Valproic Acid as a Potential Inhibitor of Plasmodium falciparum Histone Deacetylase 1 (PfHDAC1): An in Silico Approach

A new Plasmodium falciparum histone deacetylase1 (PfHDAC1) homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations were acceptable and consistent. Docking a group of hydroxamic acid histone deacetylase inhibitors and valproic acid has shown binding poses that agree well with inhibitor-bound histone deacetylase-solved structural interactions. Docking affinity dG scores were in agreement with available experimental binding affinities. Further, enzyme-ligand complex stability and reliability were investigated by running 5-nanosecond molecular dynamics simulations. Thorough analysis of the simulation trajectories has shown that enzyme-ligand complexes were stable during the simulation period. Interestingly, the calculated theoretical binding energies of the docked hydroxamic acid inhibitors have shown that the model can discriminate between strong and weaker inhibitors and agrees well with the experimental affinities reported in the literature. The model and the docking methodology can be used in screening virtual libraries for PfHDAC1 inhibitors, since the docking scores have ranked ligands in accordance with experimental binding affinities. Valproic acid calculated theoretical binding energy suggests that it may inhibit PfHDAC1.     

The Novel Class IIa Selective Histone Deacetylase Inhibitor YAK540 Is Synergistic with Bortezomib in Leukemia Cell Lines

The treatment of leukemias, especially acute myeloid leukemia (AML), is still a challenge as can be seen by poor 5-year survival of AML. Therefore, new therapeutic approaches are needed to increase the treatment success. Epigenetic aberrations play a role in pathogenesis and resistance of leukemia. Histone deacetylase (HDAC) inhibitors (HDACIs) can normalize epigenetic disbalance by affecting gene expression. In order to decrease side effects of so far mainly used pan-HDACIs, this paper introduces the novel highly selective class IIa HDACI YAK540. A synergistic cytotoxic effect was observed between YAK540 and the proteasome inhibitor bortezomib (BTZ) as analyzed by the Chou-Talalay method. The combination of YAK540 and BTZ showed generally increased proapoptotic gene expression, increased p21 expression, and synergistic, caspase 3/7-mediated apoptosis. Notably, the cytotoxicity of YAK540 is much lower than that of pan-HDACIs. Further, combinations of YAK540 and BTZ are clearly less toxic in non-cancer HEK293 compared to HL-60 leukemia cells. Thus, the synergistic combination of class IIa selective HDACIs such as YAK540 and proteasome inhibitors represents a promising approach against leukemias to increase the anticancer effect and to reduce the general toxicity of HDACIs.     

The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells’ epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time–dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G₂/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.     

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug–drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity.     

Epigenetic Targeting of Histone Deacetylases in Diagnostics and Treatment of Depression

Depression is a highly prevalent, disabling, and often chronic illness that places substantial burdens on patients, families, healthcare systems, and the economy. A substantial minority of patients are unresponsive to current therapies, so there is an urgent need to develop more broadly effective, accessible, and tolerable therapies. Pharmacological regulation of histone acetylation level has been investigated as one potential clinical strategy. Histone acetylation status is considered a potential diagnostic biomarker for depression, while inhibitors of histone deacetylases (HDACs) have garnered interest as novel therapeutics. This review describes recent advances in our knowledge of histone acetylation status in depression and the therapeutic potential of HDAC inhibitors.     

Intravitreal Injection of Liposomes Loaded with a Histone Deacetylase Inhibitor Promotes Retinal Ganglion Cell Survival in a Mouse Model of Optic Nerve Crush

Various neuroprotective agents have been studied for the treatment of retinal ganglion cell (RGC) diseases, but issues concerning the side effects of systemically administered drugs and the short retention time of intravitreally injected drugs limit their clinical applications. The current study aimed to evaluate the neuroprotective effects of intravitreally injected trichostatin A (TSA)-loaded liposomes in a mouse model of optic nerve crush (ONC) and determine whether TSA-loaded liposomes have therapeutic potential in RGC diseases. The histone deacetylase inhibitor, TSA, was incorporated into polyethylene glycolylated liposomes. C57BL/6J mice were treated with an intravitreal injection of TSA-loaded liposomes and liposomes loaded with a lipophilic fluorescent dye for tracking, immediately after ONC injury. The expression of macroglial and microglial cell markers (glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1), RGC survival, and apoptosis were assessed. We found that the liposomes reached the inner retina. Their fluorescence was detected for up to 10 days after the intravitreal injection, with peak intensity at 3 days postinjection. Intravitreally administered TSA-loaded liposomes significantly decreased reactive gliosis and RGC apoptosis and increased RGC survival in a mouse model of ONC. Our results suggest that TSA-loaded liposomes may help in the treatment of various RGC diseases.     

Biological Effect of a Hybrid Anticancer Agent Based on Kinase and Histone Deacetylase Inhibitors on Triple-Negative (MDA-MB231) Breast Cancer Cells

We examined the effects of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) combined with the vascular endothelial growth factor receptor-1/2 inhibitor (3Z)-5-hydroxy-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-2-one on MDA-MB-231 breast cancer cells (triple-negative) in the form of both a cocktail of the separate compounds and a chemically synthesized hybrid (N-hydroxy-N''-[(3Z)-2-oxo-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-5-yl]octanediamide). Comparative flow cytometric and Western blot analyses were performed on cocktail- and hybrid-treated cells to evaluate cell cycle distribution, autophagy/apoptosis modulation, and mitochondrial metabolic state in order to understand the cellular basis of the cytotoxic effect. Cell cycle analysis showed a perturbation of the rate of progression through the cycle, with aspects of redistribution of cells over different cycle phases for the two treatments. In addition, the results suggest that the two distinct classes of compounds under investigation could induce cell death by different preferential pathways, i.e., autophagy inhibition (the cocktail) or apoptosis promotion (the hybrid), thus confirming the enhanced potential of the hybrid approach vs. the combination approach in finely tuning the biological activities of target cells and also showing the hybrid compound as an additional promising drug-like molecule for the prevention or therapy of “aggressive” breast carcinoma.     

Synergistic Growth Inhibition of HT-29 Colon and MCF-7 Breast Cancer Cells with Simultaneous and Sequential Combinations of Antineoplastics and CNS Drugs

Several central nervous system (CNS) drugs exhibit potent anti-cancer activities. This study aimed to design a novel model of combination that combines different CNS agents and antineoplastic drugs (5-fluorouracil (5-FU) and paclitaxel (PTX)) for colorectal and breast cancer therapy, respectively. Cytotoxic effects of 5-FU and PTX alone and in combination with different CNS agents were evaluated on HT-29 colon and MCF-7 breast cancer cells, respectively. Three antimalarials alone and in combination with 5-FU were also evaluated in HT-29 cells. Different schedules and concentrations in a fixed ratio were added to the cultured cells and incubated for 48 h. Cell viability was evaluated using MTT and SRB assays. Synergism was evaluated using the Chou-Talalay, Bliss Independence and HSA methods. Our results demonstrate that fluphenazine, fluoxetine and benztropine have enhanced anticancer activity when used alone as compared to being used in combination, making them ideal candidates for drug repurposing in colorectal cancer (CRC). Regarding MCF-7 cells, sertraline was the most promising candidate alone for drug repurposing, with the lowest IC₅₀ value. For HT-29 cells, the CNS drugs sertraline and thioridazine in simultaneous combination with 5-FU demonstrated the strongest synergism among all combinations. In MCF-7 breast cancer cells, the combination of fluoxetine, fluphenazine and benztropine with PTX resulted in synergism for all concentrations below IC₅₀. We also found that the antimalarial artesunate administration prior to 5-FU produces better results in reducing HT-29 cell viability than the inverse drug schedule or the simultaneous combination. These results demonstrate that CNS drugs activity differs between the two selected cell lines, both alone and in combination, and support that some CNS agents may be promising candidates for drug repurposing in these types of cancers. Additionally, these results demonstrate that 5-FU or a combination of PTX with CNS drugs should be further evaluated. These results also demonstrate that antimalarial drugs may also be used as antitumor agents in colorectal cancer, besides breast cancer.     

柑橘皮提取液在硫酸介质中缓蚀性能及复配研究

采用浸泡法提取柑橘皮中缓蚀剂成分,应用电化学方法研究柑橘皮提取液和柑橘皮提取液与碘化钾的复配协同效应。结果表明,柑橘皮提取液在10%H2SO4溶液中对碳钢有良好的缓蚀性能,与碘化钾复配后,柑橘皮提取液与碘化钾存在明显的协同效应,复配后的缓蚀效率达98%以上,能同时抑制碳钢在硫酸溶液中的阴极反应和阳极反应,是一种混合型缓蚀剂,属于单分子层吸附,且反应自发进行。     

Cold Atmospheric Plasma Changes the Amino Acid Composition of Solutions and Influences the Anti-Tumor Effect on Melanoma Cells

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.     

曲古抑菌素A与罗格列酮联合应用对胃癌细胞增殖的抑制作用

目的研究组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)与过氧化物酶体增殖物激活受体γ配体罗格列酮(ROZ)联合应用对胃癌细胞SGC-7901增殖的抑制作用,并探讨其可能的作用机制。方法首先用不同浓度的TSA和ROZ分别作用于SGC-7901细胞,MTT法检测二者对细胞增殖的抑制作用,以筛选出合适的联合作用浓度。将SGC-7901细胞随机分为对照组、TSA组、ROZ组和TSA+ROZ组,加入相应的药物处理48h后,MTT法检测各组细胞增殖抑制率,并应用流式细胞仪检测细胞周期情况,RT-PCR检测PPARγ和CyclinD1基因mRNA的转录水平。结果对SGC-7901细胞增殖抑制作用不显著的低剂量TSA(40nmol/L)与ROZ联用时,能显著增强ROZ对细胞增殖的抑制作用,TSA+ROZ组G0/G1期细胞比例较单用TSA或ROZ组明显增加,且CyclinD1基因mRNA转录水平明显下降;TSA作用于细胞后,PPARγ基因mRNA转录水平较对照组明显升高,而ROZ单独作用后,细胞PPARγ基因mRNA转录水平无改变。结论TSA可明显增强ROZ对SGC-7901细胞增殖的抑制效应,提示HDAC在SGC-7901细胞内可能具有抑制PPARγ的作用。     

Combinations of Histone Deacetylase Inhibitors with Distinct Latency Reversing Agents Variably Affect HIV Reactivation and Susceptibility to NK Cell-Mediated Killing of T Cells That Exit Viral Latency

The ‘shock-and-kill’ strategy to purge the latent HIV reservoir relies on latency-reversing agents (LRAs) to reactivate the provirus and subsequent immune-mediated killing of HIV-expressing cells. Yet, clinical trials employing histone deacetylase inhibitors (HDACis; Vorinostat, Romidepsin, Panobinostat) as LRAs failed to reduce the HIV reservoir size, stressing the need for more effective latency reversal strategies, such as 2-LRA combinations, and enhancement of the immune responses. Interestingly, several LRAs are employed to treat cancer because they up-modulate ligands for the NKG2D NK-cell activating receptor on tumor cells. Therefore, using in vitro T cell models of HIV latency and NK cells, we investigated the capacity of HDACis, either alone or combined with a distinct LRA, to potentiate the NKG2D/NKG2D ligands axis. While Bortezomib proteasome inhibitor was toxic for both T and NK cells, the GS-9620 TLR-7 agonist antagonized HIV reactivation and NKG2D ligand expression by HDACis. Conversely, co-administration of the Prostratin PKC agonist attenuated HDACi toxicity and, when combined with Romidepsin, stimulated HIV reactivation and further up-modulated NKG2D ligands on HIV⁺ T cells and NKG2D on NK cells, ultimately boosting NKG2D-mediated viral suppression by NK cells. These findings disclose limitations of LRA candidates and provide evidence that NK cell suppression of reactivated HIV may be modulated by specific 2-LRA combinations.     

环己二醇二缩水甘油醚与Ca／Zn复合稳定剂协同作用及机理研究

采用环己二醇二缩水甘油醚作为辅助热稳定剂，与Ca／Zn稳定剂复配，通过刚果红热稳定测试，研究了它们相互间协同作用及机理。结果表明，环己二醇二缩水甘油醚与Ca／Zn复合稳定剂有很好的协同作用，能有效地抑制Ca／Zn复合稳定剂在使用中的“锌烧”现象；其中Ca：Zn为3：1时热稳定性最好，热稳定时间为38min；对于不同的Ca、Zn比，加入环己二醇二缩水甘油醚的配方的性能均优于环氧大豆油配方的。通过实验及傅里叶红外光谱、紫外光谱分析表明，己二醇二缩水甘油醚在PVC降解产生的HCl的作用下，其分子内环氧基团开环和醚键断裂，与PVC降解产生的HCl反应，能消除PVC降解产生的HCl；同时，环己二醇二缩水甘油醚能与硬脂酸锌在PVC降解中产生的ZnCl2发生作用，其分子内环氧基团开环，氧与Zn结合，使得ZnCl2催化PVC降解活性降低。     

Synergistic Effect of Combinatorial Treatment with Curcumin and Mitomycin C on the Induction of Apoptosis of Breast Cancer Cells: A cDNA Microarray Analysis

In order to explore the synergistic mechanisms of combinatorial treatment using curcumin and mitomycin C (MMC) for breast cancer, MCF-7 breast cancer xenografts were conducted to observe the synergistic effect of combinatorial treatment using curcumin and MMC at various dosages. The synergistic mechanisms of combinatorial treatment using curcumin and MMC on the inhibition of tumor growth were explored by differential gene expression profile, gene ontology (GO), ingenuity pathway analysis (IPA) and Signal–Net network analysis. The expression levels of selected genes identified by cDNA microarray expression profiling were validated by quantitative RT-PCR (qRT-PCR) and Western blot analysis. Effect of combinatorial treatment on the inhibition of cell growth was observed by MTT assay. Apoptosis was detected by flow cytometric analysis and Hoechst 33258 staining. The combinatorial treatment of 100 mg/kg curcumin and 1.5 mg/kg MMC revealed synergistic inhibition on tumor growth. Among 1501 differentially expressed genes, the expression of 25 genes exhibited an obvious change and a significant difference in 27 signal pathways was observed (p < 0.05). In addition, Mapk1 (ERK) and Mapk14 (MAPK p38) had more cross-interactions with other genes and revealed an increase in expression by 8.14- and 11.84-fold, respectively during the combinatorial treatment by curcumin and MMC when compared with the control. Moreover, curcumin can synergistically improve tumoricidal effect of MMC in another human breast cancer MDA-MB-231 cells. Apoptosis was significantly induced by the combinatorial treatment (p < 0.05) and significantly inhibited by ERK inhibitor (PD98059) in MCF-7 cells (p < 0.05). The synergistic effect of combinatorial treatment by curcumin and MMC on the induction of apoptosis in breast cancer cells may be via the ERK pathway.     

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.     

膦酰基羧酸的复配及协同效应研究

在优化工艺条件下合成了环境友好型缓蚀阻垢剂膦酰基羧酸(POCA),采用旋转挂片失重法、静态阻垢法对POCA及其各复合配方的缓蚀阻垢性能、协同效应进行了研究,并探讨了缓蚀机理.结果表明,60%(质量分数,下同)POCA与40% PBTC、40% POCA与60% HEDP复配后,在阻碳酸钙垢方面均有协同效应,阻垢率高达100%;40% POCA、32% PBTC、24% HEDP和4%乙酸锌复配后,具有缓蚀协同效应,缓蚀率达到92.22%,阻碳酸钙垢率为90.42%、阻磷酸钙垢率为97.60%,该复合缓蚀阻垢剂是一种以抑制阳极过程为主的混合型缓蚀剂.     

柳氮磺吡啶与维生素K3联合应用对神经胶质瘤C6细胞增殖的影响

目的观察柳氮磺吡啶(SAS)与维生素K3(VK3)联合应用对神经胶质瘤C6细胞增殖的影响,并探讨二者的作用机制。方法取对数生长期的大鼠神经胶质瘤C6细胞,分别加入不同浓度的SAS、VK3,MTT法检测各组细胞增殖水平,RT-PCR及Western blot法分别检测细胞IKBα基因mRNA的转录水平及P65蛋白的表达水平;TUNEL法检测细胞凋亡。结果SAS单独作用于C6细胞,呈剂量依赖性地抑制细胞增殖;SAS与VK3联合作用,抑制细胞增殖效果更明显。SAS与VK3联合作用4、8、12h,C6细胞IKBα基因mRNA的转录水平逐渐下降,4h时,C6细胞P65蛋白的表达水平增加,而8、12h时降低。SAS与VK3联合作用,TUNEL阳性细胞率明显高于空白对照组及SAS、VK3单独作用组。结论SAS与VK3联合应用,可通过影响NF-κB/IKBα基因的表达,抑制C6细胞增殖,二者联合应用可减少单独药物用量,减轻对正常细胞的毒性作用。     

Synergistic effects of ZrO2 or B2O3 on flame‐retarded poly (butyl methacrylate) with tricresylphosphate

The synergistic flame retarded systems consisting of ZrO₂ or B₂O₃ and tricresylphosphate(TCP) for poly(butyl methacrylate)(PBMA) are reported. The synergistic effects were studied by limited oxygen index (LOI) test, UL‐94, thermal gravimetric analysis (TGA), Infrared Spectroscopy (IR), Raman spectra and digital photos. The synergistic effect of ZrO₂ or B₂O₃ with TCP could be clearly observed by LOI. The TGA results show that ZrO₂ or B₂O₃ increases the char yields of the systems at 500^°C. The Raman spectra, IR and digital photos results provide evidence that ZrO₂ or B₂O₃ can efficiently promote the formation of charred layers composed of varying amounts of graphite and amorphous carbon. The possible mechanisms for synergy are discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells

The role of calcium ion (Ca²⁺) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca²⁺ signaling studies focused on Ca²⁺ entry routes, but rarely explored the role of Ca²⁺ extrusion. Functioning of the Na⁺/Ca²⁺ exchanger (NCX) on the plasma membrane is the major way of Ca²⁺ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca²⁺ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5–100 μM) did not affect melanoma cells or increase cytosolic Ca²⁺. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC₅₀ values of 3–20 μM. Bepridil and KB-R7943 elevated intracellular Ca²⁺ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca²⁺ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca²⁺-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.