Dichloroacetate induces autophagy in colorectal cancer cells and tumours.

BACKGROUND: Dichloroacetate (DCA) has been found to have antitumour properties. METHODS: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, (1)H and hyperpolarised (13)C-magnetic resonance spectroscopy. RESULTS: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-(13)C] pyruvate-to-lactate exchange rate (k(PL)) were decreased in DCA-treated cells, along with increased NAD(+)/NADH ratios and NAD(+). Steady-state lactate excretion and k(PL) returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD(+) and NADH. Reduced k(PL) with DCA treatment was found in HT29 tumour xenografts in vivo. CONCLUSIONS: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k(PL) and increased NAD(+)/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

Effects of negative pions and neutrons on the growth of Vicia faba bean roots.

Vicia faba bean roots have been irratiated with neutrons of various energies and with negative pi-mesons, and the effect on the ten-day growth of the roots has been determened. The neutron irratiations were made in beams of 400 and 600 MeV maximum energy, as well as with neutrons from a plutonium-beryllium source (mean energy 4.4 MeV) and from a 14 MeV neutron generator. The bean roots have also been irradiated at various points along the depth-dose curve of negative pi-mesons, including the gegion where the pions annihilate on coming to rest. The results show a maximum relative biological effectiveness (RBE) of 3.7 for 50% reduction in ten days growth for stopped negative pions and values up to 3.3 for high-energy neutrons, compared to 5.5 for 14 MeV neutrons. The biological effectiveness of high-energy neutrons and stopped pions shows a more pronounced dependence on dose than does the effect with lower-energy neutrons.

Tension due to interaction between the sliding filaments in resting striated muscle. The effect of stimulation.

1. A resting sartorius muscle of the frog or toad possesses a special kind of elasticity which is shown to be due to a component lying between the two sets of filaments. The elastic effect is seen only for very small length changes, up to about 0.2% of the muscle length, and the ;elastic limit' is then reached. If the length change then continues at a constant velocity the tension developed is maintained at a fixed level, producing a sort of frictional resistance. The component responsible is called the ;short-range elastic component', or SREC.2. It is also shown that a small part of the permanent tension of a resting muscle is probably due to ;active' interaction between the filaments. This is called the ;filamentary resting tension', or FRT. For a sarcomere length of 2.0 mu the FRT amounts to about 150 mg in a muscle weighing 100 mg.3. The stiffness of the SREC and the magnitude of the FRT are shown to be related to one another. They are both increased, and may rise to high values, by making the external solution hypertonic.4. The working hypothesis is as follows. In a resting muscle the cross-bridges on the myosin filaments are not entirely inactive, but a very small proportion of them are cross-linked with the actin filaments. The links are very stable and have a long ;life'. The elastic behaviour is due to the flexural rigidity, or spring-like properties of these bridges. The elasticity is ;short-range' because the bridges can be bent, or stretched, only a small way from the steady-state position before the contacts ;slip'. The ;filamentary resting tension' (which is present in the absence of any external length change) is attributed to an ;active process', which operates by imparting ;organized' potential energy to the participating cross-bridges, by potentiating their attachment, against their elastic resistance, to sites on the actin filaments which are displaced towards the Z line.5. It is shown that the ability of the muscle to maintain a ;frictional' resistance to a continuing, slow, length change is suppressed during a maintained tetanic contraction. The process of suppression starts during the period of the latency relaxation.6. It is suggested that the latency relaxation may be due to a reduction of the filamentary resting tension.7. The experiments with the latency relaxation were facilitated by the use of strongly hypertonic solutions. The positive twitch tension is then greatly reduced, or may be practically abolished, while the latency relaxation remains at about its normal size and is extended in duration.

The effect of temperature in the range 0-35 degrees C on the resting tension of frog's muscle.

1. It has long been known that a resting muscle shows a reversible ;rubber-like' response to a change of temperature: it develops tension when warmed and this reverses on cooling. It was recently shown (Hill, 1968) that part of the resting tension is due to a weak interaction between the sliding filaments, and it was suggested that the rubber-like tension might be caused by the same process rather than by some inert structural component. Further information on this point has now been obtained from some observations on the effect of temperature on isometric tension at temperatures not far below the level (28-35 degrees C) at which a heat contracture is produced.2. The temperature of a frog's sartorius was changed by moving it up and down in a vessel containing Ringer solution at two different temperatures. The temperature in one part was maintained constant in the range 0-20 degrees C and in the other at 5-35 degrees C. The changes was 90% complete in 12 sec.3. From 0 degrees up to about 23 degrees C the tension developed per 1 degrees C rise of temperature (DeltaP/DeltaT) is almost constant, but as the temperature approaches the level at which a heat contracture appears the value of DeltaP/DeltaT rises steeply. This behaviour indicates that the tension in question has an ;active' rather than a ;passive' origin, and this view is supported by other evidence from experiments with muscles in hypertonic solutions, described in the next paper.4. It was found that the temperature at which a muscle goes into a heat contracture depends on the length at which it is set; the critical temperature is lower in an extended muscle. A shortened muscle at a temperature not far below the critical value will therefore produce a contracture when it is stretched.

The effect of temperature on the resting tension of frog's muscle in hypertonic solutions.

1. It has long been known that the tension of a resting muscle rises when it is warmed and falls on cooling. It is shown here that treatment with hypertonic solution causes an alteration in this effect; the usual rise or fall of tension with warming or cooling is then preceded by a transient change in the opposite direction.2. The magnitude of the abnormal reversed phase does not depend in a simple, linear, manner on the temperature change. For instance, the effect for cooling from 20 to 0 degrees C is much greater than twice that for 10 to 0 degrees C.3. The results lead to the suggestion that the tension, in both normal and hypertonic muscle, comes from an active process. It is proposed that a temperature change operates by altering the concentration of an ;activating substance', and that the change of concentration becomes delayed by hypertonicity.

Resting tension and the form of the twitch of rat skeletal muscle at low temperature.

1. A study has been made of the effect of temperature on the resting tension and on the form of the isometric twitch of isolated rat skeletal muscle. The soleus and the extensor digitorum longus (EDL) were used as representatives of slow and fast types of muscle. The main interest is in the behaviour at low temperature, when rat muscle shows characteristics which are not found with frog muscle.2. At higher temperatures both soleus and EDL of the rat resemble frog muscle in showing a ;rubber-like' form of resting tension, i.e. the tension increases with warming: this has not previously been demonstrated in mammalian muscle. But at low temperatures (below 15 degrees C) the converse is observed; tension then increases as temperature falls. This ;cold tension' is much larger with soleus than with EDL. The ;cold tension' is the converse of the ;rubber-like' tension also in its dependence on length; it becomes smaller when the muscle is stretched.3. These muscles remain excitable and give consistent twitches with transverse stimulation at temperatures down to about 3 degrees C provided the exposure to cold is not prolonged beyond a few minutes. The amplitude of the twitch is reduced by cooling, e.g. at 3 degrees C it is about 30-50% of that at 25 degrees C.4. The twitch becomes very prolonged at low temperatures. With a soleus at 3-4 degrees C it may last 40 sec. The temperature coefficient of the rate constant of the relaxation phase is very high compared with that of frog's muscle.5. The rate constant of relaxation in a twitch at low temperature increases when the muscle is stretched. This is the reverse of what happens at higher temperature.6. The extreme prolongation of the twitch with cooling indicates a very large decrease in the rate of decay of the active state, and it is suggested that the increase of resting tension at low temperature is due to an accumulation of ;activator'.

Metabolic changes associated with the slowing of relaxation in fatigued mouse muscle.

1. The biochemical basis of the slowing of relaxation seen in fatigue has been examined using an isolated mouse soleus preparation. 2. Slowing of relaxation occurred during prolonged tetani under anaerobic conditions when ATP and PC fell and lactate accumulated. 3. Slowing of relaxation was also demonstrated with muscles poisoned with cyanide and iodoacetic acid when there was a fall in ATP and PC but no accumulation of lactate. During a period of anaerobic recovery following a fatiguing tetanus, relaxation became faster at a time when lactate was accumulating in the muscle. 4. It is concluded that the slowing of relaxation in fatigue is not a consequence of lactate accumulation, and a relationship is demonstrated between the ATP content of the muscle and the rate of relaxation in muscles fatigued by prolonged stimulation, 5. Rates of ATP turn-over in fresh muscle, and at intervals throughout a tetanus are consistent with the suggestion that the rate limiting step for myofibrillar ATPase may be directly related to the rate limiting step for the decay of tension during relaxation.

Heat production and chemical changes during isometric contractions of the human quadriceps muscle.

1. Development of a new thermal probe and use in conjunction with chemical analysis of needle biopsy samples, has made possible a thermodynamic study of the energetics of muscular contraction in the human quadriceps. 2. The observed rate of muscle temperature rise was proportional to the force of the contraction. During maximal contractions the rate of heat production was 54 +/-8-5 W/kg wet muscle (mean +/- s.d.). 3. The observed rates of muscle temperature rise agreed well with the rates calculated from the measured metabolite changes when standard values for the enthalpy changes of the reactions involved were used. 4. During prolonged stimulation of the quadriceps at 15/sec via the femoral nerve, the rate of heat production per unit force fell to nearly half the initial value. It is estimated that this represented a two- to fourfold increased in economy of ATP turn-over required to maintain a given force. 5. Relaxation becomes progressively slower during prolonged contractions and it is suggested that the slowing of relaxation and the increased economy of force maintenance may both be due to an increased cross-bridge cycle time in the fatigued muscle.

Scope of a technique for electrical stimulation of human brain, spinal cord, and muscle.

Brief high-voltage electrical shocks from a special low-output-resistance stimulator, delivered through electrodes on the skin, can excite human muscle directly (not by way of the nerves) and can also excite the motor cortex, the visual cortex, and the spinal cord. Possible applications of the technique include measurement in muscle disorders of the latency relaxation and of the excitability and contractility of muscle without the interposition of nerve fibres or the neuromuscular junction; measurement of conduction velocity in the pyramidal tract; and the detection of neuropathy in the nerves to the external sphincter ani.

Fatigue of long duration in human skeletal muscle after exercise.

1. After severe muscular contraction in man recovery of force is largely complete in a few minutes, but is not wholly so for many hours. The long-lasting element of fatigue is found to occur primarily for low frequencies of stimulation (e.g. 20/sec), and is much less pronounced, or absent, at high frequencies (80/sec). The twitch force is an unreliable measure of the state of fatigue. 2. The long-lasting element of fatigue is not due to depletion of high-energy phosphate nor is it due to failure of electrical activity as recorded from surface electrodes. It is probably the result of an impairment of the process of excitation-contraction coupling. Its practical importance for man could be significant as an explanation of the subjective feelings of weakness following exercise.