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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 4  |  Page : 1261-1265

(Z)-3,4,3',5'-tetramethoxystilbene, a natural product, induces apoptosis and reduces viability of paclitaxel-and cisplatin-resistant osteosarcoma cells


Department of Pharmacy, Chinese Medicine Hospital of Jiangshan City, Jiangshan, Zhejiang, China

Date of Web Publication7-Feb-2017

Correspondence Address:
Huiqing Xu
Department of Pharmacy, 38 Riverside Road, Jiangshan-324 100, Zhejiang
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.158035

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 > Abstract 

Aim of Study: Osteosarcoma is a common bone tumor and the development of drug resistance in therapy of osteosarcoma is a general rule. The natural compounds isolated from medicinal plants represent a valuable resource for anticancer therapeutics. (Z)-3,4,3', '-tetramethoxystilbene is one of them. In this work, we investigated the potential anti-cancer activities of (Z)-3,4,3' ,'5-tetramethoxystilbene in paclitaxel- and cisplatin-resistant osteosarcoma cells.
Materials and Methods: ATP assay was used to examine cell viability. Cell nuclei staining assays with Hoechst or propidium iodide (PI) were used to evaluate cell apoptosis. Xenograft tumor model was used to evaluate the in vivo anti-cancer activities of (Z )-3,4,3',5'-tetramethoxystilbene. TUNEL staining assay was used to evaluate the apoptosis of tumor cells.
Results: We found that (Z)-3,4,3',5'-tetramethoxystilbene could effectively reduce viability of both paclitaxel-and cisplatin-resistant osteosarcoma cells. Moreover, (Z)-3,4,3',5'-tetramethoxystilbene induced dramatic apoptosis in resistant cells. Importantly, (Z)-3,4,3',5'-tetramethoxystilbene significantly suppressed in vivo tumor growth of cisplatin-resistant osteosarcoma.
Conclusion: This is the first report on anti-cancer activity of (Z)-3,4,3','5- tetramethoxystilbene in resistant osteosarcoma cells. Our studies suggest that (Z)-3,4,3',5'-tetramethoxystilbene is a promising therapeutic drug for overcoming drug resistance in osteosarcoma.

Keywords: Apoptosis, osteosarcoma, resistance, viability, (Z)-3,4,3',5'-tetramethoxystilbene


How to cite this article:
Xu H. (Z)-3,4,3',5'-tetramethoxystilbene, a natural product, induces apoptosis and reduces viability of paclitaxel-and cisplatin-resistant osteosarcoma cells. J Can Res Ther 2016;12:1261-5

How to cite this URL:
Xu H. (Z)-3,4,3',5'-tetramethoxystilbene, a natural product, induces apoptosis and reduces viability of paclitaxel-and cisplatin-resistant osteosarcoma cells. J Can Res Ther [serial online] 2016 [cited 2020 Oct 25];12:1261-5. Available from: https://www.cancerjournal.net/text.asp?2016/12/4/1261/158035


 > Introduction Top


Osteosarcoma is a common type of bone tumor and is easy to develop early metastasis. Surgery treatment combined with chemotherapy has achieved certain survival rate in osteosarcoma patients. The 5-year survival rate has reached 70%.[1],[2],[3],[4] However, development of resistance to chemotherapy drugs soon after the start of treatment is common, which significantly reduces the survival rate, leading to cancer relapse and metastasis eventually.[5],[6],[7],[8],[9] Due to the dramatic influent of drug resistance on cure rate in osteosarcoma chemotherapy, there is an urgent need for improved therapeutics. Attenuating or overcoming chemoresistance will significantly contribute to treatment of osteosarcoma. Development of novel reagents to overcome drug resistance is urgent and critical in therapeutics of osteosarcoma.

Compounds isolated from medicinal plants with potential anti-cancer bioactivities represent a valuable resource for anticancer therapeutics. Many isolated natural products have been proved to be of strong anticancer activities.[10] (Z)-3, 4, 3',5'-tetramethoxystilbene is one of them, which was originally isolated from E. rigida.[11] However, the anticancer activities of (Z)-3, 4, 3',5'-tetramethoxystilbene are far from well-known. In this paper, we investigated the potential anti-cancer activities of (Z)-3, 4, 3',5'-tetramethoxystilbene in paclitaxel- and cisplatin-resistant osteosarcoma cells both in vitro and in vivo. Our studies suggest that (Z)-3, 4, 3',5'-tetramethoxystilbene is a promising therapeutic drug candidate for overcoming drug resistance in osteosarcoma.


 > Materials and Methods Top


Materials

(Z)-3, 4, 3',5'-tetramethoxystilbene was purchased from Ryan Scientific (Mount Pleasant, SC, USA). Paclitaxel, cisplatin and other reagents were obtained from Sigma (St. Louis, MO USA).

Cells and cell culture

Human osteosarcoma cancer cell line MG-63 was obtained from the American Type Culture Collection (Rockville, MD, USA). Paclitaxel- and cisplatin-resistant cell lines MG-63-R-pac and MG-63-R-cis were obtained by culturing MG-63 cells with gradually increasing doses of paclitaxel and cisplatin, respectively. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37°C in an atmosphere comprising 95% air and 5% CO2.

Cell viability assay

Osteosarcoma cells were seeded in 96-well white plates at a density of 5 × 103 cells/well, and the compounds were added at a series of indicated concentrations, followed by incubation for 72 h. A CellTiter-Glo kit (Promega, Madison, WI, USA) was used to assess cell viability. Briefly, a cell lysis/ATP detection reagent was added (30 μL/well), the cells were shaken for 10 min, and the resulting luminescence was assessed using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). All IC50 values were determined using Compusyn software.

Cell nuclei staining assay

Osteosarcoma cells were seeded onto coverslips and incubated with (Z)-3, 4, 3',5'-tetramethoxystilbeneat indicated concentrations for 72 h, then cells were fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 5% normal goat serum for 30 min, the cells were stained with Hoechst for 20 min, and then imaged using a microscope (Nikon, Japan).

Flow cytomery analysis

Osteosarcoma cells were seeded in 6-well plates at a density of 5 × 105 cells/well, and treated with (Z)-3, 4, 3',5'-tetramethoxystilbene at indicated concentrations for 72 h. Then the cells were collected, fixed with 70% ethanol and stained with propidium iodide in the presence of RNase (1 g/l), 1 g/l sodium citrate and 0.5% Triton X-100 (v/v) for 30 min in the dark. A FacsCalibur sorter (BD Biosciences) was used to collect cells for further apoptosis analysis. The percentage of hypodiploidy was taken as a measure of apoptosis and quantified using ModFIT LT software.

Immunohistochemistry

Samples of tumor tissue were fixed in phosphate-buffered formalin and embedded in paraffin. Parafilm slices were then taken at a thickness of 4 μm before mounting onto slides. The slides were then deparaffinized by three, five-minute xylene washes before being re-hydrated through the following series of alcohol washes: Two washes of 100% ethanol for 10 min each, two washes of 95% ethanol for 10 min each, and a final deionized water wash for 1 min with gentle agitation. Slides were incubated with 10 mM sodium citrate buffer (pH 6.0) and heated 1 hr. The slides were then washed three times in deionized water for 5 min per wash before a 30-min block in 5% normal goat serum. TUNEL staining was performed using a FragEL kit (Calbiochem, Billerica, MA, USA) according to the manufacturer's instructions. Then the slides were stained with Hoechst for 15 min and imaged by fluorescent microscopy.

Tumor growth suppression in vivo

Nud/nud mice were purchased from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences in Shanghai, China. MG-63-R-cis cells were injected subcutaneously into the axillary regions of selected nud/nud mice (2 × 106 cells/100μL/mouse). Cells were allowed to grow to a volume of 50 mm 3 before mice were randomized to the following experimental groups: (Z)-3, 4, 3',5'-tetramethoxystilbene (50 mg/kg), (Z)-3, 4, 3',5'-tetramethoxystilbene (100 mg/kg) and control. All groups had seven animals per condition. Tumors were measured on alternating days with a microcaliper along with body weight. Total tumor volumes were calculated as follows: (mm 3) = width × width × length × 0.5. Mice were treated with (Z)-3, 4, 3',5'-tetramethoxystilbene for 12 days and then were euthanized 12 days later at which point the tumors were collected and saved for later analysis.

Statistics

Both Student's t- test and analysis of variance (ANOVA) were performed using StatView (SAS Institute, Cary, NC, USA). P < 0.05 was considered to be statistically significant. The data shown are representative of at least three independent experiments with similar results, and are the mean values of measures taken in triplicate. Error bars indicate the standard deviation.


 > Results Top


(Z)-3, 4, 3',5'-tetramethoxystilbene reduces viability of both paclitaxel- and cisplatin-resistant osteosarcoma cells

In order to generate paclitaxel- and cisplatin-resistant cell lines, we cultured MG-63 cells with gradually increasing doses of paclitaxel and cisplatin, two frequently used drugs in osteosarcoma therapeutics. After 6 months of culture, we examined and compared the sensitivity of both MG-63-R-pac and MG-63-R-cis cell lines towards paclitaxel and cisplatin, respectively. The results showed an approximately 4-fold increase in IC50 for resistant cells compared with matched parental cells (P < 0.01), suggesting that paclitaxel- and cisplatin-resistant cell lines MG-63-R-pac and MG-63-R-cis were successfully generated [Figure 1]a and [Figure 1]b.
Figure 1: (Z)-3,4,3',5'-tetramethoxystilbene suppresses viability of both paclitaxel- and cisplatin-resistant osteosarcoma cells, (a, b) MG-63 cells were induced to become paclitaxel- (MG-63-R-pac) or cisplatin- (MG-63-R-cis) resistant cells by incubation with gradually increasing doses of paclitaxel or cisplatin. Paclitaxel or cisplatin sensitivity was then examined by cell viability assay. (c) MG-63-R-pac and MG-63-R-cis, as well as the matched parental sensitive cells were treated with (Z)-3,4,3',5'-tetramethoxystilbene (0.625 ~ 20 μM) for 72 h, followed by measurement of cell viability

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(Z)-3, 4, 3',5'-tetramethoxystilbene is a natural product originally isolated from E. rigida, which exhibits potential anti-cancer activities.[11] We examined the effects of (Z)-3, 4, 3',5'-tetramethoxystilbene on both resistant osteosarcoma cell lines and the matched sensitive lines. As shown in [Figure 1]c, (Z)-3, 4, 3',5'-tetramethoxystilbene significantly reduced viability of both sensitive and resistant osteosarcoma cell lines in a concentration-dependent manner.

(Z)-3, 4, 3',5'-tetramethoxystilbene induces apoptosis of MG-63-R-pac and MG-63-R-cis cells

We next examined (Z)-3, 4, 3',5'-tetramethoxystilbene-induced apoptosis in MG-63-R-pac and MG-63-R-cis cells. [Figure 2]a shows that the subG1 DNA content in MG-63-R-pac and MG-63-R-cis cells significantly increased after treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene compared with the control group (P < 0.01). We also observed the nuclear morphology in MG-63-R-pac and MG-63-R-cis cells after treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene. Consistently, after treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene, MG-63-R-pac and MG-63-R-cis cell nuclei had dramatic morphological changes including condensation and fragmentation, both of which are indicators of cell apoptosis [Figure 2]b and [Figure 2]c. These alterations indicate that treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene induced apoptosis in both MG-63-R-pac and MG-63-R-cis resistant osteosarcoma cells. These data are consistent with the viability suppression results showed above.
Figure 2: (Z)-3,4,3',5'-tetramethoxystilbene induces apoptosis in both paclitaxel- and cisplatin-resistant osteosarcoma cells, (a) MG-63-R-pac and MG-63-R-cis cells were treated with (Z)-3,4,3',5'-tetramethoxystilbene at the indicated concentrations for 72 h, followed by propidium iodide staining and flow cytometry analysis. (b) MG-63-R-pac and MG-63-R-cis cells were incubated with (Z)-3,4,3',5'-tetramethoxystilbene for 72 h. The nuclei were stained with Hoechst, and analyzed by fluorescent microscope. Representative images are shown. (c) The number of cells with condensed/fragmented nuclei was quantitated by counting in five random fields and the inhibition was calculated

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(Z)-3, 4, 3',5'-tetramethoxystilbene suppresses MG-63-R-cis tumor growth in vivo

To further validate our in vitro work, nud/nud mice were treated with (Z)-3, 4, 3',5'-tetramethoxystilbene (50 and 100 mg/kg), to evaluate the effect of (Z)-3, 4, 3',5'-tetramethoxystilbene (ip) on MG-63-R-cis tumor growth in mice. The results show that a low dose of (Z)-3, 4, 3',5'-tetramethoxystilbene (50 mg/kg) culminated in an intermediate degree of tumor growth suppression. In contrast, a high dose of (Z)-3, 4, 3',5'-tetramethoxystilbene (100 mg/kg) led to significant inhibition of MG-63-R-cis tumor growth [Figure 3]a, P < 0.01]. These results are consistent with TUNEL and nuclei staining data, which showed that treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene resulted in significant apoptosis as well as morphological changes of nuclei [Figure 3]b. It should be noted that administration of (Z)-3, 4, 3',5'-tetramethoxystilbene was well tolerated by mice, who presented with no measurable signs of overt toxicity or decreases in body weight (P > 0.05) [Figure 3]c. Collectively, our data indicate that (Z)-3, 4, 3',5'-tetramethoxystilbene could reduce the viability of both sensitive and resistant osteosarcoma cells in vitro and suppress tumor growth in vivo.
Figure 3: (Z)-3,4,3',5'-tetramethoxystilbene suppresses MG-63-R-cis tumor growth in vivo,(a) After inoculation of MG-63-R-cis cells, (Z)-3,4,3',5'-tetramethoxystilbene (50 and 100 mg/kg) was injected into mice every day for 12 days. The tumors were measured every other day and the tumor volumes are shown. (b) (Z)-3,4,3',5'-tetramethoxystilbene induced apoptosis of MG-63-R-cis tumor cells in vivo was determined by TUNEL assay (green), and the nuclei were stained with Hoechst (blue). (c) (Z)-3,4,3',5'-tetramethoxystilbene had no significant cytotoxic effect on mice body weight during treatments

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 > Discussion Top


Osteosarcoma is a common type of bone tumor and the number of diagnoses is increasing on a yearly basis.[1],[2],[3],[4] An advanced state of this disease is often associated with resistance to conventional chemotherapies, resulting in an extremely low 5-year patient survival rate. Resistance is developed quickly in couples of months after treatment with anti-cancer drugs like paclitaxel and cisplatin in osteosarcoma patients.[5],[6],[7],[8],[9] These clinical observations make it imperative to explore new drugs in therapy of osteosarcoma especially resistant osteosarcoma.

Natural products represent a valuable resource for anticancer therapeutics, which has drawn more and more interest recent years.[10] (Z)-3, 4, 3',5'-tetramethoxystilbene is a natural product originally isolated from E. rigida, and it exhibits potential anti-cancer activities in a series of cell lines.[11] Here, we investigated the anti-cancer activities of (Z)-3, 4, 3',5'-tetramethoxystilbene in paclitaxel- and cisplatin-resistant osteosarcoma cells both in vitro and in vivo. We found that (Z)-3, 4, 3',5'-tetramethoxystilbene could effectively suppress viability of both paclitaxel- and cisplatin-resistant osteosarcoma cells. Consistently, treatment with (Z)-3, 4, 3',5'-tetramethoxystilbene induced dramatic apoptosis of paclitaxel- and cisplatin-resistant osteosarcoma cells. Importantly, (Z)-3, 4, 3',5'-tetramethoxystilbene efficiently suppressed MG-63-R-cis tumor growth in vivo. At the same time, (Z)-3, 4, 3',5'-tetramethoxystilbene exhibited no obvious toxicity in mice at used doses.

In summary, this is the first report on (Z)-3, 4, 3',5'-tetramethoxystilbene in osteosarcoma cells especially the cells with resistance towards drugs used in osteosarcoma therapy. The capability of (Z)-3, 4, 3',5'-tetramethoxystilbene to overcome/attenuate resistance makes it a promising compound for therapy of osteosarcoma especially those already developed resistance towards anti-cancer drugs. This might open another avenue for overcoming resistance in osteosarcoma therapeutics.

 
 > References Top

1.
Zhou W, Hao M, Du X, Chen K, Wang G, Yang J. Advances in targeted therapy for osteosarcoma. Discov Med 2014;17:301-7.  Back to cited text no. 1
    
2.
Tavanti E, Sero V, Vella S, Fanelli M, Michelacci F, Landuzzi L, et al. Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma. Br J Cancer 2013;109:2607-18.  Back to cited text no. 2
    
3.
Miao J, Wu S, Peng Z, Tania M, Zhang C. MicroRNAs in osteosarcoma: Diagnostic and therapeutic aspects. Tumour Biol 2013;34:2093-8.  Back to cited text no. 3
    
4.
Yuan J, Chen L, Chen X, Sun W, Zhou X. Identification of serum microRNA-21 as a biomarker for chemosensitivity and prognosis in human osteosarcoma. J Int Med Res 2012;40:2090-7.  Back to cited text no. 4
    
5.
Zhao G, Cai C, Yang T, Qiu X, Liao B, Li W, et al. MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human osteosarcoma. PLoS One 2013;8:e53906.  Back to cited text no. 5
    
6.
Yang X, Yang P, Shen J, Osaka E, Choy E, Cote G, et al. Prevention of multidrug resistance (MDR) in osteosarcoma by NSC23925. Br J Cancer 2014;110:2896-904.  Back to cited text no. 6
    
7.
He H, Ni J, Huang J. Molecular mechanisms of chemoresistance in osteosarcoma (Review). Oncol Lett 2014;7:1352-62.  Back to cited text no. 7
    
8.
Hasei J, Sasaki T, Tazawa H, Osaki S, Yamakawa Y, Kunisada T, et al. Dual programmed cell death pathways induced by p53 transactivation overcome resistance to oncolytic adenovirus in human osteosarcoma cells. Mol Cancer Ther 2013;12:314-25.  Back to cited text no. 8
    
9.
Tsai HC, Huang CY, Su HL, Tang CH. CTGF increases drug resistance to paclitaxel by upregulating survivin expression in human osteosarcoma cells. Biochim Biophys Acta 2014;1843:846-54.  Back to cited text no. 9
    
10.
Konkimalla VB, Efferth T. Anti-cancer natural product library from traditional chinese medicine. Comb Chem High Throughput Screen 2008;11:7-15.  Back to cited text no. 10
    
11.
Zaki MA, Balachandran P, Khan S, Wang M, Mohammed R, Hetta MH, et al. Cytotoxicity and modulation of cancer-related signaling by (Z)- and (E)-3,4,3',5'-tetramethoxystilbene isolated from Eugenia rigida. J Nat Prod 2013;76:679-84.  Back to cited text no. 11
    


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  [Figure 1], [Figure 2], [Figure 3]



 

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