TCA/KREBS/CITRIC ACID CYCLE

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Reflection of the week 11

Hello there Biochem people.

Week 11!!!! Can you believe it?

We have reached the penultimate week for the blog and I must say that I am saddened and disappointed. I am not sure how time flew by so quickly but I must say that this journey was not as painful as anticipated. I believe that Sir has done what I did not think was possible, MAKE ME ENJOY BIOCHEM.

I will not lie to you, it has been a lot of work this semester, but I did have fun and the activities were very intriguing, kept me on my toes, and interested. Nevertheless, all things must come to an end and I am extremely excited to close this chapter and look forward to bigger and better things. However, that is not until next week and let us cross that bridge when we get to it. For now, I am going to focus on the week that was.

 

Week 11 was marked by the BIG EVENT. The one that we have been anticipating for weeks, the one that we thought would never come… the Mock Exam!!!!

 

Now this exam was not bad at all. The questions were fair and tested across all the topics. I was definitely prepared for worse, but I was pleasantly surprised by the ease to which I was able to answer the questions. There was only one drawback to this exam and I believe that most of us share the same sentiments, that we just needed a five minutes extra to finish that exam. In the local parlance, ‘sir put goat mouth on us’.

Why I say that? You might ask. Well because, he kept mentioning it in class that the biggest hurdle that we would face was the fact that our knowledge will not translate to the exam answers due to our inability to think quick enough and then actually finish the exam.

 

Well, I hate to say it, but he was right. I am not too disappointed though since I take it as a learning experience and know exactly what strategy to employ for the final. So, I am not ruminating on the fact that I could have done better in the exam, had I finished. I am optimistic of the finals.

 

Week 11 did not have any other remarkable event since we were all consumed with the big exam. Therefore, on to the final week of lectures, blogs and lab reports. I am eagerly anticipating the end since it means that I have successfully completed an entire semester of Biochem without being worse for wear.

 

Thus, all the best for the final week and wishing you all the success in all your remaining projects. Keep good and talk again in a bit.

Defenders of Mitochondria

Alright my Biochem peeps,

Here is my 2nd review article. It is based on the target therapies of cancer treatment. In particular, this article focuses on the drug known as dichloroacetate which targets a mitochondrial enzyme known as pyruvate dehydrogenase kinase. This enzyme inhibits the action of pyruvate dehydrogenase as well as increase the metabolism of cancer cells that facilitate cancer proliferation. This article is quite interesting and thought-provoking. It will aid your understanding of the concepts of glycolysis, Krebs, ETC and oxidative phosphorylation. It is also marvelous to read the mechanism of action of cancer cells and the intricacies of their metabolism that fosters survival and proliferation. Take a read and see for yourself.

Michelakis, Evangelos D. and Gopinath Sutendra. 2013. “Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.” Frontiers in Oncology 3(38): 1-11. doi: 10.3389/fonc.2013.00038

Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.

This article is not a study as in the previous posting, but rather delves into the strategies utilized in cancer treatment in particular targeting more efficient, effective and less destructive methods of cancer therapy. This article deals mainly with investigating the effects of inhibition of an enzyme, pyruvate dehydrogenase kinase (PDK) as well as the applications of similar techniques that promote glucose oxidation as a viable option in metabolic targeting of neoplastic cells. The authors also explore the pre-clinical and clinical evidence of the effectiveness of these inhibitors on different cancers.

Neoplastic cells have a unique metabolic property in which there is both molecular and genetic signalling that converts glucose oxidative phosphorylation in the mitochondria of cells to glycolysis in the cytosol even under normal conditions. This results in the increase in glucose consumption as well as lactate production in cancer cells. This process is referred to as the Warburg effect after Otto Warburg who published this hypothesis in 1956. This means that cancer cells depend on glycolysis as its primary energy source as well as the source of macromolecular building blocks through glycolytic intermediates.

This is initiated by the activation of hypoxia-inducible factor 1α (HIF-1α). HIF-1α is activated in hypoxic-like environments and then stimulates the expression of glycolytic enzymes, glucose transporters and mitochondrial enzymes that increase the glycolytic pathway as well as formation of lactate. One important mitochondrial enzyme that is activated is, PDK. PDK  is considered the gatekeeper of the mitochondria since it regulates the flux of pyruvate into the mitochondria to undergo oxidative phosphorylation. PDK does this by inhibition of pyruvate dehydrogenase that performs the link reaction that decarboxylates pyruvate to Acetyl-CoA to enter the Krebs cycle. This inhibition leads to pyruvate not entering the mitochondria and therefore is converted to lactate in the cytosol by lactate dehydrogenase. Oncogenes are responsible for the maintenance of hypoxic state for cancer cells to continue to activate HIF. In cancer cells, there is also a loss of p53 gene expression that was responsible for mitochondrial DNA repair and expression cytochrome c oxidase enzymes that aid in apoptosis of the cell. Loss of p53 directly induces the expression of PDK and increases the stability and transcriptional activity of HIF. Additionally, HIF also increase pro-angiogenic and metastasis-promoting factors such as VEGF and SDF-1.

With increase in glycolytic enzymes, there is an increase in glycolysis as well as these glycolytic enzymes are utilized to aid in anti-apoptotic activities, activation of transcription in neoplastic cells, inhibition of mitochondrial function. Enzymes such include, glyceraldehyde 3-phosphate dehydrogenase and hexokinase. An isoform of hexokinase, hexokinase 2 is up regulated in neoplastic cells and then translocated to the mitochondrial transition pore, a channel responsible for the release of apoptotic factors as well as the regulation of anions the facilitate oxidative phosphorylation. Thus, hexokinase 2 facilitates resistance to apoptosis as well as increase in glycolysis in tumour cells. The glycolytic intermediates may also be funnelled to alternative pathways to produce amino acids, nucleic acids and lipids.

The mechanism of action of cancer cells lies in the alteration of the mitochondria of the cell. The mitochondria are responsible for the intrinsic apoptotic mechanisms of the cells and suppression of mitochondrial function is critical to cancer cell proliferation. One method employed by cancer cells is the alteration of the mitochondrial membrane potential of the inner mitochondrial membrane. This membrane potential is crucial to oxidative phosphorylation and electron transport chain. This membrane potential is akin to the proton motive force that is generated as hydrogen ions are pumped into the inter-membrane space of the mitochondrion during electron transport chain. This membrane potential is also critical to maintenance of intracellular calcium levels since mitochondria act like calcium sinkholes that facilitate the influx of calcium.

Neoplastic cells, inhibit glycogen synthase kinase in the cytosol of the cells that lead to the translocation of hexokinase 2 to the mitochondrial membrane. Hexokinase 2 then binds to the inner membrane and blocks and inhibits the voltage-dependent anion channel found in the mitochondrial transition pore that is responsible for the efflux of anions to maintain membrane potential of the mitochondria that is crucial for oxidative phosphorylation and ETC. This results in the hyperpolarization of the membrane and prevents the opening of the channel in the mitochondrial transition pore that is responsible for release of apoptotic factors such as mitochondrial reactive oxygen species, cytochrome c oxidase  as well as apoptotic inducing factor.

The suppression of mitochondrial functioning leads to the decrease in oxidative phosphorylation as well as the release of apoptotic factors. With diminishing functioning, the mitochondria of tumour cells begin to degenerate and further increase the proliferative potential of the neoplastic cell. However, this information can be utilized in targeting specific cancers especially solid tumours.

A drug known as dichloroacetate is able to penetrate most cell membranes and tissues that are not accessible to chemotherapy treatments such as the brain. This molecule is able to activate pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinase and its isoforms. Dichloroacetate carboxylic group forms a salt bridge with Arg154 residue of the active site of pyruvate dehydrogenase kinase. Dicholoroacetate is able to decrease the membrane potential of inner mitochondria and aid in normalization of mitochondrial function in cancer cells. However, it does not affect non-cancer cells. It functions to increase apoptotic activity in the cancer cells as well as to increase the intake of pyruvate into the mitochondria and increase more ETC./oxidative phosphorylation. This decreases the production of lactate and the alternate pathways of glycolytic intermediates.

Subsequently, there is a deactivation of HIF since HIF is highly regulated by the TCA intermediate, alpha ketogluterate. Hence, an increase in TCA cycle leads to increase production of alpha ketogluterate that facilitates the inhibition of the action of HIF. This results in decrease tumour perfusion, glucose uptake, resistance to apoptosis as well as decrease production of macromolecules essential for tumour proliferation. DCA has also shown to increase p53 activity in cancer cells leading increase activation of apoptotic action by the mitochondria.

DCA is therefore very effective at selectively targeting cancer metabolism by inhibition of pyruvate dehydrogenase kinase. Clinical evidence of this has been observed in non-small cell lung carcinoma, breast carcinoma, glioblastoma, colon cancer, prostate and endometrial cancer as well as leukaemia.

However, other therapies may mimic the action of DCA and acquire similar results. Some therapies include the inhibition of lactate dehydrogenase to facilitate the channelling of pyruvate into the mitochondria to undergo oxidative phosphorylation. Similarly, decrease activation of the M2 isoform of pyruvate kinase that is expressed in neoplastic cells may prove to be another selective target for cancer treatment.

In concluding this article, the authors note that the aforementioned techniques are not a permanent solution to cancer therapy since there is no absolute action of these inhibitors on the metabolism of cancer.

My initial thoughts of this paper, was that it was long-winded and boring. However, as I continued past the introduction, the content became more interesting and captivating. The explanations and the organization of the information laid a good base level to which anyone reading was able to easily follow. It was greatly surprised at the intricacies of cancer metabolism and the very dynamic alterations made for the survival and proliferation of these cells. I knew that the mitochondria of cells were integral to the optimal functioning of the cell, nevertheless, I had no knowledge of the specific actions involved in mitochondrial functioning.

The authors aptly summarized the pathophysiology of neoplastic cells and then linked this to the action of the small DCA molecules and its efficiency in cancer treatment through selective inhibition. The importance of pyruvate dehydrogenase kinase was also elucidated. I was also intrigued by the importance of glycolysis to cancer metabolism and how effective glycolytic pathway is to the rapid growth and sustainability of the tumour.

However, it must be stated that even though DCA and the other techniques mentioned in this paper would prove highly valuable to oncology, there is no absolute solution since neoplastic cells have the ability to adapt and evolve to avoid the actions of these target drugs. There is no certainty that these therapies will show significant reduction in tumour proliferation in cancer patients and is subjected to individual differences. These therapies also have the potential to produce effects on non-cancer cells with prolonged use and high doses. Therefore, I am hesitant to believe that DCA and similar inhibitory therapies are a permanent solution to reducing the prevalence of cancer.

Overall, I think that this is a groundbreaking and lifesaving research in oncology since it will prove to reduce the mortality and morbidity of cancer. It is quite exciting to note that this method is a good step forward in finding more advance and effective treatment for a serious medical condition. Clinical evidence thus far has been positive and with more research, the methods of cancer treatment may develop into a simple cure like penicillin.

Reference

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590642/

Word Search Mitochondria Style

Word search

ACETYL-COA     ADIPOSE        ADP                    ATP

BROWN          CITRATE        COMPLEXES              COUPLING

CYANIDE        CYCLE          DECARBOXYLATION        DINITROPHENOL ELECTROCHEMICAL  ELECTRONS        ENERGY                 FADH

FORCE          GRADIENT       GTP                    HYDROGEN

INFANTS        INNER          INTERMEMBRANE          IONS

KETOGLUTERATE  LINK           LIPOATE                MAMMALS

MATRIX         MEMBRANE       NADH                   OUTER

OXALOACETATE   OXIDATIVE      OXYGEN                 PDH

PHOSPHORYLATION PROTEINS       PROTON                 PUMP

PYRUVATE       REACTIONS      REDOX                  RESPIRATION

ROTENONE       SUCCINYL       SYNTHASE               THERMOGENIN

TRANSFER       UNCOUPLERS

 

BONUS WORD CLUE: POWERHOUSE