Calcium and ROS: A mutual interplay

AbstractCalcium is an important second messenger involved in intra- and extracellular signaling cascades and plays an essential role in cell life and death decisions. The Ca2+ signaling network works in many different ways to regulate cellular processes that function over a wide dynamic range due to the action of buffers, pumps and exchangers on the plasma membrane as well as in internal stores. Calcium signaling pathways interact with other cellular signaling systems such as reactive oxygen species (ROS). Although initially considered to be potentially detrimental byproducts of aerobic metabolism, it is now clear that ROS generated in sub-toxic levels by different intracellular systems act as signaling molecules involved in various cellular processes including growth and cell death. Increasing evidence suggests a mutual interplay between calcium and ROS signaling systems which seems to have important implications for fine tuning cellular signaling networks. However, dysfunction in either of the systems might affect the other system thus potentiating harmful effects which might contribute to the pathogenesis of various disorders

Sulforaphane reduces molecular response to hypoxia in ovarian tumor cells independently of their resistance to chemotherapy.

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1alpha protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules

Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1

Melatonin-Induced Changes in Cytosolic Calcium Might be Responsible for Apoptosis Induction in Tumour Cells

Background/Aims: Melatonin is a hormone transferring information about duration of darkness to the organism and is known to modulate several signaling pathways in the cells, e.g. generation of endoplasmic reticulum stress, oxidative status of the cells, etc. Melatonin has been shown to exert antiproliferative and cytotoxic effects on various human cancers. We proposed that this hormone can differently affect tumour cells and healthy cells. Methods: We compared the effect of 24 h melatonin treatment on calcium transport (by fluorescent probes FLUO-3AM and Rhod-5N), ER stress (determined as changes in the expression of CHOP, XBP1 and fluorescently, using Thioflavin T), ROS formation (by CellROX® Green/Orange Reagent) and apoptosis induction (by Annexin-V-FLUOS/propidiumiodide) in two tumour cell lines – ovarian cancer cell line A2780 and stable cell line DLD1 derived from colorectal carcinoma, with non-tumour endothelial cell line EA.hy926. Results: Melatonin increased apoptosis in both tumour cell lines more than twice, while in EA.hy926 cells the apoptosis was increased only by 30%. As determined by silencing with appropriate siRNAs, both, type 1 sodium/calcium exchanger and type 1 IP3 receptor are involved in the apoptosis induction. Antioxidant properties of melatonin were significantly increased in EA.hy926 cells, while in tumour cell lines this effect was much weaker. Conclusion: Taken together, melatonin has different antioxidative effects on tumour cells compared to non-tumour ones; it also differs in the ability to induce apoptosis through the type 1 sodium/calcium exchanger, and type 1 IP3 receptor. Different targeting of calcium transport systems in tumour and normal, non-tumour cells is suggested as a key mechanism how melatonin can exert its anticancer effects. Therefore, it might have a potential as a novel therapeutic implication in cancer treatment

Endogenous H2S producing enzymes are involved in apoptosis induction in clear cell renal cell carcinoma

Abstract Background Knowledge about the expression and thus a role of enzymes that produce endogenous H2S - cystathionine-β-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase - in renal tumors is still controversial. In this study we aimed to determine the expression of these enzymes relatively to the expression in unaffected part of kidney from the same patient and to found relation of these changes to apoptosis. To evaluate patient’s samples, microarray and immunohistochemistry was used. Methods To determine the physiological importance, we used RCC4 stable cell line derived from clear cell renal cell carcinoma, where apoptosis induction by a mixture of five chemotherapeutics with/without silencing of H2S-producing enzymes was detected. Immunofluorescence was used to determine each enzyme in the cells. Results In clear cell renal cell carcinomas, expression of H2S-producing enzymes was mostly decreased compared to a part of kidney that was distal from the tumor. To evaluate a potential role of H2S-producing enzymes in the apoptosis induction, we used RCC4 stable cell line. We have found that silencing of cystathionine-β-synthase and cystathionine γ-lyase prevented induction of apoptosis. Immunofluorescence staining clearly showed that these enzymes were upregulated during apoptosis in RCC4 cells. Conclusion Based on these results we concluded that in clear cell renal cell carcinoma, reduced expression of the H2S-producing enzymes, mainly cystathionine γ-lyase, might contribute to a resistance to the induction of apoptosis. Increased production of the endogenous H2S, or donation from the external sources might be of a therapeutic importance in these tumors