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Title: IMPORTANT NOTICE (DCA)
Source: DCA Website
URL Source: http://www.thedcasite.com/
Published: Oct 13, 2007
Author: Staff
Post Date: 2007-10-13 19:00:41 by Horse
Keywords: None
Views: 145
Comments: 5

Two agents from the FDA visited us on Tuesday,July 17, 2007 and ordered that we stop making and selling DCA. Unfortunately, the site www.buydca.com no longer offers DCA for sale.

It is against US government law to sell substances with the suggestion that they are cancer treatments unless they are approved by the FDA.

DCA can still be obtained from pharmacies with a prescription and from chemical companies.

To keep you informed and abreast of new developments and potential cancer treatments, www.thedcasite.com will continue.

Thank you for all your support.

Jim Tassano & the DCA Team

Welcome to The DCA Site

This site is to help inform people of the exciting research done on DCA by scientists at the University of Alberta. In January 2007, a team of scientists at the University of Alberta published a paper in the scientific journal, Cancer Cell, describing the discovery that a simple, cheap molecule, DCA, worked to reactivate the apoptosis mechanism of cancer cells, causing rapid shrinkage of tumors in rats. Mitochondrial reactivation represents an entirely new approach to treating cancer.

The tumors shrank 70% in three weeks.

DCA is not patentable as a molecule. There is no incentive for private companies to spend the millions of dollars needed to get DCA approved as a cancer treatment by the FDA. This is where we, all of us, need to step in. We intend for TheDCAsite.com to be the meeting place, for the world, where all the important information on DCA is gathered and shared.

This site gives the results of the Michelakis research, a look at the possible side effects of DCA and the prospects of it being used by YOU. We have posted the best summaries we can reach on dosages, side effect concerns and additional measures to take to help you fight cancer. Questions are answered and a chat room is provided.

To avoid any legal issues, we must state we are not doctors and cannot give medical advice.

DCA has not been approved by the Food and Drug Administration for the treatment of cancer. It can be prescribed "off-label" by your physician.

Please note that DCA is not active against all cancers. An excellent analysis by "Willis", which is supported by the limited feedback we are getting, indicates that sarcomas, for example, are not controlled by DCA. However, many cancers are definitely responding to DCA. Non small cell lung cancer patients, even in Stage 4 are showing good responses. We are seeing lymphatic, bladder and other cancers responding very well to DCA.

DCA is serious medicine. Study the material on the site. Read our dosage page.

We want to encourage everyone to use the Chat Room or ask the questions you want answered. We strongly recommend you join and register as a chat room user.

The site was first uploaded late on February 5th. The chat room was added February 8.

Poster Comment:

This is from their Library Page:

Chat Room Support Papers: JohnA 1 Control (pdf) (doc) JohnA 2 Patient A (pdf) (doc)

Case Studies: 1. Skin Cancer and DCA 2. Brain Cancer reduction, scans before and after starting DCA 3. Italian scan results: shrinkage of lung tumor and complete regression of brain and liver tumors 4. Oracle's wife's reports: shrinkage of non-small cell lung and lymph node tumors 5. Bile duct tumor reduced by 50 percent.

Library: Metabolic Targeting as an Anticancer Therapy (discusses DCA and the need for other metabolic attacks on cancer)

Growth Properties of Colonic Tumor Cells are a Function of the Intrinsic Mitochondrial Membrane Potential, BG Heerdt, MA Houston, LH Augenlicht, 2006.

Montbriand papers on Herbs and Natural Substances (natural substances and cancer)

Antioxidants and Cancer: A Brief Discussion on Controversies, Contradictions and Paradoxes by MJ Gonzalez et. al.

The Long-term Survival of a Patient With Pancreatic Cancer With Metastases to the Liver After Treatment With the Intravenous alpha-Lipoic Acid/Low-Dose Naltrexone Protocol by Burton M. Berkson, Daniel M. Rubin, and Arthur J. Berkson (email: rubin@rubinmedical.com.)

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This is from the same website. Please note that apoptosis means cell death.

SUMMARY The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing to apoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity.Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria- NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

SIGNIFICANCE The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolicelectrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

RESULTS Cancer Mitochondria Are Hyperpolarized and Have Suppressed Oxidative Metabolism, Both of which Are Reversed by DCA We studied DJm in three human cancer cell lines: A549 (non-small-cell lung cancer), M059K (glioblastoma), and MCF-7 (breast cancer), and compared them with healthy, noncancerous human cell lines: small airway epithelial cells (SAEC), fibroblasts, and pulmonary artery smooth muscle cells (PASMC). All cancer cell lines had significantly more hyperpolarized DJm compared to normal cells (increased fluorescence of the DJm- sensitive positive dye tetramethyl rhodamine methyl ester; TMRM). Incubation of all three types of cancer cells with DCA (48 hr) reversed the hyperpolarization and returned the DJm to the level of the normal cells (n  80 cells, 10 plates/cell line/group). In contrast, DCA did not alter the DJm of the SAEC(Figure 2A), fibroblasts, and PASMC(not shown). The DCA effects on mitochondrial DJm occurred as quickly as 5 min and were dose dependent (Figure 2B). Because the intracellular distribution of TMRM can theoretically be affected by differences in the plasma-membrane potential (Em), we used a protonophore (carbonylcyanide- ptrifluoromethoxyphenylhydrazone; FCCP) and showed that in all cancer cell lines it decreased DJm in a dosedependent manner, eventually depolarizing untreated and DCA-treated cells at the same low DJm (see Figure S1 in the Supplemental Data available with this article online). This suggests that the TMRM signal was not confounded by DCA effects on the Em, but was rather due to true changes in the DJm. To determine the effect of DCA on metabolism, we measured glycolysis (Gl), glucose oxidation (GO), and fatty acid oxidation (FAO) in A549 cells (10 plates/experiment, n = 3). DCA significantly increased GO rates (+23%), with a concomitant decrease in both Gl and FAO (Figure 2C). As expected from the DCA-induced shift of pyruvate metabolism away from lactate and toward acetyl- CoA and the Krebs cycle, the lactic-acid levels in the culture medium of the DCA-treated cells decreased (Figure 2D, n = 10 plates/group) and the intracellular pH increased (Figure 2E, n = 5 plates, 60 cells/group). DCA Causes Efflux of Proapoptotic Factors from Mitochondria and Increases ROS Production Whereas the untreated cancer cells (A549) showed cytochrome c and apoptosis-inducing factor (AIF) restricted to the mitochondria (colocalized with mitotracker red), in the DCA-treated cells cytochrome c was diffusely present in the cytoplasm and AIF was translocated to the nucleus (Figure 3A), both indicating induction of apoptosis. DCA increased H2O2 production in a dose-dependent manner; this increase was inhibited by rotenone, suggesting that it was based on complex I of the ETC (Figure 3B, n = 5 plates/group). We also measured NADH levels in isolated mitochondria and showed that DCA increased the intramitochondrial NADH (Figure 3C, 5 plates/experiment, n = 5). The DCA-induced decrease in DJm was limited by the VDAC (an important component of the mitochondrial transition pore; MTP) inhibitor 40-diisothiocyano- 2,20-disulfonic acid stilbene (DIDS; 0.5 mM) (Granville and Gottlieb, 2003) (Figure 3D, n = 5, 60 cells/ group). To determine whether cancer cells are less dependent on the ETC and oxidative phosphorylation, we studied the effects of low-dose cyanide (a complex-IV inhibitor and a well- known poison for normal cells). Cyanide’s effects on mitochondria (as measured by DJm) were much less pronounced in cancer compared to the DCAtreated cells (Figure 3E, n = 5, 60 cells/group). DCA Activates Kv Channels in Cancer Cells by an H2O2-Dependent Mechanism Using whole-cell patch clamping, we showed that in all untreated cancer cell lines the outward K+ current (Ik) was small and essentially voltage independent. DCA increased the Ik significantly in all cancer cell lines, but did not alter the Ik in the noncancerous SAEC (Figure 4A), PASMC, or fibroblasts (not shown) (Figure 4A, n = 7–8/ group). The increase in Ik occurred as early as 5 min and persisted after 48 hr of DCA exposure. Most of the increased Ik was voltage dependent and blocked by 4-aminopyridine, a specific Kv channel inhibitor. The increased Ik caused hyperpolarization of the plasmamembrane Em. DCA also decreased cell capacitance, an electrophysiologic surrogate of cell size/volume, consistent with the cell shrinkage that characterizes early apoptosis (Figure 4A). The DCA-induced increase in Ik was blocked by intracellular catalase, delivered through the patch pipette (i.e., due to H2O2), and by rotenone (i.e., due to complex I-produced H2O2), but not by thenoyltrifluoroacetone (TTFA; an inhibitor of complex II of the ETC) (Figure 4B, n = 5). It was also not blocked by the human ether-a-go-gorelated gene (HERG) inhibitor E4031 (50 nM) (Wang et al., 2002) (not shown). The activation of Kv channels by DCA resulted in a decrease in intracellular K+, due to efflux of K+ down its concentration gradient. When this gradient was diminished by the addition of KCl, the intracellular K+-lowering effects of DCA were inhibited (Figure 4C, n = 20). DCA Decreases [Ca2+]i and Increases Kv1.5 Expression via Inhibition of NFAT1 We have recently shown that DCA increases the expression of Kv1.5 in PASMC (McMurtry et al., 2004). Only one study has linked Kv1.5 with cancer: Kv1.5 expression (but not other Kv channels) correlates with tumor grade in human gliomas, that is, the higher the grade, the lower the Kv1.5 (Preussat et al., 2003). We used quantitative real-time polymerase chain reaction (n = 5) and immunoblots and showed that Kv1.5 is significantly increased in DCA-treated A549 cancer cells (Figure 4D), in contrast to Kir2.1, a K+ channel from a different family (not shown). To study whether Kv1.5 expression correlates with human tumor grade, we used archived tumors from a cohort of 30 consecutive patients with non- small-cell lung cancer. We measured both Kv1.5 and survivin expression in each tumor sample and correlated their levels with histologic tumor grade by blinded readers. Survivin, a marker of resistance to apoptosis and tumor aggressiveness, has recently been shown to regulate mitochondria-dependent apoptosis in both cancer (Dohi et al., 2004) and vascular tissues (McMurtry et al., 2005). Survivin correlated positively with tumor grade while Kv1.5 correlated negatively (the higher the Kv1.5, the lower the grade), and no differences in the expression of Kir2.1 were seen (Figure S2). Because NFAT (nuclear factor of activated T lymphocytes) inhibits both apoptosis (Pu et al., 2003) and the expression of Kv1.5 in myocardial cells (Rossow et al., 2004), we speculated that this could also occur in cancer. Increases in [Ca2+]i activate calcineurin, which dephosphorylates NFAT, allowing its translocation to the nucleus where it regulates gene transcription (Macian, 2005). We hypothesized that the DCA-induced activation of Kv1.5 leads to plasmalemmal hyperpolarization, inhibiting voltage-gated Ca2+ channels (which are active even in nonexcitable cells; Dietl et al., 1995), decreasing [Ca2+]i, thereby inhibiting NFAT and enhancing Kv1.5 expression (Figure 1). As predicted, DCA-treated A549 cells have lower [Ca2+]i compared to untreated cells (Figure 5A). The decrease in [Ca2+]i occurs within 5 min and is sustained after 48 hr of DCA exposure. At both time points, DCA’s effects on [Ca2+]i are inhibited by 4-aminopyridine and rotenone and are mimicked by t-butyryl-H2O2, suggesting that they involve opening of Kv channels by complex I-derived H2O2 (n  20 plates/group). The 4- aminopyridine-induced increase in [Ca2+]i in the DCA-treated cells is inhibited by lanthanum (n = 6), a blocker of Ca2+ entry into the cell (Figure 5A), confirming an operative voltage-dependent pathway of Ca2+ entry. NFAT1 activation (defined by its translocation to the nucleus) is evident in the majority of untreated A549 cells, and in these cells Kv1.5 expression is low (Figure 5B). In contrast, in DCA-treated cells, NFAT1 is mostly cytoplasmic and Kv1.5 expression is proportionately increased. As with Ik (Figure 4B) and [Ca2+] i (Figure 5A), the effects of DCA on NFAT1 were mimicked by t-butyryl- H2O2 (Figure 5B). We then treated A549 cells with VIVIT, a competing peptide that selectively blocks NFATregulated mechanisms due to its ability to block calcineurin docking on NFAT, preventing its activation without inhibiting calcineurin’s catalytic site (Aramburu et al., 1999). The VIVIT-treated cells show displacement of NFAT1 from the nucleus and increased Kv1.5 expression (Figure 5B). The same was true for cells treated with cyclosporine, a nonspecific inhibitor of calcineurin (Figure S3A). In human non-small-cell lung tumors costained with anti-Kv1.5, anti-NFAT1, and 40,6-diamidino-2-phenylindole (DAPI), low-grade histology was associated with high Kv1.5 expression and low NFAT1 expression, most of which was cytoplasmic; the reverse pattern was seen in high-grade tumors (Figure 5C). The effects of NFAT1 might be potentiated by a positive feedback loop: low Kv1.5 / depolarized plasma membrane / Ca2+ influx / high [Ca2+]i / activated NFAT / low Kv1.5. NFAT inhibition effectively breaks this loop because VIVIT decreases [Ca2+]i (n = 5 plates/ group, Figure 5D). DCA Induces Mitochondria- Dependent Apoptosis and Decreases Proliferation In Vitro DCA increases annexin expression, causes a 6-fold increase in the percentage of TUNEL-positive nuclei, and activates both caspase 3 and 9 in A549 cells (Figures 6A and 6D). Eliminating highly proliferative cells by the induction of apoptosis, and by decreasing [Ca2+]i levels, DCA decreases indices of proliferation (Figures 6B and 6D) including BrdU incorporation and expression of proliferating cell nuclear antigen (PCNA). In addition, DCA decreases the expression of survivin (Figure 6B). DCA-induced apoptosis proceeds by two pathways, one in the mitochondria, where depolarization activates mitochondria-dependent apoptosis, and the other at the plasmalemmal level, where activation/upregulation of Kv1.5 channels decreases [K+]i, activating caspases. To determine the relative importance of the two mechanisms, we compared the apoptosis induced by DCA to the apoptosis induced by a primary increase in Kv1.5 expression, using adenoviral gene transfer (Figures 6C and 6D). Compared to the adenovirus carrying green fluorescent protein (GFP) only, the adenovirus carrying GFP and cloned human Kv1.5 (Pozeg et al., 2003) significantly increased the percentage of TUNEL-positive cells. However, although the increase in Ik achieved with the gene transfer was higher than the increase achieved by DCA (Figure 6C; compare with Figure 4A), the increase in apoptosis achieved by the gene transfer was significantly less than that achieved by DCA (Figure 6D). We then measured the DCA-induced apoptosis in the presence of 4-aminopyridine (5 mM), a blocker of the whole Kv family. In addition to the A549 cells we also studied glioblastoma, an excitable cell type in which Kv channels might be more important in apoptosis regulation compared to the epithelial A549 cells. In the presence of 4-aminopyridine, DCA induced68%of the apoptosis (% TUNEL-positive cells) induced by DCA alone (Figure 6D). Similarly, in glioblastoma cells, DCA + 4- aminopyridine induced 62% of the apoptosis induced by DCA alone (Figure S4). Moreover, 4-aminopyridine did not limit DCA’s ability to cause efflux of cytochrome c from mitochondria, initiating mitochondriabased apoptosis (Figure S4). These data underlie the preponderant importance of the mitochondrial component of DCA’s proapoptotic actions. That NFAT1 is a distal mediator in DCA’s anticancer effects was supported by the fact that VIVIT increased apoptosis and decreased proliferation in a manner similar to DCA (Figure S3B). For imaging studies, we studied four random fields per slide for 30 slides/group, and for patch clamping, 6–8 cells/group. Molecular Inhibition of PDK2 Mimics DCA To confirm that inhibition of PDK is the major mechanism for the effects of DCA, we determined whether molecular inhibition of PDK2 by siRNA mimics DCA. We chose PDK2 because it is the only ubiquitously expressed isoenzyme; PDK1 and 3 are restricted in the heart and testis, respectively, and PDK4 is mostly expressed in skeletal muscle and heart. PDK2 is the most active of all and also has the lowest Ki for DCA (0.2 mM) (Bowker-Kinley et al., 1998). siRNA for PDK2 inhibited the expression of PDK2 in a dose-dependent manner, inhibiting mRNA up to 80% and protein expression (measured by both immunoblots and immunohistochemistry) by 70% (Figure S5). We tested three commercially available PDK2 siRNAs, which all inhibited the gene in a similar manner. Scrambled siRNA for PDK2 as well as siRNA for PDK1 did not decrease PDK2 expression (Figure S5). Whereas the scrambled siRNA had no effect on A549 cells, PDK2 siRNA decreased DJmand increased mitochondrial ROS in a manner identical to DCA (Figures 7A and 7B, n  20 plates/group). DCA added to siRNA-treated cells had no additional effects (data not shown). Inhibition of PDK2 by siRNA also increased apoptosis and decreased proliferation in cancer cells (Figure 7C, n  30 plates/group). To further prove that DCA activates PDH by inhibiting PDK, we immunoprecipitated PDH and showed that DCA increased the nonphosphorylated fraction (i.e., active) of the catalytic subunit (E1a) (Figure 7D).

DCA in the Drinking Water Induces Apoptosis and Decreases Tumor Growth In Vivo

We studied nude athymic rats implanted subcutaneously with 3 3 106 A549 cells. The rats had free access to water with or without DCA (75 mg/l). In the first set of experiments (protocol a), 21 animals were divided into three groups: untreated controls (n = 5), DCA-prevention rats (n = 8), which received DCA just after cell injection for 5 weeks, and DCA- reversal rats (n = 8), which received DCA 2 weeks post-cell injection for 3 more weeks. The untreated rats rapidly developed tumors with a constant exponential tumor growth (Figure 8A). Both DCA-treated groups had a significant decrease in tumor size, measured by tumor weight at sacrifice and maximal diameter using calipers; in some rats, in vivo magnetic resonance imaging allowed us to visualize the tumors in vivo and calculate their volume. The decrease in tumor growth by DCA was associated with an increase in apoptosis (TUNEL) and a decrease in proliferation (PCNA) (Figure 8B). There was an inverse correlation between apoptosis and tumor size in the treated rats (Figure 8B). Kv1.5 was upregulated and survivin was downregulated in the DCA- treated rats (Figure 8C), confirming our in vitro data (Figures 4 and 6). In a second set of experiments (protocol b), we studied whether the effects of DCA were sustained for longer periods of time and whether DCA would have a similar effect in more advanced tumors.Wefollowed three groups of rats (n = 6/group) for 12 weeks: an untreated control group, a prevention group (rats given DCA at the time of tumor cell injection), and a reversal group where rats were given DCA at week 10 for 2 weeks. As in protocol a, at all times rats in the prevention group had significantly smaller tumors compared to the untreated controls; DCA at week 10 inhibited tumor growth immediately, with a significant decrease even after 1 week of treatment. DCA therapy did not have any toxic effects, as measured by several blood tests (Figure 8D; also see McMurtry et al., 2004).

DISCUSSION Here we show that a metabolic-electrical remodeling (hyperpolarized mitochondria, downregulated Kv channels) regulates the apoptosis resistance that characterizes multiple human cancers. DCA, a small molecule that targets mitochondria, reverses this remodeling, inducing apoptosis and decreasing cancer growth in vitro and in vivo. These beneficial effects occur without affecting noncancerous cells or eliciting systemic toxicity. DCA treatment significantly increases glucose oxidation (which only occurs in functional mitochondria), indicating that the well-recognized, metabolic cancer signature (aerobic glycolysis) is reversible, rather than a consequence of permanent mitochondrial damage. DCA exerts its beneficial effects by two pathways, both of which induce apoptosis: first, by mitochondrial depolarization and efflux of proapoptotic mediators, and second, by an increase in Kv channel expression/function. DCA increases Kv channel expression by inhibiting NFAT1, a calcium-sensitive transcription factor that regulates cell- differentiation programs in many cell types but which has previously been unexplored in cancer. The mitochondria-NFAT-Kv pathway in cancer offers several new candidate targets for proapoptotic therapy that would be predicted to have high therapeutic selectivity. Glycolysis and Cancer: Not Just an Epiphenomenon It is now well accepted that most cancers have a glycolytic phenotype. Warburg suggested, but did not prove, that this was due to ‘‘abnormal mitochondria’’ (Warburg, 1930); that is, cancer cells are forced to use inefficient, nonmitochondrial means of generating ATP. Our data suggest that this apparent mitochondrial ‘‘dysfunction’’ is in fact reversible. Oxidative metabolism in cancer could be actively suppressed; the resultant shift to glycolysis may lead to apoptosis resistance (Plas and Thompson, 2002), offering a survival advantage in the transformed cells (Gatenby and Gillies, 2004). This suggests that a novel way to reverse apoptosis resistance might be to undo this etabolic/mitochondrial remodeling. We show that the glycolytic phenotype in cancer is easily altered by promoting oxidative phosphorylation (Figure 2C). This is associated with mitochondrial depolarization, which facilitates apoptosis and inhibits tumor growth. All the human cancer cell lines studied had more negativeDJmcompared to several noncancerous cell lines (Figure 2A), suggesting that this might be a hallmark of malignancy. Although apoptosis is not always associated with mitochondrial depolarization, our data are in agreement with the observation that cationic lipophilic drugs preferentially accumulate to tumor mitochondria (Don and Hogg, 2004). In addition, the positively charged rhodamine-based dyes (like TMRM) have been tried as ‘‘carriers’’ for selective delivery of drugs in cancer. More than 200 carcinomas were screened and were shown to accumulate rhodamine much more than noncarcinoma cells; these findings were first reviewed in 1988 (Chen, 1988), and although the mechanism was not clear then, it likely reflects the more negative DJmof cancer compared to noncancerous cells. Our work directly shows that this relative increase inDJmis associated with increased resistance to apoptosis, and its ‘‘normalization’’ increases apoptosis and decreases cancer growth. Furthermore, it has just been shown that DJm of colon cancer cells predicts the aggressiveness of the tumor cells, that is, the more hyperpolarized the DJm, the more aggressive and metastatic the tumor (Heerdt et al., 2005), in agreement with our proposal. Studying DJm in fresh tumor specimens might be a convenient means to predict resistance to proapoptotic chemotherapies, with important implications in clinical decision making.

How Does DCA Alter Metabolism, Depolarize Mitochondria, and Initiate Apoptosis? The shift in the metabolism of pyruvate away from lactate and toward acetyl-CoA and the Krebs cycle (Figures 2C and 2D), caused by DCA or molecular inhibition of PDK2 (Figure 7), increases the intramitochondrial production of the electron- donor NADH (Figure 3C), a substrate of the ETC complex I, leading to increased complex I-basedROS production (Figures 3Band 7B) (Kushnareva et al., 2002). A sustained increase in the ROS production can cause oxidative damage in the ETC, particularly complex I. This megacomplex is the most sensitive of all ETC complexes to ROS damage because it is by far the largest (46 subunits), and has at least nine ROS-sensitive iron-sulfur centers and seven mitochondrial DNA- encoded subunits (Brandt, 2006), which are very susceptible to oxidative damage. The ROS-induced complex-I dysfunction can limit the efflux of H+, decreasing DJm. Upon sustained and significant decrease in DJm, the voltage- sensitive MTP opens (Zamzami and Kroemer, 2001), allowing the efflux of many proapoptotic factors and the initiation of apoptosis (Figures 3A, 6, and 8B). This further increases the production of mitochondrial ROS, likely einforcing a positive feedback loop enhancing apoptosis (Zamzami and Kroemer, 2001). This ‘‘complex I-centered’’ proposed mechanism has a precedent in congenital mitochondrial syndromes and neurodegenerative diseases. Patients with congenital complex-I deficiency have decreasedDJmand increased ROS production (Pitkanen and Robinson, 1996). Inhibition of complex I in cell lines is associated with decreasedDJm and increased ROS production in a dose-dependent manner, that is, the higher the percent complex-I inhibition, the higher the ROS and the lower theDJm (Barrientos and Moraes, 1999). A similar mechanism where dose-dependent inhibition of complex I leads to dose-dependent efflux of cytochrome c and apoptosis (Clayton et al., 2005) is proposed in the pathogenesis of neurodegenerative diseases such as Parkinson’s, where complex-I dysfunction and ROS-mediated oxidative damage are well described (Bao et al., 2005; Schon and Manfredi, 2003). Inhibition of VDAC limited the DCA-induced decrease in DJm (Figure 3D). VDAC (along with the adenine nucleotide translocase) is involved in the translocation of ADP (a substrate for the F1F0-ATPase) from the cytoplasm into the mitochondria. Inhibition of the VDAC would thus inhibit the function of the F1F0-ATPase, which would lead to accumulation of H+ in the intermembrane mitochondrial space, promoting hyperpolarization of the DJm, thus limiting the depolarizing effects of DCA. This is supported by findings from Thompson’s group (Vander Heiden et al., 1999), although the role of VDAC in the regulation of DJmand initiation of apoptosis remains controversial (Shimizu et al., 1999) (reviewed in Granville and Gottlieb, 2003), and some of these mechanisms might only be relevant to specific experimental conditions, such as growth-factor withdrawal (Vander Heiden et al., 1999). An additional intriguing possibility is that, because DCA is itself an anion (see structure in Figure 1), it likely enters the mitochondria via the VDAC, explaining, at least in part, why its inhibition limits the effects of DCA on DJm. Unexpectedly, but consistent with its therapeutic benefit, DCA decreased the expression of survivin, an inhibitor of apoptosis, both in vitro and in vivo (Figures 6 and 8). Survivin has recently emerged as a major antiapoptotic oncoprotein. The mechanism by which survivin is downregulated is unclear. Recent observations describing the direct involvement of a mitochondrial survivin pool in the suppression of apoptosis suggest that survivin might participate in the mitochondrial remodeling of cancer (Dohi et al., 2004; McMurtry et al., 2005).

A Mitochondria-NFAT-Kv Channel Axis in Cancer Is Normalized by DCA, Contributing to the Proapoptotic and Antiproliferative Effects of DCA The apoptosis resistance in cancer likely involves multiple mechanisms. The current findings highlight the contribution of Kv channel inhibition/downregulation, due to impaired mitochondrial signaling, to this resistance. Closing of K+ channels or decreasing their expression results in an increase in [K+]i which increases the tonic inhibition that cytosolic K+ exerts on caspases. Kv1.5 gene transfer directly activated apoptosis in A549 cells (Figures 6C and 6D). Functional inhibition of all Kv channels by 4- aminopyridine limited the DCA-induced apoptosis by 32% in A549 cells and by 38% in glioblastoma cells (Figure 6D; Figure S4), suggesting that although the majority of apoptosis in DCA-treated cells is a direct result of efflux of proapoptotic mediators from cancer cells, the secondary effects on Kv channels also play an important role. The precise role ofK+ channels in cancer remains unclear, and although K+ channel opening promotes apoptosis in several tumors, the opposite result has also been noted (reviewed in Wang, 2004). Perhaps this relates to the type of tumor or the well- known diversity of K+ channel families. SpecificK+ channels are nowemerging as important regulators of apoptosis in different cell types. For example,HERG, a Kv channel, mediates H2O2-dependent apoptosis in various cancer cell lines (i.e., low HERG expression reduces apoptosis and enhances proliferation) (Wang et al., 2002). Kv1.5 regulates apoptosis in PASMC (Remillard and Yuan, 2004) and is downregulated in the proliferative and apoptosis- resistant vascular media in pulmonary hypertension (McMurtry et al., 2004, 2005; Pozeg et al., 2003). A teleologicaladvantageofKv1.5asa regulator ofapoptosis incancer, and a factor which focused our attention on this channel, is its very short turnover time, less than 8 hr from transcription to functional expression (Levitan et al., 1995). We show that cancer cells are deficient in ETC complex I-based production of H2O2, a Kv1.5 channel opener (Figure 3B). Perhaps more importantly, Kv1.5 is downregulated in cancer cell lines (Figure 4D), and Kv1.5 expression correlates inversely with histologic grade in a cohort of patients with non-small-cell lung cancer (more aggressive tumors have less Kv1.5) (Figure S2). We have identified NFAT1 as an important transcription factor responsible for this Kv1.5 downregulation (more aggressive cancers have more activated NFAT1) (Figure 5C). The cellular environment in cancer is favorable for NFAT activation. A549 cells have increased [Ca2+]i (Figure 5A), a direct activator of calcineurin and thus NFAT (Macian, 2005). This increase in [Ca2+]i is, at least in part, due to the increased Ca2+ influx that results from the Kv channel deficiency (Figure 4). In addition, calcineurin is inhibited by increased ROS levels (Namgaladze et al., 2005); thus, the low mitochondrial ROS in cancer (Figures 3B and 7B) promote NFAT activation. Furthermore, the acidotic environment in cancer (due to aerobic glycolysis) (Figure 2E) would further promote NFAT activation (Komarova et al., 2005). It is remarkable that all of these mechanisms are reversed by DCA, which increases ROS, increases pH, and decreases [Ca2+]i, explaining its impressive effects on NFAT (Figure 5B). The upregulation of Kv1.5 by a drug that directly affects mitochondrial function suggests the presence of the mitochondria-NFAT-Kv1.5 axis, which is suppressed in cancer. Our work suggests that potential effects on Kv channels should be considered in cancer therapies targeting the mitochondria.

Metabolic Modulation in Cancer by DCA: Possibility of Prompt Translation to Clinical Oncology

Our work suggests that metabolic modulators could be beneficial in human cancer, either alone or in combination with traditional chemotherapies, as apoptosis sensitizers. By targeting a fundamental and unique property of cancer cells, this approach may combine efficacy and selectivity. DCA (in clinically relevant doses; Stacpoole et al., 2006) was effective in preventing and inhibiting tumor growth in established tumors both early (week 2) and late (week 10) in their development (Figure 8A). DCA’s effects in the reversal protocols were immediate, with significant effects even after 1 week of treatment. The relative specificity of DCA to target a metabolic (mitochondria) and electric (K+ channels) remodeling was confirmed by microarray experiments, where pathway analysis revealed a short list of altered mitochondrial apoptosis cell cycle and ion channel genes (Supplemental Results; Figure S6). A very attractive property of DCA is its selectivity, evident by the lack of any systemic toxicity in this (Figure 8D) and other recent animal (McMurtry et al., 2004) and human studies (Stacpoole et al., 2006). DCA’s ability to ‘‘restore’’ DJm might explain why it is effective preferentially in cells that have very high DJm, such as cancer cells, but has no effects in normal cells (epithelial, fibroblasts, or PASMC). Preferential expression of PDK might also contribute to its selectivity. In a recent study of nonsmall- cell lung cancer specimens, cancer cells had ncreased PDK2 and decreased PDH expression (compatible with a glycolytic phenotype) compared to neighboring nonmalignant cells (Koukourakis et al., 2005). The small size of DCA results in excellent tissue penetration after oral intake, including the central nervous system (Stacpoole et al., 2003), relevant to the difficult-to-treat glioblastoma, one of the tumors that we studied in vitro. In addition, DCA decreases tumor lactic-acid production and increases intracellular pH (Figure 2E); future studies need to address the hypothesis that this will decrease tumor invasiveness and metastatic potential (Gatenby and Gillies, 2004). Our work identifies the mitochondria-NFAT-Kv channel axis and PDK as critical components of the metabolicelectrical remodeling that characterizes many human cancers and offers a tantalizing suggestion that DCA may have selective anticancer efficacy in patients. The very recent report of the first randomized long-term clinical trial of oral DCA in children with congenital lactic acidosis (at doses similar to those used in our in vivo experiments) showing that DCA was well tolerated and safe (Stacpoole et al., 2006) suggests a potentially easy translation of our work to clinical oncology.

The Truth of 911 Shall Set You Free From The Lie

Horse  posted on  2007-10-13   19:22:29 ET  Reply   Trace   Private Reply  

WTF is the FDA doing in Canada?

Sweet Jesus.

Join the Ron Paul Revolution

Lod  posted on  2007-10-13   19:27:11 ET  Reply   Trace   Private Reply  

.....Through its participation in the Trilateral Cooperation Charter and with almost zero Congressional oversight, the FDA is secretly pushing with all its administrative might for the implementation of Codex Alimentarius among North American Union nations Canada, the U.S. and Mexico.

The FDA is currently acting as though a North American Union were already in place, even though there has NEVER been any implementing legislation passed by Congress to allow this rogue agency to do what it’s doing.

If Canada, Mexico and the U.S. are to officially merge financially and politically as the North American Union (NAU), the current laws in place that protect our health freedoms, such as the Dietary Supplement and Health and Education Act of 1994 (DSHEA), will be superseded by the NAU and Codex regulations.

At the core of this issue is national sovereignty. It is bad enough that we have unelected representatives from the FDA representing the interests of big pharma and the bio-tech food industry rather than the citizens of the U.S. But if we lose our sovereignty as a nation, the rights of the consumer will, in effect, be replaced with the regulatory schemes intended to promote "free trade"—which is a euphemism for "fascist exploitation of resources and markets."

Under Codex, consumer rights of fair access to what the FDA refers to as "complimentary and alternative modalities/medicine (CAM)" will cease to exist and will be replaced with a regulatory scheme that omits administrative remedies for relief from arbitrarily-imposed restrictions on how we choose to maintain our own health......."

From the April 2007 Idaho Observer:

Advancing NAU greatest threat to health freedom

www.proliberty.com/observer/20070415.htm

AllTheKings'HorsesWontDoIt  posted on  2007-10-13   21:11:13 ET  Reply   Trace   Private Reply  

Thanks for the proliberty linage - I'd not heard of them.

Join the Ron Paul Revolution

Lod  posted on  2007-10-13   21:18:20 ET  Reply   Trace   Private Reply  

Thanks for the proliberty linage - I'd not heard of them.

I think I've run across them before. They came up after a search on CODEX CANADA FDA, because I figured this had something to do with CODEX. I should have highlighted the following:

At the core of this issue is **** national sovereignty. It is bad enough that we have unelected representatives from the FDA representing the interests of big pharma and the bio-tech food industry rather than the citizens of the U.S. But if we lose our sovereignty as a nation, the rights of the consumer will, in effect, be replaced with the regulatory schemes intended to promote "free trade"—which is a euphemism for **** "fascist exploitation of resources and markets."

Under Codex, consumer rights of fair access to what the FDA refers to as "complimentary and alternative modalities/medicine (CAM)" will cease to exist and will be replaced with a regulatory scheme that omits administrative remedies for relief from arbitrarily-imposed restrictions on how we choose to maintain our own health......."

From the April 2007 Idaho Observer:

Advancing NAU greatest threat to health freedom It may be the greatest threat to FREEDOM, PERIOD.

If anyone was still wondering how the NAU was supposed to affect us, here's a good example. There's another example over here:

The Veterans Disarmament Act -HR 2640, section 102 HAS "PASSED" IN THE HOUSE

....two legal terms are radically redefined in the Veterans Disarmament Act to carry out this vicious attack on Veterans' gun rights.

One term relates to who is classified a "mental defective." Forty years ago that term meant one was adjudicated "not guilty" in a court of law by reason of insanity. But under the Veterans Disarmament Act, "mental defective" has been stretched to include anyone whom a psychiatrist determines might be a tiny danger to self or others.

The second term is "adjudicate." In the past, one could only lose one's gun rights through an adjudication by a judge, magistrate or court -- meaning conviction after a trial. Adjudication could only occur in a court with all the protections of due process, including the right to face one's accuser.

Now, adjudication in HR 2640 would include a finding by "a court, commission, committee or other authorized person" (namely, a psychiatrist).

Forget the fact that people with PTSD have the same violent crime rate as the rest of us. Vietnam vets with PTSD have had careers and obtained permits to carry firearms concealed.

It will now be enough for a psychiatric diagnosis (a "determination" in the language of the bill) to get a veteran barred ­for life ­ from owning guns.....

freedom4um.com/cgi-bin/readart.cgi?ArtNum=63785

I noted over here how now they just need to pass a law declaring conspiracy theorists, homophobes, or anti-Semites to the list of mental defectives. I'm going back to add a couple more like CHRISTIANS [after all, our country WAS founded on the Satanic Noahide Laws, according to those traitors on the hill, and Christians are mental defects as far as they are concerned...that should cover about 80-90% of us, and after Cheney and those other demons unleash their rogue nuke next week, and throw the whole country into terror and despair, that should take care of the rest of us.....this nation 100% occuppied by post-traumatic-stress mental defectives....never mind that the JEWSONTHEHILL have said all non-Jews and deniers of Jewish Supremacy will be beheaded, and the "other authorized person" who could declare us "mentally defective" could be a JEWISH PHARISEE-RABBI....that's not mentally

defective...nosiree....that's NORMAL when white is black and up is down and Lucifer is the God of ZOG.]

We will all have to conform to the ROCKEFELLER/ROTHSCHILD/SATANICBLOODLINE-UN's idea of what is "normal". See:

THE UN PLAN FOR YOUR MENTAL HEALTH

"....Dr. Shalala and other self-proclaimed "change agents" do tell us that

the old ways "a person thinks, feels, and acts" must change. They don't fit the new global ideology or the consensus process. According to Professor Benjamin Bloom, called the Father of OBE, the new "purpose of education and the schools is to change the thoughts, feelings and actions of students."7 So it's not surprising that UNESCO's Commission on Culture and Development in its report, Our Creative Diversity, wrote that -

"The challenge to humanity is to adopt new ways of thinking, new ways of acting, new ways of organizing itself in society, in short, new ways of living." 8

Referring to the management and monitoring of its education program, the Kansas State Board of Education made a similar statement, "QPA [Quality Performance Accreditation] is a process which demands new thinking, new strategies, new behavior, and new beliefs."

"New beliefs" and "new thinking" are key to the global management system. Adults as well as children must trade truth, facts, and logical thinking for myths, UN values, and the consensus process. The masses must learn to see themselves, not as individuals, but as part of a greater whole: a group, a community, the planet. Finally, the United Nations is nearing its goal: ***** to create a unified, socialist world made up of compliant world citizens ready to adapt to what ***** Al Gore called "a wrenching transformation of society." 9

THE WORLD HEALTH ORGANIZATION (WHO)

In 1946, the first head of WHO laid the foundation for today's mental health program. Having implied that Christian parents indoctrinated "their defenseless children" with "poisonous certainties" that cause war and conflict, Canadian psychiatrist Brock Chisholm, added,

"The problem is no longer the germ of diphtheria, but rather the attitudes of parents who are incapable of accepting and using proven knowledge...

"Surely the training of children in home and schools should be of at least as great public concern as their vaccination.... Individuals who have emotional disabilities of their own--guilts, fears, inferiorities--are certain to project their hates on to others... [S]uch reaction now becomes a dangerous threat to the whole world....

"We must be prepared to sacrifice much.... ****** If it cannot be done gently, it may have to be done roughly or even violently..." 10

Half a century has passed since Dr. Chisholm called for UN controls that would abolish divisive Christian "certainties". Today, WHO commands an international networking agency that helps nations around the world change and monitor the ways their people think, choose, and act. Called Nations for Mental Health, it links each member state to the UN agenda and promises to steer each nation toward the UN goal:

"Governments will be assisted to formulate, implement, monitor and evaluate mental health policies.

"Mental health policies should enable all individuals whose mental health is disturbed or whose psychological balance may be compromised to obtain services adapted to their needs, and to promote the optimal development of the mental health of the population." 11

To develop "the mental health of the population" and to prevent dissent and conflict from blocking progress, everyone must participate in the consensus process. Assessments for all -- young and old -- will show who might be "at risk" of not meeting the new mental standards for healthy communities....."

www.crossroad.to/text/articles/MentalHealth2-99.html

[ see how this will work: any bozo who is an "authorized person" (that will of course include anyone in the Homeland Gestapo) could at any time declare any one of us a "mental defective" in need of the government's "help"..... Lawsuit: ICE drugging {Haldol} detainees set for deportation: From October last year to the end of April this year, she said 56 received psychotropic medications during the removal process. Of those, 33 detainees received medication "because of combative behavior with the imminent risk of danger to others and/or self," she said....."First, I am aware of, and deeply concerned about reports that past practices may not have conformed to ICE detention standards," Myers said.....She added no detainee should be "involuntarily medicated without court order," except in emergency situations.....But both Soeoth [A CHRISTIAN MINISTER SEEKING ASYLUM] and Diouf say they had not exhibited any combative behavior.... A government report says he was medicated *** because he did not follow orders. http://www.freedom4um.com/cgi-bin/readart.cgi?ArtNum=63794 . Komrade Chertoff is going to require us to swear allegiance to ZOG in order to get a job, travel within or without the country. If you can't work, you can't eat, and if you don't carry a ZOG card, you must be a terrorist, and if anyone helps you, they must be terrorists, too, and subject to losing everything THEY have. Furthermore, if you don't have a ZOG card, you will be deported. WHERE TO? I don't know, but since Christians are citizens of heaven, I guess that means they will just SEND YOU THERE. See how it all works? They want to cast us out of the land God has given US for a possession, and that the name of Israel [the Church] not be heard anymore.]

They are so slick....

"...In short, the 'house of world order' will have to be built from the bottom up rather than from the top down. It will look like a great 'booming, buzzing confusion,' to use William James' famous description of reality, but an end run around national sovereignty, eroding it piece by piece, will accomplish much more than the old-fashioned frontal assault." -- Richard N. Gardner, in Foreign Affairs (April 1974)"

Two examples of how "the freedoms 'they' hate us" for, will have the final nail driven into them as they are merged into the coffin of NAU. Speaking of coffins, I've read some sites calling that little girl in the mural at Denver Airport Jewish. I don't know why....she was holding a B-I-B-L-E. More red herring, deceptive, balderdash on the part of the Magogians.

========================================================================

Psa 83:1 ¶ [[A Song [or] Psalm of Asaph.]] Keep not thou silence, O God: hold not thy peace, and be not still, O God.

Psa 83:2 For, lo, thine enemies make a tumult: and they that hate thee have lifted up the head.

Psa 83:3 They have taken crafty counsel against thy people, and consulted against thy hidden ones.

Psa 83:4 They have said, Come, and let us cut them off from [being] a nation; that the name of Israel [THE CHURCH] may be no more in remembrance.

Psa 83:5 For they have consulted together with one consent: they are confederate against thee.....

www.blueletterbible.org/kjv/Psa/Psa083.html#4

'Course, God is against them as well....see verse 9

www.blueletterbible.org/tsk_b/Psa/83/9.html

**** see here:

www.blueletterbible.org/tsk_b/Jdg/7/2.html

see verses 1-27 [edited] www.blueletterbible.org/kjv/Isa/Isa010.html#26

sorry to babble on...too much coffee and this ticked me off first thing in the morning.

oh...I almost posted this before something made me check one more little related verse:

Isa 9:4 For thou hast broken the yoke of his burden, and the staff of his shoulder, the rod of his oppressor, as in the day of Midian.

Isa 9:5 For every battle of the warrior [is] with confused noise, and garments rolled in blood; but [this] shall be with burning [and] fuel of fire. [oh, my http://www.blueletterbible.org/tsk_b/Isa/9/5.html / http://www.blueletterbible.org/kjv/Rev/Rev020.html#9 ]

Isa 9:6 For unto us a child is born, unto us a son is given: and the government shall be upon his shoulder: and his name shall be called Wonderful, Counsellor, The mighty God, The everlasting Father, The Prince of Peace.

Isa 9:7 Of the increase of [his] government and peace [there shall be] no end, upon the throne of David, and upon his kingdom, to order it, and to establish it with judgment and with justice from henceforth even for ever. The zeal of the LORD of hosts will perform this.

http://www.blueletterbible.org/tsk_b/Isa/9/7.html

now it all comes together.

AllTheKings'HorsesWontDoIt  posted on  2007-10-14   12:32:32 ET  Reply   Trace   Private Reply  

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