Cutaneous Papilloma and Squamous Cell Carcinoma Therapy Utilizing Nanosecond Pulsed Electric Fields (nsPEF)

Abstract

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7–30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

“…Cytotoxic efficiency of nsPEF against multiple cancer types has been demonstrated both in vitro [1]–[7] and in vivo [1], [5], [6], [8]–[10]. Interestingly, cancer cells reportedly were more vulnerable than matching normal cell lines [7].…”

“…As a result, the vast majority of studies into nsPEF-induced cell death focused solely on the apoptotic death pathway. Indeed, various types of cells exposed at lethal nsPEF doses expressed such manifestations of apoptosis as caspase activation, poly-ADP ribose polymerase (PARP) cleavage, cytochrome C release into the cytoplasm, and internucleosomal DNA fragmentation [4], [6], [12], [14]. The only type of necrosis considered in these studies was the so-called “secondary necrosis” (a final cell destruction following the apoptotic process in vitro ).…”

The cytotoxic synergy of nanosecond electric pulses and low temperature leads to apoptosis

“…Several studies reported necrosis due to the colloid-osmotic cell swelling as a predominant mechanism of cell death after exposure to nsEP5610212223. This mechanism could be blocked by isosmotic addition of a nanopore-impermeable solute (such as sucrose) to the growth medium32.…”

“…More recently, several groups including ours reported that cells exposed to nsEP swell and may die because of necrosis within the first several hours after the treatment5610212223. Necrosis is caused by the presence of long lived nanopores and colloid-osmotic imbalance which leads to cell swelling and membrane rupture.…”

“…In early studies, the massive externalization of phosphatidylserine (PS) in response to nsEP was interpreted as a sign of apoptosis789, and apoptosis was considered the prevalent mechanism of cell death induced by nsEP. Indeed, several groups have shown apoptosis in nsEP-treated cells using different apoptotic hallmarks such as activation of caspase, DNA fragmentation, cytochrome C release in the cytoplasm, and poly-ADP ribose polymerase (PARP) cleavage681011. However, later studies revealed that nsEP open pores in the plasma membrane12131415 and cause an increase in intracellular calcium concentration, thus inducing scramblase activation and PS externalization1617.…”