Reduced Cancer Cell Viability Research Articles

Abstract 3241: IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro

Abstract The 50% inhibitory concentration (IC50) is a widely used measure of how effective a given anti-cancer therapeutic is at reducing cancer cell viability in vitro. It is known that IC50 of a given therapeutic agent is dependent on the seeding density of the treated cells (in that more densely-seeded cells tend to be more resistant to therapeutic agents). We performed experiments using the CellTiter Glo cell viability assay to assess the cytotoxicity of various chemotherapeutic drugs towards various cancer cell lines. To elucidate the role the extracellular environment of densely-seeded cells plays in cancer cell chemoresistance, we tested conditioned media from cells cultured at high density on cells seeded at low densities. The resulting impact on the IC50 of the low-density cells was determined. The IC50 of the various therapeutic agents tested was cell density-dependent in each cancer cell line examined. Based on this analysis, we propose the IC50-seeding density slope (ISDS), which relates seeding density to the effectiveness of a drug and that we believe could be a standardized quantitative measurement of how a given cancer cell line responds to a given therapeutic treatment. Our results from the conditioned media experiments consistently indicated that the environment of densely-seeded tumor cells conferred chemoresistance to sparsely-seeded cells, suggesting that extracellular cytokines and growth factors may be involved in density-dependent chemoresistance mechanisms. We are currently exploring the effect of serum deprivation on in vitro chemoresistance, in order to further understand how the presence of extracellular nutrients impacts observed IC50 trends. In addition, we are examining the role that cell-cell contact, integrin and FGF signaling pathways play in giving rise to density-dependent chemoresistance. Moreover, to analyze the manner in which conditioned media gives rise to chemoresistance and how this effect relates to intracellular survival pathways, we are performing cytokine profiling experiments. Overall, we describe density-dependent IC50 variations as a hallmark of cancer cells and propose that ISDS may serve as a standardized method of assessing chemosensitivity. Citation Format: Ujwal Punyamurtula, Shengliang Zhang, Thomas Brown, Arielle De La Cruz, Alexander Raufi, Jillian Strandberg, Lindsey Carlsen, Lanlan Zhou, Wafik El-Deiry. IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3241.

Abstract 619: Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential

Abstract Re-activation of tumor-reactive T cells with so-called immune checkpoint inhibitors (ICIs) has translated into significant clinical breakthroughs. Specifically, antibodies directed against CTLA-4 and PD-L1/PD-1 have yielded long-term remission and cure in many solid as well as hematological malignancies. However, as not all cancer types respond to current ICI therapies, the quest for novel strategies to re-enable anti-tumor immunity by blocking immune checkpoints or by activating prominent co-stimulatory receptors is crucial. The field of cancer immunology has developed rapidly over the past decades, with numerous studies adding up to the complexity of immune-related interactions taking place in the tumor microenvironment. Compounds or combinations of compounds designed to target traditional or novel pathways require early evaluation of effectiveness on in vitro assays using primary immune cells or representative mouse models. On top of classical T cell bioassays, expanding on current methods and the development novel strategies to assess the modulation of cancer-related immunological networks is a continuously important process. One classical approach is the functional evaluation of compounds in a mixed lymphocyte reaction (MLR). Here, the potency of drug candidates to promote physiological T cell responses can be evaluated. Antigen-specific immune responses can be further assessed in Cytomegalovirus (CMV) recall- or in Staphylococcal enterotoxin B (SEB) activation assays. Additionally, it might also be beneficial to generate tumor-associated antigen-specific T cell pools or clones to be used in functional assays. The classical MLR assay could be expanded on by e.g., inclusion of specific tumor cell populations or T regulatory (Treg) cells, as evaluating reduced cancer cell viability, or reversing loss of immune effector cell functionality can be of interest. Furthermore, real-time evaluation of cancer cell killing by T cells upon addition of drug candidates could also provide pivotal information on the compound’s functional dynamics. With our deepening understanding of cancer-related immunological networks it becomes clear that different drug candidates may require customized and fine-tuned T cell bioassays to assess their full therapeutic potential. Moreover, quality controlled primary immune cells are an essential factor for the robustness of these assays and to yield reproducible and consistent results. Collectively, taking in mind these aspects will ease decision making and accelerate the drug discovery pipeline. Citation Format: Thibaut J. Janss, Martijn Vlaming, Simon Lefevre, Johan Arnold, Ellen Boelen, Sofie Pattijn. Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 619.

Fermented Soy Drink (Q-CAN® PLUS) Induces Apoptosis and Reduces Viability of Cancer Cells

This study tested the ability of a fermented soy product to induce tumor cell toxicity and to assess if this was due to fermentation of soy, and to the genistein content. Four cancer cell lines were cultured without additive, with fermented soy (Q-CAN® PLUS), nonfermented soy, or genistein, and cell viability was examined at 24 h, 48 h, and 72 h. The sensitivity of the cell lines to apoptosis by Q-CAN PLUS was tested with the Annexin V assay. All cell lines demonstrated a dose and time response reduction in tumor cell viability with exposure to Q-CAN PLUS (IC50 at 24 h 3.8 mg/mL to 9 mg/mL). Unfermented soy did not show reduction in viability of any cell line within the same concentration range. The IC50 of genistein for each of the cell lines was significantly greater than for Q-CAN PLUS. All four tumor cell lines demonstrated apoptosis in response to Q-CAN PLUS. Q-CAN PLUS reduces viability and increases apoptosis of cancer cells in a concentration- and fermentation-dependent manner. Taking into consideration the IC50 of genistein and the concentration of genistein in Q-CAN PLUS, the genistein content of Q-CAN PLUS is not responsible for the majority reduction in tumor cell viability. This suggests that fermentation of soy results in the production of metabolites that reduce cancer cell viability and induce cellular apoptosis, and play a major role in addition to any effects produced by their genistein content.

Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer

BackgroundLRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited.MethodsIn silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2’-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system.ResultsAcross breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2’-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer.ConclusionsOur study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Tetralol derivative NNC-55-0396 induces glioblastoma cell death by activating IRE1α, JNK1 and calcium signaling

Mibefradil and NNC-55–0396, tetralol derivatives with a proven –ability to block T-type calcium channels in excitable cells, reduce cancer cell viability in vitro, causing cell death. Furthermore, they reduce tumor growth in preclinical models of Glioblastoma multiforme (GBM), a brain tumor of poor prognosis. Here we found that GBM cells treated with cytotoxic concentrations of NNC-55–0396 paradoxically increased cytosolic calcium levels through the activation of inositol triphosphate receptors (IP3R) and ER stress. We used pharmacological inhibitors and gene silencing to dissect the cell death pathway stimulated by NNC-55-0396 in GBM cell lines and biopsy-derived cultures. Calcium chelation or IP3R inhibition prevented NNC-55–0396-mediated cytotoxicity, indicating that ER calcium efflux is the cause of cell death. Upstream of calcium mobilization, NNC-55–0396 activated the IRE1α arm of the Unfolded Protein Response (UPR) resulting in the nuclear translocation of pro-apoptotic CHOP. Consistent with these findings, silencing IRE1α or JNK1 rescued the cell death elicited by NNC-55–0396. Therefore, we demonstrate that activation of IRE1α and calcium signaling accounts for the cytotoxicity of NNC-55–0396 in GBM cells. The delineation of the signaling pathway that mediates the abrupt cell death triggered by this compound can help the development of new therapies for GBM.

Dehydrocrenatidine Induces Liver Cancer Cell Apoptosis by Suppressing JNK-Mediated Signaling

Liver cancer is a leading cause of death worldwide. Despite advancement in therapeutic interventions, liver cancer is associated with poor prognosis because of highly lethal characteristics and high recurrence rate. In the present study, the anticancer potential of a plant-based alkaloid namely dehydrocrenatidine has been evaluated in human liver cancer cells. The study findings revealed that dehydrocrenatidine reduced cancer cell viability by arresting cell cycle at G2/M phase and activating mitochondria-mediated and death receptor-mediated apoptotic pathways. Specifically, dehydrocrenatidine significantly increased the expression of extrinsic pathway components (FAS, DR5, FADD, and TRADD) as well as intrinsic pathway components (Bax and Bim L/S) in liver cancer cells. In addition, dehydrocrenatidine significantly increased the cleavage and activation of PARP and caspases 3, 8, and 9. The analysis of upstream signaling pathways revealed that dehydrocrenatidine induced caspase-mediated apoptosis by suppressing the phosphorylation of JNK1/2. Taken together, the study identifies dehydrocrenatidine as a potent anticancer agent that can be use clinically to inhibit the proliferation of human liver cancer cells.

Effects of H2O2 Treatment Combined With PI3K Inhibitor and MEK Inhibitor in AGS Cells: Oxidative Stress Outcomes in a Model of Gastric Cancer.

Gastric cancer is worldwide the fifth and third cancer for incidence and mortality, respectively. Stomach wall is daily exposed to oxidative stress and BER system has a key role in the defense from oxidation-induced DNA damage, whilst ErbB receptors have important roles in the pathogenesis of cancer. We used AGS cells as an aggressive gastric carcinoma cell model, treated with H2O2 alone or combined with ErbB signaling pathway inhibitors, to evaluate the effects of oxidative stress in gastric cancer, focusing on the modulation of ErbB signaling pathways and their eventual cross-talk with BER system. We showed that treatment with H2O2 combined with PI3K/AKT and MEK inhibitors influenced cell morphology and resulted in a reduction of cancer cell viability. Migration ability was reduced after H2O2 treatment alone or combined with MEK inhibitor and after PI3K/AKT inhibitor alone. Western blotting analysis showed that oxidative stress stimulated EGFR pathway favoring the MAPKs activation at the expense of PI3K/AKT pathway. Gene expression analysis by RT-qPCR showed ErbB2 and OGG1 increase under oxidative stress conditions. Therefore, we suggest that in AGS cells a pro-oxidant treatment can reduce gastric cancer cell growth and migration via a different modulation of PI3K and MAPKs pathways. Moreover, the observed ErbB2 and OGG1 induction is a cellular response to protect the cells from H2O2-induced cell death. In conclusion, to tailor specific combinations of therapies and to decide which strategy to use, administration of a chemotherapy that increases intracellular ROS to toxic levels, might not only be dependent on the tumor type, but also on the molecular targeting therapy used.

Cannabidiol and Other Phytocannabinoids as Cancer Therapeutics.

Preclinical models provided ample evidence that cannabinoids are cytotoxic against cancer cells. Among the best studied phytocannabinoids, cannabidiol (CBD) is most promising for the treatment of cancer as it lacks the psychotomimetic properties of delta-9-tetrahydrocannabinol (THC). In vitro studies and animal experiments point to a concentration- (dose-)dependent anticancer effect. The effectiveness of pure compounds versus extracts is the subject of an ongoing debate. Actual results demonstrate that CBD-rich hemp extracts must be distinguished from THC-rich cannabis preparations. Whereas pure CBD was superior to CBD-rich extracts in most in vitro experiments, the opposite was observed for pure THC and THC-rich extracts, although exceptions were noted. The cytotoxic effects of CBD, THC and extracts seem to depend not only on the nature of cannabinoids and the presence of other phytochemicals but also largely on the nature of cell lines and test conditions. Neither CBD nor THC are universally efficacious in reducing cancer cell viability. The combination of pure cannabinoids may have advantages over single agents, although the optimal ratio seems to depend on the nature of cancer cells; the existence of a ‘one size fits all’ ratio is very unlikely. As cannabinoids interfere with the endocannabinoid system (ECS), a better understanding of the circadian rhythmicity of the ECS, particularly endocannabinoids and receptors, as well as of the rhythmicity of biological processes related to the growth of cancer cells, could enhance the efficacy of a therapy with cannabinoids by optimization of the timing of the administration, as has already been reported for some of the canonical chemotherapeutics. Theoretically, a CBD dose administered at noon could increase the peak of anandamide and therefore the effects triggered by this agent. Despite the abundance of preclinical articles published over the last 2 decades, well-designed controlled clinical trials on CBD in cancer are still missing. The number of observations in cancer patients, paired with the anticancer activity repeatedly reported in preclinical in vitro and in vivo studies warrants serious scientific exploration moving forward.

Withalutin, a new cytotoxic withanolide from Athenaea velutina (Sendtn.) D’Arcy

Bioassay-guided fractionation of dichloromethane extract from Athenaea velutina leaves led to the isolation of three withanolides, all being reported for the first time in this species. They were identified as withacnistin (1), withacnistin acetate (2) and a new withanolide, designated as withalutin (3). The structures were established by spectral data analysis, including MS, 1D and 2D NMR. In addition, in silico ADMET studies were employed to understand the pharmacokinetic properties of these withanolides. The withanolides isolated from A. velutina reduced cancer cell viability with IC50 values ranging from 1.52 to 5.39 µM. In silico prediction revealed that withanolides have good gastrointestinal absorption or oral bioavailability properties; and are not likely to be mutagenic or hepatotoxic. These findings revealed that A. velutina is an important source of cytotoxic withanolides.

Efficacy of MDX-124, a novel anti-annexin-A1 antibody, in preclinical models of pancreatic cancer.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.

Closed‐Loop Temperature‐Controlled Magnetic Hyperthermia Therapy with Magnetic Guidance of Superparamagnetic Iron‐Oxide Nanoparticles

AbstractHyperthermia therapy eliminates cancer cells by heating them above physiological temperatures. Superparamagnetic iron oxide nanoparticles (SPIONs) are promising for targeted hyperthermia therapy because of their excellent heating efficiency, biocompatibility, and active magnetic navigation. When exposed to a high‐frequency alternating magnetic field (AMF), SPIONs dissipate heat to damage cancer cells. Accurate temperature control is crucial for safe and efficient hyperthermia therapy. A closed‐loop temperature controller to control the temperature and heating rate by adjusting AMF’s strength, thereby enabling controllable hyperthermia therapy. Under a low‐frequency rotating magnetic field (RMF), the SPIONs form chains which can be actively manipulated towards the target by adjusting RMF’s direction, followed by AMF exposure for hyperthermia therapy. The SPIONs are precisely manipulated in a microfluidic chip and rat brain vessels ex vivo, highlighting the potential for targeted position control. Last, in vitro, and in vivo hyperthermia treatments are performed on human prostate cancer cells (PC3) and a PC3 xenograft mouse model using the proposed temperature controller, with tracking errors under 0.5°C and significant reduction in cancer cell viability and tumor volume. The magnetic locomotion with RMF, and the controlled heating using AMF, show the feasibility of using SPIONs for targeted hyperthermia therapy.

Effect of S14161 Small Molecule and Glaucium Flavum Extract on A549 Cancer Cells

Background: Lung cancer is the fifth most common cancer in Iran. Due to the side effects of common cancer treatments, everyone has turned to herbal remedies and new treatments. This study aimed to compare the effect of S14161 small molecule and Glaucium flavum extract on the induction of apoptosis in A549 cancer cells. Materials and Methods: In this experimental study, the A549 cell line was treated with different concentrations of G. flavum and S14161 on days 1, 3, and 5. Also, half maximal inhibitory concentrations (IC 50) for both G. flavum and S14161 were measured. In addition, the quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to investigate the effects of S14161 and G. flavum extract on the expressions level of Bax, Bad, P53, and Bcl2 genes. Results: Results showed that both the combination of S14161 and G. flavum extract resulted in cell death and reduced cancer cell viability. Nevertheless, the viability rate was greater by S14161, and this small molecule significantly increased the expression of Bax, P53, and Bad apoptotic genes and decreased the expression of the Bcl2 gene, which shows the induced apoptotic death and lethal effect of S14161 in comparison with G. flavum extract. Conclusion: Our study showed that S14161 had fewer IC50 and caused cell death by inhibiting the PI3K/AKT pathway, and G. flavum caused cancer cell death due to its alkaloid compounds. Therefore, both compounds are recommended as drug candidates for the treatment of lung cancer.

Comparison of the Effect of S14161 Small Molecule and Glaucium Flavum Extract on A549 Cancer Cells

Background: Lung cancer is the fifth most common cancer in Iran. Due to the side effects of common cancer treatments, everyone has turned to herbal remedies and new treatments. This study aimed to compare the effect of S14161 small molecule and Glaucium flavum extract on the induction of apoptosis in A549 cancer cells. Materials and Methods: In this study, the A549 cell line was treated with different concentrations of S14161 small molecule and G. flavum extract compounds. MTT assay was performed to determine the half-maximal inhibitory concentration (IC50) and compare the viability of treated cells on days 1, 3, and 5. Also, the real-time polymerase chain reaction assay was used to investigate the expressions of pro- and anti-apoptotic genes. Results: MTT results showed that both the combination of S14161 and G. flavum extract resulted in cell death and reduced cancer cell viability. However, the viability rate was greater by S14161, and this small molecule significantly increased the expression of Bax, P53, and Bad apoptotic genes and decreased the expression of the Bcl2 gene, which shows the induced apoptotic effect of S14161 in comparison with G. flavum. Conclusion: The results of this study showed that S14161 had fewer IC50 and caused cell death by inhibiting the PI3K/AKT pathway, and Glaucium flavum caused cancer cell death due to its alkaloid compounds. Therefore, both compounds are recommended as drug candidates for the treatment of lung cancer.[GMJ.2021;10:e2151]

Assessing the antiumer and anti-inflammatory potentials of Calotropis procera latex

Since time immemorial medicinal plants have been used in healthcare to treat different human disease. The ideals of medicinal plants are highlighted due to the utility of the common-factor approach to engage other health promoters in propagating. One of these medicinal plants is Calotropis procera which is a species of tree in the family Apocynaceae. Calotropis procera is widespread in tropical Africa, including the Indian Ocean islands and the northern parts of South Africa. The latex is toxic and can cause rash, blisters and serious inflammations in sensitive persons and it may lead to blindness. Several side effects may be caused due to ingestion large doses of latex like burning in the throat, irritation of the stomach, nausea, vomiting, diarrhea, tremors, vertigo and convulsions. This study aimed to evaluate burns healing effects and inhibitory concentration IC50 of Calotropis procera latex on cervical cancer cell line, SiHa. Cytotoxicity analysis was performed by MTT assay in addition to determine the burns healing effect of the latex by determining the day requiring to heal the burn skin of albino male mice. The results of burns healing effects declared that the burns required 12 days to heal in comparison with positive (sliver sulfadiazine) and negative control which required 16 and 18 days for healing respectively. Also, the results revealed that the IC50 of latex was 146.8 % in comparison of ambiguous percentage of normal cell line WRL68 with reduction in cancer cell viability ranging from 95.87± 0.20to 52.35 ±3.31 for 6.2 to 400 µg\ml respectively.

Mass transfer of anti‐cancer drug delivery to brain tumors by a multiple emulsion‐based implant

AbstractThe advanced use of a pH‐responsive biomaterial‐based injectable liquid implant for effective chemotherapeutic delivery in glioblastoma multiforme (GBM) brain tumor treatment is presented. As an implant, we proposed a water‐in‐oil‐in‐water multiple emulsion with encapsulated doxorubicin. The effectiveness of the proposed therapy was evaluated by comparing the cancer cell viability achieved in classical therapy (chemotherapeutic solution). The experimental study included doxorubicin release rates and consumption for two emulsions differing in drop sizes and structures in the presence of GBM‐cells (LN229, U87 MG), and a cell viability. The results showed that the multiple emulsion implant was significantly more effective than classical therapy when considering the reduction in cancer cell viability: 85% for the emulsion‐implant, and only 43% for the classical therapy. A diffusion–reaction model was adapted to predict doxorubicin release kinetics and elimination by glioblastoma cells. CFD (computational fluid dynamics) simulations confirmed that the drug release kinetics depends on multiple emulsion structures and drop sizes.

Evaluation of Anticancer Effects and Molecular Mechanisms of Pomegranate Peel Extract in the Mouse Cellular Model (4T1) of Breast Cancer

Background: Breast cancer is the most common female cancer that, despite recent progress, its existing therapies do not have sufficient efficiency, and scientists are trying to find complementary therapies. Medicinal plants, such as pomegranate (Punica granatum L.), attract a great deal of attention in this regard. Objectives: The present study aimed to assess the anticancer effects of pomegranate peel alcoholic extract (PPE) in the mouse cellular model (4T1) of breast cancer and investigate the related molecular mechanism of antiproliferative effects of the extract through the evaluation of the expression level of apoptosis genes (ie, BAX and BCL2). Methods: For the accomplishment of the study objectives, after the preparation of PPE, an evaluation of cellular cytotoxicity was carried out using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. According to the MTT assay, the cells were treated for 24 h with selective doses of the extract. Then, ribonucleic acid extraction, complementary deoxyribonucleic acid synthesis, and gene expression analysis of BAX and BCL2 using real-time polymerase chain reaction were performed in this study. Results: The results showed that PPE reduced cancer cell viability in a dose- and time-dependent manner. The molecular analysis indicated that observed cell death could be due to an increase in the BAX/BCL2 ratio. Conclusions: Overall, PPE can be proposed as a potential complementary therapy for breast cancer. However, further studies on animal models and clinical trials are needed to verify the clinical usage of such complementary drugs.

Biological Potential of Silver Nanoparticles Mediated by Leucophyllum frutescens and Russelia equisetiformis Extracts.

Awareness about environmental concerns is increasing, specially the pollution resulting from nanoparticles (NPs) production, which has led to great interest in the usage of biogenic agents for their fabrication. The current investigation used eco-friendly organic phytomolecules from Leucophyllum frutescens and Russelia equisetiformis leaves extract for the first time in the fabrication of silver NPs from silver ions and further an assessment of their biological activities was performed. The leaves extract from both plant sources were used as capping and reducing agents and added to AgNO3. The mixtures were observed for colour changes, and after a stable dark brown colour was obtained, the NPs were separated and further investigated using dynamic light scattering, transmission electron microscopy and energy-dispersive X-ray spectroscopy. The Fourier transform infrared spectroscopy technique was employed to determine the active organic ingredients in the plant extracts. The prepared NPs were tested against three cell lines (two cancer ones and one normal control) and the effects observed using TEM and confocal laser scanning microscopy (LSM). Antibacterial activity against two Gram positive and two Gram negative species was examined and the synergistic effect of the ampicillin-NPs conjugate was studied. Findings showed successful conversion of Ag ions into L-AgNPs and R-AgNPs achieved using L. frutescens and R. equisetiformis extracts, respectively. A mean size of 112.9 nm for L-AgNPs and 151.7 nm for R-AgNPs and negative zeta potentials were noted. TEM analysis showed spherical NPs and EDS indicated Ag at 3 keV. Reduction in cancer cell viability with low half-maximal inhibitory concentrations was noted for both tested NPs. Structural changes and apoptotic features in the treated cancer cell lines were noted by TEM and cell death was confirmed by LSM. Furthermore, higher antibacterial activity was noticed against Gram positive compared with Gram negative bacteria as well as high synergistic effect was noted for the Amp-NPs conjugate, specially against Gram positive bacteria. The current investigation has thus developed an eco-friendly NPs synthesis route by applying plant extracts to efficiently produce NPs endowed with potential cytotoxic and antibacterial capacity, which therefore could be recommended as new approaches to overcome human diseases with minimal environmental impact.

Allium willeanum Holmboe exerts anticancer activities on metastatic breast cancer cells MCF-7 and MDA-MB-231

Allium species are medically important plants, rich in bioactive molecules with antitumoral properties. In this study, we aimed to investigate the molecular composition and in vitro anticancer activities of Allium willeanum Holmboe, an endemic Allium species of the island of Cyprus. GC-MS analysis of ethanolic extracts of A. willeanum H. bulb (AWB) showed bioactive molecules, octadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester (21.99 %), hexadecanoic acid (20.42 %), pentadecanoic acid (9.19 %), 1,2-benzenedicarboxylic acid, diethyl ester (8.79 %), with known anticancer activities. AWB exerted significant reduction in mitochondria dependent metabolic activity as a measure of cell growth on MCF-7 (weakly metastatic) and MDA-MB-231 (strongly metastatic) human breast cancer (BCa) cell lines with a more prominent effect on highly metastatic BCa cells. Both trypan blue and lactate dehydrogenase (LDH) cytotoxicity assays quantitatively revealed that exposure to AWB extract significantly reduced cancer cell viability in both tested cell types. Differential activation of caspases in the tested cell lines indicated faster apoptotic activity on MDA-MB-231 cells compared to MCF-7. A significant reduction in cell motility was demonstrated upon AWB exposure of MCF-7 and MDA-MB-231 cells. AWB bioactive molecules collectively act on cancer cells to exert significant cytotoxic, apoptotic activities on both MCF-7 and MDA-MB-231 cells and significantly reduce their lateral motility, elucidating AWB as a promising agent to further be utilized in anticancer studies.

Design, Synthesis and Biological Evaluation of New 5‐(2‐Nitrophenyl)‐1‐aryl‐1H‐pyrazoles as Topoisomerase Inhibitors

Abstract5‐(2‐Nitrophenyl)‐1‐aryl‐1H‐pyrazoles are designed as topoisomerase (Topo) inhibitors, synthesised and assessed for their anticancer properties against breast (MDA‐MB‐231 and MCF7), lung (A549), and colorectal (HCT116) cancer cell lines. All the compounds induced significant cytotoxicity at low micromolar concentration. The compound 5e exerted potential anticancer effects on breast cancer cell lines at a low micromolar level (IC50<2 μM), and showed negligible toxicity towards normal cells. Compound 5 e reduced reactive oxygen species (ROS) level in breast cancer cells, altered mitochondrial membrane potential and induced the cell cycle arrest at the G2/M phase. This was accompanied by downregulation of oncogenic p‐Akt and upregulation of p‐PTEN along with modulation of apoptotic markers suggesting multiple mechanisms to reduce cancer cell viability. Finally, the topoisomerase inhibition assay revealed the inhibitory activity of 5 e against Topo I and Topo II.

Reengineering Tumor Microenvironment with Sequential Interleukin Delivery.

Some cytokines can reengineer anti-tumor immunity to modify the tumor micro-environment. Interleukin-27 (IL-27) can partially reduce tumor growth in several animal models, including prostate cancer. We hypothesized that addition of IL-18, which can induce the proliferation of several immune effector cells through inducing IFNγ could synergize with IL-27 to enhance tumor growth control. We describe our findings on the effects of IL-27 gene delivery on prostate cancer cells and how sequential therapy with IL-18 enhanced the efficacy of IL-27. The combination of IL-27 followed by IL-18 (27→18) successfully reduced cancer cell viability, with significant effects in cell culture and in an immunocompetent mouse model. We also examined a novel chimeric cytokine, comprising an IL-27 targeted at the C-terminus with a short peptide, LSLITRL (27pepL). This novel cytokine targets a receptor upregulated in tumor cells (IL-6Rα) via the pepL ligand. Interestingly, when we compared the 27→18 combination with the single 27pepL therapy, we observed a similar efficacy for both. This efficacy was further enhanced when 27pepL was sequenced with IL-18 (27pepL→18). The observed reduction in tumor growth and significantly enriched canonical pathways and upstream regulators, as well as specific immune effector signatures (as determined by bioinformatics analyses in the tumor microenvironment) supported the therapeutic design, whereby IL-27 or 27pepL can be more effective when delivered with IL-18. This cytokine sequencing approach allows flexible incorporation of both gene delivery and recombinant cytokines as tools to augment IL-27’s bioactivity and reengineer efficacy against prostate tumors and may prove applicable in other therapeutic settings.