Neurobio-logy of multiple myeloma and its therapeutical use - results of the pilot study with a control arm.

BACKGROUND: Myeloma cells, occupying a bone marrow niche, are influenced not only by neighbouring stroma cells but also by signals from the axons of sympathetic nervous system. The nervous system is directly involved in the process of myeloma progression. Among other cancers, patients with myeloma suffer the most difficult distress generating intensive adrenergic signals, causing its further progression. There is a question arising from these facts regarding whether psychological interventions, modulating a function of the nervous system, can further improve outcomes of myeloma treatments. We focus on interactions between myeloma cells and the nervous system. PATIENTS AND METHODS: Twelve patients with monoclonal gamapathy of indetermined significance (MGUS) or myeloma have participated in this study; eight in the interventional arm with the intervention of forgiveness therapy and four in the control arm. The patients were in various phases of their treatment, from active observation to immuno-chemotherapy and autologous stem cell transplant. Two major types of parameters were measured during the intervention: parameters of the activity of the disease (MGUS or myeloma) and psycho-neuro-immunological parameters of the patient, such as psychological depression, anxiety, and anger by the validated test PROMIS), as well as activity of the autonomic nervous system by heart rate variability, and immune profile by flow cytometry of peripheral blood. RESULTS: Patients who completed the forgiveness intervention showed improvement of depression, anxiety, and anger measured by PROMIS above population average, significant expansion of physiological plasma cells CD138+38+ (P = 0.04), B memory lymphocytes CD27+ (P = 0.02), and dendritic plasmacytoid cells CD123+ (P = 0.03). Parameters of heart rate variability such as parasympatic nervous system (PNS) index, sympatic nervous system (SNS) index, stress index, standard deviation of NN intervals (SDNN) and root mean square of the successive differences (RMSSD) had improved in a majority of patients. CONCLUSION: An intervention centered on forgiveness therapy was able to improve distress, reduce adrenergic signals in the autonomic nervous system, and restore parameters of the immune profile of patients with plasma cell dyscrasia who suffered from chronic stress caused by repressed anger and unforgiveness. Integrative treatment of myeloma can improve the quality of life of patients and thus affect the efficiency of immuno-chemotherapy. New randomised trials are warranted to test the integrative treatment of myeloma that might be able to improve overall survival.

Complex changes of circadian proteins expression in inflammatory bowel disease.

OBJECTIVES AND BACKGROUND: Recently, a possible role of circadian system in the pathogenesis of various gastrointestinal disorders gained an attention. The association of circadian system with immune system activity and reciprocal connection with intestinal microbiota indicate possible links with inflammatory bowel diseases (IBD). METHODS: The retrospective study provided a semiquantitative immunohistochemical analysis of the expression of 8 core circadian proteins (BMAL1, BMAL2, PER1, PER2, PER3, CLOCK, NPAS2 and TIMELESS) in the epithelial cells of intestinal mucosa in 24 patients with Crohn's disease (CD) and 26 patients with ulcerative colitis (UC). Samples from patients without history of IBD served as the control. The BMAL1 protein expression in intramucosal inflammatory cells was explored as well. RESULTS: The expression of 5 core circadian proteins (BMAL1, PER1, PER3, TIMELESS and NAPS2) was decreased in mucosal epithelium of patients with IBD in comparison with the control samples, whereas the expression of BMAL1 and PER1 was more noticeably decreased in UC patients and PER3, TIMELESS and NPAS2 in CD patients. There was a decreased BMAL1 expression in intramucosal inflammatory cells of IBD patients. CONCLUSION: Decreased core circadian proteins expression in colonic mucosa and in intramucosal inflammatory cells of IBD patients indicated a circadian rhythm deregulation as contributing factor in the development of IBD. To our knowledge, this is so far the most extensive immunohistochemical analysis performed on the samples of IBD patients evaluating the changes in circadian protein expression in the intestinal mucosa (Tab. 1, Fig. 2, Ref. 31).

Preparation, properties and anticancer effects of mixed As4S4/ZnS nanoparticles capped by Poloxamer 407.

Arsenic sulfide compounds have a long history of application in a traditional medicine. In recent years, realgar has been studied as a promising drug in cancer treatment. In this study, the arsenic sulfide (As4S4) nanoparticles combined with zinc sulfide (ZnS) ones in different molar ratio have been prepared by a simple mechanochemical route in a planetary mill. The successful synthesis and structural properties were confirmed and followed via X-ray diffraction and high-resolution transmission electron microscopy measurements. The morphology of the particles was studied via scanning electron microscopy and transmission electron microscopy methods and the presence of nanocrystallites was verified. For biological tests, the prepared As4S4/ZnS nanoparticles were further milled in a circulation mill in a water solution of Poloxamer 407 (0.5wt%), in order to cover the particles with this biocompatible copolymer and to obtain stable nanosuspensions with unimodal distribution. The average size of the particles in the nanosuspensions (~120nm) was determined by photon cross-correlation spectroscopy method. Stability of the nanosuspensions was determined via particle size distribution and zeta potential measurements, confirming no physico-chemical changes for several months. Interestingly, with the increasing amount of ZnS in the sample, the stability was improved. The anti-cancer effects were tested on two melanoma cell lines, A375 and Bowes, with promising results, confirming increased efficiency of the samples containing both As4S4 and ZnS nanocrystals.

Potential biomarkers of exosomal cargo in endocrine signaling.

Cancer represents one of the leading cause of morbidity and mortality worldwide, with approximately 14 million new cases every year and more than 8 million cancer related deaths. In men, lung cancer is the most frequently diagnosed cancer type, followed by the prostate, colorectal, and gastric cancer; in woman, the most frequent cancer is the breast cancer, followed by the colorectal, lung, cervical, and stomach cancer. During the second half of the twentieth century the efforts to evaluate the importance of the solid tumor cells present in the circulating blood have been made. Similarly, long time ago in 1948, extracellular nucleic acids (circulating free DNA) floating around in human blood plasma were discovered. Exosomes were disclosed as the last component of this "triumvirate" present in the blood and applicable for diagnostics. The exosomal cargo consists of bioactive molecules from donor cells that can be transferred to recipient cells and modulate their intracellular signaling. Thus, exosomes can provide autocrine (local signals between the same cell type), paracrine (local signals between different cell types), and endocrine (distant signals between any types of cells) type of signals.

Realgar (As4S4) nanoparticles and arsenic trioxide (As2O3) induced autophagy and apoptosis in human melanoma cells in vitro.

The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms.

Proteasome inhibition leads to altered signaling in the proteome of cisplatin-resistant human ovarian carcinoma cell line.

To address a precise view into molecular mechanisms of apoptotic signaling pathways after single- or combinatory treatments with specific NF-κB- or proteasome inhibitors and/or cisplatin (CDDP), flow cytometry and western blotting of the cell proteome in human ovarian chemosensitive- and CDDP-resistant cell lines were used. We report here that proteasome inhibition (but not NF-κB inhibition) caused marked alterations in the cell proliferation and cell cycle, as well as in the levels of signaling anti- and pro-apoptotic proteins PARP, NF-κB, IκB-α, Bcl-2, Bax, and lysosome-associated LAMP-1 and ATP-7B molecules in particular proteome fractions. These findings refer to the possibility of regulation of CDDP resistance, inclusive the capacity of lysosomes to export CDDP in certain human ovarian cancer cells by proteasome inhibition.

Sulphide signalling potentiates apoptosis through the up-regulation of IP3 receptor types 1 and 2.

AIM: To investigate an interaction between the calcium and sulphide signalling pathways, particularly effects of the slow H2 S release donor morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate (GYY4137) on the expression of inositol 1,4,5-trisphosphate receptors (IP3 R) with the possible impact on the apoptosis induction in HeLa cells. METHODS: Gene expression, Western blot analysis, apoptosis determination by Annexin-V-FLUOS and drop in mitochondrial membrane potential by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC1) and immunofluorescence were used to determine differences in control and GYY4137-treated HeLa cells. RESULTS: In HeLa cell line, GYY4137 (10 µm) up-regulated expression of the IP3 R1 and IP3 R2, but not IP3 R3 on both mRNA and protein levels. Concurrently, cytosolic calcium increased and reticular calcium was depleted in concentration-dependent manner, partially by the involvement of IP3 R. Depletion of calcium from reticulum was accompanied by increase in endoplasmic reticulum (ER) stress markers, such as X-box, CHOP and ATF4, thus pointing to the development of ER stress due to GYY4137 treatment. Also, GYY4137 treatment of HeLa cells increased the number of apoptotic cells. CONCLUSION: These results suggest an involvement of H2 S in both IP3 -induced calcium signalling and induction of apoptosis, possibly through the activation of ER stress.

Immunity profile in breast cancer patients.

OBJECTIVES: Despite the multifactorial pathogenesis of malignant transformation, it is assumed that deficiency in some immune mechanisms plays a considerable role in its development. BACKGROUND: Chronically activated immune cells exert tumour-promoting effects directly by influencing the proliferation and survival of neoplastic cells, as well as by indirect modulation of neoplastic microenvironments in favour of tumour progression. PATIENTS AND METHODS: We refer to results of two separate investigations that aim to monitor the immune functions in patients with breast cancer. In the first investigation, we compare the picture of basic cellular immunity profile of patients in early stage of breast cancer with those suffering from advanced disease; in the second one, we compare the production of Th1-cytokines in patients in different stages of breast cancer and atopic healthy controls. RESULTS: We recognized that the totals of T-lymphocytes and T-helpers were lower and the expression of HLADR on T-lymphocytes were higher in patients with advanced disease; the expression of IL-2 and LFN-gamma by T-lymphocytes was decreased in metastatic breast cancer patients, however IL-2 production was increased in patients in early stage of disease. CONCLUSION: We conclude that the role of immune system in cancer development is ambivalent as it may be not only protective, but also harmful (Tab. 1, Fig. 3, Ref. 22). Full Text (Free, PDF) www.bmj.sk.

Advanced detection and measurement of cells on membrane from peripheral blood by laser scanning cytometry (LSC) in early stage breast cancer patients.

BACKGROUND: The aim of our study was the potential detection of circulating tumour cells (CTCs) in early stage breast cancer patients. Our approach was cell microfiltration through polycarbonate membrane as a concentration method suitable for CTC selection in peripheral blood. The isolated cells on membrane were further analysed by laser scanning cytometry. METHODS: Sixteen patients were enrolled in the study, of which 13 had early stage breast carcinoma and 3 patients had metastatic breast carcinoma. The analyses were performed from 9 ml of peripheral blood, in one patient blood was drawn twice. Blood samples were taken after adjuvant chemotherapy but prior to adjuvant radiotherapy. The control group consisted of 12 clinically healthy subjects. CONCLUSIONS: In the control group 3 subjects out of 12 had 1 CTC, the mean CTC numbers being 0.25 +/- 0.45. In the early stage breast cancer patients 0-36 CTCs were detected (mean 13.9 +/- 12.9 CTCs. 10 patients out of 13 had more than 2 CTCs (62%). The detection and measurement of cells on membrane is a simple and reproducible method of detection of CTCs in peripheral blood. Sensitivity of the method is 88.5%. Detection of CTCs seems to be a promising method for the monitoring of adjuvant therapy in early stage breast cancer patients and for the identification of high risk patients in whom elevated numbers of CTCs are persisting following the termination of adjuvant therapy (Tab. 1, Fig. 4, Ref. 35). Full Text (Free, PDF) www.bmj.sk.

Modulation of markers associated with aggressive phenotype in MDA-MB-231 breast carcinoma cells by sulforaphane.

Metastasis as a complex process involves loss of adhesion, migration, invasion and proliferation of cancer cells. Sulforaphane (SFN) is one of naturally occurring cancer chemopreventive isothiocyanates found in cruciferous vegetables, consumption of which has been associated with reduced risk of cancer. In this study, we describe effect of SFN on various aspects determining invasive behavior of MDA-MB-231 human breast carcinoma cells. We studied modulation of molecules associated with epithelial to mesenchymal transition (EMT), hypoxic marker CA IX and mitochondrially located peripheral benzodiazepine receptor (PBR) using flow cytometry, gene expression of matrix metalloproteinases MMP1, 3, 7, 9, 14, transcription factors POU5F1 and Twist1 mRNA by RT PCR, and cytokine production by multiplex bead assay. SFN downregulated PBR and vimentin expression in a dose dependent manner, but significantly affected neither HIF-1alpha, nor CA IX protein expression, nor VEGF and GLUT1 mRNA levels. Among studied MMPs, MMP7 and MMP14 mRNA were downregulated while no apparent effect on MMP1, MMP3 and MMP9 was observed. Further, we found significant down regulation of Twist1 and POU5F1, transcription factors that mediate EMT and the self-renewal of undifferentiated embryonic stem cells. SFN reduced also the production of pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha, IFN-gamma, immunomodulating cytokine IL-4 and growth factors involved in angiogenesis PDGF and VEGF. Our study shows that SFN efficacy is associated with the reversal of several biological characteristics connected with EMT or implicated in the matrix degradation and extracellular proteolysis, as well as with reduced production of pro-inflammatory cytokines and pro-angiogenic growth factors in MDA-MB-231 cells.

The effect of myocardial revascularization on malignant ventricular arrhythmias in coronary artery disease.

BACKGROUND: Malignant ventricular arrhythmia in coronary artery disease (CAD) is a severe life-threatening disease and a risk factor for sudden cardiac death. Myocardial revascularization influences the arrhythmogenic substrate of the malignant ventricular arrhythmia in the secondary prevention of sudden cardiac death. Its effectivity remains controversial. OBJECTIVES: The aim of this study is to assess the inducibility of sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) in patients after myocardial revascularization and to compare the effectivity of complete and incomplete revascularization. PATIENTS: Fifty patients with documented sustained VT or VF and CAD were examined in our department. RESULTS: Conservatively treated patients were significantly older than revascularized patients (68 +/- 8 versus 62 +/- 9 years, p<0.05). We registered a trend towards a lower inducibility of malignant ventricular arrhythmias in the revascularized group and completely revascularized subgroup, but without statistical significance. Incompletely revascularized patients comprised only of men (100% versus 66.6%, p<0.05). Fewer ICDs were implanted in the completely revascularized group (55.6% versus 92.3%, p<0.05). CONCLUSION: Myocardial revascularization has little effect on the inducibility of malignant ventricular arrhythmias after myocardial revascularization. Complete revascularization significantly decreases the need of ICD implantation when compared to incomplete one (Tab. 3, Fig. 4, Ref. 24). Full Text (Free, PDF) www.bmj.sk.

Administration of isothiocyanate (E-4IB) and cisplatin leads to altered signalling and lysosomal export in human ovarian carcinoma sensitive- and cisplatin-resistant cells.

The aim of this study was to compare the effect of a new synthetic isothiocyanate derivative, ethyl 4-isothiocyanatobutanoate (E-4IB) and cisplatin (CDDP) in CDDP-sensitive human ovarian carcinoma cell line (A2780) and its resistant subline (A2780/CP). In parental cells, in comparison to untreated cells, sequential administration of both compounds led to higher exosomal dye (LysoTracker Green DND-26) retention and to alterations of mitogen-activated protein kinases (MAPKs), JNK, ERK and p38, or Akt kinase accompanied by changes in several anti- and pro-apoptotic molecules and lysosomal protein LAMP-1, as detected by Western blotting. On the contrary, variant A2780/CP cells were resistant to CDDP- or to combined sensitizer (E-4IB)/inducer (CDDP)-related apoptosis induction and exerted minor changes in the levels of these molecules.

BioBran-augmented maturation of human monocyte-derived dendritic cells.

UNLABELLED: BioBran, enzymatically modified arabinoxylan from rice bran was tested for its possible effects on in vitro maturation of human dendritic cells (DC). Immature DC (iDC) derived from plastic-adhered, IL-4 and GM-CSF treated peripheral monocytes (Mo) were further cultured with cytokine maturation mix 1 (CMM1; TNF-alpha, IL-1beta and IL-6) or CMM2 (LPS and IFN-gamma) to induce their maturation into mature DC (matDC1 or matDC2, respectively). Different concentrations of BioBran (10, 100, 400 and 1000 microg/ml) were applied in the presence or absence of relevant CMM to assess the effects of BioBran on DC maturation processes. BioBran induced maturation of iDC, as these cells cultured with IL-4/GM-CSF/BioBran down-regulated CD14 and CD1a antigens on cell surface and significantly increased expression of maturation marker CD83. The increase of surface density of costimulatory molecules CD80 and CD86 on iDC in the presence of BioBran was also observed. In addition, BioBran induced functional maturation of iDC, confirmed by decreased endocytic activity of iDC. Further emore, BioBran enhanced maturation potential of cytokine mixes, as both matDC1 and matDC2 exposed to BioBran completely lost CD14 and upregulated CD83, CD80 and CD86 antigens, in comparison to DC matured with the relevant CMM alone. BioBran also increased CD123 antigen expression on all DC subsets. Interestingly, matDC2 matured in the presence of BioBran (400microg/ml) expressed higher levels of CD123 and lower levels of CD11c cell surface antigens, the phenotype represented by CD11cdim CD123bright plasmacytoid DC population. These data demonstrate that BioBran is a potent enhancer of DC maturation and suggest that BioBran might be a useful agent to create the environment that favours DC maturation. KEYWORDS: Dendritic cell, maturation, BioBran, buffy coat.

Frequency of the three most common polymorphisms in the MDR1 gene in Slovak population.

UNLABELLED: The human multidrug resistance gene (MDR1) is encoding the transmembrane transporter P-glycoprotein (P-gp) which plays an important role in the efflux of various drugs and thus is potentially influencing the drug-treatment outcome. It has been indicated that the level of P-gp activity may be affected by the presence of single nucleotide polymorphisms (SNP) in the gene which led to the studies estimating MDR1-SNP frequencies in various populations. Here, we have investigated the occurrence of seven SNP in the MDR1 gene for the first time in Slovak population using multiplex SNaPshot genotyping method. The allelic frequencies of the most common gene variants, i.e. 1236C>T, 2677G>T, 2677G>A and 3435C>T were estimated to be 42.5%, 43.5%, 2%, and 44.5%, respectively. We found that the most prevalent haplotype in Slovak population is 1236C-2677G-3435C occurring in 42.2% of individuals. Our preliminary data show that it is reasonable and feasible to utilize MDR1 genotypes and haplotypes in Slovak patients, e.g. those with acute myeloid leukemia, in order to adjust the individual effective drug dosage and predict the patient's response to the treatment as well as the treatment outcome. KEYWORDS: MDR1 gene, P-glycoprotein, polymorphisms, MDR1 haplotypes, Slovak population.

Expression of new prognostic markers, peripheral-type benzodiazepine receptor and carbonic anhydrase IX, in human breast and ovarian carcinoma cell lines.

Peripheral benzodiazepine receptor (PBR), a mitochondrial protein involved in cell proliferation and differentiation, and carbonic anhydrase IX (CA IX), an intrinsic marker of hypoxia, have been studied in the panel of human breast (MCF-7, BT- 20, MDA-MB-453, MDA-MB-231) and ovarian (A2780, A2780/CP, A2780/ADR, CH1, SKOV-3) carcinoma cell lines that differ by malignant progression. The expression of both antigens was detected by staining with the PBR-specific 8D7 and CA IX-specific M75 monoclonal antibodies and quantitated by flow cytometry. PBR was related to mitochondrial mass and CA IX to the cell density. Breast carcinoma cell lines showed higher relative fluorescence intensity of PBR expression than ovarian cell lines, with the exception of A2780/CP cisplatin-resistant subline that was comparable to highly invasive MDA-MB-231 breast line. Among the breast cell lines, PBR expression increased with their invasive potential. The ovarian cell lines showed greater variability in fluorescence intensities and the expression of PBR did not correlate with the amount of mitochondria. Mitochondrial PBR density disclosed significant difference between cisplatin-sensitive (low PBR density) and -resistant (high PBR density) ovarian cell lines. MTT test showed higher sensitivity of 2 breast cell lines MCF-7 and MDA-MB-231 (IC50 < 75 microM) to PBR ligand PK 11195 than all examined ovarian cell lines (IC50 > 90 microM, in chemo- and radio- resistant lines IC50 > 110 microM). Growth inhibitory effect of PK 11195 did not correlate with the amount of PBR and was mediated probably by another, PBRindependent mechanisms. The expression of CA IX was only marginal in majority of tested cell lines in subconfluent conditions and was inducible by high cell density. More than 5% of positive cells in sparse culture have been found in MDA-MB-231 and MDA-MB-453 breast cell lines while more than 15% of A2780/ADR adriamycin-resistant ovarian cells were positive for CA IX expression under the same conditions. Our data indicate that PBR expression in breast and ovarian carcinoma cell lines is not proportional to the amount of mitochondria and should be expressed relatively to the cell mitochondrial mass. This assessment allows establishing high PBR density as a measure of aggressiveness (invasion in breast and resistance in ovarian cancer). Observation of relatively high CA IX expression in A2780/ADR cells evokes the assumption that multidrug resistance might be connected with selection advantage towards CA IX expressing cells.

Isothiocyanate E-4IB induces MAPK activation, delayed cell cycle transition and apoptosis.

INTRODUCTION: Epidemiologic studies point towards a significant correlation between the dietary intake of isothiocyanate-containing foods and the reduced risk for cancer. METHODS AND RESULTS: In the current investigation, we examined the consequence of activating of signalling pathways during the release the cells from the block at G(1)/S boundary by synthetic isothiocyanate E-4IB. Using synchronized leukaemic HL60 cells, we show that activation of mitogen-activated protein kinases ERK1/2, c-Jun N-terminal kinase and p38 signalling pathways by E-4IB are coupled with delayed transition through the cell cycle and rapid cell cycle arrest resulted in diminished mitochondrial membrane potential culminating in apoptosis. These events were accompanied by histone deacetylase inhibition, increase of double strand DNA breaks detected by histone H2AX phosphorylation and up-regulation of cell cycle regulatory protein p21 and phosphorylation of CDC25C phosphatase. CONCLUSION: These findings suggest that the activation of mitogen-activated protein kinases signalling pathways, followed by the induction cell cycle arrest and apoptosis, might be responsible for anticancer activities of E-4IB.

Isothiocyanate iberin modulates phase II enzymes, posttranslational modification of histones and inhibits growth of Caco-2 cells by inducing apoptosis.

The aim of presented study was to further investigate the concentration-dependent changes induced by isothiocyanate iberin (IBN) in human colon carcinoma Caco-2 cells. The concentrations of IBN below IC(50) value (18 microM, 72 h) triggered the augmentation of mRNA levels for phase II detoxification GSTA1 and UGT1A1 enzymes and antioxidant thioredoxin reductase 1 gene in cells treated for 24 h. In addition a significant increase of acetylated H4 histone was detected. The mRNA induction peaked at IC(50) value and returned to level of control cells at 40 microM concentration of IBN. The cell cycle changes, gamma-H2AX stainability and the increase of phospho-H3 mitotic marker were induced at concentrations above IC(50) value. Appearance of Annexin V positive apoptotic cells and sub-G1 fragmented DNA as well as decrease of mitochondrial transmembrane potential confirmed cytotoxic effect of IBN observed in MTT assay. The predominance of necrotic cells and profound positivity of gamma-H2AX took place at the highest concentration of IBN. Thus, IBN represents the effective member of natural chemopreventive isothiocyanate family with which apoptotic potential can by employed to eliminate tumor cells.

Sensitisation for cisplatin-induced apoptosis by isothiocyanate E-4IB leads to signalling pathways alterations.

A new synthetic isothiocyanate (ITC) derivative, ethyl 4-isothiocyanatobutanoate (E-4IB), appeared to be an effective modulator of cellular proliferation and potent inducer of apoptosis. In cooperation with cisplatin, this compound exerted synergistic effects in human ovarian carcinoma A2780 cells. In the present study we investigated in more detail E4IB-sensitisation for cisplatin-induced apoptosis. Sequential administration of both cytostatic agents led to increased intracellular platinum accumulation, glutathione level depletion and mitochondrial membrane potential dissipation. These events were accompanied with poly (ADP-ribosyl) polymerase cleavage, stimulation of caspase-3 activity, upregulation of p53, FasL and Gadd45alpha, cyclin B1 downregulation and an increase in mitogen-activated protein kinases JNK, ERK and p38 phosphorylation as well as PI3K level alterations. The presented results might have implications for developing new strategies aimed at therapeutic benefit of natural or synthetic ITCs in cooperation with various anticancer drugs.

Diverse resveratrol sensitization to apoptosis induced by anticancer drugs in sensitive and resistant leukemia cells.

Naturally occurring dietary compound resveratrol (RES), possessing chemopreventive and cytostatic properties, has been shown as potent sensitizer for apoptosis induced by a variety of anticancer drugs. Cell cycle analysis in sensitive promyelocytic leukemia HL60 cell line and its multidrug-resistant variant HL60/VCR (P-gp positive) treated with RES resulted in cell cycle arrest in S-phase in both cell variants. Flow cytometry measurements showed diverse activities of RES in combination with anticancer drugs doxorubicin (DOX), cycloheximide (CHX), busulfan (BUS), gemcitabine (GEM) and paclitaxel (PTX), in some cases resulting in apoptosis induction, preferentially at the expense of S-phase. Thus, RES could become a candidate to enhance the efficacy of combination anticancer therapy in a variety of human cancer cells inclusive leukemias.

Garlic-derived organosulfides induce cytotoxicity, apoptosis, cell cycle arrest and oxidative stress in human colon carcinoma cell lines.

Organosulfur compounds (OSC) from garlic, especially allicin (ALI), diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are recognized as a group of potential chemopreventive agents. In this study, we examined the effects of OSC on human Caco-2 and HT-29 colon carcinoma cell lines. Apoptosis induction (Annexin-V-FITC/PI, fluorescein diacetate/PI, sub-G1 fraction), modulation of DNA cell cycle (G2/M arrest, phospho-H3 mitotic marker), transmembrane mitochondrial potential (JC-1) and intracellular GSH amount (monochlorobimane assay) were measured by flow cytometry and fluorimetry. Our results showed that order of OSC-induced cell death in Caco-2 and HT-29 cells increased in the range as follows: ALI < DAS = DADS < DATS and ALI = DAS < DADS < DATS, respectively. Both cell lines used are relatively resistant to OSC induced cytotoxicity, because compound concentrations required to obtain significant effect are in high micromolar range. ALI was less toxic than equimolar doses of other OSC tested with the exception of GSH modulation and G2/M arrest in Caco-2 cells. DADS-treated HT-29 cells and both DATS-treated cell lines exhibit inverse correlation between p-H3 positivity and compound concentration due to higher apoptotic rate. These results show the correlation of sulfur atoms number in OSC with their capacity in apoptosis induction and support the role of redox-sensitive "sulfhydryl switches" in maintaining intracellular redox milieu.