Metformin as an Alternative Radiosensitizing Agent to 5FU During Neoadjuvant Treatment for Rectal Cancer.
二甲双胍作为直肠癌新辅助治疗期间 5FU 的替代放射增敏剂。
- 作者列表："Fernandes JM","Jandrey EHF","Koyama FC","Leite KRM","Camargo AA","Costa ÉT","Perez RO","Asprino PF
BACKGROUND:Neoadjuvant chemoradiation for locally advanced rectal cancer combining 5FU to radiation increases tumor regression compared to radiation alone. However, it occurs at the cost of significant treatment-related toxicity. Patients with rectal cancer using metformin have been associated with improved response to radiotherapy. OBJECTIVE:To evaluate the radiosensitizing effects of metformin in vitro and in vivo and compare it to standard combination of radiation/5FU. DESIGN:Colorectal cancer cell lines SW480, HT29, and HCT116 were used as models. Cell viability was compared under treatments with radiation, radiation/5FU, metformin, radiation/metformin, and radiation/5FU/metformin. Nude mice were injected subcutaneously with SW480 cells and treated for one week with radiation/5FU, metformin, radiation/metformin or radiation/5FU/metformin. Tumor volume was evaluated for 4 weeks after treatment completion. The phosphorylation status of key proteins of the PI3K/Akt/mTOR pathway was determined by immunoblots. SETTINGS:Experimental study in vitro and in vivo. PATIENTS:Animal model/cell lines. MAIN OUTCOME MEASURES:The end point was to investigate how metformin compares to 5FU as a radiosensitizer. RESULTS:All cell lines significantly decreased cell viability after treatment with radiation/metformin when compared to radiation alone. Radiation/metformin was superior to radiation/5FU in SW480 (37% vs 74%; p<0.001). In HT29 and in HCT116 radiation/metformin was inferior to radiation/5FU (40%vs13.8%, p<0.001 and 40%vs7%, p<0.001; respectively), mainly due to increased 5FU toxicity (≤20% of cell viability). In vivo assays indicated that radiation/metformin treatment was comparable to radiation/5FU (557vs398mm, p>0.05), and that the addition of metformin to the standard radiation/5FU did not improve tumor response (349mm, p>0.05). Metformin exerted strong PI3K/Akt/mTOR pathway inactivation effects after 24-hour exposure (increasing pAMPK p<0.01, decreasing pAkt, p<0.01; and pS6, p<0.05). LIMITATIONS:In vitro and in vivo CRT regimens cannot be directly translated to human delivery methods. CONCLUSIONS:Metformin enhances tumor response to radiation in vitro and in vivo. Metformin is an attractive alternative radiosensitizing agent to be considered in future studies/trials. See Video Abstract at http://links.lww.com/DCR/B219.
背景: 与单纯放疗相比，局部晚期直肠癌新辅助放化疗联合 5FU 放疗可增加肿瘤消退。然而，它的发生是以显著的治疗相关毒性为代价的。直肠癌患者使用二甲双胍与放疗反应改善相关。 目的: 评价二甲双胍的体内外放射增敏作用，并与放射/5FU 标准组合进行比较。 设计: 以结直肠癌细胞系 SW480 、 HT29 和 HCT116 为模型。比较放射、放射/5FU 、二甲双胍、放射/二甲双胍和放射/5FU/二甲双胍处理下的细胞活力。裸鼠皮下注射 SW480 细胞，用放射/5FU 、二甲双胍、放射/二甲双胍或放射/5FU/二甲双胍治疗一周。治疗完成后评估肿瘤体积 4 周。通过免疫印迹确定 PI3K/Akt/mTOR 通路关键蛋白的磷酸化状态。 设置: 体外和体内实验研究。 患者: 动物模型/细胞系。 主要观察指标: 终点是研究二甲双胍与 5FU 作为放射增敏剂的比较。 结果: 与单纯放疗相比，放疗/二甲双胍处理后，所有细胞系均显著降低细胞存活率。SW480 中放疗/二甲双胍优于放疗/5FU (37% vs 74%; p<0.001)。在 HT29 和 HCT116 辐射/二甲双胍低于辐射/5FU (分别为 40% vs13.8 %，p<0.001 和 40% vs7 %，p<0.001),主要是由于增加了 5FU 毒性 (≤ 20% 的细胞活力)。体内试验表明，辐射/二甲双胍治疗与辐射/5FU 相当 (557vs398mm，p>0.05),在标准放疗/5FU 基础上加用二甲双胍未改善肿瘤反应 (349毫米，p>0.05)。二甲双胍在暴露 24 小时后发挥了强烈的 PI3K/Akt/mTOR 通路失活作用 (增加 pAMPK p<0.01，降低 pAkt，p<0.01; 和 pS6，p<0.05)。 局限性: 体外和体内 CRT 方案不能直接转化为人类给药方法。 结论: 二甲双胍在体外和体内增强肿瘤对辐射的反应。二甲双胍是未来研究/试验中考虑的有吸引力的替代放射增敏剂。请参阅视频摘要 http://links.lww.com/DCR/B219 。
METHODS::Aims: Radiotherapy is predominantly used as one of the treatment modalities to treat local tumor in colorectal cancer (CRC). Hindrance in disease treatment can be attributed to radio-tolerance of cancer stem cells (CSCs) subsistence in the tumor. Understanding the radio-resistant property of CSCs might help in the accomplishment of targeted radiotherapy treatment and increased disease-free survival. Telomeric RAP1 contributes in modulation of various transcription factors leading to aberrant cell proliferation and tumor cell migration. Therefore, we investigated the role of RAP1 in maintaining resistance phenotype and acquired stemness in radio-resistant cells.Main Methods: Characterization of HCT116 derived radio-resistant cell (HCT116RR) was performed by cell survival and DNA damage profiling. RAP1 silenced cells were investigated for DNA damage and expression of CSC markers through western blotting and Real-time PCR post-irradiation. Molecular docking and co-immunoprecipitation study were performed to investigate RAP1 and KLF4 interaction followed by RAP1 protein status profiling in CRC patient.Key findings: We established radio-resistant cells, which showed tolerance to radiotherapy and elevated expression of CSC markers along with RAP1. RAP1 silencing showed enhanced DNA damage and reduced expression of CSC markers post-irradiation. We observed strong physical interaction between RAP1 and KLF4 protein. Furthermore, higher RAP1 expression was observed in the tumor of CRC patients. Dataset analysis also revealed that high expression of RAP1 expression is associated with poor prognosis.Significance: We conclude that higher expression ofRAP1 implicates its possible role in promoting radio-resistance in CRC cells by modulating DNA damage and CSC phenotype.
METHODS::Cancer stem-like cells are rare immortal cells within tumor, which are thought to play important roles in ionizing radiation (IR) therapy-resistance. Quercetin is a natural flavonoid with potential anti-cancer properties without significant cytotoxicity in normal tissues. In this study, we demonstrated that quercetin-IR combination treatment exhibited more dramatic anti-cancer effect than either quercetin or IR treatment alone via targeting colon cancer stem cells (CSCs) and inhibiting the Notch-1 signaling. These effects were further verified by in vivo studies which showed remarkable decrease of the CSCs markers and the expression of Notch-1 signaling proteins in human colon cancer xenografts in nude mice. Co-treatment with quercetin and low dose of radiation significantly reduced the expressions of all five proteins of γ-secretase complex in HT-29 and DLD-1 cells. In addition, ectopic expression of the Notch intracellular domain (NICD) partly reversed the inhibition effects by the combination therapy. In conclusion, our results indicated that the combination of quercetin (20 μM) and IR (5Gy) might be a promising therapeutic strategy for colon cancer treatment by targeting colon cancer stem-like cells and inhibiting the Notch-1 signaling. In future studies, we intend to further explore the potential therapeutic efficacy of the quercetin-radiation combination treatment in clinical trials.
METHODS:OBJECTIVES:Long-term prevention of metastatic disease remains a challenge in locally advanced rectal cancer, and robust pretreatment prognostic factors for metastatic progression are lacking. We hypothesized that detecting circulating tumor-specific DNA (ctDNA) based on hypermethylation of the neuropeptide Y gene (meth-ctDNA) could be a prognostic marker in the neoadjuvant setting; we examined this in a secondary, explorative analysis of a prospective trial. MATERIALS AND METHODS:Serum samples were prospectively collected in a phase III trial for locally advanced rectal cancer. Positivity for and fractional abundance of meth-ctDNA in baseline samples were estimated. Overall survival (OS) and the rate of distant metastases were compared between meth-ctDNA positive and negative patients; other prognostic factors were controlled for in multivariate Cox regression. Importance of quantitative load was examined by considering the fractional abundance of meth-ctDNA relative to total circulating DNA. RESULTS:Baseline serum samples were available for 146 patients. In total, 30 patients had presence of meth-ctDNA, with no correlation with cT (P=0.8) or cN (P=0.6) stages. Median follow-up was 10.6 years for OS and 5.1 years for freedom from distant metastases. Patients with meth-ctDNA had significantly worse 5-year OS (47% vs. 69%), even when controlling for other prognostic factors (hazard ratio=2.08; 95% confidence interval, 1.23-1.51). This seemed mainly driven by disparity in the rate of distant metastases (55% vs. 72% at 5 y, P=0.01); hazard ratio=2.20 (95% confidence interval, 1.19-4.07, P=0.01) in multivariate analysis. Increased quantitative load was highly significant for worse outcomes. CONCLUSIONS:Meth-ctDNA could be a potential prognostic marker in the neoadjuvant setting and may, if validated, identify patients at increased risk of distant metastases.