Multidrug-Resistant Profiles in Non-Small Cell Lung Carcinoma Patient-Derived Cells: Implications for Personalized Approaches with Tyrosine Kinase Inhibitors.

The impact of tyrosine kinase inhibitors (TKIs) on multidrug resistance (MDR) in non-small cell lung carcinoma (NSCLC) is a critical aspect of cancer therapy. While TKIs effectively target specific signaling pathways of cancer cells, they can also act as substrates for ABC transporters, potentially triggering MDR. The aim of our study was to evaluate the response of 17 patient-derived NSCLC cultures to 10 commonly prescribed TKIs and to correlate these responses with patient mutational profiles. Using an ex vivo immunofluorescence assay, we analyzed the expression of the MDR markers ABCB1, ABCC1, and ABCG2, and correlated these data with the genetic profiles of patients for a functional diagnostic approach. NSCLC cultures responded differently to TKIs, with erlotinib showing good efficacy regardless of mutation burden or EGFR status. However, the modulation of MDR mechanisms by erlotinib, such as increased ABCG2 expression, highlights the challenges associated with erlotinib treatment. Other TKIs showed limited efficacy, highlighting the variability of response in NSCLC. Genetic alterations in signaling pathways associated with drug resistance and sensitivity, including TP53 mutations, likely contributed to the variable responses to TKIs. The relationships between ABC transporter expression, gene alterations, and response to TKIs did not show consistent patterns. Our results suggest that in addition to mutational status, performing functional sensitivity screening is critical for identifying appropriate treatment strategies with TKIs. These results underscore the importance of considering drug sensitivity, off-target effects, MDR risks, and patient-specific genetic profiles when optimizing NSCLC treatment and highlight the potential for personalized approaches, especially in early stages.

Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells.

Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay's capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.

Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells.

Identification of the signature molecular profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute-phase protein, and its high expression has been linked with unfavorable clinical outcome in different types of cancer; however, data on its involvement in therapy resistance are still insufficient. We analyzed SERPINA1 mRNA expression in three different multidrug-resistant (MDR) cell lines-U87-TxR, NCI-H460/R, and DLD1-TxR-and in U87 cells grown in alginate microfibers as a 3D cellular model of glioblastoma. Expression of IL-6 as a major modulator of SERPINA1 was also analyzed. Additionally, AAT protein expression in MDR cells was analyzed by immunofluorescence. SERPINA1 gene expression and AAT protein expression were significantly upregulated in all the tested MDR cell lines compared with their sensitive counterparts. Moreover, SERPINA1 was significantly upregulated in 3D models of glioblastoma, previously found to have upregulated drug-resistance-related gene expression compared with 2D cells. With the exception of NCI-H460/R, in all cell lines as well as in a 3D model of U87 cells, increase in SERPINA1 expression correlated with the increase in IL-6 expression. Our results indicate that AAT could be utilized as a biomarker of therapy resistance in cancer; however, further studies are needed to elucidate the mechanisms driving AAT upregulation in therapy resistance and its biological significance in this process.

On optimal temozolomide scheduling for slowly growing glioblastomas.

Background: Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes. Methods: Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study. Results: Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients. Conclusions: In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs.

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.

A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.

3D cultures of cancer cells enable better mimicking of physiological conditions compared to traditional monolayer 2D cultures. Here we describe alginate scaffold-based model that can be used in both static and biomimetic conditions for studying drug sensitivity in cancer cells and multidrug resistance (MDR) mechanisms. This 3D culture model resembles in vivo conditions and provides relevant and reproducible results. It is easy to set up and allows for facile manipulation for downstream analyses. All these remarkable features make this 3D culture model a promising tool in drug discovery and cancer cell biology research.

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.

Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells.

BACKGROUND: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. METHODS: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives-Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306-were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. RESULTS: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. CONCLUSION: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.

Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability.

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.

Novel TrxR1 Inhibitors Show Potential for Glioma Treatment by Suppressing the Invasion and Sensitizing Glioma Cells to Chemotherapy.

Currently, available glioblastoma (GBM) treatment remains ineffective, with relapse after initial response and low survival rate of GBM patients. The reasons behind limited capacities for GBM treatment are high tumor heterogeneity, invasiveness, and occurrence of drug resistance. Therefore, developing novel therapeutic strategies is of utmost importance. Thioredoxin reductase (TrxR) is a novel, promising target due to its overexpression in many cancer types and important role in cancer progression. Previous research on Ugi-type Michael acceptors-inhibitors of TrxR showed desirable anticancer properties, with significant selectivity toward cancer cells. Herein, two TrxR inhibitors, 5 and 6, underwent in-depth study on multidrug-resistant (MDR) glioma cell lines. Besides the antioxidative effects, 5 and 6 induced cell death, decreased cell proliferation, and suppressed invasion and migration of glioma cells. Both compounds showed a synergistic effect in combination with temozolomide (TMZ), a first-line chemotherapeutic for GBM treatment. Moreover, 5 and 6 affected activity of P-glycoprotein extrusion pump that could be found in cancer cells and in the blood-brain barrier (BBB), thus showing potential for suppressing MDR phenotype in cancer cells and evading BBB. In conclusion, investigated TrxR inhibitors are effective anticancer compounds, acting through inhibition of the thioredoxin system and perturbation of antioxidative defense systems of glioma cells. They are suitable for combining with other chemotherapeutics, able to surpass the BBB and overcome MDR. Thus, our findings suggest further exploration of Ugi-type Michael acceptors-TrxR inhibitors' potential as an adjuvant therapy for GBM treatment.

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.

Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. METHODS: Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. RESULTS: Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. CONCLUSIONS: Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies.

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.

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.

Association of CCND1 overexpression with KRAS and PTEN alterations in specific subtypes of non-small cell lung carcinoma and its influence on patients' outcome.

Cyclin D1 is one of the major cellular oncogenes, overexpressed in number of human cancers, including non-small cell lung carcinoma (NSCLC). However, it does not exert tumorigenic activity by itself, but rather cooperates with other altered oncogenes and tumor suppressors. Therefore, in the present study, we have examined mutual role of cyclin D1, KRAS, and PTEN alterations in the pathogenesis of NSCLC and their potential to serve as multiple molecular markers for this disease. CCND1 gene amplification and gene expression were analyzed in relation to mutational status of KRAS gene as well as to PTEN alterations (loss of heterozygosity and promoter hypermethylation) in NSCLC patient samples. Moreover, the effect of these co-alterations on patient survival was examined. Amplified CCND1 gene was exclusively associated with increased gene expression. Statistical analyses also revealed significant association between CCND1 overexpression and KRAS mutations in the whole group and in the groups of patients with adenocarcinoma, grade 1/2, and stage I/II. In addition, CCND1 overexpression was significantly related to PTEN promoter hypermethylation in the whole group and in the group of patients with squamous cell carcinoma and lymph node invasion. These joint alterations also significantly shortened patients' survival and were shown to be an independent factor for adverse prognosis. Overall results point that cyclin D1 expression cooperates with KRAS and PTEN alterations in pathogenesis of NSCLC, and they could serve as potential multiple molecular markers for specific subgroups of NSCLC patients as well as prognostic markers for this type of cancer.