Barbara J.S. Sanderson

Mutagenic damage to mammalian cells by therapeutic alkylating agents

Cytotoxic alkylating agents used as therapeutics include nitrogen mustards, ethyleneimines, alkyl sulfonates, nitrosoureas and triazenes. Their reactivity with DNA, RNA and proteins can cause cell death. Side-effects of treatment include tissue toxicity and secondary malignancies, likely due to the genetic damage induced. The full mutagenic potential of alkylating agents may only be realised after they undergo metabolic activation, principally by cytochromes P450. Mutagenicity is related to the ability of alkylating agents to form crosslinks and/or transfer an alkyl group to form monoadducts in DNA. The most frequent location of adducts in the DNA is at guanines. Expressed mutations involve different base substitutions, including all types of transitions and transversions. The mutational spectra of alkylating agents on mammalian cells is distinct from that induced in bacterial cells, reflecting the different codon usage by bacteria and differences in DNA repair and replication enzymes. Mutations are induced by busulfan, chlorambucil (CAB), cyclophosphamide (CP, or its metabolite), dacarbazine, mechlorethamine, melphalan, mitomycin-C (MMC), nitrosoureas and thiotepa. Although dose-dependent, the relationship is not always linear. The molarities at which alkylating agents induce cell killing and mutations vary over three orders of magnitude. The mutagenic efficiency, of alkylating agents also varies, with some agents inducing three times more mutations for equivalent cell killing. The induction of micronuclei, sister chromatid exchanges, or chromosome aberrations is variable, but has been observed for CP, CAB, MMC, melphalan and triethylenemelamine. There is insufficient information to determine whether any synergistic effects of alkylating agents used in combination will influence the cytotoxic and mutagenic damage equally. Understanding the potential synergy of alkylating agents at the cellular and molecular level should allow improvement of the therapeutic efficacy of alkylating agents without increasing the unwanted mutation induction.

Mutagenic and carcinogenic properties of platinum-based anticancer drugs.

Cisplatin (DDP) is currently one of the most effective drugs for the treatment of cancer. It causes primarily intrastrand DNA-DNA cross-links, and is highly mutagenic and carcinogenic in both in vitro and in vivo experimental models. There is, however, considerable variability between the response seen in different cellular systems, probably at least partly because of the different cellular DNA repair capacities. A number of analogues of cisplatin have been developed and one of these, carboplatin (CDDCA), is also in widespread clinical use. Although it is somewhat less toxic, there is no evidence that its mode of action differs from that of cisplatin. A limited amount of mutagenicity data suggests that it has similar mutagenic and carcinogenic consequences as the parent drug. Many further analogues of cisplatin are now in clinical trials, and some of these appear to have different DNA repair responses (and therefore possibly the development of clinical resistance). Although some (e.g., iproplatin and spiroplatin) are less mutagenic than either cisplatin or carboplatin, these appear to be the ones least likely to achieve wide use. There are insufficient data on several of the most promising clinical analogues (e.g., DWA2114R and ACDDP) to judge their relative mutagenic and carcinogenic potential. Detailed studies on the DNA repair and mutagenicity characteristics of these compounds will not only provide clinically relevant data, but may also aid in the selection of further useful antitumour agents in this series.

Molecular basis of X-ray-induced mutation at the HPRT locus in human lumphocytes

Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X-radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations.

Cytotoxic effect of xanthones from pericarp of the tropical fruit mangosteen (Garcinia mangostana Linn.) on human melanoma cells

Mangosteen (Garcinia mangostana Linn.) is a tropical tree from South East Asia and its fruit pericarp is a well-known traditional medicine. In this study, the cytotoxic effect of three xanthone compounds (α-mangostin, γ-mangostin, and 8-deoxygartanin) from mangosteen pericarp was investigated using the human melanoma SK-MEL-28 cell line. Significant dose-dependent reduction in % cell viability was induced. γ-Mangostin and 8-deoxygartanine at 5 μg/ml increased the cell cycle arrest in G(1) phase (90% and 92%) compared with untreated cells (78%). All compounds induced apoptosis, of the highest being α-mangostin at 7.5 μg/ml that induced 59.6% early apoptosis, compared to 1.7% in untreated cells. The apoptotic effect of α-mangostin was via caspase activation and disruption of mitochondrial membrane pathways as evidenced by 25-fold increased caspase-3 activity and 9-fold decreased mitochondrial membrane potential when compared to untreated cells. In conclusion, these xanthones, especially α-mangostin, are potential candidates as anti-melanoma agents.

Anti-skin cancer properties of phenolic-rich extract from the pericarp of mangosteen (Garcinia mangostana Linn.).

Skin cancers are often resistant to conventional chemotherapy. This study examined the anti-skin cancer properties of crude ethanol extract of mangosteen pericarp (MPEE) on human squamous cell carcinoma A-431 and melanoma SK-MEL-28 lines. Significant dose-dependent reduction in% viability was observed for these cell lines, with less effect on human normal skin fibroblast CCD-1064Sk and keratinocyte HaCaT cell lines. Cell distribution in G(1) phase (93%) significantly increased after 10 μg/ml of MPEE versus untreated SK-MEL-28 cells (78%), which was associated with enhanced p21(WAF1) mRNA levels. In A-431 cells, 10 μg/ml MPEE significantly increased the sub G(1) peak (15%) with concomitant decrease in G(1) phase over untreated cells (2%). In A-431 cells, 10 μg/ml MPEE induced an 18% increase in early apoptosis versus untreated cells (2%). This was via caspase activation (15-, 3- and 4-fold increased caspse-3/7, 8, and 9 activities), and disruption of mitochondrial pathways (6-fold decreased mitochondrial membrane potential versus untreated cells). Real-time PCR revealed increased Bax/Bcl-2 ratio and cytochrome c release, and decreased Akt1. Apoptosis was significantly increased after MPEE treatment of SK-MEL-28 cells. Hence, MPEE showed strong anti-skin cancer effect on these two skin cancer cell lines, with potential as an anti-skin cancer agent.

Ultrafine Quartz-Induced Damage in Human Lymphoblastoid Cells in vitro Using Three Genetic Damage End-Points.

ABSTRACT Respirable quartz is a potential human lung carcinogen. The mechanisms involved in this carcinogenesis, however, remain unclear, especially for the ultrafine particles (diameter <100 nm). The aim of the present study was to investigate the effects caused by ultrafine quartz (UF-quartz) in a human cell culture model. Genotoxicity and cytotoxicity induced by UF-quartz were investigated through the cytokinesis block micronucleus assay (CBMN), the Comet assay, the HPRT assay, the population growth assay, and the 3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. WIL2-NS cells were incubated for 10h with 0, 60, and 120 μg/mL UF-quartz. Significant decreases in percent of cell survival in the MTT assay were seen at higher doses, for example, 83%, and 64% relative survival at 60 μg/mL and 120 μg/mL, respectively. Only slight population regrowth was observed, with the population sizes recovering slightly by day 4 after quartz particles were removed. A significant increase in the frequency of micronucleated binucleated cells (MNed BNCs) was seen with 120 μg/mL quartz, from approximately 5 in 1000 BNCs in controls to 12 in 1000 BNCs. A significant reduction in the nuclear division index was observed by the CBMN assay, indicating inhibition of cell division by high-dose UF-quartz. A dose-dependent increase in induced HPRT-gene locus mutant frequency with increasing dose of UF-quartz was observed by the HPRT assay. No significant difference was found in DNA strand breakage as detected by the Comet assay. Collective findings suggest that UF-quartz can cause cytotoxicity and genotoxicity to human lymphoblasts in this model system.

Extracts from Calendula officinalis Offer in Vitro Protection Against H2O2 Induced Oxidative Stress Cell Killing of Human Skin Cells

The in vitro safety and antioxidant potential of Calendula officinalis flower head extracts was investigated. The effect of different concentrations (0.125, 0.5, 1.0, 2.0 and 5.0% (v/v)) of Calendula extracts on human skin cells HaCaT in vitro was explored. Doses of 1.0% (v/v) (0.88 mg dry weight/mL) or less showed no toxicity. Cells were also exposed to the Calendula extracts for either 4, 24 or 48 h before being exposed to an oxidative insult (hydrogen peroxide H2O2) for 1 h. Using the MTT cytotoxicity assay, it was observed that two independent extracts of C. officinalis gave time‐dependent and concentration‐dependent H2O2 protection against induced oxidative stress in vitro using human skin cells. Pre‐incubation with the Calendula extracts for 24 and 48 h increased survival relative to the population without extract by 20% and 40% respectively following oxidative challenge. The antioxidant potential of the Calendula extracts was confirmed using a complimentary chemical technique, the DPPH● assay. Calendula extracts exhibited free radical scavenging abilities. This study demonstrates that Calendula flower extracts contain bioactive and free radical scavenging compounds that significantly protect against oxidative stress in a human skin cell culture model. Copyright

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

Venom from the sea anemone, Heteractis magnifica, has multiple biological effects including, cytotoxic, cytolytic and hemolytic activities. In this study, cytotoxicity induced by H. magnifica venom was investigated using the crystal violet assay on human breast cancer T47D and MCF7 cell lines and normal human breast 184B5 cell line. Apoptosis was also assayed via Annexin V-flourescein isothiocyanate and propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle progression and mitochondria membrane potential were studied via flow cytometry following PI and JC-1 staining respectively. H. magnifica venom induced significant reductions in viable cell numbers and increases in apoptosis in T47D and MCF7 in dose-dependent manners. A significant apoptosis-related increase in the sub G1 peak of the cell cycle in both breast cancer cell lines was also observed. Moreover, treatment by venom cleaved caspase-8, caspase-9, and activated caspase-3. Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways. Consequently, H. magnifica venom has potential for the development of a breast cancer therapeutic.

Altered mRNA Expression Related to the Apoptotic Effect of Three Xanthones on Human Melanoma SK-MEL-28 Cell Line

We previously demonstrated that α-mangostin, γ-mangostin, and 8-deoxygartanin have significant cytotoxic effects on human melanoma SK-MEL-28 cell line. The current study revealed the underlying mechanisms. α-Mangostin (7.5 μg/mL) activated caspase activity, with a 3-fold and 4-fold increased caspase 8 and 9 activity, respectively. The molecular mechanisms were investigated by qRT-PCR for mRNA related to cell cycle arrest in G1 phase (p21WAF1 and cyclin D1), apoptosis (cytochrome C, Bcl-2, and Bax), and survival pathways (Akt1, NFκB, and IκBα). α-Mangostin significantly upregulated mRNA expression of cytochrome C and p21WAF1 and downregulated that of cyclin D1, Akt1, and NFκB. γ-Mangostin significantly downregulated mRNA expression of Akt1 and NFκB and upregulated p21WAF1 and IκBα. 8-Deoxygartanin significantly upregulated the mRNA expression of p21WAF1 and downregulated that of cyclin D1 and NFκB. The three xanthones significantly inhibited the mRNA expression of the BRAF V600E mutation. Moreover, α-mangostin and γ-mangostin significantly downregulated Akt phosphorylation at Ser473. In conclusion, the three xanthones induced an inhibitory effect on SK-MEL-28 cells by modulating the molecular targets involved in the apoptotic pathways.