Novel combination therapy for melanoma induces apoptosis via a gap junction positive feedback mechanism

Abstract

Metastatic melanoma cells overexpressing gap junctions were assayed for their ability to propagate cell death by a novel combination therapy that generates reactive oxygen species (ROS) by both 1) non-thermal plasma (NTP) and 2) tirapazamine (TPZ) under hypoxic conditions. Results demonstrate additive-to-synergistic effects of combination therapy compared to each agent individually. NTP induces highly localized cell death in target areas whereas TPZ partially reduces viability over the total surface area. However, when high gap junction expression was induced in melanoma cells, effects of combination NTP+TPZ therapy was augmented, spreading cell death across the entire plate. Similarly, in vivo studies of human metastatic melanoma in a mouse tumor model demonstrate that the combined effect of NTP+TPZ causes a 90% reduction in tumor volume, specifically in the model expressing gap junctions. Treatment with NTP+TPZ increases gene expression in the apoptotic pathway and oxidative stress while decreasing genes related to cell migration. Immune response was also elicited through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response with fewer side effects than current therapies. Interestingly, the gap junction protein, Cx26 was upregulated following treatment with NTP+TPZ and these gap junctions were shown to maintain functionality during the onset of treatment. Therefore, we propose that gap junctions both increase the efficacy of NTP+TPZ and perpetuate a positive feedback mechanism of gap junction expression and tumoricidal activity. Our unique approach to ROS induction in tumor cells with NTP+TPZ shows potential as a novel cancer treatment.

Hypoxia and Extracellular Acidification as Drivers of Melanoma Progression and Drug Resistance

“…While monotherapy with tirapazamine, a DNA-damaging compound, failed to elicit a significant anticancer response, promising results were obtained using combination therapy with NTP (non-thermal plasma), also called ‘cold plasma’ ( Table 1 ). NTP alone strongly induced ROS production and subsequent apoptotic response, whereas together with tirapazamine it displayed even higher effectiveness, resulting in 90% reduction in tumor volume in a murine melanoma model [ 142 ]. Another hypoxia-activated prodrug with DNA-alkylating activity, TH-302, also reduced tumor volume in combination with a standard chemotherapeutic (temozolimide) or inhibitor of multiple receptor tyrosine kinases (sunitinib), especially, when melanoma cells were first pre-treated with hypoxia-targeting drug ( Table 1 ) [ 143 , 149 ].…”

The pro- and anti-tumoral properties of gap junctions in cancer and their role in therapeutic strategies

“…Bagati et al reported additive-to-synergistic effects of a NTP combination therapy with the DNA-damaging agent tirapazamine in in vitro and in vivo metastatic melanoma cells, which underline the potential of NTP to improve cancer therapy via GJ modulation [ 163 ]. The authors observed that when high Cx26-GJs expression was induced in these cells, the combinatorial effects of NTP + tirapazamine therapy was augmented, spreading cell death.…”

“…The authors observed that when high Cx26-GJs expression was induced in these cells, the combinatorial effects of NTP + tirapazamine therapy was augmented, spreading cell death. The presence of Cx26-GJs facilitated RONS cell penetration and signaling, while increased Cx26 protein expression and amplified tumoricidal activity [ 163 ]. Furthermore, they also observed an immune response through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response [ 163 ].…”

“…The presence of Cx26-GJs facilitated RONS cell penetration and signaling, while increased Cx26 protein expression and amplified tumoricidal activity [ 163 ]. Furthermore, they also observed an immune response through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response [ 163 ]. Using a modified non-thermal helium plasma torch, the same research group showed that Cx43-GJs also contributes to NTP-induced cell death in melanoma cells [ 164 ].…”

The Development of Nonthermal Plasma and Tirapazamine as a Novel Combination Therapy to Treat Melanoma In Situ

“…ROS production via NTP has a selectively cytotoxic effect within melanoma as compared to normal keratinocytes in vitro [ 19 ]. NTP has been demonstrated to have a cytotoxic effect on cancer cells from in vitro and in vivo experiments [ 20 , 21 , 22 , 23 ]. More recently, NTP has been utilized in phase I clinical trials which showed successful parameters for safety following surgical resection in solid tumors [ 24 ].…”