To gain insight into molecular mechanisms and biological processes underlying the treatments with representative anti-cancer anthracycline/anthracenedione drugs DNR, DOXO and MTX, we have used CEM T-lymphoblastic leukemia cells and investigated protein fingerprints of the drug effects employing combination of zoomed 2DE with fluorescent protein stain and MALDI-TOF/TOF mass spectrometry. The CEM T-lymphoblastic leukemia cells have been considered as suitable model of hematological malignancies as well as tumor cells sensitive to various anti-cancer drugs [18
]. Several previous studies focused on the effects of DOXO or DNR with mostly applied 24 h or 48 h treatments and low micromolar concentrations of drugs, which may correspond to relevant clinical doses [11
]. In our study, we designed proteomic experiments focused on earlier time intervals in order to reliably monitor protein alterations that precede induction of apoptosis and minimize its impact on observed protein changes. Using individual half time to onset of apoptosis (TA50
), corresponding 10 times IC50
doses of the drugs instead of the same time interval for all treatments allowed us to optimize comparable stage of all used anti-cancer treatments. Whilst for four out of five drugs TA50
ranged from 120 min to 150 min, the longest 250 min interval was confirmed for DOXO and even this was still at least 6 times shorter than what was used in previously published studies [11
To date, the effect of DOXO treatment on different cancer cell lines has mainly been studied by proteomic techniques [11
]. To extend current observations and with the view to help translation of molecular findings toward improvements in clinical use, we focused on the effects of several clinically relevant representatives of the group of anthracycline/anthracenedione drugs. Hence, comprehensive proteome map of model T-lymphoblastic leukemia cells and its alterations after DNR, DOXO and MTX drug treatments were monitored and evaluated either by pair comparison to relevant untreated control or multivariate classification of drug treated and untreated samples.
In order to emphasise proteins specific for response toward anthracycline/anthracenedione drugs among all identified differentially abundant proteins, we performed in the same design, analysis of the effects of two additional anti-cancer drugs, CisPt and TAX, taken from distinct groups of chemotherapeutics, and compared protein alterations to those found after DNR, DOXO and MTX. As expected, using this step we marked the proteins affected and shared in anti-cancer response of such drug treatments. These proteins belong to the enzymes critical for cellular metabolism such as G6PD, the enzyme producing pentose sugars essential for nucleic acid synthesis; PHGDH, the enzyme involved in syntheses of purines and amino acids; NDUFS1, core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). More interestingly and corroborating our findings are the observations that many of these “promiscuous” general anti-cancer response proteins are the ones already known to play a vital role in various human cancers. For example PPME1 that demethylates protein phosphatase 2A was recently described as tumor suppressor [19
]. TRA2B or HNRNPA2B1 regulating repair of double strand breaks have elevated levels in various cancers [20
] and changed in levels by anti-cancer treatments as shown here. HNRNPA2B1 has been even assigned as proto-oncogene [21
]. Further evidence is presented by KHSRP regulating transcription and mRNA processing which was shown to support migration in liver cancer cells [22
]. Additionally, involvement of multifunctional protein PPIA in cancer progression has been described [23
]. Interestingly, several cytoskeleton regulating proteins including CFL1 [24
] and EZR [25
] were associated with invasion and metastasis and ARHGDIB was linked to the development of chemoresistance [26
]. These proteins, although non-specific as regards used drugs and functioning in various biological processes, most probably present important targets underlying anti-cancer mechanisms and possibly play role of anchor molecules which may connect different pathways in a very complex regulation of cancer cell processes. Despite their importance, the major aim of this study has been to identify specific proteins typical for the response to anthracycline/anthracenedione drugs DNR, DOXO and MTX and to characterize similarities in the effects of these structurally very close drugs.
In total, we found several tens of proteins with significantly changed levels at early time intervals after DNR, DOXO and MTX treatments which corresponded only to 1%–2% of the total number of spots detected. According to Gene Ontology classification of biological processes the highest representation of identified proteins for all three drugs belongs to metabolic processes of nucleic acids or proteins and cellular processes involved mainly in cytoskeleton organisation. It corresponds to well-known observations that metabolic alterations on glucose consumption and biosynthetic activity of nucleotides, amino acids and lipids are the changes for sustaining cell proliferation in cancer cells. Typical evidence of this fact is the Warburg effect, the conditions when the cancer cells switch from oxidative phosphorylation to glycolysis to produce ATP and set of enzymes such as lactate dehydrogenase and pyruvate dehydrogenase play crucial role [27
]. Evidently and surprisingly, we observed in our study such changes in CEM T-lymphoblastic leukemia cells at very early time intervals after anti-cancer DNR treatment. The most probable explanation of this behaviour is adaptive effort of tumor cells to make even stronger the essential mechanisms supporting cancer growth. Regulation of metabolic enzymes offers new directions for anti-cancer treatments and lactate dehydrogenase which catalyses the final step in the glycolytic cascade constitutes a relatively new anti-cancer target [28
]. Nevertheless, design of the combination of the enzymes or even their isoforms and development of specific inhibitors that would eliminate robustness of cancer cells is not a simple task.
In addition to changes in energy metabolism, DNR treatment of CEM cells leads to the decrease of two heterogeneous nuclear ribonucleoproteins which are involved in RNA processing but we also observed increase of TARDBP which is homologous to the heterogeneous nuclear ribonucleoproteins. The higher level of this protein was further confirmed using Western blot. The TARDBP has been identified as a cause of neuropathology in a wide spectrum of neurodegenerative diseases, including amyotrophic lateral sclerosis. Using Drosophila model for proteinopathy associated with TARDBP, it was shown that increasing human wild-type TARDBP expression is sufficient to cause neurotoxicity in vivo
]. The protein may also be involved in microRNA biogenesis, apoptosis and cell division [30
]. The finding of increased level of TARDBP in CEM leukemic cells after anti-cancer DNR treatment let us hypothesise that it might significantly contribute to the toxicity toward the tumor cell and positively influence outcome of anti-cancer response. Higher levels of this protein may also result from its decreased clearance, which was shown as mediated by lower activity of ubiqutin-proteasome system and autophagosome in synergy [31
]. Hence, the link between the level of TARDBP and activity of ubiqutin-proteasome system and autophagosome is another good example underlying importance of these cellular mechanisms in regulation of carcinogenesis or response of cancer cell to anti-cancer treatment.
Doxorubicin, the other member of anthracyclines, also affected metabolic and cellular biological processes in CEM leukemic cells and majority of targeted proteins were exclusively specific for this drug and increased in cells after drug treatment. Among them, the role of spermidine synthase is aimed to redox regulation of tumor cell followed by anti-cancer treatment. Overproduction of spermidine increases resistance to oxidative stress with spermidine serving as a free-radical scavenger in vitro
as well as in vivo
]. Hence, increase of spermidine synthase in DOXO treated cancer cell may present regulatory response which may increase resistance of cancer cell.
EEF2 translates growth and stress impulses to the regulation of protein synthesis by catalyzing ribosomal translocation step during translation elongation. However, phosphorylation of EEF2 by EEF2 kinase inactivates this factor which indicates that EEF2 kinase could be promising anti-cancer target. Interestingly, using pharmacological inhibition of EEF2 kinase demonstrated that anti-cancer activity of widely accepted inhibitor and anti-proliferation agent against different cancer cells was more correlated with induction of EEF2 phosphorylation than inhibition of EEF2 kinase activity. In addition, stronger induction of EEF2 phosphorylation mediated by AMPK activators and mTOR inhibitor was linked to more effective cancer cell growth inhibition. Accordingly, EEF2 phosphorylation appears to be mediated through multiple pathways thus alarming the need of combinatory inhibition of EEF2 kinase in anti-cancer therapy [33
]. In our study, we identified EEF2 in two protein spots from 2DE (). The more basic and less abundant spot was increased after DOXO treatment and may represent non-phosphorylated form, whilst the more acidic and more abundant protein spot may be representative of phosphorylated form increased by DOXO and MTX. The presence of more abundant/phosphorylated form might contribute to anti-cancer effect of DOXO and MTX, whilst the less abundant basic/non-phosphorylated form would have a role in regulation of protein synthesis and sustaining cancer cell growth.
One of a few examples of proteins decreased in level after DOXO treatment was found to be metabolic protein KHDRBS1. In case of human breast tumors it was shown that phosphorylation of this protein regulated its intracellular localization and anti-proliferative properties were blocked by phosphorylation [34
]. Therefore, in addition to quantitative changes observed in this study, it would be necessary to investigate its post-translationally modified forms and localization as regards contribution to anti-cancer effect of DOXO.
Among the proteins of cellular processes affected by DOXO, we observed increase of UBA1 controlling ubiquitin conjugation pathway, and MCM7 having a role in DNA strand elongation involved in DNA replication. MCM7 is a known component of minichromosome maintenance complex which is the putative replicative helicase in eukaryotic cells and demonstrated to be efficient and sensitive marker to assess disease progression in the uterine cervix [35
], prognosis of patients with non-small cell lung cancer [36
], or Hodgkin lymphoma [37
]. Comprehensive comparative analysis of pre-replication complex proteins in transformed and normal cells indicated that cellular transformation was associated with an overexpression and increased chromatin association of the pre-replication complex proteins including MCM7 [38
]. From this point of view, increased level of MCM7 at early time interval after anti-cancer DOXO treatment may reflect other adaptive mechanisms of cancer cell contributing to the transformation of cell.
Transport proteins appeared to be an important group of proteins responding to DOXO treatment. They included SAR1B involved in protein transport from endoplasmic reticulum to Golgi, and cytoplasmic activation/proliferation-associated protein-1, CAPRIN1, stress granule associated protein. These findings may suggest possible role of induction of endoplasmic reticulum stress associated with proteotoxic stress. Subsequently, such stress stimulates either apoptosis of cancer cell which is involved in anti-cancer effects or autophagy as a cytoprotective, stress-induced adaptive pathway following disruption of protein homeostasis [39
]. CAPRIN1 may also regulate the transport and translation of mRNAs of proteins with impact on cell proliferation and negative regulation of translation. The protein is putative target of miR-16 thus linking miRNA to the regulation of cell proliferation [40
]. Overexpression of CAPRIN1 induced phosphorylation of eukaryotic translation initiation factor 2 alpha resulted in global inhibition of protein synthesis [41
]. This may be synergistic with above mentioned role of phosphorylated EEF2 in suppression of protein synthesis as a part of anti-cancer effect of DOXO.
The majority of MTX induced protein alterations were moderate metabolic changes. Interesting, EIF2S3L which functions in the early steps of protein synthesis, PSMA5 and RUVBL1 with the roles in transcriptional regulation, DNA replication and probably DNA repair, were observed. Evidently, decrease of protein level mediated by decrease of EIF2S3L may play an important role in MTX anti-cancer effect. Furthermore, protein homeostasis which is controlled by ubiquitin–proteasome system as mentioned above seemed to be critical mechanisms for cancer cell. Pharmacologic inhibitors of the proteasome promote tumor cytotoxicity and clinical studies have showed improvement in patient survival. Despite success of the proteasome inhibitor bortezomib in the treatment of the hematologic malignancy such as multiple myeloma, treatment of the more complex solid tumors has been less successful [42
]. Our results document that MTX similarly to DOXO exploit proteins of ubiquitin-proteasome system to trigger or modulate cancer cell stress response to anti-cancer treatment in order to induce either apoptosis or autophagy.
RUVBL1 is a highly conserved AAA(+) ATPase whose expression as well as expression of its homolog RUVBL2 was high in different cancers. In case of human hepatocellular carcinoma silencing of RUVBL2 reduced cell growth and increased apoptosis whilst overexpression enhances tumorigenicity [43
]. The level of RUVBL1 was significantly increased in CEM cells treated with MTX, and Western blot analysis confirmed a significantly increased level not only after MTX but also DOXO drug treatment. The question remains whether RUVBL1 at an increased level is involved in promotion of tumorigenicity in CEM T-lymphoblastic cells similarly as described in the study on human hepatocellular carcinoma.
It was possible to see that very selective group of MTX treatment are the proteins of immune system process and response to stimuli, namely chaperones thus indicating significant contribution of protein folding and stress response in tumorigenesis and anti-cancer treatment [44
]. Furthermore, these chaperone proteins may be involved in presentation of tumor antigens for direct recognition of tumor by T cells [45
] or as autoantigens which can give raise to the production of autoantibodies [47
]. As regards anthracyclines, Fucikova et al.
] investigated the effectiveness of anthracyclines to induce immunogenic cell death in human tumor cell lines and primary tumor cells. The data demonstrated induction of immunogenic cell death in sensitive human tumor cells including human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells treated by anthracyclines as anti-cancer drugs. Our findings of increased chaperone proteins after MTX treatment corroborate such published data and support the role of chaperons in tumor immunity.
This study has shown that each of the studied anti-cancer anthracycline/anthracenedione drugs possess typical proteins or protein variants which are specifically changed in level by individual drugs despite of their very close structural similarity which is currently used for their grouping within chemotherapeutic drugs. However, the design of our study allowed us to evaluate and classify proteome maps of all tested anti-cancer drugs to characterize the similarities that would link drug responses. Importantly, the observation of significant decrease of LDHB after treatment of anthracyclines DNR and DOXO as well as anthracenedione MTX thus underlies common anti-cancer effect of this group of drugs directed to the energy metabolism of cancer cell. Nevertheless, it has been important to be aware of the fact, as shown in several examples mentioned above, that the given drug may affect preferentially certain isoform/species of an individual protein hence, in many cases the specific role of such protein isoform/species may play decisive role compared to the quantitative change at the total level of a given protein.
Furthermore, we found several proteins common in DOXO and MTX, among them mainly those directed to the regulation protein synthesis as well as purine and amino acid biosynthesis including MTHFD1 whose increase after treatment by DOXO and MTX was confirmed by Western blot. Regulation of SFRS3 appeared to be a new emerging role because it was recently described as a proto-oncogene critical for cell proliferation and tumor induction and maintenance. It was highly expressed in various cancers and its reduction, mediated by RNAi, resulted in G2/M arrest, growth retardation, and apoptosis [49
]. Accordingly, decreased level of SFRS3after DOXO and MTX treatments offers a new mechanism contributing to anti-cancer activities common to anthracycline/anthracenedione drugs.
Compared to a group of proteins linking the effect of DOXO and MTX, there were only a few proteins shared between DNR and DOXO or MTX thus indicating the distinct position of DNR among the anthracycline/antracenedione drugs. This finding was further corroborated by principal component analysis showing DNR sequestered from DOXO and MTX as well as other treatments such as CisPt and TAX in the first three components covering in total 71% of variances of the whole experimental set. Interestingly, there were two proteins, PSPC1 and HNRNPH3 which were shared between DNR/MTX and DNR/DOXO treatments, respectively, with surprisingly high fold changes observed. PSPC1 is required for the formation of nuclear paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNAs [50
]. It belongs to the family of proteins of the Drosophila behavior/human splicing (DBHS) which are predominately nuclear and influence various biological processes, including carcinogenesis. The significant increase of PSPC1 after DNR and MTX treatments points to possible important role of nuclear paraspeckles in anti-cancer activities of anthracycline/anthracenedione drugs.