Effects of zinc status on cell cycle and viability: An in vitro study in monocytes (THP-1) and muscle (Rhabdomyosarcoma) cells

Abstract

Monocyte and muscle cells are considered Zn reservoirs and sensitive to Zn fluctuations, especially in terms of viability. The current study aimed to understand the effect of Zn sufficiency and deficiency on THP-1 monocyte and rhabdomyosarcoma (RD) muscle cell lines. Zinc sufficiency was maintained by supplementing 25 µM of Zn, whereas varying degrees of deficiency were created with intracellular Zn chelator-TPEN in serum-free medium. Cell viability was assessed by MTT assay and the Zn deficiency effect on cell cycle stage was determined through flow cytometry analysis. Zn sufficiency has no-observable effect on cell viability, however, Zn deficiency has a significant positive (P < 0.05) effect on cell death. Cell-cycle analysis has shown a significant higher percentage of THP-1, and RD cells were arrested at Sub-G1 stage in zinc deficiency. Results suggest that cells have the tendency of adaptation to sub-optimal zinc depletion. Further, subnormal level of zinc affected THP-1 and RD cell viability by increasing the cell death at the Sub-G1 stage.

Introduction

Zinc plays a pivotal role in cell viability, proliferation, and differentiation as well as acts as a cofactor for many enzymes and hormones.[1] The adult human body contains ∼2–3 g of zinc, of which skeletal muscle is the major reservoir (60%), followed by bone (∼30%), liver, skin (∼5%), and other tissues (2%–3%).[2] Zinc deficiency (ZD) is a frequent dietary problem and accompanies many chronic diseases as a single element nutritional deficiency to understand changes at the cellular and molecular level.[3] The ZD is multifactorial, of which low dietary intake is predominant, wherein chronic diseases such as gastrointestinal disorders, diarrhea, renal disease, sickle cell anemia, cirrhosis, and cystic fibrosis lead to suboptimal zinc status.[4]

Above mentioned conditions ultimately lead to moderate to severe form of ZD thereby elevation of glucocorticoids, which in turn causes thymic atrophy, acceleration of apoptosis in thymocytes, and reduced lymphopoiesis.[5] ZD results in compromised immune response. In contrast, myeloid progenitor cells have showed enhanced cycling with a higher percentage of cells in S or G2/M noted for both mild ZD (MZD) (33%) and severe ZD (SZD) mice (56%) compared with the zinc-adequate mice.[6] Conversely, ZD in the neuroblastoma IMR-32 cells arrested the cell cycle at the G0/G1 phase, and induced apoptosis.[7]

Interestingly, cells of the immune system were able to adopt to the stress of suboptimal zinc levels, with the changes in gene expression for cytokines, DNA repair enzymes, and signaling molecules.[8] Endogenous zinc levels appear to be critical to inducing autophagy under conditions of oxidative stress. Autophagy is a necessary preceding event for lysosomal membrane permeabilization and cell death in oxidative injury.[9] Zinc depletion or chelation of intracellular Zn by agents like N,N,N',N'-Tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) blocked both vacuole formation and zinc accumulation in the vacuole and inhibited lysosomal activation and lysosomal membrane permeabilization.[10]

Further, zinc depletion in fibroblasts and neuroblastoma cells leads to apoptosis. In contrast, high levels of zinc with exogenous zinc supplementation inhibited prostate cancer initiation and/or progression through cell cycle arrest, programmed cell death, or necrosis.[11] The role of zinc in chemotherapy revealed that zinc supplementation sensitized prostate cancer cells to paclitaxel-induced apoptosis. Conversely, this effect of paclitaxel was reduced at lower zinc levels (~8 μM).[11] It is well known that the ZnT-1 transporter plays key role in zinc trafficking across the cell, any changes in zinc status affect the zinc levels via ZnT-1 and also cellular physiology. However, very scanty literature is available on the zinc depletion effect on cell proliferation, and cell viability. In view of the above-cited information, the current study aimed to understand the zinc sufficiency and subnormal zinc status on cell cycle fate and cell viability.

Materials and Methods

Human monocyte – THP-1 and muscle cell-rhabdo myosarcoma (RD) were obtained from ATCC, USA. Cell culture medium, RPMI-1640 and DMEM, FBS, L-Glutamine solution, Tryphan blue, TPEN, and MTT were obtained from Sigma-Aldrich, Bangalore, India. Antibiotic–antimycotic solution, sterile disposable cultureware was obtained from Life Technologies, California, USA.

THP-1 & RD cell culture and maintenance

THP-1 and RD cells were cultured in RPMI-1640 and DMEM medium containing 10% FBS, 1% glutamax, and 1% antibiotic–antimycotic. Cells were maintained in a humidified chamber at 37°C with 5% CO₂ in the logarithmic growth phase.

Cell viability

Cells in the logarithmic growth phase at a concentration ~ 10⁵–10⁶ cells/mL were used for experiments. Cell viability was assessed by the dye exclusion method. Briefly, cells were harvested, washed with PBS, and then cells were diluted at 1:10 using 0.4% Trypan Blue solution. The cells were incubated for 1–2 min at room temperature and enumerated the viable cells at 10x magnification (EVOS FL Imaging system).

Pre-treatment preparation of cells

The treatment procedures of either incubation with Zn for sufficiency or with TPEN for deficiency were undertaken in serum-free medium condition. THP-1 cells in suspension were counted approximately 0.9 × 10⁶ cells/mL and were seeded in T25 flasks for the cell cycle experiments and for MTT assay ~ 10,000 cells in 96 well plate for 4 h. Adherent RD cells were trypsinized and centrifuged at 1100 rpm for 3–4 min and pellet was collected. Cells ~ 0.7 × 10⁶ cells/mL were seeded in 6 well plates for the cell cycle experiment and ~ 10,000 cells in 96 well plate for MTT assay. Cells in plates were shifted to serum-free medium for 4 h. THP-1 and RD cells maintained in “serum-free medium” for 4 h, without exposure to either Zn or TPEN were used as control.

Zinc supplementation and depletion (TPEN) treatments

Zinc sufficiency conditions were created by exposing the THP-1 and RD cells with 25 µM of zinc as zinc sulfate complexed with bovine serum albumin (BSA) in serum-free medium for 4 h.[12] A range of ZD was created by treating THP-1 and RD cells with increasing concentrations of TPEN (2.5–15 µM), as an intracellular zinc chelator, in serum-free media of 4 h. The vehicle controls were BSA for zinc sufficiency and dimethyl sulfoxide (DMSO) for ZD (as TPEN solubilized in DMSO). Each treatment was conducted in 3 separate independent experiments.

Quantification of viable cells by MTT assay

The viability of THP-1 and RD cells exposed to Zn (25 µM) and TPEN (2.5–15 μM) were determined by Mosmans’s MTT assay as per manufacturer’s protocol. Briefly, ~10,000 cells from each treatment were transferred to 96 well plate, then added the MTT solution (per well 10 µl at a concentration of 5 mg/mL) was followed by incubation of 96 well plate in the dark for 4 h at room temperature.[13] Colored reaction product solubilized in DMSO and read at 570 nm (Synergy HT, Winooski, USA).

Cell cycle analysis by flow cytometry

Posttreatment of Zn sufficiency and depletion experiments, cells were suspended in PBS (cold), followed by fixing in methanol (absolute)/not < 30 min. Cells were subsequently washed with 0.5% Tween-20 in PBS and 2% FCS in PBS. Then, cells were re-suspended in PBS with 2% FCS containing 40 mg/mL RNase-A and incubated for 20 min at 37°C. Finally, cells were washed with 2% FCS in PBS and re-suspended in PBS containing Propidium Iodide (20 mg/mL) to stain the nucleus. PI fluorescence (relative DNA content per cell) was measured by a flow cytometer (BD FACS Aria-II, New Jersey, U.S.A). The flowjo10.7 software (Ashland, Oregon, U.S.A) was employed to assess cell cycle distributions viz. sub-G1, G1, S, and G2/M fractions considering DNA content i.e., 2N and 4N DNA content per cell. Whereas the cells with ˂2N DNA content were considered apoptotic cells.

Statistical analysis

Statistical analysis was performed by one-way ANOVA, post hoc Dunnetts using IBM SPSS Software (Version 22). Values are expressed as mean ± standard error of n = 3 (independent experiments) values. Significance levels were considered at P ˂ 0.05.

Results

Zinc and TPEN effect on cell viability

The viability of THP-1 and RD cells treated with zinc and TPEN for 4 h in serum-free media were given in Figure 1. “No remarkable cytotoxic effects” were noted upon exposure to 25 μM zinc sulfate [Figure 1]. Similarly, no obvious changes in viability of THP-1 and RD cells were noted upon treatment with 2.5 µM and 5 μM TPEN when compared to control. However, a significant (P < 0.05) increase in cytotoxic effect was noted upon higher concentrations of TPEN at 10 μM and 15 μM with the viability of 33% and 9.2% in THP-1 cells, whereas 47.19% and 9.2%, respectively, compared to control [Figure 1].

Figure 1.

Cell viability – MTT assay. Cell Viability was determined by MTT assay. THP-1 and Rhabdomyosarcoma cells were incubated with the indicated concentration of either zinc or TPEN in a serum-free medium for 4 h. Results are expressed as percentage viability, with 100% viability of control cells. Data sets are mean ± standard error of n = 3 independent experiments. Different superscripts are significantly different at P < 0.05

Zinc deprivation on cell cycle progression in THP-1 and Rhabdomyosarcoma cells

The data of cell cycle stages of human leukocytic monocyte (THP-1) cells treated with TPEN (2.5–15 μM) for 4 h were given in Figure 2. THP-1 cells were arrested in the G1 phase compared with control and the number of cells decrease in the S phase with the extent of zinc depletion from 2.5 to 15 μM TPEN [Figure 2]. A low dose of TPEN (2.5 μM) had arrested 57% of cells in the S stage [Figure 2b], whereas 5 µM TPEN resulted in the arrest of 45% of cells in the S stage [Figure 2c] compared with the control [Figure 2a]. Further higher concentrations of TPEN (10 and 15 µM) have not shown dose-dependent increase, except 15% and 13% of cells at the S stage compared with controls [Figure 2d and e].

Figure 2.

Cell cycle histogram of THP-1 cells. The cell cycle distribution pattern of THP-1, control cells (a) and subjected to zinc depletion, i.e., treatment with varying concentrations of TPEN viz. 2.5 μM (b), 5.0 µM (c), 10 µM (d), and 15 μM (e) followed by cell cycle analysis through flow-cytometry with propidium iodide. The numbers in each panel are the percentage of cells at each stage of treatment

Cell cycle stages of human RD cells treated with TPEN (2.5–15 μM) for 4 h were given in Figure 3. RD cells were arrested in the G1 phase compared with control and the number of cells increased in the G1 phase with the extent of zinc depletion from 2.5 to 15 μM TPEN. A low dose of TPEN (2.5 μM) had arrested 48% of cells in the G1 stage [Figure 3b], whereas 5 µM TPEN resulted in the arrest of 56% of cells in the G1 stage [Figure 3c] compared with control [Figure 3a]. Further higher concentrations of TPEN (10 and 15 µM) had not shown dose-dependent increase except in 56% of cells at the G1 stage compared with controls [Figure 3d and e].

Figure 3.

Cell cycle histogram of rhabdomyosarcoma (RD) cells. The cell cycle distribution pattern of RD, control cells (a) and subjected to zinc depletion, i.e., treatment with varying concentrations of TPEN viz. 2.5 µM (b), 5.0 µM (c), 10 µM (d) and 15 µM (e) followed by cell cycle analysis through flow-cytometry with propidium iodide. The numbers in each panel are the percentage of cells at each stage of treatment

Discussion

The current study is more related to acute zinc depletion through intracellular Zn chelator (TPEN) and investigated the cell viability and proliferation. It has been postulated that the imbalance of the cell cycle signals or failure to arrest the cell cycle may trigger the apoptotic program.[14] Cells respond to DNA damage by arresting the cell cycle at the G1 or G2 phase, a process that allows the time for repairing the DNA lesions.[15] Present study results confirm an association between the degree of Zn deficiency and stage of cell arrest, wherein low and higher percentages of THP-1 and RD cells accumulated in S and G1 stages with increased concentration of TPEN. Notably, at a higher dose of TPEN (15 µM) ~13% of THP-1 cells were arrested in the S phase, while about 56% of cells were arrested in the G1 stage compared to the control.

It is concurrent that severe forms of zinc depletion (10 and 15 µM TPEN) affected the cell viability of both THP-1 and RD cells. The contrasting results of MTT and cell cycle assay in cells could be speculated that MZD (low dose of TPEN) leads to the arrest of cells in the G1 stage, thus initiating repairing process, whereas SZD (higher concentrations of TPEN) resulted in impaired cell repairing (DNA) process, thereby increased cell death, noted through MTT assay.

It has been well known that p21 overexpression in cells, induces G1, G2, or S phase arrest.[16] Whereas p-21-deficient cells display a defect in DNA damage-induced G1 and G2 arrest.[17] Notably, the stabilization of p-21 prevents apoptosis by inducing cell cycle arrest thereby interacting with caspase-3.[18] Nonetheless, mild DNA damage further evident from MTT assay at lower concentrations of TPEN (2.5 and 5 µM) suggests that cells expressed p-21 protein which halted the cell cycle progression at G1 stage thereby repairing the damage and prevented cell death. One possibility is that p21 may bring its associated active cyclins CDK complexes to the repair sites through its interaction with PCNA.

It was well-established fact that at a higher degree of DNA damage (under un-repairable condition), p-21 protein undergoes cleavage thus triggering the apoptosis.[19] Our previous published research on SaOS-2 cells with TPEN has showed contrasting effect on cell death where a mild dose of 5 µM TPEN zinc depletion triggered cell death.[13] Studies on SaOS-2 and THP-1 showed that zinc transporters and metallothionein play key role in maintaining the zinc levels in cellular levels and in turn regulate the viability.[20] Current study results are in line with this phenomenon wherein THP-1 and RD cells treated with high dose TPEN (10 and 15 µM) had created an irreparable damage hence cells cleaved or degraded the p21 levels as it prevents the cell cycle progression and make it a way for cell repair. The limitations of the present study are the lack of data on p21 degradation/caspase activity and levels for severe form of zinc depletion.

In summary THP-1 and RD cells treated with TPEN, an intracellular zinc chelator, resulted in cell cycle arrest at S and G1 phase and further higher doses of TPEN-induced apoptosis. Whereas mild zinc depletion has no effect on cell viability.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.