Synergistic Effect of Endogenous and Exogenous Aldehydes on Doxorubicin Toxicity in Yeast

Anthracyclines are frequently used to treat many cancers including triple negative breast cancer, which is commonly observed in African-American women (AA), and tend to be more aggressive, carry worse prognoses, and are harder to manage because they lack molecular targets. Although effective, anthracyclines use can be limited by serious side effects and eventually the development of drug resistance. In S. cerevisiae, mutants of HOM6 display hypersensitivity to doxorubicin. HOM6 is required for synthesis of threonine and interruption of the pathway leads to accumulation of the threonine intermediate L-aspartate-semialdehyde. This intermediate may synergize with doxorubicin to kill the cell. In fact, deleting HOM3 in the first step, preventing the pathway to reach the HOM6 step, rescues the sensitivity of the hom6 strain to doxorubicin. Using several S. cerevisiae strains (wild type, hom6, hom3, hom3hom6, ydj1, siz1, and msh2), we determined their sensitivity to aldehydes and to their combination with doxorubicin, cisplatin, and etoposide. Combination of formaldehyde and doxorubicin was most effective at reducing cell survival by 31-fold–39-fold (in wild type cells) relative to doxorubicin and formaldehyde alone. This effect was dose dependent on doxorubicin. Cotreatment with formaldehyde and doxorubicin also showed increased toxicity in anthracycline-resistant strains siz1 and msh2. The hom6 mutant also showed sensitivity to menadione with a 2.5-fold reduction in cell survival. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy.


Introduction
Doxorubicin is one of the most effective anticancer agents [1]. Doxorubicin is an anthracycline antibiotic that is used to treat a variety of cancers including hematological cancers, carcinomas, and sarcomas [2][3][4]. This particular anthracycline antibiotic composes the major therapeutic alternative in breast cancer [5][6][7]. One of the three primary mechanisms of action for doxorubicin is its intercalation into DNA which directly affects transcription and replication [8]. The second mechanism of action is inhibition of topoisomerase II activity by stabilizing the DNA-topoisomerase II alpha complex, effectively preventing the religation portion of the ligation-religation reaction that topoisomerase II catalyzes [9]. Doxorubicin also generates free radicals as it cycles between its quinone and semi-quinone structures during metabolic reactions and thereby generates reactive oxygen species (ROS) [10].
Although extremely effective, anthracyclines are subject to drug resistance and deleterious side effects [11,12]. Some tumor cells develop modifications that confer resistance to anthracyclines [13]. Tumor cells that have elevated levels of glutathione peroxidase are less affected by anthracycline generation of ROS [12,[14][15][16]. Another feature of cells that aid in doxorubicin resistance is decreased topoisomerase II activity [12,14,15]. Less topoisomerase II equates to less DNA double strand breaks. Lastly, some tumor cell populations manifest improved ability to repair DNA breaks; therefore, DNA replication proceeds uninhibitedly, and the tumor not only survives but continues to grow [12,14,15].
A major and currently insurmountable complication with anthracyclines use is the development of cardiomyopathy that 2 BioMed Research International can manifest following a single dose as early as 24 hours after exposure [12,17] or many years later after successful treatment [12,17]. Doxorubicin induced dilative cardiomyopathy and congestive heart failure is a serious and potentially fatal adverse effect. Dilative cardiomyopathy and the subsequent development of congestive heart failure (CHF) are refractory to common cardiovascular therapy [18]. The toxic injury to the heart after doxorubicin treatment is a result of doxorubicin mechanism of action, the generation of ROS [12,18]. The increase in ROS due to doxorubicin treatment occurs with the redox cycling between the quinone and hydroquinone rings and carbonyl reduction of moieties within doxorubicin structure [19,20]. The heavy production of ROS overwhelms the ability of antioxidizing enzymes to deal with them [19,20].
Because doxorubicin effectively treats a wide variety of cancers [21,22] and patient quality of life is improved when doxorubicin is included in the treatment regimen [23], significant efforts are being directed at discovering modalities to hypersensitize cells to doxorubicin [1,24,25]. We previously reported a genome-wide genetic screening in S. cerevisiae that identified 71 deletion strains displaying varying levels of sensitivity to doxorubicin. The screen revealed that inactivation of the HOM6 gene caused an accumulation of the L-aspartate-semialdehyde metabolite and increased the sensitivity of the hom6 mutant to doxorubicin. To further investigate the contribution of the L-aspartate semialdehyde intermediate in the sensitization of cells to doxorubicin, we evaluated the survival of threonine biosynthesis mutant strains to doxorubicin. We extended this work by evaluating the sensitivity of different yeast deletions strains to various exogenously added aldehydes, either alone or in combination with doxorubicin and other cytotoxic stressors that mediate the action of these agents, such as oxidative stress, and DNA damage. Our results indicate that formaldehyde synergizes with doxorubicin to kill wild type S. cerevisiae cells and increases the sensitivity of doxorubicin resistant strains to doxorubicin.

Sensitivity of Strains to Aldehydes, Chemotherapeutic
Agents, and Cytotoxic Stressors. The concentration of the drugs used for strain exposure was determined experimentally using the wild type parental strain, BY4741. Briefly, single colonies were grown overnight in liquid YPD media, at 30 ∘ C with shaking. Cells were then washed and resuspended in UltraPure sterile water. Strains were then separated into control and treatment groups and exposed to aldehydes alone and combined with cytotoxic chemotherapeutic agent for 3 hours. After exposure, cells were again washed and suspended in sterile water. 10-fold serial dilutions were spotted onto YPD agar plates and incubated at 30 ∘ C. Heat shock treatment was performed by plating serial dilutions of the strains and the plates were incubated at 37 ∘ C for 3 days. Cell growth was monitored daily and colonies were counted at day 3. Survival was calculated relative to the corresponding untreated control and sensitivity was determined relative to the survival of the wild type strain. All trials (3 minimum) involved testing independent colonies for each cytotoxic agent or stressor.

Statistical
Analysis. Data analysis and graphing were performed using the GraphPad Prism 4 software package. Specific analysis for each experiment is indicated in each figure legend. In most cases, the mean of at least three experiments is plotted together with the standard deviation. Differences between mean values and multiple groups were analyzed by one-way analysis of variance (ANOVA). Statistical significance was set at < 0.05.

Defects in Threonine Biosynthesis Sensitize Cells to Doxorubicin.
To determine the cause of increased sensitivity to doxorubicin in genes involved in amino acid biosynthesis, we performed survival assays on mutant strains of genes required for threonine biosynthesis. The genes HOM3, HOM2, HOM6, THR1, and THR4 encode for enzymes that catalyze the sequence of reactions in the threonine biosynthesis pathway (Figure 1(a)). As seen in Figure 1(b), the S. cerevisiae mutant strains hom3, hom2, hom6, thr1, and thr4 were all individually inactivated and all show some sensitivity to doxorubicin when exposed to doxorubicin containing media (YPD + doxo) compared to the media lacking doxorubicin (YPD). The hom6 strain was the most sensitive to doxorubicin with the subsequent strains (thr1 and thr4) in the pathway being slightly less sensitive than the hom6 but more than the hom3  and hom2 (Figure 1(b)). To determine why the hypersensitivity occurred after the inactivation of the HOM6 gene, which encodes for the enzyme homoserine dehydrogenase, we inactivated the upstream gene HOM3 (Figure 1(c)). The hom6 strain with the inactivated HOM3 gene was denoted by hom6hom3 and showed decreased sensitivity to doxorubicin when replica plated onto YPD + doxo media compared to the YPD media (Figure 1(c)). In Figure 1(d), the survival of the hom3 strain decreased to 67% with 20 M doxorubicin from the 100% survival of the control (a 1.5-fold sensitivity increase). The treatment of the hom6 strain with 20 M doxorubicin reduced the survival to 25% when compared to its control but the survival of the hom6hom3 strain was reduced to only 80%. The sensitivity of the hom3 and hom6hom3 strains was increased by only 1.5-and 1.3-fold, respectively; however, the sensitivity of the hom6 strain was increased to doxorubicin by 4-fold. Inactivation of the HOM6 gene may have caused an accumulation of the toxic metabolite Laspartate semialdehyde.

Treatment with Formaldehyde Increases the Sensitivity of Wild Type S. cerevisiae to Doxorubicin.
To explore the role of aldehydes on cells treated with doxorubicin, we used the wild type strain to determine if the aldehyde intermediate sensitized wild type cells to doxorubicin. As seen in Figure 2(a), when exposed to formaldehyde (2 mM) alone the strain shows some sensitivity, whereas when exposed to doxorubicin (10 M, 50 M, and 150 M) alone, the wild type strain showed dose-dependent reduction in growth (Figure 2(a)). The exposure of the wild type cells to the combination of formaldehyde (2 mM) and doxorubicin (10 M, 50 M, and 150 M) resulted in increased sensitivity relative to formaldehyde or doxorubicin alone (Figure 2(a)). Treatment with formaldehyde (2 mM) reduced the survival of the wild type strain to 78% (Figure 2(b)). However, in the presence of doxorubicin (10 M), formaldehyde reduced the viability of the strain by 50-fold relative to the untreated strain and around 31-fold and 39-fold relative to doxorubicin alone and formaldehyde alone, respectively (Table 1). Cotreatment   of the wild type strain with formaldehyde (2 mM) and doxorubicin (150 M) reduces the viability of the strain by ∼110-fold relative to formaldehyde alone. Our data shows that the cotreatment of formaldehyde (2 mM) and doxorubicin (10 M, 50 M, and 150 M) has a synergistic effect on the sensitivity of the wild type cells.

Effects of Cotreatment with Formaldehyde and Doxorubicin on Wild
Type, siz1, and msh2. To further characterize the synergistic effect of formaldehyde and doxorubicin, a survival assay with cotreatment of formaldehyde (2 mM) and increasing concentrations of doxorubicin was performed in the strains siz1 and msh2, which are deficient in septin sumoylation and mismatch repair, respectively. The siz1 and msh2 strains were selected because previous studies showed that they are resistant to doxorubicin [8]. The siz1 and msh2 strains are indeed less sensitive to doxorubicin compared to the wild type ( Figure 3) when subjected to the cotreatment of formaldehyde (2 mM) and increasing concentrations of doxorubicin. However, the increased toxicity of doxorubicin is apparent within each strain. The viability of the siz1 and msh2 strains was reduced most notably with formaldehyde (2 mM) and doxorubicin (10 M and 50 M) combined, than with either as a single treatment (Figure 3(a)). The survival of siz1 after exposure to formaldehyde (2 mM) and doxorubicin (10 M) alone and combined was 65.2%, 53.9%, and 18.8%, respectively (Figure 3(b)). The survival of msh2 after exposure to formaldehyde (2 mM) and doxorubicin (10 M) alone and combined was 34.9%, 22.8%, and 4.8%, respectively (Figure 3(b) and Table 2). Our data shows that the combination of formaldehyde and doxorubicin has a synergistic effect on wild type cells (≥31-fold increase) (Table 1); however, the effect is synergistic on siz1 and msh2 strains (Table 3). According to Table 3, the fold increase in sensitivity relative to formaldehyde alone is 3.5 (siz1) and 5.2 (msh2). Although less pronounced than that of wild type, our data demonstrate that treatment of siz1 and msh2 strains with formaldehyde (2 mM) overcomes some of their resistance to doxorubicin.

The hom6 Mutants Are Sensitive to ROS Generation.
Menadione or vitamin K3 is a naphthoquinone derivative that is hepatically converted to menaquinone, an active form of vitamin K2. This agent was used in this study to determine the effect of increased ROS on the hom6 mutant. The structure of menadione includes quinone ring [28] and, like doxorubicin, will induce oxidative stress by ROS generation [29]. Wild type, sod1 (superoxide dismutase mutant), and hom6 mutants were exposed to menadione, 6.6 mM (Figure 4(a)). The sod1 strain served as the positive control because it cannot produce the antioxidant superoxide dismutase. As expected, sod1 was the most sensitive to menadione induced ROS (12.7% survival) (Figure 4(b)). The survival of the hom6 mutant was 27.3% (Figure 4(b)). The data shows that there was an increase in fold sensitivity relative to the wild type strain of 5.5 and 2.5 for the sod1 and hom6 mutants, respectively ( Table 4).
The results indicate that the hom6 mutant is sensitive to the generation of ROS.

The hom6 Mutant Was Not Sensitive to DNA Double Strand Breaks.
Etoposide is an antineoplastic compound that, like doxorubicin, binds to topoisomerase II and DNA and then induces DNA double strand breaks to kill the cell. The hom6, rad52, and ydj1 mutants were spotted onto synthetic complete (S.C.) media with and without etoposide, 0.5 mM. The rad52 (homologous recombination) and ydj1 (protein repair) mutants grew less following etoposide exposure; however, the hom6 mutant was not sensitive to DNA double strand breaks ( Figure 5(a)). The data shows that  rad52 and ydj1 survival were 43% and 73% after exposure to etoposide, respectively ( Figure 5(b)). The fold sensitivity of the rad52 and ydj1 mutants relative to the wild type is 2.3 and 1.4, respectively ( Table 5). The sensitivity of the hom6 mutant is 0.8-fold relative to the wild type and is statistically insignificant (Table 5). This was expected because the rad52 strain is unable to repair DNA damage by homologous recombination and the ydj1 mutant is unable to repair protein damage; therefore, it is reasonable that they would be sensitive to a drug that induces DNA double strand breaks. However, the results indicate that DNA double strand breaks alone are unable to induce cell death in the hom6 mutant.

Discussion
This study has demonstrated that the accumulation of Laspartate semialdehyde enhances the toxicity of doxorubicin to the cell. Previous work in our laboratory identified a deletion of the HOM6 gene which hypersensitizes cells to doxorubicin, suggesting that the threonine biosynthetic pathway can serve as a novel target for cell sensitization to cytotoxic chemotherapy [1]. Mutants of the HOM6 gene confer significant increased sensitivity to doxorubicin. We extended this observation by investigating the role of aldehydes in the response of cells to anthracyclines.  We tested all the nonessential mutants of the threonine biosynthetic pathway (Figure 1) to determine which deletion strains exhibited higher sensitivity in response to doxorubicin. Three deletion mutants, hom6, thr1, and thr4, were found to be consistently hypersensitive to doxorubicin. It is worth emphasizing that the concentration of the drugs used is selected to identify hypersensitive strains. Increasing the dose will eventually kill all strains, including the wild type, and would not allow us to discriminate those hypersensitive strains from those unaffected.
The HOM2 gene encodes for the aspartic beta semialdehyde dehydrogenase enzyme that produces the toxic metabolite L-aspartate-semialdehyde; this semialdehyde intermediate immediately precedes the strains in the threonine biosynthetic pathway which are hypersensitive to doxorubicin.
We tested the sensitivity of S. cerevisiae strains to the combination of formaldehyde (2 mM) and doxorubicin and our data showed that wild type cells are more sensitive to the combination of formaldehyde and doxorubicin than to either drug as a single agent (Figure 2). This sensitivity can be a result of the accumulation of the toxic aldehyde metabolite. Our data show that treatment with formaldehyde (2 mM) reduced the survival of the wild type strain to 78%. However, in the presence of doxorubicin, formaldehyde reduced the viability of the strain by 31-fold-39-fold (with 10 M doxorubicin) relative to doxorubicin and formaldehyde alone and by ∼110-fold relative to doxorubicin at 150 M concentration. The cotreatment of formaldehyde (2 mM) and doxorubicin (10 M) has a synergistic effect in the wild type cells.
The SIZ1 gene encodes the SUMO ligase enzyme which has been previously reported to have reduced accumulation of doxorubicin and therefore is slightly resistant to the effects of doxorubicin [1]. The msh2 strain is deficient in mismatch repair and will therefore show higher survival relative to the wild type strain when exposed to doxorubicin [1]. In addition to the development of dose-dependent cardiomyopathy, another limiting aspect of doxorubicin therapy is drug resistance. The siz1 and msh2 strains were selected to determine if cells that have developed a resistance to doxorubicin therapy could be sensitized with the addition of an aldehyde. Cotreatment with formaldehyde (2 mM) and doxorubicin (10 M) of anthracycline-resistant strains siz1 and msh2 also shows increased toxicity (Figure 3). The cotreatment of formaldehyde and doxorubicin shows a toxic synergistic effect in siz1 and msh2 strains ( Table 2). The combination at the lowest concentration of doxorubicin was just as effective as at higher concentrations at overcoming doxorubicin resistance in siz1 and msh2 cells (Figure 3). This would be advantageous in the development of protocols which would spare normal tissues from doxorubicin toxicity.
Since doxorubicin can act through the generation of ROS or the generation of DSBs, it is important to investigate which of these mechanisms is responsible for the synergistic effect between the L-aspartate semialdehyde and doxorubicin. Our strategy was to treat the hom6 mutant with a drug that shares a single similar mechanism of action with doxorubicin at a time. The agents menadione and etoposide were employed for that purpose. Menadione or vitamin K3 is a naphthoquinone derivative that is hepatically converted to menaquinone, an active form of vitamin K2. Menadione has a quinone ring [28] and, like doxorubicin, will induce oxidative stress by ROS generation [29]. Etoposide is an anticancer agent and, similar to doxorubicin, binds to topoisomerase II and DNA. The stable union induces DNA double strand breaks while simultaneously preventing strand religation and subsequently leading to cell death. Our data showed that accumulated L-aspartate semialdehyde sensitized the hom6 mutant to menadione induced ROS (Figures 4(a) and 4(b)) but not to etoposide ( Figure 5) induced DNA double strand breaks. This suggests that the synergism between accumulated L-aspartate semialdehyde within the hom6 mutant and doxorubicin is the result of combined mechanistic actions.
It has been reported that formaldehyde inhibits the cytochrome P450 enzymes CYP2C11, CYP2E1, and CYP3A2 while inducing the activity of the CYP1A2 enzyme [30]. Interestingly, the CYP3A family has been shown to metabolize doxorubicin and drugs that inhibit CYP3A results in elevated plasma concentration of doxorubicin [31]. It is also possible that formaldehyde coadministered with doxorubicin reduces the activity of CYP2C11 enzyme which results in elevated levels of doxorubicin [32][33][34].
Threonine is an essential amino acid; therefore humans must acquire this amino acid solely from dietary means. The effect of aldehyde combined with doxorubicin needs to be studied in nonessential amino acids that produce semialdehyde intermediates to further characterize the role of aldehydes in doxorubicin sensitization.

Conclusions
In conclusion, cotreatment of the wild type strain with formaldehyde and doxorubicin enhances the cytotoxic effect and reduces viability more than either as a single agent. Previous reports indicate that doxorubicin sensitive cancer cells have higher levels of endogenous formaldehyde compared to resistant cells that conversely lack elevated endogenous formaldehyde levels [35]. It was observed that wild type strain was sensitive to formaldehyde alone and to doxorubicin in a dose-dependent manner. However, the cotreatment of formaldehyde and doxorubicin on the wild type strain was significantly more cytotoxic than either formaldehyde or doxorubicin alone. The observed and calculated result indicated that formaldehyde synergizes with doxorubicin to kill wild type cells [36]. It has been reported that the central carbon in formaldehyde forms an adduct with DNA and doxorubicin and this adduct is proposed to be more toxic to cells than is doxorubicin [36]. The formaldehyde doxorubicin adduct could account for the synergistic effect seen with the cotreatment of formaldehyde and doxorubicin and this property can be exploited as a therapeutic enhancer. In fact, the pivaloyloxymethyl butyrate (AN-9) prodrug has been shown to be synergistic with doxorubicin and other anthracyclines [37]. The potential use of a combination of aldehydes and cytotoxic drugs may lead to applications intended to enhance anthracycline-based therapy by overcoming anthracycline drug resistance and reducing their toxic side effects.

Data Availability
Data will be available by contacting the corresponding author. All strains and reagents used in the studies are available upon request.

Disclosure
Part of this work was presented at the AACR Annual Meeting 2014 and 2016.