Electrochemotherapy: aspects of preclinical development and early clinical experience.

OBJECTIVE: To develop an optimized, reproducible system of electrochemotherapy, and to investigate its clinical application in patients with cutaneous or subcutaneous recurrences of inoperable or progressive disease recalcitrant to current anticancer treatments. BACKGROUND: Electrochemotherapy is the application of electric pulses to tumor tissue, rendering the cell membranes permeable to otherwise impermeant or poorly permeant anticancer drugs. This facilitates a potent local cytotoxic effect. STUDY DESIGN: The optimal parameters for electrical pulses and bleomycin concentration were obtained in vitro and then applied to tumors derived from 4 histologically distinct human cancer cell lines (7860, PC3, OE19, MCF-7) established in athymic nude mice. Comparison was made with tumors that received bleomycin alone, electric pulses alone, and untreated controls. The optimized electrochemotherapy was then applied to patients with cutaneous or subcutaneous tumors, of any histologic type, recurrent or metastatic and unresponsive to standard chemotherapy and/or radiotherapy regimens. Tumors were assessed at monthly intervals to determine response to the treatment. RESULTS: In vivo: Using the optimal parameters ascertained in vitro, all tumors treated by electrochemotherapy with bleomycin (n = 24) had significantly regressed (P < 0.001, all 4 lines) compared with control tumors (n = 72). Twelve tumors completely regressed (50%) following a single application, with 12 partial regressions (50%). Clinical: In 30 patients (111 tumors), none of the treated tumors progressed. Sixty percent of tumors (66 of 111) showed complete regression, 22% (24 of 111) partial response, and 18% (21 of 111) no change. Electrochemotherapy was more effective in smaller tumors (<3 cm), 71% (64 of 90) showing complete regression, 20% (18 of 90) partial response, and 9% (8 of 90) no change. CONCLUSIONS: Electrochemotherapy parameters optimized in vitro are applicable in vivo. This treatment is effective in athymic nude mice for all histologic types indicating a nonimmunologic mode of action. In clinical application, electrochemotherapy is an effective, safe, and reproducible therapy. Patients with cutaneous or subcutaneous tumors previously refractory to surgical intervention, systemic chemotherapy, and/or radiotherapy responded successfully irrespective of histologic type.

Antitumor efficacy of the novel chemotherapeutic agent coramsine is potentiated by cotreatment with CpG-containing oligodeoxynucleotides.

Coramsine is a novel chemotherapeutic agent isolated from Solanum linnaeanum (devil's apple). Topical treatment provides clinical benefit for skin tumors. To evaluate the potential broader applicability of the drug, its in vivo anticancer efficacy in a murine model of malignant mesothelioma and its mode of action were investigated. Systemic administration of coramsine slowed tumor growth and prolonged survival time. Importantly, the antitumor efficacy of coramsine was enhanced when treatment was combined with stimulation of innate immunity using unmethylated CpG-containing oligodeoxynucleotides (ODNs). Combination treatment further slowed tumor growth and provided a survival benefit. Coramsine seems to kill tumor cells by direct cell lysis. Using 2 different assays to detect apoptosis (caspase activation and DNA fragmentation), we found no evidence that coramsine induces any form of programmed cell death. The fact that the efficacy of coramsine is potentiated by CpG ODNs suggests that coramsine-induced cell death is an immunologic null event.

Successful application of targeted electrochemotherapy using novel flexible electrodes and low dose bleomycin to solid tumours.

Electroporation is the application of very brief electric pulses to cells or tissues to render the cell membranes transiently and reversibly permeable, facilitating cellular uptake of otherwise impermeant molecules. Flexible electrode arrays were developed which may be used with endoscopic and laparoscopic devices for delivery of therapeutic electroporation. Their efficacy in enhancing the delivery of bleomycin, an impermeant drug, was assessed in vitro and in vivo in both human and murine cancer cell lines, and growing tumours (xenografts). These flexible electrodes consistently and predictably deliver the permeabilising electric pulses requisite for in vivo electroporation, and would be suitable for electrochemotherapy of endoluminal tumours when incorporated into an endoscopic delivery system.

Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor.

The antifungal metabolite 2,4-diacetylphloroglucinol plays a major role in the biocontrol capabilities of Pseudomonas fluorescens. The phloroglucinol biosynthetic locus of P. fluorescens F113 has been isolated previously. From nucleotide sequence data, a putative regulator gene (phlF) was identified upstream and divergently transcribed from the phlACBD phloroglucinol biosynthetic genes. PhlF shows similarity to various transcriptional repressors in the EMBL database and exhibits a helix-turn-helix motif in its amino acid sequence. phlF was cloned into an expression vector and the PhlF protein product was purified. Gel retardation experiments demonstrated PhlF to be a DNA-binding protein and showed that it binds to the phlA-phlF intergenic region. Introduction of phlF into P. fluorescens F113 in multiple copies resulted in repression of phloroglucinol production in this strain. This effect was mediated at the transcription level since the expression of a phloroglucinol biosynthetic gene fusion in this background was equally repressed. Furthermore, the inactivation of phlF results in derepression of phloroglucinol production in this strain.

A novel gene, algK, from the alginate biosynthesis cluster of Pseudomonas aeruginosa.

Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. Many of the enzymes involved in alginate biosynthesis are clustered in an operon at 34 min on the P. aeruginosa chromosome. This paper reports the nucleotide sequence of a previously uncharacterized gene, algK, which lies between the alg44 and algE genes of the operon. DNA sequencing data for algK predicted a protein product of approximately 52.5 kDa which contains a putative 27 amino acid N-terminal signal sequence and a consensus cleavage and lipid attachment site for signal peptidase II. Expression of algK using either T7 or tac promoter expression systems, in vivo labelling studies with [35S]methionine, indicated that algK encodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed in Escherichia coli or P. aeruginosa, in agreement with the nucleotide sequence analysis. Results from an algK-beta-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored.