INTRODUCTION:

Cell death plays a considerable role during physiological processes of multicellular organisms, particularly during embryogenesis and metamorphosis The term programmed cell death was introduced in 1964, proposing that cell death during development is not of accidential nature but follows a sequence of controlled steps leading to locally and temporally defined self-destruction. Eventually, the term apoptosis had been coined in order to describe the morphological processes leading to controlled cellular self-destruction. Apoptosis is of Greek origin, having the meaning "falling off or dropping off. The apoptotic mode of cell death is an active and defined process, which plays an important role in the development of multicellular organisms and in the regulation and maintenance of the cell populations in tissues upon physiological and pathological conditions. It should be stressed that apoptosis is a well defined and possibly the most frequent form of programmed cell death, but that other, non-apoptotic types of cell death also might be of biological significance.

Induction of apoptotic cell death after treatment with anticancer drugs and irradiation is correlated with tumour response. In addition, failure in anticancer treatment is due to drug resistance in cancer cells, and the phenomenon of drug resistance is considered to be almost equal to resistance to apoptosis^1,2,3.

Despite the strong correlation between induction of apoptosis and increased drug and irradiation sensitivity, the early response cell death, apoptosis, is not necessarily correlated with overall tumour sensitivity^4,5.

Anticancer drug –induced cell death is closely associated with an increase in apoptosis by the caspase –dependent pathway;^6,7 however ,pancaspase inhibitor cannot completely block drug induced cell death ^8,9 suggesting that anticancer drug induced cell death also involves a caspase independent pathway.

Apoptosis:

Apoptosis can be defined as "gene-directed cellular self-destruction" or programmed cell death. Apoptotic cells can be recognised by a characteristic pattern of morphological, biochemical and molecular changes. These changes can be broadly assigned to three stages. Early stage characterized by Decreased cell size (cell dehydration) ,Altered cell membrane, Large (50kb) DNA strand breaks and Increase in cellular calcium levels. Intermediate stage characterized by DNA cleavage into 180-200bp fragments, which give the character"laddering" on a DNA gel, Further decrease in cell size, Alterations in plasma membrane symmetry and Decreased pH. Late stage with Loss of membrane function.

Significance of apoptosis:

The development and maintenance of multicellular biological systems depends on a sophisticated interplay between the cells forming the organism, it sometimes even seems to involve an altruistic behaviour of individual cells in favour of the organism as a whole. During development many cells are produced in excess, which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues. A particularly instructive example for the implication of programmed cell death in animal development is the formation of free and independent digits by massive cell death in the interdigital mesenchymal tissue ¹⁰.Other examples are the development of the brain, during which half of the neurons that are initially created will die in later stages when the adult brain is formed ¹¹ and the development of the reproductive organs ¹². Also cells of an adult organism constantly undergo physiological cell death, which must be balanced with proliferation in order to maintain homeostasis in terms of constant cell numbers. The majority of the developing lymphocytes die either during genetic rearrangement events in the formation of the antigen receptor, during negative selection or in the periphery, thereby tightly controlling the pool of highly efficient and functional but not self-reactive immune cells and at the same time keeping lymphocyte numbers relatively constant ¹³.

Taken together, apoptotic processes are of widespread biological significance, being involved in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of the immune system and in the removal of defect and therefore harmful cells. Thus, dysfunction or dysregulation of the apoptotic program is implicated in a variety of pathological conditions. Defects in apoptosis can result in cancer, autoimmune diseases and spreading of viral infections, while neurodegenerative disorders, AIDS and ischaemic diseases are caused or enhanced by excessive apoptosis ¹⁴

Morphological features of apoptosis:

Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms. Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion. This makes apoptosis distinct from another form of cell death called necrosis in which uncontrolled cell death leads to lysis of cells, inflammatory responses and, potentially, to serious health problems. Apoptosis, by contrast, is a process in which cells play an active role in their own death that is why apoptosis is often referred to as cell suicide.

Upon receiving specific signals instructing the cells to undergo apoptosis a number of distinctive changes occur in the cell. A family of proteins known as caspases is typically activated in the early stages of apoptosis. These proteins breakdown or cleave key cellular components that are required for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases can also activate other degradative enzymes such as DNAses, which begin to cleave the DNA in the nucleus.

Apoptotic cells display distinctive morphology during the apoptotic process. Figure 1 represents a trophoblast cell undergoing apoptosis. Typically; the cell begins to shrink following the cleavage of lamins and actin filaments in the cytoskeleton (A). The breakdown of chromatin in the nucleus often leads to nuclear condensation and in many cases the nuclei of apoptotic cells take on a "horse-shoe" like appearance (B). Cells continue to shrink (C), packaging themselves into a form that allows for their removal by macrophages. These phagocytic cells are responsible for clearing the apoptotic cells from tissues in a clean and tidy fashion that avoids many of the problems associated with necrotic cell death. In order to promote their phagocytosis by macrophages, apoptotic cells often undergo plasma membrane changes that trigger the macrophage response. One such change is the translocation of phosphatidylserine from the inside of the cell to the outer surface. The end stages of apoptosis are often characterised by the appearance of membrane blebs (D) or blisters process. Small vesicles called apoptotic bodies are also sometimes observed.

Fig-1 Trophoblast cell undergoing apoptosis

Factors inducing apoptosis:

There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as the expression of pro- and anti-apoptotic proteins (eg. the Bcl-2 proteins or the Inhibitor of Apoptosis Proteins), the severity of the stimulus and the stage of the cell cycle. Fig.2 illustrates some of the major stimuli that can induce apoptosis. In some cases the apoptotic stimuli comprise extrinsic signals such as the binding of death inducing ligands to cell surface receptors called death receptors. These ligands can either be soluble factors or can be expressed on the surface of cells such as cytotoxic T lymphocytes. The latter occurs when T-cells recognise damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme.

In other cases apoptosis can be initiated following intrinsic signals that are produced following cellular stress. Cellular stress may occur from exposure to radiation or chemicals or to viral infection. It might also be a consequence of growth factor deprivation or oxidative stress caused by free radicals. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria. The relative ratios of the various bcl-2 proteins can often determine how much cellular stress is necessary to induce apoptosis. Apoptotic cells can be recognized by stereotypical morphological changes: the cell shrinks, shows deformation and looses contact to its neighbouring cells. Its chromatin condenses and marginates at the nuclear membrane, the plasma membrane is blebbing or budding, and finally the cell is fragmented into compact membrane-enclosed structures, called 'apoptotic bodies' which contain cytosol, the condensed chromatin, and organelles. Figure 3 represents apoptotic versus necrotic morphology. The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue without causing an inflammatory response. Those morphological changes are a consequence of characteristic molecular and biochemical events occurring in an apoptotic cell, most notably the activation of proteolytic enzymes which eventually mediate the cleavage of DNA into oligonucleosomal fragments as well as the cleavage of a multitude of specific protein substrates which usually determine the integrity and shape of the cytoplasm ¹⁵

Apoptosis is in contrast to the necrotic mode of cell-death in which case the cells suffer a major insult, resulting in a loss of membrane integrity, swelling and disrupture of the cells. During necrosis, the cellular contents are released uncontrolled into the cell's environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue ¹⁶

Molecular mechanisms of apoptosis signalling pathways

ⅰ)Various death signals activate common signalling pathways:

Apoptosis is a tightly regulated and at the same time highly efficient cell death program which requires the interplay of a multitude of factors. The components of the apoptotic signalling network are genetically encoded and are considered to be usually in place in a nucleated cell ready to be activated by a death-inducing stimulus ^17. Apoptosis can be triggered by various stimuli from outside or inside the cell, e.g. by ligation of cell surface receptors, by DNA damage as a cause of defects in DNA repair mechanisms, treatment with cytotoxic drugs or irradiation, by a lack of survival signals, contradictory cell cycle signalling or by developmental death signals. Death signals of such diverse origin nevertheless appear to eventually activate a common cell death machinery leading to the characteristic features of apoptotic cell death.

ⅱ) Caspases are central initiators and executioners of apoptosis:

The caspases, cysteine proteases are of central importance in the apoptotic signalling network which are activated in most cases of apoptotic cell death ^18. Actually, strictly defined, cell death only can be classified to follow a classical apoptotic mode if execution of cell death is dependent on caspase activity .

The term caspases is derived from cysteine-dependent aspartate-specific proteases: their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG, and the caspases specifically cleave their substrates after Asp residues .In the cell, caspases are synthesized as inactive zymogens, the so called procaspases, which at their N-terminus carry a prodomain followed by a large and a small subunit which sometimes are separated by a linker peptide. Upon maturation, the procaspases are proteolytically processed between the large and small subunit, resulting in a small and a large subunit. The prodomain is also frequently but not necessarily removed during the activation process. A heterotetramer consisting of each two small and two large subunits then forms an active caspase. The proapoptotic caspases can be divided into the group of initiator caspases including procaspases-2, -8, -9 and –10, and into the group of executioner caspases including procaspases-3, -6, and –7. Whereas the executioner caspases possess only short prodomains, the initiator caspases possess long prodomains, containing death effector domains (DED) in the case of procaspases-8 and –10 or caspase recruitment domains (CARD) as in the case of procaspase-9 and procaspase-2. Via their prodomains, the initiator caspases are recruited to and activated at death inducing signalling complexes either in response to the ligation of cell surface death receptors (extrinsic apoptosis pathways) or in response to signals originating from inside the cell (intrinsic apoptosis pathways).

In extrinsic apoptosis pathways, e.g. procaspase-8 is recruited by its DEDs to the death inducing signalling complex (DISC), a membrane receptor complex formed following to the ligation of a member of the tumor necrosis factor receptor (TNFR) family ^19. When bound to the DISC, several procaspase-8 molecules are in close proximity to each other and therefore are assumed to activate each other by autoproteolysis^20.

Intrinsic apoptosis pathways involve procaspase-9 which is activated downstream of mitochondrial proapoptotic events at the so called apoptosome, a cytosolic death signalling protein complex that is formed upon release of cytochrome c from the mitochondria ²¹. In this case it is the dimerization of procaspase-9 molecules at the Apaf-1 scaffold that is responsible for caspase-9 activation . Once the initiator caspases have been activated, they can proteolytically activate the effector procaspases-3, -6, and -7 which subsequently cleave a specific set of protein substrates, including procaspases themselves, resulting in the mediation and amplification of the death signal and eventually in the execution of cell death with all the morphological and biochemical features usually observed ²²

Fig-2 Factors influencing apoptosis

) Mitochondria as central regulators of intrinsic apoptosis pathways:

Mitochondria play a central role in the integration and propagation of death signals originating from inside the cell such as DNA damage, oxidative stress, starvation, as well as those induced by chemotherapeutic drugs²³. Most apoptosis-inducing conditions involve the disruption of the mitochondrial inner transmembrane potential (Dy) as well as the so called permeability transition (PT), a sudden increase of the inner mitochondrial membrane permeability to solutes with a molecular mass below approximately 1.5 kDa. Concomitantly, osmotic mitochondrial swelling has been observed by influx of water into the matrix with eventual rupture of the outer mitochondrial membrane, resulting in the release of proapoptotic proteins from the mitochondrial intermembrane space into the cytoplasm²⁴. Released proteins include cytochrome c, which activates the apoptosome and therefore the caspase cascade, but also other factors such as the apoptosis-inducing factor AIF , the endonuclease endoG ²⁵.In addition to the release of mitochondrial factors, the dissipation of Dy and PT also cause a loss of the biochemical homeostasis of the cell: ATP synthesis is stopped, redox molecules such as NADH, NADPH, and glutathione are oxidized, and reactive oxygen species (ROS) are increasingly generated ^26,27. Increased levels of ROS directly cause the oxidation of lipids, proteins, and nucleic acids, thereby enhancing the disruption of Dy as part of a positive feedback ²⁸. Several possible mechanisms for PT have been proposed, but there appears to exist consent that a so-called permeability transition pore (PTP) is formed consisting of the adenin nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC) as its core components. ANT is the most abundant protein of the inner mitochondrial membrane and as a transmembrane channel is responsible for the export of ATP in exchange with ADP (antiport). Overexpression of ANT-1 in human cancer cell lines dominantly induces apoptosis with all its characteristic features whereas its closely conserved homologue ANT-2 does not, indicating a specific mechanistic role of ANT-1 in mitochondrial apoptosis events . VDAC, also called porin, is the most abundant protein of the outer mitochondrial membrane and forms a non-selective pore through the outer membrane. Indicated by direct protein-protein interactions, VDAC-ANT complexes presumably connect inner and outer mitochondrial membrane to so-called ‘contact sites’, corresponding to a close association of the two membranes and thereby possibly constituting the PT pore ^29. Since PT, loss of Dy, and release of mitochondrial proteins are of central importance in mediating and enhancing apoptotic pathways, those mitochondrial events must be kept under strict control of regulatory mechanisms which are in many ways dependent on members of the Bcl-2 family

Fig.3 Apoptotic versus necrotic morphology

) Regulatory mechanisms in apoptosis signaling:

Commonly, the activation of apoptosis is regarded to occur when a cell encounters a specific death-inducing signal such as the ligation of a death receptor by its cognate ligand or if cells are treated with a cytotoxic drug. This suggests that the apoptosis signalling pathways in viable cells are kept in an inactive state and are only turned on in response to a death stimulus. But it should be taken into account that the components of the apoptotic signalling network are genetically encoded and ready for action in most cell types. Therefore, an interesting and possibly more realistic alternative view would be as follows: all cells of a multicellular animal might be intrinsically programmed to self-destruct and indeed would die instantaneously unless cell death is continously repressed by survival signals such as provided by other cells of the organism, e.g. growth factors, hormones, nutrients. Those survival signals enhance the expression and/or activity of antiapoptotic regulatory molecules thereby keeping in check the activation of proapoptotic factors ³⁰.

a) The Bcl-2 family:

Bcl-2, an oncogene which in follicular lymphoma is frequently linked to an immunoglobulin locus by the chromosome translocation , was the first example of an oncogene that inhibits cell death rather than promoting proliferation. B cells transfected with Bcl-2 were shown to be rendered resistant towards apoptosis induced by IL-3 withdrawal: for the first time it was shown that the pathway toward tumorigenesis depends not only on the ability to escape growth control but also depends on the ability to prevent apoptosis ³¹. When homologues of Bcl-2 had been identified, it became apparent that a Bcl-2 family of proteins can be defined by the presence of conserved sequence motifs known as Bcl-2 homology domains (BH1 to BH4). In mammals, up to 30 relatives have been decribed of which some belong to a group of pro-survival members and others to a group of proapoptotic members ^32. In addition to Bcl-2 itself, there are a number of other prosurvival proteins, e.g. Bcl-XL, Bcl-w, A1, and Mcl-1, which all possess the domains BH1, BH2, BH3, and BH4. The proapoptotic group of Bcl-2 members can be devided into two subgroups: the Bax-subfamily consists of Bax, Bak, and Bok that all possess the domains BH1, BH2, and BH3, whereas the BH3-only proteins (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, BNIP3, and Spike) have only the short BH3 motif, an interaction domain that is both necessary for their killing action^33.Antiapoptotic Bcl-2 members sequester proapoptotic Bcl-2 members by bindig to their BH3 domains and thereby ultimately prevent Bax or Bak activation/ oligomerization and consequently inhibit mitochondrial proapoptotic events: overexpression of Bcl-2 or Bcl-XL potently inhibits apoptosis in response to many cytotoxic insults, among others by suppressing the generation of ROS, stabilizing Dy, preventing PT and consequently blocking the release of e.g. cytochrome c ³⁴. Besides eliciting its antiapoptotic effects on the mitochondrial level by indirectly controlling the activation of the apoptosome, Bcl-2 also appears to inhibit apoptotic pathways that are independent of Apaf-1/caspase-9 and which might depend on caspase-7 as a central effector ³⁵

5. Disease as a consequence of dysregulated apoptosis:

In the adult human body several hundred thousand cells are produced every second by mitosis, and a similar number die by apoptosis for the maintenance of homeostasis and for specific tasks such as the regulation of immune cell selection and activity . Dysregulation of apoptotic signalling can play a primary or secondary role in various diseases with insufficient apoptosis leading to e.g. cancer (cell acumulation, resistance to therapy, defective tumor surveillance by the immune system), autoimmunity (failure to eliminate autoreactive lymphocytes), persistent infections (failure to eradicate infected cells), whereas excessive apoptosis contributes to e.g. neurodegeneration (Alzheimers’ disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis), autoimmunity (uncontrolled apoptosis induction in specific organs), AIDS (depletion of T lymphocytes), and ischaemia (stroke, myocardial infarction) ³⁶. Malfunction of the death machinery results from the mutation of genes that code for factors directly or indirectly involved in the initiation, mediation, or execution of apoptosis, and several mutations in apoptosis genes have been identified as a causing or contributing factor in human diseases ³⁷.Of special interest is the involvement of defective apoptosis pathways in tumor formation, progression, and metastasis as well as the occurrence of multidrug resistance during cancer therapy. During the last years it became more and more evident that tumorigenesis is not merely the result of excessive proliferation due to the activation of oncogenes but to the same extent depends on the – frequently concurrent - impairment of apoptosis checkpoints . Intriguingly, many of the alterations that induce malignant transformation, such as oncogene-driven deregulated proliferation and invasion, actually sensitize a cell to apoptosis, and therefore only those oncogenic transformed cells will survive and become malignant which additionally acquire defects in apoptosis pathways and therefore are protected against cell death induction ^38. A transformed cell can achieve protection against apoptosis by inappropriate activation or expression of antiapoptotic proteins (which usually act as oncogenes), or by the inactivation of proapoptotic factors (which usually are tumor-suppressors).

Methods of causing cell death:

ⅰ) Spontaneous apoptotic cell death in tissue remodeling:

Apoptosis is a physiological process for cell removal that functions to balance mitosis in the development and maintenance of tissue homoeostasis ³⁹.An opposed to necrosis, apoptosis is a nontoxic model of cell death, which affects single cells in the midst of living tissues without eliciting an inflammatory response. Macrophage engulfment of apoptotic cells is known to be important in the remodeling of tissues,and it contributes to the resolution of inflammation through the removal of effete cells prior to release of noxious cellular constituents. Moreover, apoptotic cells are a partial source of self-antigens, and clearance of cell corpes is thougt to preclude the induction of autoimmune responses. Tissue homoeostasis is dependent not only on the balance between mitosis and apoptosis, but also on the rate of apoptosis vs that of cell clearance.

ⅱ) Anticancer drug induced apoptotic cell death:

Many anticancer drugs induce apoptosis,and the magnitude of cell death is well correlated with tumour response.The molecular mechanisms by which anticancer drugs induce apoptosis are mediated by mitochondrial dysfunction ,which is regulated by the balance of proapoptotic and antiapoprotic proteins in the Bcl-2 family ^40.DNA damage to the cancer cells induces the activation of proapoptotic proteins such as Bax and Bak, which translocate from the cytosol to mitochondria. The proteins are then inserted into mitochondrial membranes, where they interact with a voltage –dependent anion channel (VDAC)/ adenine nucleotide translocator (ANT) complex to release cytochrome c, resulting in the activation of caspase cascades ⁴¹ .Antiapoptotic proteins ,such as Bcl-2 and Bcl-xL inhibit the release of cytochrome c, thereby blocking the activation of caspase cascades.A functional defect of a proapoptotic protein or overexpression of an antiapoptotic protein causes resistance to apoptosis; both equally represent drug resistance in anticancer treatment.

ⅲ) Radiation induced apoptotic cell death:

Radiation induced DNA damage as well as anticancer drugs induce apoptosis in association with the therapeutic effect, and the significance of apoptosis as a process of cell loss from normal tissue and tumours has been critically evaluated. Various factors that may modulate the apoptotic response to DNA damage include the p53 status of the cell, levels and activity of the Bax and Bcl-2 families of proteins.Alteration of factors in the apoptotic pathway causes less susceptibility to apoptosis in response to DNA damage.

Methods of measuring cell death:

Apoptosis represents energy requiring spontaneous single cell death with specific morphologic and biochemical features.Apoptosis is characterized by a stereotypic pattern of morphologic features,which can be illustrated mostly by electron microscopy.DNA nd biochemical assays ,based on the specific pattern of nucleosomal fragmentation can detect apoptosis. In vitro methods for identification of apoptosis include regular cleavage of DNA into internucleosomal 180-200 base pair fragment i.e DNA laddering as opposed to DNA from necrotic cells,which appears as a smear of randomly degraded DNA; DNA fragmentation ( terminal deoxynucleotidal transferase mediated dUTP nick –end labeling) that is used in situ labeling techniques for demonstrating apoptosis in paraffin sections .In vivo detection, identification , and characterization of apoptosis are even more difficult. There has only been limited success so far in monitoring apoptosis by standard, noninvasive invivo imaging modalities such as magnetic resonance imaging and spectroscopy (MRI/MRS), computed tomography (CT), positron emission tomography (PET), and radionuclide imaging methods ^[42]. Nevertheless, since in vivo monitoring of the apoptotic process is vital in clinical situations and in guiding effective treatment for cancer patients, a somewhat more invasive but more sensitive approach using an extracellular marker such as PS may be useful.

CONCLUSION:

Malignant cells are characterized by alterations in multiple signaling pathways that promote proliferation, inhibit apoptosis, promote angiogenesis in the case of solid tumors, and enable cancer cells to invade and migrate through tissues. Recent advances in understanding the molecular mechanisms of apoptosis and non apoptotic cell death induced by anticancer treatment have provided critical information not only for understanding tumour response in terms of signal transduction pathways of cell death ,but also for creating an opportunity to design targeting therapy for promoting cell death.

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