Targeting inhibitor of apoptosis proteins (IAPs) enhances susceptibility of oral squamous carcinoma cells to cisplatin.

PURPOSE: Oral Squamous Cell Carcinoma (OSCC) is the 6th most common cancer worldwide. It is generally aggressive and closely associated with chemoresistance and poor survival. There is accumulating evidence for the involvement of inhibitors of apoptosis proteins (IAPs), including IAP1 and XIAP, in mediating chemotherapy resistance in OSCC. Various strategies for targeting IAPs have been designed and tested in recent years and several small molecule IAP inhibitors are in clinical trials as monotherapies as well as in combination with radiotherapy and chemotherapy. The purpose of this study was to evaluate and compare the efficacy and biological activity of three IAP inhibitors both as stand-alone and sensitising agents to cisplatin in a preclinical model of squamous cell carcinoma of the tongue. METHODS: Cisplatin-sensitive SCC4 and -resistant SCC4cisR cells were utilised in this study. Apoptosis was evaluated by flow cytometric analysis of Annexin V/Propidium Iodide-stained cells. Expression of IAP proteins was determined by western blotting and knockdown of cIAP1, livin and XIAP was conducted by transfection of cells with siRNA. RESULTS: We establish for the first time the therapeutic efficacy of the Smac mimetic, BV6 and the XIAP inhibitor Embelin, for OSCC. Both of these IAP targeting agents synergistically enhanced cisplatin-mediated apoptotic cell death in resistant cells which was mediated in part by depletion of XIAP. In addition, knockdown of XIAP using siRNA enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation. CONCLUSION: These findings provide pre-clinical evidence that IAP inhibition may be a valuable therapeutic option in OSCC.

Evaluation of targeting autophagy for the treatment of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is a rare and aggressive paediatric tumour that typically arises in the kidneys or central nervous system (CNS). The malignancy often affects patients under the age of three and is associated with an extremely poor survival rate, with most deaths occurring within the first year of presentation. Thus, there is an unmet and urgent medical need for novel therapeutic strategies for this malignancy. One of the major issues when treating MRT patients is the emergence of chemoresistance. Autophagy has become an area of focus in the study of chemoresistance due to its reported dual role as both a pro-survival and pro-death mechanism. The role of autophagy in the chemotherapeutic response of MRT remains largely unknown. A greater understanding of the role of autophagy may lead to the development of therapeutic strategies to enhance chemotherapeutic effect and improve the clinical outcome of MRT patients. This study evaluated the cellular response to cisplatin, a representative chemotherapeutic agent used in the treatment of MRT, and the role of autophagy in mediating cisplatin resistance. Our results demonstrated that cisplatin induced apoptosis and autophagy concomitantly in a panel of MRT cell lines. Furthermore, inhibition of caspase-induced apoptosis with Z-VAD-FMK also inhibited autophagy levels demonstrating a complex interplay between these two pathways. In addition, blocking autophagy at the early stages of the autophagic process using the pharmacological inhibitor SAR405 or through the genetic knockdown of critical autophagic protein ATG5 by siRNA did not sensitise cells to cisplatin-induced apoptosis. Collectively, these results suggest that induction of autophagy does not appear to elicit a pro-survival effect in the chemotherapeutic response of MRT cells.

Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy.

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.

Cisplatin induces autophagy-associated apoptosis in human oral squamous cell carcinoma (OSCC) mediated in part through reactive oxygen species.

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. Chemoresistance is a critical problem in OSCC leading to therapeutic failure and tumour recurrence. Recently, autophagy has acquired an emerging interest in cancer as it has been shown to be frequently activated in tumour cells treated with chemotherapeutics. Whether drug-induced autophagy represents a mechanism that allows cancer cells to survive or a pro-death mechanism associated with apoptosis remains controversial. This study evaluated the cellular response to cisplatin and the role of autophagy in mediating cisplatin resistance in OSCC cells. Our results demonstrated that cisplatin concurrently induced apoptosis and autophagy in OSCC cell lines partially through the ROS/JNK pathway. Moreover, inhibition of cisplatin-induced apoptosis abrogated autophagy, indicating a complex interplay between these pathways. Cisplatin-induced autophagy does not appear to elicit a pro-survival effect in OSCC as early-stage autophagy inhibition, using either a pharmacological inhibitor or knockdown of the key autophagy protein ATG5, did not sensitise cells to cisplatin. Additionally, autophagy did not play a role in acquired resistance to cisplatin in our novel cisplatin-resistant OSSC cell line (SCC-4cisR) obtained by pulsed stepwise exposure of SCC-4 cells to cisplatin (~14-fold change in sensitivity). There was no change in the basal levels of autophagy in the SCC-4cisR cells compared to the SCC-4 cells. Furthermore, a significant increase in cisplatin-induced autophagy was observed only in the SCC-4 cells, but not in the derived SCC-4cisR cells. Collectively, these data indicate that autophagy may not be implicated in acquired cisplatin resistance in OSCC.

Autophagy modulators for the treatment of oral and esophageal squamous cell carcinomas.

Oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) exhibit a survival rate of less than 60% and 40%, respectively. Late-stage diagnosis and lack of effective treatment strategies make both OSCC and ESCC a significant health burden. Autophagy, a lysosome-dependent catabolic process, involves the degradation of intracellular components to maintain cell homeostasis. Targeting autophagy has been highlighted as a feasible therapeutic strategy with clinical utility in cancer treatment, although its associated regulatory mechanisms remain elusive. The detection of relevant biomarkers in biological fluids has been anticipated to facilitate early diagnosis and/or prognosis for these tumors. In this context, recent studies have indicated the presence of specific proteins and small RNAs, detectable in circulating plasma and serum, as biomarkers. Interestingly, the interplay between biomarkers (eg, exosomal microRNAs) and autophagic processes could be exploited in the quest for targeted and more effective therapies for OSCC and ESCC. In this review, we give an overview of the available biomarkers and innovative targeted therapeutic strategies, including the application of autophagy modulators in OSCC and ESCC. Additionally, we provide a viewpoint on the state of the art and on future therapeutic perspectives combining the early detection of relevant biomarkers with drug discovery for the treatment of OSCC and ESCC.

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents.

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors.