Epoxylathyrane Derivatives as MDR-Selective Compounds for Disabling Multidrug Resistance in Cancer.

BACKGROUND: Multidrug resistance (MDR) has been regarded as one of the major hurdles for the successful outcome of cancer chemotherapy. The collateral sensitivity (CS) effect is one the most auspicious anti-MDR strategies. Epoxylathyrane derivatives 1-16 were obtained by derivatization of the macrocyclic diterpene epoxyboetirane A (17), a lathyrane-type macrocyclic diterpene isolated from Euphorbia boetica. Some of these compounds were found to strongly modulate P-glycoprotein (P-gp/ABCB1) efflux. PURPOSE: The main goal was to develop lathyrane-type macrocyclic diterpenes with improved MDR-modifying activity, by targeting more than one anti-MDR mechanism. STUDY DESIGN/METHODS: In this study, the potential CS effect of compounds 1-16 was evaluated against gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29) human cancer cells and their drug-resistant counterparts, respectively selected against mitoxantrone (EPG85-257RNOV; EPP85-181RNOV; HT-RNOV) or daunorubicin (EPG85-257RDB; EPP85-181RDB; HT-RDB). The most promising compounds (8, 15, and 16) were investigated as apoptosis inducers, using the assays annexin V/PI and active caspase-3. RESULTS: The compounds were more effective against the resistant gastric cell lines, being the CS effect more significant in EPG85-257RDB cells. Taking together the IC50 values and the CS effect, compounds 8, 15, and 16 exhibited the best results. Epoxyboetirane P (8), with the strongest MDR-selective antiproliferative activity against gastric carcinoma EPG85-257RDB cells (IC50 of 0.72 µM), being 10-fold more active against this resistant subline than in sensitive gastric carcinoma cells. The CS effect elicited by compounds 15 and 16 appeared to be by inducing apoptosis via caspase-3 activation. Structure-activity relationships of the compounds were additionally obtained through regression models to clarify the structural determinants associated to the CS effect. CONCLUSIONS: This study reinforces the importance of lathyrane-type diterpenes as lead molecules for the research of MDR-modifying agents.

Bacteria-mediated delivery of RNAi effector molecules against viral HPV16-E7 eradicates oral squamous carcinoma cells (OSCC) via apoptosis.

Delivery of RNAi-mediating shRNA molecules for gene silencing via bacteria, i.e. by transkingdom RNAi (tkRNAi) technology, is suggested to be a powerful alternative technique. In this work, the efficiency of bacterial delivery of shRNAs directed against HPV16-E7-specific mRNA to oral squamous carcinoma cells (OSCCs) was evaluated. E. coli were transfected with a plasmid encoding the inv locus and the Hlya gene to enable the bacteria to enter carcinoma cells and to escape from endocytotic vesicles. The bacterial penetration to the target cells was confirmed by DAPI staining. The HPV16-E7 mRNA expression in bacteria-treated OSCCs dropped to 61% of the controls as measured by qRT-PCR. Corresponding inhibition of the HPV16-E7 protein was confirmed by western blotting. The IC50 of bacteria-treated OSCCs was reduced to more than 75%. Flow cytometry assays showed higher total apoptosis and caspase-3 activation (6.6-fold and 8.4-fold respectively) in OSCCs following exposure to anti-HPV-E7  bacteria compared to anti-GFP bacteria (2-fold and 2.9-fold, respectively). In conclusion, it was demonstrated for the first time that tkRNAi technology is also useful for treatment of squamous carcinoma cells. Anti-HPV16-E7 shRNA-encoding bacteria can efficiently deliver RNAi effectors to OSCCs mediating a strong and specific gene silencing associated with triggering cell death.

Triterpenoids from Momordica balsamina with a Collateral Sensitivity Effect for Tackling Multidrug Resistance in Cancer Cells.

The collateral sensitivity effect is among the most promising strategies for overcoming multidrug resistance in cancer. In this work, 28 cucurbitane-type triterpenoids (1: -28: ), previously isolated from the African medicinal plant Momordica balsamina and its derivatives, were evaluated for their collateral sensitivity effect on three different human cancer entities, gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29), each with two different multidrug-resistant variants. One was selected for its resistance to daunorubicin (EPG85-257RDB, EPP85-181RDB, HT-29RDB) and the other was selected for its resistance to mitoxantrone (EPG85-257RNOV, EPP85-181RNOV, HT-29RNOV). On gastric cell lines, the best results were obtained for compounds 3: and 10: , which exhibited a collateral sensitivity effect together with high antiproliferative activity. In turn, on colon cancer cell lines, the best multidrug resistance-selective antiproliferative effects were observed for derivatives 11, 13: , and 15: , which showed collateral sensitivity effects against both resistant variants. Compounds 11: and 3: were also the most selective against the multidrug resistance pancreatic cells lines. Some compounds, such 6, 10, 11: and 15: , were previously found to be strong P-glycoprotein modulators, thus highlighting their potential as promising leads for overcoming multidrug resistance in cancer cells.

Exploring Jolkinol D Derivatives To Overcome Multidrug Resistance in Cancer.

Macrocyclic monoacyl lathyrane derivatives bearing a benzoyl moiety were previously found to be strong ABCB1 modulators. To explore the effects of different substituents of the aromatic moiety, 14 new compounds (1.1-1.7, 1.10, and 2.1-2.4) were prepared from jolkinol D (1), obtained from Euphorbia piscatoria, and from jolkinodiol (2), its hydrolysis derivative. Compounds 1.8 and 1.9, having aliphatic moieties, were also obtained. The reversal of ABCB1-mediated MDR was evaluated through functional and chemosensitivity assays on the human ABCB1-gene-transfected L5178Y mouse T-lymphoma cell line. Structure-activity relationships showed that addition of electron-donating groups to the aromatic moiety improved the activity. The effects on the ATPase activity of the strongest modulator (1.3) and the inactive jolkinol D (1) were also investigated and compared. Moreover, in the chemosensitivity assay, most of the compounds interacted synergistically with doxorubicin. Compounds 1.1-1.10 and 2.1-2.4 were further assessed for their collateral sensitivity effect against the human cancer cells: EPG85-257 (gastric) and EPP85-181 (pancreatic), and the matching drug-selected cells EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, and EPP85-181RNOV. The most promising ones (1.8 and 1.10) along with compound 3, previously selected, were investigated as apoptosis inducers. The compounds were able to induce apoptosis through caspase-3 activation, with significant differences being observed between the parental and resistant cells.

Jatrophane diterpenes and cancer multidrug resistance - ABCB1 efflux modulation and selective cell death induction.

BACKGROUND: Modulation of P-glycoprotein (ABCB1) and evaluation of the collateral sensitivity effect are among the most promising approaches to overcome multidrug resistance (MDR) in cancer. In a previous study, two rare 12,17-cyclojatrophanes (1-2) and other novel jatrophanes (3-4), isolated from Euphorbia welwitschii, were screened for collateral sensitivity effect. Herein, the isolation of another jatrophane (5) is presented, being the broader goal of this work to investigate the role of euphowelwitschines A (1) and B (2), welwitschene (3), epoxywelwitschene (4) and esulatin M (5) as ABCB1 modulators and/or collateral sensitivity agents. METHODS: Compounds 1-5 were evaluated for ABCB1 modulation ability through combination of transport and chemosensitivity assays, using a mouse T-lymphoma MDR1-transfected cell model. Moreover, the nature of interaction of compound 4 with ABCB1 was studied, using an ATPase assay. The MDR-selective antiproliferative activity of compound 5 was evaluated against gastric (EPG85-257) and pancreatic (EPP85-181) human cancer cells and their drug-selected counterparts (EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, EPP85-181RNOV). The drug induced cell death was investigated for compounds 4 and 5, using the annexin V/PI staining and the active caspase-3 assay. RESULTS: The jatrophanes 1-5 were able to modulate the efflux activity of ABCB1, and at 2µM, 3-5 maintained the strong modulator profile. Structure activity results indicated that high conformational flexibility of the twelve-membered ring of compounds 3-5 favored ABCB1 modulation, in contrast to the tetracyclic scaffold of compounds 1 and 2. The effects of epoxywelwitschene (4) on the ATPase activity of ABCB1 showed it to interact with the transporter and to be able to reduce the transport of a second subtrate. Drug combination experiments also corroborated the anti-MDR potential of these diterpenes due to their synergistic interaction with doxorubicin (combination index <0.7). Esulatin M (5) showed a strong MDR-selective antiproliferative activity against EPG85-257RDB and EPP85-181RDB cells, with IC50 of 1.8 and 4.8 µM, respectively. Compounds 4 and 5 induced apoptosis via caspase-3 activation. A significant discrimination was observed between the resistant cell lines and parental cells. CONCLUSIONS: This study strengthens the role of jatrophane diterpenes as lead candidates for the development of MDR reversal agents, higlighting the action of compounds 4 and 5.

Crocin suppresses multidrug resistance in MRP overexpressing ovarian cancer cell line.

BACKGROUND: Crocin, one of the main constituents of saffron extract, has numerous biological effects such as anti-cancer effects. Multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) are important elements in the failure of cancer chemotherapy. In this study we aimed to evaluate the effects of crocin on MRP1 and MRP2 expression and function in human ovarian cancer cell line A2780 and its cisplatin-resistant derivative A2780/RCIS cells. METHODS: The cytotoxicity of crocin was assessed by the MTT assay. The effects of crocin on the MRP1 and MRP2 mRNA expression and function were assessed by real-time RT-PCR and MTT assays, respectively. RESULTS: Our study indicated that crocin reduced cell proliferation in a dose-dependent manner in which the reduction in proliferation rate was more noticeable in the A2780 cell line compared to A2780/RCIS. Crocin reduced MRP1 and MRP2 gene expression at the mRNA level in A2780/RCIS cells. It increased doxorubicin cytotoxicity on the resistant A2780/RCIS cells in comparison with the drug-sensitive A2780 cells. CONCLUSION: Totally, these results indicated that crocin could suppress drug resistance via down regulation of MRP transporters in the human ovarian cancer resistant cell line.

Gene Therapeutic Approaches to Overcome ABCB1-Mediated Drug Resistance.

Multidrug resistance (MDR) to pharmaceutical active agents is a common clinical problem in patients suffering from cancer. MDR is often mediated by over expression of trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC)-transporters. This protein family includes the classical MDR-associated transporter ABCB1 (MDR1/P-gp). Inhibition of ABC-transporters by low molecular weight compounds in cancer patients has been extensively investigated in clinical trials, but the results have been disappointing. Thus, in the last decades alternative experimental therapeutic strategies for overcoming MDR were under extensive investigation. These include gene therapeutic approaches applying antisense-, ribozyme-, RNA interference-, and CRISPR/Cas9-based techniques. Various delivery strategies were used to reverse MDR in different tumor models in vitro and in vivo. Results and conclusions of these gene therapeutic studies will be discussed.

12,17-Cyclojatrophane and Jatrophane Constituents of Euphorbia welwitschii.

Euphowelwitschines A (1) and B (2), isolated from a methanolic extract of Euphorbia welwitschii, exhibit a rare combination of structural features in having a 5/8/8 fused-ring system and a 12,15-ether bridge. Moreover, the isolation of the additional new compounds welwitschene (3) and epoxywelwitschene (4) has provided insights into the biogenetic pathway of 12,17-cyclojatrophanes. The structures of 1-4 were determined by spectroscopic methods inclusive of 1D and 2D NMR experiments and X-ray crystallography for compounds 1 and 2. Preliminary information on the selective antiproliferative activity of compounds 1-4 is also described.

Expression of Estrogen Receptors in OSCC in Relation to Histopathological Grade.

BACKGROUND/AIM: Estrogen receptor (ER)-mediated pathways are involved in the pathogenesis of several tumors. Preliminary studies have demonstrated a significant effect of ER agonists and antagonists on oral squamous cell carcinoma (OSCC) cell lines. Recent results suggest that ER subtype-specific expression patterns might depend on the grade of differentiation of OSCC. Therefore, the aim of the present study was to evaluate the expression of ERα and ERß in OSCC and its correlation to histological tumor grade and gender. MATERIALS AND METHODS: Tumor sections of 25 patients (13 males and 12 females) retrieved from OSCC databases with two different histological gradings (well-differentiated, poorly differentiated) were evaluated. The detection of ERα and ERß expression in tumor cells and corresponding healthy mucosa adjacent to tumor was performed using immunohistochemistry. RESULTS: Well-differentiated OSCC showed no significant difference between the expression of ERß in tumor cells and corresponding mucosa. In poorly-differentiated OSCC the expression of ERß was significantly higher in tumor cells than in corresponding mucosa. In patients without regular alcohol and/or nicotine abuse, there was no significant difference of ERß expression in OSCC compared to corresponding healthy mucosa in contrast to patients having these risk factors. Expression of ERα was found in one tumor. CONCLUSION: ERß is the predominant ER sub-type expressed significantly higher in poorly-differentiated OSCC tumors compared to healthy mucosa adjacent to the tumor. Different expression patterns in relation to histological grade might suggest an influential role of ERß in tumor (de-) differentiation of OSCC.

Discovery of substituted 1,4-dihydroquinolines as novel class of ABCB1 modulators.

Transmembrane efflux pumps are one main cause for multidrug resistance (mdr) of cancer. One hopeful approach to combate the mdr has been the development of inhibitors of the efflux pump activity. A novel class of small-molecule inhibitors of the most important efflux pump ABCB1 (P-glycoprotein) has been discovered. Inhibitory activities are discussed in relation to substituent effects. Most active compounds have been evaluated in first bioanalytical studies to reverse the mdr of an anticancer drug. Cellular toxicity and ABCB1 substrate properties of the compounds were investigated. A cellular induction of relevant efflux pump protein expressions was not observed under inhibitor application, so that our compounds are perspective candidates for further preclinical studies.

Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer.

OBJECTIVES: P-glycoprotein (P-gp) is an efflux protein, the overexpression of which has been associated with multidrug resistance in various cancers. Although siRNA delivery to reverse P-gp expression may be promising for sensitizing of tumor cells to cytotoxic drugs, the therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. We investigated a special class of PEGylated lipid-based nanoparticles (NP), named nanolipoparticles (NLPs), for siRNA-mediated P-gp downregulation. MATERIALS AND METHODS: NLPs were prepared based on low detergent dialysis method. After characterization, we evaluated the effect of NLPs on siRNA delivery, and P-gp downregulation compared to oligofectamine™ (OFA) in vitro and in vivo. RESULTS: Our results showed a significant decrease in P-gp expression and subsequent enhancement of chemosensitivity to doxorubicin in vitro. Although the effectiveness of NLPs for in vitro siRNA delivery compared to OFA was limited, the results of in vivo studies showed noticeable effectiveness of NLPs for systemic siRNA delivery. siRNA delivery using NLPs could downregulate MDR1 in tumor cells more than 80%, while OFA had a reverse effect on MDR1 expression in vivo. CONCLUSION: The results indicated that the prepared NLPs could be suitable siRNA delivery systems for tumor therapy.

The photodynamic effect of far-red range phthalocyanines (AlPc and Pc green) supported by electropermeabilization in human gastric adenocarcinoma cells of sensitive and resistant type.

INTRODUCTION: Electroporation (EP) is commonly applied for effective drug transport thorough cell membranes based on the application of electromagnetic field. When applied with cytostatics, it is called electrochemotherapy (ECT) - a quite new method of cancer treatment. A high-voltage pulse causes the formation of temporary pores in the cell membrane which create an additional way for the intracellular drug transport. In the current work, EP was effectively merged with the already known photodynamic therapy (PDT) to selective photosensitizers' delivery to diseased tissue. The application of electroporation can reduce the dose of applied drug. RESEARCH OBJECTIVE: The aim of research was to evaluate the effectiveness of photodynamic reaction using two near infrared cyanines (AlPc and Pc green) combined with electroporation in two human gastric adenocarcinoma cell lines. MATERIALS AND METHODS: Two human cell lines - EPG85-257P (parental) and EPG85-257RDB (resistant to daunorubicin) - of gastric cancer were used. The effect of two photosensitizers (aluminum 1,8,15,22-tetrakis(-phenylthio)-29H,31H-phthalocyanine chloride and Phthalocyanine green) was investigated. The efficiency of EP parameters was assessed by propidium iodide uptake. The viability assay was applied to analyse EP, PDT and EP-PDT effect. Cyanine localization was determined by confocal microscopy. Immunocytochemical evaluation of manganese superoxide dismutase and glutathione S-transferase-pi was determined after applied therapies. RESULTS: PDT in combination with EP affected the viability of EPG85-257P and EPG85-257RDB cells negatively while both cyanine were used. The most evident changes were observed in the following concentrations: 15, 10 and 5µM. The optimal field strength for enhanced EP-PDT was 800 and 1200V/cm. AlPc distributed selectively in the lysosomes of parental cell line. CONCLUSIONS: PDT, enhanced by EP, caused decreased viability when compared to the application of PDT alone. Both phthalocyanines found to be more effective after electroporation. Due to the low concentration of light-sensitive compounds and safety of electroporation itself, a treatment plan can be an alternative therapeutic modality against gastric adenocarcinomas.

Delivery of siRNAs to cancer cells via bacteria.

RNA interference (RNAi) technology is a promising approach for efficient silencing of a particular gene for cancer gene therapy. However, the main obstacle for the development of RNAi-based therapeutic approaches is the delivery of the RNAi effector molecules to target cells. One promising strategy to surmount this challenge is the application of nonpathogenic bacteria as a delivery vector to target cells. In this chapter, the design of invasive Escherichia coli is described. The strain carries a plasmid encoding short hairpin RNAs (shRNAs), a protein (invasin) necessary for endocytotic absorption of the bacteria by target cells, and listeriolysin O required for the lysis of endocytotic vesicles within the target cells.

Discovery of 9,10-Dihydroacridines as Novel Class of ABCB1 Inhibitors.

Nonplanar 9,10-dihydroacridines were synthesized as promising C2 symmetric molecular scaffolds as inhibitors of the transmembrane efflux pump ABCB1. Within the series structure-activity relationships are discussed revealing the importance of hydrogen bond acceptor functions. A selectivity of ABCB1 inhibition is demonstrated for selected candidates and a bioanalytical study proved nontoxicity as well as missing ABCB1 substrate properties. The results encourage to further develop the promising class of ABCB1 inhibitors.

Diterpenes from Euphorbia piscatoria: synergistic interaction of Lathyranes with doxorubicin on resistant cancer cells.

Four new diterpenes were isolated from the methanolic extract of Euphorbia piscatoria, two ent-abietanes (1, 2) and two lathyrane-type macrocyclic diterpenes (3, 4), along with three known diterpenes (5-7). Their structures were characterized by spectroscopic methods, mainly 1D and 2D NMR ((1)H, (13)C, DEPT, COSY, HMBC, HMQC, and NOESY) experiments. Compound 2, with an unusual structure, might be considered intermediate in the biosynthesis of ent-abietane α,ß-unsaturated lactones, commonly found in Euphorbia species. Therefore, a possible biogenetic pathway is proposed. The MDR reversal potential of macrocyclic diterpenes 3-5 was evaluated through a drug combination assay, using the L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. Compounds 3-5 were able to enhance, synergistically, the antiproliferative activity of doxorubicin (combination indexes < 0.5). Moreover, compounds 1-6 were also assessed for their antiproliferative activity on human MDR cancer cell models, namely gastric, pancreatic, and colon. Weak antiproliferative activity was observed for compounds 1 (IC50 = 66.02 ± 7.10 µM) and 4 (IC50 = 39.51 ± 3.82 µM) on the MDR gastric cell line.

A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut). METHODS: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels. RESULTS: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells. CONCLUSIONS: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.

Macrocyclic diterpenes resensitizing multidrug resistant phenotypes.

Herein, collateral sensitivity effect was exploited as a strategy to select effective compounds to overcome multidrug resistance in cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1), isolated from Euphorbia piscatoria, and its derivatives (2-11) were evaluated for their activity on three different Human cancer entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) each with a variant selected for resistance to mitoxantrone (EPG85-257RN; EPP85-181RN; HT-29RN) and one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD). Jolkinol D (1) and most of its derivatives (2-11) exhibited significant collateral sensitivity effect towards the cell lines EPG85-257RN (associated with P-glycoprotein overexpression) and HT-29RD (altered topoisomerase II expression). The benzoyl derivative, jolkinoate L (8) demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as P-glycoprotein modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells. A regression analysis between the antiproliferative activity presented herein and the previously assessed P-glycoprotein modulatory effect showed a strong relation between the compounds that presented both high P-glycoprotein modulation and cytotoxicity.

Nuclear-cytoplasmic PARP-1 expression as an unfavorable prognostic marker in lymph node­negative early breast cancer: 15-year follow-up.

PARP-1 plays an important role in DNA damage repair and maintaining genome integrity by repairing DNA single-strand breaks (SSBs) by base excision repair (BER). The aim of the present study was to examine the expression of PARP-1 in breast cancer (BC) patients and to assess the relationship between the subcellular localization of this protein and clinicopathological characteristics. The reactivity of PARP-1 was analyzed by immunohistochemistry in a homogeneous group of 83 stage II ductal BC patients with a 15-year follow-up. Immunostaining of PARP-1 was also evaluated in 4 human BC cell lines and resistance prediction profile for 11 anticancer agents was performed using 3 models of drug-resistant cell lines. Nuclear-cytoplasmic expression (NCE) was associated with shorter overall survival, which was not statistically significant during the 10-year follow-up but became statistically significant after 10 years of observation, during the 15-year follow-up (P=0.015). Analysis performed in subgroups of patients with (N+) and without (N-) nodal metastases showed that NCE was associated with poor clinical outcome in N- patients (P=0.017). Multivariate analysis confirmed a significant impact of NCE on unfavorable prognosis in N- early BC. The presence of PARP-1 NCE may be a new potential unfavorable prognostic factor in lymph node- negative early BC.

The cell cycle arrest and the anti-invasive effects of nitrogen-containing bisphosphonates are not mediated by DBF4 in breast cancer cells.

Recent work has shown that a DBF4 analog in yeast may be a target of nitrogen-containing bisphosphonates. DBF4 is an essential protein kinase required for DNA replication from primary eukaryotes to humans and appears to play a critical role in the S-phase checkpoint. It is also required for cell migration and cell surface adhesion. The effects of Pamidronate, risedronate, or zoledronate on cell viability and DBF4 expression were measured via MTT assays and western blotting. In addition, FACS cell cycle analyses and invasion assays were conducted in cells in the presence of nitrogen-containing bisphosphonates to identify any correlations between DBF4 expression and S-phase arrest or anti-invasive effects of the bisphosphonates. Zoledronate transiently down-regulated DBF4 expression in all three cell lines in the first 24 h of the experiment, but after 72 h, DBF4 expression returned to the control levels in all treated cells. Following treatment of the tumor cells with the bisphosphonates, the number of cells in S-phase was increased. Pamidronate and zoledronate showed anti-invasive effects in BT20 cells. The anti-invasive effects of pamidronate, risedronate and zoledronate appeared after 48 h of exposure. In MDA-MB231 cells a reduction of invasiveness was only observed after 72 h of the pamidronate exposure. We finally concluded that the anti-invasive and cell cycle arrest-inducing effects of nitrogen-containing bisphosphonates are not DBF4 mediated, and other mediators are therefore needed to explain the observed complex behaviors.

In vitro analysis of the relationships between metallothionein expression and cisplatin sensitivity of non-small cellular lung cancer cells.

BACKGROUND: Cisplatin-based therapy is a pivotal type of chemotherapy for non-small cell lung cancer (NSCLC) and chemoresistance to cisplatin represents one of the most significant barriers to improving long-term clinical outcomes. MATERIALS AND METHODS: The present study aimed at examining metallothionein (MT) expression in six NSCLC cell lines as well as examining effects of exposure to cisplatin on MT expression in the most cisplatin-resistant (97/97) and the cisplatin-sensitive (DV90) cell lines. RESULTS: The most cisplatin-resistant NSCLC cell line [97/97; (IC50)=4.659 µM] exposed to the highest concentration of cisplatin (10 µM) exhibited decreased nuclear MT expression (MTn=6) compared to cells cultured in medium with a lower concentration of cisplatin (0, 1 and 5 µM) (MTn=12). A higher cytoplasmic metallothionein expression (MTc=6) was found in the 97/97 cell line exposed to the highest concentration of cisplatin (10 µM), compared to cells cultured in the medium with lower concentrations of cisplatin (0, 1 and 5 µM) (MTc=3). The most cisplatin-sensitive NSCLC cell line (DV90; IC50=0.184 µM) was characterized by a significant decrease of both nuclear and cytoplasmic MT expression with increasing cisplatin concentrations (5 vs. 10 µM). CONCLUSION: Nuclear and cytoplasmic expression of MT has no significant impact on the development of cisplatichemoresistance in NSCLC cell lines. The present study suggests that cisplatin resistance in NSCLC is metallothionein-independent.