Discovery of Novel ERα and Aromatase Dual-Targeting PROTAC Degraders to Overcome Endocrine-Resistant Breast Cancer.

Estrogen receptor α (ERα) plays a pivotal role in the proliferation, differentiation, and migration of breast cancer (BC) cells, and aromatase (ARO) is a crucial enzyme in estrogen synthesis. Hence, it is necessary to inhibit estrogen production or the activity of ERα for the treatment of estrogen receptor-positive (ER+) BC. Herein, we present a new category of dual-targeting PROTAC degraders designed to specifically target ERα and ARO. Among them, compound 18c bifunctionally degrades and inhibits ERα/ARO, thus effectively suppressing the proliferation of MCF-7 cells while showing negligible cytotoxicity to normal cells. In vivo, 18c promotes the degradation of ERα and ARO and inhibits the growth of MCF-7 xenograft tumors. Finally, compound 18c demonstrates promising antiproliferative and ERα degradation activity against the ERαMUT cells. These findings suggest that 18c, being the inaugural dual-targeting degrader for ERα and ARO, warrants further advancement for the management of BC and the surmounting of endocrine resistance.

Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer.

Endocrine-resistance remains a major challenge in estrogen receptor α positive (ERα+) breast cancer (BC) treatment and constitutively active somatic mutations in ERα are a common mechanism. There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrine-resistance. Given aberrant ERα activity, we herein report the identification of novel covalent selective estrogen receptor degraders (cSERDs) possessing the advantages of both covalent and degradation strategies. A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERα+ breast cancer cell lines including mutant ERα. Crystal structure of ERα‒29c complex alongside intact mass spectrometry revealed that 29c disrupted ERα protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11, thus enforcing a unique antagonist conformation and driving the ERα degradation. These significant effects of the cSERD on ERα homeostasis, unlike typical ERα degraders that occur directly via long side chains perturbing the morphology of H12, demonstrating a distinct mechanism of action (MoA). In vivo, 29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity. This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.

Identification of Novel Dual-Target Estrogen Receptor α Degraders with Tubulin Inhibitory Activity for the Treatment of Endocrine-Resistant Breast Cancer.

Endocrine resistance remains a significant problem in the clinical treatment of estrogen receptor α-positive (ERα+) breast cancer (BC). In this study, we developed a series of novel dual-functional ERα degraders based on a bridged bicyclic scaffold with selenocyano (SeCN) side chains. These compounds displayed potent ERα degradation and tubulin depolymerization activity. Among them, compounds 35s and 35t exhibited the most promising antiproliferative and ERα degradation activity in multiple ERα+ BC cell lines bearing either wild-type or mutant ERα. Meanwhile, compounds 35s and 35t disrupted the microtubule network by restraining tubulin polymerization, evidenced by 35t inducing cell cycle arrest in the G2/M phase. In MCF-7 and LCC2 xenograft models, compounds 35s and 35t remarkably suppressed tumor growth without noticeable poisonousness. Finally, this study provided guidance for developing new dual-target antitumor drug candidates for the ERα+ BC therapy, especially for the resistant variant.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERß degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

Discovery of estrogen receptor α targeting caged hypoxia-responsive PROTACs with an inherent bicyclic skeleton for breast cancer treatment.

In view of the potential off-target effects of antitumor drugs, including proteolysis targeting chimera (PROTAC), certain toxic effects may be caused in normal tissues. Herein, based on the characteristics of the tumor microenvironment, we reported the first estrogen receptor α (ERα) targeting hypoxia-responsive PROTACs in order to improve their safety in breast cancer treatment by introducing two hypoxia-activated groups, nitroimidazole and nitrobenzene, into the ER ligand or E3 ligand of an active PROTAC, which has certain cytotoxicity in normal cells. Bioactivity studies showed that these hypoxia-responsive PROTACs exhibited excellent hypoxic responsiveness and ERα degradation activity under hypoxic conditions, and thus improved the toxic effects of the active PROTAC in normal cells. It is expected that our caged compounds provide a new strategy for precise functional control of PROTAC drugs for breast cancer treatment.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer.

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

Development of Highly Efficient Estrogen Receptor ß-Targeted Near-Infrared Fluorescence Probes Triggered by Endogenous Hydrogen Peroxide for Diagnostic Imaging of Prostate Cancer.

Hydrogen peroxide is one of the most important reactive oxygen species, which plays a vital role in many physiological and pathological processes. A dramatic increase in H2O2 levels is a prominent feature of cancer. Therefore, rapid and sensitive detection of H2O2 in vivo is quite conducive to an early cancer diagnosis. On the other hand, the therapeutic potential of estrogen receptor beta (ERß) has been implicated in many diseases including prostate cancer, and this target has attracted intensive attention recently. In this work, we report the development of the first H2O2-triggered ERß-targeted near-infrared fluorescence (NIR) probe and its application in imaging of prostate cancer both in vitro and in vivo. The probe showed good ERß selective binding affinity, excellent H2O2 responsiveness and near infrared imaging potential. Moreover, in vivo and ex vivo imaging studies indicated that the probe could selectively bind to DU-145 prostate cancer cells and rapidly visualizes H2O2 in DU-145 xenograft tumors. Mechanistic studies such as high-resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations indicated that the borate ester group is vital for the H2O2 response turn-on fluorescence of the probe. Therefore, this probe might be a promising imaging tool for monitoring the H2O2 levels and early diagnosis studies in prostate cancer research.

Anticancer or carcinogenic? The role of estrogen receptor ß in breast cancer progression.

Estrogen receptor ß (ERß) is closely related to breast cancer (BC) progression. Traditional concepts regard ERß as a tumor suppressor. As studies show the carcinogenic effect of ERß, some people have come to a new conclusion that ERß serves as a tumor suppressor in estrogen receptor α (ERα)-positive breast cancer, while it is a carcinogen in ERα-negative breast cancer. However, we re-examine the role of ERß and find this conclusion to be misleading based on the last decade's research. A large number of studies have shown that ERß plays an anticancer role in both ERα-positive and ERα-negative breast cancers, and its carcinogenicity does not depend solely on the presence of ERα. Herein, we review the anticancer and oncogenic effects of ERß on breast cancer progression in the past ten years, discuss the mechanism respectively, analyze the main reasons for the inconsistency and update ERß selective ligand library. We believe a detailed and continuously updated review will help correct the one-sided understanding of ERß, promoting ERß-targeted breast cancer therapy.

Nitrogen deposition enhances the deterministic process of the prokaryotic community and increases the complexity of the microbial co-network in coastal wetlands.

Global nitrogen deposition has increased significantly in recent years. At present, research on the effects of different amounts and types of nitrogen deposition on soil microorganisms in coastal wetlands is scarce. In this study, based on 7 years of simulated nitrogen deposition at multiple levels (low, medium, high) and of multiple types (NH4NO3, NH4Cl, KNO3), the effects of different nitrogen deposition conditions on the diversity, community assembly processes, co-networks, and community function of soil prokaryotes in coastal wetlands were examined. The results showed that, compared with that in control, the microbial α diversity increased significantly under nitrogen deposition (P < 0.05). However, it decreased significantly in the high-NH4NO3 and high-NH4Cl treatments (P < 0.05). The deterministic process of community assembly was strengthened under the different types of nitrogen deposition. Compared with that under NH4+-N deposition, the microbial co-network under NO3--N deposition was more complex. Network stability significantly decreased under different NH4+-N deposition levels. In addition, the results of FAPROTAX functional prediction showed that microbial community functional groups associated with carbon and nitrogen cycling changed significantly (P < 0.05). In conclusion, our results emphasize that nitrogen deposition environments cause changes in soil microbial community structure and interactions, and may also affect soil carbon and nitrogen cycling, but the effects of different forms and levels of nitrogen deposition are not consistent. This study provides new insights for evaluating the changes in soil microbial communities in coastal wetlands caused by different types of long-term nitrogen deposition, and has scientific significance for assessing the ecological effects of long-term nitrogen deposition.

Estrogen receptor ß-targeted hypoxia-responsive near-infrared fluorescence probes for prostate cancer study.

Aberrant expression of estrogen receptor ß (ERß) and tumor hypoxia have been observed in castration-resistant prostate cancer (CRPC); therefore, hypoxia-responsive labeling of ERß will be beneficial for the early diagnosis and treatment of CRPC. Herein, we report the first ERß-targeted hypoxia-responsive near-infrared fluorescent probes, which showed superior ERß selectivity and favorable optical properties. These two probes exhibited excellent hypoxia responsiveness and specific mitochondrial ERß imaging ability in CRPC cells. In addition, P1 displayed strong anti-interference ability and good tumor imaging capacity in vivo, contributing to effective diagnosis of CRPC. Mechanistic studies, including high resolution mass spectrometry (HRMS) and density functional theory (DFT) calculations, showed that the introduction of a nitro group quenched the probe fluorescence by inducing a PET effect, while in the hypoxic tumor microenvironment, reduction of the nitro group blocked the PET effect and turned on the probe fluorescence. These novel ERß-targeted hypoxia-responsive near-infrared fluorescent probes may promote the study of prostate cancer.

Discovery of Novel Bicyclic Phenylselenyl-Containing Hybrids: An Orally Bioavailable, Potential, and Multiacting Class of Estrogen Receptor Modulators against Endocrine-Resistant Breast Cancer.

Breast cancer (BC) is a multifactorial disease and is prone to drug resistance during treatment. In this study, we described a new class of multifunctional estrogen receptor (ER) modulators ground on a prerogative indirect antagonism skeleton (OBHS, oxabicycloheptene sulfonate) of ER containing a phenylselenyl group. Compound 34b showed significant antiproliferative activities against tamoxifen-sensitive (MCF-7) and -resistant (LCC2) cells. Moreover, hexokinase 1 (HK1) was identified as a direct target of 34b. Further mechanism investigations proved that 34b induced apoptosis, which was associated with mitochondrial dysfunction caused by the synergistic effects of downregulating mitochondrial-bound HK1 protein and promoting reactive oxygen species generation. In vivo, 34b had a favorable pharmacokinetic profile with a bioavailability of 23.20% and exhibited more potent tumor suppression than tamoxifen both in MCF-7 and LCC2 tumor xenograft models. Collectively, our studies showed that 34b is a promising new multifunctional candidate compound for ERα+ BC treatment, particularly for tamoxifen-resistant BC.

Design and synthesis of marine sesterterpene analogues as novel estrogen receptor α degraders for breast cancer treatment.

Targeted protein degradation using small molecules is an intriguing strategy for drug development. The marine sesterterpene compound MHO7 had been reported to be a potential ERα degradation agent. In order to further improve its biological activity, two series of novel MHO7 derivatives with long side chains were designed and identified as novel selective estrogen receptor down-regulators (SERDs). The growth inhibition activity of the novel SERD compounds were significantly affected by the type and length of the side chain. Most of the derivatives were significantly more potent than MHO7 against both drug-sensitive and drug-resistant breast cancer cells. Among them, compound 16a, with IC50 values of 0.41 µM against MCF-7 cell lines and 9.6-fold stronger than MHO7, was the most potential molecule. A whole-genome transcriptomic analysis of MCF-7 cells revealed that the mechanism of 16a against MCF-7 cell was similar with that of MHO7. The estrogen signaling pathway was the most affected among the disturbed genes, but the ERα degradation activity of 16a was observed higher than that of MHO7. Other effects of 16a were confirmed similar with MHO7, which means that the basic mechanisms of the derivatives are the same with the ophiobolin backbone, i.e. the degradation of ERα is mediated via proteasome-mediated process, the induction of apoptosis and the cell cycle arrest at the G1 phase. Meanwhile, a decrease of mitochondrial membrane potential and an increase of cellular ROS were also detected. Based on these results, as a novel modified ophiobolin derived compound, 16a may warrant further exploitation as a promising SERD candidate agent for the treatment of breast cancer.

Estrogen Receptor ß-Targeted Near-Infrared Inherently Fluorescent Probe: A Potent Tool for Estrogen Receptor ß Research.

Estrogen receptor ß (ERß) is associated with many diseases, and ERß probes can help to reveal the complex role of ERß and promote the development of ERß-targeted therapy. Herein, we designed and synthesized the first ERß-targeted near-infrared (NIR) inherently fluorescent probe P5, which showed the advantages of high ERß selectivity, good optical properties, and excellent ERß imaging capability in living cells. The probe was successfully utilized to explore ERß motion characteristic, and for the first time, the diffusion coefficient of ERß was obtained. Moreover, P5 was also successfully applied to the in vivo imaging of ERß in the prostate cancer mice model. Therefore, this ERß-targeted NIR probe might be employed as a potential tool for the research of ERß and related diseases.

Fungi and cercozoa regulate methane-associated prokaryotes in wetland methane emissions.

Wetlands are natural sources of methane (CH4) emissions, providing the largest contribution to the atmospheric CH4 pool. Changes in the ecohydrological environment of coastal salt marshes, especially the surface inundation level, cause instability in the CH4 emission levels of coastal ecosystems. Although soil methane-associated microorganisms play key roles in both CH4 generation and metabolism, how other microorganisms regulate methane emission and their responses to inundation has not been investigated. Here, we studied the responses of prokaryotic, fungal and cercozoan communities following 5 years of inundation treatments in a wetland experimental site, and molecular ecological networks analysis (MENs) was constructed to characterize the interdomain relationship. The result showed that the degree of inundation significantly altered the CH4 emissions, and the abundance of the pmoA gene for methanotrophs shifted more significantly than the mcrA gene for methanogens, and they both showed significant positive correlations to methane flux. Additionally, we found inundation significantly altered the diversity of the prokaryotic and fungal communities, as well as the composition of key species in interactions within prokaryotic, fungal, and cercozoan communities. Mantel tests indicated that the structure of the three communities showed significant correlations to methane emissions (p < 0.05), suggesting that all three microbial communities directly or indirectly contributed to the methane emissions of this ecosystem. Correspondingly, the interdomain networks among microbial communities revealed that methane-associated prokaryotic and cercozoan OTUs were all keystone taxa. Methane-associated OTUs were more likely to interact in pairs and correlated negatively with the fungal and cercozoan communities. In addition, the modules significantly positively correlated with methane flux were affected by environmental stress (i.e., pH) and soil nutrients (i.e., total nitrogen, total phosphorus and organic matter), suggesting that these factors tend to positively regulate methane flux by regulating microbial relationships under inundation. Our findings demonstrated that the inundation altered microbial communities in coastal wetlands, and the fungal and cercozoan communities played vital roles in regulating methane emission through microbial interactions with the methane-associated community.

Warming reshaped the microbial hierarchical interactions.

Global warming may alter microbially mediated ecosystem functions through reshaping of microbial diversity and modified microbial interactions. Here, we examined the effects of 5-year experimental warming on different microbial hierarchical groups in a coastal nontidal soil ecosystem, including prokaryotes (i.e., bacteria and archaea), fungi, and Cercozoa, which is a widespread phylum of protists. Warming significantly altered the diversity and structure of prokaryotic and fungal communities in soil and additionally decreased the complexity of the prokaryotic network and fragmented the cercozoan network. By using the Inter-Domain Ecological Network approach, the cross-trophic interactions among prokaryotes, fungi, and Cercozoa were further investigated. Under warming, cercozoan-prokaryotic and fungal-prokaryotic bipartite networks were simplified, whereas the cercozoan-fungal network became slightly more complex. Despite simplification of the fungal-prokaryotic network, the strengthened synergistic interactions between saprotrophic fungi and certain prokaryotic groups, such as the Bacteroidetes, retained these phyla within the network under warming. In addition, the interactions within the fungal community were quite stable under warming conditions, which stabilized the interactions between fungi and prokaryotes or protists. Additionally, we found the microbial hierarchical interactions were affected by environmental stress (i.e., salinity and pH) and soil nutrients. Interestingly, the relevant microbial groups could respond to different soil properties under ambient conditions, whereas under warming these two groups tended to respond to similar soil properties, suggesting network hub species responded to certain environmental changes related to warming, and then transferred this response to their partners through trophic interactions. Finally, warming strengthened the network modules' negative association with soil organic matters through some fungal hub species, which might trigger soil carbon loss in this ecosystem. Our study provides new insights into the response and feedback of microbial hierarchical interactions under warming scenario.

Effects of simulated nitrogen deposition on soil microbial community diversity in coastal wetland of the Yellow River Delta.

Due to the enhancement of human activities on the global scale, the total amount of atmospheric nitrogen (N) deposition and the rate keep increasing, which seriously affect the structure and function of terrestrial ecosystems. In order to study the effects of N deposition on the soil structure and function of coastal saline wetlands, we established a long-term nitrogen deposition simulation platform in 2012 in the Yellow River delta (YRD). Herein, we analyzed the composition and diversity of the soil microbial community under different N deposition treatments (LNN, MNN and HNN, which stand for 50 kg N ha-1 yr-1, 100 kg N ha-1 yr-1, and 200 kg N ha-1 yr-1) and in a water-only control (CK). The results showed that with the increasing level of N deposition, α-diversity (Shannon and Simpson indices) decreased significantly, and the composition of the microbial community changed. At the phylum level, compared with CK, the relative abundance of Chloroflexi increased significantly under the treatment of HNN (P = 0.002), but the relative abundance of Chlorobi (P = 0.013) and Verrucomicrobia (P = 0.035) decreased significantly. At the genus level, compared with CK, the relative abundance of Bacillus (P = 0.01) and Halomonas (P = 0.042) increased significantly with HNN treatment. Bacillus and Nitrococcus showed a significant correlation with soil NH4+-N. The results suggest that the response of microorganisms to N deposition treatments varied by the concentration, and the deposition of a high concentration would increase the nutrients in the soil, but reduce the diversity of soil microorganisms, causing a negative impact on the coastal wetland ecosystem of the YRD.

Elevated atmospheric CO2 reduces yield-scaled N2 O fluxes from subtropical rice systems: Six site-years field experiments.

Increasing levels of atmospheric CO2 are expected to enhance crop yields and alter soil greenhouse gas fluxes from rice paddies. While elevated CO2 ( E CO 2 ) effects on CH4 emissions from rice paddies have been studied in some detail, little is known how E CO 2 might affect N2 O fluxes or yield-scaled emissions. Here, we report on a multi-site, multi-year in-situ FACE (free-air CO2 enrichment) study, aiming to determine N2 O fluxes and crop yields from Chinese subtropical rice systems as affected by E CO 2 . In this study, we tested various N fertilization and residue addition treatments, with rice being grown under either E CO 2 (+200 µmol/mol) or ambient control. Across the six site-years, rice straw and grain yields under E CO 2 were increased by 9%-40% for treatments fertilized with ≥150 kg N/ha, while seasonal N2 O emissions were decreased by 23%-73%. Consequently, yield-scaled N2 O emissions were significantly lower under E CO 2 . For treatments receiving insufficient fertilization (≤125 kg N/ha), however, no significant E CO 2 effects on N2 O emissions were observed. The mitigating effect of E CO 2 upon N2 O emissions is closely associated with plant N uptake and a reduction of soil N availability. Nevertheless, increases in yield-scaled N2 O emissions with increasing N surplus suggests that N surplus is a useful indicator for assessing N2 O emissions from rice paddies. Our findings indicate that with rising atmospheric CO2 soil N2 O emissions from rice paddies will decrease, given that the farmers' N fertilization is usually sufficient for crop growth. The expected decrease in N2 O emissions was calculated to compensate 24% of the simultaneously observed increase in CH4 emissions under E CO 2 . This shows that for an agronomic and environmental assessment of E CO 2 effects on rice systems, not only CH4 emissions, but also N2 O fluxes and yield-scaled emissions need to be considered for identifying most climate-friendly and economically viable options for future rice production.

Establishment of evaluation criteria for the development of high quality ERα-targeted fluorescent probes.

ERα-targeted fluorescent probes are important tools for ERα study. In order to develop high quality ERα-targeted probes, a sound and complete evaluation system is essential but has not been established yet. Herein, we set up a series of evaluation criteria for ERα-targeted fluorescent probes including ERα binding affinity, fluorescence quantum yield, cytotoxicity, ERα tracking capacity, ERα selectivity and ERα labeling ability. To verify the practicability of the evaluation criteria, we designed and synthesized two ERα-targeted fluorescent probes and fully characterized their properties based on the proposed evaluation criteria. It showed that the probes exhibited better performance. Moreover, we applied the probes in MCF-7 cells to study the ERα motion characteristics for the first time. We hope that our evaluation criteria could be helpful for the establishment of a complete evaluation system for ERα-targeted fluorescent probes.

Rational design of ERα targeting hypoxia turn-on fluorescent probes with antiproliferative activity for breast cancer.

The overexpression of estrogen receptor (ER) α is not only closely related to the development of ER+ breast cancer, but is also an important biomarker for clinical diagnosis and treatment. Herein, we report several ERα targeting hypoxia turn-on fluorescent probes with antitumor activity for breast cancer cells. Among them, probes 3 and 5 displayed good ERα targeting ability and favorable hypoxia turn-on response in MCF-7 cells. Moreover, the probes 3 and 5 exhibited good antiproliferative activity towards MCF-7 cells (IC50 = 8.5 µM, 10.3 µM) and a much lower cytotoxicity to normal cells compared with the positive control. It is expected that these novel fluorescent probes may provide useful tools for the theranostics of ER+ breast cancer.

Effects of mechanical and chemical control on invasive Spartina alterniflora in the Yellow River Delta, China.

Spartina alterniflora is one of the most noxious invasive plants in China and many other regions. Exploring environmentally friendly, economic and effective techniques for controlling Spartina alterniflora is of great significance for the management of coastal wetlands. In the present study, different approaches, including mowing and waterlogging, mowing and tilling and herbicide application, were used to control Spartina alterniflora. The results suggest that the integrated approach of mowing and waterlogging could eradicate Spartina alterniflora, the herbicide haloxyfop-r-methyl could kill almost all the Spartina alterniflora, and the integrated approach of mowing and tilling at the end of the growing season was a perfect way to inhibit the germination of Spartina alterniflora in the following year. However, no matter which control approach is adopted, secondary invasion of Spartina alterniflora must be avoided. Otherwise, all the efforts will be wasted in a few years.