Resveratrol Derivative, Trans-3, 5, 4'-Trimethoxystilbene Sensitizes Osteosarcoma Cells to Apoptosis via ROS-Induced Caspases Activation

Abstract

Numerous studies have shown that resveratrol can induce apoptosis in cancer cells. Trans-3, 5, 4'-trimethoxystilbene (TMS), a novel derivative of resveratrol, is a more potent anticancer compound than resveratrol and can induce apoptosis in cancer cells. Herein, we examined the mechanisms involved in TMS-mediated sensitization of human osteosarcoma (143B) cells to TNF-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis. Our results showed that cotreatment with TSM and TRAIL activated caspases and increased PARP-1 cleavage in 143B cells. Decreasing cellular ROS levels using NAC reversed TSM- and TRAIL-induced apoptosis in 143B cells. NAC abolished the upregulated expression of PUMA and p53 induced by treatment with TRAIL and TSM. Silencing the expression of p53 or PUMA using RNA interference attenuated TSM-mediated sensitization of 143B cells to TRAIL-induced apoptosis. Knockdown of Bax also reversed TSM-induced sensitization of 143B cell to TRAIL-mediated apoptotic cell death. These results indicate that cotreatment with TRAIL and TSM evaluated intracellular ROS level, promoted DNA damage, and activated the Bax/PUMA/p53 pathway, leading to activation of both mitochondrial and caspase-mediated apoptosis in 143B cells. Orthotopic implantation of 143B cells in mice also demonstrated that cotreatment with TRAIL and TSM reversed resistance to apoptosis in cells without obvious adverse effects in normal cells.

Figure 1

TRAIL suppresses viability and promotes apoptotic cell death in Saos-2 cells. MTT assay (a) and Trypan blue staining (20x objective lens) (b) were used to examine the survival of 143B and Saos-2 cells after treatment with 0–200 ng/mL TRAIL for 12 h. Equal volume of vehicle (0.01% BSA + PBS) was used to treat the control groups, ^∗P < 0.05; ^∗∗P < 0.01. (c) Western blotting was used to detect PRAP-1 cleavage in 143B and Saos-2 cells treated with 0–200 ng/mL TRAIL for 6 hour. (c) DAPI and TUNEL labeling were used to examine apoptosis in 143B and Saos-2 cells treated with 100 ng/mL TRAIL for 6 hours. White arrows indicate chromatin condensation in 143B and Saos-2 cells; spots of green fluorescence indicate morphological changes in 143B and Saos-2 cells (magnification ×200); additional higher-magnification images are also provided (magnification ×400).

Figure 2

TMS reverses TRAIL-resistance in 143B cells. (a) After treatment with different doses of TMS for 6 h, the survival rate of 143B cells was examined using the Trypan blue dye exclusion method (20x objective lens). (b) Survival of 143B cells, treated with 0–10 μm TMS and/or 50 ng/mL TRAIL, was assessed using the Trypan blue dye exclusion method. (c) Human osteosarcoma 143B cells and (d) normal human hFOB1.19 osteoblasts were pretreated with 50 ng/mL TRAIL and 2.5 μm TMS for 10 hours. Western blotting was used to detect the levels of cleaved PARP1. (e) DAPI and TUNEL labeling were used to evaluate the apoptosis of 143B osteosarcoma cells and normal hFOB1.19 osteoblasts after treatment with TMS and/or TRAIL for 6 h (magnification ×400).

Figure 3

Cotreatment with TRAIL and TMS can activate caspases in 143B cells. (a) Western blotting was used to detect the expression of PARP-1 and caspase cleavage in 143B cells treated with TMS and/or TRAIL for different lengths of time. The control group was treated with an equal volume of 1% DMSO + PBS+0.01% BSA. (b) Caspase cleavage was examined by Western blotting in 143B cells after treatment with 50 ng/mL TRAIL and TMS (0.5–2.5 μM) for 10 hours. (c) Caspase inhibitory agents, including zlEHDfmk, zlETDfmk, and zDEVDfmk, were used to block the caspase pathway in 143B cells. After the cells were pretreated with TMS and/or TRAIL for 10 hours, the Trypan blue dye exclusion method was used to evaluate cell survival (20x objective lens), and Western blotting was utilized to detect the expression of cleaved PARP1. ^∗∗P < 0.05.

Figure 4

TMS increases ROS levels in 143B cells. (a) Intracellular ROS levels in 143B cells were detected using fluorescence microscopy. The cell was pretreated with positive control (H₂O₂) or TMS for 50 minutes and labeled with CMH2DFCDA for 15 minutes. Considerably higher fluorescence intensity was detected in cells treated with H₂O₂ or TMS compared with that of the 0.01% BSA+ PBS-treated control group. (b) 143B cell was pretreated with different doses of TMS for various periods of time. ROS generation was measured using relative fluorescence intensity in 143B cells. ^∗∗P < 0.05. (c) After treatment with TMS (2.5, 5 μm) for various periods of time, 143B cell was labeled with CMH2DFCDA. ROS levels were analyzed at different time points according to the value of relative fluorescence intensity. (d) Pretreatment with NAC reduced ROS generation induced by TMS (2.5 μm) and (e) attenuated TMS- and TRAIL-induced apoptosis in 143B cells. Trypan blue dye exclusion method was used to evaluate cell survival (20x objective lens). ^∗P < 0.05; ^∗∗P < 0.01. Cells in the control group were treated with an equal volume of PBS+0.1% DMSO . (f) Immunoblotting were used for examining the expression of cleaved PARP-1, p53, and PUMA in 143B cells after pretreatment with NAC and treatment with TRAIL and TMS.

Figure 5

TMS induces DNA damage and facilitates phosphorylation of Ser-139 (H2AX) in 143B cells. (a) Comet assay was performed to examine DNA damage induced by treatment with TMS or TRAIL for 15 minutes. Tail moment were applied for quantifying double-strands break via calculating the tail length × percentage of tail DNA. ^∗P < 0.05. (b) 143B cell was treated with 0–10 μM TMS for 24 hours, and Western blotting was used to examine the expression of H2AX and phospho-H2AX-Ser139 in 143B cells; 0.01% DMSO was used as vehicle control. (c) 143B cell was pretreated with TMS (2.5 μM) for 0-24 hours, and immunoblotting was used to examine the expression of H2AX and phospho-H2AX-Ser139 expression in 143B cells; 0.01% DMSO was used as vehicle control.

Figure 6

TMS activates PUMA and p53 expression in osteosarcoma cells. (a) TMS (2.5 μM) was used to treat 143B cells for 0–24 h, and protein expression of phosphorylated p53 (on Ser392, Ser46, or Ser15) was examined by Western blotting; 0.01% DMSO was used as vehicle control. (b) Different doses of TMS were used to treat 143B cells for 10 h, and Western blot assay was performed to detect the expressions of P53 and PUMA. (c) Different doses of TMS and TRAIL were used to treat 143B cells for 10 h, and Western blot assay was performed to detect the expressions of P53 and PUMA. (d) Gene expression of p53 and PUMA was silenced by siRNA in 143B cells, and then, the cells were treated with TRAIL and TMS for 10 h. Western blotting was used to examine the expression of cleaved PARP1, P53, and PUMA. (e) Apoptotic cells were examined by DAPI staining after treating p53- or PUMA-silenced 143B cells with TMS and TRAIL. White arrows indicate condensed chromatin in 143B cells (magnification ×400).

Figure 7

Treatment with TMS overcomes TRAIL resistance and promotes apoptotic cell death of MG-63 cell via activating p53 and PUMA. (a) Wild-type MG-63 cell, (b) p53−/− MG-63 cell or (c) wild-type MG-63 cell, and (d) PUMA −/− MG-63 cell were pretreated by TRAIL (50 ng/mL) and TMS (2.5 μm) for 10 h. Equal volume of PBS+0.1% DMSO 0.01% + BSA was used as vehicle control. Cell survival was examined as indicated in the experimental method section. Immunoblotting was used to detect the expression of cleaved PARP1, p53, or PUMA. ^∗P < 0.01.

Figure 8

The role of BAX in TMS- and TRAIL-mediated apoptosis. (a, b) 143B Bax+/− and 143B Bax−/− cells were exposed to TMS and TRAIL for 10 h. Equal volume of 0.01% BSA + PBS+0.1% DMSO was used as vehicle control. Cell survival rate was analyzed as indicated in the experimental method section. Immunoblotting was used to examine the expression of cleaved PARP1, P53, Bax, and PUMA. ^∗P < 0.05. (c) DAPI was used as specific counterstain for apoptosis to evaluate the effects of cotreatment with TMS and TRAIL in 143B Bax+/− and 143B Bax−/− cells. White arrows indicate chromatin condensation in 143B cells evaluated by fluorescence microscopy (magnification ×400).

Figure 9

Cotreatment with TMS and TRAIL suppresses the proliferation of cancer cells in nude mice transplanted with osteosarcoma. (a) Sizes of osteosarcoma tumors in nude mice subjected to different treatment regimens. (b) Tumor volume and weight were significantly decreased in TRAIL (100 ng/g) + TMS (1 μg/g)-treated group compared with those of the other groups. No obvious changes in body weight were noted among mice subjected to different treatment regimens. ^∗∗P < 0.01; ^∗∗∗P < 0.001 versus control. (c) The expression of Bax, PUMA, and P53 was evaluated using immunohistochemistry (IHC). The scored IHC results were analyzed by multiplying the percentages of positive cells by the intensity score obtained using confocal microscopy. Signal intensity was analyzed and scored by two independent pathologists. The score of 0 indicates lack of staining, 1 indicates mild staining, and 2 indicates obvious staining. Osteosarcoma cells in five separate areas were chosen at random and analyzed according to the percentages of positively labeled cells (^∗∗P < 0.01).

Figure 10

Schematic diagram of the mechanisms underlying TRAIL and TMS-mediated apoptosis in osteosarcoma.