Normal Human Peripheral Blood Lymphocytes Research Articles

Synthesis and structures of diorganotin(IV) Schiff base complexes [R2Sn(L)Cl2] and their proliferative responses on breast cancer cells

The preparation and structural characterization in solution and the solid-state of five diorganotin(IV) complexes, namely [Me2Sn(L1)Cl2] (1), [Ph2Sn(L1)Cl2] (2), [Bn2Sn(L1)Cl2] (3), [Me2Sn(L2)Cl2] (4), [nBu2Sn(L2)Cl2] (5), [Ph2Sn(L2)Cl2] (6), and [Bn2Sn(L2)Cl2] (7), where L1 = (E)-N-(pyridin-2-ylmethylene)cyclopropanamine and L2 = (E)-N-(pyridin-2-ylmethylene)cyclohexanamine is reported. In all metal complexes the tin atoms have six-coordinate C2SnN2Cl2 environments, exhibiting two distinct configurations. Six of the seven metal complexes are related to cis-Platin concerning the distribution of the nitrogen and chlorine atoms around the metal center, with the organic substituents attached to tin occupying the axial sites. Meanwhile, the organic groups and chlorine atoms are exchanged in the diphenyltin derivative 6, giving a configuration with axial trans-oriented chlorine atoms. Aside from van der Waals contacts, the molecular arrangements in the 3D solid-state structures are controlled by weak C − H⋅⋅⋅Cl, C − H⋅⋅⋅π and/or π⋅⋅⋅π interactions, which are analyzed in a comparative manner using Hirshfeld surface and fingerprint plot analysis. Complexes 1–4, 6 and 7 were investigated by 1H, 13C and 119Sn NMR spectroscopy, with the latter confirming the hexacoordinate C2SnN2Cl2 arrangement is maintained in solution, although ligand exchange was evident for the dimethyltin complexes 1 and 4 at room temperature.The anti-proliferative activity of compounds 1–4, 6 and 7 was evaluated against the MCF-7 (human breast cancer) cell line and normal human peripheral blood lymphocytes (HPBLs) using the MTT (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Of these, the diphenyl-substituted tin compound 2 carrying a cyclopropyl substituent at the 2-pyridylimine ligand and exhibiting an orientation of the N and Cl substituents analogous to cis-Platin demonstrated the best activity with an IC50 value of 81.63 ± 1.2 µM. Importantly, the trans-complex 6 was significantly less active (IC50 = 347.68 µM). Microscopic and colorimetric studies confirmed the triggering of apoptosis for the cell death of MCF-7, consisting of a caspase-dependent intrinsic pathway as revealed by an immunoblot study.

Study of Molecular Cytogenetic Abnormalities in Lymphoma and Their Clinical Relevance

Objective: To investigate the issue of molecular cytogenetic abnormalities in lymphoma and to study their clinical relevance. Methods: First, medium-term chromosomes of healthy men and women were prepared. Normal human peripheral blood lymphocytes were stimulated with PHA, treated with MTX synchronization, blocked with colchicine, cultured for 3 days, routinely treated with hypotonicity, cells were obtained, and steam-dried method was used to drop slices for backup. Next, comparative genomic hybridization. Preparation of probes; processing of normal human mid-term split images; hybridization; washing of slices after hybridization; staining; fluorescence microscopy observation, uptake and analysis of fluorescence signals. Results: Lymphoma CGH test results; Relationship between CGH abnormalities and lymphoma histological subtypes:Hodgkin's lymphoma CGH abnormalities, diffuse large B-cell lymphoma (DLBCL) CGH abnormalities, follicular lymphoma (FL) CGH abnormalities, and follicular lymphoma (FL) CGH abnormalities; Correlation between CGH test results and lymphoma clinical features: CGH test results and lymphoma patients, the type of CGH abnormality correlated with the clinical features of lymphoma, simple CGH abnormalities and complex CGH abnormalities correlated with the clinical features of lymphoma patients, and CGH abnormalities at specific chromosomal sites correlated with the clinical features of lymphoma. Conclusion:Conventional model analysis examines both chromosomal translocations and gene deletions or spreads, fails to observe the full picture of the lymphoma genome, and fails to detect new chromosomal abnormalities.

Bamboo leaf extract ameliorates radiation induced genotoxicity: An in vitro study of chromosome aberration assay

IntroductionBamboo species are a rich source of antioxidative phytochemicals, hence can be studied for radioprotective efficacy. This study aims to determine the antioxidant activity and radioprotective effects of leaf extracts from four Indian bamboo species, Phyllostachys parvifolia, Bambusa arundinacea, Bambusa vulgaris and Dendrocalamus strictus, against γ-radiation. MethodsAntioxidant activity of bamboo leaf extract (BLE) was estimated by various assay such as 2,2-diphenyl-1-picrylhydrazyl assay, Hydrogen peroxide assay, Nitric oxide radical scavenging assay and Lipid peroxidation. Normal human peripheral blood lymphocytes were cultured with four concentrations of hydroalcoholic BLE (3,5,7 and 9 µg/ml) from the bamboo species for 30 mins prior to γ-radiation of 4 Gy or 6 Gy doses. Following this Chromosome Aberration (CA) assay and Dicentric Analysis (DCA) was performed to assess the degree of protection against radiation induced cytogenetic damage by the extracts. ResultsThe results indicate that all the BLE exerted potent antioxidant activity and the highest antioxidant activity was exhibited by BA and DC species. The CA and DCA assay indicated a significant reduction in the frequency of chromosomal aberrations in the cultures pre-treated with the extracts and 9 µg/ml showed the lowest genetic damage as well as least frequency of dicentric chromosomes. ConclusionBLE can be a good source of natural antioxidants. Administration of BLE prior to radiation exposure provided considerable protection in terms of reduction of in vitro radiation induced cytogenetic damage. This study also forms a basis for further analysis of the possible mode of action of the extract.

Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro.

Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of cancer research. To find an alternative cure for the disease from natural resources we selected Bacopa monniera, a perennial ethnomedicinal plant popularly used for boosting memory and mental health. We isolated four different types of dammarane saponins, namely bacopasaponins C-F (1-4) from the plant and evaluated their toxic effects on two different types of human breast cancer cell lines-a hormone-responsive MCF7 and a triple-negative MDA-MB-231. Interestingly, MTT assay revealed a dose-dependent toxic effect of all four types of bacopasaponins on both of these cell lines, 4 being the most effective with 48h-inhibitory concentration (IC50) of 32.44 and 30µM in MCF7 and MDA-MB-231 respectively. Further, 4 caused significant alterations in normal cytomorphology and induction of apoptosis in both of these cell lines after 48h of treatment. No caspase-8 activity was detected in these cell lines when exposed to 4 for 2, 24, and 48h; instead, Western blotting analysis confirmed involvement of either caspase-9 (MCF7) or both caspase-9 and caspase-3 (MDA-MB-231) in the process of apoptosis indicating the occurrence of intrinsic mode. Additionally, at comparable effective doses to cancer, bacopasaponins showed much less toxicity in normal human peripheral blood lymphocytes (≥ 85% cell survival). Overall, the findings project bacopasaponin F, a natural constituent of Bacopa monniera, as an efficient and safer alternative for breast cancer therapeutics.

Chitosan-gold nanoparticles trigger apoptosis in human breast cancer cells in vitro

Despite profound advancement in the field of cancer treatment, the disease is still one of the deadliest in the world. In this aspect, nanotechnology is emerging as a highly promising area to be focussed upon. In this study, we investigated the cytotoxic effect of synthesized chitosan-functionalized gold nanoparticles (G1) having a diameter of 15–20 nm on hormone-responsive MCF7 and hormone therapy-resistant MDA-MB-231 breast cancer cell lines. We found significant mortality of these two cancer cell types with the IC50 as low as ~ 11 ppb just after 48 h of treatment without exhibiting any cytotoxic effect on normal human peripheral blood lymphocytes. In both cancer cell lines, G1 induced severe cytomorphological alterations and reactive oxygen species overload with subsequent activation and nuclear translocation of master stress-regulator Nrf2 as an antioxidant response. Consequently, nuclear fragmentation and subsequent apoptotic cell death were evident that progressed primarily through activation of the Bax–Caspase9–Caspase3–PARP1 axis being concomitant with p53–p21 mediated cell cycle arrest. Moreover, disturbed homeostasis of cellular elements like copper, chlorine, potassium, sulfur, selenium and calcium further strengthened our findings. Therefore, G1 can be concluded as highly effective against two major types of breast cancer cells without any significant toxic effect in normal cells which might popularize it as a potent candidate for breast cancer therapeutics warranting further research.

In Vitro Antigenotoxic, Antihelminthic and Antioxidant Potentials Based on the Extracted Metabolites from Lichen, Candelariella vitellina.

Lichens have recently received great attention due to their pharmacological potentials. The antigenotoxic potential of C. vitellina extract (25 and 50 µg/mL) was assessed in normal human peripheral blood lymphocytes (HPBL) against Mitomycin C (MMC) co-treatments. Flow cytometric analyses of cell cycle distribution, as well as apoptosis (Annexin V/PI), revealed that the extract had significantly (p ≤ 0.05) ameliorated the MMC toxicity by reducing the apoptotic cells and normalized the cell cycle phases. C. vitellina exhibited antigenotoxicity by ameliorating the diminished mitotic index and DNA single-strand breaks caused by MMC. Herein, the hydromethanolic extract (80%) of Candelariella vitellina (Japan) lichen, exhibited very low cytotoxicity towards normal human peripheral lymphocytes (HPBL) with IC50 >1000 µg/mL. In order to explore the antihelminthic effect, Echinococcus granulosus protoscoleces were used in vitro. Eosin staining revealed significant (p ≤ 0.05) dose and time-dependent scolicidal effects of the extract confirmed by degenerative alterations as observed by electron scan microscopy. Furthermore, primary and secondary metabolites were investigated using GC-MS and qualitative HPLC, revealing the presence of sugars, alcohols, different phenolic acids and light flavonoids. Significant antioxidant capacities were also demonstrated by DPPH radical-scavenging assay. In conclusion, the promising antigenotoxic, antihelminthic and antioxidant potentials of C. vitellina extract encourage further studies to evaluate its possible therapeutic potency.

Evaluation of the Interaction between Malignant and Normal Human Peripheral Blood Lymphocytes Under Cocultivation and Separate Cultivation

Using the Comet assay, the peculiarities of the interaction between malignant and normal human peripheral blood lymphocytes under cocultivation and separate cultivation were investigated. A decrease in Tail Moment was observed against an increase in the frequency of cells in the state of apoptosis in the culture of lymphocytes from conditionally healthy volunteers (bystander cells) under the influence of blood cells from patients with CLL (inductor cells). A statistically significant (p < 0.001) reduction both in the frequency of cells with high levels of DNA damages and apoptotic activity was established in the population of inductor cells under the influence of the bystander cells. The results obtained indicate the realization of both direct (effect of cells-inductors on bystander cells) and rescue (effect of bystander cells on cells-inductors) TIBE phenomenon.

Antagonistic Effects of CAPE (a Component of Propolis) on the Cytotoxicity and Genotoxicity of Irinotecan and SN38 in Human Gastrointestinal Cancer Cells In Vitro.

The incidence of gastrointestinal cancers is increasing every year. Irinotecan (CPT-11), a drug used in the treatment of colorectal cancer and gastric cancer, is metabolized by carboxylesterases to an active metabolite, SN-38, which is more cytotoxic. CAPE (caffeic acid phenethyl ester) is an active component of propolis, which has a high antibacterial, antiviral, and antineoplastic potential. This study analyses the impact of CAPE on the cytotoxic (MTT assay), genotoxic (comet assay) and proapoptotic (caspase-3/7 activity) potential of irinotecan and its metabolite SN-38 in cultures of gastrointestinal neoplastic cells (HCT116, HT29, AGS). Cytotoxicity and genotoxicity activities of these compounds were carried out in comparison with human peripheral blood lymphocytes (PBLs) in vitro. The antioxidant potential of CAPE was investigated in relation H2O2-induced oxidative stress in the both neoplastic cells and PBLs. CAPE expressed cytotoxic, genotoxic, and pro-apoptotic activity against AGS, HCT116, and HT29 tumor cells. CAPE, in the presence of different concentrations of irinotecan or SN38, decreased the cytotoxicity, genotoxicity, and pro-apoptotic activity in these cell lines, but it has no such action on normal human peripheral blood lymphocytes.

Cytotoxic effect of green synthesized silver nanoparticles in MCF7 and MDA-MB-231 human breast cancer cells in vitro

With the incessant rise in the cancer burden worldwide it is a dire need to develop anticancer agents that will offer negligible or no side effects and at the same time will be economically feasible. In this study, we utilized the principle of green chemistry where tyrosine and chitosan were used as reducer and stabilizer respectively to synthesize biocompatible silver nanoparticles. They were characterized by ultraviolet–visible spectroscopy, transmission electron microscopy and dynamic light scattering technique and found to be spherical with average diameter of 13–22 nm. Their toxicity was evaluated in MCF7 and MDA-MB-231 human breast cancer cell lines. MTT assay revealed excellent cytotoxic effect with IC50 values as low as 6.4 and 6.56 ppb respectively after 48 h of treatment. Intriguingly, they showed minimum toxicity in normal human peripheral blood lymphocytes at these effective concentrations. Cytomorphological alteration, ROS generation (DCFDA analysis) and nuclear fragmentation (Hoechst staining) were pronounced in both cancer cell lines following treatment. These nanoparticles also promoted expression and nuclear translocation of Nrf2 as an antioxidant response which was revealed by Western blot and immunofluorescence studies respectively. ‘Apoptosis assay’ confirmed the presence of apoptosis and ‘Caspase-8 activity assay’ revealed absence of the extrinsic apoptosis pathway. Western blot data (upregulation of p21, Bax/Bcl2 ratio, Caspase-9, Caspase-3 and cleaved PARP1) established the occurrence of intrinsic apoptosis pathway following cell cycle arrest. To conclude, the green synthesized silver nanoparticles are cytotoxic to cancer cells and can be considered as effective and safe cytotoxic agents in breast cancer therapeutics.

Foeniculum Vulgare and Pelargonium Graveolens Essential Oil Mixture Triggers the Cell Cycle Arrest and Apoptosis in MCF-7 Cells.

Fennel (Foeniculum vulgare) and rose geranium (Pelargonium graveolens) oils are known for their various biological effects including anticancer properties. This study aimed to evaluate the anticancer mechanism of fennel and geranium oils combined treatment on MCF-7 cells. The GC-MS method for essential oil characterization as well as the in vitro cytotoxicity, morphological changes, real-time PCR and immunocytochemical investigation for apoptosis-related markers, in addition, to flow cytometric cell cycle distribution analysis were done. The major constituents of both essential oils were anethole (55.33 %) and estragole (11.57 %) for fennel essential oil. However, cintronellol (34.40 %) and geraniol (8.67 %) were identified in geranium oil. The results revealed an IC50 of 220±5.7 and 60±2.1µg/ml for fennel and geranium oils, respectively. The mechanistic anticancer properties were investigated throughout the 70, 50, and 25µg/ml of oils mixture. The marked apoptotic morphology and the flow cytometric cell cycle distribution analysis in addition to the levels of apoptosisrelated makers such as p53, caspase-3, mir-21, mir-92a, Bcl-2, and ki-67 confirmed that fennel and geranium oils combination induced cell cycle arrest and apoptosis in MCF-7 cells. Moreover, the oils mixture did not exert any significant (P<0.01) toxicity on normal human peripheral blood lymphocytes in vitro. The findings showed that the mixture of oils exerted selective cytotoxicity towards MCF-7 cells through induction of cell cycle arrest and apoptosis which may be triggered by the synergistic effect between the active ingredients of fennel and geranium oils.

Apoptosis induction in lung and prostate cancer cells through silver nanoparticles synthesized from Pinus roxburghii bioactive fraction.

The current study was carried out to synthesize silver nanoparticles (AgNPs) via bioactive fraction of Pinus roxburghii needles using a simple, cost-effective, and eco-friendly green chemistry method. As butanol fraction of P. roxburghii exhibited maximum anticancer activity on lung adenocarcinomas (A549) as compared to other fractions therefore, butanol fraction was used to synthesize silver nanoparticles (PNb-AgNPs). The characterization studies by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (SAED) confirmed the synthesis of the nanoparticles. The field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) analysis showed the spherical structure of nanoparticles with an average diameter of approximately 80nm. Interestingly, PNb-AgNPs exhibited significant cytotoxicity towards both A549 and prostatic small cell carcinomas (PC-3) with IC50 values of 11.28 ± 1.28μg/ml and 56.27 ± 1.17μg/ml, respectively, while lacking toxicity against normal human breast epithelial cells (fR2) and human peripheral blood lymphocytes (PBL). Further, enhanced reactive oxygen species generation, mitochondrial depolarization, apoptotic cell population (sub-G1) and DNA fragmentation observed in cancer cells were treated with PNb-AgNPs. Apoptosis was demonstrated by caspase-3 and PARP-1 activation in PNb-AgNPs-pretreated cancer cells. These results strongly suggest that PNb-AgNPs are capable of inducing cancer cell death and could act as a therapeutic nanoformulation for cancer.

Cytotoxic and mutagenic effects of green silver nanoparticles in cancer and normal cells: a brief review

Applications of silver nanoparticles (AgNPs) have revolutionized the medicinal industry. Due to small size (1–100 nm) these nanoparticles are able to cross cell and nuclear membrane and induce cyto/genotoxicity. Green synthesized (plant based) AgNPs (GSAgNPs) have emerged as alternative antimicrobial agent to chemically synthesized nanoparticles assuming their methods of synthesis will be environment friendly and non toxic. In this review we report that the GSAgNPs were found to be cytotoxic and genotoxic to different cancer cell lines. They affected cell proliferation caused apoptosis and cell death of human cancer cells but did not cause such damages to normal human peripheral blood lymphocyte cells. This property can be utilized in cancer therapeutics to overcome the major drawbacks of chemotherapeutics with surface modifications of the nanoparticles by adding functionalized AgNPs.

HPLC Analysis of Phenolic Acids, Antioxidant Activity and in vitro Effectiveness of Green and Roasted Caffea arabica Bean Extracts: A Comparative Study.

Coffee is a popular drink; it is one of the most commercialized food products and a rich source of biologically active compounds that are important for human health. This study aimed to prove the anticancer activity of Green Coffee (GC) and Roasted Coffee (RC) bean aqueous extracts (Coffea arabica) on breast cancer adenocarcinoma cell line (MCF-7) and the safety of both extracts on normal Human Peripheral Blood Lymphocytes culture (HPBL). Total phenolic content for GC and RC extracts was measured and result of both extracts were (0.308±0.016 & 0.233±0.013mg/g) respectively. The phenolic acids were screened by HPLC at the wavelength of 254& 278 and 300 nm and 5-caffeoylquinic acids (Chlorogenic acid), the predominant form of phenolic acids, was identified in GC and RC samples. Ferric Reducing Antioxidant Power (FRAP) as well as the free radical scavenging activity (DPPH) proved the antioxidant properties of both extracts. The DPPH IC50 mean values of GC and RC extracts were (2.4±0.08, 2.3±0.16 µg/ml) respectively. Cytotoxicity of both extracts on MCF-7 cells were evaluated by neutral red uptake assay which showed the IC50 mean values (377±5.7,500±8.1 µg/ml) for GC and RC extracts respectively. The safety of both extracts (0, 125, 250, 500 µg/ml) on HPBL was evaluated in vitro using trypan blue exclusion method and DNA single strand breaks (alkaline comet assay). Result revealed non-significant cytotoxic difference (P<0.001) between cultures especially at lower doses of GC and RC extracts except the highest dose of GC and RC extract which showed slightly significant damage (P<0.001). This study proved that GC and RC aqueous extracts were found to be selectively cytotoxic in vitro to cancerous cells (MCF-7 cell line) causing cell death with no cytotoxicity on normal human lymphocytes especially at lower doses.

Alkaline Phosphatase Activity of Normal and Malignant Human Lymphocytes.

Bone marrow aspirates form patients with multiple myeloma were obtained and lymphocytes were isolated using density gradient centrifugation. The APase activity of the samples ranged from 12 to 88nmol/0.2 × 106 cells as compared to the very low activity present in normal human peripheral blood lymphocytes PBL. Neither the unstimulated nor the mitogen stimulated lymphocytes from peripheral blood of normal humans showed appreciable APase activity. The lymphocytes from bone marrow of multiple myeloma patients as well as myeloma cell lines-RPMI 8226 and U266 B1 express APase activity constitutively. The results of the present investigation are discussed in the light of existing literature.

DNA Topoisomerase IB as a Potential Ionizing Radiation Exposure and Dose Biomarker.

In radiation exposure scenarios where physical dosimetry is absent or inefficient, dose estimation must rely on biological markers. A reliable biomarker is of utmost importance in correlating biological system changes with radiation exposure. Human DNA topoisomerase ІB (topo І) is a ubiquitous nuclear enzyme, which is involved in essential cellular processes, including transcription, DNA replication and DNA repair, and is the target of anti-cancer drugs. It has been shown that the cellular activity of this enzyme is significantly sensitive to various DNA lesions, including radiation-induced DNA damages. Therefore, we investigated the potential of topo I as a biomarker of radiation exposure and dose. We examined the effect of exposure of different human cells to beta, X-ray and gamma radiation on the cellular catalytic activity of topo I. The results demonstrate a significant reduction in the DNA relaxation activity of topo I after irradiation and the level of the reduction was correlated with radiation dose. In normal human peripheral blood lymphocytes, exposure for 3 h to an integral dose of 0.065 mGy from tritium reduced the enzyme activity to less than 25%. In MG-63 osteoblast-like cells and in human pulmonary fibroblast (HPF) cells exposed to gamma radiation from a 60Co source (up to 2 Gy) or to X rays (up to 2.8 Gy), a significant decrease in topo I catalytic activity was also observed. We observed that the enzyme-protein level was not altered but was partially posttranslational modified by ADP-ribosylation of the enzyme protein that is known to reduce topo I activity. The results of this study suggest that the decrease in the cellular topo I catalytic activity after low-dose exposure to different radiation types may be considered as a novel biomarker of ionizing radiation exposure and dose. For this purpose, a suitable ELISA-based method for large-scale analysis of radiation-induced topo I modification is under development.

Synergistic activity of sorafenib and betulinic acid against clonogenic activity of non-small cell lung cancer cells.

The highly selective multi‐targeted agent sorafenib is an inhibitor of a number of intracellular signaling kinases with anti‐proliferative, anti‐angiogenic and pro‐apoptotic effects in various types of tumors, including human non‐small cell lung cancer (NSCLC). Betulin displays a broad spectrum of biological and pharmacological properties, including anticancer and chemopreventive activity. Combination of drugs with different targets is a logical approach to overcome multilevel cross‐stimulation among key signaling pathways in NSCLC progression. NSCLC cell lines, A549, H358 and A427, with different KRAS mutations, and normal human peripheral blood lymphocyte cells, were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatments on viability (MTS test), proliferation and apoptotic susceptibility based on flow cytometry, alterations in signaling pathways by western blotting and colony‐forming ability. The combination of sorafenib with betulinic acid had a strong effect on the induction of apoptosis of different NSCLC cell lines. In addition, this combination was not toxic for human peripheral blood lymphocytes. Combination treatment changed the expression of proteins involved in the mitochondrial apoptosis pathway and induced apoptotic death by caspase activation. Importantly, combination treatment with low drug concentrations tremendously reduced the colony‐forming ability of A549, H358 and A427 cells, as compared to both compounds alone. In this study, we showed that combination therapy with low concentrations of sorafenib and betulinic acid had the capacity to induce high levels of cell death and abolish clonogenic activity in some NSCLC cell lines regardless of KRAS mutations.

Selective induction of DNA damage, G2 abrogation, and mitochondrial apoptosis by leaf extract of traditional medicinal plant Wrightia arborea in K562 cells.

Plants have proved to be an important source of anti-cancer drugs. Wrightia arborea, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. This study evaluates the antiproliferative/apoptotic potential of Wrightia arborea leaf extracts, prepared in different organic solvents, on cancer cell lines. MTT assay, light and fluorescence microscopy, flow cytometry, DNA laddering, alkaline comet assay, and western blotting were some of the techniques used for evaluation. Combinations of camptothecin, either with CHK1 inhibitor-PD407824 or with W. arborea leaf extract, were deployed to determine the G2 abrogating potential of the extract. The chloroform extract (WAC) selectively killed K562 cells, without affecting cancerous MCF-7, Hep G2 cells, and normal human peripheral blood lymphocytes. Cell death was characterized by observation of apoptotic bodies, increased Ca2+ and ROS, phosphatidyl serine externalization, mitochondrial membrane depolarization, DNA laddering, increased sub-G1 population, and altered expression of caspase 3, -9, and PARP. WAC also induced DNA damage, alterations in key G2/M phase protein expression, cell cycle perturbation, and potent G2 abrogation. The present study showed that W. arborea leaf extract, WAC, is capable of selectively killing leukemic cancer cells leaving normal lymphocytes unaffected. Our results indicate that this is effectuated through DNA damage and G2 abrogation leading to mitochondrial apoptosis. Taken together, this report contributes toward a better understanding of the anticancer properties of this traditional medicinal plant extract possessing valuable bioactive constituents which can serve as a bioresource for promising complimentary/alternative/chemopreventive therapeutics.

Identification of IgH gene rearrangement and immunophenotype in an animal model of Epstein-Barr virus-associated lymphomas.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma. Because the susceptible hosts of EB virus are limited to human and cotton-top tamarins (Saguinus oedipus), there have been no appropriate animal models until the lymphoma model induced by EBV in human peripheral blood lymphocyte (hu-PBL)/SCID chimeric mice was reported. However, it is still controversial whether the EBV-associated lymphoma induced in hu-PBL/SCID mice is a monoclonal tumor. In this study, we transplanted normal human peripheral blood lymphocytes (hu-PBL) from six donors infected with EBV into SCID mice to construct hu-PBL/SCID chimeric mice. The induced tumors were found in the mediastinum or abdominal cavity of SCID mice. Microscopic observation exhibited tumor cells that were large and had a plasmablastic, centroblastic or immunoblastic-like appearance. Immunophenotyping assays showed the induced tumors were LCA-positive, CD20/CD79a-positive (markers of B cells), and CD3/CD45RO-negative (markers of T cells). A human-specific Alu sequence could be amplified by Alu-PCR. This confirmed that induced tumors were B-cell lymphomas originating from the transplanted human lymphocytes rather than mouse cells. EBER in situ hybridization detected positive signals in the nuclei of the tumor cells. Expression of EBV-encoded LMP1, EBNA-1, and EBNA-2 in the tumors was significantly positive. PCR-based capillary electrophoresis analysis of IgH gene rearrangement revealed a monoclonal peak and single amplification product in all six cases of induced tumors. This indicated that EBV can induce monoclonal proliferation of human B lymphocytes and promotes the development of lymphoma. J. Med. Virol. 88:1804-1813, 2016. © 2016 Wiley Periodicals, Inc.

Chemical characterization of Rosa damascena Miller var. trigintipetala Dieck essential oil and its in vitro genotoxic and cytotoxic properties

Rosa damascena Miller var. trigintipetala Dieck (Taif rose) is considered as one of the most important economic products of Taif governorate, KSA. In this study, the different components of Taif rose essential oil were characterized using GC-MS technique. In addition, the in vitro cytotoxic, genotoxic and anticancer effects of the oil were investigated toward normal human peripheral blood lymphocytes and two kinds of human cancer cell lines; HepG2 and MCF7. GC-MS analysis showed that the acyclic monoterpene alcohols had the highest content (254.7 mg/g oil). The major components of the oil that have relative percentage > 5% were β-citronellol, geraniol, nerol, linalool, phenylethyl alcohol and nonadecane. The results showed that the oil was safe at a dose < 50 μg/mL when applied to normal human blood lymphocytes monitored by 7-AAD assay and DNA index determination. The oil exerted anticancer activity against HepG2 and MCF7 with IC50 = 13.03 ± 0.8 and 16.44 ± 1.4 μg/mL respectively. In conclusion, the essential oil of Taif rose can be used as an effective therapeutic natural agent after more toxicological in vitro and in vivo studies.

Zerumbone induces mitochondria-mediated apoptosis via increased calcium, generation of reactive oxygen species and upregulation of soluble histone H2AX in K562 chronic myelogenous leukemia cells.

Zerumbone, a natural cyclic sesquiterpene, is known to exhibit selective toxicity toward various cancer cells. Sustained efforts to explore the potential of new agents for effective therapy are critical in the context of development of drug resistance especially in cancers like chronic myelogenous leukemia (CML). The present study evaluated the effect of zerumbone on CML-K562 cells. The cell viability of zerumbone-treated K562 cells was detected by MTT assay, and morphological changes were observed by light microscopy and scanning electron microscopy (SEM). Staining with Hoechst 33258, acridine orange/ethidium bromide, and AnnexinV-FITC were used to detect apoptosis. Intracellular reactive oxygen species (ROS), Ca(2+), and changes in mitochondrial membrane potential were measured using Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Fluo-3AM, and Rhodamine-123, respectively. Western blot analysis was carried out to detect key proteins involved in apoptosis. Zerumbone inhibited K562 cell proliferation with an IC50 value of 3.5 μg/mL and colony formation capability (P < 0.001). Interestingly, zerumbone did not affect the growth of normal human peripheral blood lymphocytes (hPBLs). Distinct morphological changes observed by light microscopy and fluorescent staining with Hoechst-33258, AO/EtBr, annexin V-FITC, and cytotoxicity evaluation by comet assay indicated induction of DNA damage and apoptosis. This was further confirmed by demonstration of pro-caspase-3, -9 activation and Poly(ADP-ribose) polymerase (PARP) cleavage on western blots. Apoptosis induction was found to be mitochondria mediated, involving increased free intracellular Ca(2+), ROS, and upregulation of soluble histone H2AX. Our results suggest that zerumbone holds promise as a potential candidate drug for CML.