STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.     

A reversible calix[4]arene armed phenolphthalein based fluorescent probe for the detection of Zn2+ and an application in living cells

A reversible and easy assembled fluorescent sensor based on calix[4]arene and phenolphthalein (C4P) was developed for selective zinc ion (Zn²⁺) sensing in aqueous samples. The probe C4P demonstrated high selective and sensitive detection towards Zn²⁺ over other competitive metal ions. Interaction of Zn²⁺ with a solution of C4P resulted in a considerable increment in emission intensity at 440 nm (λ_ex = 365 nm) due to the suppression of photoinduced electron transfer (PET) process and the restriction of C=N isomerization . The binding constant (K_a) of C4P with Zn²⁺ was calculated to be 4.50 × 10¹¹ M^?2 and also the limit of detection of C4P for Zn²⁺ was as low as 0.108 μM (at 10^?7 M level). Moreover, the fluorescence imaging in the human colon cancer cells suggested that C4P had great potential to be used to examine Zn²⁺ in vivo.     

Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na⁺ and partially dependent on extracellular and intracellular Ca²⁺ and protein kinase activity. Analyzing the generalized fractal dimension (D _q ) and its Legendre transform f(α) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a “simple” dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a “complex” dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

Mitochondrial Calcium Uniporter Activity Is Dispensable for MDA-MB-231 Breast Carcinoma Cell Survival

Calcium uptake through the mitochondrial Ca²⁺ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca²⁺ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca²⁺ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca²⁺ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca²⁺ uptake mechanisms.     

Phenothiazine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on' sensor for Zn2+ and bioimaging studies in live cells

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528 nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608 nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89 × 10^?8 M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.     

(−)‑Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells

Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (−)‑oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that OLCT is unable to induce Ca²⁺ mobilization in non-tumoral cells. By contrast, OLCT induces Ca²⁺ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24–72 h downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT impairs the ability of breast cancer cells to proliferate and migrate via downregulation of TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.     

The effect of a hyposmotic shock and purinergic agonists on K(Rb) efflux from cultured human breast cancer cells

The effect of a hyposmotic shock and extracellular ATP on the efflux of K⁺(Rb⁺) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K⁺(Rb⁺) from MDA-MB-231 cells via a pathway which was unaffected by Cl⁻ replacement. Apamin, charybdotoxin or removing extracellular Ca²⁺ had no effect on volume-activated K⁺(Rb⁺) efflux MDA-MB-231 cells. An osmotic shock also stimulated K⁺(Rb⁺) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K⁺(Rb⁺) release from MCF-7 cells. ATP-stimulated K⁺(Rb⁺) efflux was only inhibited slightly by replacing Cl⁻ with NO₃⁻. Removal of external Ca²⁺ during treatment with ATP reduced the fractional efflux of K⁺(Rb⁺) in a manner suggesting a role for cellular Ca²⁺ stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K⁺(Rb⁺) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K⁺(Rb⁺). UTP also stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K⁺(Rb⁺) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K⁺ efflux pathways are separate entities. However, there may be a degree of ‘crosstalk’ between the two pathways.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

Application of 2,4,5-tris(2-pyridyl)imidazole as ‘turn-off’ fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging

The 2,4,5-tris(2-pyridyl)imidazole ( L ) molecule has been evaluated as a probe for dual sensing of Hg²⁺ and Cu²⁺ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg²⁺ and Cu²⁺ ions, which is comprehensible under long UV light. The probe can detect Cu²⁺ ion in the pH range 3–11 and Hg²⁺ ion in pH 6–8. The limit of detection for Cu²⁺ (0.77 μM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu²⁺/Hg²⁺) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na₂EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu²⁺ and Hg²⁺ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory–time-dependent density functional theory (DFT–TDDFT) calculations support experimental observations, and d-orbitals of Cu²⁺/Hg²⁺ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu²⁺ and Hg²⁺ ions in living cells.     

Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells

The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2⁺) and MDA-MB-231 (Her2^?). IC₅₀ values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 μg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 μg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2⁺ cancer.     

An SAR study of hydroxy-trifluoromethylpyrazolines as inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader assay

The proteins Orai1 and STIM1 control store-operated Ca²⁺ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca²⁺ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration

Background

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer.

Methods and results

We found that PCI inhibits cathepsin L with an inhibition rate (k₂) of 3.0 × 10⁵ M⁻¹ s⁻¹. Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells.

Conclusions

Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L.

General significance

Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.     

Neuropeptide-induced modulation of carcinogenesis in a metastatic breast cancer cell line (MDA-MB-231<Superscript>LUC+</Superscript>)

Background

Metastatic cancer to bone is well-known to produce extreme pain. It has been suggested that the magnitude of this perceived pain is associated with disease progression and poor prognosis. These data suggest a potential cross-talk between cancer cells and nociceptors that contribute not only to pain, but also to cancer aggressiveness although the underlying mechanisms are yet to be stablished.

Methods

The in vitro dose dependent effect of neuropeptides (NPs) (substance P [SP], calcitonin gene-related peptide and neurokinin A [NKA]) and/or its combination, on the migration and invasion of MDA-MB-231^LUC+ were assessed by wound healing and collagen-based cell invasion assays, respectively. The effect of NPs on the expression of its receptors (SP [NK1] and neurokinin A receptors [NK2], CALCRL and RAMP1) and kininogen (high-molecular-weight kininogen) release to the cell culture supernatant of MDA-MB-231^LUC+, were measured using western-blot analysis and an ELISA assay, respectively. Statistical significance was tested using one-way ANOVA, repeated measures ANOVA, or the paired t-test. Post-hoc testing was performed with correction for multiple comparisons as appropriate.

Results

Our data show that NPs strongly modify the chemokinetic capabilities of a cellular line commonly used as a model of metastatic cancer to bone (MDA-MB-231^LUC+) and increased the expression of their receptors (NK1R, NK2R, RAMP1, and CALCRL) on these cells. Finally, we demonstrate that NPs also trigger the acute release of HMWK (Bradykinin precursor) by MDA-MB-231^LUC+, a molecule with both tumorigenic and pro-nociceptive activity.

Conclusions

Based on these observations we conclude that NPs exposure modulates this breast cancer cellular line aggressiveness by increasing its ability to migrate and invade new tissues. Furthermore, these results also support the pro nociceptive and cancer promoter role of the peripheral nervous system, during the initial stages of the disease.

     

Progranulin Stimulated by LPA Promotes the Migration of Aggressive Breast Cancer Cells

Abstract

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p < 0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p < 0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.     

Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp. strain HZM-1

A zinc-resistant bacterium, Brevibacterium sp. strain HZM-1 which shows a high Zn²⁺-adsorbing capacity, was isolated from the soil of an abandoned zinc mine. Kinetic analyses showed that Zn²⁺ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM. The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn²⁺ biosorbents. The cells could also adsorb heavy metal ions such as Cu²⁺. HZM-1 cells could remove relatively low levels of the Zn²⁺ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions. Bound Zn²⁺ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn²⁺-adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH. Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn²⁺ from natural environments. The cells could grow in the presence of significant concentrations of ZnCl₂ (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn²⁺-contaminated aqueous systems. Received: 1 February 2000 / Received revision: 31 March 2000 / Accepted: 1 May 2000     

The effect of bee bread (Perga) with chemotherapy on MDA-MB-231 cells

Bee bread (BB) is a bee product like propolis and honey. It is the main food for larvae and bees producing royal jelly in the hive. It also known as Perga. As with other bee products, it is increasingly popular due to its antioxidant properties. The aim of this study was to examine the effects of BB on MDA-MB-231 breast cancer cells and the effects on these cells when administered together with Doxorubicin (DOX) and Cisplatin (CDDP), used in cancer treatment. The proliferation of the cells was determined by applying 5 mg/mL BB together with different concentrations of DOX and CDDP. In addition to these studies, the effect of DOX+BB and CDDP+BB combinations on the migration of MDA-MB-231 cells was determined by the wound healing method. The expression levels of Bid and Bcl-2 were determined by RtqPCR. According to these studies, as expected, BB did not show a significant toxic effect on MDA-MB-231 cells at different concentrations. BB significantly suppressed the effect of DOX and CDDP on the proliferation of MDA-MB-231 cells. BB with DOX and CDDP suppressed the proapoptotic Bid gene while overexpressing the anti-apoptotic Bcl-2 gene, separately. Interestingly, BB blocked the migration of MDA-MB-231 cells by 50% even after 72 h. As a result, BB significantly reduced the toxicity of DOX and CDDP on MDA-MB-231 cells. The most interesting result of the study is that BB prevented the migration of cancer cells.

     

Synthesis and photophysical investigation of Schiff base as a Mg2+ and Zn2+ fluorescent chemosensor and its application

In view of the fluorescent switching properties and anti-fatigue properties of diarylethene, a diarylethene fluorescent chemosensor for the immediate detection of zinc ion (Zn²⁺) and magnesium ion (Mg²⁺) in acetonitrile was synthesized in this article. The structure of 1o was determined by performing spectroscopy and elemental analysis. The presence of Zn²⁺ or Mg²⁺ made the chemosensor 1o show an obvious “turn-on” fluorescent signal (bright yellow-green for Mg²⁺ and bright cyan for Zn²⁺). The fluorescent change caused by the 1:1 binding of 1o and Zn²⁺ or Mg²⁺ might be due to hindering the excited-state intramolecular proton transfer (ESIPT) process, which were bolstered by Benesi–Hildebrand analysis, Job's plot curves, proton nuclear magnetic resonance (¹H-NMR) titration and mass spectrometry. The limits of detection were acquired from the standard curve plots for Mg²⁺ at 44.6 nM and for Zn²⁺ at 14 nM. Based on the fluorescent behaviors, a logic gate was constructed with the emission intensity at 528/518 nm as output signal, the ultraviolet-visible (UV-vis) lights, Mg²⁺/Zn²⁺ and EDTA as input signals. Exogenous Zn²⁺ and Mg²⁺ fluorescent bioimaging were performed on Hela cells with 1o , indicating its potential application in biodiagnostic analysis. In particular, 1o was manufactured into test paper, and Zn²⁺ or Mg²⁺ can be conveniently, efficiently and qualitatively identified by the fluorescent color variation of the test strips.     

Differences in intracellular mobile zinc levels affect susceptibility to plasma-activated medium-induced cytotoxicity

There is growing evidence that plasma-activated medium (PAM), which is prepared by non-thermal plasma (NTP) irradiation of cell-free medium, is a beneficial tool for cancer therapy. PAM has been reported to preferentially kill cancer cells; however, its mechanism is not fully understood. Since PAM contains reactive oxygen species (ROS) and reactive nitrogen species, the anti-cancer effects of PAM are thought to be attributed to oxidative stress induced by these reactive molecules. Oxidative stress has been shown to release zinc (Zn²⁺) from intracellular Zn²⁺ stores and provoke Zn²⁺-dependent cell death. We have previously demonstrated that intracellular free Zn²⁺ plays a critical role in PAM-induced cell death in human neuroblastoma SH-SY5Y cells. In this study, we found that normal human fibroblasts were less susceptible to PAM cytotoxicity compared with SH-SY5Y cells. PAM decreased intracellular NAD⁺ levels in both cells, whereas the depletion of ATP and mitochondrial ROS generation was hardly observed in fibroblasts. Intracellular mobile Zn²⁺ contents of fibroblasts were lower than those of SH-SY5Y cells. PAM suppressed the activity of aconitase, which is a tricarboxylic acid cycle enzyme, only in SH-SY5Y cells, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn²⁺ chelator, counteracted the suppression. The combination treatment with PAM and Zn²⁺ augmented PAM-induced ATP depletion, mitochondrial ROS generation, and cytotoxicity in fibroblasts. These findings suggest the possibility that cells with high intracellular mobile Zn²⁺ are susceptible to PAM cytotoxicity. Therefore, we concluded that the differences in mobile Zn²⁺ levels affect PAM-induced cellular responses.