Treating Cancer Through Metastasis Neutralization and Possible Activation

While cancer in any form is potentially dangerous to a patent, it is widely acknowledged that only a small percentage of primary tumors are threatening to the life of a patient in the interim. A vast majority of cancer patient deaths occur due to cancer metastasis. Metastasis is a complex pathway of molecular interactions that produce an end result that involves the departure of a group of tumor cells from the primary tumor into the bloodstream and eventual invasion into other tissues resulting in the formation of additional tumors. Not surprisingly the process is governed by a number of complex pathways that are not fully understood. Despite this lack of knowledge regarding metastasis, is it clear that one of the best strategies for addressing cancer would be to create a therapeutic regimen that would prevent metastasis from occurring in the first place allowing physicians ample time to eradicate the primary tumor with no legitimate threat of reoccurrence.

Of the agents thought to be involved in cancer metastasis chemokine receptor CXCR4 is a promising agent of study and potential therapeutic target. Chemokines are a group of low molecular weight cytokines that induce chemotaxis, most of the time as chemoattractants, largely in leukocytes, endothelial and epithelial cells.1 Chemokines are commonly classified into CC, XC, CXC or CX3C designations based on the positioning of their respective conserved cysteine residues.1 One of the key normal functions of chemotaxis is to facilitate the movement of pro-inflammatory cells to the site of inflammation, including immune cells, normally after some form of injury. CXCR4 is an attractive target because of both strong anecdotal and experimental evidence regarding its overall expression and role in tumor malignancy and metastasis.1-5

CXCR4 functions as a G protein-coupled receptor (GPCR) that principally binds stromal cell-derived factor 1 (SDF-1), which is also known as CXCL12. With regards to cancer, CXCR4 plays a significant role in directional migration though activation of actin polymerization6,7 as well as invasion and adhesion, which influence the overall level of aggression for a tumor. There is also some evidence suggesting that CXCR4 plays a role in angiogenesis as well.4,5

CXCR4 can undergo four major changes to influence its functionality: homo or heterodimerization (with CCR2, CCR5, CXCR7 or CD4), phosphorylation, glycosylation, or sulfation.8-14 Unfortunately limited information is known about functional changes associated with dimerization in cancer for almost all studies involving CXCR4 dimerization relate to HIV, but it is thought that such changes enhance CXCL12 binding. It is also believed that dimerization typically occurs internally before CXCR4 is expressed on the cell surface, typically as oligomers. This oligomeric structure persists in the plasma membrane.14

Phosphorylation occurs principally at serine residue number 339 (Ser339) after exposure to either CXCL12, epidermal growth factor (EGF), or phorbol ester and it is believed that phosphorylation may also occur to a much smaller extent at Ser324, Ser325, and Ser330.12 Phosphorylation is important for increasing the probability of receptor internalization and secondary messenger activation. On a side note mono-ubiquitination occurs at Lys327, Lys331 or Lys333.13 Glycosylation of human born CXCR4 only appears to occur at Asn11 and seems to serve no unique function other than stabilizing CXCL12 binding (lack of glycosylation reduces binding efficiency).1

Sulfation, which takes place primarily at Tyr21 and does not appear to occur on two other potential sites (Tyr7 and Tyr12),15 may the most interesting modification regarding CXCL12 binding probability and functionality.16 When CXCL12 binds to CXCR4 there is a specific site interaction between sulfated Tyr21 on CXCR4 and Arg47 on CXCL12.15 One of the reasons that sulfation appears important is that one study demonstrated that while highly metastatic NPC cells and non-metastatic NPC cells expressed similar levels of CXCR4, both via mRNA and protein, only high levels of sulfinated CXCR4 resulted in high metastatic potential.17

While there are a number of tyrosine sulfation pathways, with regards to CXCR4 one of the more prominent interactions involves the action of the latent membrane protein 1 (LMP1). While LMP1 concentration changes are not universal to CXCR4 activity increases, LMP1 interacts with EGF receptors, which is thought to be one of the early steps in inducing Tyrosylprotein sulfotransferase-1 (TPST-1) dependent tyrosine sulfation of CXCR4.15 However, there is the lingering question of whether sulfation is the chicken or the egg, is it a chief agent in how CXCR4 activation influences the potency of cancer or is it a result of that activation?

CXCR4 activation also significantly increases the overall expression of matrix metalloproteinases (MMP), especially MMP-2, MMP-3, MMP-7 and MMP-9.18,19 One of the major triggers for this action could involve the activated CXCR4 guiding Bone marrow-derived cells (BMDCs) to their pre-metastatic niche, which then triggers BMDCs to initiate the release various metastatic positive elements including various MMPs most notably MMP-7 and MMP-9.20,21 CXCR4 is also involved in the homing of cells into the endosteal HSC,22 which facilitates the expression of SUMO-specific protease 1 that regulates MMP-9 as well.23

As stated above one of the key steps in metastasis is the proteolytic degradation of the extracellular matrix (ECM) in which various 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.24-26 While there are up to 26 known MMPs (MMP-1, MMP-2, MMP-3, …) only a few have demonstrated significant roles in both cancer growth and cancer metastasis. Of these select few MMPs that play a prominent role in cancer, MMP-3 and MMP-7 appear to be the most important.

MMP-7, (a.k.a. matrilysin) is the smallest MMP and is commonly expressed in epithelial tumor cells instead of interstitial cells27 and has numerous substrates in the ECM namely collagen fibers, laminin, gelatin, proteoglycan and elastin, etc.28,29 MMP-7 is commonly over-expressed in various types of cancer including, but not limited to non-small cell lung, pancreatic, oral squamous cell carcinoma, colorectal, prostate, stomach and papillary thyroid carcinoma.28,30-34 In addition to its ECM degradation role, MMP-7 can also breakdown cell surface proteins, which aids cancer cell proliferation through the regulation of apoptosis and angiogenesis as well as help evade immune system detection.34-36

Furthermore, as mentioned above, MMP-7 is thought to increase expression of MMP-2 and MMP-9 to aid in ECM degradation and other pro-cancer actions.37,38 Finally not surprisingly MMP-7 levels increase in response to decreased blood glucose levels for this increase is tied to a low quality or deteriorating principal environment for the tumor, which should trigger elements responsible for assisting metastasis like MMP-7. However, while MMP-7 appears to be the most active MMP in most cancers, its activation may only be a downstream event caused by MMP-3.

An early action taken by a member of the MMP family typically involves MMP-3 cleaving decorin, releasing growth factor-beta and cleaving transforming growth-factor-alpha (TGF-a), which activates the MAP-kinase pathway.39 This activation can later activate MMP-7, which as previously mentioned leads to the activation of MMP-2 and MMP-9.40 In addition to activating other MMPs like -2, -7 and -9, MMP-3 can also promote genomic instability and epithelial–mesenchymal transition (EMT) through the activation of Rac1b, which stimulates both the production and release of intercellular mitochondrial superoxide.41,42 MMP-3 appears to be the dominant expression route for Rac1b.41

Due to the important role MMPs play in inducing both metastasis and possible anti-apoptosis protection, a number of researchers have thought of MMP inhibition as a promising treatment option. However, clinical trials investigating the viability of MMP inhibitors, commonly known as tissue inhibitors of metalloproteinases or TIMPs, have not proven successful.43 One of the major theories behind this failure is that during tumor development MMPs have different roles depending on tumor progression and the other molecules present in the tumor microenvironment. Some information has demonstrated anti-tumor effects for certain MMPs, most notably MMP-3, MMP-8, MMP-9, MMP-12, and a newer MMP, MMP-26, which may be a natural protectorate MMP.43-47

Clearly this dual behavior makes targeting MMPs directly difficult for therapeutic reasons, as demonstrated clinically, thus it could be more important to focus on important MMP triggers like CXCR4, which appear to activate MMPs during a time when their interaction with the tumor microenvironment will produce a net negative for the patient.

CXCR4 also can activate the P110-beta isoform of PI3K resulting in the eventual synthesis of phosphatidylinositol (3,4,5)-triphosphate, which leads to the phosphorylation of protein kinase B/Akt and mTOR pathways most notably activation of p70S6K and 4E binding protein 1.7,48,49 Not surprisingly mTOR inhibitor, rapamycin, reduces the extent of p70S6K and 4E binding protein 1 activation in a CXCL12/CXCR4 environment.7,19,50 Furthermore CXCR4 also activates elements in the Src family of protein tyrosine kinases, which aid the activation of focal adhesion elements like Crk, paxillin, and tyrosine kinase/Pyk2.51

For a long time CXCR4 was thought to be a unique target for CXCL12 until CXCR7 was identified, potentially complicating the role of CXCR4. Similar to CXCR4, CXCR7 is expressed at a much higher rate in malignant cancer cells versus normal cells and binds CXCL12 with high affinity.10,52 However, despite the significant similarities between CXCR4 and CXCR7, CXCR7 does not appear to play a meaningful role in cancer development or metastasis.53,54 The role of CXCR7 appears to involve the migration of primordial germ cells or interneurons.55 Its increased expression simply may be the result of dramatically increased levels of CXCL12 in the localized environment. Interestingly enough CXCR7 may prove to be a possible therapeutic element as an indirect natural CXCL12 competitive inhibitor of sorts for every CXCL12 molecule that binds to CXCR7 is no longer available to bind to CXCR4.

Earlier it was mentioned that CXCR4 might have a relationship in tumor growth and/or angiogenesis. If true, then this result is complicated because cancer growth at established tumor sites is more rapid in the absence of CXCR4 rather than its presence.53 Therefore, it may be that while CXCR4 assists growth immediately after invasion, its continued presence becomes detrimental for the tumor because it helps induce metastasis, thereby diverting resources like recruited and differentiated endothelial cells or progenitors from the original tumor towards elements that will be involved in the metastasis or even the attraction of metastatic elements already in the bloodstream.

CXCR4 also appears to interact with another potential important factor in cancer metastasis, macrophage migration inhibitory factor (MIF). MIF is a pro-inflammatory cytokine that plays an important role in inflammation and immune response and is expressed at a higher than normal rate during numerous cancer stages like cell proliferation, angiogenesis and anti-tumor immune interaction.56-58 High MIF concentrations have also been associated with poor outcomes in lymphoma, melanoma and colon cancer.59,60 CXCR4 interacts with MIF through the formation of a MIF receptor complex with CD74, which further enhances MIF-stimulated AKT activation.61

There is some thought that when MIF lacks its traditional activation pathway it requires caspase-1 activity for proper secretion.62 Also Golgi-associated protein p115 may be essential for the transport of MIF from the perinuclear ring to the plasma membrane and then out of the cell.63 In addition to aiding metastasis, MIF is also thought to apply some level of apoptosis resistance to cancer cells, favoring those with androgen-dependency over those with androgen-independence, but that resistance may be tied to CXCR4 interaction.64

With CXCR4 having its “fingerprints” over a number of pro-cancer processes, fortunately an additional element that makes it an attractive therapeutic target is its natural role in the body. In non-cancerous tissue CXCR4 is expressed on hematopoietic cells like CD34+ HSC, B-lymphocytes, neutrophils, monocytes, macrophages, and microglia, etc.65 CXCR4 or CXCL12 knockouts in mice result in impaired hematopoiesis through reduced hematopoietic stem cell (HSC) trafficking, which results in heart and brain defects as well as vascularization commonly producing embryonic death;66 in adults CXCR4 is important in HSC homing for the bone marrow microenvironment and lymphocyte trafficking.65 However, most of the time CXCR4 expression in normal cells is low, unless the body has been recently injured. Therefore, treatments that limit CXCL12/CXCR4 pathway activation should result in limited negative side effects for healthy non-injured individuals.

Another potential benefit from CXCR4 inhibition could involve reducing the probability of chemotherapy agent resistance, including Docetaxel (DTX) resistance. Some research suggests that the CXCL12-CXCR4 pathway interacts with p21-activated kinase 4 (PAK4)-induced LIM domain kinase 1 (LIMK1) via phosphorylation to reduce the ability of DTX to destabilize microtubules, which typically results in cell cycle arrest during the G2/M phase.67 Basically CXCR4 activation provides additional protection against cell death for tumors when exposed to DTX. This result suggests that LIMK1 could have a role similar to microtubule-associated protein (MAP) depending on whether or not it is phosphorylated. Therefore, this chemotherapy resistant pathway has two principal inhibition targets in CXCR4 or PAK4 to negatively influence prospective chemo resistance.

Existing potential therapies involving CXCR4 have focused largely on inhibiting the binding capacity of CXCR4 most notably either through the use of AMD3100, a specific CXCR4 antagonist, or synthetic peptide TM4.14,68,69 AD3100 (a.k.a. (Plerixafor) is a small molecule with two cyclam rings connected by a phenylene linker that have nitrogens on each ring that have charge-charge interactions with carboxylate groups on CXCR4, which inhibits CXCL12 binding.70-72

Plerixafor is most commonly used as a pre-treatment element for chemotherapy where, as mentioned, CXCR4 disruption reduces the probability of hematopoietic stem cells homing to bone marrow, thus it increases their circulation in the blood stream allowing for their collection for transplantation after chemotherapy regimens.73,74 Plerixafor has also proven promising as an anti-cancer treatment via its ability to reduce cancer cell chemotherapy resistance by either neutralizing the CXCL12-CXCR4 pathway or reducing the physical attachment of various micro-environment critical cells, similar to what is expected for a CXCR4 inhibitor.

An interesting side effect in plerixafor treatment is that surface expression of CXCR4 increases both in vitro and in vivo.65 One possible explanation for this outcome could be that principal signals that induce CXCR4 expression continue while plerixafor prevents CXCL12 from binding CXCR4; CXCL12 binding leads to internalization and activation of secondary pathways. When there is no CXCL12 binding there is no CXCR4 internalization, but the pathways governing CXCR4 expression towards the cell surface continue, thus explaining the overall increase in CXCR4 expression. If this tendency is accurate then long-term treatment with plerixafor alone may not be beneficial because the increased surface expression will substitute for the CXCR4 “removed” by plerixafor interaction. Basically plerixafor works well alone in the short-term, but may not work well alone in the long-term, which may be the same fate of all substrate based CXCR4 inhibitors.

Apart from preventing metastasis, treating the primary tumor is also an important task, especially when surgical options are unavailable. One promising potential therapeutic agent that can influence both primary tumors and metastasis is salinomycin (SAL), which has demonstrated effective ability to kill cancer via a perceived mixture of apoptotic and autophagic cell death in breast, prostrate, brain, blood, liver, pancreatic and lung cancers with no immediate lethal toxicity.75-77 Initially it was reported that SAL was toxic to certain neuronal cells (dorsal root ganglion in mice) at 1 uM, but this toxicity was neutralized when paired with factors that inhibited mitochondrial Na+/K+ exchangers with no resultant change in cancer cell cytotoxicity.78,79

One of the chief advantages of SAL in treating cancer is that it uses a different methodology apart from more common chemotherapy drugs like Doxorubicin, Cisplatin, Gemcitabine, Temozolamide, Tratsuzumab, Imatinib, etc.75,80-82 SAL has a preference for targeting cancer stem cells (CSCs), which reduces the probability of cancer reoccurrence after its primary removal.75 CSCs are important to address in treatment because they are commonly thought of as another element responsible for driving the core of cancer metastasis after responding to various signal triggers as well as driving cancer recurrence after the primary tumor is eliminated due to their ability to more frequently resist anti-cancer therapies. Thus, addressing CSCs, either directly or indirectly, is a critical part to addressing both cancer itself as well as its metastasis.

Another interesting behavioral aspect of SAL is amplified effectiveness under hypoxia or starvation conditions. This result makes sense on two different levels: first, it is thought that a means in which SAL triggers cell death is through damage to the mitochondria, in part to being a potassium ionophore, promoting hyperpolarization in the mitochondria, which decreases ATP availability and triggers caspase-3, 8 and 9, a consequence worsened by starvation conditions.76,83 Second, its effectiveness against CSCs is enhanced by the reaction of the tumor to hypoxia. In hypoxia the primary tumor will begin to focus an effort to metastasize due to the negative environment that currently exists; one step in this process requires the recruitment and creation of CSCs which reduce resource availability for the primary tumor, yet those CSCs are more effectively eliminated by SAL versus the cells that comprise the primary tumor.

The ability of SAL to “cooperative” with other anti-cancer drugs directly is questionable for most of the benefit from co-therapy between SAL and given drug x appears indirect.84 This result is somewhat interesting because there is some evidence to suggest that SAL can also function as an efflux pump inhibitor, which is commonly operated by a p-glycoprotein;85-87 efflux pumps increase the ability of cancer cells to remove chemotherapy agents before the inducement of cell death, so inhibiting them would increase tumor susceptibility to these chemotherapy agents. However, Metformin (METF), which is though to lower circulating insulin levels and stimulate AMPK-mediated suppression of mTOR, along with having some anti-cancer properties in thyroid, prostate, gastric, breast and glioblastoma,88-90 seems to have some form of direct enhancing cooperative relationship with SAL.84

This combination activity results in the “unspecific” inhibition of EGFR and HER2/HER3 leading to reduced concentrations of AKT and ERK1/2 via an unknown mechanism.84 However, it seems appropriate to suggest that based on SAL activity when acting alone that this “inhibition” is born from a reduction in available receptors due to cancer cell or associated cell death.

Another mechanism for inducing cancer death that includes SAL interaction is autophagy. In its most basic form autophagy involves the “self-digestion” of intracellular elements via the vacuolar lysosomal degradation pathway to recycle cytoplasmic constituents.91 Autophagy is typically used to prevent the accumulation of damaged proteins and organelles, largely born from cell damage due to outside agents; for cancer it would be anti-cancer drugs. This process also reduces the production potential of reactive oxygen species (ROS) that negatively impact cell survival.

There is reason to believe that SAL can interfere with autophagy in cancer cells by inhibiting lysosomal activity driven by cathepsins.85 Interestingly enough this activity occurs without impacting the lysosomal compartment.85 This aspect of SAL interaction is not surprising due to structural similarities with both nigericin and monensin, which have similar behavior as anti-porters themselves, but the lack of change in pH of the lysosomes from SAL treatment belies a different pathway.

Also the behavior of SAL runs contrast to ATG7 expression where ATG7 acts as a protectorate of sorts ensuring the proper functionality of autophagy. For breast cancer cells and more than likely other forms of cancer, aldehyde dehydrogenase 1 positive cells (ALDH+) promote autophagy.85,92 Thus, SAL directly works against ALDH as well as competes to “thwart” ATG7 autophagy protection. Therefore, ATG7 in some instances is able to neutralize the dual ability of SAL to kill cancer cells via induction of apoptosis while inhibiting autophagy inhibition. Therefore, inhibiting the activity of ATG7 may provide a useful co-therapy with SAL to significantly neutralize the ability of cancer cells to build resistance to SAL-related apoptotic activity.

Another popular method of action for SAL against cancerous agents is thought to be its interaction with Wnt signaling and its relation to b-catenin. The interaction between Wnt and b-catenin begins when Wnt binds to frizzled (Fzd) receptor and then that complex binds to lipoprotein receptor-related protein 5 or 6 (LRP5/6) co-receptors leading to a ternary complex that typically exists at the cell surface.93 The presence of this complex can trigger phosphorylation of either LRP leading to the recruitment of axin, which then undergoes endocytosis.93 The end result of this entire process is the breakdown or inactivation of the Adenomatous polyposis coli (APC)-Axin complex, which is responsible for b-catenin elimination.

This processes is important because b-catenin accumulation leads to its nuclear translocation and can even increase expression of Wnt genes via those tumors located at the invasive front, which have more interaction with growth factors and cytokines including hepatocyte growth factor.94 This interaction may even create a positive feedback loop of sorts.94,95 This nuclear translocation of b-catenin is thought to play some role in tumor cells experiencing cell-cycle arrest and EMTs via the loss of E-cadherin expression creating some form of cancer stem cell, with increased migration/metastasis potential.96,97

SAL interferes with the Wnt pathway by degrading the LRP6 protein and possibly LRP5 protein, which obviously reduces the probability that they form a complex with Wnt and are later phosphorylated activating the complex.85 LRP protein importance is further supported by a level of suppression in breast cancer tumor growth after treatment with a LRP antagonist, Mesoderm development (Mesd).98,99

However, SAL is not a cure-all when it comes to this supposed pro-cancer pathway, for Wnt and its associated complex is not the only significant destruction inhibition interaction experienced by b-catenin. Expression of platelet-derived growth factors (PDGF) can induce the tyrosine phosphorylation of p68 via c-Abl kinase.100 After phosphorylation p68 can bind b-catenin and inhibit GSK3b mediated phosphorylation reducing the probability that it is eliminated, thereby increasing the probability of b-catenin nuclear location.100 It is also thought that EGF and TGF-b can induce p68 phosphorylation via receptor tyrosine kinases.100,101

Another possible strategy to deal with LRP protein complex interaction involves the inhibition of vacuolar H+-adenosine triphosphatase using an agent like archazolid.102 LRP6 phosphorylation and internalization appears to require V-ATPase. The general role of V-ATPase is transport of both intracellular and extracellular organelles near the plasma membrane. Not surprisingly it also pumps protons leading to the acidification of vesicles, which promotes endocytosis.103

Another element in cancer development that has garnered attention for metastasis is the role of carcinoma-associated fibroblasts (CAFs). Tumor invasion is heavily influenced by the tumor microenvironment, especially the types of non-tumor cells. Various types of fibroblast recruitment lead to the production of soluble factors and extra-cellular matrix (ECM) remodeling usually through actin changes and cell migration born from MMPs, Rho targeted via ubiquitination and SUMO pathways104 as well as global DNA hypomethylation and recruitment of mesenchymal stromal cells; these changes increase the viability of future invasion.105-108

The cancer stroma is typically populated by various concentrations of fibroblastic cell groups that make up CAFs and are commonly divided into myofibroblast (MFs) and non-myofibroblast populations (non-MFs).105 MF populations have received much more attention than non-MF populations more than likely due to the diversity of the non-MF populations. It is thought that CAFs differ significantly from normal fibroblasts and myoblasts, but there is little information regarding the extent of these differences.105,109

CAFs are heavily involved in various pro-cancer pathways like tumor necrosis factor alpha (TNFa), IL-1 and IL-6,105,110,111 which lead to promoting invasion, immune suppression and angiogenesis through the secretion of SDF-1, TGF-b, hepatocyte growth factor (HGF), PDGFs, or vascular endothelial growth factors (VEGFs) principally driven by FSP-1- or PDGF receptor alpha-positive stromal fibroblasts.112-116

Not surprisingly if CAFs are thought to play a role in all of these pro-cancer processes, targeting them would prove useful for developing effective therapies. A number of proposals have been made regarding PDGF receptor inhibitors, SUMO inhibitors, Met receptor inhibitors or HGF inhibitors; however, on its face it is difficult to envision how to effectively target the “right” CAFs due to the widely diverse population of cells within the stroma. Interestingly enough some possibly contradictory research could prove some insight.

As mentioned above CAFs in the stroma are typically defined as either MF or non-MF, but both of these groups can be activated and/or transformed as well. One particular group to question is activated non-transformed MFs, which express alpha-smooth muscle actin (a-SMA). There are some that believe that these cells have “anti-cancer” activity instead of “pro-cancer” activity. Support for this mindset comes from studies of early and late stages of pancreatic cancer outcomes, clinical correlation between high a-SMA levels and improved survival on a general level, and studies of resected tumors.117-120 Also there is some question to whether or not a-SMA positive MF cells increase hyaluronic acid concentration.121 The anti-cancer attributes of MFs appear to stem from aiding both innate and adaptive immune response via increased fibrosis.117

Whether or not these MFs are pro-cancer or anti-cancer elements is important to deduce because anti-cancer therapies tend to kill indiscriminately around the principal tumor and its microenvironment, including a-SMA positive MFs. If these particular MFs are anti-cancer then these drugs are inherently less effective because while they are killing cancerous elements they are also killing anti-cancer elements.

The final possible important element to CAFs and their role in cancer is their ability to produce exosomes. For example in breast cancer CAFs secrete Cd81+ exosomes that can induce the planar cell polarity (PCP) signaling pathway targeting Wnt and influencing the polarity of carcinoma cells.122 Internalization of these exosomes also promotes Wnt11-PCP induction via autocrine through Frizzled receptor signaling leading to increased probability of pulmonary metastases.112 Targeting these exosomes may be a valid therapeutic strategy for reducing cancer potency.

As mentioned above directly targeting CAFs via therapies may be difficult, but one potential candidate could be TNF receptor associated factor 6 (TRAF6). At least for squamous cell carcinoma (SCC), TRAF6 plays a role in enabling nuclear factor kappa beta (NF-kB) signaling to activate a number of downstream pathways for CAFs, like Akt, Src-family kinases, IKK, IL-1beta and p38 and can regulate the formation of Cdc42-dependent F-actin microspikes.105,107,123 While the role of Cdc-42 is exactly unclear, TNFa plays a large role in promoting invasion in SCC and TRAF6 plays a significant role in producing sufficient TNFa concentration. The reduction of either one of these pathways significantly reduces cancer invasion, possibly due to the K63 ubiquitin ligase activation associated with TRAF6.124

The final issue when addressing cancer metastasis is developing a strategy to promote delivery of the anti-cancer agents to increase the probability of positive action, especially through ensuring proximity action. A reason behind this strategy is that some agents, like the rather useful SAL, demonstrate low quality aqueous solubility, which restricts their ability to be injected through a more standard IV strategy.125 Not surprisingly nanoparticles have become the most attractive vessel for transporting anti-cancer agents to cancer sites.

Overall nanoparticles are advantageous due to their low to non-immunogenic activity reducing complications and increasing their lifespan in the bloodstream, their natural and generally safe biodegradability and biocompatibility and their general design flexibility for producing the right type of particle for the given job. For example some nanoparticle structures use polypeptides with elastin and hydrophilic properties in effort to produce immune-tolerant elements. These elements are commonly referenced with the acronym, iTEP.126 However, nanoparticles need a form of “navigation” system to reach the appropriate target. The two most common targeting strategies are the use of antibodies or the use of aptamers.

Antibody targeting in some respects is the “old reliable” while aptamer targeting is somewhat new. Aptamers are comprised either of oligonucleic acid (DNA or RNA) or a peptide that are able to bind a specific target molecule. The major advantages of aptamers are their molecular specificity, their lack of immunogenicity, and their low molecular weights. These two latter advantages along with ease of production have further increased the popularity of aptamers versus antibodies regarding therapeutic targeting strategies including those dealing with cancer. A number of aptamers have already been developed for use in cancer treatment.127,128

Regardless of navigation methodology, the drug delivery vessel must have the right navigation point. One molecule that has drawn interest for potential cancer targeting ability is hyaluronic acid (HA). HA typically binds to CD44, a receptor commonly over-expressed on numerous types of tumors.129 Furthermore HA is frequently broken down by tumor cells by hyaluronidases (Hyals), which is widely thought to experience concentration increases in various cancers including prostate, bladder, colorectal, brain and breast due to the presence of increased low weight HA fragments found in these tumors versus normal cells.130-133

The general process of HA catabolism involves binding to CD44 resulting in its breakdown into smaller elements by Hyal-2 while still on the cell surface forming what is known as a caveolae. This caveolae eventually becomes an endosome that fuses with lysosomes resulting in the further degradation of HA fragments into tetrasaccharides by Hyal-1.134,135 Based on this process one could theorize that a self-assembled nanoparticle comprised of HA would serve as an effective means of drug delivery to the tumor site both tracking and through its degradation, a belief that has been supported with early empirical results.129

In addition to HA, it is widely thought that cluster of differentiation 133 (CD133) is a positive stem cell marker for both normal and cancerous tissue and is thought to be a critical agent in identifying CSCs. For example it is common for CD133+ cancer cells to form mammospheres that can initiate tumor growth in non-tumor cells. Due to the importance of CD133 expressing cells, an RNA aptamer (A15) has already been developed that binds to CD133 for use as a “tracking” marker of sorts and some groups have already explored the idea of using A15 as a drug delivery targeting agent.136

However, it must be noted that while CD133 expressing cells appear to be the most important in the CSC pool, tumors do produce CSCs that express other surface receptors while not expressing CD133. For example in osteosarcoma, CD133, CD117 and Stro-1 are all considered to be legitimate CSC markers.48 Additionally there is some evidence to support the idea that CSCs can convert to non-CSCs and back again.137 Therefore, while targeting CD133 is clearly an appropriate strategy for treating CSCs, it may not be the only targeting strategy necessary to eliminate CSCs.

The type of nanoparticle is only part of the issue involving drug delivery. Another important element is whether or not the principal drug should have other elements encapsulated with it to increase efficacy. For example SAL delivery involves a charged hydrophobic drug trapped inside a hydrophobic core of micelle-like nanoparticle; these interacting charges can increase destabilization potential, leading to ineffective drug application, which has been seen in past studies.125,126 Therefore, this charge interaction needs to be neutralized.

An early candidate for cooperation with SAL stability was N,N-dimethyloctadecylamine (DMOA) due to its similar hydrophobic nature, yet positive charge which is obviously counter to the negative charge of SAL. Unfortunately DMOA proved too toxic for this role.126 Fortunately it has a less toxic analogue in N,N-dimethylhexylamine. However, this reduced toxicity comes at the price of reduced hydrophobic strength due to a shorter hydrocarbon chain.126 Thus, researchers have added alpha-tocopherol as a second hydrophobic agent to enhance internal hydrophobicity to increase stability, which appears to work well.126

In the end while metastasis is still a process with a number of question marks associated with its occurrence and action there does appear to be certain elements that have important roles in its successful occurrence and function regardless of these question marks. First, it is quite clear that any therapy will have to involve some form of drug cocktail to cover multiple metastasis pathways including treatment of the principal tumor via either drugs or surgery. With this strategy in mind one interesting combination would involve the use of SAL in HA nanoparticles, some form of CXCR4 inhibitor, something like Plerixafor should be sufficient when not applied by itself, and a standard chemotherapy drug like Docetaxel.

Another possible addition to this cocktail could be an anti-angiogenesis drug. In recent treatment history anti-angiogenesis drugs have had a negative history of being useful anti-cancer agents despite the sound theoretical reasoning that reducing growth resources should reduce cancer growth potential. The failure of anti-angiogenesis drugs more than likely occurred due to the induced hypoxia environment increasing rates of metastasis. However, this increased metastasis may be a benefit when the anti-angiogenesis drug is used in combination with a CXCR4 inhibitor and/or SAL, which could speed cancer death by eliminating the metastatic elements versus attempting to eliminate the principal tumor. Of course this combination and the possible positive outcome is only theoretical, without appropriate empirical evidence the addition of an anti-angiogenesis agent may not provide a benefit, similar to how it functions currently.

While promising gains have been made in recent years in immunotherapy-based techniques to combat cancer, it is important to acknowledge that overall there is no magic bullet, but the above potential cocktail should be able to overlap the important negative cancerous element of both primary tumor elimination through multiple destruction pathways and metastasis neutralization via elimination of CSCs as well as the major pathways the drive the preparation and activation of metastasis itself. This method alone or in combination with a proven effective immunotherapy technique could provide a legitimate anti-cancer therapy for various stages of cancer development.



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A Theory Behind the Relationship Between Processed Foods and Obesity

While there has been a general slowing in the progression of global obesity, especially in the developed world, there has yet to be a reversal of this detrimental trend. A recent study has suggested that one aspect of influence regarding obesity progression lies with the consumption of foods that have incorporated emulsifiers and how they interact with intestinal bacteria including increasing the probability of developing negative metabolic syndromes in mice.1 Based on this result understanding the digestive process may be an important element to understanding how emulsifiers and emulsions may influence weight outcomes.

An emulsion is a mixture of at least two liquids where multiple components are immiscible, a characteristic commonly seen when oil is added to water resulting in a two-layer system where the oil floats on the surface of the water before it is mixed to form the emulsion. However, due to this immiscible aspect most emulsions are inherently unstable as “similar” droplets join together once again creating two distinct layers. When separated these layers are divided into two separate elements: a continuous phase and a droplet phase depending on the concentrations of the present liquids. Due to their inherent instability most emulsions are stabilized with the addition of an emulsifier. These agents are commonly used in many food products including various breads, pastas/noodles, and milk/ice cream.

Emulsifier-based stabilization occurs by reducing interfacial tension between immiscible phases and by increasing the repulsion effect between the dispersed phases through either increasing the steric repulsion or electrostatic repulsion. Emulsifiers can produce these effects because they are amphiphiles (have two different ends): a hydrophilic end that is able to interact with the water layer, but not the oil layer and a hydrophobic end that is able to interact with the oil layer, but not the water layer. Steric repulsion is born from volume restrictions from direct physical barriers while electrostatic repulsion is based on exactly its namesake electrically charged surfaces producing repulsion when approaching each other. As previously mentioned above some recent research has suggested that the consumption of certain emulsifiers in mice have produced negative health outcomes relative to controls. Why would such an outcome occur?

A typical dietary starch, which is one of the common foods that utilize emulsifiers is composed of long chains of glucose called amylose, a polysaccharide.2 These polysaccharides are first broken down in the mouth by chewing and saliva converting the food structure from a cohesive macro state to scattered smaller chains of glucose. Other more complex sugars like lactose and sucrose are broken down into their glucose and secondary sugar (galactose, fructose, etc.) structures.

Absorption and complete degradation begins in earnest through hydrolysis by salivary and pancreatic amylase in the upper small intestine with little hydrolyzation occurring in the stomach.3 There is little contact or membrane digestion through absorption on brush border membranes.4 Polysaccharides break down into oligosaccharides that are then broken down into monosaccharides by surface enzymes on the brush borders of enterocytes.5 Microvilli in the entercytes then direct the newly formed monosaccharides to the appropriate transport site.5 Disaccharidases in the brush border ensure that only monosaccharides are properly transported, not lingering disaccharides. This process differs from protein digestion, which largely involves degradation in gastric juices comprised of hydrochloric acid and pepsin and later transfer to the duodenum.

Within the small intestine free fatty acid concentration increases significantly as oils and fats are hydrolyzed at a faster rate than in the stomach due to the increased presence of bile salts and pancreatic lipase.3 It is thought that droplet size of emulsified lipids influences digestion and absorption where the smaller sizes allow for gastric lipase digestion in the duodenal lipolysis.6,7 The smaller the droplet size the finer the emulsion in the duodenum leading to a higher degree of lipolysis.8 Not surprisingly gastric lipase activity is also greater in thoroughly mixed emulsions versus coarse ones.

Typically hydrophobic interactions are responsible for the self-assembly of amphiphiles where water molecules react to a disordered state gaining entropy as the hydrophobes of the amphiphilic molecules are buried in the cores of micelles due to repelling forces.9 However, in emulsions the presence of oils produce a low-polarity interaction that can facilitate reverse self-assembly10,11 with a driving force born from the attraction of hydrogen bonding. For example lecithin is a zwitterionic phospholipid with two hydrocarbon tails that form reverse spherical or ellipsoidal micelles when exposed to oil.21 Basically emulsions could have the potential to significantly increase the hydrogen concentration of the stomach.

This potential increase in free hydrogen could be an important aspect to why emulsions produce negative health outcomes in model organisms.1 One of the significant interactions that govern the concentrations and types of intestinal bacteria is the rate of interspecies hydrogen transfer between hydrogen producing bacteria to hydrogen consuming methanogens. Note that non-obese individuals have small methanogen-based intestinal populations whereas obese individuals have larger populations where it is thought that the population of methanogen bacteria expands first before one gains significant weight.13,14 The importance behind this relationship is best demonstrated by understanding the biochemical process involved in the formation of fatty acids in the body.

Methanogens like Methanobrevibacter smithii enhance fermentation efficiency by removing excess free hydrogen and formate in the colon. A reduced concentration of hydrogen leads to an increased rate of conversion of insoluble fibers into short-chain fatty acids (SCFAs).13 Proprionate, acetate, butyrate and formate are the most common SCFAs formed and absorbed across the intestinal epithelium providing a significant portion of the energy for intestinal epithelial cells promoting survival, differentiation and proliferation ensuring effective stomach lining.13,15,16 Butyric acid is also utilized by the colonocytes.17 Formate also can be directly used by hydrogenotrophic methanogens and propionate and lactate can be fermented to acetate and H2.13

Overall the population of Archaea bacteria in the gut, largely associated to Methanobrevibacter smithii, is tied to obesity with the key factor being availability of free hydrogen. If there is a lot of free hydrogen then there is a higher probability for a lot of Archaea, otherwise there is a very low population of Archaea because there is a limited ‘food source’. Therefore, the consumption of food products with emulsions or emulsion-like characteristics or components could increase available free hydrogen concentrations, which will change the intestinal bacteria composition in a negative manner that will increase the probability that an individual becomes obese. This hypothesis coincides with existing evidence from model organisms that emulsion consumption has potential negative intestinal bacteria outcomes. One possible methodology governing this negative influence is how the change in bacteria concentration influences the available concentration of SCFAs, which could change the stability of stomach lining.

In addition to influencing hydrogen concentrations in the gut, emulsions also appear to have a significant influence on cholecystokinin (CCK) concentrations. CCK plays a meaningful role in both digestion and satiety, two components of food consumption that significantly influence both body weight and intestinal bacteria composition. Most of these concentration changes occur in the small intestine, most notably in the duodenum and jejunum.18 The largest influencing element for CCK release is the amount and level of fatty acid presence in the chyme.18 CCK is responsible for inhibiting gastric emptying, decreasing gastric acid secretion and increased production of specific digestive enzymes like hepatic bile and other bile salts, which form amphipathic lipids that emulsify fats.

When compared against non-emulsions, emulsion consumption appears to reduce the feedback effect that suppresses hunger after food intake. This effect is principally the result of changes in CCK concentrations versus other signaling molecules like GLP-1.19 Emulsion digestion begins when lipases bind to the surface of the emulsion droplets; the effectiveness of lipase binding increases with decreasing droplet size. Small emulsion droplets tend to have more complex microstructures, which produce more surface area that allow for more effective digestion.

This higher rate of breakdown produces a more rapid release of fatty acids as the presences of free fatty acids in the small intestinal lumen is critical for gastric emptying and CCK release.20 This accelerated breakdown creates a relationship between CCK concentration and emulsion droplet size where the larger the droplet size the lower the released CCK concentration.21 One of the main reasons why larger emulsions produce less hunger satisfaction is that with the reduced rate of CCK concentration and emulsion breakdown there is less feedback slowing of intestinal transit. Basically the rate at which the food is traveling through the intestine proceeds at a faster rate because there are fewer cues (feedback) due to digestion to slow transit for the purpose of digestion.

As alluded to above the type of emulsifier used to produce the emulsion appears to be the most important element to how an emulsion influences digestion. For example the lipids and fatty acid concentrations produced from digestion of a yolk lecithin emulsion were up to 50% smaller than one using polysorbate 20 (i.e. Tween 20) or caseinate.7 Basically if certain emulsifiers are used the rate of emulsion digestion can be reduced potentially increasing the concentration of bile salts in the small intestine, which could produce a higher probability for negative intestinal related events.

Furthermore studies using low-molecular mass emulsifiers (two non-ionic, two anionic and one cationic) demonstrated three tiers of TG lipolysis governed by emulsifier-to-bile salt ratio.3 At low emulsifier-bile ratios (<0.2 mM) there was no change in solubilization capacity of micelles whereas at ratios between 0.2 mM and 2 mM solubilization capacity significantly increased, which limited interactions between the oil and destabilization reaction products reducing oil degradation.3 At higher ratios (> 2 mM) emulsifier molecules remain in the adsorption layer heavily limiting lipase activity, which significantly reduces digestion and oil degradiation.3

Another possible influencing factor could be change in glucagon concentrations. There is evidence suggesting that increasing glucagon concentration in already fed rats can produce hypersecretory activity in both the jejunum and ileum.22-24 It stands to reason that due to activation potential of glucagon-like peptide-1 (GLP-1) in consort with CCK, glucagon plays some role. However, there are no specifics regarding how glucagon directly interacts with intestinal bacteria and the changes in digestion rate associated with emulsions.

The methodology behind why emulsions and their associated emulsifiers produce negative health outcomes in mice is unknown, but it stands to reason that both how emulsions change the rate of digestion and the present hydrogen concentration play significant roles. These two factors have sufficient influence on the composition and concentration of intestinal bacteria, which have corresponding influence on a large number of digestive properties including nutrient extraction and SCFA concentration management. SCFA management may be the most pertinent issue regarding the metabolic syndrome outcomes seen in mice born from emulsifiers.

It appears that creating emulsions that produce smaller drop sizes could mitigate negative outcomes, which can be produced by using lecithin over other types of emulsifiers. Overall while emulsifiers may be a necessary element in modern life to ensure food quality, instructing companies on the proper emulsifier to use at the appropriate ratios should have a positive effect on managing any detrimental interaction between emulsions and gut bacteria.



Citations –

1. Chassaing, B, et Al. “Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome.” Nature. 2015. 519(7541):92-96.

2. Choy, A, et Al. “The effects of microbial transglutaminase, sodium stearoyl lactylate and water on the quality of instant fried noodles.” Food Chemistry. 2010. 122:957e964.

3. Vinarov, Z, et Al. “Effects of emulsifiers charge and concentration on pancreatic lipolysis: 2. interplay of emulsifiers and biles.” Langmuir. 2012. 28:12140-12150.

4. Ugolev, A, and Delaey, P. “membrane digestion – a concept of enzymic hydrolysis on cell membranes.” Biochim Biophys Acta. 1973. 300:105-128.

5. Levin, R. “Digestion and absoption of carbohydrates from molecules and membranes to humans.” Am. J. Clin. Nutr. 1994. 59:690S-85.

6. Mu, H, and Hoy, C. “The digestion of dietary triacylglycerols.” Progress in Lipid Research. 2004. 43:105e-133.

7. Hur, S, et Al. “Effect of emulsifiers on microstructural changes and digestion of lipids in instant noodle during in vitro human digestion.” LWT – Food Science and Technology. 2015. 60:630e-636.

8. Armand, M, et Al. “Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract.” American Journal of Clinical Nutrition. 1999. 70:1096e1106

9. Njauw, C-W, et Al. “Molecular interactions between lecithin and bile salts/acids in oils and their effects on reverse micellization.” Langmuir. 2013. 29:3879-3888.

10. Israelachvili, J. “Intermolecular and surface forces. 3rd ed. Academic Press; San Diego. 2011.

11. Evans, D, and Wennerstrom, H. “The colloidal domain: where physics, chemistry biology, and technology meet.” Wiley-VCH: New York. 2001.

12. Tung, S, et Al. “A new reverse wormlike micellar system: mixtures of bile salt and lecithin in organic liquids.” J. Am. Chem. Soc. 2006. 128:5751-5756.

13. Zhang, H, et, Al. “Human gut microbiota in obesity and after gastric bypass.” PNAS. 2009. 106(7): 2365-2370.

14. Turnbaugh, P, et, Al. “An obesity-associated gut microbiome with increased capacity for energy harvest.” Nature. 2006. 444(7122):1027–31.

15. Son, G, Kremer, M, Hines, I. “Contribution of Gut Bacteria to Liver Pathobiology.” Gastroenterology Research and Practice. 2010. doi:10.1155/2010/453563.

16. Luciano, L, et Al. “Withdrawal of butyrate from the colonic mucosa triggers ‘mass apoptosis’ primarily in the G0/G1 phase of the cell cycle.” Cell and Tissue Research. 1996. 286(1):81–92.

17. Cummings, J, and Macfarlane, G. “The control and consequences of bacterial fermentation in the human colon.” Journal of Applied Bacteriology. 1991. 70:443459.

18. Rasoamanana, R, et Al. “Dietary fibers solubilized in water or an oil emulsion induce satiation through CCK-mediated vagal signaling in mice.” J. Nutr. 2012. 142:2033-2039.

19. Adam, T, and Westerterp-Plantenga, M. “Glucagon-like peptide-1 release and satiety after a nutrient challenge in normal-weight and obese subjects.” Br J Nutr. 2005. 93:845–51.

20. Little, T, et Al. “Free fatty acids have more potent effects on gastric emptying, gut hormones, and appetite than triacylglycerides.” Gastroenterology. 2007. 133:1124–31.

21. Seimon, R, et Al. “The droplet size of intraduodenal fat emulsions influences antropyloroduodenal motility, hormone release, and appetite in healthy males.” Am. J. Clin. Nutr. 2009. 89:1729-1736.

22. Young, A, and Levin, R. “Diarrhoea of famine and malnutrition: investigations using a rat model. 1. Jejunal hypersecretion induced by starvation.” Gut. 1990. 31:43-53.

23. Youg, A, Levin, R. “Diarrhoea of famine and malnutrition: investigations using a rat model. 2. Ileal hypersection induced by starvation.” Gut. 1990. 31:162-169.

24. Lane, A, Levin, R. “Enhanced electrogenic secretion in vitro by small intestine from glucagon treated rats: implications for the diarrhoea of starvation.” Exp. Physiol. 1992. 77:645-648.

Tapping into Brown Fat?

One of the impending health threats in the future is the increasing rate of obesity in the United States as well as the rest of the world. While the rate of obesity has ebbed slightly in the last year the number of obese individuals is still increasing in absolute terms due to population growth and greater access to food choices in developing countries. Numerous rationalities have been given to explain this increase ranging from too much food and not enough exercise to changing bacterial concentrations in the intestinal tract. Certainty the type of bacteria in one’s intestine affects the ability to process fats, carbohydrates and proteins from various food sources.1,2 However, despite the claims of numerous food products and their positive probiotic messages there is no proof that any of these products have a net positive benefit in managing weight. Gastro bypass surgery has a mixed history of success and for some patients has serious side effects. Therefore, the only truly viable proven method for consistently controlling weight is physical exercise and an appropriate diet.

Unfortunately numerous individuals do not enjoy exercise, so it would behoove many to develop a methodology that increased its effectiveness. Numerous “entrepreneurs” have made attempts at “increasing” exercise effectiveness with various pieces of equipment or programs, yet almost all of them are questionable in their viability. What needs to be addressed is a biochemical methodology that has influence in a majority of individuals. One strategy may be to tap into the unique properties of brown adipose tissue (a.k.a. brown fat).

There are two key elements to the fat burning capacity of brown fat. First, brown fat have multiple mitochondria versus the single mitochondria possessed by white fat which allows for greater rates of metabolism along with an increased lipid concentration. Also brown fat releases norepinephrine which reacts with lipases to breakdown fat into triglycerides and later to glycerol and non-esterified fatty acids and finally CO2 and water acting in as a positive feedback mechanism, in a sense.3,4 Second, brown fat contains uncoupling protein 1 (UCP-1).3 UCP-1 is responsible for dissipating energy, which leads to the decoupling of ATP production and mitochondrial respiration.3 Basically UCP-1 returns protons after they have been pumped out of the mitochondria by the electron transport chain where the protons are released as heat instead of producing energy.

The shiver response involves the activation of the primary motor in the posterior hypothalamus when inhibitory signals from the anterior hypothalamic-preoptic are overridden by temperature dependent sensory information from the skin and spinal cord.5 In response to this sensory information muscles begin to vibrate (i.e. shake) resulting in the production of heat as a byproduct to the activity. The key physiological role brown fat plays in mammals, especially those who do not have a shiver response, is it operates as a thermogenic organ where mitochondrial respiration is uncoupled from ATP production.4 The shiver response relies on volume of produced heat versus heat per unit, without the ability of volume brown fat augments the heat per unit amount, due to the lack of ATP production instead focusing on heat alone, in an attempt to compensate.

For a number of years it was thought that humans lost their brown fat after infancy, but numerous PET and CT studies have confirmed that adults do have concentrations of brown fat most notably in the supraclavicular area.6,7 Therefore, one could increase metabolic activity by activating brown fat in some individuals. It is important to note that global patterns of obesity are not viewed as complete enough to determine whether obesity rates are higher in warmer countries versus colder countries to provide a form of empirical support for increased brown fat despots relative to surface temperature; however, any correlation due to ambient temperatures would probably be weak because of adaptation of the shiver response.

While cold is the most common method for activating brown fat, it is not the only method to accelerate the mitochondrial activity of brown fat. Catecholamines have a similar activation effect largely because of their involvement in the fight or flight response. It makes sense that the more efficient energy producers in the body would be activated when facing a stressful life-or-death situation. Unfortunately beta blockers, which are commonly used to control high blood pressure, a common symptom in obese individuals, block catecholamines and reduce the probability of activating brown fat through catecholamine interaction. Epinephrine and caffeine also show promise in increasing brown fat activation, but epinephrine derived from drugs have too many side effects and caffeine consumption could result in too many additional calories counteracting the increase in brown fat activation.

As noted earlier norepinephrine appears especially important in triggering additional brown fat activation resulting in an increased rate of oxygen consumption and fatty acid release. Thus, the consumption of foods high in norepinephrine precursors could aid in increasing brown fat efficiency probability. Another advantage to utilizing brown fat as an augmented element to increased metabolic activity is that its decoupled nature from ATP production should result in limited to no additional oxidative free radical production from its specific mitochondria. Therefore, activation of this methodology should not increase possibilities for cellular damage and increase the rate of aging in a given subject.

Still, many questions remain before those next steps can be taken. For one thing, why is it that obese people tend to have very little brown fat compared with lean people? One possibility is that as brown fat can “eat” white fat, brown fat could also become white fat. If brown fat is not utilized it may experience apoptotic consequences. An aspect of this die-off could occur because brown fat chief role is its thermogenesis response, but more obese individuals have greater thermal insulation due to their fat content, thus have more difficulty initiating brown fat activation due to external temperature changes and competition with the shiver response.

It is also probably worth remembering how researchers discovered that adults retained stores of brown fat in the first place: they were studying head and neck scans of patients with cancer and noticed that in addition to tumor sites, certain parts of the neck also showed higher rates of glucose consumption.8 This additional rate of glucose consumption was theoretically driven by additional brown fat because brown fat appeared to be at greater concentrations in cancer patients versus individuals without cancer/tumors.8 One explanation for this additional brown fat could be that the excess energy requirements for cells that have shed their growth limitations demand conversion of white fat into brown fat through the methodology discussed above. If this theory is accurate then brown fat follows tumor development not that other way around, thus increasing brown fat stores will not increase the probability that an individual develops cancer.

While some individuals believe that one way to utilize brown fat is to develop a “cold sauna” that individuals can simply sit in, wouldn’t it be better to combine brown fat activation with white fat “activation”? There still is limited information on what type of exposure regiment is optimal for brown fat activation. For example is it better to be exposed to high intensity cold at low volume (-50 degrees F for 30 seconds) or low intensity cold at high volume (20 degrees F for 5 minutes)? Note that brown fat activates as long as the temperature is cold enough whether it is acute cold or eventual chronic cold; however, prolonged exposure to certain cold conditions can produce negative results for exposed skin, so determining a differentiated methodology would be advisable. Also gyms or cold saunas would have to set up a stepwise exposure protocol allowing users to move gradually from initial temperatures to final temperatures because a near instant temperature change from say 70 degrees F to 0 degrees F would be detrimental to the body, especially those with health problems.

Outside this methodology one could have short-term cold exposure (40 degrees F for 10 minutes) and then engage in cardiovascular exercise. The cold exposure will stimulate the brown fat with the later exercise continuing the brown fat stimulation and initiating some level of white fat loss. Another possibility would be the increased rate of norepinephrine release increasing the probability of white fat becoming brown fat due to increased activation rates along with the release of norepinephrine possibly reducing pain associated with exercising (acting as a biological pain killer). Overall with available brown fat stores still in adults, no risk for increased cancer development and the ability to produce more brown fat when routinely activated it seems that gyms should think about installing “cold saunas” not necessarily for solitary fat burning, but instead as a preparation element for a more complete calorie and fat burning workout.


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Citations:

1. Backhed, F, et, Al. “The gut microbiota as an environmental factor that regulates fat storage.” PNAS. 2004. 101(44): 15718–23.

2. Cani, P, et, Al. “Role of gut microflora in the development of obesity and insulin resistance following high-fat diet feeding.” Pathologie Biologie. 2008. 56:305–309.

3. van Marken Lichtenbelt, W, et Al. “Cold-activated brown adipose tissue in healthy men.” The New England Journal of Medicine. 2009. 360:1500-08.

4. Lowell, B, and Spiegelman, B. “Towards a molecular understanding of adaptive thermogenesis.” Nature. 2000. 404:652-60.

5. http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=& term=Shivering

6. Hany, T, et Al. “Brown adipose tissue: a factor to consider in symmetrical tracer uptake in the neck and upper chest region.” Eur J. Nucl Med. Mol Imaging. 2002. 29:1393-8.

7. Nedergaard, J, Bengtsson, T, and Cannon, B. “Unexpected evidence for active brown
adipose tissue in adult humans.” Am. J. Physiol. Endocrinol. Metab. 2007. 293:E444-
E452.

8. Rousseau, C, et Al. “Brown fat in breast cancer patients: analysis of serial (18)F-FDG PET/CT scans.” Eur J. Nucl Med. Mol Imaging. 2006. 33:785-91.

Tackling Obesity One Step at a Time

Obesity has steadily become a significant problem in modern society, especially in the United States. Note that it is clear obesity is a growing problem even without resorting to statistics which is good because the BMI stat which typically drives obesity identification is flawed in its inability to differentiate between fat weight and muscle weight. While there are a number of reasons postulated for this rampant increase in over-weight individuals, the most disconcerting issue is despite the existence of viable solutions obesity rates continue to climb. It is rational to conclude that there are two chief elements behind this continued expansion of obesity.

The first element involves the psychological reality that most people shy away from the physical exertion required to stay fit. Sadly for these individuals biochemistry has not advanced to the point where weight can be selectively and effectively controlled by simply taking some drug. Even common surgical options like lap bands and gastro-bypass surgeries have not stemmed the problems and have caused there own set of problems. The only proven methodology to avoid becoming obese is proper diet and exercise; however, these elements especially the latter demand effort and time. Note that while there is mounting evidence to suggest the involvement of stomach bacteria in calorie absorption and its role in increasing weight, this research is still in its infancy and has not yielded any therapeutic strategy, thus it is useless to scapegoat obesity with stomach bacteria.

The second element involves proper direction. Unfortunately capitalism has provided an unnecessary obstacle in preventing obesity in that the social environment has been flooded with strategies for dieting and weight control that run the gamut from eating almost nothing but carbohydrates to eating almost no carbohydrates. Most of these various strategies have only a small level of biochemical backing and instead are pushed in effort to make money for the individuals sponsoring or backing the particular diet methodology. The lack of clarity in most of these plans leads to ‘higher than should-be’ failure rates which foster greater frustration in those that fail reducing the probability that they invest their time, money and psyche in future attempts at weight loss.

Another problem related to this second element is availability. More often than is realized even if an individual would prefer to eat healthy food, that food is either supply unavailable or economically unavailable. One of the most noted issues is that of ‘food deserts’, regions (rural or urban) which lack a variety of food selection largely because of a lack of supermarkets or farmer’s markets. While there have been some noteworthy start-up efforts to address this issue, the problem of food deserts seems too large for small entrepreneur-driven individuals to solve without government assistance. If individuals want to get serious about dealing with food deserts and even domestic hunger then state governments need to conduct audits in their states to identify where these deserts are and how to divert food items from more plentiful regions.

Returning to the first element one would inherently think that pride should be a driving element in warding off obesity, but there is evidence to suggest that a number of individuals who are obese or even just over-weight do not view their weight as a problem.1 In those periods when they are concerned the concern tends to manifest as disgust rather than an affront to pride; this disgust can lead to rash short-term crash dieting instead of long-term change in behavior, which commonly results in long-term failure. However, if weight is not viewed as a problem any form of self-motivation to address weight becomes less likely including issues surrounding overall health and physical well-being, thus such a reality demands a secondary strategy. For most a form of familial intervention is also not a likely option based on this same psychological premise. With these two effective options no longer available a new incentive must be provide to drive motivation to live a healthier life.

The best form of incentive is typically some form of monetary award. Unfortunately indirect or future awards, largely those that can be calculated from healthy behavior, do not motivate effectively. The uncertainty of the future forces probabilistic arguments instead of direct yes/no arguments. For example one can make the rational argument that eating a certain assortment of food may reduce the probability of acquiring cancer by x%, but most people want a more definitive response, ‘if food x is eaten then I will or will not get cancer’.

Another problem is the incompatibility with probability figures in a deterministic reality. Getting cancer is a deterministic yes/no issue, so one seems to save the same amount regardless of what their personal cancer percentage turns out to be. Basically one does not receive more money for a lower cancer percentage, just a greater probability of receiving any money (through savings by not having to treat the cancer); this concept is rather confusing and further makes it difficult to see the benefit from a financial perspective. Finally the fact that this savings occurs over the course of a lifetime and not in an immediate lump-sum further reduces its usefulness as an incentive. Note that when does a lottery winner ever take the lifetime annuity option over the lump-sum option? With these incompatibilities with typical human psychology it is not difficult to understand why people still have difficulties undertaking healthy actions even when the resources to facilitate them are available.

With the ineffectiveness of arguing ‘you should do this because it will reduce your probability of getting cancer, macrodegeneration, osteoporosis, etc.’ a more direct incentive is required. Typically most argue that the most effective incentive is cash. Not only is the distribution of cash immediate, but it is also flexible. However, that flexibility is also a problem. Most people would like to assume that individuals would use capital in an effective manner that most helps their existence, but if such a contention were true a vast majority of the people that are in debt would not be in debt. The inability to predict what an individual will do with a monetary award creates inherent complexity with such an incentive program, especially when individuals have a wide variety of resources available from which to select. For example it is easier to predict what a person in Somalia will do with 30 dollars than a person in the United States. Therefore, distributing cash in any type of incentive program with the single target goal of improving societal physical health through weight loss seems inefficient.

With the elimination of cash as a possible option, the award mechanism for incentive will most likely take the form of a ‘gift’ card. However, to ensure a restricted flexibility, the ‘gift’ card would only be useable at certain retailers. In fact if one were willing the best possibility would be to establish a retailer designed specifically for interaction with these ‘gift’ cards. By establishing a specific retailer, the government can control the role of supply and production. For example if so desired the supply of merchandize could only include items that are manufactured in the U.S. by U.S. companies. The interaction medium for this retailer(s) must include both an online and an off-line component because not everyone who would take advantage of the program would have online access. The off-line component can be something as simple as mail order involving the U.S. Post Office.

Now that the general incentive agent has been established, the next element is how the individual would acquire this incentive. The overall goal of this program is to stem and hopefully in time reverse the growing rate of obesity in the U.S. The hope is that eventually the program would pay for itself by reducing the amount of money spent in healthcare by increasing overall societal health. The spending reduction should be seen in both Medicare payouts and private insurance payouts as well as through increased tax revenues by increasing production through reduction of sick days and other health related circumstances that lead to missed or unproductive work days. There are two chief elements that influence overall health which can be affected at a reasonable certainty and level of effort, food consumption and exercise. Due to the reward element of this program as well as lingering consistency questions involving the availability of food supplies, using exercise as the defining element seems to make more sense.

One may argue that exercise in a vacuum is not an appropriate strategy to drive good overall health. On its face this opposition is understandable, but there is value in exercise regardless of food consumption on two levels. First, the obvious benefit is that any amount of exercise can neutralize some of the ill effects from improper eating. Second, the less obvious benefit is biological memory. While still in its infancy there are theories which suggest that individuals who frequently exercise have a higher level of something (maybe fat-burning enzyme activity), even beyond simple muscle mass correlations, which control weight gain. On a related side note it is possible that this theory and the role of stomach bacteria may be associated with each other in that greater exercise increases efficiency of energy use, which decreases the demand to absorb calories which selects for stomach bacteria that absorb fewer calories. Thus the more exercise an individual performs the better his/her body is able to control weight even while resting. Finally the application of exercise is important because something needs to be done to address the overall weight problem.

If exercise will be the evaluation medium what method will be used for the evaluation? In the past such a program would commonly demand a participating individual to travel to a specific location, a special gym for example, where activity could be tracked by volunteers. However, computers have eliminated this change of venue demand where more simple aspects of exercise such as distance traveled, rpms, heart rate, etc. can be measured, tracked and saved where ever the device is being used. Not having to travel should significantly increase the probability of both participation in the program and continuation with the program. The requirement of this vital stat information demands the use of some form of machine. The machine in question needs to be simple while also involving a methodology that allows for ample heart rate increase. In effort to limit stress on the body it seems that an elliptical machine would be superior to a treadmill.

The elliptical machine used in this program would be specially designed with a microchip that documents the use of the device and would be used to determine rewards. The rules governing the payout need to be transparent and clearly stated. Three salient factors would demand a clear fixed unambiguous incentive price, an age limit and personal identification. The following is one possible example describing the use of the device:

Acquisition of the device is dependent on receiving a physical from a participating physician. The reason for this element is that it would be unfair to set a standard baseline on the device without taking into consideration the current physical health of the participant. For example to hold someone that is 350 pounds to the same exercise demand as a 185 pound individual is counter productive. The standard of measurement will use a rpm floor and because of the potential volume of participants there needs to be a ceiling on how much exercise will be counted towards the overall program over a given time period. The ceiling is required to control the total cost of the program as well as protect individuals from overzealous exercising which would be detrimental to overall health in attempt to acquire more monetary rewards. The machine should visually and audibly inform the user when this limit is reached and should reset at 12:00 am each day. One possibility for the limit would be 45 minutes at or above the rpm floor in a 24 hr period. One point of discussion would be whether the individual would have to maintain the appropriate speed at or above the price floor for the entire 60 seconds or if just the average rpm over that 60 second period would have to be at or above the rpm floor to be given credit for 1 minutes of exercise under the incentive program.

The rpm floor is designed to ensure appropriate benefit from the exercise so the monetary incentives awarded are driving the accomplishment of the overall goal. Basically one should have to actually physically push his/her body for the time spent exercising to count towards incentive, no ‘dogging it’; otherwise the entire point of the incentive program is meaningless because once again it only resorts to an individual’s pride as the driving factor and as stated that strategy is clearly not working. Establishing the correct rpm floor is one of the principle reasons a physical is required before an individual can acquire this device. Pursuant to this rpm floor the device will have a kill-switch after some amount of time (10-12 months) where an individual will have to have another physical in order to recalibrate the floor. As inefficient as it would be to expect a 350 pound individual to meet the rpm requirements assigned to a 185 pound individual, the same goes for an individual that was once 350 pound continuing to meet that rpm requirement even though he/she now only weighs 290 pounds.

The incentive price should be tied directly to the total minutes exercised at or above the specific assigned rpm floor. Initial blind thought sets an incentive price of 2 cents per minute. This rate would establish a reward of 90 cents a day and $328.50 a year. Some may argue on its face that such an incentive is too small to facilitate meaningful exercise. The counterargument is that the overall level of work required to acquire these funds is so insignificant that the seemingly small value can be viewed as appropriate. For example because the device is at the participant’s home there is an improved probability of multi-tasking in that one could be exercising and watching television, reading a book, listening to music or even having a conversation. The primary reason for establishing a meager value is concern regarding the sheer volume potential of the program. Suppose 200 million elect to participate in this program and hit 50% of their total potential as a group, such a scenario would result in 32.85 billion dollars in incentives per year.

Finally each individual should have a specific ID code that recognizes that individual, especially if only one elliptical device is distributed per household. One could argue that there could be unscrupulous behavior regarding this ID code where a healthier individual could exercise at an easier rpm floor. While true, a ‘safety measure’ could be established where if an individual did not noticeably improve while in the program after two physicals that individual would be blacklisted from the program permanently.

Overall while the idea presented above still have some more specific details to flesh-out, it is obvious that something needs to be done about the growing weight problem in the U.S. and individual pride clearly is not enough of a driving factor.

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1. Powell, T, et, Al. “Body Size Misperception: A Novel Determinant in the Obesity Epidemic.” Arch Intern Med. 2010; 170(18): 1695-1697.