| [Objective]Transferrin receptor (TfR) plays a very important role in the acquisition process of iron for cells. It is highly and stably expressed in tumor cell surface and malignant tissues. So it is a specific target for tumor biotherapeutics. Anti-TfR monoclonal antibody (mAb) can specially recognize the outer segment of TfR on the tumor cell and interfere in the intake of iron. Therapeutic strategies have been designed to target at TfR so as to interfere with tumor iron metabolism. In previous studies, we have demonstrated the anti-tumor effect of antibodies against TfR. Nevertheless, the anti-TfR mAb treatment resulted in the upward adjustment of hypoxia inducible factor-la (HIF-la) and HIF transcription targets, which may increase tumor angiogenesis and to contribute to tumor progression and metastasis. And blocking of iron intake via the TfR resulted in the upward adjustment of vascular endothelial growth factor (VEGF). VEGF stimulates angiogenesis and increases the permeability of blood vessels. In previous experiments, we observed that HIF-la inhibition was related to the prevention of cell proliferation and induced cell apoptosis in vitro and in vivo. To imagine, when use of anti-TfR mAb and anti-tumor drug in inhibiting both cell proliferation and expression of HIF-1 A, may be of therapeutic use. In this study, we investigated the anti-tumor effects of anti-TfR mAb alone or in combination with sinomenine hydrochloride in vitro and vivo, as well as the potential mechanism. This provides a new insight into how tumor cells overcome the interference of iron intake to survive and a new therapeutic strategy to develop TfR mAb combined with Sinomenine hydrochloride for liver cancer. [Methods]1. Apoptosis assayHepG2 cells were planted in 6-well plants at a density of 1×105cells/well and then incubated with Sinomenine hydrochloride at different concentrations. After 24 hours,48 hours and 72 hours, tumor cells were harvested and washed with ice-cold PBS three times and incubated with a combination of 10μg/mL FITC-Annexin V and 50μg/mL PI (Roche Diagnostics, Mannheim, Germany) at room temperature for 30min. Analysis was performed by flow cytometry. After treated by TfR mAb, sinomenine hydrochloride alone or in combination for 72 hours, HepG2 cells were harvested and tested by the above method.2. Cell cycle analysisAfter treated by TfR mAb, sinomenine hydrochloride alone or in combination for 72 hours, HepG2 cells were harvested and fixed with 70% ethanol at 4℃for 2 hours, and then stained with 50μg/mL propidium iodide contained 0.25 mg/mL RNase A at room temperature for 30 minutes. Analysis was performed by FCM.3. Cell proliferation analysisHepG2 cells were respectively treated with anti-TfR mAb alone, sinomenine hydrochloride alone or in combination for 72 hours after being marked with carboxyfluorescein diacetate (CFSE). The same concentration of non-specific mouse IgG was used as an isotype control. Tumor cells were harvested and washed with ice-cold PBS and detected by FCM.4. Western blot for detection of COX-2 and VEGF expressionsSamples containing 100μg of protein were added into 12% SDS-PAGE. The proteins were transferred on blotting grade nitrocellulose membrane. Blots were probed with primary antibodies in anti-COX-2 antibody (1:200) and anti-VEGF antibody (1:200) for overnight at 4℃. The membrane washed in 1×PBST three times for 5 min each followed by incubation with the secondary antibody horseradish peroxidase goat anti-rabbit IgG antibodies (1:5000) for 1h at room temperature. The blots were exposed to autoradiography films and developed. A laser density scan was performed on the films. The ratio between cox-2/VEGF and the correspondingβ-actin density integrating represented the protein express level. All protein were isolated and Western blots were repeated thrice.5. Statistical analysisStatistical significance of the data was calculated by Student's t-test or ANVOA. A significance level of p<0.05 was chosen. [Results and Conclusions]These results suggested that the anti-TfR mAb or Sinomenine hydrochloride could induce apoptosis, inhibit proliferation and affect cell cycle. Compared with the individual effects of TfR mAb or sinomenine hydrochloride, the synergetic effect on the growth inhibitory effects and induced apoptosis of tumor cells had been shown when TfR mAb and sinomenine hydrochloride were used simultaneously. The protein expressions of COX-2 and VEGF in HepG2 treated with TfR mAb alone were increased along with the increased dosage of TfR mAb, whereas COX-2 expression was dramatically decreased in HepG2 treated with sinomenine hydrochloride alone in its increased dosage. Furthermore, we demonstrated suggested that the inhibitory effects on COX-2 and VEGF expression of HepG2 by the combined use of sinomenine hydrochloride and TfR mAb are more prominent. In vivo, we discovered that the anti-TfR mAb or sinomenine hydrochloride could extend the life span of the nude mice with liver tumor and decrease the growth velocity of the tumor volume. To sum up, these results showed that the combined use of sinomenine hydrochloride and TfR mAb may exert synergetic inhibitory effects on human hepatoma cell HepG2 in a COX-2 dependent way. This provides a new insight into how tumor cells overcome the interference of iron intake to survive and a new therapeutic strategy to develop TfR mAb combined with Sinomenine hydrochloride for liver cancer.
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