to manage suspected adverse drug reactions. In such cases, the dose can be reduced to 400 mg once daily or 400 mg every other day.
Sorafenib rappresenta la terapia di prima linea per i malati di epatocarcinoma. ? stato osservato che la sequenza di trattamento costituita da sorafenib seguito da regorafenib, migliora la sopravvivenza dei pazienti con tumore del fegato avanzato, come dimostrato dai risultati dello studio clinico di fase III, RESORCE, che ha valutato la sopravvivenza dei pazienti affetti da carcinoma epatocellulare (HCC) trattati con regorafenib in seguito alla progressione della patologia durante il trattamento con sorafenib. [Bruix J. et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebocontrolled, phase 3 trial. The Lancet 2017, 389: 56-66]. Sorafenib is the first-line therapy for patients with hepatocellular carcinoma. ? The treatment sequence of sorafenib followed by regorafenib has been observed to improve survival of patients with advanced liver cancer, as demonstrated by the results of the phase III clinical study, RESORCE, which evaluated the survival of patients with hepatocellular carcinoma ( HCC) treated with regorafenib following disease progression while on sorafenib. [Bruix J. et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebocontrolled, phase 3 trial. The Lancet 2017, 389: 56-66].
Regorafenib monoidrato, 4-[4-({[4-cloro-3-(trifluorometil)fenil] carbamoil}ammino)-3-fluorofenossi]-N-metilpiridina-2-carbossammide monoidrato, ed altre forme farmaceuticamente accettabili, sali ed esteri di regorafenib, ? disponibile in commercio con il nome STIVARGA<?>. Regorafenib ? insolubile in acqua e moderatamente solubile in solventi organici. Regorafenib ? descritto nelle domande di brevetto internazionali WO 2004/113274, WO 2005/000284 e WO 2005/009961. Regorafenib monohydrate, 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide monohydrate, and other pharmaceutically acceptable forms, salts and esters of regorafenib, ? commercially available under the name STIVARGA<?>. Regorafenib ? insoluble in water and sparingly soluble in organic solvents. Regorafenib ? described in international patent applications WO 2004/113274, WO 2005/000284 and WO 2005/009961.
Tuttavia, va segnalato che l'esperienza pluriennale nella somministrazione di composti come sorafenib e regorafenib ha prodotto risultati deludenti in termini di sopravvivenza globale. However, it should be noted that many years of experience in the administration of compounds such as sorafenib and regorafenib has produced disappointing results in terms of overall survival.
Allo scopo di fornire alternative vantaggiose in grado di superare i problemi correlati alla somministrazione di questi due farmaci attualmente disponibili sono state sviluppate nuove formulazioni. In order to provide advantageous alternatives able to overcome the problems related to the administration of these two currently available drugs, new formulations have been developed.
La domanda internazionale WO 2018/150302 descrive formulazioni a rilascio controllato comprendenti nanoparticelle polimeriche di sorafenib o dei suoi derivati. International application WO 2018/150302 describes controlled release formulations comprising polymeric nanoparticles of sorafenib or its derivatives.
Tuttavia, anche le nuove formulazioni di sorafenib, non sono in grado di risolvere i problemi di efficacia e di citossicit? del principio attivo. However, even the new formulations of sorafenib are unable to solve the problems of efficacy and cytotoxicity. of the active ingredient.
Un?intensa attivit? di ricerca di nuove terapie e il miglioramento delle terapie esistenti ? attualmente in corso, ma finora non ha prodotto scoperte clinicamente rilevanti. An intense activity? of research for new therapies and the improvement of existing therapies ? currently underway, but has so far yielded no clinically relevant breakthroughs.
Un approccio seguito ? quello della terapia di combinazione per sviluppare nuovi usi di farmaci esistenti. La terapia di combinazione rappresenta un?attraente opzione terapeutica in tutti quei casi in cui una terapia comporta importanti effetti avversi, poich? un eventuale effetto sinergico tra pi? principi attivi pu? favorire l'impiego di dosaggi inferiori o la somministrazione in pazienti che non migliorano, o peggiorano, dopo l?inizio della terapia di prima linea, in quanto l?associazione di farmaci con meccanismi d?azione diversi potrebbe interferire con i molteplici aspetti fisiopatologici della malattia migliorando l?esito della terapia. A followed approach? that of combination therapy to develop new uses for existing drugs. Combination therapy represents an attractive therapeutic option in all those cases in which a therapy involves important adverse effects, since it a possible synergistic effect between pi? active principles can? favor the use of lower dosages or the administration in patients who do not improve, or worsen, after the start of first-line therapy, as the combination of drugs with different mechanisms of action could interfere with the many physiopathological aspects of the disease by improving the outcome of therapy.
? stato ipotizzato che la tecnologia del microRNA potrebbe migliorare la terapia con sorafenib [Yang et al. "MicroRNA-34a targets Bcl-2 and sensitizes human hepatocellular carcinoma cells to sorafenib treatment Technol Cancer Res Treat.2014;13:77-86]. ? It has been hypothesized that microRNA technology could improve sorafenib therapy [Yang et al. "MicroRNA-34a targets Bcl-2 and sensitizes human hepatocellular carcinoma cells to sorafenib treatment Technol Cancer Res Treat.2014;13:77-86].
La domanda di brevetto internazionale WO 2015/131115 descrive che alcune combinazioni di sorafenib e microRNA sono particolarmente efficaci nell'inibire e prevenire la proliferazione delle cellule tumorali del fegato. L'effetto consiste, se non in una sinergia vera e propria, in un maggiore effetto additivo, che ? specifica per sorafenib e miR-34 e miR-215 nel cancro al fegato (ad es., HCC). Questa scoperta ? coerente con altre osservazioni dell?effetto di miR-34 e miR-215 su pi? tipi di cellule tumorali epatiche, ma non si estende necessariamente direttamente ad altri microRNA, agenti chemioterapici e/o tumori. Inoltre, l?effetto osservato non si estende a tutti i rapporti sorafenib e miR-34 o miR-215: alcuni rapporti sono approssimativamente additivi e altri sono antagonisti. International patent application WO 2015/131115 describes that some combinations of sorafenib and microRNA are particularly effective in inhibiting and preventing the proliferation of liver cancer cells. The effect consists, if not in a real synergy, in a greater additive effect, which? specific for sorafenib and miR-34 and miR-215 in liver cancer (e.g., HCC). This discovery? consistent with other observations of? effect of miR-34 and miR-215 on pi? liver cancer cell types, but does not necessarily extend directly to other microRNAs, chemotherapeutic agents, and/or tumors. Also, the observed effect does not extend across all ratios of sorafenib to miR-34 or miR-215: some ratios are approximately additive and others are antagonistic.
La domanda di brevetto internazionale WO 2017151044 descrive un effetto inibitorio e preventivo della proliferazione di cellule epatiche cancerose determinato dalla somministrazione di alcune combinazione di sorafenib e specifici profarmaci fosforamidati della troxocitabina e suggerisce che l'interazione possa essere estesa anche al trattamento delle metastasi. The international patent application WO 2017151044 describes an inhibitory and preventive effect of the proliferation of cancerous liver cells determined by the administration of some combinations of sorafenib and specific phosphoramidate prodrugs of troxocytabine and suggests that the interaction can also be extended to the treatment of metastases.
La domanda di brevetto internazionale WO 2017/223433 riguarda composizioni e metodi per trattare il carcinoma epatocellulare basato sulla combinazione di sorafenib e 2-desossiglucosio. International patent application WO 2017/223433 relates to compositions and methods for treating hepatocellular carcinoma based on the combination of sorafenib and 2-deoxyglucose.
In generale, considerata la bassa sopravvivenza dei pazienti dall'inizio del trattamento, l'elevato grado di eterogeneit? dei meccanismi molecolari alla base dello sviluppo e della progressione di questo particolare tipo di tumore, e la resistenza ai due farmaci in uso in questo periodo, i.e. sorafenib e regorafenib, ? fortemente sentita la necessit? di sviluppare nuovi approcci terapeutici, metodi e composizioni focalizzati verso pi? meccanismi responsabili del complesso quadro funzionale dei pazienti. In general, given the low survival of patients from the start of treatment, the high degree of heterogeneity of the molecular mechanisms underlying the development and progression of this particular type of tumor, and the resistance to the two drugs in use in this period, i.e. sorafenib and regorafenib, ? strongly felt the need to develop new therapeutic approaches, methods and compositions focused on more? mechanisms responsible for the complex functional picture of patients.
Si ritiene che il cross-talk tra le cellule tumorali epiteliali e il microambiente circostante, in particolare i fibroblasti associati al cancro (CAF), svolga un ruolo chiave nella progressione dell'HCC e di conseguenza eserciti un'influenza critica sull'esito clinico. The cross-talk between epithelial tumor cells and the surrounding microenvironment, particularly cancer-associated fibroblasts (CAFs), is thought to play a key role in the progression of HCC and consequently exert a critical influence on the clinical outcome.
L'origine dei CAF ? ancora oggetto di studio e di dibattito. Si ritiene che vari tipi di cellule intraepatiche subiscano cambiamenti fenotipici, differenziandosi in CAF durante l'evoluzione fibrogenica infiammatoria e progressiva della malattia epatica cronica, nonch? durante lo sviluppo dell'HCC. In questo microambiente, i CAF possono essere programmati fenotipicamente da cellule maligne adiacenti e, a loro volta, aumentano la proliferazione e la diffusione delle cellule HCC, probabilmente attraverso la deposizione o la secrezione di molecole, tra cui le proteine della matrice extracellulare (ECM). In effetti, i CAF sono i principali responsabili della deposizione di ECM, compresi collageni fibrillari come glicoproteine noncollagene di tipo I e III come fibronectina, laminina, ialuronano, elastina e proteoglicani. The origin of CAFs? still the subject of study and debate. Various intrahepatic cell types are believed to undergo phenotypic changes, differentiating into CAF during the inflammatory and progressive fibrogenic evolution of chronic liver disease, as well as during the development of HCC. In this microenvironment, CAFs can be phenotypically programmed by adjacent malignant cells and, in turn, enhance HCC cell proliferation and spread, possibly through the deposition or secretion of molecules, including extracellular matrix (ECM) proteins. . In fact, CAFs are primarily responsible for the deposition of ECMs, including fibrillar collagens such as type I and III noncollagen glycoproteins such as fibronectin, laminin, hyaluronan, elastin, and proteoglycans.
I proteoglicani (PG) costituiscono una classe di proteine ad alto peso molecolare fortemente glicosilate, ampiamente espresse in tutti i tessuti fibrotici, in particolare nella cartilagine, dove hanno una funzione di lubrificazione che consente lo scorrimento delle articolazioni. Numerosi studi attribuiscono ruoli ambigui a determinati PG nella progressione del cancro. Il biglicano solubile pu? interagire con diverse combinazioni di recettori di superficie multipli, inclusi i Toll Like Receptors (TLR) 2 e 4, CD14 e CD44, che a loro volta influenzano processi come l'autofagia, l'angiogenesi, la crescita e la migrazione cellulare e promuovono anche la progressione o la soppressione del tumore. Proteoglycans (PGs) are a class of highly glycosylated high molecular weight proteins, widely expressed in all fibrotic tissues, particularly in cartilage, where they have a lubricating function that allows the joints to slide. Numerous studies attribute ambiguous roles to certain PGs in cancer progression. The soluble biglycan can? interact with different combinations of multiple surface receptors, including Toll Like Receptors (TLR) 2 and 4, CD14 and CD44, which in turn influence processes such as autophagy, angiogenesis, cell growth and migration, and also promote tumor progression or suppression.
? stato osservato che lo ialuronano e il versicano (VCAN), attraverso il legame degli stessi recettori, sono in grado di promuovere la proliferazione delle cellule tumorali e la capacit? metastatica. ? It has been observed that hyaluronan and versican (VCAN), through the binding of the same receptors, are able to promote the proliferation of tumor cells and the ability? metastatic.
Alcuni PG, come biglycan e VCAN, sono sovraespressi dal fattore di crescita trasformante beta (TGF-?). Some PGs, such as biglycan and VCAN, are overexpressed by transforming growth factor beta (TGF-?).
? stato riportato che TGF-? promuove un fenotipo pi? invasivo e aggressivo nel HCC. ? It has been reported that TGF-? promotes a more phenotype? invasive and aggressive in HCC.
Uno studio clinico multicentrico ha dimostrato che l'inibitore della via del TGF-?, galunisertib, ? efficace in pazienti con HCC avanzato [Faivre S. et al. Liver Int.2019; Aug;39: 1468-147]. A multicenter clinical study demonstrated that the TGF-? pathway inhibitor, galunisertib, ? effective in patients with advanced HCC [Faivre S. et al. Liver Int.2019; Aug;39: 1468-147].
Inoltre, in modelli sperimentali preclinici, ? stato dimostrato che l'inibizione della segnalazione della TGF? riduce l'aggressivit? dell'HCC, riducendo l'espressione di CD44. Furthermore, in preclinical experimental models, ? been shown that inhibition of TGF signaling? reduce aggression of HCC, by reducing the expression of CD44.
La lubricina o Proteoglicano 4 (PRG4) ? una glicoproteina mucinosa secreta dai fibroblasti sinoviali e dai condrociti articolari posti in superficie, ? il principale componente lubrificante del liquido sinoviale. Lubricin or Proteoglycan 4 (PRG4) ? a mucinous glycoprotein secreted by synovial fibroblasts and articular chondrocytes located on the surface, ? the main lubricating component of synovial fluid.
La forma ricombinante di PRG4 umano ha una attivit? antinfiammatoria caratterizzata dalla sua capacit? di competere con lo ialuronano legandosi al recettore CD44 [Al-Sharif A. et al. Lubricin/proteoglycan 4 binding to CD44 receptor: a mechanism of lubricin?s suppression of proinflammatory cytokine induced synoviocyte proliferation. Arthritis Rheumatol. 2015;67: 1503?1513]. L'effetto a valle del coinvolgimento del CD44 da parte del PRG4 ? l'inibizione della traslocazione nucleare di NF?B indotta da IL-1? e TNF-? nei sinoviociti da pazienti con artrite reumatoide o osteoartrite [Alquraini A, et al. The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes. Arthritis Res Ther.2017; 19: 89]. Il ruolo antinfiammatorio autocrino del PRG4 sui fibroblasti sinoviali ? mediato dalla sua capacit? di inibire la degradazione dell'inibitore citosolico kappa B alfa (I?B-?) in modo CD44-dipendente. Relativamente ai TLR, il PRG4 umano ricombinante (rhPRG4) si lega e regola l'attivazione indotta dall'agonista di TLR2 e TLR4 [Iqbal SM, et al. Lubricin/proteoglycan 4 binds to and regulates the activity of toll-like receptors in vitro. Sci Rep. 2016; 6: 18910, Alquraini A, et al. The interaction of lubricin/proteoglycan-4 (PRG4) with toll-like receptors 2 and 4: an anti-inflammatory role of PRG4 in synovial fluid. Arthritis Res Ther.2015; 17: 353]. The recombinant form of human PRG4 has an activity anti-inflammatory characterized by its ability to compete with hyaluronan by binding to the CD44 receptor [Al-Sharif A. et al. Lubricin/proteoglycan 4 binding to CD44 receptor: a mechanism of lubricin?s suppression of proinflammatory cytokine induced synoviocyte proliferation. Arthritis Rheumatol. 2015;67:1503?1513]. The downstream effect of PRG4's involvement of CD44? the inhibition of IL-1-induced NF?B nuclear translocation? and TNF-? in synoviocytes from patients with rheumatoid arthritis or osteoarthritis [Alquraini A, et al. The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes. Arthritis Res Ther.2017; 19:89]. The autocrine anti-inflammatory role of PRG4 on synovial fibroblasts? mediated by his ability to inhibit the degradation of cytosolic inhibitor kappa B alpha (I?B-?) in a CD44-dependent manner. With respect to TLRs, recombinant human PRG4 (rhPRG4) binds to and regulates agonist-induced activation of TLR2 and TLR4 [Iqbal SM, et al. Lubricin/proteoglycan 4 binds to and regulates the activity of toll-like receptors in vitro. Ski Rep. 2016; 6:18910, Alquraini A, et al. The interaction of lubricin/proteoglycan-4 (PRG4) with toll-like receptors 2 and 4: an anti-inflammatory role of PRG4 in synovial fluid. Arthritis Res Ther.2015; 17:353].
Molecole ricombinanti di lubricina e loro usi come lubrificanti, agenti antiadesivi e/o integratori intra-articolari per, ad esempio, articolazioni sinoviali, menisco, tendini, peritoneo, pericardio e pleura sono descritti nella domanda internazionale WO 2005/016130. Recombinant lubricin molecules and their uses as lubricants, anti-adhesive agents and/or intra-articular supplements for, for example, synovial joints, meniscus, tendons, peritoneum, pericardium and pleura are disclosed in international application WO 2005/016130 .
Sommario dell'invenzione Summary of the Invention
In un primo aspetto l?invenzione fornisce l?associazione di sorafenib e/o regorafenib con rhPRG4 per uso medico. In a first aspect the invention provides the association of sorafenib and/or regorafenib with rhPRG4 for medical use.
In un secondo aspetto l?invenzione fornisce l?associazione di sorafenib e/o regorafenib con rhPRG4 per uso nel trattamento di epatocarcinoma e di metastasi epatiche. In a second aspect the invention provides the association of sorafenib and/or regorafenib with rhPRG4 for use in the treatment of hepatocellular carcinoma and liver metastases.
In un terzo aspetto l?invenzione fornisce la composizione che comprende tale associazione e il suo uso nel trattamento di epatocarcinoma e di metastasi epatiche. In a third aspect, the invention provides the composition comprising this combination and its use in the treatment of hepatocellular carcinoma and liver metastases.
Breve descrizione delle figure Brief description of the figures
Figura 1 - Grafico che mostra il livello di espressione di mRNA di quattro sequenze CD44-shRNA (da A a D), rispetto alla sequenza ControlshRNA (V) in cellule HLE e HLF dopo trasduzione lentivirale e selezione di puromicina per silenziamento stabile dell'espressione di CD44. Figure 1 - Graph showing mRNA expression level of four CD44-shRNA sequences (A to D), versus ControlshRNA sequence (V) in HLE and HLF cells after lentiviral transduction and puromycin selection for stable expression silencing of CD44.
Figura 2 - Grafico che mostra i risultati del test di adesione cellulare condotto su cellule delle linee HLE e HLF. Figure 2 - Graph showing the results of the cell adhesion test conducted on cells of the HLE and HLF lines.
Figura 3 - Grafico che mostra il numero di cellule presenti nel campo microscopico e relativa immagine fotografica nel saggio di migrazione condotto su cellule delle linee HLE e HLF. Figure 3 - Graph showing the number of cells present in the microscopic field and relative photographic image in the migration assay conducted on cells of the HLE and HLF lines.
Figura 4 - Grafici che mostrano il risultato dell?esperimento di proliferazione cellulare e dati di citofluorimetria a flusso su cellule di linee cellulari che esprimono o non esprimono CD44. Figure 4 - Graphs showing the result of the cell proliferation experiment and flow cytometry data on cells of cell lines expressing or not expressing CD44.
Figura 5 - Grafici che mostrano il dato di proliferazione cellulare e dati di citofluorimetria a flusso in cellule trattate con e senza sorafenib e regorafenib. Figure 5 - Graphs showing cell proliferation data and flow cytometry data in cells treated with and without sorafenib and regorafenib.
Figura 6 - Immagini di immunofluorescenza e grafici di citometria a flusso (esempio 6). Figure 6 - Immunofluorescence images and flow cytometry graphs (example 6).
Figura 7 - Grafico che mostra la percentuale di inibizione di crescita cellulare della linea HLF determinato da sorafenif e regorafenib in presenza ed in assenza di PRG4. Figure 7 - Graph showing the percentage of cell growth inhibition of the HLF line determined by sorafenif and regorafenib in the presence and in the absence of PRG4.
Figura 8 - Pannello 8A: Grafico che mostra la sopravvivenza percentuale dei pazienti in funzione del livello di espressione dell?mRNA di CSP4, HSPG2, PRG4 e VCAN. Pannello 8B: Analisi Western Blot della proteina PRG4. Pannello 8C: Immunofluorescenza su tessuti tumorali HCC che mostrano la localizzazione di PRG4 e aSMA. Figure 8 - Panel 8A: Graph showing the percentage survival of patients as a function of the expression level of CSP4, HSPG2, PRG4 and VCAN mRNA. Panel 8B: Western blot analysis of PRG4 protein. Panel 8C: Immunofluorescence on HCC tumor tissue showing PRG4 and aSMA localization.
Figura 9 - Analisi dell?espressione dell?mRNA dei geni aSMA, CSP4, HSPG2, PRG4 e VCAN in CAF e campioni di HCC ex vivo trattati con TGFb, l'inibitore TGFbRI, LY2157299 (galunisertib), o entrambi, per 48 ore. Figure 9 - Analysis of mRNA expression of aSMA, CSP4, HSPG2, PRG4, and VCAN genes in CAF and ex vivo HCC samples treated with TGFb, the TGFbRI inhibitor, LY2157299 (galunisertib), or both, for 48 hours.
Descrizione dettagliata dell'invenzione Detailed description of the invention
Scopo della presente invenzione ? fornire composizioni e metodi utili per il trattamento del cancro, in particolare del carcinoma epatocellulare. L'invenzione si basa sull'inattesa osservazione che la combinazione di rhPRG4 e sorafenib e/o regorafenib ? in grado di aumentare sinergicamente l'efficacia dei due farmaci antitumorali. Purpose of the present invention? to provide compositions and methods useful for the treatment of cancer, especially hepatocellular carcinoma. The invention is based on the unexpected observation that the combination of rhPRG4 and sorafenib and/or regorafenib ? able to synergistically increase the efficacy of the two anticancer drugs.
Tale sinergia ? stata validata dalla sperimentazione condotta e riportata in questa descrizione. Such synergy? been validated by the experimentation conducted and reported in this description.
Pertanto, in un primo aspetto l'invenzione riguarda la combinazione di sorafenib e/o regorafenib con rhPRG4 e il loro uso nel trattamento del carcinoma epatocellulare e di metastasi epatiche, derivate da tumore colorettale, cancro esofageo, tumore polmonare, melanoma, cancro pancreatico e dello stomaco e tumore al seno, o in forme di cancro resistenti al trattamento con sorafenib e/o regorafenib. Therefore, in a first aspect the invention relates to the combination of sorafenib and/or regorafenib with rhPRG4 and their use in the treatment of hepatocellular carcinoma and liver metastases, derived from colorectal cancer, oesophageal cancer, lung cancer, melanoma, pancreatic cancer and stomach and breast cancer, or in cancers resistant to treatment with sorafenib and/or regorafenib.
L?uso terapeutico della associazione secondo la presente invenzione, e delle composizioni che la comprendono, ? utile in particolare per il trattamento di quelle forme di epatocarcinoma che sono resistenti al trattamento con gli agenti terapeutici di prima linea sorafenib e/o regorafenib. The therapeutic use of the association according to the present invention, and of the compositions comprising it, is particularly useful for the treatment of those forms of hepatocellular carcinoma that are resistant to treatment with the first-line therapeutic agents sorafenib and/or regorafenib.
Un secondo aspetto riguarda le composizioni farmaceutiche comprendenti le nuove combinazioni terapeutiche di sorafenib e/o regorafenib con rhPRG4 ed il loro uso per il trattamento del carcinoma epatocellulare e di metastasi epatiche, derivate da tumore colorettale, cancro esofageo, tumore polmonare, melanoma, cancro pancreatico e dello stomaco e tumore al seno, in mammiferi, preferibilmente in pazienti umani, che ne hanno bisogno, o che sono affetti da forme di cancro resistent a sorafenib e/o regorafenib, A second aspect concerns the pharmaceutical compositions comprising the new therapeutic combinations of sorafenib and/or regorafenib with rhPRG4 and their use for the treatment of hepatocellular carcinoma and liver metastases, derived from colorectal cancer, oesophageal cancer, lung cancer, melanoma, pancreatic cancer and stomach and breast cancer, in mammals, preferably in human patients, who need it, or who have forms of cancer resistant to sorafenib and/or regorafenib,
e che possono vantaggiosamente avvalersi di un approccio di terapia di combinazione, cio? della somministrazione di principi attivi in associazione, La composizione secondo la presente invenzione ? un prodotto terapeutico comprendente una quantit? clinicamente efficace di sorafenib e/o regorafenib e una quantit? clinicamente efficace di rhPRG4 ed eccipienti farmaceuticamente accettabili. La composizione pu? ulteriormente comprendere agenti chemoterapici aggiuntivi. and that they can advantageously make use of a combination therapy approach, that is? of the administration of active ingredients in combination. The composition according to the present invention ? a therapeutic product comprising a quantity? clinically effective sorafenib and / or regorafenib and a quantity? clinically effective of rhPRG4 and pharmaceutically acceptable excipients. The composition can further include additional chemotherapeutic agents.
Come qui utilizzata, l'espressione "terapia di combinazione" o "somministrazione in combinazione" non ? limitata alla sola situazione in cui sorafenib e/o regorafenib e rhPRG4 sono formulati in un?unica forma di dosaggio, come ad esempio una compressa o una sospensione orale, sebbene queste forme di dosaggio offrano indubbi vantaggi in termini di convenienza del dosaggio, rispetto al protocollo terapeutico da parte del paziente e accuratezza della dose. L?invenzione prevede anche che sorafenib, regorafenib e rhPRG4 siano somministrati in rispettive unit? di dosaggio; tale modalit? di somministrazione permette al medico che prescrive il trattamento una maggiore possibilit? di modulazione e calibrazione del dosaggio secondo le necessit? del paziente. As used herein, the terms "combination therapy" or "combination administration" are not limited to the single situation where sorafenib and/or regorafenib and rhPRG4 are formulated in a single dosage form, such as a tablet or an oral suspension, although these dosage forms offer clear advantages in terms of convenience of dosage, compared to the treatment protocol by the patient and dose accuracy. The invention also provides that sorafenib, regorafenib and rhPRG4 are administered in respective units. of dosage; this mode? of administration allows the doctor who prescribes the treatment a greater possibility? of modulation and calibration of the dosage according to the needs? of the patient.
La quantit? e/o il programma di dosaggio di sorafenib possono seguire linee guida clinicamente approvate o sperimentali. In alcune forme di realizzazione la dose di sorafenib ? circa 200, 400, 600 o 800 mg/die, somministrato oralmente. The quantity? and/or the sorafenib dosing schedule may follow clinically approved or experimental guidelines. In some embodiments, the dose of sorafenib is ? about 200, 400, 600, or 800 mg/day, given orally.
La quantit? e/o il programma di dosaggio di regorafenib possono seguire linee guida clinicamente approvate o sperimentali. In alcune forme di realizzazione la dose di regorafenib ? circa 160, 320, 480 o 640 mg/die, somministrato oralmente. The quantity? and/or the regorafenib dosing schedule may follow clinically approved or experimental guidelines. In some embodiments, the dose of regorafenib is ? about 160, 320, 480, or 640 mg/day, given orally.
La composizione farmaceutica per uso nella terapia di combinazione secondo la presente invenzione utilizza la forma ricombinante della proteina PRG4 umana completa. Rientrano nell'ambito dell'invenzione anche le isoforme della proteina nativa umana, frammenti, regioni e domini della glicoproteina umana ricombinante o nativa, e sequenze peptidiche con varazioni aminoacidiche che mostrano un grado di omologia pari ad almeno l'80% rispetto alla sequenza aminoacidica della forma ricombinante o nativa della proteina PRG4 umana completa, o dei suoi frammenti, regioni e domini. The pharmaceutical composition for use in combination therapy according to the present invention utilizes the recombinant form of the complete human PRG4 protein. Also within the scope of the invention are native human protein isoforms, fragments, regions and domains of recombinant or native human glycoprotein, and peptide sequences with amino acid variations exhibiting a degree of homology of at least 80% to the amino acid sequence. of the recombinant or native form of the complete human PRG4 protein, or its fragments, regions and domains.
Nell?uso terapeutico secondo l'invenzione, la via di somministrazione per il rhPRG4 non ? limitata, sebbene la via di elezione ? quella comune per farmaci di natura proteica/peptidica ad alto peso molecolare (? 150 kDa) come gli anticorpi monoclonali umanizzati (come ad esempio bevacizumab, trastuzumab, ecc.), ossia quella intravenosa per perfusione. Tuttavia, possono essere impiegate anche altre modalit? di somministrazione differenti, quali ad esempio la somministrazione sottocutanea, endovenosa, intramidollare, intraarticolare, intramuscolare o intraperitoneale, alla stregua di forme di somministrazione approvate per alcuni anticorpi monoclonali nel trattamento di alcune neoplasie. La somministrazione pu? essere anche per via orale. In the therapeutic use according to the invention, the route of administration for rhPRG4 is not limited, although the way of election ? the common one for high molecular weight (? 150 kDa) protein/peptide drugs such as humanized monoclonal antibodies (such as for example bevacizumab, trastuzumab, etc.), i.e. the intravenous one for perfusion. However, other modalities can also be employed? different administration methods, such as for example subcutaneous, intravenous, intramedullary, intraarticular, intramuscular or intraperitoneal administration, as well as approved administration forms for some monoclonal antibodies in the treatment of some malignancies. The administration can also be orally.
In alcune forme realizzative il rhPRG4 ? somministrato ogni 2 o 3 settimane per via intravenosa secondo un dosaggio da 1 a 50 mg/kg, in regime di combinazione con sorafenib e/o regorafenib secondo le modalit? stabilite dal medico oncologo. In some embodiments the rhPRG4 ? administered every 2 or 3 weeks intravenously at a dosage of 1 to 50 mg/kg, in a combination regimen with sorafenib and/or regorafenib according to the modalities? established by the oncologist.
In alcune forme realizzative il trattamento ? somministrato quotidianamente per un periodo da 1 a 4 settimane consecutive, nella somministrazione del trattamento possono essere previsti periodi di interruzione: una settimana di somministrazione e una settimana libera (totale 14 giorni), due settimane di somministrazione e una settimana libera (totale 21 giorni), una settimana di somministrazione e due settimane libere (totale 21 giorni), tre settimane di somministrazione e una settimana libera (totale 28 giorni), una settimana di somministrazione e tre settimane libere (totale 28 giorni), due settimane di somministrazione e due settimane libere (totale 28 giorni). In some embodiments the treatment ? administered daily for a period of 1 to 4 consecutive weeks, periods of interruption may be foreseen in the administration of the treatment: one week of administration and one week off (total 14 days), two weeks of administration and one week off (total 21 days) , one week on and two weeks off (total 21 days), three weeks on and one week off (total 28 days), one week on and three weeks off (total 28 days), two weeks on and two weeks off free (total 28 days).
Quando non sono somministrati in un'unica forma di dosaggio sorafenib e/o regorafenib e rhPRG4 possono essere somministrati sostanzialmente simultaneamente o separatamente in tempi diversi a distanza di alcune ore nella stessa giornata o a giorni alterni. When not administered in a single dosage form sorafenib and/or regorafenib and rhPRG4 may be administered substantially simultaneously or separately at different times several hours apart on the same day or on alternate days.
Quando la somministrazione in combinazione di sorafenib e/o regorafenib e rhPRG4 ? sfalsata o alternata, pu? avvenire con cicli separati di sorafenib e/o regorafenib e di rhPRG4. Ad esempio, i cicli settimanali di sorafenib e/o regorafenib giornalieri possono essere intervallati da uno, due, tre, cinque o sette cicli di somministrazione giornaliera di rhPRG4. When the combination of sorafenib and/or regorafenib and rhPRG4? staggered or alternating, pu? occur with separate courses of sorafenib and/or regorafenib and rhPRG4. For example, weekly cycles of daily sorafenib and/or daily regorafenib may be interspersed with one, two, three, five, or seven cycles of daily rhPRG4 dosing.
Il dosaggio dei componenti della terapia di combinazione e la durata del trattamento secondo la presente invenzione sono adattati dal medico a seconda delle condizioni e della risposta del paziente. Generalmente, pazienti con basso peso corporeo, come soggetti giovani o geriatrici ricevono un dosaggio inferiore. The dosage of the components of the combination therapy and the duration of the treatment according to the present invention are adapted by the physician depending on the patient's condition and response. Generally, patients with low body weight, such as young or geriatric subjects receive a lower dose.
Le terapie di combinazione dell'invenzione non sono specificamente limitate ad alcun corso o regime particolare e possono essere impiegate separatamente o in combinazione con altre modalit? terapeutiche (ad es. chemioterapia o radioterapia). Allo stesso modo, la presente invenzione pu? essere utilizzata come terapia adiuvante (ad esempio, quando combinata con la chirurgia). In varie forme di realizzazione, il soggetto viene anche trattato mediante resezione chirurgica, etanolo percutaneo o iniezione di acido acetico, chemioembolizzazione transarteriosa, ablazione a radiofrequenza, ablazione laser, crioablazione, radioterapia stereotassica con radiazioni a fascio esterno focalizzato, radioterapia interna selettiva, iodio intra-arteriososomministrazione di lipiodolo e/o ultrasuoni focalizzati ad alta intensit?. The combination therapies of the invention are not specifically limited to any particular course or regimen and can be used separately or in combination with other modalities. therapeutics (e.g. chemotherapy or radiotherapy). Likewise, the present invention can be used as adjuvant therapy (for example, when combined with surgery). In various embodiments, the subject is also treated by surgical resection, percutaneous ethanol or acetic acid injection, transarterial chemoembolization, radiofrequency ablation, laser ablation, cryoablation, stereotactic radiotherapy with external focused beam radiation, selective internal radiotherapy, intraiodine -arterial administration of lipiodol and/or high intensity focused ultrasound.
La combinazione di sorafenib e/o regorafenib e rhPRG4 pu? essere utilizzata come terapia adiuvante, neoadiuvante, concomitante, o palliativa, come terapia di prima linea, terapia di seconda linea o terapia crossover. The combination of sorafenib and/or regorafenib and rhPRG4 can be used as adjuvant, neoadjuvant, concomitant, or palliative therapy, such as first-line therapy, second-line therapy, or crossover therapy.
In alcune forme realizzative il trattamento basato sulla somministrazione dell?associazione secondo l?invenzione pu? comprendere la somministrazione di almeno un ulteriore agente chemoterapico aggiuntivo. In some embodiments, the treatment based on the administration of the combination according to the invention can comprising the administration of at least one additional additional chemotherapeutic agent.
L?inaspettato sinergistico effetto antiproliferativo dimostrato dalla combinazione di rhPRG4 e sorafenenib e/o regorafenib pu? determinare nei pazienti di HCC un significativo benefico effetto terapeutico. I dati della sperimentazione condotta di seguito riportata dimostrano che la proteina CD44, proteina transmembrana, monomerica, altamente glicosilata la cui funzione ? quella di legare l'acido ialuronico ed altre glicoproteine della matrice extracellulare, ? necessaria perch? l?rhPRG4 sinergizzi con i due farmaci antitumorali per bloccare la proliferazione delle cellule HCC, come dimostrato dall?evidenza che le cellule con bassa o nessuna espressione di CD44 sono meno sensibili al PRG4. The unexpected synergistic antiproliferative effect demonstrated by the combination of rhPRG4 and sorafenib and/or regorafenib may determine a significant beneficial therapeutic effect in HCC patients. The data of the experimentation conducted below demonstrate that the CD44 protein, a highly glycosylated, monomeric, transmembrane protein whose function is ? that of binding hyaluronic acid and other glycoproteins of the extracellular matrix, ? necessary why? rhPRG4 synergizes with the two anticancer drugs to block HCC cell proliferation, as demonstrated by evidence that cells with low or no expression of CD44 are less sensitive to PRG4.
Altrettanto sorprendentemente ? stata trovata l?associazione tra il livello di espressione di PRG4 e la sopravvivenza dei pazienti di HCC, indicazione che la molecola pu? svolgere un?attivit?, diretta o indiretta, limitante il tumore. Equally surprisingly ? the association was found between the expression level of PRG4 and the survival of HCC patients, an indication that the molecule can perform an activity, direct or indirect, limiting the tumour.
L'invenzione sar? ora descritta attraverso degli esempi che dimostrano che sorafenib e/o regorafenib in combinazione con rhPRG4 hanno una maggiore efficacia nel trattamento del carcinoma epatocellulare. Will the invention be now described through examples demonstrating that sorafenib and/or regorafenib in combination with rhPRG4 have greater efficacy in the treatment of hepatocellular carcinoma.
ESEMPI EXAMPLES
Esempio 1 - Colture cellulari Example 1 - Cell culture
Le linee cellulari HLE e HLF sono state acquistate da JCRB Cell Bank (Giappone). Le linee cellulari Hep3B e PLC/PRF/5 sono state acquistate da ATCC (USA). Tutte le linee cellulari sono state coltivate in DMEM (Dulbecco's Modified Eagle Medium) integrato con sodio piruvato, antibiotico, antimicotico, Hepes e 10% siero bovino fetale (FSB) (Thermo Fisher Scientific). Le cellule sono state testate per l'assenza di contaminazione da micoplasma utilizzando il MycoFluor ? Mycoplasma Detection Kit (Thermo Fisher Scientific). HLE and HLF cell lines were purchased from JCRB Cell Bank (Japan). Hep3B and PLC/PRF/5 cell lines were purchased from ATCC (USA). All cell lines were grown in DMEM (Dulbecco's Modified Eagle Medium) supplemented with sodium pyruvate, antibiotic, antifungal, Hepes and 10% fetal bovine serum (FSB) (Thermo Fisher Scientific). Were the cells tested for the absence of mycoplasma contamination using MycoFluor ? Mycoplasma Detection Kit (Thermo Fisher Scientific).
Esempio 2 - Silenziamento stabile del gene CD44 mediante trasduzione cellulare Example 2 - Stable silencing of the CD44 gene by cellular transduction
Le linee cellulari HLE e HLF sono state trasdotte con particelle lentivirali che recano il controllo non-targeting (V) o specifiche sequenze shRNA (short hairpin RNA) per il CD44 (da A a D). Cellule che presentavano stabilmente il silenziamento del gene CD44 sono state selezionate con puromicina dicloridrato (Thermo Fisher Scientific), secondo le istruzioni del produttore (OriGene Technologies, Inc., Rockville, MD 20850, USA). La sequenza di controllo negativo (V) e la sequenza B di controllo positivo CD44-shRNA sono state utilizzate in tutti gli esperimenti. La figura 1 mostra l?efficienza del silenziamento, attraverso i dati dell?analisi qPCR, di quattro sequenze CD44-shRNA (da A a D), rispetto alla sequenza Control-shRNA (V) in cellule HLE e HLF dopo trasduzione lentivirale e selezione con puromicina per silenziamento stabile dell'espressione di CD44. L?espressione del gene GAPDH ? stata utilizzata come controllo quantitativo e i dati sono normalizzati rispetto al Control-shRNA. Esempio 3 - Saggio di adesione cellulare Per verificare se rhPRG4 si lega al CD44 ? stato eseguito un saggio di adesione cellulare sulle cellule delle linee cellulari HLE e HLF ottenute come descritto all'esempio 1 in seguito a silenziamento stabile dell'espressione genica del CD44 ottenuto come descritto nell'esempio 2. HLE and HLF cell lines were transduced with lentiviral particles bearing the non-targeting control (V) or specific CD44 short hairpin RNA (shRNA) sequences (A to D). Cells stably exhibiting CD44 gene silencing were selected with puromycin dihydrochloride (Thermo Fisher Scientific), according to the manufacturer's instructions (OriGene Technologies, Inc., Rockville, MD 20850, USA). The negative control sequence (V) and the CD44-shRNA positive control sequence B were used in all experiments. Figure 1 shows the silencing efficiency, using qPCR analysis data, of four CD44-shRNA sequences (A to D), compared to the Control-shRNA sequence (V) in HLE and HLF cells after lentiviral transduction and selection with puromycin for stable silencing of CD44 expression. The expression of the GAPDH gene ? was used as a quantitative control and the data are normalized against the Control-shRNA. Example 3 - Cell adhesion assay To verify if rhPRG4 binds to CD44 ? A cell adhesion assay was performed on the cells of the HLE and HLF cell lines obtained as described in Example 1 following stable silencing of the CD44 gene expression obtained as described in Example 2.
Il saggio ? stato eseguito come descritto. [Bergamini C. et al. Hepatology 2007; 46: 1801-9]. In breve, 5x 10<4 >cellule sono state diluite in 100 ?l di terreno DMEM senza siero (+ 0,5% BSA) e seminate su pozzetti di una piastra a 96 pozzetti in un caso rivestiti con rhPRG4 o fibronectina (FN) e in un altro caso non rivestiti; quindi incubati a 37 ?C, 5% CO2 per 30 minuti. A ciascuna piastra ? stato aggiunto un pari volume di PFA al 4% (pH 7,2 in PBS) e poi le piastre sono state poste immediatamente in agitazione per alcuni secondi per consentire la miscelazione. Trenta minuti dopo, il mezzo di coltura ? stato rimosso e le cellule aderenti sono state colorate con crystalviolet per 10 minuti. Dopo un abbondante lavaggio, le cellule sono state lasciate asciugare e, il giorno successivo, sono state solubilizzate con 100 ?l di SDS all'1% in acqua. L'assorbanza ? stata letta a 595 nm e proporzionalmente correlata al numero di cellule aderenti. I dati sono la media ? DS degli esperimenti in triplicato. The wise ? was performed as described. [Bergamini C. et al. Hepatology 2007; 46:1801-9]. Briefly, 5x 10<4> cells were diluted in 100 µl serum-free DMEM medium (+0.5% BSA) and seeded into wells of a 96-well plate in one case coated with rhPRG4 or fibronectin (FN) and in another case uncoated; then incubated at 37 ?C, 5% CO2 for 30 minutes. To each plate? An equal volume of 4% PFA (pH 7.2 in PBS) was added and then the plates were immediately stirred for a few seconds to allow for mixing. Thirty minutes later, the culture medium is was removed and adherent cells were stained with crystal violet for 10 min. After abundant washing, the cells were left to dry and, the following day, they were solubilized with 100 µl of 1% SDS in water. The absorbance? was read at 595 nm and proportionally correlated with the number of adherent cells. Is the data the average? SD of the triplicate experiments.
I risultati della sperimentazione dimostrano che le cellule in cui il gene CD44 ? stato silenziato hanno una ridotta capacit? di aderire alla superficie rivestita di rhPRG4 rispetto alle cellule di controllo e alla capacit? di adesione alla fibronectina (Figura 2). The results of the experiment show that the cells in which the CD44 gene? been silenced have a reduced capacity? to adhere to the rhPRG4-coated surface compared to control cells and the ability? adhesion to fibronectin (Figure 2).
Esempio 4 - Inibizione della migrazione in cellule HCC da parte di rhPRG4 solubile senza il coinvolgimento di CD44 Example 4 - Inhibition of migration into HCC cells by soluble rhPRG4 without the involvement of CD44
Il test ? stato eseguito come descritto [Rani B. et al. World J Gastroenterol 2014; 20: 4128-40]. In breve, 1,5 x10<4 >cellule sono state sospese in 200 ml di terreno DMEM privo di siero, contenente 0,5% di albumina di siero bovino (BSA), posti nella camera superiore del transwell, la cui membrana era stata precedentemente rivestita con fibronectina sulla superficie inferiore e lasciate migrare per 16 ore in presenza/assenza di rhPRG4 (25 ?g/ml) diluito in mezzo DMEM senza siero 0,5% BSA nella camera inferiore. Le cellule sono state quindi fissate con paraformaldeide e colorate con crystalviolet. Per ciascuna membrana sono stati fotografati cinque campi e contato il numero di cellule per campo. I dati sono la media ? DS di cinque campi microscopici scelti a caso. T-test (spaiato, a due code): * p <0,05; ** p <0,01; *** p <0,001. The test ? was performed as described [Rani B. et al. World J Gastroenterol 2014; 20:4128-40]. Briefly, 1.5 x10<4 > cells were suspended in 200 ml of serum-free DMEM medium, containing 0.5% bovine serum albumin (BSA), placed in the upper chamber of the transwell, whose membrane had been previously coated with fibronectin on the bottom surface and allowed to migrate for 16 hours in the presence/absence of rhPRG4 (25 µg/ml) diluted in serum-free DMEM medium 0.5% BSA in the bottom chamber. The cells were then fixed with paraformaldehyde and stained with crystal violet. For each membrane, five fields were photographed and the number of cells per field counted. Is the data the average? SD of five randomly selected microscopic fields. T-test (unpaired, two-tailed): *p < 0.05; ** p < 0.01; *** p < 0.001.
Il risultato dell'esperimento ha mostrato che il rhPRG4 solubile (25 ng/ml) compromette la migrazione delle cellule sulla fibronectina, ma il silenziamento del CD44 non influisce sulla motilit? cellulare (Figura 3). The result of the experiment showed that soluble rhPRG4 (25 ng/ml) impaired cell migration on fibronectin, but CD44 silencing did not affect cell motility. cell phone (Figure 3).
Esempio 5 - Proliferazione cellulare delle cellule con sorafenib e regorafenib in presenza/assenza di rhPRG4. Example 5 - Cell proliferation of cells with sorafenib and regorafenib in the presence/absence of rhPRG4.
Per indagare il ruolo biologico dell'interazione PRG4/CD44, cellule delle linee cellulari HCC CD44 positive HLE, HLF e le cellule Hep3B e PLC/PRF/5 HCC CD44 negative sono state trattate con sorafenib e regorafenib in presenza ed in assenza di rhPRG4. Tutte le cellule HCC sono state coltivate per 72 ore in presenza/assenza di rhPRG4 e sorafenib o regorafenib alla concentrazione di 2,5 ?M, valore inferiore del valore IC50 (circa 5 ?M) nelle stesse condizioni sperimentali. To investigate the biological role of PRG4/CD44 interaction, cells of CD44 positive HCC cell lines HLE, HLF and CD44 negative Hep3B and PLC/PRF/5 HCC cells were treated with sorafenib and regorafenib in the presence and absence of rhPRG4. All HCC cells were cultured for 72 hours in the presence/absence of rhPRG4 and sorafenib or regorafenib at a concentration of 2.5 µM, lower than the IC50 value (about 5 µM) under the same experimental conditions.
Il saggio di proliferazione cellulare ? stato eseguito come descritto in precedenza da Bergamini et al. In breve, in 100 ?l di terreno completo, 3x 10<3 >cellule sono state seminate nei pozzetti di una piastra da 96 pozzetti e lasciate per tutta la notte per permettere la completa adesione al substrato. Il giorno successivo (al tempo t = 0 ore), il terreno ? stato rimosso e sostituito con 100 ?l di terreno fresco in presenza/assenza di sorafenib (2,5 ?M), regorafenib (2,5 ?M), rhPRG4 (da 12,5 a 100 ?g/ml). Dimetilsolfossido (DMSO) e PBS 0,01% Tween-20 sono stati utilizzati rispettivamente come agenti veicolanti di sorafenib/regorafenib e rhPRG4. Le cellule in pozzetti in triplicato per ciascuna linea cellulare sono state fissate a t=0 con paraformaldeide al 4% (PFA, pH 7,6, 10 min di incubazione), poi colorate con crystal violet e lavate accuratamente per rimuovere la colorazione in eccesso. Dopo 72 ore, le cellule sono state fissate aggiungendo 100 ?l di PFA al 4% direttamente al mezzo (concentrazione finale di PFA al 2%, 20 min di incubazione e colorate. Poi, 100 o 200 ?l di 1% SDS sono stati aggiunti ai pozzetti e le piastre sono state poste in agitazione fino al completo rilascio di colorante dalle cellule. La densit? ottica ? stata misurata alla lunghezza d'onda di 595 nm. I dati sono la media ? DS di esperimenti condotti in triplicato normalizzati. T-test (spaiato, a due code): * p <0,05; ** p <0,01; *** p <0,001. The cell proliferation assay ? was performed as previously described by Bergamini et al. Briefly, in 100 µl of complete medium, 3x 10<3> cells were seeded into wells of a 96-well plate and left overnight to allow for complete adhesion to the substrate. The next day (at time t = 0 hours), the soil ? was removed and replaced with 100 µl of fresh medium in the presence/absence of sorafenib (2.5 µM), regorafenib (2.5 µM), rhPRG4 (12.5 to 100 µg/ml). Dimethyl sulfoxide (DMSO) and PBS 0.01% Tween-20 were used as the carriers of sorafenib/regorafenib and rhPRG4, respectively. Cells in triplicate wells for each cell line were fixed at t=0 with 4% paraformaldehyde (PFA, pH 7.6, 10 min incubation), then stained with crystal violet and washed thoroughly to remove excess staining. After 72 hours, cells were fixed by adding 100 µl of 4% PFA directly to medium (final concentration of 2% PFA), 20 min incubation and stained. Then, 100 or 200 µl of 1% SDS was were added to the wells and the plates were stirred until complete release of dye from the cells.The optical density was measured at a wavelength of 595 nm.The data are the mean ± SD of normalized triplicate experiments. T-test (unpaired, two-tailed): * p < 0.05; ** p < 0.01; *** p < 0.001.
Il risultato dell'esperimento ha mostrato che il rhPRG4 da solo non altera notevolmente la proliferazione cellulare ma, quando accoppiato con sorafenib o regorafenib, migliora, a concentrazioni comprese tra 12,5 e 100 ?g/ml, fortemente e sinergicamente la loro efficacia sulle cellule HLE e HLF. L'effetto sinergico dell'associazione rhPRG4-farmaco antitumorale ? stato osservato anche sulla proliferazione di cellule Hep3B, anche se in misura inferiore a quello ottenuto con le cellule HLE, HLF, ma solo alle concentrazioni pi? elevate di rhPRG4. Invece, le celle PLC/PRF/5 non hanno risposto (Figura. 4). The result of the experiment showed that rhPRG4 alone does not significantly alter cell proliferation but, when coupled with sorafenib or regorafenib, at concentrations between 12.5 and 100 ?g/ml, it strongly and synergistically improves their efficacy on HLE and HLF cells. The synergistic effect of the rhPRG4-antitumor drug association? was also observed on the proliferation of Hep3B cells, although to a lesser extent than that obtained with HLE, HLF cells, but only at the lowest concentrations? elevated rhPRG4. Instead, the PLC/PRF/5 cells did not respond (Figure 4).
Per verificare la correlazione tra CD44 e PRG4 e il ruolo di tale eventuale interazione, il saggio di proliferazione di 72 ore in presenza/assenza di rhPRG4 (a concentrazioni comprese tra 0 e 100 mg/ml), senza o con, sorafenib o regorafenib (alla concentrazione fissa di 2,5 mM) ? stato condotto sulle cellule HCC di controllo-shRNA e CD44-shRNA. La capacit? di entrambi i farmaci di inibire la proliferazione cellulare ? stata significativamente ridotta nelle cellule CD44 silenziate (da p <0,05 a p <0,001 a seconda della concentrazione di rhPRG4), rispetto alle cellule di controllo, dimostrando la necessit? che le cellule HCC esprimano CD44 per consentire il legame del PRG4 con la membrana e la successiva diminuzione della proliferazione delle cellule HCC in vitro, indotta da sorafenib e regorafenib (Figura 5). Questa diminuzione dell?efficacia dei due farmaci nelle cellule con CD44 silenziato esposte a rhPRG4 ? coerente con la ridotta sensibilit? al rhPRG4 delle linee cellulari HCC PLC/PRF/5 e Hep38 a basso contenuto di CD44. La diminuita efficacia del farmaco che si verifica nelle cellule con CD44 silenziato esposte a rhPRG4 ? coerente con la ridotta sensibilit? al rhPRG4 delle linee cellulari PLC/PRF/5 a bassa espressione di CD44 esposte al farmaco come evidente dalla Figura 4. I dati sono la media ? DS di esperimenti condotti in triplicato normalizzati. T-test (spaiato, a due code): * p <0,05; ** p <0,01; *** p <0,001. To verify the correlation between CD44 and PRG4 and the role of this possible interaction, the 72-hour proliferation assay in the presence/absence of rhPRG4 (at concentrations between 0 and 100 mg/ml), without or with, sorafenib or regorafenib ( at the fixed concentration of 2.5 mM) ? was conducted on control-shRNA and CD44-shRNA HCC cells. The capacity? of both drugs to inhibit cell proliferation ? was significantly reduced in silenced CD44 cells (p < 0.05 to p < 0.001 depending on rhPRG4 concentration), compared to control cells, demonstrating the need for that HCC cells express CD44 to allow membrane binding of PRG4 and subsequent decrease of HCC cell proliferation in vitro, induced by sorafenib and regorafenib (Figure 5). This decreased efficacy of the two drugs in CD44-silenced cells exposed to rhPRG4? consistent with the reduced sensitivity? to rhPRG4 of HCC PLC/PRF/5 and Hep38 cell lines with low CD44 content. The decreased drug efficacy that occurs in CD44-silenced cells exposed to rhPRG4? consistent with the reduced sensitivity? to the rhPRG4 of PLC/PRF/5 cell lines with low expression of CD44 exposed to the drug as evident from Figure 4. The data are the mean ? SD of experiments performed in triplicate normalized. T-test (unpaired, two-tailed): *p < 0.05; ** p < 0.01; *** p < 0.001.
Esempio 6 - Effetto di PRG4 secreto in un mezzo di coltura condizionato di CAF stimolati con TGF. Example 6 - Effect of PRG4 secreted in a conditioned culture medium of TGF-stimulated CAFs.
Per confermare ulteriormente il ruolo del PRG4 ? stata riprodotta in vitro la cascata biologica che rispecchia una situazione pi? fisiologica. Fibroblasti associati a tumore (CAF, Cancer Associated Fibroblast) sono stati isolati come descritto in precedenza [Mazzocca et al. Hepatology 2011; 54: 920-30]. In breve, subito dopo la resezione chirurgica, i campioni di tumore HCC e peritumorali sono stati ridotti in pezzi di 0,5-1 cm e lasciati nella MACS Tissue Storage Solution (Miltenyi Biotec). I tessuti sono stati quindi ulteriormente tagliati in pezzi di dimensioni pi? piccole (1-2 mm), lavati tre volte in soluzione salina bilanciata Hanks (HBSS) e quindi incubati in HBSS in presenza di collagenasi di tipo IV (Thermo Fisher Scientific) e 3 mM CaCl2 a 37 ?C sotto leggera rotazione per 4 ore. Al termine di questa fase, la dissociazione ? stata facilitata meccanicamente pipettando i tessuti digeriti con una pipetta da 50 ml con orifizio di grandi dimensioni. Le cellule galleggianti sono state raccolte e lavate tre volte con HBSS e seminate in condizioni di coltura normali in IMDM 20% FBS. I campioni di tessuto decantato e parzialmente digerito sono stati sottoposti a un secondo ciclo di digestione con collagenasi. Le cellule dissociate risultanti sono state lavate con HBSS e coltivate in IMDM 20% FBS. Queste cellule hanno subito pochi (<5) passaggi per consentire la perdita di cellule epiteliali, cellule immunitarie e di cellule non aderenti. Per garantire la purezza dei preparati di CAF, sono state eseguite analisi di immunofluorescenza e citometria a flusso per valutare l'espressione dei marcatori mesenchimali (vimentina, aSMA, CD90). Cellule non fibroblastiche contaminanti (per lo pi? epatociti cancerosi, colangiociti o macrofagi), quando presenti, erano in quantit? molto minime e sono state valutate utilizzando anticorpi contro EpCAM, CD133, CD45, OV-6, CK19 e CD11b (Figura 6). To further confirm the role of PRG4? been reproduced in vitro biological cascade that reflects a situation more? physiological. Cancer associated fibroblasts (CAFs) were isolated as previously described [Mazzocca et al. Hepatology 2011; 54:920-30]. Briefly, immediately after surgical resection, HCC and peritumoral tumor specimens were cut into 0.5-1 cm pieces and left in MACS Tissue Storage Solution (Miltenyi Biotec). The fabrics were then further cut into smaller sized pieces. small (1-2 mm), washed three times in Hanks balanced salt solution (HBSS) and then incubated in HBSS in the presence of collagenase type IV (Thermo Fisher Scientific) and 3 mM CaCl2 at 37 ?C under gentle rotation for 4 hours . At the end of this phase, the dissociation ? was mechanically facilitated by pipetting the digested tissues with a 50 mL large-orifice pipette. The floating cells were harvested and washed three times with HBSS and seeded under normal culture conditions in IMDM 20% FBS. The decanted and partially digested tissue samples were subjected to a second round of collagenase digestion. The resulting dissociated cells were washed with HBSS and cultured in IMDM 20% FBS. These cells have undergone few (<5) passages to allow for loss of epithelial cells, immune cells, and non-adherent cells. To ensure the purity of the CAF preparations, immunofluorescence and flow cytometry analyzes were performed to evaluate the expression of mesenchymal markers (vimentin, aSMA, CD90). Contaminant non-fibroblastic cells (mostly cancerous hepatocytes, cholangiocytes or macrophages), when present, were in large quantities. very minimal and were evaluated using antibodies against EpCAM, CD133, CD45, OV-6, CK19 and CD11b (Figure 6).
I CAF sono stati trattati per 48 ore in presenza ed in assenza di fattore di crescita trasformante beta 1 (Transforming Growth Factor beta 1 o TGF?1, di seguito riportato anche come TGF) (Peprotech) alla concentrazione finale di 5 ng/ml in mezzo IMDM completo (+ 20% FBS), quindi lavati tre volte con terreno privo di siero e incubati in terreno privo di siero per altre 48 ore per l'arricchimento dell'insieme delle proteine secrete. Il terreno condizionato ? stato quindi raccolto, concentrato utilizzando un dispositivo centricon (cut-off 3 kDa, Merck-Millipore) e incubato con microbiglie magnetiche pre-lavate in PBS anti-PRG4 o isotipo (anti-pY397 FAK), secondo le istruzioni del produttore (SureBeads Protein B, Bio-Rad). Il terreno CAF condizionato impoverito (controllo) o non impoverito di PRG4 ? stato quindi analizzato per la concentrazione di proteine e utilizzato per investigare se le cellule HCC mantengono la capacit? di proliferare in presenza di 1,5 ?M di sorafenib e regorafenib e/o 20 ?g/ml (concentrazione di proteine totali) di terreno condizionato da CAF stimolati con TGF, impoverito o non impoverito di PRG4 da TGF. Il saggio ha mostrato che il mezzo condizionato da CAF stimolati da TGFb ha significativamente (p <0,01) aumentato l'efficacia di sorafenib e regorafenib sulle cellule HLF, rispetto al mezzo condizionato impoverito di PRG4 (Figura 7). Questa ? una chiara indicazione che l?interazione CD44/PRG4 aumenta l'efficacia di sorafenib e regorafenib. The CAFs were treated for 48 hours in the presence and absence of transforming growth factor beta 1 (Transforming Growth Factor beta 1 or TGF?1, hereinafter also referred to as TGF) (Peprotech) at the final concentration of 5 ng/ml in complete IMDM medium (+ 20% FBS), then washed three times with serum-free medium and incubated in serum-free medium for another 48 hours for enrichment of the secreted protein pool. The conditioned ground? was then collected, concentrated using a centricon device (3 kDa cut-off, Merck-Millipore), and incubated with pre-washed magnetic beads in PBS anti-PRG4 or isotype (anti-pY397 FAK), according to the manufacturer's instructions (SureBeads Protein B, Bio-Rad). Is the conditioned CAF soil depleted (control) or not depleted of PRG4? was then analyzed for protein concentration and used to investigate whether HCC cells retain the ability to proliferate in the presence of 1.5 µM of sorafenib and regorafenib and/or 20 µg/ml (total protein concentration) of TGF-stimulated CAF-conditioned medium, depleted or non-depleted of PRG4 by TGF. The assay showed that TGFb-stimulated CAF conditioned medium significantly (p < 0.01) increased the efficacy of sorafenib and regorafenib on HLF cells, compared with PRG4-depleted conditioned medium (Figure 7). This ? a clear indication that the CD44/PRG4 interaction increases the efficacy of sorafenib and regorafenib.
Esempio 7 - L'espressione di PRG4 nei tessuti tumorali HCC correla positivamente con la sopravvivenza globale. Example 7 - PRG4 expression in HCC tumor tissues correlates positively with overall survival.
Mediante analisi dell'espressione genica con microarray sono stati determinati i livelli di espressione dell?mRNA di diversi proteoglicani (PG), tra cui condroitin solfato proteoglicano 4 (CSPG4), perlecano (HSPG2), versicano (VCAN) e PRG4 nei tessuti tumorali e peritumorali accoppiati da un gruppo di 78 pazienti con HCC prospetticamente reclutati. The mRNA expression levels of several proteoglycans (PGs), including chondroitin sulfate proteoglycan 4 (CSPG4), perlecan (HSPG2), versican (VCAN), and PRG4 in tumor tissues were determined by microarray gene expression analysis. matched peritumorals from a group of 78 prospectively recruited HCC patients.
All'arruolamento nello studio i pazienti hanno espresso in forma scritta il loro consenso allo studio; lo studio ? stato approvato dal Comitato Etico Locale, Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari (Bari, Italia); numero di protocollo: 254; data di rilascio: febbraio 2012. Upon enrollment in the study, the patients expressed their consent to the study in writing; I study ? been approved by the Local Ethics Committee, University Hospital Consortium Policlinico di Bari (Bari, Italy); protocol number: 254; release date: February 2012.
L'analisi microarray ? stata eseguita come descritto in precedenza [Villa E. et al Gut 2016; 65: 861-9]. In breve, l'RNA totale ? stato isolato dai tessuti epatici non tumorali (NT) e tumorali (T) ottenuti in un gruppo di pazienti arruolati prospetticamente alla prima identificazione di HCC, utilizzando Trizol (lnvitrogen, Carlsbad, CA, USA). L'RNA ? stato processato utilizzando Whole Human Genome Microarray Kit, 4x44k (Agilent Technologies, Palo Alto, CA). Le procedure di marcatura e ibridazione sono state eseguite secondo le istruzioni fornite da Agilent, utilizzando il Quick Amp Labelling Kit e il protocollo di analisi dell'espressione genica basata su microarray a un colore. Dopo la conversione dell'RNA in cDNA, questo ? stato convertito in cRNA e marcato con Cy3-CTP. Una volta purificato, il cRNA ? stato ibridato con Agilent Whole Human Genome Oligo Microarrays 4x44K. Dopo la quantificazione del segnale e la normalizzazione dei risultati utilizzando un metodo lineare a bassa intensit?, i dati sono stati importati nel software Resolver (Rosetta Biosoftware, Kirkland, WA) per la gestione dei dati, il controllo di qualit? e l'analisi. I dati sull'espressione genica sono disponibili sul sito web Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) con il numero di accesso: GSE54236. Microarray analysis ? was performed as previously described [Villa E. et al Gut 2016; 65:861-9]. In short, total RNA ? was isolated from non-tumor (NT) and tumor (T) liver tissues obtained in a group of patients prospectively enrolled at the first identification of HCC, using Trizol (lnvitrogen, Carlsbad, CA, USA). The RNA? was processed using Whole Human Genome Microarray Kit, 4x44k (Agilent Technologies, Palo Alto, CA). Labeling and hybridization procedures were performed according to Agilent-supplied instructions, using the Quick Amp Labeling Kit and one-color microarray-based gene expression analysis protocol. After conversion of RNA to cDNA, this ? converted to cRNA and labeled with Cy3-CTP. Once purified, the cRNA ? been hybridized with Agilent Whole Human Genome Oligo Microarrays 4x44K. After quantification of the signal and normalization of the results using a low-intensity linear method, the data were imported into Resolver software (Rosetta Biosoftware, Kirkland, WA) for data management, quality control and analysis. and the analysis. Gene expression data is available on the Gene Expression Omnibus website (www.ncbi.nlm.nih.gov/geo) under accession number: GSE54236.
L'analisi statistica ? stata condotta secondo il metodo Kaplan-Meier per stimare la probabilit? cumulativa della sopravvivenza globale dei pazienti con HCC. Il T-test ? stato utilizzato per l'analisi statistica della qPCR e gli esperimenti di colture cellulari in vitro. Statistical analysis? was conducted according to the Kaplan-Meier method to estimate the probability? cumulative overall survival of patients with HCC. The T-test ? been used for statistical analysis of qPCR and in vitro cell culture experiments.
I valori di mRNA di qualsiasi gene PG nei tessuti peritumorali sono stati sottratti dai valori nei campioni tumorali associati della controparte per ottenere indici di espressione genica del tumore netti normalizzati. I pazienti sono stati stratificati in base ai livelli di questi indici al di sopra o al di sotto dei valori mediani. Sorprendentemente, ? stato trovato che i pazienti con livelli di espressione pi? elevati di PRG4 sopravvivevano pi? a lungo, come mostrato dall'analisi di Kaplan-Meier, rispetto a quelli con livelli di espressione pi? bassi (p = 0.000), mentre CSPG4, HSPG2 e VCAN non hanno mostrato alcuna correlazione significativa con la sopravvivenza (Figura 8A). La presenza della proteina PRG4 nei campioni di due fegati normali e in 14 tumori e campioni non cancerosi circostanti accoppiati da 14 pazienti con HCC ? stata determinata mediante la tecnica di Western blot, nota agli esperti del settore, i cui risultati sono mostrati in Figura 8B The mRNA values of any PG gene in the peritumoral tissues were subtracted from the values in the counterpart tumor associated samples to obtain normalized net tumor gene expression indices. Patients were stratified according to the levels of these indices above or below the median values. Surprisingly, ? was found that patients with expression levels pi? high PRG4 survived pi? long, as shown by Kaplan-Meier analysis, compared to those with lower expression levels? low (p = 0.000), while CSPG4, HSPG2 and VCAN showed no significant correlation with survival (Figure 8A). The presence of PRG4 protein in samples from two normal livers and in 14 tumor and surrounding non-cancerous paired samples from 14 HCC patients ? was determined by the Western blot technique, known to those skilled in the art, the results of which are shown in Figure 8B
La sub-localizzazione tissutale di PRG4 nel tumore HCC, la sua espressione ? stata visualizzata mediante immunofluorescenza (Figura 8C). Il PRG4 ? distribuito principalmente nel compartimento stromale del tumore, essendo per lo pi? rilevabile in prossimit? di cellule muscolari lisce alfa positive all'actina (aSMA+). Meno frequentemente, il PRG4 si localizza nelle aree parenchimali negative all'aSMA. Il PRG4 ? prodotto prevalentemente in aree di intensa attivit? secretoria dei miofibroblasti. The tissue sublocalization of PRG4 in HCC tumor, its expression ? was visualized by immunofluorescence (Figure 8C). The PRG4? mainly distributed in the stromal compartment of the tumor, being mostly? detectable in the vicinity? of actin-positive alpha smooth muscle cells (aSMA+). Less frequently, PRG4 localizes to aSMA-negative parenchymal areas. The PRG4? produced mainly in areas of intense activity? myofibroblast secretory.
Esempio 8 - Espressione del PRG4 in CAF di HCC in seguito a stimolazione con TGF Example 8 - PRG4 expression in CAF of HCC following stimulation with TGF
Il coinvolgimento delle cellule stromali nel determinare l'espressione di PRG4 in HCC, ? stato verificato analizzando l'espressione dell?mRNA di PRG4 in CAF e campioni di HCC ex vivo trattati con TGFb, l'inibitore TGFbRI, LY2157299 (galunisertib), o entrambi, per 48 ore (Figura 9). Il risultato dell'analisi ha mostrato che TGFb ha migliorato significativamente l'espressione di PRG4 sia nei CAF che nei campioni di HCC (p <0,05), mentre LY2157299 ha compensato questo effetto (p <0,05). ? interessante notare che LY2157299 ha anche sottoregolato l'espressione di PRG4 in campioni ex vivo rispetto al controllo. TGFb1 ha anche aumentato significativamente i livelli di espressione di HSPG2 e VCAN nei tessuti coltivati di HCC (p <0,05) e VCAN in CAF (p <0,01), ma non CSPG4 nei CAF e nei campioni ex vivo, n? HSPG2 nei CAF (Figura 9). The involvement of stromal cells in determining PRG4 expression in HCC, ? was verified by analyzing PRG4 mRNA expression in CAF and ex vivo HCC samples treated with TGFb, the TGFbRI inhibitor, LY2157299 (galunisertib), or both, for 48 hours (Figure 9). The result of the analysis showed that TGFb significantly enhanced PRG4 expression in both CAF and HCC samples (p < 0.05), while LY2157299 compensated for this effect (p < 0.05). ? Interestingly, LY2157299 also downregulated PRG4 expression in ex vivo samples compared to control. TGFb1 also significantly increased the expression levels of HSPG2 and VCAN in cultured tissues of HCC (p < 0.05) and VCAN in CAF (p < 0.01), but not CSPG4 in CAF and ex vivo samples, n? HSPG2 in CAFs (Figure 9).
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