Chen Weiguo, Yan Chunyin, Hou Jianquan, Pu Jinxian, Ouyang Jun, Wen Duangai
Department of Urology, The First Affiliated Hospital of Suzhou University, Jiangsu, China.
Urol Oncol. 2008 Jul-Aug;26(4):397-405. doi: 10.1016/j.urolonc.2007.08.010. Epub 2008 Jan 14.
All-trans retinoic acid (ATRA) has been shown to inhibit the growth of many malignancies by altering gap junctional intercellular communication (GJIC) and the expression of connexin (Cx) 43. Here, we report that the alteration of GJIC by ATRA may directly enhance the bystander effect (BE) of suicide gene therapy against prostate cancer in vitro and in vivo.
PC-3 cells were exposed to different concentrations of ATRA for varying lengths of time in culture. Flow cytometry was performed to measure Cx43-positive cells and the GJIC function of the cells was examined with the scrape-loading dye transfer assay. Cells were treated with ATRA in combination with an adenovirus/ganciclovir (Ad-TK/GCV) system encoding herpes simplex virus-thymidine kinase, and the BE was assessed in the treatment of androgen-independent prostate cancer both in vitro and in vivo. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to assess the expression of Cx43 mRNA and protein in tumor tissues.
ATRA significantly increased the amount of Cx43-positive cells in a time- and dose-dependent manner (P < 0.05). GJIC functions were enhanced 3- to 5-fold in the presence of ATRA, although ATRA did not augment GCV toxicity of PC-3 cells. In the mixing assay, ATRA significantly increased cell killing when the ratio of TK-positive cells in the coculture ranged from 30% to 60% compared with ATRA-untreated cell (P < 0.05), and attained 50% cell killing cells when the ratio of TK-positive cell was 30%, but the same result did not appear until the ratio of TK-positive cell was up to 60% in the ATRA-untreated cell. Mice treated with a combination of ATRA and GCV had significantly smaller Ad-TK infected tumors than those treated with GCV or ATRA alone after 3-weeks of therapy (P < 0.05). However, from the fourth-week of therapy, there was no difference in tumor growth inhibition between GCV treatment and GCV + ATRA treatment (P > 0.05), as two tumors in the latter group started to grow more quickly than tumors in the control group. This phenomenon was not found in other groups.
ATRA could enhance the efficiency of cell killing in suicide gene therapy against prostate cancer by strengthening the BE in vitro and in vivo. Induction of Cxs and GJIC by ATRA might provide an element of selectivity to suicide gene therapy. Future studies should focus on safety and tailoring this cooperative therapy to the patient.
全反式维甲酸（ATRA）已被证明可通过改变间隙连接细胞间通讯（GJIC）和连接蛋白（Cx）43的表达来抑制多种恶性肿瘤的生长。在此，我们报告ATRA对GJIC的改变可能直接增强自杀基因疗法在体外和体内对前列腺癌的旁观者效应（BE）。
在培养中，将PC-3细胞暴露于不同浓度的ATRA中不同时间长度。进行流式细胞术以测量Cx43阳性细胞，并通过刮擦加载染料转移试验检测细胞的GJIC功能。将细胞用ATRA与编码单纯疱疹病毒胸苷激酶的腺病毒/更昔洛韦（Ad-TK/GCV）系统联合处理，并在体外和体内评估其对雄激素非依赖性前列腺癌治疗的旁观者效应。进行半定量逆转录聚合酶链反应（RT-PCR）和免疫组织化学以评估肿瘤组织中Cx43 mRNA和蛋白的表达。
ATRA以时间和剂量依赖性方式显著增加Cx43阳性细胞的数量（P<0.05）。在存在ATRA的情况下，GJIC功能增强了3至5倍，尽管ATRA并未增加PC-3细胞的GCV毒性。在混合试验中，当共培养中TK阳性细胞的比例在30%至60%范围内时，与未用ATRA处理的细胞相比，ATRA显著增加细胞杀伤（P<0.05），并且当TK阳性细胞的比例为30%时达到50%的细胞杀伤，但在未用ATRA处理的细胞中，直到TK阳性细胞的比例高达60%时才出现相同结果。在治疗3周后，用ATRA和GCV联合处理的小鼠的Ad-TK感染肿瘤明显小于单独用GCV或ATRA处理的小鼠（P<0.05）。然而，从治疗的第四周开始，GCV治疗和GCV+ATRA治疗之间在肿瘤生长抑制方面没有差异（P>0.05），因为后一组中的两个肿瘤开始比对照组中的肿瘤生长得更快。在其他组中未发现此现象。
ATRA可通过在体外和体内增强旁观者效应来提高自杀基因疗法对前列腺癌的细胞杀伤效率。ATRA诱导Cxs和GJIC可能为自杀基因疗法提供一种选择性要素。未来的研究应关注安全性并针对患者定制这种联合疗法。
