ALA-Dependent Cancericides - Chlorophyll Biosynthesis and Technological Applications - page 440

19.4.1 Inhibition of DNA Synthesis by Oph

Many anti-tumor agents cause cells to pause during the cell cycle and induce
growth arrest prior to cell death (Dive and Hickman 1991 ; Strasser et al. 1994 ;
Yonish-Rouach et al. 1991 ). We therefore investigated whether ALA, Oph or a
combination of the two reagents could reduce cell proliferation. This was done by
measuring rates of DNA synthesis. All doses of Oph, but not ALA, caused a significant
decrease in DNA synthesis after 3 h of incubation, as evidenced by reduced
3 H-thymidine incorporation into the cells. Proliferation of MLA 144 cells was inhibited
64 % at 0.75 mM Oph, a dose that was previously shown to be non-toxic and to
enhance Proto accumulation in the presence of ALA in vitro (Rebeiz et al. 1992 ). The
level of inhibition was similar when ALA was used in combination with Oph, at all
doses tested. Treatment of MLA 144 cells with ALA alone at any concentration did not
significantly alter the rate of 3 H-thymidine incorporation. DNA synthesis was also
inhibited after 6 h and 24 h incubation with Oph (Rebeiz et al. 2001 ).

19.4.2 Reduction of Cell Proliferation by Proto
and Non-chelating Isomers of Oph

Ortel et al. ( 1993 ) reported that the metal chelator desferrioxamine inhibited cell
proliferation by chelating cellular iron, and this chelation suppressed progression
through the cell cycle. To test the possibility that inhibition of cell growth by Oph
could be due to its iron-chelating properties, two non-chelating positional-isomers
of Oph, 1,7-Oph and 4,7-Oph, were used in a DNA synthesis assay. Exogenous
Proto, which has been shown to inhibit the proliferation of cells by binding to
(M-PBR) in mitochondria (Verma and Snyder 1988 ), was used as a positive control.
Both Oph isomers inhibited 3 H-thymidine incorporation in MLA 144 cells in a
dose-dependent manner similar to Oph (Rebeiz et al. 2001 ). As expected from
previous work on exogenous Proto (Verma and Snyder 1988 ), growth arrest was
induced at micromolar concentrations. Surprisingly, exogenous Proto was 10-30
fold more efficient at inhibiting DNA synthesis than Oph or its isomers (p

0.05).
The positional isomer 1,7-Oph was two to four fold more effective than Oph in
causing growth arrest of MLA 144 cells (p

<

0.05). These results indicated that the
iron-chelating property of Oph was not responsible for inducing cell growth arrest.

<

19.4.3 Cell Viability and Membrane Permeability of MLA
144 Cells Treated with ALA, Oph or Proto

Apoptotic cells still have intact plasma membranes, and may appear viable, while
necrotic cells quickly lose membrane intactness (Cohen et al. 1992 ). It was