G361 Cells Research Articles

Cytoglobin attenuates melanoma malignancy but protects melanoma cells from ferroptosis.

Cutaneous malignant melanoma is the most aggressive and the deadliest form of skin cancer. There are two types of limitations which universally exist in current melanoma therapy: Adverse effects and reduced efficiency. Cytoglobin (CYGB), an iron hexacoordinated globin, is highly enriched in melanocytes and frequently epigenetically silenced during melanoma genesis. The present study aimed to explore its potential role as a biomarker for ferroptosis treatment. It was observed that B16F10 and A375 melanoma cells with loss of CYGB expression were highly sensitive to ferroptosis inducers RSL3 and erastin, whereas G361 melanoma cells with highly enriched CYGB were resistant to RSL3 or erastin. Ectopically overexpressed CYGB rendered B16F10 and A375 cells resistant to RSL3 or erastin, accompanied by decreased proliferation and epithelial‑mesenchymal transition (EMT). By contrast, knockdown of CYGB expression made G361 cells sensitive to ferroptosis induction but induced proliferation and EMT progression of G361 cells. Mechanistically, CYGB‑induced resistance of melanoma cells to ferroptosis may have been associated, in part, with i) Suppression of EMT; ii) upregulation of glutathione peroxidase 4 expression; iii) decrease of labile iron pool. In vivo study also demonstrated that CYGB overexpression rendered xenograft melanoma much more resist to RSL3 treatment. Based on these findings, CYGB is a potential therapeutic biomarker to screen the melanoma patients who are most likely benefit from ferroptosis treatment.

Preliminary study on cytotoxicity of selegiline on different cancer cell lines: exploration of the induction of ROS-independent apoptosis in breast cancer cells.

The concept of drug repurposing is now widely utilized by biomedical scientists for drug discovery. An example of this is the use of selegiline (SEL), a monoamine oxidase inhibitor that was initially used for the management of depression but is now being considered for another purpose. This study compares the cytotoxic effects of SEL on different cancer cells. Further, the study explores the molecular mechanism of cell death, validating the possibility of its repurposing for cancer. Preliminary analysis of network pharmacological data was conducted in silico, followed by in vitro cytotoxicity tests on PC12, G361, MDA-MB231, MCF7, THP-1, and Hela cells under normoxic and hypoxic conditions, using the MTT assay. The mechanism of cell death was then confirmed by performing DAPI and FITC-conjugated Annexin V and Propidium Iodide (PI) staining assays. Additionally, ROS levels and PKC phosphorylation were also evaluated. In silico analysis has revealed that SEL is associated with ten genes linked to different cancer types. Specifically, SEL was most cytotoxic to neuronal pheochromocytoma, triple-negative human epithelial breast cancer cells, and ER+ and PR+ breast cancer cells. Furthermore, it was observed that this cell death occurred through ROS-independent apoptosis pathways. In addition, SEL was found to inhibit the phosphorylation of PKC, which may contribute to cell death. SEL induces apoptosis in breast cancer cells independently of reactive oxygen species and inhibits the phosphorylation of protein kinase C, which merits further exploration.

Synthesis and photodynamic activity of new 5-[(E)-2-(3-alkoxy-1-phenyl-1H-pyrazol-4-yl)ethenyl]-2-phenyl-3H-indoles.

A series of new indole-pyrazole hybrids 8a-m were synthesized through the palladium-catalyzed ligandless Heck coupling reaction from easily accessible unsubstituted, methoxy- or fluoro-substituted 4-ethenyl-1H-pyrazoles and 5-bromo-3H-indoles. These compounds exerted cytotoxicity to melanoma G361 cells when irradiated with blue light (414 nm) and no cytotoxicity in the dark at concentrations up to 10 µM, prompting us to explore their photodynamic effects. The photodynamic properties of the example compound 8d were further investigated in breast cancer MCF-7 cells. Evaluation revealed comparable anticancer activities of 8d in both breast and melanoma cancer cell lines within the submicromolar range. The treatment induced a massive generation of reactive oxygen species, leading to different types of cell death depending on the compound concentration and the irradiation intensity.

Tumoral EIF4EBP1 regulates the crosstalk between tumor-associated macrophages and tumor cells in MRTK

Malignant renal rhabdoid tumor (MRTK) is an aggressive and rare malignancy primarily affecting infants and young children. The intricate interactions within the Tumor Microenvironment (TME) are crucial in shaping MRTK's progression. This study elucidates the significance of tumor-associated macrophages(TAMs) within this milieu and their interplay with eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) in tumor cells, collectively contributing to MRTK's malignant advancement. Through comprehensive analysis of clinical samples and the TARGET database, EIF4EBP1 emerges as a central macrophage-associated gene with robust prognostic implications. Elevated EIF4EBP1 expression correlates with poor prognosis and heightened infiltration of TAMs. Functional validation demonstrates that EIF4EBP1 knockdown in G401 cells significantly attenuates self-proliferation, migration, and invasion. Moreover, EIF4EBP1 regulates macrophage recruitment and M2 polarization through the ERK/P38 MAPK-MIF axis. Notably, M2 macrophages reciprocally foster the malignant behavior of MRTK tumor cells. This study unveils the pivotal role of EIF4EBP1 in propelling MRTK's malignant progression, unraveling a complex regulatory network involving EIF4EBP1 and TAMs. These findings underscore EIF4EBP1 as a promising biomarker and highlight its therapeutic potential in MRTK management.

Nephroblastoma-specific dysregulated gene SNHG15 with prognostic significance: scRNA-Seq with bulk RNA-Seq data and experimental validation

Wilms tumor (WT) is the most common malignancy of the genitourinary system in children. Currently, the Integration of single-cell RNA sequencing (scRNA-Seq) and Bulk RNA sequencing (RNA-Seq) analysis of heterogeneity between different cell types in pediatric WT tissues could more accurately find prognostic markers, but this is lacking. RNA-Seq and clinical data related to WT were downloaded from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Small nucleolar RNA host gene 15 (SNHG15) was identified as a risk signature from the TARGET dataset by using weighted gene co-expression network analysis, differentially expressed analysis and univariate Cox analysis. After that, the functional mechanisms, immunological and molecular characterization of SNHG15 were investigated at the scRNA-seq, pan-cancer, and RNA-seq levels using Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), ESTIMATE, and CIBERSORT. Based on scRNA-seq data, we identified 20 clusters in WT and annotated 10 cell types. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing M2 macrophages as hubs for intercellular communication. In addition, in vitro cellular experiments showed that siRNAs interfering with SNHG15 significantly inhibited the proliferation and migration of G401 cells and promoted the apoptosis of G401 cells compared with the control group. The effect of siRNAs interfering with SNHG15 on EMT-related protein expression was verified by Western blotting assay. Thus, our findings will improve our current understanding of the pathogenesis of WT, and they are potentially valuable in providing novel prognosis markers for the treatment of WT.

Characterization and Biological Activities of the Ulvan Polysaccharide-Rich Fraction Obtained from Ulva rigida and Ulva pseudorotundata and Their Potential for Pharmaceutical Application.

Seaweed from the genus Ulva (Ulvales, Chlorophyta) has a worldwide distribution and represents a potential biomass source for biotechnological applications. In the present study, we investigated the ulvan polysaccharide-rich fraction (UPRF) isolated from two Ulva species (U. rigida and U. pseudorotundata), naturally occurring on the Spanish Mediterranean coast. Chemical characterization of UPRFs was performed in order to explore the polysaccharides' composition. Biological assessments of UPRFs were compared by antioxidant activity and in vitro toxicity tests in the human cell lines: HCT-116 (colon cancer), G-361 (malignant melanoma), U-937 (leukemia), and HaCaT cells (immortalized keratinocytes). Chemical analysis revealed that both UPRFs presented rhamnose as the major relative sugar constituent, followed by glucose in U. rigida and xylose in U. pseudorotundata. Both also presented glucuronic acid, galactose, ribose, and mannose as the remaining monosaccharides. Similar antioxidant activity was obtained, where we observed increased activity in response to increased polysaccharide concentrations. Both UPRFs presented moderate toxicity against HCT-116 cell lines and a selectivity index ≥ 3, suggesting a good potential for use in pharmaceutical products.

Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl β-methoxyacrylate group and a strobilurin analogue bearing modified β-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Doxycycline hydrochloride inhibits the progress of malignant rhabdoid tumor of kidney by targeting MMP17 and MMP1 through PI3K-Akt signaling pathway

ObjectiveTo identify therapeutic targets for malignant rhabdoid tumors of kidney (MRTK) and to investigate the effects and underlying mechanism of doxycycline hydrochloride on these tumors. MethodsGene expression and clinical data of MRTK were retrieved from the TARGET database. Differentially expressed genes (DEGs) and prognostic-related genes (PRGs) were selected through a combination of statistical analyses. The functional roles of MMP17 and MMP1 were elucidated through RNA overexpression and intervention experiments. Furthermore, in vitro and in vivo studies provided evidence for the inhibitory effect of doxycycline hydrochloride on MRTK. Additionally, transcriptome sequencing was employed to investigate the underlying molecular mechanisms. Results3507 DEGs and 690 PRGs in MRTK were identified. Among these, we focused on 41 highly expressed genes associated with poor prognosis and revealed their involvement in extracellular matrix regulatory pathways. Notably, MMP17 and MMP1 stood out as particularly influential genes. When these genes were knocked out, a significant inhibition of proliferation, invasion and migration was observed in G401 cells. Furthermore, our study explored the impact of the matrix metalloproteinase inhibitor, doxycycline hydrochloride, on the malignant progression of G401 both in vitro and in vivo. Combined with sequencing data, the results indicated that doxycycline hydrochloride effectively inhibited MRTK progression, due to its ability to suppress the expression of MMP17 and MMP1 through the PI3K-Akt signaling pathway. ConclusionDoxycycline hydrochloride inhibits the expression of MMP17 and MMP1 through the PI3K-Akt signaling pathway, thereby inhibiting the malignant progression of MRTK in vivo and in vitro.

IMP3 Immunohistochemical Expression Is Related with Progression and Metastases in Xenografted and Cutaneous Melanomas

Introduction: Insulin-like growth factor-II messenger RNA-binding protein-3 (IMP3) over-expression is a predictor of tumor recurrence and metastases in some types of human melanoma. Our objective was to evaluate the immunohistochemical expression of IMP3 and other molecules related to tumor prognosis in melanoma-xeno-tumors undergoing treatment. We test the effect of radiotherapy (RT) and mesenchymal stromal cells (MSCs) treatment, analyzing the tumorigenic and metastatsizing capacity in a mice melanoma xenograft model. Materials and Methods: We inoculated A375 and G361 human melanoma cell lines into NOD/SCID gamma mice (n = 64). We established a control group, a group treated with MSCs, a group treated with MSCs plus RT, and a group treated with RT. We assessed the immunohistochemical expression of IMP3, E-cadherin, N-cadherin, PARP1, HIF-1α, and the proliferation marker Ki-67. Additionally, we performed a retrospective study including 114 histological samples of patients diagnosed with malignant cutaneous superficial spreading melanoma (n = 104) and nodular melanoma (n = 10) with at least 5 years of follow-up. Results: Most morphological and immunohistochemical features show statistically significant differences between the 2 cell lines. The A375 cell line induced the formation of metastases, while the G361 cell line provoked tumor formation but not metastases. All three treatments reduced the cell proliferation evaluated by the Ki-67 nuclear antigen (p = 0.000, one-way ANOVA test) and reduced the number of metastases (p = 0.004, one-way ANOVA test). In addition, the tumor volumes reduced in comparison with the control groups, 31.74% for RT + MSCs in the A357 tumor cell line, and 89.84% RT + MSCs in the G361 tumor cell line. We also found that IMP3 expression is associated with greater tumor aggressiveness and was significantly correlated with cell proliferation (measured by the expression of Ki-67), the number of metastases, and reduced expression of adhesion molecules. Conclusions: The combined treatment of RT and MSCs on xenografted melanomas reduces tumor size, metastases frequency, and the epithelial to mesenchymal transition/PARP1 metastatic phenotype. This treatment also reduces the expression of molecules related to cellular proliferation (Ki-67), molecules that facilitate the metastatic process (E-cadherin), and molecules related with prognosis (IMP3).

Cytotoxic activity of TRPV4 antagonist RN-1734 in G-361 human melanoma cancer cell line

Purpose: Intracellular calcium (Ca2+) signaling plays a role in many cellular events, such as cell proliferation and differentiation, gene transcription, oxidative stress, the antioxidant system, and apoptosis. Transient receptor potential vanilloid 4 (TRPV4) channels are non-selective cation (Ca2+) channels. The present study aims to investigate the cytotoxic activity of RN-1734, a transient receptor potential vanilloid 4 (TRPV4) antagonist, in the G361 human melanoma cancer cell line. 
 Materials and Methods: The effects of RN-1734 on G361 cell viability at concentrations of 1, 5, 25, 50, and 100 μM were measured using the 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) method. Total antioxidant status (TAS) and total oxidant status (TOS) levels were determined using a ready-made commercial kit, after which oxidative stress index (OSI) values were calculated. To determine the apoptotic effects of RN-1734, Bcl-2, Bax, and p53 expression levels, caspase-3 and -8 activities were examined via quantitative real-time PCR analysis. 
 Results: G361 cell viability significantly decreased to 82.72, 72.81, 56.36, 39.16 and 18.96% in RN-1734 groups (1, 5, 25, 50 and 100 μM) compared to the control group (100.00%). At IC50 concentration (39.48 μM), RN-1734 application (3.35 mmol/g prot.-TAS, 45.87 μmol/g prot.-TOS, and 1501.97 AU-OSI) increased the TAS level (2.17 mmol/g prot.) and decreased the TOS level (55.41 μmol/g prot.) and OSI value (3142.76 AU) compared to the control group. 
 Conclusion: Our findings show that RN-1734 may be a novel therapeutic approach to treating melanoma by decreasing the cell viability of G361 human melanoma cancer cells.

Design and synthesis of novel 1,2,4-triazolo[4,3-b]pyridazine derivatives with anti-cancer activity

Novel 1,2,4-triazolo[4,3-b]pyridazine derivatives (7a–d, 8a–m and 9a–i) were designed and synthesized in good to moderate yields. The synthesized compounds were characterized by spectroscopic studies (NMR and IR) and the fluorine coupled 13C NMR data for each synthesized series have also been explained. The compounds were evaluated for their in-vitro anti-proliferative activity against K562, MV4-11, G361 and HCC827 human cancer cell lines. Almost all the synthesised compounds were active against MV4-11 below 25 µM, especially 8l which showed activity with IC50 of 1.5 µM. Furthermore, compound 8l displayed anticancer activity against all four cancer cell lines tested with IC50 values ranging from 1.5 to 7.6 µM. In general, most of the compounds did not show any significant anti-proliferative activity against K562, G361 and HCC827 cells. Preliminary investigation of their effect on proliferation and viability of MV4-11 cells was carried out with 8l and the results indicate that the cells undergo a cell death with biochemical signs of apoptosis.

A sequential recovery extraction and biological activity of water-soluble sulfated polysaccharides from the polar red macroalgae Sarcopeltis skottsbergii

Algal biomass is comprised of a variety of biochemical components that make it a viable raw material for use in a variety of applications using a biorefining process. From the residual supernatant generated by high-pressure homogenization for the extraction of the R-phycoerythrin pigment, two soluble polysaccharides (neutral and acid fractions) were obtained. The composition of total carbon (C), hydrogen (H), nitrogen (N), and sulfur (S), gas chromatography–mass spectrometry (GC–MS), Fourier transform infrared (FTIR-ATR) spectroscopy, antioxidant capacity (ABTS) and antiproliferative in vitro and in vivo activities were evaluated. The two polysaccharides, which had a relatively high sulfate content, exhibited significant antioxidant activity in superoxide radical assay (ABTS) and high anti-proliferative effects on G-361, U-937, HCT-116 cancer cells. High selectivity index (>3) was observed for the acid extract on G-361 and U-937 cell lines, but a significant inhibitory effect on the growth of 1064sK healthy fibroblast cells was measured too. On the other hand, neutral fraction polysaccharides, with the lowest sulfate content and relatively lower radical scavenging rates, showed scarce antitumor activity, a high selectivity index on G-361 melanoma cancer cells and non-toxicity on healthy cells. This differential behavior was verified by testing toxicity and lethality of both polysaccharide extracts in and in vivo zebrafish embryo acute toxicity test. Following this method, the LC50 of the neutral fraction resulted much higher (1.477 mg mL−1, p ≈ 0.0000) than that of the acid fraction (0.080 mg mL−1, p ≈ 0.0000) at 72 h post-fertilization. During this period, a slight growth delay and/or rare teratogenic effects were also induced by these fractions at concentrations lower than their LC50. Overall, this study indicates that the antitumor, antioxidant and toxicity effects of polysaccharides may be related to the combined effects of sulfate and galactose contents from the residual supernatant bioprocess. The nutraceutical potential of the acid fractions is thus finally discussed.

BKM120 inhibits malignant rhabdoid tumor of the kidney through induction of apoptosis and G0/G1 phase arrest

Malignant rhabdoid tumor of the kidney (MRTK) has an inferior prognosis and is insensitive to radiotherapy and chemotherapy. Search for novel, potent medicinal agents is urgent. Herein, data on the gene expression and clinical characteristics of malignant rhabdoid tumors (MRT) were retrieved from the TARGET database. Prognosis-related genes were identified by differential analysis and one-way cox regression analysis, and prognosis-related signalling pathways were identified by enrichment analysis. The prognosis-related genes were imported into the Connectivity Map database for query, and BKM120 was predicted and screened as a potential therapeutic agent for MRTK. A combination of high-throughput RNA sequencing and Western blot verified that the PI3K/Akt signaling pathway is associated with MRTK prognosis and is overactivated in MRTK. Our results outlined that BKM120 inhibited the proliferation, migration, and invasion ability of G401 cells and induced apoptosis and cell cycle G0/G1 phase arrest. In vivo, BKM120 inhibited tumor growth and had no significant toxic side effects. Western blot and immunofluorescence results confirmed that BKM120 could reduce the expression of PI3K and p-AKT, critical proteins of the PI3K/Akt signaling pathway. BKM120 inhibits MRTK by inhibiting PI3K/Akt signalling pathway to induce apoptosis and cell cycle G0/G1 phase arrest, which is anticipated to give the clinical treatment of MRTK a new direction.

Abstract LB017: Potential role of kinesin family member C1 (KIFC1) in melanoma

Abstract According to the Surveillance, Epidemiology, and End Results (SEER) analyses, based on 2017-2019 data, approximately ~2.1 percent of men and women will be diagnosed with melanoma at some point during their lifetime. Melanoma can be fatal if not surgically removed at an early stage, with the 5-year survival being a dismal 29.8% for a stage IV melanoma. Melanoma mortality is primarily due to the distant organ metastases and its resistance to treatment, even with newly developed immunotherapy and/or targeted therapies (BRAF and MEK inhibitors). Thus, the discovery of novel melanoma targets is crucial as they may be able to provide alternative treatment options or be used alone or in combination with current therapies. KIFC1 (kinesin family member C1; also known as HSET, an ortholog in Drosophila melanogaster), a nonessential kinesin motor protein, has an established role in centrosome clustering which is utilized by cancer cells to evade apoptosis and mitotic arrest. This occurs when replicating cells harbor additional centrosomes, a hallmark of cancer. The requirement of KIFC1 in mitosis of cancer cells harboring abnormal centrosomes, but not that of normal cells, makes it a potential drug target for cancer management. The goal of this study was to determine the role and significance of KIFC1 in melanoma. Differential expression analysis of GTEX and TCGA RNA-seq data identified significantly higher levels of KIFC1 in both primary and metastatic melanoma as compared to normal skin. In addition, higher expression of KIFC1 was found to be associated with poorer overall survival of melanoma patients. Furthermore, a quantitative protein estimation with the automated ProteinSimple Jess capillary western blotting system demonstrated higher KIFC1 expression in several melanoma cell lines when compared to normal melanocytes. In addition, we also found significantly increased expression of KIFC1 mRNA in mouse melanoma tumors obtained from the genetically engineered BrafV600E/PtenNULL mice, as compared to the normal skin. Next, we determined the effect of a small molecule KIFC1 inhibitor, AZ82 on A375, G361 and Hs294T melanoma cells. AZ82 is an ATP-competitive inhibitor that binds the KIFC1-microtubule complex, blocking the release of ADP and negating its role as a motor protein. Employing RealTime-Glo MT cell viability assay as well as the endpoint XTT growth assay, we found that AZ82 treatment resulted in a significant growth inhibition of human melanoma cells at 2-3.5 µM concentrations. Additionally, AZ82 treatments resulted in a significant decrease in clonogenic survival in all three human melanoma cell lines. Collectively, our data suggests a potential role of KIFC1 in melanoma progression, and merits further investigation to determine the roles and functional and therapeutic significance of KIFC1 in melanoma. Citation Format: Davis S. Mau, Mary A. Ndiaye, Gabriella R. Zaemisch, Chandra K. Singh, Jess Vera, Sean R. McIlwain, Nihal Ahmad. Potential role of kinesin family member C1 (KIFC1) in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB017.

Abstract 831: Selective targeting of pediatric rhabdoid tumor using G401-CLENs: Cytotoxicity studies

Abstract Background: Rhabdoid tumor is an aggressive soft tissue sarcoma that arises in the kidney, liver, peripheral nerves, and miscellaneous soft parts throughout the body. Rhabdoid tumors usually occur in infancy or during early childhood and the most common sites for metastasis are lungs, abdominal lymph nodes, liver, and brain. Current treatment options include surgery, radiation therapy, high-dose chemotherapy, and stem cell transplant but the overall survival rate is approximately 23%. Nano-sized liposomes are drug-delivery vehicles used to enhance the delivery of drugs to target cells. The composition, size, and overall charge of nanoliposomes determine targeting and influence the degree of drug uptake. The objective of this study was to develop G401-CLENs prepared from lipid extracts derived from rhabdoid tumor cells and conventional lipid components, and to evaluate the optimized drug loaded preparations of rhabdoid-CLENs in vitro. Methods: Rhabdoid tumor cell line, G401, was the chosen model for this study. G401 cells were cultured and expanded in vitro for cellular extraction purposes. The G401 cell membrane lipid extract (LE) was included in the preparation of G401-CLENs. Additional conventional lipid ingredients include DOPC, DOTAP, and DPPE-PEG5000. DPPE-rhodamine lipids were employed for fluorescence studies only. Chemo agents' doxorubicin and vinblastine were used in cell viability and cytotoxicity assays to evaluate formulation efficacy. Phase I of evaluation included physiochemical characterization and cellular uptake studies of preparations of CLENs, including 0 to 20 mol% of G401 LE content. Phase II of evaluation included the use of Hoechst33342/propidium iodide cell viability and cytotoxicity assays with varied amounts of doxorubicin and vinblastine loaded CLENs added to the G401 cells. The control cell line used in the study represents another solid tumor, SK-N-DZ (neuroblastoma). Results: The optimal composition for G401-CLENs was DOPC/DPPE-PEG5000/LE (93/2/5). The average size and zeta potential was 198 ± 16 nm (n=5) and -12 ± 1.2 mV (n=5), respectively. Cell selectivity studies revealed an increase in the uptake of the optimized G401-CLENs by G401 cells when compared to SK-N-DZ control cells. In general, the inclusion of DOTAP increased overall cellular uptake, but diminished selective uptake of G401-CLENs. Results support a significant cellular uptake of preparations consisting of 5 mol% LE compared to 0, 10, and 20 mol% G401 LE content. Doxorubicin loaded in G401-CLENs decreased rhabdoid tumor cell viability in a dose-dependent manner compared to doxorubicin loaded in conventional liposome preparations. Additional cytotoxicity studies are currently underway. Conclusion: To this date, G401-CLENs represents a promising selective drug-targeting vehicle for the treatment of rhabdoid tumors. Citation Format: Varun Kudchadker, Dongxiao Chen, Yeji Seo, Robert Campbell. Selective targeting of pediatric rhabdoid tumor using G401-CLENs: Cytotoxicity studies [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 831.

Abstract 3089: XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies

Abstract Enhancer of zeste homolog 2 (EZH2) is the histone methyltransferase component of polycomb repressive complex 2 (PRC2) that can regulate gene expression by H3K27 trimethylation (H3K27Me3). EZH2 also mediates PRC2-dependent non-histone protein methylation or directly methylates non-histone proteins to regulate gene expression independent of PRC2. EZH2 plays an important role in regulation of cell cycle, autophagy, apoptosis and cell senescence. Mutated or overexpressed EZH2 associates with cancer initiation, progression, drug resistance and immunity regulation in cancer. XNW5004 is a rationally designed selective EZH2 inhibitor which potently inhibits both wild type and mutant EZH2. XNW5004 significantly suppresses level of H3K27Me3, arrests cell cycle in G1/S phase and promotes apoptosis in tumor. XNW5004 exhibits strong antitumor activity in EZH2-mutant lymphoma xenograft models, such as Karpas422 (Y641N), Pfeiffer (A677G) and SU-DHL-10 (Y641F). Since inhibition of EZH2 synthetically lethal in SWI/SNF subunits mutated cancers, XNW5004 exhibits dose-dependent efficacy in solid tumor models such as G401 (SMARCB1 deficient) and COV434 (SMARCA2/SMARCA4 deficient) cancer cell lines and xenografts. In prostate cancer cells, EZH2 can bind to the promoter of androgen receptor (AR) gene and activate AR expression. In PARPi-treated cancer cells, inhibition of PARP (poly-ADP-ribose polymerase) diminishes PARylation of EZH2 to result in reactivation of EZH2 and drug resistance to PARPi. In tumor microenvironment, inhibition of EZH2 promotes differentiation of T cells and NK cells, enhances antigen presentation ability of dendritic cells, and increases cytokine/chemokine secretion to recruit T cells into tumor tissue. Based on the above mechanisms, XNW5004 could potentially improve efficacy of ARi, PARPi and anti-PD-1. We have verified the synergistic effects of those combinations in multiple solid tumor xenograft models. In pharmacokinetic studies, XNW5004 has excellent oral bioavailability and exposure. XNW5004 shows good toxicological profile as well, has a wide therapeutic window in rats and dogs, and the risk of clinical safety was controllable. In summary, XNW5004 is a potent EZH2 inhibitor with promising efficacy, pharmacokinetic and safety properties. XNW5004 is expected to be a potent therapeutic candidate in cancer treatment and is now in Phase II clinical trial for both liquid and solid tumors. Citation Format: Yonghan Hu, Wei Peng, Zhe Zhang, Zhenwei Wu, Xi Chen, Yan Huang, Xin Li, Cungang Liu, Xiaojun Liu, Qifeng Shi, Xufang Wang, Wengui Wang, Haiyang Wei, Yuchuan Wu, Jun Xian, Linfeng Xu, Lina Zhang, Jinfeng Zhao, Meijie Le, Jing Qiang. XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies [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 3089.

Abstract 829: Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is a malignancy of the neuroendocrine system affecting infants. NB is highly resistant to drug therapy, which leads to ineffective treatments and increased refractory. Current treatments have low specificity to targeted NB cells resulting in unwanted side effects. A drug delivery system that can overcome these problems is urgently needed. Incorporating drugs in optimized lipid nanoparticles such as liposomes have proven to be an effective method for drug targeting. To achieve better cellular selectivity, lipid membrane extract (LE) is derived from NB cells using a modified Dyer and Blight lipid extraction procedure. The objective of the study was to determine an optimized cell membrane lipid-extracted nanoliposomes (CLENs) for the NB cell model while considering physicochemical properties (i.e. size and molecular charge) and composition of the preparation. Methods: The neuroblastoma cell line, SK-N-DZ, was used for drug selectivity study. Expanded culture of SK-N-DZ was employed for cellular extraction purposes. The method for lipid membrane extraction was previously reported by Alharbi and Campbell, (AAPS Open 4,5(2018)). SK-N-DZ lipid extract was the main ingredient to prepare SK-N-DZ CLENs. Phase I of the study is to prepare CLENs with various compositions of DOPC, cholesterol, DPPE-PEG5000 and SK-N-DZ lipid extract. DPPE-Rhodamine was used for fluorescence and cellular uptake studies. Phase II of the study evaluated the effect of SK-N-DZ lipid extraction inclusion on drug incorporation, cellular uptake and selectivity, and cytotoxicity. Invitrogen alamarBlue HS in vitro assay was used to determine cell viability for cytotoxicity studies. SK-N-SH (brain tissue), BT-20 (breast tissue) and G401 (kidney tissue) cell lines served as negative controls. Results: The results from the cellular uptake optimization studies revealed that the optimized composition and ratio for SK-N-DZ CLENs was DOPC/Cholesterol/LE/PEG (65/10/20/5). The average particle size and value for zeta potential was 164 nm +/- 1.2 and -7.92 +/- 1.55 mV, respectively. The inclusion of SK-N-DZ lipid membrane extract (2, 10, 20 and 40 mol%) in preparation of CLENs increased cellular uptake by SK-N-DZ cells compared to corresponding control (no LE included). No difference observed in cellular uptake of preparations containing 20 and 40 mol% of LE. The additional inclusion of cholesterol (10, 20, 40 mol%) increased overall cellular uptake of preparations compared to CLENs without the additional cholesterol content. Overall, the inclusion of PEG (5, 10, and 20 mol%) in SK-N-DZ CLENs increased their uptake by the target cell population. Conclusion: In conclusion, the inclusion of SK-N-DZ lipid membrane extract in the nanoliposome preparations increased their overall cellular uptake by SK-N-DZ target cells. Additional cellular selectively and cytotoxicity studies are currently underway. Citation Format: Kevin Bumanglag, Thi Tran, Robert Campbell, Yishan Li. Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells [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 829.

Determination of Selected Isoquinoline Alkaloids from Chelidonium majus, Mahonia aquifolium and Sanguinaria canadensis Extracts by Liquid Chromatography and Their In Vitro and In Vivo Cytotoxic Activity against Human Cancer Cells.

The search for new substances with cytotoxic activity against various cancer cells, especially cells that are very resistant to currently used chemotherapeutic agents, such as melanoma cells, is a very important scientific aspect. We investigated the cytotoxic effect of Chelidonium majus, Mahonia aquifolium and Sanguinaria canadensis extracts obtained from different parts of these plants collected at various vegetation stages on FaDu, SCC-25, MCF-7, and MDA-MB-231 cancer cells. Almost all the tested extracts showed higher cytotoxicity against these cancer cells than the anticancer drug etoposide. The highest cytotoxicity against the FaDu, SCC-25, MCF-7 and MDA-MB-231 cancer cell lines was obtained for the Sanguinaria candensis extract collected before flowering. The cytotoxicity of extracts obtained from different parts of Chelidonium majus collected at various vegetation stages was also evaluated on melanoma cells (A375, G361 and SK-MEL-3). The highest cytotoxic activity against melanoma A375 cells was observed for the Chelidonium majus root extract, with an IC50 of 12.65 μg/mL. The same extract was the most cytotoxic against SK-MEL-3 cells (IC50 = 1.93 μg/mL), while the highest cytotoxic activity against G361 cells was observed after exposure to the extract obtained from the herb of the plant. The cytotoxic activity of Chelidonium majus extracts against melanoma cells was compared with the cytotoxicity of the following anticancer drugs: etoposide, cisplatin and hydroxyurea. In most cases, the IC50 values obtained for the anticancer drugs were higher than those obtained for the Chelidonium majus extracts. The most cytotoxic extract obtained from the root of Chelidonium majus was selected for in vivo cytotoxic activity investigations using a Danio rerio larvae xenograft model. The model was applied for the first time in the in vivo investigations of the extract's anticancer potential. The application of Danio rerio larvae xenografts in cancer research is advantageous because of the transparency and ease of compound administration, the small size and the short duration and low cost of the experiments. The results obtained in the xenograft model confirmed the great effect of the investigated extract on the number of cancer cells in a living organism. Our investigations show that the investigated plant extracts exhibit very high cytotoxic activity and can be recommended for further experiments in order to additionally confirm their potential use in the treatment of various human cancers.

The Assessment of the Phototoxic Action of Chlortetracycline and Doxycycline as a Potential Treatment of Melanotic Melanoma-Biochemical and Molecular Studies on COLO 829 and G-361 Cell Lines.

Melanoma is still one of the most dangerous cancers. New methods of treatment are sought due to its high aggressiveness and the relatively low effectiveness of therapies. Tetracyclines are drugs exhibiting anticancer activity. Previous studies have also shown their activity against melanoma cells. The possibility of tetracycline accumulation in pigmented tissues and the increase in their toxicity under the influence of UVA radiation creates the possibility of developing a new anti-melanoma therapy. This study aimed to analyze the phototoxic effect of doxycycline and chlortetracycline on melanotic melanoma cells COLO 829 and G-361. The results indicated that tetracycline-induced phototoxicity significantly decreased the number of live cells by cell cycle arrest as well as a decrease in cell viability. The simultaneous exposure of cells to drugs and UVA caused the depolarization of mitochondria as well as inducing oxidative stress and apoptosis. It was found that the combined treatment activated initiator and effector caspases, caused DNA fragmentation and elevated p53 level. Finally, it was concluded that doxycycline demonstrated a stronger cytotoxic and phototoxic effect. UVA irradiation of melanoma cells treated with doxycycline and chlortetracycline allows for the reduction of therapeutic drug concentrations and increases the effectiveness of tested tetracyclines.

Upregulation of AKR1C3 in Sodium Butyrate Treated G361 Cell.

Upregulation of AKR1C3 in Sodium Butyrate Treated G361 Cell.