Secondary structure of 11 S globulin in aqueous solution investigated by FT-IR derivative spectroscopy.

Secondary structure of 11 S globulin, a major storage protein of soybean seeds, has been investigated in aqueous solution by FT-IR spectroscopy. Conformational changes in the native protein upon thermal and chemical denaturation have been monitored by observing changes in the frequency position and peak intensity of the various bands. The frequency of the Amide I band of the native protein shifts by 4 cm-1 from 1,643 cm-1 to 1,647 cm-1 when denatured, while the corresponding intensity of the Amide I band compared to the native protein, decreases by 30 and 67%, respectively, for the urea and thermally denatured proteins, indicating gross conformational changes in the secondary structure. Trifluoroethanol, an alpha-helix promoter shifts the Amide I band from 1,643 cm-1 to 1,651 cm-1, typical of alpha-helix, with a corresponding increase in intensity by 14% relative to the native protein. Derivative spectroscopy, allowing resolution of overlapping bands, shows that the native protein mainly consists of beta-sheet, beta-turns and disordered structure with very little alpha-helix. On denaturation, beta-sheet disappeared almost completely with urea, while this is less so with thermal denaturation.

Intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer.

We report on an antitumor treatment involving electrogene therapy (EGT), a newly developed in vivo gene transfer method using electroporation. We carried out in vivo EGT in a subcutaneous model of CT26 colon carcinoma cells, using plasmid DNAs encoding interleukin 12 (IL-12) subunits. For this purpose, we developed two IL-12 expression systems: a cotransfer system using a plasmid encoding the IL-12 p40 subunit and a plasmid encoding the IL-12 p35 subunit, and a single-vector system using a plasmid expressing a p40-p35 fusion protein. Both transfer systems significantly inhibited the growth of CT26 tumor. Immunohistochemical analysis of IL-12 EGT-treated tumors revealed enhanced infiltration of CD8(+) cells into the tumor tissue, while reverse transcriptase-polymerase chain reaction confirmed the increased expression of interferon gamma within treated tumors. The same IL-12 EGT applied to the nude mouse model was not effective, suggesting the critical role of T cell infiltration in this treatment. The inhibitory effects revealed in experiments in which previously treated mice were rechallenged with a second inoculation of CT26 tumor cells suggested that IL-12 EGT may also establish partial systemic antitumor immunity. The growth of IL-12 EGT-treated Renca tumors, a renal cell carcinoma, was also significantly inhibited. These findings suggest that EGT of the IL-12 gene has the potential to be an effective anticancer gene therapy.

Electrochemotherapy--a novel method of cancer treatment.

A critical review is presented on a novel method of treating cancer by a combination of an electric field with chemotherapeutic agents. The work described here summarizes the current state of the technique known as electrical impulse chemotherapy (EIC) or electrochemotherapy (ECT). The review discusses in vitro results with specific cell lines, in vivo work on animals and clinical results on patients with squamous cell carcinomas of the head and neck. In all cases, it has been found that uptake of various drugs by the tumor cells can be increased markedly by EIC/ECT. Partial responses and complete cures have been observed without any damaging side-effects, provided the field strength is kept sufficiently low. ECT followed by injection of a low dose of interleukin-2 (IL-2) or IL-2 secreting cells has shown better results than ECT alone. There appears to be a systemic effect and a strong indication that an immune response may be elicited by this method of treatment. Finally, we discuss the challenges involved in hardware requirements for EIC/ECT and its future prospects for both drug delivery and gene therapy.

Electroporation therapy of solid tumors.

The curative effects of some chemotherapeutic drugs are impeded by their poor permeation through the cell membrane. This limitation can be overcome by a novel approach called electroporation therapy (EPT), electrochemotherapy (ECT), or electrical impulse chemotherapy (EIC). The method involves application of brief electrical pulses, which destabilize the cell membrane barrier, allowing intracellular access of chemotherapeutic drugs that otherwise would not be able to penetrate the cell membrane effectively. EPT makes it possible to lower the drug dose, thereby relieving the patient of adverse side effects associated with conventional chemotherapy. Even with the lower drug dose, EPT has shown significantly higher efficacy than has conventional chemotherapy. The method is currently being evaluated clinically for treating various cancer indications using the anticancer drugs bleomycin or cisplatin. This article provides a historical perspective and current insights into this new modality of cancer treatment, including basic physical, biological, and medical facts about EPT; computer-assisted development of electrical pulse generators and electrodes necessary to create effective electrical fields in the treatment area; results of cancer cell and tumor treatments in vitro, in animals, and in humans; safety aspects of EPT; potential combined delivery of chemotherapeutic drugs and biological agents to reduce or eliminate metastatic disease; and intracellular delivery of DNA by electroporation for cancer gene therapy.

Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas.

Electroporation, a standard laboratory method of introducing exogenous molecules into cells, has been gaining importance as a very effective non-viral physical technique of gene delivery. In this study, we have used subcutaneous model of the C6 rat glioma cells and established an optimal condition to obtain very high gene expression in tumor tissues using both reporter and functional genes. Tumors grown on the flanks of Wistar rats are exposed and directly injected with plasmid DNA having the constructs of luciferase, green fluorescent protein and, the fragment of the diphtheria toxin, DT-A. The tumors are then subjected to square wave pulses from an electroporator. Gene expression is found to be several orders of magnitude higher when the tumors are pulsed with the optimized electrical parameters compared to the controls. For luciferase, the enhancement is approximately 135-fold, for the green fluorescent protein, gene expression is seen over a wide area within the sections examined, as contrast to a few punctate dots in the control specimens, and finally, DT-A shows massive death in the tumor tissue. A special circular array of six needles through which pulses are delivered with rotating electric field is found to be highly efficient in transferring genes inside the tumor. Direct injection of plasmid DNA followed by electroporation allows very high in vivo gene transfer and its subsequent expression into tumor tissues. This method may be applicable to any solid tumor.

Secondary structural changes in globular protein induced by a surfactant: Fourier self-deconvolution of FT-IR spectra.

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C = O bond shifts from 1639 cm-1 to 1652 cm-1. On the other hand, the position of the Amide II band remains fairly unchanged. Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alerts the beta sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm-1 indicates generation of some beta turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders. The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.

Electroporation enhances therapeutic efficacy of anticancer drugs: treatment of human pancreatic tumor in animal model.

BACKGROUND: A new method of cancer treatment called electroporation therapy (EPT) which uses pulsed electric fields in combination with a chemotherapeutic agent is being developed to treat human pancreatic tumors. Such a combination has been found to increase the cytotoxic effect of the drug to tumor cells. METHODS: Human pancreatic tumors (Pan-4-JCK) were implanted subcutaneously onto nude mice. The animals were treated with EPT using bleomycin, mitomycin C or carboplatin as a single agent, and their effect on tumor growth was monitored over a period of 89 days. RESULTS: The tumors treated with either the drug or pulse alone showed increased tumor growth. However, tumors treated with EPT using any one of the three drugs showed significant to complete regression of tumors. Among the three drugs used, the order of efficacy was: bleomycin >> mitomycin C > carboplatin. CONCLUSIONS: These results are sufficient to warrant limited clinical trials of EPT for pancreatic cancers.

Electroporation therapy of human larynx tumors HEp-2 implanted in nude mice.

BACKGROUND: Electroporation therapy (EPT) uses reversible membrane permeabilization of cells by electrical pulses for intracellular delivery of poorly permeating drugs like bleomycin. This treatment modality has been found to significantly increase the cytotoxic effect of the drug to tumor cells. METHODS: Tumors of human epidermoid carcinoma of larynx (HEp-2) were xenografted subcutaneously in nude mice. EPT consisted of intratumoral injection of belomycin followed by 6 x 100 microseconds square wave electrical pulses of 1130 Volts. The effect of treatment on tumor growth was monitored over a period of 67 days. RESULTS: Complete regression of the tumors was observed in 83% of the treated mice 67 days after treatment. These findings were confirmed by histopathological analysis of tumor samples from the treated sites, which showed complete absence of tumor cells. CONCLUSIONS: The results indicate that electroporation therapy is very effective and has potential for treating laryngeal tumors clinically.

Electroporation therapy: a new approach for the treatment of head and neck cancer.

Electroporation can deliver exogenous molecules like drugs and genes into cells by pulsed electric fields through a temporary increase in cell membrane permeability. This effect is being used for the treatment of cancer by intratumoral injection of low dosage of an otherwise marginally effective chemotherapeutic drug, bleomycin. Application of a pulsed electric field results in substantially higher uptake of the drug and enhanced killing of the cancer cells than is possible by conventional methods. The MedPulser, a new treatment system for local electroporation therapy (EPT) of head and neck tumors was developed and is described in this paper. EPT with bleomycin has been found to be very effective in killing cancer cells in vitro, in mouse tumor xenografts in vivo, and in tumors in humans. Ten head and neck cancer patients with recurring or unresponsive tumors were enrolled in a Phase I/II clinical trial. Treatment of the entire tumor mass in each of eight patients resulted in five complete responses confirmed by biopsy and MRI, and three partial responses (> or = 50% shrinkage). Two additional patients who received partial treatment of their tumor mass had local response where treated, but no overall lesion remission. Duration of the complete responses ranges from 2-10 months to date. All patients tolerated the treatment well with no significant local or systemic adverse effects.

Treatment of human pancreatic tumors xenografted in nude mice by chemotherapy combined with pulsed electric fields.

Cancer of the pancreas is currently the fifth leading cause of cancer related deaths with a five year survival of less than 1% In the United States (1). It is one of the most difficult cancers to treat, since it is hard to detect in the early stages. The patients remain asymptomatic until late in the course of the disease. An excellent review of pancreatic carcinoma has appeared (2). Despite the progress made in our understanding of the biology of this cancer (3), the final outcome for this disease has remained extremely poor. Conventional chemotherapeutic agents have not been very effective for human pancreatic adenocarcinoma (4). Use of intratumoral chemotherapy in combination with monoclonal antibodies have been reported to produce better response rate and also reduced toxicity (5,6). Smith and colleagues (7) have recently shown that an injectable gel with a sustained release profile can inhibit tumor growth in vivo in human pancreatic cancer xenografts. This was demonstrated in nude mice with BxPC-3 xenografts using fluorouracil, cisplatin, and doxorubicin with a consequent size reduction of the tumors between 72% and 79%, compared to the controls at day 28 after the first treatment. Although these figures are impressive, by any standard, no cure was reported.

Ex Vivo Stromal Cell Electroporation of Factor IX cDNA for Treatment of Hemophilia B.

Hemophilia B is an X-linked genetic disorder that typically results from chronic circulating deficiency of blood coagulation factor IX (FIX) (1). While the occurrence of hemophilia B is significantly less frequent than hemophilia A (factor VIII, deficiency) it has received special attention as a model for gene therapy. This is because hemophilia B is one of the least complicated genetic diseases from the point of view of demonstrating the proof of principle of a gene therapy protocol. Specifically, hemophilia B is a single gene recessive disorder and a wide range of tissues can be targeted for FIX gene delivery and strict regulation of FIX expression is not required. In addition, the 2.8 kb FIX cDNA is much smaller than the 9 kb FVIII cDNA, and FIX expression in transfected mammalian cells has been less problematic than FVIII expression (2). Since clinical severity of bleeding episodes closely corresponds to a patient's FIX activity, achieving even partial restoration of normal FIX levels in the bloodstream can alleviate internal bleeding. Individuals with FIX levels less than 1% of normal experience severe symptomatic episodes but providing roughly 5% of normal levels (i.e., 250 ng/mL plasma) can significantly reduce the frequency and severity of bleeding episodes and reduce long term complications (3). Treatment of hemophilia B primarily relies on intravenous injections of FIX protein purified from pooled human plasma, or very recently, on newly developed recombinant FIX. Treatment is applied typically only when bleeding episodes have occurred or are expected, for example, in case of a trauma or surgery. Although the risk of viral transmission of HIV and hepatitis viruses has been largely eliminated the absolute safety of any product derived from blood cannot be guaranteed. Furthermore, supplies of factor concentrates are limited and costs (especially if prophylactic treatment is being considered) are high. Thus, the application of gene therapy to hemophilia, whereby long-term correction of factor IX deficiency might be achieved, would be extremely useful.

Treatment of cancer using pulsed electric field in combination with chemotherapeutic agents or genes.

Electroporation is a standard laboratory technique originally developed for in vitro transfer of molecules into cells. It involves application of electrical pulses ranging from micro- to milliseconds that create transient pores in the cell membrane allowing intracellular access of exogenous molecules. This technique has been successfully applied to regress tumors in animal models by combining electroporation with chemotherapeutic agents--a process known as electrochemotherapy (ECT) which substantially enhance cytotoxicity of some antineoplastic agents. Recently ECT has moved into clinical arena and patients with cutaneous tumors and head and neck cancers have been treated very effectively with ECT. Parallel to ECT, a technique has also been developed which makes it possible to inject plasmid DNA and combine it with in vivo electroporation--electro--genetherapy (EGT)--to deliver in a highly efficient manner both marker and functional genes into target tissue and achieve gene expression. Thus, in vivo electroporation is contributing to the development of a new strategy for cancer treatment with both drugs and genes.

Effective Electroporation Therapy of Human Pancreatic Tumors Implanted in Nude Mice.

Electroporation therapy (EPT), also known as electrochemo-therapy (ECT), of a poorly differentiated human pancreatic carcinoma (Panc-3) implanted subcutaneously in nude mice significantly enhanced the cytotoxicity of bleomycin to tumor cells. A single treatment of intratumoral injection of bleomycin followed by 6 × 99 µs square wave electrical pulses of 1370 V resulted in complete tumor regression in 68% and partial regression (>80%) in 20% of the treated mice on day 28 following treatment. No palpable tumor was observed in 64% of the mice even 120 days after treatment. Histological studies of tissue samples taken from tumor sites 120 days after treatment in the D+E+ group showed complete absence of tumor cells.

Fourier transform infrared spectroscopy for the characterization of a model peptide-DNA interaction.

To better understand the structural basis of protein-DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29-residue synthetic peptide binds nonspecifically to heterogeneous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100-200 microM produces 50% saturation of heterogeneous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second-derivative spectra resolve the amide I band of native peptide into components located at 1627 (beta-strand), 1658 (alpha-helix), and 1681 (turn or beta-strand) cm-1, with a distinct shoulder at 1647 cm-1 (disordered structure). Assignment of the 1681 cm-1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and beta-strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm-1 (turn) is no longer seen; a new band appears at 1675 cm-1; the 1627 cm-1 band (beta-strand) is considerably reduced in intensity; the position of the alpha-helical (1658 cm-1) component remains unchanged; the shoulder at 1647 cm-1 (disorder) disappears. The new vibration at 1675 cm-1 is characteristic of beta-strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm-1 (bound); the relative intensities of vAS and the PO2- symmetric stretch (vS) are altered upon peptide binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Sustained local delivery of heparin to the rabbit arterial wall with an electroporation catheter.

Current methods of local drug delivery frequently fail to achieve a prolonged therapeutically effective tissue drug level without producing vascular trauma. A novel double-balloon catheter system, incorporating electroporation technology, has been designed and tested to deliver heparin into rabbit carotid arteries in an overstretch balloon injury model in vivo. Following arterial injury, fluoresceinated heparin was delivered into the volume between the two inflated balloons, and the artery was subjected to an electrical pulse. Catheter deployment and endovascular electrical pulsing were well-tolerated in all animals (N = 21) without adverse hemodynamic and histological changes. Periodic arterial blood samples revealed no abnormalities in the clotting profile or any gross morphological changes in the blood cells up to 8 hr after treatment. Much stronger heparin fluorescence was detected throughout the vessel layers for at least 12 hr in the pulsed samples compared to the control. Histochemical staining of the tissue showed intracellular localization of heparin. Endovascular electroporation may provide better retention and higher therapeutic efficacy than can be achieved by conventional systemic delivery of heparin at clinically safe concentrations.

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene.

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.