Contributed equally
This is the first study to demonstrate that benzo(a) pyrene (BaP) was able to enhance the production of parathyroid hormone-related protein (PTHrP) by human non-small cell lung cancer H460 cells. Such effect would further contribute to bone metastasis of lung cancer by increasing osteoclastogenesis. This study is also the first to reveal that tricetin (TCN), a flavonoid derivative found in Myrtaceae pollen and
Lung cancer is one of the common cancers in the world, and also one of the leading causes of cancer-related deaths worldwide (
Polycyclic aromatic hydrocarbons (PAHs), are formed by the incomplete combustion of organic matter. Benzo(a) pyrene (BaP) is the most commonly measured and studied PAH. They usually present in the environment at detectable levels in many types of uncooked food, and cooking process could generate PAHs in food. Several studies have been conducted to determine the levels of exposure to PAHs from representative human diet and the proportion of the overall burden of environmental exposure to PAHs that is attributable to diet (
Tricetin (TCN) (5,7,3′,4′,5′-pentahydroxyflavone), a flavonoid derivative found in Myrtaceae pollen and
TCN was obtained from Extrasynthese (Genay, France), dissolved in dimethyl sulfoxide (DMSO) (Sigma- Aldrich, St. Louis, MO, USA), and stored at −20°C. Control cultures received the carrier solvent (0.1% DMSO). All chemicals used were in their purest form available commercially.
Human non-small cell lung cancer H460 cells were obtained from the American Type Culture Collection (HTB-177) (Manassas, VA, USA) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (both from Gibco-BRL, Gaithersburg, MD, USA). Human primary osteoblasts were obtained from Lonza (Walkersville, MD, USA) and cultured in osteoblast medium (OBM) (Lonza).
To obtain the various conditioned media (CM), H460 cells (2×106/100 mm dish) were treated with various concentrations of BaP (Sigma-Aldrich) for 6 h. After treatment, the medium was replaced and the supernatant harvested and filtered (0.22 mm) after 24 h of incubation.
Supernatants from osteoblasts and H460 cells were collected. Levels of osteoprotegerin (OPG), macrophage colony-stimulating factor (M-CSF), RANKL and IL-8 were assessed and quantified using the DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). PTHrP levels were determined by an ELISA kit (Abnova Corp., Taipei, Taiwan).
Monocytes were purified from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Mononuclear cells were isolated from blood by Ficoll-Hypaque gradient (GE Healthcare UK, Ltd., Buckinghamshire, UK). CD14+ monocytes were purified using CD14+ monoclonal antibody-conjugated magnetic beads (MACS MicroBeads; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Osteoclasts were generated by culturing CD14+ monocytes in medium containing 20% vehicle control-CM-cultured osteoblasts or BaP-H460-CM-cultured osteoblasts presented in 100 ng/ml M-CSF and 50 ng/ml RANKL (R&D Systems) for 14–21 days. The medium was replaced with fresh medium containing M-CSF and RANKL every 5 days.
Osteoclast formation was measured by quantifying cells positively stained by TRAP (Sigma-Aldrich). Osteoclasts were deemed TRAP-positive by light microscopy that revealed staining of multinuclear (>3 nuclei) cells. The TRAP-positive cells and the number of nuclei per TRAP-positive cells in each well were counted. The bone resorption activity of osteoclasts was assessed by a 48-well plate bone resorption assay (Cosmo Bio Co., Ltd., Tokyo, Japan), under the same culture conditions as described above.
The Institutional Review Board (IRB) of Kaohsiung Medical University Chung-Ho Memorial Hospital (Kaohsiung, Taiwan) approved the study protocol and all of the participants provided written informed consent in accordance with the Declaration of Helsinki (IRB nos.: KMUH-IRB-990345, KMUH-IRB-20110377 and KMUH-IRB-20130054).
The TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used for RNA isolation while cDNA was prepared using an oligo(dT) primer and reverse transcriptase (Takara Bio, Inc., Shiga, Japan) following standard protocols. The RT-qPCR was performed using SYBR-Green on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each PCR reaction mixture contained 200 nM of each primer, 10 μl 2X SYBR-Green PCR Master Mix (Applied Biosystems), and 5 μl cDNA and RNase-free water for a total volume of 20 μl. The RT-qPCR was conducted with a denaturation step at 95°C for 10 min, then for 40 cycles at 95°C for 15 sec, and 60°C for 1 min. All PCRs were performed in triplicate and normalized to internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The relative expression level was determined using the 2−ΔΔCT method.
H460 cells were transfected with 20 nM non-target or PTHrP siRNA pool (Dharmacon, Inc., Lafayette, CO, USA) by DharmaFECT 4 Transfection Reagents, according to the manufacturer’s instructions. After 24 h transfection, the medium was changed to whole medium and the cells were treated with BaP. The PTHrP changes were measured by RT-qPCR.
Data are expressed as means ± standard errors. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (p<0.05) between the means of the test groups were analyzed by Student’s t-test.
To investigate the effects of BaP on PTHrP secretion by human non-small cell lung cancer cells, BaP was added to the culture medium of H460 cells to the concentration of 10 μM for 6 h. After washing, cells were cultured with new medium for another 24 h, the conditioned medium of BaP-treated H460 (BaP-H460-CM) was harvested and PTHrP levels of these CM were assessed. After exposure to BaP, the production of PTHrP in human non-small cell lung cancer H460 cells was increased (
Osteoclastogenesis is regulated by PTHrP by altering the ratio of osteoclastogenesis activator (M-CSF and RANKL)/inhibitor (OPG) produced by osteoblasts (
Human osteoblasts were cultured with the CM prepared to assess the effects of BaP on the interaction between non-small cell lung cancer and osteoblasts (
To assess the effect of BaP on non-small cell lung cancer-mediated osteoclastogenesis, TRAP and bone resorption assays were tested. H460-CM increased osteoclastogenesis induced by H460 cells, and such effect was strengthened when H460 cells were pre-treated with BaP (
Since PTHrP was reported to be able to increase IL-8 expression of cancer cells (
To confirm the role of PTHrP in the upregulation of IL-8 induced by BaP, H460 cells were transfected with PTHrP siRNA. The expression of PTHrP mRNA in H460 cells was decreased ~78% when the cells were transfected with PTHrP siRNA (
To access the effects of TCN on BaP-mediated non-small cell lung cancer bone metastasis, BaP-induced PTHrP and IL-8 secretion by non-small cell lung cancer H460 cells were tested again, both PTHrP and IL-8 secretion were decreased by 1 μM TCN treatment (
Bone metastasis is a devastating event for lung cancer patients because once it occurs, the morbidity and mortality will increase (
PTHrP, a potent activator of osteoclastic bone resorption, is an important pathologic factor for hypercalcemia among cancer patients (
OPG is a soluble decoy receptor for RANKL, with the ability to decrease osteoclastogenesis (
Many studies have reported that non-small cell lung cancer expresses high levels of IL-8, which enhances both osteoclastogenesis and bone resorption (
Current therapy for bone metastases have limited efficacy and are only palliative. The side-effects of these treatments, renal toxicity and osteonecrosis of the jaw potentially will decrease the quality of life of these lung cancer patients (
Our data show that TCN, a flavonoid derivative found in Myrtaceae pollen and
In conclusion, there are two novel findings in this study: it is the first study to demonstrate that BaP increases the stimulatory effect of human non-small cell lung cancer on osteoclastogenesis and their activity of osteoclast bone resorption directly by PTHrP/IL-8 and by interfering in the osteoblast-osteoclast interaction, and it is also the first to reveal that TCN, a flavonoid derivative found in Myrtaceae pollen and
This study was supported by grants from the National Science Council (NSC 101-2628-B-037-001-MY3; NSC 101-2320-B-037-043-MY3), the Ministry of Science and Technology (MOST 103-2320-B-037-006-MY3; MOST 103- 2314-B-037-052; MOST 103-2320-B-037-032), the Kaohsiung Medical University ‘Aim for the Top 500 Universities Grant, Grant no. KMU-DT103008’, the Kaohsiung Medical University ‘Aim for the Top Universities Grant, Grant nos. KMU-TP103A19 and KMU-TP103A20’, and the Kaohsiung Medical University Hospital Research Foundation (KMUH101-1M08). The authors thank the Center for Research Resources and Development of Kaohsiung Medical University for its support with the instrumentation.
Benzo(a)pyrene (BaP) increased parathyroid hormone-related protein (PTHrP) expression in human non-small cell lung cancer. (A) The effects of 10 μM BaP on PTHrP levels in H460 cells. (B) BaP increased PTHrP expression in a dose-dependent manner. H460 cells treated with 10 μM BaP or various concentrations of BaP for 6 h. After washing and a 24-h culture, the conditioned media (CM) of BaP-treated H460 cells (BaP-H460-CM) were harvested, and PTHrP levels in these CM were then assessed by enzyme-linked immunosorbent assay (ELISA). Each value was the mean ± SD of three independent experiments. *P<0.05, significant difference between the control and test groups.
Benzo(a)pyrene (BaP) reinforces the interference of human non-small cell lung cancer in osteoblasts. (A) Flow chart of the production of control-CM, H460-CM, and BaP-H460-CM. BaP (10 μM) enhanced the stimulatory effect of H460 cells on the expression of (B) macrophage colony-stimulating factor (M-CSF) and (C) receptor activator of nuclear factor κB ligand (RANKL) in osteoblast. (D) BaP (10 μM) potentiated the inhibitory effect of human non-small cell lung cancer on osteoprotegerin (OPG) expression in osteoblasts. H460 cells treated with or without 10 mM BaP for 6 h. After washing and a 24-h culture, the conditioned media (CM) of non-treated or BaP-treated H460 cells (H460-CM and BaP-H460-CM) were harvested and stored at −80°C. Osteoblasts were treated with various CM for 24 h. The levels of M-CSF, RANKL or OPG in the supernatants of osteoblasts were assessed by enzyme-linked immunosorbent assay (ELISA). Each value was the mean ± SD of three independent experiments. *Significant difference with control-CM treatment, #significant difference with H460-CM treatment, p<0.05.
Benzo(a)pyrene (BaP) strengthens the stimulatory effect of human non-small cell lung cancer on osteoclastogenesis and bone resorption. (A) BaP (10 μM) increased the effect of H460 cells on osteoclastogenesis and (B) their bone resorption activity. Osteoclastogenesis was determined by TRAP staining (osteoclast: >3 nuclei, TRAP-positive). CD14+ monocytes were treated with H460-CM and BaP-H460-CM (20%) cultured with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) for 14–21 days. Media were replaced every 5 days. Osteoclasts were then stained by TRAP. Multinucleated (>3 nuclei) TRAP-positive cells are defined as osteoclasts. The pit area was determined by AlphaEaseFC software. Each value was the mean ± SD of three independent experiments. *Significant difference with control-CM treatment, #significant difference with H460-CM treatment, p<0.05.
Benzo(a)pyrene (BaP) upregulates the expression of IL-8 by increasing parathyroid hormone-related protein (PTHrP) in human non-small cell lung cancer. (A) The effect of 10 μM BaP on IL-8 levels in H460 cells. (B) BaP upregulated IL-8 expression in a dose-dependent manner. (C) The efficiency of PTHrP siRNA transfection. (D) Inhibition of PTHrP decreased the upregulation of IL-8 by BaP in H460 cells. The IL-8 levels were assessed by enzyme-linked immunosorbent assay (ELISA), and the efficacy of siRNA was determined by real-time polymerase chain reaction (RT-qPCR). H460- or siRNA-transfected H460 cells treated with BaP (10 μM or various concentrations) for 24 h. PTHrP levels were determined by ELISA. Each value is the mean ± SD of three independent experiments. *P<0.05, significant difference between the control and test groups.
Tricetin (TCN) decreases the effects of benzo(a)pyrene (BaP) on parathyroid hormone-related protein (PTHrP), IL-8, and receptor activator of nuclear factor κB ligand (RANKL) expression. TCN (1 μM) decreased the upregulation of BaP on (A) PTHrP and (B) IL-8 in H460 cells. (C) TCN decreased the effects of BaP on RANKL expression of osteoblasts. For (A) and (B), H460 cells were pre-treated with TCN for 1 h, then incubated with BaP (10 μM) for another 6 h for PTHrP, or 24 h for IL-8 analysis. After washing and a 24-h culture, the culture media were collected and PTHrP levels were assessed by enzyme-linked immunosorbent assay (ELISA). (C), H460 cells were pre-treated with TCN for 1 h, then incubated with BaP (10 μM) for another 6 h. After washing and a 24-h culture, the culture media were collected, then added to osteoblasts for another 24 h. The levels of RANKL in the supernatants of osteoblasts were assessed by ELISA. Each value is the mean ± SD of three independent experiments. *P<0.05, or significant difference between the control and test groups.
Tricetin (TCN) decreased the effects of benzo(a)pyrene (BaP) on human non-small cell lung cancer bone metastasis events. (A) Flow chart of production of control-CM, H460-CM, BaP-H460-CM, TCN + BaP-H460-CM or various osteoblast conditioned media (CM). TCN decreased the effects of BaP on (B) osteoclastogenesis and (C) bone resorption. For (B) and (C), H460 cells were pre-treated with TCN for 1 h, then incubated with BaP (10 μM) for another 6 h. After washing and a 24-h culture, the culture medium (20%) was collected, then added to CD14+ monocytes presenting in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) for a 14–21 days. The osteoclastogenesis and bone resorption was conducted as described above. Each value is the mean ± SD of three independent experiments. *P<0.05, or significant difference between the control and test groups.
Scheme of proposed tricetin (TCN)-inhibited benzo(a)pyrene (BaP)-induced human non-small cell lung cancer bone metastasis.