Nanosecond pulsed electric field inhibits cancer growth followed by alteration in expressions of NF-κB and Wnt/β-catenin signaling molecules

Abstract

Cancer remains a leading cause of death worldwide and total number of cases globally is increasing. Novel treatment strategies are therefore desperately required for radical treatment of cancers and long survival of patients. A new technology using high pulsed electric field has emerged from military application into biology and medicine by applying nsPEF as a means to inhibit cancer. However, molecular mechanisms of nsPEF on tumors or cancers are still unclear. In this paper, we found that nsPEF had extensive biological effects in cancers, and clarified its possible molecular mechanisms in vitro and in vivo. It could not only induce cell apoptosis via dependent-mitochondria intrinsic apoptosis pathway that was triggered by imbalance of anti- or pro-apoptosis Bcl-2 family proteins, but also inhibit cell proliferation through repressing NF-κB signaling pathway to reduce expressions of cyclin proteins. Moreover, nsPEF could also inactivate metastasis and invasion in cancer cells by suppressing Wnt/β-Catenin signaling pathway to down-regulating expressions of VEGF and MMPs family proteins. More importantly, nsPEF could function safely and effectively as an anti-cancer therapy through inducing tumor cell apoptosis, destroying tumor microenvironment, and depressing angiogenesis in tumor tissue in vivo. These findings may provide a creative and effective therapeutic strategy for cancers.

Figure 1. NsPEF induces cancer cells death and proliferative inhibition in vitro.

(A) Schedule of cancer cells exposed to nsPEF with different intensities for viable cells count and CCK-8 assay. (B) The rate of viable cells/control was detected by counting trypan blue negative cells at 0 h, 0.5 h, 1 h, and 2 h post pulses with different intensities. ***p<0.001. (C and D) Numbers of viable cells were calculated after exposure to nsPEF with different intensities for 24 h and 48 h respectively. *p<0.05, ***p<0.001. (E) According to CCK-8 assay, inhibition rates of cells proliferation induced by nsPEF with different intensities were detected. **p<0.01.

Figure 2. NsPEF induces cancer cells apoptosis via dependent-mitochondria intrinsic apoptotic pathway in vitro.

(A) Morphology changes of cancer cells exposed to nsPEF with different intensities were observed by cell transmission electron microscopy (TEM). N: nuclear changes including nuclear shrinkage, nuclear membrane blebbing and nuclear lysis. M: mitochondria degeneration or vacuolar degeneration. (B) Single cell apoptosis of cancer cells exposed to nsPEF with different intensities was calculated by cell TUNEL assay. Original magnification, 400×. ***p<0.001. (C) Cell DNA fragmentation of cancer cells exposed to nsPEF was observed by apoptosis DNA ladder assay. (D) Apoptosis rates of cancer cells exposed to nsPEF with different intensities were tested with PI and Annexin-V staining assay by flow cytometry. Results were analyzed by FlowJo 7.6.1 Software. *p<0.05, **p<0.01, ***p<0.001. (E) Protein expressions of anti-/pro-apoptosis Bcl-2 family, Cytochrome C and Caspase-3 in cancer cells after exposure to nsPEF with different intensities were detected by Western-blot assay. Relative intensity of each protein was analyzed by Photoshop CS4 software. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. NsPEF inhibits cell proliferation through repressing NF-κB signaling pathway to reduce expressions of Cyclin proteins in vitro.

(A) Cell cycle of cancer cells exposed to nsPEF with different intensities was examined with PI staining assay by flow cytometry, and results were analyzed by FlowJo 7.6.1 Software. *p<0.05, **p<0.01. (B) Protein expressions of NF-κB signaling pathway including IKK-α, IKK-β IκB–α, p65 and p-p65 in cancer cells after exposure to nsPEF with different intensities were detected by Western-blot assay. (C) Protein expressions of Cyclin proteins including Cyclin D1 and Cyclin A in cancer cells after exposure to nsPEF with different intensities were detected by Western-blot assay. Relative intensity of each protein was analyzed by Photoshop CS4 software. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. NsPEF inactivates cancer cells metastasis and invasion by inhibiting Wnt/β-Catenin signaling pathway to down-regulate expressions of VEGF and MMPs family proteins in vitro.

(A) Migration ability of cancer cells exposed to nsPEF was tested by trans-well assay. The migrated cells exposed to nsPEF were stained purple by 0.1% crystal violet solution, observed under light microscope for 40 or 400 magnifications, and counted for statistical analysis. Original magnification, 40× & 400×. ***p<0.001. (B) Invasion ability of cancer cells exposed to nsPEF was detected using matrigel invasion assay. After nsPEF treatment, the cells that possessed invasion ability penetrated through the matrigel, were stained purple by 0.1% crystal violet solution, and were counted for statistical analysis. Original magnification, 40× & 400×. ***p<0.001. (C) Protein expressions of Wnt/β-Catenin signaling pathway including hDPR1, β-Catenin and c-Myc in cancer cells after exposure to nsPEF with different intensities were detected by Western-blot assay. (D) Protein expressions of MMPs family and VEGF in cancer cells after exposure to nsPEF with different intensities were detected by Western-blot assay. Relative intensity of each protein was analyzed by Photoshop CS4 software. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. NsPEF functions safely and effectively as an anti-tumor therapy in vivo.

(A) Weights of tumor-bearing mice with or without nsPEF at different time after injecting tumor cells were recorded. (B) Growth curve of tumor with or without nsPEF at different time after injecting tumor cells was shown. (C) Visual comparison of tumor with or without nsPEF through in vivo imaging of MRI for tumor-bearing mice was performed. (D) Objective comparison of most tumors in most tumor-bearing mice was performed.

Figure 6. NsPEF induces tumor apoptosis, destroyed tumor microenvironment and depressed angiogenesis in tumor tissue in vivo, and its molecular mechanism analysis.

(A) Representative tumor histology. Tumor sections were stained with H and E. Original magnification, 100× & 400×. Cell shrinkage, angiorhagia, and leucocytes infiltration were found. (B) Ultrastructure for tumor tissue with or without nsPEF was observed by TEM. N: nuclear changes including nuclear shrinkage and apoptotic body formation. M: mitochondria degeneration. (C) Tumor apoptosis was detected by TUNEL assay, and apoptosis relative proteins including Cytochrome C, Caspase-9 and Caspase-3 were determined by immunohistochemistry staining (IHC). IHC results were analyzed by Image Pro-Plus software. IOD, integrated optical density. hpf, high-powered field. Original magnification, 400×. ***p<0.001. (D) Protein expressions of β-Catenin, VEGF and CD34 were detected by IHC staining. **p<0.01, ***p<0.001. IHC results were analyzed by Image Pro-Plus software. IOD, integrated optical density. hpf, high-powered field. Original magnification, 400×. (E) Molecular mechanism analysis of nsPEF inhibiting cancer growth in vitro and in vivo.