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. 2012 Sep 2;62(3):447–454. doi: 10.1007/s00262-012-1337-y

Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells

Yi Hong 1,2, Juan Yang 1, Xin Shen 1,2, Huifen Zhu 1, Xiaoxu Sun 1, Xue Wen 1, Jing Bian 1, Heyu Hu 1, Lu Yuan 1, Juan Tao 3, Ping Lei 1, Guanxin Shen 1,✉
PMCID: PMC11028739  PMID: 22941037

Abstract

Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.

Keywords: Hepatoma, Transferrin receptor, Monoclonal antibody, Sinomenine hydrochloride

Introduction

Transferrin receptor (TfR) plays a very important role in the iron acquisition process of cells and is highly and stably expressed on the tumor cell surface and in malignant tissues [1–3]. Consequently, TfR is a specific target in tumor biotherapeutics [2, 6]. Anti-TfR monoclonal antibody (mAb) can specifically recognize the outer segment of TfR on the tumor cell and interfere with the intake of iron [2–4]. Therapeutic strategies have been designed to target TfR so as to interfere with tumor iron metabolism. In previous studies, we have demonstrated the anti-tumor effect of antibodies against TfR [7–9]. Nevertheless, anti-TfR mAb treatment resulted in an elevation in hypoxia-inducible factor-1α (HIF-1α) and HIF transcription targets, which may increase tumor angiogenesis and contribute to tumor progression and metastasis. Blocking iron intake via TfR resulted in the upward adjustment of VEGF [1]. VEGF stimulates angiogenesis and enhances the permeability of blood vessels [10, 11]. In previous experiments, we observed that HIF-1α inhibition was related to the prevention of cell proliferation and induced cell apoptosis in vitro and in vivo [12, 13]. The combination of anti-TfR mAb and anti-tumor drug may be of therapeutic use in inhibiting both cell proliferation and HIF-1α expression.

Sinomenine is a type of alkaloid extracted from the rhizome of Sinomenium acutum [14]. Its structure is similar to morphine, and its hydrochloride is often used in the clinic [15]. Previous studies have demonstrated the anti-inflammatory, immunosuppression, anti-angiogenesis, and anti-rheumatic effects of sinomenine hydrochloride [16–18]. Recently, a number of studies have focused on the anticancer activity of sinomenine hydrochloride, such as in lung cancer [19]. Besides, Du et al. [20] showed that Sinomenium acutum could combine with human serum transferrin and possibly be transported by transferrin.

The above findings indicate that sinomenine hydrochloride may affect the normal function of transferrin and the growth of cells. It has been reported that sinomenine hydrochloride may reduce the expression of cyclooxygenase-2 (COX-2) mRNA and protein in LPS-stimulated PC-12 cells [21]. COX-2 and its major physiologic product prostaglandin E2 (PGE2) have been shown to elevate HIF-1α expression in human lung epithelial and colon cell lines [22]. A reduction in COX-2 expression might inhibit VEGF, Flt-1, kinase insert domain receptor (KDR), angiopoietin-1, tie-2, and metalloproteinase-2 (MMP2) [23]. However, to date, it is still difficult to say whether there is synergistic effect between anti-TfR mAb and sinomenine hydrochloride in the inhibition of tumor cell growth.

In the present study, we investigated the potential synergy between anti-TfR mAb and sinomenine hydrochloride in the treatment of the TfR over-expressing human liver carcinoma cell line HepG2 in vitro and the potential mechanism involved.

Materials and methods

Anti-TfR mAb preparation

Murine monoclonal antibody against TfR was prepared as described previously [24]. Briefly, the hybridoma cells secreting TfR mAb were inoculated into nude mice (BALB/c-nu, SLAC Laboratory Animal Company, Shanghai, China) to induce the ascites for the preparation of anti-TfR mAb. The antibodies were purified via DEAE Sephadex A-50 chromatography and identified using SDS-PAGE.

Apoptosis assay

A total of 1 × 105 HepG2 cells were incubated with sinomenine hydrochloride (Zhengqing Pharmacy Company, Hunan, China) at increasing concentrations from 10 to 200 μg/mL for 24, 48, and 72 h. After washing three times, tumor cells were incubated with a combination of 10 μg/mL FITC–Annexin V and 50 μg/mL PI (Roche Diagnostics, Mannheim, Germany) at room temperature for 30 min. Analysis was performed using flow cytometry (FCM, LSRII, BD Biosciences, San Jose, USA).

Optimal concentration and time were determined based on the results of a preliminary experiment and the concentration of sinomenine, and anti-TfR mAb used to carry out experiments was 100 μg/mL, respectively. The same concentration of non-specific mouse IgG (Boster Immunoleader, Wuhan, China) was used as an isotype control. After 72 h, the same method was used to obtain the data and carry out the analysis.

Cell cycle analysis

After treatment with anti-TfR mAb and/or sinomenine hydrochloride for 72 h, HepG2 cells were harvested and fixed with 70 % ethanol at 4 °C for 2 h. Then, cells were stained with 50 μg/mL propidium iodide contained 0.25 mg/mL RNase A at room temperature for 30 min. Analysis was performed using FCM.

Cell proliferation analysis

HepG2 cells were treated with anti-TfR mAb alone, sinomenine hydrochloride alone, or in combination with anti-TfR mAb for 72 h, after being marked with carboxyfluorescein diacetate (CFSE: Sigma-Aldrich, Shanghai, China). Tumor cells were harvested and washed with ice-cold PBS and detected using FCM. The same concentration of non-specific mouse IgG was used as an isotype control.

Western blot

A total of 1 × 105 HepG2 cells were incubated with sinomenine hydrochloride at concentrations of 0, 10, 50, 100, 150, and 200 μg/mL and/or anti-TfR mAb at concentrations of 0, 10, 50, 100, 200, and 300 μg/mL for 72 h. Cells were washed three times with PBS and then resuspended in ice-cold Triton X-100 reagent (Invitrogen, Shanghai, China) with protease inhibitor mixture (Sigma-Aldrich, Shanghai, China) and finally lysed by means of sonication. The lysates were cleared of insoluble material by centrifugation at 15,000 rpm for 10 min at 4 °C. The supernatant was collected, and aliquots were stored until use in sterile microcentrifuge tubes at −80 °C. Protein content was measured using the bicihonimic acid protein assay kit (Beyotime, Nantong, China).

Aliquots of protein corresponding to 100 μg were mixed with SDS-PAGE sample buffer and heated in a hot water bath for 10 min. The samples were resolved on a 12 % SDS-PAGE and then transferred onto blotting grade nitrocellulose membrane (Millipore, Beijing, China). The membrane was treated with 5 % non-fat dry milk and 0.1 % PBS-tween 20 (milk-PBST) for 2 h at room temperature in order to block the non-specific sites on the membrane. Blots were probed with anti-COX-2 antibody (1:200, Cell Signal, Beverly, MA, USA) and anti-VEGF antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, USA) at 4 °C overnight. The membrane was washed in PBST three times for 5 min each followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000, Boster Immunoleader, Wuhan, China) for 1 h at room temperature. The membrane was washed in PBS-T five times for 5 min each session. Visualization of hybridization was carried out using chemiluminescence reagent (Beyotime, Haimen, China). The blots were exposed to autoradiography films (Kodak X-OMAT BT, USA) and developed. A laser density scan was performed on the films. The ratio between COX-2/VEGF and the corresponding integrated optical density of β-actin represented the protein expression level. All protein isolations and western blots were repeated a minimum of three times.

Statistical analysis

The data were statistically analyzed using Student’s t test or ANVOA. A P value of <0.05 was considered as being statistically significant.

Results

HepG2 cell apoptosis induced by sinomenine hydrochloride and/or anti-TfR mAb in vitro

When HepG2 cells were treated with sinomenine hydrochloride at different concentrations (0, 10, 30, 50, 70, 100, 150, and 200 μg/mL) for 24, 48, and 72 h, there was a clear increase in the percentage of apoptotic cells (Fig. 1a, b). The percentage of cells that underwent apoptosis increased in a dose-dependent and time-dependent manner (P < 0.05). This result indicated that sinomenine hydrochloride could promote the apoptosis of HepG2 cells.

Fig. 1.

Fig. 1

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Apoptosis of HepG2 cells induced by sinomenine hydrochloride and/or anti-TfR mAb. a HepG2 cells were treated with different concentrations of sinomenine hydrochloride for 24, 48, and 72 h. The apoptosis rate was determined by means of PI/Annexin V staining. The Annexin V positive cells were regarded as apoptotic cells. Representative data from three independent experiments were presented. b The percentage of apoptotic cells was statistically analyzed. Data represent the means and standard deviation of three independent experiments. c Apoptosis of HepG2 cells was induced by anti-TfR mAb alone administered for 24, 48, and 72 h. The apoptosis rate was determined by means of PI/Annexin V staining. The Annexin V positive cells were regarded as apoptotic cells. d The percentage of apoptotic cells was calculated. e HepG2 cell apoptosis induced by treatment with the concentrations of anti-TfR mAb and sinomenine hydrochloride, administered individually and in combination for 72 h analyzed using FCM. f Data from experiments were statistically analyzed and are shown as the mean ± SD

When HepG2 cells were treated with anti-TfR mAb at different concentrations (0, 50, 100, 200, and 300 μg/mL) for 24, 48, and 72 h, the apoptosis percentage increased (Fig. 1c, d), but the increase was limited.

When sinomenine hydrochloride and anti-TfR mAb were used in combination, more HepG2 cells underwent obvious apoptosis (P < 0.05, Fig. 1e, f). The data indicated that sinomenine hydrochloride combined with TfR mAb had a synergistic effect in relation to apoptosis induction in HepG2 cells.

In vitro effects of the combination of sinomenine hydrochloride and anti-TfR mAb on the cell cycle in HepG2 cells

The synergistic effects of sinomenine hydrochloride and anti-TfR mAb on the cell cycle in HepG2 cells were evaluated using FCM. The results indicated that the G1 and G2 phases were obviously decreased as compared with that of isotype control cells (P < 0.01). There was a significant decrease in the durations of the G1 and G2 phases in cells treated simultaneously with the two agents as compared with cells treated with the sinomenine hydrochloride or anti-TfR mAb alone (P < 0.01). The S phase of cells treated simultaneously with the two agents was increased relative to that of cells treated with sinomenine hydrochloride or anti-TfR mAb alone (P < 0.01) (Fig. 2a, b).

Fig. 2.

Fig. 2

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Effects of sinomenine hydrochloride and/or anti-TfR mAb on the cell cycle in HepG2 cells. a The cell cycle of tumor cells treated with anti-TfR mAb and/or sinomenine for 72 h. b Cell cycle phase distribution of HepG2 cells treated with anti-TfR mAb and/or sinomenine. Data are shown as the mean ± SD of three independent experiments

The synergic effect of sinomenine hydrochloride and anti-TfR mAb on growth inhibition

To verify whether or not sinomenine hydrochloride and anti-TfR mAb inhibited cell growth, sinomenine hydrochloride and/or anti-TfR mAb treated HepG2 cells were marked with CFSE and analyzed using FCM. The cell proliferation index of HepG2 cells treated with the combination of the two agents was lower than in those treated with either agent alone (P < 0.05; Fig. 3a, b).

Fig. 3.

Fig. 3

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Combined effects of anti-TfR mAb and sinomenine related to the inhibition of the proliferation of HepG2 cells. Proliferation of HepG2 cells was detected after treatment with the anti-TfR mAb and/or sinomenine as indicated for 72 h. b Statistical analysis of the inhibition of the proliferation of HepG2 cells. Data represent the mean and standard deviation of three independent experiments

Sinomenine hydrochloride enhanced the inhibitory effects of anti-TfR mAb on human hepatoma cells through down-regulation of the expression of COX-2 and VEGF

To explain the mechanisms of the biological effects of sinomenine hydrochloride and anti-TfR mAb detailed above, the expressions of the apoptosis-regulating proteins COX-2 and VEGF were studied. As shown in Fig. 4a, the expression of COX-2 and VEGF was increased in line with increased concentrations of anti-TfR mAb. Figure 4b shows that the expression of COX-2 was decreased in line with increased concentrations of sinomenine hydrochloride. Figure 4c shows that the expression of COX-2 and VEGF was lower in HepG2 cells treated with sinomenine hydrochloride and anti-TfR mAb in combination, than in cells treated with sinomenine hydrochloride or anti-TfR mAb alone. This finding suggested that the combined use of sinomenine hydrochloride and anti-TfR mAb might result in synergistic inhibition of HepG2 cells dependent on the down-regulation of COX-2 and VEGF expression.

Fig. 4.

Fig. 4

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In vitro effects of the combined use of sinomenine hydrochloride and anti-TfR mAb on the expression of COX-2 and VEGF in HepG2 cells. a Lanes 1–6 HepG2 cells were treated with TfR mAb (0, 10, 50, 100, 200, and 300 μg/mL). COX-2 and VEGF expression were detected using western blot (left) with specific antibodies. Data are expressed as the mean ± SD of three independent experiments (right). b Lane 1–6 HepG2 cells were treated with sinomenine hydrochloride (0, 10, 50, 100, 150, and 200 μg/mL). Data are shown as the mean ± SD of three independent experiments (right). c Lane 1 control group; lane 2–3 HepG2 cells were treated with sinomenine hydrochloride (50 and 100 μg/mL); lane 4 HepG2 cells were treated with anti-TfR mAb (100 μg/mL); lane 5 HepG2 cells were treated with anti-TfR mAb (100 μg/mL) and sinomenine hydrochloride (50 μg/mL); lane 6 HepG2 cells were treated with anti-TfR mAb (100 μg/mL) and sinomenine hydrochloride (100 μg/mL). Data are shown as the mean ± SD of three independent experiments (right)

Discussion

TfR is a kind of transmembrane glycoprotein related to intra-cellular iron intake and control of cell growth. Transferrin (Tf) releases its iron, and TfR is then recycled back to the surface. By virtue of this unique intracellular behavior, molecules conjugated to Tf may be efficiently delivered into cells, with TfR functioning through multiple internalization cycles. The growth inhibitory property of anti-TfR antibodies or transferrin receptor-targeted hybrid peptide has been appreciated [3–9, 25]. As for the mechanisms of anti-TfR antibody-mediated growth inhibition, different antibodies had shown different modes of action in different models. In our study, we demonstrated that the anti-TfR mAb could induce cell apoptosis, inhibit cell proliferation, and affect the cell cycle. However, after the HepG2 cells were treated with anti-TfR mAb, the intake of intracellular iron was blocked, which may cause an increase in the transcription and translation of COX-2. It will be transmitted into the cell nucleus, affect downstream signal molecules, and modulate cell proliferation and differentiation. Some studies have shown that COX-2 might mediate tumor angiogenesis and growth. In addition, the expression of COX-2 is related to the degree of malignancy of the tumor [23, 26–28]. The pathway by which COX-2 mediates tumor angiogenesis may enhance the mediator expression of vascularization such as VEGF, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) [29]. It can increase the expression of COX-2 to stimulate the formation of activation products such as TAX2, PGE2, prostacyclin (PGI2) [30], activate bcl-2, Akt, and others. Hence, anti-TfR mAb still has some limitations with regard to its anti-tumor effects.

Sinomenine hydrochloride is used to treat autoimmune disease in the clinic, especially rheumatic arthritis. Some studies have shown that sinomenine could selectively inhibit COX-2 instead of COX-1 in the treatment of rheumatic arthritis [31, 32]. In the present study, we investigated the anti-tumor effects of anti-TfR mAb alone or in combination with sinomenine hydrochloride in vitro. The results demonstrated that if HepG2 cells were treated with sinomenine hydrochloride, the percentage of apoptosis cells increased in a dose-dependent and time-dependent manner. However, relative to the individual effects of anti-TfR mAb or sinomenine hydrochloride on the HepG2 cells, the synergy between these two agents resulted in a clear increase in apoptosis, inhibition of cell proliferation, and arrest in the S/G2 phase.

To explore the mechanism of this synergy, the expressions of COX-2 and VEGF were further evaluated after HepG2 cells were treated with sinomenine hydrochloride and anti-TfR mAb in combination. Results showed that the expression of both COX-2 and VEGF was elevated with increased concentrations of anti-TfR mAb but was reduced with increased concentrations of sinomenine hydrochloride. When the two agents were administered together, the inhibitory effects on COX-2 and VEGF expression in HepG2 cells were more prominent than when they were administered separately. This finding suggested that their synergistic effect on apoptosis and proliferation might depend on the COX-2 pathway. At the same time, the expression of the downstream signal molecule VEGF was obviously decreased. These changes affected apoptosis, proliferation, and the cell cycle in the tumor cells.

The apoptotic process in cells is complicated and is controlled by multiple factors. Besides the proteins mentioned above, many proteins related to apoptosis have yet to be identified. Further studies should be carried out with regard to the molecule mechanism by which sinomenine hydrochloride and anti-TfR mAb induce cell apoptosis.

In summary, the above findings suggested that the simultaneous application of anti-TfR mAb and sinomenine hydrochloride contributed to synergy with regard to growth inhibition and the promotion of apoptosis in tumor cells. This effect relies on down-regulation of COX-2 and VEGF expression in tumor cells. Finally, our study also provides a basis for the combined application of sinomenine hydrochloride and anti-TfR mAb in future clinical trials involving human hepatoma.

Acknowledgments

We thank Yue Zhang and Shuang Zou for their kind assistance in this study. This work was supported by funding from the National Natural Science Foundation of China (No. 81102531 to Xin Shen), the Important National Science and Technology Specific Projects (2009ZX09301-014 to Guanxin Shen), and the Young Foundation of Hubei Provincial Department of Education (Q20101804 to Yi Hong).

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Yi Hong and Juan Yang contributed equally to the study and share first authorship.

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