Cancer and apoptosis

By Pasan Wijesinghe.

Introduction

Apoptosis, programed cell death is an active genetically controlled pathway which is responsible for the cell death, development and function of multicellular organisms. In healthy adult human 1011-1012 cells are produce daily. Therefore, apoptosis is needed for maintaining a constant cell number. Apoptosis contains cell shrinkage, chromatin condensation and nuclear and cell fragmentation. Apoptosis is a gene directed program. Specially Bcl-2 proteins and p53 genes are connected with its mechanisms. Therefore, mutations in genes can leads to cancers and progression of the cancers. Due to the mutations, they will be lost their ability to undergo apoptosis. E.g. down regulation of p53 gene inactivate the p53 gene and reduce apoptosis in many human cancers. Reduce apoptosis affect to the balance between cell death, cell divisions and cell deaths can also occur without receiving a signals to die. Apoptosis plays a major role in carcinogenesis and also in cancer treatments.

Mechanisms of apoptosis.

Caspase are initiators and executioners in apoptosis. Caspases can be activated by three pathways. Intrinsic(mitochondria)pathway and extrinsic (death receptor) pathway are the common initiation pathways. Both pathways lead to execution phase of apoptosis. Intrinsic endoplasmic pathway is the third pathway which is less well-known.

Extrinsic death receptor pathway.

Initiate with death ligand bind to a death receptor. Example type 1 TNF receptor (TNFR1) and related proteins Fas (CD95) their ligands TNF and Fas ligand. (Fasl). Death receptor have an intracellular death domain. It consists with Fas-associated death domain(FADD), TNF receptor-associated death domain(TRADD) and caspase8 cysteine protease. Binding of death ligand to the death receptor form a binding site for the adaptor protein. This complex is called death-inducing signaling complex(DISC). It initiates the activation of pro-caspase8. Activated form of the caspase 8 initiates apoptosis by cleaving executioner caspases.

Intrinsic mitochondrial pathway.

Initiated within the cell. Hypoxia, genetic damages, high concentration of cytosolic Ca2+and sever oxidative stress are triggers of the initiation of intrinsic pathway. Increased permeability of mitochondria and pro-apoptotic molecules like cytochrome-c in to the cytoplasm. This pathway regulated by BCL-2 family of proteins. BCL-2 gens can be observed at the chromosomal breakpoints of the translocation of chromosome 18 to 14 in follicular non-Hodgkin lymphoma. Two groups of BCL2 proteins present. Pro-apoptotic proteins, (e.g. Bak, Bax, Bad, Bcl-Xs, Bik, Bim, Bid, and Hrk). They promote release of cytochrome-c by mitochondria. Other group of proteins is Anti-apoptotic proteins, (e.g. Bcl-x, Bcl-w, Bfl-1, Bcl-2 and Mcl-1). They regulate apoptosis by blocking the release of cytochrome-c by the mitochondria. Initiation of the apoptosis determine by the balance between the Pro- and Anti- apoptotic proteins. Second mitochondria-derived activator of caspase (Smac), apoptosis inducing factor (AIF), Low Pi, IAP binding proteins (DIABLO). And Omi/high temperature requirement protein A(HtrA2). Cytochrome-c activates caspase3 by forming apotosome. Apotosome made out from Apaf-1, caspase9 and cytochrome-c. Samc/DIABLO or Omi/HtrA2 promote activation of the caspases by binding to inhibitor of apoptosis proteins (IAPs). That binding leads disruption of interaction between IAPs and caspase 3 or 9.

Common pathway (Execution phase).

Involves activation of caspases. Caspase9 is the upstream caspase for intrinsic pathway and caspase8 is the upstream caspase for extrinsic pathway. Both pathways converge to caspase3. Caspase3 cleaves inhibitor of the caspase-activated deoxy ribonuclease. Inhibitor of caspase-activated deoxy ribonuclease responsible for nuclear apoptosis. Protein kinases, cytoskeletal proteins, DNA repair proteins and inhibitory subunits of endonuclease family induce cleavage by downstream caspases. They will effect on the cell cycle, signaling pathways and to cytoskeleton and those will be contributed to morphological changes in apoptosis.

Intrinsic endoplasmic reticulum pathway.

This pathway known as caspase12 and mitochondria dependent pathway. ER injured by cellular stress (hypoxia, free radical) cause unfolding of proteins and reduced protein synthesis and TNF receptor associated factor2 (TRAF2) which is known as adaptor protein dissociates from procaspase 12.

  Figure number1:- Mechanisms of apoptosis

Resistance to apoptosis and mechanisms.

Reduce caspase functions, impaired death receptor signaling and disrupt the balance between Pro-apoptotic and Anti-apoptotic proteins cause resistance in apoptosis. Inhibitors of apoptosis proteins(IAPS), p53 protein and Bcl-2 proteins regulate the apoptosis.

Bcl-2 proteins located outer membrane of the mitochondria. Through dimerization form ion channels and pores. Important for the permeability of the mitochondria membrane. Can be divided in to three groups according to their functions and BH domain. Mcl-1, A1/Bfl-1, Bcl-XL are Bcl-2 proteins which contains all four BH domains. They protect cell from apoptosis stimuli. Second group of the Bcl-2 proteins made up BH-3 proteins only. They are pro-apoptotic and activated due to the cellular stresses and initiate apoptosis. Bax, Bok are examples for third group of Bcl-2 proteins and they are also pro-apoptotic proteins. Disruption of the balance between Anti- and Pro- apoptotic Bcl-2 proteins cause dys-regulated apoptosis. And overexpressing of some Bcl-2 proteins cause drug resistance in tumor cells and prevent undergoing apoptosis. Frame shift mutation in Bax(G)8 impair the apoptosis and it can lead to cancer cells to resist anti-cancer treatments. Overexpress of Bcl-2 increase the activity of NF- κB transcriptional activity in cancers. Bcl-2 down regulation control the expression of PAKT in cancer. Mlc-1 protein can block apoptosis.

The p53 gene located at the short arm of chromosome 17 and it is important in cell development, differentiation, cell cycle, DNA recombination, chromosomal segregation, gene amplification and cellular senescence. The p53 mutation is silent mutation, which deletion mutant of p53 (∆122p53) and it will reduce the apoptosis. It can leads to cancers also. Expression of BAX is regulate by p53. And also p53 control the Bcl-2 family proteins.

Signal transduction, activity of the cytokines and apoptosis also regulate by IAPs proteins. They inhibit the caspase activity by binding their BIR domains to the active site of the caspases. In some cancers IAP overexpressed. Expression of cIAP-2 can leads to drug resistance also.

Caspases can be divide mainly in to caspase 1 (-1,-4, -5,-13and -14) which involve in processing of cytokines and caspase2 (-2,-3,-6,-7,-8,-9and-10) which play a major role in apoptosis. Caspase2 can again divide in to initiator caspases which important for the initiation of the apoptotic pathway and effector caspases which important for the cleavage cellular compartments. Silent mutations, frameshift mutations, missense mutations, deletion mutations can be seen in caspases. Those mutations in caspases cause for reduce of apoptosis and can leads to different types of cancers. And also impairment in caspase function and low level of caspases can leads to decrease in apoptosis.eg: in stage ii colorectal cancers down regulation of caspase -9 can observed. Breast cancer cells survival contributed by the loss of caspase -3 functions. Downregulate of caspases help to growth and the development of the tumor cells.

In extrinsic pathway, ligand of the death receptors and death receptors are important. DR3 (apo-3), DR4 (TRAIL-1), DR5, (TRIL-2), DR6, EDAR are some examples of the death receptors. Death domain possess by those receptors. Signaling cascades activation cause due to the attachment of molecules to the death domain when it triggered by a death signal. Reduce apoptosis and impaired signaling due to the defects, impairment of the function of the receptors, abnormalities and downregulation of the receptors and also due to reduce death signals. Down regulation of the receptors can also leads to drug resistances. Eg: in lukaemia drug resistance was found due to downregulation of CD95. Changes which occur in the expression of death receptors and ligands can leads to imbalance of the apoptosis.

Target apoptosis for treatment in cancer

Abnormalities in apoptosis pathway can use as target of cancer treatments. By that it can restore the apoptotic signaling pathways and eliminate cancer cells. Some use to target the Bcl-2 proteins to inhibit them. Oblimersen sodium, small molecule inhibitors of Bcl-2 and BH3 mimetics are the agents use for that. Oblimersen sodium has the chemosensitising effect. Sodium butyrate, feneretinide are the small molecule inhibitors of Bcl-2 which affect gene expression. Gossypol, ABT-737 act on the proteins. ATF4, ATF3 are BH-3 mimetics which inhibit anti-apoptotic proteins and bind to Mcl-1 and inhibit. Some Bcl-2 specific si-RNA use to inhibit the expression of the Bcl anti-apoptotic genes. P53 based gene therapy, drug therapy and immunotherapy use as treatments.

 In gene therapy, genetically engineered wild- p53 gene introduce to the body to show sesitise tumor cells. In drug therapy small molecules used. CP-31398 get attach to DNA and destabilize the DNA-p53 and restore the p53 mutant gene. MI-219 inhibit the cell proliferation and do selective apoptosis by disrupt the MDM-p53 interaction. Tenovins decrease the growth of the tumor by activating the p53. Give vaccine which contain replication-defective adenoviral vector with wild-type human p53 in immunotherapy.

Designing of the novel drugs IAPs are the best drug targets. Specially XIAP which inhibit the apoptosis. Targeting XIAP can divide into Antisense and siRNA approaches. In Antisense, inhibition of the XIPA cause to control the tumor by radiotherapy. Radiation sensitivity of the cancer cells increased by the siRNA targeting to XIAP. Targeting survivn with use of siRNA diminish radio resistance, inhibit cell proliferation and induce apoptosis. Cyclin-dependent kinase inhibitors and Hsp90 inhibitors are small molecules which are the target of surviving in cancer therapy. Pepetidic and non –peptidic small molecules act as IAP inhibitors. Restore the activites of casepase-9 by smac mimetics 2, 3 by binding with the XIAP and cIAP-1/2.

Drugs can design to activate the caspases. Apoptin and small molecules caspase activators are used for activate caspases and also to increase drug sensitivity of the cancers. Selectively induce the apoptosis of malignant cells by apoptin. Increased the drug sensitivity of the cancer cells and lower the caspases activation energy by small molecules caspases activators. And caspase based gene therapy also use for the treatment in cancers.

Conclusion and future directions

Defects and abnormalities in apoptotic pathways play a major role in induce cancers. Therefore many treatment methods test through the apoptotic pathways. Molecular base understand of the apoptosis and its mechanisms can useful to develop treatments for the cancers. Study the chemistry of the apoptosis mechanisms and mutations will be important for the better understand about reduce apoptosis and cancers. Cancer cells produce higher entropy and also can see negative entropy flow for their survival. Tumor cells withdraw energy from the healthy tissues. Therefor it has a different of the magnitude of the entropy production in normal cells and cancer cells. If the tumor cells can’t withdraw energy from a healthy normal cells, then it can’t survival. Apoptosis happened. Mutations which occurs in apoptotic pathways may be leads to the entropy and energy changes. When there is a cancer cells then the temperature is low in there due to low metabolic rate. Gibbs energy of the cancer cells have to be negative value. Therefore if we can change the energy of the cancer cells, then we can reduce cancer and also increase apoptosis. But it is not easy. Hope that future studies will be pay more attentions about that also.

   Pasan maduranga wijesinghe.

(Undergraduate student, Institute of chemistry Ceylon)

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