Resistance to conventional chemotherapy continues to be a challenge in PDAC. Acquired resistance to apoptosis, which is prevalent in PDAC, is a critical pathway that promotes resistance to conventional chemotherapy [17
]. Therefore, targeting apoptosis resistance has emerged as an attractive novel cancer therapeutic strategy [26
]. Small molecules with proapoptotic activity are particularly advantageous, as they are membrane permeable, inhibit antiapoptotic molecules, augment efficacy of current therapeutics and minimize side effects of single agent therapy. Smac mimetics represent a novel class of anticancer drugs that are currently undergoing clinical evaluation [28
]. We studied the combination treatment effects of a novel Smac mimetic, JP1201, in combination with the deoxycytidine analogue gemcitabine and the cell-mitosis inhibitor docetaxel in experimental pancreatic cancer.
Human PDAC cells lines have been shown to display marked heterogeneity towards Gem [29
]. A similar heterogeneity regarding Gem sensitivity was seen in our four lines tested. Nevertheless, we observed that JP inhibited the proliferation of all four PDAC cell lines, and that the combination of JP + Gem had additive effects. Antitumor activity of Smac mimetics is mediated through induction of apoptosis. We therefore explored if proliferation inhibition of PDAC cells after combination therapy is in part due to induction in apoptosis. Detection of early apoptotic cells by annexin V/PI staining demonstrated that JP and Gem moderately induced apoptosis, and JP + Gem had an additive effect. Smac mimetic-induced apoptosis involves caspase activation that cleaves PARP-1, a DNA repair enzyme, to produce 89 kDa or 24 kDa cleavage product. We observed a dramatic increase in the 89 kDa C-terminus cleavage product of PARP-1 after JP treatment indicating the involvement of caspases in JP-induced apoptosis. While Gem treatment for 12 hours caused an increase in annexin V positive cells, no PARP-1 cleavage was detected after 24 hours of Gem treatment; this finding is likely related to the fact that 24 hours incubation may not be enough to cause detectable levels of PARP-1 cleavage. Based on in vitro
additive anti-proliferative and proapoptotic effects of JP and Gem together, we examined effects of these agents on in vivo
animal survival and observed that the JP + Gem combination significantly increases the animal survival compared with controls or monotherapy. Our findings corroborate a recently published report [23
] that the Smac mimetic JP1201 enhances chemotherapy response of Gem in PDAC cell lines; accordingly, the JP-mediated increase in antiproliferative response after Gem was greatest in Panc-1 cells followed by MIA PaCa-2, BxPC-3 and AsPC-1 cells. Correspondingly, a 30% reduction in tumor weight was observed in our orthotopic Panc-1 tumor experiment compared to a 50% reduction in orthotopic MIA PaCa-2 tumors in the previous study [23
]. In addition, a significant improvement in animal survival was observed with JP + Gem treatment in AsPC-1 xenografts compared with JP or Gem alone similar to the previous study [23
]. Of note, the lack of a significant reduction in tumor growth (Figure ) by Gem alone in the present study is likely merely a result of the small number of animals in that tumor growth experiment, and does not pose a conflict with the significant improvement in animal survival seen after the same treatment (Figure ). Altogether, our results can thus support a more generalizable phenomenon in this context, as JP combination benefits have been obtained in four PDAC cell lines, and with other cytotoxic agents beyond gemcitabine.
Smac mimetics have been shown to enhance antitumor effects of several agents including cisplatin [30
] and TRAIL [23
] in different cancer types. Docetaxel is a clinically well established anti-mitotic chemotherapy treatment for several cancers including breast, ovarian and non-small cell lung cancer [32
]. We explored the combination treatment effects of JP with other chemotherapy agents such as doxorubicin and docetaxel in experimental pancreatic cancer. In vitro
studies showed that JP significantly enhanced antiproliferative effects of Dox and DT in all four PDAC cell lines tested. In addition JP and DT combination had significant enhanced effect on tumor regression and animal survival in pancreatic cancer xenografts. These results indicate that a potential clinical benefit to Smac-mimetic combination therapies does not appear to be chemotherapeutic agent specific, and that such approach may carry a wide range of indications.
Small molecule Smac mimetics have been shown to be particularly advantageous in overcoming chemotherapy resistance when resistance occurs through modulation of the NFkB-IAP pathway. Since several pancreatic cancer cell lines and tumors have been shown to overexpress IAPs (16-18), treatment of PDAC with JP in combination with other chemotherapeutic agents shows specific promise for becoming effective for this disease. Our present study supports this notion through preliminary, preclinical evidence. The potential to render traditionally non-effective agents more effective for clinical PDAC therapy is particularly intriguing in this context.