Non-malignant Cell Line Research Articles

SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP–galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin–Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell–cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

Nanoarchitectonics of (110) directed polyethylene glycol stabilized cerium nanoparticles for UV filtering applications

The use of zinc oxide (ZnO) and titanium oxide (TiO2) nanoparticles in ultraviolet (UV) filters can cause serious health issues due to their genotoxicity and cytotoxicity effect. Recent research on cerium oxide (CeO2) nanoparticles has demonstrated their biocompatible nature, excellent antioxidant and ultraviolet–visible (UV–Vis) absorption properties. CeO2-based nanocomposite materials or nano-architectures can offer uniform absorptive properties, therapeutic skin effect and easy skin application. In this study, cerium oxide-polyethylene glycol (CeO2-PEG) nano-architecture was prepared by hydrothermal processing with the aim to improve stability, antioxidant properties, and enhance UV absorption for UV filtering applications. The morphology study of the developed nanocomposite revealed the formation of polymer bands with well-dispersed CeO2 nanoparticles. The nanocomposite structure has exhibited excellent UV absorption in the UV range 200–400 nm due to decrease in optical band gap in comparison to the naked CeO2 NPs. The evaluation of the photocatalytic activity (PCA) of the nanocomposite structure against P25 photocatalyst and ZnO nanoparticles showed no dye (crystal violet) degradation on UV irradiation. Hence, PCA assay verified the absence of ROS generation and increased stability of the nanostructure under UV irradiation. The investigation of antioxidant properties of the Ce-PEG nanocomposite by a dichlorofluorescein (DCF)-based assay revealed sufficient intracellular ROS scavenging in comparison to N-acetylcysteine (NAC) and H2O2 only assays. In vitro toxicity evaluation of Ce-PEG nanocomposite towards non-malignant human keratinocyte cell line (HaCaT) revealed a non-significant cell mortality ~ 10% of control at a dosage of 50 mg L−1. The nanocomposite structure also exhibited excellent UV protection for HaCaT cells under UV irradiation conditions compared to P25. Our findings suggest that the developed nanocomposite combined the antioxidant and UV absorption properties of CeO2 and skin emollient role of (PEG), and hence offered a novel biocompatible and multifunctional structure for sunscreens and therapeutic skin product.Graphical abstract

MgAl and ZnAl-Hydrotalcites as Materials for Cosmetic and Pharmaceutical Formulations: Study of Their Cytotoxicity on Different Cell Lines.

The knowledge about the effect of hydrotalcites (HTlcs), largely used in pharmaceutics, on non-malignant cell lines is limited. The effect of MgAl-HTlc-and ZnAl-HTlc- (NO3−/Cl−/CO32−) on the cell viability of HaCat, fibroblasts and HepG2 was studied by MTT assay. Cells were incubated either with HTlc suspensions in the culture media and with the supernatant obtained from the suspension being centrifuged. MgAl-HTlcs suspensions resulted in being cytotoxic. As SEM and TEM analyses showed the presence of sub-micrometric particles in all the MgAl-HTlc examined, it could be hypothesized that this fraction can be internalized into cells reducing the viability. MgAl-HTlc-NO3 is the most cytotoxic probably due to the additional effect of NO3− anions. ZnAl-HTlcs are cytotoxic, especially for HaCat and HepG2 cells (viability <60% at all the concentrations assayed). The effect is attributable both to the sub-micrometric fraction (identified by TEM) and to the high Zn2+ levels found in the culture medium by ICP-OES analysis, suggesting that ZnAl-HTlcs are less stable than MgAl-HTlc in the used media. The obtained results suggest that it is very important to perform ad hoc studies in order to evaluate HTlc safety before to be introduced in a formulation.

Abstract 5451: New organoselenium compounds with cytotoxic activity in vitro and in vivo towards colon cancer

Abstract Colon cancer is currently the third most common diagnosed cancer and the second leading cause of cancer death worldwide. The inclusion of a selenium atom (Se) in organic molecules has been shown to be a valid approximation in the design of novel chemotherapeutic agents with antitumoral activity. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) have gained interest in recent years in terms of their chemopreventive and chemotherapeutic use for cancer treatment whether they are evaluated alone, as a combination therapy, or in the form of structurally modified analogs. Considering these findings, we have designed five novel NSAID derivatives containing Se in their structure. The cytotoxicity and selectivity of the new compounds were evaluated in a panel of 10 cancer cell lines including breast, prostate, lung, and colon cancer, and in two non-malignant cell lines. Compound 5 was the most cytotoxic derivative with IC50 values below 10 µM in all the cancer cell lines tested, and it was also the most selectively toxic towards cancer cells while sparing the non-malignant cell lines. The efficacy of compound 5 in vivo was assessed in a subcutaneous colon cancer xenograft mouse model and it showed a significant inhibition of the tumor growth. Taken together, this work supports that the novel Se-NSAID combination provides a feasible frame to develop new agents with potent antitumoral properties Citation Format: Sandra Ramos-Inza, Cesar Aliaga, Asif Raza, Ignacio Encío, Arun Sharma, Carmen Sanmartín, Daniel Plano. New organoselenium compounds with cytotoxic activity in vitro and in vivo towards colon cancer [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 5451.

Syntheses and high selective cytotoxicity of dehydroabietylamine C-ring nitration derivatives

To find more effective anticancer agents, a series of novel dehydroabietylamine (DA) derivatives were synthesized, focusing on C-ring nitro modifications and C-18 imide introduction. Their cytotoxic activities against human tumor cell line HeLa (cervix), MCF-7 (breast), A549 (lung), HepG2 (liver), and nonmalignant cell line HUVEC (umbilical vein) in vitro were screened. The C-18 imide heterocyclic compounds 1, 2, and C-ring 14-nitro substituted 14 exhibited moderate to good cytotoxic activities and significant selectivity towards malignant cell lines. More importantly, they were significantly less cytotoxic to nonmalignant cells (HUVEC) than the parent compound and positive control doxorubicin hydrochloride (DOX). Meantime the mechanism of cytotoxicity of DA derivatives was studied. Annexin V-FITC/PI double-staining analysis suggested that cytotoxicity of compounds 2 and 14 was associated with early apoptosis induction. The interaction between compounds and DNA (herring sperm DNA) was studied using absorption spectral analysis and ethidium bromide (EB) fluorescence displacement experiments, the results exhibited that the binding of the compound to DNA was in the intercalative mode. The structure-activity relationship discussion implied that introduction of the nitro-group, especially the 14-nitro group, can significantly improve the cytotoxicity of dehydroabietylimide compounds. The relatively high cytotoxicity and significant high selectivity of compounds 2 and 14 indicated that they were particularly noteworthy. NO released amounts indicated that the amounts of NO released by the compounds bearing nitro-group were quite well associated positive correlation with their cytotoxic activity, which provide a new strategy for structure design of DA anticancer agents in the future.

Chlorinated benzothiadiazines inhibit angiogenesis through suppression of VEGFR2 phosphorylation

Angiogenesis inhibitors are a critical pharmacological tool for the treatment of solid tumors. Suppressing vascular permeability leads to inhibition of tumor growth, invasion, and metastatic potential by blocking the supply of oxygen and nutrients. Disruption of the vascular endothelial growth factor (VEGF) signaling pathway is a validated target for the design of antiangiogenic agents. Several VEGFR2 inhibitors have been clinically approved over the past years. Structural analysis of these clinical VEGFR2 inhibitors highlighted key functional group overlap with the benzothiadiazine core contained in a library of in-house compounds. Herein we ascribe anti-angiogenic activity to a series of chlorinated benzothiadiazines. Selected compounds show significant activity to completely ameliorate VEGF-induced endothelial cell proliferation by suppression of VEGFR2 phosphorylation. The scaffold is devoid of activity to inhibit carbonic anhydrases and generally lacks cytotoxicity across a range of cancer and non-malignant cell lines. Assay of activity at 468 kinases shows remarkable selectivity with only four kinases inhibited > 65% at 10 µM concentration, and with significant activity to inhibit TNK2/ACK1 and PKRD2 by > 90%. All four identified kinase targets are known modulators of angiogenesis, thus highlighting compound 17b as a novel angiogenesis inhibitor for further development.

Investigations of Bis(alkylthiocarbamato)copper Linkage Isomers

Linkage isomers are coordination compounds with the same composition but different donor atoms, resulting in distinct physical and electronic structures. A pair of linkage isomers, CuL555 and CuL465, derived from phenylglyoxal bis(ethylthiocarbamate) were synthesized, isolated, and characterized by structural, electrochemical, and spectroscopic methods. The isomers are stable in solution under ambient conditions, but CuL465 converts to CuL555 in acid, consistent with quantum-chemical calculations. The complexes were screened against a lung adenocarcinoma cell line (A549) and a nonmalignant lung fibroblast cell line (IMR-90) to evaluate the antiproliferation activity. CuL555 and CuL465 possessed EC50 values of 0.113 ± 0.030 and 0.115 ± 0.038 μM for A549 and 1.87 ± 0.29 and 0.77 ± 0.22 μM for IMR-90, respectively.

Expression analysis and function of mitochondrial genome-encoded microRNAs

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

In vitro Susceptibility of Human Cell Lines Infection by Bovine Leukemia Virus.

Evidence of the presence of bovine leukemia virus (BLV) in human beings and its association with breast cancer has been published in the literature, proposing it as a zoonotic infection. However, not enough evidence exists about transmission pathways nor biological mechanisms in human beings. This study was aimed at gathering experimental evidence about susceptibility of human cell lines to BLV infection. Malignant and non-malignant human cell lines were co-cultured with BLV-infected FLK cells using a cell-to-cell model of infection. Infected human cell lines were harvested and cultured for 3 to 6 months to determine stability of infection. BLV detection was performed through liquid-phase PCR and visualized through in situ PCR. Seven out of nine cell lines were susceptible to BLV infection as determined by at least one positive liquid-phase PCR result in the 3-month culture period. iSLK and MCF7 cell lines were able to produce a stable infection throughout the 3-month period, with both cytoplasmic and/or nuclear BLV-DNA visualized by IS-PCR. Our results support experimental evidence of BLV infection in humans by demonstrating the susceptibility of human cells to BLV infection, supporting the hypothesis of a natural transmission from cattle to humans.

GC-MS and LC-QTOF-MS Phytochemical analysis, antiproliferative and antioxidant activities of stems extracts of Retama monosperma grown in Algeria

Retama species have been used in traditional Algerian medicine for a very long time. In this study, extracts of Algerian Retama monosperma stem were examined for their phytochemical composition by GC-MS and LCQTOF- MS analysis, then screened for their antioxidant activities and antiproliferative effects. Antioxidant activity has been evaluated in-depth using DPPH radical-scavenging activity test, ABTS•+ radical cation scavenging activity, total antioxidant activity and the β-carotene bleaching test. Antiproliferative activity was assessed by using four human cancer cell lines: A549, HEK-293, SKOV-3, HeLa and a non-malignant human cell line MRC-5. Interestingly, our results revealed a high amount of phenolic compounds and alkaloids in the methanolic extract. Indeed, the rates of total phenolics, flavonoids, flavonols and tannins were higher in the methanolic extract rather than aqueous extract. As a result, a higher antioxidant activity in all tests performed was registered in the methanolic extract. Remarkably, the methanolic extract exerted the highest antiproliferative activity against A549 and HeLa cells (IC50 = 85.42 ± 3.52 and 90.28 ± 6.75 μg/mL respectively) compared to aqueous extract. In a direct line, the chloroform, ethyl acetate and hexane fractions were able to inhibit cell growth in a dose-dependent manner and showed a significant antiproliferative activity in A549 cells. Moreover, a considerable activity on SKOV-3 (IC50 = 17.92 μg/mL) has been observed using the chloroform fraction. It should be noted that no cytotoxic effect has been detected by the extracts on the non-malignant human cell line MRC-5. In conclusion, these results underlined the antioxidant and antiproliferative potential of Algerian Retama monosperma.

Systematic In Vitro Evaluation of a Library of Approved and Pharmacologically Active Compounds for the Identification of Novel Candidate Drugs for KMT2A-Rearranged Leukemia

Patients whose leukemias harbor a rearrangement of the Mixed Lineage Leukemia (MLL/KMT2A) gene have a poor prognosis, especially when the disease strikes in infants. The poor clinical outcome linked to this aggressive disease and the detrimental treatment side-effects, particularly in children, warrant the urgent development of more effective and cancer-selective therapeutics. The aim of this study was to identify novel candidate compounds that selectively target KMT2A-rearranged (KMT2A-r) leukemia cells. A library containing 3707 approved drugs and pharmacologically active compounds was screened for differential activity against KMT2A-r leukemia cell lines versus KMT2A-wild type (KMT2A-wt) leukemia cell lines, solid tumor cells and non-malignant cells by cell-based viability assays. The screen yielded SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), that limited the viability of 7 out of 11 KMT2A-r leukemia cell lines including 5 out of 7 lines derived from infants, without affecting KMT2A-wt leukemia cells, solid cancer lines, non-malignant cell lines, or peripheral blood mononuclear cells from healthy controls. The compound also significantly inhibited growth of leukemia cell lines with a CALM-AF10 translocation, which defines a highly aggressive leukemia subtype that shares common underlying leukemogenic mechanisms with KMT2A-r leukemia. SID7969543 decreased KMT2A-r leukemia cell viability by inducing caspase-dependent apoptosis within hours of treatment and demonstrated synergy with established chemotherapeutics used in the treatment of high-risk leukemia. Thus, SID7969543 represents a novel candidate agent with selective activity against CALM-AF10 translocated and KMT2A-r leukemias that warrants further investigation.

Comparison of UVC Sensitivity and Dectin-2 Expression Between Malignant and Non-malignant Cells

Rapid spread of COVID-19 resulted in the revision of the value of ultraviolet C (UVC) sterilization in working spaces. This study aimed at investigating the UVC sensitivity of eighteen malignant and nonmalignant cell lines, the protective activity of sodium ascorbate against UVC, and whether Dectin-2 is involved in UVC sensitivity. Various cell lines were exposed to UVC for 3 min, and cell viability was determined using the MTT assay. Anti-UV activity was determined as the ratio of 50% cytotoxic concentration (determined with unirradiated cells) to 50% effective concentration (that restored half of the UV-induced loss of viability). Dectin-2 expression was quantified using flow cytometry. The use of culture medium rather than phosphate-buffered saline is recommended as irradiation solution, since several cells are easily detached during irradiation in phosphate-buffered saline. Oral squamous cell carcinoma cell lines showed the highest UV sensitivity, followed by neuroblastoma, glioblastoma, leukemia, melanoma, lung carcinoma cells, and normal oral and dermal fibroblasts. Human dermal fibroblasts were more resistant than melanoma cell lines; however, both expressed Dectin-2. Sodium ascorbate at micromolar concentrations eliminated the cytotoxicity of UVC in these cell lines. Normal cells are generally UVC-resistant compared to corresponding malignant cells, which have higher growth potential. Dectin-2 protein expression itself may not be determinant of UVC sensitivity.

Sida tuberculata: In vitro cytotoxicity and in vivo anti-inflammatory effect

Ethnopharmacological relevanceSida tuberculata R. E. Fries (Malvaceae) is a pioneer species considered a weed in farm fields in Southern Brazil. Widely distributed in South Brazil, S. tuberculata is popularly used to treat inflammatory conditions. Aims of the studyThe current study aimed to assess the in vitro cytotoxic and in vivo anti-inflammatory properties of S. tuberculata. Materials and methodsInitially, extracts obtained from leaves (STLE) and roots (STRE) were submitted to cytotoxicity tests using human leukocytes (non-malignant cell line) and HepG2 and MCF-7 (tumor cell lines). In sequence, anti-inflammatory properties were investigated against carrageenan-induced peritonitis model. ResultsIn vitro analyses displayed a significant decrease in human leukocytes viability without genotoxic damage. IC50 results from tumor cells presented significant decrease in cell viability, slightly more pronounced for STRE. In addition, STLE significantly inhibited the inflammatory and oxidative parameters (TBARS, NPSH, SOD, MPO activity, cell influx, and cytokines release). ConclusionOur findings indicate S. tuberculata extracts have cytotoxic potential more pronounced on tumor cell lines, as well as leaves extract shows a significant reduction in acute inflammation process, as already reported for Sida genus and specifically for this species.

How does direct current atmospheric pressure glow discharge application influence on physicochemical, nutritional, microbiological, and cytotoxic properties of orange juice?

We proposed an innovative and economic method for rapid production of functionalized orange juice (OJ) with excellent nutritional properties, prolonged shelf life, and safe consumption. To reach this goal, we have employed direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD) in a highly-throughput reaction-discharge system. It was found that controlled FLC-dc-APGD-treatment of OJ lead to increase the concentration of selected metals and phenolic compounds.The so-obtained OJ had the same qualitative composition of fragrance as the untreated one, however, its shelf life was prolonged up to 26 days. Furthermore, OJ exposed to FLC-dc-APGD-treatment did not exhibit any cytotoxic properties towards non-malignant human intestinal epithelial cell lines. On the other hand, the induction of cell cytotoxicity was observed in human colorectal adenocarcinoma cells line after FLC-dc-APGD-treated OJ application. We truly believe that produced by us functionalized OJ might be a tempting alternative to classic, non-treated by FLC-dc-APGD OJ.

Chlovalicin B, a Chlorinated Sesquiterpene Isolated from the Marine Mushroom Digitatispora marina.

As part of our search for bioactive metabolites from understudied marine microorganisms, the new chlorinated metabolite chlovalicin B (1) was isolated from liquid cultures of the marine basidiomycete Digitatispora marina, which was collected and isolated from driftwood found at Vannøya, Norway. The structure of the novel compound was elucidated by spectroscopic methods including 1D and 2D NMR and analysis of HRMS data, revealing that 1 shares its molecular scaffold with a previously isolated compound, chlovalicin. This represents the first compound isolated from the Digitatispora genus, and the first reported fumagillin/ovalicin-like compound isolated from Basidiomycota. Compound 1 was evaluated for antibacterial activities against a panel of five bacteria, its ability to inhibit bacterial biofilm formation, for antifungal activity against Candida albicans, and for cytotoxic activities against malignant and non-malignant human cell lines. Compound 1 displayed weak cytotoxic activity against the human melanoma cell line A2058 (~50% survival at 50 µM). No activity was detected against biofilm formation or C. albicans at 50 µM, or against bacterial growth at 100 µM nor against the production of cytokines by the human acute monocytic leukemia cell line THP-1 at 50 µM.

Abstract P140: Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer

Abstract Breast cancer (BC) continues to pose a major health burden. In 2020 alone, over 2 million people were diagnosed with BC globally and of these 684 996 people succumbed to the disease. Cancer cell drug resistance and the notion of transcription factors as undruggable targets has slowed progress for novel BC therapies. The transcription factor, TBX2, is commonly over-expressed in breast cancer with various hormone receptor statuses where it functions as a powerful pro-proliferative factor by directly repressing p21, PTEN, CST-6, and NDGR1 expression. Furthermore, TBX2 mediates Cisplatin drug resistance by inducing an S-phase cell cycle arrest allowing for cisplatin-induced DNA damage repair and breast cancer cell survival. Targeting TBX2 with DNA-damaging chemotherapies therefore emerges as a promising anti-cancer strategy to treat BC. To this end, we performed a reverse affinity screen to fish for marine-derived compounds with DNA-damaging- and TBX2 binding- activity and identified the antibiotic Chromomycin A5 (CMA5) as a promising agent. This project confirms the binding affinity of CMA5 for TBX2 by thermophoresis and fully characterizes the in vitro anticancer activity of CMA5 in TBX2-driven BC cells. Short-term and long-term selective cytotoxicity of CMA5 were investigated in two TBX2-driven BC cell lines (MCF-7 and T47D) and a non-malignant breast epithelial cell line (MCF-12A) using MTT and clonogenic assays. Our results show that CMA5 exhibited potent (IC50 ≤6.5 nM) and selective (SI ~ 3.3 ) cytotoxicity against BC cells. Importantly, CMA5 inhibited TBX2 protein levels and co-treatment of the BC cells with CMA5 and the proteasome 26S inhibitor MG132 rescued TBX2 levels. These results show that CMA5 targets TBX2 for protein degradation. Furthermore, whereas knocking down TBX2 using shRNA blocked the cytotoxicity of CMA5 (IC50 increased by 5-fold), ectopically over-expressing TBX2 increased the cytotoxic activity of CMA5 (IC50 decreased by 1.2-fold). These results suggest that CMA5 cytotoxicity is dependent, in part, on the presence of TBX2. To further explore the significance of TBX2 inhibition by CMA5 we investigated the impact of CMA5 on expression levels of the TBX2 target genes p21, PTEN, CST-6 and NDGR1 by quantitative real-time PCR analyses. The results revealed that all four genes were upregulated in the presence of CMA5 treatment which suggested that CMA5 inhibits BC cell viability and survival by targeting TBX2 and thus releasing its ability to repress these tumor suppressors. To elucidate the mechanism(s) underpinning the cytotoxic activity of CMA5 we investigated its effects on several markers of DNA damage (yH2AX expression) and apoptosis (cleaved caspase 3, 7, 8, 9 and PARP) by western blotting and immunocytochemistry. Our results show that CMA5 induced DNA damage and killed cells, in part, by both the intrinsic and extrinsic apoptotic programmed cell death pathways. Overall, this study identifies CMA5 as a promising anti-breast cancer agent because of its capability to induce DNA damage and to inhibit the oncogenic TBX2. Citation Format: Claire Bellis, Suparna Chakraborty, Mihlali Mlaza, Bianca Del Bianco Sahm, Paula Rezende Teixeira, Leticia Costa-Lotufo, Sharon Prince. Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P140.

Comparison of FTIR spectra of different breast cell lines to detect spectral biomarkers of pathology

Many efforts have been made for several years to make Fourier Transform Infrared (FTIR) micro-spectroscopy usable in the cytological diagnostic. In fact, some events arising when cells undergo a disease, as alterations in the relative content of biochemical cellular components and variations of the proteins secondary structure, lead to modifications of the FTIR spectra, as changes of relative intensity and/or spectral shifts of specific peaks. In this study, we compare FTIR spectra measured for three different breast cell lines: the non-malignant MCF10A cell line, the malignant non-metastatic MCF7 cell line and the metastatic MDA cell line. The major differences of average absorption spectra involve a blue-shift of amide I and II bands and a larger relative intensity of nucleic acid bands in MCF7 spectra with respect to the other ones. Curve fitting also provided important information about spectral markers able to discriminate the three cell lines. In fact, the ratio of sum of the areas of peaks at 2852 and 2922 cm−1 with respect to the sum of the areas of peaks at 2873 and 2958 cm−1 is able to differentiate the non-malignant, malignant and metastatic cells among them. These results suggest that FTIR micro-spectroscopy may be a potentially useful tool for breast cancer diagnosis at cytological level.

Ciprofloxacin and Levofloxacin as Potential Drugs in Genitourinary Cancer Treatment-The Effect of Dose-Response on 2D and 3D Cell Cultures.

Introduction: Introducing new drugs for clinical application is a very difficult, long, drawn-out, and costly process, which is why drug repositioning is increasingly gaining in importance. The aim of this study was to analyze the cytotoxic properties of ciprofloxacin and levofloxacin on bladder and prostate cell lines in vitro. Methods: Bladder and prostate cancer cell lines together with their non-malignant counterparts were used in this study. In order to evaluate the cytotoxic effect of both drugs on tested cell lines, MTT assay, real-time cell growth analysis, apoptosis detection, cell cycle changes, molecular analysis, and 3D cultures were examined. Results: Both fluoroquinolones exhibited a toxic effect on all of the tested cell lines. In the case of non-malignant cell lines, the cytotoxic effect was weaker, which was especially pronounced in the bladder cell line. A comparison of both fluoroquinolones showed the advantage of ciprofloxacin (lower doses of drug caused a stronger cytotoxic effect). Both fluoroquinolones led to an increase in late apoptotic cells and an inhibition of cell cycle mainly in the S phase. Molecular analysis showed changes in BAX, BCL2, TP53, and CDKN1 expression in tested cell lines following incubation with ciprofloxacin and levofloxacin. The downregulation of topoisomerase II genes (TOP2A and TOP2B) was noticed. Three-dimensional (3D) cell culture analysis confirmed the higher cytotoxic effect of tested fluoroquinolone against cancer cell lines. Conclusions: Our results suggest that both ciprofloxacin and levofloxacin may have great potential, especially in the supportive therapy of bladder cancer treatment. Taking into account the low costs of such therapy, fluoroquinolones seem to be ideal candidates for repositioning into bladder cancer therapeutics.

Securinine induces mitotic block in cancer cells by binding to tubulin and inhibiting microtubule assembly: A possible mechanistic basis for its anticancer activity

AimAnalysis of the anticancer and antimitotic activity of the plant derived alkaloid securinine along with its effect on the organization of cellular microtubules as well as its binding with purified goat brain tubulin in-vitro. Materials and methodsThe cytotoxicity of securinine on different cell lines was conducted using SRB assay. The effect of securinine on the cellular microtubules was analyzed using immunofluorescence microscopy. The binding of securinine on purified goat brain tubulin was evaluated using fluorescent spectroscopy. Key findingsSecurinine effectively prevented the proliferation of cervical, breast and lung cancer cells with an IC50 of 6, 10 and 11 μM respectively and induced minimal toxicity in HEK cell line. Securinine at concentrations higher than IC50 induced significant depolymerization in interphase and mitotic microtubules and it suppressed the reassembly of cold depolymerized spindle microtubules in HeLa cells. In the wound healing assay, securinine effectively suppressed the migration of HeLa cells to close the wound. Securinine bound to tubulin with a Kd of 9.7 μM and inhibited the assembly of tubulin into microtubules. The treatment with securinine induced a mitochondrial dependent ROS response in HeLa cells which enhanced the cytotoxic effect of securinine. The result from gene expression studies indicates that securinine induced apoptosis in MCF-7 cells through p53 dependent pathway. SignificanceConsidering the strong anticancer and anti-metastatic property and low toxicity in non-malignant cell lines, we suggest that securinine can be used as a chemotherapeutic drug either alone or in combination with other known anticancer molecules.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients’ asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.