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Table of Contents
ORIGINAL ARTICLE
Year : 2018  |  Volume : 29  |  Issue : 2  |  Page : 64-72

A novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human bladder cancer cells


Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan

Date of Web Publication30-Apr-2018

Correspondence Address:
Tefu Tsai
Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/UROS.UROS_2_17

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  Abstract 

Background: Quinazolines, which process a wide spectrum of biological properties such as antibacterial, antifungal, antivirus, and anticancer activities, are considered one of the most important heterocycles in medicinal chemistry. Here, we described for the first time the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazoline-4-one) which emits green fluorescent in the cytosol and exhibits phototoxicity toward human bladder cancer (BC) cells under blue-light exposure. Materials and Methods: Human BC cells (5637 and T24) and immortalized uroepithelial cell (SV-HUC1) were utilized in this study. To trace the localization of MJ-56, MitoTracker and LysoTracker were applied. The cell viability with or without blue light exposure were monitored by WST-1 reagent, direct recording, and clonogenic assays. The apoptosis induction in MJ-56 treated cells was detected. Results: MJ-56 emits green fluorescent in the cytosol. Vital staining of mitochondria or lysosomes demonstrated that the MJ-56 fluorescent was not located in either organelles. MJ-56 treatment for 24 h did not cause significant loss of cell viability in BC cells. However, treatment of 0.125 μM MJ-56 for 1 h and exposed to blue light for 15 mins significantly reduced cell viability. Interestingly, our results showed that MJ-56 has minimal impact on SV-HUC1 even with the blue-light exposure. The caspase 3/7 activities in BC cells treated with MJ-56 and exposed to blue light were significantly increased 1 h post-treatment. However, the DNA fragmentation cannot be detected at 1, 6, or 24 h posttreatment due to the loss of viable cells. Conclusions: MJ-56 exhibits phototoxicity toward BC cells with minimal impact on uroepithelial cells, indicating a novel therapeutic agent against BC. The mechanism underlying MJ-56-induced cell death as well as the translational studies warrants further investigation.

Keywords: Acridine orange, autophagy, bladder cancer, photodynamic therapy


How to cite this article:
Chen HE, Lin JF, Hwang TI, Lin YC, Chou KY, Hour MJ, Tsai T. A novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human bladder cancer cells. Urol Sci 2018;29:64-72

How to cite this URL:
Chen HE, Lin JF, Hwang TI, Lin YC, Chou KY, Hour MJ, Tsai T. A novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human bladder cancer cells. Urol Sci [serial online] 2018 [cited 2018 May 24];29:64-72. Available from: http://www.e-urol-sci.com/text.asp?2018/29/2/64/229910


  Introduction Top


Bladder cancer (BC) is the ninth most common cancer in Taiwanese males according to the Taiwan Cancer Registry Annual Report in 2012.[1] In the United States, BC is the sixth most commonly diagnosed cancer with estimated 79,030 new cases and 16,870 deaths in 2017.[2] The risk factors of BC include cigarette smoking, occupational exposure to chemical compounds, including aniline dyes, benzidine compounds, and analgesic abuse, and chronic irritation.[3] Almost 70%–80% of patients with bladder tumors present with low-grade, superficial, or nonmuscle-invasive BC (NMIBC).[4] Transurethral resection of bladder tumor combined with intrabladder chemotherapy or immune therapy, such as Bacillus Calmette–Guérin infusion, is the standard procedure performed for initial diagnosis, staging, and treatment for NMIBC.[5] Neoadjuvant followed by radical cystectomy or adjuvant cisplatin-based chemotherapy or chemoradiation for bladder preservation is used in the management of locally advanced disease. Platinum-based chemotherapeutic regimens are used in advanced disease. The treatment with gemcitabine and cisplatin is favored for its moderate side effects, including granulocytopenia, nausea, and vomiting, compared to the combination of methotrexate, vinblastine, doxorubicin, and cisplatin.[6] Despite these efforts in managing NMIBC, approximately 50%–70% of NMIBCs will recur and about 10%–20% will progress to muscle-invasive BC (MIBC).[7] Furthermore, half of all patients with locally advanced BC die from metastatic disease in 2 years.[8] Therefore, novel therapeutic approaches are warranted in the treatment of BC.

The most frequent molecular signature detected in low-grade BC is the upregulation of receptor tyrosine kinase-Ras pathway which includes the overexpression of fibroblast growth factor receptor-3 (FGFR3). It has been reported that up to 70% of low-grade BC is associated with FGFR3, 30%–40% with HRAS, and 10% with PIK3CA mutation.[9],[10],[11] In high-grade BC, the most frequent reported contributors to the tumor progression are the deletion or mutation of tumor suppressor genes p53 and pRB, both are critical cell cycle regulators.[12],[13] Loss of PTEN and p16 is also commonly detected in high-grade BC.[14],[15] It has been demonstrated that EGFR overexpression, while modest in NMIBC, is evident in MIBC in several studies.[16],[17] Another proto-oncogene, c-MET, has been demonstrated to be overexpressed in more than 60% of locally advanced and metastatic BC and is linked to poor survival.[18],[19] The network of c-MET is purposed to serve as a novel prognostic marker for predicting BC risk of developing aggressive disease.[20] In addition, matrix metalloproteases (MMPs) regulated by p38 mitogen-activated protein kinase (MAPK) in BC were reported to correlate with tumor grade and invasion.[21],[22]

Benzopyrimidine derivatives, also known as quinazolinones, are compounds with a wide spectrum of biological activities, such as anticancer, anti-inflammatory, antitubercular, and antibacterial effects.[23] Quinazolinone compounds have been shown to exhibit antimetastatic effects in human prostate cancer cells through the suppression of MAPK, AKT, AP-1, and nuclear factor-κB, leading to the inhibition of MMP-2 and MMP-9.[24] Recently, one of the novel quinazoline derivatives, MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazolin-4-one), has been demonstrated to reduce the metastasis of human colorectal cancer cells via dual inhibition of EGFR and c-MET activation.[25]

Therefore, given the evidence that activation of MAPK, EGFR, and c-MET is associated with BC progression, and taking into account that the effectiveness of MJ-56 against BC is unclear, this study aimed to investigate the anticancer effects of MJ-56 in BC cell lines.


  Materials and Methods Top


Cell lines and compounds

Human BC (urothelial cell carcinoma) cells, 5637 (Grade II) and T24 (Grade III); and immortalized uroepithelial cell, SV-HUC1, were purchased from Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). These cells have been profiled with short tandem repeat-polymerase chain reaction (PCR) at BCRC for authentication and routinely checked with mycoplasma contamination using a PCR-based method. The 5637 and T24 cells were cultured in RPMI-1640 medium, with SV-HUC1 in F-12 medium containing 10% fetal bovine serum (FBS) as described.[26] All the culture media, FBS, and supplements including antibiotics were purchased from Thermo-Fisher Scientific (San Jose, CA, USA). MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazolin-4-one) was obtained as a generous gift from Professor Hour Mann-Jen (School of Pharmacy, China Medical University, Taichung, Taiwan) and dissolved in dimethyl sulfoxide (DMSO) prior to use.

Cell viability assay

The cell viability in 5637, T24, and SV-HUC1 cells treated with indicated concentrations of MJ-56 was detected at 24 h posttreatment using WST-1 reagent (Roche Diagnostics) following the manufacturer's instructions and determined as the percentage of the control. Data were obtained from at least three separate experiments with each condition that assayed in eight replicate wells.

Subcellular localization with fluorescent imaging

Cells were seeded in glass-bottomed chamber slides (SPL Life Sciences, Pocheon, Korea) for 24 h prior to the treatment of 1.25 μM MJ-56. In some experiments, 50 nM MitoTracker Red (Thermo-Fisher Scientific), or 50 nM LysoTracker Red DND-99 (Thermo-Fisher Scientific) was added simultaneously with MJ-56 to detect the location of mitochondria and lysosomes, respectively. The nucleus was also detected by staining with 150 nM DAPI nucleic acid stain solution (Thermo-Fisher Scientific). Cells were stained for 5 min in the cell culture incubator, and all the treatment processes were conducted with avoidance of environmental light. After 3-time washes with phosphate-buffered saline (PBS), complete medium was added to each well and the slides were immediately wrapped with aluminum foil and subjected to fluorescent imagining. A Nikon inverted microscope Eclipse Ti-E (Nilon, Kobe, Japan) which was equipped with 130 W fluorescence light source with proper filter sets for detecting MitoTracker, LysoTracker, and DAPI was used. Images were processed with Nikon NIS-Elements Advanced Research software (Nikon Instruments, Tokyo, Japan). Representative photographs from at least three independent experiments with similar results were shown.

Blue-light irradiation

We used a homemade device equipped with six blue-light light-emitting diode (LED) bulbs with peak wavelength of 443.7 nm and 55.28 lumen per LED, as the blue-light source. The LED produces less heat compared to the traditional mercury lamp. Six LED bulbs corresponding to the center of each well of a 6-well plate were welded to an electronic board that was attached to the ceiling of a cardboard box with the dimension of 15 cm × 12 cm × 6 cm. The bottom space of the box is accessible for various types of conventional culture plastic wares. Once the cells were treated with indicated concentrations of MJ-56 for 1 h, the culture plates, dishes, or slides were placed in the box and the cells were exposed to blue light for 30 s. After the exposure, the medium was immediately replaced with complete medium. Moreover, the cell viability was detected with WST-1 immediately or at 4, 6, and 12 h posttreatment.

Clonogenic survival

Clonogenic assay was performed for evaluating the long-term effects of MJ-56 with blue-light exposure. To determine clonogenic ability, cells seeded in 6-well plates were receiving DMSO (control), 1.25 μM MJ-56 for 1 h with or without the 30 s exposure to blue light. Afterward, cultures were rinsed with PBS and cultured in complete medium for 7 days. The cells were stained with 0.01% (w/v) in dH2O for 60 min and the images were taken by an EPSON V300 Photo Scanner (Taipei, Taiwan). Representative photographs from three independent experiments with similar results were shown.

Detection of apoptosis

Apoptotic induction in cells treated with MJ-56 with or without blue-light exposure was detected by (a) activation of caspase-3/7 activity, (b) detection of the cleaved form of poly (ADP-ribose) polymerase (PARP) or caspase 3 using Western blot, and (c) DNA fragmentation assays using flow cytometry as described.[26] In brief, (a) activation of caspase 3/7 in cells treated with indicated concentrations of MJ-56 with or without blue-light exposure for 24 h was assayed using (H-Asp-Glu-Val-Asp)2-Rhodamine 110 ([(Z-DEVD)2-R110], Bachem, Torrance, CA, USA) substrate as described,[27] (b) cells subjected to the indicated treatment were harvested. The cytosolic protein fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo-Fisher Scientific). Antibodies against cleaved caspase-3 (c-Casp3), PARP (c-PARP), cytochrome C (Cyto C; all from Cell Signaling Technology, Danvers, Massachusetts, USA) and β-actin (as internal control, Sigma-Aldrich) were utilized. Subsequent immunoblotting procedures were performed using a chemiluminescence kit (Merck Millipore, Temecula, CA, USA) as per the manufacturer's instruction. Representative blots that had been repeated for three times with similar results were shown. The intensity of immunoreactive bands was determined using ImageJ software (V.1.8.0, US National Institutes of Health, Bethesda, MD, USA).[28] Results are expressed as mean of fold ± standard deviation (SD) compared to the control, and (c) cells with indicated treatment immediately after treatment or growth in complete medium for further 6 or 24 h were subjected to DNA fragmentation assays using APO-Direct apoptosis detection kit from BD Biosciences (San Jose, CA, USA). Samples were analyzed using an Accuri C5 flow cytometer (BD Biosciences, Bedford, MA, USA), and 10,000 events were collected for each sample.

Statistical analysis

All experiments were performed for at least three times. The data were analyzed by Student's t-test for statistical significance. Results were expressed as the means ± SD. P values were considered significant if <0.05. Statistical analyses were carried out using the SigmaPlot Version 10.0 (Systat Software, San Jose, CA, USA).


  Results Top


MJ-56 treatment failed to decrease cell viability in bladder cancer cells

It has been demonstrated that quinazolinone derivatives possess anticancer activities in osteogenic sarcoma cells.[29] A novel quinazolinone derivative, MJ-56, was synthesized and shown to inhibit the migration and invasion of HT29 colorectal cancer cells through the inhibition of c-MET and EGFR.[25] Chen et al. demonstrated that MJ-56 suppressed the migration and invasion of HT29 in the concentrations ranging from 5 to 15 μM.[25] We therefore determine the cell viability in SV-HUC1, 5637, and T24 cells treated with MJ-56 ranging from 0 to 20 μM. As shown in [Figure 1]. MJ-56 slightly decreased the cell viability in these cells, even when treated with 20 μM MJ-56 for up to 24 h. Although previous reports show that 5 μM MJ-56 suppressed the migration and invasion of colorectal cancer cells, MJ-56 did not exhibit severe cytotoxicity toward BC cells.
Figure 1: The cell viability of cells treated with various concentrations of MJ-56 for 24 h. Cells were seeded in 96-well plates for 24 h prior to the administration of indicated concentrations of MJ-56 for another 24 h. The cell viability was detected using WST-1 reagents. Data were from three independent experiments with quadruplicated wells and presented as mean of percentage ± standard deviation compared to control. *, P < 0.05

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MJ-56 emits green fluorescent and induces phototoxicity in human bladder cancer cells

After intensive literature search, we found that many quinazolinone derivatives were synthesized for fluorescent imaging.[30],[31] We therefore are interested in finding whether MJ-56 emitted fluorescent in the cells. As demonstrated in [Figure 2]. cells were treated with 1.25 μM MJ-56 for 5 min and examined immediately under fluorescent microscopy. We found that MJ-56 emitted green fluorescent (fluorescein isothiocyanate, FITC channel) when excited with blue light. To determine the subcellular localization of MJ-56, MitoTracker Red or LysoTracker Red DND-99 and DAPI were co-stained with 1.25 μM MJ-56. As shown in [Figure 2]. MJ-56 did not co-localize with either mitochondria or lysosomes in BC cells. Therefore, the subcellular localization of MJ-56 warrants further investigation. However, we notice that cells treated with MJ-56 and exposed to blue light during microscopic examination exhibited disrupted membrane in the later time point (data not shown). Therefore, it raises a question that whether MJ-56 induces phototoxicity in human BC cells. To test this hypothesis, we developed a homemade device containing six LED bulbs as blue-light source. We treated 5637 and T24 cells with 0–20 μM MJ-56 for 24 h prior to a 30 s exposure of blue light. As shown in [Figure 3]a and [Figure 3]b, the cell viability only decreased slightly in cells treated with high concentrations of MJ-56 without blue-light exposure. However, the cell viability was significantly suppressed in the lowest concentration of MJ-56 (1.25 μM) with blue-light exposure. We next investigated whether MJ-56 also induced phototoxicity in immortalized urothelial SV-HUC1 cells. The SV-HUC1, 5637, and T24 cells were treated with 1.25 μM MJ-56 for 1 h [Figure 3]c or 24 h [Figure 3]d prior to a 30 s exposure of blue light. One hour MJ-56 treatment and blue-light exposure did not significantly decrease the cell viability of SV-HUC1 compared to 5637 or T24 cells. However, the 24 h incubation of SV-HUC1 with 1.25 μM MJ-56 with blue-light exposure decreased the cell viability to 70.74 ± 1.26%. However, the effect of MJ-56 treatment with blue-light exposure was more profound in BC cells compared with SV-HUC1. To determine whether lowering the concentration and shortening the treatment period affect the phototoxicity induced by MJ-56 in BC cells, 5637 [Figure 3]e and T24 [Figure 3]f cells were treated with 0–1.25 μM MJ-56 for just 1 h and exposed to blue light for 30 s. The cell viability was then detected immediately after treatment. As depicted in [Figure 3]e and [Figure 3]f, a dose-dependent phototoxicity was observed in cells treated with MJ-56 in lower concentrations, indicating its effectiveness against BC cells.
Figure 2: MJ-56 emitted green fluorescent in the cytosol. (a and b) 5637 and (c and d) T24 cells seeded in glass-bottomed chamber slides were treated with 1.25 μM MJ-56, 50 nM MitoTracker Red (a and c), or 50 nM LysoTracker Red DND-99 (b and d) with 150 nM DAPI nucleic acid stain solution for 5 min in the incubator. Staining medium was replaced with complete medium prior to the imagining using fluorescent microscopy. Representative photographs for each condition that repeated three times with similar results were shown. Scale bar, 10 μm

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Figure 3: MJ-56 treatment with blue-light exposure significantly reduced cell viability in bladder cancer cells. (a and b) The 5637 or T24 cells were treated with 0–20 μM MJ-56 for 1 h and exposed to blue light for 30 min. The medium was replaced with fresh complete medium. The cells were further incubated for 24 h and the cell viability was detected. To test the cytotoxicity of MJ-56 in SV-HUC1, cells were treated with 1.25 μM MJ-56 for 1 h and exposed to blue light for 30 min. Cell viability was detected at (c) 1 h posttreatment and (d) 24 h posttreatment. (e and f) The cell viability of 5637 and T24 cells treated with 0–1.25 μM MJ-56 with blue-light exposure was detected at 24 h posttreatment. Data shown represent the means of quadruplicate measurements for each condition and were repeated three times. The results are presented as percentage of control (means ± standard deviation); *, P < 0.05

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To get more insight on MJ-56 phototoxicity toward BC cells, we investigated the cellular morphology in a real-time fashion by using an image recorder (CytoSmart system, Lonza). Cells were treated with 1.25 μM MJ-56 for 1 h and exposed to blue light for 30 s, medium was refreshed, and the cells were recorded in the incubator continuously for 36 h. As shown in [Figure 4], 5637 [Figure 4]a and T24 [Figure 4]b cells treated with MJ-56 and exposed to blue light went into a steady state immediately after treatment, while the control cells continued to move, grow, and proliferate [Supplementary Videos 1 and 2]. The results further emphasized that MJ-56 exhibits severe phototoxicity in human BC cells. In order to understand the long-term effect of the phototoxicity induced by MJ-56, clonogenic assays were employed. As demonstrated in [Figure 5], the phototoxicity induced by MJ-56 nearly wipes out all the cancer cells at 7 days posttreatment. In summary, MJ-56 did not exhibit severe cytotoxicity toward human BC cells. However, when combined with blue-light exposure, low concentrations of MJ-56 possessed significant phototoxicity against human BC cells. The impact of MJ-56-induced phototoxicity could be controlled by lowering the concentration and shortening the treatment duration in normal urothelial cells.
Figure 4: Time lapse cell morphological images of MJ-56-treated cells with blue-light exposure recorded by the CytoSmart system. Cells were incubated with refreshed medium after indicated treatment. Representative photographs of (a) 5637 and (b) T24 cells treated with 1.25 μM MJ-56 with blue-light exposure for 30 s for 0, 6, 12, 18, 24, 30, and 36 h were shown. The composed videos for each cell line were provided as Supplementary Videos 1 and 2. Representative results from three independent experiments with similar results were shown

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Figure 5: Clonogenic assay in MJ-56-treated cells with or without blue-light exposure. 5637 (upper panel) and T24 (lower panel) cells were plated in 6-well plates, treated with dimethyl sulfoxide (control) or 1.25 μM MJ-56 for 1 h with or without blue-light exposure for 30 s, then replaced with complete medium and incubated for 7 days prior to the staining of crystal violet. After staining, the whole plate was scanned with a photo scanner. In addition, twenty photographs in different fields were taken using GeneFlash gel documentation system (Syngene) from each plate, and the colonies were counted assisted by the Photoshop software. Data were from three independent experiments with similar results and were presented as mean ± standard deviation; *, P < 0.05

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MJ-56 with blue-light exposure induces dose- and time-dependent apoptosis in the remaining cancer cells.

According to [Figure 3], the cell viability of 5637 and T24 cells, which were treated with 1.25 μM MJ-56 for 1 h and exposed to blue light for 30 s, reduced to 32.41 ± 1.23 and 28.56 ± 2.47%, respectively. The total treatment duration for these cells was around 2 h and 30 s, including MJ-56 treatment for 1 h, blue-light exposure for 30 s, and incubation with WST-1 for another hour. It is unlikely that MJ-56 induced phototoxicity-induced apoptotic programed cell death in such a short time. Instead, necrotic cell death in these cancer cells treated with MJ-56 and blue-light exposure should be responsible for the phototoxicity caused by MJ-56 and needed further investigation. Nevertheless, we found that there are still 30% cells at 24 h posttreatment, but the live imaging showed steady-state cells on the surface of culture vessels for at least 36 h [Figure 4], and the total cell lost was detected by clonogenic assays at 7 days posttreatment [Figure 5]. It is possible that the MJ-56 treatment induces apoptotic cell death in the remaining cells. We therefore detected the apoptotic induction in the posttreated cells. The results showed that the caspase 3/7 activity was increased dose dependently in the remaining cells compared to control at 24 h posttreatment [Figure 6]a. Apoptotic marker proteins, including cleaved-PARP, cleaved caspase 3 (c-Casp3), and cytochrome C, were readily detected in a time-dependent fashion [Figure 6]b. In addition, when the level of DNA fragmentation was detected in cells treated with 1.25 μM MJ-56 for 1 h and exposed to blue light for 30 s, and immediately subjected to the assays, no significant elevation of DNA fragments was detected [Figure 6]c. However, increased level of DNA fragmentation was detected at 6 and 24 h posttreatment, suggesting that apoptotic induction in the remaining cells was responsible for the loss of cells at later stages of the treatment.
Figure 6: Dose- and time-dependent elevation of apoptosis in MJ-56-treated cells with blue-light exposure. (a) Blue-light exposure increased caspase 3/7 activity in 5637 and T24 cells treated with indicated concentrations of MJ-56. Cells were seeded in 96-well plates for 24 h prior to the treatment. Caspase 3/7 activity was detected at 24 h posttreatment and expressed as percentage of control cells. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05. (b) Increased level of cleaved peroxisome proliferator-activated receptor and cleaved caspase-3 (c-Casp3) in MJ-56-treated T24 cells with blue-light exposure. Lane 1: control; Lane 2: 1.25 μM MJ-56; Lane 3: 30 s of blue-light exposure; Lane 4: cells treated with 1.25 μM MJ-56 with 30 s of blue-light exposure. Lane 5: same condition as Lane 4, but the cells were further incubated for 6 h in refreshed complete medium; Lane 6: same condition as Lane 4, but further incubated in refreshed medium for 24 h. The cytosolic protein was extracted from each condition and subjected to the detection of cleaved peroxisome proliferator-activated receptor, caspase-3, and cytosolic cytochrome C. β-actin served as loading control. Representative blots from three independent experiments with similar results were shown. (c) Increased DNA fragmentation in MJ-56-treated cells with blue-light exposure. 5637 and T24 cells treated with 1.25 μM of MJ-56 for 1 h and exposed to blue light for 30 s. Cells were then cultured in refreshed medium for 0, 6, and 24 h. Afterward, cells were collected for the DNA fragmentation assay (BD) followed by the detection of fluorescent intensity using flow cytometry. Data were obtained from 10,000 events and presented as the fold of incorporated fluorescent. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05

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  Discussion Top


BC is a common and costly disease. The economics of BC draw a lot of attentions.[32] The burden of BC care is due to the lack of new technologies (urine-based tests) and therapeutic regimens (intravesical chemotherapy, adjuvant immunotherapy, and even immune-cell based therapy).[33] Therefore, maintaining interests and investment in BC research are required to ensure further advancements.

We reported in the current study for the first time that a novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human BC cells. It has been demonstrated that quinazolinone derivatives not only possess anticancer activity against many cancer cell linesin vitro or in vivo, but also induce cell death through apoptosis or autophagy.[34],[35],[36] More recently, MJ-56 was demonstrated to inhibit the migration and invasion of HT-29 colorectal cells.[25] Interestingly, the researchers found that MJ-56 exhibits antimigratory and anti-invasive properties by inhibiting the activation of EGFR and c-Met kinase, both are important marker proteins during BC tumorigenesis. We thus investigated the cytotoxicity induced by MJ-56 in human BC cells. However, the doses that inhibit cancer cell migration and invasion did not reduce the cell viability in SV-HUC1, 5637, and T24 cells [Figure 1]. Although increasing the concentration of MJ-56 may reduce the cell viability of BC cells, we believed that it may also cause significant side effectsin vivo in the future experiments. We therefore searched the literature and found that several quinazoline derivatives were designed and developed to serve as fluorescent markers. For example, Zhang et al. reported the development of a quinazoline derivative which functioned as a fluorescent chemical sensor for Fe 3+.[30] It is possible that MJ-56 also function as a fluorescent dye; therefore, we stained the cells with 1.25 μM MJ-56 for 5 min and examined the fluorescence using the three conventional fluorescent channels (DAPI, FITC, and Rodamin). To our surprise, MJ-56 did emit green fluorescent (under FITC channel) when excited with blue light [Figure 2]. We also found that the proliferation was inhibited in cells treated with MJ-56 and blue-light exposure (under fluorescent microscopy) when leaving them back into the incubator. Therefore, it is likely that MJ-56 exhibits phototoxicity toward BC cells. To test our hypothesis, we first investigated the subcellular localization of MJ-56 by using organelle-tracking dyes such as MitoTracker and LysoTracker. Since MJ-56 emitted green fluorescent, MitoTracker Red and LysoTracker Red DND-99 were selected to trace cytosolic mitochondria and lysosomes, respectively. However, as depicted in [Figure 2], MJ-56 was not co-localized with these tracking dyes, suggesting that MJ-56 was not attracted by the mitochondria or lysosomes [Figure 2]. A previous report by Yang et al. demonstrated that another quinazoline derivative, MJ-29 (6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone), functioned as a microtubule-targeting agent.[34] It is possible that MJ-56 with structural similarity to MJ-29 also targets microtubules within the cells. However, the exact target of MJ-56 in BC cells warrants further investigation. To test whether MJ-56 induces phototoxicity in BC cells, we established a treatment protocol involving treating cells with indicated concentrations of MJ-56 for 1, 6, or 24 h. Immediately after treatment, the cells were exposed to a homemade device with six blue-light LED bulbs for 30 s. After refreshing the medium, the cell viability was determined immediately by adding WST-1 reagents and incubated for another hour. In some experiments, posttreatment cells were incubated for another 6 or 24 h with refreshed medium prior to the determination of cell viability. The significant phototoxicity induced by MJ-56 with blue-light exposure was detected in cells at 24 h posttreatment [Figure 3]a and [Figure 3]b. However, the impact of phototoxicity toward immortalized SV-HUC1 urothelial cells was controlled when the treatment duration was shortened from 24 h [Figure 3]d to 1 h [Figure 3]c. When reducing the concentration of MJ-56 or shortening the treatment duration from 24 h [Figure 3]a and [Figure 3]b to 1 h [Figure 3]e and [Figure 3]f, a clear dose-responsive pattern was observed in BC cells. Therefore, it is possible to manipulate the MJ-56-induced phototoxicity by optimal determination of treatments' dose and durations. Furthermore, we found that the proliferation of MJ-56-treated, blue-light exposed cells was completely stopped when using a cell recorder [Figure 4], but the cells were completely wiped out at 7 days posttreatment [Figure 5]. A programed cell death is unlikely to occur in the very short treatment time according to our protocol that cells only received MJ-56 for 1 h. Most of the rapid cell deaths in MJ-56-treated, blue-light-exposed cells may be due to necrotic cell death. We further determined induced apoptosis in the remaining cells at 0, 6, and 24 h posttreatment. The results [Figure 6] showed that MJ-56 with blue-light exposure possesses a dose- and time-dependent induction of apoptosis in the remaining cells. Therefore, even the remaining cancer cells that did not die from necrotic cell death were compromised with the phototoxicity induced by MJ-56 with blue-light treatment.

The limitation of the current report is that the phototoxicity induced by MJ-56 was only detected in thein vitro cultured cells. The subcellular localization, the exact target of MJ56, and the mechanism involving the induced phototoxicity in BC cells are still unclear. However, our results provide the first evidence that a quinazoline derivative, MJ-56, exhibits phototoxicity toward BC cells and has minimal impact on SV-HUC1 uroepithelial cells when the treatment conditions, including the dose of MJ-56 and treatment duration, are optimized. Furthermore, unlike other cancer types, the MJ-56 with blue-light treatment for human BC could be easily adapted for regular intravesical infusion with the current existing cystoscope (narrow-band image) that introduces fluorescent light source into the bladder. Therefore, intravesically introduced MJ-56 with blue-light treatment not only avoids systematic side effects as other chemotherapeutic drugs do, but also provides targeted and controlled effects on BC cells with minimal impact on the adjacent normal cells. We plan to investigate the mechanisms of cell death involved in MJ-56-induced phototoxicity by using not only culture cells but also BC orthotopic model for the possible development of novel therapeutic methods against human BC.

Conclusion

In this study, we described for the first time that MJ-56, a novel quinazoline derivative, emitted green fluorescent and exhibited phototoxicity under blue-light exposure in human BC cells. Further investigation is warranted to explore the possibility of utilizing MJ-56 in the treatment of BC.

Financial support and sponsorship

The research leading to these results received funding from Shin-Kong WHS Memorial Hospital (grand no. SKH-8302-103-0201, SKH-8302-103-0202, and SKH-8302-103-NDR-06) and Ministry of Science and Technology, Taiwan (grand no. NSC102-2314-B-341-003-MY3).

Conflicts of interest

There are no conflicts of interest.

 
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