HL60 human promyelocytic leukemia cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere containing 5/95% CO₂/air at 37°C. All cell culture reagents were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Cells were exposed to blue light (wavelength: 456 nm; storage battery power supply: 12 V; radiation power: 0.25 mW/cm² (Jiangmen Weigu Lighting Technology Co., Ltd., Jiangmen, China) for 10–56 h and to 50 µM of PTX (Jiangsu Yew Pharmaceutical Co., Ltd., Jiangsu, China) for 24 h. Cell viability was analyzed using a quantitative colorimetric assay and the Cell Counting Kit-8 (CCK-8; BestBio, Shanghai, China) according to the manufacturer's instructions.

Cells were exposed to blue light and 50 µM of PTX for 24 h, after which the concentrations of LDH in the culture supernatants were detected using an in vitro Toxicology Assay kit (C0017; Beyotime Biotechnology, Jiangsu, China). Data were expressed in terms of cell mortality, according to the instruction manual.

Cells were exposed to blue light and 50 µM of PTX for 24 h. Subsequently, suspended cells were incubated with Annexin V-FITC and propidium iodide for 10 min at 25°C in darkness, and then analyzed using a Muse^™ Cell Analyzer (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions.

Cells were exposed to blue light and 50 µM of PTX for 24 h, and subsequently incubated with 2 µM of 5,5′, 6,6′-tetrachloro-1,1′, 3,3′tetraethylbenzimidazol-ylcarbocyanine iodide (JC-1; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 20 min at 25°C in darkness. Changes in the levels of red and green fluorescence were detected using a Muse^™ Cell Analyzer (EMD Millipore).

Cells were exposed to blue light and 50 µM of PTX for 24 h, followed by 10 µM of 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) at 37°C for 10 min in darkness. Changes in the levels of green fluorescence were detected using a Muse^™ Cell Analyzer (EMD Millipore).

The animal experimental protocol was approved by the Animal Ethics Committee of Jilin University, and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 8023, 1978 revision). Five-week-old male BALB/c nude mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in groups of three in cages exposed to a 12-h light/dark cycle (lighted from 7:00 a.m. to 7:00 p.m.) at 23±1°C. Water and food were available ad libitum.

HL60 cells were harvested from mid-log-phase cultures, and 0.1 ml of cell suspension (1×10⁸ cells/ml) was subcutaneously (s.c.) injected into the right abdomen of each mouse. When the tumor diameters reached 3–5 mm, the mice were randomly divided into two groups (n=3) and exposed to normal (CTRL) or blue light for nine days. The body weights and tumor dimensions were measured daily, and the tumor volumes (mm³) were calculated as follows: Length × (width)² ×0.5. Finally, the mice were sacrificed via injection with 200 mg/kg of pentobarbital, after which the tumors were dissected and liver and kidney tissues were collected.

Hematoxylin and eosin (H&E) staining was used to assess hepatic and renal histology. Briefly, liver and kidney tissues were fixed in 4% paraformaldehyde for 24 h, and subsequently dehydrated in a stepwise manner using an ethanol gradient (50, 70, 80, 90, 95 and 100%). The dehydrated samples were immersed in xylene for 30 min and incubated overnight in paraffin at 65°C. Once embedded, the specimens were cut into 4-µm-thick slices using a microtome (Leica, Wetzlar, Germany) and placed on microscopy slides. The sections were deparaffinized with fresh xylene for 10 min, hydrated using an ethanol gradient (100, 90, 80 and 70%) and washed thrice with distilled/deionized water. After staining with H&E, the sections were examined using an IX73 inverted microscope with a ×40 objective (Olympus, Tokyo, Japan).

Tumor tissues were lysed using RIPA buffer (Sigma-Aldrich; Merck KGaA) containing a 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) and 2% PMSF (Sigma-Aldrich; Merck KGaA). Forty micrograms of protein per sample were separated on a 12% SDS-PAGE gel. The proteins were then transferred electrophoretically to 0.45 µm nitrocellulose membranes, which were then incubated overnight with primary antibodies against Bcl-2, Bax, Bcl extra-long (Bcl-xL), cytochrome c, cleaved caspases-3 and −9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C (all dilutions: 1:1,000). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 4 h. An enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, UK) was then used to detect the labeled protein bands. The band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

The data are expressed as means ± standard deviations (SD). Statistical significance was detected using a one-way variance analysis (ANOVA), followed by Dunn's test, using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

We first determined the optimal experimental blue light dose by controlling the exposure duration. Blue light reduced the viability of HL60 cells in a time-dependent manner from 12 to 56 h of exposure (P<0.001; Fig. 1A). Both PTX and a 24-h exposure to blue light reduced cell viability by >50% (P<0.001; Fig. 1B). The cell mortality rate was determined by LDH release, which was enhanced significantly in HL60 cells exposed to blue light for 24 h (Fig. 1C).

Apoptosis rates of 81.8 and 68.3% were observed among cells exposed to PTX (50 µM) and blue light, respectively, for 24 h (Fig. 2A). Moreover, compared with the control cells, PTX or blue light treatment led to MMP depolarization rates of 72.5% (63.65 vs. 17.45%) and 65.6% (50.85 vs. 17.45%) (Fig. 2B), which further promoted the apoptosis of HL60 cells.

In addition, ROS-mediated late mitochondrial events (13) were found to be strongly enhanced in HL60 cells exposed to blue light and PTX treatment (Fig. 2C).

Blue light exhibited significant antitumor activity against HL60-xenografted tumors in a nude mouse model. A 9-day exposure to blue light strongly suppressed tumor growth without affecting the body weights of the mice, indicating that blue light has few side effects in normal tissues (Fig. 3A-C). A histopathological study in which few pathologic changes were observed between the livers and kidneys of blue-light exposed and non-exposed mice further confirmed the safety of blue light therapy (Fig. 3E and F). Sixteen days after tumor implantation, the tumors began to grow rapidly in the non-treated control mice, reaching a mean tumor volume of 975.5±283.4 mm³. In contrast, tumor growth was strongly inhibited in blue light-treated mice, beginning on day 7 (P<0.05; Fig. 3D).

Compared with the non-treated mice, the tumor tissues of the blue light-treated mice exhibited significantly reduced expression of Bcl-2 and Bcl-xL and enhanced expression of Bax, cytochrome c and cleaved caspases-3 and −9 (P<0.05; Fig. 4). The Bcl-2/Bax ratio was 13.8% higher in the tumor tissues of the blue light-exposed mice, compared to the non-exposed mice (Fig. 4).