FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs.

BACKGROUND: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis. METHODS: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value. RESULTS: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients. CONCLUSIONS: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.

Elevated expression of complement factor I in lung cancer cells associates with shorter survival-Potentially via non-canonical mechanism.

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

The Influence of Bacteriophages on the Metabolic Condition of Human Fibroblasts in Light of the Safety of Phage Therapy in Staphylococcal Skin Infections.

Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages-vB_SauM-A, vB_SauM-C and vB_SauM-D-high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic.

Synthesis of 3-(2-Alkylthio-4-chloro-5-methylbenzenesulfonyl)-2-(1-phenyl-3-arylprop-2-enylideneamino)guanidine Derivatives with Pro-Apoptotic Activity against Cancer Cells.

The untypical course of reaction between chalcones and benzenesulfonylaminoguanidines led to the new 3-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-2-(1-phenyl-3-arylprop-2-enylideneamino)guanidine derivatives 8-33. The new compounds were tested in vitro for their impact on the growth of breast cancer cells MCF-7, cervical cancer cells HeLa and colon cancer cells HCT-116 by MTT assay. The results revealed that the activity of derivatives is strongly related to the presence of hydroxy group in the benzene ring at the 3-arylpropylidene fragment. The most cytotoxic compounds 20 and 24 displayed mean IC50 values of 12.8 and 12.7 µM, respectively, against three tested cell lines and were almost 3- and 4-fold more active toward MCF-7 and HCT-116 when compared with non-malignant HaCaT cells. Furthermore, compound 24 induced apoptosis in cancer cells and caused a decrease of mitochondrial membrane potential as well as an increase of cells in sub-G1 phase in contrast to its inactive analog 31. The strongest activity against the most sensitive HCT-116 cell line was found for compound 30 (IC50 = 8 µM), which was 11-fold more effective in the growth inhibition of HCT-116 cells than those of HaCaT cells. Based on this fact, the new derivatives may be promising leading structures for the search for agents for the treatment of colon cancer.

The Acquisition of Complement-Dependent Cytotoxicity by the Type II Anti-CD20 Therapeutic Antibody Obinutuzumab.

Rituximab, a prototypic anti-CD20 mAb, and the third-generation anti-CD20 mAb obinutuzumab differ in their ability to activate the complement system. According to recent studies, this contrast stems from the architecture of the antigen-antibody complex formed by these two mAbs that facilitates (rituximab) or disables (obinutuzumab) further oligomerization, leading to engagement of the initial classical complement pathway component C1q. We examined whether a gain-of-function C2 variant that acts downstream of C1q and enforces the formation of complement convertase resistant to physiological decay can impact complement activation by obinutuzumab. Co-application of the C2 variant with obinutuzumab and human serum resulted in complement-dependent cytotoxicity equal to or higher than attainable for rituximab. This effect was observed either in serum or hirudin-anticoagulated whole blood. Long-term (24 h) overall cytotoxicity of obinutuzumab was improved in target cells of moderate sensitivity to complement but diminished in cells of low sensitivity. Our results demonstrate that the ability of complement activation of a given antibody is not ultimately determined at the stage of initial interactions with its target antigen but is modulable at later stages of the cascade and that the benefit of the acquisition of this new effector mechanism by obinutuzumab depends on the target cell characteristics.

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.

In Silico Designed Gain-of-Function Variants of Complement C2 Support Cytocidal Activity of Anticancer Monoclonal Antibodies.

The molecular target for the classical complement pathway (CP) is defined by surface-bound immunoglobulins. Therefore, numerous anticancer monoclonal antibodies (mAbs) exploit the CP as their effector mechanism. Conversely, the alternative complement pathway (AP) is spontaneously induced on the host and microbial surfaces, but complement inhibitors on host cells prevent its downstream processing. Gain-of-function (GoF) mutations in the AP components that oppose physiological regulation directly predispose carriers to autoimmune/inflammatory diseases. Based on the homology between AP and CP components, we modified the CP component C2 so that it emulates the known pathogenic mutations in the AP component, factor B. By using tumor cell lines and patient-derived leukemic cells along with a set of clinically approved immunotherapeutics, we showed that the supplementation of serum with recombinant GoF C2 variants not only enhances the cytocidal effect of type I anti-CD20 mAbs rituximab and ofatumumab, but also lowers the threshold of mAbs necessary for the efficient lysis of tumor cells and efficiently exploits the leftovers of the drug accumulated in patients' sera after the previous infusion. Moreover, we demonstrate that GoF C2 acts in concert with other therapeutic mAbs, such as type II anti-CD20, anti-CD22, and anti-CD38 specimens, for overcoming cancer cells resistance to complement attack.

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein.

The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2.

A tale of the monoclonal anti-CD20 antibodies, in tribute to prof. Waclaw Szybalski (1921-2020).

Technical advances that lead to the era of targeted therapeutics demanded several milestones that were reached in the second half of the previous century. Professor Waclaw Szybalski was the first one to perform a stable gene transfer in eukaryotic cells. To do so, he used his own designed system consisting of HPRT-deficient cells and HAT selective medium. Moreover, the first-ever hybridoma cells were also constructed by Waclaw Szybalski's team. These spectacular achievements made him not only a forerunner of gene therapy, but also became a foundation for immunotherapy, as hybridoma and their selection by the HPRT-HAT system turned into a crucial technical step during production of monoclonal antibodies (mAbs). Herein, we present a story of anti-CD20 mAbs, one of the most successful lines of anticancer drugs. When looking back into history, the prototypic mAb rituximab was considered the biggest step forward in the therapy of B-cell malignancies. Nowadays, the second and third generations of anti-CD20 mAbs are approved in clinical use and numerous breakthrough studies on immune effector mechanisms were conducted with the aforementioned immunotherapeutics as a model.

Monitoring of the Complement System Status in Patients With B-Cell Malignancies Treated With Rituximab.

Rituximab is a pioneering anti-CD20 monoclonal antibody that became the first-line drug used in immunotherapy of B-cell malignancies over the last twenty years. Rituximab activates the complement system in vitro, but there is an ongoing debate on the exact role of this effector mechanism in therapeutic effect. Results of both in vitro and in vivo studies are model-dependent and preclude clear clinical conclusions. Additional confounding factors like complement inhibition by tumor cells, loss of target antigen and complement depletion due to excessively applied immunotherapeutics, intrapersonal variability in the concentration of main complement components and differences in tumor burden all suggest that a personalized approach is the best strategy for optimization of rituximab dosage and therapeutic schedule. Herein we critically review the existing knowledge in support of such concept and present original data on markers of complement activation, complement consumption, and rituximab accumulation in plasma of patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas (NHL). The increase of markers such as C4d and terminal complement complex (TCC) suggest the strongest complement activation after the first administration of rituximab, but not indicative of clinical outcome in patients receiving rituximab in combination with chemotherapy. Both ELISA and complement-dependent cytotoxicity (CDC) functional assay showed that a substantial number of patients accumulate rituximab to the extent that consecutive infusions do not improve the cytotoxic capacity of their sera. Our data suggest that individual assessment of CDC activity and rituximab concentration in plasma may support clinicians' decisions on further drug infusions, or instead prescribing a therapy with anti-CD20 antibodies like obinutuzumab that more efficiently activate effector mechanisms other than complement.

Novel N-(aryl/heteroaryl)-2-chlorobenzenesulfonamide derivatives: Synthesis and anticancer activity evaluation.

A new series of N-(aryl/heteroaryl)-2-chlorobenzenesulfonamide derivatives 4-21 have been synthesized, and evaluated at the National Cancer Institute (USA) for their in vitro activities against a panel of 60 different human cancer cell lines. Among them, compounds 16, 20 and 21 exhibited remarkable cytotoxic activity against numerous human cancer cell lines. We found that sulfonamide derivative 21 appeared to be more selective than compounds 16 and 20. In comparison to compounds 16 and 20 it showed higher cytotoxic activity against A549 non-small cell lung adenocarcinoma and HCT-116 colon carcinoma cells and was less toxic to HEK-293 human embryonic kidney cells and HaCaT immortalized human keratinocytes. Treatment of A549 and HCT-116 cells with compound 21 resulted in the G0/G1-cell cycle arrest with a concomitant increase in p53 and p21 protein levels. Moreover, compound 21 led to ATP depletion and disruption of the mitochondrial membrane potential in both studied cell lines. Our results suggest that 2,4-dichloro-N-(quinolin-8-yl and/or 1H-indazol-7-yl)benzenesulfonamides serve as novel promising anticancer agents.

Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies.

Monoclonal antibodies ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) which are approved for usage in patients with chronic lymphocytic leukemia (CLL), efficiently activate the classical complement pathway. However complement is an exhaustible component and high doses of its activators may deplete complement-dependent cytotoxicity (CDC) potential, thus reducing the effect of repeated mAb dosing. Widely used method to measure CDC activity of patients' serum is hemolytic assay (CH50) on sheep erythrocytes. Despite its simplicity, such CH50 assay may not reflect pivotal interactions between patient serum and human complement inhibitors on the surface of target cells. We propose calcein release assay performed on tumor cells similar to those targeted by therapeutic antibodies as an alternative method. We analyzed serum samples collected from 12 patients participating in the clinical study, receiving s.c. 30 mg alemtuzumab three times per week combined with i.v. ofatumumab at an initial dose of 300 mg in week 3 further escalated to 2000 mg every other week. All serum samples were measured by hemolytic assay on sheep erythrocytes as well as using calcein release assay on CD20-positive Raji cells. Our data show that results obtained from both assays are related to each other at the level of the whole group (n = 96 samples, Spearman r = 0.504, p < .001) but may substantially differ when analyzing individual patients. Furthermore, by using CDC assay on Raji cells, we found that in the presented clinical study CDC serum potential was not significantly affected when measured before consecutive administrations in most of the patients.

Defective apoptosis of U937 cells induced by benzyl isothiocyanate (BITC).

Isothiocyanates precursors (ITCs), including benzyl isothiocyanate (BITC), are considered as cancer chemopreventive agents. ITC derivatives were tested in clinical trials (NCT00005883, NCT01265953, NCT01790204) and preclinical studies aimed to inhibit tumor growth and modulation of their microenvironment. Although efficacy of ITCs was demonstrated with several leukemic cell lines, the final steps of BITC-induced apoptosis were not completely elucidated in the literature. Therefore, we focused on morphological and biochemical events occurring upon treatment of U937 leukemia cells with BITC. Micromolar concentrations of BITC induced cytotoxicity in U937 cells, with major features resembling the hallmarks of apoptosis: phosphatidylserine exposure, low mitochondrial membrane potential, and presence of PARP cleavage by caspases. Disassembly to apoptotic bodies, a final step of classic apoptosis, was not observed. While tracing the signalling pathways, our results showed increased levels of BAG-1 and PUMA proteins, but in contrast to other models of ITCs-induced apoptosis, downregulation of Mcl-1 protein was not noticed. Additionally, BITC-induced dying U937 cells released lower levels of chemoattractants, such as IL-8 and MCP-1, when compared to cells undergoing classical apoptosis. This may disrupt clearance of cell debris by macrophages in vivo (efferocytosis), and in turn affect the inflammatory response. In summary, BITC inhibits tumor growth which makes it a good candidate for supporting cancer treatment. However, atypical apoptosis of leukemic U937 cells induced with BITC may affect the ability of phagocytes to effectively scavenge cellular debris, which poses a question of BITC effectiveness as a chemopreventive agent for leukemias.

Clinical and Biological Significance of ESR1 Gene Alteration and Estrogen Receptors Isoforms Expression in Breast Cancer Patients.

The amplification of estrogen receptor alpha (ERα) encoded by the ESR1 gene has been described as having a prognostic role in breast cancer patients. However, increased dosage of the ESR1 gene (tested by real-time PCR) is also observed in ER-negative breast cancers, which might suggest the expression of alternative isoforms of ERα (other than classical ERα of 66 kDa). In the current work, we have investigated the ESR1 gene dosage in 402 primary breast cancer patients as well as the expression of ERα isoforms-ERα66 and ERα36-on mRNA and protein levels. The obtained results were correlated with clinicopathological data of the patients. Results showed that increased ESR1 gene dosage is not related to ESR1 gene amplification measured by fluorescent in situ hybridization (FISH), but it correlates with the decreased expression of ERα66 isoform (p = 0.01). Interestingly, the short ER isoform ERα36 was expressed in samples with increased ESR1 gene dosage, suggesting that genomic aberration might influence the expression of that particular isoform. Similarly to ESR1 increased gene dosage, high ERα36 expression was linked with the decreased disease-free survival of the patients (p = 0.05), which was independent of the status of the classical ERα66 level in breast tumors.

Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics.

Anti-CD20 monoclonal antibodies (mAbs) rituximab and ofatumumab are potent activators of the classical complement pathway, and have been approved for the treatment of B-cell malignancies. However, complement exhaustion and overexpression of complement inhibitors by cancer cells diminish their therapeutic potential. The strategies of targeting membrane complement inhibitors by function-blocking antibodies and the supplementation with fresh frozen plasma have been proposed to overcome tumour cell resistance. We present a novel approach, which utilizes gain-of-function variants of complement factor B (FB), a component of alternative C3/C5 convertases, which augment mAb-activated reactions through a positive feedback mechanism called an amplification loop. If complement concentration is limited, an addition of quadruple gain-of-function FB mutant p.D279G p.F286L p.K323E p.Y363A (or selected single mutants) results in significantly increased complement-mediated lysis of ofatumumab-resistant tumour cells, as well as the complete lysis of moderately sensitive cells. Importantly, this effect cannot be achieved by further increasing ofatumumab concentration. Potentiation of cytotoxic effect towards moderately sensitive cells was less apparent at physiological serum concentration. However, an addition of hyperactive FB could compensate the loss of cytotoxic potential of serum collected from the NHL and CLL patients after infusion of rituximab. Residual levels of rituximab in such sera, in combination with added FB, were able to efficiently lyse tumour cells. We suggest that the administration of gain-of-function variants of FB can restore cytotoxic potential of complement-exhausted serum and maximize the therapeutic effect of circulating anti-CD20 mAbs.

Synthesis, molecular structure, and metabolic stability of new series of N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-1-(5-phenyl-1H-pyrazol-1-yl)amidine as potential anti-cancer agents.

A series of new N'-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-1-(5-phenyl-1H-pyrazol-1-yl)amidine derivatives have been synthesized and evaluated in vitro by MTT assays for their antiproliferative activity against cell lines of colon cancer HCT-116, cervical cancer HeLa and breast cancer MCF-7. The studied compounds display selective activity mainly against HCT-116 and HeLa cells. Thus, five compounds show selective cytotoxic effect against HCT-116 (IC50 = 3-10 µM) and HeLa (IC50 = 7 µM). Importantly, the noticed values of IC50 for four compounds are almost 4-fold lower for HeLa than non-malignant HaCaT cells. More-in-depth biological research revealed that the treatment of HCT-116 and HeLa with active compound resulted in increased numbers of cells in sub-G1 phase in a time dependent manner, while non-active derivative does not influence cell cycle. Metabolic stability assays using liver microsomes and NADPH provide important information on compounds susceptibility to phase 1 biotransformation reactions.

The Designer Drug 3-Fluoromethcathinone Induces Oxidative Stress and Activates Autophagy in HT22 Neuronal Cells.

Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Although many synthetic cathinones have lost their legal status in many countries, their abuse still continues worldwide. Recently, they have been reported to exert neurotoxic effects in vitro and in vivo. The molecular mechanisms of their action have not been fully elucidated. Recently, they have been linked to the induction of oxidative stress, autophagy, and apoptosis. The aim of this study was to investigate whether 3-fluoromethcathinone (3-FMC), a synthetic cathinone, is able to induce oxidative stress, autophagy, and apoptosis in HT22 immortalized mouse hippocampal cells. We found that treatment of HT22 cells with this compound results in a concentration-dependent increase in the intracellular production of reactive oxygen species. Moreover, 3-FMC induced concentration-dependent conversion of cytosolic LC3-I to membrane-bound LC3-II and formation of autophagic vacuoles. Additionally, the level of p62/SQSTM1 protein decreased after 3-FMC treatment, suggesting that accumulation of autophagic vacuoles resulted from activation rather than inhibition of autophagy. Our results also showed that 3-FMC at millimolar concentration is able to induce caspase-dependent apoptotic cell death in HT22 cells. Our findings suggest that abuse of 3-FMC may disturb neuronal homeostasis and impair functioning of the central nervous system.

MiR-192 and miR-662 enhance chemoresistance and invasiveness of squamous cell lung carcinoma.

OBJECTIVES: Overexpression of miR-192, miR-192* and miR-662 was previously found to correlate with poor prognosis of early-stage squamous cell lung cancer (SCC) patients. In this study, we investigated the relevance of these miRNAs to cancer cell biology and chemoresistance. MATERIALS AND METHODS: MiRNA expression profile was analysed in 10 non-small cell lung cancer (NSCLC) cell lines using RT-qPCR. H520 and H1703 cells were transfected with miRNA inhibitors (anti-miR-192, -192* and -662) for functional studies. Chemoresistance to cisplatin and etoposide was evaluated using MTT colorimetric assay. H520 cells were subjected to 3D soft-agar colony formation assay and H1703 cells to wound healing assay. Whole transcriptome analysis was used to assess the effect of miR-192 and miR-662 inhibition on gene expression. RESULTS: SCC cell lines, H520 and H1703, differed in miRNA expression and phenotypic features. MiR-192 and miR-662 inhibition decreased clonogenicity and motility of SCC cells. MiR-192 and miR-662 inhibition sensitized SCC cells to etoposide but not to cisplatin. Whole transcriptome analysis revealed genes regulated by miR-192 and miR-662 in SCC, relevant to maintaining chemoresistance, invasiveness, epithelial-mesenchymal transition (EMT) and immune evasion. CONCLUSIONS: We showed for the first time that miR-192 and miR-662 have functional role in SCC cells. Our findings suggest that targeting these miRNAs may impact both chemoresistance and invasiveness of SCC, and add to the evidence linking these aspects of tumour biology. Overexpression of miR-192 and miR-662 might be useful as a marker of resistance to etoposide.