Vascular-targeted photodynamic therapy (PDT) selectively disrupts vascular function by inducing oxidative damages to the vasculature, particularly endothelial cells. Although effective tumor eradication and excellent safety profile are well demonstrated in both preclinical and clinical studies, incomplete vascular shutdown and angiogenesis are known to cause tumor recurrence after vascular-targeted PDT. We have explored therapeutic enhancement of vascular-targeted PDT with PI3K signaling pathway inhibitors because the activation of PI3K pathway was involved in promoting endothelial cell survival and proliferation after PDT. Here, three clinically relevant small-molecule inhibitors (BYL719, BKM120, and BEZ235) of the PI3K pathway were evaluated in combination with verteporfin-PDT. Although all three inhibitors were able to synergistically enhance PDT response in endothelial cells, PDT combined with dual PI3K/mTOR inhibitor BEZ235 exhibited the strongest synergism, followed in order by combinations with pan-PI3K inhibitor BKM120 and p110α isoform-selective inhibitor BYL719. Combination treatments of PDT and BEZ235 exhibited a cooperative inhibition of antiapoptotic Bcl-2 family protein Mcl-1 and induced more cell apoptosis than each treatment alone. In addition to increasing treatment lethality, BEZ235 combined with PDT effectively inhibited PI3K pathway activation and consequent endothelial cell proliferation after PDT alone, leading to a sustained growth inhibition. In the PC-3 prostate tumor model, combination treatments improved treatment outcomes by turning a temporary tumor regrowth delay induced by PDT alone to a more long-lasting treatment response. Our study strongly supports the combination of vascular-targeted PDT and PI3K pathway inhibitors, particularly mTOR inhibitors, for therapeutic enhancement. Mol Cancer Ther; 16(11); 2422–31. ©2017 AACR.

Photodynamic therapy (PDT) combines a photosensitizer, laser light matching the absorption of photosensitizer, and oxygen to produce reactive oxygen species (ROS)-dependent therapeutic effects (1). Vascular-targeted PDT is a PDT regimen that preferentially targets abnormal blood vessels through targeted delivery of photosensitizer molecules to the vasculature (2). As a modality for disrupting existing blood vessels, vascular-targeted PDT has been clinically used for the treatment of diseases with abnormally activated vascularization including age-related macular degeneration (AMD) and port-wine stain birthmarks (3, 4). It is also being actively explored for the treatment of various types of cancers.

Vascular-targeted PDT has been particularly investigated for treating localized prostate cancer, a disease that is in a confined gland and readily accessible by laser fibers. Preclinical studies demonstrate that vascular-targeted PDT with photosensitizers such as verteporfin and Tookad effectively causes tumor destruction by inducing endothelial cell injuries (5–7) and consequent disruption of vascular function (8–10). Multicenter clinical trials have confirmed the clinical efficacy of vascular-targeted PDT and its excellent safety profile (11). A recent phase III randomized clinical trial showed that, compared with active surveillance (the standard treatment for low-risk localized prostate cancers), vascular-targeted PDT with photosensitizer padeliporfin (Tookad-soluble, WST11) significantly prevented cancer progression and increased the number of cancer-free patients at 24 months after PDT (12). However, despite such promising outcomes, tumor recurrence was detected after vascular-targeted PDT with verteporfin or Tookad in animal tumor models (10, 13). Close to 40% prostate cancer patients treated with padeliporfin-PDT had residual tumors in the treated lobes (14), and about 30% patients still showed disease progression after PDT (12). Clearly, a therapeutic enhancement strategy is needed for vascular-targeted PDT.

We have shown that PDT response can be significantly enhanced by PI3K pathway inhibitors (15, 16). As the most frequently altered cell signaling pathway in human cancers, the PI3K signaling pathway plays an important role in promoting tumor cell survival and progression (17). PI3K enzymes convert phosphatidylinositol-4,5-biphosphate (PIP2) in the cell membrane to phosphatidylinositol-3,4,5-triphosphate (PIP3), which acts as a second message to activate downstream cell signaling molecules such as Akt (protein kinase B) and mTOR by inducing a cascade of protein phosphorylation. Because abnormal activation of the PI3K pathway is often involved in tumorigenesis and progression, this pathway has become a well-explored cancer therapeutic target (17). A variety of selective and potent inhibitors targeting 4 different isoforms of class I PI3Ks (p110α, β, γ, δ) and downstream signaling components have been developed and are showing promising results in clinical trials.

The interaction between PDT and these newly developed PI3K pathway inhibitors is not well studied. Although beneficial effects were reported in previous studies combining PDT with early PI3K inhibitors such as wortmannin and LY294002 (18–20), these first-generation PI3K inhibitors are now considered of little clinical relevance due to the lack of selectivity for PI3K enzymes (21). With the availability of clinically relevant inhibitors targeting different signaling molecules in the PI3K pathway, it is important to evaluate their interactions with PDT in order to identify optimal combination treatments.

In this study, we assessed combination treatments of verteporfin-PDT and three types of PI3K pathway inhibitors. These inhibitors, which have all progressed to clinical trials, include p110α isoform-selective inhibitor BYL719 (22), pan-PI3K inhibitor BKM120 that inhibits all four isoforms of class I PI3Ks (23), and dual PI3K/mTOR inhibitor BEZ235 that inhibits all four isoforms of class I PI3Ks and mTOR kinases (24). Although all three inhibitors were able to synergistically enhance PDT response, PDT combined with BEZ235 showed the strongest synergism. PDT combined with BEZ235 increased endothelial cell apoptosis and caused sustained inhibition of cell proliferation, leading to a greater and more long-lasting therapeutic response than each individual treatment both in vitro and in vivo. Our results demonstrate the importance of using clinically relevant PI3K pathway therapeutics, particularly the mTOR inhibitors, for improving vascular-targeted PDT.

Reagents

Verteporfin (benzoporphyrin derivation in a lipid formulation) was kindly provided by QLT Inc. A stock PBS solution of verteporfin was reconstituted according to the manufacturer's instruction and stored at 4°C in the dark until use. BEZ235 was purchased from LC Laboratories, and BKM120 and BYL719 were kindly provided by Novartis. BEZ235, BKM120 and BYL719 were dissolved in DMSO, sterilized through filtration using 0.22-μm pore size filters, and stored in a −80°C freezer.

Cell culture

SV40 immortalized mouse endothelial cells (SVEC) and PC-3 human prostate tumor cells were obtained from ATCC in October 2010, expanded and frozen in the liquid nitrogen. Cells were not tested for mycoplasma. Cell culture was maintained in the RPMI1640 (with glutamine) medium supplemented with 9% FBS (Hyclone) and 100 U/mL penicillin/streptomycin (Mediatech) at 37°C in a 5% CO2 incubator. Cells less than 20 passages were used for experiments.

PDT treatment

Cells were incubated with verteporfin for 15 minutes and then exposed to 5 mW/cm2 irradiance of 690-nm light for 100 seconds with verteporfin in cell culture media. Light intensity was measured by an optical power meter (Thorlabs Inc.). Light treatment was performed using a diode laser system (High Power Devices Inc.). For combination treatments, PI3K inhibitors (dissolved in DMSO) was added into cell culture media at 1 hour before PDT. The final concentration of DMSO was less than 0.1%.

Cell viability assay

Cell viability at different time points after treatment was examined by Alamar blue (resazurin from Sigma) assay where resazurin dye is metabolically converted to fluorescent resorufin by live cells. SVEC cells were implanted in multi-well cell culture plates, allowed to attach for overnight, and treated with PI3K inhibitors alone, PDT alone, and combination treatments. At various time points after treatments, cells were incubated with Alamar blue (180 μg/mL) for 4 hours and fluorescence intensity was detected by a BioTek Synergy microplate reader with excitation at 540 ± 20 nm and emission at 620 ± 20 nm. Cell viability was calculated by normalizing the fluorescence intensity of treated cells to that of control cells. Each experiment was repeated at least three times.

Verteporfin uptake

Effects of PI3K inhibitors on the intracellular uptake of verteporfin were determined by flow cytometry. SVEC cells were incubated with 400 ng/mL verteporfin for 15 minutes. Cells were trypsinized, rinsed with PBS, and resuspended in PBS for the analysis of verteporfin fluorescence with a FACSCalibur flow cytometer (BD Biosciences) in the FL3 channel (488 nm excitation, 650 nm long pass emission). The fluorescence intensity of 20,000 cells in each sample was recorded. To determine the effects of PI3K pathway inhibitors on verteporfin uptake, inhibitors were added at 45 minutes prior to adding verteporfin and verteporfin fluorescence was measured 15 minutes later. Experiments were repeated three times.

Annexin V staining for apoptosis

Apoptotic cells were stained by Alexa Fluor 488-labeled Annexin V (Life Technologies) and quantified with flow cytometry. SVEC cells were seeded in 60-mm cell culture dishes and allowed to grow to reach about 70% confluency. Cells were treated with PI3K inhibitors alone, PDT alone, and combination treatments. At various times after treatment, treated and untreated control cells were trypsinized and resuspended in Annexin V binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5 mmol/L CaCl2, pH 7.4) at a concentration of 1 × 106 cells per mL. Floating cells collected from cell culture media were also included for the Annexin V staining. According to manufacturer's instruction, 100-μL cell suspension was incubated with 3 μL of Alexa Fluor 488-labeled Annexin V for 15 minutes at room temperature with constant rotation for apoptotic cell staining. Cell fluorescence was measured with a FACSCalibur flow cytometer (BD Biosciences) in the FL1 channel (488 nm excitation, 530 ± 15 nm emission). For each sample, 20,000 cells were measured and the percentage of apoptotic cells (Annexin V-positive) was quantified. Experiments were repeated three times.

Western blot analysis

Cells at 70%–80% confluence were treated and lysed on ice with the NP40 lysis buffer supplemented with protease and phosphatase inhibitors at various time points after treatment, as described previously (15). Cell lysates were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). Blots were incubated with a primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Primary antibodies include phospho-AktSer473 (p-Akt), Akt, phospho-ribosome protein S6Ser235/236 (p-S6), S6, PARP, caspase-3 (casp-3), caspase-9 (casp-9), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL), myeloid cell leukemia-1 (Mcl-1), and β-actin antibodies (from Cell Signaling Technology). Blots were treated with Super Signal West Dura extended duration substrate (Thermo Scientific) and immunoreactive bands were captured with a GE Amersham Imager 600 (GE Healthcare Bio-Sciences). The density of immunoreactive bands was quantified with the NIH ImageJ software.

Tumor model and treatments

Subcutaneous PC-3 human prostate tumors in male athymic nude mice (NCR, nu/nu, 6–8 weeks old) from Charles River Laboratories were used. Tumor implantation and PDT treatment were described previously (25). Tumors at a size of about 5 mm in diameter were used for experiments and randomly distributed to different groups. BEZ235 was orally administered at a dose of 40 mg/kg body weight once a day for 24 days. For vascular-targeted PDT, PC-3 tumors were externally illuminated with an irradiance of 50 mW/cm2 light at 690 nm for 600 seconds, resulting in a total light dose of 30 J/cm2, at 15 minutes after intravenous injection of 0.5 mg/kg dose of verteporfin. Animals were anesthetized by intraperitoneal injection of a mixture of ketamine (120 mg/kg) and xylazine (12 mg/kg) during PDT treatment. BEZ235 was given at 1 hour before PDT for the combination therapy. Tumor size was measured every 4 days after treatment and tumor volume was calculated using the formula of 0.52 × length × width × height. Animal body weight was also monitored to indicate treatment-associated toxicity. All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC).

Statistical and combination index analysis

Two-way ANOVA test followed by multiple comparisons was used to determine statistical difference. Statistical significance was accepted at P < 0.05. The mode of interaction (antagonistic additive, synergistic) between PDT and PI3K inhibitors was determined using CompuSyn software based on the Chou–Talalay method for drug combination (26).

Effects of PI3K inhibitors on endothelial cell viability and PI3K pathway signaling

Effects of BYL719 (a p110α isoform–selective inhibitor), BKM120 (a pan-PI3K inhibitor), and BEZ235 (a dual pan-PI3K/mTOR inhibitor) on cell viability and PI3K pathway signaling were evaluated in SVEC endothelial cells. Figure 1 shows that different types of PI3K inhibitors had significantly different effects on the cell viability (Fig. 1A) and PI3K signaling (Fig. 1B). BEZ235 potently reduced SVEC cell viability at all concentrations examined (P < 0.001). Its inhibitory effect on cell viability was much stronger than BKM120, which was slightly effective only at higher concentrations (250 and 500 nmol/L). BYL719 showed no significant inhibition on cell viability at all doses examined. The reduction in cell viability induced by different types of PI3K inhibitors appeared to correlate with the inhibition on PI3K pathway activity shown by Western blot analysis. Treatment of SVEC cells with BEZ235 (500 nmol/L) led to almost complete dephosphorylation of two PI3K downstream signaling molecules Akt and S6 for up to 24 hours, indicating a potent and sustained inhibition of PI3K pathway signaling. Compared with BEZ235, less inhibition of PI3K signaling was induced by BKM120 and pathway activity started to recover at 24 hours after treatment. BYL719 induced the least inhibitory effect on PI3K pathway signaling, as substantial p-Akt and p-S6 signal remained detectable after treatment.

Figure 1.

Effects of PI3K inhibitors on endothelial cell viability and PI3K pathway signaling. A, SVEC cells were treated with different PI3K inhibitors for 48 hours, and cell viability was determined by Alamar blue assay. Data are presented as mean ± SD from at least three independent experiments. **, P < 0.01; ***, P < 0.001, compared with untreated control. *** shown on the right of dose–response curve indicates P < 0.001 at all concentrations examined. B, SVEC cell lysates were collected at various time points after treatments with PI3K inhibitors and probed for PI3K pathway signaling molecules by Western blot analysis.

Figure 1.

Effects of PI3K inhibitors on endothelial cell viability and PI3K pathway signaling. A, SVEC cells were treated with different PI3K inhibitors for 48 hours, and cell viability was determined by Alamar blue assay. Data are presented as mean ± SD from at least three independent experiments. **, P < 0.01; ***, P < 0.001, compared with untreated control. *** shown on the right of dose–response curve indicates P < 0.001 at all concentrations examined. B, SVEC cell lysates were collected at various time points after treatments with PI3K inhibitors and probed for PI3K pathway signaling molecules by Western blot analysis.

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Effects of PI3K pathway inhibitors on verteporfin-PDT and verteporfin uptake

Effects of three types of PI3K inhibitors on cell viability reduction induced by verteporfin PDT were examined in SVEC cells. Figure 2A and B shows the cell viability after PDT (200 or 400 ng/mL verteporfin) alone and PDT in combination with PI3K inhibitors. BEZ235 combined with either 200 or 400 ng/mL verteporfin PDT caused significantly more reduction in cell viability than PDT alone, indicating therapeutic enhancement. BKM120 was also able to significantly increase the effectiveness of 400 ng/mL verteporfin PDT, and 200 ng/mL verteporfin PDT at 250 and 500 nmol/L concentrations. BYL719 only increased the response of PDT with 400 ng/mL verteporfin, but not PDT with 200 ng/mL verteporfin.

Figure 2.

Verteporfin-PDT in combination with PI3K inhibitors synergistically reduced endothelial cell viability. Effects of PDT with 200 (A) or 400 (B) ng/mL verteporfin alone or PDT in combination with PI3K inhibitors on SVEC cell viability determined by Alamar blue assay at 48 hours after treatment. Data are presented as mean ± SD from at least three independent experiments. **, P < 0.01; ***, P < 0.001, compared with PDT alone, which is the data point at 0 concentration. *** shown on the right of dose–response curve indicates P < 0.001 at all concentration examined. Combination index analysis showing the mode of interaction between each PI3K inhibitor and PDT with 200 (C) or 400 (D) ng/mL verteporfin. Combination index of 1 indicates additive interaction and combination indexes of less than 1 and more than 1 are synergistic and antagonistic, respectively. **, P < 0.01; ***, P < 0.001, compared with the index of 1.

Figure 2.

Verteporfin-PDT in combination with PI3K inhibitors synergistically reduced endothelial cell viability. Effects of PDT with 200 (A) or 400 (B) ng/mL verteporfin alone or PDT in combination with PI3K inhibitors on SVEC cell viability determined by Alamar blue assay at 48 hours after treatment. Data are presented as mean ± SD from at least three independent experiments. **, P < 0.01; ***, P < 0.001, compared with PDT alone, which is the data point at 0 concentration. *** shown on the right of dose–response curve indicates P < 0.001 at all concentration examined. Combination index analysis showing the mode of interaction between each PI3K inhibitor and PDT with 200 (C) or 400 (D) ng/mL verteporfin. Combination index of 1 indicates additive interaction and combination indexes of less than 1 and more than 1 are synergistic and antagonistic, respectively. **, P < 0.01; ***, P < 0.001, compared with the index of 1.

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To determine the mode of interaction between PDT and three types of PI3K inhibitors, combination index analysis was performed using ComboSyn software based on the Chou–Talalay method for drug interaction assessment. According to this method, combination index of 1 indicates additive interaction, and indexes of less than 1 and more than 1 are synergistic and antagonistic, respectively (26). Combination index analysis demonstrates a strong synergistic interaction between BEZ235 and PDT with 200 ng/mL verteporfin (Fig. 2C). However, 200 ng/mL verteporfin PDT combined with either BKM120 or BYL719 only showed additive effects. Synergism was detected when 400 ng/mL verteporfin PDT was combined with any one of three PI3K inhibitors, except with BYL719 at 500 nmol/L concentration (Fig. 2D). Notably, PDT combined with BEZ235 exhibited the strongest synergism, followed in order by combinations with BKM120 and BYL719.

Effects of PI3K inhibitors on verteporfin intracellular uptake were determined by flow cytometry. Figure 3 shows that BEZ235 caused a slight, but statistically significant, increase in the uptake of verteporfin whereas BKM120 and BYL719 had no significant effect on verteporfin uptake.

Figure 3.

Effects of PI3K inhibitors on verteporfin intracellular uptake in SVEC cells. Cells were incubated with 400 ng/mL verteporfin for 15 minutes, trypsinized and resuspended in PBS for the flow cytometry analysis of verteporfin fluorescence. PI3K inhibitors were added at 45 minutes before adding verteporfin. Data are presented as mean ± SD from three independent experiments. ***, P < 0.001, compared with cells without PI3K inhibitor treatment, which was normalized to 100%.

Figure 3.

Effects of PI3K inhibitors on verteporfin intracellular uptake in SVEC cells. Cells were incubated with 400 ng/mL verteporfin for 15 minutes, trypsinized and resuspended in PBS for the flow cytometry analysis of verteporfin fluorescence. PI3K inhibitors were added at 45 minutes before adding verteporfin. Data are presented as mean ± SD from three independent experiments. ***, P < 0.001, compared with cells without PI3K inhibitor treatment, which was normalized to 100%.

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BEZ235 combined with verteporfin-PDT enhanced mitochondria-mediated apoptosis

Because BEZ235 combined with verteporfin-PDT exhibited the strongest synergism, this combination was studied further to determine therapeutic enhancement mechanisms. Annexin V staining was performed to assess whether combination treatments increased endothelial cell apoptosis. Figure 4A shows flow cytometer histograms of Annexin V staining at 1, 4, and 24 hours after BEZ235 (12.5 nmol/L) alone, 400 ng/mL verteporfin PDT alone, and combination treatments. Quantification of apoptotic cells based on positive Annexin V staining is shown in Fig. 4B. BEZ235 alone did not induce apoptosis in SVEC cells, whereas PDT alone caused a robust endothelial cell apoptosis. The combination of BEZ235 and PDT induced significantly more cell apoptosis than PDT alone at 4 and 24 hours after treatment.

Figure 4.

Combination treatments of BEZ235 (12.5 nmol/L) and PDT (400 ng/mL verteporfin) enhanced endothelial cell apoptosis. SVEC cells were treated as indicated and stained with Alexa Fluor 488-labeled Annexin V at 1, 4, and 24 hours after treatment. A, Annexin V staining histograms after different treatments. B, The percentage of apoptotic cells (Annexin V-positive) was quantified and shown. Data shown are mean ± SD from three independent experiments. ^^, P < 0.01; ^^^, P < 0.001, compared with controls at corresponding time points. *, P < 0.05; ***, P < 0.001, compared with PDT alone.

Figure 4.

Combination treatments of BEZ235 (12.5 nmol/L) and PDT (400 ng/mL verteporfin) enhanced endothelial cell apoptosis. SVEC cells were treated as indicated and stained with Alexa Fluor 488-labeled Annexin V at 1, 4, and 24 hours after treatment. A, Annexin V staining histograms after different treatments. B, The percentage of apoptotic cells (Annexin V-positive) was quantified and shown. Data shown are mean ± SD from three independent experiments. ^^, P < 0.01; ^^^, P < 0.001, compared with controls at corresponding time points. *, P < 0.05; ***, P < 0.001, compared with PDT alone.

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To examine effects of treatments on PI3K signaling and apoptotic pathways, SVEC cells were lysed at 1–24 hours after treatment and probed for p-S6, caspase-9 (marker of intrinsic mitochondrial apoptotic pathway), caspase-3 (a general executioner caspase), PARP (a substrate of activated executioner caspases), and Bcl-2 family proteins by Western blot analysis (Fig. 5A). Western blot band intensity was quantified and shown in Fig. 5B–H. BEZ235 alone induced a potent and continuous inhibition of p-S6, whereas PDT alone caused a transient upregulation of p-S6 followed by a downregulation at 24 hours after treatment (Fig. 5B). Combination treatments with BEZ235 completely inhibited PDT-induced p-S6 upregulation. PDT alone activated the cleavage of caspase-9 (Fig. 5C), caspase-3 (Fig. 5D), and PARP (Fig. 5E), indicating mitochondria-mediated apoptosis, whereas BEZ235 alone had no effect on the activation of apoptotic pathway. PDT in combination with BEZ235 resulted in significantly more mitochondria-mediated apoptosis than PDT alone, as indicated by greater caspase-9, caspase-3, and PARP cleavage after the combination therapy. PDT alone increased the level of antiapoptotic Bcl-2 family protein Mcl-1 (Fig. 5F), which was statistically significant by 4 hours after treatment. PDT-induced Mcl-1 elevation was completely suppressed by BEZ-235. Bcl-2 level did not show significant change after either individual or combination treatments (Fig. 5G). PDT alone induced Bcl-XL level reduction, although this was not statistically significant (Fig. 5H). There was no significant difference in the Bcl-XL level between PDT alone and combination treatments.

Figure 5.

Combination treatments of BEZ235 and verteporfin-PDT enhanced mitochondrial apoptosis in endothelial cells. A. Effects of BEZ235, PDT, and combination treatments on PI3K signaling and apoptotic pathway markers. SVEC cells were treated with BEZ235 (12.5 nmol/L) alone, PDT (400 ng/mL verteporfin) alone, and combination treatments. Cells were lysed at 1 to 24 hours after treatments and probed for various markers by Western blot analysis. Control cells received no treatment. The quantification of Western blot band intensity showing changes of p-S6/S6 (B), caspase-9 cleavage (C), caspase-3 cleavage (D), PARP cleavage (E), Mcl-1/actin (F), Bcl-2/actin (G), and Bcl-XL/actin (H) after treatments. Band intensity ratios of p-S6/S6, cleaved/total (uncleaved plus cleaved), and each Bcl-2 family protein/actin were normalized to untreated control and shown. Data represent mean ± SE from at least three independent experiments. ^, P < 0.05; ^^, P < 0.01; ^^^, P < 0.001, compared with untreated control, which was normalized to 100%. *, P < 0.05; ***, P < 0.001, compared with PDT alone.

Figure 5.

Combination treatments of BEZ235 and verteporfin-PDT enhanced mitochondrial apoptosis in endothelial cells. A. Effects of BEZ235, PDT, and combination treatments on PI3K signaling and apoptotic pathway markers. SVEC cells were treated with BEZ235 (12.5 nmol/L) alone, PDT (400 ng/mL verteporfin) alone, and combination treatments. Cells were lysed at 1 to 24 hours after treatments and probed for various markers by Western blot analysis. Control cells received no treatment. The quantification of Western blot band intensity showing changes of p-S6/S6 (B), caspase-9 cleavage (C), caspase-3 cleavage (D), PARP cleavage (E), Mcl-1/actin (F), Bcl-2/actin (G), and Bcl-XL/actin (H) after treatments. Band intensity ratios of p-S6/S6, cleaved/total (uncleaved plus cleaved), and each Bcl-2 family protein/actin were normalized to untreated control and shown. Data represent mean ± SE from at least three independent experiments. ^, P < 0.05; ^^, P < 0.01; ^^^, P < 0.001, compared with untreated control, which was normalized to 100%. *, P < 0.05; ***, P < 0.001, compared with PDT alone.

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BEZ235 combined with verteporfin-PDT resulted in a sustained inhibition of PI3K pathway signaling and cell proliferation

Effects of PDT alone, BEZ235 alone and combination treatments on cell proliferation and PI3K signaling pathway marker p-S6 were monitored daily for up to 5 days after treatment. PDT with either 200 or 400 ng/mL verteporfin significantly inhibited endothelial cell proliferation (Fig. 6A and B). However, SVEC cells resumed proliferation shortly after PDT, particularly after the lower dose PDT. The level of p-S6 was increased after PDT with either 200 or 400 ng/mL verteporfin, which occurred sooner after the lower dose PDT (Fig. 6C). Comparing the time course of cell proliferation versus p-S6 signaling showed that p-S6 signaling activation occurred prior to cell proliferation increase, suggesting the involvement of PI3K signaling activation in cell regrowth after PDT. PDT in combination with BEZ235 resulted in a complete inhibition of PDT-induced p-S6 elevation and continuous inhibition of SVEC cell proliferation.

Figure 6.

Combination treatments of BEZ235 and verteporfin-PDT resulted in sustained inhibition of endothelial cell proliferation and tumor growth. A–C, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on endothelial cell proliferation and the PI3K signaling. SVEC cells were treated with 200- (A) or 400- (B) ng/mL verteporfin PDT alone, BEZ235 (12.5 nmol/L) alone, and combination treatments. Cell proliferation was determined by Alamar blue assay every day for 5 days after treatment. Data were normalized to untreated control on Day 1. Data are presented as mean ± SD from at least three independent experiments. Cells lysates were prepared at corresponding time points after each individual and combination treatments and probed for p-S6/S6 by Western blot analysis (C). D and E, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on PC-3 tumor growth (D) and animal body weight (E). Mice bearing subcutaneous PC-3 tumors were treated as indicated and tumor volume was monitored every 4 days. Each group included six to eight animals.

Figure 6.

Combination treatments of BEZ235 and verteporfin-PDT resulted in sustained inhibition of endothelial cell proliferation and tumor growth. A–C, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on endothelial cell proliferation and the PI3K signaling. SVEC cells were treated with 200- (A) or 400- (B) ng/mL verteporfin PDT alone, BEZ235 (12.5 nmol/L) alone, and combination treatments. Cell proliferation was determined by Alamar blue assay every day for 5 days after treatment. Data were normalized to untreated control on Day 1. Data are presented as mean ± SD from at least three independent experiments. Cells lysates were prepared at corresponding time points after each individual and combination treatments and probed for p-S6/S6 by Western blot analysis (C). D and E, Effects of verteporfin-PDT alone, BEZ235 alone, and combination treatments on PC-3 tumor growth (D) and animal body weight (E). Mice bearing subcutaneous PC-3 tumors were treated as indicated and tumor volume was monitored every 4 days. Each group included six to eight animals.

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BEZ235 combined with vascular-targeted PDT resulted in a more durable treatment response in prostate tumors

Effects of vascular-targeted PDT alone, BEZ235 alone and combination treatments on tumor growth were examined in the PC-3 prostate tumor model (Fig. 6D). BEZ235 (40 mg/kg, daily, orally) alone significantly inhibited PC-3 tumor growth. Although PDT alone (30 J/cm2 light at 15 minutes after intravenous injection of 0.5 mg/kg verteporfin) was effective in inhibiting tumor growth, tumor regrowth occurred after an initial decrease in tumor volume. The combination of BEZ235 and PDT caused a significantly greater reduction in tumor volume and more sustained inhibition of tumor growth than each individual treatment. There was no significant difference in the body weight of animals in different treatment groups (Fig. 6E).

Vascular-targeted PDT with photosensitizer verteporfin has been approved for AMD and is being intensively investigated as a cancer therapy (27). We previously reported the intravascular localization of verteporfin and PDT-induced vascular effects including vascular permeability increase, thrombus formation, and vascular dysfunction, which lead to tumor destruction (8, 28). However, as we and others have shown, incomplete occlusion of blood vessels after PDT as well as subsequent angiogenesis-mediated neovascularization often result in vascular function resumption and disease recurrence (13, 29, 30). Because endothelial cell survival and proliferation are primarily involved in these post-PDT events that lead to tumor recurrence, we attempt to target prosurvival signaling pathways in endothelial cells for therapeutic enhancement of vascular-targeted PDT. We have shown that verteporfin-PDT in combination with PI3K pathway inhibitors enhanced endothelial cell death and increased the inhibition of endothelial cell proliferation (15, 16). As more PI3K pathway inhibitors targeting different signaling molecules/nodes are developed for clinical applications, we performed this study to evaluate the effects of three types of PI3K pathway inhibitors on verteporfin-PDT to identify the optimal combination treatments.

We found that inhibitors targeting different PI3K signaling molecules exhibited distinct effects on endothelial cell viability and PDT response. Although p110α is considered the most important PI3K isoform in endothelial cells (31), p110α isoform-selective inhibitor BYL719 at concentrations used in this study was unable to inhibit SVEC cell survival and only partially inhibited PI3K signaling, suggesting that endothelial cells resort to other isoforms for downstream signaling when p110α is blocked. We have shown that SVEC cells also express p110β isoform, albeit at a lower level than p110α (15). Inhibiting all isoforms of class I PI3Ks by a pan-inhibitor BKM120 did lead to more effective inhibition of SVEC cell viability and stronger inhibition of PI3K signaling. However, it is the dual PI3K/mTOR inhibitor BEZ235 that caused the most effective inhibition of cell viability and the strongest PI3K signaling suppression. The same trend was also found in the enhancement of PDT response by PI3K inhibitors. BEZ235 showed the highest efficacy for enhancing PDT response and BYL719 had the lowest effectiveness. These results indicate the redundancy of different PI3K isoforms in endothelial cell signaling and the necessity of inhibiting all isoforms for better cell growth inhibition and PDT enhancement. More importantly, our data suggest the importance of mTOR signaling in determining endothelial cell survival and PDT response. We are currently determining how selective inhibition of mTOR only affects the PDT response.

Strong synergism between BEZ235 and verteporfin-PDT in cell viability reduction prompted us to determine whether combination treatments enhanced cell death. BEZ235 alone is not effective for inducing apoptosis in tumor cells (32). As shown in this study, it did not induce apoptosis in endothelial cells either. In fact, inability to induce cell death is a major problem limiting the clinical application of PI3K inhibitors as cancer monotherapy because tumor cells are able to survive the cytostatic effect and develop drug resistance eventually (33). In contrast, verteporfin-PDT alone effectively induced endothelial cell apoptosis through mitochondria-mediated pathway, as shown in the current and previous studies (25, 34). Importantly, our data demonstrate that combining cytostatic BEZ235 with cytotoxic PDT significantly enhanced therapeutic lethality by enhancing mitochondrial apoptosis pathway, which provides a strong rationale for the combination therapy.

Probing for antiapoptotic Bcl-2 family proteins revealed an interesting mechanistic cooperation between PDT and BEZ235 in enhancing endothelial cell apoptosis. Mcl-1 level was significantly upregulated after PDT, a possible consequence of increased protein translation due to PDT-induced p-S6 activation. By strongly suppressing p-S6 and consequent Mcl-1 protein translation, BEZ235 effectively inhibited PDT-induced Mcl-1 upregulation. Because increased Mcl-1 may protect cells from mitochondrial apoptosis, inhibition of PDT-induced Mcl-1 upregulation by BEZ235 likely led to increased mitochondrial apoptosis after the combination therapy. This proposed mechanism, yet to be corroborated, is supported by some published results. For instance, inhibition of Mcl-1 by BEZ235 has been shown to sensitize tumor cells to undergo apoptosis induced by chemotherapy (32). Increase of Mcl-1 level was also reported after PDT with photofrin in tumor cells, and inhibition of Mcl-1 elevation by celecoxib was shown to enhance apoptosis (35). Antiapoptotic protein Bcl-XL was found reduced after verteporfin-PDT. Bcl-XL is known as a target of PDT with phthalocyanine Pc 4 for the initiation of apoptosis (36). Bcl-XL level reduction after verteporfin-PDT suggests its involvement in verteporfin-PDT-mediated apoptosis as well. However, Bcl-XL is unlikely to play an important role in the increased apoptosis after combination treatments because its level was not modulated by BEZ235.

In addition to promoting cell death, our results demonstrate that BEZ235 combined with verteporfin-PDT resulted in more sustained inhibition of endothelial cell proliferation and durable treatment response, which provides another advantage to this combinatorial approach. Although verteporfin-PDT alone effectively inhibited endothelial cell viability and tumor growth, its therapeutic effects were nevertheless transient. Thus, increasing treatment response durability is an important issue that needs to be addressed for vascular-targeted PDT. Fine adjustments of PDT dosimetry parameters such as light and photosensitizer doses are known to increase PDT response and treatment durability by optimizing photochemical reactions in the tumor microenvironment (37–39). Rational combinations of PDT and other treatment modalities have been shown to enhance treatment outcomes (40). Our results here demonstrate that the activation of PI3K pathway signaling after PDT represents a therapeutic target for the enhancement of vascular-targeted PDT. We found that the PI3K pathway signaling was activated after sublethal and therapeutic doses of PDT with 200 and 400 ng/mL verteporfin, respectively. The activation of PI3K signaling led to subsequent endothelial cell proliferation, which occurred sooner after the sublethal dose of PDT. Blocking the PI3K signaling activation with BEZ235 transformed a transient growth inhibition induced by PDT alone to a sustained suppression of endothelial cell proliferation. The durable therapeutic response as a result of cooperative inhibition of PI3K signaling provides another rationale for the combination of PDT and PI3K pathway inhibitors.

A pilot study in the PC-3 prostate tumor model confirmed that PDT combined with BEZ235 led to a more durable tumor response than individual treatment, demonstrating the effectiveness of this therapeutic enhancement approach in vivo. We previously showed that endothelial cells were more sensitive to PDT in combination with BEZ235 than PC-3 prostate tumor cells (15, 16). Thus, enhanced tumor response after the combination therapy is presumably attributed to the enhanced vascular response. In addition to targeting tumor vasculature, PDT combined with BEZ235 also enables a direct inhibition of PC-3 tumor cells. The PC-3 cells do not express PTEN, a negative regulator of the PI3K pathway that terminates the pathway signaling by converting PIP3 to PIP2 (15). Because of the loss of PTEN function, PC-3 cells exhibit activated PI3K signaling and are responsive to PI3K pathway inhibitors such as BEZ235 (16). Because mutations in the PI3K pathway including the loss of PTEN tumor suppressor and activating mutations in PI3Ks often occur in human cancers particularly prostate and breast cancers (33), combining vascular-targeted PDT with a PI3K inhibitor ensure the maximum eradication of tumor cells by targeting both tumor vasculature and tumor cells.

It is interesting to note that BEZ235 increased verteporfin uptake in SVEC cells, which provides additional benefit for the combination therapy. Although the exact mechanism remains to be determined, increased verteporfin uptake induced by BEZ235 is likely due to the interaction with ATP-binding cassette subfamily G2 (ABCG2) transporters. As a type of efflux pump involved in transporting a variety of exogenous substances and endogenous metabolites out of cells, the ABCG2 transporter is known for pumping out verteporfin (41). PDT with verteporfin has been shown to cause ABCG2 transporter downregulation (42), further suggesting the association between ABCG2 transporters and verteporfin. BEZ235 was recently identified as an ABCG2 substrate as well (43). Thus, competing for the same efflux transporter possibly led to increased verteporfin uptake when it was combined with BEZ235. Furthermore, some small molecule kinase inhibitors have been shown to inhibit ABCG2 activity by directly inhibiting transporter ATP-binding sites, thereby reducing the efflux of ABCG2 substrates (44). A kinase inhibitor erlotinib was recently shown to increase verteporfin intracellular uptake (45). It is not yet known whether BEZ235 directly inhibits ABCG2 activity. Interestingly, BYL719 and BKM120 had no effect on the uptake of verteporfin. Because PDT in combination with BYL719 or BKM120 also showed synergistic effects, it suggests that increased verteporfin uptake is just an additional benefit, but not indispensable, for the combination of verteporfin-PDT and PI3K pathway inhibitors.

In summary, we have shown in this study synergistic interactions between verteporfin-PDT and three newly developed PI3K therapeutics that are in clinical trials. Particularly, PDT combined with a dual PI3K/mTOR inhibitor BEZ235 exhibited the strongest synergism in reducing endothelial cell viability. The combination therapy overcomes the limitation of being cytostatic for BEZ235 alone and having short-lived effectiveness for PDT alone through a cooperative inhibition of PI3K signaling and apoptotic pathways. As a result, PDT in combination with BEZ235 led to more lethal and long-lasting therapeutic effects. These results demonstrate the importance of PI3K signaling pathway in determining PDT treatment outcomes, and support the use of clinically relevant PI3K pathway inhibitors for enhancing vascular-targeted PDT.

No potential conflicts of interest were disclosed.

Conception and design: D. Kraus, B. Chen

Development of methodology: D. Kraus, B. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Kraus, P. Palasuberniam, B. Chen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Kraus, B. Chen

Writing, review, and/or revision of the manuscript: D. Kraus, P. Palasuberniam, B. Chen

Study supervision: B. Chen

We would like to thank Novartis for providing PI3K pathway inhibitors, QLT Inc. for providing verteporfin, and Dr. Babasola Fateye, Dr. Xue Yang, and Matthew Mansi for assistance and helpful discussions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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