Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2019.10756 mmr-20-06-5064 Articles Antitumor effects and mechanisms of 1,25(OH)2D3 in the Pfeiffer diffuse large B lymphoma cell line HanJing 1 2 TangYonghong 2 ZhongMeizuo 2 WuWenlin 3 Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China Department of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong 510000, P.R. China Correspondence to: Professor Meizuo Zhong, Department of Oncology, Xiangya Hospital, Central South University, 88 Xiangya Road, Changsha, Hunan 410011, P.R. China, E-mail: zhongmeizuo_csu@csu.edu.cn Dr Wenlin Wu, Department of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, 9 Jinsui Road, Tianhe, Guangzhou, Guangdong 510000, P.R. China, E-mail: wenlinwu@csu.edu.cn 122019 17102019 20 6 5064 5074 08102018 06082019 Copyright: © Han et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma in China. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been shown to possess significant antitumor potential and is degraded by 25-hydroxyvitamin D-24-hydroxylase (CYP24A1). In the present study, the role of CYP24A1 and autophagy, and their underlying mechanisms in the anticancer effects of 1,25(OH)2D3 in DLBCL cells, were investigated. It was found that the levels of CYP24A1 in DLBCL lymph node tissues were higher than in hyperplasia lymphadenitis tissue. Moreover, the expression of CYP24A1 was positively associated with the Ann Arbor stage and the International Prognostic Index in patients with DLBCL, and negatively associated with the clinical response to treatment. Patients >60 years of age were found to have a higher level of CYP24A1. 1,25(OH)2D3 inhibited the proliferation of the Pfeiffer DLBCL cell line and increased the G1 phase population of Pfeiffer cells. Rapamycin (RAPA) in combination with 1,25(OH)2D3 increased the G1 phase distribution of Pfeiffer cells. Furthermore, RAPA blocked the increase of CYP24A1 and vitamin D receptor (VDR) expression induced by 1,25(OH)2D3. 1,25(OH)2D3 induced the formation of autophagosomes, increased the expression of autophagy related protein light chain (LC)3II/LC3I and reduced the expression of the ubiquitin binding protein P62. In addition, 1,25(OH)2D3 decreased the phosphorylation of AKT and mammalian target of RAPA (mTOR), and downstream targets eukaryotic translation imitation factor 4E-binding protein 1 and ribosomal protein S6 kinase β-1 in Pfeiffer cells. The results from the present study suggested that CYP24A1 may be a novel prognostic indicator for DLBCL. 1,25(OH)2D3 inhibited proliferation and induced autophagy of Pfeiffer cells. In addition, 1,25(OH)2D3 increased the G1 phase population of Pfeiffer cells. These effects may be mediated by inhibition of the AKT/mTOR/PI3K signaling pathway. RAPA increased the cell cycle arrest induced by 1,25(OH)2D3 by blocking the upregulated expression of CYP24A1 and VDR.

 1,25(OH)2D3 autophagy DLBCL mTOR CYP24A1 Introduction

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma (1) and accounts for ~40% of newly diagnosed cases of non-Hodgkin lymphoma annually in China (2). DLBCL is an aggressive form of lymphoma, with a highly variable clinical manifestation, histology, outcome and prognosis (1). Despite the fact that patients with DLBCL who receive comprehensive treatment, including radiotherapy, chemotherapy and combined therapy with molecular targeted therapy, have a high chance of achieving partial or complete remission, some patients are resistant to first line therapy or relapse, leading to reduced survival rates, psychological and physical pain (1). Furthermore, the medical cost of DLBCL is high (1). Therefore, identifying new strategies for the treatment of DLBCL is needed.

B lymphocyte receptor (BCR) signaling is important for B cell lymphoma proliferation (3). BCR signaling can activate the PI3K signaling pathway, leading to the production of phosphatidylinositol 3,4,5-triphosphate, which initiates a large number of signaling cascades, including those involving serine/threonine kinase AKT (4). This can lead to uncontrolled growth by inhibiting the function of the cell cycle inhibitors p21 and p27 (5–7). AKT can indirectly activate mammalian target of rapamycin (mTOR), a serine/threonine kinase downstream of the PI3K/AKT pathway (8). Activated mTOR can increase the phosphorylation levels of the translational repressor eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase β-1 (p70S6K), which increases mRNA translation, protein synthesis and cell proliferation (9,10). Therefore, mTOR is able to influence cell cycle progression and the homeostasis of metabolism, and enhance cell growth. The PI3K/AKT-mTOR axis also plays an important role in the negative regulation of autophagy (11). Previous studies have reported the aberrant activation of the PI3K/AKT/mTOR pathway, and its role in controlling tumor cell proliferation and survival in DLBCL (4,8). Moreover, patients with DLBCL with active PI3K/AKT/mTOR signaling experience a more rapid deterioration, with poor treatment response and shortened survival times (12). The suppression of active PI3K/AKT/mTOR signaling has been studied, and several inhibitors have been discovered; mTOR inhibitors have been shown to be effective in preclinical and clinical settings for the treatment of autoimmune diseases, cancer and infectious diseases, such as HIV (13–24). Furthermore, dual inhibitors of PI3K and mTOR, including GSK2126458A and NVP-BEZ235, are being developed for cancer that show promising chemotherapeutic profiles in different settings (25–27). However, the mTOR inhibitor rapamycin (RAPA) and other analogues, such as everolimus, exhibit various side effects, including stomatitis, rashes, myelosuppression and metabolic complications, such as hyperglycemia, hypertriglyceridemia, hypercholesterolemia and fatigue (28,29). In the case of serious side effects, dose adjustment, interruption or permanent discontinuation are required to reduce the toxicity of these medications (28). The combination of RAPA or other analogues with additional medications to reduce toxicity may be an effective way to treat cancer.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has an important role in bone homeostasis and calcium metabolism (30,31). Furthermore, 1,25(OH)2D3 has been shown to have antiproliferative and proapoptotic effects, and promote differentiation in cancer cells, including pancreatic, breast and prostate cancer (32–38). 1,25(OH)2D3 inhibits the mTOR signaling pathway by stimulating the expression of DNA damage-inducible transcript 4 (DDIT4), a potent suppressor of mTOR activity (39). This may a novel strategy for the treatment of cancer. 1,25(OH)2D3 induces autophagy in SH-SY5Y cells to attenuate rotenone-induced neurotoxicity (40) and inhibits the proliferation of keratinocytes by suppressing the mTOR signaling pathway (41). However, whether 1,25(OH)2D3 inhibits the progression of DLBCL by suppressing the mTOR signaling pathway is unclear.

The concentration of 1,25(OH)2D3 available in tissues depends on the balance between its synthesis and degradation, carried out by 25-hydroxyvitamin D-1 α hydroxylase and 25-hydroxyvitamin D-24-hydroxylase (CYP24A1), respectively. A previous study found that the basal expression of CYP24A1 was higher in several tumor types, including colon, breast, lung, ovarian and cervical tumors (42). The high expression of CYP24A1 was found to be associated with the increased expression of replication licensing factors and tumor progression (43–47). However, little is known concerning the relationship between the clinical features of patients with DLBCL and the expression of CYP24A1, or how the antitumor effect of 1,25(OH)2D3 in DLBCL is influenced by the expression of CYP24A1.

The present study aimed to investigate the relationship between the clinical features of patients with DLBCL and the expression of CYP24A1. In addition, the roles of the PI3K/AKT/mTOR signaling pathway and autophagy in the anticancer effects of 1,25(OH)2D3 in DLBCL cells were investigated.

 Materials and methods <sec> <title>Reagents

1,25(OH)2D3 and RAPA were purchased from Sigma-Aldrich; Merck KGaA. 3-Methyladenine was purchased from Selleck Chemicals. The FITC Annexin V Apoptosis Detection kit, propidium iodide (PI) and ribonuclease A (RNase A) were purchased from BD Biosciences. The Cell Counting Kit-8 (CCK-8) was purchased from 7Sea Biotech. The ECL Western kit was purchased from Advansta, Inc. The following primary antibodies were purchased from Cell Signaling Technology, Inc.: mTOR (cat. no. 2972), phosphorylated (p)-mTOR (Ser2448; cat. no. 2971), AKT (cat. no. 4691), p-AKT (Ser473; cat. no. 4060), p-4EBP1 (Thr37/46; cat. no. 2855), 4E-BP1 (cat. no. 9452), p-p70S6K (Thr389; cat. no. 9234), p70S6K (cat. no. 2708), ubiquitin binding protein P62 (P62; cat. no. 8025), LC3I/II (cat. no. 4108). The goat monoclonal antibody against CYP24A1 was purchased from Abcam (cat. no. ab189322). A rabbit monoclonal antibody against the vitamin D receptor (VDR) was purchased from Santa Cruz Biotechnology (cat. no. sc-13133), Inc. α-Tubulin was purchased from ProteinTech Group (cat. no. 11224-1-AP), Inc. Horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (cat. no. A-11001) and HRP-conjugated goat-anti-rabbit IgG (cat. no. A-11034) were obtained from Thermo Fisher Scientific, Inc. 1,25(OH)2D3 was dissolved and stored in ethanol and RAPA was dissolved and stored in DMSO at −20°C. A biotinylated goat-anti-mouse antibody (cat. no. PV-9003) was purchased from Origene Technologies, Inc. A fluorescein-conjugated goat anti-rabbit secondary antibody (cat. no. Andy Fluor™ 594) was purchased from GeneCopoeia, Inc. Fetal calf serum was purchased from Biological Industries.

 Clinical specimens

In total, 57 formalin-fixed (overnight at 4°C), paraffin-embedded (FFPE) DLBCL lymph node tissues from diagnostic biopsies (age, 31–70 years; male:female, 30:27) and 21 FFPE lymphnoditis lymph node tissues from diagnostic biopsies (age, 35–66 years; male:female, 12:9) were collected. All the samples were obtained between January 2010 to June 2013 from Xiangya Hospital. The Institutional Ethical Review Board of Xiangya Hospital approved the present study and written informed consent was obtained from all patients for the use of their biopsy samples. No patient had received any antitumor treatments before the biopsy sample was collected. The Ann Arbor staging system was used to assess the stage of the patient and the International Prognostic Index (IPI) was used to assess the risk of the patient (48). The serum lactate dehydrogenase (LDH) level was measured by clinical laboratory staff using the Hitachi 7170 biochemical analyzer (Hitachi, Ltd.).

 Cell culture

The human Pfeiffer DLBCL cell line was purchased from the American Type Culture Collection (cat. no. ATCC® CRL-2632™) and cultured in RPMI-1640 (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and streptomycin (HyClone; GE Healthcare Life Sciences) at 37°C in a humidified 5% CO2 incubator. Cells were passaged every 2–3 days to maintain a density between 1–2×106 cells/ml.

 Immunohistochemistry

Sections (thickness, 4 mm) obtained from the 57 FFPE DLBCL specimens were deparaffinized, rehydrated and the endogenous peroxidase activity was blocked using 3% H2O2 in methanol for 15 min at room temperature. The sections were microwaved for 4 min with trisodium citrate dihydrate solution (0.125%, pH 6.0) and then soaked with PBS three times for 5 min for citrate-mediated high-temperature antigen retrieval. Non-specific binding was blocked by pre-incubating with 5% BSA for 30 min at room temperature. Sections were incubated with anti-CYP24A1 (1:300) at 4°C overnight and then incubated with a biotinylated secondary antibody (1:5,000) for 30 min at room temperature. Sections were incubated with a streptavidin-HRP complex (Origene Technologies, Inc.) at room temperature for 5 min and 3,3′-diaminobenzidine (Origene Technologies, Inc.) at room temperature for 5 min, sections were then counterstained with hematoxylin at room temperature for 20. Positive cells were counted in 10 randomly selected fields with a ×40 objective (Olympus CX23; Olympus Corporation). All sections were scored by two pathologists. The staining index was calculated as the product of staining intensity (Score: 0, no staining; 1, weak/light yellow; 2, moderate/yellow-brown; 3, strong/brown). The number of stained cells was scored as 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). The sum of the intensity and number scores was used as the final staining score (0–7). Low expression was defined as a final staining score of ≤3. High expression was defined as a final staining score >3.

 Cell viability assay

Pfeiffer cells in exponential phase growth were plated at a density of 1×104 cells/well in 96-well plates. The wells were divided into three groups (five wells/group): Control group (0.1% ethanol); 10 nM 1,25(OH)2D3 group; 100 nM 1,25(OH)2D3 group. The concentrations of 1,25(OH)2D3 used were similar to previous studies (49,50). Cytotoxicity was determined using the CCK-8 assay, according to the manufacturer's instructions. After 48 h of treatment, cells were incubated with 10 µl CCK-8 reagent for a further 3 h. The optical density of the samples was determined using a microplate reader at 450 nm. Each experiment was performed in triplicate.

 Cell cycle analysis

Pfeiffer cells were plated into 6-well plates at a density of 3×105 cells/well and divided into six groups: Control group (0.1% ethanol); 10 nM 1,25(OH)2D3; 100 nM 1,25(OH)2D3; 10 nM RAPA; combination group (10 nM or 100 nM 1,25(OH)2D3 + 10 nM RAPA). After incubation for 48 h, the cells were washed twice with PBS and fixed with ice-cold 70% ethanol at −20°C overnight. Subsequently, the cells were centrifuged at 111.8 × g for 5 min at 4°C, washed once with PBS and the DNA was labeled with 0.5 ml cold PI solution (0.1% Triton X-100, 0.1 mM EDTA, 50 µg/ml RNase A, 50 µg/ml PI in PBS) on ice for 30 min in the dark. The cell cycle distribution was determined using a FACSCalibur flow cytometer (BD Biosciences) using ModFit LT software version 3.0 (Verity Software House).

 LC3II immunofluorescence staining

Cells were fixed for 15 min with 4% paraformaldehyde at 4°C and permeabilization with 0.25% Triton X-100 in PBS for 5 min at room temperature. After blocking with fetal calf serum for 1 h at room temperature, the slides were incubated with an LC3I/II primary antibody (1:200) overnight at 4°C. The slides were then incubated with a fluorescein-conjugated goat anti-rabbit secondary antibodies (1:1,000) for 1 h at 37°C. The nuclei of cells were stained using DAPI (1 µg/ml) at room temperature. The fluorescent staining was imaged using a confocal microscope (IX71; Olympus Corporation). In total, 10 random fields were selected for analyzed (magnification, ×400 and ×600).

 Transmission electron microscopy

To morphologically observe the induction of autophagy in 1,25(OH)2D3 treated Pfeiffer cells, ultrastructural analysis was performed. After treatment with 100 nM 1,25(OH)2D3 for 48 h, cells were washed twice with PBS and fixed with ice-cold glutaraldehyde (3% in 0.1 M cacodylate buffer, pH 7.4) for 30 min. Cells were post-fixed in 1% osmium tetroxide at room temperature for 2 h and embedded in Epon812 (SERVA Electrophoresis GmbH) before being cut (60 nm) and stained with 2% uranyl acetate/2.5% lead citrate and incubated for 15 min at room temperature. The formation of autophagosomes was assessed using the Tecnai electron microscope (FEI; Thermo Fisher Scientific, Inc.) at a magnification of ×1,700 and ×5,000.

 Western blot analysis

Proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology). Protein concentrations were determined using the bicinchoninic acid method. Proteins (30 µg) were separated using 6% SDS-PAGE under reducing conditions and then transferred onto PVDF membranes. The membranes were blocked in 5 mg/ml BSA for 2 h at room temperature. The following primary antibodies were used: VDR (1:1,000), CYP24A1 (1:1,000), AKT (1:1,000), p-AKT (1:1,000), mTOR (1:1,000), p-mTOR (1:1,000), LC3II (1:1,000) and P62 (1:1,000), P70S6K (1:1,000), p-p70S6K (1:1,000), 4EBP1 (1:1,000) and p-4EBP1 (1:1,000). α-Tubulin (1:5,000) was used as a loading control. The primary antibodies were incubated with the membranes overnight at 4°C. HRP-conjugated secondary antibodies (1:5,000) were incubated with the membranes at room temperature for 1–2 h. Protein bands were visualized using ECL and the Chemi Doc™ MP Imaging System (Bio-Rad Laboratories, Inc.). Band densitometry was assessed using ImageJ software 1.8.0 (National Institutes of Health).

 Statistical analysis

All statistical analysis was performed using SPSS 22.0 software (IBM Corp.). Data are present as the mean ± SD. Data were analyzed using the χ2 test, Student's t-test, or one- or two-way ANOVA and the Tukey's test when applicable. Data were representative of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Expression of CYP24A1 is different in patients with DLBCL and lymphnoditis

To examine whether the expression of CYP24A1 protein in DLBCL and lymphnoditis tissues, immunohistochemistry was performed on the 57 paraffin-embedded DLBCL lymph node tissue sections and 21 lymphnoditis lymph node tissue sections to evaluate the expression of CYP24A1 (Fig. 1A-D). High levels of CYP24A1 expression were detected in 9.5% of patients with lymphnoditis (n=21), whereas high levels of CYP24A1 expression were detected in 35.1% of patients with DLBCL (n=57). Low CYP24A1 expression was detected in 90.5% of patients with lymphnoditis, while low CYP24A1 expression was detected in 74.9% of patients with DLBCL. The expression levels of CYP24A1 between patients with lymphnoditis and DLBCL was significantly different (P<0.001). The patients with DLBCL were stratified into different groups, using factors such as age and sex, to investigate associations with the expression of CYP24A1 (Table I). There was no significant different in the expression of CYP24A1 between the Ann Arbor stage I/II patients and the Ann Arbor stage III/IV patients (P=0.28), however, the expression of CYP24A1 was significantly higher in patients with a higher IPI (P<0.05). Serum LDH levels were also significantly associated with CYP24A1 expression. The patient whose clinical treatment response was stable or showed complete remission had a lower level of CYP24A1 (P<0.05). The expression of CYP24A1 was significantly higher in patients over the age of 60 (P<0.05).

 1,25(OH)<sub>2</sub>D<sub>3</sub> inhibits the proliferation of Pfeiffer cells

To investigate the growth inhibitory properties of 1,25(OH)2D3, CCK-8 assays were performed, which is based on the ability of viable cells to reduce CCK-8 to formazan crystals. As shown in Fig. 1E, Pfeiffer cell proliferation was significantly inhibited by 10 nM (P<0.01) and 100 nM 1,25(OH)2D3 (P<0.001). The rate of inhibition was calculated as 16.8±3.4% for cells treated with 10 nM 1,25(OH)2D3 and 28.2±2.9% for cells treated with 100 nM 1,25(OH)2D3 for 48 h.

 1,25(OH)<sub>2</sub>D<sub>3</sub> induces cell cycle arrest in Pfeiffer cells

To determine whether 1,25(OH)2D3 induces cell cycle arrest in Pfeiffer cells, the effect of 1,25(OH)2D3 on the cell cycle distribution was analyzed using PI staining. Pfeiffer cells were treated with different concentrations of 1,25(OH)2D3 for 48 h and then subjected to cell cycle analysis. As shown in Fig. 2A and B, 22.70±0.40% of cells in the control group were in the G1 phase, while the cells treated with 100 nM 1,25(OH)2D3 showed a significant increase in the proportion of cells in the G1 phase (26.11±0.64%; P<0.05). In addition, whether 1,25(OH)2D3 increased the G1 phase arrest induced by RAPA was investigated. It was found the addition of RAPA increased the number of cells in the G1 phase compared with 1,25(OH)2D3 treatment alone (Table II; Fig. 2A and B).

1,25(OH)2D3 induced the expression of CYP24A1 mediated by VDR, which is the receptor of 1,25(OH)2D3, and CYP24A1 causes the degradation of 1,25(OH)2D3. This is an important regulatory mechanism to control the level of 1,25(OH)2D3 (30,31). In order to investigate whether RAPA increases the cell cycle arrest induced by 1,25(OH)2D3 by reducing the degradation of 1,25(OH)2D3, the levels of CYP24A1 and VDR were determined in Pfeiffer cells after exposure to either 1,25(OH)2D3 or RAPA using western blot analysis. It was found that RAPA suppressed the expression of CYP24A1 and VDR induced by 1,25(OH)2D3 (Fig. 2C and D). This suggested that RAPA increased the cell cycle arrest in Pfeiffer cells induced by 1,25(OH)2D3 by suppressing the expression of CYP24A1 and VDR.

 1,25(OH)<sub>2</sub>D<sub>3</sub> induces autophagy in Pfeiffer cells

In order to investigate whether 1,25(OH)2D3 regulates autophagy in Pfeiffer cells, the induction of autophagy was analyzed. The induction of autophagy in Pfeiffer cells after treatment with or without 1,25(OH)2D3 (10 nM, 100 nM) for 48 h was analyzed. The extent of autophagosome formation is associated with the modification of LC3I to LC3II and the degradation of P62. A significant increase in LC3II/LC3I and a reduction in P62 was induced by 100 nM 1,25(OH)2D3, as assessed using western blot analysis (Fig. 3C and D). Furthermore, the aggregation and localization of LC3 plays an important role in the maturation and transport of the autophagosome; therefore, immunofluorescence experiments were performed to observe changes in LC3 aggregation. A notable increase in green LC3 puncta was observed in the 100 nM 1,25(OH)2D3 treatment group compared with the control group (Fig. 3B). In addition, it was found that 100 nM 1,25(OH)2D3 enhanced the formation of autophagosomes, as observed using transmission electron microscopy (Fig. 3A). These findings indicated that 1,25(OH)2D3 activates autophagy in Pfeiffer cells.

 1,25(OH)<sub>2</sub>D<sub>3</sub> inhibits the AKT/mTOR signaling pathway

Having established that 1,25(OH)2D3 inhibited proliferation and induced autophagy in Pfeiffer cells, the molecular mechanisms underlying these biological effects were investigated. Activation of the AKT/mTOR signaling pathway increases the proliferation of Pfeiffer cells and is one of the major targets in the process of autophagy (51). Western blot analysis showed that p-AKT and p-mTOR were downregulated after 1,25(OH)2D3 exposure. The activation of downstream targets of mTOR, including p70S6K and 4EBP1, were also decreased following 1,25(OH)2D3 treatment (Fig. 3E and F).

 Discussion

In the present study, it was found that the levels of CYP24A1 in DLBCL lymph node tissues were higher than in lymphnoditis tissues. There was no significant difference in the expression of CYP24A1 between Ann Arbor stage I/II patients and Ann Arbor stage III/IV patients (P=0.28), however, the expression of CYP24A1 was significantly higher in patients with a higher IPI. Moreover, CYP24A1 expression was negatively associated with clinical treatment response. Patients over the age of 60 had a higher level of CYP24A1 expression. Furthermore, it was found 1,25(OH)2D3 inhibited proliferation and increased the G1 phase population of Pfeiffer cells. RAPA led to a further increase in the G1 population and decreased the expression of CYP24A1 and VDR induced by 1,25(OH)2D3. 1,25(OH)2D3 induced the formation of autophagosomes and increased the levels of the autophagy related protein LC3II/LC3I and reduced the expression of P62. In addition, 1,25(OH)2D3 decreased the phosphorylation of AKT, mTOR, and its downstream targets 4EBP and p70S6K in Pfeiffer cells.

To the best of our knowledge, the present study was the first to show higher levels of the vitamin D-degrading enzyme CYP24A1 in DLBCL tissues compared with lymphnoditis tissues, which was used as a normal control for the lymphoma tissues. CYP24A1 degrades 1,25(OH)2D and 25(OH)D, which is a substrate required for the synthesis of 1,25(OH)2D. Therefore, CYP24A1 is an important factor in determining the local concentration of 1,25(OH)2D and regulates the antiproliferative effect of 1,25(OH)2D (52). The higher expression of CYP24A1 may be one explanation for the significantly lower serum concentration of 25(OH)D in patients with DLBCL (53). In addition, previous studies have described the moderate impact of 1,25(OH)2D on the proliferation of certain DLBCL cell lines, including DOHH2 and K442 (50,49). The present study found higher levels of CYP24A1 expression in DLBCL lymph node tissues, suggesting that the actual concentration of vitamin D available and the concentration of 1,25(OH)2D in these tissues may be low, and that this may attenuate the antitumor effects of 1,25(OH)2D in vivo. The increased expression of CYP24A1 was also found in some solid tumors, including colon, lung, prostate, thyroid, breast and ovarian cancer cells (43–47). However, little is known regarding the mechanism underlying the increased expression of CYP24A1 in these cancer cells. Understanding the mechanism that leads to the upregulation of CYP24A1 in tumors may provide strategies for targeting CYP24A1 in the future. A combination of CYP24A1 inhibitors, such as liarozole, genistein [a soy isoflavone that directly inhibits CYP24A1 activity and increases the sensitivity of cells to 1,25(OH)2D] or ritonavir (which inhibits the expression of CYP24A1), may increase the level and calcemic activity of 1,25(OH)2D, increasing the risk of hypercalcemic side effects (54,55). Structural non-calcemic action analogs of calcitriol that resist CYP24A1 may be more biologically active and more useful in cancer therapy and for the treatment of DLBCL, which expresses a high level of CYP24A1.

In the present study, it was found that 1,25(OH)2D3 inhibits the proliferation of DLBCL Pfeiffer cell line. In a previous study into the antiproliferative effect of 1,25(OH)2D3 in other DLBCL cell lines, 1,25(OH)2D3 was found to have an antiproliferative effect on the Pfeiffer cell line (50).

CYP24A1, which degrades 1,25(OH)2D3 and 25(OH)D, is important for lowering the levels of 1,25(OH)2D3 and 25(OH)D. Vitamin D deficiency is a risk factor for autoimmune diseases, such as rheumatoid arthritis and system lupus erythematosus (SLE) (56,57). A recent study reported that vitamin D deficiency and the upregulation of CYP24A1 have a combined role in the transition to SLE (58). Patients with autoimmune diseases, in particular those mediated by B lymphocytes, have a higher risk of developing DLBCL, with a more aggressive nature, possibly due to the misregulated production of several cytokines, including interleukin (IL)-6, IL-10 and tumor necrosis factor-α, that contribute to the pathogenesis of DLBCL (59–62). 1,25(OH)2D3 also plays an important role in the regulation of immune function by inhibiting the production of cytokines (63,64). In future research, whether the increased production of cytokines contribute to the pathogenesis of DLBCL, and whether this is associated with the upregulation of CYP24A1, should be investigated. Furthermore, whether inhibiting the production of cytokines using 1,25(OH)2D3 is beneficial for the treatment of some cases of DLBCL should be investigated in in vivo studies.

Autophagic cell death is a survival mechanism to deal with metabolic stress and is a caspase-independent mechanism of cell death (65). Nevertheless, previous studies have indicated that autophagy and apoptosis may be interconnected process (66,67). The present study found that 1,25(OH)2D3 induced autophagy in Pfeiffer cells. These results suggested that 1,25(OH)2D3 induced cell killing in a caspase-independent autophagy-mediated manner in Pfeiffer cells.

Activation of the PI3K/AKT/mTOR signaling pathway suppresses autophagy (68,69). The data from the present study showed that 1,25(OH)2D3 decreased the phosphorylation levels of AKT and mTOR in Pfeiffer cells, consistent with a previous study that found that 1,25(OH)2D3 is involved in mTOR signaling (70). These results suggested that 1,25(OH)2D3 induces autophagy in Pfeiffer cells by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, the PI3K/AKT/mTOR signaling pathway also increases mRNA translation, protein synthesis and cellular proliferation (9,10). The aberrant activation of mTOR is frequently associated with poorer prognosis and has been well described in non-Hodgkin lymphoma (4,71–73). Suppressing mTOR in Pfeiffer cells may extend the therapeutic applications of 1,25(OH)2D to the treatment of DLBCL. A previous study reported that 1,25(OH)2D3 inhibits the mTOR signaling pathway by stimulating the expression of DDIT4, a potent suppressor of mTOR activity (39). The present study showed that 1,25(OH)2D3 decreased the phosphorylation of downstream factors in the PI3K/AKT/mTOR signaling pathway in Pfeiffer cells, including 4EBP and p70S6K. p70S6K is an important regulator of protein synthesis; blocking the activation of p70S6K, for example using Saquinavir-NO, interrupts protein synthesis, leading to decreased cancer cell proliferation (74–77). The inhibition of Pfeiffer cell proliferation by 1,25(OH)2D3 may be due to the decreased phosphorylation of p70S6K. In future studies, the anticancer effects of other p70S6K inhibitors should be investigated in DLBCL, and their potential synergism with 1,25(OH)2D3 should be tested.

A previous study reported that the mTOR signaling inhibitor RAPA and its analogs increase the antitumor effects of 1,25(OH)2D in breast cancer cells and acute myelogenous leukemia (78,79). This effect of RAPA and its analogs may be due to the increased expression, or nuclear translocation, of VDR (79). Consistent with these previous studies, the data from the present study showed that 1,25(OH)2D3 increased the G1 phase population of Pfeiffer cells and that this was potentiated by RAPA. However, it was found that RAPA blocked the increase in VDR and CYP24A1 expression induced by 1,25(OH)2D3. 1,25(OH)2D3 induced the expression of CYP24A1, mediated by VDR, which is the receptor of 1,25(OH)2D3, and CYP24A1 causes the degradation of 1,25(OH)2D3. This is an important autoregulatory mechanism of 1,25(OH)2D3. RAPA may potentiate the effect of 1,25(OH)2D3 by reducing its degradation.

In conclusion, the results of the present study suggested that the expression of CYP24A1 may be a novel prognostic indicator for DLBCL. 1,25(OH)2D3 inhibited the proliferation, and induced the autophagy, of Pfeiffer cells. In addition, 1,25(OH)2D3 increased the G1 phase population of Pfeiffer cells. These effects may be mediated by inhibiting the PI3K/AKT/mTOR signaling pathway. RAPA may potentiate the cell cycle arrest caused by 1,25(OH)2D3 by inhibiting the expression of CYP24A1 and VDR.

 Acknowledgements

Not applicable.

 Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 81570200) and was partially supported by the fund from Guangzhou Institute of Pediatrics/Guangzhou Women and Children's Medical Center (grant no. YIP-2018-005). The funders had no role in the study concept, study design, data analysis, interpretation or reporting of the results. The authors had full control of the data and information submitted for publication.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

WW and JH contributed to the design of the study and were involved in performing the experiments, data analysis and the preparation of the manuscript. YT contributed to the data analysis. MZ contributed to data analysis and manuscript preparation. All authors contributed to the data interpretation and approved the final version of the manuscript. All authors read and approved the manuscript.

 Ethics approval and consent to participate

The Institutional Ethical Review Board of the Xiangya Hospital approved the present study, and written informed consent was obtained from all patients for the use of their biopsy samples.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 Abbreviations 1,25(OH)2D3

1,25-dihydroxyvitamin D3

 4EBP

eukaryotic translation imitation factor 4E-binding protein 1

 CYP24A1

25-hydroxyvitamin D-24-hydroxylase

 DLBCL

diffuse large B cell lymphoma

 IPI

International Prognostic Index

 LC3

Protein light chain 3

 mTOR

mammalian target of rapamycin

 FFPE

Paraffin-embedded

 RAPA

rapamycin

 VDR

vitamin D receptor

 References LiSYoungKHMedeirosLJDiffuse large B-cell lymphomaPathology507487201810.1016/j.pathol.2017.09.00629167021 LiJMWangLShenYXiaZGChenYChenQSChenYZengXYYouJHQianYShenZXRituximab in combination with CHOP chemotherapy for the treatment of diffuse large B cell lymphoma in Chinese patientsAnn Hematol86639645200710.1007/s00277-007-0320-817572895 HeFWangLHuXBYinDDZhangPLiGHWangYCHuangSYLiangYMHanHNotch and BCR signaling synergistically promote the proliferation of Raji B-lymphoma cellsLeuk Res33798802200910.1016/j.leukres.2008.09.01618937977 UddinSHussainARSirajAKManogaranPSAl-JomahNAMoorjiAAtizadoVAl-DayelFBelgaumiAEl-SolhHRole of phosphatidylinositol 3′-kinase/AKT pathway in diffuse large B-cell lymphoma survivalBlood10841784186200610.1182/blood-2006-04-01690716946303 LiangJZubovitzJPetrocelliTKotchetkovRConnorMKHanKLeeJHCiaralloSCatzavelosCBenistonRPKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrestNat Med811531160200210.1038/nm76112244302 Héron-MilhavetLFranckhauserCRanaVBerthenetCFisherDHemmingsBAFernandezALambNJOnly Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 bindingMol Cell Biol2682678280200610.1128/MCB.00201-06169826991636765 BrazilDPYangZZHemmingsBAAdvances in protein kinase B signalling: AKTion on multiple frontsTrends Biochem Sci29233242200410.1016/j.tibs.2004.03.00615130559 CaiSLTeeARShortJDBergeronJMKimJShenJGuoRJohnsonCLKiguchiKWalkerCLActivity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioningJ Cell Biol173279289200610.1083/jcb.200507119166361472063818 CantleyLCThe phosphoinositide 3-kinase pathwayScience29616551657200210.1126/science.296.5573.165512040186 ChangFLeeJTNavolanicPMSteelmanLSSheltonJGBlalockWLFranklinRAMcCubreyJAInvolvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: A target for cancer chemotherapyLeukemia17590603200310.1038/sj.leu.240282412646949 NicolettiFFagonePMeroniPMcCubreyJBendtzenKmTOR as a multifunctional therapeutic target in HIV infectionDrug Discov Today16715721201110.1016/j.drudis.2011.05.00821624501 XuZZXiaZGWangAHWangWFLiuZYChenLYLiJMActivation of the PI3K/AKT/mTOR pathway in diffuse large B cell lymphoma: Clinical significance and inhibitory effect of rituximabAnn Hematol9213511358201310.1007/s00277-013-1770-923636313 HansCPWeisenburgerDDGreinerTCGascoyneRDDelabieJOttGMüller-HermelinkHKCampoEBrazielRMJaffeESConfirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarrayBlood103275282200410.1182/blood-2003-05-154514504078 OttGZiepertMKlapperWHornHSzczepanowskiMBerndHWThornsCFellerACLenzeDHummelMImmunoblastic morphology but not the immunohistochemical GCB/nonGCB classifier predicts outcome in diffuse large B-cell lymphoma in the RICOVER-60 trial of the DSHNHLBlood11649164925201010.1182/blood-2010-03-27676620736456 MahadevanDChioreanEGHarrisWBVon HoffDDStejskal-BarnettAQiWAnthonySPYoungerAERensvoldDMCordovaFPhase I pharmacokinetic and pharmacodynamic study of the pan-PI3K/mTORC vascular targeted pro-drug SF1126 in patients with advanced solid tumours and B-cell malignanciesEur J Cancer4833193327201210.1016/j.ejca.2012.06.02722921184 BendellJCRodonJBurrisHAde JongeMVerweijJBirleDDemanseDDe BuckSSRuQCPetersMPhase I, dose-escalation study of BKM120, an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumorsJ Clin Oncol30282290201210.1200/JCO.2011.36.136022162589 ChengYZhangYZhangLRenXHuber-KeenerKJLiuXZhouLLiaoJKeihackHYanLMK-2206, a novel allosteric inhibitor of Akt, synergizes with gefitinib against malignant glioma via modulating both autophagy and apoptosisMol Cancer Ther11154164201210.1158/1535-7163.MCT-11-060622057914 DoniaMManganoKAmorosoAMazzarinoMCImbesiRCastrogiovanniPCocoMMeroniPNicolettiFTreatment with rapamycin ameliorates clinical and histological signs of protracted relapsing experimental allergic encephalomyelitis in Dark Agouti rats and induces expansion of peripheral CD4+CD25+Foxp3+ regulatory T cellsJ Autoimmun33135140200910.1016/j.jaut.2009.06.00319625166 BagherpourBSalehiMJafariRBagheriAKiani-EsfahaniAEdalatiMKardiMTShaygannejadVPromising effect of rapamycin on multiple sclerosisMult Scler Relat Disord264045201810.1016/j.msard.2018.08.00930219744 NguyenQDMerrillPTClarkWLBankerASFardeauCFrancoPLeHoangPOhnoSRathinamSRThurauSIntravitreal sirolimus for noninfectious uveitis: A Phase III sirolimus study assessing double-masKed uveitis TReAtment (SAKURA)Ophthalmology12324132423201610.1016/j.ophtha.2016.07.02927692526 SteelmanLSMartelliAMCoccoLLibraMNicolettiFAbramsSLMcCubreyJAThe therapeutic potential of mTOR inhibitors in breast cancerBr J Clin Pharmacol8211891212201610.1111/bcp.12958270596455061784 LeonardiGCFalzoneLSalemiRZanghìASpandidosDAMccubreyJACandidoSLibraMCutaneous melanoma: From pathogenesis to therapy (Review)Int J Oncol52107110802018295328575843392 DoniaMMcCubreyJABendtzenKNicolettiFPotential use of rapamycin in HIV infectionBr J Clin Pharmacol70784793201010.1111/j.1365-2125.2010.03735.x211754333014061 NicolettiFLapentaCDonatiSSpadaMRanazziACacopardoBManganoKBelardelliFPernoCAquaroSInhibition of human immunodeficiency virus (HIV-1) infection in human peripheral blood leucocytes-SCID reconstituted mice by rapamycinClin Exp Immunol1552834200910.1111/j.1365-2249.2008.03780.x190768262665676 CaporaliSAlvinoELacalPMLevatiLGiuratoGMemoliDCapriniEAntonini CappelliniGCD'AtriSTargeting the PI3K/AKT/mTOR pathway overcomes the stimulating effect of dabrafenib on the invasive behavior of melanoma cells with acquired resistance to the BRAF inhibitorInt J Oncol4911641174201610.3892/ijo.2016.359427572607 YuYYuXMaJTongYYaoJEffects of NVP-BEZ235 on the proliferation, migration, apoptosis and autophagy in HT-29 human colorectal adenocarcinoma cellsInt J Oncol49285293201610.3892/ijo.2016.350727176231 ChangZShiGJinJGuoHGuoXLuoFSongYJiaXDual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagyInt J Mol Med3114491456201310.3892/ijmm.2013.135123588698 PaplomataEZelnakAO'ReganREverolimus: Side effect profile and management of toxicities in breast cancerBreast Cancer Res Treat140453462201310.1007/s10549-013-2630-y23907751 LewSChamberlainRSRisk of metabolic complications in patients with solid tumors treated with mTOR inhibitors: Meta-analysisAnticancer Res3617111718201627069150 MarcinkowskaEWallaceGRBrownGThe use of 1α,25-dihydroxyvitamin D3 as an anticancer agentInt J Mol Sci17E729201610.3390/ijms1705072927187375 HolickMFVitamin D and bone healthJ Nutr126(Suppl 4)1159S1164S199610.1093/jn/126.suppl_4.1159S8642450 Abu El MaatyMAWölflSVitamin D as a novel regulator of tumor metabolism: Insights on potential mechanisms and implications for anti-cancer therapyInt J Mol Sci18E2184201710.3390/ijms1810218429048387 BarretoSGNealeREVitamin D and pancreatic cancerCancer Lett36816201510.1016/j.canlet.2015.06.03026276715 CastronovoCCastronovoVNikkelsAPeulenOVitamin D anti-cancer activities: Observations, doubts and certaintiesRev Med Liege704955002015(In French)26727838 DuffyMJMurrayASynnottNCO'DonovanNCrownJVitamin D analogues: Potential use in cancer treatmentCrit Rev Oncol Hematol112190197201710.1016/j.critrevonc.2017.02.01528325259 LiMXLiLFZhangLXiaoZGShenJHuWZengQChoCHVitamin D and cancer stem cells in the gastrointestinal tractCurr Med Chem24918927201710.2174/092986732466617021411063328201969 ZhangXHarbeckNJeschkeUDoisneau-SixouSInfluence of vitamin D signaling on hormone receptor status and HER2 expression in breast cancerJ Cancer Res Clin Oncol14311071122201710.1007/s00432-016-2325-y28025696 AhnJParkSZunigaBBeraASongCSChatterjeeBVitamin D in prostate cancerVitam Horm100321355201610.1016/bs.vh.2015.10.01226827958 LisseTSLiuTIrmlerMBeckersJChenHAdamsJSHewisonMGene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signalingFASEB J25937947201110.1096/fj.10-172577211232973042839 JangWKimHJLiHJoKDLeeMKSongSHYangHO1,25-Dyhydroxyvitamin D3 attenuates rotenone-induced neurotoxicity in SH-SY5Y cells through induction of autophagyBiochem Biophys Res Commun451142147201410.1016/j.bbrc.2014.07.08125078626 Datta MitraARaychaudhuriSPAbriaCJMitraAWrightRRayRKundu-RaychaudhuriS1α,25-Dihydroxyvitamin-D3-3-bromoacetate regulates AKT/mTOR signaling cascades: A therapeutic agent for psoriasisJ Invest Dermatol13315561564201310.1038/jid.2013.323314787 GröschelCTennakoonSKállayECytochrome P450 Vitamin D hydroxylases in inflammation and cancerAdv Pharmacol74413458201510.1016/bs.apha.2015.03.00226233913 Tannour-LouetMLewisSKLouetJFStewartJAddaiJBSahinAVangapanduHVLewisALDittmarKPautlerRGIncreased expression of CYP24A1 correlates with advanced stages of prostate cancer and can cause resistance to vitamin D3-based therapiesFASEB J28364372201410.1096/fj.13-236109240819046188232 SunHWangCHaoMSunRWangYLiuTCongXLiuYCYP24A1 is a potential biomarker for the progression and prognosis of human colorectal cancerHum Pathol50101108201610.1016/j.humpath.2015.11.00826997443 SakakiTYasudaKKittakaAYamamotoKChenTCCYP24A1 as a potential target for cancer therapyAnticancer Agents Med Chem1497108201410.2174/1871520611313999030723869781 OsanaiMLeeGHCYP24A1-induced vitamin D insufficiency promotes breast cancer growthOncol Rep3627552762201610.3892/or.2016.507227600601 HuNZhangHCYP24A1 depletion facilitates the antitumor effect of vitamin D3 on thyroid cancer cellsExp Ther Med16282128302018302336626143870 NgAKWeissLLaCasceASChapter 74 - Hodgkin's LymphomaClinical Radiation Oncology3rdGundersonLLTepperJEW.B. SaundersPhiladelphia15271543201210.1016/B978-1-4377-1637-5.00074-2 KozielewiczPGraftonGKutnerACurnowSJGordonJBarnesNMNovel vitamin D analogues; cytotoxic and anti-proliferative activity against a diffuse large B-cell lymphoma cell line and B-cells from healthy donorsJ Steroid Biochem Mol Biol16498105201610.1016/j.jsbmb.2015.10.01526485664 HickishTCunninghamDColstonKMillarBCSandleJMackayAGSoukopMSloaneJThe effect of 1,25-dihydroxyvitamin D3 on lymphoma cell lines and expression of vitamin D receptor in lymphomaBr J Cancer68668672199310.1038/bjc.1993.40683986901968598 KlionskyDJAbdallaFCAbeliovichHAbrahamRTAcevedo-ArozenaAAdeliKAgholmeLAgnelloMAgostinisPAguirre-GhisoJAGuidelines for the use and interpretation of assays for monitoring autophagyAutophagy8445544201210.4161/auto.19496229664903404883 ChungIKarpfARMuindiJRConroyJMNowakNJJohnsonCSTrumpDLEpigenetic silencing of CYP24 in tumor-derived endothelial cells contributes to selective growth inhibition by calcitriolJ Biol Chem28287048714200710.1074/jbc.M60889420017244627 DrakeMTMaurerMJLinkBKHabermannTMAnsellSMMicallefINKellyJLMaconWRNowakowskiGSInwardsDJVitamin D insufficiency and prognosis in non-Hodgkin's lymphomaJ Clin Oncol2841914198201010.1200/JCO.2010.28.6674207138492953973 IkezoeTBandobashiKYangYTakeuchiSSekiguchiNSakaiSKoefflerHPTaguchiHHIV-1 protease inhibitor ritonavir potentiates the effect of 1,25-dihydroxyvitamin D3 to induce growth arrest and differentiation of human myeloid leukemia cells via down-regulation of CYP24Leuk Res3010051011200610.1016/j.leukres.2005.12.00816457885 LyLHZhaoXYHollowayLFeldmanDLiarozole acts synergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activityEndocrinology14020712076199910.1210/endo.140.5.669810218956 AdoriniLPennaGControl of autoimmune diseases by the vitamin D endocrine systemNat Clin Pract Rheumatol4404412200810.1038/ncprheum085518594491 Agmon-LevinNTheodorESegalRMShoenfeldYVitamin D in systemic and organ-specific autoimmune diseasesClin Rev Allergy Immunol45256266201310.1007/s12016-012-8342-y23238772 YoungKAMunroeMEGuthridgeJMKamenDLNiewoldTBGilkesonGSWeismanMHIshimoriMLKellyJGaffneyPMCombined role of vitamin D status and CYP24A1 in the transition to systemic lupus erythematosusAnn Rheum Dis76153158201710.1136/annrheumdis-2016-20915727283331 KleinsternGAverbuchMAbu SeirRPerlmanRBen YehudaDPaltielOPresence of autoimmune disease affects not only risk but also survival in patients with B-cell non-Hodgkin lymphomaHematol Oncol36457462201810.1002/hon.249829469175 BarcelliniWRizzardiGPBorghiMONicolettiFFainCDel PapaNMeroniPLIn vitro type-1 and type-2 cytokine production in systemic lupus erythematosus: Lack of relationship with clinical disease activityLupus5139145199610.1177/0961203396005002098743127 NarazakiMKishimotoTThe two-faced cytokine IL-6 in host defense and diseasesInt J Mol Sci19E3528201810.3390/ijms1911352830423923 DlouhyIFilellaXRoviraJMagnanoLRivas-DelgadoABaumannTMartínez-TrillosABalaguéOMartínezAGonzález-FarreBHigh serum levels of soluble interleukin-2 receptor (sIL2-R), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) are associated with adverse clinical features and predict poor outcome in diffuse large B-cell lymphomaLeuk Res592025201710.1016/j.leukres.2017.05.01428544905 Ysmail-DahloukLNouariWAribiM1,25-dihydroxyvitamin D3 down-modulates the production of proinflammatory cytokines and nitric oxide and enhances the phosphorylation of monocyte-expressed STAT6 at the recent-onset type 1 diabetesImmunol Lett179122130201610.1016/j.imlet.2016.10.00227717877 YangMXuJYuJYangBGanHLiSLiXAnti-inflammatory effects of 1,25-dihydroxyvitamin D3 in monocytes cultured in serum from patients with type 2 diabetes mellitus and diabetic nephropathy with uremia via Toll-like receptor 4 and nuclear factor-κB p65Mol Med Rep1282158222201510.3892/mmr.2015.448226497229 LockshinRAZakeriZCaspase-independent cell deathsCurr Opin Cell Biol14727733200210.1016/S0955-0674(02)00383-612473346 MatsuiYTakagiHQuXAbdellatifMSakodaHAsanoTLevineBSadoshimaJDistinct roles of autophagy in the heart during ischemia and reperfusion: Roles of AMP-activated protein kinase and Beclin 1 in mediating autophagyCirc Res100914922200710.1161/01.RES.0000261924.76669.3617332429 TakagiHMatsuiYHirotaniSSakodaHAsanoTSadoshimaJAMPK mediates autophagy during myocardial ischemia in vivoAutophagy3405407200710.4161/auto.428117471015 MaiuriMCTasdemirECriolloAMorselliEVicencioJMCarnuccioRKroemerGControl of autophagy by oncogenes and tumor suppressor genesCell Death Differ168793200910.1038/cdd.2008.13118806760 WullschlegerSLoewithRHallMNTOR signaling in growth and metabolismCell124471484200610.1016/j.cell.2006.01.01616469695 LisseTSHewisonMVitamin D: A new player in the world of mTOR signalingCell Cycle1018881889201110.4161/cc.10.12.15620215588083154412 PeponiEDrakosEReyesGLeventakiVRassidakisGZMedeirosLJActivation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphomaAm J Pathol16921712180200610.2353/ajpath.2006.051078171486791762462 RudeliusMPittalugaSNishizukaSPhamTHFendFJaffeESQuintanilla-MartinezLRaffeldMConstitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphomaBlood10816681676200610.1182/blood-2006-04-015586166451631895501 HasselblomSHanssonUOlssonMTorénLBergströmANilsson-EhleHAnderssonPOHigh immunohistochemical expression of p-AKT predicts inferior survival in patients with diffuse large B-cell lymphoma treated with immunochemotherapyBr J Haematol149560568201010.1111/j.1365-2141.2010.08123.x20201946 Maksimovic-IvanicDFagonePMcCubreyJBendtzenKMijatovicSNicolettiFHIV-protease inhibitors for the treatment of cancer: Repositioning HIV protease inhibitors while developing more potent NO-hybridized derivatives?Int J Cancer14017131726201710.1002/ijc.3052927870005 RothweilerFMichaelisMBrauerPOtteJWeberKFehseBDoerrHWWieseMKreuterJAl-AbedYAnticancer effects of the nitric oxide-modified saquinavir derivative saquinavir-NO against multidrug-resistant cancer cellsNeoplasia1210231030201010.1593/neo.10856211702663003137 Maksimovic-IvanicDMojicMBulatovicMRadojkovicMKuzmanovicMRisticSStosic-GrujicicSMiljkovicDCavalliELibraMThe NO-modified HIV protease inhibitor as a valuable drug for hematological malignancies: Role of p70S6KLeuk Res3910881095201510.1016/j.leukres.2015.06.01326220866 PearceLRAltonGRRichterDTKathJCLingardoLChapmanJHwangCAlessiDRCharacterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1)Biochem J431245255201010.1042/BJ2010102420704563 GuoLSLiHXLiCYZhangSYChenJWangQLGaoJMLiangJQGaoMTWuYJSynergistic antitumor activity of vitamin D3 combined with metformin in human breast carcinoma MDA-MB-231 cells involves m-TOR related signaling pathwaysPharmazie70117122201525997252 YangJIkezoeTNishiokaCNiLKoefflerHPYokoyamaAInhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D(3) to induce growth arrest and differentiation of AML cells in vitro and in vivoExp Hematol38666676201010.1016/j.exphem.2010.03.02020382200

CYP24A1 is differently expressed in patients with DLBCL and lymphnoditis, and 1,25(OH)2D3 inhibits the proliferation of Pfeiffer cells. Sections of paraffin-embedded lymph node tissues from the diagnostic biopsies of 20 patients with lymphnoditis and 57 patients with DLBCL were processed for immunohistochemical staining with an antibody against CYP24A1. Representative images of CYP24A1 immunohistochemical staining in lymphnoditis samples at (A) ×200 and (C) ×400 magnification. Representative images of CYP24A1 immunohistochemical staining in DLBCL lymph node tissue at (B) ×200 and (D) ×400 magnification. Scale bar, 100 µm. (E) Pfeiffer cells were cultured with 1,25(OH)2D3 (10–100 nM) for 48 h. After treatment, cell viability was determined using a Cell Counting Kit-8. Pfeiffer cell proliferation was significantly inhibited by 10 and 100 nM 1,25(OH)2D3. The rate of inhibition was calculated to be 16.8±3.4% for cells treated with 10 nM 1,25(OH)2D3 and 28.2±2.9% for cells treated with 100 nM 1,25(OH)2D3 for 48 h. Each experiment was performed in triplicate. **P<0.01, ***P<0.001 vs. control. CYP24A1, 25-hydroxyvitamin D-24-hydroxylase; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; OD, optical density; DLBCL, diffuse large B cell lymphoma.

Cell cycle distribution of Pfeiffer cells after treatment with 1,25(OH)2D3 and RAPA, and the expression of CYP24A1 and VDR in Pfeiffer cells. (A) Pfeiffer cells were cultured with 10 or 100 nM 1,25(OH)2D3 and RAPA (10 nM). After 48 h, the cell cycle distribution was analyzed. The figure shows representative examples of three independent experiments. (B) Quantification of the G1 population of Pfeiffer cells. (C) After treatment with 1,25(OH)2D3 and/or RAPA for 48 h, cells were harvested and the protein levels of CYP24A1 and VDR were analyzed via western blotting. A representative of three independent experiments is shown. (D) Quantification of western blotting results. *P<0.05 vs. control; #P<0.05 vs. 100 nM 1,25(OH)2D3; &P<0.05 vs. 10 nM RAPA. VDR, vitamin D receptor; CYP24A1, 25-hydroxyvitamin D-24-hydroxylase; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RAPA, rapamycin.

Analysis of autophagy and the AKT/mTOR signaling pathway in Pfeiffer cells after treatment with 1,25(OH)2D3. (A) Representative transmission electron microscope images [magnification, ×1,700 (left) and ×5,000 (right)] of the ultrastructure of Pfeiffer cells after treatment with or without 100 nM 1,25(OH)2D3 for 48 h. (B) Changes in the level and localization of LC3 examined by confocal microscopy after treatment with or without 100 nM 1,25(OH)2D3 for 48 h. Scale bar =100 µm. (C) Western blot analysis of the expression of the autophagy related proteins LC3 and P62 after treatment with 100 nM 1,25-D3 for 48 h and (D) the quantification. (E) Pfeiffer cells were cultured without or with 1,25(OH)2D3 for 1 h. The phosphorylation levels of AKT/mTOR signaling proteins were evaluated using western blotting. A representative example of three independent experiments is shown. (F) Quantification of western blotting. *P<0.05 vs. control. LC3, protein light chain 3; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; mTOR, target of rapamycin; p-, phosphorylated; 4EBP, eukaryotic translation imitation factor 4E-binding protein 1; p70SK6, ribosomal protein S6 kinase β-1; P62, ubiquitin binding protein P62.

Clinical features and CYP24A1 expression of patients with diffuse large B cell lymphoma.

CYP24A1 expression
Parameter Low High P-value
Age, years
  <60 32 11 0.008a
  ≥60   5   9
Sex
  Male 18 12 0.37
  Female 19   8
Ann Arbor stage
  I/II 10   3 0.28
  III/IV 27 17
LDH
  Normal 32 11 0.015a
  Elevated 5   9
IPI
  0–2 18   8 0.008a
  3–5 19 20
Clinical treatment response
  CR/PR 26   8 0.028a
  SD/PD 11 12

The χ2 test was used to compare the distribution of clinical features between low- and high-level CYP24A1 expression.

P<0.05. CYP24A1, 25-hydroxyvitamin D-24-hydroxylase; LDH, serum lactate dehydrogenase; IPI, International Prognostic Index; CR, complete remission; PR, partial remission; SD, stable disease; PD, progressive disease.

Cell cycle distribution of Pfeiffer cells treated with 1,25(OH)2D3 and RAPA.

Group G1 phase (%)
Control 22.70±0.40
10 nM 1,25(OH)2D3 22.92±0.93
100 nM 1,25(OH)2D3 26.11±0.64a
10 nM RAPA 35.74±0.55a,b
10 nM RAPA + 10 nM 1,25(OH)2D3 37.13±0.75a,b
10 nM RAPA + 100 nM 1,25(OH)2D3 42.75±0.61a,c

The differences between G1 phase populations were analyzed using the Tukey's post hoc test.

P<0.05 vs. control

P<0.05 vs. 100 nM 1,25(OH)2D3

P<0.05 vs. 10 nM RAPA. RAPA, rapamycin; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3.