(T1430-01-08) Sensitization of Paclitaxel-Induced Apoptosis by Echinops giganteus Extract via Downregulation of PI3K/Akt/NF-κB Pathway and Enrichment of TIMP-3 in Cervical Cancer

Purpose: Paclitaxel is a well-known chemotherapeutic agent in cervical cancer treatment. However, the acquired resistance and toxicity limits its therapeutic efficacy. Recently, evolving interest has focused on natural products to assess their augmenting efficacy in combination with chemotherapeutics. In this study, we report that the methanolic extracts of Echinops giganteus (EG), a widely used plant in traditional medicine in Africa and Asia, can efficiently sensitize tumor cells to paclitaxel treatment.

Methods: Evaluation of the sensitivity of 2D HeLa cells towards various concentrations of paclitaxel, EG extract or a combination of paclitaxel and EG extract was done after 48 h of treatment using the MTT viability assay. Cell viability of 3D spheroids was assayed based on ATP production as described previously [1]. A live/dead assay was performed to confirm the anti-proliferative effects of paclitaxel, EG extract and their combination on 2D and 3D HeLa cell cultures. Furthermore, analysis of the apoptotic and necrotic cell death was evaluated with the Annexin V assay. The cell cycle analysis was carried out as previously reported [2]. To further evaluate the effect of the combined therapy on tumor angiogenesis, HeLa cells were cultured on the CAM model as pre.previously described [3]. Chromatin immunoprecipitation coupled with quantitative Polymerase Chain Reaction (ChIP-qPCR) assay was performed to explore the direct effect of the combined therapy of EG and paclitaxel on the regulation of cervical cancer-related oncogenes.

Results: Paclitaxel and EG exhibited the IC50 values at the concentration of 20 nM and 32.38 µg/mL, respectively. On the other hand, paclitaxel at a concentration 3 nM and 5 µg/ml concentration of EG induced only 9% and 14% cell growth inhibition, respectively. On the contrary, a combination dose of 3 nM paclitaxel and 30 µg/mL EG significantly induced 88% cytotoxicity. More interestingly, a combination of 0.8 nM paclitaxel and 5.3 µg/mL EG caused 50% cell growth inhibition. EG synergistically enhances the cytotoxic activity compared to paclitaxel alone towards HeLa cells, mainly by upregulation of p53 and p21Cip1/Waf1, consequently arresting the cell cycle at S and G2/M phases. Our results reveal that this combination induces the intrinsic apoptotic pathway at the cellular level through a change in cytochrome C phosphorylation leading to an elevated BAX/Bcl-2 ratio, disruption of mitochondrial membrane potential and caspase activation. Furthermore, the evaluation of the signaling pathways revealed that this synergism is dependent on the downregulation of MDR1 via inhibition of PI3K/Akt/NF-κB signaling pathway. Additionally, the combined therapy causes overexpression of TIMP-3 and downregulation of COX-2 and CXCL8, potentially inhibiting cell migration and angiogenesis. Moreover, combining EG and paclitaxel significantly could markedly restrain the migration of HeLa cells and frustrate the outgrowth of epithelial cells to form microvessels in the  the ex-vivo rat aortic ring assay.

Conclusion: This study is the first to demonstrate the potential synergism between EG extract and paclitaxel. This synergism is dependent on targeting multiple mechanisms and signaling pathways, ultimately inhibiting the proliferation, migration, angiogenesis and tumorigenesis of cervical cancer cells. Thus, our findings pave the way for future studies to validate this combination through in vivo studies.

References: 1. Fey SJ, Wrzesinski K, Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line, Toxicological Sciences, 2012;127(2):403–411.
2. Selvendiran K, Tong L, Vishwanath S, Bratasz A, Trigg NJ, Kutala. et al. EF24 induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by increasing PTEN expression. J Biol Chem. 2007;282:26809–26818.
3. Dai J, Lin Y, Duan Y, Li Z, Zhou D, Chen W, Wang L, Zhang QQ. Andrographolide Inhibits Angiogenesis by Inhibiting the Mir-21-5p/TIMP3 Signaling Pathway. Int J Biol Sci 2017;13(5):660-668.


Fig. 1 A combination of EG and paclitaxel augments the cytotoxic activity in HeLa cervical cancer cells in vitro. HeLa cells were treated with different concentrations of (A) EG extract, (B) Paclitaxel or (C) in combination and cell viability was assessed by the MTT assay after 48h. (D) The bar graph indicates the effect of paclitaxel and EG, alone or in combination, on 2D HeLa cells. (E) 3D HeLa spheroids were exposed to different concentrations of paclitaxel and EG, alone or in combination, for 48h and their ATP content determined by the CellTiter-Glo luminescent cell viability assay from Promega. (F) A bar graph represents the quantification of live and dead HeLa cells after different treatments; data analysis was performed using the ImageJ and GraphPad Prism 5 software, and ** indicates p < 0.01 and *** indicates p < 0.001. . (G) Assessment of the synergistic effect between EG and paclitaxel towards 2D and 3D HeLa cells by calculation of the combination index (CI) which depending on a quantitative measure of dose–effects relationships to explore the combined effects of different compounds, where additive effect (CI = 1), synergism (CI < 1), or antagonism (CI > 1). (H and I) A live/dead assay is performed after treating HeLa 2D and 3D cultures for 48h. Green fluorescence denotes viable cells stained with calcein-AM, while red fluorescence represents dead cells stained with propidium iodide. Images represent four random fields. All values expressed in mean ± standard deviation (SD) and analyzed using the one-way analysis of variance (ANOVA) at a significance level of p < 0.05.


Fig. 2. EG enhances the antiproliferative effect of paclitaxel by activation of caspases, cell cycle arrest, downregulation of MDR1 and elevation of the Bax/Bcl-2 ratio. Evaluation of the enhancement of caspase activation after treatment of HeLa cells with various concentrations of paclitaxel, EG extract, alone or in combination using Caspase-Glo 3/7 and 9 Assay kit, (A) caspase 3/7 and (B) caspase 9. (C) Representative bar graphs indicating the percentages of cells in G0/G1, S and G2/M phases of the cell cycle after 48 h of HeLa cells treatment of paclitaxel, EG extract, alone or in combination. The mRNA expression levels of (D) MDR1 (E) p21Cip1/Waf1 and (F) Bax/Bcl-2 ratio was detected using qRT-PCR after treatment of HeLa cells with indicated concentrations of paclitaxel, EG extract, alone or in combination for 48 h. β-actin was used as an internal control. Data was analyzed using the Graphpad Prism 5 software. ***: p < 0.001. Chromatin Immunoprecipitation (ChIP) analysis was performed using the True MicroChIP kit based on two histone modifications: histone 4 acetylation (H4Ac) as a representative for gene activation correlating with active promoters and the tri-methylation at lysine-27 of histone H3 (H3K27me3) as epigenetic marker for repression of gene expression. Following immunoprecipitation with antibodies for these epigenetic targets, qPCR analysis for the studied genes was performed. (G) Heat map for representative Z-score for each gene to evaluate the enrichment upregulation and downregulation for the immunoprecipitated chromatin. (J) Interactive genes network analysis by STRING PPI 11.0 database using medium confidence. Line thickness indicates the strength of related genes and shared pathways in tumorigenesis and cell death mechanisms.


Fig. 3. The combined therapy of paclitaxel and EG significantly inhibits cell migration and microvessel formation in an ex-vivo rat aortic ring assay. Moreover, it plays a protective role in paclitaxel-associated hemolytic anemia. (A) The migration ability of HeLa cells was determined using the wound healing (scratch) assay over 24 and 48 h. Images were processed using the Image J software. (B) The bar graphs represent the analysis of the wound closure in response to the different treatment regimens. ** indicates p < 0.01. (C) A rat aortic assay was used to detect the direct effect of the combined therapy on the outgrowth of epithelial microvessels. The thoracic aorta was isolated, cut into segments and placed on Matrigel to initiate microvessel growth. After 10 days of treatment with paclitaxel, EG extract, alone or in combination. Photographs of representative rings for assays performed in triplicate. Arrows indicate the reduction of microvessel formation. (D) Data analysis for microvessels outgrowth (% of control) from the aortic ring, using GraphPad Prism 5 and *** indicates p < 0.001. (E) Hemolysis assay for quantification of toxicity with paclitaxel, EG extract, alone or in combination. Lysis (%) of red blood cells in female mice (n= 3, 8 weeks old) in blood samples collected post-sacrifice using serial dilutions of the combined therapy, quantified using fluorescence spectrophotometer at 540 nm wavelength. The hemolysis reduced considerably over combining paclitaxel with EG and its dilutions 10x and 100x, confirming the protective role of EG extract in paclitaxel-associated hemolytic anemia; Data analyzed using GraphPad Prism 5 and *** indicates p < 0.001