Due to the unique structures of arvanil and olvanil, the drugs combine certain properties of both cannabinoids and vanilloids, which makes them able to stimulate both TPRV1 and CB1 receptors and causes them to be interesting agents in the setting of carcinoma treatment. The aim of this study was to investigate the cytotoxic and anti-proliferative effects of arvanil and olvanil when administered alone and in combination with cisplatin (CDDP) and mitoxantrone (MTX), using various primary  and metastatic  human malignant melanoma cell lines. The results indicate that both arvanil and olvanil inhibited (dose-dependently) the viability and proliferation of various malignant melanoma cells, as demonstrated by MTT and BrdU assays. The safety profile of both arvanil and olvanil tested in human keratinocytes (HaCaT) and normal human melanocytes (HEMa–LP) revealed that neither arvanil nor olvanil caused significant cytotoxicity in HaCaT and HEMa–LP cell lines in LDH and MTT assays. Isobolographically, it was found that both arvanil and olvanil exerted additive interactions with MTX and antagonistic interactions with CDDP in the studied malignant melanoma cell lines. In conclusion, the combinations of arvanil or olvanil with MTX may be considered as a part of melanoma multi-drug therapy; however, the combination of these compounds with CDDP should be carefully considered due to the antagonistic interactions observed in the studied malignant melanoma cell lines. Cutaneous melanoma is a malignant neoplasm deriving from melanin-producing cells. In 2020, the standardized incidence rate of melanoma reached 3.0 in women and 3.8 in men, which represents 324,625 new melanoma cases and 1.7% of total new cases of the most common cancers [The treatment of melanoma in the early stages include wide excision of nevus, with lymph node dissection and stereotactic irradiation if necessary; in the metastatic stage, the treatment is systemic therapy above all . After nCannabinoids together with their two main receptors, the CB1 and CB2 receptors, have recently gained attention as potential anticancer agents. Similar interest has been placed in the determination of the antineoplastic properties of transient receptor potential cation channel subfamily V (TRPV) members, especially TRPV1.Ki = 0.25–2.6 μM) than olvanil (Ki = 1.6 μM); moreover, it is a stronger inhibitor of the anandamide membrane transporter than olvanil (IC50 = 3.6 μM vs. IC50 = 9 μM) [Arvanil and olvanil are structural “hybrids” because they present features of both groups as they express structural features of anandamide and capsaicin . Arvanil = 9 μM) ,11,12. S = 9 μM) . The las = 9 μM) ,12,14.Lipophilicity of the TPRV1 agonists is directly related to the kinetics of TRPV1 activation and their pungency, but not to their potency. Arvanil is more pungent than olvanil ,16. ArvaMitoxantrone (MTX), an anthracenedione antineoplastic agent, manifests its cytotoxic properties by inhibiting catalytic activity of topoisomerase II (the group of enzymes regulating replication and transcription of DNA) and intercalating to DNA and causing drug-stabilized cleavage complexes . As a reSince molecular mechanisms of the action of arvanil and olvanil differ from the molecular mechanisms of the CDDP and MTX, this study was designed to evaluate the two drug combinations of arvanil with CDDP or MTX and olvanil with CDDP or MTX and compare the results to find out which of the tested hybrids could be more effective and favorable in the treatment of melanoma. The main hypothesis was that these drugs exert synergistic anti-proliferative interactions in the primary and metastatic melanoma cell lines .To determine the types of interactions between arvanil/olvanil and CDDP or MTX, isobolographic analysis was performed, which is considered to be the gold standard in the evaluation of types of drug–drug interactions in cancer studies ,26. More50) values for arvanil and olvanil in various melanoma cell lines are presented in 50 values for CDDP and MTX in various malignant melanoma cell lines have been determined earlier [Arvanil and olvanil reduced the viability of various human melanoma cell lines  in a concentration-dependent manner, when applied separately , Table 1 earlier .The quantification of the cytotoxicity of both arvanil and olvanil in malignant melanoma cell lines, normal keratinocytes, and normal human melanocytes was performed in the LDH test. The diagrams show that the cytotoxicity of arvanil and olvanil in various malignant melanoma cell lines grows in a concentration-dependent manner while they present no significant impact on normal human keratinocytes and melanocytes .In the BrdU test, both arvanil and olvanil inhibited the proliferation of all tested melanoma cell lines  and normal human keratinocytes (HaCaT) . Arvanil50 values for arvanil and olvanil when used separately, mixtures of the two cannabinoid ligands with either CDDP or MTX (at a fixed drug dose ratio of 1:1) in the melanoma cell lines  were examined in the MTT assay. All the mentioned cell lines were incubated with different increasing concentrations of arvanil or olvanil and CDDP or MTX. The log-probit analysis of concentration-response anti-proliferative effect produced by the respective two-drug mixtures allowed the calculation of the experimentally derived IC50mix values for the combinations studied in the MTT assay. The obtained results presented the concentration-dependent reduction in cancer cell viability  exerted antagonistic interactions in the A375 cell line A’. The Ihe drugs A’. In cohe drugs B’. In thhe drugs B’. The che drugs C’. The Ihe drugs C’. In cohe drugs D’. The Ihe drugs D’.50mix value for this combination did not differ from the upper IC50add value  exerted additive interactions in the A375 cell line A’. The Ihe drugs A’. An adhe drugs B’. In thhe drugs B’. The che drugs C’. The I and MTX C’. The c and MTX D’. The Ihe drugs D’.Results from this study confirmed that both arvanil and olvanil inhibited, in a concentration dependent manner, the proliferation of malignant melanoma cell lines. Both arvanil and olvanil when combined with CDDP produced antagonistic interactions in the MTT assay, whereas the combinations of arvanil and olvanil with MTX exerted additive interactions in various melanoma cell lines. Both cannabinoid ligands exerted quite similar types of interactions in the in vitro MTT assay, when combined with CDDP and MTX, as that of cannabidiol in various melanoma cell lines .2+ response and cell death in high-grade astrocytoma in vitro. In an animal model of astrocytoma, it reduced tumor growth and prolonged life of the experimental mice. A suggested mechanism of arvanil’s antineoplastic activity was the stimulation of the endoplasmic reticulum stress pathway by activating transcription factor-3 (ATF3) [Previously, it was observed that arvanil caused TRPV1-dependent Ca3 (ATF3) . Moreove3 (ATF3) . Arvanil3 (ATF3) ,31.Arvanil and olvanil present anti-invasive activity, which is not dependent on CB1 and TRPV receptors in the human small-cell lung cancer (DMS 114 and DSM 53) cell lines. The mechanism of this favorable effect was mediated by the activation of the MAPK pathway . ArvanilIn the case of olvanil, it inhibited human breast cancer cell line EFM-19 proliferation and its anti-proliferative action was enhanced by palmitoylethanolamide (PEA). It was suggested that the possible mechanism of this phenomenon may be related to the potentiation of activity of VR1 receptors . Olvanil50 values in all the tested in vitro cell lines. However, it was found that olvanil exerted more favorable effects when combined with CDDP in two melanoma cell lines  than arvanil did. In this case, arvanil produced antagonistic interactions when combined with CDDP, while olvanil exerted additive interactions when combined with CDDP in these two cell lines. The observed difference remains unexplained at present. Since the molecular mechanisms of action of both ligands are quite similar, the exerted interaction should also be similar. However, some differences observed in in vitro studies must be explained as a result of activation of different pathways involved in the anti-proliferative effects of CDDP and arvanil or olvanil. Of note, some differences in the potency of arvanil and olvanil to both CB1 and VR1 receptors should be kept in mind while explaining the observed effects in these two cell lines (SK–MEL 28 and FM55M2). Another fact deserves more attention. In primary melanoma cell lines (A375 and FM55P), olvanil exerted antagonistic interactions, whereas in the metastatic cell lines (SK–MEL 28 and FM55M2), olvanil produced additive interactions when combined with CDDP. Perhaps more advanced studies will shed light on these combinations. Since antagonistic interactions between drugs are not favorable during anti-cancer treatments, arvanil should not be combined with CDDP. From a pharmacological viewpoint, one drug inactivates the second one, and finally, the resulting combination has an antagonistic nature. In contrast, the combinations of MTX with both ligands (arvanil and olvanil) deserve a pre-clinical recommendation as favorable combinations during anti-cancer treatments.Comparison of effects produced by the two cannabinoid and vanilloid receptor ligands  in this study revealed that the more efficacious agent in relation to the anti-proliferative effect was arvanil due to its lower ICAs mentioned in the introduction, the first line of treatment of melanoma is based on surgical excision of the neoplasm. However, the application of multidrug therapy can be considered when there exists a high risk of appearance of neoplastic transformation in various places or when metastatic foci are expected to be present. In such cases, multidrug treatment should slow down the progression of melanoma. This is the reason to pre-clinically test various combination of arvanil and olvanil with CDDP and MTX. A general rule in oncology is to combine drugs producing the anticancer effects via different molecular mechanisms activating various pathways so the drugs could mutually cooperate in inhibiting the proliferation of melanoma cells.Additionally, various cannabinoids may improve quality of life for oncologic patients, including these who suffer from melanoma . One of 2+ channels through a biochemical pathway that involves intracellular Ca2+-calmodulin and calcineurin, therefore producing desensitization of nociceptors in nociceptive neurons [Arvanil and olvanil also produce an anti-nociceptive effect that is independent on CB1 or TPRV1 receptor activation ,8,41,42.2. The cells were grown to 80% confluence.Two cell lines, A375  and SK–MEL 28 , were purchased from the American Type Culture Collection  and cultured in Dulbecco’s Modified Eagle’s Medium—high glucose (DMEM) and Eagle’s minimal essential medium (EMEM) , respectively. In contrast, another primary (FM55P) and metastatic (FM55M2) malignant melanoma cell lines were purchased from the European Collection of Cell Cultures  and cultured in RPMI—1640 Medium (Sigma-Aldrich). All culture media were supplemented with 10% Fetal Bovine Serum  and 1% of penicillin/streptomycin (Sigma-Aldrich). Cultures were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO2+ and Mg2+. The arvanil and olvanil  were dissolved in ethanol as stock solutions in concentration of 5 mg/mL. The drugs were dissolved to the respective concentrations with culture medium before use.Mitoxantrone (MTX—Sigma-Aldrich) was dissolved in DMSO as stock solutions. Cisplatin (CDDP—Sigma-Aldrich) was dissolved in phosphate buffered saline (PBS) with Ca4 cells/mL, 2 × 104 cells/mL, 2 × 104 cells/mL, and 2 × 104 cells/mL, respectively. On the next day, the culture medium was removed and cells were exposed to serial dilutions of arvanil, olvanil, CDDP, and MTX in fresh culture medium. Cell viability was assessed after 72 h by means of the MTT test, in which the yellow tetrazolium salt (MTT) was metabolized by viable cells to purple formazan crystals. The 72-h incubation time is the average doubling time for all tested melanoma cell lines. In the case of the SK–MEL 28 line, doubling time is 17.5 h [A375, SK–MEL 28, FM55P, and FM55M2 cells were placed on 96-well plates  at a density of 3 × 10s 17.5 h ; for thes 17.5 h , but in s 17.5 h ,45,46. A4/mL), SK–MEL 28 (3 × 104/mL), FM55P (2 × 104/mL), FM55M2 (2 × 104/mL), and normal human keratinocytes HaCaT (1 × 104/mL) cells were placed on 96-well plates (Nunc). After 24 h, cells were washed in PBS, and then exposed to increasing concentrations of arvanil or olvanil in the proper fresh culture medium. The cytotoxicity was estimated by measuring cytoplasmic lactate dehydrogenase (LDH) activity released from damaged cells after exposure to arvanil or olvanil for 72 h. LDH assay was performed according to the manufacturer’s instructions (Cytotoxicity Detection KitPLUS LDH) . Concisely, after the collection of 50 µL of cell medium from each well, the 50 µL of reaction mixture (freshly prepared) was added and incubated for 30 min at RT. The next step was adding 25 µL of Stop solution to each well on the 96-well plate. Finally, absorbance was measured at two different wavelengths, one being the “measurement wavelength” (492 nm) and the other “reference wavelength” (690 nm) using a microplate spectrophotometer . Maximum LDH release (positive control) was achieved by the addition of lysis buffer to untreated control cells. The average values of the culture medium background were subtracted from all values of experimental wells. The percentage of death cells was calculated in relation to the maximum LDH release.Optimized amounts of A375 (2 × 104/mL), SK–MEL 28 (3 × 104/mL), FM55P (2 × 104/mL), and FM55M2 (2 × 104/mL) cells were placed on a 96-well plate (Nunc) (100 μL/well). On the following day, the cancer cells were treated with increased concentrations of arvanil and olvanil for 48 h. After that, 10 µL/well BrdU Labeling Solution (100 µM) was added and cells were re-incubated for an additional 24 h at 37 °C. Then, the culture medium was removed and cells were fixed in FixDenat solution (200 µL/well) . The working solution of anti-BrdU antibody coupled with horseradish peroxidase (anti-BrdU-POD) were subsequently added (100 µL/well)  and detected using tetramethylobenzidine substrate (TMB) (100 µL/well) . To stop the enzymatic reaction, a total of 1 M (25 µL/well) sulfuric acid was added. The quantitation was performed spectrophotometrically at 450 nm using a microplate spectrophotometer .Cell Proliferation Elisa, BrdU Kit  was performed by following the manufacturer’s instructions. Optimized amounts of A375  of arvanil and olvanil were calculated [50add) for the two-drug mixture of (arvanil or olvanil) with CDDP or MTX, which theoretically should inhibit 50% of cell viability, as demonstrated earlier [50mix (at the fixed ratio of 1:1) were determined in malignant melanoma cell lines measured in vitro by the MTT assay.Due to log-probit analysis, it was possible to transform percentage of inhibition of cell viability into probit and concentrations of arvanil and olvanil when administered singly  into logarithm of concentrations as reported earlier . Subsequlculated . Linear lculated ,49,50,51lculated ,53,54,5550 values for the drugs. Of note, calculation of the IC50 from sigmoidal Hill and linear log-probit equations provide the same results and both methods are used equivalently in toxicological studies [Because arvanil and olvanil have never been tested in melanoma cell lines , the selection of their concentrations in the MTT assay was purely empirical in this study. Although a few concentrations of the tested substances in the MTT assay were plotted graphically , many mo studies ,59,60. H studies ,61. Of n studies . This wa studies ,63,64. G. The experimentally derived IC50mix values for the mixture of arvanil or olvanil with CDDP and arvanil or olvanil with MTX were statistically compared with their respective theoretically additive IC50add values by the use of the unpaired Student’s t-test with Welch correction, as described earlier [The statistical analysis of data was performed by means of GraphPad Prism 8.0 Statistic Software. One-way analysis of variance (ANOVA test) for multiple comparisons followed by Tukey’s significance test was used. The data are expressed as the mean ± standard error (SEM)  earlier .We have found, for the first time, that arvanil and olvanil possess anti-viability effects in various malignant melanoma cell lines. Isobolographic analysis of the interactions of both arvanil and olvanil with either CDDP or MTX provided evidence that the more favorable combinations were those with MTX. Antagonistic interactions of arvanil and olvanil with CDDP votes against their recommendation in further experimental and clinical studies. However, more favorable profiles for the combinations of olvanil with CDDP were observed in metastatic cell lines , while in primary malignant melanoma cell lines , the combination of olvanil with CDDP exerted antagonistic interactions. Further experiments are needed to elucidate differences in activity of combinations in primary and metastatic melanoma cell lines.


AKAP4 gene were demonstrated to lyse AKAP4+ myeloma cells. Seven of the 12 candidate epitopes predicated by the BIMAS and SYFPEITH algorithms were able to bind HLA-A*0201 in the T2 binding assay, of which only two peptides were able to induce CTL cytotoxicity in the co-culture of peptide-loaded human mature dendritic cells and the autologous peripheral blood mononuclear cells (PBMCs) from the same HLA-A*0201 donor. The AKAP4 630–638 VLMLIQKLL was identified as the strongest CTL epitope by the human IFN-γ ELISPOT assay. Finally, the VLMLIQKLL-specific CTLs can lyse the HLA-A*0201+AKAP4+ myeloma cell line U266 in vitro, and inhibit tumor growth in the mice bearing U266 tumors in vivo. These results suggest that the VLMLIQKLL epitope could be used to develop cancer vaccine or T-cell receptor transgenic T cells (TCR-T) to kill myeloma cells.Multiple myeloma (MM) is a malignant plasma cell disorder affecting mainly the elderly population. Revolutionary progress in immunotherapy has been made recently, including monoclonal antibodies and chimeric antigen receptor T cell (CAR-T) therapies; however, the high relapse rate remains problematic. Therefore, combination therapies against different targets would be a reasonable strategy. In this study, we present a new X-chromosome encoded testis-cancer antigen (CTA) AKAP4 as a potential target for MM. AKAP4 is expressed in MM cell lines and MM primary malignant plasma cells. HLA-A*0201-restricted cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transduced with an adenovirus vector encoding the full-length  The past decade witnessed great progress in multiple myeloma (MM) management, including new agents such as proteasome inhibitors , 2 and iThe high response rate of BCMA CAR-T cell therapy in refractory and relapsed myeloma patients made it a “superstar” in recent years , 8, but The combination of flow cytometry cell sorting and next-generation sequencing (NGS) technology has accelerated research into cancer antigens . One of AKAP4), is a CTA gene located at Xp11.22  as a naturally processed HLA-A*0201-specific epitope. CTLs induced by this peptide can effectively kill HLA-A*0201+) and RPMI-8226 (HLA-A*0201-); a human myeloid leukemia monocyte cancer cell line, THP-1 (HLA-A*0201+); a lymphoma cell line, RAMOS (HLA-A*0201-); and the T2 cells (HLA-A*0201+), a TAP-deficient human hybrid B/T lymphoblastic cell line; cells were maintained in RPMI-1640  or DMEM (THP-1) medium (Euro Clone) supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 10 mM HEPES, 100-U/ml penicillin, and 100 U/ml streptomycin , at 37°C in a 5% CO2 atmosphere.The following cell lines were used in this study: the human MM cell lines U266 (HLA-A*0201http://www.bimas.cit.nih.gov/molbio/hla_bind/) and SYFPEITHI (http://www.syfpeithi.de/bin/mhcserver.dll/epitopeprediction.htm) websites. The CMV peptide NLVPMVATV served as positive control in the T2 stabilization assays. All peptides were synthesized by Sangon  with a purity of 98% verified by mass spectrometry analysis.Twelve HLA-A*0201 restricted peptides from the AKAP4 protein sequence were selected based on an integration of their score using the HLA peptide binding prediction algorithms available at BIMAS (5 cells were incubated with 3 mg/ml β2-microglobulin (Sigma-Aldrich) and 50 μM peptide in a final volume of 200 μl for 4 h at 37°C. Cells were then washed and stained with the PE-conjugated HLA-A*0201 monoclonal antibody (BioLegend) before cytometry evaluation . As described elsewhere  overnight and then cocultured with peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of IL-2 (10 ng/ml). Peptide-induced CTLs were harvested after 5 days. Monoclonal antibodies used for DC and CTLs analysis included CD11c, CD80, CD86, CD3, CD4, and CD8 (BD Pharmingen).All the relevant health donors and patients in this study signed the informed consent. Our study complied with the Helsinki Declaration and was approved by the Ethics Committee of the First Hospital of Peking University, with the approval number of 2021-323 from the Human Ethics Committee. The CTLs were generated based on the protocol described elsewhere . Briefly5 PBMCs and 5 × 104 autologous DCs were co-cultured for 24 h under the stimulation of different peptides. These cells were then harvest for ELISPOT assay. The spots were developed according to the program, and each well was analyzed by a Dual color plate-reader.Human IFN-γ precoated ELISPOT kit  was used for ELISPOT assay according to the instructions. PBMCs were isolated from the peripheral blood of healthy donors, and some cells were used to induce the formation of DC, while others were stimulated by a mixture of IL-2 and AKAP4 peptides . On the fifth day, these cells were harvested and plated. Briefly, 2 × 105/ml. The cultured CTL cells and labeled target cells were laid into a 96-well concave bottom plate at a fixed effector–target ratio for 6 h. Cells were then washed twice using PBS and stained with 7-AAD antibody before examined using flow cytometry.First, the target cells are labeled with CFSE . One microliter of CFSE was rapidly added to 1 ml of PBS resuscitated cells , and stained at room temperature for 10 min in the absence of light. Complete medium was then added to stop staining. Cells were centrifuged at room temperature for 5 min. The complete medium was then used to resuscitate the cells at a cell density of 1× 105 U266 cells in the axilla (day 0). The No. 8 peptide-induced CTLs (5 × 106 per mice) or PBS was injected intravenously on day 1 and day 7. Tumors were measured along two orthogonal axes  every 5 days and tumor volume was calculated by the following formula: volume = a × b × b/2.NTG mice were purchased from SPF Biotechnology Co. Ltd. (Beijing). and bred in the animal breeding facilities at Peking University First Hospital under specific pathogen-free conditions. The experiments were approved by the Ethics Committee of Peking University First Hospital . CTLs were induced by No. 8 peptide following the above-mentioned procedure. NTG mice were inoculated subcutaneously (s.c.) with 5 × 10t-test was used. When comparing more than two groups, two-way ANOVA was first used to test the whole groups; if significant, unpaired t-test was then used to test all possible two groups. *p-value < 0.05 was considered significant. Statistical analyses were performed using SPSS 20.0 software.Wilcoxon rank sum test was used to assess difference in the data that do not meet the normality test. When comparing more than two groups, non-parametric Friedman test was first used to test the whole groups; if significant, Kruskal–Wallis rank sum test or Wilcoxon rank sum test was then used to test all possible groups. In the data that meet the normality test, unpaired We examined the expression of AKAP4 by flow cytometry in common myeloma cell lines, including U266 and RPMI-8226, control cell lines THP-1 and RAMOS, and primary myeloma cells. The expression of AKAP4 was confirmed in both cell lines (U266 and RPMI-8226) and patient samples  to detect the ability of high affinity peptides identified in the T2 binding assays to induce CTLs to secrete IFN-γ. Specifically, PBMCs were isolated from healthy donors. Part of the cells were induced by cytokines to form DC and then were co-incubated with mixed high-affinity peptides. These peptide-loaded DCs were then co-cultured with the rest of the PBMCs to induce peptide-specific CTLs. In the ELISPOT experiment, we found that numbers of interferon-γ-secreting CTLs induced by different peptide-loaded DCs differed a lot. The positive control CMV peptide induced more than 400 spots per well. Among the 7 candidate peptides, peptide No. 8 (VLMLIQKLL), followed by peptide No. 5, induced the highest number of interferon-γ-secreting CTLs (+ HLA-A*0201-), THP-1 (AKAP4- HLA-A*0201+), and RAMOS (AKAP4- HLA-A*0201-) cells were significantly lower than that to U266 cells, which are AKAP4+ and HLA-A*0201+.To further test whether peptide No. 8 could induce CTL-specific killing to myeloma cell lines, we carried out a tumor cell line killing assay. The killing efficiently is positively correlated with the effector-to-target (E:T) ratios, the specific killing ability to U266 cells reached ~64% at an E:T ratio of 25:1, while the killing efficiency was only about 27% and 5% at the E:T ratios of 5:1 and 1:1, respectively  is a real antigenic epitope. Therefore, it is promising that the VLMLIQKLL epitope can be used as another myeloma antigen peptide to develop DC vaccine , which showed the strongest immunogenicity, could induce specific CTLs that showed significant anti-myeloma activity both  vaccine , TCR-T .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.


One of the most significant current discussions in educational psychology is academic passion. This research aimed to investigate the mediating role of academic passion in the relationship between goal orientation and academic self-regulation with students’ academic burnout. However, so far, there has been little discussion about these variables together. The statistical population of the present study includes all undergraduate English students of Islamic Azad University, Tabriz, whose strength is equal to 598 people, and the sample size is determined by the convenience sampling method and Cochran’s formula (248 participants). Data collection methods in this study include four questionnaires of the academic passion; goal orientation; self-regulatory and academic burnout. The Pearson test results showed that the correlation coefficient of academic burnout with goal orientation, self-regulatory, and academic passion is statistically significant at 0.05%. These variables are inconsistent with academic burnout, and with increase in these variables, academic burnout significantly decreases. The correlation coefficient of goal orientation with self-regulatory variables and academic passion is statistically significant at the level of 0.05%. These variables are consistent with goal orientation, self-regulated learning, and academic passion, and with increase in the aforementioned variables, they significantly rise. Goal-oriented and self-regulated goal orientation can predict academic passion. Academic passion has a direct and significant relationship with academic self-regulation and goal orientation. Finally, the obtained fit indices of the general model have the desired fit with the collected data. The implication is that academic passion plays an important role in creating a positive learning environment that will lead to effective learning and teaching. It may influence student learning. Passion inspires and stimulates, and teaching with passion promotes learning. Students are the cornerstone of the education system in every society, and one of the most important tasks of this system is to invest in learners’ achievements. Researchers are constantly attempting to identify variables influencing learners’ academic performance . There hAnother factor that can be related to learning is academic passion. Students’ academic passion is a variable that is vital to learning to the extent that it is mentioned as an essential learning factor. Academic passion refers to the amount of energy a learner spends doing their academic work, as well as the amount of effectiveness and efficiency achieved. Academic passion has been mentioned as a psychological capital of students and their direct efforts to learn, acquire skills, and desire to improve their level of success, which can lead to effective participation in university activities, participation in-class activities, adaptation to university culture, and appropriate relationship with teachers and other students .One of the most prominent issues in self-regulation is the issue of academic goals and goal orientations. In general, goal orientation theories have been developed to explain the behavior of progress, especially in educational settings and in learning and learners’ performance in completing academic assignments. Goal orientation is one of the psychological variables that Academic self-regulation is another variable that can play a role in the relationship between academic passion and academic burnout that has a mediating role. Bandura’s social cognitive theory provided an appropriate theoretical framework for developing self-regulated learning. According to contextual factors and behaviors, each variable provides the necessary opportunity to control student learning. Self-regulation means that students can design, control, and direct their learning process. They tend to learn and evaluate the whole learning process and think that this process, in turn, reduces student fatigue and burnout . Self-reIn education, students develop self-confidence in different ways, and those who are more confident are more involved in academic activities. These beliefs also affect persons’ way of thinking, feeling, motivating, or behaving. A strong sense of academic self-regulation and goal orientation improves positive individual attitudes and better student participation in academic activities .The present study intends to examine the structural model of the relationship between goal orientation and academic self-regulation with academic burnout while exploring the direct effects to answer the question of whether academic passion plays a mediating role in the relationship between goal orientation and academic self-regulation with academic burnout?Academic passion can be associated with academic self-regulation. Students’ academic passion is a variable that is vital to learning to the extent that it is mentioned as an important learning factor. Academic passion refers to the amount of energy a learner spends doing their academic work, as well as the amount of effectiveness and efficiency achieved .In various humanities books, “motivation” is used, instead of “passion.” It has been said that the keyword of motivation in multiple sources is that human actions are of two types: One is voluntary, and the other is motivational. Voluntary actions are actions that a person performs consciously and with order and intention. Students’ passion for education is another important variable that is vital for learning, and it is considered an important harbinger of learning. This variable is also the focal point of most theories related to academic failure . AcademiA study by Another variable is goal orientation. One of the primary and noteworthy factors in developing educational programs is determining and specifying the goals that educational activities are selected and arranged to achieve . In addiAcademic self-regulation is another variable that can play a mediating role in the relationship between academic passion and academic burnout. Bandura’s social cognitive theory provided an appropriate theoretical framework for developing self-regulated learning, according to which contextual factors and behaviors in each individual provide the necessary opportunity to control student learning. Self-regulation means that students can design, control, and direct their learning process . They teOne factor that affects students’ academic performance and has recently been studied in universities is academic burnout . BurnoutStudies on the relationship between academic passion and academic burnout showed a negative correlation between academic passion and burnout . ResearcIn addition to academic goal orientation, academic self-regulation is another variable that can mediate the relationship between academic passion and academic burnout. Bandura’s social cognitive theory provided an appropriate theoretical framework for developing self-regulated learning, according to which contextual factors and behaviors in each individual provide the necessary opportunity to control student learning.The present study intends to answer the question of the relationship between goal orientation and academic self-regulation with academic burnout by examining the structural model of the relationship between goal orientation and academic self-regulation with academic burnout. Does academic passion play a mediating role?Response: It was added.Hypothesis No. 1: Goal orientation has a negative and significant effect on the academic burnout of English language undergraduate students in Tabriz.Hypothesis No. 2: Self-regulation has a negative and significant effect on the academic burnout of English language undergraduate students in Tabriz.Hypothesis No. 3: Goal orientation has a negative and significant effect on academic burnout with the mediating role of the academic passion of undergraduate English students in Tabriz.Hypothesis No. 4: Self-regulation has a negative and significant effect on academic burnout with the mediating role of the academic passion of English language undergraduate students in Tabriz.The method of the present study is descriptive , and the correlational research design is structural equation modeling. This study examines the relationships between variables in a causal model. The statistical population of this study included students at Islamic Azad University, Tabriz, in the academic year 2021–2022 who were studying at the university.The burnout questionnaire was developed by The 14-item questionnaire of The scale was developed by Reliability or reliability of an instrument is its degree of stability in measuring everything measured, that is, the extent to which the measuring instrument gives the same results in the same conditions .The standard questionnaire of academic passion has been designed and developed by The statistical population of the present study includes all undergraduate English students of Islamic Azad University, Tabriz, whose strength is equal to 598 people, and the sample size is determined by the convenience sampling method and Cochran’s formula for a limited population as follows:To use structural equations, factor validity is required, the results of which are presented in According to To analyze the data in two sections of descriptive statistics and inferential statistics, spss 25 and Amos 24 software were used. First, the descriptive statistics section presented the central indicators and the dispersion of research variables. The results are shown in The descriptive statistics in To evaluate the normality of the research variables, the Kolmogorov–Smirnov normality test is used, the results of which are as follows:Based on the results of To investigate the correlation of research variables, the Pearson test was used, the results of which are as follows :Pearson test results showed that the correlation coefficient of academic burnout with goal orientation is −0.805, self-regulatory is −0.709, and academic passion is −0.744, which is statistically significant at the level of 0.05%. A negative sign indicates that these variables are inconsistent with academic burnout, and with increasing these variables, academic burnout decreases significantly. The correlation coefficient of goal orientation with self-regulatory variables is 0.697, and academic passion is 0.702, which is statistically significant at the level of 0.05%. A positive sign indicates that these variables are homologous with goal orientation, and with increase in these variables, goal orientation rises significantly. The correlation coefficient of self-regulatory with academic passion is 0.672 and is statistically significant at the level of 0.05%. A positive sign indicates that this variable is in line with the self-regulatory, and with increase in academic passion, self-regulatory increases significantly.The research model is as follows –3:The model was fitted in Amos24 software.According to the software output, the calculated value of 2χ is equal to 2.867, which is less than three of its degree of freedom, that is, 1. The low value of this index indicates a slight difference between the conceptual model and the observed research data. The RMSEA value is equal to 0.057. The GFI, NFI, IFI, RFI, and CFI are similar to 1.000, 1.000, 1.000, 0.989, and 1.000, respectively, which indicate a high fit. In the following, the research hypotheses are examined.The results of this hypothesis are presented in t-test statistics, which is equal to 8.912 and higher than 1.96, it can be concluded with a 99% probability of goal orientation. It has a negative and significant effect on academic burnout) β = −0.475, p-value = 0.001). In other words, in exchange for increasing one unit of goal orientation, academic burnout decreases by −0.475 units. The coefficient of determination, which is equal to the second power of the path coefficient, is 0.23, which shows that 0.23% of the changes in academic burnout are due to goal orientation.The results of path analysis in The results of this hypothesis are presented in t-test statistics, which is equal to 3.464 and higher than 1.96, it can be concluded with 99% probability. Self-regulation has a negative and significant effect on academic burnout . In other words, in exchange for increasing a self-regulatory unit, academic burnout decreases by −0.187 units. The coefficient of determination, which is equal to the square of the coefficients of the path coefficient, is 0.035, which indicates that 3.5% of academic burnout changes are due to self-regulation.The results of path analysis in A bootstrap test was used to test this hypothesis. The results of this hypothesis are presented in p-value = 0.006). In other words, in exchange for increasing a unit of goal orientation, academic burnout decreases with the mediating role of academic desire of 0.129 units.The results of path analysis in A bootstrap test was used to test the hypothesis. The results of this hypothesis are presented in p-value = 0.012). In other words, in exchange for increasing a self-regulatory unit, academic burnout decreases with the mediating role of −0.101 academic achievement.The results of path analysis in This research aimed to investigate the mediating role of academic passion in the relationship between goal orientation and academic self-regulation with students’ academic burnout among students at Islamic Azad University, Tabriz. The results of the structural model test showed that the collected data fit well with the conceptual model and the results in general, while confirming the mediating role of academic passion, it showed that goal orientation and self-regulation are negatively correlated with academic burnout. Internal academic passion is negatively correlated with academic apathy, and external passion is positively correlated with academic burnout. Academic passion also mediates the relationship between goal orientation, burnout, and academic self-regulation. Regarding the relationship between academic passion and academic burnout, the literature  showed aBased on the self-regulated learning theory and metacognitive processes, students’ procedures constitute self-regulation . Based oFindings showed that academic passion plays a mediating role in the relationship between goal orientation variables and academic self-regulation. These findings are consistent with the results of research by The desire to learn is vital to the extent that it is considered an important learning precursor. On the other hand, more academically, successful students feel competent. They have higher self-regulation because of this sense of adequacy in specific assignments, more interest in the task, an inner desire to master it, and a deep understanding of the subject. Therefore, these students are more likely to pursue the goal of dominating a tendency. By contrast, students who believe in low self-regulation and believe that they cannot perform a particular course’s activities or assignments are more likely to get a passing grade in that course. These students avoid appearing incompetent in the eyes of others. Their main goal is simply to prevent failure and unfavorable judgment. Because they study with external motivation, they only memorize the lessons and do not use advanced and deep cognitive strategies.The purpose of the current study was to investigate academic passion as an essential element in the study that can be guaranteed by strengthening and improving students’ academic success. This study showed that goal orientation has a negative and significant effect on the academic burnout of English language undergraduate students; self-regulation has a negative and significant effect on the academic burnout of English language undergraduate students; goal orientation has a negative and significant effect on academic burnout with the mediating role of the academic passion of undergraduate English students, and self-regulation has a negative and significant effect on academic burnout with the mediating role of the academic passion of English language undergraduate students. Further experimental investigations are needed to estimate academic passion, self-regulation, goal orientation, and academic burnout. Considering that the structural equation modeling method has been used to evaluate the suitability of the proposed model, the conclusion of cause and effect must be done with caution.The small study sample of students was a limitation of the present study, which should be observed in generalizing the results of caution. However, based on the appropriateness of the conceptual model in the present study, it is suggested that professors try to strengthen the inner motivation among the students and also make the academic results meaningful for the students by providing accurate and appropriate feedback, they establish a relationship between the amount of effort, the amount of questioning, the amount of participation of students and their successes, which leads to an increase in academic orientation and academic self-regulation, and as a result, reducing academic burnout. According to the findings of the present study, it is suggested that future researchers should investigate the role of gender modulators in the relations of the present conceptual model or other variables, such as the theory of learning strategies and academic help-seeking variables, in the theoretical model of the present study. Finally, it is suggested to those in charge of education that they hold formal and informal training courses, such as workshops, regarding the factors involved in students’ academic burnout, to provide a basis for students’ promotion.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants OR patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.The author confirms being the sole contributor of this work and has approved it for publication.The 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.


Glycine max (L.) Merr.) seeds imbibed for 3 h to 15 h which remained attached to the cotyledons during imbibition would grow normally after 24 h of desiccation and re-imbibition on wet filter paper. By contrast, when the embryonic axes excised after 3 h imbibition of seeds were kept on wet filter paper for 12 h to 16 h, their growth was significantly retarded after 24 h of desiccation and subsequent re-imbibition. Numerous lipid droplets were observed lining the plasma membrane and tonoplasts in radicle cells of desiccation tolerant embryonic axes before and after desiccation treatment. By contrast, the lipid droplets lining the plasma membrane and tonoplasts became very sparse in radicle cells that were placed for longer times on wet filter paper before desiccation. We observed a clear correlation between the amount of lipid droplets lining plasma membranes and the ability to grow after desiccation and re-imbibition of the excised embryonic axes. In addition to the reduction of lipid droplets in the cells, a gradual increase in starch grains was observed. Large starch grains accumulated in the radicle cells of those axes that failed to grow further.Desiccation tolerance allows plant seeds to remain viable during desiccation and subsequent re-hydration. In this study, we tried to develop an experimental system to understand the difference between desiccation tolerant and desiccation sensitive radicle cells by examining excised embryonic axes after re-desiccation and subsequent imbibition under various regimes. Embryonic axes excised from soybean ( Seed desiccation tolerance is the characteristic of orthodox seeds; the term refers to tolerance of repeated dehydration and implies the ability of cells to rehydrate successfully. Seeds of orthodox species gain tolerance of desiccation towards the end of seed maturation; it is generally lost during germination with the onset of radicle elongation. A complex nexus of physiological, genetic, and molecular mechanisms is involved in survival under desiccation in orthodox seeds .At the early stage of germination, orthodox seeds can tolerate repeated desiccation and re-hydration. This characteristic contributes to the survival of germinated seeds under unexpected adverse conditions and is considered an important ecological trait , which iDuring desiccation and hydration of seeds, several types of changes occur inside cells and some of them can be lethal. It is necessary to prevent lethal cellular injury if the seed is to be desiccation tolerant. Hence a tolerant seed must have some distinctive features to accomplish this. At least three main defensive systems have been characterized ,2: (i) aOne of the most important cell components which can be severely affected during desiccation and re-hydration is the plasma membrane. Damage to plasma membranes during the re-hydration of seeds has been examined by measuring electrolyte leakage ,14. NumeRecently, several studies have indicated that triacylglycerols (TAG) have a role in the protection of cells under stress conditions. Lipids are the main structural elements of cellular membrane systems, and as such are often the most significant casualties of the adverse impacts of abiotic environmental stresses, including low temperature and desiccation; they suffer first because of their high susceptibility to peroxidation damage and degradation ,21. BiolThe major storage reserve substances of the soybean seed are proteins and lipids . The oilAs storage materials, starch grains are minor components in soybean seeds, both in cotyledons and embryonic axes; small starch grains are occasionally observed in the plastids of cotyledon cells. Usually only after the emergence of hypocotyl and cotyledons into light, starch grains develop in the chloroplasts of those cells.Glycine max (L.) Merr. cv maple arrow) are germinated for a shorter period of time, such as 6 h, before drying, they show dehydration tolerance, which means after re-imbibition the seeds usually grow. If, however, they germinate for a more extended period (36 h), they lose the ability to tolerate desiccation [We aimed to investigate the relationship between lipid droplets lining plasma membranes and the desiccation tolerance of the cells after repeated desiccation and re-hydration on embryonic axis radicle cells of soybean seeds. Soybean is one of the most important cultivated crops which produce desiccation-tolerant seeds and intact embryonic axes can be excised after 3 h imbibition of seeds. It is known that during germination, imbibed seeds have the ability to survive repeated desiccation and re-hydration only for a limited period and this ability is lost once radicles start elongating ,25. Wheniccation . Pea andiccation .Storage lipids in cotyledons are converted into sugar via the glyoxylate cycle  and sucrThe main purpose of this research is to investigate the role of lipid droplets in keeping soybean seed cells alive during the processes of desiccation and subsequent imbibition under deliberately rather artificial conditions. Embryonic axes were excised from cotyledons in order to eliminate the constant influence and nutrition supply from cotyledons and to focus on cellular events especially in the radicle cells of embryonic axes.We thus designed an experimental system by which desiccation and re-hydration damage and localization of lipids could be investigated cytologically in radicle cells of embryonic axes. The distribution of lipid droplets lining plasma membranes would seem to indicate a protective function in the maintenance of cell membrane integrity during the processes of desiccation and re-hydration.As a framework for the investigation, we established an experimental system  in whichi h) were desiccated (d) and re-imbibed (ri) (i sample). All of them grew normally and differences depending on imbibition time were not recognized (fi sample). In the case of the fi sample, the growth of embryonic axes after desiccation (d) and re-imbibition (ri) decreased gradually as they were kept longer on the wet filter paper   i–t.d) and re-imbibition (ri). The growth of 3 i h (control without desiccation and re-imbibition), and 3 i h -d -ri and 15 i h -d -ri, embryonic axes was similar up to 3 days (d) and re-imbibition (ri) was suppressed gradually in fi samples, when they were kept on wet filter paper for longer  before desiccation (d) (The length of the axis and its hypocotyl and radicle were compared during growth after desiccation (o 3 days . By conttion (d) .i) and re-imbibed (ri) radicle cells of embryonic axes excised from 3 h to 18 h imbibed seeds (fi) after excision from the cotyledons, the intensity of the lipid staining at the cell membrane was gradually reduced before desiccation (d) (ri) of the samples kept on filter paper (fi) for longer periods  along the plasma membrane (stained with FM 1-43) in imbibed (ed seeds a–h. We ction (d) i,j,m,n a periods o,p.fi samples when they were kept on the wet filter paper for 12 h and 16 h.The percentage of the length of the intense Nile red stained portion at the plasma membrane was measured and compared . The leni h, 15 i h), numerous lipid droplets were lining the plasma membrane and some lipid droplets in the cytoplasm surrounded small vacuoles or protein bodies (ri) following desiccation (d) (fi) before desiccation (d), the amount of lipid droplets along the plasma membrane and surrounding small vacuoles was reduced significantly. Interestingly, numerous starch grains were observed in plastids inside the radicle cells, instead  c. Howeve instead d.i h, 8 i h, 12 i h, 15 i h), starch grains were very sparse and only small starch grains were observed (fi) after excision from the imbibed seeds  Merr.cv. Shiratori) seeds were excised after 3 h to 15 h imbibition in tap water with aeration at 23 °C (imbibition step: i). Excised embryonic axes after 3 h imbibition of seeds were kept on wet filter paper in Petri dishes for further imbibition for up to 18 h at 23 °C (extra imbibition on filter paper: fi). For desiccation of the sample, the imbibed embryonic axes were kept on dry filter paper and left for 24 h at 23 °C (desiccation step: d). For re-imbibition of the desiccated samples, they were put on wet filter paper at 23 °C, 16 h light/8 h dark condition (25 μmol/m2s at light condition) for up to 3 days to observe the growth of the axes (re-imbibition step: ri).Embryonic axes of soybean  for 2 h at room temperature and at 4 °C overnight. They were rinsed with the same buffer and post-fixed in 2% OsO4 in 0.05 M potassium phosphate buffer (pH 6.8) for 2 h at room temperature, dehydrated in an acetone series and embedded in Spurr’s resin. Ultrathin sections (silver–gold) were stained with aqueous 2% uranyl acetate for 10 min followed by lead citrate for 5 min and observed with a Hitachi H-7500 TEM at an accelerating voltage of 80 kV.Free hand sections were cut from within the 1 mm tip portion of the radicles with a razor blade. The sections were stained with iodine solution for 10 min, rinsed in distilled water and observed by light microscope (Nikon ECLIPSE 50i). Estimation of the starch occupied area per cell was performed using Image J.Continued imbibition of excised embryonic axes from imbibed soybean seeds affected radicle cells and caused gradual loss of desiccation tolerance. Abundant lipid droplets lining the plasma membrane appeared to be gradually reduced and this reduction was accompanied by an increase of starch grains in the radicle cells. The consequent dramatic decrease in lipid droplets lining plasma membranes corresponded with the loss of desiccation tolerance.The strong correlation between the presence of lipid droplets and desiccation tolerance, persistent under the artificial conditions in our experiments, strengthens the case for a significant role for lipid droplets in the protection of plasma membranes during desiccation and re-hydration.


Background: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. Methods: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. Results: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD’s catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. Conclusions: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells. Cutaneous melanoma, a melanocyte-originated skin cancer, ranks among the most aggressive human cancers, and its worldwide incidence has been steadily increasing over the last two decades ,2. PatieV600E melanoma in order to respond to MAPK inhibitors [Multiple mechanisms involved in BRAFi resistance have been identified, including genetic and epigenetic ones . Treatmehibitors . Moreovehibitors ,21. Therhibitors , proteomhibitors ,24. Morehibitors ,26.In the present study, we applied a mass spectrometry-based proteomic approach coupled with a multi-denaturation protocol on two different human melanoma cell lines resistant to the BRAFi vemurafenib, along with their drug-sensitive counterparts, in order to reveal structural alterations in cellular components  potentially related to drug resistance. The two cell lines were selected as being representative of two different phenotypes of aggressiveness . Specifically, a differential denaturation protocol (TRIDENT) was applied to both the parental and resistant melanoma cell extracts in order to achieve their proteomic profiles and to also characterize them from a structural point of view . These pFor the present study, resistant cell lines were generated by chronic exposure of SK-MEL-28 and A375 cells to vemurafenib. Two independent sublines (namely VR2 and VR3) for SK-MEL-28 were generated and validated as previously described . The acqTo characterize proteins potentially associated with BRAFi resistance, a first level analysis was initially carried out on the total identified proteins, i.e., taking into account the three different denaturation protocols. The mass spectrometry analysis revealed a total of 1001, 970, 1181, and 1001 non-redundant proteins expressed in A375, A375-VR1, SK-MEL-28, and SK-MEL-28-VR3, respectively A. As preTo provide further insights on the protein–protein interaction (PPI) network, the STRING online tool was used. The specific proteins expressed in parental  and in rSince the total proteins analysis revealed many differences between parental and resistant cell lines, most of which were potentially related to cells heterogeneity, a detailed analysis to increase the probability of identifying a more specific resistance-related target was carried out. Different sensitivity to denaturing protocols of protein lysates from resistant and parental cells was analyzed using the TRIDENT approach, which may highlight specific protein-protein interactions, PTMs and/or intra-residues interactions that are otherwise difficult to investigate . We firsGO analysis was performed as previously described, and results were characterized by functional categories , biologiTherefore, DLD expression in our melanoma cell lines was also evaluated by Western blot analysis in two additional resistant cell lines (namely A375-VR2 and SK-MEL-28-VR2). DLD is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes oxidation of dihydrolipoamide into lipoamide with the parallel reduction of NAD+ (nicotinamide adenine dinucleotide) to NADH (FAD is an intermediary in the electron transfer from dihydrolipoamide to NAD+). CPI-613 octanoic acid or devimistat) A is a liOn the basis of the comparison between peptides found in the parental and resistant cells , it can The major aptitude of DLD of the parental A375 cell line for undergoing post-translational modifications such as succynilation/acetylation/glycosylation, as well as explaining the lack of recognition of these peptides, is also in line with the lower response of this cell to the inhibitor in vitro (see next paragraph). The predicted glycosylation on N38 could interfere with the binding of FAD and with the possible interactions between the inhibitor and the residues in pocket D (responsible for the perturbation of the binding channel). N58 glycosylation, in a similar way, could represent a problem for inhibitor binding in pocket H, as well as for protein dimerization. Even more serious problems arise from the acetylation/succinylation of the peptide 2 terminators, which, being directly involved with the inhibitor binding, can interfere with its interaction in pockets E or D. Peptide 4 is identified only in A375 cells after treatment with DENT3 denaturation. The N-terminal cleavage site Lys265 is well exposed, and is neither annotated as being nor predicted to be a target of acetylation. The C-terminal cleavage site, Arg280, is in a region accessible to the solvent, although it is located in a groove that may not be well exposed to larger molecules. The arginine follows Gly279, a residue involved in the binding of NAD. Moreover, the arginine is followed by a second arginine and a proline, a condition that makes cleavage by trypsin difficult . In factOn the basis of the analysis of the interaction between the DLD and rE2b and E3BP A, the foSince in silico analysis suggested several differences between resistant and parental cells in terms of DLD structure and its interaction with the inhibitor (CPI-613), these data were integrated with in vitro experiments . Parental and resistant cells were treated with 50 and 100 µM CPI-613 for 48 h. As shown in Cell cycle distribution through flow cytometry was used to investigate the possible mechanism of cell growth inhibition by CPI-613. Exposure to 100 μM CPI-613 resulted in an alteration of cell cycle distribution in melanoma cells . InteresThe development of immune checkpoint inhibitors and molecularly targeted drugs has revolutionized the treatment of patients with metastatic melanoma. However, primary resistance and, especially in the case of targeted therapy with tyrosine kinase inhibitors , acquireIn the present study, to identify potential mechanisms of resistance to BRAFi, the proteomic profiles of two extensively studied human melanoma cell lines, namely A375 and SK-MEL-28, and their vemurafenib-resistant sublines were compared. The novelty of our approach lies in taking into account both the expression and the structural-functional behavior of the proteins. This was achieved by applying the TRIDENT protocol, an approach developed primarily to optimize the electrophoretic discrimination of serum proteins related to different sensitivity to denaturation of proteins or protein complexes . In factThe bioinformatics analysis revealed that the proteomic patterns of resistant cell lines showed significant differences in terms of molecular function when compared to parental ones, moving from a “proliferative” phenotype  towards a more “functional” phenotype . To exploit the features of the TRIDENT approach, further bioinformatics analyses were carried out, firstly employing the DENT1 protocol. Notably, of the 31 specific proteins identified in resistant cells in both cell lines, only 14 were connected in the STRING analysis (PPI network), and these were therefore selected for subsequent analyses see . When coDLD was also found to be markedly over-expressed in all of the resistant sublines under investigation compared to their parental counterparts. It is noteworthy that a pivotal role of this enzyme in melanoma progression was recently suggested by Yumman and colleagues, who demonstrated that DLD down-regulation induced autophagy and inhibited proliferation of melanoma cells . In partCPI-613 (devimistat) is one of the best-characterized DLD inhibitors , demonstThe combination of TRIDENT and proteomic approaches carried out in the present study show that PTMs and other tridimensional features could provide useful tools and information for the identification of novel potential targets for new therapies in BRAFi-resistant melanomas. In this work, we focused our attention on the DLD, but since it is involved in several important metabolic routes, including mitochondrial α-ketoacid dehydrogenase complexes, in addition to the glycine cleavage system, in-depth studies aimed at better defining which functions would be affected in resistant melanoma cells should be further undertaken.Roswell Park Memorial Institute medium (RPMI-1640), Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), glutamine, penicillin  and streptomycin , dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), propidium iodide (PI), and all other reagents were obtained from Sigma Chemicals . Fetal calf serum (FCS) was obtained from HyClone . Vemurafenib (PLX4032) was obtained from Selleck Chemicals , and it was dissolved in DMSO to a stock concentration of 20 mM. 6,8-bis(benzylthio)-octanoic acid (CPI-613), obtained from Tocris Bioscience , was dissolved in DMSO (50 mM stock concentration). 2 humidified atmosphere. Vemurafenib-resistant (VR) variants of each cell line  were derived from original parental cell lines according to a previously published procedure [The BRAF-mutant (V600E) A375 and SK-MEL-28 human melanoma cell lines  were obtrocedure  with slirocedure . Resistarocedure ,56,57.w/v mannitol solution, sonication for 60 min then followed by dilution 1:1 with sample buffer (SB) without bromophenol blue, and boiling at 100 °C for 2.5 min). The last two protocols were selected for their stronger denaturation activity. All three pre-treated samples were mixed in 1:1 ratio with SB , 10% glycerol (v/v), 5% 2-bmercaptoethanol (v/v) and 0.0125% bromophenol blue (w/v)), and then heated for 7 min in a thermoblock pre-heated at 95 °C . The product of each denaturation protocol was then subjected to SDS-PAGE. The same amount of total protein extracts  were loaded onto a 4–15% Mini-PROTEAN® TGX™ pre-cast polyacrylamide gel , and the electrophoretic profile was evaluated using a colloidal blue staining kit  according to the manufacturer’s instructions. Then, each protein band was cut, and the proteins were reduced (dithiothreitol), alkylated (iodoacetamide), and digested overnight with bovine trypsin sequencing grade  as described elsewhere [Gel-based proteome analysis was performed on A375, SK-MEL-28, and their first established resistant sublines (A375-VR1 and SK-MEL-28-VR3). Cell lysates were prepared and denatured according to the TRIDENT protocol, as previously described . Brieflylsewhere . The peplsewhere . http://david.abcc.ncifcrf.gov/) (accessed on 15 December 2021) [http://string-db.org) (accessed on 20 February 2022) [The DAVID  online tool (er 2021)  was useder 2021) . The Seary 2022)  was used® TGX™ pre-cast polyacrylamide gel (Bio-Rad) and blotted onto Nitrocellulose using Trans-Blot® Turbo™ Transfer Starter System (Bio-Rad). After blocking for 1 h in 10% BSA in tris-buffered saline Tween buffer, membranes were probed with antibodies specific for DLD (Sigma-Aldrich) and actin . Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse secondary antibodies  were used, followed by visualization with a Clarity Western ECL Substrate Kit (Bio-Rad). Chemiluminescence was revealed by means of a FluorChem System , and digitized images were used for purposes of densitometric quantification.Whole cell lysates were separated on 4–15% Mini-PROTEANhttps://www.ebi.ac.uk/pdbe/pisa/) (accessed on 15 July 2022) was used to detect the interaction at the protein-protein interface. Visualization of the 3D structures was performed using the open source PyMOL Molecular Graphics System (https://sourceforge.net/projects/pymol/) (accessed on 15 July 2022), Version 2.0 Schrödinger, LLC. A visualization of the sequence chain with a schematization of the secondary structure, functional amino acids, and the identified fragments based on the PDBsum diagram (http://www.ebi.ac.uk/pdbsum/) (accessed on 15 July 2022) was produced. Other information was retrieved from the UniProt database (http://www.uniprot.org) (accessed on 15 July 2022) and using the ScanProsite tool (https://prosite.expasy.org/scanprosite/) (accessed on 15 July 2022).The protein structures were obtained from the Protein Data Bank (PDB) . Differe4 cells per well) and treated with CPI-613 (50 and 100 µM) for 48 h or vehicle as a control, and then harvested and counted using a Neubauer-modified chamber. For cell cycle distribution analysis, cells were exposed to 100 µM CPI-613 for 24 h. Cells were harvested and fixed in 80% cold ethanol, then washed and incubated with 200 µg/mL ribonuclease A (Life Technologies) for 30 min at 37 °C and 50 µg/mL PI. Samples were analyzed with a FACSCanto Becton Dickinson Instrument  and FACSDiva software (5.0.3 version).To perform the proliferation studies, melanoma cells were seeded onto 12-well plates . The statistical significance of the differences was determined by two-tailed https://clinicaltrials.gov/) (accessed on 28 October 2022), supporting its low in vivo toxicity.The metabolic plasticity of melanoma cells is considered to be of paramount importance in explaining the acquisition of resistance to antineoplastic therapies. The use of energy sources alternative to glucose  is linked to high mitochondrial oxidative metabolism in targeted-therapy-resistant melanoma . Therefo


Cell lines were checked for sensitivity to BH3 mimetics by BH3 profiling and metabolic assays and monotherapy was only partially successful. Doxorubicin was synergistic with a BCL-XL inhibitor and BCL2/XL/W inhibitor navitoclax. Tamoxifen that targets the estrogen receptor β present in the mitochondria of the cell lines, could induce cell death, and was synergistic with several BH3 mimetics including/as well as navitoclax. In conclusion, targeting the anti-apoptotic pathway by the triple inhibitor navitoclax in combination with doxorubicin or tamoxifen is a promising treatment strategy in HL.The treatment of young patients with Hodgkin lymphoma (HL) is often successful but a significant proportion of patients suffers from late toxicity. In the current era there are new opportunities for less toxic and more targeted treatment options. In this respect, the anti-apoptotic pathway is an attractive target since Hodgkin tumor cells abundantly express components of this pathway. We measured the effect of BH3 mimetics that interfere with anti-apoptotic proteins in cell lines, also in combination with the standard of care chemotherapeutic doxorubicin and the recently discovered preclinically active tamoxifen. Several anti-apoptotic BCL-2 family proteins were expressed in each case ( Hodgkin lymphoma has a very good prognosis with cure rates of more than 85% with current chemotherapybased regimens for young patients. However, since many patients are in a young age group (15–35 years) late side effects of treatment are a serious problem and current research is focusing on less toxic and more specific treatment alternatives to improve long term survival of these patients.One of the hallmarks of the tumor cells in HL is the escape from apoptosis. The expression of several anti-apoptotic proteins has been described, and with the expression of BCL-2 predicting poor outcome , the treThe treatment of patients with obatoclax mesylate, a specific BCL-2 inhibitor, was not successful and was stopped . TherefoRecently we have shown that B-cells, specifically diffuse large B cell lymphoma (DLBCL) cells, express estrogen receptor β (ERβ) that is located in the mitochondria and functions as protection against apoptosis. Treatment with the ERβ inhibitor tamoxifen can cause apoptosis in these DLBCL cells . It has In 84 HL patients, expression of BCL-2, BCL-XL, BCL-W, and MCL-1 was tested by immunohistochemistry on TMA A. In 54%The expression of the anti-apoptotic proteins was tested on 5 HL cell lines in comparison with DLBCL cell line U2932, by western blot B. SUPHD1Next, we checked the expression of ER in HL. mRNA levels were low for ESR1 compared to breast cancer cell line MCF7 in the HL cell lines. For ESR2, although at lower levels than in DLBCL cell line U2932, mRNA was present in the cell lines, with the highest level in DEV A. ERβ waWe started with the combination with doxorubicin as it was already shown to be synergistic with venetoclax  and is uNext, we investigated the combinational effect of tamoxifen and apoptosis inhibitors. SUPHD1 was the most sensitive to tamoxifen and was treated with 15 µM tamoxifen, while the other cell lines were treated with 20 µM. For DEV, there was limited effect of tamoxifen. In the dynamic BH3 profiling after tamoxifen treatment, SUPHD1 showed a BAD response, KMH2 showed NOXA, HRK and BAD, L428 BIM and HRK, L1236 BIM and BAD, and DEV showed no response A. With tWe set out to check expression of all anti-apoptotic proteins in HL and the sensitivity of HL cell lines to BH3 mimetics in combination therapy. We found that BCL-2 is expressed in 60% of cases, which is the lowest percentage in HL of the anti-apoptotic proteins tested; interestingly, BCL-2 is not expressed in NLPHL. Our numbers compare well with the paper from Rassidakis who reported 65% in NS and 47% in MC . BCL-XL For the sensitivity of the HL cell lines to BH3 mimetics, SUPHD1 is BCL-2 sensitive, L428 and DEV are BCL-XL sensitive, while KMH2 and L1236 are not sensitive. Recently it was described that KMH2 was sensitive to venetoclax  and ABT-In the combination therapy with doxorubicin, we see a strong navitoclax and BCL-XLi response, except for L1236, which has a very limited response to doxorubicin. The triple inhibitor ABT-737 has been published to be synergic with doxorubicin in KMH2 .Like in other B cells, ERβ is expressed in Hodgkin lymphoma. Expression of ERβ was already shown in L428 in 2006  and in aThe combination of tamoxifen with venetoclax was shown to be synergistic for ERβ positive breast cancer . In HL cWe see the strongest effect of doxorubicin with the BCL-XL inhibitor and navitoclax and think the combination of doxorubicin with navitoclax and also of tamoxifen with navitoclax could be effective treatment options in HL. In the cell lines, there is a limited effect of the BH3 mimetics alone, but we did observe an effect with combination therapy. The side-effects, especially thrombocytopenia, as well as the introduction of venetoclax as a specific BCL-2 inhibitor, have somewhat halted the introduction of navitoclax. The thrombocytopenia is shown to be reversible , and a cGiven the current high cure rates with standard first line chemotherapy and radiotherapy in young HL, it would be logical to test effects of navitoclax and doxorubicin in patients who had multiple relapses or elderly that are unfit to tolerate more intensive treatment regimens. If effective, combined use of these drugs in the future might enable the elimination of toxic drugs that are used in first line treatment like bleomycin and procarbazine. The combination of tamoxifen and navitoclax is likely to be even less toxic and might serve as a treatment option for elderly of frail patients that have a dismal prognosis with current treatment options.In conclusion, HL patients express multiple anti-apoptotic proteins in the HRS cells to protect them from apoptosis. In the cell lines, we are able to show that the combination therapy with a triple inhibitor as BH3 mimetic in combination with either tamoxifen or doxorubicin can be used as treatment in HL.2 in a humidified chamber. They were cultured 3 times per week. The identity of the cell lines was checked periodically by STR profiling, and cell lines were regularly tested for mycoplasma infection.The HL cell lines L1236 and KMH2 were cultured in RPMI1640 with 10% fetal bovine serum , 1% penicillin-streptomycin , and 1% glutamine (Lonza BioWhittaker). The HL cell line DEV was cultured in RPMI1640 with 20% FBS, and L428 was cultured in RPMI1640 supplemented with 5% FBS. Cell line SUPHD1 was cultured in McCoy’s medium supplemented with 20% FBS. All cell lines are cultured at 37˚C in 5% COImmunohistochemistry was performed with antibodies against BCL-2 , BCL-XL , BCL-W , MCL-1  and ERβ  on paraffin-embedded tissue sections after antigen retrieval . Staining was visualized using HRP-labeled secondary antibodies (Dako) and 3,3′-diaminobenzidine . Appropriate positive and negative controls were performed for each staining. The cases were stained and scored on a tissue micro-array (TMA). The TMA contained a total of 84 HL cases with 67 cases of NS HL, 7 cases of MC, 1 NOS HL, and 9 NLPHL. Patient material was acquired in accordance with international regulations and professional guidelines . This project has been approved by the internal review board of the UMCG under RR#201800551.A nCounter Custom Codeset consisting of capture and reporter probes was hybridized to 100 ng of RNA for 16 h at 65 °C. RNA samples were mixed with 4 µL capture probe-hybridization buffer mix and 1 µg of capture probes. After hybridization, the sample was loaded on a nCounter SPRINT Cartridge and then processed on the nCounter SPRINT™ Profiler.Expression data were analyzed using Nanostring’s nSolver analysis software (version 3.0.). The raw counts were normalized against the geometric mean of internal positive controls to compensate for technical variability. Next, the data were normalized against the geometric mean of the housekeeping genes GAPDH, POLR2A, and WDR55.Cytospins of cell lines were made, dried for 20 min, and fixed with 4% paraformaldehyde for 10 min. Mitochondrial staining was performed using TOMM20 (1:200) for 1 h and TRITC labeled secondary antibody (1:100) + AB serum for 30 min, followed by FLTX1  staining for 4 h and DAPI (1:1000) staining overnight at 4 °C.4 cells were incubated with increasing concentrations of BH3-mimetics ) and A1155463 (BCL-XLi) all from Selleckchem, VWR, PA, USA) in combination with tamoxifen (Selleckchem) for 48 h or doxorubicin (Selleckchem) for 72 h. AlamarBlue (Thermo Fisher Scientific) was added eight hours prior to read-out . IC50 data were calculated with GraphPad Prism.6 × 102EDTA/1% Triton X-100/0.5% sodium deoxycholate/0.1% SDS in dH20) with 1% phenylmethanesulphonyl fluoride (PMSF) for 30–45 min on ice. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit . Lanes were loaded with 40 μg protein and electrophoresis and blotting were carried out according to standard protocols. Staining was performed with primary antibodies for BCL-2 , BCL-XL, BCL-W, MCL-1 , ERβ , ERα (Abcam), and GAPDH  overnight at 4 °C in 5% milk.Cells were washed with ice cold PBS and lysed in RIPA buffer  for 30 min at 4 °C. After staining, cells were washed with and suspended in MEB 90.15 M Mannitol, 0.01 M HEPES, 0.15 M KCl, 1 mMEGTA, 1 mM EDTA, 0.1%BSA, and 5 mM Succinate, pH7.5) at 3–5 × 106 cells/mL. Cells were incubated with peptides  in 0.001% digitonin at room temperature for 60 min in the dark. The reaction was stopped and cells were fixed by addition of 4% formaldehyde  in PBS for 10 min at room temperature in the dark. To neutralize the formaldehyde N2 buffer  was added for at least 5 min. Cells were stained overnight for intracellular cytochrome-c in PBS containing 1% BSA in PBS, 2% Tween 20 (P7949 Sigma Aldrich) and 1:400 Alexa Fluor 488 anti-cytochrome-c antibody  at 4 °C. Flow cytometry analysis was performed on a FACS (BD Biosciences). Data were normalized to PUMA2A (negative control) and alamethicin (positive control). The dynamic BH3 profile (Δ Cytochrome C) was calculated by subtracting the percentage treated Cyt C from percentage untreated Cyt C [Cell lines were incubated at 1.0 × 10t-test. Combination index was calculated using observed/expected, results under 0.7 are considered antogonistic, between 0.7 and 1.3 are additive and over 1.3 are seen as synergistic. * indicates p ≤ 0.05, ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001.Data were analyzed using GraphPad Prism  and tested for significant differences using a paired