Enhancing PDT efficacy in NMIBC: Efflux inhibitor mediated improvement of PpIX levels and efficacy of the combination of PpIX-PDT and SO-cleavable prodrugs

Abstract

Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) has shown limited efficacy in non-muscle invasive bladder cancer (NMIBC). To improve PDT efficacy, we developed singlet oxygen-cleavable prodrugs. These prodrugs, when combined with PpIX-PDT, induce cancer cell death through both PDT and drug release mechanisms. Inhibition of PpIX efflux was reported to be an effective strategy to improve PpIX-PDT in certain cancer cells. Our main goal was to investigate whether adding an efflux inhibitor to the combination of PpIX and prodrugs can improve the PpIX levels in bladder cancer cells and the release of active drugs, thus improving the overall efficacy of the treatment. We treated bladder cancer cell lines with lapatinib and evaluated intracellular PpIX fluorescence, finding significantly increased accumulation. Combining lapatinib with prodrugs led to significantly reduced cell viability compared to prodrugs or PpIX-PDT alone. The effect of lapatinib depended on the expression level of the efflux pump in bladder cancer cells. Interestingly, lapatinib increased paclitaxel (PTX) prodrug uptake by 3-fold compared to prodrug alone. Adding an efflux inhibitor (e.g., lapatinib) into bladder instillation solutions could be a straightforward and effective strategy for NMIBC treatment, particularly in tumors expressing efflux pumps, with the potential for clinical translation.

Graphical Abstract

Hexyl ester of 5-aminolevulinic acid (HAL) is converted into PpIX (protoporphyrin IX) within the mitochondria, and an efflux pump inhibitor is used to prevent the efflux of PpIX, leading to its accumulation. This accumulation, followed by photoactivation, results in the generation of reactive oxygen species (ROS) causing cell death. Additionally, there is a protecting group linked to chemotherapy drugs (PG-L-D) which upon activation, releases more active drug, enhancing cell death. Adding an efflux inhibitor to the combination of PpIX and prodrugs improved the PpIX levels in bladder cancer cells and the release of active drugs enhanced cell death.

INTRODUCTION

Cancer is the second leading cause of death in humans after cardiovascular disease (1, 2). The number of new cancer cases worldwide expected to increase to 28 million by 2040. The most recent GLOBOCAN report shows that bladder cancer (BC) accounts for 3% of all cancer diagnoses globally, and is particularly widespread in developed countries (3). Nearly 75% of newly diagnosed bladder cancers are classified as non-muscle-invasive bladder cancers (NMIBC), and about 25% are classified as muscle-invasive bladder cancers (MIBC) (4, 5). The current standard of care for NMIBC tumors is transurethral resection of bladder tumor (TURBT), along with immediate intravesical chemotherapy. Though this treatment is effective, it is not always curative. Patients often require regular retreatment and follow-up because progression rates are about 45% for high-risk cancers, such as submucosal invasion and/or carcinoma in situ (6–8). It is, therefore, important to develop new intravesical treatment regimens.

Photodynamic therapy (PDT) has been shown to be a highly effective treatment modality in treating both malignant and non-malignant diseases (9, 10). In PDT, cancer cells are killed by using light with a specific wavelength in combination with a photosensitizer. A photosensitizer can be administered systemically or locally, intratumorally, intraperitoneally, topically, or intravesically. Laser illumination activates a nontoxic photosensitizer. When activated at the triplet state, photosensitizers transfer their excited energy to molecular oxygen, converting it to singlet oxygen. The production of singlet oxygen (SO) by lasers has the potential to have a cytotoxic effect on tumors and their associated vessels, causing necrosis or apoptosis (11–13). PDT can be a selective treatment because cancer cells accumulate photosensitizers in large amounts compared to normal cells (14, 15). In 2010, the US FDA approved hexyl aminolevulinic acid (HAL) as a PDT agent for the diagnosis of non-muscle invasive bladder cancer. As a prodrug, HAL itself lacks photosensitive activity. As part of the heme biosynthesis pathway, it is converted to protoporphyrin IX (PpIX), a heme precursor metabolite that has photosensitizing and fluorescent properties (12, 16).

PDT using PpIX has emerged as a promising approach for NMIBC, exploiting the selective accumulation of PpIX in cancer cells and its activation by light to induce cancer cell death. However, the therapeutic efficacy of PpIX-PDT, where PpIX is over prodrug in cancer cells by -5-ALA (called ALA-PDT) or its ester prodrugs (e.g., hexyl ester of 5-ALA (HAL, called HAL-PDT), alone was not strong enough (17). We have developed singlet oxygen-cleavable prodrugs of anticancer drugs, which release anticancer drugs by singlet oxygen generated by PpIX upon illumination in cancer cells. Cancer cells are killed by both PpIX-PDT effect and chemo-drug effect by the released anticancer drugs from the prodrugs in cancer cells (18).

Both anticancer effects should be dependent on the PpIX concentration in cancer cells. However, it has been reported that an ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the efflux of PpIX from cancer cells. Dr. Chen’s group has reported that inhibiting its activity showed an effective therapeutic strategy in increasing aminolevulinic acid, ALA-PpIX fluorescence and PDT response (19–23). The ABCG2 inhibitors, fumitremorgin C (FTC), and its derivative Ko143 enhanced the ALA-PpIX fluorescence and PDT effects in ABCG2-expressing cells (24–26). Cancer cell lines with high ABCG2 activities responded well to Ko143 for increase of PpIX and PDT, but not those with low ABCG2 activities (22, 23). Previous studies have supported the effectiveness of combining ABCG2 inhibitors and ALA (23). Richard Howley and his team conducted a study to determine the impact of combining ABCG2 inhibitors with ALA on human renal cell carcinoma cell lines. The study revealed that inhibiting ABCG2 significantly increased the intracellular PpIX in RCC cell lines with high ABCG2 activity, but not in cells with low activity. Additionally, they found a strong positive relationship between ABCG2 activity and the response to ABCG2 inhibition for PpIX enhancement across RCC cell lines. However, FTC and Ko143 cannot be used in vivo due to their neurotoxicity and serum instability, respectively (27, 28).

Some kinase inhibitors used in the treatment of cancer are ABCG2 substrates, which act as competitive inhibitors of the transporter (29). Inhibitors of ABCG2, such as lapatinib (30), gefitinib (31) and imatinib (32) increased ALA-PpIX fluorescence and PDT effects, suggesting that these agents could enhance the therapeutic benefit of PpIX-PDT. As reported in previous literature, lapatinib has unique and noteworthy capabilities. Compared to other kinase inhibitors, lapatinib increased intracellular PpIX levels while reducing efflux (19).

Based on the above background, we hypothesized that the addition of an efflux pump inhibitor to our combination treatment (PpIX-PDT + SO-cleavable prodrug) can enhance its anticancer effects by increasing the PpIX concentration in cancer cells. While the PpIX-PDT effect can be improved directly by the higher PpIX concentration, the chemo-drug effect can be indirectly boosted by the increased activation yield of the prodrugs to anticancer drugs by the higher PpIX concentration. We aimed to investigate the impact of lapatinib on PpIX levels in bladder cancer cells and on the subsequent phototoxic effect of the combination treatment. If proven effective, this multiple combination therapy could be readily translatable to the clinic because all the drugs for this new combination treatment are mixed and then administered directly to a bladder, intravesically. Thus, systemic toxicities, if any with the efflux pump inhibitors, can be avoided using local administration.

MATERIALS AND METHODS

Materials:

Lapatinib was purchased from Ambeed, Inc. IL, USA. Pheophorbide A was purchased from Cayman Chemicals, Michigan, USA. Ko143 and PpIX were procured from MedChemExpress, NJ, USA. Hexyl ester of 5-aminolevulinic acid (HAL) was purchased from Chem-Impex International, IL, USA. Prodrugs (Rh-L-PTX (18) and Rh-L-SN-38 (33)) were synthesized and purified as described. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), Trypan blue, EDTA, Triton X-100 were procured from Thermo Fischer Scientific, MA, USA. Protease cocktail inhibitor was purchased from Sigma-Aldrich, USA.

Cell lines:

Dr. Steven Salman (University of Toledo) provided AY-27 cells. RT-112 and RT-4 cells were purchased from the American Type Culture Collection (ATCC, USA). Roswell Park Memorial Institute (RPMI-1640) growth medium supplemented with 10% FBS and 1% antibiotic defined as complete media was used to grow and maintain cell lines at 37°C and 5% CO₂. Penicillin streptomycin, RPMI-1640, and Trypsin-EDTA were procured from Thermo Fisher Scientific, USA. Fetal bovine serum (FBS) was purchased from Bio-Techne, MN, USA.

Methods:

Real-time PCR analysis:

As per the manufacturer’s instructions, total RNA was isolated from the AY-27, RT-112, and RT-4 cells using a total RNA isolation kit (Omega Bio-Tek Inc. GA, USA). In each sample, total RNA concentration was measured and purity was verified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) was obtained from 200 ng of RNA using the iScript cDNA synthesis kit (Bio-Rad, CA, USA). Real-time PCR was carried out with iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). A 40-cycle reaction was conducted with denaturation at 95°C for 10 min, annealing at 58°C for 30 sec and extension at 72°C for 45 sec. Data was exported from the qPCR instrument to the Bio-Rad CFX manager software and analyzed for ABCG2 mRNA expression. Table S1 provides primer sequences. A housekeeping gene, GAPDH, was used as a standard for normalizing the levels of expression.

In vitro evaluation of ABCG2 transporter activity:

ABCG2 activity was measured in various bladder cancer cell lines as described in a previous study (23). Bladder cancer cells were seeded at a density of 50,000 cells/well in 1 mL of complete cell culture medium in each well of 12 well plates and incubated for 48 h. The old medium was replaced with fresh medium containing 500 nM pheophorbide A (PA an ABCG2 substrate), in combination with 1μM Ko143, an ABCG2 inhibitor and incubated for 2 h. Two hours after incubation in a CO₂ cell culture incubator at 37 °C, the drug-containing medium was discarded from each well. Then, cells were washed with 500 μL of PBS twice. The cells were lysed in PBS containing 0.1% sodium dodecyl sulfate (SDS). Using a pipette, the cell lysates were gently mixed in the solution. The lysates were transferred into 1.5 mL microcentrifuge tubes and spun at 2000 RCF for 5 min at room temperature (RT). The supernatant was collected, and 200 μL of each sample was added to a 96-well plate. PA fluorescence was measured using a microplate spectrofluorometer with excitation/emission wavelengths of 400/675 nm. Using the formula below, ABCG2 transporter activity was determined. A high value indicates an increase in ABCG2 activity.

$$\mathit{\text{ABCG}}2\mathit{\text{activity}}=\frac{{\mathit{\text{Flu}}}_{\mathit{\text{PA}}+Ko143}-{\mathit{\text{Flu}}}_{Ko143}}{{\mathit{\text{Flu}}}_{\mathit{\text{PA}}}-{\mathit{\text{Flu}}}_{\mathit{\text{Ctl}}}}$$

Where Flu_PA+Ko143 indicates fluorescence of samples treated with pheophorbide A in combination with Ko143, Flu_Ko143 indicates fluorescence of samples treated with Ko143, Flu_PA indicates fluorescence of samples treated with pheophorbide A, and Flu_PA fluorescence of DMSO control samples.

ABCG2 influence:

Each well of a 12-well plate was seeded with 50,000 cells in 1 mL of complete cell culture medium and incubated for 48 h. Fresh medium was added containing (a) DMSO solvent control, (b) 0.5 mM HAL alone, (c) 1 μM Ko143 alone, (d) 0.5 mM HAL in combination with 1 μM Ko143, (e) Lapatinib (1 μM), (f) 0.5 mM HAL in combination with Lapatinib (1 μM). After 2 h incubation, the drug-containing medium was removed from each well. Cells were rinsed with PBS twice. To each well, cell lysis buffer (1 mL) containing 0.1% Triton X-100, 100 μM EDTA, and a protease inhibitor cocktail was added to lyse the cells. After gently mixing the solution, the lysate was centrifuged for 5 min at RT at 2000 RCF. The supernatant was collected and 200 μL of each sample was added to 96-well plate. PpIX fluorescence was determined using a spectrofluorometer with an excitation/emission wavelength of 405/635 nm.

Intracellular PpIX formation assay:

The procedure was performed according to the previous reported literature (30). Briefly, AY-27, RT-112, and RT-4 cells were cultured in a 6-well plate at a density of 100,000 cells/well in a 2 mL complete media and incubated for 48 h. The cells were treated with HAL alone (0.5 mM), lapatinib only, and HAL in combination with lapatinib at a concentration of 0.01 μM to 100 μM for 2 h at 37 °C. Control cells received vehicle alone without drug. Next, cells were washed twice with PBS (1 mL) and lysed in DMSO (1 mL). The lysed samples were collected and centrifuged at 2000 RCF for 5 min at RT to remove cellular debris. Finally, the supernatants were collected for PpIX measurement using a SpectraMax Gemini EM microplate spectrofluorometer (Molecular devices, US) at a PpIX excitation and emission wavelength of 405 and 635 nm. The effect of lapatinib on PpIX fluorescence was determined using the following equation.

$$\mathit{\text{PpIX}}\mathit{\text{Flu}}=\frac{{\mathit{\text{Flu}}}_{\mathit{\text{HAL}}+\mathit{\text{Lap}}}-{\mathit{\text{Flu}}}_{\mathit{\text{Lap}}}}{{\mathit{\text{Flu}}}_{\mathit{\text{HAL}}}}\times 100$$

Where Flu_HAL+Lap is the fluorescence of HAL in combination with lapatinib, Flu_Lap is the fluorescence of lapatinib only and Flu_HAL is the fluorescence of HAL alone.

In vitro phototoxicity and dark toxicity using 635 nm laser:

A cell density of 10,000 cells per well was used for seeding AY-27, RT-112 and RT-4 cells in 96-well plates and incubated for 24 h. After incubation, cells were exposed to HAL alone, lapatinib only and HAL + Lapatinib for 2 h at a lapatinib concentration of 0.01 μM-100 μM. MTT assays were also performed for parent drugs (SN38 and paclitaxel (PTX), prodrugs (Rh-L-PTX and Rh-L-SN-38) at a drug concentration of 0.05–2.5 μM, and prodrugs in combination with lapatinib at a concentration of 10 μM. All treatment groups were incubated for 2 h. Next, old media were removed and 200 μL of fresh complete media were added to each well. For phototoxicity, plates were illuminated with a 635 nm diode laser at 10 mW/cm² for 20 min. After the illumination, plates were incubated for an additional 48 h. MTT reagent was added to the cells (5 mg/mL in 1x phosphate buffer) and incubated for 4 h. Following this, 150 μL of DMSO was added to each well to dissolve the formazan crystals, and absorbance was determined at 570 nm and 630 nm using a microplate spectrofluorometer., GraphPad prism software (Version 9.0.2) was used to calculate IC₅₀ values.

For dark toxicity study, AY-27, RT-112 and RT-4 cells were seeded in 96-well plates at a cell density of 10000 cells/well and incubated for 24 h. After incubation, cells were exposed to HAL alone, Lapatinib alone and HAL + Lapatinib for 2 h and 48 h at a lapatinib concentration of 0.01 μM-100 μM. Next, old media were removed and 200 μL of fresh complete media were added to each well. After 48h, cells were treated with MTT reagent (5 mg/mL in 1x phosphate buffer) and incubated for 4 h. To dissolve the formazan crystals, 150 μL of DMSO was added to each well, and then absorbance was measured at 570 nm and 630 nm using a microplate spectrophotometer (Molecular devices).

Prodrug uptake study using fluorescence imaging:

This study utilized a fluorescence method to determine whether lapatinib enhanced the prodrug uptake by the cancer cells. The microscopic approach was employed to visualize and measure the intracellular concentrations. AY-27 cells (50,000 cells/well) were seeded on 12-well plates in 1 mL of complete RPMI growth medium and were incubated for 48 h at 37 °C in 5% CO₂. Then, 1 mL of 5 μM Rh-L-PTX and Rh-L-SN-38 in combination with lapatinib (1 μM and 10 μM) was added in each well and incubated at 37 °C for 2 h. After 2 h, the drug solution was removed and washed 3 times with PBS, and then immediately imaged on a Leica SP8 microscope with filters (ext. 510–545 nm and em. 570–600 nm). Next, the fluorescence images of AY-27 cancer cells were processed in the Image J software (version 2.9.0/1.53t), and rhodamine fluorescence intensity was measured.

Time-dependent prodrug uptake study using spectrophotometer:

A total of 50,000 AY-27 cells were seeded in each well of a 12-well plate in their respective growth medium (1 mL) and incubated at 37 °C in 5% CO₂ for 48 h. To each well, 1 mL of both prodrugs (5 μM) and prodrugs in combination with lapatinib (1 and 10 μM) were added and incubated for 10, 30, 60, 90, 120, 150, and 180 min. The plate was removed from the incubator after each time point, and the medium was removed and washed with PBS. The cells were then detached by incubating 0.5 mL of trypsin (0.25%) for 5 min. Fresh 1 mL of media was added and centrifuged to obtain the cell pellet. The cell pellet was digested with 120 mL of DMSO and mixed well. In 96-well plates, 100 μL of samples were added to each well to read using a plate reader with ext. 535 nm, em. 587 nm for Rh-L-PTX and Rh-L-SN-38. The spectrum scans were conducted at 535 nm for excitation, and 570 nm – 700 nm for emission. Based on the standard curve prepared from Rh-L-PTX and Rh-L-SN-38 in DMSO, the intracellular concentration was calculated.

Data collection and statistics:

The data analysis was performed using GraphPad Prism (version 8.0.2). The experiments were performed in triplicates. The results are presented as mean and standard deviation (mean + SD). A two-tailed student t-test was used to determine the significance of differences between the two groups. A p-value of less than 0.05 was considered statistically significant. The symbols, *, ** and *** represent p-values < 0.05, 0.01, and 0.001, respectively.

RESULTS

In vitro ABCG2 evaluation:

The mRNA expression of ABCG2 (Figure 1A) and transporter activities (Figure 1B) were determined in bladder cancer cell lines. The mRNA expression of the ABCG2 transporter in the bladder cancer cell lines was quantified using real-time PCR, with ABCG2 mRNA levels normalized to those of GAPDH. The data revealed that ABCG2, a principal ABC transporter implicated in non-muscle invasive bladder cancer (NMIBC), was expressed at significantly higher levels in AY-27 cells than in RT-112 and RT-4 cells. As illustrated in Figure 1A, the mean mRNA expression level of ABCG2 was approximately 50% and 98% higher in AY-27 cells compared to RT-4 and RT-112 cell lines respectively. We observed a strong positive correlation between ABCG2 activity and mRNA expression, as determined by Pearson correlation analysis (Figure 1C). AY-27 cells had the highest ABCG2 mRNA expression and showed the strongest ABCG2 activity compared to RT-112 and RT-4 cell line.

Figure 1.

(A) mRNA expression of ABCG2 transporter in bladder cancer cell lines, as determined via reverse transcription-quantitative PCR analysis. GAPDH was used as an internal reference. (B) ABCG2 transporter activity of bladder cancer cell lines and data are presented as mean ± SD from at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with AY-27 by t-test. (C) A Pearson correlation analysis has indicating a positive correlation between the activity of ABCG2 and the mRNA expression. (D) lapatinib and Ko143 induced intracellular PpIX increase with ABCG2 activity in bladder cancer cell lines, and Data are mean ± SD from at least 3 independent experiments. ns-not significant, *P < 0.05, **P < 0.01, ***P < 0.001, compared with HAL (0.5 mM) by t-test.

To assess the cellular response to the treatment with the transporter inhibitors (Ko143 and Lapatinib), the levels of PpIX in cell lysates following HAL + inhibitor treatment were compared with those observed after treatment with HAL alone (Figure 1D). Bladder cancer cells demonstrating heightened ABCG2 activity, AY-27 cells, exhibited a pronounced response to Ko143 and lapatinib treatment, as evidenced by elevated PpIX concentrations compared to cells treated with HAL alone. In contrast, cell lines such as RT-112 and RT-4, which exhibit lower ABCG2 activity, showed no significant response to the treatment.

Intracellular PpIX formation assay:

The mean fluorescence intensity after various treatments is shown in Figure 2, Figures S1, S2. Compared to treatment with HAL alone, the combination of HAL and Lapatinib resulted in significantly higher PpIX fluorescence in AY-27 cells (Figure 2A & B). Specifically, AY-27 cells showed a notable dose-dependent enhancement in PpIX fluorescence, far exceeding that observed in RT-112 cells and RT-4 cells (see Figure 2C, S1, and S2). This enhancement amounted to a threefold increase in fluorescence intensity when compared to the fluorescence observed with HAL treatment alone.

Figure 2.

(A) PpIX formation assay. Mean fluorescence measured after 2 h treatment with HAL alone, HAL + lapatinib treatments in AY-27 cell line. Data are presented as mean ± SD from at least 3 independent experiments. ns-not significant, *P < 0.05, **P < 0.01, ***P < 0.001, compared with HAL (0.5 mM) by t-test. (B) PpIX fluorescence spectra of AY-27 cell lysates were measured with a spectrofluorometer, and (C) Normalized to the fluorescence of HAL treatment alone to show the percent change in cell lysates of bladder cancer cell lines.

In vitro phototoxicity and dark toxicity:

Effects of lapatinib at various concentration on photo- and dark toxicity of PpIX-PDT, with and without HAL (0.5 mM), were assessed with AY-27 bladder cancer cells (Figure 3). Intracellular PpIX, generated with or without HAL, was used as a photosensitizer illuminated with a 635 nm laser (Figure 3A). At the same concentration of lapatinib, the cells treated with HAL + L (Lapatinib) killed more than the cells treated with only L, presumably due to enhanced PpIX formation by the addition of HAL. Lapatinib alone at a lower concentration (0.01–1 μM) did not show significant phototoxicity. It did show significant phototoxicity at higher concentrations (28 and 15% cell viability at 10 and 100 μM, respectively). Since L itself cannot be phototoxic with 635 nm, this L-concentration-dependent phototoxicity might be coming from L intrinsic toxicity (dark toxicity), consistent with the dark toxicity data (Figure 3B). Without illumination, lapatinib itself showed concentration-dependent toxicity. However, HAL (0.5 mM) did not increase the toxicity of L in most of the L concentrations.

Figure 3.

(A) Phototoxicity of HAL-PDT, lapatinib and HAL-PDT in combination with lapatinib treatments in AY-27 cell line. Treated groups were illuminated at 10 mW/cm2 for 20 min using 635 nm laser *P < 0.05, **P < 0.01, ***P < 0.001, ns-not significant, compared with HAL only by t-test. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with HAL only by t-test. (B) dark toxicity of HAL-PDT, lapatinib and HAL-PDT in combination with lapatinib treatments in AY-27 cell line ns- not significant HAL+ lapatinib compared with the lapatinib by t-test.

Effects of lapatinib (10 μM) on the combination treatment with HAL-PDT and mitochondria-targeting and SO-cleavable prodrugs of PTX and SN-38 (Rh-L-PTX or Rh-L-SN-38) were assessed with AY-27 cells (Figure 4). Interestingly, the addition of lapatinib significantly killed more cells in the combination with Rh-L-PTX (30% → 14 % cell kill) (Figure 4A) while its effect was not significant in the combination with Rh-L-SN-38 (20 % → 17% cell kill) (Figure 4B). Considering the increased PpIX by lapatinib (10 μM) by 300%, the result of the combination with Rh-L-SN-38 was not expected. There could be a difference in the effects of lapatinib on these two prodrugs.

Figure 4.

Phototoxicity comparison between PTX (A) and SN-38 (B) prodrugs and with prodrugs in combination with lapatinib. All the treatments groups were illuminated at 10 mW/cm² for 20 min using 635 nm laser. Statistical significance by t-test *P < 0.05, **P < 0.01, ***P < 0.001, ns-not significant.

Additionally, we compared the phototoxicities of HAL-PDT in combination with the parent drugs (PTX and SN-38) or the prodrugs (Rh-L-PTX and Rh-L-SN-38) at the different concentrations (0.05–2.5 μM). *The cells were exposed to all the drugs (HAL, parent drugs, and prodrugs) for 2 h and then washed with the complete media. In both cases, the prodrug was better than the parent drug in killing cancer cells with HAL-PDT (Figure 5 and Table S2). The results are somewhat counterintuitive if we only consider the prodrug activation with the total concentration (1 μM) (Figure 4A and B).

Figure 5.

Phototoxicity of parent and prodrugs in combination with HAL-PDT in AY-27 cell line illuminated at 10 mW/cm2 using 635 nm laser for 20 min. Statistical significance *P < 0.05 HAL + Rh-L-PTX compared with HAL + PTX , ##P < 0.01 HAL + Rh-L-SN38 compared with HAL + SN-38 by t-test.

Uptake of the prodrugs with and without lapatinib:

Fluorescence microscopy was used to elucidate whether lapatinib has a positive impact on the intracellular uptake of the prodrugs. Following 2 h incubation of prodrugs alone or with lapatinib, AY-27 cells were imaged for fluorescence (Figure 6A). Lapatinib increased the intracellular PTX prodrug. The maximum fluorescence intensity for Rh-L-PTX was observed as ~140 A.U. and for Rh-L-PTX in combination with lapatinib (10 μM) was ~271 A.U. (Figure 6B). The uptake of Rh-L-SN-38 in combination with lapatinib was not improved in combination with lapatinib (Figure 6A and C).

Figure 6.

(A) Uptake study in cancer cells. Fluorescence microscopic images treated with prodrugs and in combination with lapatinib. The line graphs represent the fluorescence intensity plot profile across the indicated cut lines in AY-27 cells treated with PTX prodrug (B) and SN-38 prodrug (C) in combination with lapatinib.

Intracellular prodrug concentration after 2 h incubation with HAL was determined using a quantitative method. We have observed that a 2-fold increase in PTX prodrug in combination with lapatinib compared to PTX prodrug only. Intracellular Rh-L-PTX concentration was 34 μM after 2 h of incubation and for Rh-L-PTX in combination with lapatinib (10 μM) was 110 μM (Figure 7A and S3). In contrast, the intracellular concentration of Rh-L-SN-38 was 174 μM (Figure 7B and S4). There was only a 10 % increase in intracellular concentration of Rh-L-SN-38 when treated in combination with lapatinib. The results suggest that Rh-L-PTX is pumped out of the cell by a ABC transporter but Rh-L-SN-38 is not, which is consistent with reported information that lapatinib inhibits both ABCG2 and ABCB1 (34).

Figure 7.

In vitro drug uptake of Rh-L-PTX (A) and Rh-L-SN-38 (B) in combination with lapatinib in AY-27 cell line. Wash represents replacing the drug solution with complete media. “Wash” represents the time of replacing the drug solution with complete media.

DISCUSSION

The objective of this study was to investigate if adding lapatinib, an efflux inhibitor, to the PpIX PDT and the combination of PpIX and prodrugs can improve the PpIX levels, thus improving the overall efficacy of these treatments for NMIBC. While the enhancement of PpIX concentration by lapatinib in various cancer cells has been reported by Dr. Chen’s lab. Its impact on bladder cancer cells and the combination treatment with PpIX-PDT and SO-cleavable prodrug has not been reported.

Dr. Chen’s group reported that expression levels of ABCG2 and its activity are necessary for enhancing PpIX concentration (19, 21, 30). Thus, we determined ABCG2 expression and activity and correlated those with intracellular PpIX levels in three bladder cancer cells (one rat (AY-27) and two human (RT-112 and RT-4) bladder cancer cell lines). Impacts of Ko143 and lapatinib were evaluated on intracellular PpIX. Those inhibitors greatly increased PpIX in AY-27 cells, with high ABCG2 mRNA expression, but did not have any significant increase in RT-112 cells, with very low ABCG2 mRNA expression. On the other hand, RT-4 cells, expressing moderate ABCG2 mRNA, did not show any significant PpIX increase by these two inhibitors. For translating this strategy, further study is needed to investigate the relation between ABCG2 expression and PpIX increase by efflux pump inhibitors. Identifying biomarkers associated with PpIX levels is crucial to the selection of patients for optimal treatment outcomes.

Developing new combination therapies in systemic treatments is complicated due to various factors such as increased systemic side effects, drug-drug interactions, issue in ADME of certain drugs and more complicated dosing schedule of multiple drugs (35–38). However, the combination treatments for local therapies could be more straightforward. For the bladder instillation treatments, additional drugs can be simply added to the instillation solution, with minimal concerns about the complications due to the systemic absorption of drugs. For this strategy, efflux pump inhibitors can be simply added to the instillation solution of HAL and prodrug. For example, a potent ABCG2 inhibitor, Ko143, cannot used in vivo because of its metabolic instability (39, 40). However, in bladder instillation, the metabolic instability may be a less concern.

The observed enhanced phototoxicity from combining PpIX-PDT with Rh-L-PTX and Rh-L-SN-38 prodrugs over their parent drugs (PTX and SN-38) (Figure 4) contradicts expectations since prodrugs are generally less toxic than their parent counterparts and their activation would be 100% at best. Intuitively, the use of parent drugs, at equimolar concertation with prodrugs, should augment HAL-PDT’s phototoxic effects more effectively than prodrugs. However, this anomaly could be attributed to two key factors: 1) the efficacy of drug-induced cell death is more significantly influenced by the amount of drug within the cells rather than the total amount present in the culture medium; and 2) prodrug activation takes place within cells, making the concentration of prodrugs inside the cells a crucial factor. It is plausible that the intracellular release of drugs from prodrugs due to HAL-PDT is higher than the uptake of parent drugs from the medium, explaining the observed results.

CONCLUSION

The study presents a promising advancement in the treatment of non-muscle invasive bladder cancer (NMIBC) by integrating an efflux inhibitor, lapatinib, with photodynamic therapy (PDT) and prodrug administration. Our findings indicate a significant enhancement in intracellular PpIX accumulation and more effective therapeutic effect of PpIX-PDT and its combination with prodrugs within bladder cancer cells when lapatinib is employed. This improvement is attributed to lapatinib’s ability to inhibit efflux pump activity, thereby increasing the intracellular concentration of PpIX, a critical photosensitizer in PDT, and facilitating greater uptake of certain prodrug (Rh-L-PTX).

Importantly, our research demonstrates a substantial decrease in cell viability in bladder cancer cell lines treated with the combination of lapatinib, prodrugs, and PpIX-based PDT compared to treatments utilizing prodrugs or PDT alone. This effect was particularly pronounced in cell lines expressing high levels of the efflux pump, indicating the importance of efflux pump expression in the efficacy of this combination treatment. Furthermore, lapatinib tripled the uptake of paclitaxel prodrug in comparison to prodrug treatment without the efflux inhibitor.

These results underscore the potential of utilizing efflux inhibitors like lapatinib as part of a combination therapy for NMIBC. The approach not only enhances the efficacy of PpIX-based PDT but also increases the effectiveness of prodrug treatments. Given the translational potential of this strategy, particularly due to the clinical availability of efflux inhibitors and the simplicity of their addition to bladder instillation solutions, our study paves the way for novel, more effective intravesical treatment regimens for NMIBC. Future investigations are warranted to further explore the therapeutic advantages of this combination therapy with more human bladder cancer cells.

ABBREVIATIONS

ABCG2

ATP-binding cassette super-family G member 2

AY-27

Rat bladder cancer cell line

DMSO

Dimethyl sulfoxide

GADPH

Human-specific glyceraldehyde 3-phosphate dehydrogenase

HAL

Hexyl ester of 5-aminolevulinic acid

IC₅₀

Half maximal inhibitory concentration

MIBC

Muscle-invasive bladder cancer

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide

NMIBC

Non-muscle invasive bladder cancer

PBS

Phosphate-buffered saline

PDT

Photodynamic therapy

PpIX

Protoporphyrin IX

PTX

Paclitaxel

qPCR

Real-time quantitative polymerase-chain-reaction

RT

Room temperature

RT-112

Human urinary bladder transitional cell carcinoma

RT-4

Human urothelial bladder cancer cell line

SN38

7-ethyl-10-hydroxycamptothecin

UV

Ultraviolet