Photodynamic therapy is one of the most patient friendly and promising anticancer therapies. The active ingredient is irradiated protoporphyrin IX, which is produced in the body that transfers energy to the oxygen-triggering phototoxic reaction. This effect could be enhanced by using iron chelators, which inhibit the final step of heme biosynthesis, thereby increasing the protoporphyrin IX concentration. In the presented work, we studied thiosemicarbazone derivative, which is a universal enhancer of the phototoxic effect. We examined several genes that are involved in the transport of the heme substrates and heme itself. The results indicate that despite an elevated level of ABCG2, which is responsible for the PpIX efflux, its concentration in a cell is sufficient to trigger a photodynamic reaction. This effect was not observed for 5-ALA alone. The analyzed cell lines differed in the scale of the effect and a correlation with the PpIX accumulation was observed. Additionally, an increased activation of the iron transporter MFNR1 was also detected, which indicated that the regulation of iron transport is essential in PDT. Generally, cancer therapies have the most side effects of any of the therapies that are used today. These side effects often create serious psychological and medical problems. Therefore, therapies that are safe for the patient should be developed. One such therapy is photodynamic therapy (PDT), which is one of the few anticancer methods that is relatively safe for the patient ,2,3. The2 [2 and focused on the transporters that are involved in the heme biosynthesis pathway intermediates: flux and the phototoxic effects , which plays a crucial role in the delivery of a prodrug (5-ALA) to a cell ,10. PepT2 emerged as the most promising in terms of its applicability in ALA-PDT because it increased the PpIX levels in most of the tested cell lines, did not exhibit any antiproliferative properties [Using iron chelators in ALA-PDT to increase the efficiency of this therapy is not new. However, the number of chelators that can be used in ALA-PDT is limited and their mechanism of action is poorly understood. In our previous work, we investigated the ability of novel iron chelators from the TSC group to increase the PpIX accumulation on different cell lines with the heterogeneous expression of heme biosynthesis and degradation pathway genes. Of the TSC derivatives that were tested, derivative operties  and had operties .2. The first of these was ABCG2, which is one of the most studied transporters in this context and is involved in PpIX efflux from the cell, whose increased expression correlated with a low PpIX accumulation as well as a low PDT efficacy [2 compared to the cells they had been treated with 5-ALA alone , i.e., Hs683 (3.8-fold increase) and A549  in 75 cm2 flasks . The cells were cultured in standard conditions of 37 °C in a humidified atmosphere at 5% CO2. All of the cell lines were screened for Mycoplasma contamination.The HCT 116 (colon cancer), MCF-7 (breast cancer) and A549 (lung cancer) cell lines were purchased from the American Type Culture Collection , while the Hs683 (glioma) cell line was kindly provided by Prof. Gabriela Kramer-Marek from the Institute of Cancer Research . All of the cell lines were cultured in Dulbecco’s Modified Eagle’s Medium , which had been supplemented with 12% foetal bovine serum  and an antibiotic blend , derivative 2 (25 µM) and a combination of 5-ALA and derivative 2 in a manner in which their final concentrations matched the concentrations of 5-ALA and derivative 2 alone. After another 24 h of incubation, the total RNA was isolated from the cells with TRIzol Reagent  according to the manufacturer’s protocol. Reverse transcription was performed on 2 µg total RNA using a GoScript™ Reverse Transcriptase kit  and Oligo(dT)23 Primers . Quantitative RT-PCR was performed in a CTX96 Touch™ Real-Time PCR Detection System  using SYBR® Green Master Mix , the appropriate primer pairs and a cDNA template. The primers that were used in this study were designed using Primer 3 online software (−ΔΔCT (Livak and Schmittgen) method. The experiments were repeated at least three times in triplicate.Cells were seeded in 3-cm plastic Petri dishes  at a density of 4 × 10software  and synt6 cells/dish for 24 h at 37 °C. Next, 5-ALA (1 mM), TSC derivative 2 (25 µM) and a combination of 5-ALA and derivative 2 were added and incubated for 24 h in the dark at 37 °C. After incubation, the cells were washed with PBS and lysed with a 1% Triton X-100 solution in PBS. The lysates were then sonicated and centrifuged at 10,000× g for 10 min. The free heme concentration was measured using a Hemin Assay Kit  according to the manufacturer’s protocol. The heme concentration was calculated from the calibration curve of three independent experiments.The cells were seeded at a density 1 × 102 (25 µM) and a combination of 5-ALA and derivative 2 were added and incubated for 24 h in the dark at 37 °C. After the incubation, the cells were washed three times with a medium without phenol red and then irradiated or with an LED red light (634 ± 5 nm) at a dose 12 J/cm2. The cells were further incubated in the dark under 5% CO2 at 37 °C for 24 h. After irradiation, the cell viability was determined using an MTS assay. Briefly, the medium was removed and 100 µL of DMEM without phenol red and 20 µL of CellTiter 96® AQueous One Solution  was added and then incubated for approximately 1 h at 37 °C. Simultaneously, the same experiment was performed with irradiation step being omitted in order to show dark cytotoxicity. The results are presented as the mean ± standard deviation of three independent experiments and the cell viability was calculated using the GraphPad Prism 5 software .The cells were seeded into a 96-well plate at a density of 11,000 cells/well for 24 h at 37 °C. Then, the medium was removed and 5-ALA (1 mM), derivative p value of 0.05 or less was considered significant.All of the experiments were repeated three times in independent experiments and the results are presented as the mean ± standard deviation. All of the data were analyzed using GraphPad Prism 5 software . A two-way ANOVA followed by Tukey’s test was used to examine the significance of any differences between more than two groups. A two-tailed 2 is particularly interesting in this case due to its strong photocytotoxic effect, which was obtained on most of the tested cell lines in combination with 5-ALA. Despite the significant increases in ABCG2 gene expression, the observed therapeutic effect on most of the cell lines that were included in this work was substantial. This indicates that TSC derivative 2 is an excellent candidate for increasing the efficiency of ALA-PDT. We also showed that the expression level of the ABCG2 gene correlated with the phototoxic effect for most of the cell lines. The absence of this effect for the A549 lineage may be due to the increased expression of MFNR1. Surprisingly, this study does not explain the strong phototoxic effect that was observed for the Hs683 line and the reasons for this remain an open question. Because of the still insufficient knowledge about the mechanisms of ALA-PDT therapy, the results presented here are a necessary addition to fill in the existing gaps. At the same time, our study still remains an interesting area for further research.In conclusion, the use of iron chelators from the thiosemicarbazone group as enhancers is a promising approach for improving the efficiency of ALA-PDT. The TSC derivative


PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines  were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha ( In precision medicine, variants in oncogenes and cancer-suppressor genes can be analyzed using next-generation sequencing (NGS), and appropriate molecular targeted drugs can be more effectively chosen for cancer treatment based on the results of NGS analysis ,2. HowevPDGFRA) gene using the CRISPR/Cas9 system, which has been identified as potentially pathogenic .Genomic DNA was extracted using the Wizard SV Genomic DNA Purification System . The DNA fragment surrounding the target site was amplified using the primers listed in Genomic DNA was extracted using the Wizard SV Genomic DNA Purification System (Promega) and sequenced using a MiSeq sequencer . Briefly, the sequencing sites were amplified from the genomic DNA using the KOD One PCR Master Mix (TOYOBO) and the primers listed in p < 0.05.The data were plotted and analyzed using R (version 4.1.0) and ggplot2 (version 3.3.3). Three or more replicates were performed for all experiments, and all data are presented as the means ± standard error. Statistical significance was determined using an unpaired one-tailed Student’s t-test, and the results were considered statistically significant at


Sarcophyton ehrenbergi afforded cembrene diterpenoids, sarcoehrenbergilid D–F (1–3). Chemical structures were established by modern spectroscopic techniques with absolute stereochemistries determined by circular dichroism (CD) and time-dependent density functional theory electronic CD calculations (TDDFT-ECD). Cytotoxicity activities for 1–3 were evaluated against three human cancer cell lines: lung (A549), colon (Caco-2) and liver (HepG2).A solvent extract of the soft coral  Sarcophyton ehrenbergi afforded cembrene diterpenoids, sarcoehrenbergilid D–F (1–3).A solvent extract of the soft coral  The molecular formula C21H32O5 was determined by high-resolution electron ionization mass spectrum (HREIMS) .Compound 1 was isolated as a white powder with an optical rotation of 25D = −5.4 . The molecular formula (C20H30O5) was detected by high resolution electron ionization (HREIMS) spectrum . HREIMS analysis exhibited a molecular ion peak at m/z 350.2094 [M]+  The IR spectrum showed characteristic bands at νmax 3445 cm−1 and 1747 cm−1 for hydroxyl and keto groups, respectively. The 13C NMR spectrum revealed twenty carbon signals . Compound 3 was isolated as a colorless oil with a negative optical rotation of [α]25D = −10.8 . The molecular formula of C20H30O5 was detected by high resolution electron ionization (HREIMS) analysis .Stereochemistry was established based on coupling constants and NOESY experiments . NOESY cCD curve . Accordiνmax 3445 cm−1 and 1747 cm−1 for hydroxyl and keto groups, respectively. The 13C NMR spectrum  as well as 2D NMR  . Additionally, several carbon signals showed downfield chemical shift in comparison of sarcophyolide E: δC 37.0/36.2 (C-5), 73.1/71.8 (C-12), 123.1/121.7 (C-15), and 175.5/174.9 (C-17), respectively. The carbon signals at δC 163.9 (C-1) and 36.3 (C-13) showed upfield chemical shift in comparison with sarcophyolide E [δC 165.8 (C-1) and 37.3 (C-13)].The IR spectrum showed characteristic bands at spectrum  showed 2nd HMBC)  spectrosrelation  as well δH 3.09 dd, J = 7.5, 2.5) and H-11  established an alpha linkage for the ether bridge between C-7 and C-11. The NOE correlations between H-3 and the γ-lactone-(H-2) as well as vicinal coupling constant indicated a trans-geometry for H-2 and H-3 of the olefinic bond .The relative configuration for 3 was established based on coupling constants and NOESY experiments . A NOE cnic bond . As experotation . These d25 values of 23.3, 27.3, and 25.4 μM, respectively. Compound 2 and 3 showed weaker activity toward liver (HepG2) human cancer cell lines with IC25 values of 22.6 and 31.8 μM, respectively. The treated human colon cancer cells (Caco-2) cell viability was over 100% for all the isolated compounds (IC25 > 100 μM). Since primary necrosis is not easily differentiated from secondary necrosis that occurs with apoptosis,27 the mode of action will not be considered. To differentiate these distinct biological events requires apoptotic assays accompanying necrosis measurements. A combined necrosis/apoptotic time-course will be presented in a subsequent study to elaborate on mode of action.Isolated metabolites 1–3 were tested for cytotoxic activity toward lung (A549), colon (Caco-2) and liver (HepG2) human cancer cell lines based on an MTT reduction assay . Compoun3.3.1.Circular dichroism was measured on JASCO 810 spectropolarimeter. HREIMS data were collected on a JEOL JMS-700 instrument . NMR spectra were recorded on a Bruker 500 NMR spectrometer (Japan). JASCO P-2200 polarimeter and JASCO FT/IR-6300 spectrometer was used for optical rotation and infrared measurements, respectively.254)  were used for purification and monitoring spotting, respectively. A H2SO4 : MeOH (1 : 9) spraying reagent was used for spot visualization after heating. HPLC purification was performed using Shimadzu HPLC-RID-10A with YMC-Pack ODS-A analytical (250 × 4.6 mm i.d.) and preparative (250 × 20 mm i.d.) columns  for separation.Normal-phase silica gel 60 (230–400) column chromatography (CC) as well as aluminum TLC plates  was deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.3.3.2Cl2 : MeOH  at room temperature (3 L × 4 times). Isolation protocol was performed as described previously by Hegazy et al., 2017  were extracted with CHl., 2017  to affor3.3.1α]25D +10.8 ; FT-IR (KBr) νmax: 3435, 2941, 1748, 1462, and 1224 cm−1; 1H and 13C NMR data, see m/z 346.2127 [M − H2O]+ .White powder; [3.3.2α]25D −5.4 ; FT-IR (KBr) νmax: 3433, 2938, 1743, 1446, and 1218 cm−1; 1H and 13C NMR data, see m/z 350.2094 [M]+ .White powder; [3.3.3α]25D −10.8 ; FT-IR (KBr) νmax: 3441, 2932, 1742, 1448, and 1229 cm−1; 1H and 13C NMR data, see m/z 332.1993 [(M − H2O)+] .White amorphous powder; [3.4.3.4.12 at 37 °C.Three human cancer cell lines, A549 , Caco-2 (colon adenocarcinoma) and HepG2 (hepatocellular carcinoma) (ATCC®) were assayed with the purified compounds. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% , 1% penicillin and incubated in 5% CO3.4.23 cells per well; 96-well plate in a volume of 100 μL). To generate concentration-dependent curves, sample concentration was varied  for a total well volume of 200 μL for 48 h. MTT solution (5 mg ml−1) was added (100 μL per well) for 90 min before measurements.28,29 After medium removal, dark blue formazan crystals formed in viable cells were dissolved in 100 μL of DMSO, followed by shaking for 10 min (200 rpm). The absorbance was recorded at 492 nm using a microplate reader  for cell viability measurement. IC25 values were expressed as a concentration of tested compound that inhibits 50% cell growth in comparison with a vehicle control (quadrate to octuplet treatment) by non-linear regression model analyses using GraphPad Prism® v 6.0 software.The cytotoxicity of tested compounds was investigated by a MTT assay. Cell lines were seeded and incubated overnight allowing cell adhesion to the plate well  calculations with incorporating a polarizable continuum model (PCM) using methanol as a solvent were carried out at the B3LYP/6-31G* level of theory to calculate the first fifty excitation states. Electronic circular dichroism (ECD) spectra were finally generated using SpecDis 1.71  by appl4.S. ehrenbergi soft coral. The isolated compounds were tested against three human cancer cell lines, which resulted in 2 being the most potent compound against lung A549 cancer cell. The absolute stereochemistry of 1–3 were confirmed by comparing experimental and TDDFT-simulated ECD spectra.Cembrene diterpenoids (1–3) were isolated and identified from the There are no conflicts to declare.RA-009-C9RA04158C-s001


The fatal clinical course of human glioblastoma (GBM) despite aggressive adjuvant therapies is due to high rates of recurrent tumor growth driven by tumor cells with stem-cell characteristics . The aldehyde dehydrogenase 1 (ALDH1) family of enzymes has been shown to be a biomarker for GSCs, and ALDH1 seems to be involved in the biological processes causing therapy resistance. Ferroptosis is a recently discovered cell death mechanism, that depends on iron overload and lipid peroxidation, and it could, therefore, be a potential therapeutic target in various cancer types. Since both ALDH1 and ferroptosis interact with lipid peroxidation (LPO), we aimed to investigate a possible connection between ALDH1 and ferroptosis. Here, we show that RSL3-induced LPO and ferroptotic cell death revealed RSL3-sensitive and -resistant malignant glioma cell lines. Most interestingly, RSL3 sensitivity correlates with ALDH1a3 expression; only high ALDH1a3-expressing cells seem to be sensitive to ferroptosis induction. In accordance, inhibition of ALDH1a3 enzymatic activity by chemical inhibition or genetic knockout protects tumor cells from RSL3-induced ferroptotic cell death. Both RSL-3-dependent binding of ALDH1a3 to LC3B and autophagic downregulation of ferritin could be completely blocked by ALDH inhibition. Therefore, ALDH1a3 seems to be involved in ferroptosis through the essential release of iron by ferritinophagy. Our results also indicate that ferroptosis induction might be a particularly interesting clinical approach for targeting the highly aggressive cell population of GSC. The reason for the devasting clinical prognosis of human malignant gliomas despite aggressive adjuvant therapies is the high rate of recurrent tumor growth, most likely due to an extremely resistant tumor cell population with stem-cell characteristics . AldehydALDH1 seems to be involved in detoxification of aldehydes that result from lipid peroxidation (LPO) in glioma cells . High leThe functional interrelationship between these two important regulators in tumor biology could be of interest in glioma CSCs since LPO seems to be functional in both ferroptosis and ALDH1. This could also have clinical implications. Inhibition of ALDH1-dependent resistance against chemotherapy and induction of ferroptosis could theoretically lead to more effective adjuvant therapies. Here, we investigated the functional role of ALDH1 in ferroptosis in human malignant glioma cell lines.2). Isolated primary cell lines were cultured in primary cell culture medium, which included 500 mL of RPMI-1640 , 10 mL of B27 , 5 mL of N2 Supplement , 5 mL of nonessential amino acids , 10 mL of L-glutamine , 10 μg of fibroblast growth factor 2 , 150 ng of transforming growth factor beta 2 , and 1 μg of epidermal growth factor . GBM stem-like cell line X01  [Human glioblastoma cell lines U87MG , LN229 , and T98G  were purchased from the American Type Culture Collection  and cultured in DMEM with 10% FBS  under standard cell culture conditions -2A-GFP (PX458) (Addgene plasmid #48138) using ALDH1A3 single-guided RNA as previously described .Glioblastoma specimens  were coLN229, U87MG, and T98G cells were seeded in 96-well plates (5000 cells/well). Treatment was performed at different concentrations for 24 h, while controls received 0.5% DMSO, only. The proportion of viable cells was determined using the 3--2,5-diphenyltetrazolium bromide  assay following the manufacturer’s recommendations. Absorbance was examined using an Infinite F200 pro Microplate Absorbance Reader .For cell death detection, LDH release was quantified using a cytotoxicity detection (LDH) kit  and was conducted following the manufacturer’s instructions. Absorbance was examined using an Infinite F200 pro Microplate Absorbance Reader at 490 nm.® 581/591 C11-Reagent  for 30 min and washed with PBS. Cells were either fixed by 30% PFA to perform fluorescence imaging or trypsinized into single cells to perform fluorescence-activated cell sorting (FACS). Fluorescence images were taken by a Zeiss fluorescence microscope  and analyzed using software Image J . FACS results were analyzed using FlowJo .Cells were seeded on cover slides and treated 24 h before performing the lipid peroxidation assays. Cells were stained by BODIPY2, and protease inhibitor cocktail). Then, 30 μg of protein was taken out from the cell lysate as the input sample. Anti-ALDH1a3  and anti-IgG  were added to the lysate for immunoprecipitation (containing 30 μg of protein), and then incubated at 4 °C overnight. Next, 2 mg of protein-G-Sepharose beads were added to each immunoprecipitation sample for 3 h in 4 °C. After washing, Sepharose beads were collected and diluted with SDS sample buffer , and then heated at 100 °C in 5 min. Input samples and immunoprecipitation samples were fractionated by 10% SDS-page gels and read out by Western blotting with anti-LC3b .Glioblastoma cell lines LN229 and T98G were treated and collected after 24 h in lysis buffer  treatment for 2 weeks.ALDH1a3 plasmid was obtained from OriGENE (MG222097). Briefly, PDCL#9 cells (ALDH1a3 negative) were transfected with ALDH1a3 plasmid using lipofectamine 2000 . After 48 h, ALDH1a3-overexpressing clones were selected by G418  was used to perform the analysis, and p-values < 0.05 were regarded as statistically significant.Three independent experiments of each assay were conducted to validate results. A To discover RSL3-induced cell death in glioblastoma, we treated tumor cells of three established glioma cell lines , three primary GBM cell lines , and GSC-enriched glioma cell line X01 with different concentrations of RSL3. We observed the RSL3 dose-dependent loss of cell viability, but it was different between glioblastoma cell lines A. There The type of RSL-3-induced cell loss was most likely ferroptosis, since concomitant treatment with ferrostatin-1, a specific ferroptosis inhibitor, and with deferoxamine (DFO), an iron chelator, completely abolished RSL3-induced cell death B.Induction of lipid peroxidation (LPO) after RSL3 treatment was measured using BODIPY staining C. Using These results indicate that the RSL3-induced cell death in glioma cell lines is a ferroptosis-like cell death.We next verified cell death according to LDH release A. Most iTo prove the role of the ALDH1a3 subtype as a glioma stem cell (GSC) marker, we performed sphere formation assays of ALDH1a3 wildtype and corresponding knockout glioma cell lines. Our results showed that ALDH1a3 knockout cells lost their ability to form spheres or formed much smaller spheres compared to their ALDH1a3 wildtype counterparts C.To further investigate the role of ALDH1a3 in RSL3-induced cell death, we tested the effect of inhibition of ALDH1 enzymatic activity by the specific inhibitor diethylaminobenzaldehyde (DEAB) in ALDH1a3 high-expression  and low-expression cell lines (T98G and PDCL#9). DEAB at least partially reversed cell death induced by RSL3 A. These These results show that ALDH1a3 seems to be involved in RSL3-induced ferroptotic cell death, and that it might play a role upstream of LPO.Autophagy is involved in the induction of ferroptosis, and ALDH1 also seems to play a role in autophagy. We, therefore, investigated the effect of autophagy inhibition on ferroptosis in glioma cell lines. The concomitant treatment with the specific autophagy inhibitor bafilomycin A1 rescued glioma cells from RSL3-induced LPO and ferroptotic cell death A–C. MoreThe fatal clinical course of human glioblastoma despite aggressive adjuvant therapies is due to highly resistant tumor cells, i.e., glioma stem cells (GSC), which express ALDH1 , and ALDHere, we show that RSL3-induced ferroptotic cell death revealed RSL3-sensitive and -resistant malignant glioma cell lines. Most interestingly, the RSL3 sensitivity correlated with ALDH1a3 expression; only high-ALDH1a3-expressing cells seemed to be sensitive to ferroptosis induction. In accordance, inhibition of ALDH1a3 enzymatic activity by DEAB protected cells from RSL3-induced cell death.Ferroptosis is a novel cell death mechanism distinct from necrosis or apoptosis . So far,Since autophagy is involved in both ferroptosis  and ALDHAutophagy plays an important role in ferroptosis . FerroptThese data are also relevant for clinical consequences, although our results do not support the hypothesis that ferroptosis and concomitant ALDH1 inhibition could increase tumor cell death under standard treatment conditions. However, tumor cells highly expressing ALDH1 represent the most aggressive subpopulation of GSC within GBM. Moreover, it has been shown that the number of ALDH1a3-expressing cells dramatically increases in recurrent tumor growth , eventua


Oesophageal adenocarcinoma (OAC) is a leading cause of cancer mortality in the United Kingdom with a 5-year survival rate of approximately 15%. Major contributors to poor outcome are late diagnosis and chemotherapy resistance, and while targeted therapies have benefitted certain cancer settings, they have had limited success in OAC. An understanding of the mechanisms mediating chemotherapy resistance could identify novel targets with the potential to improve standard treatments. This paper aimed to identify mediators of both OAC cancer cell pro-survival signalling and chemotherapy resistance, as well as determine potential small molecular compounds to counteract this. Gene set enrichment analysis of transcriptional data generated a gene-list with significant differential expression between responder and non-responder OAC patients. Gene functionality assessment using siRNA screening showed that targeting SRC had an anti-tumour effect in OAC cells with the potential to enhance chemotherapy treatment. In parallel to this, a compound screen showed the Src inihibitor dasatinib sensitised OAC cells to chemotherapy. Together, these findings suggest targeting SRC as a novel therapeutic strategy in OAC.Drug resistance limits the effectiveness of oesophageal adenocarcinoma (OAC) chemotherapies, leading to a poor prognosis for this disease. Elucidation of the underlying resistance mechanisms is key to enabling the identification of more effective treatments. This study, therefore, aims to identify novel therapeutic and/or chemotherapy sensitising drug targets in OAC. Transcriptional data from a cohort of 273 pre-treatment OAC biopsies, from patients who received neoadjuvant chemotherapy followed by surgical resection, were analysed using gene set enrichment analysis (GSEA) to determine differential gene expression between responding and non-responding OAC tumours. From this, 80 genes were selected for high-throughput siRNA screening in OAC cell lines with or without standard chemotherapy treatment. In parallel, cell viability assays were performed using a panel of FDA-approved drugs and combination index (CI) values were calculated to evaluate drug synergy with standard chemotherapy. Mechanisms of synergy were investigated using western blot, propidium iodide flow cytometry, and proliferation assays. Taken together, the screens identified that targeting Src, using either siRNA or the small molecule inhibitor dasatinib, enhanced the efficacy of chemotherapy in OAC cells. Further in vitro functional analysis confirmed Src inhibition to be synergistic with standard OAC chemotherapies, 5-fluorouracil (5-FU), and cisplatin (CDDP). In conclusion, a compound screen together with a functional genomic approach identified Src as a potential chemosensitising target in OAC, which could be assessed in a clinical study for poor prognosis OAC patients. Incidence rates of OAC have progressively increased by six-fold since the 1970s, with the United Kingdom reported to have one of the highest global incidence rates together with the poorest survival rates ,3. HowevResistance to chemotherapy is a major problem in the treatment of cancer, hence the clinical urgency to identify key molecular targets. The expression or mutational status of these targets may constitute novel predictive biomarkers of drug response or resistance. In addition, these molecular determinants of drug resistance may provide novel therapeutic strategies for enhancing the clinical effectiveness of current chemotherapeutics.The aim of this study was to apply high-throughput technologies such as siRNA and compound screens to identify molecular determinants of drug resistance in OAC. We discovered pathways and genes differentially regulated between chemotherapy non-responders and responders by applying gene set enrichment analysis (GSEA) to the transcriptional data from 273 pre-treatment OAC biopsies . A funct2. All cell lines were routinely examined for mycoplasma contamination using PlasmoTestTM .OE33 (Cat. No. 96070808), FLO-1 (Cat. No. 11012001), SKGT4 (Cat. No. 11012007), and OE19 (Cat. No. 96071721) OAC cell lines were obtained from Public Health England. MFD-1 cells were a kind gift from Professor Tim Underwood, University of Southhampton ..15].30(72 h) CDDP or 5-FU treatment  reduced OE33 viability with either knockdown alone or in combination with CDDP (p < 0.05) decreased cell viability (p < 0.05) decreased viability (p < 0.05) decreased cell viability  doses of CDDP. Following 72 h treatment, cell viability was measured using the Cell Titer Glo® assay. A total of 97 compounds, either alone or in combination with CDDP, were found to decrease cell viability by ≥80% in OE33 cells and/or FLO-1 cells  doses following 72 h treatment  were assessed across a panel of OAC cell lines: OE33, FLO-1, SKGT4, OE19, and MFD-1 a. This sreatment . Howeverreatment b. Knockdreatment c. Of notFor Src inhibitor investigations, three OAC cell lines were selected based on high (OE33), medium (FLO-1), and low levels (MFD-1) of total-Src and phospho-Src Y416 a. InhibiGiven the poor response rates in OAC patients that undergo neoadjuvant chemotherapy, we asked if Src inhibition could improve sensitivity to standard chemotherapy. CDDP is the main focus of this study as platinum-based chemotherapy is the main active DNA damaging agent used to treat OAC.30(72 h) treatment (p < 0.001), FLO-1 (p < 0.01), and MFD1 (p < 0.05) cells when combined with CDDP (Dasatinib/CDDP combination experiments were assessed by MTT assays following 72 h ~ICreatment . Dasatinith CDDP a. To ideith CDDP b.p < 0.01), FLO-1 (p < 0.05), and MFD-1 (p < 0.05) cell viability  doses of CDDP and dasatinib (p < 0.05) compared with either treatment alone, confirming an increase in cell death in this cell line , FLO-1 (p < 0.05), and MFD-1 (p < 0.05) cells compared with either treatment alone  doses for 72 h (p < 0.05). No significant change in FLO-1 cell viability was observed following combination treatment. To identify dasatinib/5-FU drug interactions, CI values were generated as before. From this, the CI values showed moderate synergy of dasatinib/5-FU in the OE33 and MFD-1 cell lines, with most CI values ranging between 0.4 and 0.8, and antagonistic (CI > 1) in the FLO-1 cells (p < 0.01), FLO-1 (p < 0.05), and MFD-1 (p < 0.05) cell viability compared with either treatment alone  dose of 4 µM CDDP compared with 1 µM in the original OE33  cell line (30(72 h), the sub-line was labelled OE33 CDDP-resistant (CDDP-R) and, together with the OE33 parentals, this cell line pair was used to investigate the effect of Src inhibition on acquired CDDP resistance.An OE33 CDDP-resistant sub-line was generated by drugging OE33 cells continuously with incremental increased doses of CDDP over ~6 months. The OE33 sub-line was found to have an ~ICell line . Given t30(72 h) dose of 1 µM CDDP and cell viability was measured by MTT following 72 h treatment. Indeed, this combination treatment gave a significant reduction (p < 0.05) in OE33 CDDP-R cell viability to <70%, thus restoring CDDP sensitivity to that of the OE33 parental cells (30(72 h) dose of CDDP resulted in additional loss of OE33 CDDP-R cell viability  that consistently caused decreased cell viability alone or when combined with cisplatin or 5-FU. In parallel, a drug screen was performed to identify compounds that decrease OAC cell viability, focusing on compounds that had increased efficacy when combined with cisplatin. This parallel screen approach identified Src and several of its interactants as a potential pathway mediating platinum sensitivity in OAC. Further validation showed that Src inhibitors synergised with CDDP and 5-FU in multiple OAC settings in vitro. These results suggest that clinical targeting of Src in OAC could improve outcomes to neoadjuvant chemotherapy treatment.Src is a non-receptor tyrosine kinase and a central nodal protein within multiple signalling pathways. It has a key role in driving tumourigenesis through mediating tumour survival, proliferation, angiogenesis, and invasion . Indeed,Increased Src kinase activity has been reported in endoscopic tissue samples of Barrett’s oesophagus, the premalignant precursor of OAC (3–4-fold) and OAC (6-fold), compared with normal oesophagus control samples . Src phoOur study has several strengths compared with previous attempts to identify mediators of chemo-resistance to neoadjvant chemotherapy in OAC. Firstly, the large gene expression dataset of pre-chemotherapy biopsies with comprehensive clinicopathological information used in this study provides a high-powered discovery cohort. Secondly, the extensively validated parallel screen approach lends robustness to the finding of Src as a potential therapeutic target. Finally, the in vitro validation of Src inhibiton in parental and drug-resistant cell lines provides a strong rationale for further study of this target.There are, however, limitations to our study. These include the use of a single siRNA and a single cell line to select potential candidate genes in the primary siRNA screen. Moreover, as the candidate gene list was limited to 80 genes, this likely overlooked other potential, novel targets. In addition, future work should include in vivo validation of the in vitro findings from the siRNA screen and the compound screen. During this study, the standard of care for neoadjuvant chemotherapy for resectable gastric or gastro-oesophageal junction adenocarcinoma in the United Kingdom changed from epirubicin, CDDP, and 5-FU/capecitabine (ECF/ECX) to a combination of 5-FU, leucovorin, oxaliplatin, and docetaxel (FLOT). While FLOT is still platinum-based, future work should include taxane-based chemotherapy in novel target selection. Finally, the selected OAC cell line panel used in this study may not be fully representative of this highly heterogeneous disease, thus further validation in a broader panel may be required.In summary, our study suggests that Src inhibitors could enhance the OAC response to cisplatin and 5-FU. Further investigation using in vivo or near-patient models is required to validate the current findings followed by a re-appraisal of the efficacy of Src inhibition as a clinical strategy in OAC. Development of a companion biomarker may also be necessary to select sensitive patients for this combination treatment approach in clinical trials.In conclusion, this functional genomic screening approach identified Src as a novel chemosensitization target in OAC with a parallel FDA compound screen also identifying the Src inhibitor, dasatinib. Further functional analysis showed the ability of Src inhibitors to synergise with, and overcome resistance to, standard OAC chemotherapies. This lends support to a combined therapeutic strategy of chemotherapy and Src inhibition to improve the current poor response rates to neoadjuvant chemotherapy in OAC.


IntroductionIt has been shown that the expression of the epidermal differentiation marker, Cornulin, declines with the progression of squamous cell carcinomas of several tissue types.ObjectivesThis study aims to examine Cornulin expression at the cellular level in various cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. This can pave the way for the utilization of this novel biomarker as a diagnostic and prognostic indicator for oral cancer and help guide treatment options.Study designWestern blotting was performed to measure Cornulin expression levels using standardized cell lysates from four different cell lines representing the successive steps of OSCC progression. Specifically, primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells were used to represent normal oral keratinocytes, DOKs, locally invasive OSCC cells, and metastatic OSCC cells, respectively.ResultsCornulin expression was found to be downregulated with the progression from normal to premalignant to malignant cells. Quantitative analysis revealed that Cornulin is significantly downregulated by 3.4 folds in DOK cells, by 23.7 folds in SCC25 cells, and by 5.2 folds in Detroit 562 cells compared to normal gingival keratinocytes. Interestingly, Cornulin was upregulated by 4.5 folds in the metastatic Detroit 562 cell line compared to the locally invasive SCC25 cells.ConclusionAltogether, Cornulin proved to be differentially expressed at the cellular level in cell lines representative of the successive steps of OSCC progression. Specifically, we documented a gradual decrease in Cornulin expression with the progression from normal to premalignant to malignant cells. Notably, there is a significant increase in the expression of Cornulin in the metastatic cell line Detroit 562 compared to the locally invasive cell line SCC25, suggesting a possible correlation with the biological behavior and unique characteristics of these two different phenotypes. Oral cancer consists of a family of neoplasms that could arise in any part of the oral cavity, pharyngeal region, or salivary glands . Oral sqCRNN gene, is a 495 amino acid protein belonging to the family of S100 fused-type proteins, is located on chromosome 1q21 locus, and has tumor suppressor characteristics [Cornulin, encoded by the eristics , esophageristics , cutaneoeristics , and OSCeristics . This stCell cultureA total of four different cell lines were used to model the successive steps of OSCC progression. The squamous cell carcinoma 25 (SCC25) human OSCC cell line, Detroit 562 metastatic pharyngeal squamous cell carcinoma (SCC), and primary gingival epithelial cells were acquired from American Type Culture Collection (ATCC) , and the dysplastic oral keratinocytes (DOKs) were acquired from the European Collection of Authenticated Cell Cultures (ECACC)  via Millipore Sigma . All cell lines were cultured and maintained in their respective specialized media according to vendors’ instructions.Western blotThe four cell lines were separately harvested for their total protein content and the protein concentration was determined using the BCA protein assay . The cell lysate was prepared for the SDS-PAGE electrophoresis in radioimmunoprecipitation assay buffer (RIPA buffer) containing dithiothreitol (DTT), and the samples were then boiled for 5 minutes. An XCell SureLock system and Novex precast protein gels  were used for the SDS-PAGE electrophoresis, and the gel was run at two different settings: the first at 60 V for 40 minutes and the second at 140 V for 1 hour and 35 minutes. Once complete, the wet transfer was set up using a Mini-PROTEAN Tetra system  and Immobilon-P PVDF transfer membranes . This was carried out at 100 V for 1 hour in a pre-chilled transfer buffer.The resulting membranes were then blocked using 5% milk dissolved in 1x tris-buffered saline with 0.01% Tween (TBST). Membranes were blocked for 2 hours. The membranes were then incubated with the primary antibody in blocking buffer overnight at 4°C. A 1:100 dilution was used for the anti-Cornulin mouse monoclonal antibody . Membranes were then washed and incubated with the secondary antibodies for 1 hour at room temperature. A 1:5,000 dilution was used for the mouse IgGκ light chain binding protein secondary antibody . Subsequent labeling of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as a loading control. A 1:10,000 dilution was used for GAPDH mouse monoclonal antibody . Membranes were subsequently imaged using a BioRad ChemiDoc XRS+ system (Bio-Rad) and Amersham ECL western blotting detection reagent .Quantitative analysist-test.The images produced by the BioRad ChemiDoc system (Bio-Rad) were analyzed using ImageJ software . The pixel intensity of each sample was assessed compared to the GAPDH control and plotted as a fold difference in protein expression of Cornulin compared to GAPDH. Sample sets of three repeats were compared to each other using an unpaired Based on existing evidence on the role of Cornulin as a tumor suppressor gene product, we put forward a hypothesis predicting a gradual decrease in Cornulin expression in correlation to the stepwise progression from normal oral keratinocytes to premalignant keratinocytes to invasive and metastatic malignant OSCC keratinocytes Figure .To test our hypothesis, we utilized established human cell lines including primary gingival keratinocytes to model the normal oral mucosa, DOKs to represent the premalignant lesion known as leukoplakia, SCC25 cell line to represent locally invasive SCC cells, and Detroit 562 cell line as a representative for the metastatic phenotype Table .Western blot proteomic analysis on the four cell lines confirmed that Cornulin expression declines with the progression from normal oral keratinocytes to premalignant dysplastic keratinocytes to malignant OSCC cells Figure . StatistCornulin was first referred to as C1Orf10 and is known to be expressed in esophageal, oral, anal, cervical, and skin squamous epithelia ,13. It iSignificant differences in Cornulin expression were observed in dysplastic keratinocytes (DOK), locally invasive cancerous cells (SCC25), and metastatic cancerous cells (Detroit 562) in comparison to the normal gingival keratinocytes. Interestingly, a statistically significant increase in Cornulin expression was noted between the locally invasive SCC25 cells and the metastatic Detroit 562 neoplastic cells, possibly pointing to a role for Cornulin in the process of metastasis or perhaps as a result of the natural selection of few cells with metastatic abilities and higher than average Cornulin levels from the heterogeneous cell population of the primary tumor with variable Cornulin expression levels.A limitation of this study is the reliance on established human cell lines to represent the steps in progression of oral squamous carcinoma lesions. Although in vitro models for tumor progression, such as the immortalized, dysplastic, and cancer cell lines used in this study, have the advantages of fast growth rate , unlimitIn the clinical practice, OSCC, leukoplakia, and several non-cancerous oral lesions are difficult to distinguish from each other by physical examination alone ,18. ThisThis study has demonstrated significant quantitative differences in the expression of Cornulin across the successive steps of OSCC progression. These findings allude to the potential utility of Cornulin as a predictive and prognostic indicator. Follow-up studies to monitor Cornulin levels throughout the clinical course of disease and relative survival rate data are needed to firmly establish this promising biomarker in the clinical setting to supplement current pathological assessment and prognosis for patients afflicted with oral cancer.


A transfection vector that can home in on tumors is reported. Whereas previous vectors that allow moderately cell selective gene transfection used larger systems, this small‐molecule approach paved the way for precise structure‐activity relationship optimization. For this, biotin, which mediates cell selectivity, was combined with the potent DNA‐binding motif tetralysine‐guanidinocarbonypyrrol via a hydrophilic linker, thus enabling SAR‐based optimization. The new vector mediated biotin receptor (BR)‐selective transfection of cell lines with different BR expression levels. Computer‐based analyses of microscopy images revealed a preference of one order of magnitude for the BR‐positive cell lines over the BR‐negative controls. Picky for cancer cells: A small‐molecule transfection vector containing a strong DNA binder and a targeting moiety shows low toxicity and a tenfold selectivity for cell lines expressing biotin receptors (BR). The binder contains the tailor‐made guanidinocarbonypyrrol (GCP) motif. A tetrapeptide of only four GCP‐modified lysine residues is usually sufficient to achieve transfection. Addition of an appropriate linker deactivates the vector. Further attachment of a biotin label allowed BR‐rich cancer cell lines to be targeted. For example, Wang and co‐workersIn more general terms, such a tradeoff between selectivity and efficiency is to be expected, as increasing the number of targeting moieties per DNA binder is likely to reduce DNA‐binding capacity. A small‐molecule approach in which the cellular recognition unit is combined with a very strong DNA binder of similar size addresses this issue. Due to the strong binding interaction less DNA binder can be used per targeting moiety, thus increasing the relative amount of targeting moiety in a DNA‐vector polyplex. Furthermore, a small‐molecule approach allows for addressing hydrophobicity issues by synthesis.1 can condense DNA into sufficiently packed polyplexes, which are taken up preferably by biotin receptor‐positive cell lines  in HeLa and HEK293T cells. HeLa cells expose a high number of biotin receptors on their cellular surface (BR+), whereas 293T cells do not (BR−).1. Just as reported for PEI, lysosomal degradation can possibly be avoided by the proton sponge effect.First, we compared the transfection ability of 1 led to a decent expression of mCherry in HeLa cells whereas 2 did not appear to transport genetic material at all and PEI showed the highest transfection efficiency  HeLa cell line compared to the biotin‐receptor‐deficient (BR−) HEK293T cell line, and b) that the structurally similar, but biotin‐lacking control vector 2 was completely ineffective, point to a key role of the biotin moiety in mediating DNA uptake. However, a simple difference in the ability of the two vectors to condense DNA, which is of key importance for the passage through the cell membrane,1 and 2 in solution was measured by DLS. Stock solutions of the peptides in DMSO were titrated to a solution of plasmid DNA. For both vectors a decrease in size was observed with increasing vector concentrations.As illustrated in Figure y Figure , left. I1 with pH 2B‐mCherry DNA was confirmed by AFM  and two BR− cell lines .[2 was chosen as an all‐negative and PEI as an all‐positive control . After staining cellular nuclei with Hoechst 33342 the percentage of transfected cells was calculated using CellProfiler®. The results are summarized in the heatmap shown in Figure In Figure M Figure B.[26] ll line A49 and tw+ and BR− cell lines. It is immediately apparent that 1 is a whole order of magnitude more efficient in terms of transfection for the two BR+ cell lines A549 and HeLa compared to the three BR− cell lines CHO, 293T and HCT. On the other hand, the two controls PEI and 2 are almost indiscriminate in their ability/inability to transfect all cell lines. Although PEI does not seem to prefer any of the two groups of cell lines, its efficiency abates one order of magnitude for A549 cells. This is hardly surprising, as this cancer cell line is known for its resistance towards transfection.+ cell line is transfected even more efficiently with the small‐molecule vector 1.The rows in Figure 1 in a concentration dependent manner  should be addressed to the authors.Supporting InformationClick here for additional data file.


The present research investigates the causal explanation of academic enthusiasm based on the interaction between teachers and English learners: self-regulation, academic hope, and academic engagement among English language learners.The implementation method of this descriptive research and research design was structural equation correlation. The research’s statistical population included intermediate Zanjan city learners  who were studying in the academic year 2021-2022. This research used multi-stage cluster random sampling to test the proposed model. The following questionnaires were used to collect data: Academic enthusiasm ; Teacher-student interaction questionnaire (Moray and Zurich); Academic hope questionnaire (Khormai and Kameri); Self-Regulation Questionnaire (Bouffard) and academic engagement (Zarang). Lisrel software was used for data analysis and evaluation of the proposed model using structural equation modeling.The results of the present study showed that teacher-student interaction, academic self-regulation, academic engagement, and academic hope are related to students’ academic enthusiasm. The results of the present study also support the role of mediators of academic self-regulation, academic engagement, and academic hope in the causal relationships between teacher-student interaction and academic enthusiasm.Based on this, it can be concluded that with the improvement of teacher-student interaction, the level of academic self-regulation, academic engagement, and academic hope of students increases, and these factors together increase their academic enthusiasm. Therefore, it is suggested that to increase academic engagement, academic self-regulation, and academic hope in the students in the education system, attention should be paid to the position of the students’ academic enthusiasm. In recent years, the approach of positivist psychology concerning human talents and capabilities has attracted the attention of psychologists. One of the effective capabilities in education, referred to as strategies for dealing with academic issues and challenges, is academic enthusiasm. Students face various obstacles, challenges, and pressures in their daily academic lives. They face poor grades, threatening self-esteem, and diminished motivation, which provide them with a source of stress . One of Self-regulation is one of the scopes in which its application is of great importance and leads to students’ academic success. The theory of self-regulatory learning was proposed by Another effective factor that plays an important role in students’ academic enthusiasm is academic hope . Hope isAcademic engagement is a concept that was first used to explain academic failure and has been used as a basis for reformist actions in education . AcademiAcademic enthusiasm refers to behaviors that are related to learning and academic achievement . In theoAcademic enthusiasm includes how institutions organize their learning opportunities, resources, and services in such a way as to encourage learners to participate in such activities and gain benefits . ConcernIn other models, academic enthusiasm is considered an instrument consisting of behavioral, cognitive, and motivational dimensions . A positSelf-regulation means self-motivated thoughts, feelings, and actions designed to achieve personal goals . From thAcademic hope is a belief or expectation of education, a belief by which one expects to achieve positive educational results . The revAcademic engagement is a structure that was first used to explain the failure of academic achievement and was proposed as a basis for reformist measures in the field of education . AcademiOne of the factors related to the school and participation in the education process, which plays an effective role in the students’ academic enthusiasm and is related to academic self-regulation, academic hope, and academic engagement is the interaction between the teacher and the student. The teaRegarding the necessity of this research, it can be said that the educational environment is one of the environments that require adaptation and adaptability, in fact, the challenges and pressures of the educational environment, on the one hand, and the rapid cognitive and emotional changes on the other hand - the need for learners increases it to have it. In general, academic enthusiasm can protect the learner against academic difficulties as a protective factor. Based on this, it is necessary for researchers in the field of education to identify the factors that affect academic enthusiasm and the process of influencing academics. In this regard, considering the relationship between academic enthusiasm and academic self-regulation - academic hope and student-teacher interaction on the one hand, and the relationship between academic self-regulation, academic hope, and academic enthusiasm, on the other hand, shows the study of causal explanation of academic enthusiasm based on the interaction of teachers and English language learners: self-regulation, academic hope, and academic engagement among English language learners.1) The interaction between teachers and language learners significantly affects the academic enthusiasm of English language learners.(H2) Self-regulation significantly affects the academic enthusiasm of English language learners.(H3) Academic hope significantly affects the academic enthusiasm of English language learners.(H4) Academic engagement significantly affects the academic enthusiasm of English language learners.(H5) The interaction of teachers and language learners has a significant effect on the academic enthusiasm of English language learners with the mediating role of self-regulation, academic hope, and academic engagement.. Using this method, the direct and indirect effects of the variables in the assumed model can be investigated. In this research, Lisrel software was used for data analysis.The research’s statistical population included intermediate Zanjan city learners  who were studying in the academic year 2021–2022. The total number of language training centers was 24 (14 for girls and 10 for boys). This research used multi-stage cluster random sampling to test the proposed model. In this way, first, among the 24 intermediate-level institutions in Zanjan city . Four boys’ institutions were randomly assigned (4 classes were randomly selected from each institution for research and answering questions). In the current study, all intermediate-level language learners, whose number was 412, were investigated by the census method. Of these, 25 people did not want to answer, 26 questionnaires were incomplete, and a total of 361 correct questionnaires were examined. To carry out the research, first, the necessary permission was obtained to conduct the research and distribute the questionnaires in intermediate-level institutions in Zanjan city. In the next stage, questionnaires were distributed among the learners while making the necessary arrangements with Zanjan institutions within 1 month by referring to the institutions and informing managers and officials about the research objectives. The average time to answer the questionnaires was 45 min.In this research, the following questionnaires were used to collect data:Academic enthusiasm : This questionnaire has 15 items and measures it with a five-point Likert scale. This questionnaire has a subscale of behavioral desire with items 1, 2, 3, and 4, emotional desire with items 5, 6, 7, 8, 9, and 10, and cognitive desire with items 11, 12, 13, 14, and 15. In terms of the psychometric properties of the instrument, thusiasm : This quTeacher-student interaction questionnaire: This questionnaire consists of 17 items that have three subscales of communication (8 questions), trust (5 questions), and alienation (4 questions). Questions are graded on a four-point scale  on a scale of 1 to 4, respectively. In Also, in Academic hope questionnaire , Normed Fit Index (NFI), Incremental Fit Index (IFI), Relative Fit Index (RFI), and Comparative Fit Index (CFI) indices are equal to 0.940, 0.965, 0.966, 0.893, and 0.965, respectively, and is greater than the critical point of 0.9, which indicates a high fit. In the following, the research hypotheses are examined.According to the software output, the calculated value of The results of the relationship analysis are presented in H1: Teacher-student interaction significantly affects English students’ academic enthusiasm.p = 0.001; β = 0.428) In other words, in exchange for increasing one unit of teacher-language interaction, the academic enthusiasm of English language learners increases by 0.428 units.The results of path analysis in H2: Self-regulation significantly affects English students’ academic enthusiasm. The results of this hypothesis are presented in p = 0.028; β = 0.151) In other words, in exchange for increasing a self-regulatory unit, the academic enthusiasm of English students increases by 0.151 units.The results of path analysis in H3: Academic hope significantly affects English students’ academic enthusiasm.t-test statistics which is equal to 3.289 and greater than 1.96, can be concluded with 99% probability that academic hope has a positive and significant effect on academic enthusiasm of English language learners  In other words, in exchange for an increase in one unit of academic hope, English students’ academic enthusiasm increases by 0.197 units.The results of path analysis in H4: Academic engagement significantly affects the academic enthusiasm of English language learners.p = 0.001; β = 0.208) In other words, in exchange for an increase in one unit of academic engagement, English students’ academic enthusiasm increases by 0.208 units.The results of path analysis in H5: Teacher-student interaction significantly affects English students’ academic enthusiasm with the mediating role of self-regulation, academic hope, and engagement.p = 0.013; β = 0.455). In other words, in exchange for increasing one unit of teacher-student interaction, academic enthusiasm with the mediating role of self-regulation, academic hope, and academic engagement increases by 0.455 units.The results of the bootstrap test in This research aimed to investigate the relationship between teacher-student interaction and academic enthusiasm with the mediation of academic self-regulation, academic engagement, and academic hope within the framework of a causal model using structural equation modeling. In general, the values of the fitness indices showed that the proposed model in this method has a good fit and can predict the changes in academic enthusiasm.The results of the present study showed that teacher-student interaction is related to academic enthusiasm both directly and indirectly through academic hope and academic self-regulation. This finding is with the results of  are aligAlso, according to the research findings, academic hope is directly related to academic enthusiasm. This finding is with the results by On the other hand, the research results showed that academic self-regulation is directly related to academic enthusiasm. This finding is with the results of the research of The coefficients of the effects of academic engagement on academic enthusiasm were significant. The results of this study were aligned with the research of In addition, the research results showed that student-teacher interaction is directly related to academic hope. This finding is consistent with the results of Also, the research results showed that teacher-student interaction is directly related to academic self-regulation. This finding is with the results of the research of One of the important findings of the research was that the variables of academic self-regulation and academic hope play an important role in the relationship between student-teacher interaction and students’ academic enthusiasm. The results of some studies  show theThe purpose of the current study was to determine the causal explanation of academic enthusiasm based on the interaction of teachers and English language learners: self-regulation, academic hope, and academic engagement among English language learners. Our findings suggest that: The interaction between the teacher and the students has a significant effect on the academic enthusiasm of the English language students; self-regulation significantly affects the academic enthusiasm of English language learners.; academic hope significantly affects English language learners’ academic enthusiasm; academic engagement significantly affects English language learners’ academic enthusiasm; the interaction between the teacher and the students significantly affects the academic enthusiasm of the English language learners with the mediating role of self-regulation, academic hope, and academic engagement.Despite the important results of the present study, due to the limitations of this study, caution should be exercised in generalizing the results. Among the limitations of the current research, it can be mentioned that the statistical population of the research is limited to intermediate students, the lack of proof of the cause and effect relationship in the structural equation method, and the lack of confidence in the answers of the participants due to the self-report nature of the research tools. It is suggested that in future research, the role of other social and individual factors effective in academic enthusiasm should be investigated to test the assumptions raised in related theories, identify each factor’s contribution and examine the consequences of academic enthusiasm in the life of knowledge. Students and other groups of society should be addressed from different angles. Also, to test similar and generalized patterns, other samples with different ages and educational levels should be used. Also, considering the effect of teacher-student interaction on academic enthusiasm, it is suggested to hold effective communication skills training workshops for teachers and students during the academic year. On the other hand, considering the effect of academic hope and academic self-regulation on students’ academic enthusiasm, it is suggested that the educational system consider programs to improve the level of students’ academic hope, self-regulation, and academic engagement.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved 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.


NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb‐M’, which is the inhibitor that switches off the entire RUNX family by alkylating agent–conjugated pyrrole‐imidazole (PI) polyamides, inhibited T‐cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX‐binding sequences (TGTGGT). Chb‐M’ also suppressed the expression of NFATC2 and pro‐inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb‐M’ showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD‐associated cytokines including tissue necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor were reduced in the peripheral blood of Chb‐M’ injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX‐NFATC2 axis can be a novel therapeutic target against GVHD.Patients with refractory graft‐versus‐host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt‐related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating  Approximately 20%–60% of HSCT recipients develop GVHD despite advances in immunosuppressive therapy. In addition, the prognosis for patients with steroid‐refractory acute GVHD (SR‐aGVHD) is dismal, with a survival rate of only 5%‐30% . RuxolitNuclear factor of activated T cells (NFAT) is a family of transcription factors identified in activated T cells and promotes the expression of multiple cytokines and other regulatory molecules . Among NCSF2) plays an emerging role across a range of inflammatory diseases . Cy. CyCSF2)seases  thymidine uptake assay. Compared to control, Chb‐M’ significantly reduced alloreactive T cell proliferation  , 39, 40, by RUNX  and NFAT by RUNX  has been by RUNX , have no by RUNX , 43, ourRunx1 or Runx3 knockout mouse model is known to result in over‐immunity rather than suppression of the immune system. Deletion of Runx1 in naive CD4+ T cells caused spontaneous cellular activation and cytokine production that eventually led to a catastrophic autoimmune inflammatory disease [Runx3 knockout mice spontaneously develop inflammatory bowel disease with enhanced expression of both Th1 and Th2 signature cytokines [Runx knockout model. Thus, we would like to emphasize that targeting all RUNX family can be effective in reducing suppressing effector T cell activation. We acknowledge that targeting whole RUNX family may also have a negative effect on T cell differentiation in humans, considering that mature thymocyte were virtually absent by reducing core‐binding factor β levels in mice, in which the activity of all three Runx proteins should be affected [In the vivo models that use our xenogeneic GVHD model in which human peripheral human T cells were injected into NOG mice, we showed that RUNX family inhibition by Chb‐M' inhibited T cell proliferation and GVHD phenomenon Figures  and 5. S disease . Besidesytokines . A possiaffected . This isRunx3‐deficient mice CD8+ T cells in vitro [FOXP3 in Tregs [We acknowledge that the study has several limitations. First, we only analyzed the effect of Chb‐M' on GVHD and not the graft‐versus‐leukemia (GVL) effect. The challenge in the current era is to prevent GVHD while sparing GVL effect. Donor CD8 T cells play a dominant role in mediating the GVL effect via cytotoxic T lymphocyte activity . An essein vitro  and in vin vitro . Thus, tin vitro . Secondlin vitro  and alsoin Tregs , we did in Tregs . The effNFATC2 expression and various cytokine gene expressions in T cells. Considering Chb‐M’ is expected to have therapeutic effects through a mechanism different from other existing GVHD drugs, the first priority is to test whether it is effective as an additional treatment for refractory GVHD in the clinical settings. Further studies are awaited to validate this new strategy against GVHD.In conclusion, we have shown that targeting the whole RUNX family suppresses The authors have no conflicting financial interests.Hirohito Kubota performed experiments, analyzed data and wrote the manuscript. Tatsuya Masuda, Mina Noura,Kana Furuichi and Masahiro Hirata performed experiments and analyzed data. Hidefumi Hiramatsu, Takahiro Yasumi, Tatsutoshi Nakahata, Yoichi Imai, Junko Takita and Souichi Adachi participated in discussions and the interpretation of data and results and commented on the research direction. Hiroshi Sugiyama synthesized and designed the PI polyamides. Yasuhiko Kamikubo designed and initiated the study, supervised research, and gave the final approval for submission.Supporting InformationClick here for additional data file.


Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy and lacks effective therapeutic targets. Trametinib is considered to be a promising potential indirectly targeted KRAS inhibitor in PDAC. However, the clinical outcomes were poor. JQ1 displayed a significant synergistic effect when combined with chemotherapy or potential targeted therapy in pancreatic cancer. The impact of Trametinib and JQ1 combination treatment in PDAC remains to be fully elucidated.KRAS mutant pancreatic cancer cell lines. The cytotoxic effects of drugs either alone or in combination were evaluated using a luminescent cell viability assay. Immunoblot analysis was carried out to investigate changes in p62 and autophagy.Methods: The efficacy of trametinib and JQ1 on cell proliferation and cytotoxicity was assayed in 7 KRAS alterations, irrespective of the mutational loci of KRAS and the aberrant status of the other driver genes. The synergistic effects of combination treatment of trametinib and JQ1 were observed in both trametinib-resistant and trametinib-sensitive cells. In trametinib-sensitive PDAC cells, the combined treatment definitely inhibited p62 expression compared with trametinib alone, while LC3 expression at high levels changed little. In trametinib-resistant PDAC cells, the combination of MEK/BET inhibitor dramatically decreased p62 expression compared with single agent, while p62 expression increased after anti-autophagic therapy was added.Results: We found that either trametinib or JQ1 alone inhibited the proliferation of some pancreatic cancer cell lines with KRAS mutant PDAC cells. Combination therapeutic synergism may induce different cell death modes in different pancreatic cancer subtypes.Conclusions: Blocking RAS downstream signaling and epigenetic pathway synergistically increases the antiproliferative activity in  Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with a rapid incidence rate worldwide . ApproxiKRAS activating alterations, which play an essential role in PDAC initiation and maintenance [KRAS seems to be a desirable approach for specifically treating PDAC patients with KRAS mutations. However, with the exception of KRAS p.G12C specific inhibitors (a mutation merely accounts for 1% of PDAC patients), various attempts to directly inhibit KRAS have been unsuccessful [Studies have revealed that up to 90% of PDAC patients harbor oncogene ntenance . Directlccessful . As an accessful . Trameticcessful . The faiJQ1, an epigenetic reader protein BET inhibitor of bromodomain-containing protein 4 (BRD4) has emerged as a potential modulation agent . In pancKRAS mutant pancreatic cancer and address the potential mechanism.In this study, we aimed to explore the impact of trametinib and/or JQ1 on KRAS and TP53 mutations. AsPC-1 also had SMAD4 and CDKN2A alterations. PSN1 and CFPAC-1 had SMAD4 copy number variation (CNV) loss alterations, while Mia PaCa-2, PANC-1, HuP-T3, and HuP-T4 carried CDKN2A CNV loss alterations. Then we treated all PDAC cell lines with a decreasing concentration gradient of trametinib. Cytostatic responses were observed in all PDAC cell lines, but the effectiveness was totally different in different cell lines from the fitting curve , the combination of trametinib and JQ1 substantially reduced the percentage of cell viability, in AsPC-1 cells matching the multiplicative expectation and in PSN1 cells exceeding that which would be expected if monotherapy effects were multiplied  [Trametinib, an MEK1 and MEK2 kinase inhibitor, blocked ERK phosphorylation which downregulated MYC protein causing G1 cell cycle arrest and inducing apoptosis . JQ1 a se arrest . Combinecription , 16. Rec (NSCLC) , 18. HowKRAS alterations, irrespective of the mutational loci of KRAS and the mutational status of the other driver genes. Further studies demonstrated synergistic effects of the combination treatment of trametinib and JQ1 in both trametinib-resistant and trametinib-sensitive cell lines. It was shown that the BET inhibitor not only further enhanced the sensitivity of trametinib in trametinib-sensitive PDAC cells, but also improved the sensitivity of trametinib in trametinib-resistant PDAC cells. Finally, we preliminarily explored the mechanisms mediating the synergistic effects of the combination therapy in PDAC.In our study, we found that either trametinib or JQ1 alone could inhibit the proliferation of some pancreatic cancer cell lines with In the trametinib-sensitive PDAC cell line, the combined treatment definitely inhibited MYC, leading to an increase in p62 expression compared with trametinib alone, while LC3 expression at high levels changed little. It was elucidated that the synergistic effect of MEK/BET inhibitors mainly induced apoptosis in trametinib-sensitive cells, despite slight protective autophagy. In the trametinib-resistant PDAC cell line, the combination of MEK inhibitor and BET inhibitor dramatically decreased p62 expression compared with single drug, while p62 expression increased after anti-autophagy therapy was added. This result revealed that the synergistic effect of combination therapy mainly elicited autophagy-dependent cell death in trametinib-resistant cells. P62/SQSTM1, a ubiquitin-binding multifunctional protein linked to the extrinsic apoptosis pathway promoting programmed cell death, binds directly to LC3 family proteins to negatively regulate autophagy as a marker to study autophagic flux . Autophain vivo. Animal assays to evaluate the safety and immune effect of trametinib/JQ1 combination therapy have been confirmed in other tumors [KRAS downstream pathway combined with an anti-epigenetic BET inhibitor has a favorable synergistic effect in KRAS mutant PDAC cells. In addition, KRAS wild-type pancreatic cancer and the detailed regulatory molecular mechanism of different cell death modes induced by combined therapy should be explored and confirmed in the future. Finally, the mechanism of trametinib/JQ1/HCQ combined treatment is complex, and the antiproliferative effect and the cell death mode need strict designed experiments to be further evaluated.Our study also has some limitations. We have not tested the synergistic effect of combination therapy r tumors , 18. We KRAS mutant pancreatic cancer cells. Combination therapeutic synergism induces autophagy-dependent cell death in some pancreatic cancer subtypes. This suggests that trametinib and JQ1 can be viewed as potential combination therapeutic options for PDAC patients with KRAS alterations. Treatment containing anti-autophagic regimens requires screening suitable pancreatic cancer patients, which needs to be further verified.In summary, our findings show that blocking RAS downstream signaling and epigenetic pathway synergistically increases the antiproliferative activity in The human pancreatic cancer cell lines AsPC-1, MIA PaCa-2, PANC-1, HuP-T4, HuP-T3, PSN1, and CFPAC-1 were purchased from American Type Culture Collection (ATCC) and provided by Suzhou Truway Biotechnology Inc. All cell genetic information was analyzed and downloaded from the Cancer Cell Line Encyclopedia (CCLE) or Catalogu of Somatic Mutations in Cancer (COSMIC) database. Cells were cultured in RPMI 1640, McCoy's 5a, MEM or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under the recommended conditions. The ATCC has performed morphological, cytogenetic and DNA profile analyses for characterization of these cell lines. The cell passages were limited to 15 generations for all experiments in this study. Mycoplasma contamination was excluded using the antibiotic mycoplasmincin (InvivoGen) and was periodically examined using a MycoFluor Mycoplasma Detection Kit .Trametinib , JQ1 , and hydroxychloroquine  were dissolved in DMSO.http://www.graphpad.com/scientific-software/prism/). The software build-in analyses “nonlinear regression (curve fit)” and equation “log (inhibitor) vs. response-variable slope” were used for the data analysis and IC50 calculation.Cell viability assays were carried out using the CellTiter-Glo® Luminescent Cell Viability Assay . Cells were seeded into 96-well cell culture plates at a density of 5000 cells per well in 100 μL of culture medium and treated with the indicated drugs at various concentrations. After 72 h of incubation, the cells were lysed with CellTiter Glo reagent , and the luminescence signals produced by ATP molecules from live cells were measured using a SPARK microplate reader  after 30 min of incubation at room temperature. The dose–response curve was fitted based on the relative survival cell percentage in nonlinear fitness (curve fit) using GraphPad Prism 8 software (Cultured cells were extracted with RIPA buffer containing protease inhibitors and a phosphatase inhibitor cocktail (Roche). The protein concentration was determined by the BCA assay (Pierce). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore) and analyzed by immunoblotting. The antibodies used were as follows: LC3A/B antibody  and SQSTM1/p62 antibody  were purchased from Cell Signaling Technology, and β-actin  was purchased from GenScript.All statistical analyses were performed using GraphPad Prism 8 . Student’s unpaired t-tests were used to compare two independent groups before and after different treatments as appropriate. P values less than 0.05 were considered to be statistically significant.Supplementary Tables


Complexes 1–8 were assessed on a panel of cell lines including HT29 colon, U87 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, SJ-G2 glioblastoma, MIA pancreas, the ADDP-resistant ovarian variant, and the non-tumour-derived MCF10A breast line. The in vitro cytotoxicity results confirmed the superior biological activity of the studied complexes, especially those containing 4-fluorophenylacetic acid and 4-bromophenylacetic acid ligands, namely 4 and 6, eliciting an average GI50 value of 20 nM over the range of cell lines tested. In the Du145 prostate cell line, 4 exhibited the highest degree of potency amongst the derivatives, displaying a GI50 value of 0.7 nM, which makes it 1700-fold more potent than cisplatin (1200 nM) and nearly 7-fold more potent than our lead complex, SS56ME (4.6 nM) in this cell line. Notably, in the ADDP-resistant ovarian variant cell line, 4 (6 nM) was found to be almost 4700-fold more potent than cisplatin. Reduction reaction experiments were also undertaken, along with studies aimed at determining the complexes’ solubility, stability, lipophilicity, and reactive oxygen species production.A new series of cytotoxic platinum(IV) complexes ( The str3H6O or DCM to afford a cloudy solution that is primarily due to the presence of an unwanted by-product, dicyclohexylurea (DCU)  . The solea (DCU) , NMR (Fiea (DCU) , and ESIea (DCU)  experimeSSPHEN and SS56ME) and platinum(IV) (SS(IV)(OH)2PHEN and SS(IV)(OH)256ME) complexes were synthesised as previously described 2+2+2+[Pt (SSPHEN and SS56ME) and IV(HL)(AL)(OH)2]2+2+2++: m/z = 674.15; experimental: m/z = 675.15.SS(IV)(4-CPA)(OH)]2++: m/z = 702.18; experimental: m/z = 703.18.SS(IV)(4-FPA)(OH)]2++: m/z = 658.18; experimental: m/z = 658.18.SS(IV)(4-FPA)(OH)]2++: m/z = 686.21; experimental: m/z = 686.21.SS(IV)(4-BPA)(OH)]2++: m/z = 718.10; experimental: m/z = 719.09.SS(IV)(4-BPA)(OH)]2++: m/z = 746.13; experimental: m/z = 747.13.SS(IV)(4-IPA)(OH)]2++: m/z = 766.08; experimental: m/z = 766.08.SS(IV)(4-IPA)(OH)]2++: m/z = 794.12; experimental: m/z = 794.12.1–8, 4-CPA, 4-FPA, 4-BPA, and 4-IPA were also tested for reference and comparison. All test agents were prepared in DMSO (30 mM stock solutions) and stored at −20 °C until use. All cell lines were cultured in a humidified atmosphere of 5% carbon dioxide at 37 °C. The cancer cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% foetal bovine serum, 10 mM sodium bicarbonate penicillin (100 IU mL−1), streptomycin (100 µg mL−1), and glutamine (4 mM). The non-cancer MCF10A cell line was cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum, supplemented with penicillin (50 IU mL−1), streptomycin (50 µg mL−1), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), L-glutamine (2 mM), epidermal growth factor (20 ng mL−1), hydrocortisone (500 ng mL−1), cholera toxin (100 ng mL−1), and insulin (10 μg mL−1). Cytotoxicity was determined by plating cells in duplicate in 100 mL medium at a density of 2500–4000 cells per well in 96 well plates. On day 0 (24 h after plating) when the cells were in logarithmic growth, 100 μL of medium with or without the test agent was added to each well. After 72 h, the GI50 was evaluated using the MTT  assay, and absorbance was read at 540 nm. An eight-point dose response curve was produced from which the GI50 value was calculated, representing the drug concentration at which cell growth was inhibited by 50% based on the difference between the optical density values on day 0 and those at the end of drug exposure [50 values presented for all precursor platinum(II) and platinum(IV) scaffolds, including cisplatin, oxaliplatin, and carboplatin were obtained from a previous study using the aforementioned method [Cell growth assays were performed at the Calvary Mater Newcastle Hospital, Waratah, Newcastle, NSW 2298, Australia. The cell lines tested were: HT29 colon, U87 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, SJ-G2 glioblastoma, MIA pancreas, the ADDP-resistant ovarian variant, and the non-tumour derived MCF10A breast line. In addition to exposure . The GI5d method .50 drug concentration for each complex. The plates were directly scanned to measure fluorescence (relative fluorescence units (RFU)) at different time points using the Glo-Max®-Multimode microplate reader  at an excitation/emission of 485/535 nm. To generate the positive control (20 μM tert-butyl hydroperoxide (TBHP)), cells were washed with 1X kit buffer, and stained with DCFDA (25 μM) for 45 min; this was removed and TBHP was added in phenol red free media, and the resulting solution was scanned as described above.To investigate the presence of ROS in treated cells, a DCFDA/H2DCFDA-cellular ROS Assay Kit  was used, as previously described ,91,92,931–8) incorporating halogenated PAA derivatives, 4-CPA, 4-FPA, 4-BPA, and 4-IPA, were confirmed by HPLC, NMR, UV, CD, and ESI-MS. NMR was used to monitor the reduction behaviour of the complexes in the presence of AsA. HPLC provided insights into the stability and lipophilicity of the complexes. All complexes proved to be soluble in aqueous solution, and surprisingly stable for 36 h at 37 °C in 10 mM PBS (~7.4 pH). As for the lipophilicity results, the phen derivatives were less lipophilic than the 5,6-Me2phen derivatives. The order of increasing lipophilicity of the complexes also parallels the order of increasing size of the halogens incorporated into the axial ligands (F < Cl < Br < I). No correlation was demonstrated between lipophilicity and cytotoxicity of the complexes. The in vitro results also confirmed that 1–8 surpassed the potency of cisplatin and its derivatives in all of the cell lines tested. Notably, 4 and 6 demonstrated strong selectivity towards the cell lines HT29 colon, Du145 prostrate, MIA pancreas, ADDP-resistant ovarian, and MCF10A, with GI50 values ranging between 0.7 and 11 nM. In the ADDP-resistant ovarian variant cell line, 4 and 6 were almost 4700-fold more potent than cisplatin. There was some correlation between ROS potential and cytotoxicity in the HT29 colon cell line. The most biologically active complex, 4, demonstrated the most significant production of ROS, while the least cytotoxic complex, 3, demonstrated the lowest observable production of ROS. In summary, the findings of this study are a testament that modifying certain structural features is essential in tuning the biological activity of novel therapeutic molecules. Furthermore, stability and reduction experiments on the studied complexes in human blood plasma or serum using NMR are scheduled in the near future.The structure and purity of eight novel platinum(IV) complexes  complexes, February 2022, Western Sydney University, Sydney, Australia.