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Endothelial-to-mesenchymal transition (EndMT) is a complex biological process by which endothelial cells lose their endothelial cell characteristics and acquire mesenchymal cell properties under certain physiological or pathological conditions. Recently, it has been found that EndMT plays an important role in the occurrence and development of fibrotic cardiovascular diseases. In this review, we first summarize the main induction pathways involved in EndMT process. In addition, we discuss the role of EndMT in fibrotic cardiovascular diseases and its potential implication in new therapeutic interventions.

Circular RNAs (circRNAs), one type of non-coding RNA, were initially misinterpreted as nonfunctional products of pre-mRNA mis-splicing. Currently, circRNAs have been proven to manipulate the functions of diverse molecules, including non-coding RNAs, mRNAs, DNAs and proteins, to regulate cell activities in physiology and pathology. Accumulating evidence indicates that circRNAs play critical roles in tumor genesis, development, and sensitivity to radiation and chemotherapy. Radiotherapy and chemotherapy are two primary types of intervention for most cancers, but their therapeutic efficacies are usually retarded by intrinsic and acquired resistance. Thus, it is urgent to develop new strategies to improve therapeutic responses. To achieve this, clarification of the underlying mechanisms affecting therapeutic responses in cancer is needed. This review summarizes recent progress and mechanisms of circRNAs in cancer resistance to radiation and chemotherapy, and it discusses the limitations of available knowledge and potential future directions.

Metastasis is still the primary cause of cancer-related mortality. However, the underlying mechanisms of cancer metastasis are not yet fully understood. Currently, the epithelial-mesenchymal transition, metabolic remodeling, cancer cell intercommunication and the tumor microenvironment including diverse stromal cells, are reported to affect the metastatic process of cancer cells. Calcium ions (Ca2+) are ubiquitous second messengers that manipulate cancer metastasis by affecting signaling pathways. Diverse transporter/pump/channel-mediated Ca2+ currents form Ca2+ oscillations that can be decoded by Ca2+-binding proteins, which are promising prognostic biomarkers and therapeutic targets of cancer metastasis. This paper presents a review of the advances in research on the mechanisms underlying cancer metastasis and the roles of Ca2+-related signals in these events.

Due to its Ca(2+) buffering capacity, the mitochondrion is one of the most important intracellular organelles in regulating Ca(2+) dynamic oscillation. Mitochondrial calcium uniporter (MCU) is the primary mediator of Ca(2+) influx into mitochondria, manipulating cell energy metabolism, ROS production, and programmed cell death, all of which are critical for carcinogenesis. The understanding of the uniporter complex was significantly boosted by recent groundbreaking discoveries that identified the uniporter pore‐forming subunit MCU and its regulatory molecules, including MCU‐dominant negative β subunit (MCUb), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), mitochondrial calcium uptake (MICU) 1, MICU2, and MICU3. These provide the means and molecular platform to investigate MCU complex (uniplex)‐mediated impaired Ca(2+) signalling in physiology and pathology. This review aims to summarize the progress of the understanding regulatory mechanisms of uniplex, roles of uniplex‐mediated Ca(2+) signalling in cancer, and potential pharmacological inhibitors of MCU.

Esophageal cancer (EC) is the 6th leading cause of cancer mortality worldwide with poor prognosis, hence more effective chemotherapeutic drugs for this deadly disease are urgently needed. We previously reported that high expression of Orai1, a store-operated Ca2+entry (SOCE) channel, was associated with poor survival rate in EC patients and Orai1-mediated intracellular Ca2+ oscillations regulated cancer cell proliferation. Previous studies suggested that Orai1-mediated SOCE is a promising target for EC chemotherapy. Here, we evaluated the anti-cancer effect of a novel SOCE inhibitor, RP4010, in cultured EC cells and xenograft models. Compared to other previously reported SOCE channel inhibitors, RP4010 is more potent in blocking SOCE and inhibiting cell proliferation in EC and other cancer cells. Treatment with RP4010 resulted in reduction of intracellular Ca2+ oscillations, caused cell cycle arrest at G0/G1 phase in vitro, decreased nuclear translocation of nuclear factor kappa B (NF-κB) in vivo and in vitro, and inhibited tumor growth in vivo. Taken together, data demonstrated the therapeutic potential of RP4010 in EC patients via inhibition of SOCE-mediated intracellular Ca2+ signaling.

BackgroundAngiogenesis is a key prerequisite for wound healing. The conditioned medium following culture of umbilical cord mesenchymal stem cells (UCMSCs) has a potential to promote angiogenesis, but the efficacy is very low. Autophagy is an important process in protein recycling and a contributor for cell exocrine, which maybe stimulate the release of cytokines from UCMSCs to the medium and enhance the pro-angiogenic efficacy of the conditioned medium.MethodsAutophagy in UCMSCs was induced by 100 nM, 1 µM and 10 µM rapamycin for 6-hour and then detected by LC-3 immunofluorescence staining. After induction, the cells were washed with PBS for 3 times and cultured in fresh medium without rapamycin for additional 24-hour. And then, the conditioned medium was collected for the following experiments. The angiogenic effects of different groups of conditioned medium were verified by in vitro and in vivo tube formation assays in the matrigel-coated plates and matrigel plaques injected in mouse inguinal areas. Finally, the expressions of angiogenic factors including VEGF, FGF-1, FGF-2, TGF-α, MMP-3, MMP-9, PDGF-α, PDGF-β, HIF-1α and Ang II in the autophagic and control UCMSCs were measured by q-PCR assay.ResultsRapamycin induced autophagy of UCMSCs in a dose dependent manner, but the conditioned medium in 100 nM rapamycin-induced group was with the best pro-angiogenic efficacy. Thus, this group of medium was viewed as the optimal conditioned medium. The in vivo tube formation assay showed that angiogenesis in matrigel plaques injected daily with the optimal conditioned medium was more obvious than that injected with the control conditioned medium. Further, the expressions of VEGF, FGF-2, PDGF-α, MMP-9 and HIF-1α were markedly increased in UCMSCs following treatment with 100 nM rapamycin.ConclusionAppropriate autophagy improves the pro-angiogenic efficacy of the conditioned medium, which might be utilized to optimize the applications of UCMSCs-derived conditioned medium in wound healing and tissue repair.Trial registrationNot applicable.

Background: The incidence and symptoms of Alzheimer’s disease (AD) have significant gender differences, but its mechanisms are not completely understood. Recent studies showed that NLRP1 inflammasome was markedly overexpressed in females under some other pathological states such as nodular melanoma. Whether NLRP1 signals are also expressed with a gender difference in AD has not been elucidated. This study was designed to investigate the expressions of NLRP1 signals including NLRP1, Capase-1 and IL-1β in brains of different sexual APP/PS1+/- mice. Methods: 30-week-old male and female APP/PS1+/- mice were used in this study. Amyloid-β (Aβ) plaques were stained with Congo red dye and cell apoptosis in brains was detected by TUNEL staining. The expressions of NLRP1, Capase-1 and IL-1β were measured by immunofluorescent staining and Western-blotting assay. RESULTS: The numbers of Aβ plaques in cortex and hippocampus and neuronal apoptosis in cortex of female APP/PS1+/- mice increased 4 and 2 folds than that in males respectively (P

Various types of nanoparticles have been proposed for targeted drug delivering, imaging, and tracking of therapeutic agents. However, highly biocompatible nanoparticles with structure-induced fluorescence and capability to conjugate with biomarkers and drugs remain lacking. This research proposes and synthesizes fluorescent nanoparticles (f-PNPs) assembled by cyclic peptides to combine imaging and drug delivering for esophageal cancer (EC). To achieve tumor targeting, f-PNPs are first conjugated with RGD moieties to selectively target EC cells via αvβ3 integrin; the nanoparticles are then embedded with epirubicin (EPI). Cell viability assays and analysis of tissue histology reveal that EPI-loaded RGD-f-PNPs (RGD-f-PNPs/EPI) led to significantly reduced cardiotoxicity and improved anti-tumor activity compared to EPI alone. Moreover, the drug delivery to tumor sites and therapeutic responses could be monitored with near-infrared fluorescence using RGD-f-PNPs/EPI. This unique nanoparticle system may lead to potential approaches for bioorganic fluorescence-based delivering, imaging, and drug release tracking., Biocompatible nanoparticles for targeted drug delivery, imaging and tracking of therapeutic agents are sought-after. Here, the authors report cyclic peptide nanoparticles with structure induced fluorescence that can conjugate with biomarkers and carry drugs for application in esophageal cancer.

Background/Aims: Multidrug resistance (MDR) induced by the ABC transporter subfamily B member 1 (ABCB1) and subfamilyG member 2 (ABCG2) limits successful cancer chemotherapy and no commercially available MDR modulator is used in the clinic. In the current study, we aimed to investigate the effects of PCI29732 on the enhancement of chemotherapeutic agents. Methods: Cell cytotoxicity and reversal effect were measured with MTT assay. Additionally, flow cytometry was employed to detect the accumulation and efflux of the drugs. We investigated the interaction of PCI29732 and the substrate binding sites of ABCG2 was investigated via the photo-labeling of ABCG2 with the [125I] iodoarylazidoprazosin. The vanadate-sensitive ATPase activity of ABCG2 was measured to identify whether the drug affected the ATPase activity. RT-PCR and Western blot were utilized to analyze mRNA and protein expression respectively. Results: Here, we found that PCI29732 significantly enhanced the efficacy of substrate chemotherapeutic agents in ABCG2-overexpressing cells and also in xenografts harboring the H460/MX20 cell that overexpress ABCG2, but not in their parental sensitive cells and ABCB1-overexpressing cells. Mechanistically, the intracellular accumulations of doxorubicin and Rhodamine 123 were increased in ABCG2-overexpressing S1-MI-80 cells with the presence of PCI29732. PCI29732 stimulated the ATPase activity of ABCG2 at low concentrations. However at the high concentrations, PCI29732 inhibited the ATPase activity, and competed with [125I]-iodoarylazidoprazosin for photo-affinity labeling of ABCG2. PCI29732 did neither alter the mRNA or protein expression levels of ABCG2 nor the phosphorylation levels of AKT and ERK1/2. Conclusion: Our study demonstrates that PCI29732 inhibits the function of ABCG2 by competitively binding to the ATP-binding site of ABCG2 and enhances the anti-tumor efficacy of substrate chemotherapeutic agents, This findings encourages the development of combinational chemotherapy for the treatment of ABCG2- overexpressing cancer patients.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.—Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

NLRP3 inflammasome is an intracellular protein complex that initiates cellular injury via assembly of NLRP3, ASC and caspase-1 in response to microbial infection and sterile stressors. The importance of NLRP3 inflammasome in immunity and human diseases has been well documented. Up to now, targeted inhibition of the assembly of NLRP3 inflammasome complex and of its activation was thought to be therapeutic strategy for associated diseases. Recent studies show that a host of molecules such as NIMA-related kinase 7 (Nek7) and DEAD-box helicase 3 X-linked (DDX3X) and a large number of biological mediators including cytokines, microRNAs, nitric oxide, carbon monoxide, nuclear factor erythroid-2 related factor 2 (Nrf2) and cellular autophagy participate in the activation and inactivation of NLRP3 inflammasome. This review summarizes current understanding of the molecular basis of NLRP3 inflammasome activation and inactivation. This knowledge may lead to development of new therapies directed at NLRP3 inflammasome related diseases.

The intracellular calcium ions (Ca2+) act as second messenger to regulate gene transcription, cell proliferation, migration and death. Accumulating evidences have demonstrated that intracellular Ca2+ homeostasis is altered in cancer cells and the alteration is involved in tumor initiation, angiogenesis, progression and metastasis. Targeting derailed Ca2+ signaling for cancer therapy has become an emerging research area. This review summarizes some important Ca2+ channels, transporters and Ca2+-ATPases, which have been reported to be altered in human cancer patients. It discusses the current research effort toward evaluation of the blockers, inhibitors or regulators for Ca2+ channels/transporters or Ca2+-ATPase pumps as anti-cancer drugs. This review is also aimed to stimulate interest in, and support for research into the understanding of cellular mechanisms underlying the regulation of Ca2+ signaling in different cancer cells, and to search for novel therapies to cure these malignancies by targeting Ca2+ channels or transporters., Graphical abstract Ca2+ plays vital roles in normal cell physiology, such as gene transcription, cell proliferation and migration. Abnormal Ca2+ signaling by virtue of altered channel expression or activation contributes to carcinogenesis and promotes tumor development. Targeting the dysregulated Ca2+ channels/transporters/pumps may provide a promising chemotherapy for cancer treatment.fx1

Intracellular Ca2+ signals, such as oscillations, regulate proliferation, migration and other cellular events in cancer cells. Zinc (Zn), a major dietary micronutrient, plays important roles in chemoprevention. However, the molecular mechanisms underlying Zn-preventive function as well as the cross-talk with Ca2+ signaling are still unclear. We previously demonstrated that Orai1, a store-operated Ca2+ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to adjacent normal tissues from patients, and the elevated expression of Orai1 is strongly associated with poor prognosis. The present study shows that Zn can greatly inhibit cell proliferation and Orai1-mediated SOCE in ESCC cells. We also found that the inhibitory function of Zn on SOCE is redox sensitive; reducing agent DTT could recover the full activity of Orai1-mediated SOCE. Based on the topology information of Orai1, we hypothesized that a histidine residue in the linker region between two transmembranes of Orai1 (H113) as well as three cysteine residues may play critical roles in the Zn-inhibitory effects. The data showed that, in the cells containing H113A-mutated Orai1, the Zn-inhibitory effect is vanished whereas Zn treatment reduces SOCE in the cell expressing wildtype Orai1. Similarly, the ESCC cells expressing any of three mutations in cysteine residues (C126A, C143A and C195A) of Orai1 displayed partial loss of the Zn-inhibitory function. These results suggest that the H113 and three cysteine residues likely bind Zn, which modulate Zn-inhibitory functions on the Orai1-mediated SOCE in ESCC cells.This study reveals how dietary Zn inhibits SOCE and thus Zn and Ca2+ signals may cross-talk, which in turn regulates cancer cell proliferation and migration in esophageal epithelia. Further investigations are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2+ signaling in ESCC.

Background: Esophageal cancer is the 6th leading cause of cancer mortality worldwide with poor prognosis and 5-year survival rate as low as 18%. It is an unmet need to develop more effective chemotherapeutic drugs to improve the treatment outcome. We previously reported that the expression of Orai1, a calcium channel, was elevated in esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, and the high expression is associated with poor survival rate in patients. Orai1-mediated store-operated calcium entry (SOCE) has been demonstrated to contribute to cancer cell proliferation, migration, and invasion in ESCC and other cancers. RP4010 is a potent inhibitor for Orai1 channel (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. Here we evaluated the anticancer effect of RP4010 in cultured ESCC cells and xenograft animals. Materials and Method: Orai channel inhibitory function was analyzed in ESCC cells loaded with fluorescent calcium indicator Fura-2. The antiproliferative effect was evaluated by MTT assay in various human ESCC cell lines, e.g., KYSE-30, KYSE-150, KYSE-790, and KYSE-70. Quantitative real-time RT-PCR, Western blot, and flow cytometry were used to analyze the changes in cell cycle, changes in mRNAs and proteins of some key molecules in calcium signaling pathway. The antitumor effects of RP4010 were monitored in ESCC xenografted nude mice. Result: Compared to commonly used Orai1 channel blockers, such as BTP-2, RP4010 is more potent in blocking Orai1-mediated SOCE and cell proliferation in ESCC cells. The IC50 in cell viability analysis for RP4010 were calculated as 1.2, 1.4, 1.4, and 5.5 μM for KYSE-30, KYSE-70, KYSE-150, and KYSE-790, respectively, while IC50 for BTP-2 was 17.7-17.8 μM for these cells. Treatment of RP4010 (10 μM) halted intracellular calcium oscillations, inhibited calcium-dependent activation of Akt/NF-κB and ERK1/2 pathways, and resulted in cell cycles arrested at G0/G1 phase. Interestingly, RP4010 did not induce significant cell death even at rather high concentrations. In vivo study revealed that the mice tolerated daily I.P. injection of 20 mg/kg RP4010 without body weight loss or noticeable adverse effects. At this dose, RP4010 significantly reduced tumor growth compared to vehicle treated control group. Conclusion: R4010 is a novel potent inhibitor for Orai1-mediated SOCE with demonstrated potential in animal models of esophageal cancer. Data indicate a therapeutic potential of the molecule in ESCC. Citation Format: Chaochu Cui, Yan Chang, Xiaoli Zhang, Kumar V. Penmetsa, Srikant Viswanadha, Liwu Fu, Zui Pan. Targeting Orai1-mediated store-operated Ca2+ entry by a novel compound RP4010 for antiproliferative activity against esophagus squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B062.

Intracellular Ca2+ signals, including oscillations, regulate proliferation, migration, and other cellular events in cancer cells. Zinc (Zn), an essential micronutrient, has been studied for its chemopreventive effects in several cancers, including esophageal cancer. Although there is a growing research interest in the cross-talk between Ca2+ and Zn signaling, it remains elusive as to how Zn prevents tumor growth and whether Ca2+ signaling is involved. Our previous report demonstrated that Orai1, a store-operated Ca2+ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to normal tissues, and that the elevated expression of Orai1 is strongly associated with poor prognosis in patients. In the current study, we show that physiological levels of Zn can significantly suppress cell proliferation in KYSE-150, a human ESCC cell line. We also show that Zn is able to inhibit Orai1-mediated SOCE and intracellular Ca2+ oscillations, both which are known as proliferation signals. Based on the topology information of Orai1, we hypothesized that the histidine residue in linker region between transmembrane 1 and 2 of Orai1 (H113) as well as three cysteine residues may play a critical role in Zn-inhibitory effects. Using a point mutation approach, we exhibit that the Zn-inhibitory effects on both SOCE and cell proliferation are vanished in KYSE-150 cells containing Orai1 H113A mutant. Furthermore, KYSE-150 cells expressing Orai1 with any mutation in cysteine residues (C126A, C143A and C195A) display significant loss of Zn-inhibitory functions. These results indicate that the four amino acid residues are likely to be involved in Zn-inhibitory effects on SOCE and cell proliferation. Taken together, our data suggest that dietary Zn may inhibit Orai1-mediated SOCE and intracellular Ca2+ signaling, which in turn suppresses cell proliferation in ESCC. Further studies are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2+ signaling in ESCC. Citation Format: Sangyong Choi, Chaochu Cui, Yanhong Luo, Jae-Kyun Ko, Jianjie Ma, Liwu Fu, Irina Korichneva, Zui Pan. Zinc inhibits Orai1-mediated Ca2+ signals and proliferation in esophageal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2612.