Protocol for whole-mount immunofluorescence staining of ECM gel-embedded innervated pancreatic organoids.

The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting.

Targeting Periostin Expression Makes Pancreatic Cancer Spheroids More Vulnerable to Natural Killer Cells.

Pancreatic cancer (PaCa) characteristically has a dense tumor microenvironment, which results in poor patient prognosis. Pancreatic stellate cells (PSCs) are the most abundant cells in the PaCa microenvironment and the principal source of collagen. Periostin, a matricellular protein, is produced specifically by PSCs and promotes the aggressiveness of PaCa cells by facilitating extracellular collagen assembly. Here, we aimed to decrease extracellular collagen assembly by suppressing periostin, thereby increasing the cytotoxic activity of natural killer (NK) cells. Periostin expression was suppressed in PSCs (called PSC-P) using CRISPR-Cas9. PaCa cells (BxPC-3) were co-cultured with PSC and PSC-P cells in a 3D environment to form tumor spheroids mimicking the tumor microenvironment. The extracellular collagen production of spheroids was evaluated by Masson's trichrome staining. The cytotoxic activity of NK-92 cells was analyzed by flow cytometry and confocal microscopy via CD107a staining. Cell death in BxPC-3 cells was evaluated by measuring Annexin-V and PI positivity using flow cytometry. As a result, periostin suppression decreased extracellular collagen and increased the infiltration of NK-92 cells into spheroids, and induced cell death in PaCa cells. In conclusion, we suggest that periostin might be a therapeutic target for PaCa and further analysis is warranted using in vivo models for proof-of-concept.

Evidence for heterogeneity in response to treatment in mammary tumors of dogs as happens in humans.

Tumors are formed by various clones developed over a long time. This gives rise to a heterogeneous nature. This heterogeneity is the hardest challenge in the treatment of cancers because it is the main reason for drug resistance. This is a well-known fact in human cancer. Therefore, we have reasoned that if the tumor heterogeneity in canine mammary gland tumors (CMGTs) could be shown by an ex vivo assay, which will be used first time in veterinary oncology practice, this could be used further in clinics. To achieve this, twenty-six patients were included in the study. Tumor tissues were obtained from animals during routine surgery. Tumor cells were isolated and seeded ex vivo. The cells were exposed to anticancer drugs that are clinically used. Seven days after the treatment, chemosensitivity has luminometrically been assayed by ATP-tumor chemosensitivity assay (ATP-TCA). It has clearly been shown that all the tumor tissues have responded to treatment differently, implying that heterogeneity exists in mammary tumors. There has also been found that there was a weak to moderate statistically significant correlation between tumor size and drug index. However, there has been no correlation between drug index and metastasis to lymph nodes. Hyperplasic areas had relatively higher PCNA values. The results of our study demonstrate the heterogeneity in responses to in vitro drugs. Clinical trials based on test results and follow-up studies with large numbers of animals are needed to prove that such chemotherapeutic activity assessment tests can be clinically useful in predicting drug responses in CMGTs.

Tumor Chemosensitivity Assays Are Helpful for Personalized Cytotoxic Treatments in Cancer Patients.

Tumor chemosensitivity assays (TCAs), also known as drug response assays or individualized tumor response tests, have been gaining attention over the past few decades. Although there have been strong positive correlations between the results of these assays and clinical outcomes, they are still not considered routine tests in the care of cancer patients. The correlations between the assays' results (drug sensitivity or resistance) and the clinical evaluations (e.g., response to treatment, progression-free survival) are highly promising. However, there is still a need to design randomized controlled prospective studies to secure the place of these assays in routine use. One of the best ideas to increase the value of these assays could be the combination of the assay results with the omics technologies (e.g., pharmacogenetics that gives an idea of the possible side effects of the drugs). In the near future, the importance of personalized chemotherapy is expected to dictate the use of these omics technologies. The omics relies on the macromolecules (Deoxyribonucleic acid -DNA-, ribonucleic acid -RNA-) and proteins (meaning the structure) while TCAs operate on living cell populations (meaning the function). Therefore, wise combinations of TCAs and omics could be a highly promising novel landscape in the modern care of cancer patients.

The Role of LncRNAs in Translation.

Long non-coding RNAs (lncRNAs), a group of non-protein coding RNAs with lengths of more than 200 nucleotides, exert their effects by binding to DNA, mRNA, microRNA, and proteins and regulate gene expression at the transcriptional, post-transcriptional, translational, and post-translational levels. Depending on cellular location, lncRNAs are involved in a wide range of cellular functions, including chromatin modification, transcriptional activation, transcriptional interference, scaffolding and regulation of translational machinery. This review highlights recent studies on lncRNAs in the regulation of protein translation by modulating the translational factors (i.e, eIF4E, eIF4G, eIF4A, 4E-BP1, eEF5A) and signaling pathways involved in this process as wells as their potential roles as tumor suppressors or tumor promoters.

ncRNA therapy with miRNA-22-3p suppresses the growth of triple-negative breast cancer.

Deregulation of noncoding RNAs, including microRNAs (miRs), is implicated in the pathogenesis of many human cancers, including breast cancer. Through extensive analysis of The Cancer Genome Atlas, we found that expression of miR-22-3p is markedly lower in triple-negative breast cancer (TNBC) than in normal breast tissue. The restoration of miR-22-3p expression led to significant inhibition of TNBC cell proliferation, colony formation, migration, and invasion. We demonstrated that miR-22-3p reduces eukaryotic elongation factor 2 kinase (eEF2K) expression by directly binding to the 3' untranslated region of eEF2K mRNA. Inhibition of EF2K expression recapitulated the effects of miR-22-3p on TNBC cell proliferation, motility, invasion, and suppression of phosphatidylinositol 3-kinase/Akt and Src signaling. Systemic administration of miR-22-3p in single-lipid nanoparticles significantly suppressed tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC models. Evaluation of the tumor response, following miR-22-3p therapy in these models using a novel mathematical model factoring in various in vivo parameters, demonstrated that the therapy is highly effective against TNBC. These findings suggest that miR-22-3p functions as a tumor suppressor by targeting clinically significant oncogenic pathways and that miR-22-3p loss contributes to TNBC growth and progression. The restoration of miR-22-3p expression is a potential novel noncoding RNA-based therapy for TNBC.

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation, migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-ß/Src signaling in breast cancer cells.

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin ß1 and ß3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target.

Eukaryotic elongation factor-2 kinase (eEF2K), an atypical member of alpha-kinase family, is highly overexpressed in breast, pancreatic, brain, and lung cancers, and associated with poor survival in patients. eEF2K promotes cell proliferation, survival, and aggressive tumor characteristics, leading to tumor growth and progression. While initial studies indicated that eEF2K acts as a negative regulator of protein synthesis by suppressing peptide elongation phase, later studies demonstrated that it has multiple functions and promotes cell cycle, angiogenesis, migration, and invasion as well as induction of epithelial-mesenchymal transition through induction of integrin ß1, SRC/FAK, PI3K/AKT, cyclin D1, VEGF, ZEB1, Snail, and MMP-2. Under stress conditions such as hypoxia and metabolic distress, eEF2K is activated by several signaling pathways and slows down protein synthesis and helping cells to save energy and survive. In vivo therapeutic targeting of eEF2K by genetic methods inhibits tumor growth in various tumor models, validating it as a potential molecular target. Recent studies suggest that eEF2K plays a role in tumor microenvironment cells by monocyte chemoattractant protein-1 (MCP-1) and accumulation of tumor-associated macrophages. Due to its clinical significance and the pivotal role in tumorigenesis and progression, eEF2K is considered as an important therapeutic target in solid tumors. However, currently, there is no specific and potent inhibitor for translation into clinical studies. Here, we aim to systematically review current knowledge regarding eEF2K in tumor biology, microenvironment, and development of eEF2K targeted inhibitors and therapeutics.

Cancer Stem Cells: Root of the Evil.

Cancer stem cells (CSCs) have been one of the most attractive research areas over the last two decades due to their important roles in aggressive tumor behaviors. Although CSCs are usually a small subpopulation of tumors (less than 0.1%), these cells determine the fate of cancer patients because of their roles in poor prognoses. Accumulating evidence has shown that CSCs are the major responsible cell population in tumor development, growth, metastasis, and therapy resistance. The self-renewal and differentiation capacity of CSCs contribute to the initiation and maintenance of tumor growth. Several reports indicate that CSCs share numerous molecular characteristics with mesenchymal cells, so CSCs are accepted as leading players in metastasis. In addition, these cells possess a higher intrinsic resistance to chemotherapies and radiotherapies than bulk cancer cells. Therefore, therapeutic approaches in recent years have focused on effectively targeting CSCs. Here, we have reviewed the current literature on the resistant mechanisms of CSCs, their roles in metastasis, and the roles of non-coding RNAs in the regulation of aggressive CSC characteristics and novel therapy approaches against CSCs.

A novel 1,4-naphthoquinone-derived compound induces apoptotic cell death in breast cancer cells.

Breast cancer is the most-diagnosed cancer type among women. The triple-negative subtype is an especially aggressive type of breast cancer. Although chemotherapy is almost the only option for the treatment of triple-negative breast cancer (TNBC), currently used chemotherapeutics are not effective enough, considering the poor survival rate of patients. Therefore, novel compounds need to be developed to improve survival rates. It has been known that quinonic compounds, which are found in nature, have antibacterial, antifungal, and antitumorigenic properties. Naphthoquinones are members of the quinone family and are widely used in research due to their promising properties. In this study, we evaluated the cytotoxic activity of a novel naphthoquinone-derived compound (1,4-naphthoquinone (1,4-NQ)) against two different breast cancer cells: a hormone-responsive cell line (MCF-7) and a triple-negative cell line (MDA-MB-231). As a result, 1,4-NQ decreased cell viability in both tested cell lines in a dose-dependent manner. Increased apoptotic markers (presence of pyknotic nuclei, annexin-V positivity, caspase 3/7 activity, and decreased mitochondrial membrane potential) and DNA damage were especially observed in MDA-MB-231 cells after treatment with the compound. Considering the promising cytotoxic effect of the compound, 1,4-NQ needs further evaluation as a potential candidate for the treatment of TNBC.

Unfolded Protein Response is Involved in Trans-Platinum (II) Complex-Induced Apoptosis in Prostate Cancer Cells via ROS Accumulation.

BACKGROUND: Prostate cancer is one of the most common cancer types and it is the sixth leading cause of cancer-related death in men worldwide. Even though novel treatment modalities have been developed, it still a lifethreatening disease. Therefore novel compounds are needed to improve the overall survival. METHODS: In our study, it was aimed to evaluate the anti-cancer activity of newly synthesized Platinum (II) [Pt(II)] complex on DU145, LNCaP and PC-3 prostate cancer cell lines. The cytotoxic activity of Pt(II) complex was tested by SRB and ATP cell viability assays. To detect the mode of cell death; fluorescent staining, flow cytometry and western blot analyses were performed. RESULTS: The Pt(II) complex treatment resulted in a decrease in cell viability and increasing levels of apoptotic markers (pyknotic nuclei, annexin-V, caspase 3/7 activity) and a decrease in mitochondrial membrane potential in a dose dependent manner. Among cell types, tested PC-3 cells were found to be more sensitive to Pt(II) complex, demonstrating elevation of DNA damage in this cell line. In addition, Pt(II) complex induced Endoplasmic Reticulum (ER) stress by triggering ROS generation. More importantly, pre-treatment with NAC alleviated Pt(II) complex-mediated ER stress and cell death in PC-3. CONCLUSION: These findings suggest an upstream role of ROS production in Pt(II) complex-induced ER stressmediated apoptotic cell death. Considering the ROS-mediated apoptosis inducing the effect of Pt(II) complex, it warrants further evaluation as a novel metal-containing anticancer drug candidate.

Dual Suppressive Effect of miR-34a on the FOXM1/eEF2-Kinase Axis Regulates Triple-Negative Breast Cancer Growth and Invasion.

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225-41. ©2018 AACR.

A promising natural product, pristimerin, results in cytotoxicity against breast cancer stem cells in vitro and xenografts in vivo through apoptosis and an incomplete autopaghy in breast cancer.

Several natural products have been suggested as effective agents for the treatment of cancer. Given the important role of CSCs (Cancer Stem Cells) in cancer, which is a trendy hypothesis, it is worth investigating the effects of pristimerin on CSCs as well as on the other malignant cells (MCF-7 and MDA-MB-231) of breast cancer. The anti-growth activity of pristimerin against MCF-7 and MCF-7s (cancer stem cell enriched population) cells was investigated by real time viability monitorization (xCELLigence System®) and ATP assay, respectively. Mode of cell death was evaluated using electron and fluorescence microscopies, western blotting (autophagy, apoptosis and ER-stress related markers) and flow cytometry (annexin-V staining, caspase 3/7 activity, BCL-2 and PI3K expressions). Pristimerin showed an anti-growth effect on cancer cells and cancer stem cells with IC50 values ranging at 0.38-1.75µM. It inhibited sphere formation at relatively lower doses (<1.56µM). Apoptosis was induced in MCF-7 and MCF-7s cells. In addition, extensive cytoplasmic vacuolation was observed, implying an incompleted autophagy as evidenced by the increase of autophagy-related proteins (p62 and LC3-II) with an unfolded protein response (UPR). Pristimerin inhibited the growth of MCF-7 and MDA-MB-231-originated xenografts in NOD.CB17-Prkdcscid/J mice. In mice, apoptosis was further confirmed by cleavage of PARP, activation of caspase 3 and/or 7 and TUNEL staining. Taken together, pristimerin shows cytotoxic activity on breast cancer both in vitro and in vivo. It seems to represent a robust promising agent for the treatment of breast cancer. Pristimerin's itself or synthetic novel derivatives should be taken into consideration for novel potent anticancer agent(s).

Cytotoxic and apoptotic effects of the combination of palladium (II) 5,5-diethylbarbiturate complex with bis(2-pyridylmethyl)amine and curcumin on non small lung cancer cell lines.

Metal-based chemotherapeutics such as cisplatin are widely used treatment of lung cancer which is the major cause of cancer-related mortality worldwide. Recent studies demonstrated that novel metal-based compounds have strong cytotoxic activity in a similar way as cisplatin. Therefore, metal-based compounds have been synthesized and investigated in order to determine their cytotoxic activities. It has been also reported curcumin, which has been derived from turmeric plant, has powerful cytotoxic effect on various cancer cell lines. In the light of these data, it has been investigated the cytotoxic effects of combination of curcumin (0.78-100µM) and palladium (II) 5,5-diethylbarbiturate complex with bis(2-pyridylmethyl)amine [Pd(II) complex] (0.39-50µM) against non small lung cancer cell lines, A549 and H1299. It has been found that combination of Pd(II) complex and curcumin enhanced the cytotoxic activity and apoptotic cell death at 48h, compared to single use of each agent, only in H1299 cell line (combination index <1). Apoptosis was evident by annexin v staining positivity, increased caspase 3/7 activity and the presence of pyknotic nuclei. Pro-apoptotic genes of TNFRSF10A and HRK were found to be involved in apoptotic cell death. In conclusion, the application of this combination may be regarded as a novel and effective approach for the treatment of lung cancer due to its promising cytotoxic and apoptotic effect.

The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts.

The MTT assay is one of the often used cell viability/cytotoxicity assays. However, when the methanol extracts of plants are used to test their cytotoxic potential, interference may occur, resulting in false-positive viability results. Therefore, in this study, the reliability of the MTT assay was investigated in the case of plant use. The methanol extracts of three different plants (Hypericum adenotrichum, Salvia kronenburgii, and Pelargonium quercetorum) were tested in breast cancer cell lines (MCF-7 and MDA-MB-231) using the MTT assay and the results were compared to the ATP assay, which is a much more sensitive and reliable assay due to its interference-free feature. Additionally, decreased cell density was confirmed with phase-contrast microscopy and fluorescence staining (Hoechst 33342 dye). Although both of the viability/cytotoxicity assays are considered as metabolic assays, viabilities (in %) in the MTT assay were found to be strikingly higher when compared to the results with the ATP assay. Even in the case of total death, the MTT assay still produced artificial/false increases in viability. The morphology-based evaluation of viability/cytotoxicity by phase-contrast microscopy and Hoechst 33342 staining were greatly compatible with the ATP assay results. Overestimated (false) viabilities in the MTT assay suggests a serious interference between the MTT assay itself and the extracts used. Some ingredients of plants may have reducing activity (like the dehydrogenase activity of the cells) that converts the MTT compound into the colored formazan that is the principle of the assay. Therefore, the MTT assay may not be a suitable assay for some plant extracts, urging great caution when plants are used.

Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents.

BACKGROUND: To overcome the hurdles of cisplatin, majorly its toxicity and resistance, there has been extensive search for alternative anti-cancer metal-based compounds. Here, three Cu(II)-complexes, Cu(Sal-Gly)(phen), Cu(Sal-Gly)(pheamine), Cu(Sal-Gly)(phepoxy) are characterized for their interaction with DNA, cytotoxicity and mechanism of action. METHODS: The binding ability of the complexes to Calf-Thymus DNA was evaluated by competition fluorescence studies with thiazole-orange, UV-Vis and circular dichroism spectroscopic titrations. Cytotoxicity was evaluated by MTT analysis. The DNA damage was analyzed through cleavage of supercoiled DNA via agarose gel-electrophoresis, and 8-oxo-guanidine and É£H2AX staining in cells. Apoptosis was detected via DNA condensation/fragmentation, mitochondrial membrane potential, Annexin V staining and caspase 3/7 activity. Formation of reactive oxygen species was determined by DCFDA- and GSSG/GSH-analysis. RESULTS: Binding constants to DNA were evaluated as 1.7×106 (Cu(Sal-Gly)(phen)), 2.5×106 (Cu(Sal-Gly)(pheamine)) and 3.2×105 (Cu(Sal-Gly)(phepoxy)). All compounds induced DNA damage. Apoptosis was the main form of cell death. There was an increase in ROS, which is most likely responsible for the observed DNA-damage. Although the compounds were cytotoxic to all tested cancer cell lines, only Cu(Sal-Gly)(pheamine) displayed significantly lower toxicity towards non-cancer cells, its associated phenotypes differing from the other two Cu-complexes. Thus, Cu(Sal-Gly)(pheamine) was further assayed for molecular changes in response to drug treatment using a custom designed RT-qPCR array. Results showed that Harakiri was significantly upregulated. Presence of p53 was not required for apoptosis in response to Cu-complexes. CONCLUSIONS AND GENERAL SIGNIFICANCE: These Cu-complexes, namely Cu(Sal-Gly)(pheamine), may be considered promising anticancer agents with activity in cancer cells even with deficient p53 status.

A trans-platinum(II) complex induces apoptosis in cancer stem cells of breast cancer.

Recent accumulating evidence has supported the notion that tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (CSCs), are responsible for tumor initiation, maintenance as well as drug resistance. Therefore, targeting the CSCs along with the other cancer cells has been the most important topic during the last decade. In the present study, we evaluated the cytotoxic activity of trans-[PtCl2(2-hepy)2] [2-hepy=2-(2-hydroxyethyl) pyridine] complex and the mechanism of cell death in breast CSCs. Stemness markers, Oct-4 and Sox2, were determined in mammospheres by western blotting. Cytotoxicity was assessed using the ATP viability assay. Cell death was fluorescently visualized and further confirmed by flow cytometry as well as gene expression analysis. The Pt(II) complex significantly reduced the cell viability, prevented mammosphere formation and disrupted mammosphere structures in a dose-dependent manner (0-100µM). The mode of cell death was apoptosis and it was shown by the presence of caspase 3/7 activity, Annexin V-FITC positivity, decreased mitochondrial membrane potential and increased expressions of pro-apoptotic genes (TNFRSF10A and HRK). Interestingly, necroptosis was also observed by the evidence of increased MLKL expression. In conclusion, the Pt(II) complex seems to be a highly promising anticancer compound due to its promising cytotoxic activity on CSCs. Therefore, it deserves in vivo further studies for the proof-of-concept.

Validation data supporting the characterization of novel copper complexes as anticancer agents.

Three copper(II) complexes, Cu(Sal-Gly)(phen), Cu(Sal-Gly)pheamine, Cu(Sal-Gly)phepoxy were synthesized and characterized for their anticancer properties and mechanism of action (Acilan et al., in press) [1]. Here, we provide supporting data on colon cancer cell lines complementing our previous findings in cervix cells. This paper also contains a data table for the fold changes and p-values of all genes analyzed in this study via a custom RT-qPCR array. All compounds induced DNA damage (based on 8-oxo-guanidine, ɣH2AX staining in cells) and apoptosis (based on elevated DNA condensation/fragmentation, Annexin V staining, caspase 3/7 activity and mitochondrial membrane depolarization) in HCT-116 colon cancer cells. The increase in oxidative stress was also further confirmed in these cells. Further interpretation of the data presented here can be found in the article entitled "Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents" (Acilan et al., in press) [1].

Design, Synthesis, Biological Evaluation, and Antioxidant and Cytotoxic Activity of Heteroatom-Substituted 1,4-Naphtho- and Benzoquinones.

In the present paper, we report the synthesis, characterization, and biological evaluation as antifungal, antibacterial, antioxidant, and cytotoxic/anticancer agents of N-, S-, O-substituted-1,4-naphtho- and 2,5-bis(amino-substituted)-1,4-benzoquinone derivatives. In the synthesized compounds, antimicrobial activity at low concentrations against Escherichia coli B-906, Staphylococcus aureus 209-P, and Mycobacterium luteum B-917 bacteria and Candida tenuis VKM Y-70 and Aspergillus niger F-1119 fungi in comparison with controls was identified. 2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-naphthoquinone 9a was the most potent, with a minimum inhibitory concentration value of 3.9 µg/mL against test culture M. luteum. The synthesized compounds were screened for their antioxidant capacity using the cupric-reducing antioxidant capacity (CUPRAC) method. 2,2'-[1-(2-Aminoethyl)piperazin-1-yl]-3,3'-dichloro-bis(1,4-naphthoquinone) 10 showed the highest antioxidant capacity, with a 0.455 CUPRAC-trolox equivalent antioxidant capacity (TEAC) coefficient. Other parameters of antioxidant activity (scavenging effects on OH(·), O2(·ï¼), and H2O2) of these compounds were also determined. The cytotoxic activity of the compounds was investigated by employing the sulforhodamine B cell viability assay against A549 (lung), MCF-7 (breast), DU145 (prostate), and HT-29 (colon) cancer cell lines. Compound 10 exhibited the most powerful cytotoxic activity at a concentration of 20 µM against all cell lines. In addition to the strongest antioxidant activity of compound 10, it also had lowest IC50 values (<3 µM), warranting further in vivo studies due to its anticancer activity.

Addition of niclosamide to palladium(II) saccharinate complex of terpyridine results in enhanced cytotoxic activity inducing apoptosis on cancer stem cells of breast cancer.

Wnt signaling is one of the core signaling pathways of cancer stem cells (CSCs). It is re-activated in CSCs and plays essential role in the survival, self-renewal and proliferation of these cells. Therefore, we aimed to evaluate the cytotoxic effects of palladium(II) complex which is formulated as [PdCl(terpy)](sac)2H2O and its combination with niclosamide which is an inhibitor of Wnt signaling pathway associated with breast cancer stem cells. Characteristic cell surface markers (CD44(+)/CD24(-)) were determined by flow cytometry in CSCs. ATP viability assay was used to determine the cytotoxic activity. The mode of cell death was evaluated morphologically using fluorescence microscopy and biochemically using M30 ELISA assay as well as performing qPCR. Our study demonstrated that the combination of niclosamide (1.5 µM) and Pd(II) complex (12.5, 25 and 50 µM) at 48 h has enhanced cytotoxic activity resulted from the induction of apoptosis (indicated by the presence of pyknotic nuclei, increments in M30 and over expression of proapoptotic genes of TNFRSF10A and FAS). Importantly, the addition of niclosamide resulted in the suppression of autophagy (proved by the decrease in ATG5 gene levels) that might have contributed to the enhanced cytotoxicity. In conclusion, the application of this combination may be regarded as a novel and effective approach for the treatment of breast cancer due to its promising cytotoxic effect on cancer stem cells that cause recurrence of the disease.