Decreased TSPAN14 Expression Contributes to NSCLC Progression.

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression.

New Therapeutic Strategy for Overcoming Multidrug Resistance in Cancer Cells with Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) often interact with the multidrug resistant (MDR) phenotype of cancer cells. In some cases, TKIs increase the susceptibility of MDR cancer cells to chemotherapy. As the overexpression of membrane transporter P-glycoprotein (P-gp) is the most common alteration in MDR cancer cells, we investigated the effects of TKI pyrazolo[3,4-d]pyrimidines on P-gp inhibition in two cellular models comprising sensitive and corresponding MDR cancer cells (human non-small cell lung carcinoma and colorectal adenocarcinoma). Tested TKIs showed collateral sensitivity by inducing stronger inhibition of MDR cancer cell line viability. Moreover, TKIs directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was proposed by molecular docking simulations. TKIs reversed resistance to doxorubicin and paclitaxel in a concentration-dependent manner. The expression studies excluded the indirect effect of TKIs on P-gp through regulation of its expression. A kinetics study showed that TKIs decreased P-gp activity and this effect was sustained for seven days in both MDR models. Therefore, pyrazolo[3,4-d]pyrimidines with potential for reversing P-gp-mediated MDR even in prolonged treatments can be considered a new therapeutic strategy for overcoming cancer MDR.

Development and Validation of a Long-Term 3D Glioblastoma Cell Culture in Alginate Microfibers as a Novel Bio-Mimicking Model System for Preclinical Drug Testing.

BACKGROUND: Various three-dimensional (3D) glioblastoma cell culture models have a limited duration of viability. Our aim was to develop a long-term 3D glioblastoma model, which is necessary for reliable drug response studies. METHODS: Human U87 glioblastoma cells were cultured in alginate microfibers for 28 days. Cell growth, viability, morphology, and aggregation in 3D culture were monitored by fluorescent and confocal microscopy upon calcein-AM/propidium iodide (CAM/PI) staining every seven days. The glioblastoma 3D model was validated using temozolomide (TMZ) treatments 3 days in a row with a recovery period. Cell viability by MTT and resistance-related gene expression (MGMT and ABCB1) by qPCR were assessed after 28 days. The same TMZ treatment schedule was applied in 2D U87 cell culture for comparison purposes. RESULTS: Within a long-term 3D model system in alginate fibers, U87 cells remained viable for up to 28 days. On day 7, cells formed visible aggregates oriented to the microfiber periphery. TMZ treatment reduced cell growth but increased drug resistance-related gene expression. The latter effect was more pronounced in 3D compared to 2D cell culture. CONCLUSION: Herein, we described a long-term glioblastoma 3D model system that could be particularly helpful for drug testing and treatment optimization.

In vitro biomimetic models for glioblastoma-a promising tool for drug response studies.

The poor response of glioblastoma to current treatment protocols is a consequence of its intrinsic drug resistance. Resistance to chemotherapy is primarily associated with considerable cellular heterogeneity, and plasticity of glioblastoma cells, alterations in gene expression, presence of specific tumor microenvironment conditions and blood-brain barrier. In an attempt to successfully overcome chemoresistance and better understand the biological behavior of glioblastoma, numerous tri-dimensional (3D) biomimetic models were developed in the past decade. These novel advanced models are able to better recapitulate the spatial organization of glioblastoma in a real time, therefore providing more realistic and reliable evidence to the response of glioblastoma to therapy. Moreover, these models enable the fine-tuning of different tumor microenvironment conditions and facilitate studies on the effects of the tumor microenvironment on glioblastoma chemoresistance. This review outlines current knowledge on the essence of glioblastoma chemoresistance and describes the progress achieved by 3D biomimetic models. Moreover, comprehensive literature assessment regarding the influence of 3D culturing and microenvironment mimicking on glioblastoma gene expression and biological behavior is also provided. The contribution of the blood-brain barrier as well as the blood-tumor barrier to glioblastoma chemoresistance is also reviewed from the perspective of 3D biomimetic models. Finally, the role of mathematical models in predicting 3D glioblastoma behavior and drug response is elaborated. In the future, technological innovations along with mathematical simulations should create reliable 3D biomimetic systems for glioblastoma research that should facilitate the identification and possibly application in preclinical drug testing and precision medicine.

Advanced technological tools to study multidrug resistance in cancer.

The complexity of cancer biology and its clinical manifestation are driven by genetic, epigenetic, transcriptomic, proteomic and metabolomic alterations, supported by genomic instability as well as by environmental conditions and lifestyle factors. Although novel therapeutic modalities are being introduced, efficacious cancer therapy is not achieved due to the frequent emergence of distinct mechanisms of multidrug resistance (MDR). Advanced technologies with the potential to identify and characterize cancer MDR could aid in selecting the most efficacious therapeutic regimens and prevent inappropriate treatments of cancer patients. Herein, we aim to present technological tools that will enhance our ability to surmount drug resistance in cancer in the upcoming decade. Some of these tools are already in practice such as next-generation sequencing. Identification of genes and different types of RNAs contributing to the MDR phenotype, as well as their molecular targets, are of paramount importance for the development of new therapeutic strategies aimed to enhance drug response in resistant tumors. Other techniques known for many decades are in the process of adaptation and improvement to study cancer cells' characteristics and biological behavior including atomic force microscopy (AFM) and live-cell imaging. AFM can monitor in real-time single molecules or molecular complexes as well as structural alterations occurring in cancer cells induced upon treatment with various antitumor agents. Cell tracking methodologies and software tools recently progressed towards quantitative analysis of the spatio-temporal dynamics of heterogeneous cancer cell populations and enabled direct monitoring of cells and their descendants in 3D cultures. Besides, novel 3D systems with the advanced mimicking of the in vivo tumor microenvironment are applicable to study different cancer biology phenotypes, particularly drug-resistant and aggressive ones. They are also suitable for investigating new anticancer treatment modalities. The ultimate goal of using phenotype-driven 3D cultures for the investigation of patient biopsies as the most appropriate in vivo mimicking model, can be achieved in the near future.

Parvifloron D from Plectranthusstrigosus: Cytotoxicity Screening of Plectranthus spp. Extracts.

The Plectranthus genus is commonly used in traditional medicine due to its potential to treat several illnesses, including bacterial infections and cancer. As such, aiming to screen the antibacterial and cytotoxic activities of extracts, sixteen selected Plectranthus species with medicinal potential were studied. In total, 31 extracts obtained from 16 Plectranthus spp. were tested for their antibacterial and anticancer properties. Well diffusion method was used for preliminary antibacterial screening. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the five most active acetonic extracts (P. aliciae, P. japonicus, P. madagascariensis var. "Lynne", P. stylesii, and P. strigosus) were determined. After preliminary toxicity evaluation on Artemia salina L., their cytotoxic properties were assessed on three human cancer cell lines (HCT116, MCF-7, and H460). These were also selected for mechanism of resistance studies (on NCI-H460/R and DLD1-TxR cells). An identified compound-parvifloron D-was tested in a pair of sensitive and MDR-Multidrug resistance cancer cells (NCI-H460 and NCI-H460/R) and in normal bronchial fibroblasts MRC-5. The chemical composition of the most active extract was studied through high performance liquid chromatography with a diode array detector (HPLC-DAD/UV) and liquid chromatography-mass spectrometry (LC-MS). Overall, P. strigosus acetonic extract showed the strongest antimicrobial and cytotoxic potential that could be explained by the presence of parvifloron D, a highly cytotoxic diterpene. This study provides valuable information on the use of the Plectranthus genus as a source of bioactive compounds, namely P. strigosus with the potential active ingredient the parvifloron D.

Cytotoxic Activity of Royleanone Diterpenes from Plectranthus madagascariensis Benth.

Cytotoxicity screenings have identified Plectranthus plants as potential sources of antitumor lead compounds. In this work, several extracts from Plectranthus madagascariensis were prepared using different solvents (acetone, methanol, and supercritical CO2) and extraction techniques (maceration, ultrasound-assisted, and supercritical fluid extraction), and their chemical composition was detailed using high-performance liquid chromatography with a diode array detector. The cytotoxic activity of the major compounds identified, namely, rosmarinic acid (1) and abietane diterpenes 7α,6ß-dihydroxyroyleanone (2), 7α-formyloxy-6ß-hydroxyroyleanone (3), 7α-acetoxy-6ß-hydroxyroyleanone (4), and coleon U (5), was evaluated in a battery of human cancer cell lines, including breast (MDA-MB-231, MCF-7), colon (HCT116), and lung (NCI-H460, NCI-H460/R) cancer, and also in healthy lung (MCR-5) cells. Royleanone (3) was isolated for the first time from P. madagascariensis, and its full spectroscopic characterization (proton and carbon nuclear magnetic resonance) was accomplished. A high selectivity for lung cancer cells was observed for royleanones (2, 4) with selectivity indexes of 4.3 and 3.2, respectively. The observed results combined with literature data allowed the establishment of important structure-activity relationships for substituted royleanone abietanes, such as the requirement for an electron-donating group at positions 6 and/or 7 in the abietane skeleton, and an improved cytotoxic effect for substituents with log P values between 2 and 5.

Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance.

New 6-triazolyl-substituted sulfocoumarins were described as potent inhibitors of the transmembrane human carbonic anhydrase isoforms, CAIX and CAXII. These membrane associated enzymes that maintain pH and CO2 homeostasis are involved in cancer progression, invasion, and resistance to therapy. Recently, it was shown that CAXII expression associates with the expression of P-glycoprotein in multidrug resistant cancer cells. CAXII regulates P-glycoprotein activity by maintaining high intracellular pHi. In this study, the activity of three new sulfocoumarins was evaluated in three sensitive and corresponding multidrug resistant cancer cell lines with increased P-glycoprotein expression (non-small cell lung carcinoma, colorectal carcinoma and glioblastoma). Compound 3 showed the highest potential for cancer cell growth inhibition in all tested cell lines. Flow cytometric analyses showed that compound 3 induced intracellular acidification, cell cycle arrest in G2/M phase and necrosis in non-small cell lung carcinoma cells. Compound 3 demonstrated irreversible, concentration- and time-dependent inhibition of P-glycoprotein activity in multidrug resistant non-small cell lung carcinoma cells. The suppression of P-glycoprotein activity was accompanied with increased P-glycoprotein expression suggesting a compensatory mechanism of multidrug resistant cancer cells. In addition, compound 3 was able to sensitize multidrug resistant non-small cell lung carcinoma cells to doxorubicin. Overall, results imply that compound 3 has multidrug resistance modulating effect through intracellular acidification and subsequent inhibition of P-glycoprotein activity.

A New Strategy for Glioblastoma Treatment: In Vitro and In Vivo Preclinical Characterization of Si306, a Pyrazolo[3,4-d]Pyrimidine Dual Src/P-Glycoprotein Inhibitor.

Overexpression of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transporters in multidrug resistant (MDR) cancer cells is responsible for the reduction of intracellular drug accumulation, thus decreasing the efficacy of chemotherapeutics. P-gp is also found at endothelial cells' membrane of the blood-brain barrier, where it limits drug delivery to central nervous system (CNS) tumors. We have previously developed a set of pyrazolo[3,4-d]pyrimidines and their prodrugs as novel Src tyrosine kinase inhibitors (TKIs), showing a significant activity against CNS tumors in in vivo. Here we investigated the interaction of the most promising pair of drug/prodrug with P-gp at the cellular level. The tested compounds were found to increase the intracellular accumulation of Rho 123, and to enhance the efficacy of paclitaxel in P-gp overexpressing cells. Encouraging pharmacokinetics properties and tolerability in vivo were also observed. Our findings revealed a novel role of pyrazolo[3,4-d]pyrimidines which may be useful for developing a new effective therapy in MDR cancer treatment, particularly against glioblastoma.

Modulation of Antioxidant Potential with Coenzyme Q10 Suppressed Invasion of Temozolomide-Resistant Rat Glioma In Vitro and In Vivo.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

Association of Overexpressed MYC Gene with Altered PHACTR3 and E2F4 Genes Contributes to Non-small Cell Lung Carcinoma Pathogenesis.

BACKGROUND: C-Myc is one of the major cellular oncogenes overexpressed in non-small cell lung carcinoma (NSCLC). Its deregulated expression is necessary but not sufficient for malignant transformation. We evaluated expression of MYC gene in NSCLC patients and its association with alterations in the genes previously identified to be related to NSCLC pathogenesis, PHACTR3 and E2F4. METHODS: We analyzed MYC gene expression by qRT-PCR in 30 NSCLC patients' samples and paired normal lung tissue. MYC expression was further statistically evaluated in relation to histopathological parameters, PHACTR3 and E2F4 gene alterations and survival. Alterations in aforementioned genes were previously detected and identified based on AP-PCR profiles of paired normal and tumor DNA samples, selection of DNA bands with altered mobility in tumor samples and their characterization by the reamplification, cloning and sequencing. RESULTS: MYC expression was significantly increased in NSCLC samples and its overexpression significantly associated with squamous cell carcinoma subtype. Most importantly, MYC overexpression significantly coincided with mutations in PHACTR3 and E2F4 genes, in group of all patients and in squamous cell carcinoma subtype. Moreover, patients with jointly overexpressed MYC and altered PHACTR3 or E2F4 showed trend of shorter survival. CONCLUSIONS: Overall, MYC is frequently overexpressed in NSCLC and it is associated with mutated PHACTR3 gene, as well as mutated E2F4 gene. These joint gene alterations could be considered as potential molecular markers of NSCLC and its specific subtypes.

Dual Inhibitors as a New Challenge for Cancer Multidrug Resistance Treatment.

BACKGROUND: Dual-targeting in cancer treatment by a single drug is an unconventional approach in relation to drug combinations. The rationale for the development of dualtargeting agents is to overcome incomplete efficacy and drug resistance frequently present when applying individual targeting agents. Consequently, -a more favorable outcome of cancer treatment is expected with dual-targeting strategies. METHODS: We reviewed the literature, concentrating on the association between clinically relevant and/or novel dual inhibitors with the potential to modulate multidrug resistant phenotype of cancer cells, particularly the activity of P-glycoprotein. A balanced analysis of content was performed to emphasize the most important findings and optimize the structure of this review. RESULTS: Two-hundred and forty-five papers were included in the review. The introductory part was interpreted by 9 papers. Tyrosine kinase inhibitors' role in the inhibition of Pglycoprotein and chemosensitization was illustrated by 87 papers. The contribution of naturalbased compounds in overcoming multidrug resistance was reviewed using 92 papers, while specific dual inhibitors acting against microtubule assembling and/or topoisomerases were described with 55 papers. Eleven papers gave an insight into a novel and less explored approach with hybrid drugs. Their influence on P-glycoprotein and multidrug resistance was also evaluated. CONCLUSION: These findings bring into focus rational anticancer strategies with dual-targeting agents. Most evaluated synthetic and natural drugs showed a great potential in chemosensitization. Further steps in this direction are needed for the optimization of anticancer treatment.

Anticancer properties of the abietane diterpene 6,7-dehydroroyleanone obtained by optimized extraction.

AIM: 6,7-dehydroroyleanone (DHR) is a cytotoxic abietane present in the essential oil of Plectranthus madagascariensis. METHODS/RESULTS: Different extraction parameters were tested, and its extraction optimization was accomplished with a Clevenger apparatus-based hydrodistillation. After isolation, its effect on microtubules, P-glycoprotein and caspases was assessed on several cell lines and the compound was coupled with hybrid nanoparticles. The results show that DHR does not interfere with microtubule formation, but evades the resistance mechanisms of P-glycoprotein. Strong activation of caspases-3 and -9 indicates that DHR is able to induce apoptosis by triggering the intrinsic cell death pathway. Moreover, the assembly of DHR with hybrid nanoparticles was able to potentiate the effect of DHR in cancer cells. CONCLUSION: DHR seems to be a promising starting material with anticancer properties to further be explored.

Jatrophane diterpenoids with multidrug-resistance modulating activity from the latex of Euphorbia nicaeensis.

Seven previously undescribed jatrophane diterpenoids, nicaeenin A-G, with eight known jatrophane diterpenoids, namely euphodendrophanes A-C, F, N, O, Q, S, were isolated from latex of Euphorbia nicaeensis collected in Serbia. The chemical structures of the compounds were determined by spectroscopic analysis including 1D and 2D NMR and HRESIMS experiments. All but one of the previously undescribed jatrophanes, showed significant potential to inhibit P-glycoprotein (P-gp) activity in two MDR cancer cells (NCI-H460/R and DLD1-TxR). The most powerful were nicaeenin F and nicaeenin G. Moreover nicaeenin G significantly stronger sensitized NCI-H460/R cells to DOX than Dex-VER.

Anti-invasive effects of CXCR4 and FAK inhibitors in non-small cell lung carcinomas with mutually inactivated p53 and PTEN tumor suppressors.

Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer. At the time of diagnosis, a large percentage of NSCLC patients have already developed metastasis, responsible for extremely high mortality rates. CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate such invasive cancer behavior. Their expression is downregulated by p53 and PTEN tumor suppressors which are commonly co-inactivated in NSCLC patients and contribute to metastasis. Therefore, targeting CXCR4 or FAK seems to be a promising strategy in suppressing metastatic spread of p53/PTEN deficient NSCLCs. In this study, we first examined the invasive characteristics of NSCLC cells with suppressed p53 and PTEN activity using wound healing, gelatin degradation and invasion assays. Further, changes in the expression of CXCR4 and FAK were evaluated by RT-qPCR and Western Blot analysis. Finally, we tested the ability of CXCR4 and FAK inhibitors (WZ811 and PF-573228, respectively) to suppress the migratory and invasive potential of p53/PTEN deficient NSCLC cells, in vitro and in vivo using metastatic models of human NSCLC. Our results showed that cells with mutually inactive p53 and PTEN have significantly increased invasive potential associated with hyperactivation of CXCR4 and FAK signaling pathways. Treatments with WZ811 and PF-573228 inhibitors significantly reduced migratory and invasive capacity in vitro and showed a trend of improved survival in vivo. Accordingly, we demonstrated that p53/PTEN deficient NSCLCs have extremely invasive phenotype and provided a rationale for the use of CXCR4 or FAK inhibitors for the suppression of NSCLC dissemination.

Targeting CXCR4 and FAK reverses doxorubicin resistance and suppresses invasion in non-small cell lung carcinoma.

BACKGROUND: Current high lung cancer mortality rates are mainly due to the occurrence of metastases and therapeutic resistance. Therefore, simultaneous targeting of these processes may be a valid approach for the treatment of this type of cancer. Here, we assessed relationships between CXC chemokine receptor type 4 (CXCR4) and focal adhesion kinase (FAK) gene expression levels and expression levels of the drug resistance-related genes ABCB1 and ABCC1, and tested the potential of CXCR4 and FAK inhibitors to reverse doxorubicin (DOX) resistance and to decrease the invasive capacity of non-small cell lung carcinoma (NSCLC) cells. METHODS: qRT-PCR was used for gene expression analyses in primary lung tissue samples obtained from 30 NSCLC patients and the human NSCLC-derived cell lines NCI-H460, NCI-H460/R and COR-L23. MTT, flow cytometry, cell death and ß-galactosidase activity assays were used to assess the in vitro impact of CXCR4 and FAK inhibitors on DOX sensitivity. In addition, invasion and gelatin degradation assays were used to assess the in vitro impact of the respective inhibitors on metastasis-related processes in combination with DOX treatment. RESULTS: We found that ABCB1 over-expression was significantly associated with CXCR4 and FAK over-expression, whereas ABCC1 over-expression was associated with increased FAK expression. We also found that CXCR4 and FAK inhibitors strongly synergized with DOX in reducing cell viability, arresting the cell cycle in the S or G2/M phases and inducing senescence. Additionally, we found that DOX enhanced the anti-invasive potential of CXCR4 and FAK inhibitors by reducing gelatin degradation and invasion. CONCLUSIONS: From our data we conclude that targeting of CXCR4 and FAK may overcome ABCB1 and ABCC1-dependent DOX resistance in NSCLC cells and that simultaneous treatment of these cells with DOX may potentiate the anti-invasive effects of CXCR4 and FAK inhibitors.

New Approaches With Natural Product Drugs for Overcoming Multidrug Resistance in Cancer.

Resistance to chemotherapeutic drugs is one of the main obstacles to effective cancer treatment. Multidrug resistance (MDR) is defined as resistance to structurally and/or functionally unrelated drugs, and has been extensively investigated for the last three decades. There are two types of MDR: intrinsic and acquired. Tumor microenvironment selection pressure leads to the development of intrinsic MDR, while acquired resistance is a consequence of the administered chemotherapy. A central issue in chemotherapy failure is the existence of heterogeneous populations of cancer cells within one patient and patient-to-patient variability within each type of cancer. Numerous genes and pathways contribute to the development of MDR in cancer. Point mutations, gene amplification or other genetic or epigenetic changes all affect biological functions and may lead to the occurrence of MDR phenotype. Similar to the characteristics of cancerogenesis, the main features of MDR include abnormal tumor vasculature, regions of hypoxia, aerobic glycolysis, and a lower susceptibility to apoptosis. In order to achieve a lethal effect on cancer cells, drugs need to reach their intracellular target molecules. The overexpression of the efflux transporter P-glycoprotein (P-gp) in MDR cancer cells leads to decreased uptake of the drug and intracellular drug accumulation, minimising drug-target interactions. New agents being or inspired by natural products that successfully target these mechanisms are the main subject of this review. Two key approaches in combating MDR in cancer are discussed (i) finding agents that preserve cytotoxicity toward MDR cancer cells; (ii) developing compounds that restore the cytotoxic activity of classic anticancer drugs.

Lower antioxidative capacity of multidrug-resistant cancer cells confers collateral sensitivity to protoflavone derivatives.

PURPOSE: Multidrug resistance (MDR) may develop due to a series of adaptive responses under a new stress conditions, such as chemotherapy. Novel strategies are urgently needed to fight MDR in cancer, and chemotherapeutics that are selective for MDR cancer cells offer a promising approach. Certain protoflavones were previously found to have potential in this regard. METHODS: Cytotoxicity of six protoflavones was assessed in different P-glycoprotein overexpressing MDR cancer cell lines and in their non-MDR counterparts. The impacts of compound 5, 6-methylprotoflavone previously published and a new derivative, 6-bromoprotoflavone (compound 6), on the cell cycle distribution were evaluated, and 6 was also studied for its potential to regulate the intracellular antioxidative capacity. RESULTS: Protoflavones showed a significant cytotoxicity against all cancer cell lines and selectivity toward MDR cancer cells adapted to conventional chemotherapeutics. Inverse sensitivity versus MDR selectivity pattern was observed. Treatment with H2O2 showed that MDR cancer cells are more vulnerable to oxidative stress. Compounds 5 and 6 significantly decreased the portion of MDR cells in the G1 phase. The levels of reactive oxygen and nitrogen species (ROS/RNS) between MDR and non-MDR cells significantly differed upon exposure to 6, accompanied by changes in the glutathione (GSH) levels and in the expression of manganese superoxide dismutase (MnSOD), glutathione-S-transferase π (GST π) and hypoxia-inducible factor-1α (HIF-1α). CONCLUSIONS: Our results suggest that MDR cancer cells can be more vulnerable to the pro-oxidative activity of protoflavones due to an impaired antioxidative defense that might arise during the adaptation processes provoked by chemotherapy.

Chemo-protective and regenerative effects of diarylheptanoids from the bark of black alder (Alnus glutinosa) in human normal keratinocytes.

Medicinal plants are recognized from ancient times as a source of diverse therapeutic agents and many of them are used as dietary supplements. Comprehensive approaches are needed that would identify bioactive components with evident activity against specific indications and provide a better link between science (ethno-botany, chemistry, biology and pharmacology) and market. Recently, the bark of black alder (Alnus glutinosa) appeared at market in the form of food supplement for treatment of different skin conditions. This study aimed to evaluate protective effects of two diarylheptanoids isolated from the bark of black alder: platyphylloside, 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-ß-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-ß-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2) towards doxorubicin damaging activity. To that end, we employed HaCaT cells, non-cancerous human keratinocytes commonly used for skin regenerative studies. Diarylheptanoids significantly antagonized the effects of doxorubicin by lowering the sensitivity of HaCaT cells to this drug. Compound 2 prevented doxorubicin-induced cell death by activating autophagy. Both 1 and 2 protected HaCaT cells against doxorubicin-induced DNA damage. They significantly promoted migration and affected F-actin distribution. These results indicate that chemo-protective effects of diarylheptanoids may occur at multiple subcellular levels. Therefore, diarylheptanoids 1 and 2 could be considered as protective agents for non-cancerous dividing cells during chemotherapy.

Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin.

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-α (HIF-1α) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain.