Extremely low frequency variable electromagnetic fields affect cancer and noncancerous cells in vitro differently: Preliminary study

The exposure to extremely low frequency electromagnetic field (ELF-EMF) may result in various changes at the cellular level. To identify the effect of ELF-EMF exposure on viability of cells, cancer cells (U87-MG; 143B) and noncancerous cells (BJ; HEK) in exponential growth phase were exposed or sham-exposed to different values of frequency (2, 20, 30, 50 and 60 Hz), different shapes (sinusoidal, square and triangular) and time of exposure (0.5, 1, 2, 3 h) to electromagnetic field. After exposure, viability of cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We found a different effect of exposition of cancer and noncancerous cells to ELF-EMF on viability of cells. This preliminary study revealed that electro magentic field(EMF) might serve as a potential tool for manipulating viability of cells.     

Antimelanoma effect of manool in 2D cell cultures and reconstructed human skin models

Melanoma is the most aggressive and lethal type of skin cancer, characterized by therapeutic resistance. In this context, the present study aimed to investigate the cytotoxic potential of manool, a diterpene from Salvia officinalis L., in human (A375) and murine (B16F10) melanoma cell lines. The analysis of cytotoxicity using the XTT assay showed the lowest IC₅₀ after 48 h of treatment with the manool, being 17.6 and 18.2 µg/ml for A375 and B16F10, respectively. A selective antiproliferative effect of manool was observed on the A375 cells based on the colony formation assay, showing an IC₅₀ equivalent to 5.6 µg/ml. The manool treatments led to 43.5% inhibition of the A375 cell migration at a concentration of 5.0 µg/ml. However, it did not affect cell migration in the B16F10 cells. Cell cycle analysis revealed that the manool interfered in the cell cycle of the A375 cells, blocking the G2/M phase. No changes in the cell cycle were observed in the B16F10 cells. Interestingly, manool did not induce apoptosis in the A375 cells, but apoptosis was observed after treatment of the B16F10 cells. Additionally, manool showed an antimelanoma effect in a reconstructed human skin model. Furthermore, in silico studies, showed that manool is stabilized in the active sites of the tubulin dimer with comparable energy concerning taxol, indicating that both structures can inhibit the proliferation of cancer cells. Altogether, it is concluded that manool, through the modulation of the cell cycle, presents a selective antiproliferative activity and a potential antimelanoma effect.     

The influence of experimentally generated turbulence on the Mash01 unicellular Microcystis aeruginosa strain

Phytoplankton experience a continuously changing fluid environment and the response to this is reflected at individual and community levels. The large-scale motions of winds, waves and artificial circulations are coupled by turbulence to the viscous small-scale environment of the phytoplankton cell. To investigate the significance of turbulence in the ecology of Microcystis aeruginosa, cultures were exposed to turbulent conditions using a vertically oscillating grid for a period of 7 days under controlled laboratory conditions. M. aeruginosa was exposed to a range of turbulent intensities, by adjusting the frequency of oscillation from 1 to 4 Hz. To improve the resolution of scale between turbulence phenomena and phytoplankton, flow cytometry and fluorescent probes were used to assess the response of M. aeruginosa. Metabolic activity and cell viability were monitored daily in both the turbulent cultures and quiescent control cultures using the FDA and Sytox green fluorescent probes, respectively. Initially, low turbulence levels generated by the grid at frequencies of 1 and 2 Hz stimulated metabolic activity, and did not affect cell viability compared to the control quiescent cultures. However, higher levels of turbulence generated by the grid at frequencies of 3 and 4 Hz were deleterious to metabolic activity and viability. Metabolic activity significantly decreased and over 85 % of cells were nonviable after 96 h at a grid oscillation of 4 Hz. It was concluded that due to the long lag time (>96 h) and high intensities needed to exert a deleterious effect, small-scale turbulence is unlikely to be a significant factor controlling M. aeruginosa compared to large scale motion which lead to changes in light and nutrient conditions.     

Difference in frequency spectrum of extremely-low-frequency effects on the genome conformational state of AB1157 and EMG2 E. coli cells

The effect of weak extremely-low-frequency (ELF) magnetic fields (sinusoidal, 30 μT amplitude) on the genome conformational state (GCS) of E. coli mutant and wild type cells was studied by using the method of anomalous viscosity time dependency (AVTD) in the 6–37 Hz frequency range. We confirmed the existence of three resonance frequencies of 8.9, 15.5, and 29.4 Hz when mutant cells of K12 AB1157 strain were exposed. In the same frequency range, the wild type K12 EMG2 cells displayed only two effective windows, with resonance frequencies of 8.3 and 27 Hz. The resonance frequencies differed significantly (P < .001–.000001) in the strains studied, whereas other resonance parameters did not. It was concluded that mutations in the AB1157 strain resulted in a significant rearrangement in the ELF action spectrum, including the appearance of a new resonance. © 1996 Wiley-Liss, Inc.     

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.     

Effects of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) on glioblastoma cells (U87)

The impact of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) at various frequencies and amplitudes was investigated on cell cycle, apoptosis and viability of the Glioblastoma Multiforme (GBM) cell line (U87), in vitro. The GBM is a malignant brain tumor with high mortality in humans and poorly responsive to the most common type of cancer treatments, such as surgery, chemotherapy and radiation therapy. U87 cells with five experimental groups (I–V) were exposed to various ELF-PEMFs for 2, 4 and 24 h, as follows: (I) no exposure, control; (II) 50 Hz 100 ± 15 G; (III) 100 Hz 100 ± 15 G; (IV) 10 Hz 50 ± 10 G; (V) 50 Hz 50 ± 10 G. The morphology properties, cell viability and gene expression of proteins involved in cell cycle regulation (Cyclin-D1 and P53) and apoptosis (Caspase-3) were investigated. After 24 h, the cell viability and Cyclin-D1 expression increased in Group II (30%, 45%), whereas they decreased in Groups III (29%, 31%) and IV (21%, 34%); P53 and Caspase-3 elevated only in Group III; and no significant difference was observed in Group V, respectively, compared with the control (p < 0.05). The data suggest that the proliferation and apoptosis of human GBM are influenced by exposure to ELF-PEMFs in different time-dependent frequencies and amplitudes. The fact that some of the ELF-PEMFs frequencies and amplitudes favor U87 cells proliferation indicates precaution for the use of medical devices related to the MFs on cancer patients. On the other hand, some other ELF-PEMFs frequencies and intensities arresting U87 cells growth could open the way to develop novel therapeutic approaches.     

In vitro effect of carbonic anhydrase inhibitor acetazolamide on cell viability,migration and colony formation of colorectal cancer cells

Acidification of extracellular medium in malignant tumors increases the invasive behaviors of cancer cells. In normal healthy tissues, acid production is catalyzed by carbonic anhydrases. Some of the carbonic anhydrase enzymes are overexpressed in certain types of cancer. The present study aimed to investigate the effect of acetazolamide, a potent carbonic anhydrase inhibitor, on in vitro cultivated cancer cells. Three different assays (MTT test, wound healing and clonogenic assay) were performed using human colorectal adenocarcinoma cells (SW620) to evaluate the suppressive effect of acetazolamide, on the colorectal cancer cells migration ability, colony formation and cell viability. The dose-dependent (1–1000 μM) reducing effect of acetazolamide on the cell viability was more significant within the first 48 h. This inhibitory effect of acetazolamide was found to be decreased at 72 h, and affects cells migration ability of cells at 24 and 48 h. Acetazolamide was observed to inhibit the cell viability, migration and colony formation ability of cells, depending on dose.     

Effective Skin Cancer Treatment by Topical Co-delivery of Curcumin and STAT3 siRNA Using Cationic Liposomes

The aim of the present study was to evaluate the effectiveness of iontophoretic co-delivery of curcumin and anti-STAT3 siRNA using cationic liposomes against skin cancer. Curcumin was encapsulated in DOTAP-based cationic liposomes and then complexed with STAT3 siRNA. This nanocomplex was characterized for the average particle size, zeta-potential, and encapsulation efficiency. The cell viability studies in B16F10 mouse melanoma cells have shown that the co-delivery of curcumin and STAT3 siRNA significantly (p < 0.05) inhibited the cancer cell growth compared with either liposomal curcumin or STAT3 siRNA alone. The curcumin-loaded liposomes were able to penetrate up to a depth of 160 μm inside the skin after iontophoretic (0.47 mA/cm²) application. The in vivo efficacy studies were performed in the mouse model of melanoma skin cancer. Co-administration of the curcumin and STAT3 siRNA using liposomes significantly (p < 0.05) inhibited the tumor progression as measured by tumor volume and tumor weight compared with either liposomal curcumin or STAT3 siRNA alone. Furthermore, the iontophoretic administration of curcumin-loaded liposome-siRNA complex showed similar effectiveness in inhibiting tumor progression and STAT3 protein suppression compared with intratumoral administration. Taken together, cationic liposomes can be utilized for topical iontophoretic co-delivery of small molecule and siRNA for effective treatment of skin diseases.     

Ethanol withdrawal tremor potentiates the tremorogenic action of nicotine

The tremorogenic effect of nicotine was studied in control rats and in rats withdrawn for 16-48 h from six to nine days' ethanol administration. The frequency and the intensity of tremor were measured electronically. A single dose of nicotine 5 mg/kg caused shortlasting (2-3 min) convulsions within 1 min after injection in control rats. Tremor occurred first after five repeated injections of nicotine (5 mg/kg) at half-hour intervals. This tremor encompassed peak frequencies of 6, 10 and 15 Hz. Hexamethonium at a dose of 10 mg/kg did not inhibit the tremor but eliminated the highest peak frequency (15 Hz) and tended to increase the intensity. Propranolol 5 mg/kg completely abolished the nicotine-induced tremor in control rats. Rats withdrawn from repeated ethanol administration showed a marked hypersensitivity to the tremorogenic action of nicotine so that a single dose of 1 mg/kg of nicotine caused clear tremor. The frequency spectra of this tremor showed peak frequencies at 6, 10 and 12 Hz. Hexamethonium did not change these frequencies. Furthermore, it tended to increase the intensity of nicotine-induced tremor in ethanol-withdrawn rats. Propranolol did not inhibit the tremor although it eliminated the 12 Hz peak frequency. The results suggest that the hypersensitivity to the tremorogenic action of nicotine in ethanol-withdrawn rats is not mediated by a sympathetic beta-adrenergic mechanism.     

Optimization of Electric Pulse Amplitude and Frequency In Vitro for Low Voltage and High Frequency Electrochemotherapy

During standard electrochemotherapy (ECT), using a train of 1,000 V/cm amplitude rectangular pulses with 1 Hz frequency, patients experience an unpleasant sensation and slight edema. According to the patients, muscle contractions provoked by high amplitude (about 1,000 V/cm) and low repetition frequency (1 Hz) pulses are the most unpleasant and painful sensations. Recently, ECT using low voltage and higher repetition frequency (LVHF) has been shown to be an effective tool for inhibiting tumor growth. The aim of the present study was to optimize electric pulse amplitude and repetition frequency for LVHF ECT by sampling the different sets of pulse parameters on cell viability and permeabilization. In ECT, a reversible effect based on high permeabilization is desirable. For this purpose, we used bleomycin to evaluate the permeabilization of K562 and MIA-PACA2 cells caused by low voltage (50–150 V/cm) and higher repetition frequency (4–6 kHz) electric pulses. We show that the reversible effect with electropermeabilization of the cells caused by LVHF ECT is accessible; this interaction is more effective for electric pulses with 70 V/cm amplitude.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

Dynamic characteristics of the human resonance EEG responses to rhythmic photostimulation

The development of the resonance EEG responses of the left and right occipital areas was studied in right-handed men during prolonged (12 or 120 s) rhythmic, photostimulation with the intensity of 0.7 J and frequencies of 6, 10, and 16 Hz. Analysis of the EEG fine spectral structure was applied to compare the accumulated baseline EEG spectra and EEG spectra during photostimulation, to observe the dynamics of the short-term spectra and to detect power changes in the EEG narrow spectral band sharply coincident with the stimulation frequency. The more pronounced EEG responses to photostimulation were observed in subjects with the initially low EEG baseline, α-rhythm. Two-minute flash trains produced a substantial increase in the EEG power within the stimulation frequency with superposed oscillatory processes with different periods. These fluctuations are considered a reflection of intricate interaction between the adaptive and resonance EEG responses to the presented intermittent stimulation. Under 12-s stimulation the resonance EEG responses are steadily recorded within the first 3 s of stimulation and immediately after the flash cessation EEG power at the stimulation frequency returns to the initial level. The resonance EEG responses were more pronounced in the right hemisphere than in the left one, especially, at the stimulation frequencies of 6 and 16 Hz. With increasing the stimulation frequency, the maximum of resonance EEG responses was reached earlier. Under the stimulation frequency of 6 Hz, the maximal response was recorded 9–12 s after the beginning of flashes, at the frequencies of 10 and 16 Hz, it was recorded within 3–6 and 3 s, respectively.     

Free‐Radical‐Scavenging,Antityrosinase, and Cellular Melanogenesis Inhibitory Activities of Synthetic Isoflavones

In this study, we examined the potential of synthetic isoflavones for application in cosmeceuticals. Twenty‐five isoflavones were synthesized and their capacities of free‐radical‐scavenging and mushroom tyrosinase inhibition, as well as their impact on cell viability of B16F10 murine melanoma cells and HaCaT human keratinocytes were evaluated. Isoflavones that showed significant mushroom tyrosinase inhibitory activities were further studied on reduction of cellular melanin formation and antityrosinase activities in B16F10 melanocytes in vitro. Among the isoflavones tested, 6‐hydroxydaidzein ( 2 ) was the strongest scavenger of both ABTS ^{. +} and DPPH ^. radicals with SC₅₀ values of 11.3±0.3 and 9.4±0.1 μM , respectively. Texasin ( 20 ) exhibited the most potent inhibition of mushroom tyrosinase (IC₅₀ 14.9±4.5 μM ), whereas retusin ( 17 ) showed the most efficient inhibition both of cellular melanin formation and antityrosinase activity in B16F10 melanocytes, respectively. In summary, both retusin ( 17 ) and texasin ( 20 ) exhibited potent free‐radical‐scavenging capacities as well as efficient inhibition of cellular melanogenesis, suggesting that they are valuable hit compounds with potential for advanced cosmeceutical development.     

Frequency-dependent interference by magnetic fields of nerve growth factor-induced neurite outgrowth in PC-12 cells

We have shown that 50 Hz sinusoidal magnetic fields within the 5-10 micro Tesla (μT) rms range cause an intensity-dependent reduction in nerve growth factor (NGF) stimulation of neurite outgrowth (NO) in PC-12 cells. Here we report on the frequency dependence of this response over the 15-70 Hz range at 5 Hz intervals. Primed PC-12 cells were plated in collagen-coated, 60 mm plastic petri dishes with or without 5 ng/ml NGF and were exposed to sinusoidal magnetic fields for 22 h in a CO₂ incubator at 37 °C. One 1,000-turn coil, 20 cm in diameter, generated vertically oriented magnetic fields. The dishes were stacked on the center axis of the coil to provide a range of intensities between 3.5 and 9.0 μT rms. The flux density of the ambient DC magnetic field was 37 μT vertical and 19 μT horizontal. The assay consisted of counting over 100 cells in the central portion (radius ≤0.3 cm) of each dish and scoring cells positive for NO. Sham exposure of cells treated identically with NGF demonstrated no difference in the percentage of cells with NO between exposed and magnetically shielded locations within the incubator. Analysis of variance demonstrated flux density-dependent reductions in NGF-stimulated NO over the 35-70 Hz frequency range, whereas frequencies between 15 Hz and 30 Hz produced no obvious reduction. The results also demonstrated a relative maximal sensitivity of cells at 40 Hz with a possible additional sensitivity region at or above 70 Hz. These findings suggest a biological influence of perpendicular AC/DC magnetic fields different from those identified by the ion parametric resonance model, which uses strictly parallel AC/DC fields. © 1995 Wiley-Liss, Inc.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

Anti-proliferative and cytotoxic activity of pentadactylin isolated from <Emphasis Type="Italic">Leptodactylus labyrinthicus</Emphasis> on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.