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Methylselenocysteine Promotes Etoposide Cytotoxicity by Enhancing Homotypic Gap Junctions Composed of Connexin 26
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摘要:
目的 探究硒-甲基硒代半胱氨酸（methylselenocysteine, MSC）对缝隙连接蛋白（connexin, Cx）26构成的同型缝隙连接（gap junction, GJ）功能的影响及其对化疗药物细胞毒性的调控。 方法 以转染并稳定表达Cx26的Tet-on HeLa细胞为工具细胞，MTT法观察MSC对细胞生长的影响；细胞接种荧光示踪法测定MSC对GJ功能的影响；Western blot检测MSC对Cx26蛋白表达的影响；标准细胞集落形成分析法观察化疗药物的细胞毒性；在此基础上，分析MSC对化疗药物细胞毒性的影响及其与调控GJ的关系。 结果 多西环素（doxycycline, Dox）可诱导Tet-on HeLa细胞Cx26表达并形成有功能的GJ。MSC在50 μmol/L内对细胞生长无明显影响，无毒浓度的MSC可以浓度依赖性增强GJ，并可在纳摩尔级发挥作用。该效应与其诱导Cx26蛋白的表达有关。在三种不同作用机制的常见化疗药物中，依托泊苷（etoposide, Eto）在有无GJ形成的情况下表现一定的细胞毒性差异。MSC与Eto联合应用对细胞集落形成的抑制作用强于Eto单药，且该效应只在有GJ形成的HeLa细胞中发生。 结论 MSC可以通过增强Cx26组成的GJ提高Eto的细胞毒性，提示硒化物联合化疗在肿瘤治疗中具有一定的潜在价值。 -
关键词:
- 硒-甲基硒代半胱氨酸 /
- 缝隙连接蛋白 /
- 缝隙连接 /
- 依托泊苷
Abstract:Objective To investigate the effect of methylselenocysteine (MSC) on the function of homotypic gap junction (GJ) composed of connexin (Cx) 26 and its regulation of chemotherapeutic drug cytotoxicity. Methods The Tet-on HeLa cells transfected with and stably expressing Cx26 were used as the tool cells. Effects of MSC on cell growth, GJ function, and Cx26 protein expression were examined by MTT method, parachute assay, and Western blot analysis, respectively. The cytotoxicity of chemotherapeutic drugs was determined by standard colony-forming assay, and the relationship between MSC's effect on cytotoxicity of these chemotherapeutic drugs and its regulation of GJ was further analyzed. Results In Tet-on HeLa cells, doxycycline (Dox) can induce the expression of Cx26, which could then form functional GJs. Within a concentration range of 50 μmol/L, MSC had no significant effect on HeLa cell growth. Non-toxic concentrations of MSC can enhance GJs in a concentration-dependent manner and exert its effect at the nanomolar level. This effect was associated with an induction of Cx26 protein expression by MSC. Among the three common chemotherapeutic agents with different mechanisms of action, etoposide (Eto) presented cytotoxicity differences between HeLa cells cultured at low density (nonconfluent, no GJ formed) and high density (confluent, GJ formed). What's more, the inhibitory effect of Eto combined with MSC on HeLa cell colony formation was stronger than that of Eto alone, and this effect occurred only in HeLa cells with GJ formation. Conclusion MSC can potentiate the cytotoxicity of Eto by enhancing the GJs composed of Cx26, indicating that combined strategy of selenide and chemotherapy shows potential value in the treatment of malignant tumors. -
Key words:
- Methylselenocysteine /
- Connexin /
- Gap junction /
- Etoposide
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图 1 Dox诱导Cx26蛋白表达和功能性GJ形成
Figure 1. Dox induced Cx26 protein expression and functional GJ formation
A: Expression of Cx26 protein in Tet-On HeLa cells can be induced by Dox as shown by Western blot assay. B: Fluorescence transmission between HeLa cells in different culture conditions detected by parachute assay. HeLa cells were cultured with or without Dox (1 μg/mL) for 48 h. n=4.
图 2 MSC对转染并稳定表达Cx26的HeLa细胞生长的影响
Figure 2. Effect of MSC on the growth of HeLa cells transfected with and steadily expressing Cx26
The HeLa cells were pre-induced with Dox (1 μg/mL) for 48 h, and then treated with MSC for 48 h. n=4, ${\bar x \pm s } $.
图 3 MSC对转染并稳定表达Cx26的HeLa细胞GJ功能的影响
Figure 3. Effect of MSC on GJ function in the HeLa cell line transfected with and steadily expressing Cx26
Representative images of fluorescent dye diffusion from donor cells to adjacent and distant cells in each group. HeLa cells were all induced by Dox (1 μg/mL) for 48 h before the above treatment.
图 4 MSC对HeLa细胞Cx26蛋白表达的影响
Figure 4. Effect of MSC on Cx26 protein expression in HeLa cells
A: Cells were treated with MSC at varying concentrations for 48 h; B: cells were treated with 100 nmol/L MSC for varying periods of time. n=3.
图 5 MSC对HeLa细胞中Eto的细胞毒性（细胞集落形成率）的影响
Figure 5. Effect of MSC on the cytotoxicity of etoposide in HeLa cells (surviving fraction)
Effect of MSC (100 nmol/L) in combination with etoposide at different concentrations on the clonogenic survival of cells at either high or low cell density. HeLa cells were treated with Dox (1 μg/mL) for 48 h to induce the expression of Cx26 protein. * P< 0.05, vs. Eto single-agent group. n=4.
表 1 不同浓度MSC作用4 h对Cx26组成的GJ功能（GJ增强率）的影响
Table 1. Effect of MSC for 4 h at different concentrations on the function of GJs composed of Cx26 (GJ enhancement rate)
MSC concentration/(nmol/L) n GJ enhancement rate ($\bar x \pm s $) 0.001 4 0.14±0.02 0.01 4 0.19±0.02 0.1 4 0.26±0.04 1 4 0.33±0.04 10 4 0.36±0.02 100 4 0.46±0.05 1000 4 0.41±0.02 
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表 2 100 nmol/L MSC作用不同时间对Cx26组成的GJ功能（荧光传递细胞数）的影响
Table 2. Effect of MSC treatement at 100 nmol/L for different periods of time on the function of GJs composed of Cx26 (number of fluorescent spread cells)
Control group DMSO group MSC treatment group ($\bar x \pm s $) 4 h 24 h 48 h 0.98±0.05 1 1.46±0.05* 1.52±0.04* 1.56±0.06* Dye spread normalized to DMSO group. * P<0.05, vs. DMSO group. n=4.
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表 3 不同浓度MSC作用48 h对HeLa细胞Cx26蛋白表达的影响
Table 3. Effect of MSC treatment for 48 h at different concentrations on Cx26 protein expression in HeLa cells
Group n Cx26 protein expression ($\bar x \pm s $) Not induced 3 0.12±0.03* Control 3 0.95±0.05 DMSO 3 1.00 MSC (10 nmol/L) 3 1.38±0.01* MSC (100 nmol/L) 3 3.11±0.18*, # MSC (1000 nmol/L) 3 2.09±0.41* ATRA 3 2.41±0.61* Scanning density normalized to DMSO group. * P<0.05, vs. DMSO group; # P<0.05, vs. MSC (10 nmol/L) group.
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表 4 100 nmol/L MSC作用不同时间对HeLa细胞Cx26蛋白表达的影响
Table 4. Effect of MSC treatement at 100 nmol/L for different periods of time on on Cx26 protein expression in HeLa cells
Group n Cx26 protein expression ($\bar x \pm s $) Not induced 3 0.12±0.03* Control 3 0.95±0.05 DMSO 3 1.00 MSC 4 h 3 1.89±0.12* MSC 24 h 3 2.65±0.19*, # MSC 48 h 3 3.11±0.18*, # Scanning density normalized to DMSO group. * P<0.05, vs. DMSO group; # P<0.05, vs. MSC 4 h group.
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表 5 GJ的形成对转染并稳定表达Cx26的HeLa细胞中化疗药物细胞毒性（细胞集落形成率）的影响
Table 5. Effect of GJ formation on the cytotoxicity of chemotherapeutic agents in HeLa cells transfected with and steadily expressing Cx26 (surviving fraction)
Cell culture group Cisplatin 5-Fluorouracil Etoposide 2.5 μmol/L 5 μmol/L 0.25 mmol/L 0.5 mmol/L 2 μmol/L 4 μmol/L High-density 0.84±0.05 0.67±0.03 0.88±0.11 0.76±0.05 0.66±0.03 0.46±0.04 Low-density 0.83±0.06 0.78±0.04 0.81±0.04 0.63±0.06 0.72±0.03 0.58±0.02* All HeLa cells were treated with Dox (1 μg/mL) for 48 h to induce the expression of Cx26 protein. * P<0.05, vs. high-density culture group. n=4, $\bar x \pm s $.
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表 6 有无Dox诱导下MSC对HeLa细胞中Eto细胞毒性（细胞集落形成率）的影响
Table 6. Effect of MSC on the cytotoxicity of etoposide in HeLa cells with or without Dox induction (surviving fraction)
Dox MSC Eto 2 μmol/L Eto 4 μmol/L Eto Eto+MSC Eto Eto+MSC + 0.97±0.07 0.67±0.03* 0.54±0.03*, # 0.47±0.03* 0.34±0.03*, # − 0.99±0.04 0.74±0.03* 0.72±0.01* 0.62±0.02* 0.63±0.04* Dox: doxycycline; MSC: methylselenocysteine; Eto: etoposide. * P<0.05, vs. MSC group; # P<0.05, vs. Eto single-agent group. n=3, $\bar x \pm s $.
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