Use of a Genetically Engineered E. coli Overexpressing β-glucuronidase Accompanied by Glycyrrhizic Acid, A Natural and Anti-inflammatory Agent, for Directed Treatment of Colon Carcinoma in a Mouse Model.
- 作者列表："Afkhami-Poostchi A","Mashreghi M","Iranshahi M","Matin MM
:Bacteria-directed enzyme prodrug therapy (BDEPT), is an emerging alternative directed and tumor-specific approach. The basis of this method is the conversion of a non-toxic prodrug by a bacterial enzyme to a toxic drug within the tumor-microenvironment (TME). In the present study, the therapeutic efficacy of BDEPT was investigated based on the ability of Escherichia coli DH5α-lux/βG in activation of glycyrrhizic acid (GL), a natural and non-toxic compound purified from licorice, to glycyrrhetinic acid (GA) only in TME. To do so, the anti-bacterial effects of GL on bacteria and the cytotoxic effects of the produced GA on survival rate of CT26 mouse colon carcinoma cells were evaluated. The IC50 values of the produced GA and cisplatin were determined as 210 μM and 100 μM, respectively. Comparing these values to GL treatment (1305 μM) indicates that bacteria could have efficiently activated GL to GA to inhibit the growth of tumor cells. Afterward, the anti-cancer effects of bacteria used in combination with GL was investigated in a mouse model of colon carcinoma. Results were indicative of targeted homing and even proliferation of luminescent bacteria in TME. Moreover, combined treatment greatly inhibited tumor growth. Histopathological analysis of dissected tissues also demonstrated increased apoptosis rate in tumor cells after combined treatment and interestingly, showed no obvious damage to the spleen and liver of treated mice. Accordingly, this BDEPT approach could be considered as an effective alternative tumor-specific therapy utilizing prodrug-activating enzymes expressing from tumor-targeting bacteria to allow the development of new tumor-specific pharmacotherapy protocols.
: 细菌导向的酶前体药物治疗 (brept)，是一种新兴的替代导向和肿瘤特异性方法。该方法的基础是在肿瘤微环境 (TME) 内通过细菌酶将无毒前药转化为有毒药物。在本研究中，基于大肠杆菌 dh5 α-lux/β g 对甘草酸 (GL) 的活化能力研究了 BDEPT 的治疗效果, 一种从甘草中纯化的天然无毒化合物，仅在 TME 中为甘草次酸 (GA)。为此，评价了 GL 对细菌的抗菌作用和产生的 GA 对 CT26 小鼠结肠癌细胞存活率的细胞毒作用。所产生的 GA 和顺铂的 IC50 值分别测定为 210 μ m 和 100 μ m。将这些值与 GL 处理 (1305 μ m) 进行比较表明，细菌可以有效激活 GL 到 GA 来抑制肿瘤细胞的生长。随后，在结肠癌小鼠模型中研究了细菌与 GL 联合使用的抗癌作用。结果表明 TME 中发光细菌的靶向归巢甚至增殖。此外，联合治疗大大抑制了肿瘤的生长。解剖组织的组织病理学分析也显示联合治疗后肿瘤细胞凋亡率增加，有趣的是，对治疗小鼠的脾脏和肝脏无明显损伤。因此，这种 BDEPT 方法可以被认为是一种有效的替代肿瘤特异性疗法，利用肿瘤靶向细菌表达的前体药物激活酶，以允许开发新的肿瘤特异性药物治疗方案。
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.