Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

Abstract

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

Optimising intratumoural treatment of head and neck squamous cell carcinoma mouse models with EBC-46

“…No significant histological change was noted following the subcutaneous injection of EBC-46 into normal skin from BALB/c Foxn1 nu mice [10]. To confirm this was also the case with NOD/SCID mice, Section 4.2.1 was replicated again using normal skin from NOD/SCID mice.…”

“…Specifically, EBC-46, a novel diterpene ester and PKC activator discovered by QBiotics Pty. Ltd (Queensland, Australia), has recently been found to successfully ablate subcutaneous xenografts of 2 different human HNSCC cell lines in nude mice by intratumoural injection [10]. This body of work aimed to optimise intratumoural injection of HNSCC mouse models with EBC-46 in conjunction with further investigation into the drug's mechanism of action, to facilitate the progression of EBC-46 into clinical trials.…”

“…Foxn1 nu mice [10]. Localised hemorrhagic necrosis appeared to be the overt mechanism of action, however the molecular subtleties behind this and the potential to exploit them for enhancing efficacy still need to be investigated.…”

Anticorpos conformacionais para PKCs clássicas e suas aplicações

“…A presença da PKC ativa promove a fosforilação do substrato, a ligação deste ao domínio FHA2 e por consequência uma redução na medida de FRET (J. D. . Os demais métodos existentes para mensurar a atividade das PKCs, como ensaios de fracionamento celular ou radioativos, os quais permitem mensurar a fosforilação dos substratos pela incorporação de  32 -P-ATP e ensaios de ELISA que utilizam peptídeo substrato e anticorpos contra a forma fosforilada do peptídeo substrato, envolvem o preparo de extratos proteicos e por isso não são adequadas para a realização de estudos de dinâmica de ativação e concomitantemente localização das PKCs ativas (Boyle et al, 2014;Sando et al, 2003). Nesse sentido, a determinação do papel das PKCs ao nível endógeno e sem a necessidade de transfecção de células com sondas, pode ser estabelecido com a utilização do anti-C2Cat.…”

“…Esses estudos revelaram o comportamento dinâmico das PKCs no interior das células e sua distribuição espaço-temporal, além de mostrar que estímulos específicos levam à ativação de uma determinada PKC, revelando assim diferenças funcionais entre as isoenzimas das PKCs (Sakai et al, 1997;Shirai et al, 1998). Adicionalmente a atividade das PKCs pode ainda ser mensurada por ensaios radioativos de incorporação de  32 -P-ATP ou por ensaios de ELISA que utilizam um peptídeo substrato e anticorpos específicos para formas fosforiladas desse peptídeo (Boyle et al, 2014;Sando et al, 2003). Ainda, a atividade das PKCs pode ser mensurada após a imunoprecipitação desta quinase seguida pela adição de ativadores (lipídios e/ou Ca 2+ ) e de substratos proteicos (por exemplo, histona ou mielina básica).…”