Disruption of caspase-independent cell proliferation pathway on spheroids (HeLa cells) treated with curcumin

Curcumin is an antiproliferative phytochemical extracted from <i>Curcuma longa L</i> and which has been studied in preclinical drug screening using cell monolayers and animal models. However, several limitations of these culture systems may be overcome by performing screening with three-dimensional (3-D) cell culture. The aim of this study was to investigate the effects of curcumin on cytotoxicity and genotoxicity as well as spheroid growth using cervical adenocarcinoma HeLa cell spheroids by performing RT-PCR mRNA expression of genes involved in cell death (<i>CASP3</i>, <i>CASP8</i>, <i>CASP9</i>, <i>PARP1</i>, <i>BBC3</i>, <i>BIRC5</i>, <i>BCL2</i>, <i>TNF</i>), autophagy (<i>BECN1, SQSTM1</i>), cell cycle regulation (<i>TP53</i>, <i>C-MYC</i>, <i>NF-kB</i>, <i>CDKN1A</i>, <i>m-TOR</i>, <i>TRAF-2</i>), DNA damage repair (<i>H2AFX</i>, <i>GADD45A, GADD45G</i>), oxidative stress (<i>GPX1</i>), reticulum stress (<i>EIF2AK3, ERN1</i>), and invasion (<i>MMP1</i>, <i>MMP9</i>) was investigated. Curcumin was cytotoxic in a concentration-dependent manner. Curcumin-treated spheroids exhibited lower proliferative recovery and cell proliferation attenuation, as observed in the clonogenic assay. Further, no marked genotoxicity was detected. Curcumin-treated spheroids displayed reduced expression of <i>BECN1</i> (2.9×), <i>CASP9</i> (2.1×), and <i>PARP1</i> (2.1×) mRNA. <i>PARP1</i> inhibition suggested disruption of essential pathways of proliferation maintenance. Downregulated expression of <i>CASP9</i> mRNA and unchanged expression of <i>CASP3/8</i> mRNA suggested caspase-independent cell death, whereas downregulated expression of <i>BECN1</i> mRNA indicated autophagic disruption. Therefore, curcumin exhibits the potential for drug development with antiproliferative activity to be considered for use in cancers.

姜黄素（Curcumin）是一种从姜黄（Curcuma longa L）中提取的抗增殖植物化学物，既往多通过单层细胞培养与动物模型开展临床前药物筛选研究。但此类培养体系存在诸多局限，可通过三维（3D）细胞培养筛选予以克服。本研究以宫颈腺癌HeLa细胞球体为模型，旨在探究姜黄素对细胞毒性、遗传毒性及球体生长的影响，并通过逆转录聚合酶链反应（RT-PCR）检测相关基因的mRNA表达水平，所涉基因涵盖细胞死亡（CASP3、CASP8、CASP9、PARP1、BBC3、BIRC5、BCL2、TNF）、自噬（BECN1、SQSTM1）、细胞周期调控（TP53、C-MYC、NF-kB、CDKN1A、m-TOR、TRAF-2）、DNA损伤修复（H2AFX、GADD45A、GADD45G）、氧化应激（GPX1）、内质网应激（EIF2AK3、ERN1）及侵袭（MMP1、MMP9）相关基因。实验结果表明，姜黄素以浓度依赖性方式发挥细胞毒性作用。经姜黄素处理的细胞球体表现出更低的增殖恢复能力与细胞增殖抑制效果，该结果在克隆形成实验中得到验证。此外，未检测到明显的遗传毒性。姜黄素处理组的细胞球体中，BECN1（2.9倍）、CASP9（2.1倍）与PARP1（2.1倍）的mRNA表达水平显著下调。PARP1抑制实验提示，细胞增殖维持的关键通路遭到破坏。CASP9 mRNA表达下调而CASP3/8 mRNA表达无显著变化，表明存在不依赖半胱氨酸天冬氨酸蛋白酶的细胞死亡途径；而BECN1 mRNA表达下调则提示自噬过程受损。综上，姜黄素具备抗增殖活性的药物开发潜力，有望用于癌症治疗。