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Sciedu.caJournal of Solid Tumors 2016, Vol. 6, No. 1 BRIEF REPORT
Studies on antitumor activity spectrum of doxycycline Bo Chen1, 3, Hong-gang Zhou2, 3, Wei Wang2, 3, Wen-guang Gu2, 3, Dong Zhao2, 3, Peng Wang∗3 1College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China 2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China3Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China Received: January 28, 2016 Accepted: February 25, 2016 Online Published: March 16, 2016 In this study, in vitro (21 kinds of cell lines) and in vivo (four kinds of tumor-bearing mouse models) experiments were performed to determine the anticancer effect of doxycycline. This drug may elicit a strong inhibitory effect on cancer cells and improve the survival condition of mice. This study also preliminarily investigated the inhibitory effect of doxycycline on different kinds of tumor cells.
Key Words: Doxycycline, Cancer cell, Antitumor, Cell proliferation Novel efficacy of "old" drugs have been extensively investi- All of the cell lines were cultured in a medium supplemented gated for use as new treatments. For instance, antibiotics can with 10% (v/v) heat-inactivated (56◦C, 30 min) fetal calf be used as anticancer drugs.Four to five classes of drugs, serum (Hyclone, USA) and maintained at 37◦C in humid- such as tetracyclines, can also be used to eradicate 12 cancer ified air containing 5% CO2. PC-3 cells were cultured in cell lines.Tetracyclines are cytotoxic to tumor cells.
F12K medium. NCI-H446, A549, PLC/PRF/5, SMMC-7721, As a semi-synthetic tetracycline, doxycycline inhibits matrix MuM2B, MuM2C, MCF-7, SGC-7901, SH-SY5Y, K562, metalloproteinase (MMP) activation, prevents cell prolifera- and HL60 cells were cultured in RPMI1640 medium. HepG- tion, and downregulates DNA-dependent protein kinase.
2, MHCC97H, MHCC97L, LOVO, A875, A375, MDA-MB-231, PANC-1, ASPC-1, and HeLa cells were cultured in 2. MATERIALS AND METHODS DMEM (high glucose) medium.
Cell viability was determined via an MTT assay. The cells (5 Doxycycline was purchased from Sangon Biotech (Shanghai, × 103 cells/well) were seeded in 96-well culture plates. TheChina). Cyclophosphamide was procured from Alfa-Aesa cells were incubated overnight. Afterward, they were treated (Ward Hill, USA). All of the cell lines were supplied by Kaiji with various doxycycline concentrations (0.00, 0.39, 0.78, Biotech (Nanjing, China). Cell culture media were obtained 1.56, 3.12, 6.25, 12.5, 25.00, 50.00, and 100.00 µM). After from Hyclone (Waltham, USA). Mice were purchased from 48 h of incubation, 20 µl of MTT was added at 37◦C for 4 h, the Academy of Military Medical Sciences of the Chinese and cell viability was determined. Then, 150 µl of dimethyl PLA (Beijing, China).
sulfoxide was added to dissolve the formazan crystals. Opti- ∗Correspondence: Peng Wang; Email: [email protected]; Address: Tianjin Key Laboratory of Molecular Drug Research, Tianjin Interna- tional Joint Academy of Biomedicine, Tianjin, China.
Published by Sciedu Press Journal of Solid Tumors 2016, Vol. 6, No. 1 cal density was determined at 570 nm by using a microplate 50 dose of doxycycline for different cancer cell reader (MultiskanTM FC, Thermo Scientific, Waltham, MA, linesUSA).
Five- to six-week-old male C57BL/6 and BAlB/c nu/nu mice were prepared for mouse and human tumorigeneses, respec- tively. They were maintained in a specific pathogen-free ani- mal care facility in accordance with institutional guidelines.
Tumor xenografts were established by subcutaneously inject- ing 1 × 107 cells suspended in phosphate buffered salineinto the flank. The mice were randomly divided into five groups (n = 10/group) 1 day after the tumor cells were inocu- Lung cancer cells lated. After the tumors reached an approximate volume of 100 mm3 (approximately 6 weeks after injection), the mice Hepatoma carcinoma cell were treated with 60, 30, or 15 mg/kg doxycycline, 20 mg/kg cyclophosphamide, and saline via oral gavage once a day.
Their body weights were measured at different time points Breast cancer cells after the tumor cells were inoculated. Tumor diameters were also measured every day. Tumor volumes were calculated ac- cording to the following equation: V = ab2/2, where a is the tumor length and b is the tumor width. Seven weeks after the treatment was administered, all of the mice were euthanized, and xenografts were resected and measured. The inhibitory Pancreatic cancer rates were calculated as (%) = (1 − T i/Ci) × 100%, where Ti is the tumor volume of the treated group and Ci is the Human embryonic kidney HEK 293 tumor volume of the control group. Changes in weight were determined as (%) =(Tw/Cw − 1) × 100%, where Tw is thebody weight of the treated group and Cw is the body weight Gastric cancer cells of the control group.
Cervical cancer cells Data were expressed as means ± standard deviation. Compar- isons between the groups were performed through one-way ANOVA, followed by Bonferroni post hoc test (SPSS version 17.0, SPSS Inc., Chicago, IL, USA). The significance level Prostate cancer cells was set at P < .05.
Note. Each experiment was performed in triplicate. Results show the means of the three experiments, and the error bars represent standard deviation. We analyzed the changes in the tumor volume and bodyweights of the mice treated with doxycycline and then com- Performing the MTT assay, we determined the effect of the pared them with those observed in the control group. The 48 h doxycycline treatment on the cell viability of various inhibition effect of doxycycline on the tumor cells was very cancer cell lines. We measured IC50 of the different cell evident compared with that of the positive drug control group lines to doxycycline. Doxycycline significantly inhibited the (see Table 2). Doxycycline could significantly inhibit the proliferation of these cells in a dose-dependent manner (see proliferation of B16 melanoma cells, Lewis lung cancer cells, Table 1). IC50 of most cells was less than 5 µM, which was MCF-7 breast cancer cells, and NCI-H446 human small cell quite low. This result demonstrated that many cancer cell lung cancer cells.
lines were very sensitive to doxycycline.
The body weight of the doxycycline-treated mice was sig- Tumor formation ability is an important indicator of cancer nificantly higher than that of the control group. The body cell malignancy. Therefore, we investigated the inhibitory weights of the mice in the low-, middle-, and high-dose effect of doxycycline on tumor growth in vivo by using four groups respectively treated with 15, 30, and 60 mg/kg doxy- tumor-bearing mouse xenograft models.
cycline were also significantly higher than those of the mice Journal of Solid Tumors 2016, Vol. 6, No. 1 in the cyclophosphamide-treated group.
caused few side effects. Doxycycline also elicited remark-able anti-tumor effects. Therefore, this drug can be applied Doxycycline increased the body weight of the mice and im- to treat and prevent cancer.
proved their survival conditions. This finding indicated that doxycycline exhibited a very good antitumor activity and Table 2. The changes of tumor volume and body weight compared with control group Observation
Cyclophosphamide -14.3 -7.1 -14.0 tor activities.Therefore, this drug elicits an antitumor Doxycycline influences cell adhesion processes and the activ- effect through the combined action of various targets.
ity of focal adhesion kinase; for instance, this drug prevents Our study revealed that doxycycline elicits a remarkable in- cell adhesion during migration.Doxycycline induces the hibitory effect on cancer cells. In our animal experiments, membrane expression of VE-cadherin on endothelial cells the inhibitory rates of doxycycline were higher than those of and prevents vascular hyperpermeability.Besides, doxy- cyclophosphamide. Doxycycline could also improve thecycline also inhibits metabolism and metastasis by interfering survival condition of mice. Therefore, doxycycline is a with MMPs and E-cadherin levels.Previous study had promising anticancer agent because it inhibits cancer cell proved decreasing MMP activity could inhibit cancer cell proliferation, induces less toxic effects and causes fewer side invasion and metastasis, decreasing of E-cadherin expression effects than other drugs do.
is sufficient to confer metastatic ability to breast cancer.Doxycycline increases E-cadherin levels, decreases vimentin CONFLICTS OF INTEREST DISCLOSURE protein expression, which are two markers of invasiveness The author declares that there is no conflict of interest state- and metastasis, and inhibits EMT-related transcription fac- ment.
tumor types: Treating cancer like an infectious disease. Oncotarget.
 Ribeiro JA, Silva F, Pereira CM. Electrochemical study of the anti- 2015; 6(7): 4569-84. PMid:25625193. cancer drug daunorubicin at a water/oil interface: Drug lipophilic- ity and quantification. Analytical chemistry. 2013; 85(3): 1582-90.
 Richards C, Pantanowitz L, Dezube BJ. Antimicrobial and non- antimicrobial tetracyclines in human cancer trials. Pharmacological  Wang L, Meng Q, Wang C, et al. Dioscin restores the activity of the Research. 2011; 63(2): 151-6. PMid:20951804. anticancer agent adriamycin in multidrug-resistant human leukemia K562/adriamycin cells by down-regulating MDR1 via a mechanism  Lokeshwar BL. Chemically modified non-antimicrobial tetracy- involving NF-κB signaling inhibition. Journal of natural products.
clines are multifunctional drugs against advanced cancers. Phar- 2013; 76(5): 909-14. PMid:23621869. macological Research. 2011; 63(2): 146-150. PMid:21093590.
 Chaoul N, Fayolle C, Desrues B, et al. Rapamycin impairs anti-  Lamb R, Fiorillo M, Chadwick A, et al. Doxycycline down-regulates tumor CD8+ T-cell responses and vaccine-induced tumor eradica- DNA-PK and radiosensitizes tumor initiating cells: Implications for tion. Cancer Research. 2013; 75(16): 3279-91. PMid:26122844.
more effective radiation therapy. Oncotarget. 2015; 6(16): 14005- 25. PMid:26087309.  Wu W, Dong Y, Gao J, et al. Aspartate-modified doxorubicin on its N-terminal increases drug accumulation in LAT1-overexpressing  Sun T, Zhao N, Ni C, et al. Doxycycline inhibits the adhesion and tumors. Cancer scienc. 2015; 106: 747-56. PMid:25867020. migration of melanoma cells by inhibiting the expression and phos- phorylation of focal adhesion kinase (FAK). Cancer letters. 2009;  Lamb R, Ozsvari B, Lisanti CL, et al. Antibiotics that target mi- 285(2): 141-50. PMid:19482420. tochondria effectively eradicate cancer stem cells, across multiple Published by Sciedu Press Journal of Solid Tumors 2016, Vol. 6, No. 1  Fainaru O, Adini I, Benny O, et al. Doxycycline induces membrane cal research communications. 2009; 384(1): 6-11. PMid:19379710.
expression of VE-cadherin on endothelial cells and prevents vascu- lar hyperpermeability. The FASEB Journal. 2008; 22(10): 3728-35.
 Zhao Y, Yan Q, Long X, et al. Vimentin affects the mobility and invasiveness of prostate cancer cells. Cell biochemistry and function.
 Chen Z, Wang Y, Liu W, et al. Doxycycline inducible Kruppel- 2008; 26(5): 571-7. PMid:18464297. like factor 4 lentiviral vector mediates mesenchymal to epithelial transition in ovarian cancer cells. Plos one. 2014; 9(8): e105331.
PMid:25137052.  Qin Y, Zhang Q, Lee S, et al. Doxycycline reverses epithelial-to- mesenchymal transition and suppresses the proliferation and metas-  Baranwal S, Alahari SK. Molecular mechanisms controlling E- tasis of lung cancer cells. Oncotarget. 2015; 6(38): 40667-79.
cadherin expression in breast cancer. Biochemical and biophysi-
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