Treatment with Uncaria tomentosa Promotes Apoptosis in B16-BL6 Mouse Melanoma Cells and Inhibits the Growth of B16-BL6 Tumours.

Uncaria tomentosa is a medicinal plant native to Peru that has been traditionally used in the treatment of various inflammatory disorders. In this study, the effectiveness of U. tomentosa as an anti-cancer agent was assessed using the growth and survival of B16-BL6 mouse melanoma cells. B16-BL6 cell cultures treated with both ethanol and phosphate-buffered saline (PBS) extracts of U. tomentosa displayed up to 80% lower levels of growth and increased apoptosis compared to vehicle controls. Treatment with ethanolic extracts of Uncaria tomentosa were much more effective than treatment with aqueous extracts. U. tomentosa was also shown to inhibit B16-BL6 cell growth in C57/bl mice in vivo. Mice injected with both the ethanolic and aqueous extracts of U. tomentosa showed a 59 ± 13% decrease in B16-BL6 tumour weight and a 40 ± 9% decrease in tumour size. Histochemical analysis of the B16-BL6 tumours showed a strong reduction in the Ki-67 cell proliferation marker in U. tomentosa-treated mice and a small, but insignificant increase in terminal transferase dUTP nick labelling (TUNEL) staining. Furthermore, U. tomentosa extracts reduced angiogenic markers and reduced the infiltration of T cells into the tumours. Collectively, the results in this study concluded that U. tomentosa has potent anti-cancer activity that significantly inhibited cancer cells in vitro and in vivo.

Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells.

Exposure to specific electromagnetic field (EMF) patterns can affect a variety of biological systems. We have shown that exposure to Thomas-EMF, a low-intensity, frequency-modulated (25-6 Hz) EMF pattern, inhibited growth and altered cell signaling in malignant cells. Exposure to Thomas-EMF for 1 h/day inhibited the growth of malignant cells including B16-BL6 mouse melanoma cells, MDA-MB-231, MDA-MB-468, BT-20, and MCF-7 human breast cancer and HeLa cervical cancer cells but did not affect non-malignant cells. The Thomas-EMF-dependent changes in cell proliferation were mediated by adenosine 3',5'-cyclic monophosphate (cAMP) and extracellular-signal-regulated kinase (ERK) signaling pathways. Exposure of malignant cells to Thomas-EMF transiently changed the level of cellular cAMP and promoted ERK phosphorylation. Pharmacologic inhibitors (SQ22536) and activators (forskolin) of cAMP production both blocked the ability of Thomas-EMF to inhibit cell proliferation, and an inhibitor of the MAP kinase pathway (PD98059) was able to partially block Thomas-EMF-dependent inhibition of cell proliferation. Genetic modulation of protein kinase A (PKA) in B16-BL6 cells also altered the effect of Thomas-EMF on cell proliferation. Cells transfected with the constitutively active form of PKA (PKA-CA), which interfered with ERK phosphorylation, also interfered with the Thomas-EMF effect on cell proliferation. The non-malignant cells did not show any EMF-dependent changes in cAMP levels, ERK phosphorylation, or cell growth. These data indicate that exposure to the specific Thomas-EMF pattern can inhibit the growth of malignant cells in a manner dependent on contributions from the cAMP and MAP kinase pathways. Bioelectromagnetics. 39;217-230, 2018. © 2017 Wiley Periodicals, Inc.

The effects of electromagnetic fields on B16-BL6 cells are dependent on their spatial and temporal character.

Exposure to low intensity, low frequency electromagnetic fields (EMF) has effects on several biological systems. Spatiotemporal characteristics of these EMFs are critical. The effect of several complex EMF patterns on the proliferation of B16-BL6 mouse melanoma cells was tested. Exposure to one of these patterns, the Thomas-EMF, inhibited cell proliferation and promoted calcium uptake. The Thomas-EMF is coded from a digital-to-analog file comprised of 849 points, which provides power to solenoids and can be set to alter timing, intensity, and duration of variable EMF. Setting the point duration to 3 ms generated a time-varying EMF pattern which began at 25 Hz and slowed to 6 Hz over a 2.5 s repeat. Exposing B16-BL6 cells to Thomas-EMF set to 3 ms for 1 h/day inhibited cell proliferation by 40% after 5 days, while setting the point duration to 1, 2, 4, or 5 ms had no effect on cell proliferation. Similarly, exposing cells to Thomas-EMF set to 3 ms promoted a three-fold increase in calcium uptake after 1 h, while the other timings had no effect. Exposure to Thomas-EMF for as short as 15 min/day slowed cell proliferation, but exposure for 1 h/day was optimal. This corresponded to the effect on calcium uptake where uptake was detected after 15 min exposure and was maximal by 1 h of treatment. Studies show that the specific spatiotemporal character of EMF is critical in mediating their biological activities. Bioelectromagnetics. 38:165-174, 2017. © 2016 Wiley Periodicals, Inc.

Biophoton emissions from cell cultures: biochemical evidence for the plasma membrane as the primary source.

Photon emissions were measured at ambient temperature (21°C) in complete darkness once per min from cultures of 10(6) cells during the 12 h following removal from 37°C. The energy of emission was about 10(-20) J/s/cell. Of 8 different cell lines, B16-BL6 (mouse melanoma cells) demonstrated the most conspicuous emission profile. Acridine orange and ethidium bromide indicated the membranes were intact with no indication of (trypan blue) cell necrosis. Treatments with EGF and ionomycin produced rapid early (first 3 h) increases in energy emission while glutamine-free, sodium azide and wortmanin-treated cells showed a general diminishment 3 to 9 h later. The results suggested the most probable origin of the photon emission was the plasma cell membrane. Measures from cells synchronized at the M- and S-phase supported this inference.

Treatment with flaxseed oil induces apoptosis in cultured malignant cells.

Flaxseed oil is widely recognized for its exceptional nutritional value, high concentration of fiber-based lignans and large amounts of ω-fatty acids. It is one of a generic group of functional foods that is often taken by cancer patients as a potential treatment. We have examined the anti-cancer effects of flaxseed oil by studying its direct effects on cancer cell growth in vitro. Treatment of a variety of cancer cell lines with flaxseed oil decreased their growth in a dose-dependent manner while non-malignant cell lines showed small increases in cell growth. Cells treated with a mixture of fatty acids, including α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid and lignans including enterodiol and enterolactone was also able to decrease the growth of cancer cells. Treatment of B16-BL6 murine melanoma and MCF-7 breast cancer cells with flaxseed oil induced apoptosis as determined by changes in cell morphology, annexin V staining, DNA fragmentation and/or caspase activation. In addition, treatment with flaxseed oil also disrupted mitochondrial function in B16-BL6 and MCF-7 cells. These results indicate that flaxseed oil can specifically inhibit cancer cell growth and induce apoptosis in some cancer cells and suggests it has further potential in anti-cancer therapy.

Frequency of Immune Cell Subtypes in Peripheral Blood Correlates With Outcome for Patients With Metastatic Breast Cancer Treated With High-Dose Chemotherapy.

BACKGROUND: The frequency of circulating leukocytes has been shown to be a prognostic factor in patients being treated for different types of cancer. In breast cancer, tumor-infiltrating leukocytes may predict patient outcome, but few studies have investigated such associations for circulating leukocytes. PATIENTS AND METHODS: Multiparametric flow cytometry was used to examine the immunophenotypes of circulating peripheral blood mononuclear cells for 88 patients with metastatic breast cancer, which was then correlated to breast cancer-specific survival. Patients had been treated either with high-dose cyclophosphamide-containing regimens (group 1, n = 51 patients) or high-dose paclitaxel-containing regimens (group 2, n = 37 patients). RESULTS: The frequency of peripheral blood CD14+ monocytes indicated prognosis for patients in group 1 (but not group 2), while higher levels of CD11c+ dendritic cells indicated a better prognosis for patients in group 2 (but not group 1). The frequency of a number of different CD4+ or CD8+ T cell subtypes also predicted prognosis for patients in group 2. For example, patients in group 2 with a higher frequency of circulating CD4+ or CD8+ naive T cells (CD45RA+CD95-CD27+CD28+) showed a poorer prognosis. In contrast, T cells were not associated with prognosis for patients in group 1. CONCLUSION: Circulating leukocytes can predict clinical outcome for patients with breast cancer. Prediction of clinical outcome in this cohort of metastatic breast cancer patients was specific to the type of chemotherapy, and this finding is likely to apply to other therapies.

Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) Sensitizes THP-1 Cells to Radiation-induced Cell Death.

BACKGROUND: Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae), known as Cat's Claw or Uña de gato, is a traditionally used medicinal plant native to Peru. Some studies have shown that U. tomentosa can act as an antiapoptotic agent and enhance DNA repair in chemotherapy-treated cells although others have shown that U. tomentosa enhanced apoptosis. OBJECTIVE: To determine if treatment with U. tomentosa can significantly enhance cell death in THP-1 cells exposed to ionizing radiation. MATERIALS AND METHODS: THP-1 monocyte-like cells were treated with ethanolic extracts of U. tomentosa in the presence or absence of bacterial lipopolysaccharide and then exposed to ionizing radiation. Cell proliferation was assessed by MTT and clonogenic assays and the effects on cell cycle measured by flow cytometry and immunoblotting. Changes in cell signaling were determined by immunoblotting and cytokine ELISA and activation of apoptosis measured by caspase activation and DNA fragmentation analysis. RESULTS: Treatment of THP-1 cells with U. tomentosa had a small effect on cell proliferation. However, when the U. tomentosa-pretreated cells were also subjected to 5-9 Gy ionizing radiation, they showed a significant decrease in cell proliferation and increased cellular apoptosis as measured by DNA fragmentation and caspase activation. Treatment with U. tomentosa also decreased the expression of Cyclin E and Cyclin B, key regulators of normal cell cycle progression, and decreased the phosphorylation of various stress-activated, cell survival proteins including p38, ERK, and SAP/JNK kinase. CONCLUSIONS: These results suggest that U. tomentosa could be useful in enhancing cell death following anticancer therapies including ionizing radiation. SUMMARY: Treatment of THP-1 cells with Uncaria tomentosa increases their susceptibility to X-rays. The combination of Uncaria tomentosa and X-ray exposure strongly inhibits cell signaling and promotes apoptosis. Abbreviations Used: LPS: Lipopolysaccharide, TNF: Tumor necrosis factor: IL-1, Interleukin-1: SDS: Sodium dodecylsulphate, TBS: Tris-buffered saline.

Inhibition of cancer cell growth by exposure to a specific time-varying electromagnetic field involves T-type calcium channels.

Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca(2+) influx which could be blocked by inhibitors of voltage-gated T-type Ca(2+) channels. Blocking Ca(2+) uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca(2+) influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.

Digitized quantitative electroencephalographic patterns applied as magnetic fields inhibit melanoma cell proliferation in culture.

Weak (1 µT) physiologically patterned magnetic fields produce changes in behavioral, physiological, and cellular activity. In the present experiments 12 temporal samples of the electroencephalographic anomaly and normal activity of a person (SLH) whose proximity reliably affected the brain activity of others were extracted from QEEG data, digitized, and presented as equivalent magnetic field patterns to B16 mouse melanoma cells. Only two of the patterns, both originating from the primary source (right temporal lobe) of the EEG anomaly reduced the cell growth by one-third compared to the other patterns extracted from his QEEG or sham field exposures. In previous experiments these EEG transients were also associated with marked increases in photon emissions from the right side of SLH's head. The results suggest that the intrinsic complexity of electroencephalographic patterns of some people, when amplified appropriately and applied as computer-generated magnetic fields in the three spatial planes, could diminish cancer cell growth.

Photon emissions from human brain and cell culture exposed to distally rotating magnetic fields shared by separate light-stimulated brains and cells.

Light flashes delivered to one aggregate of cells evoked increased photon emission in another aggregate of cells maintained in the dark in another room if both aggregates shared the same temporospatial configuration of changing rate, circular magnetic fields. During the presentation of the same shared circumcerebral magnetic fields increases in photon emission occurred beside the heads of human volunteers if others in another room saw light flashes. Both cellular and human photon emissions during the light flashes did not occur when the shared magnetic fields were not present. The summed energy emissions from the dark location during light stimulation to others was about 10(-11) W/m(2) and calculated to be in the order of 10(-20) J per cell which is coupled to membrane function. These results support accumulating data that under specific conditions changes in photon emissions may reflect intercellular and interbrain communications with potential quantum-like properties.

Growth of injected melanoma cells is suppressed by whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields.

PURPOSE: To measure the effect of exposure to a specific spatial-temporal, hysiologically-patterned electromagnetic field presented using different geometric configurations on the growth of experimental tumours in mice. METHODS: C57b male mice were inoculated subcutaneously with B16-BL6 melanoma cells in two blocks of experiments separated by six months (to control for the effects of geomagnetic field). The mice were exposed to the same time-varying electromagnetic field nightly for 3 h in one of six spatial configurations or two control conditions and tumour growth assessed. RESULTS: Mice exposed to the field that was rotated through the three spatial dimensions and through all three planes every 2 sec did not grow tumours after 38 days. However, the mice in the sham-field and reference controls showed massive tumours after 38 days. Tumour growth was also affected by the intensity of the field, with mice exposed to a weak intensity field (1-5 nT) forming smaller tumours than mice exposed to sham or stronger, high intensity (2-5 microT) fields. Immunochemistry of tumours from those mice exposed to the different intensity fields suggested that alterations in leukocyte infiltration or vascularisation could contribute to the differences in tumour growth. CONCLUSIONS: Exposure to specific spatial-temporal regulated electromagnetic field configurations had potent effects on the growth of experimental tumours in mice.

Cell adhesion and cancer: is there a potential for therapeutic intervention?

Carcinogenesis involves a disruption in adhesion molecule expression and tissue architecture, and tumour invasion requires adhesion-dependent migration into surrounding tissues. Therefore, a variety of peptide and antibody-based reagents that block integrins, cadherins, immunoglobulin superfamily and selectin adhesion molecules have been developed to treat cancers. Therapeutics directed at adhesion molecules can block interactions between tumour cells, endothelial cells and immune cells to prevent tumour cell invasiveness and metastasis. Blocking the adhesion molecules that facilitate the invasion of tumours by endothelial cells and immune cells can prevent tumour-associated angiogenesis and the recruitment of immune-mediated growth factors which are required for tumour growth and spread. In addition, targeted therapies using anticancer agents attached to antibodies or peptides directed as tumour-specific adhesion molecules are being developed.