Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death.

The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the 'two-in-one' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.

[Comparative effects of menthol and icilin on the induced contraction of the smooth muscles of the vas deferens of normal and castrated rats].

Non-specific TRPM8 agonist menthol was shown to inhibit voltage- and agonist-evoked contractions of the smooth muscle (SM) of rat vas deferens. Here we compared the action of menthol with the action of more specific TRPM8 agonist icilin on depolarization- (60 mM KCl), carbachol-(CCh) and noradrenalin-(Nor)-evoked contractions of the SM strips from the prostatic and epididymal portions of the vas deferens of normal and castrated (60-137 days) rats. Inhibitory action of menthol (100 microM) and icilin (10 microM) on the amplitude of KCl-, CCh- and Nor-induced contractions of normal as well as castrated rats was similar consisting about 50%, despite castration per se strongly potentiated CCh- and Nor-evoked contractions compared to the control animals. In the epididymal portion of the control animals menthol suppressed KC 1- and CCh-evoked contractions by 46 +/- 5% and 32 +/- 3% and icilin by only 14 +/- 4% and 6 +/- 7%, respectively, whilst after castration both compounds became virtually ineffective. Considering that TRPM8 may localize in the sarcolemma and sarcoplasmic reticulum (SR) membrane and that menthol can also block voltage-gated calcium channels (VGCCs), our data indicate that in the prostatic portion TRPM8 modulates contractility by primarily decreasing the SR Ca2+ stores content, whilst in the epididymal one by both decreasing the SR filling and supporting Ca2+ entry. Drop in the circulation androgens as a result of castration changes the menthol- and icilin-mediated modulation of the rat vas deferens SM contractility via the decrease of the expression of L-type VGCCs and increase of the expression of TRPM8.

Acceleration of keratinocyte differentiation by transient receptor potential vanilloid (TRPV6) channel activation.

BACKGROUND: Numerous studies have demonstrated the beneficial effect of Avène Thermal Spring Water (TSW) in dermatological diseases but the molecular mechanisms remain unknown. The objective of the present study was to evaluate the effect of Avène TSW on the morphological and molecular features related to the more advanced status of differentiation of human keratinocytes. MATERIAL AND METHODS: Normal human keratinocytes (NHK) were differentiated in medium powder reconstituted with Avène TSW and assessed by RT-PCR and immunohistochemistry. Calcium entry was measured by a Fura-2 AM probe. TRPV6 channel were detected by immunohistochemistry, RT-PCR and western blot. RESULTS: Treatment of NHK with Avène TSW led to an enhanced constitutive calcium entry that resulted in the increased expression of involucrin and cytokeratins 1 and 10. This enhanced constitutive calcium entry in Avène TSW-treated keratinocytes was mediated by the TRPV6 calcium channel. Moreover, Avène TSW-mediated calcium entry was due to the increase in TRPV6 expression as well as the channel abundance at the cell membrane. CONCLUSIONS: An other mechanism of action of Avène TSW is described. Avène TSW treatment induced an enhanced constitutive calcium entry mediated by TRPV6 channel leading to the acceleration of human keratinocytes differentiation.

Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells.

The molecular nature of calcium (Ca(2+))-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca(2+) entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca(2+) influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca(2+)-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.

[Gender differences in cold sensitivity: role of hormonal regulation of TRPM8 channel].

We have studied the gender differences in the perception of cutaneous cold stimuli in the innocuous temperature range (5-30 degrees C) in mice and rats. In the behavioral tests using two variable temperature plates technique female subjects displayed lower threshold for the sensation of cooling temperatures in the range of 15-25 degrees C compared to males. Patch-clamp experiments carried out on dorsal root ganglion (DRG) neurons from male and female rats maintained in the short-term cultures in the presence of testosterone or 17beta-estradiol, respectively, have revealed gender- and hormone-related differences in the electrophysiological properties of cold/menthol-sensitive TRPM8 channel: average density of menthol-activated I(TRPM8) current density in females' DRG neurons was higher compared to males', and the steady-state voltage-dependent activation curve of TRPM8 in females was shifted towards hyperpolarized potentials compared to males. These distinctive TRPM8 properties vanished upon withdrawal of testosterone and 17beta-estradiol from the culture mediums. We conclude that the observed differences in the behavioural sensitivity to innocuous cold and in functional properties of TRPM8 cold receptor are due to differential regulation of TRPM8 by sex steroid hormones, testosterone and/or 17beta-estradiol.

Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry.

Accumulating data point to K(+) channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K(+) channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK(Ca1)) in human PCa as well as their involvement in cell proliferation. We showed that IK(Ca1) mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK(Ca1) potassium channel suppressed PCa cell proliferation. The activation of IK(Ca1) hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK(Ca1) channel is likely to promote carcinogenesis in human prostate tissue.

[Expression of the cold receptor TRPM8 in the smooth muscles of the seminal ejaculatory ducts in rats].

By means of real-time RT-PCR and immunofluorscent staining combined with confocal microscopy we show for the first time the expression ofmRNA and protein ofthe cold/menthol-sensitive cationic channel, TRPM8, in the smooth muscle cells (SMC) from the epididimal and prostatic portions of the rat vas deferens. Expression of TRPM8 mRNA correlated with the expression ofmRNA for androgen receptor (AR): orchidectomy of the animals resulted in the enhancement of the expression of both mRNAs, which likely reflects specific for the vas deferens compensatory response to the decreasing levels of circulating androgens. TRPM8 protein in the SMC from both parts of the vas deferens primarily localized outside the plasma membrane (PM), however, in the SMC from prostatic portion we observed higher TRPM8 protein targeting specifically the endoplasmic reticulum and PM, where it can form functional cold/menthol-sensitive cationic channel capable of modulating contractile activity of the smooth muscle.

[Modulation of the smooth muscle contractions of the rat vas deferens by TRPM8 channel agonist menthol].

TRPM8 is nonselective, Ca2- permeable cationic channel, which is activated by innocuous cold and by chemical drugs imitators of cooling, menthol, icilin and cucalyptol. TRPM8 expression was detected in the smooth muscle cells of the rat vas deference with preferential localization of the TRPM8 protein to the membrane of sarcoplasmic reticulum (SR). In the present work we have studied the effects of TRPM8 channel agonist, menthol, on the contractions of the smooth muscle strips of the epididimal and prostatic portions of the rat vas deferens evoked by potassium rich (KCl) Krebs solution and by muscarinic or adrenergic agonists carbachol (CCh) or noradrenalin (Nor). Menthol (0.1-1 mmol/l) per se virtually unaffected the basal tone, but inhibited in a dose-dependent manner KCl-, CCh- and Nor-evoked contractions of both parts of the vas deference by 30-50%. Blockade of the Ca2+ -ATPase of the SR with cyclopiazonic acid (CPA, 10 micromol/l) enhanced inhibitory action of menthol on KCl-induced contractions, but slightly decreased inhibition by menthol of agonist-induced ones. Nonspecific TRPM8 blocker, capsazepine (10 micromol/l), did not eliminate, but augmented inhibitory action of menthol on all types of contractions. Our data propose that menthol inhibits contractions via two mechanisms: partial blockade of Ca2+ entry via the voltage-gated, L-type calcium channels and a decrease of the calcium storage capacity of the SR. The latter mechanism at least in part is mediated by the SR-resident TRPM8 channel, which by activation of menthol leads to the enhancement of passive leak of Ca2+ from the SR and reduction in the amount of the releasable calcium during activation of contractions.

TRPV6 channel controls prostate cancer cell proliferation via Ca(2+)/NFAT-dependent pathways.

The transient receptor potential channel, subfamily V, member 6 (TRPV6), is strongly expressed in advanced prostate cancer and significantly correlates with the Gleason >7 grading, being undetectable in healthy and benign prostate tissues. However, the role of TRPV6 as a highly Ca(2+)-selective channel in prostate carcinogenesis remains poorly understood. Here, we report that TRPV6 is directly involved in the control of prostate cancer cell (LNCaP cell line) proliferation by decreasing: (i) proliferation rate; (ii) cell accumulation in the S-phase of cell cycle and (iii) proliferating cell nuclear antigen (PCNA) expression. We demonstrate that the Ca(2+) uptake into LNCaP cells is mediated by TRPV6, with the subsequent downstream activation of the nuclear factor of activated T-cell transcription factor (NFAT). TRPV6-mediated Ca(2+) entry is also involved in apoptosis resistance of LNCaP cells. Our results suggest that TRPV6 expression in LNCaP cells is regulated by androgen receptor, however, in a ligand-independent manner. We conclude that the upregulation of TRPV6 Ca(2+) channel in prostate cancer cells may represent a mechanism for maintaining a higher proliferation rate, increasing cell survival and apoptosis resistance as well.

Ion channels in death and differentiation of prostate cancer cells.

Plasma membrane ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. Enhanced proliferation, aberrant differentiation, and impaired ability to die are the prime reasons for abnormal tissue growth, which can eventually turn into uncontrolled expansion and invasion, characteristic of cancer. Prostate cancer (PCa) cells express a variety of plasma membrane ion channels. By providing the influx of essential signaling ions, perturbing intracellular ion concentrations, regulating cell volume, and maintaining membrane potential, PCa cells are critically involved in proliferation, differentiation, and apoptosis. PCa cells of varying metastatic ability can be distinguished by their ion channel characteristics. Increased malignancy and invasiveness of androgen-independent PCa cells is generally associated with the shift to a 'more excitable' phenotype of their plasma membrane. This shift is manifested by the appearance of voltage-gated Na(+) and Ca(2+) channels which contribute to their enhanced apoptotic resistance together with downregulated store-operated Ca(2+) influx, altered expression of different K(+) channels and members of the Transient Receptor Potential (TRP) channel family, and strengthened capability for maintaining volume constancy. The present review examines channel types expressed by PCa cells and their involvement in metastatic behaviors.

Differential role of TRP channels in prostate cancer.

A major clinical problem with PC (prostate cancer) is the cell's ability to survive and proliferate upon androgen withdrawal. Indeed, deregulated cell differentiation and proliferation, together with the suppression of apoptosis, provides the condition for abnormal tissue growth. Here, we examine the differential role of TRP (transient receptor potential) channels in the control of Ca(2+) homoeostasis and growth of PC cells.

Calcium signalling and cancer cell growth.

Cancer is caused by defects in the mechanisms underlying cell proliferation and cell death. Calcium ions are central to both phenomena, serving as major signalling agents with spatial localization, magnitude and temporal characteristics of calcium signals ultimately determining cell's fate. There are four primary compartments: extracellular space, cytoplasm, endoplasmic reticulum and mitochondria that participate in the cellular Ca2+ circulation. They are separated by own membranes incorporating divers Ca2(+)-handling proteins whose concerted action provides for Ca2+ signals with the spatial and temporal characteristics necessary to account for specific cellular response. The transformation of a normal cell into a cancer cell is associated with a major re-arrangement of Ca2+ pumps, Na/Ca exchangers and Ca2+ channels, which leads to the enhanced proliferation and impaired ability to die. In the present chapter we examine what changes in Ca+ signalling and the mechanisms that support it underlie the passage from normal to pathological cell growth and death control. Understanding this changes and identifying molecular players involved provides new prospects for cancers treatment.

Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl- current associated with neuroendocrine differentiation of prostate cancer epithelial cells.

Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant, swelling-activated Cl- current (I(Cl,swell)), associated with NE differentiation of androgen-dependent LNCaP prostate cancer epithelial cells. NE-differentiating regimens, namely, chronic cAMP elevation or androgen deprivation, resulted in generally augmented I(Cl,swell) and enhanced RVD. This occurred as a result of both the increased endogenous expression of ClC-3, which is a volume-sensitive Cl- channel involved, as we show, in I(Cl,swell) in LNCaP (lymph-node carcinoma of the prostate) cells and the weaker negative I(Cl,swell) control from Ca2+ entering via store-dependent pathways. The changes in the RVD of NE-differentiated cells generally mimicked those reported for Bcl-2-conferred apoptotic resistance. Our results suggest that strengthening the mechanism that helps to maintain volume constancy may contribute to better survival rates of apoptosis-resistant NE cells.

[Calcium-dependent programmed cell death in prostate cancer]. / Kal'tsiieva zalezhnist' zaprohramovanoï klitynnoï zahybeli pry raku prostaty.

In the present review we describe the major molecular determinants of calcium homeostasis in prostate cancer cells and establish their role in the transformation to apoptosis-resistant cell phenotypes typical of advanced androgen-independent prostate cancer. We show that the hallmark of such transformation is complete loss of apoptosis pathway associated with endoplasmic reticulum calcium store depletion.

2-APB inhibits volume-regulated anion channels independently from intracellular calcium signaling modulation.

It has previously been suggested that volume-regulated anion channels (VRACs) and store-operated channels (SOCs) interact with each other according to their expected colocalization in the plasma membrane of LNCaP cells. In order to study interactions between these two channels, we used 2-aminoethoxydiphenyl borate (2-APB) as a regular SOC inhibitor. Surprisingly 2-APB reduced VRAC activity in a dose-dependent manner (IC(50)=122.8 microM), but not 2,2-diphenyltetrahydrofuran (a structural analog of 2-APB). This effect was also present in keratinocytes. We conclude that 2-APB is an inhibitor of the VRAC family, and is also a potent tool to study the SOC-VRAC interaction in LNCaP cells.

Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells.

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.

Store-operated Ca2+ channels in prostate cancer epithelial cells: function, regulation, and role in carcinogenesis.

Ca2+ homeostasis mechanisms, in which the Ca2+ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca2+ channels (SOCs)--a primary Ca2+ entry pathway for non-excitable cells--for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with "classical" Ca2+ release-activated Ca2+ channels (CRAC), but have a higher single channel conductance (3.2 and 12pS in Ca2+- and Na+-permeable modes, respectively). They are subject to feedback inhibition via Ca2+-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca2+-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.

Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells.

Although the prostate gland is a rich source of alpha1-adreno- (alpha1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP3-sensitive Ca2+ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations (PK(25) > PCs(4.6) > PLi(1.4) > PNa(1.0)), and the following permeability sequence for divalent cations: PCa(1.0) > PMg(0.74) > PBa(0.6) > PSr(0.36) > PMn(0.3). They are 4.3 times more permeable to Ca2+ than Na+ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, alpha1-ARs and m1-AChRs are functionally coupled to Ca2+-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.

A new human gene KCNRG encoding potassium channel regulating protein is a cancer suppressor gene candidate located in 13q14.3.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K+ channels. Using the patch-clamp technique we determined that KCNRG suppresses K+ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K+ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

[Effect of neuroendocrine cell differentiation of the human prostatic neoplasm cell line LNCaP on the characteristics of volume-sensitive chloride content]. / Vplyv neiroendokrynnoho dyferentsiiuvannia klityn raku prostaty liudyny liniï LNCaP na kharakterystyky ob'iemchutlyvoho khlornoho strumu.

By means of patch-clamp technique we examined changes in volume-regulated chloride current (ICl,swell) at neuroendocrine differentiation of androgen-dependent LNCaP prostate cancer cells. In those cells with neuroendocrine differentiation resulted from an increase in the intracellular cAMP, ICl,swell became much faster in response to applying external hypotonic solution and cell swelling. Changes in final rectification and voltage-dependent inactivation were not detected, as compared to the control cells. The differentiation also diminished ICl,swell blockade by Ca2+ transported via store-operated channels (SOC). On the base of our data we suggest that potentiation of the current at neuroendocrine differentiation, at least in part, resulted from a decrease in an inhibitory effect of Ca2+, transported into a cell through SOC, on volume-sensitive chloride current. Accelerated current in those cells might be induced by cytoskeleton rearrangement at the neuron-like growth.