Spermidine-Induced Autophagy Regulates the Survival of HeLa Cells

Spermidine, a type of natural polyamines, participates in various biological processes (Igarashi and Kashiwagi, 2000). Many studies indicate that cellular polyamines can modulate normal and cancer cell survival (Rajeeve et al., 2013; Gomes et al., 2017). Most of the studies acknowledge the role of spermidine as a facilitator of cell survival. However, it is reported that spermidine also has a role in promoting cell death (Chen et al., 2018; Schultz et al., 2018). The controversial effect of spermidine on survival of cancer cells is not fully clarified yet. Thus, the effect of spermidine on viability of cervical cancer HeLa cells is investigated in this study.

The basis of these diverse cellular responses to spermidine is currently not known, but the induction of autophagy may serve as a great contributor. Autophagy is known to be a cellular degradation pathway that is responsible for the breakdown of damaged or redundant macromolecules at critical circumstances such as cellular stress (Ryter et al., 2018). LC3 (microtubule-associated protein light chain 3), typically characterized as an autophagosome marker, is one of the most widely monitored autophagy-related proteins in mammalian cells (Jiang and Mizushima, 2015). Besides, the p62 protein can be incorporated into the autophagosome and be degraded in autolysosomes, thus decreased p62 levels are associated with autophagy activation (Pankiv et al., 2007).

Although autophagy mostly serves a cytoprotective function against certain stresses such as mitochondrial membrane potential (MMP) collapse, it has also been linked to cell death (Chen et al., 2016; Henson et al., 2017). In certain conditions, autophagy may activate cell death, and depending on context, autophagy may promote survival of a given cell species (Das et al., 2012; Classen et al., 2018). Despite the mounting knowledge about autophagy, the mechanisms through which the autophagic machinery regulates cancer cell survival are not entirely understood. It has been reported that spermidine induces autophagy in cultured yeast, immune cells, nematodes and flies (Eisenberg et al., 2009; Morselli et al., 2011). Thus, the effects of external spermidine on autophagy and cell viability are investigated, using HeLa cell line as a model.

The present study explores the effects of spermidine on autophagy and viability and their relationships in HeLa cells. It reveals that spermidine induces autophagy and decreases viability of HeLa cells. Moreover, 3-MA pretreatment affects the autophagy process dramatically and restores the viability of HeLa cells, indicating autophagy is a cause of decreased cell survival. The purpose of this study is to illuminate the complexity of the role of spermidine in cancer cell survival and delineate the function of autophagy in this process.

Spermidine, 3-methyladenine (3-MA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were obtained from Sigma-Aldrich Inc.

HeLa cells, obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), were cultured in DMEM (Gibco) supplemented with 10% FBS (Royabio), 100 IU/ml of penicillin & streptomycin at 37°C in a 5% CO2 incubator.

Cell viability was carried out using MTT assay as our previous study (Gu et al., 2018). Briefly, HeLa cells were seeded into 96-well plates (5000 cells/well) and treated with spermidine (0.1, 0.5, 1 mM) for 8 h. Then, MTT was added to each well at a final concentration of 0.5 mg/mL and further incubated for 4 h. Media were removed and 150 mL DMSO was added to dissolve the formazan crystals. The absorbance was detected at 570 nm using a spectrophotometer (Bio-Rad, UltramarkTM Microplate System).

Meanwhile, the morphology of HeLa cells was visualized with an inverted fluorescence microscope (Axio Observer Z1, Carl Zeiss) after the same treatment as above.

LDH, released into the culture media by damaged cells, was measured using LDH assay kits (Beyotime Biotechnology) according to the manufacturer’s protocol. Briefly, after exposure to spermidine, 100 μL LDH reaction solutions were added to the supernatants of HeLa cells (10 μL/well) and incubated for 30 min. The absorbance was quantified at 450 nm using a spectrophotometer (Bio-Rad, UltramarkTM Microplate System).

HeLa cells were treated with 0.5 mM spermidine for 8 h and then fixed with 1 ml of 2.5% glutaraldehyde. The samples were sent to the electron microscope center of Lanzhou University for evaluation using a transmission electron microscope (JEOL JEM-1230). Micrographs were taken using a Gatan Erlangshen ES500W camera.

The western blotting was performed as our previous study (Gu et al., 2018). Briefly, proteins were separated by SDS-PAGE and transferred to a PVDF membrane, and then incubated with the following primary antibodies: rabbit anti-LC3 and anti-p62 (1:1000, L7543 and P0067, Sigma-Aldrich Inc.). The samples were incubated with HRP conjugated secondary antibodies and developed with the Immobilon Western Chemiluminescent reagents (Millipore).

HeLa cells were incubated with 1 μg/ml JC-1 (excitation, 507 nm; emission, 530 nm) at 37°C for 30 min. Cells with healthy mitochondria were detected by measuring the increase in the red fluorescence due to JC-1aggregates. While cells that have depolarized Δψm emitting JC-1 monomers were detected by increase in the green fluorescence. The fluorescence intensity was monitored by flow cytometer (BD Biosciences).

Statistical analyses were performed using SPSS 19.0 (IBM, USA) and graphed with Origin 8.0 (Origin Lab, USA). Multiple comparisons were performed by ANOVA with LSD post hoc test. Differences were considered significant at p < 0.05.

The effect of spermidine on the survival of HeLa cells was investigated by MTT assay. As shown in Figure 1A, 0.1, 0.5, 1.0 mM spermidine significantly reduced the cell viability to 83.92%, 65.45% and 67.80%, respectively when compared to control (p<0.05).

Next, we defined whether spermidine led to a change in morphology of HeLa cells. According to Figure 1B, spermidine caused deterioration of HeLa cells as indicated by more cytoplasmic vacuolation compared to control.

To confirm the adverse effect of spermidine on HeLa cells, we measured the LDH levels in the supernatant. After 8 h of treatment, LDH levels were significantly increased in supernatants of cells at 0.5 and 1.0 mM spermidine (Fig. 2).

As demonstrated in Figure 3, 0.5 and 1.0 mM spermidine significantly decreased the MMP relative to control (p<0.05). The mitochondrial uncoupler CCCP as a positive control showed more potent effect.

Autophagy induction was evaluated by observing the formation of autophagic vacuole. As shown in Figure 4A, 0.5 mM spermidine encapsulated the cytosolic contents within autophagosome.

To confirm the effect of spermidine on autophagy, we examined the conversion of LC3I to LC3II as the amount of LC3II is clearly correlated with the number of autophagosomes. HeLa cells were exposed to 0.5 mM spermidine for different time-points (0, 2, 4, 6, 8, 10 h) and starvation for 5 h as a positive control. Spermidine significantly increased the ratio of LC3II / LC3I at 4, 6 and 8 h (p<0.05) with the most dramatic effect at 8 h (Fig. 4B).

In addition to LC3, receptor protein p62 was used as an autophagy marker. p62 expression was reduced by 10% and 38% when HeLa cells were treated with 0.5 and 1.0 mM spermidine for 8 h (p<0.05) (Fig. 4C).

Autophagy inhibitor 3-MA (5 mM) cotreated with 0.5 mM spermidine significantly restored cell viability when compared to spermidine alone (p<0.05). 5.0 mM 3-MA alone slightly decreased cell viability (Fig. 5).

Excessive accumulation or depletion of spermidine may disrupt many cellular functions, including mitochondrial integrity, leading to oxidative stress, autophagy or apoptosis (Igarashi and Kashiwagi, 2000; Stefanelli et al., 2000; Thomas and Thomas, 2001). In this study, we found that exogenous supply of spermidine reduced the survival, caused cell damage and compromised MMP of Hela cells. Meanwhile, spermidine elicited autophagy and inhibition of autophagy by 3-MA restored the viability, indicating that induction of autophagy is essential for the decreased survival of Hela cells.

It is well accepted that spermidine is a strong inducer of autophagy (Eisenberg et al., 2009; Madeo et al., 2010; Morselli et al., 2011). The consequence of promoting autophagy depends on multiple factors, including extent of induction, duration, and cellular context. Although the multiple roles of autophagy in cancer require further clarification, it is obvious that autophagy is directly involved in many important physiological processes such as response to stress, and cell death pathways in cancer cells (Alva et al., 2004; Navarro-Yepes et al., 2014). Autophagy is generally a stress-responsive, survival mechanism. However, it may also be a cell death mechanism (autophagic cell death) (Levine and Yuan, 2005; Yu et al., 2006). The present study showed that direct induction of autophagy through spermidine is sufficient to inhibit cell survival and collapse MMP of Hela cells. Although it is found that addition of spermidine could increase the survival of human peripheral blood mononuclear cells (PBMC), this effect was obtained at relatively low concentrations (around 20 nM) (Eisenberg et al., 2009). When applied at millimolar concentrations as in this study, spermidine efficiently elicited autophagy and decreased the survival of Hela cells. These differences may be either due to cell-type-specific differences in the metabolic pathway of spermdine or to differences in the sensitivity to cell death.

Accumulating evidence demonstrates interesting links between autophagy and cancer cell survival (Kovacs et al., 2012; Shen and Codogno, 2012). In this study, spermidine led to autophagy activation as well as cell survival inhibition. Remarkably, inhibition of autophagy by 3-MA restored the viability of Hela cells. Therefore, autophagy seems to be an important player in the life and death of Hela cells. It is interesting to note that a recent study confirmed this by demonstrating that spermidine induced growth inhibition and apoptosis by autophagic activation in Hela cells (Chen et al., 2018).

Unraveling the complex molecular regulation of autophagy is pivotal in understanding the mechanism of cancer cell survival. Recent progress in autophagy research has been led by the identification of a number of autophagy-related proteins. Among these proteins, LC3 has been extensively studied (Kabeya et al., 2000). We found that the conversion of LC3-I to LC3-II was obviously increased when Hela cells were exposed to spermidine. In addition to LC3, p62 is also served as a protein marker of autophagy, which links LC3 and ubiquitinated substrates (Pankiv et al., 2007; Jiang and Mizushima, 2015). As expected, induction of autophagy by spermidine is accompanied by decreased level of p62 in Hela cells.

This study clearly shows that autophagy fulfills the critical functions in the death of Hela cells. However, a complete understanding of the autophagic process and its crosstalk with cell death pathways represents an evolving story that will only be clarified with additional research.

This work was supported by National Natural Science Foundation of China (31300941) and Fundamental Research Funds for the Central Universities (lzujbky-2018-kb18).

The authors declare that there is no conflict of interests regarding the publication of this article.