The Comparison of Anticancer Efficiency of Methylene Blue- and Aluminum Phthalocyanine-Mediated Sonophotodynamic Therapy on Prostate Cancer Cells In Vitro

Abstract

Prostate cancer ranks highest in male diagnoses and second in cancer-related deaths. Conventional treatments necessitate exploration of new modalities due to their side effects. Sonophotodynamic therapy (SPDT) represents a potential anticancer approach that integrates both sonodynamic and photodynamic therapies to improve the efficacy of cancer treatments. This study aims to evaluate and compare the mechanisms and anticancer efficacy of photodynamic therapy, sonodynamic therapy, and SPDT using methylene blue (MB) and aluminum phthalocyanine (AlPc) in the androgen-sensitive and androgen-insensitive prostate cancer cell lines. Cells were cultivated using different concentrations of MB and AlPc, followed by the exposure to ultrasound and/or light irradiation. Cell metabolic activity was assessed using the MTT assay, which evaluates mitochondrial enzyme function as an indicator of viability rather than clonogenic survival. Additionally, apoptosis was evaluated using Hoechst staining and Western blot analysis of apoptotic proteins, while reactive oxygen species (ROS) and antioxidant levels were determined through biochemical assays. Results showed significant proliferation inhibition, with SPDT exhibiting the highest efficacy. MB demonstrated superior efficiency compared to AlPc. The treatment groups displayed a greater quantity of apoptotic cells, indicating elevated levels of caspase-3, caspase-8, PARP, and Bax proteins, whereas levels of caspase-9 and Bcl-2 were lower compared to the control groups. Additionally, the treatments resulted in increased levels of ROS and malondialdehyde, while antioxidant activities were diminished. In summary, SPDT mediated by MB and AlPc presents promising potential for treating prostate cancer and may significantly contribute to apoptotic mechanisms.

Keywords

photodynamic therapy sonodynamic therapy sonophotodynamic therapy methylene blue aluminum phthalocyanine prostate cancer cells

Introduction

Prostate cancer is characterized by uncontrolled cell proliferation and remains one of the most frequently diagnosed malignancies among men worldwide, representing a major cause of cancer-related morbidity and mortality.¹ Current treatment strategies-including radiotherapy, surgery, radiotherapy, chemotherapy, and hormone-based therapies—can be effective, particularly at early stages of the disease.² However, these approaches are often associated with substantial adverse effects, limited efficacy in advanced or resistant tumors, and a significant reduction in patients’ quality of life.^3,4 These limitations underscore the urgent need for alternative therapeutic modalities that are both effective and less invasive.

In recent years, photodynamic therapy (PDT) and sonodynamic therapy (SDT) have emerged as promising noninvasive anticancer approaches. PDT relies on light-activated sensitizers to generate reactive oxygen species (ROS), particularly singlet oxygen, leading to selective tumor cell death.^5,6 Despite its clinical success, PDT is limited by shallow light penetration, restricting its effectiveness primarily to superficial tumors. SDT addresses this limitation by using low-intensity ultrasound (US) to activate sonosensitizers, allowing deeper tissue penetration and broader therapeutic applicability.⁷ The combination of PDT and SDT—referred to as sonophotodynamic therapy (SPDT)—has gained increasing attention due to its synergistic potential, enabling enhanced anticancer efficacy while reducing the required sensitizer dose.⁸

The therapeutic efficacy of SPDT critically depends on the choice of sensitizer, as different compounds exhibit distinct physicochemical properties, cellular uptake behaviors, subcellular localizations, and ROS-generating capacities. Among the available sensitizers, methylene blue (MB) and aluminum phthalocyanine (AlPc) represent two fundamentally different classes with unique advantages. MB is a cationic, hydrophilic dye with well-documented cellular permeability, mitochondrial interaction, and redox activity and has been widely investigated in photodynamic and sonodynamic applications.^9–11 In contrast, AlPc is a second-generation, hydrophobic phthalocyanine derivative with strong absorption in the red region, high photostability, and enhanced tissue penetration, making it particularly attractive for clinical PDT.¹²

Although both MB and AlPc have demonstrated anticancer activity in PDT- and SDT-based approaches,^13,14 direct comparative studies evaluating their relative efficacy and mechanistic effects within a unified SPDT framework remain scarce, particularly in prostate cancer models. Moreover, prostate cancer exhibits marked biological heterogeneity, with hormone-dependent and hormone-independent phenotypes responding differently to therapeutic interventions. Therefore, a systematic comparison of these sensitizers across different prostate cancer cell lines is essential for optimizing SPDT strategies and guiding future translational applications.

In this study, we aimed to compare the anticancer efficacy and underlying mechanisms of MB- and AlPc-mediated PDT, SDT, and SPDT in androgen-sensitive (LNCaP) and androgen-insensitive (PC3) prostate cancer cell lines. By integrating metabolic viability assays, apoptosis analysis, oxidative stress evaluation, and molecular profiling of apoptotic pathways, this work provides a comprehensive assessment of how sensitizer selection influences SPDT outcomes. To our knowledge, this is the first study to directly compare MB and aluminum phthalocyanine within the context of SPDT in prostate cancer cells.

Material and Methods

Cell culture

Human prostate cancer cell lines PC3 and LNCaP were obtained from the American Type Culture Collection (ATCC, USA). Although mycoplasma testing was not routinely performed, all cultures were maintained aseptically in RPMI-1640 medium with 10% Fetal Bovine Serum, 1% penicillin-streptomycin, and 1% l-glutamine at 37°C in 5% CO₂. Cells were harvested at 80–90% confluence for experiments.

Sonophotodynamic treatment

Before treatment, cells were cultured overnight and randomly assigned to experimental groups: control, MB, AlPc, US, light, US + light, MB + SDT, AlPc + SDT, MB + PDT, AlPc + PDT, MB + SPDT, and AlPc + SPDT. For metabolic activity assays, cells were seeded in 48-well plates (1 × 10⁵ cells/mL) and incubated for 24 h, followed by sensitizer exposure at 0–50 µM for 4 h to allow uptake.

US treatment was applied using a Sonidel SP100 (1.0 MHz, 0.5 W/cm², 60 s) and light irradiation with an ABET solar simulator (0.5 mW/cm², 60 s). In SPDT groups, light exposure began 30 min after US. After all treatments, cells were transferred to fresh medium and incubated for another 24 h before analysis.

Cytotoxicity

The cytotoxic effects of MB and AlPc under PDT, SDT, and SPDT conditions were assessed using the MTT assay. After treatment, 40 µL of MTT solution was added to each well and incubated for 4 h at 37°C, followed by dissolution with 100 µL of Dimethyl Sulfoxide. Optical density (OD) was measured at 560–620 nm using a microplate reader (Thermo Multiskan Spectrum, USA).

Cell metabolic activity was calculated as [(OD_sample − OD_blank)/(OD_control − OD_blank)] × 100. All experiments were performed independently in triplicate, and results are expressed as mean ± SD. Although the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay reflects mitochondrial enzyme activity rather than clonogenic survival, it reliably indicates treatment-induced metabolic inhibition.

HOPI staining of apoptosis and biochemical analyses

After cytotoxicity assessment, treatment-induced cell death mechanisms were examined using Hoechst 33342/propidium iodide (HOPI) staining, biochemical assays, and Western blot analysis. After treatment, cells were stained with 5 µL HOPI for 1 h in the dark, then 30 µL of the suspension was placed on slides and analyzed under a fluorescence microscope (Olympus BX51, Japan). Viable, apoptotic, and necrotic cells were quantified with ImageJ (NIH, USA).

Apoptosis was further confirmed by Western blot analysis of caspase-3, caspase-8, PARP, Bax, and Bcl-2. Oxidative stress markers—malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD)—were measured using commercial ELISA kits according to the manufacturer’s instructions. Results represent the mean ± Standard Deviation (SD) of three independent experiments.

Western blot analysis

After treatment, cells were trypsinized, washed with Phosphate-Buffered Saline (PBS), and lysed in buffer containing 20 mM Tris-HCl (pH 7.4), 1% Sodium Dodecyl Sulfate (SDS), and protease inhibitors (Sigma-Aldrich, Germany). Protein concentrations were determined by Bicinchoninic Acid (BCA) assay, and 20 µg of protein per lane was separated on 12% Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to Polyvinylidene Difluoride (PVDF) membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies against Bcl-2, Bax, Poly (ADP-ribose) Polymerase (PARP), cleaved caspase-3, caspase-8, and caspase-9 (Cell Signaling Technology, USA), followed by (Horseradish Peroxidase) HRP-conjugated secondary antibodies.

Signals were detected using Enhanced Chemiluminescence (ECL) and quantified with ImageJ (NIH, USA). Each protein was analyzed on a separate gel and normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Data were expressed as mean ± SD from three independent experiments within the linear detection range.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software, USA). The data represent the mean ± SD from three independent experiments conducted in triplicate. Group differences were analyzed by one-way ANOVA followed by the Dunnett’s post hoc test, with a p value of ≤0.05 considered significant.

Results

Effects of SPDT on cytotoxicity

No significant differences in metabolic activity were observed in PC3 or LNCaP cells treated with MB, AlPc, light, or US alone (Fig. 1). In contrast, combination treatments—particularly MB-PDT and MB-SPDT—markedly reduced metabolic activity compared to controls (Fig. 2).

FIG. 1.

The cytotoxic efficiency of a) MB against aganist PC3 cells b) MB onto LNCaP cells c) AlPc onto PC3 cells d) AlPc onto LNCaP cells.

FIG. 2.

The cytotoxic efficiency of SDT, SPT and SPDT treatments with control groups The effects of MB-mediated treatments on cell viabilities of a) PC3 cells and b) LNCaP cells; the effects of AlPc-mediated treatments on cell viabilities of c) PC3 cells and d) LNCaP cells.

In PC3 cells, the viability decreased to 40.3% (MB-SDT), 4.4% (MB-PDT), and 5.7% (MB-SPDT), while in LNCaP cells, the viability decreased to 67.2%, 45.6%, and 7.1%, respectively. AlPc treatments showed similar trends, reducing viability to 62.4% (SDT), 12.5% (PDT), and 8.7% (SPDT) in PC3 and 68.5%, 19.1%, and 12.9% in LNCaP. These findings confirm the synergistic cytotoxicity of SPDT over single-modality treatments in both prostate cancer cell lines.

Activation of apoptosis of SPDT

Apoptosis induction was assessed by HOPI staining and confirmed by Western blot of apoptosis-related proteins. HOPI staining showed a marked increase in apoptotic cells across all treated groups compared to controls (Figs. 3 and 4).

FIG. 3.

Rate of apoptosis in the cells and fluorescence microscope images of HOPI (Hoechst 33342 and propidium iodide) staining at ×10 magnification. a) HOPI images of PC3 cells after MB-mediated treatments with untreated control groups. b) HOPI images of LNCaP cells after MB-mediated treatments with untreated control groups. c) The effects of MB-mediated treatments on the rate of apoptosis in -PC3 cells d) The effects of MB-mediated treatments on the rate of apoptosis in LNCaP cells.

FIG. 4.

Rate of apoptosis in the cells and fluorescence microscope images of HOPI (Hoechst 33342 and propidium iodide) staining at ×10 magnification. a) HOPI images of PC3 cells after AlPc-mediated treatments with untreated control groups. b) HOPI images of LNCaP cells after AlPc-mediated treatments with untreated control groups. c) The effects of AlPc-mediated treatments on the rate of apoptosis in PC3 cells d) The effects of AlPc-mediated treatments on the rate of apoptosis in LNCaP cells.

In PC3 cells, apoptosis reached 44.5% (MB-SDT), 74.8% (MB-PDT), and 84.7% (MB-SPDT), while in LNCaP cells, apoptosis reached 47.2%, 72.6%, and 78.9%, respectively. AlPc-based treatments yielded similar trends, with apoptosis rates of 48.8%, 61.1%, and 82.3% in PC3 cells and 44.1%, 73.5%, and 82.4% in LNCaP cells.

ROS and oxidative stress response

ROS generated during PDT or SDT can trigger apoptosis or necrosis by inducing lipid peroxidation and oxidative damage. In this study, both prostate cancer cell lines showed marked increases in ROS and MDA levels after PDT, SDT, and SPDT. ROS production, measured by an ELISA-based assay, was expressed as fluorescence intensity relative to controls. Concurrently, antioxidant defenses—including SOD, CAT, and reduced GSH—were significantly reduced in all treated groups (Tables 1 and 2), indicating that SPDT-induced oxidative stress plays a major role in treatment-related cytotoxicity.

Table 1.

Biochemical Measurements of Androgen-Insensitive and Androgen-Sensitive Cells After Methylene Blue-Mediated Treatments

	Control	MB	MB + SDT	MB + PDT	MB + SPDT
Part I: PC3 cells
ROS (% level)	2.69 ± 0.33	4.69 ± 0.46	39.65 ± 3.10***	81.40 ± 3.63***	96.53 ± 4.59***
MDA (nmol/mg)	2.04 ± 0.06	2.47 ± 0.16	3.98 ± 0.06**	8.37 ± 0.14***	8.76 ± 0.11***
GSH (nmol/mL)	0.94 ± 0.09	0.83 ± 0.10	0.72 ± 0.20	0.46 ± 0.12*	0.26 ± 0.06***
SOD (% inhibition)	76.90 ± 7.66	52.72 ± 1.24	36.46 ± 1.84*	29.67 ± 3.27**	2.90 ± 1.62***
CAT (pg/mL)	0.16 ± 0.04	0.15 ± 0.04	0.11 ± 0.06***	0.08 ± 0.03***	0.05 ± 0.09***
Part II: LNCaP cells
ROS (% level)	8.13 ± 0.73	16.02 ± 1.80	55.31 ± 6.46⁺⁺⁺	81.64 ± 4.20⁺⁺⁺	96.21 ± 4.16⁺⁺⁺
MDA (nmol/mg)	2.92 ± 0.54	4.48 ± 0.17	5.37 ± 1.14⁺	7.85 ± 1.95⁺⁺	11.74 ± 0.94⁺⁺⁺
GSH (nmol/mL)	0.51 ± 0.08	0.42 ± 0.02	0.35 ± 0.07	0.29 ± 0.03⁺⁺	0.05 ± 0.03⁺⁺
SOD (% inhibition)	78.71 ± 10.41	68.15 ± 2.54	48.65 ± 3.45⁺⁺	31.91 ± 2.84⁺⁺⁺	22.82 ± 7.56⁺⁺⁺
CAT (pg/mL)	0.17 ± 0.03	0.14 ± 0.02⁺⁺	0.13 ± 0.07⁺⁺⁺	0.09 ± 0.09⁺⁺⁺	0.07 ± 0.03⁺⁺⁺

The data was represented as mean ± standard error of mean. Differences in between variances were compared by One-way ANOVA test with Dunnett’s test applied as a post-hoc test. P-values equal or less than 0.05 were considered as statistically significant (*,⁺p < 0.05, **,⁺⁺p < 0.01, ***,⁺⁺⁺p < 0.001) in comparison to untreated control.

CAT, catalase; GSH, glutathione; LNCaP, androgen-sensitive; MDA, malondialdehyde; PC3, androgen-insensitive; PDT, photodynamic therapy; ROS, reactive oxygen species; SDT, sonodynamic therapy; SOD, superoxide dismutase; SPDT, sonophotodynamic therapy.

Table 2.

Biochemical Measurements of Androgen-Insensitive and Androgen-Sensitive Cells After Aluminum Phthalocyanine-Mediated Treatments

	Control	AlPc	AlPc + SDT	AlPc + PDT	AlPc + SPDT
Part I: PC3 cells
ROS (% level)	2.69 ± 0.33	5.46 ± 0.99	25.27 ± 6.63***	63.86 ± 4.70***	77.92 ± 2.30***
MDA (nmol/mg)	2.04 ± 0.06	3.30 ± 0.35	4.91 ± 0.73**	7.58 ± 0.68***	17.83 ± 12.41***
GSH (nmol/mL)	0.94 ± 0.09	0.91 ± 0.04	0.79 ± 0.08	0.57 ± 0.07*	0.31 ± 0.03***
SOD (% inhibition)	76.90 ± 7.66	52.05 ± 4.88	42.28 ± 9.35*	25.65 ± 10.93**	15.43 ± 3.93***
CAT (pg/mL)	0.16 ± 0.04	0.15 ± 0.02	0.13 ± 0.01***	0.08 ± 0.01***	0.06 ± 0.02***
Part II: LNCaP cells
ROS (% level)	8.13 ± 0.73	8.87 ± 2.83	39.30 ± 8.15⁺⁺⁺	74.65 ± 5.60⁺⁺⁺	94.21 ± 4.52⁺⁺⁺
MDA (nmol/mg)	2.92 ± 0.54	4.97 ± 1.25	6.73 ± 0.20⁺	8.54 ± 1.74⁺⁺	11.03 ± 0.76⁺⁺⁺
GSH (nmol/mL)	0.51 ± 0.08	0.36 ± 0.22	0.31 ± 0.16	0.22 ± 0.15⁺⁺	0.07 ± 0.03⁺⁺
SOD (% inhibition)	78.71 ± 10.41	69.62 ± 13.26	48.11 ± 9.22⁺⁺	31.22 ± 12.29⁺⁺⁺	24.51 ± 6.71⁺⁺⁺
CAT (pg/mL)	0.17 ± 0.03	0.15 ± 0.04⁺⁺	0.14 ± 0.06⁺⁺⁺	0.12 ± 0.02⁺⁺⁺	0.10 ± 0.01⁺⁺⁺

AlPc, aluminum phthalocyanine.

Western blot assessment of apoptosis-related proteins

Apoptosis-related protein expression after MB and AlPc treatments was analyzed by Western blot (Figs. 5 and 6). Both sensitizers increased cleaved caspase-8, caspase-3, PARP, and Bax levels while reducing Bcl-2 and procaspase-9 in PC3 and LNCaP cells. Band intensities were normalized to GAPDH within the linear detection range.

FIG. 5.

Western blot analysis of the expression levels of apoptosis-related proteins (caspase-8, caspase-3, caspase-9, PARP, Bcl-2, Bax) after MB-mediated treatments on prostate cancer cell lines. Western blot images of a) PC3 cells, b) LNCaP cells relative protein expression levels in c) PC3. Fold change of apoptotic protein expressions normalized to GAPDH, which served as a loading control. Comparison was performed by the One-way ANOVA test with Dunnett’s multiple comparison test applied as a post-hoc test. P-values equal or less than 0.05 were considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001) versus untreated control. d) LNCaP cells. Fold change of apoptotic protein expressions normalized to GAPDH, which served as a loading control. Comparison was performed by the One-way ANOVA test with Dunnett’s multiple comparison test applied as a posthoc test. P-values equal or less than 0.05 were considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001) versus untreated control.

FIG. 5.

Continued.

FIG. 6.

Western blot analysis of the expression levels of apoptosis-related proteins (caspase-8, caspase-3, caspase-9, PARP, Bcl-2, Bax) after AlPc-mediated treatments on prostate cancer cell lines. Western blot images of a) PC3 cells, b) LNCaP cells. c) relative protein expression levels in PC3 cells. Fold change of apoptotic protein expressions normalized to GAPDH, which served as a loading control. d) relative protein expression levels in LNCaP cells. Fold change of apoptotic protein expressions normalized to GAPDH, which served as a loading control.

FIG. 6.

Continued.

These results confirm activation of apoptotic pathways by MB- and AlPc-mediated therapies, with SPDT eliciting the strongest response. Notably, MB-SPDT induced the highest caspase activation and Bax/Bcl-2 modulation, demonstrating a synergistic enhancement of apoptosis compared to single-modality treatments.

Discussion

This study aimed to compare the anticancer efficacy of MB and AlPc under PDT, SDT, and combined SPDT therapies in prostate cancer cell lines. PDT and SDT have emerged as promising alternatives to conventional treatments, while SPDT synergistically integrates both modalities to overcome their individual limitations and enhance efficacy.

Although MB- and AlPc-mediated PDT and SDT have been investigated in various cancers, no direct comparison has previously been reported for prostate cancer. This study therefore provides the first comparative evaluation of MB- and AlPc-mediated SPDT in prostate cancer cells. Results showed that both sensitizers significantly reduced metabolic activity under SDT, PDT, and SPDT, with AlPc generally exhibiting higher residual viability than MB.^15,16 Moreover, MB-mediated SPDT caused an almost complete loss of metabolic activity in both PC3 and LNCaP cells, highlighting its superior cytotoxic potential. These findings corroborate previous reports of MB-SDT-induced viability loss in sarcoma cells and further demonstrate the enhanced efficacy of SPDT.¹⁷ When US was applied at 0.24 W/cm² for 30 s, the reduction in cell viability was more pronounced in MB- or AlPc-mediated SPDT groups than in SDT alone. Gomes et al. reported that MB-PDT decreased antioxidant levels, increased lipid peroxidation, and affected PC3 cell viability and migration, with autophagy acting as a protective response to oxidative stress and potentially triggering alternative death pathways such as necroptosis.¹³ Fan et al. demonstrated that an MB-conjugated A9 aptamer nanoplatform selectively targets PSMA-positive LNCaP cells without cytotoxicity in the absence of light and can enable combined photothermal and PDT under 785 nm NIR activation.¹⁸ Rodrigues et al. showed that nanoemulsified AlPc rapidly accumulates in mitochondria, lysosomes, and the endoplasmic reticulum of tumor cells, effectively eradicating mouse mammary adenocarcinomas and preventing metastasis.¹⁹ Mello et al. developed a third-generation photosensitizer by combining aluminum phthalocyanine with Amazon oil-based solid lipid nanoparticles, which exhibited high cytotoxicity in melanoma cells through caspase-3 activation.²⁰ A study on Caco-2 cells showed that a PEGylated Cu–Au nanoparticle—conjugated aluminum phthalocyanine complex induced extensive apoptosis, primarily through ROS generation.²¹ Recent studies show that drug delivery systems can enhance PDT efficacy in prostate cancer. For example, a monoamine oxidase-A inhibitor—zinc phthalocyanine conjugate reduced cell proliferation and inhibited migration and metastasis under PDT.²² In our previous studies, we found that the SPDT effect using TiO2 nanoparticles caused a significant decrease in cell viability compared to PDT and SDT groups.²³ Consistent with previous findings, MB-mediated SPDT caused an almost complete loss of cell viability in prostate cancer cells. HOPI staining confirmed a marked increase in apoptotic cells in MB- and AlPc-mediated SDT, PDT, and SPDT groups, whereas control and single-modality treatments showed minimal apoptosis. These observations indicate that PDT and SPDT primarily induce apoptosis-driven cell death, supported by the reduced cell density seen in HOPI images and consistent with MTT results.

Western blot analysis further revealed the activation of apoptotic signaling, with caspase-3 cleavage representing a key event initiating programmed cell death.²⁴ Apoptosis is a preferred cell death mechanism due to its minimal immune side effects. Studies have shown that SDT can induce apoptosis through various mechanisms, including the mitochondrial pathway.²⁵ Similarly, our results showed increased caspase-8 levels after treatment compared to controls. Consistent with previous studies, both PDT and SDT modulate apoptosis-related proteins, while ROS and singlet oxygen generation further trigger apoptosis by disrupting mitochondrial permeability.²⁵ Our findings indicate that the effects of MB and AlPc are comparable to those of other sensitizers, with a primary function of increasing ROS production after treatment. Further, our findings revealed a notable elevation in MDA levels, accompanied by a decline in SOD, CAT, and GSH levels. Similarly, in a study using MB in the treatment of ovarian cancer and human lung adenocarcinoma cells in the literature, it was shown that SDT significantly increased the intracellular ROS level.²⁶ SDT and PDT induce oxidative stress by elevating intracellular ROS and reducing antioxidant enzyme activity, thereby activating apoptotic pathways. This study is the first to compare MB- and AlPc-mediated PDT, SDT, and SPDT in prostate cancer cells, demonstrating significant reductions in viability and enhanced apoptosis. MB proved more potent in PDT, while SPDT showed the greatest overall efficacy. Elevated ROS and MDA levels, coupled with reduced SOD, CAT, and GSH, confirm redox-mediated cytotoxicity. Increased cleaved caspase-3, caspase-8, PARP, and Bax, along with decreased Bcl-2 and procaspase-9, indicate activation of both intrinsic and extrinsic apoptotic pathways, with MB-SPDT exerting the strongest effect.

Although promising, the 4-h sensitizer incubation may not reflect in vivo pharmacokinetics, and MTT data cannot replace clonogenic assays. Future studies should include animal models and broader prostate cell line panels to validate these findings. Overall, MB- and AlPc-mediated SPDT significantly enhance cytotoxic and apoptotic effects compared with PDT or SDT alone, supporting its potential for prostate cancer therapy. However, a limitation of this study is that mycoplasma testing was not routinely performed on the cell lines prior to experiments. Although strict aseptic culture conditions were maintained to prevent contamination, future studies should ensure routine screening to fully rule out mycoplasma interference.

Conclusion

This study compared the effects of MB- and AlPc-mediated SDT, PDT, and combined SPDT therapies in prostate cancer cell lines—hormone-independent PC3 and hormone-dependent LNCaP. Both sensitizers significantly reduced cell metabolism and induced apoptosis, with MB-SPDT showing the strongest response. Western blot confirmed caspase-dependent apoptosis, marked by increased cleaved caspase-3, caspase-8, PARP, and Bax, and decreased Bcl-2.

Overall, SPDT mediated by MB and AlPc significantly increased oxidative stress and apoptosis, indicating its potential as an alternative or adjunctive therapeutic strategy for prostate cancer, pending further validation through in vivo and pharmacokinetic studies.

Author Disclosure Statement

Mehran Aksel: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing—original draft. Ozlem Bozkurt Girit: Methodology, Investigation, Data curation, Writing—review & editing. Ali Ozmen: Formal analysis, Validation, Writing—review & editing. Mehmet Dincer Bilgin: Conceptualization, Supervision, Project administration, Writing—review & editing. All authors read and approved the final version of the manuscript.

No competing financial interests exist.

Footnotes

Funding Information

This work was supported by the Adnan Menderes University Scientific Research Fund (TPF-15068) and TÜBİTAK (114S491).

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