Role of the auxiliary ligand in determining the genotoxicity and mode of cell death of thiosemicarbazone Pd(II) complexes.

A series of Pd(II) complexes of the general formula [PdX(NNS)] (X = Cl, Br, I, NCS and phenyl-tetrazole-thiolato; NNS = 2-quinolinecarboxyaldehyde-N4-phenylthiosemicarbazone) was tested against four malignant cell lines for their antiproliferative properties and the outcomes were compared to those seen in normal mouse splenocytes. Various auxiliary ligands were substituted in order to investigate the impact of the character of the ligand on the cytotoxicity of this class of Pd(II) complexes. The iodo complex was the most cytotoxic compound towards the Caco-2 cell line in this study. The improved apoptosis and necrosis cell modes were in accordance with the fragmentation results of DNA, which revealed increased fragmentation terminals, especially in isothiocyanate and tetrazole-thiolato complexes. After 24 hours, at half the IC50 of each complex, the complex-treated cells exhibited considerable genotoxicity when compared to the corresponding non-treated control especially in the case of isothiocyanate and tetrazole-thiolato complexes.

Enhancement of the antitumor effect of 5-fluorouracil with modulation in drug transporters expression using PI3K inhibitors in colorectal cancer cells.

AIMS: 5-Fluorouracil (5-FU) represents the cornerstone for colorectal cancer therapy. However, resistance to its action is a major hindrance. This study aimed to investigate the effectiveness of suppressing the activity of PI3K/Akt/mTOR signaling pathway on the chemosensitivity of colorectal cancer cells to 5-FU, as well as to delineate the possible underlying cellular mechanisms and the expected modulation in the expression of specific ABC drug transporters. MAIN METHODS: HCT116 and Caco-2 cells were incubated with 5-FU, LY294002, or PI-103 individually or in combination. Cell viability was monitored using MTT assay. The expression of a panel of drug transporters was evaluated by RT-PCR. Immunofluorescence staining was applied to evaluate the expression pattern of phospho-AKT, phospho-mTOR, and ABGG2. HPLC evaluated the enhancement in the 5-FU cellular uptake. Cell apoptosis was detected by flow cytometry, and cell morphological changes following treatment were inspected under a fluorescence microscope. Additionally, the migration ability of cells following our suggested treatment combination was examined by wound healing assay. KEY FINDINGS: The results reveal a notable enhancement in the cytotoxicity of a low dose of 5-FU when combined with a PI3K inhibitor (LY294002 or PI-103). This enhancement was influenced by the significant reduction in the expression of p-AKT and p-mTOR and was also mediated by a significant suppression in the expression of ABCG2 and ABCC5. Consequently, we detected an increase in the cellular uptake and concentration of 5-FU in cells treated with this combination rather than a single 5-FU treatment. Our Suggested combination treatment also induced cell apoptosis and reduced the migration ability of cells. SIGNIFICANCE: Our data provide evidence that survival signaling pathways represent distinctive targets for the enhancement of chemotherapeutic sensitivity. The antitumor efficacy of 5-FU is enhanced when combined with a PI3K inhibitor, and this effect was mediated by alterations in the expression of specific drug transporters.

Lung cancer risk in workers occupationally exposed to polycyclic aromatic hydrocarbons with emphasis on the role of DNA repair gene.

OBJECTIVE: Workers in secondary aluminum production plants are occupationally exposed to polycyclic aromatic hydrocarbons (PAHs). We aimed to monitor the concentrations of PAHs in air and in serum of workers at two secondary aluminum production plants. We also investigated the potential risk of lung cancer development among PAHs exposed workers with emphasis on the role of A1AT mutation and APEX1 gene polymorphisms. METHODS: This study included 177 workers from administrative departments and production lines. Blood samples were obtained for estimation of benzo(a)pyrene diol epoxide albumin adduct (BPDE-Alb adduct), anti-Cyclin-B1 marker (CCNB1) and squamous cell carcinoma antigen (SCCAg). Genes' polymorphism for human apurinic/apyrimidinic endonuclease (APEX1) and alpha-1-anti-trypsin (A1AT) gene mutation were detected. RESULTS: There was a significant increase in the level of BPDE-Alb adduct among exposed workers in comparison to non-exposed group. Moreover, 41.67% of exposed workers in El Tebbin had BPDE-Alb adduct level ≥ 15 ng/ml versus 29.6% of workers in Helwan factory. There was a significant increase in tumor markers (SCCAg and CCNB1) among workers whose BPDE-Alb adduct ≥ 15 ng/ml. There was a significant increase in the level of BPDE-Alb adducts in exposed workers carrying homozygous APEX1 genotype Glu/Glu. Furthermore, exposed workers with the Glu/Glu genotype had high tumor markers levels. There was a significant increase in levels of BPDE-Alb adducts in workers carrying A1AT mutant allele. Moreover, workers with mutant A1AT genotype had significantly high tumor markers (SCCAg and CCNB1) levels. CONCLUSION: Therefore, we conclude that aluminum workers may be at a potential risk of lung cancer development due to PAHs exposure. Although PAHs concentrations in air were within the permissible limits, yet evidence of DNA damage was present as expressed by high BPDE-albumin adduct level in exposed workers. Also, elevation of tumor markers (SCCAg and CCNB1) in exposed workers points to the importance of periodic biological monitoring of such workers to protect them from cancer risk.

Cytocompatibility of MG-63 osteosarcoma cells on chitosan/hydroxyapatite/lignin hybrid composite scaffoldin vitro.

This study aims at fabricating promising cytocompatible hybrid biocomposite scaffolds from chitosan (CS), hydroxyapatite (HAP) and lignin (L) for bone tissue engineering by using freeze-drying technique. Different ratios of HAP to L (50:0, 37.5:12.5, 25:25 and 12.5:37.5) were taken to determine the optimum ratio for obtaining a composite with superior properties. The mechanical and biological properties of the resulting composites were investigated. The mechanical results showed that the prepared composite with a ratio of 25:25 of HAP/L exhibited a remarkable enhancement in the mechanical properties compared to the others. Additionally, it was found from thein vitroresults that the addition of L enhanced the water uptake value of the resulting scaffolds indicating their increased hydrophilicity. As a result, a significant increase in the attachment and proliferation of MG-63 cell line (osteoblast like cells) was observed in composite scaffolds with L over the scaffold without L (CS/HAP). From these results, it could be suggested that the prepared composite scaffold with 25:25 of HAP/L is very promising biomaterials in bone tissue-engineering as it exhibited a better mechanical and biological properties than the other prepared composites.

Phthalimide Analogs Enhance Genotoxicity of Cyclophosphamide and Inhibit Its Associated Hypoxia.

Cyclophosphamide (CP) is a mutagen that is used in cancer chemotherapy, due to its genotoxicity and as an immunosuppressive agent. Thalidomide (TH) is another cancer chemotherapeutic drug. In this study, the cytogenotoxicity and hypoxia modulatory activities of two phthalimide analogs of TH have been evaluated with/without CP. Both analogs have increased CP-stimulated chromosomal aberrations than those induced by TH, including gaps, breaks/fragments, deletions, multiple aberrations, and tetraploidy. The analogs have elevated the cytotoxic effect of CP by inhibiting the mitotic activity, in which analog 2 showed higher mitosis inhibition. CP has induced binucleated and polynucleated bone marrow cells (BMCs), while micronuclei (MN) are absent. TH and analogs have elevated the CP-stimulated binucleated BMCs, while only analogs have increased the CP-induced polynucleated BMCs and inhibited the mononucleated BMCs. MN-BMCs were shown together with mononucleated, binucleated, and polynucleated cells in the CP group. Both analogs have elevated mononucleated and polynucleated MN-BMCs, whereas in presence of CP, TH and analogs have enhanced mononucleated and binucleated MN-BMCs. The analogs significantly induce DNA fragmentation in a comet assay, where analog 1 is the strongest inducer. The treatment of mice with CP has resulted in a high hypoxia status as indicated by high pimonidazole adducts and high HIF-1α and HIF-2α concentrations in lymphocytes. Analogs/CP-treated mice showed low pimonidazole adducts. Both analogs have inhibited HIF-1α concentration but not HIF-2α. Taken together, the study findings suggest that both analogs have a higher potential to induce CP-genotoxicity than TH and that both analogs inhibit CP-hypoxia via the HIF-1α-dependent mechanism, in which analog 1 is a more potent anti-hypoxic agent than analog 2. Analog 1 is suggested as an adjacent CP-complementary agent to induce CP-genotoxicity and to inhibit CP-associated hypoxia.

Evaluation of lapatinib cytotoxicity and genotoxicity on MDA-MB-231 breast cancer cell line.

Lapatinib, one of the tyrosine kinase inhibitors (TKIs), is used to reduce epidermal growth factor family proteins overexpression. This study aims to assess the cytotoxic and genotoxic effects of lapatinib on the triple negative breast cancer cell line "MDA-MB-231". We investigated the cytotoxicity of lapatinib by MTT assay, mode of cell death using apoptosis-necrosis assay, DNA damage using micronucleus test, EGFR protein expression by immunocytochemistry, and assessed its effect on EGFR (7p11.2 locus) and TP53 (17p13 locus) genes using interphase-FISH technique. Lapatinib induced cytotoxicity on MDA-MB-231 cell line by elevating the concentration and its IC50 value was 32.5 µM after 24 h. Lapatinib increased apoptotic cells and micronuclei in binucleated cells gradually by increasing the concentration for 24 h. The EGFR protein expression was reduced by double fold that expressed in non-treated cells. Lapatinib enhanced deletion of EGFR gene signals highly significantly from the lowest concentration. Alternatively, lapatinib amplified signals of TP53 gene effectively by raising the concentration. In conclusion, lapatinib induced cytotoxic and genotoxic effects on MDA-MB-231 cell line. However, laptinib reduced the EGFR protein expression and EGFR signals, it raised the apoptotic cells and TP53 gene signals, which triggered extensive DNA damage. Therefore, lapatinib is an effective TKI in triple negative breast cancer cells as elucidated by its mode of cell death.

Flavonoid fraction of Cajanus cajan prohibited the mutagenic properties of cyclophosphamide in mice in vivo.

Cajanus cajan (L.) is a Pigeon pea cultivated in tropical and subtropical areas. It contains many bioactive components. The present study aimed to assess the antimutagenic efficacy of a flavonoid fraction of Cajanus cajan (FFCC) to reduce cytotoxicity and genotoxicity induced by cyclophosphamide (CP). We assessed genotoxic and cytotoxic effects using chromosome aberration, in mouse bone-marrow cells and spermatocytes, cell viability and DNA damage, in mouse bone-marrow cells. Animals received FFCC at concentrations 50,100 and 200 mg/kg b wt by oral gavage, and injected simultaneously with CP (20 mg/kg b wt) for 24 h. The results revealed that FFCC was safe and its effect was normal compared to control group. Moreover, we observed significant inhibition of CP-induced chromosome abnormalities in both, somatic and germ, cells (p ≪ 0.05) after concurrent administration of different concentrations of FFCC and CP. FFCC reduced chromosome aberrations by 14.29%, 25.21% and 28.57% in somatic cells, and 25.35%, 35.21% and 49.29% in germ cells after simultaneous treatment with CP respectively. Additionally, FFCC improved the cell viability of bone-marrow cells in a concentration-dependent manner when administered concurrently with CP. Similarly, FFCC diminished DNA damage (p ≪ 0.05) in CP-treated animals. The inhibitory index of tail DNA (%) reached 90.6% at the highest concentration of FFCC when administered simultaneously with CP. In conclusion, the flavonoid extract improved cell viability and protected animal cells from the cytotoxic and genotoxic effects exhibited by CP. Cajanus cajan flavonoids might contain the antioxidant bioactivity that effectively lessened chromosome aberrations and DNA damage induced by mutagenic agents.

Photodynamic therapy using 5-aminolevulinic acid triggered DNA damage of adenocarcinoma breast cancer and hepatocellular carcinoma cell lines.

Targeting cancer cells with photosensitizer (PS) excited by appropriate laser irradiation to release singlet oxygen as a photodynamic therapy (PDT) remains a challenge. This research aimed to assess the cytogenetic potential of 5-aminolevulinic acid (5-ALA) activated with laser irradiation (5-ALA/PDT) to damage the intact DNA of adenocarcinoma breast cancer cell line (MCF-7) and hepatocellular carcinoma cell line (HepG2). Cancer cells were treated with 0.5 and 1 mM 5-ALA for 4 h, the precursor of the photochemical protoporphyrin IX (PpIX), and then exposed to laser irradiation at 633 nm and 0.25 W for 4 min before incubation for 24 h. Cytotoxicity of cancer cells was assessed using trypan blue exclusion assay. Genotoxicity was recorded by micronucleus test and comet assay. Both 5-ALA and laser irradiation, separately, were nontoxic on cancer cell lines, however, 5-ALA/PDT enhanced cell death in a concentration-dependent manner. Also, 5-ALA/PDT generated high percentages of micronuclei in MCF-7 and HepG2 cell lines as recorded in binucleated cells. Similarly, the mean percentages of DNA damage and tail moment ratio were intensified extremely in cancer cell lines treated with 5-ALA/PDT rather than non-treated cells or cells treated by 5-ALA or laser irradiation separately. In conclusion, the singlet oxygen of 5-ALA targets DNA of cancer cells when activated by laser irradiation. Therefore, photodynamic therapy is an applicable process to damage DNA effectively during M-phase and prohibit cancer cells proliferation.

Gum Arabic-encapsulated gold nanoparticles for a non-invasive photothermal ablation of lung tumor in mice.

BACKGROUND: In our previous work, we have extensively evaluated the physiochemical characteristics of Gum Arabic-encapsulated gold nanoparticles (GA-AuNPs; 15-18nm) and reported their effectiveness in stopping the tumor initiation via inhibiting the pre-neoplastic lesions in liver. OBJECTIVE: The rationale of this study is to detect the efficiency of using GA-AuNPs in photothermal application as a non-invasive technique against lung tumor. We investigated the cytotoxicity of GA-AuNPs on A549 cells, and then studied their apoptotic, anti-inflammatory, lipid peroxidation and anti-neovascular effect in in vivo model using a chemically-induced lung cancer in mice. The histopathological changes due to GA-AuNPs were investigated. RESULTS: In the presence of laser irradiation, GA-AuNPs had a considerable cytotoxicity against A549 cells. The treatment of lung tumor-bearing mice with GA-AuNPs followed by laser exposure enhanced the apoptotic pathway and this was obvious from the histopathological investigations and the elevations in cytochrome-c, death receptor 5 and the subsequent upregulation of caspase-3, we also reported a significant reduction in the levels of the inflammatory mediator TNF-α and the angiogenesis inducer VEGF. An induction of lipid peroxidation was also reported upon treatment with either GA or GA-AuNPs. CONCLUSION: GA-AuNPs showed no cytotoxicity in the absence of light, however the combination of GA-AuNPs with laser induced cell death in lung tumor tissues with a reduction in the inflammation and angiogenesis together with an elevation in lipid peroxidation, suggesting the potential use of these functionalized nanoparticles as a promising photothermal non-invasive treatment modality.

Cytotoxic effect of ferrimagnetic glass-ceramic nanocomposites on bone osteosarcoma cells.

This work pointed out the anti-cancer effect of ferrimagnetic glass ceramic nanocomposites (CaO-ZnO-Fe2O3-SiO2), which contain high amount of magnetite (∼60%), crystallite size <100nm, and different nucleating agents on bone cancer Saos-2 cells. The cell viability was inhibited by FH and FW to <50% and <25%, respectively, with/without magnetism, and both also reduced mitochondrial transmembrane potential (ΔYm), with/without magnetism (no influence of magnetism). Histone deacetylase (HDAC) activity was inhibited by FH, FW, and FHPNT, with/without magnetism. FHP3/magnetism resulted in HDAC inhibition. In absence of magnetism, FH and FW increased both necrotic and apoptotic cell death, while FW/magnetism induced late apoptosis. DNA fragmentation was increased by FH- and FW-treatment, with/without magnetism. At the same time, FW and FH/magnetism can efficiently induce the intrinsic apoptotic pathway in Saos-2 cells, whereas FW with/without magnetism and FH/magnetism enhanced cytochrome-C release. Similarly, caspase-7 activity was elevated by FH and FW, with/without magnetism. However, the presence of P2O5 in the composition of the nanocomposites inhibited their apoptotic properties and diminished their anti-cancer activity.

Three further triterpenoid saponins from Gleditsia caspica fruits and protective effect of the total saponin fraction on cyclophosphamide-induced genotoxicity in mice.

Three triterpenoidal saponins were isolated from the saponin fraction derived from a Gleditsia caspica Desf. methanolic fruit extract. The isolated saponins were identified as gleditsiosides B, C, and Q based on spectral data. The saponin-containing fraction was evaluated in vivo for genotoxic and antigenotoxic activities. The fraction caused no DNA damage in Swiss albino male mice treated with a dose of 45 mg/kg body weight for 24 h, although it significantly inhibited the number of chromosomal aberrations induced by cyclophosphamide (CP) in bone marrow and germ cells when applied before or after CP administration. The inhibitory indices in chromosomal aberrations were 59% and 41% for bone marrow and 48% and 43% for germ cells, respectively. In addition, the saponin fraction was found to reduce the viability of the human tumor cell line MCF-7 in a dose-dependent manner with an extrapolated IC50 value in the range of 220 µg/mL.

Photodynamic therapeutic activity of indocyanine green entrapped in polymeric nanoparticles.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic modality involving the use of a photosensitizer agent activated by light of appropriate wavelength to selectively destroy tumor cells. Indocyanine green (ICG) is a promising photosensitive agent for PDT of tumor cells. The main disadvantage of using ICG in PDT is the instability of ICG in aqueous solutions. Encapsulating ICG dye in a biocompatible matrix based on PEBBLE technology showed an improvement of aqueous stability comparing with free ICG dye. The main objective of this study is to investigate the photodynamic effect of ICG-ormosil PEBBLEs on two different cell lines: human breast adenocarcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2). METHODS: ICG-embedded ormosil PEBBLEs were synthesized based on a sol-gel process, and characterized by transmission electron microscopy and other fluorescence tests. The cell viability was evaluated by MTT and trypan blue assays. Apoptosis, necrosis, and DNA damage (comet assay), were evaluated by fluorescence microscopic tests. RESULTS: The results declared that ICG-ormosil PEBBLEs and free ICG both have the same cytotoxic and phototoxic effect on MCF-7 and HepG2 cell lines, where the apoptotic mode of cell death is preferentially occurred in case of PDT using ICG-ormosil PEBBLEs. Both ICG and ICG-ormosil PEBBLEs induced DNA damage after laser exposure. These results would suggest that entrapping ICG in Polymeric nanoparticles forming ICG-ormosil PEBBLEs improve the aqueous stability of the photosensitizer and in the same time retain its photodynamic activity, suggesting that it is preferred to use ICG-ormosil PEBBLEs instead of free ICG dye.

Molecular cytogenetic evaluation of the efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma MCF-7 cells.

BACKGROUND: Photodynamic therapy (PDT) is used for the treatment of many types of predominantly epithelial cancers. Photosensitizer is taken up by fast growing tumor cells more actively than by other body cells and is activated by light, generating reactive oxygen species that cause cell death by necrosis or apoptosis. This study aimed to evaluate the efficacy of PDT with indocyanine green (ICG) through the investigation of TP53, HER-2 and TOP2A genes signals as breast cancer gene markers by interphase fluorescence in situ hybridization (nuc-FISH). METHODS: The photosynthetizer ICG (200 µM) was applied to breast cancer cell line MCF-7 cells (adenocarcinoma) in combination with laser irradiation (807 nm) exposure for 20 min and then incubated for 12, 24 and 48 h. Cell viability was evaluated using trypan blue. The signals for nuc-FISH was investigated and counted for probes specific for the genes TP53 (17p13), HER-2 (17q11.2-q12), and TOP2A (17q21-q22), and BAC-probes RP11-746M1 in 17p11.2 and RP11-403E9 in 17q11.2. RESULTS: The cell viability of MCF-7 did not reduced significantly when the cells were treated with ICG (200 µM) or exposed to laser irradiation for 20 min followed by incubation for 24 h. ICG/PDT treatment with laser irradiation exposure for 20 min reduced the cell viability after incubating cells for 12, 24 and 48 h highly significantly in a time dependent manner. For nuc-FISH analysis, TP53, HER-2, TOP2A, RP11-746M1 and RP11-403E9 signals did not reduce or increase in a significant manner when the cells were treated with ICG or exposed to laser irradiation for 20 min then incubated for 24h. PDT enhanced amplification of TP53 signals from nuc ish 17p13(TP53×2) to nuc ish 17p13(TP53×3) or nuc ish 17p13(TP53×4). However, the signals of HER-2 gene, TOP2A gene and BAC probes were reduced highly significantly when MCF-7 cells were treated with PDT with all time intervals. CONCLUSION: ICG/PDT and laser induced cytotoxic effect in MCF-7 cells. Also, PDT enhanced TP53 gene amplification, and reduced HER-2, TOP2A, and BAC probes RP11-746M1 and RP11-403E9 signals. Therefore ICG/PDT can be used for breast cancer treatment. It has the potential to induce apoptotic effect and reduce HER-2 and TOP2A genes propagation. Further in vivo studies are needed to evaluate ICG/PDT as a promising therapeutic approach for breast cancer.

Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage'.

Honey isolate Bacillus subtilis M was cultivated in shake flasks and in 16-l bioreactor cultures to investigate cell growth, bio-metabolites production kinetics and bioprocess scalability. The respective maximal levan and levansucrase productions of 59.5 g/l and 74.1 U/ml were achieved in bioreactor cultures under pH controlled condition (pH=7.0) after only 24 h. Crude levan (levE) was isolated, characterized and fractionated into F1, F2, and F3 with different molecular weight (21.8, 13.118, 9.53 kDa). (1)H NMR and (13)C NMR spectroscopy proved that LevE and their fractions were mainly ß-(2, 6)-linked levan-type polysaccharide. The cancer chemo-preventive activity indicated that the levE and its fraction 3 were promising inhibitors of cytochrome P-450 1A activity, inducers of glutathione-S-transferase activity in Murine hepatomaHepa1c1c7cells and possessed highest radical scavenging affinity to both ROO and OH. They inhibited the induced-DNA fragmentation. None of the tested samples triggered apoptosis or necrosis in splenocytes, except F2.

Anti-genotoxic effect of the Sargassum dentifolium extracts: prevention of chromosomal aberrations, micronuclei, and DNA fragmentation.

The alga Sargassum dentifolium (Turner) C. Agardh, belongs to Sargassaceae, is a brown seaweed in red sea shores in Egypt. This work aimed to extract different water-soluble polysaccharide extracts (E1, E2, and E3) from S. dentifolium and to investigate their protective effect against cyclophosphamide (CP)-induced genotoxicity. Mice bone marrow cells (BMCs) were collected and analyzed for the chromosomal aberration, micronucleated BMCs (MN-BMCs), the mitotic index, DNA fragmentation by comet assay, and histone deacetylases (HDACs), and radical scavenging capacity of extracts was evaluated by the oxygen radical absorbance capacity assay. The results indicated that E2 and E3 significantly inhibited CP-induced multiple chromosomal aberrations, where E1 and E3 significantly suppressed the number of CP-induced formation of tetraploidy. The extracts prohibited the cytotoxic effect of CP and recovered the mitotic activity, whereas E1 possessed the highest recovery and mitosis. In absence of MN, CP induced formation of bi- and poly-nucleated BMCs. E1 prohibited CP-induced formation of bi-nucleated BMCs, while E2 and E3 prohibited CP-induced formation of poly-nucleated BMCs. CP-induced MN-BMCs were accompanied with mono-, bi- and poly-nucleated cells. E1 and E3 remarkably suppressed mono-nucleated MN-BMCs, while E2 inhibited bi-nucleated MN-BMCs. All the extracts significantly inhibited the CP-induced formation of poly-nucleated MN-BMCs. CP-induced DNA fragmentation was inhibited by all extracts, where E1 was the strongest inhibitor as concluded from the comet tail moment. All the extracts were strong OH scavengers, while only E3 was ROO scavenger. The results revealed a drastic decline in HDACs activity by E1 and E3. In conclusion, S. dentifolium polysaccharide extracts E1 and E3 possessed a potential anti-genotoxic and a promising anti-mutagenic activity.