Ethyl Acetate Fraction from Hedyotis Diffusa Plus Scutellaria Barbata Inhibits the Progression of Breast Cancer via Targeting LMO1 and AKT/Mtor Signaling Pathway

BACKGROUND: Traditional Chinese medicines are widely used in cancer treatment. Scutellaria barbata and Hedyotis diffusa herb pair (SH) has an anticancer effects in various tumors. However, the specific mechanism of SH in breast cancer remains unclear. METHODS: In the present research, we investigated the effect and regulatory network of SH in in breast cancer. CCK8, colony formation, transwell, wound healing and flow cytometry analysis were used for the detection of cell function. RESULTS: Ethyl acetate fraction from SH at an equal weight ratio (EA11) could inhibit the proliferation, migration and invasion of MCF7 and MDA-MB-231 cells. It also induced apoptosis in these two cell lines by downregulating Bcl2 and upregulating Bax and Cleaved-Caspase3. SH reduced the activation of the AKT/mTOR signaling pathway and the expression of p70S6K. Sequencing results showed that LMO1 was significantly downregulated in SH-treated cells compared with control cells. Importantly, overexpression of LMO1 attenuated the inhibitory effect of SH on cell proliferation and invasion and induced inflammatory tumor microenvironment. CONCLUSION: In conclusion, the SH herb pair inhibited the proliferation and metastasis through downregulating LMO1 expression and reducing the activation of the AKT/mTOR signaling pathway. LMO1 has the potential as a new target in the treatment of breast cancer.

Green merger and acquisition decision driven by environmental regulation and its impact on green innovation: evidence from Chinese heavily polluting listed enterprises.

How heavily polluting enterprises achieve sustainable development has become an urgent issue to be solved. Based on the data of Chinese heavily polluting listed enterprises during the period of 2010-2020, this paper adopts the probit method to examine how heterogeneous environmental regulations affect green merger and acquisition (GMA) decision, and propensity score matching and difference-in-differences method to explore innovation performance of GMA. The findings are as follows: (1) Command-and-control environmental regulation (CMCER) can promote GMA, while there exists a U-shaped relationship between market-based environmental regulation (MBER) and GMA; (2) from a dynamic perspective, it turns out that GMA can promote green innovation (GI) in the first and second post-acquisition year, but this effect disappears in the third year; (3) compared with CMCER, MBER has a more pronounced positive effect on the relationship between GMA and GI; (4) heterogeneous analysis indicates that the above GMA performance is more persistent when the acquirer is state-owned, with high media attention, with high internal control or engages in vertical GMA. The findings further enrich the literature on GMA driver and performance and provide references for optimizing ER policies and promoting corporate sustainable development.

M&A goodwill and corporate technological innovation: The mediating moderating effect of stock pledges.

Identifying whether and how merger and acquisition (M&A) goodwill influences corporate technology innovation is a critical topic in the context of growing M&A activities. Based on the panel data of 2,634 Chinese non-financial listed companies from 2007 to 2019, this paper adopts multiple linear regression and the Bootstrap method to investigate the impact of M&A goodwill on corporate technological innovation and the mechanism through which M&A goodwill affects technological innovation. The results show that: (1) There is a significant negative correlation between M&A goodwill and technological innovation input, with financial constraints acting as the main channels. (2) Non-private corporations enjoy a higher level of financing capacity in terms of debt and equity compared with private corporations, resulting in the absence of the disincentive effect of M&A goodwill on corporate technological innovation inputs, despite the fact that M&A activities also cause massive resource consumption. (3) Stock pledges by controlling shareholders are effective in alleviating the negative impact of M&A goodwill on technological innovation inputs. Furthermore, our results highlight that simple stock pledge measures capture a significant influence of alleviating financing constraints, thereby decreasing the adverse impact of M&A, and hence need to be adequately considered in future M&A and innovation studies. While enriching the literature on the economic consequences of M&A and the research on stock pledge, this paper also provides micro-evidence for an in-depth understanding of the effect of M&A goodwill on technological innovation.

Does the Environmental Regulation Intensity and ESG Performance Have a Substitution Effect on the Impact of Enterprise Green Innovation: Evidence from China.

Against the background of green and sustainable development strategy, it is an effective way to carry out green innovation to cope with the increasing intensity of government environmental regulation for enterprises. Nevertheless, the regulatory role of ESG performance has been ignored. Based on panel data from Chinese listed companies from 2010 to 2019, this paper mainly studies whether the environmental regulation intensity and ESG performance have a substitution effect on the impact of green innovation by constructing a double fixed effect model. The empirical results showed that first, positive ESG performance is conducive to promoting green innovation. Second, there is a U-shaped relationship between the intensity of environmental regulation and high-quality green innovation, which reflects the effect of "offset before compensation". With the increasing intensity of environmental regulation, high-quality green innovation tends to crowd out low-quality green innovation, which further improves the practical test of the "Porter Hypothesis". Third, the positive ESG performance showed a negative regulatory effect between environmental regulation intensity and enterprise green innovation, which means that environmental regulation intensity and ESG performance have a substitution effect, and the effect is heterogeneous in different enterprises. This paper makes a beneficial exploration on how environmental regulation intensity and ESG performance affect enterprise green innovation, and demonstrates the regulatory role of ESG performance between environmental regulation intensity and green innovation, which reveals the impact of macro environmental policies on the green innovation behavior of micro subjects, and contributes to the further improvement of ESG concept and green innovation theory.

The effective path of green transformation of heavily polluting enterprises promoted by green merger and acquisition-qualitative comparative analysis based on fuzzy sets.

Green merger and acquisition (GMA) is becoming a growing tendency for heavily polluting enterprises in recent years; however, the realization path of green transformation through GMA is still unexplored. Taking 48 Chinese heavily polluting enterprises that had GMA in 2018 as the research object, this paper constructs the "M&A attributes, Organizational characteristics, and External environment" (M-O-E) framework, by using the method of fuzzy-set qualitative comparative analysis (fsQCA) to reveal the configurations of conditions that lead to high levels of green innovation performance. The results show that the high green technology innovation performance after GMA of heavily polluting enterprises is the outcome of multiple antecedents, and no singular antecedent is sufficient for achieving it. Besides, there are three equivalent configurations of conditions to achieve green transformation: professional buyer, internal leading, and internal-external linkage. Among them, the professional buyer configuration highlights that the combination of M&A experience and M&A scale is of great importance, the internal leading configuration emphasizes that the existence of environmental awareness and organizational resources is the core conditions, and the internal-external linkage configuration requires simultaneous efforts of M&A experience and government environmental regulations. Our research contributes to the understanding of green transformation in heavily polluting enterprises from a configurational perspective, and provides a practice-oriented guide to achieve green transformation for the government and heavily polluting enterprises.

Evolutionary game analysis of coal enterprise resource integration under government regulation.

Resource integration of coal enterprises is conducive to reducing pollution and carbon emissions, thus alleviating environmental problems such as global warming. Government regulation has a great influence on enterprise behavior. Therefore, it is necessary to analyze the strategies of government and coal enterprises in resource integration. Based on the perspective of government regulation, this paper discusses how to guide and restrict coal enterprises to conduct resource integration behavior, and whether the government supervises this behavior. First, through empirical research, government regulations of coal enterprises are given practical policy implications. Second, using evolutionary game and simulation technology, from the perspective of government regulation, we explore the complex behavioral interaction mechanism between the dominant and inferior coal enterprises, the mechanism between the government and coal enterprises, and analyze the impact of key factors on the dynamic evolution process. Finally, the sensitivity analysis of the selected parameters is discussed in details, which provides useful decision-making suggestions for the government and enterprises. In addition, this paper further analyzes the impact of different government policies on coal enterprises' green innovation strategies. Results demonstrate that (1) when the power gap between enterprises is large, the probability of dominant enterprises choosing resource integration converges to 1, while the probability of inferior enterprises converges to 0. Therefore, government regulations are invalid for inferior enterprises; (2) the combination of government regulations can help improve the efficiency of coal enterprises' strategic choices. With the increase in the intensity of government rewards and punishments, the probability of enterprise resource integration evolves from 0 to 1; (3) excessive government regulations make the choice between the government and coal companies tend to swing, because the probability of the two is between 0 and 1. Therefore, excessive government regulations cannot effectively achieve resource integration and government regulation. (4) The government subsidy strategy is less effective than the government's pollution penalty strategy in promoting the green innovation of enterprises. Our research shows that the government should choose different policy combinations and intensities to regulate resource integration according to the market power of coal enterprises, which provides theoretical reference and practical guidance for the government to regulate corporate resource integration behavior.

Impact of Monetary Policy Uncertainty on R&D Investment Smoothing Behavior of Pharmaceutical Manufacturing Enterprises: Empirical Research Based on a Threshold Regression Model.

R&D investment is the source of technological innovation of pharmaceutical enterprises, but it will be restricted by the funding level, especially in the context of major public health emergencies occurring more frequently, therefore exploring the impact of monetary policy uncertainty on the R&D investment smoothing behavior of pharmaceutical manufacturing enterprises has important theoretical and practical value. Based on the relevant data of Chinese pharmaceutical manufacturing enterprises from 2012 to 2018, this paper studies the impact of monetary policy uncertainty on R&D investment smoothing behavior of pharmaceutical enterprises, and investigates whether there is a threshold effect. First, our results demonstrate that the empirical test results of this article support the hypothesis of R&D investment smoothing behavior of pharmaceutical manufacturing enterprises. Second, there is a negative correlation between monetary policy uncertainty and R&D investment smoothing behavior, and the shorter the period is, the higher the financing constraints of pharmaceutical enterprises are, and the more obvious the negative correlation is. Third, financing constraints have a single threshold effect on the R&D investment smoothing behavior of pharmaceutical manufacturing enterprises, with a threshold of -13.7693. Moreover, this conclusion can better promote the virtuous circle of the real economy of financial and pharmaceutical manufacturing enterprises. It is recommended that pharmaceutical manufacturing enterprises establish and improve the enterprise R&D reserve system, reduce the risk of R&D investment, play the role of R&D smoothing, and realize the sustainable development of enterprise R&D.

Treatment of bilateral granulomatous lobular mastitis during lactation with traditional Chinese medicine: A case report.

BACKGROUND: Granulomatous lobular mastitis (GLM) is a type of benign chronic inflammatory disease that poses therapeutic challenges to healthcare providers. The diagnosis of GLM relies on tissue biopsy, and incorrect treatment may lead to delayed diagnosis, considerable aesthetic damage, and even mastectomy. CASE SUMMARY: We report the case of a 37-year-old Chinese woman who was lactating and had GLM in both breasts. At the time of treatment, the right breast had a mass of approximately 15 cm × 11 cm, which was hard and had poor mobility. Multiple skin ulcerations and pus spills were also observed on the surface of the breast. The left breast had a mass of about 13 cm × 9 cm, which was hard and had poor mobility. CONCLUSION: Herein, we report a case of bilateral GLM in a lactating woman that was successfully treated with traditional Chinese medicine (TCM), without the requirement for surgery or other treatments. Therefore, TCM may have advantages in the nonsurgical treatment of GLM.

Economic Policy Uncertainty, Environmental Regulation, and Green Innovation-An Empirical Study Based on Chinese High-Tech Enterprises.

As the continuous changes in environmental regulations have a non-negligible impact on the innovation activities of micro subjects, and economic policy uncertainty has become one of the important influencing factors to be considered in the development of enterprises. Therefore, based on the panel data of Chinese high-tech enterprises from 2012-2017, this paper explores the impact of heterogeneous environmental regulations on firms' green innovation from the perspective of economic policy uncertainty as a moderating variable. The empirical results show that, first, market-incentivized environmental regulation instruments have an inverted U-shaped relationship with innovation output, while voluntary environmental regulation produces a significant positive impact. Second, the U-shaped relationship between market-based environmental regulation and innovation output becomes more pronounced when economic policy uncertainty is high. However, it plays a negative moderating role in regulating the relationship between voluntary-based environmental regulation and innovation output. This paper not only illustrates the process of technological innovation by revealing the intrinsic mechanism of environmental regulation on firm innovation, but also provides insights for government in environmental governance from the perspective of economic policy uncertainty as well.

Economic policy choice of governing haze pollution: evidence from global 74 countries.

Haze pollution not only has a huge impact on economic development but also seriously damages the health of residents, which has attracted the attention of many countries and scholars. The geographical detector model and the panel quantile regression model are used in combination to analyze the socio-economic driving factors of haze pollution from 2010 to 2015 for 74 significantly representative countries. The main results are as follows: (1) Industrial structure is the main factor affecting the haze concentration, followed by economic growth and research and development (R&D) intensity. (2) Government influence and industrial structure will significantly aggravate haze pollution, whereas the energy intensity and economic growth have an inhibitory effect on haze concentration. Countries with severe haze pollution should focus on upgrading their industrial structure and avoiding energy rebound. (3) Urbanization, foreign investment, and R&D intensity have different effects on the haze concentration among countries with different pollution levels. Specifically, the relationship between economic growth and pollution is inverted N-shaped in countries with medium haze concentration, whereas in other countries, it is positive N-shaped. Countries should actively leverage the agglomeration effect of high-density urban populations and focus on the introduction of high-quality foreign capital.

The impact of heterogeneous environmental regulation on innovation of high-tech enterprises in China: mediating and interaction effect.

It is important for China's green innovation productivity and sustainable development to study the effect of heterogeneous environmental regulation on microenterprise innovation activities. Based on the panel data of high-tech enterprises in China from 2012 to 2017, the article studies the incentive effect of heterogeneous environmental regulation on technological innovation and the mediation of innovation input and explores whether different types of environmental regulations have interactive effects on enterprise innovation. The results reveal that compared with the command-controlled environmental regulation, the incentive effect of market-incentive environmental regulation and voluntary environmental regulation on enterprise innovation is more significant, where the innovation input fully plays its role as a mediating effect. Further research finds that there is an interactive effect between command-controlled environmental regulation and other two regulatory tools, but no interactive effect between market-incentive environmental regulation and voluntary environmental regulation, which shows that the control-based regulatory tools and more flexible regulatory tools have a complementary effect on enterprise innovation. Through revealing the internal mechanism of environmental regulation on enterprise innovation, the article displays the process of technological innovation, and it also finds that flexible regulation tools and the combination of rigid and flexible tools are more conducive to encourage enterprises to carry out innovation activities.

How to Evaluate Provincial Ecological Civilization Construction? The Case of Jiangsu Province, China.

Ecological civilization is a transcendence of industrial civilization. The improvement of China's ecological civilization system can not only provide developing countries with an empirical reference for ecological civilization construction, but also make a huge contribution to the progress of global ecological civilization. This paper focuses on improving the provincial ecological civilization evaluation system. First, the Provincial Ecological Civilization Construction Evaluation Index System is established according to the Green Development Index System issued by the Chinese government in 2016, and then the applicability of the Spatio-Temporal Factor Analysis (STFA) is verified as the evaluation method of the provincial ecological civilization construction level. Further, taking Jiangsu Province, China as an example, this paper evaluates the level of ecological civilization construction in each city of Jiangsu Province, and analyzes the main factors affecting ecological civilization based on the evaluation results. Finally, according to the relationship between the ecological civilization construction and economic quality of each city, the four-quadrant method is applied to divide Jiangsu Province into four types of regions to help each city position itself in ecological civilization construction. This paper mainly draws the following conclusions: (1) Due to its simple operation and accurate evaluation results, STFA is applicable in evaluating the provincial ecological civilization construction level. (2) Taking Jiangsu Province as an example, it is found that the level of ecological civilization construction in the southwestern region is higher than that of the northeast coastal region in Jiangsu. Three inequality indices are applied to measure the regional inequality of ecological civilization construction among 13 cities, which is relatively high in recent years. (3) By analyzing the results of STFA, it is found that economic quality and natural protection are not only the main factors affecting the ecological civilization construction in Jiangsu province, but also the main reasons for the regional heterogeneity of provincial ecological civilization. (4) For the four types of regions, suggestions are proposed according to the characteristics of each region to help improve the provincial ecological civilization construction level. This paper not only improves China's ecological civilization construction system, but also provides information for the provincial ecological civilization construction in developing countries.

How does vertical integration promote innovation corporate social responsibility (ICSR) in the coal industry? A multiple-step multiple mediator model.

This paper aims to demonstrate the impact of coal enterprises' vertical integration on the performance of innovation corporate social responsibility (ICSR) and to elaborate its specific transmission path through financing structure and market power. This paper takes the data of A-share listed coal company from 2008 to 2017 as samples, uses input-output table method to measure the degree of vertical integration, and explores the relationship between the four factors through multiple linear regression and Bootstrap method. We found that: (1) the vertical integration of coal enterprises has positive incentives for the promotion of ICSR. (2) Financing structure and market power play a chain intermediary role in this process of incentive. (3) In areas with high marketization process, vertical integration has more significant incentive effect on ICSR. The paper extends the research on the relationship between vertical integration and innovation, which provides a reference for the improvement of China's supply-side reform and corporate social responsibility (CSR) fulfillment policy. It is helpful to promote the sustainable development of the coal industry, stimulate the innovation vitality of enterprises, and improve the fulfillment of CSR.

How Can We Improve the Transformation Success Rate of Research Results in the Pharmaceutical Industry? The Game Theoretic Model of Technology Transfer Subjects.

University-industry technology transfer (UITT) plays an important role in the construction of the national pharmaceutical innovation system. The speculations of a faculty inventor may hinder the successful transfer of pharmaceutical research results. This paper divides the specific process of the transformation of pharmaceutical research results into two parts: (1) an evolutionary game between faculty inventors and universities; and (2) a Stackelberg game between faculty inventors and pharmaceutical companies. Further, we carry out numerical simulations to analyze the impact of transformation success rate, income distribution coefficient, and a faculty inventor's future working years on the transformation of pharmaceutical research results. The findings indicated that whether a combination of action strategies of faculty inventors and universities can evolve to the optimal equilibrium is determined by many factors, such as the technological transaction price of the pharmaceutical company and the reward or the income obtained by the faculty inventor. The transformation success rate and the income distribution coefficient are the key factors that affect the faculty inventor's will and the behavior of the pharmaceutical company. The conclusions of this paper contribute to the research on how we can improve the success rate of research results and avoid resource waste, and provide a decision-making reference for the management of pharmaceutical research results in universities.

Antitumor Effects of Ruyiping on Cell Growth and Metastasis in Breast Cancer.

Background: Ruyiping is an effective traditional Chinese herbal medicine formula for preventing postoperative recurrence and metastasis of breast cancer. However, the exact function and underlying mechanism of Ruyiping in breast cancer remain unclear. Materials and Methods: After breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with Ruyiping, the CCK8, colony formation, wound-healing, and transwell invasion assays were used to examine cell proliferation, migration, and invasion, respectively. Flow cytometry was performed to examine the effect of Ruyiping on cell cycle distribution. Western blot was performed to examine the expression of related proteins, and the activity of MMP9 was detected using Gelatin zymography assay. Results: Ruyiping treatment significantly inhibited cell proliferation and viability of MDA-MB-231 and MDA-MB-468 cells. Ruyiping was also revealed to trigger cell cycle arrest at the G2 phase in MDA-MB-231 and MDA-MB-468 cells. Moreover, Ruyiping suppressed the migration and invasion abilities of MDA-MB-231 and MDA-MB-468 cells in vitro. Furthermore, Ruyiping blocked the activity of MMP9 in MDA-MB-231 and MDA-MB-468 cells. Additionally, western blotting showed that Ruyiping attenuated epithelial-to-mesenchymal transition (EMT) of breast cancer through downregulation of N-cadherin, Vimentin, Snail1, and Snail2 and upregulation of E-cadherin. The authors observed that the components of Ruyiping Pseudobulbus Cremastra seu pleiones polysaccharide and curcumol showed significant suppression in the growth and invasion of breast cancer cell. Conclusions: The observations of this study suggest the antitumor properties of Ruyiping in cell growth and invasion of breast cancer, which are modulated by induction of cell cycle arrest and reduction of MMP9 and EMT.

Multi-targeting chemoprevention of Chinese herb formula Yanghe Huayan decoction on experimentally induced mammary tumorigenesis.

BACKGROUND: Development of safe and effective chemopreventive agents is a winning strategy in reducing the morbidity and mortality of breast cancer. The current study was to investigate the mechanism-based chemopreventive potential of a Chinese herb formula Yanghe Huayan (YHHY) Decoction on the classical 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary carcinogenesis model. METHODS: Female Sprague-Dawley rats at 42 days of age were orally administered with a human equivalent dose of YHHY Decoction at 0.02 ml/g (10 mg/ml) once daily, starting 1 wk. before and 4 wks following DMBA treatment. Mammary tumor occurrence was monitored every day. The length of time before palpable tumor is examined is defined as tumor-free survival time. High performance liquid chromatography (HPLC) analyses were adopted to identify major chemical compositions of the decoction. Following bioinformatics data mining and experimental analyses were performed to demonstrate the underlying mechanism of action. RESULTS: DMBA animals receiving YHHY Decoction exhibited a significant delay (P = 0.014) and in some animals prevention (P = 0.046) of tumor occurrence without obvious toxicity. Oncogenic myc activation was significantly suppressed in the DMBA-induced rats by the YHHY treatment. Eight major chemical compositions of the decoction were identified and were shown to interfere with multiple tumorigenic pathways simultaneously in the mammary tumors, including inducing tumor apoptosis and up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2; suppressing abnormal cell proliferation and the MAPK/ERK, PI3K/AKT signalings; blocking neo-angiogenesis and the VEGF/KDR signaling, and inhibiting oxidative stress in the mammary tumors. CONCLUSION: The multi-components and multi-targeting properties of the YHHY Decoction support its use as a potent chemopreventive drug in breast cancer.

Yanghe Huayan decoction inhibits the capability of trans-endothelium and angiogenesis of HER2+ breast cancer via pAkt signaling.

Background: Yanghe Huayan Decoction (YHD), a traditional Chinese medicine, is one of the most common complementary medicine currently used in the treatment of breast cancer (BC). It has been recently linked to suppress precancerous lesion and tumor development. The current study sought to explore the role of YHD on trans-endothelium and angiogenesis of BC. Methods: HER2+ BC cells were treated with YHD, Trastuzumab, or the combination in vitro and in vivo to compare the effects of them on trans-endothelium and angiogenesis features. The present study also investigated the potential molecular mechanism of YHD in inhibiting angiogenesis of BC. Results: YHD significantly suppressed the invasion and angiogenesis of BC cells via elevated pAkt signaling. Administration of YHD in vivo also strikingly repressed angiogenesis in tumor grafts. Conclusion: YHD could partially inhibit and reverse tumorigenesis of BC. It also could inhibit Akt activation and angiogenesis in vitro and in vivo Its effect was superior to trastuzumab. Thus it was suitable for prevention and treatment of BC.

Modulation of NRF2 and UGT1A expression by epigallocatechin-3-gallate in colon cancer cells and BALB/c mice.

BACKGROUND: Green tea is an important source of flavonoids in human diets and epidemiological data correlate green tea consumption with a reduced cancer risk. Given its complicated properties at effective concentrations, we put epigallocatechin-3-gallate (EGCG) that previously reported on its anti-proliferative activities against several cancer cell lines on our research agenda to further examine the mechanism of its chemopreventive potential. METHODS: RNA interference (RNAi) expression vector pSilencer 3.1-H1 was used to construct recombinant nuclear factor erythroid 2 related factor 2 (Nrf2)-targeting RNAi plasmids. EGCG (5 microg/ml) was added into the culture fluid of cells before and after transfection. RT-PCR and Western blotting were used to detect the expression of uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A in cells. Forty male BALB/c mice were assigned to four groups: a normal unexposed control and three groups treated with varying doses of EGCG. Four weeks later, the mice were sacrificed, and their colon tissues were subjected to mRNA and protein expression of Nrf2 and UGT1A via RT-PCR and Western blotting analysis. RESULTS: EGCG up-regulated the expression of Nrf2 and increased the level of UGT1A in cells. The blockade of Nrf2 activity via RNA intervention largely attenuated the induction of UGT1A expression by EGCG. In mice, the mRNA and protein levels of Nrf2 and UGT1A detected by RT-PCR and Western blotting increased (both P < 0.05 compared with the control). This increase in Nrf2 expression also had a positive correlation with an increased UGT1A expression. CONCLUSIONS: EGCG mediated its effect in part by inducing the NRF2 signaling pathway and increasing UGT1A expression. Both in vitro and in vivo studies demonstrated the role of NRF2 and UGT1A expression in the potential use of EGCG as a possible chemopreventive agent and supported further study of EGCG for cancer treatment.

[The role of NF-E2-related factor 2 in the induction of uridine 5'-diphosphate-glucuronosyltransferase 1A and its isoforms by epigallocatechin gallate in colon cancer cells].

OBJECTIVE: To investigate the role of human transcription factor NF-E2-related factor 2 (Nrf2) in the induction of the gene expression of uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A and its isoforms by epigallocatechin gallate (EGCG). METHODS: (1) Human colon carcinoma cells Caco-2 and HT-29 were cultured. Immunocytochemistry, western blotting and confocal laser microscopy were used to detect the protein expression of Nrf2. Twenty samples of colon carcinoma with surrounding normal tissues were collected during endoscopic course. (2) RNA interference expression vector pSilencer 3.1-H1 was used to construct four Nrf2-trageting plasmids: pSilence-Nrf2-A, B, C, and D and a control pSilence-CON. Cells were transfected with pSilence-Nrf2 for 48 hours to observe the effects of transient transfection. Cells were stably transfected with pSilence-Nrf2-B for 4 weeks and re-named as Caco-2-siNrf2 and HT-29-siNrf2 (siNrf2 cells), and others stably transfected with blank plasmid pSilencer 3.1-H1 were used as controls. (3) EGCG was added into the culture fluid of cells before and after the stably transfection. RT-PCR was used to detect the mRNA expression of Nrf2, UGT1A, UGT1A8 and UGT1A10 in cells and the samples of human colon cancer tissue. RESULTS: (1) The expressions of UGT1A8 and UGT1A10 mRNA were significantly lower than that in the surrounding healthy mucosa. (2) The mRNA expression of Nrf2, UGT1A8, and UGT1A10 increased by 1.8-9.2 times after the addition of EGCG (all P < 0.05). Immunocytochemistry, western blotting and immunofluorescence demonstrated a significant increase of Nrf2 protein expression in the nucleus after treatment with EGCG. (3) SalIenzyme digestion and DNA sequencing confirmed that pSilence-Nrf2-A, B, C, and D and pSilence-CON were all successfully constructed. The inhibition rate of Nrf2 gene expression was above 80% 48 h after transfection with pSilence-Nrf2-B, and that was no significant difference after transfection with pSilence-CON (P > 0.05). There was specific inhibition of Nrf2 in Caco-2-siNrf2, HT-29-siNrf2 cells (both P < 0.01). (4) The basal levels of UGT1A8 and UGT1A10 mRNA expression in the Caco-2-siNrf2 and HT-29-siNrf2 cells were lower by 15%-65% in comparison with those in control, and the induction of genes by EGCG was largely attenuated in them (all P > 0.05). CONCLUSION: Nrf2 is localized in the cytoplasm of non-stimulated cells, and EGCG triggered its rapid nuclear accumulation. Suppression of Nrf2 gene expression results in down-regulation of the constructive expression of UGT genes and their induction by EGCG. EGCG induces the expression of UGT1A, UGT1A8 and UGT1A10 genes via a Nrf2-dependent mechanism.