Cancer-cell Death

If funding is obtained, this research would attempt to confirm the findings of an unofficial study that a specific combination of frequency, amplitude and phasing of a wave can cause cancer cells to die.  

BACKGROUND – APOPTOSIS

The body can correct errors.  A built-in “auto destruct mechanism,” limits survival and expansion of a harmful or malignant cell, such as a cancer cell. This process, called ‘apoptosis’, is a key research strategy against cancer.  All experiments in an unofficial study showed an increase in acetylated p53, meaning that apoptosis, an increase in the rate of cell death, is likely to have occurred by the method employed.  Technical data and outcomes follow. 

Testing on flasks of cancer cells

Through a microscope – round blobs are dead cancer cells

 

 

 

Materials and methods for Western Blotting detecting levels of total and acetylated at K320 p53.

U2OS human osteosarcoma cells were seeded in 100mmx20mm plates and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% foetal calf serum (FCS) and 1% 10U/ml penicillin and streptomycin. The cells were subjected to frequency and amplitude wave therapy for 0 (control), 30, 60, 90 and 120 minutes/plate. The same experiment (with variations in phasing) was repeated four times.

Following the wave therapy procedure cells were washed three times with PBS buffer (Sigma) and lysed in TNN lysis buffer (50mM Tris-HCl, pH 7.4, 120mM NaCl, 5mM EDTA, 0.5% Igepal), containing 1mM dithiothreitol (DTT), 1mM phenyl-methylsulphonyl fluoride (PMSF) and a protease inhibitor mixture (Rosche). Cell extracts were incubated at 4°C with rotation for 20 minutes. After establishing protein content of the sample supernatants, the normalised samples containing equal amounts of  protein were subjected to a 10% SDS-PAGE gel and blotted on nitrocellulose membranes. Non-specific antibody binding was blocked by incubating the membranes overnight with 5% non-fat milk in PBS.

The primary antibodies used were the p53-specific acetylated at lysine 320 rabbit polyclonal antibody (diluted 1:2000, Upstate) and the p53-specific mouse monoclonal antibody DO-1 (diluted 1:15000, Santa Cruz Biotechnology, Santa Cruz, CA). These were introduced into 0.1% Tween 20 in PBS containing 2.5% non-fat milk in which membranes were incubated at 4°C overnight. After washing three times with PBS/ 0.1% Tween 20, membranes were left to incubate in 0.1% Tween 20 in PBS 2.5% non-fat milk containing goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated antibodies (1:1600). Three washes with PBS Tween/ 0.1% Tween 20 followed and chemiluminescent signal was detected by subjecting the membranes to the ECL reagent (Amersham Biosciences).

 
Above is a copy of slides taken from the five plates of cancer cells.  0 minutes is the control plate, plus plates that had 30, 60, 90 and 120 minutes of wave therapy.

Top rows show total p53 protein level
Total p53 erratic, maybe with an inclination to decline over time. 
There is no indication of whether this is active or inactive p53, so this measure is of no benefit.

Bottom rows show acetylated p53
All experiments show an increase in acetylated p53. 
This shows that apoptosis, an increase in the rate of cell death, is likely to have occurred. 
CREATE uses similar methods to try to increase the death rate of cancer cells in patients.  

Funding of this research study may validate these initial findings, and confirm that such therapeutic approaches may help resolve cancer.