SRB Assay Research Articles

HPLC-PDA Method for Quantification of Bioactive Compounds in Crude Extract and Fractions of Aucklandia costus Falc. and Cytotoxicity Studies against Cancer Cells.

Aucklandia costus Falc. (Synonym: Saussurea costus (Falc.) Lipsch.) is a perennial herb of the family Asteraceae. The dried rhizome is an essential herb in the traditional systems of medicine in India, China and Tibet. The important pharmacological activities reported for Aucklandia costus are anticancer, hepatoprotective, antiulcer, antimicrobial, antiparasitic, antioxidant, anti-inflammatory and anti-fatigue activities. The objective of this study was the isolation and quantification of four marker compounds in the crude extract and different fractions of A. costus and the evaluation of the anticancer activity of the crude extract and its different fractions. The four marker compounds isolated from A. costus include dehydrocostus lactone, costunolide, syringin and 5-hydroxymethyl-2-furaldehyde. These four compounds were used as standard compounds for quantification. The chromatographic data showed good resolution and excellent linearity (r2 ˃ 0.993). The validation parameters, such as inter- and intraday precision (RSD < 1.96%) and analyte recovery (97.52-110.20%; RSD < 2.00%),revealed the high sensitivity and reliability of the developed HPLC method. The compounds dehydrocostus lactone and costunolide were concentrated in the hexane fraction (222.08 and 65.07 µg/mg, respectively) and chloroform fraction (99.02 and 30.21 µg/mg, respectively), while the n-butanol fraction is a rich source of syringin (37.91 µg/mg) and 5-hydroxymethyl-2-furaldehyde (7.94 µg/mg). Further, the SRB assay was performed for the evaluation of anticancer activity using lung, colon, breast and prostate cancer cell lines. The hexane and chloroform fractions show excellent IC50 values of 3.37 ± 0.14 and 7.527 ± 0.18 µg/mL, respectively, against the prostate cancer cell line (PC-3).

Rutin Inhibits Hepatic and Pancreatic Cancer Cell Proliferation by Inhibiting CYP3A4 and GST

Abstract: Background: Digestive cancer is among the major causes of mortality and morbidity worldwide. Rutin, a bioactive secondary metabolite belonging to flavonoids and distributed in many fruits and vegetables has shown anti-proliferative, anti-cancer, and neuroprotective activities. In this study, the antiproliferative, antioxidant and apoptotic activities of rutin on hepatic and pancreatic cancer cell lines were investigated. Materials and Methods: The effect on cellular viability was monitored by SRB assay. Increasing activity of caspases (3/7) was used as an indicator of apoptosis. Additionally, the anti-inflammatory and antioxidant activities of rutin were evaluated after measuring amount of prostaglandin E2 (PGE2) produced and through DPPH free radical scavenging assays, respectively. Moreover, the inhibitory effect on both CYP3A4 and GST enzymes has also been evaluated. Results: According to the data presented here, rutin has anti-proliferative effect and raises the number of caspases 3/7 in investigated cell lines. Conclusion: HepG-2 cells showed the highest cellular growth inhibition, followed by BxPC-3, Huh-7, MiaPaCa-2, Suit-2, and the normal cell line HPDE. Rutin also inhibited the cyctochrome P450 enzyme (CYP450 3A4) and glutatihione-S-transferase activity, with dose-dependent inhibition. Furthermore, suppression of PGE2 synthesis in BxPC-3 cells supported rutin's anti-inflammatory action (high COX-2 expression). Keywords: Rutin, Pancreatic and Liver cancer, Apoptosis and Anti-inflammatory, Antioxidant.

Targeting HE4 for treatment of endometrial cancer using a novel small molecule inhibitor.

e17616 Background: HE4 expression is upregulated in endometrial cancer and has been shown to have prognostic significance in endometrial cancer patients. High-throughput screening and lead optimization led us to discover UR-238, a novel small molecule inhibitor of HE4. The purpose of this study was to investigate the in vitro and in vivo effects of UR-238 in treatment of endometrial cancer. Methods: UR-238 was identified by focused medicinal chemistry. Six endometrial cancer cell lines (ECC1, AN3CA, HEC1A, MFE296, MFE280, and KLE) were grown in cell culture and treated with 8 concentrations of UR-238 (125 nM, 250 nM, 375 nM, 500 nM, 625 nM, 750 nM, 875 nM, and 1000 nM) or DMSO (control). Cell proliferation was assessed using SRB assays at 24 and 48 hours. Effects of UR-238 on the cell cycle were assessed by flow cytometry and related cell-cycle proteins were targeted via Western blots. An ELISA was performed to evaluate UR-238 treatment effect on HE4 expression. An AN3CA xenograft mouse model was used to assess in vivo effects of UR-238. Mice were treated with vehicle control or UR-238 (5 mg/kg, IP, 3 days/week) for 20 days (N=5/group). Tumor volume and weight were evaluated over time using a repeated measures ANOVA. Results: UR-238 resulted in decreased cell proliferation in a dose-dependent manner in all 6 endometrial cancer cell lines. UR-238 increased expression of p21 and p27 and induced M/G2 cell cycle arrest. UR-238 also downregulated HE4 expression. In vivo treatment with UR-238 of AN3CA expressing xenograft mice caused a significant reduction (p=0.02) in mean tumor volume compared with vehicle control after 20 days (control mean 1068.13 mm3, SE 239.76; UR-238 mean 265.62 mm3, SE 68.82). Conclusions: UR-238 inhibits cell proliferation in endometrial cancer cell lines and exhibits antitumorigenic effects in xenograft mouse models. Future studies are warranted as UR-238 may be a promising therapeutic agent for treatment of endometrial cancer.

In Vitro Antiproliferative Efficacy, Antioxidant Activity and LC MS Based Metabolite Profiling of Premna Barbata Stem Bark

Premna barbata Wall. ex Schauer is used traditionally as folkloric medicines for the treatment of differentpathological conditions. The first reported constituent from the bark of the plant was an iridoid glycoside premnosidic acid having antioxidant activity. Other species of this genus i.e., Premna latifolia, Premna tomentosa has shown to have antioxidant and cytotoxic activities. Despite the ethnomedicinal uses, no scientific evidence in support of antitumor activity on the stem bark of Premna barbata is reported so far. Hence, the current work aims to assess anticancer potentiality of different extracts of P. barbata on various cancer cell lines. Different extracts i.e., Petroleum ether extract (PBPE), Chloroform extract (PBCE), Ethyl acetate extract (PBEE) and Alcoholic extract (PBAE) were prepared and on each extract in-vitro antiproliferative activity was performed by using SRB assay. The most potent extract i.e., PBEE was then evaluated for antioxidant activity. Qualitative phytochemical investigation of different extracts indicates the presence of proteins, carbohydrates, steroids/triterpenoids, alkaloids, glycosides, flavonoids, and phenolic constituents. Ethyl acetate extract of Premna barbata gives potent cytotoxic activity in all cancer cell lines but more selectively to human colon cancer cell line (COLO-205) with GI 50 44.6 μg/ml. The phenolic and flavonoid content in ethyl acetate extract was 3.43±0.09 mg GE/g and 4.28±0.18mg QE/g respectively. Nineteen compounds were observed in positive (+) ESI mode chromatograms when LC-MS analysis was carried out. The LC-MS analysis by positive ionization mode, the predicted compounds such as Geniposidic acid (synonym: Premnosidic acid) and Rutin were detected. The cytotoxicity observed on cancerous cell lines in our study may be due to the presence of observed compounds. So, it can be concluded that Premna barbata stembark has remarkable cytotoxic activity against different tumor cell lines but the effect is more on colon cell lines as compared to others.

Isoryanodane diterpene derivatives from Barringtonia macrocarpa

From the MeOH residue of Barringtonia macrocarpa branches and leaves, one new isoryanodane diterpene, barringisol (1), and two new isoryanodane diterpene glucosides, barringisosides A and B (2 and 3), were obtained using various chromatographic isolations. The structural characterization was confirmed by spectroscopic methods including 1D, 2D NMR and HR-ESI-QTOF-MS. This is the first isolation of isoryanodane diterpene derivatives from Barringtonia species. Moreover, the in vitro cytotoxicity of 1−3 on three human cancer cell lines (HepG2, LNCaP and MCF7) was also accessed using SRB assays.

Recovery and separation of glycyrrhizic acid from Natural Deep Eutectic Solvent (NADES) extract by macroporous resin: adsorption kinetics and isotherm studies

Natural Deep Eutectic Solvents (NADESs) have emerged as a green and sustainable alternative to conventional organic solvents to extract bioactive compounds. However, the recovery of bioactive compounds from the NADES extracts is challenging, restricting their large-scale applications. The present work investigated the recovery of glycyrrhizic acid (GA) from choline-chloride/lactic acid NADES extract using macroporous resins. GA possesses a wide spectrum of biological activities, and it is extracted from the well-known herb Glycyrrhiza glabra. During resin screening, DIAIONTM SP700 showed high adsorption and desorption capacities. The adsorption kinetics study demonstrated that the adsorption of GA on SP700 followed Pseudo First-order kinetic model. Moreover, the adsorption behaviors were elucidated by the Freundlich isotherm using a correlation coefficient based on a static adsorption study at different temperatures and pH. Furthermore, the thermodynamic parameters, for instance, the change of Gibbs free energy (ΔG*), entropy (ΔS*), and enthalpy (ΔH*), showed that the adsorption process was spontaneous, favorable and exothermic. In addition, the sample after macroporous resin treatment, which is enriched with GA exhibited good anticancer potential analyzed by SRB assay. The regenerated NADES solvent was recycled twice, keeping more than 90% extraction efficiency, indicating good reusability of NADES in the GA extraction process by using macroporous resin.

Biological Evaluation of Graphene Quantum Dots and Nitrogen-Doped Graphene Quantum Dots as Neurotrophic Agents.

Over time, developments in nano-biomedical research have led to the creation of a number of systems to cure serious illnesses. Tandem use of nano-theragnostics such as diagnostic and therapeutic approaches tailored to the individual disease treatment is crucial for further development in the field of biomedical advancements. Graphene has garnered attention in the recent times as a potential nanomaterial for tissue engineering and regenerative medicines owing to its biocompatibility among the several other unique properties it possesses. The zero-dimensional graphene quantum dots (GQDs) and their nitrogen-doped variant, nitrogen-doped GQDs (N-GQDs), have good biocompatibility, and optical and physicochemical properties. GQDs have been extensively researched owing to several factors such as their size, surface charge, and interactions with other molecules found in biological media. This work briefly elucidates the potential of electroactive GQDs as well as N-GQDs as neurotrophic agents. In vitro investigations employing the N2A cell line were used to evaluate the effectiveness of GQDs and N-GQDs as neurotrophic agents, wherein basic investigations such as SRB assay and neurite outgrowth assay were performed. The results inferred from immunohistochemistry followed by confocal imaging studies as well as quantitative real-time PCR (qPCR) studies corroborated those obtained from neurite outgrowth assay. We have also conducted a preliminary investigation of the pattern of gene expression for neurotrophic and gliotrophic growth factors using ex vivo neuronal and mixed glial cultures taken from the brains of postnatal day 2 mice pups. Overall, the studies indicated that GQDs and N-GQDs hold prospect as a framework for further development of neuroactive compounds for relevant central nervous system (CNS) purposes.

Phytochemical profile, antioxidant, cytotoxic and anti-inflammatory activities of stem bark extract and fractions of Ailanthus excelsa Roxb.: In vitro, in vivo and in silico approaches

This study aimed to assess the phytochemical composition, in vitro antioxidant, cytotoxicity, and in vivo anti-inflammatory activities of the methanolic extract of Ailanthus excelsa (Simaroubaceae) stem bark and its fractions. Quantitative phytochemical analysis revealed that methanolic extract and all fractions contained a high level of flavonoids (20.40–22.91 mg/g QE), phenolics (1.72–7.41 mg/g GAE), saponins (33.28–51.87 mg/g DE), and alkaloids (0.21–0.33 mg/g AE). The antioxidant potential was evaluated in vitro using a range of assays, i.e., DPPH•, ABTS radical scavenging ability, and total antioxidant capacity. The chloroform and ethyl acetate fractions showed stronger antioxidant activity than the methanol extract. In vitro cytotoxic activity was investigated in three human tumor cell lines (A-549, MCF7 and HepG2) using the SRB assay. In addition, the in vivo anti-inflammatory effect was assessed by carrageenan-induced paw edema in rats. The chloroform fraction showed a more pronounced effect by effectively controlling the growth with the lowest GI50 and TGI concentrations. The human lung cancer cell line (A-549) was found to be more sensitive to the chloroform fraction. Furthermore, the chloroform fraction exhibited significant anti-inflammatory activity at a dose of 200 mg/kg in the latter phase of inflammation. Besides, methanol extract and ethyl acetate fraction revealed a significant cytotoxic and anti-inflammatory effects. The chloroform fraction of stem bark showed a strong anti-inflammatory effect in experimental animals and significant COX-2 inhibitory potential in the in vitro experiments. GC-MS analysis of chloroform fraction identified the phytochemicals like caftaric acid, 3,4-dihydroxy phenylacetic acid, arachidonic acid, cinnamic acid, 3-hydroxyphenylvaleric acid, caffeic acid, hexadeconoic acid, and oleanolic acid. The in-silico results suggest that identified compounds have better affinity towards the selected targets, viz. the BAX protein (PDB ID: 1F16), p53-binding protein Mdm-2 (PDB ID: 1YCR), and topoisomerase II (PDB ID: 1QZR). Amongst all, caftaric acid exhibited the best binding affinity for all three targets. Thus, it can be concluded that caftaric acid in combination with other phenolic compounds, might be responsible for the studied activity. Additional in vivo and in vitro studies are required to establish their exact molecular mechanisms and consider them as lead molecules in developing of valuable drugs for treating oxidative stress-induced disorders, cancers, and inflammations.

Abstract LB201: A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer

Abstract Background: Colorectal cancer (CRC) has been a difficult-to-treat cancer with a significant proportion of microsatellite stable (MSS-CRC) patients who do not respond to immunotherapy. Currently, various strategies are being investigated to sensitize MSS-CRC tumors by converting them to immunologically “hot” tumors and improve responsiveness to immunotherapy. In this study, we demonstrate a novel strategy of combining a repurposed FDA-approved compound, Pimavanserin tartrate (PVT), with a Listeria monocytogenes (Lm)-based vaccine targeting Interferon-Stimulated Gene 15(ISG15), Lm-LLO-ISG15, against MSS-CRC tumors. Our findings demonstrate that both agents synergize to produce CD8+ T cell-dependent therapeutic efficacy in immunotherapy-resistant MSS-CRC tumors. Methods: The direct killing effect of PVT in vitro was confirmed by cytotoxic SRB assay. BALB/c mice bearing CT26, a murine model of MSS-CRC, were randomly assigned to receive either PBS, PVT, Lm-LLO-ISG15, or PVT+Lm-LLO-ISG15. Mice were monitored for tumor growth kinetics to study the anti-cancer effect of each monotherapy and the combination therapy. Changes in immune-related gene and protein expression were analyzed by qPCR and Western blot, respectively. Various immune cell subsets in the tumors and spleens were evaluated by multi-color flow cytometry and immunohistochemistry. In addition, organs such as the liver, kidney, heart, lungs, spleen, brain, and pancreas were collected for safety analysis. Results: PVT efficiently induced apoptosis and directly killed both human and mouse MSS-CRC cell lines in vitro. Further, PVT treatment enhanced T-cell activation and induced ISG15 expression, thus, sensitizing the tumor cells to targeting by the CTL-mediated immunotherapy, Lm-LLO-ISG15. While each monotherapy failed to demonstrate anti-tumor efficacy, treatment with the combination therapy (PVT + Lm-LLO-ISG15) significantly controlled tumor burden and extended the median survival. The therapeutic efficacy of the combination approach was associated with a higher infiltration of CD8+ T cells in the TME and an increased population of effector memory T cells (CD4+CD44hiCD62Llo) in the spleen. The anti-tumor efficacy of combination therapy on subcutaneous CT26 tumor growth was completely abrogated after the depletion of CD8+ T cells. Moreover, the safety profile of dual therapy was comparable to that of each monotherapy and PBS. Conclusions: MSS-CRCs are considered “immunologically cold” tumors that represent great challenges for any standalone therapies. In this study, we evaluated, for the first time, the therapeutic efficacy of a novel combination of PVT and Lm-LLO-ISG15 in an aggressive MSS-CRC mouse model. Combination therapy demonstrated a synergistic effect that led to a significant reduction of tumor burden and an extension of median survival. While the underlying mechanism for synergism requires further examination, our findings strongly suggest that combining PVT and Lm-LLO-ISG15 could be a promising approach for MSS-CRC patients. Citation Format: Shreyas Ramchandra Gaikwad, Hong My Nguyen, Sanjay K. Srivastava, Laurence Wood. A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer [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 LB201.

Evaluation of cashew gum-polyvinyl alcohol (CG-PVA) electrospun nanofiber mat for scarless wound healing in a murine model

Modern-day treatment for burns and wounds demands scarless healing which is becoming a challenging clinical problem. Thus, to alleviate such issues, it becomes essential to develop biocompatible and biodegradable wound dressing material for skin tissue regeneration, which could heal the wound in a very short span leaving no scars. The present study focuses on the development of nanofiber of Cashew gum polysaccharide-Polyvinyl alcohol using electrospinning. The prepared nanofiber was optimized based on uniformity of fiber diameter (FESEM), mechanical property (Tensile Strength), and optical contact angle (OCA) and was subjected to evaluation of: antimicrobial activity against Streptococcus aureus and Escherichia coli, hemocompatibility, and in-vitro biodegradability. The nanofiber was also characterized using different analytical techniques including thermogravimetric analysis, Fourier-transform infrared spectroscopy, and X-ray diffraction. The cytotoxicity was also investigated on L929 fibroblast cells using an SRB assay. The in-vivo wound healing assay showed accelerated healing in comparison to untreated wounds. The in-vivo wound healing assay and histopathological slides of regenerated tissue confirmed that the nanofiber has the potential to accelerate healing properties.

Artemisia sieberi Besser essential oil inhibits the growth and migration of breast cancer cells via induction of S-phase arrest, caspase-independent cell death and downregulation of ERK

Ethnopharmacological relevanceArtemisia sieberi Besser is a medicinal herb that has been traditionally used across the Middle East for the treatment of cancer. Further pharmacological studies on its extracts revealed that they possess cytotoxic activity against certain cancer cells, however, there were no studies conducted on the anticancer potential of Artemisia sieberi essential oil (ASEO). Aim of the studyTo evaluate the anticancer potential of ASEO, elucidate the oil's mode of action for the first time and investigate its chemical composition. Materials and methodsArtemisia sieberi was collected from Hail, Saudi Arabia, and its essential oil was obtained via hydrodistillation. The oil's activity against HCT116, HepG2, A549 and MCF-7 cells was assessed using SRB assay, while its anti-metastatic potential was assessed via a migration assay. Cell-cycle analysis and apoptosis assay were conducted via flow cytometry, while protein expression levels were investigated using Western blotting. The oil's chemical constituents were identified using GCMS. ResultsASEO exerted its highest cytotoxic activity against MCF-7 with an IC50 value of 38.7 μg/ml. Further studies showed that the oil inhibited MCF-7 cells' migration, induced S-phase arrest and apoptosis. Western blot analysis showed no change in the expression level of caspase-3 after treatment, indicating the induction of caspase-independent apoptosis-like cell death in MCF-7. Treatment of MCF-7 with the oil resulted in downregulation of the protein expression levels of total ERK and its downstream target, LC3, indicating that any potential activation of the ERK signalling pathway during the cancer cells' growth would be inhibited. Finally, GCMS analysis identified the oil's major components as cis-crysanthenyl acetate (48.56%), davanone (10.28%), 1,8-cineole (6.81%) and caryophyllene diepoxide (5.34%), whereby it is suggested that these compounds might be responsible for the oil's bioactivity. ConclusionASEO possessed in vitro anticancer activity and modulated the ERK signalling pathway. This is the first study to explore the anticancer potential of ASEO in detail and reflects the significance of investigating essential oils from medicinal plants that have been traditionally used against cancer. This work might pave the way for further in vivo studies that could result in developing the oil into a natural effective anticancer treatment.

Abstract 3104: Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1

Abstract Introduction. Activating the immune system against cancer is becoming an increasingly effective therapy option that can result in dramatic and durable responses in several cancer types. One approach to achieve the reactivation of endogenous antitumor T cells is by blocking PD-1/PD-L1 immune checkpoints expressed on T cells and other leukocytes. However, only limited cancer patients (15-25%) respond to anti-PD-1/PD-L1 immunotherapy. One of the most pressing current clinical challenges is to convert nonresponsive, “cold” tumors to responsive, “hot” tumors. Interestingly, after DNA-damaging chemotherapy, the immune environment may be changed from “cold” tumors to “hot” tumors by increasing the tumor mutation burden and the generation of neoantigens on the surface of cancer cells. Therefore, we surmised that a molecule capable of inducing promutagenic DNA and block PD-1/PD-L1 could not only induce neoantigens but also synergistically enhance immune response against the targeted tumor. Using an approach developed in our laboratory termed the “combi-targeting” strategy, we designed and synthesized a series of “combi-molecules” programmed to generate the promutagenic species and a small molecule capable of blocking PD-1/PD-L1. Material and methods. Melanoma cell line B16-F10 was used to determine IC50 of the new molecules with SRB assay. Homogenous time-resolved fluorescence (HTRF) binding assay was used to determine the IC50 inhibition of PD-1/PD-L1. Drug metabolism in extracted cells was measured by LC-MS. Results. We discovered a structure activity relationship of the combi-molecules based on the substitution of the side chain of the alkylating agent and the PD-1/PD-L1 scaffold. By altering the scaffolds of the combi-molecules from sulfonamides to biphenyl derivatives, we optimized binding to PD-1/PD-L1 from millimolar to micromolar levels. In vitro growth inhibitory analysis showed that the combi-molecules with biphenyl scaffold were 24-fold more potent in B16-F10. Importantly, analysis of intracellular metabolites of the combi-molecules revealed three main metabolites that can only result from the release of the shortlived promutagenic species. Conclusions. The biphenyl scaffold is optimal for maintaining strong PD-1/PD-L1 binding potency and enough potent to contribute to cell death. Hydrolytic cleavage of the alkylating agent is an indirect evidence of the formation of the alkylating species required to induce promutagenic lesions. Citation Format: Ana Belen Fraga Timiraos, Caterina Facchin, Nadia Babaa, Anne-Laure Larroque-Lombard, Bertrand Jean-Claude. Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1 [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 3104.

Abstract 2669: RRM2 inhibition synergizes with PARP inhibitor in ovarian cancer cells

Abstract The use of Poly(ADP-ribose) polymerase inhibitors (PARPi) has been applied for the treatment of epithelial ovarian cancer (EOC) patients with homologous recombination (HR)-deficient disease, such as BRCA1/2 mutations. However, 40 - 70% of patients do not initially respond to PARPi in ovarian cancer with non-BRCA mutations, and acquired resistance has been demonstrated clinically. Ribonucleotide reductase (RNR, which consists of two RRM1 subunits and two RRM2 subunits) is the rate-limiting enzyme in the biosynthesis of dNTPs for DNA replication and DNA damage repair. As targeting RRM2 has been applied for many cancer therapies clinically or in clinical trials, this study hypothesizes that the inhibitory of RRM2 arguments PARPi potency against ovarian cancer. To this goal, this study evaluates the therapeutic effects of RRM2 inhibitor Osalmid (a resveratrol analog) alone or combined with PARPi by employing a panel of ovarian cancer cell lines using SRB assay. The effects on apoptosis and cell-cycle arrest were analyzed using flow cytometry. The cellular thermal shift assay was conducted to explore if Osalmid interacts with RRM2. DNA double strand break marker, ϒH2AX, was detected in compound-treated cells using Western blot. The results provide a new synthetic lethality by combining RRM2 inhibitor and PARPi. The combination induced apoptosis and caused the cell-cycle arrest. The results show that Osalmid binds to RRM2 directly. This study provides both a novel RRM2 inhibitor from a natural compound and a new understanding of the dNTP metabolic demands and may lead to urgently-needed strategies for the treatment of ovarian cancer. Citation Format: Chi-Wei Chen. RRM2 inhibition synergizes with PARP inhibitor in ovarian cancer 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 2669.

Abstract 1647: In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice

Abstract Introduction. Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumours with an average survival rate of 12-18 months. The existing treatment (chemotherapy with temozolomide (TMZ)+radiotherapy) increases the median survival by 2-3 months. One protein, the epidermal growth factor receptor (EGFR) is expressed in 57.4% of GBM and half of them expresses its aggressive mutant, EGFRvIII, whose expression is associated with resistance to apoptosis, activation of DNA repair and ultimately resistance to chemotherapy. In the context of a strategy termed “combi-targeting”, we developed molecules designed to block EGFRvIII while being capable of inducing DNA damage. In vivo analysis suggests that the first prototype of such a class ZR2002 loses its DNA damaging function through metabolic oxidation. Recently, we surmised that a molecule designed to be metabolically stable may require a lower dose to be active in vivo. To test this hypothesis, we measured the cytotoxicity of the new combi-molecule, called "AF143", in a panel of glioblastoma cell lines and then we determined its efficacy and drug metabolism in an in vivo model of glioblastoma derived from human glioblastoma stem cells (GSC) with known resistance to TMZ. Material and Methods. Three glioblastoma cells line (U87-wt, U87-EGFR and U87-EGFRvIII) and the human GSC (1123IC7R) were used to determine IC50 of AF143 and ZR2002 with SRB or alamarBlue assay. Xenograft of tumors were obtained by intracranial injection of 1123IC7R-Luc GSC into the head of NSG female mice. One group of mice was treated with ZR2002 (N=8), the other with AF143 (N=7), the last with vehicle (N=8). Mice received 10 total doses: 2 cycles of 1 dose (100mg/kg) daily for 5 consecutive days followed by 2 days off, by oral gavage. The tumour burden was measured by bioluminescent imaging and drug metabolism by LC-MS. Body weights, mortality and toxicity were monitored. Survival curves and statistical analysis calculated. Results. In vitro growth inhibitory analysis showed that AF143 was 2-fold more potent than ZR2002 in U87-EGFR and 1123IC7R GSC and 3-fold more potent in U87-EGFRvIII. In vivo analysis of AF143 given alone showed significant antitumor activity (p&amp;lt;0.05) after 5 doses treatment, while ZR2002 induced an apparent tumour delay (p&amp;gt;0.05). Remarkably, AF143 and ZR2002 treatment significantly prolonged the overall survival of mice compared with the control group (p= 0.0004 for AF143 and p=0.04 for ZR2002). Analysis of drug metabolism revealed that in contrast to ZR2002, AF143 remained intact after 2h treatment, with moderate levels of N-acetyl and N6-dealkylated metabolites. Conclusion. The results in toto suggest that the intact structure of AF143 is stable to metabolism, can cross the blood brain barrier and induce antitumour activity in a EGFRvIII expressing glioblastoma stem cell model. Citation Format: Caterina Facchin, Ana Belen Fraga Timiraos, Brian Meehan, Nadia Babaa, Lata Adnani, Anne-Laure Larroque, Janusz Rak, Bertrand J Jean-Claude. In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice [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 1647.

Effect of Protein Repellent Addition to Self-Healing Experimental Resin Composite on Microcosm Biofilm and Cytotoxicity

Purpose: To investigate the effect of addition of MPC protein repellent agent to selfhealingexperimental resin composite on the self-healing efficacy, microcosm biofilmand the cytoxicity. Material and Methods: Polyurea formaldehyde microcapsuleswere synthesized by Insitu Polymerization Technique. MPC and the microcapsules areadded to the prepared experimental resin composite with different MPC concentrations.Protein repellence essay was examined by the Micro bicinchoninic Acid method.Biofilm models formed of streptococcus mutans growth was prepared and visualized byconfocal laser electron microscope to investigate the live \ dead assay over the studiedspecimens. The cytotoxicity of the specimens was examined via SRB assay. Fracturetoughness was examined using universal testing machine till fracture. Self-healingefficacy is evaluated. Results: In protein deposition, it was found that the 0%MPChas the highest value while the least value was found in the 10% group. In live/dead bacterial assay, it was found that the 0%MPC has the highest living bacterialvalue and the lowest dead bacterial value while the 7.5% group has the lowest livingbacterial value and the highest dead bacterial value. In the mechanical evaluation,the virgin fracture toughness, the highest facture toughness was found in the controlspecimens while the least fracture toughness was found in 10% group, the selfhealingefficacy results showed that the highest self-healing value was found in the0%MPC while the least self-healing efficacy value was found in the control group.Conclusion: Innovative self-healing composite with protein repellence give rise fora great variety of dental biomaterials with more promising results and long clinicalserviceability

Effect of Protein Repellent Addition to Self-Healing Experimental Resin Composite on Microcosm Biofilm and Cytotoxicity

Purpose: To investigate the effect of addition of MPC protein repellent agent to selfhealingexperimental resin composite on the self-healing efficacy, microcosm biofilmand the cytoxicity. Material and Methods: Polyurea formaldehyde microcapsuleswere synthesized by Insitu Polymerization Technique. MPC and the microcapsules areadded to the prepared experimental resin composite with different MPC concentrations.Protein repellence essay was examined by the Micro bicinchoninic Acid method.Biofilm models formed of streptococcus mutans growth was prepared and visualized byconfocal laser electron microscope to investigate the live \ dead assay over the studiedspecimens. The cytotoxicity of the specimens was examined via SRB assay. Fracturetoughness was examined using universal testing machine till fracture. Self-healingefficacy is evaluated. Results: In protein deposition, it was found that the 0%MPChas the highest value while the least value was found in the 10% group. In live/dead bacterial assay, it was found that the 0%MPC has the highest living bacterialvalue and the lowest dead bacterial value while the 7.5% group has the lowest livingbacterial value and the highest dead bacterial value. In the mechanical evaluation,the virgin fracture toughness, the highest facture toughness was found in the controlspecimens while the least fracture toughness was found in 10% group, the selfhealingefficacy results showed that the highest self-healing value was found in the0%MPC while the least self-healing efficacy value was found in the control group.Conclusion: Innovative self-healing composite with protein repellence give rise fora great variety of dental biomaterials with more promising results and long clinicalserviceability

Anticancer Activity of Methanol extract of Limnophila repens and Argyeia cymosa by Using SRB Assay

Introduction: Plants have a special place in the treatment of cancer. It is estimated that plant-derived compounds constitute more than 50% of anticancer agents. In this present study I attempted an experiment to find out the anti-cancer activity of selected plants Limnophila repens and Argyeia cymosa by using SRB Assay. Material method: I have collected the plants, dried very well and extracted with methanol crude methanol extract of the both plants tested for its anti-cancer activity. Results and disscussionThe anticancer activity of methanolic extracts of Argyreia cymosa and Limnophila repens was determined by sulforhodamine B colorimetric assay. The results of the cytotoxicity of extracts from both plant extracts analysed and The Methanol extract of Limnophila repens extract showed comparable activity to the standard compound, i.e., Adriamycin on Ishikawa and SCC-29B cell lines, respectively. This extract showed TGI, and GI50 was 38.9 and &lt;10 µg/ ml on Ishikawa cell lines and 58.12, &lt;10 and &lt;10 µg/ ml of LC50, TGI and GI50 activity on SCC-29B cell lines respectively. The Methanol extract of Argyeia cymosa showed GI50 was &gt;80 µg/ ml on Ishikawa cell lines and &lt;10 and &lt;10 µg/ ml of, TGI and GI50 activity on SCC-29B cell lines .Estimations based on GI50 values shows that MELR was more active against Ishikawa and SCC-29B cell lines than MEAC on Ishikawa (human endometrial adenocarcinoma) and SCC-29B (human oral cancer) cell lines.

Pyrogallol from Spirogyra neglecta Inhibits Proliferation and Promotes Apoptosis in Castration-Resistant Prostate Cancer Cells via Modulating Akt/GSK-3β/β-catenin Signaling Pathway.

Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer associated with poor survival rates. The high proliferation and metastasis rates have made CRPC one of the most challenging types of cancer for medical practitioners and researchers. In this study, the anti-cancer properties and inhibition of CRPC progression by S. neglecta extract and its active constituents were determined using two CRPC cell lines, DU145 and PC3. The ethyl acetate fraction of S. neglecta (SnEA) was obtained using a solvent-partitioned extraction technique. The active constituents of SnEA were then determined using the HPLC technique, which showed that SnEA mainly contained syringic acid, pyrogallol, and p-coumaric acid phenolic compounds. After the determination of cytotoxic properties using the SRB assay, it was found that pyrogallol, but not the other two major compounds of SnEA, displayed promising anti-cancer properties in both CRPC cell lines. SnEA and pyrogallol were then further investigated for their anti-proliferation and apoptotic induction properties using propidium iodide and Annexin V staining. The results showed that SnEA and pyrogallol inhibited both DU145 and PC3 cell proliferation by inducing cell cycle arrest in the G0/G1 phase and significantly decreased the expression of cell cycle regulator proteins (cyclin D1, cyclin E1, CDK-2, and CDK-4, p < 0.001). SnEA and pyrogallol treatments also promoted apoptosis in both types of CRPC cells through significantly downregulating anti-apoptotic proteins (survivin, Bcl-2, and Bcl-xl, p < 0.001) and upregulating apoptotic proteins (cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP-1, p < 0.001). Mechanistic study demonstrated that SnEA and pyrogallol inactivated the Akt signaling pathway leading to enhancement of the active form of GSK-3β in CRPC cell lines. Therefore, the phosphorylation of β-catenin was increased, which caused degradation of the protein, resulting in a downregulation of β-catenin (unphosphorylated form) transcriptional factor activity. The current results reflect the potential impact of S. neglecta extract and pyrogallol on the management of castration-resistant prostate cancer.

In-vitro Anticancer Activity of Various Plant Extracts

Herbal medicinal plants have been explored for a variety of pharmacological properties but still a large number of phytoconstituents are still unexplored. The manuscript aims to assess the anticancer property of Leucas cephalotes (Roth) Spreng, Acalypha indica L. and Lantana camara L. extracts on MDA-MB-231, A-549, PC-3 and Hep-G2 cell lines. The ethanol extract of the selected plants was explored for anticancer activity by SRB assay. Since L. camara exhibited promising activity, this plant’s successive extraction in diff erent extracts (n-hexane, chloroform, ethanol and hydroalcoholic) was further studied. All the extracts were screened for anticancer activity by SRB assay on MDA-MB-231 and A-549 cell lines. Additionally, the apoptotic assay was carried out on n-hexane and chloroform extracts on A-549 cell lines. GCMS also analyzed both extracts for characterization of the phytoconstituents. Obtained data indicate that n-Hexane and chloroform extracts of L. camara showed promising anticancer activity on MDA-MB-231 and A-549 cell lines. Both extracts also induced apoptosis in A-549 cell line. GC-MS data of L. camara extracts revealed the presence of 25 and 19 compounds in n-hexane and chloroform extract, respectively. The present study shows that L. camara has strong anticancer properties against lung cancer cell lines compared to breast, prostate, and hepatoma cancer cell lines

Bioactive hybrid membrane-based cellulose acetate/bioactive glass/hydroxyapatite/carbon nanotubes nanocomposite for dental applications

The present work aimed to fabricate a set of hybrid bioactive membrane in the form of bio-nanocomposite films for dental applications using the casting dissolution procedures. The formulation of the targeted materials was consisting of cellulose acetate/bioactive glass/hydroxyapatite/carbon nanotubes with a general abbreviation CA-HAP-BG-SWCNTs. The nanocomposites were characterized using XRD, FTIR, SEM-EDX and Raman spectroscopy. XRD, FTIR and SEM characters confirm the nanocomposites formation with good compatibility. The fabricated materials had a semi crystalline structure. The mechanical and thermal properties, as well as contact angle and bioactivity of the fabricated nanocomposites were investigated. The SEM images for showed beehive-like architectures with a thicker frame for the second material. All fabricated materials showed good thermal behaviors. Furthermore, the agar diffusion antimicrobial study showed that the prepared nanocomposites do not exhibit an antibacterial activity against five pathogenic bacterial strains. Additionally, cytotoxicity of a dental nanocomposite filling agent was evaluated. Vero normal cells were incubated with test materials for 72h at 37 °C and 5% CO2. Cell viability was detected using a SRB assay. All nanocomposites were mildly to non-cytotoxic to Vero cells at high concentration in contrast to the inhibitory effect of doxorubicin which was added at 10-fold lower concertation than the nanocomposites. Hence, the proposed nanocomposite is promising candidates for dental applications.