Hyp with a purity of 95% and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cell culture medium (RPMI 1640) and penicillin-streptomycin were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from GE Healthcare Life Sciences (Logan, UT, USA). The Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kyushu, Japan). Rabbit anti-human antibodies against C-Jun N terminal kinase (JNK) (cat. no. 9252, 1:1,000), phosphorylated (p) JNK (cat. no. 4668, 1:1,000), caspase 9 (cat. no. 9508, 1:1,000), caspase 3 (cat. no. 9665, 1:1,000), and cleaved caspase 3 (cat. no. 9664, 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse anti-human anti-GAPDH monoclonal antibody (cat. no. E021010, 1:1,000) was supplied by EarthOX Company (San Francisco, CA, USA). Goat anti-rabbit secondary antibody (cat. no. 103349, 1:12,000) was purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA). RIPA buffer was purchased from Sigma-Aldrich. SDS-PAGE gel and PVDF membrane were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Glutaraldehyde, osmium tetroxide, lead citrate and uranyl acetate were purchased from Rongbai Biological Technology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade.

The K562 human CML cell line K562 was purchased from the Cell Resource Center, Institute of Chinese Academy of Medical Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C, in a 5% CO₂ humidified atmosphere. Exponentially growing cells were used for the following experiments, with a cell density of ~4×10⁵ cells/ml. The stock solution of Hyp (0.8 µg/ml) was prepared by dissolving the compound in DMSO, which was then maintained in the dark at −4°C. The Hyp working solution for each experiment was freshly diluted with DMSO priot to its addition to the K562 suspension; and the final DMSO concentration in all cultures was 0.1%.

A BHY-Y50 W matrix LED lamp (Baihuixiang Electric Co., Ltd., Shenzhen, China), with a maximum power of 50 W, was used as a light source, having a maximum peak value of 595.18 nm (measured by the Department of Physics, Wenzhou University, Wenzhou, Zhejiang, China). The light intensity of the led lamp was adjustable between 0.1 and 40 mW/cm². The K562 suspensions were incubated with Hyp or DMSO (as a control) for 5 h; and were then exposed and irradiated under a matrix led lamp for 4 min.

Cell viability was measured using a CCK8 assay. To examine the effectiveness of Hyp-PDT at moderate light intensity, the cells were seeded into 96-well plates, cultured overnight and exposed to a Hyp solution (0.4 µg/ml); using a similar in vitro Hyp-PDT procedure as that performed previously in the U937 human leukemic monocyte lymphoma cell line (9). Irradiation was performed for 4 min with 0.1, 0.3 and 0.5 mW/cm² light intensities on the sample surface. Subsequently, 10 µl CCK8 solution was added to each well prior to irradiation, 5 min following irradiation, and 4, 8 and 16 h following irradiation, respectively. The cells were incubated for an additional 2 h at 37°C. The optical density (OD) was then measured at 450 nm using a microplate reader (550; Bio-Rad Laboratories, Inc.). Cell viability was calculated as follows: Cell viability = (OD_test − ODblank) / ODcontrol − ODblank) × 100%.

In order to determine the effects of the different Hyp concentrations at various irradiation durations on the K562 cells, a CCK8 assay was performed with a series of Hyp concentrated solutions (0, 0.2, 0.4 and 0.8 µg/ml, respectively) and at various irradiation durations (0, 2, 4 and 8 min, respectively), using a light intensity of 0.3 mW/cm² at different time-points (pre-irradiation, and 5 min, 4, 8 and 16 h post-irradiation).

PDT with 4 min irradiation at a light intensity of 0.3 mW/cm² was applied to the Hyp-treated K562 cells (0.4 µg/ml) for 5 h, and the control cells were treated with DMSO only for 5 h. Cell lysates were then prepared, and total proteins were extracted at various time-points (prior to irradiation, and 4, 8, and 16 h post-irradiation). Western blot analysis was then performed. In brief, the K562 cells were gently washed thee times with cold phosphate-buffered saline (PBS) and then lysed with radioimmunoprecipitation assay lysate buffer containing 1 mmol/l phenylmethylsulfonyl fluoride and 10 mmol/l of NaF for 30 min on ice. The cell lysates were then centrifuged at 7,200 × g for 15 min at 4°C and the supernatants were collected. Equal quantities of protein (40 µg) were separated by SDS-PAGE (8%) and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline with Tween (0.05%; TBST) for 1 h at room temperature. After washing for 15 min with TBST 3 times, the membrane was immunoblotted with antibodies against JNK, p-JNK, caspase 9, caspase 3 and cleaved caspase 3 at 4°C overnight. Subsequently, the membrane was incubated with the secondary antibody for 2 h at room temperature after washing for 15 min with TBST 3 times. The protein of interest was identified using an enhanced chemiluminescence system (GE Healthcare Life Sciences). All experiments were repeated thee times.

Cell morphology was examined under a light microscope and a transmission electron microscope. The K562 cells were first observed under a Leica DMR-HC (Leica Microsystems GmbH, Wetzlar, Germany) upright research microscope, and microscopic images were captured using Openlab imaging software, version 5.5.2 (PerkinElmer, Inc., Waltham, MA, USA). For transmission electron microscopic observation, the cells were fixed with 2% glutaraldehyde and 1% osmium tetroxide; and dehydrated in graded ethanol solutions. Following embedding in TAAB 812 epoxy resin (TAAB Laboratories Equipment, Ltd., Aldermaston, UK) ultrathin 80 nm sections were produced using a Reichert Ultracut E Ultramicrotome (Leica, Vienna, Austria). Following staining with 1% lead citrate and 2% alcoholic uranyl acetate, the sections were examined under a Philips CM-12 electron microscope (Philips, Amsterdam, Netherlands) at 80 kV.

Experimental data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The data are expressed as the mean ± standard deviation of three experiments. The differences between two groups were assessed using Student's t-test; P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A shows the results of the cell viability assessment. The cells were incubated in 0.4 µg/ml Hyp for 5 h, followed by 4 min of irradiation using an LED lamp (maximum peak value, 595.18 nm) at 0.1, 0.3 and 0.5 mW/cm² adjusted light intensities at different time-points (Pre-irradiation; 5 min, and 4, 8 and 16 h post-irradiation). Hyp-PDT with 0.3 and 0.5 mW/cm² light intensities significantly decreased cell viability at different time-points post-irradiation (all P<0.01). However, the decrease in cell viability was only observed 4 h post-irradiation with a 0.1 mW/cm² light intensity (P<0.05). Furthermore, cell viability at low light intensity (0.1 mW/cm²) was marginally increased following longer incubation periods (8 and 16 h post-irradiation); however, the differences were not statistically significant. According to the results above, Hyp-PDT with 0.3 mW/cm² light intensity exhibited significant but moderate cell damage and was selected for the subsequent experiments. No significant changes were observed in the control cells at different time-points (Fig. 1B).

Under PDT conditions of 0.3 mW/cm² light intensity, the cell viability of the samples was analyzed at a series of Hyp concentrations (0, 0.2, 0.4 and 0.8 µg/ml) durations of irradiation (0, 2, 4 and 8 min) and time-points (pre-irradiation; 5 min post-irradiation; and 4, 8 and 16 h post-irradiation). As shown in Fig. 2, neither Hyp without irradiation (Fig. 2A) nor irradiation without Hyp, had any effects on the K562 cells. Overall, Hyp-PDT was observed to affect cell viability in a dose- and irradiation-time-dependent manner; with the lowest cell viability (5%) observed in the cells treated with 0.8 µg/ml Hyp under 8 min of irradiation. However, the cells treated with 0.2 µg/ml Hyp resulted in minimal effects on cell viability (Fig. 2B and C); although moderate effects were observed after 8 min of irradiation (Fig. 2D).

As shown in Fig. 3, morphological observation under a phase-contrast microscope confirmed the cytotoxic effect of Hyp-PDT on the K562 cells. Translucent spheroids with uniform profiles, growing in isolated or scattered colonies, were observed in the control K562 cells treated with DMSO alone (Fig. 3A). Following Hyp-PDT (Fig. 3B and C), the cells began to float on the culture medium and started to lose cell-to-cell contact. It also appeared that the cells underwent cell death and cell intensity decreased, compared with the control cells (Fig. 3D).

Transmission electron microscopy analysis demonstrated normal cell morphology in the control K562 cells treated with DMSO alone (Fig. 4A). The absence of irradiation predominantly reflected intact cell and nuclear membranes and an evident nucleus with nuclear material. Normal organelles were identified in the cytoplasm, consisting of mitochondria, endoplasmic reticulum and lysosomes (Fig. 4B). Following Hyp-PDT (0.4 µg/ml Hyp; 5 h drug light duration; 4 min irradiation with 0.3 mW/cm² light intensity), the cells presented typical apoptotic cell characteristics, including chromatin condensation, agglomeration at the central nuclear area or gathering at the periphery to form crescents, a concentrated cytoplasm, bubble-like protrusions on the cell surface and the formation of apoptotic bodies (Fig. 4C). Apoptotic changes were more frequent over time, and numerous cells exhibiting loss of intracellular detail were clearly observed 16 h after Hyp-PDT (Fig. 4D).

Following incubation for 5 h with 0.4 µg/ml Hyp and irradiating for 4 min with 0.3 mW/cm² light intensity, the expression of cleaved caspase-9 began to appear 4 h post-irradiation; which significantly increased 8 and 16 h post-irradiation. The appearance of the processed fragments was accompanied by a decrease in total capsase-9. Cleaved caspase-3 was observed 8 h post-irradiation and was significantly upregulated at 16 h, with a corresponding decrease of total caspase-3 at 16 h post-irradiation (Fig. 5). Treatment with either Hyp or irradiation alone had no effect on the expression levels of total or cleaved caspase-9 or caspase-3 in the control K562 cells.

In order to investigate the effect of Hyp-PDT on JNK, the protein expression level of JNK and its phosphorylated products were determined prior to irradiation, and 4, 8 and 16 h post-irradiation, respectively. As shown in Fig. 6, Hyp-PDT significantly promoted JNK phosphorylation, the expression of which peaked at 4 h post-irradiation with a subsequent marginal decrease. No significant changes in the protein levels of total JNK were observed in the Hyp-alone treated and control cells.