?-Aminolevulinic acid (ALA) is recognized as the starter in the biosynthesis of the heme group, the structural basis of cytochromes, chlorophylls, biliary pigments, and other porphyrins. In mammals, ALA is synthesized within the mitochondria by the condensation of glycine with succinyl CoA. It is the first intermediary in the biosynthesis of protoporphyrin IX (PpIX) and, consequently, of the heme group. It is known that PpIX is present at low concentrations in normal cells and in high concentrations in tumor cells, because in cancerous cells, the enzyme ferrochelatase which converts protoporphyrin IX to heme has been found to be reduced, whereas the opposite situation has been found for porphobilinogen deaminase. In view of these characteristics, it has been proposed that PpIX might be involved in a new therapy for cancer. This therapy is called photodynamic therapy (PDT). PDT is a relatively selective local treatment that has been applied to a considerable number of tumors (glioblastoma multiforme, certain intraperitoneal cancers, early-stage lung cancers, and refractory in situ carcinoma of the urinary bladder) in order to define the role of PDT in cancer treatment for both palliation and complete local tumor eradication. PDT requires the simultaneous presence of a photosensitizer, light, and oxygen. The sensitizers accumulate specifically in tumor cells and are photoactivated by local exposure to light of the appropriate wavelength. By means of a mechanism involving hydrogen and electron transfer, the excited sensitizer reacts directly or indirectly with the oxygen of the tissue to yield singlet oxygen- and toxic-oxygen species, products that lead to malignant cells death. The sensitizers most widely used in clinical studies have been the hematoporphyrin derivative (HpD), commonly known as Photofrin I, and dihematoporphyrin ether/ester (DHE), known as Photofrin II. Both of these derivatives are considered to be first-generation sensitizers. Another sensitizer is a benzoporphyrin derivative (BPD), which belongs to the second generation. However, all these photosensitizers are highly unstable and have a clinical disadvantage related to their capacity to produce a prolonged period of skin photosensitivity. For this reason, Kennedy et al. proposed, in 1990, the development of a PDT in which ALA could be administered externally. This proposal was based on the fact that this chemical acts in certain types of malignant cells to form protoporphyrin IX (PpIX). Malignant cells tend to accumulate PpIX, which causes endogenous photosensitization. This photosensitization is induced in cells of the epidermis but not of the dermis; therefore, photosensitization of the skin is eliminated. In this context, ALA has been used for treatment of basal cell carcinoma, superficial squamous cell carcinoma, and actinic keratosis. It has been used in tumor models including models for amelanotic melanomas, cancer, and colonic tumors. There have also been reports of its use in the treatment of patients with oral cavity epidermal carcinoma, esophagus high-degree dysplasias, breast cancer explants, and bladder cancer.

PDT has been used in the treatment of patients with cervical cancer, utilizing both the hematoporphyrin derivative and sodium porfimer; however, only partial benefits have been obtained from this therapy. In Mexico, cervical cancer is considered a public health problem, due to the fact that 4,500 deaths annually from cervical cancer in women between the ages of 20 and 65 years have been reported; it is considered to be, therefore, one of the principal causes of death among Mexican women.

Currently, the types of treatment used for cervical cancer include cryosurgery, electrosurgery, laser therapy, hysterectomy, radiotherapy, and chemotherapy. Nonetheless, these treatments are only partially effective, due to the fact that the tumor is, in most cases, recurrent. For this reason, it is necessary to seek new therapies.

In developed countries, the new therapy, PDT, has been applied for the treatment of many types of cancer, with encouraging results. Nevertheless, there have been limited reports concerning the use of PDT in the treatment of patients with cervical cancer, and there are no reports using PDT with ALA. Therefore, in this work we have examined the effect of ALA on two cervical cancer cell lines, one obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), and one isolated from a Mexican female to find out whether cervico-uterine cell lines accumulate PpIX induced by ALA. (The two cell lines have a fast-growth rate.)

Two cervico-uterine cancer cell lines were used in the present study. HeLa cells are a line derived from human epidermoid cervical carcinoma obtained from ATCC, and CaLo cells are a cell line from the epidermoid cervical carcinoma of a Mexican female in clinical stage tumor IV. NHCE cells were derived from the adenomyosis of a Mexican female who underwent a hysterectomy; these cells were used as an experimental control in the study.

Cells were seeded in round-bottomed, 6-well plates at a seeding density of 1.8 × 10⁶ cells per well. HeLa and CaLo cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, and NHCE cells were cultured in Dulbecco’s modified Eagle’s medium (MEM) supplemented with 20% fetal bovine serum. Both media had free phenol red and were supplemented with 0.3 mg/mL of L-glutamine, 0.11% sodium bicarbonate, 50 U/mL of benzylpenicillin and 50 ?g/mL of gentamycin sulfate and pH 7.4. The cells were incubated at 37°C in humidified 95% O₂/5% CO₂.

ALA was obtained from the Sigma Chemical Co. (St. Louis, MO, USA) as a hydrochloric salt (98% pure). It was dissolved in phosphate buffer solution (PBS) at a concentration of 5 mg/mL before each experiment. Cells were cultured to a subconfluent monolayer and were exposed to different concentrations of ALA: 0, 50, 150, 300, 500, 750, and 1,000 ?g/mL of medium, and incubated for 24 h in order to find the concentration of ALA that produces the greater accumulation of PpIX, and to evaluate the effects on cell viability. When the optimal concentration of ALA was obtained, a second experiment was done to determine the exposition time that induced the highest levels of intra- and extracellular concentrations of PpIX per hour over a 24-h period. Each experiment was done with at least two sets of experiments, each performed in duplicate.

The culture of NHCE cells from cervical biopsies was done following a procedure described by Freshney with some modifications. Cervical biopsies were placed aseptically in RPMI-1640 medium supplemented with 10% fetal bovine serum, 0.5 ?g/mL of hydrocortisone, 50 U/mL of benzylpenicillin, and 50 ?g/mL of streptomycin and were stored for 1–4 days at 4°C. In the laboratory, the biopsies were washed twice with PBS in Petri dishes (20 × 100 mm). In an empty Petri dish, the tissue was cut up using two scalpel knives with #65 miniblades until the pieces were approximately 2 mm in width. Any connective tissue attached to the sample was removed, and the pieces were transferred to a centrifuge tube. The pieces were pelleted at 2,000 rpm for 10 min and resuspended in 10 mL of fresh MEM supplemented with 20% fetal bovine serum, 0.5 ?g of hydrocortisone, and gentamycin. This last procedure was repeated using fresh growth medium. The medium was discarded and replaced with 1 mL of fresh medium. Then, 0.5 mL was taken from a suspension of basal epithelial cells and added to culture bottles with 2.5 mL of fresh medium. The cultures were incubated at 37°C in humidified 95% O₂/5% CO₂ until some of the explants or cells became attached, being accomplished in 21 days. The cells were subcultivated when they reached one-half or two-thirds confluence. The cells were exposed to ALA in the second subculture.