(W1030-04-23) ELC-Cu Nanoparticles Induce Cell Death by Mediating Mitochondria Oxidative Stress and Trigger an Innate Immune Response for Cancer Therapy

Purpose: Elesclomol (ELC) has been used in clinical trials for anticancer therapy. Several research studies showed that its anticancer effect significantly increased when ELC and Cu (II) ions formed an ELC-Cu complex of a 1:1 molar ratio. ELC has been reported to exert greater anticancer activity with Cu (II) ions, which makes codelivery of ELC and Cu (II) ions in one delivery system a promising strategy for cancer therapy [1][2]. Herein, a D-a-tocopherol polyethylene glycol 1000 succinate/Chondroitin sulfate-Cholic acid (TPGS/CS-CA) based nanoparticle was developed for codelivering ELC and Cu (II) ions with a tumor-immunotherapeutic effect.

Methods: ELC-Cu loaded TPGS/CS-CA nanoparticles were prepared and screened for an optimal formulation by a thin film dispersion/hydration method. The nanoparticles of various formulations were characterized by DLS, UV-vis spectrometer, and AFM. The cell viabilities of ELC and ELC-Cu nanoparticles in six cell lines based on MTT assay after 48 h incubation were evaluated by Gen5 Cytation plate reader. The mitochondria membrane potential changes of different formulations in DU145TXR cells based on JC-1 staining were characterized by Gen5 Cytation Imager. The accumulations of intracellular ROS of different formulations based on fluorescence intensity in DU145TXR and A549TXR cells were measured by Gen5 Cytation. The ubiquitin accumulation and expression of ER stress of different formulations in DU145TXR cells were investigated by western blot analysis. The mtDNA levels of different formulations in DU145TXR cells were measured by PCR. The quantifications of the secretion levels of cytokines based on RNA expression levels in Raw 264.7 macrophages treated with fresh medium mixed with DU145TXR medium were measured by PCR. The medium from DU145TXR cells was pretreated with different formulations. The intracellular uptake of rhodamine in DU145TXR cell of ELC-Cu nanoparticles or lapatinib was measured by flow cytometry. The P-glycoprotein (P gp) expression level in DU145 and DU145TXR cells were investigated by western blot analysis. The mRNA expression levels of P gp, MRP1, and BCRP of different formulations in DU145TXR cells were measured by PCR. The student’s t-test was used for statistical analysis: *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Results: The ELC-Cu complex in DMSO and ELC-Cu nanoparticles showed a UV-vis absorbance at 630 nm, which was different from ELC nanoparticles or Cu (II) ions. The particle size of ELC-Cu nanoparticles was 16.4 nm by DLS. The cell viabilities of six cell lines showed that only ELC-Cu nanoparticles exerted a dose-dependent cytotoxicity in six cell lines, which indicated that the complexation of ELC and Cu (II) ions in nanoparticles increased cytotoxicity when compared to ELC nanoparticles alone. The IC50 of ELC-Cu nanoparticles in DU145TXR cells was 0.81 mM, whereas the viability of ELC nanoparticles was around 100%. Both ELC nanoparticles and ELC-Cu nanoparticles increased mitochondria ROS levels in DU145TXR and A549TXR. However, the ROS levels of ELC-Cu nanoparticles were higher than ELC nanoparticles. The ROS level of ELC-Cu nanoparticles was 1.6 folds compared to ELC nanoparticles in DU145TXR cells and 1.9 folds in A549TXR cells. The JC-1 staining results showed that only ELC-Cu nanoparticles decreased mitochondria membrane potentials in DU145TXR cells. The ubiquitin and ER stress western blot analysis showed that only ELC-Cu free drug and ELC-Cu nanoparticles induced ubiquitin accumulation and CHOP expression in DU145TXR cells. In the in vitro simulation of immune response study, the secretion of TNFa and IL-6 in Raw 264.7 macrophages increased significantly in ELC-Cu nanoparticles and LPS groups. The IL-6 expression level of ELC-Cu nanoparticles was 6.6 folds compared to ELC nanoparticles and the control group. The in vitro innate response data indicated that ELC-Cu nanoparticles increased mitoROS to release mtDNA from DU145TXR cells. Then the released mtDNA could induce Raw 264.7 macrophages to release cytokines which could trigger an innate immune response. In the in vitro drug resistance study, rhodamine cellular uptake only showed in the lapatinib group. Neither the ELC nanoparticle group nor the ELC-Cu nanoparticle group increased intracellular uptake of rhodamine. The western blot and PCR analysis showed similar results. The P gp, MRP1, and BCRP expression of different formulations in DU145TXR cells did not show significant difference. These drug resistance data indicated that ELC and ELC-Cu are not the substrates of P gp.

Conclusion: We report here a novel TPGS/CS-CA based ELC-Cu nanoparticle with effective anticancer efficacy and innate immune response induction. Future in vivo study needs to be performed to evaluate the anticancer activity of ELC-Cu nanoparticles on mice. Overall, the novel TPGS/CS-CA based ELC-Cu nanoparticle has been demonstrated as an efficient delivery system for greater anticancer therapy with innate tumor-immunotherapeutic effect.

References: [1].Faria, A.R., Silvestre, O.F., Maibohm, C. et al. Cubosome nanoparticles for enhanced delivery of mitochondria anticancer drug elesclomol and therapeutic monitoring via sub-cellular NAD(P)H multi-photon fluorescence lifetime imaging. Nano Res. 12, 991–998 (2019). https://doi.org/10.1007/s12274-018-2231-5
[2].Tsvetkov P, Detappe A, Cai K, Keys HR, Brune Z, Ying W, Thiru P, Reidy M, Kugener G, Rossen J, Kocak M, Kory N, Tsherniak A, Santagata S, Whitesell L, Ghobrial IM, Markley JL, Lindquist S, Golub TR. Mitochondrial metabolism promotes adaptation to proteotoxic stress. Nat Chem Biol. 2019 Jul;15(7):681-689. doi: 10.1038/s41589-019-0291-9. Epub 2019 May 27. Erratum in: Nat Chem Biol. 2019 Jun 4;: PMID: 31133756; PMCID: PMC8183600.

Acknowledgements: This project was supported by Auburn University start-up fund (F. Li), Launch Innovation Award (F, Li).


Figure 1. (A) The mitochondria ROS accumulation of different formulations in DU145TXR and A549TXR cells. (B) The changes of mitochondria membrane potentials in DU145TXR cells.