FaDu Cell Lines Research Articles

Curcumin-Triterpene Type Hybrid as Effective Sonosensitizers for Sonodynamic Therapy in Oral Squamous Cell Carcinoma.

Sonodynamic therapy (SDT) is a non-invasive therapeutic modality in cancer treatment that combines low-intensity ultrasound (US) and sonosensitizers. Tumor cells are destroyed through the synergistic effects of ultrasound and a chemical sonosensitizer. This study focused on the synthesis and in vitro evaluation of the sonodynamic effect of natural curcumin, triterpene oleanolic acid, and their semi-synthetic derivatives on tongue cancer SCC-25 and hypopharyngeal FaDu cell lines. The combination of the tested compounds with sonication showed a synergistic increase in cytotoxicity. In the group of oleanolic acid derivatives, oleanoyl hydrogen succinate (6) showed the strongest cytotoxic effect both in the SCC-25 and FaDu cell lines. Comparing curcumin (4) and its pyrazole derivative (5), curcumin showed a better cytotoxic effect on SCC-25 cells, while curcumin pyrazole was more potent on FaDu cells. The highest sonotherapeutic activity, compared to its individual components, was demonstrated by a structural linker mode hybrid containing both curcumin pyrazole-oleanoyl hydrogen succinate units within one complex molecule (7). This study can be beneficial in the context of new perspectives in the search for effective sonosensitizers among derivatives of natural organic compounds.

Design and Synthesis of an Hsp90 and HDAC Dual Inhibitor as Antitumor Agent

Background: Cancer incidence and mortality have been increasing, and cancer is still the leading cause of death all over the world. Therefore, expanding the arsenal of anticancer drugs with high efficiency and low toxicity is still one of the most challenging tasks. As a branch of antitumor drug design and discovery, dual-targeting drug candidates draw extensive attention. Objective:: In this work, we try to construct a multitarget drug candidate and evaluate its antitumor effects. Methods: Hsp90 and histone deacetylase were selected as two targets to design a dual targeting inhibitor w11. Enzyme inhibition work, cell viability assay, and docking simulation were carried out to evaluate the activity of the compound. Results: w11 could inhibit the activity of Hsp90α and HDAC6 with the IC50 of 50.1 nM and 8.1 nM, respectively. In cell viability assay, five human tumor cell lines Eca-109, FaDu, HN6, MCF-7 and MDAMB- 231 were used, results showed that w11 could potently inhibit the proliferation of three human lines with IC50 values in the nM range. Molecular docking experiments proved the rationality of structure design. Conclusion: Compound w11 was a potent Hsp90 and HDAC dual inhibitor for anticancer research.

In vitro evaluation of photon and carbon ion radiotherapy in combination with cisplatin in head and neck squamous cell carcinoma cell lines.

Heavy ion radiotherapy, such as carbon ion radiotherapy (CIRT), has multiple advantages over conventional photon therapy. Cisplatin, as a classic anti-tumor drugs, has been tested and discovered as a photon radiosensitizer in several cell lines, including head and neck squamous cell carcinoma (HNSCC). Hence, the aim of our study is to evaluate whether cisplatin can sensitize CIRT towards HNSCC cell lines in vitro. Human nasopharyngeal carcinoma cell line CNE-2, human tongue squamous carcinoma cell line TCA 8113 and human hypopharynx squamous carcinoma cell line FADU were all irradiated with photon beam of 2, 4, 6, 8 Gy (physical dose) and carbon ion beam of 1, 2, 3, 4 Gy (physical dose) and treated with cisplatin. Cell survival was assessed by clonogenic survival assay. CIRT showed significantly stronger cytotoxic effect than standard photon radiotherapy. The relative biological effectiveness (RBE) of carbon ion beam at 10% survival ( ) was calculated 3.07 for CNE-2, 2.33 for TCA 8113 and 2.36 for FADU. Chemoradiotherapy (both photon radiotherapy and CIRT) was more effective than radiotherapy alone. In vitro sensitizer enhancement ratios (SERs) of cisplatin in CNE-2, TCA 8113 and FA DU cell lines after photon irradiation were 1.33, 1.14 and 1.21, while after carbon ion irradiation were 1.02, 1.00 and 0.96, showed that cisplatin sensitized photon irradiation but showed no sensitization effect in carbon ion irradiation in all tested cell lines. In conclusion, high linear energy transfer (LET) CIRT was more effective than photon irradiation to prevent the proliferation of HNSCC cell lines. Additional treatment with cisplatin could sensitize photon irradiation but showed no effect on carbon ion irradiation.

Abstract 4756: NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC)

Abstract Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4756.

Abstract 4936: Activity of carfilzomib in head and neck cancer cell lines in vitro

Abstract Background: Carfilzomib (CF) is a 2nd generation proteasome inhibitor with activity in hematologic cancer, but its study in solid tumors like squamous cell carcinoma of the head and neck (SCCHN) is limited. We investigated its antitumor activity in three SCCHN cell lines. Methods: Cells: We studied two human tongue cell lines (CAL27; SCC-25) and one pharynx line (FaDu); all HPV-negative and p53-mutant. MTT assays: We utilized MTT assays to evaluate cytotoxicity of CF, cisplatin, and their combination. The IC50 of CF and Combination Index (CI) of varying concentrations of combined CF and cisplatin were calculated. Clonogenic assays: Cells were seeded at low density and after 24 hours, cells were treated with varying concentrations of CF. Plates were incubated for 10 days. The cells were fixed and stained with crystal violet dye and colonies counted. Wound Healing assays: After cells reached 100% confluence, a scratch was created using a 200 µL micropipette. Cells were treated with CF and images were acquired at 0, 24, 48, and 72 hours post-scratch. Results: MTT assays: IC50 values as a function of exposure lengths are shown in Table 1. CAL27 was the most sensitive while SCC-25 was the most resistant. In all cell lines, the results of combined CF and cisplatin were complex: at lower doses there was evidence of antagonism, while at higher doses there was synergy. Clonogenic assays: CF significantly inhibited clonogenicity compared to DMSO control (P≤ 0.05). The surviving fraction for CF 2.5, 5, and 10 nM were 0.411. 0.151, 0.051, and 0.5, 0.339, 0.102 in CAL27 and SCC-25 cell lines, respectively. Wound Healing assays: Wound closure % after treatment with CF5 was significantly different from DMSO control (P≤0.05). Wound closure % for CF5 were 30.97, 56.75, and 28.03% compared to 94.65, 75.88, and 42.05% for control in CAL27, FaDu, and SCC-25 cell lines, respectively. Conclusion: CF exhibits antitumor effects in SCCHN in vitro. Our findings support further investigation of CF in combination with conventional agents in vitro and in vivo. IC50 values for carfilzomib in head and neck cancer cell lines Cell line 24 hours 48 hours 72 hours CAL27 18.02 (11.76- 28.22) 9.364 (7.189- 12.45) 6.401 (5.579- 7.373) FaDu 57.34 (41.98-84.90) 16.22 (13.438- 19.446) 8.237 (7.260- 9.365) SCC25 77.78 (67.18- 91.30) 12.87 (11.36- 14.64) 11.49 (10.51- 12.58) Citation Format: Abdullah S. Eldaly, Douglas B. Stairs, Mitchell Machtay. Activity of carfilzomib in head and neck cancer cell lines in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4936.

Differential activation of NOTCH1 pathway in HNSCC cell lines of different anatomical sites

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent types of cancers and the role of NOTCH1 pathway during development of HNSCC is debatable. Here, we have made an attempt to evaluate the NOTCH1 pathway status in HNSCC cell lines from different anatomical subsites. At first, mRNA expression status of NOTCH1 pathway associated genes (NOTCH1/JAG1/JAG2/HES1/HEY1/CD44/FBXW7/HIF1α/VEGF) was analysed in two HNSCC cell lines: FaDu (hypopharyngeal carcinoma) and SCC9 (tongue carcinoma) and was compared with publicly available database. Then, molecular profiling (RNA/protein) of the genes and cell cycle phase distribution analysis were done after DAPT (γ-secretase inhibitor) administration at different concentrations on the cell lines to see the differential effect, if any. High NOTCH1 pathway activation was noted in FaDu cell line than the SCC9. In cytotoxicity assay with DAPT, FaDu showed more sensitivity than SCC9. Therefore, gradual decline of the expression of NOTCH1 pathway associated genes was noted in FaDu with the increasing DAPT concentrations, leading to high S/G2-M arrest of the cell population. Contrastingly, SCC9 showed significant reduced expression of the genes at higher concentration of DAPT with comparatively low S/G2-M arrest of the cell population. The study demonstrates distinct NOTCH1 pathway signature in the HNSCC cell lines of specific sub-sites of head and neck.

Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

CDCA3 promotes the proliferation and migration of hypopharyngeal squamous cell carcinoma cells by activating the Akt/mTOR pathway

ABSTRACT Hypopharyngeal squamous cell carcinoma (HSCC) is a highly invasive and fatal tumor with a poor prognosis in head and neck tumors. It is urgent to further study the molecular mechanism of HSCC progression and identify new effective therapeutic targets. Cell division cycle-related protein 3 (CDCA3) was reported overexpressed in several cancers and involved in tumor progression. However, the biological role of CDCA3 and its potential mechanism in HSCC remain undetermined. Reverse transcription quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression levels of CDCA3 in HSCC tissue and matched peritumoral tissue. The effects of CDCA3 on cell proliferation, invasion, and migration were explored using the Celigo image cytometry assay, MTT assay, flow cytometric analysis, cell invasion, and migration assays. The results showed that CDCA3 was upregulated in HSCC tissue and FaDu cell line. Knockdown of CDCA3 inhibited the proliferation, invasion, and migration of FaDu cells and promoted apoptosis of FaDu cells. Furthermore, knockdown of CDCA3 blocked the cell cycle in the G0/G1 phase. Mechanistically, CDCA3 may play a role in tumor progression of HSCC through the Akt/mTOR signaling pathway. In summary, these results suggest that CDCA3 serves as an oncogene in HSCC and may be used as a prognostic indicator and a potential therapeutic target for HSCC.

Chemical and Biological Studies of Endophytes Isolated from Marchantia polymorpha.

Natural bioresources, predominantly plants, have always been regarded as the richest source of drugs for diseases threatening humanity. Additionally, microorganism-originating metabolites have been extensively explored as weapons against bacterial, fungal, and viral infections. However, the biological potential of metabolites produced by plant endophytes still remains understudied, despite significant efforts reflected in recently published papers. Thus, our goal was to evaluate the metabolites produced by endophytes isolated from Marchantia polymorpha and to study their biological properties, namely anticancer and antiviral potential. The cytotoxicity and anticancer potential were assessed using the microculture tetrazolium technique (MTT) against non-cancerous VERO cells and cancer cells-namely the HeLa, RKO, and FaDu cell lines. The antiviral potential was tested against the human herpesvirus type-1 replicating in VERO cells by observing the influence of the extract on the virus-infected cells and measuring the viral infectious titer and viral load. The most characteristic metabolites identified in the ethyl acetate extract and fractions obtained by use of centrifugal partition chromatography (CPC) were volatile cyclic dipeptides, cyclo(l-phenylalanyl-l-prolyl), cyclo(l-leucyl-l-prolyl), and their stereoisomers. In addition to the diketopiperazine derivatives, this liverwort endophyte also produced arylethylamides and fatty acids amides. The presence of N-phenethylacetamide and oleic acid amide was confirmed. The endophyte extract and isolated fractions showed a potential selective anticancer influence on all tested cancer cell lines. Moreover, the extract and the first separated fraction noticeably diminished the formation of the HHV-1-induced cytopathic effect and reduced the virus infectious titer by 0.61-1.16 log and the viral load by 0.93-1.03 log. Endophytic organisms produced metabolites with potential anticancer and antiviral activity; thus, future studies should aim to isolate pure compounds and evaluate their biological activities.

Stathmin1 promotes lymph node metastasis in hypopharyngeal squamous cell carcinoma via regulation of HIF‑1α/VEGF‑A axis and MTA1 expression.

Extensive neck lymph node metastasis (LNM) is an important clinical feature of hypopharyngeal squamous cell carcinoma (HSCC). Stathmin1 (STMN1) is closely associated with LNM in numerous human cancers. In the present study, the association between STMN1 and neck LNM in HSCC and the underlying molecular mechanisms were explored. First, postoperative samples of HSCC were screened and the association between STMN1 and neck LNM in HSCC was analyzed. Then, cell functional experiments were performed to assess the potential of STMN1 to promote invasion and migration. Subsequently, the potential target genes and pathways of STMN1 were predicted using bioinformatics analysis. Finally, the obtained target genes and pathways of STMN1 were validated by reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses to confirm the potential mechanisms by which STMN1 promotes LNM in HSCC. As a result, a total of 117 postoperative samples of HSCC were screened, and STMN1 was proven to be associated with neck LNM in HSCC. Further, cell functional experiments established that high expression of STMN1 could actually promote FaDu cell invasion and metastasis. Bioinformatics analysis revealed that high expression of STMN1 was associated with the activation of hypoxia inducible factor-1alpha (HIF-1α) pathway and increased expression of metastasis-associated protein 1 (MTA1). Finally, RT-qPCR and western blot analyses confirmed that STMN1 promotes the expression levels of HIF-1α/vascular endothelial growth factor (VEGF)-A and MTA1 in FaDu cell lines. In conclusion, it was found that high expression of STMN1 promoted neck LNM in HSCC and the potential mechanisms may be via regulation of the HIF-1α/VEGF-A axis and MTA1 expression.

LSD1 silencing inhibits the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.

Hypopharyngeal cancer is a subtype of the head and neck malignancies. We aimed to explore the role of lysine-specific demethylase 1 (LSD1/KDM1A) in the progression of hypopharyngeal cancer and to identify the potential mechanisms. First, LSD1 expression in head and neck squamous cell carcinoma (HNSCC) tissues and the correlation between LSD1 and the stage of HNSC were analyzed by the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Following LSD1 silencing, proliferation of pharyngeal cancer cell line FaDu cells was evaluated by cell counting kit-8 and colony formation assays. Wounding healing and transwell assays were used to measure the capacities of migration and invasion. In addition, expression of proteins related to epithelial-to-mesenchymal transition (EMT), autophagy, and pyroptosis was tested by Western blot analysis or immunofluorescence. After treatment with autophagy inhibitor 3-methyladenine (3-MA) or NLR family pyrin domain containing 3 (NLRP3) inhibitor MCC950, the malignant biological properties were measured again. High LSD1 expression was observed in HNSC tissues, which was correlated with stage. LSD1 knockdown significantly suppressed the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. Moreover, autophagy and pyroptosis were induced by LSD1 depletion, observed by the enhanced fluorescence intensity of LC3, gasdermin-D (GSDMD)-N, and apoptosis-associated speck-like protein containing a CARD (ASC), accompanied by upregulated expression of LC3II/LC3I, Beclin-1, NLRP3, cleaved-caspase 1, ASC, interleukin (IL)-1β, and IL-18 and downregulated expression of p62. Importantly, 3-MA or MCC950 addition obviously reversed the inhibitory effects of LSD1 silencing on the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. To sum up, LSD1 silencing could restrain the progression of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.

HSP90AA1 promotes lymphatic metastasis of hypopharyngeal squamous cell carcinoma by regulating epithelial-mesenchymal transition.

Lymphatic metastasis (LM) emerges as an independent prognostic marker for hypopharyngeal squamous cell carcinoma (HSPSCC), chiefly contributing to treatment inefficacy. This study aimed to scrutinize the prognostic relevance of HSP90AA1 and its potential regulatory mechanism of concerning LM in HPSCC. In a preceding investigation, HSP90AA1, a differential gene, was discovered through transcriptome sequencing of HPSCC tissues, considering both the presence and absence of LM. Validation of HSP90AA1 expression was accomplished via qRT-PCR, western-blotting(WB), and immunohistochemistry(IHC), while its prognostic significance was assessed employing Kaplan-Meier survival analysis(KMSA), log-rank test(LR), and Cox's regression analysis(CRA). Bioinformatics techniques facilitated the prediction and analysis of its plausible mechanisms in LM, further substantiated by in vitro and in vivo experiments utilizing FaDu cell lines. HSP90AA1 is substantially up-regulated in HPSCC with LM and is identified as an independent prognostic risk determinant. The down-regulation of HSP90AA1 can achieve inhibition of tumor cell proliferation, migration and invasion. Both in vivo experiments and Bioinformatics exploration hint at promoting LM by Epithelial-mesenchymal transition (EMT), regulated by HSP90AA1. HSP90AA1, by controlling EMT, can foster LM in HPSCC.This finding sets the foundation for delving into new therapeutic targets for HPSCC.

Isolation and characterization of head and neck cancer-derived peritumoral and cancer-associated fibroblasts.

Head and neck squamous cell carcinomas (HNSCC) are characterized by strong cellular and molecular heterogeneity and treatment resistance entailing poor survival. Besides cell-intrinsic properties, carcinoma cells receive important cues from non-malignant cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a major component of the TME that impact on the molecular make-up of malignant cells and have a decisive function in tumor progression. However, the potential functionality of fibroblasts within tumor-adjacent, macroscopically normal tissue remains poorly explored. Here, we isolated primary peritumoral fibroblasts (PtFs) from macroscopically normal tissue in vicinity of primary human papillomavirus-negative and -positive oropharyngeal HNSCC and compared their phenotype and functionality with matched CAFs (n = 5 pairs) and with human oral fibroblasts (hOFs). Expression patterns of CD90, CD73, CD105, smooth muscle actin, Vimentin, and S100A4 were comparable in PtFs, CAFs, and hOFs. Cell proliferation and doubling times of CAFs and PtFs were heterogeneous across patients (n =2 PtF>CAF; n = 1 CAF>PtF; n = 2 CAF=PtF) and reflected inferior growth than hOFs. Furthermore, PtFs displayed an reduced heterogeneity in cell size compared to matched CAFs, which were characterized by the presence of single large cells. Overall, conditioned supernatants from CAFs had more frequently growth-promoting effects on a panel of carcinoma cell lines of the upper aerodigestive tract carcinoma cell lines (Cal27, Cal33, FaDu, and Kyse30), whereas significant differences in migration-inducing effects demonstrated a higher potential of PtFs. Except for Kyse30, CAFs were significantly superior to hOFs in promoting proliferation, while PtFs induced stronger migration than hOFs in all carcinoma lines tested. Analysis of soluble factors demonstrated significantly increased VEGF-A production in CAFs (except in pat.8), and significantly increased PDGF-BB production in PtFs of two patients. Tube formation assays confirmed a significantly enhanced angiogenic potential of conditioned supernatants from CAFs compared to hOFs on human umbilical vascular endothelial cells (HUVECs) in vitro. Hence, matched CAFs and PtFs present in HNSCC patients are heterogeneous in their proliferation-, migration-, and angiogenesis-promoting capacity. Despite this heterogeneity, CAFs induced stronger carcinoma cell proliferation and HUVEC tube formation overall, whereas PtFs promoted migration of tumor cells more strongly.

Graphene Oxide/Chitosan Injectable Composite Hydrogel for Controlled Release of Doxorubicin: An Approach for Enhanced Intratumoral Delivery

Intratumoral (IT) injection of chemotherapeutics into needle-accessible solid tumors can directly localize the anticancer drug in the tumor site, thus increasing its local bioavailability and reducing its undesirable effects compared to systemic administration. In this study, graphene oxide (GO)-based chitosan/β-glycerophosphate (CS/GP) thermosensitive injectable composite hydrogels (CH) were prepared and optimized for the localized controlled delivery of doxorubicin (DOX). A quality-by-design (QbD) approach was used to study the individual and combined effects of several formulation variables to produce optimal DOX-loaded GO/CS/GP CH with predetermined characteristics, including gelation time, injectability, porosity, and swelling capacity. The surface morphology of the optimal formulation (DOX/opt CH), chemical interaction between its ingredients and in vitro release of DOX in comparison to GO-free CS/GP CH were investigated. Cell viability and cellular uptake after treatment with DOX/opt CH were studied on MCF 7, MDB-MB-231 and FaDu cell lines. The statistical analysis of the measured responses revealed significant effects of the concentration of GO, the concentration of CS, and the CS:GP ratio on the physicochemical characteristics of the prepared GO/CS/GP CH. The optimization process showed that DOX-loaded GO/CS/GP CH prepared using 0.1% GO and 1.7% CS at a CS: GO ratio of 3:1 (v/v) had the highest desirability value. DOX/opt CH showed a porous microstructure and chemical compatibility between its ingredients. The incorporation of GO resulted in an increase in the ability of the CH matrices to control DOX release in vitro. Finally, cellular characterization showed a time-dependent increase in cytotoxicity and cellular uptake of DOX after treatment with DOX/opt CH. The proposed DOX/opt CH might be considered a promising injectable platform to control the release and increase the local bioavailability of chemotherapeutics in the treatment of solid tumors.

Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B.

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein-DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.

Phenolic Profile, Antioxidant, Anti-Enzymatic and Cytotoxic Activity of the Fruits and Roots of Eleutherococcus senticosus (Rupr. et Maxim.) Maxim.

Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. is well-known for its adaptogenic properties in traditional Eastern medicine. It has been categorized as an endangered species due to the over-exploitation of the roots. As a result, alternatives must be found, including the usage of renewable aerial parts such as fruits. The goal of this research was to determine the phenolic compounds and the enzymatic, antioxidant, and cytotoxic activities of the intractum gained from the E. senticosus fruits and the mixture of chloroform-methanol roots extract with naringenin (3:7:5). The obtained results showed, that the intractum contained 1.02 mg/g ext. of polyphenols, 0.30 mg/g ext. of flavonoids, and 0.19 mg/g ext. of phenolic acids. In turn, the mixture of chloroform-methanol roots extract with naringenin (3:7:5) contained 159.27 mg/g ext. of polyphenols, 137.47 mg/g ext. of flavonoids, and 79.99 mg/g ext. of phenolic acids. Regarding the anti-enzymatic assay, the IC50 values for tyrosinase and hyaluronidase were equal to 586.83 and 217.44 [μg/mL] for the intractum, and 162.56 and 44.80 [μg/mL] for the mixture, respectively. Both preparations have possessed significant antioxidant activity in the ABTS, DPPH, and ferrozine tests. No cytotoxic effect on the FaDu and HEP G2 cancer cell lines was observed. Our findings support the traditional use of fruits and roots. Moreover, the results indicate also that adaptogens are rather nontoxic for normal and cancer cells, which corresponds with some hypotheses on adaptogens activity.

Downregulation of S100A9 Reverses Cisplatin-Resistance and Inhibits Proliferation and Migration in Hypopharyngeal Carcinoma.

Purpose Patients with hypopharyngeal carcinoma (HPC) often progress to an advanced clinical stage at diagnosis. Cisplatin has been widely used in first-line chemotherapy for advanced HPC. However, acquired chemotherapeutic resistance leads to recurrence, metastasis, and a poor survival rate. Therefore, identifying new drug targets to improve treatment effects is still in need. Methods To screen the differential expression genes (DEGs) and proteins (DEPs), we conducted transcriptomic and proteomic analysis on cisplatin-sensitive cell lines (FaDu) and cisplatin-resistant cell lines (FaDu/DDP) of hypopharyngeal carcinoma. DEGs and DEPs, possibly the most associated with cisplatin-resistance, were verified by real-time polymerase chain reaction (RT-PCR) and western blot (WB), respectively, and the biological function of the screened S100A9 was further tested by CCK8, wound healing, and transwell assays. Results We identified S100A9 as a target for resensitizing the response to cisplatin in an acquired resistance model. S100A9 overexpression was significantly related to cisplatin resistance. Functional studies in vitro models demonstrated that downregulation of S100A9 overcame cisplatin-resistance and inhibited proliferation and migration. Later, we verified that downregulation of S100A9 suppressed the interleukin-6 (IL6) expression and epithelial-mesenchymal transition (EMT) pathway. Conclusion In all, S100A9 plays a crucial role in cisplatin-resistance, proliferation, and migration of HPC. Targeting S100A9 may become a novel strategy for the treatment of HPC.

Cyclopiumolides A and B, unusual 13-membered macrolides from the deep sea-sourced fungus Penicillium cyclopium SD-413 with antiproliferative activities

Cyclopiumolides A (1) and B (2), first representatives of two novel biosynthetic related 13-membered macrolides featuring an uncommon verrucosidinol unit condensed with a spiculisporic acidic moiety, were identified from the fungus Penicillium cyclopium SD-413, which was obtained from the deep-sea sediments collected in the East China Sea. The structures of cyclopiumolides A (1) and B (2) were identified on the basis of extensive NMR spectroscopic and mass spectrometric data analysis. Their relative and absolute configurations were determined by quantum mechanical calculations of ECD spectra comparing with that of experimental curves and by DP4 + NMR data calculations. Compounds 1 and 2 exhibited significant cytotoxic potencies against the tumor cell lines SF126, FaDu, and TE-1 with IC50 values ranging from 5.86 to 17.05 μM. The inhibition modes and binding sites of 1 and 2 were inspected using molecular docking simulations.

SHMT2 Induces Stemness and Progression of Head and Neck Cancer.

Various enzymes in the one-carbon metabolic pathway are closely related to the development of tumors, and they can all be potential targets for cancer therapy. Serine hydroxymethyltransferase2 (SHMT2), a key metabolic enzyme, is very important for the proliferation and growth of cancer cells. However, the function and mechanism of SHMT2 in head and neck cancer (HNC) are not clear. An analysis of The Cancer Genome Atlas (TCGA) data showed that the expression of SHMT2 was higher in tumor tissue than in normal tissue, and its expression was significantly associated with male sex, aggressive histological grade, lymph node metastasis, distant metastasis, advanced TNM stage, and lymphovascular invasion in HNC. SHMT2 knockdown in FADU and SNU1041 cell lines significantly inhibited cell proliferation, colony formation, migration, and invasion. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using TCGA data revealed that SHMT2 was closely related to cancer stem cell regulation and maintenance. Furthermore, we found that silencing SHMT2 inhibited the expression of stemness markers and tumor spheroid formation compared with a control group. On the contrary, stemness markers were significantly increased after SHMT2 overexpression in HEP-2 cells. Interestingly, we found that knocking down SHMT2 reduced the expression of genes related to the Notch and Wnt pathways. Finally, silencing SHMT2 significantly reduced tumor growth and decreased stemness markers in a xenograft model. Taken together, our study suggests that targeting SHMT2 may play an important role in inhibiting HNC progression.

KDM1A inhibition increases UVA toxicity and enhances photodynamic therapy efficacy.

Lysine-specific histone demethylase 1 (KDM1A/LSD1) regulates multiple cellular functions, including cellular proliferation, differentiation, and DNA repair. KDM1A is overexpressed in squamous cell carcinoma of the skin and inhibition of KDM1A can suppress cutaneous carcinogenesis. Despite the role of KDM1A in skin and DNA repair, the effect of KDM1A inhibition on cellular ultraviolet (UV) response has not been studied. The ability of KDM1A inhibitor bizine to modify cell death after UVA and UVB exposure was tested in normal human keratinocytes and melanocytes, HaCaT, and FaDu cell lines. KDM1A was also downregulated using shRNA and inhibited by phenelzine in HaCaT and FaDu cells to confirm the role of KDM1A in UVA response. In addition, cellular reactive oxygen species (ROS) changes were assessed by a lipid-soluble fluorescent indicator of lipid oxidation, and ROS-related gene regulation using qPCR. During photodynamic therapy (PDT) studies HaCaT and FaDu cells were treated with aminolaevulinic acid (5-ALA) or HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) sodium and irradiated with 0-8J/cm2 red LED light. KDM1A inhibition sensitized cells to UVA radiation-induced cell death but not to UVB. KDM1A inhibition increased ROS generation as detected by increased lipid peroxidation and the upregulation of ROS-responsive genes. The effectiveness of both ALA and HPPH PDT significantly improved in vitro in HaCaT and FaDu cells after KDM1A inhibition. KDM1A is a regulator of cellular UV response and KDM1A inhibition can improve PDT efficacy.