Stimulation of calcium influx and CK1α by NF-κB antagonist [6]-Gingerol reprograms red blood cell longevity

Hassan S. Alamri1,2 | Jawaher Alsughayyir3 | Maaged Akiel1,2,4 | Yazeed A. Al-Sheikh5 | Ahmed M. Basudan5 | Ayed Dera6,7| Tlili Barhoumi8,9 | Abdulrahman M. Basuwdan10 | Mohammad A. Alfhili5

Abstract

Chemotherapy-induced anemia (CIA) is a major obstacle in cancer management. Although the mechanisms governing CIA are poorly understood, recent efforts have identified suicidal erythrocyte (red blood cell, RBC) death as a possible cause of CIA. [6]-Gingerol (GNG), a polyphenol extracted from Zingiber officinale plant, ex- hibits a wide array of biological activities including antimicrobial, antioxidant, anti- inflammatory, immunomodulatory, and anticancer activities, in vitro and in vivo. However, the potential toxicity of GNG to human RBCs remains unexplored. RBCs from heparinized blood were isolated by centrifugation and exposed to antitumor concentrations (10–100 µM) of GNG for 24 hr at 37°C. Hemolysis was calculated from hemoglobin leakage in the supernatant (λmax = 405 nm), while cytofluorometric analysis of eryptosis employed Annexin-V-FITC to detect phosphatidylserine (PS) exposure, forward scatter (FSC) to estimate cell volume, Fluo4/AM to measure cal- cium activity, and H2DCFDA to assess oxidative stress. Moreover, zVAD(OMe)-FMK, SB203580, necrostatin-2, staurosporin, and D4476 were used to identify signaling pathways responsive to GNG. GNG induced significant hemolysis at 100 µM, inde- pendently of extracellular calcium, and increased Annexin-V-FITC fluorescence that was thoroughly abrogated without extracellular calcium. GNG also enhanced Fluo4 fluorescence and reduced FSC, but had no significant effect on DCF fluorescence. Importantly, the presence of D4476 significantly attenuated GNG-induced hemol- ysis. In conclusion, GNG stimulates premature RBC death characterized by loss of membrane asymmetry, elevated cytosolic calcium, cell shrinkage, and casein kinase 1α activation. Blocking the activity of calcium channels or CK1α may, therefore, ame- liorate the toxic effects of GNG on RBCs.

Practical applications

This report presents a safety assessment of GNG as a chemotherapeutic agent and highlights the novel toxicity of GNG to human RBCs. Our findings provide novel insights that may lead to more efficient utilization of GNG in chemotherapy. Specifically, our data revealed the involvement of calcium channels and casein kinase 1α in mediating GNG-induced premature RBC death, and, therefore, inverse agonists

1 | INTRODUC TION

It has been estimated that over 60% of chemotherapeutic agents used for the treatment of cancer are derived from natural sources (Demain & Vaishnav, 2011; Fridlender et al., 2015). 6-Gingerol (5-hydroxy-1-(4- hydroxy-3-methoxyphenyl)-3-decanone; GNG) is a compound isolated from the rhizome of ginger (Zingiber officinale Rosc), which possesses a wide array of bioactive properties. In particular, GNG has been shown to exhibit antibacterial (Saha et al., 2013), anti-inflammatory (Chang & Kuo, 2015; Kim, Kundu, et al., 2005), antioxidant (Hegazy et al., 2016), and antiangiogenic (Kim, Min, et al., 2005) activities. More importantly, GNG induces cell death and apoptosis in a variety of tumors including leukemia (Lee & Surh, 1998), colon (Lee et al., 2008; Lin et al., 2012; Radhakrishnan et al., 2014), pancreatic (Park et al., 2006), oral, cervical (Kapoor et al., 2016), osteosarcoma (Fan et al., 2015), and hepatic tu- mors (Yang et al., 2010).
At least 75% of cancer patients develop anemia during treatment due to drug interactions (Visweshwar et al., 2018). Anemia may arise from programmed red blood cell (RBC; erythrocyte) death known as eryptosis (Lang et al., 2017). Cardinal signs of eryptotic RBCs include phosphatidylserine (PS) exposure, cell shrinkage, dysregu- lated ion influx, oxidative stress, and ceramide abundance (Pretorius et al., 2016). Moreover, dying RBCs have been demonstrated to activate an intricate signaling cascade involving classical cell death and survival mediators, such as caspase, p38 mitogen-activated protein kinase (MAPK), and casein kinase (CK) (Lang & Lang, 2015). Necroptosis also exists in RBCs and is under the regulation of recep- tor-interacting protein-1 and −3 (RIPs) (Alfhili et al., 2019).
To date, little is known about the toxicity of GNG to human RBCs, and the current study aims to evaluate its safety as a che- motherapeutic agent (Baliga et al., 2011). Our data show that GNG, at antitumor concentrations, induces hemolysis and eryptosis ac- companied by loss of membrane asymmetry, increased intracellular calcium, and cell shrinkage. Moreover, GNG-induced eryptosis, but not hemolysis, is abolished following the removal of extracellular calcium, and the cytotoxic activity of GNG against erythrocytes is regulated by CK1α.

2 | MATERIAL S AND METHODS

2.1 | Blood collection and RBC isolation

The study protocol was approved by the Institutional Review Board of King Saud University (Project No. E-20-4544). Blood samples from consented, healthy adults were obtained by venipuncture in lithium heparin vacutainer tubes, and erythrocytes were isolated by centrifu- gation and resuspended in phosphate-buffered saline (PBS).

2.2 | Chemicals and reagents

GNG (purity: 99.54%) was obtained from Solarbio Life Science (Beijing, China), and a 100 mM stock solution was prepared by dissolving 5 mg of GNG in 0.169 ml of dimethylsulfoxide (DMSO). Exposure was car- ried out at 5% hematocrit for 24 hr at 37°C in presence and absence of vehicle (0.1% DMSO) or 10–100 µM of GNG. PBS, Hank’s balanced salt solution (HBSS), calcium-free HBSS, Annexin-V-FITC, Fluo4/AM, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), zVAD(OMe)- FMK (zVAD), SB203580 (SB), D4476, staurosporin (StSp), and necrostatin-2 (Nec-2) were obtained from Solarbio. All dyes and inhib- itors were dissolved in DMSO to prepare stock solutions of 100 mM.

2.3 | Hemolysis

Control and experimental cells were pelleted by centrifugation at 13,300 ×g for 1 min at 20°C and the supernatant was assayed for hemoglobin content by photometric measurement of absorbance at 405 nm. Cells suspended in distilled water served as positive control.

2.4 | Phosphatidylserine exposure

A homogeneous 50 μl aliquot of control and treated cells was washed and resuspended in 150 μl of 1% Annexin staining solution and incu- bated at room temperature in the dark for 10 min. Cells were excited with 488 nm light and emitted FITC fluorescence was captured at 520 nm.

2.5 | Intracellular calcium

A homogeneous 50 μl aliquot of control and treated cells was washed and resuspended in 150 μl of 5 µM Fluo4 staining solution (154 mM NaCl and 5 mM CaCl2 buffer) and incubated at 37°C in the dark for 30 min. Following repeated washing to remove excess dye, cells were analyzed for Fluo4 intensity by excitation at 488 nm and capture of emitted light at 520 nm.

2.6 | Oxidative stress

A homogeneous 50 μl aliquot of control and treated cells was washed and resuspended in 150 μl of 10 µM H2DCFDA staining solution and incubated at 37°C in the dark for 30 min. Cells were washed twice and DCF fluorescence was measured at 488/521 nm Ex/Em wave- lengths, respectively.

2.7 | Statistical analysis

Data are reported as means ± SEM. In each experiment, autologous cells served as control. Multiple means were analyzed by one-way ANOVA followed by Tukey’s post hoc range test, and a cutoff P value of < .05 was used for statistical significance.

3 | RESULTS

3.1 | GNG induces hemolysis

Cell-free hemoglobin, leaked from physically damaged and hemolyzed RBCs, precipitates inflammatory and oxidative damage in various organs. To assess the hemolytic potential of GNG, cells were incubated with 0.1% DMSO (vehicle control) or 10–100 µM of GNG in HBSS for 24 hr at 37°C, and hemolysis was measured by absorbance at 405 nm relative to that of cells in distilled water. As shown in Figure 1b, although GNG induces hemolysis at 10 µM (5.1% vs. 11.6%) and 50 µM (5.1% vs. 17.2%), statistically significant levels were only observed at 100 µM (5.1% vs. 71.5%). Next, we determined the role of extracellular calcium influx in GNG-induced hemolysis. Control and experimental cells (100 µM of GNG) were incubated in HBSS and calcium-free HBSS for 24 hr at 37°C and hemoglobin was again assayed. Figure 1c demonstrates that while GNG in presence of extracellular calcium caused significant hemoly- sis (5.0% vs. 66.6%), exclusion of calcium had no significant effect on hemolysis (6.3% vs. 62.2%). Therefore, GNG-induced hemolysis is apparently not mediated through calcium influx through calcium channels from the extracellular space.

3.2 | GNG stimulates PS exposure

Eryptotic cells lose membrane asymmetry as PS is translocated to the outer membrane leaflet. We, thus, used Annexin-V-FITC to iden- tify the dead cells by flow cytometry. Our results show that 10 µM of GNG had no significant effect on Annexin-V binding (4.0% vs. 4.6%), whereas 50 µM (4.0% vs. 9.1%) and 100 µM (4.0% vs. 16.3%) significantly increased the percentage of cells with enhanced PS ex- ternalization (Figure 2). As with hemolysis, it was also of interest to examine the participation of extracellular calcium in GNG-induced PS exposure. Cells were exposed to the vehicle control or to 100 µM of GNG in HBSS and calcium-free HBSS, and Annexin-V binding was then determined. In contrast to what we observed in GNG-induced hemolysis in Figure 1c, Figure 5c demonstrates that PS exposure in presence of extracellular calcium (3.8% vs. 15.9%) was completely abrogated when extracellular calcium was removed from the me- dium (1.2% vs. 3.2%). Taken together, these data suggest that GNG stimulates eryptosis in a calcium-dependent fashion.

3.3 | GNG increases intracellular calcium

Based on the established role of calcium in regulating cell survival, and in light of the role of calcium in mediating GNG-induced membrane scrambling, it was necessary to examine possible alterations in intracel- lular calcium levels. To this end, cells were incubated for 24 hr at 37°C in presence and absence of 10–100 µM of GNG and then, stained with 5 µM of Fluo4/AM as described earlier. Figure 3d shows that Fluo4 fluorescence was not significantly increased by GNG at 10 µM (163.5 vs. 187.5) or 50 µM (163.5 vs. 237.6), and only exhibited significant el- evations at 100 µM (163.5 vs. 394.5). Similarly, the percentage of cells with enhanced Fluo4 fluorescence (Figure 3e) was not significantly af- fected by GNG at 10 µM (2.4% vs. 3.2%) and 50 µM (2.4% vs. 4.8%), but only at 100 µM (2.4% vs. 9.7%). GNG, therefore, stimulates eryptosis by increasing the intracellular calcium content.

3.4 | GNG promotes cell shrinkage

The activity of Gardos channels (calcium-responsive potassium channels) is regulated by intracellular calcium levels. Under condi- tions of calcium overload, these channels mediate excessive potas- sium chloride and water efflux leading to cell shrinkage (Pretorius et al., 2016). To investigate the effect of calcium increase on cell size, control and GNG-treated cells were subjected to FSC analysis. As is the case with Fluo4 fluorescence, Figure 4d shows that GNG only significantly reduced FSC at 100 µM (115.8 vs. 108.0), but not at 10 µM (115.8 vs. 117.0) or 50 µM (115.8 vs. 115.8). A dot plot analy- sis of the results (Figure 4e and f) relates the diminished FSC inten- sity to the enhanced Fluo4 fluorescence in upon GNG treatment. Altogether, it is discerned that GNG-induced eryptosis is associated with significant cell shrinkage following calcium overload.

3.5 | GNG does not cause oxidative stress

A profound increase in reactive oxygen species (ROS) predisposes the cell to eryptosis. Thus, we tested whether GNG-induced cell death is related to oxidative damage. Following incubation with or without 10–100 µM GNG for 24 hr at 37°C, cells were labeled with 10 µM of H2DCFDA to measure the ROS levels. As depicted in Figure 6d, GNG did not significantly increase DCF fluorescence at all concentrations tested (209.8 vs. 134.1, 10 µM; 181.6, 50 µM; and 193.8, 100 µM) and, accordingly, GNG-induced eryptosis does not seem to be mediated through oxidative stress.

3.6 | CK1α is essential to GNG-induced hemolysis

A number of pathways have been identified as regulators of eryth- rocyte death and survival. Using small molecule inhibitors, we blocked distinct signal transduction pathways and evaluated the ef- ficacy of 100 µM of GNG in inducing hemolysis. In Figure 7a, the hemolytic activity of GNG was not significantly reduced in absence or presence of caspase inhibitor zVAD (68.6% vs. 71.1%), and the role of caspases in GNG-induced hemolysis is, therefore, excluded. Likewise, Figure 7b demonstrates the lack of p38 MAPK participa- tion in GNG-induced hemolysis as evident from hemolytic values without and with SB (58.8% vs. 55.1%). Also, RIP1 is a recently rec- ognized modulator of both eryptosis and necroptosis (31,078,025), and, as shown in Figure 7c, RIP1 inhibition by Nec-2 did not signifi- cantly decrease GNG-induced hemolysis (65.8% vs. 59.6%); an ob- servation that rules out RIP1-mediated mechanisms. Furthermore, blocking PKC activity with StSp was not effective in significantly at- tenuating GNG-induced hemolysis (64.8% vs. 66.2%). However, we observed a significant decrease in hemolysis following GNG treat- ment without and with D4476 (72.6% vs. 56.3%). This identifies CK1α as a molecular target of GNG in erythrocytes.

4 | DISCUSSION

GNG exhibits a wide array of biological activities and remains a widely consumed dietary plant in traditional medicine. In light of promising anticancer potential, GNG is a candidate for chemother- apy. The current study describes a serious side effect of GNG ex- posure, which is the reprogramming of cellular longevity of human RBCs. We show, for the first time, that GNG elicits hemolysis and eryptosis at concentrations reported to inhibit tumor progression (10–50 µM). These observations, therefore, may inform prospective applications of GNG in cancer management.
Our data revealed that GNG triggers profound hemoly- sis (Figure 1b) independently of calcium availability (Figure 1c). Interestingly, this is in contrast to findings reported by Lam et al. in which GNG rather protected rat RBCs from 2,2'-azobis(2-amid- inopropane) hydrochloride-induced hemolysis (Lam et al., 2007). This discrepancy is possibly attributed to inter-species differences between rodent and man. Of note, we have recently shown that tri- closan, another phenolic compound, similarly induces hemolysis and eryptosis in human RBCs (Alfhili, et al., 2019). Extracellular hemo- globin undergoes autoxidation leading to excessive accumulation of ROS and severe tissue damage (Alfhili et al., 2019). It has also been demonstrated that hemoglobin leakage and subsequent degradation generates damage-associated molecular patterns (DAMPs), most notably heme, that promote an inflammatory immune response (Jeney, 2018). Furthermore, hemoglobin exhausts the nitric oxide (NO) reservoir responsible for the maintenance of vascular tone (Bozza & Jeney, 2020).
Our investigations also showed that GNG significantly increases the percentage of eryptotic cells (Figure 2) as evident from Annexin-V binding (Figure 2). PS externalization to the outer membrane leaflet serves as a binding site for circulating phagocytes, which eliminate dead RBCs before hemolysis ensues. An important consequence of eryptosis is the lack of elasticity and deformability of dead RBCs; an effect which predisposes to microvascular occlusion and thrombo- sis (Pretorius, 2018). Interestingly, PS exposure, but not hemolysis, was thoroughly abolished under conditions of extracellular calcium deprivation (Figure 5), which suggests the involvement of calcium channels in GNG-induced eryptosis. Accordingly, blocking calcium channel activity may ameliorate the pro-eryptotic potential of GNG. A similar behavior was noted for taurolidine (Fink et al., 2018), which also mirrors the lack of oxidative stress observed in the current study (Figure 6). This is in congruence with the antioxidant activity of GNG observed in rodents (Han et al., 2020; Hegazy et al., 2016), but con- tradicts the oxidative damage seen in cancer cells (Lin et al., 2012; Yang et al., 2010). It seems likely that GNG may exhibit differential interaction dynamics with the antioxidant system in normal and can- cer cells in both rodent and man.
Eryptosis is also regulated by calcium activity as it controls cytoskeleton proteins, membrane lipids, redox state, caspases, and ion trafficking. GNG is shown to elevate intracellular calcium (Figure 3), a distinctive feature of aged erythrocytes (Pretorius et al., 2016). Under physiological conditions, the energy required to pump cal- cium out of the cell is, in part, supplied by ATP hydrolysis (Pasini et al., 2006). Thus, GNG may deplete ATP stores; a recognized trigger of eryptosis (Pretorius et al., 2016). Notably, GNG-induced calcium elevations similarly commensurate with reduced cellular volume (Figure 4e and f), suggesting potassium Staurosporine and water efflux through calcium-dependent potassium channels (Pretorius et al., 2016).
GNG has previously been reported to influence the activity of multiple signaling molecules, including caspases (Radhakrishnan et al., 2014), p38 MAPK (Kim, Kundu, et al., 2005), and PKC (Lee et al., 2008). In our study, we sought to identify the signaling path- ways responsive to GNG using small molecule inhibitors. In contrast to the activity of GNG in nucleated cells, inhibition of caspases, p38 MAPK, PKC, and RIP1 does not appreciably attenuate the cytotoxicity of GNG (Figure 7a-d). Nevertheless, significant reduction in hemolysis was observed in D4476-treated cells (Figure 7e). CK1α is essential for eryptosis of oxidatively damaged and energy depleted erythrocytes (Zelenak et al., 2012), but, to the best of our knowledge, remains to be recognized as a molecular target of GNG in nucleated cells.
In conclusion, this report identifies GNG as a novel stimulator of premature cell death of erythrocytes, and describes the involvement of calcium signaling in the breakdown of membrane asymmetry, cell shrinkage, and ensuing hemolysis. These detrimental effects of GNG may be overridden by either blocking calcium channels or inhibiting CK1α enzymatic activity.

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