Effects of photoactivated 5-aminolevulinic acid hexyl ester on MDR1 over-expressing human uterine sarcoma cells

Toxicology Letters 181 (2008) 7–12

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Effects of photoactivated 5-aminolevulinic acid hexyl ester on MDR1 over-expressing human uterine sarcoma cells夽 Ellie S.M. Chu a , Christine M.N. Yow a,∗ , Mark Shi b,1 , Rodney J.Y. Ho b a b

Medical Laboratory Science Section, Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, China Department of Pharmaceutics, University of Washington, Seattle, WA 98195-7610, United States

a r t i c l e

i n f o

Article history: Received 4 February 2008 Received in revised form 12 June 2008 Accepted 13 June 2008 Available online 25 June 2008 Keywords: 5-Aminolevulinic acid hexyl ester Photodynamic therapy Multi-drug resistance MDR1 P-glycoprotein

a b s t r a c t The role of multi-drug resistance (MDR1) and its product, P-glycoprotein (P-gp) on 5-aminolevulinic acid hexyl ester (Hexyl-ALA) mediated phototoxicity was determined with human uterine sarcoma cells, MES-SA control and MDR1 expressing MES-SA-Dx5. MDR1 expression reduced intracellular levels of the Hexyl-ALA metabolite, protoporphyrin IX (PpIX) to a limited degree and could be reversed with a P-gp inhibitor, verapamil. P-gp expression also reduced Hexyl-ALA photosensitivity. More importantly, photoactivated Hexyl-ALA reduced at the mRNA and protein levels without altering housekeeping GAPDH mRNA. These findings suggest that Hexyl-ALA could be used to selectively reduce P-gp expression in overcoming resistance to chemotherapy agents such as doxorubicin and paclitaxel. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction A major concern for oncologists with conventional chemotherapy is the development of multi-drug resistant tumor cells, which have the ability to efflux a wide variety of chemotherapeutics (Gottesman, 2002). Over-expression of the multi-drug resistance (MDR1) transporter, P-glycoprotein (P-gp), is one of the key mechanisms of cancer drug resistance for a number of tumor cell types (Annereau et al., 2004). Verapamil and cyclosporine A, P-gp modulators, have been used to overcome this problem. A major drawback to this treatment, however, is the nonspecific toxicity to normal tissues, including the liver and kidney. The interference of P-gp inhibitors in normal tissue function prevents their use as a general strategy to overcome cancer resistance (Ozols et al., 1987; Thomas and Coley, 2003; Yahanda et al., 1992). For the past two decades, there has been considerable growth in the field of photodynamic therapy (PDT), which uses agents that can be photoactivated to suppress tumor growth (Pandey et al., 2006). Clinical and pre-clinical data indicate that some PDT agents may be effective in overcoming several neoplastic diseases, such as head

夽 Supported by the Central Earmarked Research Grant (HKSAR) (PolyU 5404/04M) and in part by NIH grant NS 39178. ∗ Corresponding author. Tel.: +852 34008575; fax: +852 23624365. 1

E-mail address: [email protected] (C.M.N. Yow). This author is a recipient of the Mary Gates Endowment Research Scholarship.

0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.06.860

and neck, bladder, skin, lung, liver and nasopharyngeal carcinomas (Moan and Peng, 2003; Qiang et al., 2008; Solban et al., 2006; Yow et al., 2007, 2008). Photodynamic therapy is based on the activation of a photosensitizer by visible light, which generates reactive oxygen species and singlet oxygen molecules for tumor cell destruction (Dougherty et al., 1998). Intracellular uptake of the photosensitizer, 5-aminolevulinic acid (ALA), can be readily converted to protoporphyrin IX (PpIX), which is a precursor for heme formation in cells (Calzavara-Pinton et al., 2007). In PDT mediated by ALA, PpIX is the compound that can be photoactivated leading to cytotoxicity. Currently, ALA-PDT is being investigated as a potential drug candidate against multi-drug resistant breast tumors with promising results (Tsai et al., 2004). However, the exact mechanism for overcoming resistance is not fully understood. More recently, the hexyl ester of ALA (Hexyl-ALA) has been investigated with the intent to enhance cell penetration through the increased hydrophobicity provided by the hexyl side chain (Fotinos et al., 2006; Godal et al., 2006). Compared to the parental ALA, Hexyl-ALA has been shown to produce higher intracellular PpIX concentrations at lower doses of photosensitizer, resulting in a similar phototoxic effect (Casas et al., 2001; Wu et al., 2006). Thus, this treatment is more efficient than PDT mediated by ALA alone. As previously reported by us, another advantage for using Hexyl-ALA is its negligible genotoxic potential in lymphocytes that had not been light irradiated, compared to the relatively high dark toxicity observed for ALA (Chu et al., 2006). Although preliminary tests have indicated that Hexyl-ALA has many advantages over ALA,

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its increased hydrophobicity could also increase the likelihood of being recognized by P-gp and thus reduce its effectiveness against MDR1 expressing tumors that are highly resistant to a significant number of drugs. The role of drug efflux transporters on ALA has been studied in a number of cell lines. In addition to MDR1, Robey et al. has demonstrated that ABCG2, another efflux transporter, plays a significant role in resistance to ALA-PDT (Robey et al., 2005). However, most studies have examined various MDR1 cell lines and have suggested that ALA is not a P-gp substrate. In gynecological malignancies, cells expressing MDR1 showed no cross-resistance to ALA mediated PDT (Rossi et al., 1996). MDR1 leukemia cells similarly exhibited no resistance to ALA-PDT and when incubated with a P-gp inhibitor, verapamil, intracellular PpIX levels did not increase (Li et al., 2001). While there is a general consensus that ALA is not a P-gp substrate, it is more important to understand the interactions between its hydrophobic derivative, Hexyl-ALA, and MDR1. However, it is not currently known whether Hexyl-ALA is a substrate, inhibitor or modulator of MDR1. Therefore, we have systematically examined whether HexylALA is a P-gp substrate and determined its effects on the drug resistant phenotype by examining MDR1 mRNA and protein expression. To do so, we chose two human uterine sarcoma cells, parental MES-SA and its MDR1 over-expressing counterpart, MESSA-Dx5. These cells are not known to express high levels ABCG2 (Wesolowska et al., 2005). Our results suggest that Hexyl-ALA or one of its downstream metabolites, PpIX (Hexyl-ALA/PpIX), is a weak P-gp substrate. We also observed that upon photoactivation, Hexyl-ALA selectively reduced MDR1 mRNA and protein levels in human uterine sarcoma cells. 2. Materials and methods 2.1. Cell culture Two human uterine sarcoma cell lines, parental MES-SA and MDR1 overexpressing MES-SA-Dx5, were purchased from ATCC. Cells were routinely cultured in monolayer in 75 cm2 tissue culture flasks (NUNC, USA) with Macoy’s 5A (Gibco BRL) culture medium, supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and antibiotics (50 IU/mL penicillin, 50 ␮g/mL streptomycin and 100 ␮g/mL neomycin) at 37 ◦ C in a humidified 5% CO2 incubator. When cell growth reached 90% confluence, they were harvested for PDT treatment using 0.25% trypsin–EDTA (Gibco BRL). 2.2. Photosensitizer The photosensitizer, Hexyl-ALA, was kindly provided by Photocure (Oslo, Norway). The stock and working solutions were prepared in phosphate-buffered saline (PBS, pH 7.4) and freshly prepared with RPMI-1640 culture medium, respectively.

(MTT assay). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) solution was added to the cells, which were incubated for 3 h. Cells were then washed with PBS and lysed with 150 ␮L DMSO (Fluka), releasing the formazan crystal. Finally, the absorbance was measured by spectrophotometer at a wavelength of 570 nm. All results were presented as the mean ± standard deviation (S.D.) in triplicate. 2.5. Inhibition of MDR1/P-gp efflux transporter with verapamil The MES-SA-Dx5 cells (2 × 105 cells/well) were seeded in 35 mm culture dishes with 5% CO2 at 37 ◦ C overnight. The control or test cells were incubated in serum free medium with either Rhodamin123 (5 ␮M), a P-gp substrate (Sigma, St. Louis, MO), for 30 min or with 30 ␮M of Hexyl-ALA for 4 h. The Rhodamin123 or Hexyl-ALAtreated cells were washed with PBS to remove unincorporated compounds. Then, these cells were incubated in fresh medium and allowed to efflux for 4 h in the presence or absence of 100 ␮M verapamil as a P-gp inhibitor (Sigma, St. Louis, MO). All the treated cells were harvested, washed, and analyzed by flow cytometry as described above. Data presented were the mean or representative value of triplicate samples. 2.6. Quantitation of MDR1 mRNA expression by real-time RT-PCR Both MES-SA and MES-SA-Dx5 cells (1 × 106 cells/dish) were either treated as dark control cells (50 ␮M Hexyl-ALA without light irradiation) or at the following lethal doses of Hexyl-ALA and light. For MES-SA cells, LD30 (30 ␮M, 1 J/cm2 ), LD50 (30 ␮M, 2 J/cm2 ) and LD70 (30 ␮M, 4 J/cm2 ) were applied for subsequent experiments. For the MES-SA-Dx5 cells, LD30 (30 ␮M, 2 J/cm2 ), LD50 (50 ␮M, 2 J/cm2 ) and LD70 (50 ␮M, 4 J/cm2 ) were used. At 24 h post-PDT, the cells were harvested and the total cellular RNA was extracted using the Purescript RNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s specifications. The concentration of RNA in extracted samples was estimated as the product of a conversion factor (40 ␮g/mL) and absorbance at 260 nm measured with a spectrophotometer (model DU7400; Beckman Coulter, Fullerton, CA, USA). Transcripts of the human MDR1 gene were quantitatively measured by realtime PCR using an Applied Biosystems (ABI) 7900HT-SDS instrument (Foster City, CA, USA). The primers and probe set for the assay were purchased from Applied Biosystems as “Taqman Gene Expression Assays® ” for MDR1 transcripts, which spans intron 23 and generates a 110 by amplicon. RNA (1 ␮g per reaction) was converted to cDNA with an ABI reverse transcription kit with random hexamer primers in a final volume of 10 ␮L. Samples were incubated for 50 min at 45 ◦ C and subsequently for 5 min at 85 ◦ C to terminate the reverse transcription reaction. For real-time PCR, 5 ␮L fivefold-diluted cDNA (equivalent to 100 ng RNA) was used for every PCR reaction in a final volume of 25 ␮L, containing 900 nM sense and anti-sense primers and 300 nM fluorogenic probe and ABI Taqman universal PCR master mix. The cycling conditions for PCR were 95 ◦ C for 10 min followed by 40 cycles each of 95 ◦ C for 15 s and 60 ◦ C for 1 min. Each sample was run in triplicate and mean/standard deviation cycle threshold values determined. MDR1 RNA copies were estimated with a validated standard of MDR1 RNA expressed with T7 cell free system (Yang et al., 2002). Inter-assay variability was controlled using reference RNAs isolated from MES-SA and MES-SA/Dx5 human sarcoma cells expressing low and high levels of MDR1, respectively. 2.7. Determination of hTERT mRNA expression in MDR cell lines at pre- and post-PDT using reverse transcription-PCR

2.3. Time course of intracellular PpIX after Hexyl-ALA exposure The cellular uptake of the photosensitizer, Hexyl-ALA, which is converted into fluorescent PpIX inside MES-SA and MES-SA-Dx5, was determined by flow cytometry (Elite; Beckman Coulter, USA). Each cell line (2 × 105 cells/well) was seeded in 35 mm culture dishes and incubated with freshly prepared Hexyl-ALA (30 ␮M) for various time intervals (4, 8, 15, 24 and 32 h). The photosensitizer was removed and the cells were trypsinized and centrifuged, then washed with PBS twice. The cell pellet was then re-suspended in 500 ␮L of PBS and subject to flow cytometry analysis. The fluorescent intensities of PpIX at each time point were measured (ex = 490 nm; em = 520 nm) with a minimum of 10,000 cells for each analysis. All results were presented at three independent experiments. 2.4. Effect of MDR1 over-expression on phototoxicity of Hexyl-ALA Phototoxicity procedures were modified from our previous study (Chu et al., 2006; Yow et al., 2000a). The MES-SA and MES-SA-Dx5 cells (3 × 104 cells/well) were incubated with varying concentrations of Hexyl-ALA (5–50 ␮M) for 4 h. Then, the cells were washed twice with PBS and incubated in fresh medium before they were irradiated with 0–4 J/cm2 of light from a 400-W quartz-halogen lamp with a heat isolation filter and a 600-nm long-pass filter. The spectral intensity of light source was basically flat from 600 to 800 nm and the total intensity was measured to be at 14 mW/cm2 using a power meter (Yow et al., 2000b). Cytotoxicity in each cell line was determined after 24 h by the tetrazolium colorimetric reduction assay

Reverse transcriptase-polymerase chain reaction (RT-PCR) was applied for the detection of human telomerase reverse transcriptase (hTERT) transcript levels (Accession no. AF018167), which is a catalytic subunit of telomerase required for tumor immortality. The MES-SA and MES-SA-Dx5 tumor cells (1 × 106 cells) were first seeded in 35 mm culture dishes and incubated with Hexyl-ALA until LD30 and LD50 was achieved after light irradiation. After 24 h post-PDT, the 2 ␮g of RNA isolated from the treated and control cell samples were reverse transcribed into cDNA using RT-PCR. Random primer (Invitrogen, USA) Moloney Murine Leukemia Virus Reverse Transcriptase RNase H Minus, point mutation (M-MLV RT(H−)) (Promega) and RNA template were added. The synthesized cDNA was further amplified with a specific hTERT forward primer (5 -TTT CCG CTA GAA GAG TGT CTG-3 ) and a reverse primer (5 -CGT CCA AGT TGT TCA CAA TCG-3 ) with a product size of 196 bp. A primer pair for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was added as a housekeeping gene of internal control (forward: 5 -CTC ATC ATC TCT GCC CCC CTC CTG-3 and reverse: 5 -CGC CTG CTT CAC CAC CTT CTTG-3 ) that synthesized a product size of 450 bp. The reaction mixture was denatured at 95 ◦ C for 10 min, followed by 45 cycles at 95 ◦ C for 45 s, 58 ◦ C for 60 s and 72 ◦ C for 60 s with the final hold for extension at 72 ◦ C for 10 min. The final amplified products were separated on a 1.5% agarose gel by electrophoresis. The 196 bp (hTERT) and 450 bp (GAPDH) products detected after ethidium bromide staining procedure were captured under UV light with a Lumi-imager F1 workstation (Roche Diagnostics, Switzerland). The band density of the hTERT PCR products from the gel was normalized by that of GAPDH PCR products using a software Quantity One® v4.6.3 (Bio-Rad).

E.S.M. Chu et al. / Toxicology Letters 181 (2008) 7–12 2.8. Modulation of P-glycoprotein expression in MES-SA-Dx5 cells at pre- and post-PDT by flow cytometric analysis The untreated control MES-SA-Dx5 cells and Hexyl-ALA-treated MES-SA-Dx5 cells at LD30 , LD50 and LD70 were harvested 24 h post-PDT. The cells were trypsinized and washed twice with PBS. Then, the cells were re-suspended in 100 ␮L PBS and stained with 20 ␮L of PE-conjugated P-glycoprotein monoclonal antibody (Immunotech; Beckman Coulter, USA) for 30 min at room temperature. Each sample with 10,000 events/counts was analyzed by flow cytometry. The fluorescent intensity was proportional to the P-gp expression on the cell surface.

3. Results 3.1. Time course of intracellular PpIX after Hexyl-ALA exposure To determine if MDR1 plays a role in Hexyl-ALA uptake, the time-dependent intracellular levels of Hexyl-ALA were evaluated in parental human uterine sarcoma cells (MES-SA) and its MDR1 over-expressing derivative (MES-SA-Dx5). The intracellular HexylALA levels were assessed by measuring PpIX fluorescence, which were shown to correlate with Hexyl-ALA concentration. The HexylALA concentration was fixed at 30 ␮M and PpIX fluorescence was measured over 32 h. As shown in Fig. 1, both MES-SA (Fig. 1A) and MES-SA-Dx5 (Fig. 1B) exhibited strong and similar fluorescent intensity after 4 h. At 8 and 15 h, there was a slightly higher PpIX fluorescence level observed in the MES-SA cells than in MES-SA-Dx5. At 24 and 32 h, there was a significant difference in fluorescent intensity between the two human uterine sarcoma cell lines. The MES-SA-Dx5 cells, with high MDR1 expression, had much lower intracellular PpIX than MES-SA, which had no detectable P-gp transporters. Collectively, this data indicates that MDR1 overexpression in human uterine sarcoma cells significantly reduced PpIX levels over time after being exposed to Hexyl-ALA.

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3.2. Effect of MDR1 over-expression on phototoxicity of Hexyl-ALA To compare the phototoxic effect of Hexyl-ALA in parental human uterine sarcoma cells (MES-SA) and its MDR1 overexpressed derivative (MES-SA-Dx5), both cells were incubated with varying concentrations of Hexyl-ALA and exposed to a similar range of light intensities. Fig. 2 shows that cytotoxicity in both cells occured in a drug and light dose-dependent manner. When light dose was held constant, the difference in phototoxicity was only apparent at greater concentrations of Hexyl-ALA (30–50 ␮M). At 50 ␮M Hexyl-ALA and 2 J/cm2 , the cytotoxic effect to MES-SA was about 80% while only 45% to MES-SA-Dx5. At lower concentrations (5–10 uM), the difference in photoactivated Hexyl-ALA toxicity was minimal between the two cells. When Hexyl-ALA concentration is held constant, MES-SA cells appear more sensitive to light irradiation than MDR1 over-expressing MES-SA-Dx5 cells. Dark toxicity was negligible in both cells. This data suggests that MDR1 overexpression in human uterine sarcoma cells significantly limits the phototoxic effect of Hexyl-ALA, especially at high concentrations. 3.3. Inhibition of P-gp/MDR1 efflux transporter with verapamil To determine whether Hexyl-ALA or one of its downstream metabolites is a P-gp substrate, we evaluated PpIX levels in cells in the presence of a validated P-gp inhibitor, verapamil. Cells were treated with Rhodamin123 (Rho123) or Hexyl-ALA and incubated 4 h in the presence or absence of 100 ␮M verapamil, a concentration known to effectively inhibit P-gp function. When Rho123-treated MES-SA-Dx5 cells were also incubated with verapamil, they retained significantly higher levels of Rho123 than those that were not treated with the P-gp inhibitor (Fig. 3A). This is evident by the increased fluorescent intensity when in the presence of verapamil. As shown in Fig. 3B, MES-SA-Dx5 cells treated with Hexyl-ALA and verapamil also exhibited a higher level of fluorescent intensity than those that had not been incubated with the P-gp inhibitor. This shift towards higher fluorescent intensity when verapamil was also present indicates that the intracellular levels of the fluorescent compound had increased when P-gp was blocked. Similar studies with MES-SA cells, which do not express P-gp, showed no change in fluorescent intensity when the cells were also incubated with verapamil (data not shown). These data suggest that Hexyl-ALA/PpIX, is a P-gp substrate in the MDR1 over-expressed human uterine sarcoma cell line. 3.4. Effects of Hexyl-ALA photoactivation on MDR1 expression

Fig. 1. Time-dependent intracellular PpIX fluorescence in human uterine sarcoma cells: MES-SA (A) and MES-SA-Dx5 (B) cells were incubated in 30 ␮M Hexyl-ALA and the intracellular levels of induced PpIX were measured over 32 h by flow cytometry.

To determine whether photoactivation of Hexyl-ALA modulates multi-drug resistance transporter function, MDR1 transcript and protein expression were measured. Our initial data indicate that the MES-SA parent cells expressed undetectable levels of membrane Pgp and MDR1 mRNA, thus these data were not presented. On the other hand, we detected 1250 copies of MDR1 mRNA/ng of total RNA in the MDR1 over-expressing MES-SA-Dx5 cells (Fig. 4). When incubated with 50 ␮M Hexyl-ALA for 4 h alone, MES-SA-Dx5 exhibited no decrease in mRNA levels. Only when Hexyl-ALA-treated MDR1 over-expressing human uterine sarcoma cells were also exposed to light was a decrease in MDR1 mRNA levels observed. This decrease in MDR1 mRNA levels was Hexyl-ALA concentration and light intensity-dependent, as we observed a 36% reduction at LD30 , greater than 90% reduction at LD50 and 93.7% reduction of MDR1 mRNA at LD70 . To further investigate the effects of Hexyl-ALA photoactivation on MDR1 cells, mRNA levels for the housekeeping gene GAPDH were observed in both parental and MDR1 over-expressing human uterine sarcoma cells. Surprisingly, there was no difference in GAPDH mRNA levels before and after treatment with Hexyl-ALA

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Fig. 2. Effects of MDR1 over-expression on phototoxicity of Hexyl-ALA. MES-SA (B) and MES-SA-Dx5 (A) cells were incubated over a range of concentrations (5–50 ␮M) for 4 h and irradiated with varying light intensities (0–4 J/cm2 ). Cytotoxicity in each cell line was determined after 24 h by the MTT assay as described in Section 2.

and light in either cell line (Fig. 5). To confirm if this decrease in mRNA levels after Hexyl-ALA photoactivation was selective, mRNA levels for hTERT, the target of another cancer drug therapy, were also investigated. Fig. 5 shows hTERT mRNA levels in both MES-SA and MES-SA-Dx5 exhibit a slight decrease after photoactivation of

Fig. 4. MDR1 mRNA levels before and after treatment with photoactivated HexylALA. MES-SA-Dx5 cells were treated with Hexyl-ALA at LD30 , LD50 , or LD70 . The MDR1 mRNA levels in the control and treated cells were measured using real-time RT-PCR 24 h post-PDT as described in Section 2. There were undetectable levels of MDR1 mRNA expression in both Hexyl-ALA-untreated and -treated MES-SA cells (data not shown).

Hexyl-ALA. This decrease in hTERT mRNA was minimal compared to the drastic reduction of MDR1 mRNA after Hexyl-ALA phototherapy and only detectable when treated with 50 ␮M of Hexyl-ALA, a relatively high concentration of photosensitizer. We next evaluated P-gp expression to determine if Hexyl-ALA photoactivation also reduced MDR1 expression at the protein level in MES-SA-Dx5. As shown in Fig. 6, when Hexyl-ALA concentrations increased, P-gp levels decreased. This is evident by the decrease in the number of cells with a high P-gp surface expression. Together, these results indicate that photoactivation of Hexyl-ALA greatly reduces MDR1 mRNA levels and the expression of P-gp proteins at the cell surface. 4. Discussion

Fig. 3. Inhibition of P-gp efflux by verapamil. MES-SA-Dx5 cells were incubated with either 5 ␮M Rhodamin123 for 30 min (A) or with 30 ␮M Hexyl-ALA for 4 h (B) to allow for uptake. Intracellular fluorescent intensities were measured by flow cytometry in both the Rhodamin123-treated cells and the Hexyl-ALA-treated cells after 4 h efflux time in the presence or absence of verapamil (100 ␮M).

While human data collected with ALA suggests that this photosensitizer shows promise in eradicating resistant tumors though PDT, its limited membrane penetration requires large doses to be effective, which may lead to dose-limiting toxicities. The more hydrophobic hexyl ester of ALA appears to enhance cell penetration and could potentially increase ALA potency while also

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Fig. 5. GAPDH and hTERT mRNA levels before and after treatment with photoactivated Hexyl-ALA. MES-SA (A) and MES-SA-Dx5 (B) cells were treated with Hexyl-ALA at LD30 or LD50 . The GAPDH and hTERT mRNA levels in the control and treated cells were measured 24 h post-PDT using RT-PCR as described in Section 2.

reducing toxicity (Palumbo, 2007). To evaluate the potential of Hexyl-ALA-PDT as a treatment for multi-drug resistant tumors, we examined the effects of MDR1 expression on cytotoxicity mediated by photoactivated Hexyl-ALA and the impact of Hexyl-ALA on MDR1 transcript and protein levels in human uterine sarcoma cells. Using parental MES-SA and the highly drug resistant, MDR1 over-expressing MES-SA-Dx5 cells, we found that photoactivation of intracellular PpIX resulted in the selective reduction of MDR1 mRNA levels and the amount of P-gp protein detected in MES-SADx5. In addition, Hexyl-ALA or PpIX (Hexyl-ALA/PpIX) appeared to be a weak substrate of P-gp and its efflux transport activity could be blocked with the P-gp inhibitor, verapamil. Data collected to date suggests that the parent photosensitizer, ALA, is not a P-gp substrate. When Robey et al. analyzed ALA as a potential P-gp substrate using MCF-7 TX200 cells, they observed a negligible increase in intracellular ALA levels incubated with a P-gp inhibitor, valspodar (Robey et al., 2005). Li et al. examined intracellular levels of the ALA metabolite, PpIX, in resistant leukemia cells and observed only small differences in fluorescence when incubated with and without a P-gp inhibitor, verapamil (Li et al., 2001). These two studies suggest that ALA uptake is not mediated by P-gp. Our data suggest that the increased hydrophobicity of Hexyl-ALA, by attachment of the hexyl ester group to ALA, may have enhanced its recognition by the P-gp transporter. The effect of MDR1 over-

Fig. 6. P-gp membrane expression before and after treatment with photoactivated Hexyl-ALA. MES-SA-Dx5 cells were treated with Hexyl-ALA at LD30 , LD50 and LD70 . Then the control and treated cells were stained with P-gp antibody (CD243) for 20 min at 24 h post-PDT followed by flow cytometry analysis (one-way ANOVA *p < 0.05 and **p < 0.01).

expression is noted only after prolonged incubation (i.e. 8–32 h), suggesting that Hexyl-ALA/PpIX is a weak substrate (Fig. 1). Abrogation of Hexyl-ALA efflux in MES-SA-Dx5 with a P-gp inhibitor verapamil (Fig. 3B) is consistent with Hexyl-ALA/PpIX being a weak P-gp substrate. Our ability to detect a small but notable shift in intracellular concentrations of the Hexyl-ALA surrogate, PpIX, when P-gp transporters were blocked with verapamil, may be due to the very high levels of P-gp transporter protein expressed on the cell surface of MDR1 over-expressing human uterine sarcoma MES-SA-Dx5 cells. These cells, which require approximately 102 -fold higher doxorubicin concentrations to produce a similar degree of cytotoxicity to its MES-SA counterpart (Harker and Sikic, 1985), may have amplified the effect of P-gp to allow identification of weak substrates such as Hexyl-ALA/PpIX. It is possible that cell lines examined to determine whether ALA is a P-gp substrate had too few P-gp transporters so that the shift in intracellular PpIX fluorescence, when blocked with a P-gp inhibitor, would be less significant and therefore could lead to an underestimation of the effect of MDR1 and its product, P-gp. Nevertheless, as a weak P-gp substrate it is likely that only highly drug resistant cancer cells, related to an extremely high level of MDR1 expression, such as MES-SA-Dx5 cells, could have a small but significant effects on intracellular PpIX levels. Given that the MDR1 effects appeared to increase with drug exposure time, it is possible that initiating photoactivation process at the earliest time after drug accumulation in cells may further reduce MDR1 effects. Regardless of the limited recognition of Hexyl-ALA/PpIX by P-gp transporters, sufficient intracellular PpIX levels were reached to provide reasonable potency against highly resistant human uterine sarcoma MES-SA-Dx5 cells. In drug resistant leukemia cells, high ferrochelatase activity has been observed to reduce phototoxicity by decreasing the intracellular levels of light-sensitive PpIX (Li et al., 2001). Ferrochelatase is the enzyme that catalyzes the last step in heme formation by inserting ferrous iron into PpIX. While it is possible, it is not known whether MDR1 over-expressing MES-SA-Dx5 and its parental MES-SA cells exhibit a different degree of this enzyme function. Assuming that ferrochelatase levels in these two cells are similar, the substantial difference in intracellular PpIX levels between MES-SA and MES-SA-Dx5 is probably mediated by P-gp efflux transport of Hexyl-ALA/PpIX, which action was reversed with a P-gp inhibitor verapamil. While it is also possible that Hexyl-ALA may be recognized by ABCG2, there is no data that suggests MESSA-Dx5 express significant levels of this protein (Wesolowska et al., 2005). Thus, the interaction of Hexyl-ALA with ABCG2, which is

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a less prevalent in multi-drug resistant cells, remains to be determined. An unexpected but significant finding was the Hexyl-ALA and light dose-dependent reduction in MDR1 mRNA levels after treatment with photoactivated Hexyl-ALA was observed in MES-SA-Dx5 cells. This data was consistent with the drastic reduction in membrane P-gp transporters following treatment with Hexyl-ALA and light (Fig. 6). To our surprise, mRNA levels of the housekeeping gene, GAPDH, were unaffected by treatment with photoactivated HexylALA in both the parental and highly drug resistant human uterine sarcoma cells, suggesting MDR1 specificity. The effects of MDR1 were detected at both the mRNA and protein levels (Figs. 4 and 6). On the other hand, another gene transcript, hTERT, a potential drug target for cancerous tissue, was partially affected by Hexyl-ALA treatment in MES-SA-Dx5 cells and to a lesser degree, in MESSA cells. It was reported in our previous study that hTERT mRNA expression level was down-regulated by hexyl-ALA treatment in medulloblastoma cells (Chu et al., 2008). Whether other proteins over-expressed in cancer cells may be suppressed by Hexyl-ALA, remains to be investigated. Nevertheless, the ability of Hexyl-ALA phototherapy to suppress MDR1 and hTERT without impacting GAPDH, which is considered a housekeeping gene, could be useful to overcome drug resistance by shutting down overactive proteins in cancerous cells. Studies to date have focused on the resistant tumor cell destruction that results after treatment with photoactivated Hexyl-ALA. Our mechanistic study data suggest that reduction of MDR1 activity by photoactivated Hexyl-ALA could play a more significant role in overcoming resistant tumors by modification of MDR1 expression. If these results can be reproduced in other cell lines, this therapy could be used in combination with other highly potent compounds, such as paclitaxel, doxorubicin, or vinblastine, to overcome MDR1-related cancer drug resistance. Also, its limited impact on housekeeping genes, such as GAPDH, could give a higher safety margin at clinical or sub-clinical concentrations. In summary, we have investigated the role of MDR1 on HexylALA accumulation in human uterine sarcoma cells and elucidated its time concentration effects on cytotoxicity and the role of HexylALA on MDR1 levels. Our results suggest that Hexyl-ALA/PpIX is a weak to poor MDR1 substrate and more importantly, photoactivation of Hexyl-ALA suppresses MDR1 levels, which may allow the resistant cells to return to a drug-sensitive phenotype. Acknowledgements This study was supported by a Grant (PolyU 5404/04M) from the Central Earmarked Research Grant of Hong Kong SAR and in part by a Grant (NS39178) from NIH. We would also thank Dr. Tot Bui for providing technical support. References Annereau, J.P., Szakacs, G., Tucker, C.J., Arciello, A., Cardarelli, C., Collins, J., Grissom, S., Zeeberg, B.R., Reinhold, W., Weinstein, J.N., Pommier, Y., Paules, R.S., Gottesman, M.M., 2004. Analysis of ATP-binding cassette transporter expression in drug-selected cell lines by a microarray dedicated to multidrug resistance. Mol. Pharmacol. 66, 1397–1405. Calzavara-Pinton, P.G., Venturini, M., Sala, R., 2007. Photodynamic therapy: update 2006. Part 1. Photochemistry and photobiology. J. Eur. Acad. Dermatol. Venereol. 21, 293–302.

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