Non-Cytotoxic Photodynamic Therapy with Talaporfin Sodium Reduces the Expression of CXCR4 and Enhances Chemotherapeutic Efficacy in Undifferentiated Gastric Cancer Cell Line HGC27

Abstract

Gastric cancer (GC), particularly the undifferentiated type, is frequently associated with peritoneal metastasis, which significantly worsens prognosis due to its resistance to conventional treatments. Photodynamic therapy (PDT) is localized treatment using a photosensitizer (PS) activated by light of a specific wavelength to generate cytotoxic reactive oxygen species that induce cell death. Severe adverse events were reported from clinical trials investigating PDT for peritoneal dissemination conducted until the early 2000s, leaving its safety and clinical effectiveness unestablished. The present study explored whether “non-cytotoxic” PDT using talaporfin sodium (TS) could enhance efficacy of chemotherapeutic agents in undifferentiated GC cell line HGC27. Cell viability was evaluated with MTT assay following TS-PDT, and the synergistic effect between non-cytotoxic TS-PDT and anticancer drug SN-38 was assessed. Changes in expression of drug resistance markers were analyzed through qRT-PCR, Western blotting, and immunocytochemistry. We found that non-cytotoxic TS-PDT enhanced the efficacy of chemotherapy in the undifferentiated GC cell line and reduced the expression of C-X-C chemokine receptor type 4, a key marker associated with GC stem-like properties. These findings highlight the potential of non-cytotoxic TS-PDT as a synergistic treatment approach. We conclude that non-cytotoxic TS-PDT could enhance drug sensitivity and offers a promising therapeutic strategy for GC.

I.  Introduction

Gastric cancer (GC) is the fifth most prevalent cancer worldwide and the fourth leading cause of cancer-related mortality [1]. In Japan, GC ranks third in both incidence and cancer-related deaths among all malignancies [2]. A major factor contributing to the poor survival rate is the frequent late-stage diagnosis, often accompanied by systemic metastases. Among these, peritoneal dissemination (PD), which is particularly common in undifferentiated GC, significantly worsens the prognosis [3]. This type of metastasis occurs when cancer cells disseminate throughout the abdominal cavity, forming numerous metastatic nodules. At this advanced stage, surgical resection is no longer feasible, leaving systemic chemotherapy as the primary treatment option. Unfortunately, the therapeutic outcomes for PD are generally inferior compared to metastases in organs such as the liver or lungs [4].

The treatment resistance observed in PD is attributed to both environmental and tumor-specific factors. Environmental barriers, such as the peritoneal-plasma barrier, impede the effective delivery of therapeutic agents to the peritoneal surface [5, 6]. Additionally, diffuse-type GC, which is strongly associated with PD, exhibits cancer stem cell-like properties. This subtype is characterized by extensive stromal fibrosis, poor vascularization, and a low rate of cancer cell proliferation, all of which contribute to significant therapeutic challenges [7]. Several cancer stem cell markers have been reported in GC, among which high expression of C-X-C chemokine receptor type 4 (CXCR4) is strongly associated with invasiveness and poor prognosis, particularly in undifferentiated GC [8–10]. CXCR4 interacts with its ligand, CXCL12, to enhance the migratory and invasive capabilities of GC cells [11]. This CXCR4-CXCL12 axis is believed to play a critical role in determining metastatic sites, including the peritoneum, lymph nodes, and bone marrow. Given its multifaceted role, CXCR4 represents a promising therapeutic target for GC, particularly in addressing PD [12].

Photodynamic therapy (PDT), a laser-based treatment modality, has been associated with significant tumor regression since the 1980s [13]. This approach involves the intravenous administration of a photosensitizer (PS) drug, which selectively accumulates in the mitochondria or lysosomes of tumor cells. When exposed to laser light at a specific wavelength, the PS triggers a cytotoxic reaction in cancer cells or the neovascular structures surrounding tumors [14]. In Japan, talaporfin sodium (TS) (Laserphyrin^®, Meiji Seika Pharma, Tokyo, Japan) is approved for the treatment of superficial lung cancer [13] and as a salvage therapy for esophageal cancer following chemoradiotherapy or radiotherapy [15]. Clinical trials investigating PDT for PD were conducted until the 2000s. These studies often combined debulking surgery with photosensitizer administration and subsequent laser irradiation [16, 17]. However, severe adverse events, including gastrointestinal perforations and fatalities, were reported, leaving its safety and clinical effectiveness unestablished.

The anti-tumor effects of PDT are mediated through multiple mechanisms, including the induction of apoptosis via damage to intracellular organelles [18], disruption of tumor vasculature [18], and activation of immune responses by tumor-sensitized lymphocytes [19]. Despite these promising outcomes, many aspects of the mechanisms of PDT remain poorly understood, and diverse findings have been published. In recent years, increasing attention has been given to the effects of PDT on cancer stem cells [20], with reports of its efficacy in various cancer types, including breast cancer [21], colorectal cancer [22], and pancreatic cancer [23]. One area of interest is the “photodynamic priming (PDP) effect,” which is a new approach that utilizes photodynamic therapy not to directly eliminate cancer cells, but to enhance the drug sensitivity of cancer cells [24, 25]. By suppressing the expression of genes related to cancer stemness or drug resistance, it reduces cancer treatment resistance, acting as a “pre-treatment” for cancer cells to improve the efficacy of subsequent therapies, such as chemotherapy. If “non-cytotoxic” PDT can enhance the chemosensitivity of PD in GC, combining intraperitoneal PDT with systemic chemotherapy could provide new hope for terminal patients facing this challenging condition.

In the present study, we investigated the anti-cancer effects of PDT and its synergistic interactions with an anti-cancer drug in HGC27 and MKN74 cell lines, representing undifferentiated and moderately differentiated GC, respectively. Using the MTT assay, we evaluated cell viability following non-cytotoxic TS-PDT. We further analyzed changes in drug resistance marker expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR), supported by Western blotting (WB) and immunocytochemistry (ICC). Our findings revealed that non-cytotoxic TS-PDT effectively reduced the expression of CXCR4, a marker associated with GC-related PD and stem-like cells [8, 9]. Additionally, this approach significantly enhanced the efficacy of an anti-cancer drug in HGC27 cells.

II.  Materials and Methods

 Cell line and culture conditions

HGC27, a human undifferentiated GC cell line, and MKN74, a human moderately differentiated GC cell line, were obtained from the Riken Cell Bank (Ibaraki, Japan). HGC27 and MKN74 were maintained in Dulbecco’s Modified Eagle’s Medium (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) and Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), respectively, in a humidified atmosphere with 5% CO₂ at 37°C. The media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich) and penicillin-streptomycin-amphotericin B (Fujifilm Wako).

 PDT experiment

The cells were cultured in the medium for 48 hr and then incubated in culture medium supplemented with TS (Meiji Seika Pharma) (Fig. 1A) for 3 hr. The cells were washed once with phosphate-buffered saline (PBS), and the medium was replaced with PBS. Subsequently, the cells were irradiated with light at 660 nm wavelength from the bottom of the well plates at 37°C for 30 min. The wavelength of 660 nm in the light source device is set as a value close to the absorption peak of talaporfin sodium at 664 nm. A light-emitting diode (LED) light source was fabricated using 100 plug-in LED arrays on a LED substrate (SPL-100-LC, Revox, Kanagawa, Japan). A light-emitting diode (LED) light source was fabricated using 100 plug-in LED arrays on a LED substrate (SPL-100-LC, Revox, Kanagawa, Japan). Considering the balance between heat generation from the LED board and light intensity, a irradiation device was designed and manufactured, resulting in a measured light intensity of 4.09 mW/cm² on the well plate surface. A heat-absorbing filter filled with water (100 mm in diameter and 20 mm in thickness) was placed between the well plate and the LED light source to minimize heat emitted from the LED (Fig. 1B) [26].

Fig. 1.

Photodynamic therapy with talaporfin sodium (TS) and diffuse light emitting diodes (LED). (A) Molecular structure of TS. (B) An LED light source was fabricated using 100 plug-in LED arrays on a LED substrate. A heat-absorbing filter filled with water was placed between the well plate and the LED light source to minimize heat emitted from the LED.

 Cytotoxicity of TS-PDT

Cell viability was examined by 3-(4,5-dimethyl1-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HGC27 or MKN74 cells were respectively seeded in a 96-well plate at a density of 5 × 10³ or 7.5 × 10³ cells/well and incubated at 37°C for 48 hr. After PDT using TS, the cells were incubated at 37°C for 24 hr. Then, after adding MTT solution (5 mg/mL) (Nacalai Tesque, Kyoto, Japan), the cells were further incubated at 37°C for 2 hr. After removal of the MTT reagent, formazan crystals were dissolved in DMSO. The resulting intracellular purple formazan was quantified with a spectrophotometer at absorbance of 562 nm using an Immuno Mini NJ-2300 microplate reader (Nalge Nunc Int. Co. Ltd., Tokyo, Japan). Survival was expressed as the percentage of live cells relative to untreated control cells. Data are expressed as the mean ± SE of three independent experiments.

 Synergistic cytotoxicity of TS-PDT and SN-38

To evaluate whether the combined use of TS-PDT and 7-ethyl-10-hydroxycamptothecin (SN-38; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) contributes to synergistic cytotoxicity, the viability of HGC27 or MKN74 cells treated by different concentrations of TS and SN-38 was measured. SN-38 is an active metabolite of irinotecan and is metabolized by carboxylesterase in vivo to exert its anticancer effects. In in vitro experiments, irinotecan itself exhibits weak anticancer activity due to the lack of metabolic conversion, making SN-38 the preferred choice for use. We selected this anticancer agent with the expectation of enhancing the effect of non-cytotoxic TS-PDP in combination with irinotecan, which is used as a standard treatment in the third-line and later for gastric cancer. HGC27 or MKN74 cells were respectively seeded in a 96-well plate at a density of 5 × 10³ or 7.5 × 10³ cells/well and incubated at 37°C for 48 hr. After PDT using TS at concentrations of 5 or 10 μM, the cells were incubated at 37°C for 3 hr. Then, the medium was replaced by freshly prepared medium containing different concentrations of SN-38 and incubated for 24 hr. Finally, a MTT assay was conducted as mentioned earlier. Cell viability was calculated from the triplicate samples of each group (n = 3) relative to that of the control group, and the half-maximal (50%) inhibitory concentration value (IC₅₀) was deduced. The combination index (ComI) was calculated based on the median-effect equation developed by Chou and Talalay [27], where

  

$$\mathrm{ComI}=\raisebox{1ex}{$\mathrm{A}$}\!\left/ \!\raisebox{-1ex}{${\mathrm{A}}_{50}$}\right.+\raisebox{1ex}{$\mathrm{B}$}\!\left/ \!\raisebox{-1ex}{${\mathrm{B}}_{50}$}\right.$$

In the formula, A₅₀ or B₅₀ is the dose of TS or SN-38 alone with inhibition at 50%. In the numerators, either A or B is the portion of TS or SN-38 in combination with the same at inhibition of 50%. According to the obtained ComI, different treatment interactions can be obtained. Specifically, ComI = 1 indicates an addictive effect in the absence of synergism or antagonism, ComI < 1 indicates synergism, and ComI > 1 indicates antagonism.

 qRT-PCR analysis

qRT-PCR was performed as reported previously [28]. Total RNA was extracted from cell lines using Reliaprep RNA Miniprep Systems (Promega, Madison, WI, USA). RNA was reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Transcript expression levels were analyzed by an ABI StepOne plus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using Fast SYBR Green (Applied Biosystems). β-actin was used calculated using the 2-ΔΔct methods. The primer pairs are listed in Table 1.

Table 1. 

Real-time PCR primer sequences

Gene symbol	Primer sequence
CXCR4	F: 5′-ACTACACCGAGGAAATGGGCT-3′
	R: 5′-CCCACAATGCCAGTTAAGAAGA-3′
CD44	F: 5′-ACCCTCCCCTCATTCACCAT-3′
	R: 5′-GTTGTACTACTAGGAGTTGCCTGGATT-3′
ABCG2	F: 5′-GCAGATGCCTTCTTCGTTATG-3′
	R: 5′-TCTTCGCCAGTACATGTTGC-3′
ABCC1	F: 5′-CATTCAGCTCGTCTTGTCCTG-3′
	R: 5′-GGATTAGGGTCGTGGATGGTT-3′
β actin	F: 5′-TCCTCCCTGGAGAAGAGCTAC-3′
	R: 5′-TCCTGCTTGCTGATCCACAT-3′

 Immunocytochemistry analysis

ICC was performed as reported previously [29, 30]. HGC27 or MKN74 cells were seeded on coverslips in 12-well plates at a density of 1 × 10⁴ or 2 × 10⁴ cells/well and incubated at 37°C for 24 hr. After PDT using 10 μM TS for 1, 3, and 24 hr, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.2% Triton X-100 in PBS at room temperature (RT) for 10 min. Cellular endogenous peroxidase activity was blocked with 0.3% H₂O₂ (Fujifilm Wako) in methanol for 30 min, and nonspecific binding sites were blocked by incubation with 500 μg/mL normal goat IgG (I9140, Sigma-Aldrich) in 1% bovine serum albumin (Sigma-Aldrich) in PBS for 30 min. Cells were incubated with CXCR4 (D4Z7W, 1:100, Cell Signaling) for 2 hr and washed in 0.075% Brij L23 (Sigma-Aldrich) in PBS. The cells were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:200, Dako, Glostrup, Denmark) for 1 hr and washed in 0.075% Brij L23 in PBS. After visualization with 3-amino-9-ethylcarbazole (Nichirei Bioscience, Tokyo, Japan), nuclei were counterstained with Mayer’s hematoxylin solution (Fuji Wako). As a negative control, normal rabbit IgG was used at the same concentration as the primary antibody in each experiment. All of these antigen-antibody reactions were performed at room temperature. Microphotographs were captured with an Olympus microscope (BX53, Tokyo, Japan). For quantitative analysis of CXCR4, at least 1000 cells were counted in random fields, and results are shown as the percentage of positive cells per total number of counted cells.

 Western blotting analysis

WB was performed as reported previously [31]. Total HGC27 cell lysates were prepared using hot 0.9% sodium dodecyl sulfate (SDS) buffer containing 15 mM ethylenediaminetetraacetic acid (EDTA), 8 mM unlabeled methionine, and a protease inhibitor cocktail. Lysates were boiled for 10 min, cooled, diluted in 0.3% SDS, and then adjusted to contain 33 mM Tris/acetate, 1.7% Triton X-100. The protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were mixed with loading buffer (0.2 M Tris-HCl, pH 8.0, 0.5 M sucrose, 5 mM EDTA, 0.01% bromophenol blue, 10% 2-mercaptoethanol, and 2.5% SDS), boiled for 5 min, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred on onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with nonfat skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris buffer, pH 7.6, and 150 mM NaCl) for 1 hr at RT, and then incubated overnight with primary antibodies against CXCR4 (D4Z7W, 1:250) or β-actin (A3854, 1:30,0000, Sigma-Aldrich). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (1:3,000, Dako) at RT for 1 hr. Protein bands were visualized with DAB, Ni, Co, and H₂O₂. β-actin was used as an internal standard in each lane for normalization of target protein expression.

 Statistical analysis

Data are expressed as means ± standard deviation (SD) from three independent experiments. Differences between experimental groups were assessed by Student t-test, with p < 0.05 considered to indicate statistical significance. All analysis were performed with StatFlex version 7 software (Artech, Osaka, Japan).

III.  Results

 TS-PDT decreased the viability of HGC27 and MKN74 cells in a dose-dependent manner

The impact of TS-PDT on cell viability was assessed using the MTT assay in GC cell lines (Fig. 2A, B). TS-PDT reduced the viability of HGC27 and MKN74 cells in a dose-dependent manner. The IC₅₀ of TS was determined to be 13.4 ± 1.0 μM for HGC27 and 17.4 ± 2.3 μM for MKN74 at 24 hr after TS-PDT. In both cell lines, TS concentrations of 10 μM or lower did not result in a significant decrease in cell viability. However, at concentrations exceeding 20 μM, cell viability was significantly reduced in both HGC27 and MKN74 (p < 0.005). At a concentration of 15 μM, a significant reduction in cell viability was also observed in both cell lines, though the degree of reduction varied (HGC27: p < 0.005, MKN74: p < 0.05).

Fig. 2.

TS-PDT decreases the viability of HGC27 and MKN74 cells in a dose-dependent manner. The viability of HGC27 (A) and MKN74 (B) cells was assessed using the MTT assay at 24 hr after TS-PDT. TS-PDT reduced cell viability in a dose-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of TS was determined to be 13.4 ± 1.0 μM for HGC27 and 17.4 ± 2.3 μM for MKN74. No significant reduction in cell viability was observed at TS concentrations of 10 μM or lower. However, significant reductions were observed at concentrations exceeding 20 μM (p < 0.005 for both cell lines). At 15 μM, significant reductions were also observed (HGC27: p < 0.005, MKN74: p < 0.05), though the degree of reduction differed between the two cell lines. Data represent the mean ± SD of at least three independent experiments.* p < 0.05, *** p < 0.005. TS-PDT, talaporfin sodium photodynamic therapy.

 SN-38 decreased the viability of HGC27 synergically at non-cytotoxic doses of TS-PDT

As shown in Fig. 2A and B, TS-PDT alone at concentrations below 10 μM had minimal impact on the viability of GC cell lines. In subsequent experiments, we investigated the synergistic effect of TS-PDT at non-cytotoxic doses (5 μM and 10 μM) in combination with SN-38. The IC₅₀ values for single-agent treatments and combinations after 24 hr were calculated from the MTT assay, with the results summarized in Table 2. For HGC27 cells, the IC₅₀ values for SN-38 alone and in combination with TS-PDT at 5 μM and 10 μM were 87.5 ± 3.6 μM, 47.8 ± 28.8 μM, and 12.3 ± 8.6 μM, respectively (Fig. 3A). Conversely, for MKN74 cells, the IC₅₀ values for SN-38 alone and in combination with TS-PDT at 5 μM and 10 μM were 10.4 ± 5.2 μM, 9.4 ± 0.5 μM, and 9.4 ± 4.3 μM, respectively (Fig. 3B). To quantitatively assess the interaction between TS-PDT and SN-38, ComI values were calculated. Notably, the ComI value for the combination of SN-38 and non-cytotoxic TS-PDT at 10 μM in HGC27 cells was less than 1, indicating a synergistic effect. In contrast, the ComI values for all other combinations in both HGC27 and MKN74 cells were greater than 1, suggesting antagonism.

Table 2. 

Drug loading efficacy and combination index

	[HGC27]		[MKN74]
	TS (5 μM) + SN38	TS (10 μM) + SN38	TS (5 μM) + SN38	TS (10 μM) + SN38
A (TS)	5 μM	10 μM	5 μM	10 μM
A50 (TS)	13.4 ± 1.0 μM	13.4 ± 1.0 μM	17.4 ± 2.3 μM	17.4 ± 2.3 μM
B (SN38)	47.8 ± 28.8 μM	12.3 ± 8.6 μM	9.4 ± 0.5 μM	9.4 ± 4.3 μM
B50 (SN38)	87.5 ± 3.6 μM	87.5 ± 3.6 μM	10.4 ± 5.2 μM	10.4 ± 5.2 μM
ComI*	1.23 ± 0.17	0.91 ± 0.11	1.37 ± 0.95	1.51 ± 0.64

* ComI (combination index) = A ⁄ A₅₀ + B ⁄ B₅₀

A₅₀ or B₅₀ is the dose of TS or SN-38 alone with inhibition of 50%. In the numerators, either A or B is the portion of TS or SN-38 in combination with the same inhibition of 50%. According to the obtained ComI, different treatment interactions can be obtained. Specifically, ComI = 1 indicates an addictive effect in the absence of synergism or antagonism. CI < 1 indicates synergism. and CI > 1 indicates antagonism.

Fig. 3.

Synergistic effect of TS-PDT and SN-38 on gastric cancer cell lines. The half-maximal inhibitory concentration (IC₅₀) values, which represent the concentration of SN-38 required to reduce cell viability by 50%, were determined for SN-38 alone and in combination with TS-PDT at non-cytotoxic doses (5 μM and 10 μM) in HGC27 (A) and MKN74 (B) cells using the MTT assay after 24 hr of treatment. In HGC27 cells, IC₅₀ values of SN-38 decreased significantly with the addition of TS-PDT at 5 μM and 10 μM (87.5 ± 3.6 μM, 47.8 ± 28.8 μM, and 12.3 ± 8.6 μM, respectively). In MKN74 cells, the IC₅₀ values of SN-38 showed minimal change in the presence of TS-PDT at 5 μM and 10 μM (10.4 ± 5.2 μM, 9.4 ± 0.5 μM, and 9.4 ± 4.3 μM, respectively). Data represent the mean ± SD of at least three independent experiments. TS-PDT, talaporfin sodium photodynamic therapy.

Comparing Figures 2A and 3A, Figure 3A shows that in the HGC27 cell experiment, without SN-38 administration, cell viability was reduced to less than 90% with TS-PDT treatment at 5 and 10 μM. In contrast, in Figure 2A, cell viability remained nearly 100% despite the same TS-PDT treatment. The experimental processes of Figures 2 and 3 differ significantly. Specifically, in Figure 2, cell viability was measured 24 hrs after TS-PDT, whereas in Figure 3, SN-38 was administered 24 hrs after TS-PDT, and cell viability was measured an additional 24 hrs later. In other words, for the samples without SN-38 treatment, cell viability was assessed 48 hrs after TS-PDT. The differences observed among the three TS-PDT groups at 0, 5 and 10 μM in Figure 3A suggest that non-cytotoxic TS-PDT may influence cancer cell proliferative activity during this period.

 Non-cytotoxic dose of TS-PDT improved anticancer effects of SN-38 for HGC27

To assess the synergistic effects of an anti-cancer drug and non-cytotoxic doses of PDT in two different GC cell types, we compared the impacts of SN-38 alone and in combination with low-dose TS-PDT in HGC27, an undifferentiated GC cell line, and MKN74, a differentiated GC cell line. In the treatment with SN-38 alone, HGC27 exhibited significantly lower cytotoxicity compared to MKN74 at concentrations above 10 μM (Fig. 4A). The cancer stem cell properties of HGC-27 have been demonstrated in previous reports through side population experiments [32], and our experimental results showing chemoresistance compared to the differentiated gastric cancer cell line MKN-74 are consistent with these findings. In contrast, when combined with non-cytotoxic TS-PDT, the treatment sensitivity of HGC27 was nearly identical to that of MKN74 (Fig. 4B). We hypothesize that the differences in the synergistic effects of non-cytotoxic TS-PDT on SN-38 across the two GC cell lines are likely due to a reduction in drug resistance in the poorly differentiated HGC27 cells, bringing their sensitivity closer to that of the well-differentiated MKN74 cells.

Fig. 4.

Comparison of SN-38 sensitivity alone and in combination with non-cytotoxic TS-PDT in gastric cancer cell lines. The cytotoxic effects of SN-38 alone (A) and in combination with non-cytotoxic TS-PDT (B) were assessed in HGC27 (undifferentiated gastric cancer cell line) and MKN74 (differentiated gastric cancer cell line) using the MTT assay. HGC27 cells exhibited significantly lower sensitivity to SN-38 alone compared to MKN74 at concentrations above 10 μM. However, when combined with non-cytotoxic TS-PDT, the sensitivity of HGC27 to SN-38 increased markedly, becoming nearly equivalent to that of MKN74. Data represent the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01. TS-PDT, talaporfin sodium photodynamic therapy.

 Non-cytotoxic dose of TS-PDT downregulated mRNA expression of cancer stemness in HGC27

Next, we investigated the impact of PDT on the drug resistance mechanism by analyzing the mRNA levels of several drug resistance-related factors in HGC27 cells treated with non-cytotoxic TS-PDT (Fig. 5). For comparison, we also assessed the gene expression of MKN74 cells without PDT treatment. We focused on genes previously implicated in drug resistance in GC cell lines, including cancer stemness markers CXCR4 and CD44, and drug efflux pump markers ABCC1 and ABCG2, the latter of which is considered both a drug efflux transporter and a cancer stem cell marker. The expression of CXCR4 and CD44 was significantly higher in HGC27 cells compared to MKN74, whereas that of ABCG2 was notably higher in MKN74 cells, with no significant difference observed for ABCC1. After non-cytotoxic TS-PDT, CXCR4 mRNA expression in HGC27 decreased significantly over time—by 47 ± 15% (p < 0.01) at 3 hr and 20 ± 17% (p < 0.01) at 24 hr—approaching the baseline expression level seen in MKN74. A similar time-dependent reduction in expression was noted for CD44 and ABCG2, with decreases of 83 ± 67% and 43 ± 51% at 3 hr, and 40 ± 53% and 23 ± 40% at 24 hr, respectively, though these reductions were not statistically significant. In contrast, ABCC1 expression remained unaffected both between HGC27 and MKN74 and over time following TS-PDT.

Fig. 5.

Impact of non-cytotoxic TS-PDT on the expression of drug resistance-related genes in HGC27 cells. mRNA levels of drug resistance-related factors were analyzed in HGC27 cells treated with non-cytotoxic TS-PDT and compared to untreated MKN74 cells. Gene expression of cancer stemness markers (CXCR4, CD44) and drug efflux transporters (ABCC1, ABCG2) was evaluated using qRT-PCR. Baseline expression levels of CXCR4 and CD44 were significantly higher in HGC27 compared to MKN74, whereas ABCG2 was higher in MKN74; however, no significant difference was observed for ABCC1. Following TS-PDT, CXCR4 expression in HGC27 decreased significantly over time, with reductions of 47 ± 15% (p < 0.01) at 3 hr and 20 ± 17% (p < 0.01) at 24 hr, approaching MKN74 baseline levels. CD44 and ABCG2 also showed time-dependent reductions of 83 ± 67% and 43 ± 51% at 3 hr, and 40 ± 53% and 23 ± 40% at 24 hr, respectively, though these changes were not statistically significant. ABCC1 expression remained unchanged. Data represent the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01. PDP, photodynamic priming; TS-PDT, talaporfin sodium photodynamic therapy.

 Non-cytotoxic dose of TS-PDT reduced CXCR4 protein expression in HGC27

To validate the intriguing observation that non-cytotoxic TS-PDT reduced the gene expression of CXCR4 in poorly differentiated GC cell lines to levels comparable to those in well-differentiated GC cells, we further examined protein expression using ICC and WB. ICC analysis revealed that CXCR4 expression in HGC27 cells was localized in the nucleus, consistent with previous findings by Wang et al. [33, 34]. The staining patterns exhibited variability in intensity, distribution, and whether the staining was diffuse or patchy, so only cells with strong, diffuse nuclear staining were considered “positive” (Fig. 6A). The positivity rate of CXCR4 in HGC27 cells before PDT was 19.0 ± 2.6%, which decreased significantly to 8.7 ± 5.3% (p < 0.05) 1 hr after non-cytotoxic TS-PDT (Fig. 6B). This reduction remained at 9.5 ± 6.4% 3 hr later, but by 24 hr, the expression level had nearly returned to baseline at 17.6 ± 4.7%. Notably, no morphological changes indicative of apoptosis or necrosis were observed in the microscopic analysis. WB analyses showed a progressive decrease in CXCR4 expression up to 24 hr after PDT (Fig. 6C).

Fig. 6.

Analysis of CXCR4 protein expression in HGC27 cells following non-cytotoxic TS-PDT. Immunohistochemical analysis of CXCR4 expression in HGC27 cells before and after TS-PDT. (A) Representative images show nuclear localization of CXCR4. No morphological changes suggesting apoptosis or necrosis were observed. (B) The positivity rate of CXCR4 decreased significantly from 19.0 ± 2.6% at baseline to 8.7 ± 5.3% (p < 0.05) 1 hr after TS-PDT and remained at 9.5 ± 6.4% at 3 hr. By 24 hr, the positivity rate had returned to near-baseline levels (17.6 ± 4.7%). (C) Western blot analysis confirmed a progressive reduction in CXCR4 protein expression up to 24 hr after TS-PDT. Data are presented as mean ± SD from at least three independent experiments. * p < 0.05. TS-PDT, talaporfin sodium photodynamic therapy.

IV.  Discussion

PDT is generally regarded as a localized treatment that capitalizes on the differential affinity of PS agents between tumor and normal tissues. To enhance the therapeutic outcomes of PDT, there have been increasing efforts to combine it with other cancer treatments [35], including chemotherapy [36–38], radiotherapy [39, 40], immunotherapy [41, 42], and enzyme inhibitors [43]. While the exact mechanisms behind this synergy remain unclear, combining PDT with chemotherapy may help overcome multi-drug resistance that arises during tumor treatment. Multi-drug resistance is linked to the overexpression of drug efflux transporters or cancer stem cell markers in tumor cells, which can be induced by the effective use of chemotherapeutic agents. For instance, Khdair et al. [44] showed that methylene blue-mediated PDT combined with adriamycin showed potent cytotoxicity against drug-resistant tumor cells, accompanied by a reduction in P-glycoprotein expression. Furthermore, combination PDT has been recognized as potentially advantageous in improving both treatment efficacy and the quality of life for advanced-stage patients suffering from conditions such as bronchial obstruction in non-small cell lung cancer [45] and unresectable cholangiocarcinoma [46].

The findings of the present study suggest that “non-cytotoxic” TS-PDT can enhance the drug sensitivity of SN-38 in undifferentiated GC cell lines, bringing their sensitivity to a level comparable to that of well-differentiated GC cells. Our results differ from previous studies on combination therapies involving PDT and anticancer drugs by showing that non-cytotoxic TS-PDT can improve the drug sensitivity of poorly differentiated GC cell lines, aligning it with the sensitivity of differentiated GC lines. To investigate the underlying mechanisms of this improvement, we evaluated several drug resistance molecules, including cancer stem cell markers and drug efflux transporters. Huang et al. [24] showed that PDT using nanoliposomal benzoporphyrin derivatives, combined with irinotecan, reduces the expression of the ABCG2 efflux transporter, thereby increasing intracellular irinotecan concentrations. They also reported that PDT inhibits survivin expression to enhance apoptosis. These mechanisms were validated in vivo using pancreatic cancer cell lines and introduced the concept of the PDP effect [25]. Although their results share similarities with ours, our study is significant as it demonstrates the PDP effect through non-cytotoxic PDT using TS, a clinically approved agent in Japan, specifically targeting GC—a disease with a high incidence in East Asia—thereby increasing its clinical relevance. Furthermore, a key aspect in which our research provides a more detailed evaluation compared to the approach of Huang et al. is the assessment of the synergistic effect between non-cytotoxic TS-PDT and chemotherapy. Notably, we objectively quantified this synergy using the combination index, offering a clear and measurable indicator of their interaction.

Undifferentiated GC cells are strongly associated with PD, with factors such as epithelial-mesenchymal transition and the expression of adhesion molecules being crucial [47]. Moreover, cancer stemness contributes to their high invasiveness and ability to survive even under hypoxic conditions [48, 49]. Gao et al. [32] conducted a side population analysis on HGC-27 cells, which we used in our experiments, and reported their cancer stem cell properties. Furthermore, Fujita et al. [8] showed that CXCR4 can be a marker for GC stem cells involved in PD. Numerous studies have indicated that CXCR4 plays a key role in cancer progression and metastasis [50–52]. In cancer cells, CXCR4 is not limited to the cell membrane but is frequently found in the cytoplasm and nucleus [32, 53]. Masuda et al. [9] reported that nuclear CXCR4 positivity is often observed in poorly differentiated GC, which tends to be large and infiltrative, resulting in poor prognosis for patients. In our ICC experiments, HGC27 cells before PDT showed nuclear CXCR4 expression, whereas no expression was found in the cytoplasm or cell membrane. Based on the findings from previous studies, it is reasonable to hypothesize that nuclear CXCR4 expression in undifferentiated GC cell lines would contribute to their resistance to therapy.

We explored the molecular mechanisms behind the enhanced drug sensitivity induced by non-cytotoxic TS-PDT and observed a significant reduction in CXCR4 expression across the qRT-PCR, ICC, and WB analyses. In qRT-PCR, although not statistically significant, CD44, a known cancer stem cell marker, exhibited a downward trend. In contrast, ABCC1, a drug efflux transporter, showed no such trend. Interestingly, ABCG2, which functions both as a cancer stem cell marker and a drug efflux transporter, exhibited a decreasing trend. These results suggest that the drug resistance-reducing effects of non-cytotoxic TS-PDT may be linked to its impact on cancer stem cell properties.

For CXCR4, discrepancies were noted in the temporal changes observed between the qRT-PCR, WB, and ICC analyses. Specifically, the qRT-PCR and WB analyses showed a progressive decrease in CXCR4 expression up to 24 hr after PDT. However, ICC showed decreases at 1 and 3 hr after PDT, with expression returning to near baseline levels by 24 hr. The differences in these results can be explained by the fact that when proteins are redistributed to different cellular compartments, such as the cytoplasm, nucleus, or membrane, ICC can detect this redistribution as an increase or decrease, whereas WB only evaluates the total amount of protein across the entire cell, which may not reflect these dynamic changes [54, 55]. Considering the present results, it is possible that after PDT intervention, CXCR4 re-synthesis occurred, leading to the observed increase in nuclear localization at 24 hr after treatment in ICC. However, as the total protein and mRNA levels in both WB and qPCR were still in a decreasing phase, this re-synthesis may have been overshadowed by the overall downward trend in protein levels. The biological mechanisms underlying the nuclear translocation and nuclear localization of CXCR4 remain largely unclear, but several studies have attempted to elucidate these processes. Xu et al. confirmed the significant implications of the putative nuclear localization sequence ‘146RPRK149’ on CXCR4 subcellular localization and metastatic potential using point-mutation assays in renal cancer cell lines [56]. Additionally, Bao et al. demonstrated that CXCR4 undergoes nuclear translocation in response to hypoxic conditions in a renal cancer cell lines [57]. These references could support our explanation under the hypothesis that the discrepancy observed between qPCR, WB, and ICC results in our study may be due to preferential migration to the nucleus triggered by some biological change associated with non-cytotoxic PDT.

PD refers to the dissemination and growth of cancer deposits within the peritoneal cavity, often representing a late-stage manifestation of cancers such as GC. The prognosis for GC-related PD is poor, with 5-year survival rates between 13% and 23%, even among selected patients who could receive cytoreductive surgery and heated intraperitoneal chemotherapy [58]. The complex anatomy of the peritoneum, with a surface area comparable to that of the external body, allows cancer deposits to cover vital intra-abdominal structures such as the small bowel, liver, and major blood vessels. Although PDT holds promise for treating PD, its narrow therapeutic window and potential serious side effects limit its clinical application. Our findings support the potential for non-cytotoxic PDT to enhance the effects of subsequent systemic chemotherapy in the peritoneal cavity while minimizing normal tissue damage. This approach could be a crucial step towards the development of cytotoxic PDT for treating PD.

From the perspective of clinical application, it is essential to carefully consider the limitations of this study, particularly the generalizability of the obtained results to in vivo environments. First, in a model mouse of undifferentiated gastric cancer, several critical factors need to be thoroughly examined, including the extent to which light reaches nodules formed within the mesenteric spaces, the penetration of the drug into peritoneal lesions following transabdominal or intravenous administration, and the potential immune-inducing effects of PDT. Second, further investigation is required regarding the design and technical aspects of an irradiation device capable of delivering diffuse intraperitoneal illumination to achieve PDP effects in vivo. Finally, this study did not assess the potential side effects of PDP on normal tissues. Therefore, conducting safety evaluations through animal experiments remains one of the most crucial future challenges.

In conclusion, non-cytotoxic TS-PDT appears to offer promising potential as a strategy to enhance the efficacy of chemotherapeutic agents such as SN-38 in treating poorly differentiated GC. Moving forward, we aim to incorporate these findings into future animal models to establish more robust evidence, with a primary goal of addressing GC-related PD.

V.  Conflicts of Interest

The authors have no conflict of interest to declare.

VI.  Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 24K11937 to KK and 21K06738 to YH. We gratefully acknowledge the Frontier Science Research Center at the University of Miyazaki for allowing us to use their facilities.

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