Gastric cancer is a leading cause of death worldwide, accounting for approximately 700,000 deaths per year. Although surgery is the mainstay of any curative treatment, recurrences and metastases are still observed in approximately two-thirds of patients [1
]. Since gastric cancer is not sensitive to radiotherapy, chemotherapy remains the most effective treatment for improving patients’ quality of life and prolonging their survival [2
Microtubules, built by α/β-tubulin dimers, are key components of the cytoskeleton and associated with multiple cell functions [3
]. During mitosis, microtubules undergo rapid polymerization and depolymerization to enable movement of chromosomes. As cell cycle progression approaches metaphase, microtubules are disrupted to form a mitotic spindle to facilitate chromosomal alignment on the metaphase plate. In this process, tubulin subunits freely exchange on the microtubules. If suchfree exchange of tubulin subunits is disrupted, the mitotic spindles will be compromised, thus interfering disturbing the cell division. Drugs, such as taxol and vinblastine, can bind tubulin and prevent its incorporation into growing microtubules. Consequently, cells undergoing division, especially those showing a rapid division, are killed. Anti-microtubule drugs constitute an important major class of anticancer drugs with broad activities both in solid tumors and hematological malignancies [4
]. Due to the clinical drug resistance observed in patients using anti-microtubule drugs, the discovery of new agents with optimal biopharmaceutical and pharmacological properties becomes the focus of numerous academic and industrial groups [7
Previous studies showed that deoxypodophyllotoxin (DPT, Figure 1
A), a naturally occurring flavolignan, inhibited microtubule assembly [8
]. It shows potent antiproliferative and antitumor properties on several cancer types [9
]. However, the in vivo
antitumor efficacy of DPT are currently indeterminate and the details of the cellular and molecular mechanisms underlying its action against gastric cancer have not been systematically investigated. The present study aims to investigate the anti-gastric cancer effects of DPT both in vitro
and in vivo
, and to further characterize its mechanism.
Natural products, especially microtubule-binding agents, play important roles in the fight against cancer. From the clinical use of vinblastine in 1961 and paclitaxel in 1992 to ixabepilone in 2007, microtubule-binding natural products have continually contributed to the development of cancer therapy. Clinically, inhibiting microtubules is the primary therapeutic strategy for the treatment of gastric cancer. Furthermore, the clinical success of the currently available microtubule-binding chemotherapeutic agentsis mainly based on their direct and strong cytotoxic effects against tumor cells. DPT, a derivative of podophyllotoxin, is a lignan with potent antimitotic and antiviral properties isolated from rhizomes of Sinopodophullum hexandrum
(Berberidaceae). Several studies indicated that DPT inhibits microtubule assembly and cell growth of several types of human cancer cell lines. DPT has been found to regulate gene expression associated with cell proliferation, cancer cell invasion and metastasis in vitro
]. In the present study, we established a xenograft model of gastric cancer in nude mice and systematically evaluated the anti-gastric cancer effects of DPT both in vitro
and in vivo
Our in vitro investigations confirmed that DPT treatment induced extensive microtubule depolymerization and disrupted the microtubule network in SGC-7901 cancer cells, compared to the effects observed in cells treated with taxol. Results from CCK-8 assay showed that DPT significantly suppressed SGC-7901 cell proliferation and viability in a dose- and time- dependent manner.
In accordance with the fact that microtubule-binding agents arrest the cell cycle at G2/M phase [15
], our results showed that DPT induced a G2/M blockade in SGC-7901 cells as indicated by flow cytometry analysis. We further investigated the molecular mechanism by which DPT stopped the cell cycle. It is well-known that cyclins and cyclin-dependent protein kinases (Cdks) are key regulators of the cell cycle progression. Cyclin B1 plays animportant role in the G2/M transitionas well as in the M phase progression. As reported, the cyclin B1 protein level substantially accumulates in G2 phase, peaks as cell division approaches metaphase, and rapidly decreased during anaphase [16
]. We observed an accumulation of cyclin B1 protein 6 h after DPT-treatment. Its cellular level considerable increased after 48 h of treatment. It is well-known that cyclin B1 accumulation is a marker of cells stopped in G2 and/or M phases of the cell cycle. Cdc2 interacts with cyclin B1 and form the maturation promoting factor (MPF) which regulates the transition from G2 to M phase [18
]. Cdc25C regulates the subsequent activation of cyclin B1/Cdc2 complex by removing the inhibitory phosphorylations of Cdc2 on Thr14/Tyr15. In the present study, the expression of Cdc2, Cdc25C regulatory proteins markedly changed after administration of DPT, implying that those proteins may be involved in DPT-induced G2/M arrest.
Cells would either undergo repair mechanisms or follow the apoptotic pathway when the arrest of cell cycle progression at G2 phase occurs. FACS analysis showed a marked accumulation of SGC-7901 cells in G2/M phase prior to the induction of a sub-G1 cell population. These data suggest that G2/M-phase arrest might be an upstream event leading to apoptosis. The fact that blockage of cell cycle progression at mitotic phase leads to apoptotic cell death has been well established [6
]. In the present study, the Annexin V/PI double-staining assay showed that the apoptotic rate of SGC-7901 cells significantly increased after DPT treatment.
The apoptosis signal is mainly regulated by the caspase family, which exist as inactive zymogens in cells and undergo a cascade of catalytic effects at the initiation of apoptosis. Activation of caspase-3 and caspase-7 leads to the cleavage and inactivation of many cellular proteins, such as lamin and PARP, therefore resulting in apoptotic cell death in many cell types [19
]. Our results showed that DPT induced activation of caspase-3 which was accompanied by PARP cleavage, indicating that the caspase apoptotic pathway is involved in the mechanism of DPT-induced cell death.
The strong tumor inhibition properties as well as the caspase-mediated apoptotic action of DPT prompted us to evaluate its efficacy and safety in vivo
. We used the well-known anti-cancer agents docetaxel and etopside as positive controls. Docetaxel was chosen due to the fact that it is widely used in the clinic as a first-line drug for gastric cancer chemotherapy and shares the similar mechanism of DPT action. On the other hand and like DPT, etopside is a derivative of phodophyllotoxin. In our preliminary experiments, DPT suppressed tumor growth at a dose of 20 mg/kg to an extent without any significant changes of the mice body weight (Figure 7
A,B). Side effects, such as hair loss, lethargy, dysphoria, or other macroscopicalvisceral pathogenic changes were not observed (data not shown). DPT at 5 and 10 mg/kg also exerted robust growth inhibitory activity in the xenograft model. In contrast, marked weight loss was observed in the mice administrated etopside. Furthermore, the effect of DPT at 10 and 20 mg/kg was more pronounced than that of docetaxel. Hence, these data clearly indicated that DPT possessed a strong anti-tumor activity in vivo
with a reasonable safety margin.
The targets of microtubule-binding agents in cancer therapy include both cancer cells and vascular endothelial cells. Angiogenesisis mediated by endothelial cells can be quantified through counting microvasculars in a given area by immunohistochemical staining (microvessel density, MVD) [20
]. Indeed, MVD has been extensively evaluated as a measure of angiogenesis [21
]. MVD in tumors derived from SGC-7901 cells xenograft significantly decreased after DPT and docetaxel treatment for three week. Consequently, our results provide the initial evidence that DPT exerts a potent anti-angiogenic effect.
Antiangiogenesis has been an attractive anticancer strategy for more than fifty years [22
]. Compounds that target the microtubule have been greatly successful in the clinic as chemotherapeutics, and this success is likely due to their ability to target cells regardless of their cell cycle stage. Preclinical and clinical studies have suggested that microtubule-binding agents might be a particularly useful class of drugs for vascular-targeted therapy [23
].There are a lot of these compounds in development that act on the vasculature, and various formulations of clinically used drugs are being developed to take advantage of these anti-angiogenic properties. Thus, DPT, as a drug that target the microtubule will continue to have a major impact in oncology not only as anti-mitotics but also as potent inhibitors of angiogenesis.
DPT and it’s hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complex were obtained from the Medicinal Chemical Institute, China Pharmaceutical University, Nanjing, China. Taxol was purchased from Guilin Huiang Biochemistry Pharmaceutical Ltd (Guilin, Guangxi, China). Docetaxol and etopside were obtained from Qilu Pharmaceutical Ltd (Jinan, Shandong, China). A stock solution (10−2 M) was prepared in DMSO and stored at −20 °C. The antibodies against β-actin and α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against cyclin B1, Cdc2, Cdc25C, PARP and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa-Fluor 488 (green)-conjugated second antibody was purchased from Invitrogen (Carlsbad, CA, USA). Cell cycle and Apoptosis Analysis Kit(s), Caspase-3 Activity Assay Kit and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Suzhou, China). FITC-Annexin V Apoptosis Detection Kit was purchased from BD Bioscience (San Diego, CA, USA). Cell Counting Kit (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan).
4.2. Cell Culture
Human gastric carcinoma SGC-7901 cell line was purchased from Type Culture Collection of Chinese Academy of Sciences, Shanghai, China. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (all reagentswere purchased from Hyclone (Logan, UT, USA) and maintained in a humidified atmosphere containing 5% CO2.
Female nude mice (6–8 week old, BALB/c) were used to establish the SGC-7901 xenograft tumor model and were purchased from the Institute of Laboratory Animal Science, Academy of Military Medical Sciences of the Chinese PLA (Beijing, China). All animal tests and experimental procedures were approved by the Ethical Committee of China Pharmaceutical University, Nanjing University, and Laboratory Animal Management Committee of Jiangsu Province (Approval ID: 2110682).
4.4. Immunofluorescence Analysis
SGC-7901 cells were plated on glass coverslips and treated with vehicle, 75 nM DPT, or with 100 nM taxol [12
] diluted in media for 6, 12 and 24 h. The cells were fixed with 4% paraformaldehyde for 30 min, permeabilized in 0.1% Triton X-100/TBS for 10 min, blocked with 5% bovine serum albumin for 1 h to reduce nonspecific staining and then incubated with primary anti-α-tubulin antibody (4 °C, overnight) and Alexa-Fluor 488-conjugated secondary antibody for 60 min. The nucleus was stained with Hoechst 33342 for 1 h. Fluorescence images were obtained using a confocal microscope (FV-1000; Olympus, Tokyo, Japan).
4.5. Cell Proliferation Analysis
Cells were seeded in 96-well plates at a density of 1500–3000 cells/well and allowed to adhere overnight and then treated with either vehicle (RPMI-1640 medium) or DPT (25, 50, 75 or 100 nM) for 6, 12, 24, 48 and 72 h. Cell proliferation was assessed using the CCK-8 assay [25
]. The inhibition rate of cell proliferation was calculated as (ODcontrol
4.6. Cell Cycle Analysis
SGC-7901 cells were treated with DPT (25, 50 and 75 nM) for 6, 12, 24 and 48 h in complete medium. The floating and adherent cells were collected, washed twice with cold PBS and centrifuged. Cells were then fixed in 70% (v/v) ethanol for 24 h at 4 °C. After centrifugation, cells were washed with cold PBS and stained according to the manufacturer’s protocol. After incubation for 30 min in the dark, cell cycle distribution was determined by flow cytometry (BD, FACSCalibur) using multi-cycle Software (ModFit LT 3.2 Mac).
4.7. Apoptosis Detection
SGC-7901 cells were treated with DPT as previously described for various lengths of time, harvested, washed with cold PBS, and stained with AnnexinV-FITC/PI according to the manufacturer’s protocol. After incubation, the apoptotic cells were measured by flow cytometry (BD, FACSCalibur) using the CELLQUEST Pro.
4.8. Western Blot Analysis
Cells were treated with DPT and cell lysates were prepared as described previously [26
]. Equal amounts of cell lysates (50 µg of protein) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidenedifluoride membranes (PVDF) (Millipore; Bedford, MA, USA). Membranes were then blocked with 5% non-fat dry milk in Tris-Buffed- Saline with Tween (TBST) for 1 h, and incubated with appropriate dilutions of primary antibodies (overnight, 4 °C) followed by horseradish peroxidase-conjugated secondary antibodies. The immunoreactive proteins were then detected by the ECL-Plus Western Blotting Detection System.
4.9. Caspase-3 Activity Assay
Caspase-3 activity was assayed by colorimetric detection of p-nitroanilidine (pNA) after cleavage of the peptide substrate, N-acetyl-Asp-Glu-Val-Asp (DEVD)-p-nitroaniline, specific for caspase-3. Gastric cancer cells were treated with DPT as described previously. Floating and adherent treated-cells were then collected and lysed in ice-cold lysis buffer for 15 min. Absorbance was measured at 405 nm with a Tecan microplate reader (Safire2, TECAN, Männedorf, Switzerland) after 12 h of incubation in a humidified atmosphere of 5% CO2 in air at 37 °C. Data are presented as mean ± SD of three independent experiments.
4.10. In Vivo Study
Xenograft model of gastric cancer was established by a subcutaneous injection (s.c.) of 3 × 106 SGC-7901 cells into the right rear flank of each mouse. Following two weeks of growth, tumor tissues were cut into multiple 3 × 3 × 3 mm3 pieces and implanted (s.c.) into the right rear flank of each mouse using a range trocar. Tumor diameters were measured with a caliper and tumor volume was calculated by the formula: Volume = (width)2 × length/2. Treatments were started after one week when the tumors reached an average volume of 100–200 mm3. Animals were randomly divided into 6 groups (n = 6) and intravenously injected with: (a) HP-β-CD; (b) 20 mg/kg docetaxel; (c) 20 mg/kg etoposide; (d) 5 mg/kg DPT; (e) 10 mg/kg DPT; (f) 20 mg/kg DPT. HP-β-CD, DPT and etoposide were administrated three times a week and docetaxel was administrated once a week. After 21 days of treatment, mice were sacrificed and tumors were weighed and excised for immunohistochemistry assay. The inhibition rate was calculated as: (tumor weight of vehicle control group − tumor weight of treatment group)/tumor weight of vehicle control group × 100%.
4.11. Quantification of Microvessel Density
Microvessel density (MVD) in tumors derived from SGC-7901 cell line xenograft was examined using a Blood Vessel Staining Kit (Millipore). Primary antibody against von Willebrand Factor was used to evaluate MVD and was performed according to the methods described previously [16
]. MVD was assessed on vWF stained slides. Endothelial cell clusters and endothelial cells which were stained brownish-yellow were regarded as a single microvessel [27
]. Microvessels were counted on five microscopic fields per specimen at ×200 magnification [28
4.12. Statistical Analysis
All data represent mean values of at least three independent experiments and are expressed as mean ± SD. Statistically significant differences were assessed via one-way ANOVA followed by Tukey’s post hoc test for multiple comparison tests. A value of p < 0.05 was considered statistically significant.