Mona-Liza Lottering

The mammalian metabolite, 2-methoxyestradiol, affects P53 levels and apoptosis induction in transformed cells but not in normal cells.

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.

Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells

The effects of arachidonic acid (AA) and gamma-linolenic acid (GLA) on cell cycle progression and apoptosis induction, using flow cytometry, were compared on normal human skin fibroblasts, strain HSF43 with wild type (wt) p53, large T antigen transformed HSF43 cells (line E8T4) with non functional p53, and on two lymphoblast cell lines, TK6 with wt p53 and WTK1 with mutant p53. AA and GLA caused similar dose (50, 75 and 100 microg/ml AA and GLA) and time dependent (24 and 48 h) induction of apoptosis in each cell line. The degrees of the response of the four cell lines were, however, different. The normal HSF43 cells were most resistant against apoptosis induction and the WTK1 cells most susceptible. The apoptosis induction appeared to be independent of functional p53. Cell cycle progression was also similarly affected by AA and GLA in the two cell types. In the fibroblast type cells (HSF43 and E8T4) S- and G2/M-phase arrests were evident after 48 h exposure to AA and GLA, and in the lymphoblast cell lines (TK6 and WTK1) the cells were arrested in the G1-phase.

In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2-10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P<0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G(1) apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.

The induction of apoptosis in human cervical carcinoma (HeLa) cells by gamma-linolenic acid

A high concentration (50 micrograms/ml) of gamma-linolenic acid (GLA) induced morphological lesions typical of apoptosis, as well as DNA fragmentation, in HeLa cells. A lower concentration of GLA (20 micrograms/ml), caused an increased proliferating cell nuclear antigen (PCNA) labelling, with 92.7% cells positive, compared to 27.7% at a concentration of 50 micrograms/ml GLA. In correlation with these results, the number of cells with degraded DNA below the G0/G1 peak increased significantly in the 50 micrograms/ml GLA-treated cells, but increased only slightly in cells exposed to the lower level of GLA. The high levels of PCNA induced by 20 micrograms/ml GLA, in both G1 and S phases, may indicate a state of DNA repair synthesis, whilst at the higher concentration of GLA, most of the cells became apoptotic. Since apoptosis is associated with the deregulation of c-Myc expression, and as the Raf-1-MAP kinase cascade activates the expression of c-Myc and c-Jun, we investigated the effects of 20 and 50 micrograms/ml GLA on the Raf-1, c-Myc and c-Jun levels, and on the activity of MAP kinase. The results showed that 50 micrograms/ml GLA lowered the activity of MAP kinase. As expected with the decreased MAP kinase activity in the cells exposed to the higher level GLA, the c-Jun levels were also lowered. The levels of c-Myc, however, were increased. It is therefore possible that the deregulated expression of c-Myc in the HeLa cells exposed to the high level of GLA (50 micrograms/ml) may contribute to the induction of apoptosis in HeLa cells.

In vitro effects of 2-methoxyestradiol on MCF-12A and MCF-7 cell growth, morphology and mitotic spindle formation

The influence of 2‐methoxyestradiol (2ME) was investigated on cell growth, morphology and spindle formation in a tumorigenic (MCF‐7) and non‐tumorigenic (MCF‐12A) epithelial breast cell line. Inhibition of cell growth was more pronounced in the MCF‐7 cells compared to the MCF‐12A cells following 2ME treatment. Dose‐dependent studies (10−5 – 10−9 M) revealed that 10−6 M 2ME inhibited cell growth by 44% in MCF‐12A cells and by 84% in MCF‐7 cells (p‐value < 0.05). 2ME‐treated MCF‐7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent or less prominent in MCF‐12A cells. 2ME had no effect on the length of the cell cycle between S‐phase and the time a mitotic peak was reached in either cell line but MCF‐7 cells were blocked in mitosis with no statistically significant alterations in the phosphorylation status of Cdc25C. Nevertheless, Cdc2 activity was significantly increased in MCF‐7 cells compared to MCF‐12A cells (p‐value < 0.05). The results indicate that 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest that may result in the induction of apoptosis. The tumorigenic MCF‐7 cells were especially sensitive to 2ME treatment compared to the normal MCF‐12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Copyright

17β-Estradiol metabolites affect some regulators of the MCF-7 cell cycle

The activity of p34(cdc2) plays a key role in the regulation of the eukaryotic cell cycle. Another cell cycle associated molecule is PCNA. We investigated the effects of 2-hydroxy-17beta-estradiol, a cell proliferator, and 2-methoxy-17beta-estradiol, a potent inhibitor of cell growth, on the levels and activity of p34(cdc2) and on the levels of PCNA, as well as on protein phosphorylation in MCF-7 cells. 2-Hydroxyestradiol increased p34(cdc2) activity at G1/S and elevated PCNA levels during S-phase. 2-Methoxyestradiol caused unscheduled activation of p34(cdc2) in S-phase and decreased levels of p34(cdc2) and PCNA during G2/M. We conclude that 2-hydroxy- and 2-methoxyestradiol have definite, though different regulatory functions during the cell cycle.

In vitro effects of 2-methoxyestradiol on cell numbers, morphology, cell cycle progression, and apoptosis induction in oesophageal carcinoma cells

The influence of 2‐methoxyestradiol (2‐ME) was investigated on cell numbers, morphology, cell cycle progression, and apoptosis induction in an oesophageal carcinoma cell line (WHCO3). Dose‐dependent studies (1 × 10−9M–1 × 10−6M) revealed that 2‐ME significantly reduced cell numbers to 60% in WHCO3 after 72 h of exposure at a concentration of 1 × 10−6M compared to vehicle‐treated cells. Morphological studies entailing light‐, fluorescent‐, as well as transmission electron microscopy (TEM) confirmed 2‐MEs antimitotic effects. These results indicated hallmarks of apoptosis including cell shrinkage, hypercondensation of chromatin, cell membrane blebbing, and apoptotic bodies in treated cells. Flow cytometric analyses demonstrated an increase in the G2/M‐phase after 2‐ME exposure; thus preventing cells from proceeding through the cell cycle. β‐tubulin immunofluorescence revealed that 2‐ME caused spindle disruption. In addition, increased expression of death receptor 5 protein was observed further supporting the proposed mechanism of apoptosis induction via the extrinsic pathway in 2‐ME‐exposed oesophageal carcinoma cells. Copyright

The effects of prostaglandin A2 on cell growth, cell cycle status and apoptosis induction in HeLa and MCF-7 cells

The effects of 20 microg/ml exogenous prostaglandin A(2) (PGA(2)) were evaluated on cell numbers in HeLa (human epithelial cervix carcinoma) and MCF-7 (human breast carcinoma) cells. In HeLa cells, PGA(2) reduced cell numbers significantly to 75% after 24 h (P < 0.05) and exposure of 48 h decreased cell numbers to 61% (P < 0.05) of the control. In MCF-7 cells, PGA(2) significantly reduced cell numbers to 48% after 24 h and to 20% after 48 h, compared to vehicle-treated control cells (P < 0.05). The anti-mitogenic effects were confirmed by morphological studies conducted after 48 h of exposure to PGA(2), when optimal effects were observed. HeLa and MCF-7 cells exposed to PGA(2), showed chromatin aggregation, cell membrane blebbing and uneven distribution of chromosomes. Cell cycle progression analysis of HeLa and MCF-7 cells, showed an increase in DNA content preceding the G(0)/G(1) peak after 48 h of exposure, which is indicative of apoptotic body formation.

Differential cytotoxic effects of gamma-linolenic acid on MG-63 and HeLa cells

Gamma-linolenic acid (GLA) inhibited cell proliferation in MG-63 and HeLa cells. Different morphological lesions were present in dividing cells; abnormal spindle formation in MG-63 cells and chromosome hypercondensation in HeLa cells. Different types of cell death were also present in interphase cells, no apoptosis but only 6% pycnosis in MG-63 cells and 90% apoptosis in HeLa cells. In MG-63 cells immunofluorescence showed segregation of nucleoli components, abnormal spindle formation and decreased labelling of microtubuli during interphase. In HeLa interphase cells prominently labelled abnormally arranged microtubuli were observed. The effects of GLA on protein synthesis in synchronized cells were determined with [35S]-methionine incorporation and SDS-PAGE. Decreased protein synthesis in both G1- and S-phase MG-63 cells was present. In S-phase HeLa cells, proteins of approximately 40, 92 and 150 kD were markedly expressed. Signalling mechanisms involved in cell proliferation and cell death may be differently affected in MG-63 and HeLa cells.

Comparative anti-mitotic effects of lithium gamma-linolenate, gamma-linolenic acid and arachidonic acid, on transformed and embryonic cells.

The effects of gamma-linolenic acid (GLA), the lithium salt of gamma-linolenic acid (LiGLA) and arachidonic acid (AA) were compared at doses of 50 microg/ml for periods of 6 and 24 h on cell cycle progression and apoptosis induction in transformed and in normal cells. In WHCO3 (oesophageal cancer) cells and on primary embryonic equine lung cells, we found LiGLA to be the most effective in apoptosis induction. After 24 h, 94% of the WHCO3 cancer cells and 44% of the primary embryonic equine lung cells exposed to LiGLA were apoptotic. The WHCO3 cancer cells were also very susceptible to the apoptosis-inducing effects of AA (56%) and GLA (44%), whereas the embryonic equine lung cells were much less affected by these two fatty acids. After 6 h exposure to all three compounds, most of the cycling WHCO3 cancer cells were blocked in S-phase. After 24 h treatment, some of the S-phase cells exposed to AA and GLA were apparently able to move into the G2/M phase, the LiGLA exposed cells were mostly apoptotic and no cycling cells were present. The primary embryonic equine lung cells were fairly resistant to the cytotoxic effects of GLA and AA. From our studies we conclude that, although LiGLA was the most toxic to the cancer cells, it is apparently less selective, compared to AA and GLA, in the killing of cancer and normal cells. It would also appear that the lithium might have added to the cytotoxic effects of LiGLA. The mechanism needs to be clarified.