Ultraviolet Radiation Promoted Hypoxia-Induced Apoptosis in HL-60 Human Promyelocytic Leukemia Cell Line

Minimal residual disease (MRD) is an important reason for the failure of autologous hematopoietic stem cell transplantation (auto-HSCT). Reducing MRD in grafts is particularly important to improve the efficacy of auto-HSCT. Previously, we reported that ultraviolet light-emitting diode (UV LED) suppressed the expression of Bcl-2 to induce apoptosis in HL-60 cells. Leukemia can lead to severe hypoxia of the bone marrow. Therefore, this study aimed to investigate the effect of UV LED on leukemia cells under hypoxia. HL-60 cells were irradiated with a UV LED (30 J/m2) and simulated under hypoxia with cobalt chloride. We found that UV LED irradiation or CoCl2 inhibited proliferation, induced apoptosis, decreased the Bcl-2/Bax ratio, and increased the levels of caspase 3, cleaved-caspase 3, and caspase 9 in HL-60 cells. In particular, the combined application of UV and CoCl2 significantly enhanced the apoptosis of HL-60 cells. In conclusion, UV LED in hypoxia exacerbated the inhibition of proliferation and induction of apoptosis and necrosis in HL-60 cells via the regulation of caspase 3/9 and the Bcl-2/Bax ratio-dependent pathway. The application of UV LEDs in hypoxia conditions may be a promising approach to kill residual drug-resistant leukemia cells in autologous grafts.


Introduction
Leukemia is the most common hematological malignancy in children and with the widespread use of chemical drugs, the prognosis of some children with leukemia has been signifcantly improved. At present, the 5-year overall survival (OS) of acute lymphocytic leukemia (ALL) patients has improved to approximately 90%. Te OS of acute myeloid leukemia (AML) has increased to approximately 60% [1]. However, 10-15% of ALL patients and nearly half of AML patients still require autologous hematopoietic stem cell transplantation (auto-HSCT) for further treatment.
Auto-HSCT was originally used as an alternative treatment for leukemia patients who did not have a suitable donor for allogeneic hematopoietic stem cell transplantation (allo-HSCT). For hematopoietic recovery or hematopoietic reconstruction, auto-HSCT can produce a better antitumor efect than chemotherapy and avoid the side efects of repeated chemotherapy such as organ damage and multidrug resistance [2].
Peripheral blood stem cells (PBSCs) have become the preferred source of stem cells for auto-HSCT, and they are usually collected in the early stages of chemotherapy [3,4]. However, at this time, autologous hematopoietic stem cell transplants tend to contain residual cells that are resistant to chemotherapy, which is known as minimal residual disease (MRD). Tis condition can easily lead to transplantation failure and recurrence after transplantation. Using various purifcation methods to reduce tumor cell contamination in transplants and increase the sensitivity of residual tumor cells in transplants to chemotherapeutic drugs has become an important measure to improve the efcacy of auto-HSCT.
Hypoxia is a common feature of the tumor microenvironment, and it could inhibit tumor cell proliferation and induce apoptosis by afecting the expression of hypoxiainduciblefactor-1α (HIF-1α) and the production of oxygen species (ROS) [5]. In in vitro studies, hypoxia is usually induced by reducing the atmospheric oxygen concentration or by using chemicals such as cobalt chloride (CoCl 2 ) and deferoxamine (DFO) [6,7].
Ultraviolet (UV), especially in the medium wave range (UVB, 280-320 nm), is an important environmental factor afecting human health. Paradoxically, UV-induced immunosuppression may have the therapeutic potential [8]. Clinically, UV has been used to treat skin diseases and tumors. UV is a powerful inducer of apoptosis by inducing DNA damage, reactivation of death receptors, and production of reactive ROS. Ultraviolet light-emitting diodes (LEDs) can replace traditional ultraviolet lamps in the felds of sterilization, water purifcation, and medical treatment [9]. Previously, we reported that irradiation with 30 J/m 2 of a 280 nm UV LED inhibited the proliferation of HL-60 cells and induced apoptosis and necrosis in vitro [10]. However, the efect of 280 nm UV LED irradiation on cells under hypoxia is not clear. In this study, we investigated the efect of 280 nm UV LED irradiation on HL-60 human leukemia cells cultured under hypoxia. Our results showed that the combination of hypoxia and ultraviolet irradiation could signifcantly inhibit the viability of HL-60 human leukemia cells.

Cell Culture and Treatment.
Promyelocytic leukemia HL-60 cells were provided by the American Type Culture Collection (Manassas, VA, USA) and cultured in Iscove's modifed Dulbecco's medium (BI, Logan, UT, USA) containing 10% fetal bovine serum (BI) and 1% penicillin (BI) at 37°C in a 5% CO 2 incubator. Cells in logarithmic growth were selected for the experiments and were divided into the control (without treatment), UV LED (cells were irradiated with 280 nm UV LED (Qingdao Ziyuan Photoelectronic Co., Ltd., Qingdao, China) at a dose of 30 J/m 2 )), CoCl 2 (cells were treated with 150 μN CoCl 2 for 48 h), UV + CoCl 2 (cells were pretreated with 280 nm UV LED at a dose of 30 J/m 2 and then treated with 150 μN CoCl 2 for 48 h), 2UV (cells were irradiated once, and then, irradiated again at the 24th hour), and 2UV + CoCl 2 groups (cells were treated with 150 μN CoCl 2 for 48 h, during which they were irradiated with UV LED twice at an interval of 24 h).
Te lamp head of the LED UV lamp corresponds to the size of one single well of a 24-well plate. HL-60 cells were resuspended with culture medium after centrifugation and seeded in a 24-well plate at a density of 1 × 10 6 cells/well. Te cells were incubated for 1 h and irradiated after the cells formed a single-cell suspension. Irradiation dose (J/m 2 ) � irradiation intensity (W/m 2 ) × time(s). Referring to previous studies, the best irradiation dose was 30 J/M 2 . Te intensity of the UV lamp was measured and adjusted every time, and according to the measurement results, when the irradiation intensity of the lamp head was 400 W/m 2 , the fnal irradiation intensity on the 24-well plate was 22.8 W/m 2 . Te cells treated with UV were irradiated in the 24-well plate, and then, the cells were pipetted evenly and then transferred to the 96-well plate. Each 24-well was repeated 3 times to reduce errors.

Cell Viability Assay. Cell viability was detected by a Cell
Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kyushu, Japan) assays. HL-60 cells were plated in a 96well plate at a density of 1 × 10 4 cells per well. Te cells were treated with cobalt chloride and/or ultraviolet light. Ten, CCK-8 solution was added to each well and the absorbance (OD) values at 450 nm were measured by a microplate reader (Multiskan FC; Termo Fisher Scientifc, Waltham, MA, USA).

Flow Cytometry.
For analysis of apoptosis, cells were stained with annexin V-fuorescein isothiocyanate (FITC)/ propidium iodide (PI) with an annexin V-FITC apoptosis detection kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions. Te samples were analyzed by FC 500 MPL fow cytometry (Beckman Coulter, CA, USA) within 1 h.

RT-qPCR.
Total RNA was isolated from cells using RNAiso Plus (TaKaRa Bio, Shiga, Japan) on ice, and then, the mRNA was reverse transcribed into cDNA using the PrimeScript ™ RT kit and gDNA eraser (TaKaRa Bio, Shiga, Japan). A 20 μL reaction system was generated with SYBR ® Premix Ex Taq II (TaKaRa Bio, Shiga, Japan), and RT-qPCR was performed according to the standard reaction conditions: an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 20 s. Te glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used as an internal reference, and the sequences of the primers (Sangen Biotechnology Shanghai Co., Ltd., Shanghai, China) are shown in Table 1. Te results were analyzed using the 2 -ΔΔCq method.
2.6. Statistical Analysis. Statistical analysis was performed using IBM SPSS V 25 and GraphPad Prism 7. Te data were shown as the mean ± standard deviation, and all experiments were repeated three times. Comparisons among groups were performed using one-way ANOVA followed by the Bonferroni correction for multiple pairwise comparisons. P < 0.05 was considered a signifcant diference.

UV LED and CoCl 2 Inhibited the Viability of HL-60 Cells.
We found that UV and CoCl 2 reduced the viability of HL-60 cells. Te cell viability ranged from high to low as follows: CoCl 2 group, UV group, CoCl 2 + UV group, 2UV group, and CoCl 2 + 2UV group (Figure 1(a)). Tese results indicated that ultraviolet radiation had a signifcant efect on inhibiting cell viability under hypoxia.
Microscopic examination showed that the cells in the control group were arranged neatly, round and transparent, with a complete morphology and high cell density. However, in the treatment group, the cells had a disorderly arrangement, the shrinkage and transparency decreased, and the number of cells was signifcantly lower than that in the control group. A certain proportion of dead cells and lysed cytoplasmic bodies were observed in the 2UV group. Te cells showed the weakest growth in the 2UV + CoCl 2 group, and a large number of dissolved cells were observed (Figures 1(b) and 1(c)).

UV LED and CoCl 2 Induced Apoptotic and Necrotic
Death of HL-60 Cells. We then observed apoptosis in the HL-60 cells treated with ultraviolet light and CoCl 2 . As expected, ultraviolet light and CoCl 2 treatment induced apoptosis of cells. When cells were exposed to both UV and CoCl 2 , the rate of apoptotic cells was higher than that of either alone. Te ratio of apoptotic cells also increased with increasing UV irradiation times. However, when the UV times were increased and CoCl 2 was added, the ratio of both apoptotic and necrotic cells increased signifcantly, indicating that 2UV + CoCl 2 mainly induced apoptosis and necrosis ( Figure 2).

Changes in Bax, Bcl-2, and Caspase 3 mRNA Levels.
To confrm the apoptosis of cells treated by ultraviolet and hypoxia, we detected the expression levels of Bcl-2, Bax, and caspase 3 mRNA using RT-qPCR. We observed that the mRNA expression of Bcl-2 was substantially lower in the CoCl 2 group, UV group, CoCl 2 + UV group, 2UV group, and CoCl 2 + 2UV group than in the control group, but the expression of Bax and caspase 3 increased signifcantly. Te expression of caspase 3 increased in ascending order in the CoCl 2 group, UV group, CoCl 2 + UV group, and 2UV group but was higher than that in the CoCl 2 + 2UV group. Te ratio of Bcl-2/Bax also decreased with the change in exposure (Figure 3).

Discussion
UV is a potent inducer of apoptosis and participates in multiple molecular pathways. UV irradiation induces DNA damage to efectively block replication and transcription [11,12]. DNA damage can lead to the activation of p53; promote the transcription of DNA repair enzymes and antiapoptotic genes, and arrest the cell cycle in the G1 phase. However, when DNA damage cannot be fully repaired, p53 triggers apoptosis and prevents damaged DNA from inducing tumorigenesis [13,14]. UV can induce the aggregation of the death receptors TNF and Fas in a ligandindependent manner and participate in apoptosis [15]. UV can also stimulate the production of ROS, RNS, and free radicals to promote apoptosis [16]. In recent years, LED UV has received extensive attention. Te 280 nm LED UV is located near the strongest UV absorption peak of DNA and nucleoprotein and has a strong killing efect [17]. Our previous study confrmed that LED UV irradiation can inhibit the proliferation of HL-60 cells and induce cell apoptosis and necrosis [10]. UV LED can induce cell cycle arrest in the G0/G1 phase in HL-60 cells [18]. However, the efect of 280 nm LED UV irradiation on HL-60 cells under the hypoxic condition of the bone marrow hematopoietic microenvironment remains to be elucidated.
Oxygen is necessary for tumor cells to maintain cell survival. Te activation of the hypoxia-induciblefactor-1 (HIF-1) signaling pathway plays an important role in the response of tumor cells to hypoxia [19]. CoCl 2 is an iron chelator. As a commonly used chemical hypoxia inducer, CoCl 2 simulates hypoxia by replacing the iron ions in hemoglobin with cobalt ions. Cobalt chloride can simulate hypoxia to induce apoptosis of diferent types of cells [20,21]. In our study, we found that after irradiating HL-60 cells with UV LED and treating HL-60 cells with CoCl 2 to simulate a hypoxic environment, HL-60 cell growth was signifcantly inhibited, and apoptosis and necrosis appeared. Te apoptotic efects of UV LED on HL-60 cells under hypoxic conditions were stronger than those of single exposure. In addition, the cell apoptosis rate under two rounds of UV LED irradiation was signifcantly higher than that under single exposure. However, HL-60 cells mainly died when an irradiation dose of 280 nm LED UV was added under hypoxic conditions.
Te endogenous mitochondrial pathway and the death receptor pathway are the main signaling pathways of apoptosis. Caspase and Bcl-2 family proteins are involved in both apoptotic pathways [22]. Te levels of Bcl-2 and Bax determine the resistance of cells to apoptosis. An elevated Bax/Bcl-2 ratio is associated with apoptosis of cells [23]. In this study, we found an increased Bax/Bcl-2 ratio in cells exposed to CoCl 2 and UV LED, consistent with the changes in apoptosis rate. Furthermore, we detected the expression of caspase 3/9, and the results showed that CoCl 2 and UV upregulated the expression of caspase 3/9. However, irradiating cells twice under hypoxic conditions downregulated protein expression levels and are consistent with the necrotic results. Tis fnding indicates that UV LED may induce apoptosis of HL-60 cells by increasing the expression of Bax, inhibiting the expression of Bcl-2, and activating caspase 3/9 under hypoxic conditions. Te results suggest that it may be related to mitochondria-mediated apoptosis.
In conclusion, the application of UV LEDs under hypoxic conditions can provide a promising approach to kill residual drug-resistant tumor cells in autologous grafts and may reduce the recurrence rate after autologous hematopoietic stem cell transplantation in leukemia patients.

Data Availability
Te data that support the fndings of this study are available from the corresponding authors upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Western blot analysis of protein levels of caspase 3, cleaved-caspase 3 and caspase 9. Te relative protein levels of Bax (c), Bcl-2 (d), caspase 3(e), cleaved-caspase 3 (f ) and caspase 9 (g) in the cells exposed to UV LED and CoCl 2 in diferent groups were compared with those of the untreated cells (set as 1). (h) Te Bcl-2/Bax protein ratio in diferent groups. * P < 0.05 vs. the control.
Journal of Oncology 7