New chemotherapy agents are warranted for head and neck squamous cell carcinoma (HNSCC), particularly for incidence-rising HPV-positive tumors. Based on the evidence of Notch pathway involvement in cancer promotion and progression, we aimed to gain insights into the in vitro antineoplastic effects of gamma-secretase inhibition in HPV-positive and -negative HNSCC models.All in vitro experiments were conducted in two HPV-negative  and one HPV-associated HNSCC cell line (SCC154). The influence of the gamma-secretase inhibitor PF03084014 (PF) on proliferation, migration, colony forming, and apoptosis was assessed.We observed significant anti-proliferative, anti-migratory, anti-clonogenic, and pro-apoptotic effects in all three HNSCC cell lines. Furthermore, synergistic effects with concomitant radiation were observable in the proliferation assay. Interestingly, effects were slightly more potent in the HPV-positive cells.We provided novel insights into the potential therapeutic relevance of gamma-secretase inhibition in HNSCC cell lines in vitro. Therefore, PF may become a viable treatment option for patients with HNSCC, particularly for patients with HPV-induced malignancy. Indeed, further in vitro and in vivo experiments should be conducted to validate our results and decipher the mechanism behind the observed anti-neoplastic effects. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is associated with low survival rates . Due to HPV-related oncogenesis revolves primarily around the HPV-associated oncoproteins E6 and E7, which promote degradation of the tumor-suppressor genes TP53 and RB1 and modulate important cell cycle processes, resulting in tumor promotion and progression  resultinHPV-positive tumors differ from HPV-negative disease with regards to genetic alternations. For example, FGFR2 and FGFR3 mutations are uniquely found in HPV-positive tumors . Due to Cisplatin is the most commonly used systemic agent treatment option for advanced HNSCC. However, the response to therapy remains highly variable among patients. One important limitation is the cisplatin resistance, which often associates with termination of treatment in HNSCC patients . FurtherThe Notch pathway is involved in cell processes such as cell proliferation, differentiation, and survival. It operates by transmitting extracellular information, depending on the cell–cell contact interactions, and plays a role in cell cycle regulation. It is often upregulated in cancer, and many Notch signaling alterations have been linked to oncogenesis , 15. PreAs no substantial improvements in chemotherapy options for HNSCC in recent years, efforts need to be given to the identification of new therapeutic agents –13. TherAll experiments were conducted in three HNSCC cell lines, two HPV-negative  and one HPV-positive cell line  , which wIn order to determine the cytototoxic effects of PF and calculate its half-maximal inhibitory concentration (IC50) value of PF, we performed a dose–response assay in 96-well plates  for each cell line. In particular, 5000 cells/well were seeded in 100 μl DMEM. After 24 h (h) of incubation, cells were exposed to gradually increasing concentrations of the inhibitor. The inhibitor was reconstituted in dimethyl sulfoxide (DMSO)  and diluted in cell culture medium . Vehicle control was conducted using 0.1% DMSO. PF treatment was followed by irradiation with 2, 4, and 8 Gray (Gy) with the YXLON device . After 72 h, the medium containing the inhibitor/vehicle control was removed and replaced with 100 μl of 56 μM resazurin  solution. FaDu and Cal27 were then incubated for 90 min (min), while SCC154 were incubated for 180 min. Measurements were performed using the TECAN Spark reader .In order to assess the anti-migratory potential of the PF, we used 24-well plates  with Ibidi inserts  to create the initial gap. For this, 70,000 Cal27 and FaDu cells were seeded in Ibidi chambers  and 250,000 SCC154 cells/ibidi chamber in starvation medium (DMEM with 1% P/S and 1% FBS). Ibidi inserts were removed after reaching 100% cell confluency. Upon removal of the Ibidi inserts, wells were washed with DPBS and introduced with starving medium containing two different inhibitor concentrations , as well as the vehicle control (0.1% DMSO). Visualizations using the TECAN Spark plate reader were utilized after 0, 24, and 48 h. Calculations and analysis of the obtained images were performed with ImageJ.Furthemore, to determine a single-cell’s ability to form colonies after treatment, we performed a clonogenic assay. In particular, 1000 SCC154 cells/well and 350 cells/well for Cal27 and FaDu were seeded in 12-well plates  containing 1 mL of medium. Treatment was performed 24 h after seeding . After 72 h of treatment, cells were incubated for 10  and 14 days (FaDu). Subsequently, wells were washed with DPBS to remove cell debris, and the visualization was performed with the TECAN Spark plate reader.Caspase 3/7 Glo Assay  was conducted to assess the pro-apoptotic effects of the inhibitor. For Cal27 and FaDu, 7.500 cells/well and 15,000 SCC154 cells/well in 100 μL DMEM were seeded into 96-well plates . After 24 h, the medium was cells were treated. After 72 h, the medium was collected and mixed in a 1:1 ratio with Caspase 3/7 Glo reagent in three conditions: blank , tested sample , and negative control (media containing vehicle control + reagent and treated cells). We prepared the reagent according to the manufacturer’s instructions (Promega). Cells were incubated for 30 min and the luminescence was measured with the TECAN Spark plate reader.In order to compare differences in cell viability between different inhibitor concentrations, we conducted a two-way ANOVA analyses. On the other hand, differences in the migration, the colony formation, and the apoptosis assays were analyzed with a one-way ANOVA. For both, multiple comparison testings were performed on all possible pairwise means. We used GraphPad Prism  for graphical representation. All figures show mean values ± standard deviation (SD). Synergistic interactions between the inhibitor and radiation therapy were evaluated using the online tool Synergy Finder using a zero-interaction potency (ZIP) model.In order to assess the cytotoxicity of the PF in HNSCC, we performed the dose–response assay. For these, 5000 cells/well were seeded into 96-well plates with in 100 μl DMEM for each cell line. After 24 h of incubation, we treated cells with increasing concentrations of the inhibitor. We observed significant anti-proliferation effects of PF in all three cell lines (Fig. We utilized a wound healing assay to assess the effects of PF on the migratory potential of three HNSCC cell lines and graphically depicted these results in Fig. Next, in order to assess the colony forming abilities, we performed the clonogenic assay with 1000 SCC154 cells/well and 350 cells/well for Cal27 and FaDu seeded into 12-well plates. Cal27 and SCC154 cells were treated with three PF concentrations and a DMSO vehicle control. In total, strong anti-clonogenic properties of PF were shown in both cell lines. These results are shown in Fig. Finally, we assessed the pro-apoptotic potential of PF with the Caspase 3/7 Glo assay. For this, 7500 Cal27 and FaDu cells/well and 15,000 SCC154 cells/well were seeded into 96-well plates in 100 μL DMEM. All three cell lines were treated the corresponding IC50 PF concentration. Notably, the treatment induced apoptosis in all three cell lines. In particular, a significant increase was observed in FaDu and SCC154 cells. These results are depicted in Fig. The prognosis in patients with advanced HNSCC remained poor in recent years. Moreover, no major advancements regarding chemotherapy were made in the last decades. Furthermore, widely used cisplatin is associated with various adverse effects, often causing a transition to another agent or treatment termination. Finally, particularly treatment of HPV-positive HNSCC is set to become a major challenge in the coming decades, due to the aforementioned global rising incidence rates , 11–13. The data on targeting the Notch pathway via GSI is sparse in HNSCC. To the best of our knowledge, only one study reported on the effects of PF in HNSCC. In particular, Zhang et al. showed that PF in combination with erlotinib suppressed cancer growth . No furtGenerally, HPV-associated HNSCC patients have a more favorable prognosis than HPV-unrelated HNSCC. In particular, 5-year survival rates are reported as high as 80% in HPV-related HNSCC. Meanwhile, the 5-year survival rate is reported around only 50% in HPV-negative HNSCC . Still, Another broadly used treatment option in HNSCC is radiation therapy . TherefoBesides cytotoxic effects, the anti-neoplastic potential of PF was further demonstrated in a migration assay. In particular, treatment with PF significantly decreased the gap closure in all cell lines. These are, to the best of our knowledge, first insights into the anti-migratory potential of PF in HNSCC. With regards to the anti-clonogenic effects of PF, its IC50 concentration seemed sufficient to disrupt colony formation in SCC154. Correspondingly, treatment with PF had a similar anti-clonogenic potential in the Cal27 cell line. Disrupting the clonogenic abilities via Notch pathway inhibition has been observed in other studies as well. For example, Lan et al. showed adverse effects on cell growth and migration by Notch inhibition in embryonic microenvironment . FurtherLastly, we provided evidence on the pro-apoptotic properties of PF in HNSCC in vitro. In particular, slightly stronger pro-apoptotic effects were shown in the HPV-positive cell line. Certainly, the mechanistic background underlining our findings remains unclear and warrants further investigations. Interestingly, HPV infection has shown to have anti-apoptotic properties  and therIn total, our study provided novel results and we gained insights into the therapeutic potential of GSI in HNSCC. However, certain limitations need to be addressed. First, we conducted all experiments solely in 2D cell cultures and these do not completely reflect the native tumor microenvironment of HNSCC patient tumors. Furthermore, the mechanistic background behind the antitumor effects of PF was not analyzed and these could be deciphered with molecular biology experiments such as Western blotting. In particular, the direct downregulation of the Notch pathway by the GSI was not assessed. Most importantly, we assessed PF antitumor properties only in one HPV-positive cell line. Therefore, in order to be able to make definitive statements on PF effects in HPV-positive HNSCC, our findings need to be validated in further HPV-dependent cell lines.


Mammalian reproduction requires the fusion of two specialized cells: an oocyte and a sperm. In addition to producing gametes, the reproductive system also provides the environment for the appropriate development of the embryo. Deciphering the reproductive system requires understanding the functions of each cell type and cell–cell interactions. Recent single-cell omics technologies have provided insights into the gene regulatory network in discrete cellular populations of both the male and female reproductive systems. However, these approaches cannot examine how the cellular states of the gametes or embryos are regulated through their interactions with neighboring somatic cells in the native tissue environment owing to tissue disassociations. Emerging spatial omics technologies address this challenge by preserving the spatial context of the cells to be profiled. These technologies hold the potential to revolutionize our understanding of mammalian reproduction.We aim to review the state-of-the-art spatial transcriptomics (ST) technologies with a focus on highlighting the novel biological insights that they have helped to reveal about the mammalian reproductive systems in the context of gametogenesis, embryogenesis, and reproductive pathologies. We also aim to discuss the current challenges of applying ST technologies in reproductive research and provide a sneak peek at what the field of spatial omics can offer for the reproduction community in the years to come.The PubMed database was used in the search for peer-reviewed research articles and reviews using combinations of the following terms: ‘spatial omics’, ‘fertility’, ‘reproduction’, ‘gametogenesis’, ‘embryogenesis’, ‘reproductive cancer’, ‘spatial transcriptomics’, ‘spermatogenesis’, ‘ovary’, ‘uterus’, ‘cervix’, ‘testis’, and other keywords related to the subject area. All relevant publications until April 2023 were critically evaluated and discussed.First, an overview of the ST technologies that have been applied to studying the reproductive systems was provided. The basic design principles and the advantages and limitations of these technologies were discussed and tabulated to serve as a guide for researchers to choose the best-suited technologies for their own research. Second, novel biological insights into mammalian reproduction, especially human reproduction revealed by ST analyses, were comprehensively reviewed. Three major themes were discussed. The first theme focuses on genes with non-random spatial expression patterns with specialized functions in multiple reproductive systems; The second theme centers around functionally interacting cell types which are often found to be spatially clustered in the reproductive tissues; and the thrid theme discusses pathological states in reproductive systems which are often associated with unique cellular microenvironments. Finally, current experimental and computational challenges of applying ST technologies to studying mammalian reproduction were highlighted, and potential solutions to tackle these challenges were provided. Future directions in the development of spatial omics technologies and how they will benefit the field of human reproduction were discussed, including the capture of cellular and tissue dynamics, multi-modal molecular profiling, and spatial characterization of gene perturbations.Like single-cell technologies, spatial omics technologies hold tremendous potential for providing significant and novel insights into mammalian reproduction. Our review summarizes these novel biological insights that ST technologies have provided while shedding light on what is yet to come. Our review provides reproductive biologists and clinicians with a much-needed update on the state of art of ST technologies. It may also facilitate the adoption of cutting-edge spatial technologies in both basic and clinical reproductive research.  Reproduction ensures the transmission of genetic and epigenetic information to the next generation and the continuity of species. The maintenance of the reproductive systems, the generation of gametes, and embryonic development are some of the central focuses of reproductive biology. A deep understanding of mammalian reproduction could facilitate the diagnosis and treatment of infertility, cancer, and other reproductive pathologies, as well as the development of contraceptives.Mammalian reproduction is regulated by numerous biological pathways and involves many cell types. For example, the development of gametes is a highly regulated process, which includes, but is not limited to, chromatin remodeling, epigenetic reprogramming, cell cycle regulation, meiosis, and cell migration . Deciphein situ hybridization (ISH) and in situ sequencing (ISS) to solid phase-based capturing technologies  technologies have emerged as tools that can not only provide the information on the abundance of mRNA molecules in the cells but also capture their spatial locations within the tissue . For readers who are interested in the current landscape of ST technologies, we have compiled a list of representative ST technologies in in situ hybridization (smFISH) (in situ hybridization (MERFISH)  .8 = 65 536). However, global profiling of hundreds or thousands of mRNA is hindered by optical crowding. To overcome this challenge, seqFISH+ was developed   has achieved nanoscale resolution (220-nm spot diameter with ∼500-nm center-to-center distance) by using spatially barcoded DNA nanoball (DNB) chips . The spaST technologies have been widely applied for the visualization of molecular spatial structures within various tissues  was discovered as a potential novel regulator of the chromatin remodeling process during male germ cell development. Furthermore, by comparing the gene expression profiles of Leydig cells , the authors showed that Leydig cells that are spatially adjacent to a subset of seminiferous tubules express a high level of 1700017N19Rik. These 1700017N19Rik-expressing Leydig cells also express the stem Leydig cell marker Nr2f2 (nuclear receptor subfamily 2 group F member 2), indicating that 1700017N19Rik may be involved in the regulation of stem Leydig cell functions. A similar analysis was performed on testicular macrophages, which identified two spatially distinct macrophage subpopulations. One population localizes in the interstitial space and the other is enriched in the peritubular space. These two populations can be distinguished by the expression of H2-Ab1  and Il1b genes that are exclusively expressed in peritubular macrophages. Besides Leydig cells and macrophages, it is likely that other testicular somatic cells, such as Sertoli cells and myoid cells, also exhibit spatially dependent gene expression patterns. However, the spindle shape of the myoid cells as well as the spatial proximity of Sertoli cell cytoplasm and that of germ cells make it challenging to capture the myoid cell-specific or Sertoli cell-specific transcriptome using Slide-seqV2. This is because one spot on the Slide-seqV2 array may capture mRNA transcripts from two to three adjacent cells. Thus, single-cell-level ST approaches may be better at resolving the spatial transcriptome in cells with irregular shapes or small sizes. For example, seqFISH, a single-cell ST approach, was employed to probe the spatial expression patterns of marker genes of human spermatogonial sub-states identified by scRNA-seq (PIWIL4 (piwi-like RNA-mediated gene silencing 4) and ETV5 (ETS variant transcription factor 5)/L1TD1 (LINE1-type transposase domain containing 1) are enriched in spatially distinct spermatogonium (SPG) subpopulations.For example, seminiferous tubules are the functional units of spermatogenesis in mammalian testes  Fig. 2AFig. 2A. C11orf88, C20orf85, FAM183A (family with sequence similarity 183 member A), and CDHR3 (cadherin-related family member 3)). ISH targeting of these four genes revealed their consistent co-expression with FOXJ1 protein  in both glandular and luminal epithelia on Days 17 and 25 of the menstrual cycle. In another study, FOXJ1 and LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5), are enriched at the luminal surface while NOTCH2 (notch receptor 2) is mainly expressed in glands in the functional layer. Furthermore, the authors found that NOTCH2 expression increases in glands moving away from the lumen while WNT7A (wnt family member 7A) expression is higher in the luminal epithelium compared with glands. By contrast, the noncanonical WNT gene WNT5A is enriched in stromal cells surrounding the glands. These findings suggest an almost mutually exclusive spatial expression pattern between the canonical and noncanonical WNT pathways in the glandular microenvironment.The human endometrium is another example in which ST technologies have been employed to reveal spatial gene expression pattens , ii. StuEzh2 (enhancer of zeste homolog 2) knockout (KO) model  using 10× Genomics Visium allowed a specific selection of epithelial cells for downstream analyses. Differential expression analysis identified up-regulated (Asb4 (ankyrin repeat and SOCS box-containing 4), Cxcl14 (chemokine (C-X-C motif) ligand 14), Dio2 , and Igfbp5 (insulin-like growth factor-binding protein 5)) and down-regulated , Mt3 , and Lcn2 ) genes in Ezh2 KO versus WT uterine epithelium.Besides the human, ST technologies have also been applied to study the mouse uterus in a uterine Hox genes, a class of homeotic transcription factors that specify the body plan, could not be explained solely by spatial restriction of a single-cell subtype. The spatial expression of Cyp26b1, a gene encoding a member of the cytochrome P450 superfamily, is restricted to the brainstem with expression observed in multiple neuronal subclusters. In another study, LCM-based Tomo-seq was applied to zebrafish embryos  and camk2g1 , that localize vegetally in all three species, suggesting their conserved function in germ cell development or dorsoventral axis development.Finally, multiple ST technologies have been used to spatially profile gene expression during embryogenesis . In one Another key feature of ST is the ability to identify spatial clustering of interacting cell populations (i.e. cell neighborhoods) within the tissue context.In the testis, developing gametes are regulated by the tissue microenvironment consisting of various somatic cell types  . TherefoDuring pregnancy, the relationship between the placenta and the decidua of the uterus is essential to nurture and protect the fetus. To this end, one study focused on the development of trophoblast cells at the human maternal–fetal interface by integrating single-nucleus RNA sequencing (snRNA-seq) with 10× Genomics Visium + ventricular zone, Slc5a7 + marginal zone, Vsnl1 (visinin-like 1)+ basal plate, Fut9 (fucosyltransferase 9)+ ventral and Hoxb8+ lateral parts of the spinal alar plate, and Pdyn (prodynorphin)+ superficial stratum of spinal basal plate. Furthermore, spatial clustering of cell types in the embryonic brain recapitulated known anatomically defined brain regions including the ventricular and mantle zones of the pallium, subpallium, midbrain, hindbrain, diencephalon, cerebellum, hypothalamus, olfactory bulb, and choroid plexus. In another study, Finally, ST technologies have also been applied to embryos for tissue structure analysis . For exaPathological states in a tissue are often associated with altered cellular microenvironments. By identifying genes with spatially patterned expressions and spatially clustered cell populations, ST is well positioned to detect and characterize such alterations.ob/ob) and WT mice identified genes with altered spatial expression patterns such as Smcp (sperm mitochondria-associated cysteine-rich protein) and Malat1 (metastasis-associated lung adenocarcinoma transcript 1). Further analysis of Slide-seqV2 data showed a significant increase in the extent of spatial mixing between haploid spermatids and other testicular cell types in ob/ob seminiferous tubules + population; PIFO (primary cilia formation)+TPPP3 (tubulin polymerization promoting protein family member 3)+ ciliated cells; LGR5+ lumenal cells; and SCGB2A2 (secretoglobin family 2A member 2)+ glandular cells. Further analysis of the SOX9+ population revealed three cell clusters: SOX9+LGR5+ cells; SOX9+LGR5− cells; and proliferative SOX9+ cells. By integrating scRNA-seq and 10× Genomics Visium data, the authors showed that SOX9+LGR5+ cells are spatially enriched in the surface epithelium; SOX9+LGR5− cells locate in the basal glands; and proliferative SOX9+ cells are spatially mapped to glands in the regenerating superficial layer. ISH experiments further confirmed the spatial distribution of proliferative SOX9+ cells by showing high expression of the proliferative marker MKI67 in the superficial layer of the endometrium during the proliferative phase  were found to have a greater SOX9+LGR5+ signal. This SOX9+LGR5+ signal is also stronger in endometrial tumors characterized by high copy number alterations and is linked with a worse prognosis (FOXJ1+ ciliated cells and LGR5+ and SOX9+ cells were found to be surrounded by KRT10 (keratin 10)+ cells both within and outside of the endometriosis lesion proper. Furthermore, ECM1 (extracellular matrix protein 1)+ and MMP11 + endometrial-type stroma cells were detected in the endometriosis lesions. CFD (complement factor D)+ peritoneal fibroblasts were separated from the lesions by a region of C7 (complement C7)+ fibroblasts scattered with FAP + cells. This study demonstrates the cellular and spatial heterogeneity in the endometriosis lesion.In the human uterus, Garcia-Alonso In the ovary, high-grade serous ovarian carcinoma (HGSC) is the most common type of ovarian cancer and is also highly chemosensitive. Platinum-based combination chemotherapy is an important treatment for this disease, but patient responses to the treatment vary significantly , iii. Fo+ tumors are more active in energy usage, metabolism, mitosis, and cell growth than myCAF– tumors, whereas signaling activities associated with cellular adhesion, apoptosis, and immune responses are down-regulated in myCAF+ tumors. These observations indicate that the presence of myCAFs may play important roles in supporting cervical cancer progression.In addition to ovarian cancer, cervical cancer also threatens the reproductive health of women worldwide. Recently, A growing number of studies, as reviewed above, have demonstrated the utility of ST technologies in revealing the biological regulation of reproductive physiology and pathology. However, challenges remain to apply ST technologies to study reproductive systems.in situ RNA polyadenylation with existing ST technologies may solve the issue  screens coupled with scRNA-seq, can examine gene functions at scale (Finally, ST technologies may offer an opportunity to dissect gene functions at scale within the native tissue context. For biological processes like gametogenesis, thousands of genes are involved, which makes it difficult to pinpoint the functional contribution of each gene. Traditionally, the Reproduction is essential for the continuation of our species, as it ensures that parental genetic and epigenetic information are passed on to the next generation. Besides producing gametes, the reproductive system also provides the environment for the appropriate development of the embryo. New genomic and computational tools offer unique opportunities to study the intricate spatiotemporal regulatory mechanisms that are required for mammalian reproduction.in vitro spermatogenesis—a technology that would have tremendous impact on fertility preservation. Second, spatially altered genes, cell types, and spatial neighborhoods under pathological conditions identified by ST technologies may be considered as markers for infertility, cancer, or other reproductive disorders. Finally, CCC through LR interactions revealed by ST technologies may also serve as new therapeutic targets for either treating reproductive disorders or developing novel contraceptive approaches.Like scRNA-seq, ST technologies hold tremendous potential for clinical applications. First, the identification of signaling pathways regulating human germ cell differentiation and proliferation will enable the development of protocols for human In summary, our review discusses the novel biological insights that have been revealed by studies using ST technologies, while also shedding light on what is yet to come. We hope that this review will provide reproductive biologists and clinicians with a much-needed update on the state of art of ST technologies. This review may also facilitate the adoption of cutting-edge spatial omics technologies in both basic and clinical reproductive research.


The activity of compounds 15 and 16 as single treatments on all cell lines was found to be non-cytotoxic. Therefore, our findings showed that the investigated compounds have a promising potential to target cancer cells, possibly via an ALDH-related pathway, and sensitize them to DOX treatment.Aldehyde dehydrogenase (ALDH) enzymes are involved in the growth and development of several tissues, including cancer cells. It has been reported that targeting the ALDH family, including the ALDH1A subfamily, enhances cancer treatment outcomes. Therefore, we aimed to investigate the cytotoxicity of ALDH1A3-affinic compounds that have been recently discovered by our group, on breast (MCF7 and MDA-MB-231) and prostate (PC-3) cancer cell lines. These compounds were investigated on the selected cell lines as single treatments and in combination with doxorubicin (DOX). Results showed that the combination treatment experiments of the selective ALDH1A3 inhibitors (compounds  Aldehyde dehydrogenases (ALDHs) are phase I oxidizing enzymes , which iFurthermore, the expression of ALDHs is linked to the cancer progression of several tissues, such as melanoma and melanoma stem cells, lung cancers, human prostate cancer, bone metastasis, hepatocellular carcinoma and adenoma, and breast cancer ,10,11,12Moreover, ALDHs are expressed in normal stem cells (NSCs) and cancer stem cells (CSCs) and, hence, have been considered as CSC biomarkers ,17. ALDH1–6, 15–19, 23–28, and 32–34, which have been investigated on several ALDH-expressing cancer cell lines and have shown promising cytotoxic results [15 was the most potent ALDH1A3 inhibitor, followed by compounds 16, 18, and 1, with remaining enzyme activities of 0.14%, 4.27%, 16.01%, and 21.07%, respectively, compared to the control enzyme activity [Recently, our group has discovered a number of compounds with ALDH-affinic properties , compoun results ,22. Thes results ,22. In pactivity , which wactivity . PC-3 haactivity , and ALDactivity , and DU1Further investigation is required to demonstrate the potential of these ALDH-affinic compounds in cancer treatment. This includes exposing additional cancer cell lines to these compounds, either as single agents or in combination with anticancer drugs to enhance their sensitivity.Although doxorubicin (DOX) is a commonly prescribed anticancer drug for a wide variety of cancer types, emerging resistance is considered a major barrier to its effective treatment outcomes. Several studies have shown that high ALDH activity and/or expression is correlated with low treatment outcomes using DOX ,26,27,28Breast cancer cell lines have been reported to express ALDH1A3 with a variety of in vitro activities, with MDA-MB-468 having the highest ALDH activity, followed by SKBR3, MDA-MB-435, BT-20, MCF7, T47D, and finally MDA-MB-231 . This acIn addition, using diethylaminobenzaldehyde (DEAB), which is a pan-ALDH inhibitor , has beeIn the present study, we aimed to investigate the cytotoxicity of DEAB and the recently discovered ALDH-affinic compounds  on severAs discussed in the introduction, breast cancer (MCF7 and MDA-MB-231) and prostate cancer (PC-3) cell lines are known to express ALDH1A3. In our study, we also investigated the expression of this enzyme in these cell lines before conducting our experiments. As shown in 1–6, 15–19, 23–28, and 32–34, which have been synthesized by our group [Cell viability was evaluated using an MTT colorimetric assay by treating MCF7, MDA-MB-231, and PC-3 cell lines for 48 h with compounds ur group , and DEA50 values presented in 24 is the most cytotoxic on all cell lines tested: MCF7, MDA-MB-231, and PC-3, with IC50 values of 24.5, 31.7, and 25.2 μM, respectively. In contrast, compounds 1–5, 15–18, 23, 34, and DEAB showed no cytotoxicity in any of the cell lines tested. Compounds 19 and 27 showed no cytotoxicity in MDA-MB-231 and PC-3 cell lines, while compound 33 was non-toxic in the MCF7 cell line, with IC50 values higher than 100 μM. The IC50s of the remaining compounds on the indicated cell lines are shown in 24.The IC15 and 16 were the most potent of these ALDH1A3 inhibitors, with IC50 values of 0.23 μM and 1.29 μM, respectively. Given that compounds 15 and 16 showed no cytotoxicity on the tested cell lines, which have been found to variably express the ALDH1A3 isoform—as previously mentioned and as our Western blot results showed  with IC50 values of 47.0, 57.9, and 50.7 μM, respectively, as presented in 19 showed an IC50 value of 91.9 μM on the MCF7 cell line, with no cytotoxicity in either MBA-MB-231 or PC-3 cell lines , MDA-MB-231, and PC-3 cell lines, as presented in Compounds e nuclei . Cinnamae nuclei  and MCF7e nuclei . Althouge nuclei , compoun6, 17, 19, 24, 28, 32, and 33) resulted in cytotoxicity in H1299 cells, which do not express the ALDH1A3 isoform. On the other hand, on A549 cells—an ALDH1A3-expressing cell line—a cytotoxic effect was reported only for compounds 23, 28, and 32 [Ibrahim et al.  reported, and 32 . The lac, and 32  that theVariable sensitivity to DOX has been reported in breast and prostate cancer cell lines ,39,40. I15, 16, 24, 27, 33, and DEAB were used in this study, in which compounds 15 and 16 have been found to be the most potent ALDH1A3 inhibitors [50 values shown in 24 showed the highest cytotoxicity on all tested cell lines, while compounds 27 and 33 appeared to be non-cytotoxic on PC-3 and MCF7 cell lines , and 33 (on MCF7), in combination with DOX. In addition, DEAB was used in combination with DOX on MCF7 and PC-3 cell lines. The concentrations used for DOX were selected based on their respective IC50 on each cell line: 5 μM and 1 μM for MCF7 and PC-3, respectively.Cell viability assays were also evaluated via MTT colorimetric assays by treating MCF7 and PC-3 cell lines for 48 h with compounds 15 and 16, concentrations of 90, 60, 30, 10, and 6 μM were used, which were below their IC50 values on both MCF7 and PC-3 cell lines. This was to investigate whether the combination treatment showed a dose-dependent sensitization or whether the effect was independent of the concentration used. For compound 24, the concentrations used  ranged above and below its respective IC50 value . As compounds 27 and 33 have expected IC50 values above 100 μM, 160 μM was used alongside concentrations below 100 μM to determine the effect of its non-cytotoxic concentrations, as with compounds 15 and 16. Similar to compounds 27 and 33, the concentrations used for DEAB were 100, 50, 25, 12.5, and 6.25 μM, which are non-cytotoxic concentrations on both cell lines.For compounds  ~25 μM,  on MCF7 One-way ANOVA followed by Tukey’s multiple comparisons analysis was conducted to analyze the results of the combination treatments on MCF7 and PC-3 cell lines when compared to DOX alone to determine any significant enhancement of cytotoxicity, as shown in 15, 16, 24 and 33, respectively, in combination with 5 μM DOX compared to 5 μM DOX treatment alone on the MCF7 cell line. The data analysis revealed a significant decrease in cell viability of the cells treated with 5 μM DOX in combination with various concentrations of compound 15 (16 (24 (33 (15 with DOX showed a significant reduction in MCF7 cell viability (16 was only observed at higher concentrations (60 μM and 90 μM), compared to compound 15, as shown in pound 15 a, with 6d 15  or potential substrates (for ALDH3A1) [24 and 33 also showed significant reduction in cell viability compared to DOX alone  , which cOX alone c,d, but 15, 16, 24, and 27, respectively, on PC-3 cells, in combination with 1 μM DOX compared to the single 1 μM DOX treatment. The results showed no decrease in the cell viability for the combination treatment of DOX with compound 15 at any of the concentrations used  in combination with DOX, compared to DOX alone. Unexpectedly, compound 15, at all tested concentrations, did not show any enhancement of the DOX cytotoxicity, compared to the single DOX treatment. A possible explanation for the differences observed between MCF7 and PC-3 cell lines results may be due to the variations in the level of ALDH expressions in these cell lines, and which may be related to variable metabolic processes. Again, further validation studies are required to support this hypothesis, which could potentially pave the way for selective targeting of certain cancer types.The results obtained from the combination treatment experiments on the PC-3 cell line showed a significant reduction in cell viability only at the highest concentration used for the compounds tested , in which the combined treatment resulted in higher cell viability than the compound alone at both concentrations used. This requires further investigation to understand why this kind of combination decreased the cytotoxic effect of the compound on both cell lines. An alternate hypothesis based on the compound 24 results could be that DOX is enhancing the activity of the ALDH-affinic compounds.In both cell lines, compound 15 and 16, the observed effects of DEAB on both cell lines may be due to DEAB’s non-selective ALDH inhibitory activity.15 and 16 are non-cytotoxic on ALDH1A3-expressing cells even though they are potent ALDH1A3 inhibitors, the enhancement of cytotoxic activity when combined with DOX might be driven by a different mechanism. Moreover, DOX might be the agent that is enhancing the cytotoxic effects of these ALDH-affinic compounds on the tested cell lines.Given that compounds The ATP-binding cassette (ABC)-superfamily is composed of several efflux pump transporters that are responsible for chemoresistance . Example15 and 16.Both hypotheses—ALDH-affinic compounds enhancing DOX sensitivity or vice versa, and the possibility of ALDH-affinic compounds being ABC substrates—require further investigation, along with further analog development related to compounds ®. MCF7 and MDA-MB-231 cells were grown in 10% (v/v) FBS/DMEM High Glucose (complete medium) and PC-3 cells were grown in 10% (v/v) FBS/RPMI-1640 (complete medium). They were incubated at 37 °C in a humidified air atmosphere of 5% CO2. All cells are adherent and were frequently washed with PBS and supplemented with fresh media for their growth.DMEM High Glucose, RPMI-1640 media, Heat Inactivated Fetal Bovine Serum (FBS), and PBS were purchased from Euroclone, Pero, Italy. Breast cancer cell lines (MCF7 (ATCC HTB-22) and MDA-MB-231 (ATCC CRM-HTB26)) and a prostate cancer cell line (PC-3 (ATCC CRL-1435)) were purchased from ATCCw/v), fat-free milk diluted in TBST  Tween 20) for 30 min at 4 °C, the membrane was incubated overnight at 4 °C with one of the following primary antibodies: anti-ALDH1A3  or anti-GAPDH antibody as a loading control . The amount of each antibody used was as per the manufacturer’s instructions. The next day, the primary antibody was removed, and the washing and blocking processes were carried out for 15 and 30 min, respectively. The secondary antibody (goat anti-rabbit IgG H&L (HRP) ) was then added for 90 min. The secondary antibody was then removed and the membrane was washed for 15 min to prepare it for imaging using a ChemiDocTM Imaging System .Samples of MCF7, MDA-MB-231, and PC-3 cells were lysed using a RIPA lysis buffer . Equal quantities of isolated proteins were then mixed with 5× Laemmli loading dye. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and electrophoretically transferred at a constant current of 25 V for 30 min using a Trans-Blot TurboTM Transfer System  onto a polyvinylidene difluoride (PVDF) membrane, Santa Cruz Biotechnology). After blocking with 3% (3 (MCF7), 9 × 103 (MDA-MB-231), or 8 × 103 (PC-3) cells/well in triplicates. Cells were incubated for 24 h to allow for cell attachment, then the media were aspirated and fresh complete media containing the compounds of interest were added. Compounds were first dissolved in DMSO  and then diluted in the complete medium, resulting in a final well concentration of 1% for DMSO for the highest concentration for any compound used. Untreated control samples were also prepared (1% DMSO/complete media). Final well concentrations for all compounds were 100, 50, 25, 12.5, and 6.25 μM. Plates were then incubated for 48 h. After 48 h incubation, the MTT colorimetric assay was performed as described previously [Cells were seeded in 96-well plates at a seeding density of 10 × 10eviously . BrieflyCell viability assays were conducted as described above in 50 values. One-way ANOVA followed by Tukey’s multiple comparisons analysis was used to compare the results of combination treatments (compounds plus DOX) with DOX alone. All statistical analyses were based on a p < 0.05 level of significance, using GraphPad Prism version 9.0.Data were presented as means and standard errors of the means (SEM). Nonlinear regression analysis was used to generate dose–response curves and determine IC15, on the MCF7 cell line. Other compounds, including DEAB—a known non-selective ALDH inhibitor—did not show any promising enhancements of the DOX cytotoxicity on either cell line. Thus, the combination treatment experiment highlighted a promising potential impact of ALDH-selective inhibition in overcoming cancer resistance to chemotherapeutic agents. Indeed, the use of these ALDH-affinic compounds with DOX showed enhancement of antiproliferative activity and may be applied to overcome DOX resistance and/or to improve DOX-based therapeutic regimens.In conclusion, several ALDH-affinic compounds were investigated as single agents and in combination with DOX on ALDH-expressing cells: breast cancer cell lines (MCF7 and MDA-MB-231) and a prostate cancer cell line (PC-3). The treatment with DOX showed enhanced cytotoxicity when combined with the selective ALDH inhibitors, particularly compound


Infrequently, WR1065 has exhibited anticancer effects when delivered via the FDA-approved cytoprotectant amifostine, which provides one WR1065/molecule extracellularly. The relative anticancer effectiveness of 4SP65, 1LP65, and amifostine was evaluated in a panel of 15 human cancer cell lines derived from seven tissues. Additional experiments assessed the capacity of 4SP65 co-treatments to potentiate the anticancer effectiveness and overcome drug resistance to cisplatin, a chemotherapeutic, or gefitinib, a tyrosine kinase inhibitor (TKI) targeting oncogenic EGFR mutations. The CyQUANT®-NF proliferation assay was used to assess cell viability after 48-h drug treatments, with the National Cancer Institute COMPARE methodology employed to characterize dose-response metrics. In normal human epithelial cells, 4SP65 or 1LP65 enhanced or inhibited cell growth but was not cytotoxic. In cancer cell lines, 4SP65 and 1LP65 induced dose-dependent cytostasis and cytolysis achieving 99% cell death at drug concentrations of 11.2 ± 1.2 µM and 126 ± 15.8 µM, respectively. Amifostine had limited cytostatic effects in 11/14 cancer cell lines and no cytolytic effects. Binary pairs of 4SP65 plus cisplatin or gefitinib increased the efficacy of each partner drug and surmounted resistance to cytolysis by cisplatin and gefitinib in relevant cancer cell lines. 4SP65 and 1LP65 were significantly more effective against TP53-mutant than TP53-wild-type cell lines, consistent with WR1065-mediated reactivation of mutant p53. Thus, 4SP65 and 1LP65 represent a unique prodrug family for innovative applications as broad-spectrum anticancer agents that target p53 and synergize with a chemotherapeutic and an EGFR-TKI to prevent or overcome drug resistance.The lack of anticancer agents that overcome innate/acquired drug resistance is the single biggest barrier to achieving a durable complete response to cancer therapy. To address this issue, a new drug family was developed for intracellular delivery of the bioactive aminothiol WR1065 by conjugating it to discrete thiol-PEG polymers: 4-star-PEG-S-S-WR1065 (4SP65) delivers four WR1065s/molecule and m-PEG Yet, among seven meta-analyses assessing the impact of amifostine on tumor response rates in cancer patients receiving radiotherapy and/or chemotherapy amino]ethanethiol WR1065), was originally developed at the Walter Reed Army Institute of Research as the major active ingredient of the FDA-approved prodrug, amifostine, to protect normal cells from radiation-induced damage 065, was . The firimatinib . For exaotherapy –21, fourWAF1, GADD45, 14-3-3σ), regulators of apoptosis , and genes involved in the control of intracellular redox metabolism   , 33. WR1, NOS-2) –36. Thus, NOS-2) . While a, NOS-2) –40 via n, NOS-2) –43 amongTP53 is the most common driver or co-driver in various cancer types, novel therapies that reactivate mutant p53 protein and/or prevent degradation of wild-type p53 have the potential to be promising agents for use alone or in combination therapies addressing the problem of drug resistance . Preclinical studies of 4SP65, 1LP65, and amifostine were conducted to define the relative capacity of each prodrug to inhibit cell growth or induce cell death in a panel of human cancer cell lines with differing TP53 gene mutation status. Series of experiments also were performed to define the degree that co-treatment with 4SP65 enhances the anticancer efficacy and overcomes drug resistance (i) to cisplatin, as a representative cytotoxic chemotherapeutic, and (ii) to gefitinib, as a representative TKI targeting epidermal growth factor receptor (EGFR) mutations, in relevant panels of human cancer cell lines.For this report, a novel family of prodrugs was designed to deliver WR1065 or other bioactive aminothiols intracellularly to both normal and diseased cells for new clinical uses including cancer therapy. The first two synthesized Burlington HC Research Group (BRG) prodrugs included (i) WR1065 conjugated via bioreducible disulfide bonds to each thiol-terminated arm of a 4-arm PEG-SH scaffold to yield 4-'star'-PEG-S-S-WR1065 (4SP65) and (ii) WR1065 conjugated to one end of m-PEG2O(CH2CH2O)nCH2CH2SH)4, 10,000 Da average; catalog#JKA7008] were purchased from Sigma-Aldrich . m-PEG6-thiol  was obtained from BroadPharm . Additional high purity chemicals used for synthesizing 4SP65 or 1LP65 were acquired by MedChem Partners. Amifostine was purchased from Moravek Biochemicals . Other pharmaceuticals or reagents including cisplatin (DDP) , difluoromethylornithine (DFMO) , gefitinib , Trypan Blue, alamarBlue™ cell viability reagent (Invitrogen), and CyQUANT®-NF cell proliferation assay kits were obtained via ThermoFisher Scientific .WR1065 dihydrochloride  and 4-arm-PEG-SH [pentaerythritol core, formula C, as shown below, is the abbreviation for the trifluoroacetic acid salt of the prodrug or conjugate 7 in 4SP65 was synthesized as the first BRG prodrug using a novel multi-step scheme described in brief in Supplementary Material and in detail in a Composition of Matter patent (WO2017087668), entitled 6-SH shown below for compound 5, 1LP65 was synthesized using the same multistep method in conjugate 6 and then the final product conjugate 7 or 1LP65 following Boc deprotection:to generate Boc-protected 1LP65 is the abbreviation for the di-trifluoroacetic acid salt of this prodrug, weighing 483 Da once dissolved in solution (® Mycoplasma PCR Detection Kit (Sigma-Aldrich) and to match previously annotated DNA fingerprints by the Vermont Integrative Genomics Resource DNA Analysis Facility.Normal human mammary epithelial cell (NHMEC) strains used to assess potential cytotoxicity of 4SP65 and 1LP65 were established from reduction mammoplasty tissue from a healthy female donor (M99005 or ‘Strain-1’)  or purch2. Mesothelioma and ovarian cancer cell lines were maintained in 50:50 DMEM /F12 medium (Lonza) supplemented with 10% (vol/vol) fetal bovine serum  and 100 U/mL penicillin-streptomycin (Corning). H1975 cells were cultured in an ATCC modified RPMI-1640 medium  with low L-glutamine, 10% FBS, and Pen-Strep. All other human cancer cell lines were grown in standard RPMI-1640 medium (Corning) supplemented with FBS and Pen-Strep.Cell culture medium components from various vendors were obtained through ThermoFisher Scientific. Normal mammary epithelial cells were cultured in Mammary Epithelium Growth Medium , containing BulletKit™ growth supplements, at 37°C in a humidified incubator with 5% COTo determine drug stability in tissue culture medium for short-term treatments, 4SP65 was added to medium containing alamarBlue reagent in 96-well plates and incubated in the absence of cells at 37°C for 72 h. After this incubation period, wells with medium containing alamarBlue alone or both alamarBlue and 4SP65 were assessed for fluorescence readings using a Tecan Infinite 200 PRO plate reader .®-NF cell proliferation assay for determining changes in cell numbers following test agent exposures, H1437 non-small cell lung cancer (NSCLC) cells were exposed for 48 h to 0 - 12.5 µM 4SP65 or to 0 - 8 µM cisplatin. After treatments, fluorescence counts were determined using alamarBlue reagent or CyQUANT® dye according to manufacturers’ instructions using a plate reader. Both logarithmic and polynomial curves were fitted to each relative cell survival curve, and R2 values were determined using trendlines and formulas calculated with Excel software.To compare the utility of the alamarBlue assay and the CyQUANTSince the BRG prodrugs are composed of WR1065 as one of the major constituents, assays were conducted to determine if factors reported to modulate the activity of amino acid and polyamine plasma membrane transport systems impacted the efficacy of 4SP65 or 1LP65 in selected cancer cell lines. HL60 cells were exposed to 4SP65 with up to 0.5 mM NaCl, MBA-MD-231 cells were tested in the presence and absence of medium supplemented with estradiol plus insulin (in the BulletKit™ for growth supplements for NHMECs), HCC38 cells were exposed to 4SP65 with and without 10 µM insulin added to the growth medium, and multiple cells lines were pretreated for 24 h with 1 mM DFMO prior to starting exposures to 4SP65.®-NF proliferation assay was used with a plate reader (i) to generate standard curves for cell numbers for each cell line, (ii) to measure the starting cell numbers at initiation of drug treatment, and (iii) to obtain fluorescence readings from cells in 96-well plates for comparisons to a standard curve for the relevant cancer cell line and quantifying cell viability after drug treatments. DNA dyes like CyQUANT are a highly reliable method for determining the effects of anticancer agents on cell proliferation . Five metrics were calculated and used to characterize the shape of the dose-response curve following exposure to a single test agent. Using cell numbers based on fluorescence measurements and standard curves, the percentage growth was calculated at each drug concentration level. The percentage of growth inhibition was calculated as follows:The experimental design followed guidelines for comparisons between drugs used by the NCI  along wiP-values <0.05 considered significant.where Tz = cell numbers or fluorescence counts at time xero, C = vehicle-exposed control cell numbers at the end of the assessment time period, and Ti = test agent-exposed cell numbers or fluorescence counts at each drug concentration. The inhibitory concentration 50% (IC50) was calculated as the concentration of the drug that reduced the growth of treated cells by 50% compared to vehicle-exposed control cells. However, IC50 values do not consider the initial cell population at time zero, leading to the development of three new special concentration parameters to improve the measurement of drug effectiveness and to enhance comparisons between drugs (see https://synergyfinder.fimm.fi) (Possible synergy between treatments with 4SP65-cisplatin or 4SP65-gefitinib drug pairs was explored further using SynergyFinder 2.0 (fimm.fi) , a standfimm.fi) . The ZIPfimm.fi) . Synergyfimm.fi) .To determine 4SP65 prodrug stability in standard growth medium, reduction of alamarBlue by 4SP65 was evaluated in a cell free system for 72 h. At this timepoint, there was no significant change in the fluorescence of alamarBlue reagent in wells containing 4SP65 compared to control wells with medium containing alamarBlue without 4SP65 (data not shown). This finding indicates that 4SP65 did not induce detectable conversion of resazurin to resorufin in the absence of cells.® dye to measure cell viability following 48-h exposures to 4SP65 , an acute promyelocytic leukemia (HL-60), two triple negative breast cancers (HCC38 and MDA-MB-231), three prostate cancers , a serous and a clear cell ovarian carcinoma , one pancreatic cancer (PANC1), and two malignant pleural mesotheliomas (HMESO1 and PPMMill). The results of cell proliferation assays demonstrated that, based on individual dose-response metrics listed in The BRG prodrug 4SP65, delivering four WR1065s/molecule intracellularly, yielded broad-spectrum TP53 mutations (not including TP53-null cell lines), H1437 cells were much less responsive to the cytostatic/cytocidal effects of 4SP65 with significantly higher values observed for each dose-response metric compared to other TP53 mutant cancer cell lines . A pilot study suggested that the low effectiveness of 4SP65 in TP53-mutant H1437 cells is a consequence of restricted active transport of the drug. This phenomenon also was observed in H460 cells and was overcome when 4SP65 was combined with cisplatin, resulting in a dramatic increase in the efficacy of 4SP65 (36-fold at the GI50 level), postulated to be due to cisplatin-induced polyamine depletion and compensatory changes in plasma membrane transport systems . TOV21G cells have wild-type TP53 but exhibit a ‘hypermutator’ genotype that clearly sets them apart from other ovarian cancer cell lines including SKOV3 and high-grade serous ovarian cancer cell lines  are reported to confer either primary resistance, or to occur as “on-target” secondary mutations causing acquired resistance, to inhibitors of the most common KRAS mutation in NSCLC, which is KRAS G12C , which are anticancer drugs that reactivate mutant p53 protein  than TP53 wild-type cell lines to 1LP65 treatment  with a 38-fold greater effect for 4SP65 (average GI50 = 1.7 µM) than amifostine (average GI50 = 64.8 µM) and a 2.1-fold greater effect for 1LP65 (average GI50 = 30.4 µM) than amifostine. Third, 4SP65 was 9.7- to 11.3-fold (averaging 10.4-fold) more potent than 1LP65 depending on the dose-response metric, with the difference increasing as drug levels were raised to achieve LC50 or LC99 effects. Thus, the potency of 4SP65 was ~2.5-fold greater than that predicted by the ratio of four WR1065s/molecule in 4SP65 versus one in 1LP65. Last, the relative potency of 4SP65 and 1LP65 as cytocidal agents compared to amifostine is immeasurable because the latter drug did not induce cytolytic effects in any cancer cell line.max) of free drug averaging from 3.8 ± 1.7 µM to 7.5 ± 6.8 µM cisplatin during routine 1 h IV infusions to differing sets of cancer patients , and the synergy scores for the combination. For H460, HMESO1, PPMMill, and SKOV3 cells, cisplatin alone induced only cytostatic effects, consistent with slowing of progressive disease. In contrast, 4SP65-cisplatin combined resulted in cytolytic effects at plasma-equivalent levels of cisplatin with significant dose-dependent gains of both cytostatic and cytolytic activities by 1.7- to 6.9-fold for cisplatin in all four cancer cell lines. Co-treatment with 4SP65-cisplatin also resulted in significant fold increases in the cytostatic and cytolytic activities by 1.3- to 36-fold in H460 and SKOV3 cells, but the modest gains of effect for 4SP65 (1.1- to 1.3-fold) in HMESO1 and PPMMill cells were not significant. In A549 cells and TOV21G cells, treatment with cisplatin alone achieved a LC50 but not a LC99 at <15 µM drug. In A549 cells, combined treatment with 4SP65-cisplatin resulted in significant gains of both cytostatic and cytolytic activities by 1.9- to 2.4-fold for 4SP65 and 1.7- to 3.5-fold for cisplatin to achieve a LC99 at 8 µM 4SP65 plus 7 µM cisplatin. In TOV21G cells, co-treatment with 4SP65-cisplatin also yielded significant gains in total growth inhibition and cytolytic effects by 2.4- to 3.2-fold for 4SP65 and 1.3- to 2.9-fold for cisplatin, reducing the LC99 values of 3.0 µM for 4SP65 alone and 17.2 µM for cisplatin alone to 0.95 µM 4SP65 plus 5.9 µM cisplatin when combined.For each cancer cell line, in vitro treatment levels corresponding to reported peak plasma levels of free drug. The highest average HSA synergy score of 36.4 on a scale of 60 was observed in H460 cells, where the potentiation of 4SP65-cisplatin combined was most prominent in the low-dose region of both drugs as shown in the heat map and in a ridge of high synergy score in the volcano plot of 45 to 57 at concentrations of <1 µM cisplatin combined with 1.5 to 4.6 µM 4SP65 , and the synergy scores for the combination.A limited set of experiments was conducted to determine if 4SP65 enhanced the activity of and overcame resistance to the first-generation TKI gefitinib in A549 cells, which are sistance . For the or more  but trou or more . For eacin vitro treatment levels corresponding to reported peak plasma trough levels of free drug at 4.50 µM and 6.55 µM after 3 and 8 days of treatment in patients with NSCLC   to both in vitro cell fate decision outcomes induced by amifostine versus the BRG prodrugs probably reflect a multiplicity of factors beyond differences in drug uptake, metabolic activation, and number of active ingredients . In normal cells, WR1065 induces a p53-dependent G1 arrest , 93, 97.Reported effects for WR1065 also provide insight into possible PCD pathways and cell death induction mechanisms by BRG prodrugs. The p53 protein has a major role in both intrinsic and extrinsic apoptosis  and thusThe results reported here place BRG prodrugs among a few anticancer agents that function to reactivate mutant p53 protein , such asAlthough available reports of WR1065 modulatory effects upon gene and protein expression profiles are few, they reveal an interesting pattern that suggests a mechanistic hypothesis for BRG-induced activities. These activities include (i) safety in normal cells, (ii) broad-spectrum anticancer activity, (iii) synergistic effects in combination with other drugs, and (iv) drug resistance reversal effects. In brief, gene expression studies in normal cells report that WR1065 alone enhances pro-survival stress-response related genes and proteins while suppressing pro-cell death components/pathways. This profile is enhanced further if WR1065-pretreated cells are exposed to a stress-inducing agent such as ionizing radiation within the appropriate time frame. These effects provide an explanation for the safety of BRG prodrugs in normal cells and for the cytoprotective effects of WR1065 in cells exposed to stress conditions. Conversely, an opposite pattern is reported in cancer cells where WR1065 alone enhances expression of genes encoding pro-cell death components and suppresses pro-survival gene expression. This altered gene expression profile is enhanced further if cancer cells are exposed to a second stress-inducing agent, resulting in reduction of barriers to PCD pathways and induction of cell death. Some of the commonly reported PCD components modulated by WR1065 include p53, caspases, BCL2 family members, and pathways including MAPK and PI3K/AKT. Although these paradoxical pro-survival/pro-death effects are unexpected, they are outcomes of cellular stress response systems, which are regulated to a large degree by the status of oxygen/sulfur redoxomes , 110. Blin vitro to WR1065 alone, ionizing radiation alone, or the combination. In the category of apoptosis and stress response genes, WR1065 alone up-regulated a few pro-death/stress response genes, down-regulated several, and had no effect upon a third group. However, when cells were exposed to WR1065 combined with ionizing radiation, the expression of most pro-PCD genes was suppressed. Similar modulation of pro-survival pathways and elements was observed in human pulmonary artery endothelial cells exposed to WR1065 and bacterial lipopolysaccharides, H2O2, or IL-6 , DW, MP, TM, BC, VW. Analysis and interpretation of data : DW, BC, VW. Administrative, technical, or material support , DW, VW. Preparation of figures and tables and writing of the manuscript, DW, TL, SR, VW. All authors contributed to the article and approved the submitted version.


IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa  and three CRISPRi  systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of  Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), which was first discovered in the bacterial immunity system , is a poNot only was Cas9 designed for gene knockout by inducing DSBs, but it was repurposed to allow for gene regulation rather than genome editing by utilizing catalytically inactive Cas9 lacking endonuclease activity (dCas9) and fusing effector proteins ,6,7,8,9.Due to their versatility and simplicity, the dCas9 systems, CRISPRa and CRISPRi, are widely used to regulate endogenous gene expression in a variety of species through in vitro and in vivo experiments ,18,19,20CRISPR/Cas9 technology in livestock species, including poultry, is continuously expanding. Through the use of these genome-editing technologies, the possibility of producing fitter, healthier, and more productive animals continues to grow ,30,31,32The limited use of the CRISPR-based toolkit in chickens warrants a systemic evaluation of the CRISPRa and CRISPRi systems with diverse effector domains and different gRNA locations for enabling effective transcriptional regulation in chickens in order to make better use of this powerful platform in avian research. Therefore, in this study, the effects of the three different types of CRISPRa  and CRISPRi  systems in chicken DF-1 cells were evaluated and compared. The effective transcriptional regulation effect was evaluated at the transcriptome level in the CRISPRa and CRISPRi DF-1 cell lines by bulk RNA sequencing.2 and 60–70% relative humidity.Chicken DF-1 cells  were maintained and sub-passaged in Dulbecco’s minimum essential medium  supplemented with 1× antibiotic-antimycotic  and 10% fetal bovine serum . DF-1 cells were incubated at 37 °C with an atmosphere of 5% COGAPDH) gene (GAPDH #1) and targeting the common region of the three CRISPRa vectors (CRISPRa #1), Cas9m4-VP64 (Addgene plasmid #47316) [5 DF-1 cells were then transfected with 1 µg of GAPDH #1, 1 µg of CRISPRa #1, and 2 µg of each CRISPRa vector using Lipofectamine 3000 according to the manufacturer’s instructions. The transfected cells were treated with Geneticin Selective Antibiotic   pathway (CRISPR/Cas9-NHEJ)-mediated genome editing strategy was applied . First,  #47316) , SP-dCas #47316) , or pcDN #47316) ,36. The 5 DF-1 cells were transfected with 200 ng of piggyBac transposon vectors containing CRISPRi components  which were gifts from Alejandro Chavez and George Church  and 50 ng of transposase vector  using the Lipofectamine 3000 (Thermo Fisher–Scientific) according to the manufacturer’s instructions [To generate the CRISPRi cell line, 2.5 × 10ructions . The traTo confirm the expression of CRISPRa and CRISPRi components in the cell lines, total RNA from each cell line was isolated using Direct-Zol RNA Mini or Microprep kit  and reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher–Scientific) . Then, tTo validate the targeted gene insertion of CRISPRa vectors, the genomic DNA of the cell lines was extracted by using Quick Extract DNA Extraction Solution  according to the manufacturer’s instructions. Then, the genomic DNAs were analyzed by PCR using knock-in-specific primers . The ampBbsI restriction enzyme  digestion and subsequent ligation [IRF7), and three gRNAs each for peroxisome proliferator-activated receptor γ (PPARG), high mobility group AT-hook 1 (HMGA1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1) were designed using the CHOPCHOP algorithm  [https://tfbind.hgc.jp/, accessed on 14 February 2022) [SMARCB1, 1.5 µg of gRNAs was used—500 ng of each gRNA . For IRF7 combination gRNA, 2 µg of gRNAs was used—400 ng of each gRNA . The transfected cells were treated with puromycin (1 µg/mL) (Thermo Fisher–Scientific) and harvested 48–72 h after transfection, followed by RNA extraction using the Direct-Zol RNA Mini or Microprep kit (ZymoResearch). Off-targets of each gRNA were determined using CRISPOR  [For transient expression of gRNAs, the vector containing guide RNA (gRNA) scaffold driven by human U6 promoter and puromycin resistant gene was synthesized . gRNAs targeting each gene or mock controls were then cloned into the synthesized vector by ligation . Five gRry 2022) . For mocry 2022) . The oliGAPDH expression using the ΔΔCt method [t-test.Total RNA was used to synthesize cDNA by using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher–Scientific). For qPCR, the PowerUp SYBR Green Master Mix and protocol (Thermo Fisher–Scientific) was used. For each reaction, 2 µL of cDNA and 1 µL of each forward and reverse primers were used for a 20 µL qPCR reaction. Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by a melting cycle. Each gene expression level was normalized to the housekeeping gene t method . All qPCp-value < 0.05.A total of 8 cDNA libraries (2 replicates each) were prepared with NEBNext Ultra™ II RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocol. Each library was sequenced at a minimum of 20 million, 150 bp paired-end reads per sample. The FASTQ read files from RNA-seq analysis have been deposited in NCBI’s Gene Expression Omnibus (GSE217310). We checked the quality of each FASTQ read file using fastQC (version 0.11.9) and trimmed the adaptor sequence with TrimGalore (version 0.6.7). The trimmed FASTQ files were aligned against bGalGal1.mat.broiler.GRCg7b (NCBI annotation release 106) reference chicken genome using STAR aligner (version 2.7.10a) . Raw reaGAPDH was targeted without disturbing gene expression (GAPDH gene (GAPDH#1) was validated by T7E1 assay and subsequent sequencing analysis   mediated gene targeting methods were adapted . To indupression . The taranalysis . The allanalysis A and anoanalysis . The RT-analysis B. These  adapted . The pig9-MeCP2) C were trll lines D.HMGA1, SMARCB1, IRF7 and PPARG were selected that have FPKM values of 107, 56, 4.5 and 1.2, respectively. Three  or five (for IRF7) gRNAs targeting near the TSS of each gene were designed and the gRNA expression vector was constructed. For HMGA1, the three gRNAs were designed in locations within −161 bp to +40 bp , and all of the gRNAs were co-transfected to the CRISPRa and CRISPRi in chicken cell lines. The quantitative PCR analysis revealed that the HMGA1 expression was significantly increased in the VP64 and VPR CRISPRa cell lines when all gRNAs were combined. VP64 displayed the highest level of gene upregulation, with a +22% gene activation. However, there was no significance among the p300, dCas9, dCas9-KRAB, or dCas9-KRAB-MeCP2 cell lines . The quantitative analysis shows that the VP64 and p300 cell lines represented significant upregulation. The VP64 cell line displayed the highest level of gene upregulation with a +62% gene activation; however, there were no significant values observed for the VPR and CRISPRi cell lines  were designed spanning from −256 to +163 for gene activation or repression. As results, the expression of IRF7 was significantly upregulated in the VP64 and VPR CRISPRa cell lines; however, there was no significant gene upregulation in the p300 CRISPRa cell line. On the other hand, in the CRISPRi cell lines, the IRF7 gene expression in dCas9 (−67%) and dCas9-KRAB (−58%) was significantly downregulated in comparison to mock control; however, there was no significance within the dCas9-KRAB-MeCP2 CRISPRi cell line  or repress (58% downregulation) the IRF7 expression in the cell lines. In CRISPRa cell lines, 259 genes were differentially expressed—200 were upregulated and 59 were downregulated (FDR < 5%) (IRF7 ((LOC107050638), in dCas9- KRAB cell line (FDR < 5%)  A. In CRI%) (IRF7 B. One poDR < 5%) .IRF7 gene modulation. The effect of IRF7 upregulation was seen in the enriched GO:biological process (BP) terms in the genes from the CRISPRa cell lines. These terms were “Extracellular matrix organization”, “Cell adhesion”, and “Collagen fibril organization”. In the KEGG pathway, “ECM-receptor interaction”, “Fatty acid metabolism”, and “PPAR signaling pathway” were considerably enriched , histone deacetylases, and SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) . While tSMARCB1 and IRF7 were further examined in CRISPRa and CRISPRi cell lines. Three gRNAs were designed for SMARCB1 and five gRNAs were designed for IRF7 in different locations, either on the positive or negative strand, to determine if different locations or directions were more efficient. For the CRISPRa system, only gRNA3 significantly increased SMARCB1 expression in the VP64 and P300 cell lines and for IRF7, gRNA2 increased gene expression significantly in the VP64 and VPR cell lines. Both of these gRNAs were located close to the TATA box. The result indicates that the location of gRNA is critical for gene regulation in dCas9-mediated gene regulation platforms, which corresponds to the previous research describing optimal gRNA location for CRISPRa (from −400 to −50 bp upstream of the TSS) [To investigate the effects of individual gRNAs on gene regulation, the TSS) .Collectively, these results indicate that the diverse effector domains for the dCas9 toolkit are applicable in the chicken system with a variable range of gene regulation. Further studies using additional effector domains that were not validated in this study, such as SunTag, SAM, LSD1 or DNMT1 and a combination of gRNAs, could help optimize chicken gene regulation.IRF7 were further tested to compare with our previous studies [IRF7-regulated DF-1 cells were further validated. IRF7 is known as the master transcription factor of the type I interferon response in mammals and birds, and IRF7 gene knockout resulted in increased virus replication with significant downregulation of antiviral activities [To test the potential use of the CRISPRa and CRISPRi systems as a platform for functional studies in chickens, activation and repression of chicken  studies ,62,63. TIRF7 by CRISPRi resulted in the downregulation of interferon stimulated genes that are crucial in antiviral activity such as 2′-5′-oligoadenylate synthetase-like (OASL), Toll-like receptor 3 (TLR3), interferon induced with helicase C domain 1 , interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (MX1), and signal transducer and activator of transcription 1 (STAT1). On the other hand, the IRF7 upregulation by CRISPRa stimulated the genes relating to cell–cell interaction such as integrin subunit α 8 (ITGA8), cadherin 11 (CDH11) and collagen type VI α 2 chain (COL6A2), suggesting the genes’ roles in viral defense by modifying cell surface moiety and also corresponding to previous research [In this research, the downregulation of research ,62,63.In summary, the CRISPRa and CRISPRi systems using diverse effectors in chicken DF-1 fibroblast cells were successfully evaluated in this study, and significant results were observed when evaluating the combination of gRNAs and individual gRNAs in both the CRISPRa and CRISPRi cell lines. With the combination of bioinformatic analysis, the parallel studies of CRISPRa and CRISPRi using diverse effector domains are expected to not only contribute to regulating specific gene expression but also help to annotate the functional elements, including enhancer and repressor regions in the chicken genome. Further studies are warranted to utilize different cell lines to address the different biological contexts of gene expression.


Mechanistic studies in HCT116 colon cancer cells revealed that congener 1t dose-dependently increased levels of cleaved-caspases 8 and 9 and cleaved-PARP, while it concentration-dependently decreased levels of CDK4, CDK6, Cdc25A, and Cyclin D and E resulting in induction of cell cycle arrest and apoptosis in colon cancer HCT116 cells. Mechanistic study also presented MET receptor tyrosine kinase as the molecular target mediating the anticancer activity of compound 1t in HCT116 cells. In silico study predicted folded p-loop conformation as the form of MET receptor tyrosine kinase responsible for binding of compound 1t. Together, the current study presents compound 1t as an interesting anticancer lead for further development.A library of 24 congeners of the natural product sulfuretin were evaluated against nine panels representing nine cancer diseases. While sulfuretin elicited very weak activities at 10 µM concentration, congener  Globally, cancer is the second leading cause of death amongst people in developed and developing countries ,2,3,4,5.2, cytokines, and NF-κB promote survival, invasion, and metastasis of cancer cells [Inflammation is an axial component in cancer diseases. While acute inflammation response can promote tumor suppression, chronic inflammation was found to create an immunosuppressive microenvironment ,20,21,22er cells . Consequer cells . Proliferation of cells follows sequential steps of cell cycle phases that involve duplication of cells’ DNA and organelles followed by cell division. Several signaling pathways are involved in initiation and regulation of these phases. Within cancer cells, genetic mutations and/or epigenetic factors usually results in dysregulation of pathways promoting and/or repressing cell cycle and proliferation ,30,31,32Since ancient history, nature has served as a major source for drug discovery and development. Indeed, natural-product-based drug discovery affords higher success rates ,39,40,41meta and para-dihydroxy substituents at ring-B and a C6-hydroxy moiety at ring-A, is a natural product obtained from Rhus verniciflua which exhibits interesting properties [Sulfuretin , an aurooperties . Interesoperties . In addioperties . Despite2, an inflammatory mediator that is correlated with both inflammation and cancer [In addition to the antiproliferative activity, sulfuretin was also reported to show potential anti-inflammatory effects through inhibition of the NF-κB signaling pathway, a common pathway for inflammation and cancer . Howeverd cancer ,46. NeveC6-hydroxy or methoxy moiety on ring-A  or C6-hydroxy moiety on ring-A (compounds 1a–1c), derivatives having a methoxy moiety on ring-B combined with C6-hydroxy moiety on ring-A (compounds 1h–1j) or C6-methoxy moiety on ring-A (compounds 1w and 1x), and trihydroxylated or trimethoxylated ring-B derivatives (compounds 1g and 1o) were found to be almost inactive at the tested concentration. Such an outcome, coupled with the found low to no activity of compounds 1k–1m possessing dimethoxy moieties at ring-B combined with a C6-hydroxy moiety on ring-A, might support the conclusion that the vicinal dihydroxy moieties at ortho- and meta-positions of ring-B combined with a C6-methoxy moiety on ring-A might be optimal for activity. However, compound 1n possessing dimethoxy moieties at both meta-positions of ring-B showed potential activity. Interestingly, its activity was higher on HL60(TB) of myeloid origin; this represents a distinct pattern that is different from compounds 1t and 1v which were more active on cell lines of lymphoblastic origin. Blood cancers are a group of diverse cancers that may originate from lymphoblastic or myeloid progenitor cells and, furthermore, might be acute or chronic. Towards near inclusive evaluation, five cell lines were employed including: (1) CCRFCEM, a childhood T acute lymphoblastic leukemia (ALL); (2) MOLT4, an acute lymphoblastic leukemia (ALL); (3) HL60(TB), an acute myeloid leukemia (AML); (4) K562, a chronic myelogenous leukemia (CML); and (5) RPMI8226, a multiple myeloma (MM). At the tested concentration, sulfuretin showed very limited activity against CCRFCEM and MOLT4 ; both arf ring-A . It was activity , but los1a–1x were profiled against lung adenocarcinoma cells , bronchoalveolar carcinoma cells (H322M), and large-cell lung carcinoma cell lines (HOP92 and H460). As shown in 1t combining polar, hydrogen-bond-donor or acceptor dihydroxy moieties at vicinal ortho- and meta-positions of ring-B with the more steric, less polar hydrogen-bond-donor methoxy moiety at C6-position of ring-A and compound 1n having a C6-hydroxy moiety ring-A but possessing the less polar dimethoxy moieties at both meta-positions of ring-B. Both compounds elicited almost the same level of activity against the bronchoalveolar carcinoma cell line H322M and the human large-cell lung carcinoma cell line H460, but not HOP92. However, compound 1t was more active against lung adenocarcinoma cell lines A549, EKVX, HOP62, and H23, suggesting different molecular targets impacted by compounds 1t and 1n. Except for compounds 1t and 1n, the activity was limited for all other compounds whether bearing a C6-hydroxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B . In regards to sulfuretin, the growth of only two cell lines (HCT15 and KM12) was weakly inhibited by almost 10% while no inhibition was observed at all against the other three cell lines at the tested concentration . In consll lines . It beca1a–1x were profiled against diverse cells including SF268, SNB75, SF295, SF539, SNB19, and U251. As shown in 1t, bearing dihydroxy moieties at vicinal ortho- and meta-positions of ring-B coupled with the less polar hydrogen-bond-donor-only methoxy moiety at the C6-position of ring-A. It showed almost 10-fold the activity of sulfuretin against the anaplastic astrocytoma SF268 cell line, triggering near 50% growth inhibition. In addition, it induced near 40% growth inhibition of SF539 and U251 glioblastoma cell lines and the SNB75 astrocytoma cell line. Like the activity pattern against blood cancers, compound 1v, incorporating a C6-methoxy moiety at ring-A as in compound 1t but with the dihydroxy moieties shifted to vicinal meta- and para-positions of ring-B, showed significant, yet less, activity relative to compound 1t against these four cell lines  or bearing a monohydroxy moiety at ring-B (compounds 1a–1c) showed low activity. All other tested derivatives of the C6-hydroxy series bearing dihydroxy, polyhydroxy or monomethoxy substituents on ring-B were virtually inactive. Collectively, the results suggest compounds 1t, 1v, and 1n as possible hits possessing significant activities against gliomas. Gliomas are common brain cancers that originate from glial cells. Considering the hematopoietic origin of glial cells ,48, it mll lines . Other cactivity . It was 1a–1x were profiled against diverse melanoma cells, including the following: LOXIMVI, M14, MDAMB435, MALME3M, SKMEL28, SKMEL5, UACC257, and UACC62. As revealed from profiling results, sulfuretin was almost ineffective. It showed no inhibition for five cell lines out of the employed eight cell lines and, furthermore, the growth inhibition found for the other three cell lines was less than 8%. In line with the activity pattern found against lung cancers, compounds 1t and 1n were the most active derivatives  showed almost no significant activity except for compound 1l that showed low activity against most of the employed melanoma cell lines. Melanoma is the most aggressive form of skin cancer. Accordingly, compounds ivatives . The metell line . Howevercompound . Conside1a–1x were profiled against IGROV1 (an endometrioid carcinoma), as well as OVCAR3, OVCAR4, OVCAR5, OVCAR8, and ADRRES . The results showed that sulfuretin was almost ineffective against all employed cell lines. As shown in 1t which triggered almost 77% growth inhibition of the high-grade ovarian serous adenocarcinoma OVCAR3 cell lines and near 63% growth inhibition of the endometrioid carcinoma IGROV1 cell line. It also inhibited other high-grade ovarian serous adenocarcinoma cell lines by varying degrees. Other compounds sharing the C6-methoxy moiety on ring-A present in compound 1t but with other hydroxy or methoxy substitution patterns on ring-B (compounds 1q–1x) were almost inactive except for minor activity for compound 1v. Except for some activity for compound 1n bearing a dimethoxy substitution pattern at both meta-positions of ring-B and, to a lesser extent, compound 1l possessing a dimethoxy substitution pattern at distal ortho- and meta-positions of ring-B, all other compounds with the C6-hydroxy moiety on ring-A were almost inactive  is the most common ovarian cancer. Meanwhile, endometrioid carcinoma is an invasive ovarian cancer. Accordingly, compounds 1a–1x were profiled against a panel composed of the following: (1) 786O, a primary ccRCC cell line; (2) CAKI1, a metastatic ccRCC cell line; (3) ACHN, a metastatic papillary renal cell carcinoma (a subtype of nccRCC); (4) RXF393, an unclassified poorly differentiated renal cell carcinoma; (5) SN12C, an unclassified renal cell carcinoma; and (6) UO31, an unclassified renal cell carcinoma. As illustrated in C6-hydroxy moiety on ring-A coupled with hydroxy functions on ring-B  could be clear-cell renal cell carcinomas  or non-clear-cell renal cell carcinomas  . Consequ1a–1x were profiled against two metastatic prostate adenocarcinoma cell lines, PC3 and DU145. As shown in 1a–1g having hydroxy groups on ring-B in conjunction with a C6-hydroxy moiety on ring-A had negligible to no effect on both metastatic DU145 and PC3 cell lines. Replacement of the hydroxy substitution patterns on ring-B by methoxy substitution patterns enabled three compounds to trigger growth inhibition of the metastatic DU145 cell line  MCF7, a metastatic invasive hormone-responsive breast adenocarcinoma; (2) BT549, a primary triple-negative invasive breast adenocarcinoma; (3) MDAMB231, a metastatic triple-negative breast adenocarcinoma; and (4) MDAMB468, a metastatic triple-negative breast adenocarcinoma. As shown in C6-hydroxy derived compounds having hydroxy substituents on ring-B did not inhibit the growth of any primary or metastatic cell lines . When both vicinal ortho- and meta-positions were substituted simultaneously by dihydroxy moieties, the activity was more enhanced (compound 1t). In addition, compound 1t, amongst the C6-methoxy derived compounds 1q–1x, showed the best inhibition of the growth of the primary triple-negative BT549 cell line and metastatic triple-negative MDAMB231 cell line. However, the growth of the triple-negative MDAMB468 cell was more inhibited by compound 1w, possessing a ortho-methoxy substituent on ring-B relative to compound 1t possessing vicinal ortho- and meta-methoxy substituents on ring-B. Such a difference in structural feature requirements suggests the involvement of different molecular targets. Collectively, the results might nominate compound 1t as a hit candidate for the development of antiproliferative compounds against metastatic hormone responsive MCF7 or triple-negative primary and metastatic triple-negative BT549 and MDAMB231 breast cancers. In addition, compound 1n might be nominated as a hit compound against primary triple-negative BT549 cells. Breast cancer could be hormone-sensitive or resistant and, in addition, primary or metastatic. Accordingly, compounds ell line . Amongstell line . As reve1t, the potency of compound 1t was first assessed against three cell lines that were available to us and included in the panels used to profile the activity that involved lung cancer A549, colon cancer HCT116, and breast cancer MDAMB231 cell lines. As shown in 1t showed potent inhibitory activity for HCT116 cell growth with a low micromolar IC50 value in the range of 8.68 μM. Consequently, the mechanism of action of compound 1t was studied employing colon cancer HCT116 cells.To assess the mechanism mediating the potential antiproliferative activity of compound 1t in HCT116 cells at different doses to explore possible apoptotic and/or necrotic activity. The results showed that compound 1t could significantly induce the apoptosis in HCT116 cells  blood cancer , a flow 16 cells A. Accord16 cells B. These 1t, analysis of HCT116 cell distribution in the different phases of cell cycle progression revealed that compound 1t dose-dependently increased the G0/G1 cell population /Akt/mammalian target of rapamycin (mTOR) pathways is involved in cancer cells’ increased motility, proliferation, migration, invasion, survival, and angiogenesis ,51,52. I1t in HCT116 cells. As evident in 1t are mediated by suppression of MET receptor tyrosine kinase activation, and invariably lead to down-regulation of the PI3K/AKT/mTOR signaling pathways. However, it is possible the suppression of activation of MET found might arise from other factors and signaling pathways rather than direct interaction with MET, and this might be explored in the future.Western blotting was used to verify whether mTOR is involved in mediating the cell cycle arrest and apoptotic effects of compound 1t with MET receptor tyrosine kinase, an in silico study was conducted employing crystal structures of folded p-loop, extended p-loop, and rearranged αC helix conformations of MET receptor tyrosine kinase . The results showed that compound 1t was able to dock perfectly into the p-folded MET receptor tyrosine kinase conformation (PDB: 7b41), showing a predicted binding mode that overlayed the reported X-ray co-crystallized ligand  revealed that the predicted binding has lower score (−6.27824) relative to the folded p-conformation and is not aligned over the co-crystalized ligand  were used to profile the anticancer activity of the natural product sulfuretin as a starting point and a library of 24 sulfuretin congeners. In contrast to the poor anticancer activity of sulfuretin at the tested concentration, compound 1t was discovered amongst tested sulfuretin congeners as a potential anticancer agent. It was found to induce death of colon cancer cells through the triggering cell cycle arrest and apoptosis, and a mechanistic study revealed MET receptor tyrosine kinase as the molecular target mediating its anticancer activity. In silico study suggested that the folded p-loop conformation of MET receptor tyrosine kinase is the conformation responsible for binding of compound 1t. Collectively, these findings suggest compound 1t as a lead compound for further development of MET receptor tyrosine kinase inhibitors with potential anticancer activity. In summary, starting from the natural product sulfuretin eliciting a modest anticancer activity, compound


This report details a case of a 16-year-old African American girl who presented with a two-year history of white spicules on her face without associated symptoms, including pruritus or pain. On physical examination, there were many 1-2 mm discreet white to yellow filamentous spicules on the mid and lower face. Histopathology scrapings showed cornified cells and calcification consistent with sebaceous filaments. Sebaceous filaments are a rare condition that presents as white-to-yellow spicules distributed in highly sebaceous areas on the face. It is caused by sebum accumulation and cornified keratinocytes surrounding hair follicles, resulting in visible excretions. Treatment of sebaceous filaments is targeted at reducing the size of sebaceous glands which subsequently decreases excretions and improves skin appearance. Despite low incidence, sebaceous filaments are likely under-reported. It is important for dermatologists to recognize and treat sebaceous filaments in patients who present with this condition to improve their appearance and quality of life. The present patient was successfully treated with topical tretinoin. Sebaceous glands are acinar exocrine glands that release sebum into the hair follicle to lubricate and protect the skin. Most sebaceous glands are part of pilosebaceous units and are predominantly found on the face, scalp, chest, and upper back . Sebum iDemodex folliculorum), follicular hyperkeratotic spicules (myeloma immunoglobulin or cryoglobulin), and acne vulgaris [Sebaceous filaments are a rare condition that presents as many white-to-yellow spicules distributed in highly sebaceous areas on the face. The condition is caused by sebum accumulation, cornified keratinocytes, and dead cells that surround hair follicles and results in visible excretions . The difvulgaris .Treatment focuses on reducing the size of sebaceous glands which subsequently decreases excretions and improves skin appearance. Here, we report the case of a 16-year-old girl who presented with sebaceous filaments and was successfully treated with topical tretinoin.A 16-year-old African American girl presented with a two-year history of white papules on her face. The patient believed these papules were acne and treated her face with over-the-counter salicylic acid washes but reported little improvement. The patient denied any pruritus, pain, or other physical symptoms. However, the patient reported that the appearance of her condition was upsetting and had a negative impact on her quality of life. The patient had a history of intellectual disability. Personal and family medical history was otherwise not relevant.Demodex folliculorum mites were seen on histopathology.Physical examination revealed multiple 1-2 mm discreet white-to-yellow filamentous spicules distributed evenly on the mid and lower face , follicular hyperkeratotic spicules (myeloma immunoglobulin or cryoglobulin), and acne vulgaris [Sebaceous filaments result from a build-up of sebum and cornified keratinocytes surrounding hair follicles . Sebaceovulgaris .Trichodysplasia spinulosa is caused by trichodysplasia spinulosa-associated polyomavirus (TSPyV) and usually presents as erythematous papules and spicules on the face in immunosuppressed patients . The preTrichostasis spinulosa is a common disorder of hair follicles that presents as darker papules on the face and extremities . DiagnosDemodex folliculorum mite and presents similarly to sebaceous filaments with plugged sebaceous hair follicles. The Demodex folliculorum mite may be in pilosebaceous units of 80-100% of patients aged 50 and over, although not all patients develop clinical symptoms [Demodex folliculorum mites [Pityriasis folliculorum results from infestation of the symptoms . Skin scum mites . TreatmeFollicular hyperkeratotic spicules are a rare disorder usually associated with underlying multiple myeloma and present as hyperkeratotic spicules on the face . The diaSebaceous glands are involved in a range of other pathologies. Conditions arising from primary involvement include steatocystoma, sebaceous adenoma, sebaceous carcinoma, sebaceoma, sebaceous hyperplasia, and nevus sebaceous . ConditiThe clinical presentation of the present case favored the diagnosis of sebaceous filaments. The patient’s papules were most abundant in highly sebaceous areas, including the nose, chin, and cheeks. The patient’s adolescent age and poor hygiene due to her intellectual disability also support sebaceous filaments. The histopathology shows cornified cells and calcification further confirming sebaceous filaments. Additionally, her face cleared after topical tretinoin, which helped support the diagnosis.Treatment for sebaceous filaments targets the sebaceous gland to reduce the size and subsequent secretions. Topical or oral retinoids, including isotretinoin, can effectively achieve this outcome and improve appearance . RetinoiDemodex folliculorum), follicular hyperkeratotic spicules (myeloma immunoglobulin or cryoglobulin), and acne vulgaris. Despite low incidence, sebaceous filaments are likely under-reported. It is important for dermatologists to recognize and treat sebaceous filaments in patients who present with this condition to improve their appearance and quality of life.This case presents sebaceous filaments in a teenager who was successfully treated with topical tretinoin. The differential diagnosis for this rare condition includes trichodysplasia spinulosa, trichostasis spinulosa, pityriasis folliculorum (