Transient Receptor Potential Channels Play a Role in the Regulation of Intercellular Calcium and Cell Vitality in BON Neuroendocrine

Tumor Cells
Christina Pucci
Supervisor: Stefan Mergler, PhD

Charit - University Medicine Berlin

Campus Virchow Hospital Biomedical Research Centre Forum IV, Room No. 01.0507 Wiedenmann Lab (Gastroenterology) Dept. of Ophthalmology Section Eletrophysiology Augustenburger Platz 1 13353 Berlin Germany

ABSTRACT
The Transient Receptor Potential (TRP) channel ion family has been implicated in many diseases, including cancer. Previous studies have demonstrated that ion channels play a role in hormone secretion of neuroendocrine carcinoid tumors.(Mergler et al., 2007; Mergler, Wiedenmann & Prada, 2003) The present study aims to characterize BON cells, a functional human pancreatic neuroendocrine tumor (NET) cell line, particularly in regards to cell vitality and responsiveness to stress. Calcium imaging shows BON cells to have a characteristic hyper-responsiveness of intracellular calcium following withdrawal of extracellular calcium. This response is ablated in the presence of the TRP and TRPV channel inhibitors, lanthanum chloride (100 !M) and ruthenium-red (20 !M), respectively. This study gives first evidence for the presence of TRP channels, in particular TRP channels of the TRPV (vanilloid) subtype in these cells. TRPV channels may play a role in the functionality of NET cell lines and may correspond to secretion patterns. However, further studies are needed to verify this hypothesis. The findings could potentially have important implications for diagnosis and treatment of these rare tumors, and may present an attractive drug target.

INTRODUCTION Neuroendocrine Tumors (NETs) represent a group of rare and heterogenous cancers that occur at the interface between the endocrine and nervous systems, frequently arising in the gasteroenteropancratic system.(Wiedenmann & Pape, 2004) They are distinct from adenocarcinomas , and their phenotype is characterized by neuroendocrine features, including their capacity to produce and secrete peptide hormones and biogenic amines, such as insulin, chromogranin A, and serotonin. Such tumors are classified as functional, as opposed to nonfunctional tumors which do not display inappropriate secretion. NETs are further classified by the point of origin, as foregut (lung, thymus, stomach, and duodenum), midgut (distal ileum and proximal colon), or hindgut (distal colon and rectum). Less than one percent of tumors originate in the pancreas. However, for many tumors, the point of origin is unknown. (Plockinger et al., 2004) NETs present a particular diagnostic challenge for the physician, as the consequences of secretion can lead to a wide range of symptoms such as weight loss, depression, and night sweats,

and may be only mild in severity. (Wiedenmann & Pape, 2004) NETs are often only diagnosed when they become clinically apparent by the inappropriate secretion of these biologically active substances. (Perren, Komminoth & Heitz, 2004) Furthermore, the onset of secretory syndromes often only occurs after metastasis has occurred, thereby making the tumor ineligible for curative surgery, and hindering the possibility for early detection of tumors.(Wiedenmann et al., 1998) The release of biogenic substances can cause the devastating sequel known as carcinoid syndrome, with characteristic symptoms of flushing, diarrhea, bronchi-constriction and cardiac valvular disease.(Creutzfeldt & Stockmann, 1987) The symptoms arising from hormone hypersecretion classically present the greatest difficulties for the patient and treatment is directly primarily towards the management of these secretion syndromes.(Moertel, 1983) Chemotherapy has proven to be largely ineffective at slowing the growth of carcinoid tumors, and is often wrought with harmful side effects. Therefore, surgical resection is still the best available therapeutic option, although recurrence is common.(Wiedenmann, 2003) The development of effective treatment regimens for patients with carcinoid tumors has been hampered in the past by the lack of effective models of carcinoid tumor.(Evers et al., 1994) These models are vital to a better understanding of genetic and molecular alterations that may contribute to pathogenesis. Experimental models also allow the assessment of the clinical behavior of these tumors, and the evaluation of the possible efficacy of new chemotherapeutic agents. For this reason, permanent cell cultures provide a good model for studying these types of tumors. BON cells are functioning neuroendocrine pancreatic carcinoma cells, originally taken from a lymph node metastasis of a 28 year old patient presenting with typical symptoms of carcinoid syndrome, such as flushing and diarrhea, due to an overproduction of serotonin. (Evers et al., 1994) First established in the 1980s, BON cells have been well characterized on the molecular level. The cells are known to contain an N-Ras point mutation, and secrete hormones such as neurotensin, and chromogranin A, serotonin (5-HT), and bombesin (a known tumor marker from gastric and neural cancers, which acts in gastrin release.) (Arany et al., 1994; von Wichert et al., 2000) Previous studies have shown that 5-HT reverses growth inhibition by TGF--1. Moreover, serotonin stimulates the BON cells and acts as an autocrine growth factor through specific receptors linked to the cyclic AMP pathway.(Ishizuka et al., 1993) BON cells also express functional IGF receptors and use IGF-1 as an autocrine growth factor which allows anchorageindependent growth, and leads to chromogranin A secretion. (von Wichert et al., 2005; von Wichert et al., 2000) Mergler et al. has recently shown that this process is mediated by Ca2+permeable cation channels. (Mergler et al., 2007; Mergler et al., 2003) It has been known for many years that spontaneous action potentials coincide with oscillations of intracellular calcium in various neuroendocrine tumor cells.(Scherubl et al., 1994) This calcium influx is long-thought to potentiate and synchronize hormone secretion in these cells. However, the precise mechanisms are still not yet fully understood. Ion channels are integral membrane proteins present in the cells of all living organisms, which mediate the flow of ions across the plasma membrane. These channels play an important role in homeostasis, as they mediate rapid changes in cells, such as muscle contraction, transport of nutrients and ions, and conduction of nerve impulses. Additionally, they also play a role in central cell processes such as proliferation and regulation of the cell cycle. (Schonherr, 2005) Genetic disorders of ion channels and their modifiers are known as channelopathies. (Nilius, Voets & Peters, 2005) These diseases due to ion channel dysfunction may be either inherited or sporadic, and there is growing evidence that they may play a role in cancer as well. (Kunzelmann, 2005) During the multi-step process of cancer, ion channels have a speculative role in supporting many stages of this progression, both early and late. (Figure 1a) (Fraser & Pardo, 2008) However,

the explicit role ion channels play in the pathogenesis of certain cancers still remains unclear. There is evidence that ion channels are involved in both mitogenesis and malignancy.(Pardo et al., 2005) A number of endogenous ion channels have been shown to be upregulated in various cancers, while some ion channels are selectively expressed in aggressive cancers and are intimately involved in metastasis. Channels may also be involved in angiogenesis. (Schonherr, 2005) Some of the most important signaling pathways altered in tumorigenesis enhance cell proliferation and inhibit apoptosis.(Prevarskaya, Zhang & Barritt, 2007c) Calcium homeostasis controls these cellular processes, as well as gene transcription and angiogenesis. (Gkika & Prevarskaya, 2009) Furthermore, the regulation of cell cycles, apoptosis, and proliferation also depends on the amplitude and temporal spatial aspects of the calcium signal. It is thought that the overexpression of plasma membrane Ca2+ channels amplifies the calcium influx with consequent promotion of Ca2+-dependent proliferative pathways.(Roderick & Cook, 2008) As calcium is an essential regulator of the cell cycle and is indispensible for cell proliferation, it is not surprising to find alterations in cancer cells. Voltage-gated L-type Ca2+channels are over-expressed in colon cancer, and these changes are paralleled by increased calcium influx and subsequently enhanced growth factor signaling.(Wang et al., 2000) Other voltage-gated channels have been shown in animal models of prostate cancer to determine lateral motility and invasive capacity in vitro and metastasis in vivo. This is due to changes of the cytoskeleton, modulation of ion fluxes, and regulation of gene transcription and enzyme activity.(Grimes et al., 1995) Nonselective cation channels have also been associated with proliferation and cancer. Most important are those of the TRP family, first discovered to play a role in drosophila photoreception of light. To date over 30 of these channels have been discovered in humans, and are subdivided into 6 subfamilies based upon structural homology: TRPC (canonical), TRPV (vanilloid),TRPA (ankyrin), TRPM (melastatin) ,TRPP (polycystin), and TRPML (mucolipin).(Gkika & Prevarskaya, 2009) These channels are exceptional in that they are polymodal, and can be activated by many types of very different gating stimuli. They are integrators for a range of external and internal signals, and therefore act as multifunctional cell sensors.(Nilius et al., 2005) Many of these channels are known to be activated be a wide range of common chemical substances, particularly those found in foodstuffs such as chili peppers, garlic, and wasabi. (Vriens, Nilius & Vennekens, 2008) (Interestingly, eating spicy foods is known to sometimes worse the symptoms of carcinoid syndrome.(Laplante, Archambault & Thibert, 1971)) Additionally, TRP channels have an essential role in heat sensation, and the temperatures at which they are activated form a gradient (figure 1b). (Tominaga, 2008) TRP channels contribute to changes in intracellular Ca2+ concentrations, either by activating as Ca2+ entry pathways in the plasma membrane or via changes in membrane polarization, thereby modulating the driving force for Ca2+ entry mediated by alternative pathways. Additionally, TRP channels are expressed on the membranes of internal Ca2+ stores, where they may act as triggers for enhanced proliferation, aberrant differentiation, and impaired ability to die, thus leading to uncontrolled expansion and the invasive characteristics of cancer. (Nilius et al., 2007) To date, most changes involving TRP receptors do not involve mutations in the TRP gene, but rather increased or decreased expression levels of the wild-type TRP protein.(Gkika & Prevarskaya, 2009) In addition to alterations of TRP channel regulation on the transcriptional and translation levels, these channels can be differentially regulated by the trafficking of the channel to the plasma membrane or in regards to protein stabilization. Alternative spicing may also play a role.(Gkika & Prevarskaya, 2009) The TRP channels have been associated with many cancers, including those of the breast, prostate, bladder, and melanoma. (Bodding, 2007) Particularly, the expression levels of members of the TRPC, TRPM, and TRPV families have been correlated to the emergence and progression

of cancer. (Prevarskaya et al., 2007c) TRPV6, a highly selective Ca2+-channel has been found to be highly overexpressed in prostate carcinoma, as opposed to being virtually undetectable in healthy tissues or benign tumors of the prostate. TRPV6 is considered a tumor marker in this disease, as its expression correlates with tumor grade and is a strong predictor of clinical outcome. (Fixemer et al., 2003) Moreover, TRPV6 is consistently overexpressed in breast, thyroid, colon, and ovarian carcinomas as well.(Zhuang et al., 2002) TRPV1, best characterized for its role in sensation, is also thought to play a role in chronic cancer pain, particularly that of the pancreas.(Hartel et al., 2006) Recently, the group of Mergler et al. has found that certain TRP channels play an essential role in mediating the secretion of hormones in neuroendocrine tumor cells, with the discovery that TRPM8 channels function in neurotensin secretion in BON cells. (Mergler et al., 2007) As TRPM8 is classically understood for its role in cold sensation, it is possible that heating receptors may play a role as well. Therefore, we wished to investigate the potential role of other TRP channels, particularly those of the TRPV family, in BON neuroendocrine tumor cells. MATERIALS AND METHODS Cell Culture The human NET cell line BON was cultured in DMEM/Ham'sF12 medium containing 10% fetal bovine serum (FBS) (all reagents from Biochrom, Berlin, Germany) at 5% CO2 and 37C. Cells were dissociated by trypsin/EDTA treatment and split at a ratio of 1:3 once or twice a week. Reagents All chemicals were purchased from Sigma (Deisenhofen, Germany) unless otherwise indicated. Fluorescence Cell Imaging Cytosolic free calcium concentrations were determined by fluorescence cell imaging, as previously described.(Grynkiewicz, Poenie & Tsien, 1985) Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. One of the most common chemical indicators is fura-2, a UV-excitable ratiometric chelating agent which binds Ca2+ using four carboxyl groups. Ratiometric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of [Ca2+]i to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. Upon Ca2+ binding, the fluorescent excitation maximum of the indicator undergoes a blue shift from 363 nm (Ca2+-free) to 335 nm (Ca2+ saturated), while the fluorescence emission maximum is relatively unchanged at ~510 nm. Calibration can be performed and absolute Ca2+ can be calculated, using a disassociation constant. The equation [Ca] = (R-Rmin)/(Rmax-R)Sf*Kd can be used to convert the Fura-2 ratio values to intracellular Ca2+ concentrations. In this, [Ca] is the Ca2+ concentration, R is the Fura-2 340/380 ratio, RMin and RMax are the 340/380 ratios in the absence of Ca2+ or in the presence of a saturating concentration of Ca2+, respectively. Sf*Kd is the product of the disassociation constant of Fura-2 (approximately 120 nM at RT) and a scaling value.(Grynkiewicz et al., 1985) To measure RMin, RMax and Sf*Kd it is necessary to perform either an in vivo or an in vitro calibration. In vivo calibration requires a combination of patch clamping and calcium imaging, which can be complicated but is also very precise. In many cases, the calculation of the exact quantitative calculation of [Ca2+]i is not necessary because qualitative change in [Ca2+]i is sufficient for the investigation. Furthermore, the absolute values of [Ca2+]i (nM) are only assessed

values because the measuring conditions are artificial (e.g. specific bath solution). (Grynkiewicz et al., 1985) There are several advantages with this method. The main advantage of using ratiometric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. This method has the further advantage that it can be combined with other methods, and that single cells can be measured. Like all fluorescent dyes, photo-bleaching may occur with prolonged exposure to light. (Almers & Neher, 1985) However, furo-2 is less susceptible to bleaching effect than other dyes such as Indo1 or quin2. (Becker & Fay, 1987) Also, care must be taken to not move the apparatus, to maintain an consistent level of fluid to retain focusing, and to avoid air bubbles. As Ca2+ can not be visualized directly in living cells, [Ca2+] has to be inferred indirectly by means of the law of mass action. Furthermore, calcium changes can be the result of internal store release as well as membrane channel activation, so further studies are needed to confirm the presence and action of specific ion channels.(Almers & Neher, 1985) In order to overcome the issue of membrane permeability, we loaded the cells with acetoxy-methyl-ester Fura-2 (Fura-2 AM), which diffuses across the cell membrane and is deesterified by cellular esterases to yield Fura-2 free acid. For these experiments, BON cells were preincubated with culture medium containing 1 !M fura-2-AM for 1525 min at 37 C. Thereafter, the cells were washed with the fresh cell culture medium in order to halt the fluorescence reaction. Fura-2 uorescence was measured at room temperature (~ 20 C) using a digital imaging system (T.I.L.L. Photonics, Munich, Germany). Cells were alternately illuminated at 340 and 380 nm, and emission was detected every 1 s at 510 nm. Changes in [Ca2+]i were monitored based on the ratio of the measured fluorescence. The ratio of the measured uorescence f340nm/f380nm represents changes in [Ca2+]i. To exclude sodium- and potassiumpermeable ion fluxes, we used a sodium- and potassium-free bath solution containing (in mM): N-methyl-D-glut mine 120, CsCl 5.4, MgCl2 1.0, glucose 10, and Hepes acid 10 (pH adjusted to 7.3). 10 mM Ca2+ was used as charge carrier. The calcium-free solution contained 1mM EGTA, in order to bind free calcium. Chemical TRP channel antagonists lanthanum chloride (100 !M) and ruthenium-red (20 !M) were used for TRP and TRPV channel inhibition, respectively. Temperature change experiments were carried out at room temperature, with the substitution of bath solution which has been chilled (14C) at the final wash-out. Data Analysis & Statistics All values are reported as means SEM. The number of replicates is indicated in each case as n, near the traces (graphs) or in the gure legends. Statistical signicance was determined with Student's t-test if the values were normally distributed (Gaussian distribution). P values of 0.05 (*) were considered signicant. Statistical tests were performed by SigmaStat version 3.5 for Windows (Systat Software, Inc.) and GraphPad Prism Software (San Diego California USA). RESULTS Experimental Design The aim of this study was to characterize BON neuroendocrine tumor cells, particularly in regards to cellular vitality. This assays the ability of the cells to respond to stress, such as Ca2+ depletion. These conditions were induced by a period of withdrawal of extracellular Ca2+, followed by a final washout with Ca2+ solution (Figure 2). The experiments were then repeated in

the presence of blockers and altered temperature conditions, in order to elucidate the effects of certain ion channels in the regulation of [Ca2+]i in BON cells. Characterization of BON Cell Vitality To detect fluctuations of [Ca2+]i levels, calcium imaging was carried out using Fura-2-AM as previously described.1,2 Figure 3 demonstrates the typical response pattern of BON neuroendocrine tumor cells when subjected to a time course of calcium withdrawal and replenishment. The measurement began in the presence of 10 mM of extracellular Ca2+ (control). At 240 seconds the solution was replaced with Ca2+ -free solution (1 mM EGTA), and at 420 seconds the final washout with solution containing 10 mM extracellular calcium was executed. The data show a typical pattern of BON cells, demonstrating calcium regulation of the cells, and the effects of extracellular calcium on [Ca2+]i levels. After washout of Ca2+-free solution, there was a sharp increase, past the baseline levels. This was then followed by a slow decrease back to baseline levels. As a control, the baseline levels of [Ca2+]i under isotonic conditions were also measured. BON cells display an characteristic pattern of [Ca2+]i oscillations under isotonic conditions, which were observed in most recordings. TRP channels are Expressed in BON NET Cells The response of intracellular calcium past baseline levels was a characteristic of the BON cells, which could perhaps be due to TRP channel activation. In order to test this, all TRP channels were unspecifically blocked by lanthanum chloride (La3+Cl), a chemical antagonist of all TRP channels. The blocker was added at a concentration of 100 !M to all solutions: pre-incubation medium containing 1 !M Fura-2, the washing solution used to stop the Fura-2 reaction, and all solutions employed during measurement. The cell vitality experiments were repeated exactly as before, in the presence of the blocker. The results clearly demonstrate the role of TRP channel activation in BON cell vitality (Figure 4). There was very little response following withdrawal of[Ca2+]i, and the hyper-response seen in figure 3 was completely suppressed. The calcium oscillations seen under isotonic baseline were also strongly repressed by the presence of the blocker, indicating the role of TRP channels in mediating this phenomenon. TRPV channel activity In order to further elucidate which subfamily of TRP channels are involved, the experiments were performed in the presence of Ruthenium Red (RuR) at a concentration of 20uM. RuR has been shown to antagonize all TRPV channels, though particularly TRPV4, a heating receptor. Figure 5 demonstrates the role of TRPV channels in BON cell vitality. In the presence of blocker, the responsiveness to Ca2+ withdrawal was more subtle. Additionally, the response to calcium replenishment at 420 seconds was clearly altered, as the slope was much slower than that seen without the presence of blocker. The level of calcium response, as indicated by the florescence ratio, was also significantly ablated, as shown by statistical analysis (Figure 5). Lastly, the baseline oscillations were nearly completely suppressed by the blocker, indicating the role of TRPV channels in this observation. Effects of Temperature on Cell Vitality Lastly, we tested the vitality of the cells in the presence of altered temperature conditions, by replacing the final washout with an identical Ca2+ solution that has been chilled to 14C. The results show that calcium recovery was not only blocked under cold conditions, but even reveal a subtle decrease, despite the addition of extracellular Ca2+ (Figure 6). This was followed by a slow increase after 500 seconds, presumably as the solution begins to approach room temperature

under experimental conditions, with an average final temperature of 18 at 600 seconds. Statistical analysis revealed the effect of the temperature change to be highly significant, in comparison to vitality experiments performed entirely at stable room temperature conditions (Figure 6). DISCUSSION Previous studies have demonstrated the validity of the BON cell line as an appropriate primary cell model for studying functional NETs.(Evers et al., 1994; Ishizuka et al., 1993) Calcium signaling has long been thought to play a role in tumorigenic processes, and more specifically in the characteristic secretion syndromes that clinically present in patients with carcinoid tumors.(Scherubl et al., 1994) The newly characterized TRP ion channels are particularly relevant to these studies. Mergler et al. have already established a clear link between several ion channels to various important secretion products that afflict patients with NETs. (Mergler et al., 2007; Mergler et al., 2003) As cold receptors have been found to regulate hormone secretion in BON cells, it is plausible that heat receptors also play a similar role, perhaps with regard to other secretion products. TRP channels mediate Cell Vitality in BON Cells The present study confirms the presence in temperature-sensing TRP channels in BON neuroendocrine tumor cells using fluorescence cell imaging. Moreover, this study indicates that these channels play a major role in the regulation of [Ca2+]i, particularly following cell stress. This study shows that BON cells have characteristically high levels of vitality and exhibit a marked increase in [Ca2+]i following periods of Ca2+ depletion. The reasons behind this compensatory hyper-response are still unclear, and may possibly be related to cell passage number. Some possibilities include a putative link to stimulating hormone secretion, conference of a growth or survival advantage of the cancer cells, or the release of calcium from intracellular stores. Further studies will be needed to elucidate these questions. TRPV Heating Receptors Play a Role in Calcium Regulation This study also shows for the first time that TRPs of the TRPV subtype family are expressed in BON neuroendocrine tumor cells. By using chemical blockers, our experiments demonstrate TRPV channels to play a significant role in mediating cell vitality and regulating levels of [Ca2+]i . However, these preliminary studies indicate that TRPV channels are only a significant component in this mechanism, and most likely act in consort with other ion channels. In addition, our studies give the first evidence of the role of TRPV channels in regulating the highly fluctuating baseline oscillations of [Ca2+]i in these cells. This characteristic property may also in itself play a role in secretion or mitogenesis, although further tests are needed to shed light on this. Lastly, we demonstrate that temperature has discernable effects on cell vitality. This is not surprising considering the integral role of various TRP channels in temperature sensation. (Tominaga, 2004) These studies indicate that cold receptors, such as TRPM8, do not alone account for calcium hyper-response, although further studies involving heating will better clarify this issue. TRP Channel Interaction and Convergence Although TRP channels are best understood in regards to their role in sensation of temperatures and taste, the specific nature of TRP channel activation is intricate, highly complex, and often overlapping. It seems likely that a combination of TRP channels are responsible for calcium regulation of BON cells; that together they orchestrate changes in the cell, which may

directly or indirectly lead to the characteristics of NET cells, such as hormone secretion and metastasis. While specific channels may play a more predominate role at certain stages of cancer pathogenesis, the overall effects are most the result of a concerted effort of a number of ion channels. For instance, it is plausible that there is a degree of functional redundancy among certain members of the TRPV family. This hypothesis is supported by their highly similar molecular structures, mechanisms of activation, and overlapping activators and inhibitors. (Tominaga, 2004; Vriens et al., 2008) Furthermore, TRPV channels have been found to form heterodimers with other family members, although usually preferentially for certain other subtypes.(Prevarskaya et al., 2007b) Although no channelopathies involving TRPA1 have yet been identified, it is possible that it may play a role in this story. Studies in only the last several months have indicated that TRPA1 and TRPV1are functionally linked and co-expressed.(Salas, Hargreaves & Akopian, 2009) Animal models have shown that TRPV1 must also be present in order for TRPA1 to exert its effects. Other studies have demonstrated that Resinferatoxin, a potent inhibitor of TRPV1 (which is widely regarded as being highly selective), also removes TRPA1 as well.(Pecze et al., 2009) The authors hypothesize that TRPM8 compensates for the loss of TRPA1 and TRPV1 doubleknockouts, which causes the observed hyper-sensitization to cold in these animal models. While some studies have implicated TRPA1 in the perception of noxious cold, along with TRPM8, the evidence is conflicting and not yet conclusive.(Prevarskaya et al., 2007c) TRP Channels in Secretion and Tumorigenesis A Putative role of TRPA1 in Serotonin Secretion It is known that TRPV ion channels regulate 5-HT biosynthesis in Caenorhabditis elegans. (Zhang et al., 2004) In higher mammals, TRPA1 shares close functional relationship to TRPV1, and there is additional support for a putative role of TRPA1 in BON cell secretion, particularly in regards to serotonin. (Salas et al., 2009) Very recent studies of the past several months have shown that TRPA1 regulates serotonin release from enterochromaffin cells. (Nozawa et al., 2009) It is widely accepted that, despite their pancreatic location, BON cells actually originate from such enterochromaffin cells. (Evers et al., 1994) Enterochromaffin cells normally function in 5-HT release from the intestines, and their metastasis to the pancreatic region could well explain the sudden appearance of carcinoid symptoms, due to inappropriate secretion outside of their native environment. TRPA1 agonists stimulate increasing [Ca2+]i concentrations, which thereby causes the release of serotonin. Interestingly, however, the effect of these TRPA1 agonists on and 5-HT release was blocked by ruthenium red. (Nozawa et al., 2009) Furthermore, studies in normal and neoplastic enterochromaffin cells have shown serotonin release to be stimulated by chemical olfactants, such as eugenol, which is found in clove. (Kidd et al., 2008) This paper does not discuss mechanisms behind olfaction detection in cells of the gut. However, other resources reveal that the effects of olfactants are mediated through their activation of TRP channels. In particular, eugenol is not only a TRPV1 agonist, but it also activates and sensitizes TRPV3. It is notable that, although it is a heat receptor, TRPV3 is also activated by methanol. Eugenol also activates TRPM8, a cold receptor, as well as TRPA1. (Vriens et al., 2008) Interestingly, olfactant mediated secretion of serotonin in KRJ-1 neoplastic EC cells is thought to be induced via GPCR-mediated ERK phosphorylation and calcium flux, and is blocked by the presence of somatastatin analogs. (Nozawa et al., 2009) Cumulatively, these studies seem to indicate the possibility of a similar combinatorial role of various TRP receptors in BON cell secretion.

Further Implications of Channel Activation in BON Cells The possibility remains that TRPV-mediated calcium influx plays a role in hormone secretion, such as Chromogranin A, bombesin, or pancreastatin. For example, fluctuating baseline oscillations in [Ca2+]i or highly responsive levels following stress may indirectly stimulate exocytosis of secretory granules. TRPV1 may play a role in mediating exocytosis of insulin in pancreatic -cells, while TRPA1 is known to act as a binding partner with Secretogranin III, a protein that mediates exocytosis of secretory vesicles when complexed with Chromogranin A.(Bae, Park & Kwon, 2003) Serotonin, ATP and various common inflammatory mediators, are known to lower the activation temperature of TRPV1 from 40C to a level below body temperature.(Prevarskaya et al., 2007c) Channel activation can be sensitized by phosphorylation, for instance by protein kinase C (PKC), which can itself be activated through GPCR pathways activated by these same inflammatory mediators. (Kunzelmann, 2005) Interestingly, both insulin and IGF-1 enhance TRPV1 expression, not only by channel activation but also by increasing expression of the TRPV1 gene.(Nilius et al., 2007) TRPV4 activation, in addition to TRPV1, has been found to be potentiated in the presence of serotonin.(Ducret et al., 2008) Cumulatively, this could indicate the possibility of TRPV channels acting with other secretion products to create an autofeedback mechanism for the cancer cells in their environment. Calcium influx by overexpressed TRP channels could also indirectly play a role in other tumorigenic effects, such as the stimulation of other Ca2+-dependent channels. For instance, Kca3.1, a calcium-activated potassium channel has been implicated in breast cancer. This channel drives cell cycle progression from G1 to S phase. (Gkika & Prevarskaya, 2008). IGF-1which is upregulated in BON cellsincreases the expression of these channels. (Ishizuka et al., 1993) It is thought that Kca3.1 activation during G1 results in a strong hyperpolarization of the cell, and increases the driving force for Ca2+ entry through the TRPV6 channel. (Gkika & Prevarskaya, 2008) IGF-1 has been associated with TRPV2 upregulation in bladder cancer.(Prevarskaya et al., 2007a) Microarrays have also shown Kca3.1 channels to be consistently over-expressed in 80% of fresh melanoma biopsies. This channel type is particularly upregulated in cases of hypoxia, a form of cell stress common in cancer. (Kunzelmann, 2005) Future Outlook Further studies are needed both to investigate these possibilities, as well as to further validate our preliminary studies. Such studies may involve use of additional chemical TRP antagonists and activators, as well as heat. As a major hurdle in ion channel study is the lack of very specific chemical modulators. Therefore the use of siRNAs is recommended in order to achieve a more specific effect. Combinations of siRNA can also be co-transfected to show interactions between the channels. Secretion studies will further elucidate the role of TRPV channels in BON cell hormone secretion, and the link to G-coupled protein receptors in mediating this process. It is proposed to verify our findings on further levels, including: gene expression (PCR), protein expression (Western Blot), localization on the PM membrane and co-expression of other channels (in-situ hybridization and confocal microscopy), and the activity of channels on the electrophysiological level (patch-clamp). Other considerations include the possible roles of splicing on channel regulation, and the ratio of phosphorylation: unphosphorylated proteins in studies regarding G-coupled protein receptors. It would then be advisable to compare our findings in BON cells with other NET cells, such as healthy cells, another well-established cell line (e.g. KRJ-1), as well as primary cell cultures from fresh patient tissues. (Modlin et al., 2006; Pfragner et al., 2009; Siddique et al., 2009)

Clinical Implications In conclusion, the findings of this study and future studies may have important implications for treatment of patients with carcinoid tumors. Although the outcome of the young patient from whom BON cells derived is not known, the prognosis according to statistics is bleak with a 5 year survival rate of less than 20%. (Pape et al., 2004) Better understanding of the mechanisms behind tumorigenesis on the molecular level can lead to more advanced therapies, and ion channel dysregulation presents an excellent drug target. The role of TRP channels in NET secretion provides an excellent drug target in palliative care, as the symptoms of hypersecretion often causes the greatest difficulties for the patient. Growth inhibition could be achieved by either blocking the channel and thereby halting its proliferation, or selective channel expression could be exploited by chemotherapy, with channel activation causing a calcium influx which leads to apoptosis of only the cancer cells.(Prevarskaya et al., 2007c) Furthermore, drugs could modulate the sensitivity of TRP channels. This could possibly potentiate other chemotherapy, thereby reducing the necessary dosage and also the side effects associated with other cancer drugs. TRP channels may also serve as a tumor marker in NETs, and may help identity recurrence or even assist in earlier diagnosis. This enhanced staging and prognosis method could contribute to a more personalized treatment program, thereby achieving a better patient outcome. Lastly, characterization of ion channel activity based upon tumor origins and localization could be used to identity the source in cases of an unknown primary. Overall, studies in this area hold great promise for cancer treatment and better understanding could potentially prolong life expectancy, improve quality of life, and ultimately cure patients with neuroendocrine tumors. Acknowledgements Sincere thanks to Dr. Stefan Mergler for his invaluable guidance, expertise, encouragement, and time throughout this project. Also to Fr. Yvonne Giesecke for her technical assistance with cell culture, as well as to the project collaborators Dr. Carsten Grtzinger, Dr. Mathias Strowski, and Prof. Bertram Wiedenmann.

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