We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/μm . RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of  The potential therapeutic advantage of particle radiotherapy was recognized in the 1940s and was based upon the physical properties of the energy deposition patterns of said particles. Since that time, particle therapy has continuously developed predominantly based upon advances in engineering, imaging, and physics. The first dedicated clinical heavy ion therapy center was opened in 1994, and this facility focused on the use of accelerated carbon ions because of the physical and biological advantages over photons and protons including steeper lateral dose penumbra at greater depths in the body, a higher LET which results in a higher relative biological effectiveness (RBE) and took into account the experience gained at the Lawrence Berkeley Laboratory where the initial results for the use of heavy charged particles as a cancer therapy took place  has been used against intracranial cancers, head and neck cancers, primary and metastatic lung cancers, gastrointestinal tumors, sarcomas, prostate cancer, breast cancer, and pediatric cancers at what has grown to become 12 carbon centers across the globe, although none currently exist in the USA. CIRT has been shown to exert a strong antitumor effect in tumors resistant to conventional photon therapy; however, some tumor sites have been less amenable to therapy over concerns for the response of adjacent normal tissues. Whereas, the efficacy of CIRT has been shown for nonsquamous tumors like mucosal melanomas , 6, aden12C ions against head and neck squamous cell carcinoma (HNSCC) of the oral cavity, larynx, pharynx, and nasopharynx in a fashion that is beneficial for sparing organs at risk such as tissues of the oral cavity, the spinal cord, or bony structures like the mandible and vertebrae has been considered and acted upon. For example, the multi-institutional in silico trial designated ROCOCO describes the benefits of particle therapy and in particular CIRT, in a trial of reirradiation for recurrent HNSCC  tumors. Furthermore, identification of patients as potentially radioresistant by omics or other analysis that requires tumor sampling, is less complicated in H&N cancers based upon ease of access to tumor tissue.p < 0.01) conferring a 9-fold greater risk of mortality at 2 years. Furthermore, using a battery of HNSCC cell lines, tumor growth and metastatic potential were determined in an orthotopic model of oral tongue cancer, including how TP53 mutations influence tumor growth and metastasis  supplemented with 10% Fetal Plus brand fetal bovine serum , penicillin/streptomycin solution . All cell lines were authenticated by genotyping and validated as negative for mycoplasma contamination by the Molecular Diagnostics Core Services at the Dana Farber Cancer Center, Boston, MA. All cell culture was conducted in incubators at 37°C in ambient 5% COAn expression microarray dataset (GEO accession number GSE 67614) that was generated from 102 tumor samples collected from patients treated with a consistent protocol of surgery followed by radiotherapy based upon a prospective trial that evaluated pathologic risk features, total combined treatment duration, and postoperative radiation therapy  was used137Cs-sourced irradiator or at the MD Anderson Cancer Center using the “NASAtron” 137Cs irradiator. Dosimetry for these sealed source irradiators was validated on an annual basis. Briefly, for the J. L. Shepherd, irradiator cells in culture were placed on a 360° platform revolving at 13 RPM, irradiated, removed from the irradiator, and immediately returned to the incubator. For the NASAtron irradiations, the source was vertically above the stage and there was no sample rotation. Both devices had dose rates of ~3.25 Gy/min.Photon irradiation was conducted at the University of Texas Southwestern Medical Center using a J. L. Shepherd sealed horizontal 12C irradiations took place at the Centro Nazionale di Adroterapia Oncologica (CNAO) facility in Pavia, Italy. Cells were irradiated in T12.5 cm flasks while immersed in a water bath at 37°C using CNAO’s clinical, therapeutic quality, pencil beam scanning 12C-ion beam. A spread-out Bragg peak (SOBP) was created to assure a homogenous ( ± 2.5%) physical dose. The beam quality has been previously characterized  Using 2% FBs for such a limited time had no effect on cell growth or radioresponse.Following incubation, cultures were rinsed with phosphate-buffered saline at a pH of 7.4 and fixed in a solution of 0.5% crystal violet and 10% methanol in water. After staining and drying, colonies were counted to determine the number of surviving cells following irradiation. Only colonies identified as having more than 50 cells per colony were scored as surviving, and the surviving fraction was determined by dividing the number of colonies by the product of the plating efficiency of the cell line multiplied by the number of cells seeded.d is the dose per fraction and a, b, and c are parameters determined using a curve fitting algorithm  Model as described in Equation 1 where lgorithm .The γ-ray survival assays were performed at least twice for each cell line. If the coefficient of variation at 2 Gy was greater than 25%, they were repeated.137Cs exposures (reference) to that same radiosensitivity value determined from 12C exposures (test) as in Equation 2.RBE values were calculated by comparing a radiosensitivity value from Radiosensitivity parameters included:10% was calculated using values generated with the RCR model as described in Equation 1.The dose at SFuation 3 .Here, D0, represents the linear portion of the RCR fit located at the distal end of the survival curve. D0 was calculated by plotting the linear portion of the RCR line of best fit in MATLAB and using a linear to calculate the slope. The relationship between the slope of the linear portion and D0 is given in Equation 4.The limiting slope, In vitro transcription was done to synthesize biotin-labeled cRNA using T7 RNA polymerase. The cRNA was column purified and then checked for size and yield using the Bio-Rad Experion system. A total of 1.5 µg of cRNA was hybridized for each array using the standard Illumina protocols with streptavidin-Cy3  being used for detection. Slides were scanned on an Illumina Beadstation. Summarized expression values for each probe sets were generated using BeadStudio 3.1 .The tumor data set was generated using Illumina Whole Genome HumanWG6 v2 arrays (GEO accession number GSE67614). The v3 Illumina Expression BeadChip was used to generate transcriptome profiles for the HNSCC cell lines. Each RNA sample with 0.5 µg of total RNA was amplified using the Illumina TotalPrep RNA amplification kit with biotin UTP (Enzo) labeling. The Illumina TotalPrep RNA amplification kit uses T7 oligo(dT) primer to generate single-stranded cDNA followed by a second-strand synthesis to generate double-stranded cDNA which is then column purified. The Illumina BeadChip expression data were background subtracted and quantile–quantile normalized across samples using the MBCB algorithm –29. Normp-values generated from an F-test using the R limma package. The top-ranked genes corresponded to those that significantly changed in more than one of the radiosensitivity groups. Feature selection was performed by incrementally expanding the gene list from 4 to 500 from the top of the gene ranking. Classification models were built using a support vector machine (SVM) algorithm and the models were validated using a 10-fold repeated cross-validation.The gene expression values from 38 HNSCC cell lines were ranked by p-values calculated from moderated t-test using the R limma package. The R limma analysis was performed to compare resistant cell lines vs. other cell lines or tumors where there was local recurrence (LR) vs. tumors where there was NED in patients treated with postoperative radiotherapy. A false-discovery rate (FDR) <0.4 was used as a cutoff for significantly enriched pathways and then interrogated against the GSEA Hallmark Pathways by Leading Edge Analysis for pathway enrichment using the metrics of p-value, FDR, and Normalized Enrichment Scores, whereas the IPA analysis focused on pathways were enriched based upon a Fisher’s test with a p-value of 0.05 used as a cutoff. Z-scores for these pathways were then calculated with negative scores representing downregulation and positive scores representing upregulation of a given pathway.Functional pathway analysis was performed using the Gene Set Enrichment Analysis (GSEA 4.0.1) and Ingenuity Pathways Analysis (IPA) online software packages. Genes were ranked by Equation 5 where Nvox is the number of voxels in an in silico tumor, iN is the number of cells in voxel i, and i,j S is the surviving fraction in voxel i at fraction j with dose d, oxygen partial pressure 2pO, and LET L.The calculation of tumor control probability (TCP) is adapted from Antonovic et al.  and is s3) was assumed to contain 108 cells distributed equally across the voxels in the in silico tumor simulation. The survival model utilized is the Repairable Conditionally Repairable (RCR) model which can account for changes in survival due to dose, LET, and partial oxygenation, as shown in Equation 6.A spherical tumor with a radius of 0.6203 cm  was determined based upon the fitted values for SF2 and SF3.5 from the survival curves. The cell lines were clustered into four groups based upon Euclidean distances using the SF3.5 values for each cell line as depicted in A collection of 38 HNSCC cell lines was tested for radioresponse To determine whether there were differences in gene expression underlying these radiosensitivity cohorts, an SVM model was trained using the most significantly changed genes. As depicted in Z-scores.GSEA and IPA were used to examine differences in Hallmark Pathways (GSEA) and canonical pathway signaling (IPA). As shown in These same pathways were then interrogated in a gene expression dataset derived from 102 flash-frozen HNSCC tumor specimens from patients treated by postoperative radiotherapy (PORT) using a consistent treatment protocol , 32, 33.12C radioresponse of five HNSCC cell lines whose SF3.5 ranged from average to resistant when compared with the larger panel of 38 cell lines to determine if 12C irradiation can overcome radioresistance. Radiation survival curves were generated using 137Cs as a low LET radiation and 12C ions were generated by the Carbon Therapy Center in Pavia, Italy (CNAO). The cell lines SqCC/Y1 and UMSCC1 were chosen to represent the range of SF3.5 in the MR group with an emphasis on the high end of response; HN31 represents the highest SF3.5 in the MS cohort; and HN5 and SCC9 represent the R cohort. Survival curves for these cell lines are seen in 12C ions was calculated at: 10% survival; by mean inactivation dose D0) for each cell line. Those values are found in Next, we examined the lgorithm ; D¯ us12C ions from the five HNSCC cell lines, TCP curves were generated based upon Antonovic etal. (12C) were based upon schedules used in recurrent H&N cancers treated at the Shanghai and Heidelberg Heavy Ion radiotherapy facilities  by reirradiation for recurrent HNSCC with CIRT developed little in the way of either acute or late severe (>Grade III) toxicity  using a 12C ions. Radioresistant HNSCC can be manifested via a number of mechanisms, the predominant mechanism being hypoxia, but resistance may also be through acquired mechanisms of enhanced DNA repair, abrogation of apoptosis or through the development of a tolerogenic immune environment. 12C ions—or for that matter other ions separately or in combination—should at least partially overcome these limitations.The comparable overall improvements in late normal tissue toxicities in H&N cancers treated with CIRT suggest the potential for overall improvements in outcome in primary and recurrent disease as well as the potential for dose escalation in tumors identified as radioresistant. Also, given the limitations of access, if patients with tumors that are radioresistant can be identified, the argument could be made to treat those patients where possible with via clonogenic survival. Basal gene expression analysis was then tested as a surrogate for clonogenic survival and found to be capable of segregating the most radioresistant cell lines from the other cell lines. Furthermore, the gene expression patterns in these radioresistant cell lines supported their classification as radioresistant. Besides identifying some of the more commonly identified genes and pathways associated with radioresistance, one of the more intriguing finds is the increased expression of the GAGE genes. These genes are not expressed in normal tissue with the exception of testes. In tumors, members of this family (GAGE1 and 2) are CD4+T cell antigens, attracting T cells into tumors. GAGE12 family members have been shown to increase both radio- and chemoresistance and metastasis  for dose equivalent, dose equivalent for 12C radiotherapy is described as GyEq, GyE, or GyRBE because there is no unit definition for dose equivalent in a deterministic setting. GyEq relies on the determination of the RBE for a given endpoint. The earliest determinations of RBE, still used today, were based upon cell survival in mostly rodent (CHO) cell lines and the human radioresistant salivary tumor cell line HSG, which was determined to be contaminated with HeLa cells (12C scattered beam at NIRS was normalized to the dose-averaged LET at 80 KeV/µm using their clinical neutron RBE of 3.0 as they were equally effective (12C RBE in a small cohort of moderate to radioresistant cell lines using multiple endpoints and then applied the survival and RBE values to model tumor control probability and asked whether individualization of RBE could be consequential when compared to a generic value.Assuming that one will be able to identify radioresistance so that patients could be triaged to receive La cells , 54. Theffective . This alffective , 30, 56.12C, because this is GyE and uses a more conventional fraction size, is comparable with the conventional 2 Gy/fraction regimen. The SabR approach with 12C ions may be more effective for carbon ions but suffers the same complications as a conventional SAbR approach within the radioresistant subset.12C physical dose and it is consequential in that it reflects the need to understand the intrinsic radioresistance for a given tumor. Those values of “underdosing” at a TCP of 70% are given in In 12C and because HNSCC outcomes have seen little improvement. At least for cell lines, gene expression was able to segregate radioresistant tumor cell lines from cell lines less resistant. One could argue that 38 cell lines do not bring enough diversity of radiosensitivity as the most radioresistant cell lines were few in number. Also, while there was overlap in the enrichment of specific pathways in common between cell lines and tumors that were treated with PORT, it is evident that much work is needed, irrespective of the gene expression analysis.In this manuscript, we have attempted to draw attention to the need for individualizing therapy based upon the intrinsic radioresponse of a tumor. Head and neck squamous cells were used because of the recent trials where HNSCC are being treated with 12C exposures. This exercise highlighted the potential for underdosing radioresistant tumors when generic rather than personalized RBEs were used, which would negate the impact of triaging patients with radioresistant tumors.Using five cell lines whose radiosensitivity ranged from moderate to resistant across the 38 cell line panel, the potential impact of intrinsic radiosensitivity was tested by determining TCP curves using various radiation regimens for both γ-ray and 12C ions with different dose and fractionation schedules and a realistic understanding of the impact of tumor hypoxia to challenge and ultimately improve upon the biophysical modeling of CIRT with the intent to ultimately personalize therapy.We understand that our TCP calculations reflect fully oxygenated tumors, did not reflect tumor response to varied LETs, etc., but our goal is more one of relative comparisons and not necessarily quantitative comparisons. What we require are data from actual tumors treated with curative intent using X-rays and The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.MS conceived of and carried out experiments, wrote the manuscript, and led the research program. AD conceived of and carried out experiments, edited the manuscript, and participated intellectually. AP was responsible for radiation physics, helped develop the beam configuration, and wrote a portion of the manuscript. DS was partly responsible for designing and carrying out experiments. MC was responsible for 12C dosimetry, developing the SOBP for CNAO, and all physics aspects of 12C irradiation. AF was the CNAO biology contact person. She helped facilitate experiments and helped with the design of our irradiation setup at CNAO. JY was responsible for collecting the panel of H&N tumor cell lines, designed and carried out experiments to determine the X-ray survival parameters for these cells, and provided DNA and RNA for omics analysis. EP carried out additional survival X-ray survival curves, plotted all data, and calculated survival fits and tumor control probability curves. BS conceived of and carried out both X-ray and 12C experiments. He was involved in the interpretation of data and wrote sections of this manuscript. LD was responsible for all omics analysis, interpretation of that data, and wrote sections of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.Funds were provided through the Department of Radiation Oncology Seed Grant Program to MS as well as the David A. Pistenmaa M.D., Ph.D. Distinguished Chair in Radiation Oncology to MS. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The annual number of cancer deaths continues increasing every day; thus, it is urgent to search for and find active, selective, and efficient anticancer drugs as soon as possible. Among the available anticancer drugs, almost all of them contain heterocyclic moiety in their chemical structure. Xanthone is a heterocyclic compound with a dibenzo-γ-pyrone framework and well-known to have “privileged structures” for anticancer activities against several cancer cell lines. The wide anticancer activity of xanthones is produced by caspase activation, RNA binding, DNA cross-linking, as well as P-gp, kinase, aromatase, and topoisomerase inhibition. This anticancer activity depends on the type, number, and position of the attached functional groups in the xanthone skeleton. This review discusses the recent advances in the anticancer activity of xanthone derivatives, both from natural products isolation and synthesis methods, as the anticancer agent through in vitro, in vivo, and clinical assays. Cancer is one of the most fatal diseases in the world. Cancer mainly occurs due to gene mutations, and the condition is worsened by other carcinogenic agents. Both gene mutations and carcinogenic agents then influence and change the cell functions and metabolism, and thereby, the replication and spread of cancer cells are uncontrolled. The cancer cells grow and multiplicate rapidly, thus crowding the other normal cells . In someThe research on anticancer drug design and development is an urgent need, because people are suffering and the number of death cases is unstoppable until active, selective, and efficient anticancer drugs are found ,6,7. CanGentiana lutea in 1821. Xanthone with an IUPAC name of 9H-xanthen-9-one is a heterocyclic compound with a dibenzo-γ-pyrone framework, with a basic molecular formula of C13H8O2 + and [M + 2]+ in 3:1 and 1:1 ratios, respectively. In contrast, elucidation of fluoro- and iodo-substituted xanthones from their MS spectrum is difficult, as both halogen atoms are only found as a single isotope. Therefore, fluoro- and bromo-substituents in xanthones can be observed using the 19F-NMR and 127I-NMR techniques, respectively xanthone structure seemed to be critical for the anticancer activity against these cell lines [In 2008, Varache-Lembège et al. synthesized several xanthone derivatives and evaluated their anticancer activity. Among the prepared xanthones, compound ectively . On the ll lines . It was ll lines .4 exhibited the best anticancer activity, as measured by inhibitory activity (3.57–20.1 µM) against the aforementioned cancer cells. For a specific MGC-803 cancer cell, compound 4 exhibited a time-and dose-dependent proliferation inhibition. For a concentration greater than 1.0 µM, MGC-803 cells viability decreased significantly, due to lower mitochondrial membrane potential and intracellular calcium leading to the apoptosis mechanism [In 2014, Yang et al. examined the anticancer activity of several synthetic compounds based on 1,3-dihydroxyxanthone against CNE, MGC-803, Bel-7402, and A549 cell lines. It was reported that mono- and dioxygen functional groups were pivotal for the inhibitory activity against protein kinases. The presence of the aminoalkoxy group showed the increment of anticancer activity, while the introduction of the bromoalkoxyl group did not improve the anticancer activity. When the terminal amino group was quaternary ammonium salt, the anticancer activity of xanthone became weaker, due to poor cytomembrane penetration. The different terminal amino substituents had different effects in the increment of anticancer activity with the order: diethylamino > dimethylamino > pyrrolidine > piperidine > morpholino. Compound echanism .5a gave the IC50 value of 25.7, 33.2, and >50.0 µM against ECA109, SGC7901, and GLC-82 cell lines, respectively. Meanwhile, compound 5b with aminoalkyl moiety gave lower IC50 values of 9.56, 13.3, and 16.1 µM against the same cancer cell lines, respectively. The presence of polar aminoalkyl moiety in compound 5b gave a higher hydrophilicity; thus, this compound could easily penetrate the cancer cell membrane, leading to anticancer activity enhancement. Additionally, the aminoalkyl moiety having a higher pKa value is easily protonated which generate a cationic charge in order to bind with the DNA through its phosphate groups. This phenomenon blocked the DNA replication which is related to the inhibitory activity against the evaluated cancer cell lines [6 was the most potent anticancer agent against the A375-C5, MCF-7, and NCI-H460 cell lines, with IC50 values of 32.2, 22.6, and 14.1 µM, respectively. They found that the effect of the growth inhibitory activity of the xanthone compound was not only affected by the nature and position of the substituent but also the enantioselective effect. The presence of aryl group on the stereogenic center of xanthone was found to enhance the inhibitory activity against cancer cell lines [R)-isomer of compound 7 gave lower IC50 value (IC50 = 24.0 µM) than the (S)-isomer (IC50 = 112 µM) against MCF-7 cancer cell line. This phenomenon was caused by a more stable complex conformation between the (R)-isomer with the DNA through crosslinking reactions than the (S)-isomer one [In 2014, Shen et al. synthesized several xanthones through the modification of the free hydroxyl group of xanthone with dimethylamine group. The inhibitory activity of these xanthones was tested against ECA109, SGC-7901, and GLC-82 cell lines. It was found that the presence of aminoalkyl moiety was critical to its anticancer activity, as well as DNA binding. Moreover, the addition of dimethylamino side-chain resulted in the enhancement of anticancer activity. Compound ll lines . On the omer one .8 exhibited the highest anticancer activity with the IC50 value of 6.18, 8.06, 4.76, 4.59, and 6.09 µM against PC-3, MDA-MB-231, AsPC-1, A549, and HCT-116 cell lines, respectively. Compound 8 promoted cell cycle arrest, induced cell apoptosis, and provided antiproliferative effect on cancer cell viability. The presence of the 4-methyl group was critical in the xanthone structure, as it exhibited a higher potent anticancer activity. In contrast, the presence of oxygen atoms on the side chain of xanthone reduced the growth inhibitory activity against these cancer cell lines. This effect may be generated from the possibility of hydrogen bonds formation with other biological macromolecules outside the active site of the protein target. Additionally, the steric effect of the substituent at the side chain may also contribute to a lower inhibitory activity [In 2016, Liu et al. synthesized xanthone derivatives bearing 3,6-disubstituted aminocarboxymethoxy moiety and evaluated them as the anticancer agent. Among the evaluated xanthones, compound activity . A similactivity .9 was found to give the most potent inhibition of the catalytic activity of topoisomerase IIα, as evaluated using DNA relaxation assay. Compound 9 inhibited DNA relaxation with the IC50 of 1.00 µM. Specifically, compound 9 acts at the DNA cleavage/ligation active side and compound 9 is able to inhibit the ability of DNA to stimulate the rate of ATP hydrolysis. Furthermore, the presence of primary and secondary amine side-chains is more important, rather than the number of primary amines in the chain, or their distance from the primary amine [Minniti et al. in 2017 investigated the anticancer activity of synthetic xanthone derivatives with various polyamine moieties, including spermine, spermidine, butanediamine, and propanediamine at C-3 position. It was reported that the presence of the secondary amine group in the side chain significantly affected the topoisomerase II-drug interaction. Compound ry amine .10 showed the highest anticancer activity against MDA-MB-231 cell line, with an IC50 value of 0.46 µM. The inhibitory activity of compound 10 against MDA-MB-231 cell line was found to be much stronger compared to DMXAA (IC50 = 48.0 µM). Additionally, compound 10 also showed potent inhibitory activity against MCF-7, HepG2, K562, and COLO-320 cell lines, with IC50 values of 3.40, 9.20, 13.4, and 10.5 µM, respectively. The inhibitory effect was found as the result of induced apoptosis of the evaluated cancer cell lines. The effect of the electron-withdrawing group introduced to the opposite aromatic ring of 1,3-dihydroxyxanthone derivatives increased the anticancer activity, while the electron-donating group showed the opposite effect. Remarkably, the xanthone derivatives showed an enhanced inhibitory activity when combined with DMXAA. This synergistic effect demonstrated a good approach for the design and development of more potent anticancer drugs [In 2017, Liu et al. examined the anticancer activity of synthetic xanthones against MCF-7, MDA-MB-231, HepG2, K562, and COLO-320 cell lines. Among the synthesized xanthones, compound er drugs .11a–b with fluoro substituents were active against A549 cells, whereas compounds 11c–d were inactive against this cancer cell line. The anticancer activity of compound 11a (IC50 = 29.9 µM) and compound 11b (IC50 = 24.9 µM) was stronger than cisplatin (IC50 = 32.4 µM) as the positive control. In contrast, the anticancer activity of non-fluorinated xanthones (IC50 = 6.14–14.0 µM) was stronger than that of fluorinated ones (IC50 = 27.2–37.4 µM) against SMMC-7721 cell line. The anticancer activity of non-fluorinated xanthones 11c–d (IC50 = 6.14–14.0 µM) was also stronger than cisplatin (IC50 = 26.3 µM) [In 2018, Zhou et al. synthesized several xanthone derivatives and evaluated their anticancer activity against A549 and SMMC-7721 cancer cell lines. Compounds 26.3 µM) .12 was reported as the best anticancer agent against MCF-7, TK-10, and UACC-62 cells with the IC50 values of 21.9, 34.3, and 20.0 µM, respectively. In addition, the compound 12 is considered to be safe for the anticancer application as no cytotoxic to the human lymphocytes (cell viability > 70%) is observed. The nature of substituents in oxygenated xanthone structure had a significant effect on its anticancer activity. For example, the 1,2-dihydroxyxanthone exhibited a weaker anticancer activity than 2-hydroxy-1-methoxyxanthone. Therefore, the suitable functional group should be considered to be a critical parameter in the design of oxygenated xanthone derivative, with high potential for anticancer activity [Recently, Pedro et al. synthesized oxygenated xanthones and evaluated their anticancer activity against MCF-7, TK-10, and UACC-62 cell lines. Additional oxygenated groups in the xanthone core gave a significant effect on the anticancer activity enhancement. Among several synthesized xanthone derivatives, compound activity .13) was one of the most active anticancer agents. The anticancer activity of compound 13 against WiDr cells gave an IC50 value of 37.8 µM. Furthermore, compound 13 was not toxic against Vero cell lines (IC50 = 2510 µM), giving a selectivity index (SI) of 66.4, which was higher than doxorubicin (49.4) [13 suppressed the mRNA cyclooxygenase-2 (COX-2) expression by 37%, with no inhibitory expression against vascular endothelial growth factor receptors. Therefore, it was concluded that compound 13 inhibited the COX-2 enzyme and started chronic inflammation in the cancer cells. A molecular docking study showed that compound 13 interacted with Tyr355 and Arg120 amino acid residues of COX-2 enzymes, yielding a binding energy of −77.0 kcal/mol [Our research group has been working on anticancer research employing xanthone derivatives since 2010. Several hydroxyxanthone derivatives including 2-hydroxyxanthone, 3-hydroxyxanthone, 1,3-dihydroxyxanthone, 1,6-dihydroxyxanthone, 3,4-dihydroxyxanthone, 3,6-dihydroxyxanthone, 1,3,6-trihydroxyxanthone, 1,5,6-trihydroxyxanthone, 3,4,6-trihydroxyxanthone, and 1,3,8-trihydroxyxanthone have been synthesized in 2010–2021 ,125,126.n (49.4) . From thkcal/mol .50 values of 206–666 µM. These IC50 values are lower than hydroxyl derivatives (786–828 µM) against the same HepG2 cancer cell line. Furthermore, the SI value of chlorinated-hydroxyxanthones was higher (5.31–22.0) than hydroxyxanthones (2.58–10.60) against HepG2 cells. On the other hand, 4-chloro-1,3-dihydroxyxanthone also gave a lower IC50 (12.5 µM) than 1,3-dihydroxyxanthone (68.0 µM) against the P388 cancer cell line. The 4,5,7-trichloro-1,3,6-trihydroxyxanthone gave a lower IC50 (5.21 µM) than 1,3,6-trihydroxyxanthone (23.5 µM) against P388 cells. Meanwhile, the 4-chloro-3,6-dihydroxyxanthone gave a lower IC50 (0.69 µM) than 3,6-dihydroxyxanthone (10.4 µM) against P388 cancer cells. The SI value of chlorinated-hydroxyxanthones were much higher (87.6–9200) than hydroxyxanthones (31.2–844) against P388 cell line. From the molecular docking study, it was found that chlorinated-hydroxyxanthones were able to inhibit the c-KIT protein through hydrogen bond interactions with Asp810, Cys809, Ile789, His790, and Leu644 [Anticancer activity of chlorinated-hydroxyxanthones has been evaluated against HepG2 and P388 cell lines. Chlorinated-hydroxyxanthones, i.e., 4-chloro-1,3-dihydroxyxanthone, 4,5,7-trichloro-1,3,6-trihydroxyxanthone and 4-chloro-3,6-dihydroxyxanthone are inactive against HepG2 cells with ICd Leu644 .14) gave no anticancer activity against HeLa (IC50 = 251 µM), T47D (IC50 = 1398 µM), and HepG2 (IC50 = 262 µM) cancer cell lines. In contrast, compound 14 gave moderate anticancer activity against P388 (IC50 = 5.21 µM) cancer cells. Furthermore, the IC50 value of compound 14 (IC50 = 15.9 µM) was lower than doxorubicin (IC50 = 25.4 µM) against Raji lymphoma cell line. The toxicity of compound 14 for Vero cell lines was reported to be in IC50 of 256 µM, yielding an SI value of 16.1. From the molecular docking study, compound 14 gave strong binding energy against Raf-1  and c-Jun-N-terminal protein kinase (c-JNK)  proteins. Compound 14 formed hydrogen bonds with Cys424, Lys431, Ser427, and Gly426. The number of the hydrogen bond interactions (4) of compound 14 with Raf-1 protein was much higher than for the native ligand (1 hydrogen bond with Cys424). Compound 14 generated additional hydrogen bonds with Met111, Glu109, and Ser34 (two hydrogen bonds) on the active site of c-JNK protein. The number of hydrogen bond interactions (4) of compound 14 with c-JNK protein was also higher than the native ligand (2 hydrogen bonds with Met111 and Glu109) [The 4,5,7-trichloro-1,3,6-trihydroxyxanthone ( Glu109) .14 and doxorubicin against Raji lymphoma cell line has also been studied. It is well-known that drug resistance can be overcome through drug combination as the mechanism of action of two/more drug compounds, which may be different from in their individual usage. Furthermore, a synergistic effect of two/more drugs could lower the dose and thus suppress the side effects to the human body [14 inhibited Raf-1 protein and activated c-JNK protein. Inhibition of Raf-1 protein led to a higher sensitivity of cancer cells to doxorubicin. On the other hand, the activation of c-JNK protein led to the translocation of pro-apoptotic protein Bax to cytoplasm and stimulated the apoptosis mechanism of cancer cells [The synergistic effect between compound man body . Combinaer cells .15a and 15b, have also been prepared from 2,4-dihydroxybenzoic acid. The compound 15a was synthesized through bromination reaction of 2,4-dihydroxybenzoic acid first, and then followed by a cyclization reaction with phloroglucinol. On the other hand, compound 15b was obtained from a bromination reaction of 1,3,6-trihydroxyxanthone. Both compounds gave moderate anticancer activity against P388 cell line, with IC50 and SI values of 6.34–10.7 µM and 43.2–74.4, respectively. The molecular docking study revealed that the anticancer activity of brominated-hydroxyxanthones was generated by hydrogen bonding interactions with His790, Cys809, Leu644, Ile789, and Asp810 on the active site of c-KIT protein [Two new brominated-hydroxyxanthones, i.e., compounds  protein .16), a famous glycosylated xanthone, was reported for its anticancer activity against colorectal cancer cells through the inhibition of bcr/abl gene expression, thus inducing cellular apoptosis. In 2013, Li et al. isolated mangiferin from mangosteen fruit, and evaluated its anticancer activity against MDA-MB-231 and BT-549 cell lines. Mangiferin exhibited poor anticancer activity against MDA-MB-231 and BT-549 cells, giving the IC50 of 299 and 274 μM, respectively. Mangiferin gave a lower expression of matrix metalloproteinase-7 (MMP-7), MMP-9, beta-catenin, and vimentin, and simultaneously gave a higher expression of E-cadherin, thus leading to the antiproliferative phenomenon [Glycosylated xanthones have also been evaluated as anticancer agents. Mangiferin  [Psorospermum febrifugum in Africa in 1980. This compound exhibited in vitro and in vivo anticancer activity against wild type and drug-resistant leukemia cells. Furthermore, good anticancer activity of psorospermin against breast and colorectal cancer cells has also been reported. The psorospermin acts as the anticancer agent through DNA intercalation between base pairs at positions 11 and 12, as well as guanine alkylation on the topoisomerase II protein with its epoxydihydrofuran moiety. The DNA alkylation on the active site of topoisomerase II protein leads to inactivation on the DNA replication of cancer cells [20) isolated from the dry latex of Garcinia hanburyi. It was reported that desoxymorellin gave IC50 values of 0.74, 0.77, and 1.15 µM against HeLa, K562, and K562/R cell lines, respectively, through the apoptosis induction mechanism [Prenylated xanthones are the most evaluated xanthones, due to their promising application as the anticancer agent. In general, prenylated xanthones exhibited higher anticancer activities than the other classes. Early in 1992, Sordat-Diserens et al. reported that compound 3.08 µM) . The anter cells . Anotherechanism ,137.Cratoxylum cochinchinense and evaluated its anticancer activity against NCI-H187. This compound showed a potential cytotoxic effect against NCI-H187 cancer cell line with an IC50 value of 1.45 µM. Remarkably, it showed no cytotoxic activity against KB and BC-549 cell lines, which indicated excellent selectivity against NCI-H187 cancer cells. It was proposed that the presence of geranyl moiety in the isolated compound corresponded to its strong anticancer activity [21), from the early ripe fruit of Garcinia mangostana. The anticancer activity of mangostenone C was evaluated against KB, BC-1, and NCI-H187 cells, giving IC50 values of 6.11, 7.70, and 8.11 µM, respectively. The anticancer activity of mangostenone C against NCI-H187 was still weaker, but comparable to α-mangostin (5.07 µM) and gartanin (IC50 = 2.72 µM). This finding means that the tetraoxygen framework exhibits stronger anticancer activity [In 2006, Laphookhieo et al. isolated a prenylated xanthone from the roots of activity . In the activity .50 of 0.38–4.45 µM [22) as the main component in gamboge, an orange-brownish resin exuded from the plant of Garcinia hanburryi, which is often used as traditional medicine for cancer treatment. The anticancer activity of gambogic amide was found to be related to the apoptotic activity against T17 cells with an IC50 of 5.00 nM. The T17 cell line was derived from the basal forebrain of SN56 cell; therefore, T17 cells were defined as TrkA stably transfected SN56 cells. The gambogic amide could selectively bind to TrKA, triggering its tyrosine phosphorylation, and preventing neuronal cell death. These phenomena yielded an agonist effect to nerve growth factor (NGF) thus giving potent neurotrophic activity. Moreover, gambogic amide could block apoptotic machinery independent of the Trk receptor, which led to its ability to trigger programmed cell death for the cancer cell lines [Gambogic acid is one of the most famous natural xanthones as an anticancer agent ,141. Gam–4.45 µM ,145,146.–4.45 µM . Gambogi–4.45 µM ,151,152.ll lines .Terminalia calcicola named as termicalcicolanones A (23) and B (24) have been reported in 2007. Termicalcicolanones A and B showed weak to moderate anticancer activity against A2780 cells, with IC50 values of 40.6 and 8.10 µM, respectively [25 gave the most promising inhibitory activity on the K-562 cell viability, with an IC50 value of 20.0 µM. Additionally, compound 25 gave a moderate inhibitory activity against HL-60 and BV-173, with an IC50 value of 7.00 and 14.0 µM, respectively. The inhibitory mechanism was suggested to come from the antiproliferative and apoptotic effects on the evaluated cancer cell lines [Two new xanthones isolated from ectively . With a ectively . A combiectively . In 2010ll lines .26) against LNCaP, 22Rv1, DU 145, and PC-3 cancer cell lines, with IC50 values of 5.90, 6.90, 22.5, and 12.7 µM, respectively. The α-mangostin could induce cancer cell apoptosis at a concentration equal to or higher than 15 µM. Meanwhile, α-mangostin can promote G0/G1 cell cycle arrest at a lower concentration (<15 µM) [50 values of 2.44–28.5 µM [In 2011, Johnson et al. evaluated the anticancer activity of pure α-mangostin ((<15 µM) . Other r–28.5 µM ,163,164.–28.5 µM ,165. Fur–28.5 µM ,166. A s–28.5 µM . The int–28.5 µM . Azevedo–28.5 µM . On the –28.5 µM .27), from the hull of mangosteen fruit. The γ-mangostin was one of the major xanthone components in the mangosteen fruit. The γ-mangostin was evaluated as the anticancer agent against HT-29 cells. Unfortunately, γ-mangostin was non-active against HT-29 cells (IC50 = 68.5 µM). This inhibitory activity occurred through the induced apoptosis mechanism [28 showed the most potent anticancer activity, with IC50 values of 14.3, 15.8, and 5.17 µM for A549, MDA-MB-435, and HCT-116, respectively. The inhibitory activity of compound 28 as an anticancer agent relies on topoisomerase I protein inhibition, which led to the DNA relaxation of cancer cells [In 2012, Chang and Yang isolated another natural xanthone, γ-mangostin , and garciniaxanthone E (31) exhibited strong anticancer activity against HL-60 cell line, with IC50 values of 2.80, 3.40, and 3.10 µM, respectively. Compound 29 and garciniaxanthone E had prenyl moiety at their structure which corresponded to a stronger anticancer activity against HL-60 cells, indicating that the prenyl groups play an important role in the growth inhibitory activity. The number of prenyl groups was also suggested to significantly increase the inhibitory activity of xanthone. However, the addition of prenyl groups at the side-chain as in garciniaxanthone E did not significantly affect its activity compared to compound 29. Garciniaxanthone E was also reported for its anticancer activity against breast, lung, liver, gastric, colorectal, and leukemia cancer cell lines through the activation of caspase proteins and inhibition of PG-E2 pathways [In 2012, Niu et al. isolated a bioactive compound-based xanthone derivative from the stem bark of pathways .32 showed the most promising anticancer activity. Compound 32 gave IC50 values of 8.90 and 4.50 µM against HeLa and MDA-MB-231 cell lines, respectively. The anticancer activity of compound 32 was twice as strong as doxorubicin and cisplatin as the positive standards. The presence of nonplanar geminal-diprenylated rings was suggested to enhance its inhibitory activity against the cancer cell lines [33 showed the highest anticancer activity, with IC50 values of 2.62, 2.10, and 16.4 µM against HepG2, A549, and U-251 cell lines, respectively. Moreover, compound 33 also showed inhibitory activity against serine/threonine protein kinase, nuclear factor kappa-B kinase subunit gamma (IKK-β), with an IC50 value of 8.02 µM. The introduction of hydrophobic moiety in the aza-caged xanthone structure leads to a stronger anticancer activity. Moreover, it was reported that the presence of substituent group with larger volume was preferred in the anticancer activity enhancement, due to preferable interaction with the putative receptors. Compound 33 exhibited the anticancer activity through the apoptosis induction mechanism [Lim et al., 2012 synthesized several xanthone derivatives owing to the prenyl group, and evaluated their anticancer activity against HeLa and MDA-MB-231 cell lines. Among the synthesized compounds, compound ll lines . In the echanism .34 was considered to give strong anticancer activity against HepG2 and A549 cell lines with IC50 values of 3.25 and 3.60 µM, respectively. Furthermore, compound 34 was also found to have good bioavailability, and it was orally active, as tested through an in vivo assay. Compound 34 was capable of giving the inhibition rate of 58% against tumor growth (hepatoma solidity/Heps) at 100 mg/kg daily oral dose for 4 days, which was much better compared to the natural product with a similar structure, gambogic acid. However, a further clinical evaluation has not been reported yet [In 2013, Zhang et al. synthesized a natural xanthone derived from Garcinia species and evaluated its anticancer activity against HepG2 and A549 cell lines. Compound rted yet .Garcinia achachairu were investigated as the anticancer agents against nasopharynx cancer cells . A new prenylated xanthone (35) showed a remarkable anticancer activity with the IC50 of 1.43, 0.73, and 2.23 µM against CNE-1, CNE-2, and SUNE1 cell lines, respectively. On the other hand, the anticancer activity of compound 36 was evaluated against U-251, MCF-7, NCI/ADR-RES, 786-0, NCI-H460, PC-3, HT-29, and HaCat cell lines, giving IC50 values of 11.2, 34.0, 8.61, 19.6, 56.2, 10.5, 58.1, and 30.6 µM, respectively, which was comparable to doxorubicin [Garcinia paucinervis plant, which yielded a new compound, paucinervin I (37). Paucinervin I showed strong anticancer activity against HL-60 cell line with the IC50 value of 1.30 µM. The anticancer activity of paucinervin I was stronger than 5-fluorouracil (IC50 = 2.37 µM). The presence of angular pyranoxanthone skeleton in paucinervin I compound seemed to play a pivotal role in its antiproliferative activity [38), from the pericarp of mangosteen fruits, and studied its anticancer activity against CNE-1, CNE-2, A549, H490, PC-3, SGC-7901, and U-87 cancer cell lines. The 7-O-demethyl mangostanin gave IC50 values of 3.35, 4.01, 4.84, 7.84, 6.21, 8.09, and 6.39 µM against CNE-1, CNE-2, A549, H490, PC-3, SGC-7901, and U-87 cancer cell lines, respectively. The 7-O-demethyl mangostanin gave a stronger anticancer activity when compared to the Hirsutanol A as the positive control (IC50 = 6.30–15.0 µM). The mechanism of action of 7-O-demethyl mangostanin was generated by the induction of the late and early-stage apoptosis of cancer cells [In 2015, several isolated xanthones from the branches of orubicin . The mecorubicin . In 2016activity . In 2017er cells .Garcinia paucinervis and evaluated their anticancer activity against HL-60, PC-3, and Caco-2 cell lines. The isolation process yielded two new xanthones, (-)paucinervin O (39) and pacinervin P (40). Compound 39 showed strong anticancer activity, with IC50 values of 0.87 and 2.06 µM against HL-60 and Caco-2 cells, respectively. Compound 39 owing a furan ring was found to be more active against three cancer cell lines compared to the compounds with a dihydrofuran ring. On the other hand, compound 40 gave a strong anticancer activity, with IC50 value of 4.66 µM against PC-3 cells [41 was found to give the strongest anticancer activity, with IC50 values of 18.6, 36.5, 52.8, and 69.6 µM against HepG2, Hep3B, SMMC-7721, and HuH-7, respectively. The mechanism of its anticancer activity was reported through interactions with caspase 3, caspase 9, and poly(adenosine diphosphate-ribose) polymerase (PARP) proteins, which participate in programmed cell death [In 2018, Jia et al. isolated xanthone derivatives from the stem of -3 cells . In 201942 with the ethylamino functional group at C-1 position was the most active anticancer agent. Compound 42 gave IC50 value of 1.90 µM, which was much lower than doxorubicin (12.0 µM) [43 showed the most promising anticancer activity. Compound 43 gave moderate to strong anticancer activity, with IC50 values of 7.90 and 3.90 µM for MCF-7 and MDA-MB-468 cells, respectively. Compound 43 showed no significant toxicity effect toward H9C2 cells (>25.0 µM), demonstrating that it could be applied as a potential candidate for an anticancer lead compound [Since a limited number of isolated and synthesized xanthone dimers and xantholignoids are found, both groups are rarely investigated as anticancer agents. On the other hand, thioxanthone is a xanthone derivative in which the oxygen atom of the pyran ring is replaced by a sulfur atom ,181. In 12.0 µM) . In 2015compound .44 showed the most potent anticancer activity, with an IC50 value of 6.10, 6.00, and 3.60 µM against MCF-7, NCI-H460, and A375-C5 cells, respectively. The anticancer activity was caused through the induced apoptosis mechanism. The modification of compound 44 to its hydrochloride salt form was found to enhance its solubility and bioavailability [45 and tested its cytotoxic anticancer activity against HT-29 cells. Compound 45 showed no anticancer activity (IC50 > 165 µM) against HT-29 cell lines. In contrast, UV light irradiation stimulated compound 45 to enhance its cytotoxic activity (IC50 = 65.5 µM). Moreover, compound 45 exhibited higher activity compared to the common chemotherapeutic agents, such as 5-fluorouracil (IC50 = 222 µM) and cisplatin (IC50 = 66.4 µM). Under UV light irradiation, compound 45 released CO2 and produced alkyl radicals, thus increasing its anticancer activity. Therefore, compound 45 has potential for its application as a theragnostic agent toward specific targeted cancer treatment [In 2016, Barbosa et al. examined the anticancer activity of synthetic thioxanthone derivatives against MCF-7, NCI-H460, and A375-C5 cell lines. Compound lability . In 2020reatment . The sumA molecular docking study of xanthone and thioxanthone derivatives against platelet-derived growth factor response (PDGFR) and epidermal growth factor receptor (EGFR) protein kinases has been evaluated in our previous study. It was found that hydroxyxanthones with halogen substituents gave stronger binding energies  than erlotinib  against EGFR protein. However, they gave weaker binding energies  than imatinib  against PDGFR protein. A similar hydrogen bond of Met769 with erlotinib was also found on the hydroxyxanthones with halogen substituents. Furthermore, the hydroxyxanthones with halogen substituents gave an additional hydrogen bond with Thr766; consequently, they gave stronger binding energy than imatinib against EGFR protein. Meanwhile, a similar hydrogen bond with Cys673 of imatinib was also found on the hydroxyxanthones with halogen substituents. However, the hydrogen bond between imatinib with Asp810 was not found in the hydroxyxanthones with halogen substituents; thus, it may be the reason why they gave weaker binding energy than imatinib against PDGFR protein . On the Rational design on the structure and anticancer activity of xanthone derivatives has been reported; however, a comprehensive and holistic point of view is still very limited. For example, it was reported that the hydroxyl group at C-1 position of xanthone was critical for the anticancer activity against MCF-7 . The 1-h50 of haloxanthones against HepG2 cancer cells can be predicted through log(IC50) = −1.198(qC7) + 9.274(qC8b) − 1.887(qC8) − 23.35(qO carbonyl) − 6.034 (R2 = 0.94), where qX represented the charge of atom X. On the other hand, it was found that IC50 of haloxanthones against MCF-7 cancer cells can be predicted through log(IC50) = 100(LUMO) + 0.36(DM) − 3.02(qC6) − 1.71(qC4) − 7.98(qC1) + 7.85 (R2 = 0.89), whereas LUMO and DM represent the lowest unoccupied molecular orbital and dipole moment, respectively [50 of xanthones as topoisomerase IIα inhibitors can be predicted through pIC50 = 2.19(DE) + 0.22(n(OH)) − 0.54(LogP) − 0.469(SIB) + 0.018(SSA) + 2.57 (R2 = 0.84), whereas DE, n(OH), LogP, SIB, and SSA represent dielectric energy, the group count of hydroxyl substituents, logarithm of the partition coefficient between n-octanol and water, shape index basic (order 3), and solvent-accessible surface area, respectively [Quantitative structure–activity relationship (QSAR) is a computational approach to rationalize the relationship between the chemical structure of drugs with their biological activity . A QSAR ectively . A QSAR ectively .50 value of xanthone derivatives against A549 cancer cell line can be predicted through log (IC50) = −39.0090(x1) − 0.8078(x2) − 1.0827(x3) − 0.0483(x4) + 0.5053(x5) − 0.0560 (R2 = 0.87), whereas x1 is a parameter of electronegativity contribution, x2 is the atom type count descriptor class, x3 is the number of carbon atoms connected with four single bonds, x4 is the number of C=C bonds, and x5 is the electrotopological state of methyl group. Meanwhile, the IC50 value of xanthone derivatives against HepG2 cancer cell line can be predicted through log IC50 = −0.6407(y1) − 0.0336(y2) − 0.1278(y3) + 0.2226(y4) + 0.5877(y5) + 0.5940 (R2 = 0.87), whereas y1 is electrotopological state of carbon atoms, y2 is the number of double bonds, y3 is the number of hydrogen bond acceptor atoms, y4 is electrotopological state of methine group, and y5 is the number of oxygen atoms. On the other hand, the IC50 value of xanthone derivatives against U251 cancer cell line can be predicted through log IC50 (µM) = 0.0948(z1) + 0.5217(z2) + 0.3687(z3) + 1.1313 (R2 = 0.86), whereas z1 is the electrotopological state of nitrogen atoms, z2 is the number of oxygen atoms, and z3 is the topological state of carbon atoms. However, a more comprehensive QSAR study involving all reported xanthone derivatives against a certain cancer cell is not available yet, as of today. Therefore, computational studies on this research are still open to be investigated by researchers in the future.A more comprehensive QSAR study on the anticancer activity of xanthone derivatives was investigated by Alam and Khan in 2018 . They evThe xanthone derivatives showed promising in vitro anticancer activity; thus, further investigation through in vivo and then clinical assays is required . The in Garcinia mangostana fruit rinds containing 81% of α-mangostin and 16% of γ-mangostin significantly reduced the tumor volume of HCT-116 on mice. The inhibitory activity was related to the induction of apoptosis and inhibition of cancer cells migration, invasion, and clonogenicity [It was reported that panaxanthone (a mixture of α-mangostin and γ-mangostin in 6:1 wt/wt) inhibited the DNA replication and induced the G1 phase cell cycle arrest of cancer cells . Subcutagenicity .max) was found to be at 1 h, with a decrement to 65% after 4 h. A similar result was reported in China for maximum α-mangostin concentration (4.20 µg/L) and Tmax (1 h) value in the human group (20 people) [max value was found to be at 2–4 h on average [A clinical study for mangosteen juice (containing 3.90 g/L of α-mangostin) to the human group (20 people) was conducted . It was  people) . In 2012 average . Faster 2 dose. At a higher dose, the DMXAA gave the side effects of visual disturbance, tremors, confusion, slurred speech, and anxiety [Detailed anticancer investigation on DMXAA has also been reported. The DMXAA leads to vascular collapse and tumor necrosis through tumor necrosis factor-α induction. Synergistic anticancer activity with chemotherapy agents such as doxorubicin, imatinib, erlotinib, and sunitinib, as well as with the cancer radiotherapy technique has been reported. DMXAA exhibited low toxicity to the normal cells with suitable pharmacokinetic parameters. Furthermore, the clinical trials of DMXAA as the anticancer agent have been evaluated in New Zealand and the United Kingdom. The phase I clinical trial of DMXAA to 63 patients gave no drug-related myelosuppression. The DMXAA was well tolerated up to 4900 mg/m anxiety . Unfortu anxiety . HoweverAs an isostere of xanthone, thioxanthones have similar structures and physicochemical properties to xanthone derivatives . NowadayThe most serious drawbacks for the utilization of xanthone derivatives as the anticancer agent are their poor solubility in water and their reactivity to be transformed into phase 2 conjugates form. Therefore, further technologies are critical to overcome these limitations ,211. It This review highlights the potential anticancer of xanthone derivatives, contributing to the design and develop of new drugs. Both isolated and synthesized xanthone derivatives have been proven to have promising anticancer activities against breast, hepatoma, cervix, colorectal, ovarian, lung, gastric, leukemia, skin, epidermoid nasopharynx, prostate, neuron, brain glioblastoma, and other cancer cells through in vitro assay. However, their further evaluation as anticancer agents through in vivo and clinical assays is still limited as of today. Furthermore, a comprehensive QSAR study for the better design of anticancer drugs based on the xanthone structure has not yet been established. These research gaps require extensive collaborations and contributions from researchers around the world to further develop active, selective, and efficient anticancer drugs based on xanthone derivatives.


Photodynamic therapy (PDT) is an anticancer therapeutic modality with remarkable advantages over more conventional approaches. However, PDT is greatly limited by its dependence on external light sources. Given this, PDT would benefit from new systems capable of a light-free and intracellular photodynamic effect. Herein, we evaluated the heavy-atom effect as a strategy to provide anticancer activity to derivatives of coelenterazine, a chemiluminescent single-molecule widespread in marine organisms. Our results indicate that the use of the heavy-atom effect allows these molecules to generate readily available triplet states in a chemiluminescent reaction triggered by a cancer marker. Cytotoxicity assays in different cancer cell lines showed a heavy-atom-dependent anticancer activity, which increased in the substituent order of hydroxyl < chlorine < bromine. Furthermore, it was found that the magnitude of this anticancer activity is also dependent on the tumor type, being more relevant toward breast and prostate cancer. The compounds also showed moderate activity toward neuroblastoma, while showing limited activity toward colon cancer. In conclusion, the present results indicate that the application of the heavy-atom effect to marine coelenterazine could be a promising approach for the future development of new and optimized self-activating and tumor-selective sensitizers for light-free PDT. S0) to its lowest singlet excited state (S1) and will undergo intersystem crossing (ISC) to triplet states (T1), now capable of sensitizing the highly cytotoxic singlet oxygen after reacting with triplet oxygen . ,7. 6,7].1 states . Howeverd states ,11.S0 → T1 ISC pathway can then enable the use of the chemiluminescent reaction of Clz as a self-excitation mechanism to directly generate triplet states able to sensitize singlet oxygen, thus leading to a light-free intracellular reaction exclusively triggered by a cancer marker (superoxide anion). One of the most effective strategies to enhance the efficiency of ISC pathways is through the heavy-atom effect  [a] pyrazin-3(7H)-one (Br-Cla)  . Accordi(Br-Cla) a, a bromectively . Br-Cla ectively ,13. GiveThus, studies with Br-Cla and the remaining brominated Clz derivatives  providedTo further understand the true potential of these Clz derivatives as novel anticancer compounds, it is also important to know how they behave toward different cancer types and assess their spectrum of application. So far, our knowledge in this regard is limited to their application to prostate and breast cancer ,13.S0 → T1 pathway present in the chemiluminescent reaction of these compounds and, hence, favor T1 chemiexcitation in the increasing order of OH < Cl < Br. If the reported anticancer activity of the compounds is indeed related to the intracellular superoxide-triggered T1 chemiexcitation, this should lead to heavy-atom-dependent enhancement of their cytotoxicity in the increasing order of OH < Cl < Br. Thus, these three derivatives were subjected to a detailed and in-depth luminometry, photophysical, and theoretical characterization of their chemiluminescent reactions, focused on the possible superoxide-induced generation of triplet excited states. Subsequently, their cytotoxicity was also evaluated for the first time toward neuroblastoma and colon cancer cell lines, with the resulting performance compared with that obtained for breast and prostate cancer. With this approach, we will be able to understand if the heavy-atom effect is indeed responsible for the anticancer activity of these molecules, supporting their identification as prototypical light-free and self-activating photosensitizers, and to understand the scope of their cytotoxicity toward different cancer types.Herein, we aimed to clarify these topics by employing a target-oriented approach. Specifically, we report the synthesis of three Cla derivatives  basis set. The T1 state was calculated by performing single-point calculations at the same level of theory, on top of the S0 IRC-obtained structures. ωB97XD is a long-range-corrected hybrid exchange-correlation functional, which provides quite good estimates for π → π * and n → π * local excitation, as well as charge transfer and Rydberg states [The nctional  and the g states . Geometrg states . This apg states .1H-NMR and high-resolution mass spectra (HR-MS) for OH-/Cl-Cla are also available in the Br-Cla was prepared following the procedure described in , while ON,N-dimethylformamide (DMF)-acetate buffer pH 5.14 (0.68%) or methanol in the presence of a superoxide anion source . The steady-state chemiluminescent and fluorescent spectra of the three Cla derivatives were measured using a Horiba Jovin Fluoromax 4 spectrofluorimeter, with an integration time of 0.1 s. Slit widths of 5 nm were used for both the excitation and emission monochromators when obtaining fluorescent spectra. Chemiluminescent spectra were obtained with a slit of 29 nm for the emission monochromators. Quartz cells with a 10 mm path length were used.Chemiluminescence kinetic measurements were performed in a homemade luminometer using a Hamamatsu HC135-01 photomultiplier tube. All reactions took place at room temperature at least in sextuplicate. The light-emitting reactions took place at room temperature at least in sextuplicate and were carried out in either 2)) The chemiluminescent and fluorescent spectra were obtained in 2 mL DMF-acetate buffer pH 5.14 (0.68%) solutions and measured using a Horiba Jovin Fluoromax 4 spectrofluorimeter (The detailed procedure is available in the The light-emitting reactions took place at ambient temperature at least in sextuplicate and were carried out in DMF-acetate buffer pH 5.14 (0.68%) or methanol , supplemented with 100 U/mL penicillin/100 µg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS), incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. For cell culture maintenance, cells were subcultured once a week and the cell medium was renewed every 2 days. Both the human prostate (PC-3) and the breast (MCF-7) cancer cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics (complete medium), and they were incubated in a 5% CO2 incubator at 37 °C.The HT-29 and SH-SY5Y cell lines were obtained from ATCC. The human colon cancer HT-29 cell line was cultured at 37 °C and 5% CO2. Controls were composed of DMSO 0.1% v/v. Cells were exposed to the different treatments for 48h. For assays involving PC-3 and MCF-7 lines, cells were treated with Br-Cla at 0.1, 1, 10, 25, 50, and 75 µM (in methanol) for 72 h [v/v. The half-maximal inhibitory concentration (IC50) value was determined for the different assays by MTT viability assay.Before each treatment, HT-29, SH-SY5Y, PC-3, and MCF-7 cells were seeded in 96-well plates in triplicate with the following densities: 5000 cells/well for PC-3 and MCF-7, 15,000 cells/well for HT-29, and 20,000 cells/well for SH-SY5Y. For assays involving HT-29 and SH-SY5Y lines, cells were treated with OH-, Cl-, and Br-Cla (in DMSO) at 0.01, 0.1, 1, 10, 20, 30, 50, 75, or 100 µM for 48 h. During the experimental period, cells were maintained at 37 °C with 5% COfor 72 h . The conAfter cell treatment with the Cla derivatives, mitochondrial function was evaluated since mitochondrial dehydrogenases of living cells can reduce the MTT (yellow) to formazans, which are purple compounds. At the end of incubation, the cell medium was removed and 100 µL of MTT solution (0.5 mg/mL in PBS) was added to each well. Cells were then incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 µL/well) was added to solubilize the formazan crystals. Absorbance was measured at 570 nm using an automated microplate reader . All conditions were performed in triplicate.p-value < 0.05.GraphPad Prism 8  was used to produce concentration-response curves by nonlinear regression analysis. MTT results are presented as the mean ± SEM for n experiments performed. All data were assessed in three independent experiences. Statistical comparisons between control and treatment groups were performed with one-way ANOVA test. Statistical significance was accepted at a Cell morphology was assessed on a Leica DMI 6000B microscope equipped with a Leica DFC350 FX camera and then analyzed with the Leica LAS X imaging software v3.7.4 .T1 chemiexcitation during their chemiluminescent reaction, which can be enhanced by the heavy-atom effect, before their synthesis and characterization. Thus, we calculated the potential energy curves for both the S0 and T1 states during the thermolysis of OH-Cla  [S0 and T1 states during the thermolysis reaction , when reaching the TS and onward, both states become degenerated in a large and flat region of the potential energy surface (PES).As consistent with previous reports for different dioxetanones (including Br-Cla) ,11,12,17reaction  is more T1 chemiexcitation, in line with previous studies for this type of system [S0 → T1 chemiexcitation pathway for OH-Cla is not expected to be efficient [This indicates that both chemiluminescent reactions possess an intrinsic pathway for f system ,11,12,18fficient . Given tT1 state with an energy higher than 0.98 eV (the required energy to convert molecular oxygen into singlet oxygen) [S0 – T1 energy gap of OH-Cla coelenteramide and found a value of 2.78 eV. Furthermore, this value is identical to what was found by us previously for Br-Cla [S0 – T1 energy gap of Br-Cla coelenteramide [It should be noted that an efficient ISC is not the only parameter determining the performance of a photosensitizer in singlet oxygen generation, as it should also have a  oxygen) . We moder Br-Cla . Thus, bteramide .Given that the in silico modeling results indicate that the chemiluminescent reactions of these Cla derivatives present intrinsic pathways for light-free generation of triplet states capable of sensitizing singlet oxygen, we then proceeded to the synthesis of OH-Cla, Cl-Cla, and Br-Cla . The int1H-NMR spectroscopy  with addition of buffer , conditions in which Clz and derivates are known to readily generate chemiluminescence by reacting with dissolved oxygen . All comS0 reaction. However, in our previous study of Br-Cla [−1). Furthermore, the S0 energetics for the thermolysis of OH-Cla and Br-Cla dioxetanones are identical . To verify this hypothesis, we also measured the fluorescence intensity of the spent reaction mixtures (after 30 min of reaction) in an aprotic solvent  in methanol. Interestingly, all compounds responded to the addition of KO2 with immediate emission of light and subsequent decay to basal levels , without affecting the chemiluminescent reaction. Thus, if the anticancer activity found before for this type of molecules ,13 is inGiven this, the potential anticancer activity of the three R-Cla compounds was evaluated in both colon cancer (HT-29) and neuroblastoma (SH-SY5Y) cell lines with a standard MTT assay for exposure times of 48h, along with changes in cell morphology analyzed by microscopy.T1 chemiexcitation. Interestingly, while Cl-Cla induced virtually no anticancer effect in these cells, there was a statistically significant difference at a concentration of 100 µM , in the same concentration range as R-Cla . This drIn neuroblastoma cells , the resT1 chemiexcitation and the subsequent intracellular generation of triplet states capable of sensitizing singlet oxygen [Given this, it is clear that there was a halogen-dependent effect on the anticancer activity of these compounds for both cancer cell lines. This is in line with our proposition that the addition of halogens to Clzs will provide them with anticancer activity, by enhancing t oxygen ,13. In ct oxygen ,13 is inHaving said that, it is also important to note that the intrinsic anticancer activity of the studied compounds (including Br-Cla) might not be as high as desired toward neuroblastoma and colon cancer cell lines, especially when compared to 5-FU. Given this, it is important to determine whether the magnitude of the obtained anticancer activity is similar across different cancer types or if it is dependent on the studied cell line.50 of Br-Cla for different cell lines. Br-Cla was chosen for its consistent toxicity in this study, contrary to OH-Cla and Cl-Cla. IC50 values were determined for neuroblastoma SH-SY5Y, prostate PC-3, and breast MCF-7 cancer cell lines  was still significantly higher than the IC50 found for MCF-7, irrespective of the duration of treatment with Br-Cla.The impact of treatment with Br-Cla on the cellular viability of PC-3 and MCF-7 cell lines can be found in 0 values . More spreatment . This vaThus, the results indicate that it is safe to state that the magnitude of the anticancer activity of Br-Cla is dependent on the cancer cell type, being higher for prostate and breast cancer than for neuroblastoma and colon cancer. Further studies are required to assess if these differences arise from (among other possibilities) higher resistance of HT-29 and SH-SY5Y cell lines to the photodynamic effect, lower efficiency of internalization of R-Cla compounds into these cell lines, or a lower generation of superoxide anion in these cells.S0 → T1 chemiexcitation pathways. The anticancer activity of these compounds was evaluated toward different cancer cell lines, and a clear halogen effect was observed with Cl-Cla and Br-Cla presenting increasing toxicities. Furthermore, it was found that the magnitude of the anticancer activity of these types of compounds is dependent on the cancer cell line, being more relevant in prostate and breast cancer than in neuroblastoma and colon cancer. Thus, the results indicate that applying the heavy-atom effect to marine Clz would be a promising strategy for designing self-activating and light-free photosensitizers. With this design strategy, the Clz system appears to be a prototypical system for optimized photosensitizers to be used in PDT strategies not limited by the need for external light sources.In conclusion, we reported the target-oriented synthesis of three Clz derivatives , to provide anticancer activity to this class of compounds through the heavy-atom effect. On the basis of this strategy, we developed novel compounds able to directly generate readily available triplet excited states with enough energy to sensitize singlet oxygen, in a chemiluminescent reaction triggered by a cancer marker. This was achieved by the introduction of the heavy-atom effect into this system, which enhanced the ISC rate of available Patent PCT/IB2019/053642 (pending)—chemiluminescent imidazopyrazinone-based photosensitizers with available singlet and triplet excited states.


N-acetylglucosaminyltransferase 2, a polylactosamine synthase, is an important member of the glycosyltransferase family. However, the biological function and regulatory mechanism of β3GNT2 in esophageal carcinoma (ESCA) is still poorly understood.Glycosyltransferases play a crucial role in various cancers. β1, 3-The Cancer Genome Atlas and Genotype-Tissue Expression databases were used for gene expression and prognosis analysis. Quantitative real-time PCR, Western blot, and immunohistochemistry were performed to detect the expression of β3GNT2 in ESCA cell lines and tissues. In vitro assays and xenograft tumor models were utilized to evaluate the impact of β3GNT2 on ESCA progression. The downstream effectors and upstream regulators of β3GNT2 were predicted by online software and verified by functional experiments.We found that β3GNT2 was highly expressed in ESCA tissues and positively correlated with poor prognosis in ESCA patients. β3GNT2 expression was closely associated with the tumor size, TNM stage, and overall survival of ESCA patients. Functionally, β3GNT2 promoted ESCA cell growth, migration, and invasion in vitro, as well as tumorigenesis in vivo. Mechanistically, β3GNT2 knockdown decreased the expression of the polylactosamine on EGFR. Knockdown of β3GNT2 also inhibited the JAK/STAT signaling pathway. Meanwhile, the JAK/STAT inhibitor could partly reverse the biological effects caused by β3GNT2 overexpression. Moreover, β3GNT2 expression was positively regulated by CREB1 and negatively regulated by miR-133b. Both CREB1 and miR-133b was involved in the β3GNT2-mediated ESCA progression.Our study, for the first time, reveals the importance of β3GNT2 in ESCA progression and offers a potential therapeutic target for ESCA.The online version contains supplementary material available at 10.1186/s11658-022-00306-y. Esophageal carcinoma (ESCA) is one of the most common cancers around the world . China iGlycosylation is a ubiquitous post-translational modification of proteins , 5. AlteN-acetylglucosaminyltransferase 2 (β3GNT2) was overexpressed in ESCA tissues and correlated with the poor prognosis of ESCA patients. We further investigated the biological function and regulatory mechanism of β3GNT2 in ESCA progression. Our results suggest that β3GNT2 may serve as a potential therapeutic target for ESCA.In this study, The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to identify differentially expressed glycosyltransferases between ESCA tissues and normal tissues. We found that β1, 3-https://tcga-data.nci.nih.gov/tcga/) and GTEx  platforms. Differentially expressed glycosyltransferases, transcription factors, miRNAs were screened using the DESeq2 R package. An adjusted P-value < 0.05 and |log2 fold-change (FC)|> 0.5 were set as the thresholds. Volcano plots and heatmaps were generated using the R package ggplot and pheatmap, respectively. A prognostic nomogram was constructed by R software with the rms package. The Receiver Operating Characteristic (ROC) curves were plotted and the area under the ROC curve was calculated using the R package pROC. Survival prediction was performed using the GEPIA database (http://gepia.cancer-pku.cn/). Gene Set Enrichment Analysis (GSEA) analysis was carried out using the GSEA software (http://www.broadinstitute.org/gsea). Co-expression analysis was determined by using the LinkedOmics platform (http://www.linkedomics.org/).RNA-seq data, miRNA-seq data, and the corresponding clinical information were obtained from the TCGA  between 2008 and 2012. The patients, including 35 males and 30 females, were aged 35–75 years with an average age of 54.62 ± 9.14. None of the patients underwent any other treatment before surgery. Samples were stored at − 80 °C until further processing. The studies involving human participants were approved by the Ethics Committee of the Hubei University of Medicine. All patients provided informed written consent.2. The pcDNA3.1/β3GNT2 plasmid, pcDNA3.1/CREB1 plasmid, empty pcDNA3.1 plasmid, a lentiviral vector expressing β3GNT2 shRNA, a lentiviral vector expressing CREB1 shRNA, control lentiviral vector, miR-133b inhibitor, inhibitor control, miR-133b mimics, and mimics control were constructed by GenePharma . All transfections were conducted with Lipofectamine 3000 reagent  according to the manufacturer’s protocol. Stable clones were selected by puromycin or G418.Human ESCA cell lines TE-1, KYSE150, and KYSE410 were purchased from Procell . These cells were cultured in RPMI-1640 medium  containing 10% FBS at 37 °C, 5% CO−ΔΔCt method. All primers were synthesized by GenePharma. Total protein was isolated using RIPA lysis buffer . Western blot was conducted as previously described [Total RNA was extracted using RNAiso Plus . For mRNA detection, cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara) and qPCR was carried out employing the SYBR-Green PCR master mix (Takara). For miRNA analysis, the One-Step PrimeScript miRNA cDNA synthesis kit (Takara) and the SYBR PrimeScript miRNA RT-PCR Kit (Takara) were used. Data were normalized to GAPDH or U6 based on the 2escribed . PrimaryTissues were fixed in formalin and embedded in paraffin. Afterward, 5 µm sections were prepared for IHC staining. PV-9000 two-step immunohistochemical kit  was used to detect the expression of β3GNT2 protein. The positive signal was visualized by DAB detection Kit (ZSGB-BIO). Slides were read by two pathologists using a Nikon Eclipse E600 microscope . IHC score = intensity score × percentage score . β3GNT2 3 per well) were seeded into a 96-well plate. Absorbance was measured at 450 nm. For colony formation assay, cells were added into a 6-well plate at a density of 5 × 103 cells/well. After 2 weeks, colonies (> 50 cells) were stained with 0.5% crystal violet (Beyotime).Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay . Cells (1 × 104 in 200 μL serum-free medium) were seeded into the upper chamber of the Transwell apparatus . Transwell chambers precoated with or without Matrigel  were used to assess cell invasion and migration, respectively. The lower chamber was filled with 600μL of complete culture medium. After incubation for 36 h, migrated or invaded cells were stained with 0.5% crystal violet.Cells . The percentage of apoptotic cells was defined as the sum of early apoptotic cells and late apoptotic cells. All experimental steps were conducted according to kit manual instructions.Lycopersicon esculentum lectin (LEL) agarose beads  were used to analyze polylactosamine chains on glycoproteins. Cell lysates (0.5 mg) were incubated with 30 μL of LEL-conjugated beads to capture the lectin-glycoprotein complexes. Precipitated glycoproteins were eluted and subjected to Western blot analysis with an anti-EGFR antibody. The EGFR in total lysates was used as the internal loading control.The wild-type or mutant β3GNT2 3′UTR sequences containing the binding sites for miR-133b were cloned into the pmirGLO vector. Besides, the fragments of β3GNT2 promoter with wild-type or mutant CERB1 binding sites were inserted into a pGL3-basic vector. All the above plasmids were constructed by GenePharma. Cells were harvested 48 h after transfection. Luciferase activities were determined using the dual-luciferase reporter assay system .The EZ-Magna ChIP™ Kit  was used for ChIP assay . Detecti7) were subcutaneously injected into nude mice. Tumor volume (0.5 × length × width2) was measured every week. Four weeks after injection, all mice were sacrificed.Female BALB/c nude mice, 4–5 weeks old, were provided by the Animal Center of Hubei University of Medicine. Transfected cells  software . Student’s t-test, Chi-square test, and one-way ANOVA were used to determine statistical significance. A correlation was analyzed using the Pearson correlation coefficient test. Overall survival curves were plotted according to the Kaplan–Meier method. A P-value < 0.05 was considered significant.Using the RNA-seq data from the TCGA and GTEx datasets, we screened differentially expressed glycosyltransferases between ESCA tissues 182 cases) and normal esophageal tissues (666 cases)  and the String database (https://www.string-db.org/) to determine the interactive relationship between β3GNT2 and EGFR   was used. Through bioinformatics analysis, one putative CREB1 binding site was identified in the β3GNT2 promoter region  Fig. A. The asml) Fig. B. Notablml) Fig. C. A posiml) Fig. D. Thus, ml) Fig. E, F. To ion Fig. G. The enion Fig. H. Dual-lion Fig. I. To expion Fig. J–M. In sSince miRNAs are important post-transcriptional modulators of gene expression, we then investigated whether β3GNT2 was regulated by specific miRNAs . Three pThe prognosis of patients with ESCA remains unsatisfactory, despite the use of multiple therapeutic approaches. Further exploration of the diagnostic and prognostic biomarkers for ESCA is warranted. Glycosyltransferases have attracted increasing interest owing to their importance in many biological processes and diseases, including cancer. In the current study, we first found that β3GNT2 expression was upregulated in ESCA. β3GNT2 upregulation was closely associated with the poor prognosis of ESCA patients. Subsequent experiments proved that β3GNT2 contributed to ESCA progression by promoting EGFR glycosylation and activating the JAK-STAT pathway. In addition, CREB1 and miR-133b were critical regulators of β3GNT2 Fig. M. Thus, β3GNTs are a family of crucial glycosyltransferases that synthesize a unique glycan structure known as polylactosamine . AbnormaWe explored the underlying mechanism of β3GNT2 in promoting ESCA progression. Bioinformatics analysis showed that β3GNT2 expression was linked to the JAK-STAT pathway. Numerous studies revealed that the JAK-STAT pathway was involved in cell proliferation, differentiation, apoptosis, angiogenesis, and immune regulation , 22. TheTo explore the potential factors leading to β3GNT2 upregulation in ESCA, the transcription factors of β3GNT2 were predicted by bioinformatics methods. CREB1 was proposed as a candidate because it had a strong positive correlation with β3GNT2 expression in ESCA. CREB1 is a transcription factor that influences multiple cellular processes, such as cell survival, differentiation, and proliferation , 29. In There are, however, several limitations to our study. First, the expression of polylactosamine chains in ESCA tissues is not examined. Second, the association between the JAK-STAT pathway and EGFR signaling has not been elucidated. The specific glycosylation sites of EGFR remain unknown. The effect of EGFR glycosylation on ESCA progression has not yet been elucidated. Additionally, the mechanism of synergy between CREB1 and miR-133b is not clearly understood. It will be interesting to address these questions in future research.Our results confirm that β3GNT2 predicts poor prognosis and acts as an oncogene in ESCA. β3GNT2 can enhance EGFR glycosylation and activate the JAK-STAT pathway. β3GNT2 is transcriptionally upregulated by CREB1. Meanwhile, miR-133b directly targets β3GNT2 and negatively regulates its expression. To our knowledge, this is the first study to explore the biological function, molecular mechanism, and regulatory network of β3GNT2 in ESCA. Thus, β3GNT2 may be a promising prognosis biomarker and a potential therapeutic target for ESCA.Additional file 1: Table S1. Screening of dysregulated glycosyltransferases in ESCA based on the TCGA and GTEx databases.Additional file 2: Fig. S1. Analysis of β3GNT2 protein expression in different ESCA cells by Western blot.Additional file 3: Table S2. Differentially expressed genes correlated with β3GNT2 in ESCA based on the LinkedOmics platform.


However, the therapy is mainly limited by severe acute and chronic graft-versus-host disease (GvHD), both being life-threatening complications after allo-HCT. GvHD develops when donor T cells do not only recognize remaining tumor cells as foreign, but also the recipient’s tissue, leading to a severe inflammatory disease. Typical GvHD target organs include the skin, liver and intestinal tract. Currently all approved strategies for GvHD treatment are immunosuppressive therapies, with the first-line therapy being glucocorticoids. However, therapeutic options for glucocorticoid-refractory patients are still limited. Novel therapeutic approaches, which reduce GvHD severity while preserving GvL activity, are urgently needed. Targeting kinase activity with small molecule inhibitors has shown promising results in preclinical animal models and clinical trials. Well-studied kinase targets in GvHD include Rho-associated coiled-coil-containing kinase 2 (ROCK2), spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) to control B- and T-cell activation in acute and chronic GvHD. Janus Kinase 1 (JAK1) and 2 (JAK2) are among the most intensively studied kinases in GvHD due to their importance in cytokine production and inflammatory cell activation and migration. Here, we discuss the role of kinase inhibition as novel treatment strategies for acute and chronic GvHD after allo-HCT.Allogeneic hematopoietic stem cell transplantation  is a potentially curative therapy for patients suffering from hematological malignancies  Pati, cGvHD) –7. GvHD , cGvHD) , 5, 8–10, cGvHD) . Acute a, cGvHD) . Acute G, cGvHD) , 11. Sta, cGvHD) , 14. Man, cGvHD) –17. TCR , cGvHD) –28. ROCK, cGvHD) , 29–32. survival –35. Basesurvival . In genesurvival . Since Gsurvival –38. Manyvia JAK1/2 and signal transducer and activator of transcription (STAT) pathways are crucial for the stimulation of inflammatory cytokine production and the activation of a variety of immune cells during GvHD onset and progression  1/2. Signaling gression .Acute GvHD: The JAK/STAT signaling pathway has high importance in aGvHD onset and progression, as STAT1 and STAT3 are activated early after disease onset. The signaling plays a pivotal role in mediating T-cell activation and changes of the T-cell phenotype  GvHD and patients with severe intestinal or skin GvHD showed impressive response to JAK1/2 inhibition. Treatment with Ruxolitinib was liked to decreased pro-inflammatory serum cytokine levels and lower numbers of activated T-cells. The overall response rate (ORR) was 81.5 % and the overall survival (OS) of steroid-refractory aGvHD patients receiving Ruxolitinib was higher than ever reported for any other pharmacological therapy . This trChronic GvHD: Besides application in aGvHD, Ruxolitinib treatment was also evaluated for the treatment of glucocorticoid-refractory cGvHD  were published recently . The triDonor T-cell activation and inflammatory cytokine secretion is a hallmark of GvHD after allo-HCT. Activation and effector functions of T-cells are tightly connected to the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling cascade, which is crucial for the regulation of T-cell survival, proliferation, cell cycle progression, differentiation and metabolism Figure 3Acute GvHD: In a preclinical study, transplantation of Mtor-deficient donor T-cells reduced aGvHD severity in mice, whereas more detailed analysis with Raptor-deficient allogeneic T-cells revealed that T-cell-mediated pathogenesis is dependent on mTORC1 but not on mTORC2 . The effect of pacritinib/sirolimus was tested in human MLRs and a xenogeneic GvHD model and consistently suppressed allogeneic T-cell proliferation and GvHD severity  , 118. IbBased on very promising preclinical and clinical trial data, ITK and BTK inhibition with ibrutinib could be a very potent therapy in chronic GvHD. However, additional detailed analysis of the underlying mechanism is necessary to improve the therapeutic success. Since clinical trials focus on cGvHD, further analysis and preclinical models are needed to investigate the role of ITK blockade in aGvHD.The non-receptor cytoplasmic spleen tyrosine kinase (Syk) was hypothesized being an important regulator of GvHD as it has functions in transmitting signals from surface receptors, including Toll-like receptors (TLRs) , Fc receAcute GvHD: In a preclinical murine aGvHD model, daily treatment with Fostamatinib led to significantly improved survival, reduced histopathology scores and reduced pro-inflammatory serum cytokine concentrations. Fostamatinib treatment did not interfere with donor lineage engraftment and immune reconstitution. Syk phosphorylation is rapidly increased upon CD3/CD28 stimulation of T-cells and higher pSyk 525/526 levels were also seen in T-cells isolated from aGvHD mice. Using luciferase transgenic T-cells, Syk inhibitor treatment was found to reduce alloreactive donor T-cell expansion in vivo. The findings were confirmed by CFSE staining, indicating reduced T-cell proliferation in vivo upon Syk inhibition. Besides blockade of T-cell proliferation, Syk inhibition also reduced T-cell migration towards CXCL12. Contrary to previous findings about the importance of Syk in T-cell lineage commitment, Fostamatinib treatment did not change the percentage of Th2 and Th17 cells after allo-HCT. Effects on T-cells could be further affected by APCs as Syk inhibition was also connected to diminished costimulatory molecule expression and reduced DC migration in vivo and in vitro. Although proliferation and effector cytokine secretion of allogeneic donor T-cells was significantly reduced by Fostamatinib, the GvL effect was preserved, as confirmed by in vivo bioluminescence imaging using luciferase transgenic leukemia cells and ex vivo killing assays. Overall, pharmacological inhibition of Syk was found being a novel treatment strategy in aGvHD by reducing T-cell expansion and costimulation while preserving anti-leukemia immunity . Another phase II trial was investigating the efficacy and tolerability of entospletinib in combination with systemic corticosteroids cGvHD as first-line therapy, however, the study was terminated (NCT02701634). Based on pre-clinical studies, it would also be interesting to evaluate effects of Syk inhibition in patients with aGvHD. However, ongoing clinical trials first focused on cGvHD, which might be due to the importance of Syk in BCR downstream signaling, whereas it is not essential for TCR downstream events.Taken together, the data derived from genetic and pharmacological approaches in pre-clinical murine GvHD models clearly indicate that Syk plays an important role in GvHD pathophysiology. Pharmacological targeting of Syk could be a novel attempt to treat GvHD. Based on these promising findings of Syk inhibition with Fostamatinib Chronic GvHD: cGvHD is often presented with dermal fibrosis and sclerosis, associated with the presence of stimulatory anti-platelet-derived growth factor receptor (PDGFR) antibodies, suggesting a direct link between skin fibrosis and PDGF signaling  levels. Calcium influx is essential for activation, maturation and effector functions of T-cells. TCR engagement leads to the activation of PLC-γ, thereby increasing the intracellular levels of inositol 1,4,5-triphosphate (IP3). Binding of IP3 to its specific receptors in turn stimulates the release of calcium from intracellular storage compartments. Continuous depletion of intracellular calcium storage stimulates the opening of cell membrane based calcium channels to enhance the influx of calcium from the extracellular compartment. The intracellular increase of Ca2+ is required to activate calcium-dependent kinases and the transcription factor calcineurin, leading to activation of nuclear factor of activated T-cells (NFAT), thereby enhancing the transcription of a variety of different genes necessary for T-cell activation and effector functions . The modulation of intracellular calcium levels was therefore hypothesized to be a potential therapeutic strategy for autoimmune diseases  family, comprising Itpka, Itpkb, Itpkc and inositol polyphosphate multikinase. The Itpk family acts as a negative regulator of Ca2+ influx through conversion of IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4) and this regulatory mechanism is known to be highly important for T-cell development and survival  and interferon γ (IFNγ) levels compared to Itpkb wildtype T-cells. However, quantitative T-cell analysis in the spleen, mesenteric lymph nodes and the small intestine and liver revealed lower numbers of Itpkb deficient T-cells compared to the WT control, whereas the proliferative capacity remained unchanged. Reduced numbers of donor Itpkb-/- T-cells could be linked to lower T-cell survival, indicated by higher abundance of active caspase 8 in CD4+ and CD8+ T-cells from Itpkb-/- donors after HCT , followed by analysis of cytokine production. Pro-inflammatory cytokine secretion was suppressed in a concentration-dependent manner. Application of OZ in a major mismatch murine aGvHD model prolonged the survival of GvHD mice, reduced GvHD scores and did result in lower serum levels of pro-inflammatory cytokines  is a major control mechanism for cellular responses, proliferation and cytokine production , 186. DiChronic GvHD: Since enhanced p38 MAPK phosphorylation was found in fibroblasts from systemic sclerosis patients and p38 MAPK blockade reduced elevated type I collagen expression, the signaling pathway was hypothesized playing a role in sclerosis pathogenesis  in donor grafts were found to accelerate acute intestinal GvHD in mice  – SFB-1479 – Project ID: 441891347, SFB TRR167, SFB850 (to RZ), by the Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984 to RZ) and by ERC Consolidator grant (681012 GvHDCure to RZ). The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding program Open Access Publishing.RZ received honoraria from Novartis, Incyte and Mallinckrodt.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.