5-Aminolevulinic acid (ALA)為光感劑前趨物質，能於體內經由血紅素生成路徑轉變成protoporphyrin IX (PpIX)，應用在光化學動力療法（photodynamic therapy, PDT）用以治療腫瘤。由於其非侵入性及對於腫瘤部位具有高選擇性，局部給予ALA 廣泛應用於皮膚癌的治療。文獻指出ALA-PpIX腫瘤選擇性可能原因除了受到ALA為代謝成為PpIX的生合成步驟中酵素活性的影響，ALA於細胞膜的穿透速率亦可能受到載體的調控。
為進一步了解ALA於皮膚細胞吸收之機轉，本研究選擇人類之基底細胞癌(basal cell carcinoma, BCC)，鱗狀細胞癌(squamous cell carcinoma, SCC)，發育障礙口腔角質細胞(dysplasia oral keratinocyte, DOK)及株化角質細胞(human immortalized keratinocyte, HaCaT)，藉由各種抑制劑探討輸送載體對皮膚腫瘤細胞吸收ALA所扮演的角色；再者，藉由評估PpIX於細胞內的蓄積量，證實調控ALA的細胞穿透，對於ALA生成PpIX之影響。本研究亦針對最常見的惡性皮膚癌之一的BCC進行ALA吸收動力學之研究。
研究結果顯示，以3H-ALA培養60分鐘內，BCC細胞內ALA蓄積量隨時間呈線性增加；藉由抑制劑試驗結果得知，β-alanine、GABA、taurine等胺基酸載體：System BETA之受質，對於BCC內ALA的蓄積量確實有顯著的抑制，其次是SCC，於DOK及HaCaT細胞則較不明顯，推測胺基酸載體System BETA參與ALA於腫瘤的細胞膜穿透機轉。隨著GABA濃度之增加，在BCC細胞內抑制ALA吸收的比例會趨近飽和；另外以能量抑制劑，亦可降低ALA進入細胞內，顯示載體輸送為一耗能之機制。此外，藉由抑制劑調控ALA於細胞膜穿透，對於ALA-生成PpIX的影響與細胞攝取ALA的程度大致上具有比例關係，證實ALA在細胞膜之穿透量是細胞內生成PpIX的速率限制步驟。
上述研究結果推論，胺基酸載體System BETA對於ALA在皮膚腫瘤細胞之吸收扮演了重要的角色，並藉此達到較高之PpIX蓄積，產生腫瘤選擇性。此外，ALA於BCC細胞株可經由被動擴散及載體輸送兩種方式進入細胞；載體輸送部份可能與System BETA中之GAT有關。

5-Aminolevulinic acid (5-ALA), a precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) in the heme synthesis pathway, has been used for photodynamic therapy (PDT) in the treatment of various tumors. Because it is noninvasive and tumor selective, topical-ALA-PDT has been widely used in skin cancer treatment. Previous studies have demonstrated that preferential accumulation of ALA-induced PpIX in tumor tissues is influenced affected by the enzyme activity in heme biosynthesis pathway. Evidences have also shown, the cellular uptake of ALA can be mediated by transporter system.
The objectives of this study were (1) to investigate the mechanism of ALA entrance into the human skin cells, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), dysplasia oral keratinocytes (DOK) and human immortalized keratinocytes (HaCaT), by measuring ALA uptake in the presence of selective transport inhibitors, and (2) to verify the effect of modulation of these transporters upon PpIX formation, and (3) to study the transport kinetics of ALA into the BCC cells.
The results showed that the cellular uptake of 3H-ALA in BCC cells was linear during the first 60 min, and was clearly inhibited by the typical substrates of amino acid transporter: System BETA, such as β-alanine, taurine, GABA. The degree of inhibition was less in SCC cells than BCC cells. In contrast, these compounds only showed slight inhibition on ALA uptake by DOK and HaCaT cells. Inhibition on ALA uptake by BCC cells was saturable with increasing concentrations of GABA. Moreover, the uptake of ALA could be inhibited by metabolic inhibitors, confirming the energy dependency of mediated-transport. Furthermore, inhibitors were found to reduce the ALA-induced PpIX accumulation in these experiments. The results demonstrated that incorporation of ALA is rate-limiting in PpIX formation.
In conclusion, amino acid transporter: System BETA may play an important role in the uptake of ALA in skin tumor cells and results in selective accumulation of ALA-induced PpIX in tumors. In addition, we demonstrated here that ALA is incorporated into human basal cell carcinoma cells by two different processes, passive diffusion and mediated transport. Inhibition studies suggested that the transporter involved in ALA incorporation is amino acid transporter: System BETA, most likely GAT.

Ackroyd R, Kelty C, Brown N, Reed M: The history of photodetection and photodynamic therapy. Photochem Photobiol 74(5):656-69, 2001.
Baron JM, Holler D, Schiffer R, Frankenberg S, Neis M, Merk HF, Jugert FK: Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes. J Invest Dermatol 116(4):541-8, 2001.
Borden LA, Smith KE, Hartig PR, Branchek TA, Weinshank RL: Molecular heterogeneity of the γ-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain. J Biol Chem 267: 21098–104, 1992.
Bottomley SS, Muller-Eberhard U: Pathophysiology of heme synthesis. Semin Hematol 25(4):282-302, 1988.
Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE: Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106(3):761-71, 1988.
Brennan MJ, Cantrill RC: The effect of delta-aminolaevulinic acid in the uptake and efflux of [3H]GABA in rat brain synaptosomes. J Neurochem 32:1781-6, 1979.
Chang SE, Foster S, Betts D, Marnock WE: DOK, a cell line established from human dysplastic oral mucosa, shows a partially transformed non-malignant phenotype. Int J Cancer 52(6):896-902, 1992.
Chiang LC, Chiang W, Yu HS, Sheu HM, Chen HY: Establishment and characterization of a continuous human basab cell carcinoma cell line from facial skin (I) cytological behavior of early passages. Kaohsiung J Med Sci 10:170-6, 1994.
Christensen HN: Role of amino acid transport and counter-transport in nutrition and metabolism. Physiol Rev 70: 43–77, 1990.
Clark JA., Amara SG: Stable expression of a neuronal γ-aminobutyric acid transporter, GAT-3, in mammalian cells demonstrates unique pharmacological properties and ion dependence. Mol Pharmacol 46: 550–7, 1994.
De Rosa FS, Bentley MV: Photodynamic therapy of skin cancers: sensitizers, clinical studies and future directives. Pharm Res 17(12): 1447-55, 2000.
Döring F, Walter J, Will J, Föcking M, Boll M, Amasheh S, Clauss W, Daniel H: Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implication. J Clin Invest 101:2761-7, 1998.
Dougherty TJ, Cooper MT, Mang TS: Cutaneous phototoxic occurrences in patients receiving Photofrin. Lasers Surg Med 10(5):485-8, 1990.
Dougherty TJ, Kaufman JE, Goldfarb A, Weishaupt KR, Boyle D, Mittleman A: Photoradiation therapy for the treatment of malignant tumors. Cancer Res 38(8):2628-35, 1978.
Elfsson B, Wallin I, Eksborg S, Rudaeus K, Ros AM, Ehrsson H: Stability of 5-aminolevulinic acid in aqueous solution. Eur J Pharm Sci 7(2):87-91, 1999.
Guastella J, Nelson N, Nelson H, Czyzyk L, Keynan S, Miedel MC, Davidson N, Lester HA, Kanner BI: Cloning and expression of a rat brain GABA transporter. Science 249: 1303–6, 1990. Gupta G, Morton CA, Whitehurst C, Moore JV, MacKie RM: Photodynamic therapy with meso-tetra(hydroxyphenyl) chlorin in the topical treatment of Bowen's disease and basal cell carcinoma. Br J Dermatol 141(2):385-6, 1999.
Henderson BW, Fingar VH: Relationship of tumor hypoxia and response to photodynamic treatment in an experimental mouse tumor. Cancer Res 47:3110-4, 1987.
Heyerdahl H, Wang I, Liu DL, Berg R, Andersson-Engels S, Peng Q, Moan J, Svanberg S, Svanberg K: Pharmacokinetic studies on 5-aminolevulinic acid-induced protoporphyrin IX accumulation in tumours and normal tissues. Cancer Lett 112(2):225-31, 1997.
Hinnen P, de Rooij FW, van Velthuysen ML, Edixhoven A, van Hillegersberg R, Tilanus HW, Wilson JH, Siersema PD: Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation: a study in patients with (pre)malignant lesions of the oesophagus. Br J Cancer 78(5):679-82, 1998.
Irie M, Terada T, Sawada K, Saito H, Inui KI: Recognition and transport characteristics of nonpeptidic compounds by basolateral peptide transporter in Caco-2 cells. J Pharmacol Exp Ther 298(2):711-7, 2001
Itoh Y, Ninomiya Y, Henta T, Tajima S, Ishibashi A: Topical delta-aminolevulinic acid-based photodynamic therapy for Japanese actinic keratoses. J Dermatol 27(8):513-8, 2000.
Kalka K, Merk H, Mukhtar H: Photodynamic therapy in dermatology. J Am Acad Dermatol 42(3):389-413, 2000
Kelty CJ, Brown NJ, Reed MW, Ackroyd R: The use of 5-aminolaevulinic acid as a photosensitiser in photodynamic therapy and photodiagnosis. Photochem Photobiol Sci. 1(3):158-68, 2002
Kennedy JC, Pottier RH, Pross DC: Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience. J Photochem Photobiol B 6(1-2):143-8, 1990.
Krieg RC, Fickweiler S, Wolfbeis OS, Knuechel R: Cell-type specific protoporphyrin IX metabolism in human bladder cancer in vitro. Photochem Photobiol 72(2):226-33, 2000 .
Krieg RC, Messmann H, Rauch J, Seeger S, Knuechel R: Metabolic characterization of tumor cell-specific protoporphyrin IX accumulation after exposure to 5-aminolevulinic acid in human colonic cells. Photochem Photobiol 76(5):518-25, 2002 .
Krogsgaard-Larsen P, Frolund B, Frydenvang K: GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects. Curr Pharm Des 6(12):1193-209, 2000
Langer S, Abels C, Botzlar A, Pahernik S, Rick K, Szeimies RM, Goetz AE: Active and higher intracellular uptake of 5-aminolevulinic acid in tumors may be inhibited by glycine. J Invest Dermatol 112(5):723-8, 1999.
Langmack K, Mehta R, Twyman P, Norris P. Topical photodynamic therapy at low fluence rates-theory and practice. J Photochem Photobiol B 60(1):37-43, 2001.
Moretti MB, Correa Garcia SR, Chianelli MS, Ramos EH, Mattoon JR, Batlle A: Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae. Int J Biochem Cell Ciol 27(2):169-73, 1995.
Moretti MB, García SC, Perotti C, Batlle A, Casas A: δ-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters. Bri J Cancer 87:471-4, 2002.
Morton CA, Brown SB, Collins S, Ibbotson S, Jenkinson H, Kurwa H, Langmack K, McKenna K, Moseley H, Pearse AD, Stringer M, Taylor DK, Wong G, Rhodes LE: Guidelines for topical photodynamic therapy: report of a workshop of the British Photodermatology Group. Br J Dermatol 146(4):552-67, 2002.
Morton CA, MacKie RM, Whitehurst C, Moore JV, McColl JH; Photodynamic therapy for basal cell carcinoma: effect of tumor thickness and duration of photosensitizer application on response. Arch Dermatol 134(2):248-9, 1998.
Morton CA, Whitehurst C, McColl JH, Moore JV, MacKie RM. CA, Whitehurst C, McColl JH, Moore JV, MacKie RM: Photodynamic therapy for large or multiple patches of Bowen disease and basal cell carcinoma. Arch Dermatol 137(3):319-24, 2001.
Nelson H, Lill H: Porters and neurotransmitter transporters. J Exp Biol 196: 213–228, 1994.
Nelson H, Mandiyan S, Nelson N: Cloning of the human brain GABA transporter. FEBS Lett 269: 181-4, 1990.
Novo M, Huttmann G, Diddens H: Chemical instability of 5-aminolevulinic acid used in the fluorescence diagnosis of bladder tumours. J Photochem Photobiol B 34(2-3):143-8, 1996
Novotny A, Xiang J, Stummer W, Teuscher NS, Smith DE, Keep RF: Mechanisms of 5-Aminolevulinic acid uptake at the choroids plexus. J Neurochem 75:321-8, 2000
Palacin M, Estevez R, Bertran J, Zorzano A: Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78(4):969-1054, 1998.
Peng Q, Warloe T, Berg K, Moan J, Kongshaug M, Giercksky KE, Nesland JM: 5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges. Cancer 79(12):2282-308, 1997.
Percy VA, Lamm MC, Taljaard JJ: Delta-Aminolaevulinic acid uptake, toxicity, and effect on [14C]gamma-aminobutyric acid uptake into neurons and glia in culture. J Neurochem 36(1):69-76, 1981.
Rheinwald JG, Beckett MA: Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res 41(5):1657-63, 1981
Rud E, Gederaas O, Hogest A, Berg K: 5-aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters. Photochem Photobiol 71(5):640-7, 2000.
Smith KE, Borden LA, Wang CH, Hartig PR, Branchek TA, Weinshank RL: Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site. Receptors Channels 2: 207–13, 1994.
Soudijn W, van Wijngaarden I: The GABA transporter and its inhibitors. Curr Med Chem 7(10):1063-79 , 2000
Tsai JC, Chen IH, Wong TW, Lo YL: In vitro/in vivo correlations between transdermal delivery of 5-aminolaevulinic acid and cutaneous protoporphyrin IX accumulation and effect of formulation. Br J Dermatol 146(5):853-62, 2002.
Weishaupt KR, Gomer CK, Doufherty TJ: Identification of singlet oxygen as the cytotoxic agent in photo-inactivation of a murine tumor. Cancer Res 36:2326-9, 1976.
Zeitouni NC, Oseroff AR, Shieh S: Photodynamic therapy for nonmelanoma skin cancers. Current review and update. Mol Immunol. 39(17-18):1133-6, 2003.