Breast cancer (BC) is the most common cancer among women worldwide. New medical treatments to face the side effects and acquired resistance of the traditional ones are needed. Many studies showed that the use of a combination of several therapeutic methods gives better results than using separate therapies. In this context, NaAsO2, sodium meta-arsenite, is promising as adjuvant treatment for solid and liquid tumours thanks to its eligibility as an anti-cancer agent. NaAsO2 is a trivalent arsenic-based compound under investigation because of its ability to inhibit cell growth. Human epidermal growth factor receptor 2 positive (HER2+) breast cancer comprises around 20-30% of all BC and is correlated with poor prognosis. Trastuzumab (Tz), a monoclonal antibody, has been used to inhibit HER2 activity, but the developing of resistance mechanisms to this drug leads the research to new therapies to use in synergy with Tz. Starting from these considerations, NaAsO2 was tested alone or in combination with Tz, at increasing doses and incubation times, for its ability of killing cancer cell line SKBR-3, overexpressing HER2 receptors, in comparison to a non-neoplastic cell line, the murine fibroblasts cell line, NIH-3T3. To study the synergic activity of the compounds, three types of experiments were performed: Exp. 1: cells pre-treated with NaAsO2 before adding Tz Exp. 2: cells pre-treated with Tz before adding NaAsO2 Exp. 3: cells co-incubated with the two agents The three conditions were tested to evaluate the time and dose dependence cytotoxicity trend by quantitative cell viability (MTT) assay. Both experiments 1 and 2 demonstrated that after the pre-treatment of cells both with NaAsO2 (exp.1) or Tz (exp.2), the cells resulted more sensitive to the subsequent treatment. In particular, the reduction of cell viability was respectively of 80-90% and around 40 % in exp. 1 and 2, respectively. Exp. 3 showed that the co-incubation led to a greater reduce, nearly 90%, of cell viability compared to the agent’s sole exposure. In all the conditions tested emerged tolerance by the healthy cells NIH-3T3, proving a specific effect towards HER2-cancer cells. At the light of these results, the experiments were focused on the co-incubation condition. Qualitative cell viability (LIVE/DEAD) assay and western blot (WB) analysis were performed. In line with MTT data, LIVE/DEAD analysis confirmed the efficacy of co-incubation in comparison with the use of NaAsO2 or Tz alone. Correspondingly, WB showed that the co-incubation triggered cell death with respect to the single treatments with a significant alteration of the activity and expression of survival or apoptosis proteins, respectively. Indeed, co-incubation determined the downregulation of survival (P-AKT/AKT) and proliferation (P-ERK/ERK) pathways, an increase of pro-apoptotic protein BAX and a decrease of the anti-apoptotic protein BCL-2, proving a concomitant action of the two anticancer agents. Although NaAsO2 action mechanism is still unclear, it is maybe related to NaAsO2 affinity for sulfhydryl groups reducing cysteines in proteins. To gain more insights on NaAsO2 and Tz synergism, Tz was incubated for 1 and 24 h with NaAsO2 both at room temperature and 37°C, and then analysed by SDS-PAGE and circular dichroism (CD). SDS-PAGE showed no fragmentation of Tz after incubation with NaAsO2 at both incubation time and CD revealed no significant modification of Tz secondary structure after NaAsO2 incubation. Other experiments are required to elucidate this synergism: for example, NaAsO2 internalization study and HER2 receptors expression analysis on the surface of BC cells, before and after the co-incubation treatment. Despite several studies are still needed, overall from these preliminary data emerge that the co-incubation exerts a toxicity effect greater towards cancer cells than agent’s sole treatment, that led to hypothesise a new approach in the BC treatment scenario.
Il tumore al seno è il tumore femminile più frequente al mondo. Sono necessari nuovi trattamenti per contrastare gli effetti collaterali e la resistenza dati da quelli tradizionali. Molti studi dimostrano come l’utilizzo di una combinazione di metodi terapeutici dia risultati migliori rispetto alle terapie utilizzate singolarmente. Il NaAsO2, Sodio meta Arsenito, è considerato un adiuvante per il trattamento di tumori solidi e liquidi grazie alla sua azione come agente antitumorale, esso è un composto trivalente a base di arsenico studiato per la sua capacità di inibire la crescita cellulare. Il tumore al seno positivo per il recettore umano per il fattore epidermico 2 (HER2) costituisce il 30% di tutti i tumori al seno ed è associato a una cattiva prognosi. Trastuzumab (Tz), un anticorpo monoclonale, è stato utilizzato per inibire l’attività di HER2 ma lo sviluppo di meccanismi di resistenza al farmaco ha fatto sì che la ricerca s’indirizzasse verso nuove terapie da usare in sinergia con Tz. Partendo da queste considerazioni, il NaAsO2 è stato testato da solo o in combinazione con Tz, a concentrazioni e tempistiche crescenti, per verificarne la tossicità sulle cellule tumorali SKBR-3, che over esprimono il recettore HER2, in comparazione con una linea di controllo non neoplastica, la linea murina di fibroblasti NIH-3T3. Per determinare l’attività sinergica sono stati fatti 3 tipi di esperimenti: Esp. 1 Cellule pretrattate con NaAsO2 prima dell’aggiunta di Tz Esp. 2 Cellule pretrattate con Tz prima dell’aggiunta del NaAsO2 Esp. 3 Cellule co incubate con entrambi gli agenti Le tre condizioni sono state testate per valutare la citotossicità tempo e dose dipendente, tramite MTT assay. Gli esp. 1 e 2 hanno dimostrato che, dopo il pretrattamento con NaAsO2 (Esp.1) o Tz (Esp.2), le cellule diventano più sensibili al trattamento successivo. Si ha una riduzione della vitalità del 90% nell’esp. 1 e del 40% nell’esp.2. L’esp. 3 ha mostrato che la coincubazione produce una maggiore riduzione della vitalità (90%) rispetto all’esposizione ai singoli agenti. In tutte le condizioni emerge la tolleranza della linea NIH-3T3, provando un effetto specifico diretto contro le cellule SKBR-3. Alla luce di ciò, ci si è focalizzati sulla condizione di co incubazione. Sono stati fatti studi di vitalità cellulare (LIVE/DEAD) e Western Blot (WB). In linea con i dati dell’MTT, il LIVE/DEAD ha confermato l’efficacia della co incubazione rispetto all’uso del NaAsO2 o del Tz da soli. Il WB ha dimostrato che la co incubazione è in grado di innescare la morte cellulare rispetto ai singoli trattamenti, alterando l’attività e l’espressione delle proteine coinvolte in sopravvivenza e apoptosi. Infatti, la co incubazione ha determinato una downregolazione dei pathways di sopravvivenza (P-AKT/AKT) e proliferazione (P-ERK/ERK), un incremento della proteina pro apopotica (BAX) e una diminuzione della proteina anti apoptotica (BCL-2), confermando un’azione combinata dei due agenti. Sebbene il meccanismo d’azione del sale sia ancora incerto, esso è probabilmente legato alla sua affinità per i gruppi solfidrilici e alla riduzione delle cisteine nelle proteine. Per indagare questa sinergia, Tz e NaAsO2 sono stati incubati per 1 e 24 h, a temperatura ambiente e a 37°C, e poi analizzati tramite SDS PAGE e dicroismo circolare (DC). L’SDS PAGE non ha riportato frammentazioni di Tz dopo l’incubazione con il sale e il DC non ha rilevato modificazioni della struttura secondaria dell’anticorpo. Sono necessari altri esperimenti per chiarire questa sinergia: studi di internalizzazione del sale o sull’espressione dei recettori HER2 sulla superficie cellulare, prima e dopo il trattamento. Sebbene siano indispensabili altri studi, da questi dati emerge che la coincubazione ha un effetto tossico maggiore rispetto ai singoli composti, ciò permette di ipotizzare un nuovo approccio nel trattamento del cancro al seno
Co-incubazione di Trastuzumab e Sodio Meta Arsenito nel trattamento del tumore al seno HER2+
ZANINI, CHIARA
2018/2019
Abstract
Breast cancer (BC) is the most common cancer among women worldwide. New medical treatments to face the side effects and acquired resistance of the traditional ones are needed. Many studies showed that the use of a combination of several therapeutic methods gives better results than using separate therapies. In this context, NaAsO2, sodium meta-arsenite, is promising as adjuvant treatment for solid and liquid tumours thanks to its eligibility as an anti-cancer agent. NaAsO2 is a trivalent arsenic-based compound under investigation because of its ability to inhibit cell growth. Human epidermal growth factor receptor 2 positive (HER2+) breast cancer comprises around 20-30% of all BC and is correlated with poor prognosis. Trastuzumab (Tz), a monoclonal antibody, has been used to inhibit HER2 activity, but the developing of resistance mechanisms to this drug leads the research to new therapies to use in synergy with Tz. Starting from these considerations, NaAsO2 was tested alone or in combination with Tz, at increasing doses and incubation times, for its ability of killing cancer cell line SKBR-3, overexpressing HER2 receptors, in comparison to a non-neoplastic cell line, the murine fibroblasts cell line, NIH-3T3. To study the synergic activity of the compounds, three types of experiments were performed: Exp. 1: cells pre-treated with NaAsO2 before adding Tz Exp. 2: cells pre-treated with Tz before adding NaAsO2 Exp. 3: cells co-incubated with the two agents The three conditions were tested to evaluate the time and dose dependence cytotoxicity trend by quantitative cell viability (MTT) assay. Both experiments 1 and 2 demonstrated that after the pre-treatment of cells both with NaAsO2 (exp.1) or Tz (exp.2), the cells resulted more sensitive to the subsequent treatment. In particular, the reduction of cell viability was respectively of 80-90% and around 40 % in exp. 1 and 2, respectively. Exp. 3 showed that the co-incubation led to a greater reduce, nearly 90%, of cell viability compared to the agent’s sole exposure. In all the conditions tested emerged tolerance by the healthy cells NIH-3T3, proving a specific effect towards HER2-cancer cells. At the light of these results, the experiments were focused on the co-incubation condition. Qualitative cell viability (LIVE/DEAD) assay and western blot (WB) analysis were performed. In line with MTT data, LIVE/DEAD analysis confirmed the efficacy of co-incubation in comparison with the use of NaAsO2 or Tz alone. Correspondingly, WB showed that the co-incubation triggered cell death with respect to the single treatments with a significant alteration of the activity and expression of survival or apoptosis proteins, respectively. Indeed, co-incubation determined the downregulation of survival (P-AKT/AKT) and proliferation (P-ERK/ERK) pathways, an increase of pro-apoptotic protein BAX and a decrease of the anti-apoptotic protein BCL-2, proving a concomitant action of the two anticancer agents. Although NaAsO2 action mechanism is still unclear, it is maybe related to NaAsO2 affinity for sulfhydryl groups reducing cysteines in proteins. To gain more insights on NaAsO2 and Tz synergism, Tz was incubated for 1 and 24 h with NaAsO2 both at room temperature and 37°C, and then analysed by SDS-PAGE and circular dichroism (CD). SDS-PAGE showed no fragmentation of Tz after incubation with NaAsO2 at both incubation time and CD revealed no significant modification of Tz secondary structure after NaAsO2 incubation. Other experiments are required to elucidate this synergism: for example, NaAsO2 internalization study and HER2 receptors expression analysis on the surface of BC cells, before and after the co-incubation treatment. Despite several studies are still needed, overall from these preliminary data emerge that the co-incubation exerts a toxicity effect greater towards cancer cells than agent’s sole treatment, that led to hypothesise a new approach in the BC treatment scenario.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/19788