Thursday, July 12, 2012

Sleep Drugs and Cancer

Sleep drugs (commonly referred to as hypnotic drugs in the pharmaceutical industry) are widely prescribed with an estimated 6-12% of U.S. adults using some form of sleep drug in 2010 and even higher estimates of use in Europe.1,2 Unfortunately one reason the overall level of consumption, both in unique use and repeat use of sleep drugs, is so high is because they do not cure insomnia instead simply reduce its effects. Basically they treat the symptoms of a chronic condition instead of treating the underlying cause. Despite not addressing the cause directly, reducing the influence of insomnia at any level is an important issue because chronic insomnia is thought to significantly increase the probability of developing psychiatric ailments and also reduces wakeful efficiency, productivity and health.2-5

Sleep drugs are commonly divided into pharmacological agents and non-pharmacological agents. While non-pharmacological agents, which range from stimulus control strategies to sleep pattern development with relaxation therapy, are viewed as an initial treatment, most research focuses on pharmacological agents, which are further sub-divided into two categories: benzodiazepines and non-benzodiazepines. The most common sleep drugs are zolpidem, temazepam, eszopiclone and zaleplon with zolpidem as the most prescribed sleep drug between 2002 and 2006 with temazepam in second place.1

Unfortunately meta-analysis has revealed some disturbing information regarding the consumption of sleep inducing drugs and overall mortality. When compared against placebo a number of trials involving commonly used sleep drugs demonstrate a significantly higher rate of cancer including pancreatic, non-melanoma skin, lymphoma, lung, colon or prostate cancer.1,6 The rate of death for those who consume these drugs increases more than three times over those who do not consume these drugs even at the smallest dosage (1-18 pills per year).1 Not surprisingly the probability of death increases as individuals increase the dosage. Also there was no significant difference between the different drugs in relation to how they increase the probability of death,1 thus it appears that these drugs operate over the same or at least a similar mechanism.

One of the more concerning issues with this increased rate of death is that the time variance is scatted; there are probability increases in both short-term and long-term rates of death. The reasoning behind the short-term death increases is currently unknown (peptic ulcers and esophageal damage due to regurgitation are leading theories), but most believe that long-term deaths increases are due to increased rates of cancer.1 In fact in one study the top third of sleep drug consumers (> 132 pills per year) had a 35% greater chance of developing cancer versus non-consumers.1 Another study monitoring 13,177 individuals taking zopiclone determined that 42% of the total deaths were due to cancer.7

The rationality behind the increased probability of cancer development has largely revolved around increasing infections and/or inflammation. The prevailing theory is that sleep drugs somehow suppress immune function.1,6 This suppression of immune function leads to reduced abnormal cell and pathogen destruction resulting in greater rates of cancer and other infections. However, this explanation may not be the only one that accurately describes the increased rates of cancer in individuals that consume sleep drugs.

Most sleep drugs are either benzodiazepines or operate with a similar mechanism of influence on GABAA receptors. Benzodiazepines are agnoists for most GABAA receptors, which increase frequency and duration of their activation. The mechanism of action increases the firing of GABAergic neurons,which reduces the firing rate of excitory neurons increasing the probability of initating sleep and its duration. Application of benzodiazepines result in sedative, anxiolytic, anti-convulsant and hypnotic characterization in the user. There are three types of benzodiazepines largely defined through their residance times: short, intermediate or long.8 Short and intermediate mechanisms are used in controlling insomnia and long mechanisms are used to control anxiety. However, because these sleep compounds are only GABA agnoists their effectiveness is still contingent on the total concentrations of GABA.

Gamma-amino butyric acid (GABA) plays three critical roles in the brain as a signaling molecule, neurotransmitter and metabolite. During development GABA guides neurite outgrowth and directionality.9 Once development of the Central Nervous System (CNS) is complete GABA then alters its function becoming the chief inhibitory neurotransmitter for both the CNS and Peripheral Nervous System (PNS). As the chief inhibitory neurotransmitter GABA plays a role in various neurodegenerative diseases most notably Temporal Lobe Epilepsy (TLE), Parkinson’s Disease (PD) and Huntington’s Disease (HD) stemming from a breakdown in critical components that govern GABA regulation.10-13 However, there is also evidence that GABA plays an important role in the development and progression of certain types of cancer.

GABA has three corresponding biological receptors, which are classified as GABAA, GABAB and GABAC (a.k.a. GABAA-rho). The ion largely associated with GABA receptors is chloride (Cl-), which drives the inhibitory action of GABA. GABAA exists in two activator based constructs, nicotinic and muscimol, and are oligomeric comprised of five different subunits from a pool of seven possible (alpha1-6, beta1-3, gamma1- 3, sigma, epsilon, pi and theta).14-16 GABAA receptors are ionotropic meaning that when an appropriate molecular agent binds the receptor forms a channel/pore in the membrane that allows an appropriate ion to pass through the membrane (direction of passage is largely governed by concentration and charge gradients).17

During development GABAA receptors are more commonly excitatory over inhibitory due to the absence of a chloride pump on the membrane that transfers chloride ions from inside the membrane to the extracellular space. Without this pump there is an excess amount of chloride ions in the cell, thus when the GABAA receptor activates chloride ions escape the neuron along the concentration gradient increasing membrane potential increasing probability of depolarization. Upon the incorporation of the chloride pump the chloride concentration gradient reverses so upon GABAA activation chloride ions flow from the extracellular space into the neuron increasing the probability of hyperpolarization. GABAB receptors are metabotropic activating a G-protein pathway. A majority of GABAB receptors are located on pre-synaptic cells and act as feedback mechanisms.

The third class of GABA receptors, GABAC, is somewhat controversial in whether or not it is uniquely different enough from GABAA to be considered a separate class.18 GABAC receptors are typically insensitive to GABAA receptor allosteric modulators like benzodiazepine and barbiturates because they are exclusively composed of a unique subunit (rho.18,19 This composition does not significantly change the functionality of GABAC receptors compared to GABAA receptors with respect to their interaction with GABA.

Each subunit in a GABAA receptor possesses four hydrophobic membrane-spanning domains.17 Studies have shown that functional GABAA receptors contain at least one alpha and one beta with one gammasubunit typically also involved; sigma, epsilon, pi and thetasubunits are thought to be assembled into GABAA receptors in place of subunits or complementary pairings.20 Overall it is rare to have a GABAA receptor comprised of subunits that lack an alpha or a beta and such a conformation will not be functional.

The importance of receptor composition is largely demonstrated in how individuals subunits are able to confer different sensitivities to GABA and its associated agonists and antagonists.21,22 For example pi subunits appear to highly sensitive to excitation by loreclezole (where non-pi subunit receptors are either unaffected or inhibited), inhibited by lanthanum and unaffected by benzodiazepine diazepam.19,23 Specificially the pi subunit has drawn interest with respects to the role of GABA in the development of cancer.24

Using cDNA libraries the pi subunit was isolated to multiple reproductive tissue (uterus, ovaries, etc.), digestive tissue (gall bladder, small intestine) and specific regions of the brain namely the hippocampus and temporal cortex; two of the major expression cell types in the brain are teratocarcinoma NT2 neuronal precursor and terminally differnetiated NT2-N cells.25,26 However, while NT2 neuronal cells express pi subunit mRNA there is some question to whether those pi subunits are actually incorported into NT2 neuronal GABA receptors.19 Unfortunately despite the apparent importance of the pi subunit, both the developmental expression of the epsilon and pi subunits have yet to be isolated, but both have been cloned.27 From cloning analysis the pi subunit has a 37% relation to the beta subunit, a 35% relation to the sigma subunit and a 33% relation to the rho subunit with very little relation on any of the other subunits.11 Most specifically the pi subunit appears to have similarities to alpha-5, beta-3 and gamma-3.19,27

This similarity of the pi subunits to these other subunits is not surprising in that the pi subunits are commonly incorporated into receptors with alpha-5beta-3 or alpha-5beta-3gamma-3 configurations.19 With respect to insomnia the amplification of benzodiazepine sensitivity is governed by the type of gamma subunit which determines extent of benzodiazepine influence with the requirement of a gamma subunit for a GABA receptor to have signiifcant affinity for benzodiazepines.16,19 Most GABA receptors in the brain have gamma-2 subunits, which demonstrate the highest gamma subunit sensivity to benzodiazepines.16 Receptors with pi subunits are thought to interfere with the ability of the gamma subunit to form the benzodiazepine binding site by either replacing the gamma subunit or blocking the interaction between the gamma subunit and alpha subunit.19,25,28 Receptor interaction with zinc is also influenced by the gamma subunit, but incorporation of the pi subunit into different receptors does not appear to interfear with zinc interaction.19

Receptors that incorporate the pi subunit have demonstrated higher GABA EC50 values, less outward rectification and larger single-channel conductance.19 There is also reason to believe that pi subunits flip activity from hyperpolarization to depolarization in cancer cells.19,24 Therefore, these changes imply longer duration firing over receptors without pi subunits. If this information is accurate then a depolarizing GABAA pi subunit receptor has an advantage over normal hyperpolarizing GABAA receptors demanding greater activity from non-pi subunit GABAA receptors for hyperpolarization and cancer limitation. The extent of pi subunit pentration in cancer cells versus non-pi subunit may also explain the somewhat contradicting results with whether or not GABA aids or hinders cancer development.

The differing action between pi subunit and non-pi subunit containing GABA receptors could offer one reason for why taking benzodiazepine based sleep aids increase cancer rates. Increasing benzodiazepine concentrations act on GABAA receptors, which lead to increased GABAA receptor expression. Increased expression rates would increase the number of mutations simply through volume changes alone (subunit mutation rates may not change, but because more subunits would be created there would be more subunit mutations). Among these subunit mutations could be gamma subunits mutating into pisubunits or pi subunits being incorporated over gamma subunits. Increasing the number of pi subunits would increase the number of GABAA receptors that depolarize instead of hyperpolarize, which would aid cancer development instead of hinder it.

Outside very specific subunit interactions like those involving the pi subunit, the relationship between cancer and GABA appears complex for GABA may influence different cancers in different ways. However, there does appear to be a general pattern of operation between GABA, cancer and the two major GABA receptors. To best understand this relationship temporal issues must be acknowledged between immature/developing cancer and mature cancer. Note that developing cancer refers to cells that have become cancerous and are starting to grow, not cells that are going through the initial mutation stages to become cancerous.

Typically the expression of GABA and its corresponding synthesizing enzyme GAD (both isoforms 65 and 67) significantly increase in concentration in the presence of neoplastic cells ((colorectal carcinoma, breast cancer, prostate cancer, glioma, pancreatic and gastric cancer).29-37 However, what does that increase mean relative to cancer growth? Based on existing evidence it appears that a reduction in GABAA receptor functionality leads to accelerated cancer growth.29,38 Such a result implies that GABA activity when binding to GABAA results in reduced cancer growth, which has been supported through reductions in membrane potentials of cancerous cells.39,40 If GABAA binding is detrimental to cancer growth then why do GABA and GAD concentrations increase in the presence of cancer versus non-cancerous cells? One explanation is that this increased expression may be a general ‘safety’ feedback mechanism designed to curtail excess (i.e. cancer) growth, especially if GABAA receptor expression decreases.38

GABAB interaction appears to play a similar role to GABAA with regards to reducing cancer growth. Activation of GABAB in a cancer cell does not kill the cancer cell, but instead arrests its growth between stages G0 and G1.41 In addition GABAB activation also reduces intra-cellular cAMP concentrations through the inhibition of adenylyl cyclase due to the activation of G-protein alpha-2.42,43 cAMP is important in cellular growth (both normal and cancerous) because it activates phosphokinase A (PKA) which among other things (i.e. ERK1/2 cascade) activates phospholamban.44 Phospholamban activation increases the rate of calcium release from the endoplasmic reticulum (ER). While some of this excess calcium is sequestered by the existing cAMP, in typical situations the calcium release from the ER exceeds the rate of sequestration by cAMP resulting in an increased level of cytosolic calcium.44 This increased cytosolic calcium concentration activates calcium dependent secondary messenger systems increasing the rate of cellular growth. Thus, the ability of GABAB activation to reduce cAMP concentrations reduces the rate of cancer growth through this particular mechanism.

While GABA does appear to influence cancer growth in a negative way regardless of which receptor it binds to it could play an even more important role in cancer migration/metastasis, albeit a slightly confusing one. The confusion in the issue of metastasis appears to come from somewhat conflicting evidence, but the confliction is not insurmountable. First, increased expression of GABA67 is seen in patients with higher Gleason scores30 (note Gleason scores are derived from examination of histological samples in an effort to track cancer progression). Initially such a result could be explained, as mentioned earlier, as a feedback mechanism from the body in effort to control cancer growth and as cancer growth increases (leading to a higher Gleason score) the body compensates further. However, what if there is another explanation, what if GABA actually assists in metastasis? This amplification of cancer metastasis seems to be in play at least for some forms of prostate and lymph node cancers where increasing GABA concentration resulted in an increase in matrix metalloproteinase (MMP) expression due to GABAB activation.30

Metastasis is a complex series of interactions leading from tumor development to detachment from its principle location and movement to a different more distant location in the body. The major events involve detachment of from the primary tumor, invasion of the stromal tissue, entrance to the bloodstream, extravasate, invasion of the new target organ and finally the formation of the metastatic colony.17,30 One of the key steps in this process is the proteolytic degradation of the extracellular matrix and in this step MMPs are critical agents.

Initially MMPs were thought to be degenerative proteases that were limited to cleaving matrix components, but that role has expanded to include the release of growth factors and other bioactive peptides localized at cleaved extracellular matrices.45-47 Most MMP action involves MMP-3 cleaving decorin which releases transforming growth factor-b leading to greater levels of angiogenesis in addition to cleaving TGF-alpha activating MAPK inducing cellular proliferation.48,49 MMPs also cleave matrix receptors that may inhibit metastasis inhibitors like E-cadherin and activate a semi-self-regulating pathway with MMP-3 activating MMP-7 and 9.50

However, there appears to be a problem with concluding that GABA increases metastasis probability through GABAB activation in that another study has demonstrated that GABA decreases metastasis probability through GABAB activation.44 In a study using SW480 colon carcinoma cells cellular locomotion (basically metastasis probability) was reduced due to reduction in cAMP concentration. One possible reason for this difference is the differing cell types, but another line of thought produces another possible solution.

First, the SW480 colon study focused on metastasis induced through norepinephrine, not MMPs. In norepinephrine induced metastasis norepinephrine activates beta-arrestin through the beta-2 adrenoceptor which activates Protein Tyrosine Kinase (PTK) which activates the key agent, protein kinase C gamma (PKC).44 Activation of PKC-gamma results in the dual activation of both inositol-1,4,5-trisphosphate and diacylglycerol.44,51 Inositol-1,4,5-triphosphate opens intracellular calcium channels increasing cAMP activation and diacylglycerol activates PKC-alpha, which has been shown to increase metastasis in cancer cells.52,53

Basically norepinephrine increases cancer proliferation by activating inositol-1,4,5-triphosphate and increases cancer metastasis probability through activating diacylglycerol. This dual activation is important because cAMP cannot activate diacylglycerol on its own, yet it does appear to augment diacylglycerol activity in that if cAMP concentration is reduced diacylglycerol does not aid metastasis. Thus GABAB is able to prevent this form of metastasis by reducing cAMP concentration through secondary messenger inhibition of adenylyl cyclase.

However, while GABAB prevents metastasis through PKC-gamma, why doesn’t the ability to increase MMP expression compensate for the PKC-gamma blocking resulting in greater metastasis versus controls? One explanation may be temporal in nature in that the colon cells were not in a high enough state of maturity to express and/or interact with the MMP that should have been generated from the GABAB activation. If this theory is accurate then GABAB may be a beneficial therapeutic agent in the early stages of cancer, but as cancer progresses its usefulness flips and it becomes more detrimental than beneficial.

Unfortunately the role of GABAA in metastasis may not be as simple. While a number of studies suggest that GABAA binding plays no role in metastasis30,41,44 other studies suggest that GABAA binding increases metastasis probability54 or decrease metastasis.55 Despite this contradiction based on currently understood GABAA behaviors and mechanisms it is hard to believe that GABAA positively influences metastasis if configured properly because of its hyperpolarizing nature. The difference between ‘neutrality’ and reducing metastasis for GABAA may also be temporal, similar to GABAB.

Early in cancer development GABAA has little influence, but has MMP concentrations increase, GABAA works to reduce those concentrations despite GABAB augmenting them.55 Another reason for this disparity may be that in some studies the tumors did not mature to the point where metastasis was at a reasonable probability of occurrence because GABAA activity arrested tumor growth, thus GABAA influenced growth, but not metastasis. Overall it appears that the theory to describe GABA receptor behavior with respect to cancer is that GABAA negatively affects cancer growth and has little influence on cancer metastasis whereas GABAB negative affects cancer growth and has a negative influence on early cancer metastasis and a positive influence on late cancer metastasis.

If the above characterization of GABA receptors with respect to cancer is taken as accurate, then it appears that ability of sleep drugs to increase cancer rates largely relies on receptor subunit configuration. Other than pi subunits47 there is reason to suspect that incorporation of theta or rho subunits also increases cancer proliferation probabilities.56,57 These receptor confirmations may be self-augmenting in that if cancer develops due to their depolarizing characteristics over hyperpolarizing the cancer mutates to produce more receptors with similar configurations explaining why rarer pi and theta subunit configurations, with even rho at times, are so prevalent in cancers.24,27,56,57

Another possibility may be that augmenting GABAA activation through agonists like benzodiazepine may cause greater GABAB activity as a result of feedback. While GABAB activation would be viewed as more cancer-preventative than cancer-aiding, recall that as cancer develops the benefit/detriment ratio for GABAB with respect to cancer prevention decreases. Thus one question to ask is if cancer rates increase with sleep drug administration or does cancer metastasis also increase relative to the increase in cancer rates?

Overall one of the chief concerns is that while rates of death are slightly lower with non-benzodiazepine sleep drugs there is little firm scientific evidence that these non-benzodiazepine treatments result in higher rates of increased sleep time or reduction in wake probability after sleep onset compared against placebo.1,58 Benzodiazepine treatments do decrease sleep latency and increase sleep duration8,58 (although there are some questions regarding the statistical significance of the latter effect despite concerns of overestimation of sleep latency and underestimation of total sleep time),58 thus taking non-benzodiazepine treatments which have less cancer promoting tendencies may not be a suitable alternative to addressing a lack of sleep. While GABA seems to prevent cancer more than aid in its development stimulation of the GABA system through artificial means could also increases the rate of mutation in the subunits which comprise GABA receptors and these mutations significantly increase the probability of generating cancer.

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