Nicotinamide Mononucleotide Versus Nicotinamide Riboside in The Protective Effects of Cisplatin-induced DNA Damage in HeLa Cells

doi:10.21203/rs.3.rs-3177159/v1

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Nicotinamide Mononucleotide Versus Nicotinamide Riboside in The Protective Effects of Cisplatin-induced DNA Damage in HeLa Cells

https://doi.org/10.21203/rs.3.rs-3177159/v1

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Background Previous studies have shown that the nicotinamide adenine dinucleotide (NAD⁺) precursors, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), protected against endogenously or exogenously induced DNA damage, however, whether the two compounds have the same or different efficacy against DNA damage is not clear. In the current study, we systematically compared the effects of NMN and NR on cisplatin-induced DNA damage in HeLa cells.

Methods The viability of cisplatin treated HeLa cells with NMN or NR were tested by Trypan blue staining. NMN and NR were added in cells before or after exposed to cisplatin, respectively. Briefly, HeLa cells were pretreated with series doses of NMN or NR (0、0.625、1.25、2.5、5 and 10 mM) for 12 h, and then challenged with 10 µM of cisplatin for the following 12 h; or, HeLa cells were treated by 10 µM of cispaltin for 12 h, and then cultured in medium with 10mM NMN or NR, the cells were harvested at 0, 8, 16, 24 and 32 h later. The DNA damage were assessed by immunofluorescent against phosphor-H2AX (γH2AX) and alkaline comet assay. The intracellular nicotinamide adenine dinucleotide (NAD+) and reactive oxygen species (ROS) were determined by according kit.

Results

Both NMN and NR could rescued cisplatin-induced cell death in a dose-dependent manner comparably. NMN and NR pretreatment decreased γH2AX levels and shortened comet tail length in a dose-dependent manner, while NR pretreatment exhibiting stronger protective effects than NMN. Although the post-cisplatin administration of NMN and NR also exhibited a protective effect against DNA damage, there were no significant differences between the two compounds. In addition, both NMN and NR can reverse the cisplatin-induced decrease of NAD⁺ and the generation of ROS, also with no significant difference between them.

Conclusion NR is more effective than NMN in maintaining DNA integrity in cisplatin-treated cells.

nicotinamide mononucleotide

nicotinamide riboside

DNA damage

cisplatin

reactive oxygen species

DNA damage that continuously happens in a living organism can impact health and modulate disease-states (4, 19, 26), while DNA damage response (DDR) dutifully protects the DNA by either removing or tolerating the damage (5, 20), thus ensuring the overall survival of the organism. Nicotinamide adenine dinucleotide (NAD⁺) is a co-substrate for NAD⁺ consuming enzymes, including Poly ADP-ribose polymerases (PARPs) and Sirtuins (Sirt), which are important players in DDR (22, 24, 1). Interestingly, it has been reported that NAD⁺ levels decrease while DNA damage levels increase during aging (17). Therefore, methods for restoring NAD⁺ levels have been explored, such as the supplementation of NAD⁺ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), which has been verified as an effective measure to combat DNA damage and improve certain disease-states of aging such as Alzheimer's disease (AD) in mice (2, 9). Currently, NMN and NR are marketed as food supplements in the United States and other countries that claim to improve glucose control, enhance energy metabolism, and reverse metabolic complications associated with aging.

There are three pathways for the biosynthesis of NAD⁺ in cells, namely, the de novo biosynthesis pathway, Preiss-Handler pathway, and salvage pathway (28, 3). The salvage pathway is the primary source of NAD⁺ in mammalian cells, in which nicotinamide (NAM) is converted to NMN under the catalysis of nicotinamide phosphoribosyltransferase (NAMPT), the key rate-limiting enzyme in mammalian NAD⁺ biosynthesis; and then NMN is converted to NAD⁺ by NMN adenylyltransferases (NMNATs) (29). On the other hand, nicotinamide riboside (NR), can be converted into NMN by nicotinamide riboside kinase (NRK). NMN and NR are natural compounds that effectively enhance NAD⁺ biosynthesis which bypass the rate-limiting enzyme NAMPT (23). Several studies have shown that exogenous NR is transported into cells through equilibrative nucleoside transporters (ENTs), while exogenous NMN has to be converted into NR by CD38 and then transported into cells via ENTs (8). These reports imply that NR might be more effective in replenishing cellular NAD⁺ than NMN to some extent. Nonetheless, a recent study identified Slc12a8 as a specific NMN transporter for NMN uptake both in vitro and in vivo (13), thus avoiding the step to be converted to NR and transported into cells by ENTs. Furthermore, it was shown that Slc12a8 in the lateral hypothalamus is important in maintaining energy metabolism and skeletal muscle functions during aging (13). Therefore, NMN and NR might have similar efficacy for their anti-aging effects.

Accumulating evidences have demonstrated that both NMN and NR supplementation can reduce the accumulation of DNA damage in aging process or other diseases (21, 9, 27). However, to date, no comparative study of the efficacy of these two compounds on protecting cells from DNA damage had been conducted. Therefore, in this study, we investigated whether there is any difference between NMN and NR in alleviating DNA damage. Cisplatin was used as the DNA damage agent. HeLa cells were cultured with either NMN or NR before challenged with cisplatin, or, exposed to cisplatin first and then cultured with NMN or NR. The DNA damage levels were evaluated by comet assay or γH2AX protein expression. As reported here, both NMN and NR can efficiently protect cells from cisplatin-induced DNA damage with no significant difference

Cell Line and Chemicals

HeLa cells were grown in Dulbecco modified Eagle medium (DMEM) medium supplemented with 10% (v/v) of FBS, 2 mMol/L glutamine, 1 mMol/L sodium pyruvate, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C under 5% CO2. NMN (#GC16971), NR (#GC44401), and Cisplatin (#GC11908) were purchased from GlpBio Company (Montclair, CA, USA).

Cell Survival Assay

Cell viability was measured by Trypan blue exclusion assay as described before (30). After cells were harvested and resuspended with fresh medium, 20 µL cell suspension and 20 µL 0.4% trypan blue solution were mixed, and then 20 µL mixture was transferred to a hemocytometer. Live cells excluded trypan blue dye whereas dead cells were stained, and they were counted under microscope. The cell viability (%) was calculated as: (number of live cells/number of total cells)×100%. Experiments were performed in triplicates.

Immunofluorescent Labeling of γH2AX

Expression of γH2AX was evaluated as described before with modifications (30). First, cells were fixed in 4% paraformaldehyde for 30 min, and then permeabilized in 0.1% Triton-X 100 and immersed three times for 5 min each in a washing solution of PBSTX (0.1%) (phosphate buffer saline 0.1 mol/L, pH 7.5, 0.1% Triton X-100) at room temperature. The slides were incubated with a blocking solution of PBSTX (0.1%) containing 1% bovine serum albumin (BSA) for 60 min at room temperature. Slides were then rinsed with PBS three times for 5 min each and were then incubated with γH2AX antibody (Cat# ab81299, Abcam) solution in a moisture chamber for 16 h at 4°C. After washing a secondary goat-anti-rabbit IgG antibody conjugated with Alexa Fluor 633 was added and incubated for another hour. Slides were then rinsed in PBS three times and nucleus was stained with DAPI at a concentration of 0.1 µmol/L for 10 min at room temperature. Images were acquired on a Zeiss LSM 710 single-photon confocal system using a multi-track configuration. The intensity of immunofluorescence was measured by ImageJ software.

Alkaline Comet Assay

Comet assay was conducted as described before with some modifications (31). In short, 75 µL of pre-warmed regular melting point agarose (0.7%) at 70°C was used as the first gel layer. 10 µL of cells (approximately 1×10⁵) were mixed with 75 µL of pre-warmed low melting point agarose (0.7%) at 37°C. 10 µL of these mixtures were used as the second gel layer. After solidification, the cometslides were dipped into a cold lysis solution (1% Triton X-100, 10% dimethyl sulfoxide and 89% lysis buffer containing 10 mmol/L Tris, 2.5 mol/L NaCl and 100 mmol/L Na2EDTA, pH 10) for an hour. After lysis, cometslides were placed in a horizontal electrophoresis chamber filled with cold alkaline buffer and incubated for 20 min in the dark for DNA unwinding, and then electrophoresis was performed (20 V, 20 min). For neutralization, cometslides were washed in PBS for 10 min. After air-drying, cometslides were stained with GelRed for scoring. The tail length was scored using ImageJ in 100 randomly selected nuclei per sample.

Measurement of NAD⁺ Levels

NAD⁺ was measured using the NAD⁺/NADH Assay Kit following the manufacturer’s instruction (Beyotime, Shanghai, China). In brief, cells were plated in six-well dishes at a density of 3×10⁵ cells/well overnight. After various treatments, cells were washed 3 times in ice-cold PBS and were extracted in 150 µL extraction buffer. 20 µL lysates were added to a 96-well plate and mixed thoroughly with an ethanol dehydrogenase working solution, incubated at 37℃ for 10 min. Chromogenic solution was then added to the plate and the mixture was further incubated at 37℃ for 30 min. The absorbance was measured at 450 nm and analyzed using a SPARK microplate multimode reader. The total concentration of NAD⁺ in cell samples was calculated according to the standard curve. All measurements were repeated at least three times in independent experiments.

2.6 Measurement of reactive oxygen species (ROS) levels

ROS was measured using the ROS Assay Kit (Beyotime, Shanghai, China). Briefly, 3×10⁵ HeLa cells were seeded in 6-well plates overnight. After various treatments, cells were washed twice with PBS and incubated with ROS detection reagent (DCFH-DA, 5 µmol/L) at 37°C for 30 min. Subsequently, the cells were washed three times with PBS. Finally, the fluorescence signal was detected using SPARK microplate multimode reader.

Statistical Analysis

Each experiment was repeated at least three times independently. Data were presented as Mean ± SD. Two-way ANOVA Tukey’s multiple comparisons test was applied to confirm the significant differences between the groups. Results were considered statistically significant with P < 0.05. Statistical evaluation between different groups was analyzed by using the R software.

Both NMN and NR Protect HeLa Cells from the Cytotoxicity of Cisplatin in a Dose-dependent Manner

First the cytotoxicity of cisplatin on HeLa cells was evaluated. HeLa cells were treated with different doses of cisplatin (0, 1.25, 2.5, 5, 10, and 20 µmol/L) for 24 h, then the cell viability was determined by Trypan blue exclusion assay. As showed in Fig. 1A, cisplatin decreased cell viability in a dose-dependent manner, and an IC50 value of 17 µmol/L was obtained (Fig. 1B). In subsequent experiments, 10 µmol/L cisplatin was chosen, because the cell viability was significantly reduced at this dose, but enough cells could still be collected.

Then the effects of NMN and NR on the viability of cisplatin-treated cells were examined. HeLa cells were co-treated with different concentrations (0, 0.625, 1.25, 2.5, 5, and 10 mmol/L) of NMN or NR and 10 µmol/L cisplatin for 12 h. The results showed that both NMN and NR enhanced the cell viabilities in a dose-dependent manner compared with cisplatin-treated cells (P < 0.05, Fig. 1C), but there was no significant difference in cell viability between NMN and NR cotreatment groups.

NR Has Better Protective Effect Against Cisplatin-induced DNA Damage Than NMN

To examine the protective effects of NMN or NR on DNA damage, HeLa cells were first pre-incubated with 0.625, 1.25, 2.5, 5, and 10 mmol/L NMN or NR for 12 h, then the cells were exposed to 10 µmol/L cisplatin with fresh medium for another 12 h. The cells were harvested and the DNA damage levels were determined by immunofluorescent γH2AX intensity and alkaline comet assay. The γH2AX immunofluorescent assay showed that pretreatment with NMN or NR decreased the γH2AX levels in a dose-dependent manner (Fig. 2A and 2B). The intensity of fluorescence was quantified by ImageJ software. At each dose, NR-treated cells had relatively lower γH2AX levels than that of NMN-treated cells (P < 0.05). The alkaline comet assay is another assay to evaluate DNA damage level through the tail length for the fragmented DNA after electrophoresis. Similarly, the tail lengths were shortened in NMN- or NR-pretreated cells in a dose-dependent manner (Fig. 2D and 2E), and NR-pretreated groups showed even shorter tail length than that of NMN-pretreated groups (P < 0.05; Fig. 2F). These results indicated that both NMN and NR mitigated cisplatin-induced DNA damage in a dose-dependent manner, while NR had a stronger protective effect against DNA damage induced by cisplatin than NMN.

NMN and NR Have Similar Effects in Promoting the Repair of Cisplatin-induced DNA Damage

When DNA is damaged, DDR is activated to repair the damage and maintain genome stability. We then investigated whether NMN and NR influenced the repair of damaged DNA. HeLa cells were first exposed to 10 µmol/L cisplatin for 12 h, and then cultured in fresh medium with 10 mmol/L NMN or NR. Hereafter, the cells were harvested every 8 h until 44 h. The DNA damage levels were assessed through γH2AX immunofluorescent microscopy and alkaline comet assay.

After cisplatin was removed, the fluorescent intensity of γH2AX decreased with time in all groups (P < 0.05; Fig. 3A). At each time point, the γH2AX fluorescent intensity was significantly lower in NMN and NR treated cells than that in PBS treated control cells, but there was no difference between NMN and NR treated groups (Fig. 3B). Similar results were obtained using the comet assay to assess DNA damage, with tail length shortened after cisplatin was removed (Fig. 3C), and both NMN- and NR-treated cells had shorter tail length than PBS treated cells (P < 0.05), while there was no difference between NMN and NR treated groups (Fig. 3D). These results indicated that both NMN and NR promoted the repair of damaged DNA with a similar efficacy.

NMN and NR Rescue Intracellular NAD⁺ Levels in Cisplatin-treated Cells

NAD⁺ is important in maintaining cellular redox status, and cisplatin is also able to decrease NAD⁺ level (12). Both NMN and NR can replenish cellular NAD⁺ levels, but whether they can restore NAD⁺ levels in cisplatin-treated cells was still not clearly documented. Therefore, HeLa cells were treated as described above, and total NAD⁺ content was measured.

As shown in Fig. 4, compared with the untreated cells, cisplatin significantly decreased intracellular NAD⁺ levels (Ρ < 0.05), while both NMN and NR increased cellular NAD⁺ levels (Ρ < 0.05). On the other hand, addition of either NMN or NR in cisplatin-treated cells partially restored NAD⁺ levels that were significantly higher than cisplatin treatment alone, while still lower than control. Furthermore, there was no significant difference between NMN and NR for their effects on increasing intracellular NAD⁺ levels.

NMN and NR Inhibit Cisplatin-induced Increase of Intracellular ROS Levels

Many studies have established that cisplatin-induced increase of ROS is a key mechanism for its DNA-damaging effect, and therefore, we were interested to know whether NMN and NR reduced the severity of DNA damage by decreasing cisplatin-induced ROS. HeLa cells were cultured in medium containing 10 µmol/L cisplatin with/without 10 mmol/L NMN or NR for 12 h, and the intracellular ROS levels were analyzed. As shown in Fig. 5, compared to control group, NMN or NR did not affect cellular ROS levels, while the ROS levels in cisplatin-treated cells were significantly increased (Ρ < 0.05). However, in cisplatin-treated cells with NMN or NR addition, intracellular ROS were significantly decreased, though still at levels that were higher than controls (P < 0.05). There was also no significant difference in ROS level between NMN/cisplatin and NR/cisplatin co-treatment groups, suggesting that NMN and NR have similar effects on intracellular ROS generation.

Maintaining proper cellular NAD⁺ level has been gradually recognized as an important approach for combating aging (6). However, as NAD⁺ cannot be absorbed directly by cells, supplementation of NAD⁺ precursors, such as NMN and NR, which are presented in natural foods, including broccoli, tomatoes, milk, etc., has been tested in cell/animal models, as well as human clinical trials with relatively satisfying results (25). Therefore, NMN and NR are now being marketed in many countries as food supplements. Nonetheless, whether these two compounds have the same efficacy is worth of further investigation, in particular, considering the huge market share and financial gain.

One key aspect for the beneficial effects of NMN and NR is protection against DNA damage. Indeed, many studies have clearly demonstrated such effects. For example, a recent study showed that administration of NMN maintained telomere length and dampened the DDR by activating Sirt1 (2). NMN administration could also significantly minimize tubular cell DNA damage and subsequent cellular senescence caused by H2O2 and hypoxia (14). Similarly, NR reduced DNA damage levels in AD mice (9, 10). These results indicated that the NAD⁺ precursors, both NMN and NR, could alleviate DNA damage, though a quantified comparison between the two has not been reported. In this study, HeLa cells were treated with NMN or NR before exposure to cisplatin, and the DNA damage levels were detected. Interestingly, the γH2AX immunofluorescent microscopy and comet assay results showed that both NMN and NR mitigated cisplatin-induced DNA damage in a dose-dependent manner, but NR had a stronger protective effect than NMN (Fig. 2).

Based on the biosynthesis pathway for NAD⁺, NMN is a more direct precursor for NAD⁺, and thus should have better efficacy than NR. On the other hand, in mammals, exogenous NR is transported into cells through ENTs, while exogenous NMN has to be converted into NR by CD38 (8), and the conversion of NMN to NR is essential for it to act as extracellular precursor of intracellular NAD⁺ in HEK293 cells (16). The extra step in cellular entrance for NMN might be the reason of its less efficient DNA damage protection. However, a recent study reported that NMN can be directly transported into cells via the transporter coded by the SLC12A8 gene in the intestine of mice (7).. If this were the case, then some other mechanisms should be looked into. Still, as whether HeLa cells as well as human intestinal cells express SLC12A8 are not yet known, the expression pattern of SLC12A8 in human tissues should be carefully examined.

As a first-line therapeutic drug against cancer, the underlying molecular mechanisms for the toxic effects of cisplatin have been well studied, and decreasing cellular NAD⁺ levels (or decreased NAD⁺/NADH ratio) is one of them (18). This imbalance in NAD⁺/NADH is coupled with the generation of excess ROS, which can lead to extensive DNA damage. Once DNA is damaged, various DNA repair systems would be activated to deal with the different types of damage. Among them, the PARP1-mediated pathway is very important for the toxic effect of cisplatin (15), as it is an early sensor of single- and double-strand breaks and catalyzes ADP-ribosylation using NAD⁺ as substrate to mediate DNA damage repair (11). As a consequence to PARP1 activation, cellular NAD⁺ levels would be decreased further, and if NAD⁺ levels were not properly restored, the exhaustion of NAD⁺ eventually causes deleterious effect on cells, such as apoptosis. Therefore, the basis for protective effects of NMN and NR lie in their ability to restore cellular NAD⁺ levels. Indeed, the results presented here showed that cisplatin decreased cellular NAD⁺ levels, while both NMN and NR could restore NAD⁺ levels (Fig. 4). As a result, cisplatin induced ROS generation was inhibited (Fig. 5), repair of damaged DNA was more effective (Fig. 3), and cell viability was rescued by both NMN and NR (Fig. 1). However, unlike the preventive effects for DNA damage (Fig. 2), there was no significant difference between NMN and NR in these analyses. The reason for such discrepancy remains elusive and needs to be investigated in depth.

Collectively, our results showed that both NMN and NR effectively protected HeLa cells against cisplatin-induced DNA damage. The two compounds have similar effect on enhancing cell viability, replenishing intracellular NAD⁺ and scavenging intracellular ROS. Interestingly, we found that NR might have a better protective (preventive) effect against cisplatin-induced DNA damage than NMN.

Author Contributions X. T., J.Y. and C. D. conceived and designed the study. S.Q., Y.Z., S.S., C.D., Y. J. and X.H. carried out the experiments. S.Q., Y.Z., X.X. and L.Y. analyzed the data. S.Q. and Y.Z. wrote the first draft of the manuscript, X.T., J.Y. and C. D. revised the manuscript. All authors have read and approved the final manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Nos. 32270186, 31971138, and 81772168), and the Natural Science Foundation of Zhejiang Province (Nos. LY23H190001 and LQ18H190003) and Hangzhou Bio-medicine and health industry development support science and technology project (Nos. 2021WJCY144, 2022WJC016) and Postgraduate research and innovation promotion program of Hangzhou Normal University (Nos. 1115B20500437).

Data Availability

Data will be available on genuine request to the corresponding author.

Ethical Approval and Consent to Participate Not applicable.

Consent for Publication Not applicable.

Competing Interest The authors declare no competing interests.

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Nicotinamide Mononucleotide Versus Nicotinamide Riboside in The Protective Effects of Cisplatin-induced DNA Damage in HeLa Cells

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Abstract

Figures

Backgroud

Materials and Methods

Cell Line and Chemicals

Cell Survival Assay

Immunofluorescent Labeling of γH2AX

Alkaline Comet Assay

Measurement of NAD⁺ Levels

Statistical Analysis

Results

NR Has Better Protective Effect Against Cisplatin-induced DNA Damage Than NMN

NMN and NR Have Similar Effects in Promoting the Repair of Cisplatin-induced DNA Damage

NMN and NR Rescue Intracellular NAD⁺ Levels in Cisplatin-treated Cells

NMN and NR Inhibit Cisplatin-induced Increase of Intracellular ROS Levels

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

Nicotinamide Mononucleotide Versus Nicotinamide Riboside in The Protective Effects of Cisplatin-induced DNA Damage in HeLa Cells

Status:

Version 1

Abstract

Figures

Backgroud

Materials and Methods

Cell Line and Chemicals

Cell Survival Assay

Immunofluorescent Labeling of γH2AX

Alkaline Comet Assay

Measurement of NAD+ Levels

Statistical Analysis

Results

NR Has Better Protective Effect Against Cisplatin-induced DNA Damage Than NMN

NMN and NR Have Similar Effects in Promoting the Repair of Cisplatin-induced DNA Damage

NMN and NR Rescue Intracellular NAD+ Levels in Cisplatin-treated Cells

NMN and NR Inhibit Cisplatin-induced Increase of Intracellular ROS Levels

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

Measurement of NAD⁺ Levels

NMN and NR Rescue Intracellular NAD⁺ Levels in Cisplatin-treated Cells