Introduction
Cancer is a multifactorial disease and the second cause of death of general population in the world. Its causality is related to lifestyle, genetic predisposition and psychological and mood states of individuals.1 The prevalence of cancer has increased in recent years, and it is therefore a health problem that demands a very high level of attention.2,3 In males, the highest rates of cancer incidence occur in the prostate, lung, bronchi, colon, rectum and urinary bladder; in females, in the breast, lung, bronchi, colon, rectum, uterus and thyroid gland. The above is an indicator that the most common types of cancer in men and women, respectively, are prostate and breast cancer.4 In children, the highest incidence of cancer is observed in the bone marrow, brain and lymph nodes.5,6
Epithelial cancer is one of the most aggressive types. Particularly in prostate cancer, the treatment will depend on the stage the disease is identified at; however, it is very common for it to progress and develop a phenotype of apoptosis resistance and, therefore, of hormone independence, which makes for cells to continue proliferating until metastasis is reached, hence the development of new therapeutic strategies that prevent the disease from reaching advanced stages being essential.
During the process of apoptosis of numerous cell types, a sustained increase in cytoplasmic Ca2+ has been defined as a determinant in the onset of programmed cell death.7 Considering this rationale, for years we have worked on the study of the mechanisms of cytoplasmic calcium homeostasis control and the impact of Ca2+ altered level in various cell and neoplastic types.
We have been able to demonstrate that an increased level of cytoplasmic calcium results in the onset of programmed cell death in Chinese hamster epithelial cell lines of prostate and ovarian cancer. In this sense, our research group discovered a nonselective cationic channel permeable to Ca2+ with 23pS-conductance, expressed under conditions of induced apoptosis by removing serum from the culture medium in androgen-independent lymph node carcinoma of the prostate (LNCaP) cells.8 In addition, it was also possible to isolate the complementary DNA (cDNA) from LNCaP cells that encodes apoptosis regulatory protein 2 (which we called ARP2), which, when overexpressed, induces apoptosis in Xenopus laevis oocytes and androgen-independent LNCaP cells, from which ARP2 was originally isolated.9 ARP2 overexpression was shown to induce Chinese hamster ovary cell apoptosis. These findings demonstrate that Ca2+ increased levels are an indispensable condition to initiate apoptosis in various epithelial cell lines, and this protein could therefore constitute an excellent tool in the future in the treatment of the disease by preventing progression to the advanced stages that culminate with a metastatic process.
Prostate cancer
Prostate cancer is the second leading cause of cancer-related death in the United States and the fifth in the world.10-12 The prevalence of this disease increases with increasing age. Prostate cancer is found during autopsy in more than half of men older than 50 years in the United States, in spite of this disease beimng the cause of death only in 3 %. It is an asymptomatic disease until the appearance of metastatic lesions that are usually discovered in bone tissue. Initial therapies for its treatment include surgery, radiation, and use of 5-alpha reductase inhibitors, which promotes the formation of more potent androgens from testosterone.13 Initial treatment methods often cause sexual, urinary and intestinal dysfunction.14
From the molecular point of view, this disease manifests itself at early and late stages. In the former, prostate cell proliferation is slow and dependent on androgens, so that during treatment with chemotherapeutic agents, the cells manage to repair the damage and continue to proliferate. Over time, cells become independent of androgenic hormones to proliferate, which culminates in the development of metastases, leading to patient death.15
Apoptosis and disease
Programmed cell death or apoptosis is an intrinsic cellular event of relevant importance in processes such as cell homeostasis, embryonic development and onset and maintenance of several diseases such as cancer and atherosclerosis.16,17 This mechanism develops through two pathways; the cell death receptor and the mitochondrial pathway.18 In turn, the apoptotic process develops in several stages: the first one is related to the stimuli that trigger programmed cell death, the second involves the signal transduction processes, in the third, effector enzymes that take care of cell disassembly, such as active caspases, participate and, finally, in the fourth, chromatin condensation, DNA degradation and apoptotic body formation occurs.18,19
There are various apoptotic pathways, such as the extrinsic or death receptor pathway, whose initiating caspase is caspase 8, and the intrinsic or mitochondrial pathway, which has cytochrome C as intermediary protein and caspase 9 as initiator. Both can converge in the set of effector caspases, mainly caspases 3 and 7, which are activated through self-processing or cascade activation, whereby caspases themselves are self-activated and activated between each other.20,21 There is another alternative pathway called the perforin-granzyme pathway,22,23 which corresponds to an important serine protease complex in apoptosis that is induced by cytotoxic T cells by activating independent caspase pathways.
Ca2+ as the second determining messenger in the apoptotic process
Variations in intracellular Ca2+ concentration promote the onset of cellular events such as the regulation of metabolism, mitosis, secretion of neurotransmitters and hormones, as well as the contraction of myofilaments, and therefore it is considered a second messenger that is determinant for cell functions.24,25 Ca2+ levels are also involved in the regulatory mechanisms of apoptotic programmed cell death.26,27 A Ca2+ level higher than basal is considered highly toxic, since it generates the activation of proteases and phospholipases that participate in cell disassembly.28 The increase in Ca2+ can occur at early and late stages of apoptosis, through the outflow of Ca2+ from the endoplasmic reticulum and Ca2+ inflow to the cytoplasm through channels activated by Ca2+ release.7,29 It is necessary taking into account that part of intracellular Ca2+ moves to the endoplasmic reticulum through the endoplasmic reticulum calcium pump and that Ca2+ is released from these stores by inositol 1,4,5 triphosphate receptors or ryanodine receptors. Additionally, in various intracellular organelles (such as the Golgi apparatus, nucleus and mitochondria) there are specialized systems for Ca2+ transport.30
Calcium enters the cell through transmembrane proteins called calcium channels.31 Calcium passes through the channels by means of different mechanisms, depending on the type of channel or voltage, or through receptors; these pathways do not require energy, unlike calcium pumps, which release cytoplasmic calcium to the exterior of the cell at the expense of the use of adenosine triphosphate.
In collaboration with Dr. Agustín Guerrero of CINVESTAV, in the laboratory of the Institute of Cellular Physiology, National Autonomous University of Mexico, for the obtainment of electrophysiological records, the membrane fixation technique (patch clamp) and single cell simultaneous Ca2+ measurements were combined, with the purpose of studying the activation of Ca2+-permeable channels using two different inducers: an ionophore (ionomycin) and serum elimination.8 The latter deprives the cells from essential nutritional components such as proteins, growth factors and vitamins, inducing cells to death.32,33
The results demonstrated the activation of a non-selective and Ca2+-permeable cationic channel with 23pS-conductance. Ca2+ increased levels induced cells to apoptosis, which demonstrated that activation of this channel promotes the development of LN-CaP cells programmed death, which in turn constitutes an important finding for programmed death induction in this cell type (Fig. 1).
Continuing with this line of research, in our group, cDNA arp 2, which codes for ARP2 of androgen-independent LNCaP cells, and which were induced to apoptosis by serum removal, was isolated, identified and characterized.9 The sequence of this protein shows homology with the splicing factor prp8 (a component of the spliceosome)34 and proapoptotic functions in different cell types (Fig. 2). The alternative-type splicing mechanism is defined as a property that prevails in higher organisms to produce multiple proteins from a simple gene.35,36 Prp8 protein ubiquitin-binding activity suggests that some splicing factors for pre-mRNA can be ubiquitinated to have an interaction with Prp8.37
Xenopus laevis frog oocytes injected with arp2 mRNA showed blistering after 12 hours of injection; these morphological changes were observed to increase when the oocytes are treated with thapsigargin (Fig. 3). It was also possible to observe that the oocytes suffered a decrease in the resting membrane potential: from a control value of -46.8 ± 6.6 mV they went to -5.9 ± 3.4 mV (5-8 oocytes, two frogs) (Fig. 4). Incubation of the cells with thapsigargin accelerated and increased morphological changes, with the loss of definition of the animal and plant poles being evident, which was observed 36 hours after injection with arp2 mRNA and eight hours after incubation with thapsigargin (Figure 3B).
The morphological changes and membrane depolarization observed in our study have been described in Xenopus laevis oocytes when injected with cytochrome C to trigger the apoptotic mechanism,38 as well as in oocytes injected with the Bcl-xs proapoptotic molecule.9,39 Thapsigargin, a drug that depletes Ca2+ intracellular stores by the specific inhibition of endoplasmic reticulum Ca2+-ATPase, elicits the activation of cytoplasmic membrane-independent voltage channels (TRP, transient receptor potential).40
This way, we have gathered sufficient evidence that ARP2 is an apoptosis promoter in androgen-independent LNCaP cells and in Chinese hamster ovary epithelial cells,41 thus favoring Ca2+ sustained increases by directly interacting with Ca2+-permeable membrane channels or by presenting a membrane channel function. On the other hand, considering the homology of the cDNA sequence that encodes ARP2 with the Prp8 splicing factor, it is not yet known whether this protein could additionally have any participation in the assembly or functioning of the spliceosome or be related to alternative splicing regulation mechanisms of mRNAs that encode proteins of the apoptotic machinery. There is a study where the Prp8 protein was observed to bind to one of the androgen receptor domains of prostate cancer cells and that, consequently, could be intervening in its functionality during the development of the disease.42 Furthermore, some splicing factors that make up the spliceosome, such as the SNW1 factor, have been reported to be linked to the development of breast cancer.43
Continuing with the project, ARP2-encoding cDNA was cloned in an expression plasmid and transfected to androgen-independent LNCaP cells and Chinese hamster ovary cells. ARP2 overexpression induced the cells to develop apoptosis, with an important impact on cell viability and effector caspases 3 and 7 activation, which are comparable results to those of starving cells treated with ionomycin41 (Fig. 5). In this study, using confocal microscopy, it was possible to demonstrate that ARP2 is initially located in the perinuclear region of cells and migrates over time to the plasma membrane41 (Fig. 5). Taking into account our results, we consider that ARP2 is inserted into the plasma membrane, with a function similar to that of a membrane channel, thus constituting a valuable target to modulate Ca2+ flow and concentration in the cytoplasm of epithelial cancer cells that show an apoptosis-resistant phenotype41 (Fig. 6).
Perspectives
The apoptosis mechanism in malignant cell resistant phenotypes has been the subject of extensive study in recent years. Due to the strong impact of cancer development, implementation of successful molecular strategies that support treatments against this disease is urgent. Our group has demonstrated that ARP2 protein overexpression induces programmed cell death in different cell types: androgen-independent lymphoid nodule prostate cancer cells, from where it was originally isolated, Chinese hamster ovary cells, embryonic human kidney cells and coronary artery endothelial cells (data not shown).
Given the collected experimental evidence, we consider that ARP2 could be contributing during its overexpression to the increase in Ca2+ intracellular levels as a messenger molecule or as a protein inserted in the membrane that favors Ca2+ inflow. In addition to being found in the membrane region, we also observed its perinuclear localization, and due to the homology with the Prp8 factor, there is the possibility that it participates in the alternative splicing mechanisms of pro-apoptotic molecules.44-46 In this sense, and taking into account that transcription through alternative splicing mechanisms are molecular processes that are highly regulated by Ca2+ intracellular level, among other factors,47,48 it is feasible to think that ARP2 might have a double function: in the control of plasma Ca2+ flow and in alternative splicing molecular mechanisms.
Based on the evidence that cell death genes are deregulated in cancer, a phenomenon that is associated with the control of their alternative splicing patterns, it is reasonable thinking that the apoptosis process occurs as a positive response to chemotherapy.49 If we take into account that anticancer drugs efficacy may also depend on apoptosis activation or on a form of cell-senescence acute induction, we consider that ARP2 protein overexpression could potentially be used as a new tool in the treatment epithelial-type cancer.