ALA-Dependent Cancericides - Chlorophyll Biosynthesis and Technological Applications - page 433

exclusion and 3 H-blue thymidine incorporation were used to determine cell viability
and proliferation, respectively. Chromatography was used to determine the type of
porphyrin species that accumulated. Flow cytometry was used to determine porphy-
rin content by monitoring the red fluorescence (longer than 630 nm) emitted from
individual cells. The fluorescence was proportional to porphyrin content. The cells
that synthesized Proto became sensitive to light and exhibited 99 % inactivation
(cell death) after 4 days of dark incubation at the optimal Proto concentration.
Then Kennedy and coworkers (Kennedy et al. 1990 ; Kennedy and Pottier 1992 ,
1994 ) used ALA to treat different types of skin cancers. In their protocol, ALA was
mixed into a paste (10 % glaxal base 35-100 mg ALA/lesion), then applied
topically to the skin. The skin was then photoirradiated with laser light. The results
appeared to be promising. However highly intense light (52 mW/h/cm 2 ) and high
concentrations of ALA were required. As a consequence, these conditions caused
some damage to healthy tissues.
Then it was reported that better ALA-dependent photoradiation therapy results
were obtained by using long chain-fatty acid esters of ALA. That in turn reduced
the hydrophilic properties of ALA and improved its penetration into treated tissues
(Kloek et al. 1996 ). The ALA esterases that converted the ALA esters into ALA
before conversion to Proto were very active in animal tissues and less active in
insect and plant tissues (Kolossov and Rebeiz 2004 ).
The rest of this chapter is devoted to the discussion of using ALA with porphyrin
modulators for the photodynamic destruction of cancer cells.

19.2 Photodestruction of Tumor Cells by Induction
of Protoporphyrin IX Accumulation by ALA
and 1,10-Orthophenanthroline

Photodynamic herbicides (Chap. 17 ) and porphyric insecticides (Chap. 18 ) are two
novel technologies that manipulate the photosensitizing capability of metabolic
porphyrins. These two novel technologies destroy undesirable plants and insects
following co-treatment with -aminolevulinic acid (ALA), a naturally occurring

5-carbon amino acid, and one of a number of tetrapyrrole biosynthesis modulators
(Rebeiz et al. 1984 , 1988 ). The amino acid and the modulator act in concert. The
amino acid serves as a building block for intracellular tetrapyrrole accumulation,
while the modulator amplifies the accumulation of harmful tetrapyrroles. In the
light, the accumulated tetrapyrroles photosensitize the formation of singlet oxygen
which kills treated plants or insects by oxidation of their cellular membranes.
In the next four sections it is shown that treatment of rapidly multiplying
immortalized cells, with ALA and 1-8-orthophenanthroline (Oph) caused the cells
to accumulate much larger amounts of Proto than untreated cells. This endogenous
Proto accumulation caused in turn rapid cell death in the light. Slower growing
cells responded to such treatments by accumulating much lower levels of Proto.

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