Figure 5. Enriched PTMs in Celecoxib-treated and untreated samples. The PTMs of Celecoxib- targets were shown by triangle and others by circle.

Discussion

Celecoxib is one of the top-selling NSAID medicines in the world [60]. Also, NSAIDs involve 5%-10% of the remedy of all prescriptions per year [37, 61]. There are some reports that show the possible indication of celecoxib with the neurodegenerative diseases associated with inflammatory processes [62-66]. Though celecoxib can pass through the BBB and access to the CNS, reports about side effects of celecoxib [67, 68] are related to cardiovascular diseases rather than the nervous system [12]. In other words, the major molecular footprints of this medicine on central nervous system (CNS) are not well-described [12]. Indeed, as we expected, we observed that most of the introduced targets of celecoxib in different databases are not related to CNS. Considering the essential role of celecoxib in the treatment of pain and inflammation, and its influence on the CNS, our study aimed to characterize protein targets of this drug especially in the nervous system.
One of the identified celecoxib-targets is Rab-2A, which is a GTPase required for protein transport from the endoplasmic reticulum to the Golgi complex by regulating COPI-dependent vesicular transport [69, 70]. This protein was common between TPP-identified targets and the PDP database (Fig. 2C). PDP is a powerful up-to-date web resource that unifies various commercial and public bioactive compound libraries [53]. To explore the role of Rab2A in detail, Sugawara et al.studied the effect of Rab2A knockdown on glucose-stimulated insulin secretion and the Golgi intermediate compartment in the corresponding cells. They reported that inactivation of Rab2A mitigated glucose-induced ER stress and inhibited apoptosis induced by ER stress through enlarging of the endoplasmic reticulum (ER)-Golgi intermediate compartment [71]. Therefore, it seems that celecoxib is associated with apoptosis by targeting Rab-2A and implicating ER stress. Providing more evidence through testing celecoxib on the same cells, insulinoma cells, to clarify the celecoxib influence on the ER stress is warranted.
Also, TPP-identified proteins were enriched in pathways related to neurodegenerative disease and cancer. Interestingly, the anticancer activity of celecoxib has been reported in various models of animal tumors, and it is proposed that this drug is beneficial for the prevention and treatment of cancer [72-74]. The molecular mechanisms of antitumoral effects of celecoxib have become a challenging issue, since some reports showed that the effect of celecoxib on cancer is apart from COX-2 inhibition, meaning that celecoxib has other targets than COX-2 [2, 75, 76]. Several components as intermediate candidates have been proposed for the anticancer effects of celecoxib, the most common of which is the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) [1, 77, 78]. Our CC enrichment analysis also disclosed that the endoplasmic reticulum lumen annotation was statistically enriched in TPP-identified proteins, such that several of the proteins involved in the pathways that regulate calcium concentrations, including ERO1A, ARSB, NOL3, STIM1, CALCR, SDF4, and BAX (Figure 4). Interestingly, it has been previously shown that celecoxib increases the intracellular concentration of calcium by inhibiting SERCA [1, 77-79] and the long-term leakage of calcium from the endoplasmic reticulum acts as a potent stimulant of ER stress, which finally leads to cell death and exerts its effect on cancer [77, 80].
Several members of the Ras-associated binding (Rab) family are obviously expressed in various cancer tissues, and dysregulation of Rab expression could be tumorigenic or tumor-suppressive [81]. The Rab family plays an essential role in multiple aspects of membrane trafficking control. Therefore, vesicle transport regulators play crucial roles in the mediation of cancer cell biology, including uncontrolled cell growth, invasion, and metastasis. The Rabs, like other members of the Ras superfamily, function as molecular switches through changes in its guanine nucleotide-binding status between the active GTP-bound and inactive GDP-bound forms. In its active, GTP-bound form, Rabs could mediate vesicular transport by allowing transport carriers or vesicles to engage specific effectors such as motor proteins and tethering factors, as well as vesicle fusion with the engagement of soluble N-ethylmaleimide sensitive factor (NSF) [82] attachment receptor (SNARE) [83, 84] proteins. Vesicle delivery and dynamics are critical for regulating cell behavior associated with cell migration/invasion and tumorigenesis. Cooperation between Rabs and effectors in mediating vesicle movement pathways has significant influences on tumor progression and malignancy. Therefore, it raises the possibility that targeting a particular trafficking system may provide a new approach to cancer treatment [85]. As shown in this study, celecoxib targeted proteins, i.e., RAB2A, RAB10, and RAB11B are notably involved in Rab protein signal transduction. As shown in Figure 4B, TPP-identified proteins are enriched in GDP binding, GTPase activity, and protein phosphatase inhibitor activity that change the GTPases and, as a result, involve in mechanisms associated with cancer. Therefore, it seems that studying the effect of celecoxib on cancer models by TPP provide more supporting evidence.
Neurodegenerative diseases are also assigned to TPP-identified targets of celecoxib as an anti-inflammatory drug. Recent studies demonstrated that neuronal inflammation is a vital trigger of neurological diseases [66], and it exacerbates disorders including Alzheimer ’s -, Parkinsons - , Huntingtons diseases, as well as amyotrophic lateral sclerosis and multiple sclerosis [62-65]. In the present study, some of the mentioned neurodegenerative disorders were enriched based on phenotypic-based biological annotations, such as schizophrenia and depression. Twelve of 44 TPP-identified celecoxib targets are involved in Alzheimer’s disease metabolism, suggesting a high possibility of celecoxib involvement in the mechanisms of this neurodegenerative disease. Notably, inflammation of the nervous system is observed in these disorders, and it is accompanied by an increase in inflammatory cytokines [86-88]. We also illustrated that celecoxib could be beneficial in treating the diseases mentioned above that are associated with inflammation by affecting the biosynthesis pathway of prostaglandins by the involvement of four identified proteins, i.e., DCTN1, PSIP1, BAX and AMPH.
Finally, we describe the importance of PTMs for the thermal stability of proteins. We show that multiple PTMs are involved in the protein thermostability. For example, acetylation, which significantly affects the life span of intracellular proteins by avoiding intracellular proteases degradation, is enriched in all TPP-identified proteins [89, 90]. Citrullination is the specific PTM identified in celecoxib treated sample (See Fig. 5). It is related to the change of arginine to citrulline, which strongly affects the structure and function of proteins in both physiological and pathological processes such as apoptosis, multiple sclerosis, and Alzheimer’s disease [91-93]. Interestingly, an important diagnostic tool in the painful inflammatory disease such as Rheumatoid arthritis is to use anti-cyclic citrullinated peptide (anti-CCP) antibodies which detect citrullination levels of the patients and NSAIDs including Celecoxib are usually prescribed for those patients [94, 95]. Our findings highlight the role of citrullinated proteins as a target of Celecoxib.

Conclusion

Although phenotypic-based screens have become increasingly popular in drug discovery, the major challenge of this approach is the mechanistic deconvolution of the putative drug action during screening. The promising TPP approach has been introduced and expanded to tackle such challenges. In the present study, targets of celecoxib within rat hippocampus were characterized using TPP as a high throughput target discovery approach.
We show that celecoxib plays an effector role in several signaling pathways and biological processes, which can be linked to various diseases such as neurodegenerative disorders and cancer. Therefore, in addition to inhibiting COX2, we illustrate that celecoxib might modify also other pathways. Our findings support the pharmaceutical reports related to the repurposing of celecoxib for cancer and neurodegenerative disorders [96-98]. It seems that celecoxib is potentially beneficial for treating cancer by inhibiting SERCA and increasing the intracellular concentration of calcium, which causes ER stress along with cell death. Another proposed mechanism is affecting the trafficking system since transport regulators play essential roles in the mediation of cancer cell biology and especially circulating tumor cells. We found a significant effect of this medicine on proteins involved in the trafficking system of cells.
On the other hand, neuronal inflammation is a major culprit of neurodegenerative diseases, proteins of which were significantly enriched in the present study. Inflammation in CNS starts by stimulation of astrocytes, and it continues with entering environmental immune cells to the brain. This process causes overproduction of cytokines, nitric oxide, active oxygen species, prostaglandins and eventually damage and cause death of neurons [36, 66, 68, 88]. Our findings support the idea of using celecoxib for neuronal inflammation due to the explored association of celecoxib targets and the inflammation.
To conclude, we identified several novelCelecoxib protein targets using TPP, which could be of interest in order to modify several pathways in CNS. Our findings provide new molecular evidence for celecoxib to be used as an add-on therapy in neurodegenerative disorders and cancer. However, more preclinical and paraclinical evidence is required to suggest the true drug repurposing potential of celecoxib.

Author contribution

 EG and RK performed experiments, contributed to data analysis, interpretation and manuscript first-draft writing. AK, MS, KG and HR contributed to sampling, performing drug treatment and protein extraction. RS and MB contributed to do mass spectrometry analysis. ZT contributed to bioinformatic data gathering. HR, RS, MJ and JT also contributed to data interpretation and manuscript writing. MJ, RJ, JT and MB conceived and commenced the project and provided direction on study design and feedback on the final results.

Acknowledgement

The authors also acknowledge Dr. Rozbeh Jafari and Dr. Farnaz Barneh for helpful comments. This study was financially supported by the National Institute for Medical Research Development of Iran (NIMAD) (Elite Grants, Grant No.964580), Academy of Finland (No. 317680) and European Research Council (No. 716063).

Competing Interests’ Statement

None

Supplementary files

Supplementary file 1: Whole soluble protein concentrations at a range of temperature and celecoxib concentrations. The horizontal axis represents temperature, and the vertical axis shows protein concentration. Each section is dedicated to a particular celecoxib concentration, DMSO-control, and water control. As demonstrated by increasing the temperature, we have a significant decrease in protein concentration.
Supplementary file 2: The distribution of the expression profile of Rab-2A, one of the TPP-identified protein targets, across the rat organs and body based on the TISSUES database.

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