TRIM24-Mediated Acetylation of STAT6 Suppresses Th2-Induced Allergic Rhinitis.

PURPOSE: Allergic rhinitis (AR) is a T helper type 2 (Th2)-mediated inflammatory disease. The E3 ligase tripartite motif-containing 24 (TRIM24) regulates the recruitment of acetyltransferase CREB-binding protein (CBP) to signal transducer and activator of transcription 6 (STAT6). CBP mediates the acetylation of STAT6 and decreases its activity. To date, the precise role of TRIM24 in AR has not been fully interpreted. Herein, our study aimed to explore the functions of TRIM24 in AR. METHODS: The expression of TRIM24 in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from patients with AR was measured. TRIM24-conditional knockout mice with TRIM24 deficiency in CD4+ T cells were generated. Wide-type (WT) AR mice and TRIM24-conditional knockout AR mice were established. Then, AR symptoms and interleukin (IL)-4 levels were compared. Further, the proliferation, activation and polarization of CD4+ T cells from WT mice and TRIM24 knockout mice after stimulation were determined. The effects of TRIM24 deficiency on STAT6 activities were also evaluated. RESULTS: Downregulated TRIM24 expression was detected in PBMCs and CD4+ T cells from patients with AR. TRIM24 conditional knockout mice had more sever AR symptoms with elevated IL-4 production. TRIM24-knockout CD4+ T cells had similar proliferation and activation when compared to WT CD4+ T cells, while they had enhanced Th2 polarization. TRIM24-knockout CD4+ T cells had decreased acetylation of STAT6 and enhanced STAT6 activities after IL-4 stimulation. The regulation of STAT6 activities by TRIM24 depended on TRIM24 N terminal RIGN domain and Lys383 acetylation site of STAT6. CONCLUSIONS: TRIM24 suppresses Th2-mediated AR by regulating the acetylation of STAT6.

Preliminary dynamic observation of wound healing after low-temperature plasma radiofrequency ablation for laryngeal leukoplakia.

BACKGROUND: Low-temperature plasma ablation (LTPA) is an emerging technique for laryngeal leukoplakia (LL). OBJECTIVE: To initially observe the healing process of trauma after LTPA for LL. MATERIALS AND METHODS: Seventeen patients who underwent LTPA for LL were collected, and the degrees of wound healing were analyzed. RESULTS: Only 1 patient in who dysbiosis of the pharyngeal flora was induced by self-administered hormone nebulization treatment during the follow-up period. In the remaining patients, the wound healing was characterized by a crater-shaped defect on the vocal folds surface with pseudo-membranes, congestion, and mild edema on postoperative day 1. These symptoms became worse on postoperative day 7. On postoperative day 15, the pseudo-membrane was fully formed and some patients had granulomatous swelling of the vocal cords. These symptoms became better and better on postoperative day 30 and day 45. On postoperative day 60, the mucosa of the vocal folds had essentially returned to normal. On postoperative day 90, the vocal folds morphology and function had recovered well. CONCLUSION: It takes 2-3 months for the wound to heal completely after LTPA for LL. SIGNIFICANCE: A proper understanding of the wound healing process can reduce unnecessary surgical and pharmacologic interventions and avoid excessive treatment.

High mobility group box 1 inhibition by BoxA attenuates ovalbumin-induced allergic rhinitis in mice.

This study was designed to evaluate the effects of BoxA on allergic rhinitis (AR). Ovalbumin (OVA)-induced AR mice model was employed and BoxA was administered to AR mice. AR symptoms, levels of cytokines and chemokines, and the expression of high mobility group box 1 (HMGB1), TLR2, and TLR4 were measured. BoxA treatment significantly ameliorated AR symptoms, decreased level of histamine, OVA-specific antibodies, suppressed the infiltration of immune cells in nasal tissues, inhibited the expression of IL-4, IL-6, IL-5, TNF-α, IL-13, IL-17, IL-2 while promoting the expression of IL-10, suppressed the expression of HMGB1, TLR2, and TLR4 in AR mice. BoxA ameliorated allergic rhinitis in mice by inhibiting HMGB1.

Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein.

The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.

The Rho family GTPases are crucial drivers of tumor growth and metastasis. However, it is difficult to develop GTPases inhibitors due to a lack of well-characterized binding pockets for compounds. Here, through molecular dynamics simulation of the RhoA protein, a groove around cysteine 107 (Cys107) that is relatively well-conserved within the Rho family is discovered. Using a combined strategy, the novel inhibitor DC-Rhoin is discovered, which disrupts interaction of Rho proteins with guanine nucleotide exchange factors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs). Crystallographic studies reveal that the covalent binding of DC-Rhoin to the Cys107 residue stabilizes and captures a novel allosteric pocket. Moreover, the derivative compound DC-Rhoin04 inhibits the migration and invasion of cancer cells, through targeting this allosteric pocket of RhoA. The study reveals a novel allosteric regulatory site within the Rho family, which can be exploited for anti-metastasis drug development, and also provides a novel strategy for inhibitor discovery toward "undruggable" protein targets.

Low-temperature radiofrequency coblation reduces treatment interval and post-operative pain of laryngotracheal recurrent respiratory papillomatosis.

Laryngeal papillomatosis is a benign disease in the larynx but with the potential to develop into significant complications as a result of its high recurrence rate. CO2 laser and radiofrequency controlled ablation (coblation) have been used to treat recurrent respiratory papillomatosis, but detailed comparisons of their respective treatment outcomes are not fully investigated. This retrospective study examines the procedure time, time interval between interventions, blood loss during operation, post-operative complications and pain scores among patients who received either CO2 laser or radiofrequency coblation interventions for laryngotracheal recurrent respiratory papillomatosis. Compared with CO2 laser intervention, radiofrequency coblation significantly reduced operation time, time interval between interventions, blood loss during operation and number of times bipolar electrocoagulation needed in each procedure. Post-operatively, pain scores after radiofrequency coblation were significantly lower than those after CO2 laser intervention. Incidence rates of post-operative complications, in terms of palate pharyngeal mucosa damage, bleeding and subcutaneous emphysema, were also significantly reduced after radiofrequency coblation. Low-temperature radiofrequency coblation is a superior intervention compared with CO2 laser against laryngotracheal recurrent respiratory papillomatosis.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 µM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 µM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

Long Noncoding RNA Linc00632 Inhibits Interleukin-13-Induced Inflammatory Cytokine and Mucus Production in Nasal Epithelial Cells.

Allergic rhinitis (AR) is an allergic disease characterized as (immunoglobulin E)-mediated type I hypersensitivity disorder. The interleukin-13 (IL-13) signaling pathway has been implicated in the pathogenesis of AR. In the present study, we investigated the regulatory role and mechanism of long noncoding RNA Linc00632 in IL-13-induced inflammatory cytokine and mucus production in nasal epithelial cells (NECs) from AR patients. We evaluated the expression of Linc00632 in nasal tissues from AR patients and in IL-13-treated NECs. We explored the role of Linc00632 in granulocyte-macrophage colony-stimulating factor (GM-CSF), eotaxin, and MUAC5AC production in IL-13-treated NECs. We searched for the potential target of Linc00632. Downregulation of Linc00632 was identified in nasal tissues of AR patients and in IL-13-treated NECs. Linc00632 inhibited IL-13-induced GM-CSF, eotaxin, and MUAC5AC production. Linc00632 targeted miR-498 and negatively regulated its expression. MiR-498 targeted IL1RN and inhibition of miR-498 suppressed IL-13-induced GM-CSF, eotaxin, and MUC5AC expression. The regulation of IL-13-induced dysfunction of NECs by Linc00632 depended on miR-498. Linc00632 inhibited IL-13-induced GM-CSF, eotaxin, and MUAC5AC production in IL-13-treated NECs by targeting miR-498.

Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors.

Transcriptional enhancer associated domain family members (TEADs) are the most important downstream effectors that play the pivotal role in the development, regeneration and tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo autopalmitoylation that is indispensable for its function making the lipid-binding pocket an attractive target for chemical intervention. Herein, through structure-based virtual screen and rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent, selective, covalent TEAD autopalmitoylation inhibitor with the IC50 value of 197 ±â€¯19 nM while it showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family, lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression. Taken together, our findings proved the validity of modulating transcriptional output in the Hippo signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in the future.

Discovery of trisubstituted nicotinonitrile derivatives as novel human GCN5 inhibitors through AlphaScreen-based high throughput screening.

The general control nonrepressed protein 5 (GCN5) is an important target for drug design and drug discovery largely owing to its pathogenic role in malignancies. Chemical probes that target GCN5 have been developed in recent decades, but their potencies are still unsatisfactory. In this study, through an in-house developed AlphaScreen-based high throughput screening platform, radioactive acetylation assays and 2D-similarity based analogue searching, we discovered DC_HG24-01 as the novel hGCN5 inhibitor with the IC50 value of 3.1 ± 0.2 µM. Further docking studies suggested that DC_HG24-01 could occupy the binding pocket of acetyl-CoA cofactor, which laid the foundation for the development of more potent hGCN5 inhibitors in the future. At the cellular level, DC_HG24-01 could retard cell proliferation and block the acetylation of H3K14 leading to cell apoptosis and cell cycle arrest at the G1 phase in MV4-11 cell lines. Taken together, the discovery of DC_HG24-01 may serve as a good starting point to accelerate the development of more potent hGCN5 inhibitors through further structural decoration and provide new insight into the pharmacological treatment of leukemia.

Discovery and biological evaluation of thiobarbituric derivatives as potent p300/CBP inhibitors.

Histone acetyltransferases (HATs) relieve transcriptional repression by preferentially acetylation of ε-amino group of lysine residues on histones. Dysregulation of HATs is strongly correlated with etiology of several diseases especially cancer, thus highlighting the utmost significance of the development of small molecule inhibitors against this potential therapeutic target. In the present study, through virtual screening and iterative optimization, we identified DCH36_06 as a bona fide, potent p300/CBP inhibitor. DCH36_06 mediated p300/CBP inhibition leading to hypoacetylation on H3K18 in leukemic cells. The suppression of p300/CBP activity retarded cell proliferation in several leukemic cell lines. In addition, DCH36_06 arrested cell cycle at G1 phase and induced apoptosis via activation of capase3, caspase9 and PARP that elucidated the molecular mechanism of its anti-proliferation activity. In transcriptome analysis, DCH36_06 altered downstream gene expression and apoptotic pathways-related genes verified by real-time PCR. Importantly, DCH36_06 blocked the leukemic xenograft growth in mice supporting its potential for in vivo use that underlies the therapeutic potential for p300/CBP inhibitors in clinical translation. Taken together, our findings suggest that DCH36_06 may serve as a qualified chemical tool to decode the acetylome code and open up new opportunities for clinical intervention.

Identification of novel inhibitors of histone acetyltransferase hMOF through high throughput screening.

The histone acetyltransferases (HATs) in mammals include GCN5 N-acetyltransferases, the MOZ, YBF2, SAS2, and TIP60 proteins, and the orphan HATs. The males absent on the first (MOF) is mainly related to acetylation of histone H4 Lys16 and has influence on downstream genes expression. However, the only inhibitor MG149 presented low activity against MOF. Besides, there was no high throughput screening platform on MOF, which limited the inhibitor discovery and functional study. In our study, we set up a high throughput screening platform based on amplified luminescent proximity homogeneous assay (ALPHA), which led us to a moderate inhibitor DC_M01. By chemical modification, we found DC_M01_7, which was the analog of DC_M01 with an IC50 value of 6 µM. DC_M01_7 significantly inhibited HCT116 cells proliferation and could also inhibit histone 4 lysine 16 acetylation in HCT116 cells. To sum up, our work will probably assist the further development of more potent MOF inhibitors and the functional study of hMOF.

Identification of small molecule inhibitors targeting the SMARCA2 bromodomain from a high-throughput screening assay.

SMARCA2 is a critical catalytic subunit of the switch/sucrose non-fermenting (SWI/SNF) chromatin remodeling complexes. Dysregulation of SMARCA2 is associated with several diseases, including some cancers. SMARCA2 is multi-domain protein containing a bromodomain (BRD) that specifically recognizes acetylated lysine residues in histone tails, thus playing an important role in chromatin remodeling. Many potent and specific inhibitors targeting other BRDs have recently been discovered and have been widely used for cancer treatments and biological research. However, hit discovery targeting SMARCA2-BRD is particularly lacking. To date, there is a paucity of reported high-throughput screening (HTS) assays targeting the SMARCA2-BRD interface. In this study, we developed an AlphaScreen HTS system for the discovery of SMARCA2-BRD inhibitors and optimized the physicochemical conditions including pH, salt concentrations and detergent levels. Through an established AlphaScreen-based high-throughput screening assay against an in-house compound library, DCSM06 was identified as a novel SMARCA2-BRD inhibitor with an IC50 value of 39.9±3.0 µmol/L. Surface plasmon resonance demonstrated the binding between SMARCA2-BRD and DCSM06 (Kd=38.6 µmol/L). A similarity-based analog search led to identification of DCSM06-05 with an IC50 value of 9.0±1.4 µmol/L. Molecular docking was performed to predict the binding mode of DCSM06-05 and to decipher the structural basis of the infiuence of chemical modifications on inhibitor potency. DCSM06-05 may be used as a starting point for further medicinal chemistry optimization and could function as a chemical tool for SMARCA2-related functional studies.

Discovery of novel BET inhibitors by drug repurposing of nitroxoline and its analogues.

The BET family of bromodomain-containing proteins (BRDs) is believed to be a promising drug target for therapeutic intervention in a number of diseases including cancer, inflammation and cardiovascular diseases. Hence, there is a great demand for novel chemotypes of BET inhibitors. The drug repurposing strategy offers great benefits to find inhibitors with known safety and pharmacokinetic profiles, thus increasing medicinal chemists' interest in recent years. Using the drug repurposing strategy, a BRD4-specific score based virtual screening campaign on an in-house drug library was conducted followed by the ALPHA screen assay test. Nitroxoline, an FDA-approved antibiotic, was identified to effectively disrupt the interaction between the first bromodomain of BRD4 (bromodomain-containing protein 4) and acetylated H4 peptide with IC50 of 0.98 µM. Nitroxoline inhibited all BET family members with good selectivity against non-BET bromodomain-containing proteins, thus it is defined as a selective BET inhibitor. Based on the crystal structure of the nitroxoline-BRD4_BD1 complex, the mechanism of action as well as BET specificity of nitroxoline were determined. Since the anticancer activity of nitroxoline against MLL leukemia, one of the BET related diseases, has not been studied before, we tested whether nitroxoline might serve as a potential repurposing drug candidate for MLL leukemia. Nitroxoline effectively inhibited the proliferation of MLL leukemia cells by inducing cell cycle arrest and apoptosis. The profound efficacy is, at least in part, due to the inhibition of BET and downregulation of target gene transcription. Our discovery of nitroxoline as a BET inhibitor suggests potential application of nitroxoline and its derivatives for clinical translation in BET family related diseases.

Discovery of novel trimethoxy-ring BRD4 bromodomain inhibitors: AlphaScreen assay, crystallography and cell-based assay.

As a member of the bromodomain and extra terminal domain (BET) protein family, BRD4 is closely related to cancers and other diseases. Small-molecule BRD4 inhibitors have already demonstrated promising potential for the therapy of BRD4-related cancers. In this study, we report the discovery and evaluation of a novel category of BRD4 inhibitors, which share a trimethoxy ring and target the first bromodomain of the human BRD4 protein. The IC50 value of the most potent compound, DC-BD-03, is 2.01 µM. In addition, a high-resolution crystal structure of the compound DC-BD-29 with the first bromodomain of BRD4 was determined, which revealed the binding mode and facilitated further structure-based optimization. These compounds exhibited anti-proliferation activity, caused cell cycle arrest, and induced apoptosis in human leukemia MV4-11 cells. Thus, the results presented in this study indicated the potential of this series of compounds as drug candidates for the therapy of BRD4-related cancers.

Identification of novel small-molecule inhibitors targeting menin-MLL interaction, repurposing the antidiarrheal loperamide.

Leukemia with a mixed lineage leukemia (MLL) rearrangement, which harbors a variety of MLL fusion proteins, has a poor prognosis despite the latest improved treatment options. Menin has been reported to be a required cofactor for the leukemogenic activity of MLL fusion proteins. Thus, the disruption of the protein-protein interactions between menin and MLL represents a very promising strategy for curing MLL leukemia. Making use of menin-MLL inhibitors with a shape-based scaffold hopping approach, we have discovered that the antidiarrheal loperamide displays previously unreported mild inhibition for the menin-MLL interaction (IC50 = 69 ± 3 µM). In an effort to repurpose this drug, a series of chemical modification analyses was performed, and three of the loperamide-based analogues, DC_YM21, DC_YM25 and DC_YM26 displayed better activities with IC50 values of 0.83 ± 0.13 µM, 0.69 ± 0.07 µM and 0.66 ± 0.05 µM, respectively. Further treatment with DC_YM21 demonstrated potent and selective blockage of proliferation and induction of both cell cycle arrest and differentiation of leukemia cells harboring MLL translocations, which confirmed the specific mechanism of action. In conclusion, molecules of a novel scaffold targeting menin-MLL interactions were reported and they may serve as new potential therapeutic agents for MLL leukemia.

Discovery of Novel Inhibitors Targeting the Menin-Mixed Lineage Leukemia Interface Using Pharmacophore- and Docking-Based Virtual Screening.

Disrupting the interaction between mixed lineage leukemia (MLL) fusion protein and menin provides a therapeutic approach for MLL-mediated leukemia. Here, we aim to discover novel inhibitors targeting the menin-MLL interface with virtual screening. Both structure-based molecular docking and ligand-based pharmacophore models were established, and the models used for compound screening show a remarkable ability to retrieve known active ligands from decoy molecules. Verified by a fluorescence polarization assay, five hits with novel scaffolds were identified. Among them, DCZ_M123 exhibited potent inhibitory activity with an IC50 of 4.71 ± 0.12 µM and a KD of 14.70 ± 2.13 µM, and it can effectively inhibit the human MLL-rearranged leukemia cells MV4;11 and KOPN8 with GI50 values of 0.84 µM and 0.54 µM, respectively.

Functional interplay between the RK motif and linker segment dictates Oct4-DNA recognition.

The POU family transcription factor Oct4 plays pivotal roles in regulating pluripotency and somatic cell reprogramming. Previous studies have indicated an important role for major groove contacts in Oct4-DNA recognition; however, the contributions of the RK motif in the POUh domain and the linker segment joining the two DNA-binding domains remain poorly understood. Here, by combining molecular modelling and functional assays, we find that the RK motif is essential for Oct4-DNA association by recognizing the narrowed DNA minor groove. Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4. These findings uncover a self-regulatory mechanism for specific Oct4-DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family. Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine.

Discovery of two aminoglycoside antibiotics as inhibitors targeting the menin-mixed lineage leukaemia interface.

Menin functions as an oncogenic cofactor of mixed lineage leukaemia (MLL) fusion proteins in leukaemogenesis. The menin-MLL interface is a potential therapeutic target in acute leukaemia cases. In this study, approximately 900 clinical compounds were evaluated and ranked using pharmacophore-based virtual screening, the top 29 hits were further evaluated by biochemical analysis to discover the inhibitors that target the menin-MLL interface. Two aminoglycoside antibiotics, neomycin and tobramycin, were identified as menin-MLL inhibitors with binding affinities of 18.8 and 59.9 µM, respectively. The results of thermal shift assay validated the direct interactions between the two antibiotics and menin. The results of isothermal titration calorimetry showed that the equilibrium dissociation constant between menin and neomycin was approximately 15.6 µM. We also predicted the binding modes of inhibitors at the menin-MLL interface through molecular docking analysis. The results indicated that neomycin and tobramycin competitively occupy the binding site of MLL. This study has shed light on the development of powerful probes and new therapies for MLL-mediated leukaemogenesis.

[Aggressive fibromatosis of the head and neck (A report of two cases and literature review)].

OBJECTIVE: To evaluate the clinic manifestation, pathologic behavior, therapy and prognosis of rare aggressive fibromatosis in the head and neck. METHOD: Two cases of aggressive fibromatosis were analyzed and relevant literatures were reviewed. RESULT: Aggressive fibromatosis was characterized as infiltrative, locally aggressive and tended to recur after surgical resection. Pathology showed fibroblastic monoclonal proliferation. Fibromatosis was composed of well-differentiated fibroblasts and myofibroblasts, lacking cytological features of malignancy and scanty or absent mitotic activity. Complete surgical excision of aggressive fibromatosis was considered to be the only effective method of cure by most authorities. Chemotherapy and radiotherapy can be used together with surgery in recurrence or unsatisfactory surgical margin. In our study, one patient recurred after the first operation, and after another operation, the patient did not recur after 6 months follow up, and the other one did not recur after 6 months follow up. CONCLUSION: The diagnosis of aggressive fibromatosis depended on pathological examination. Radical removal was an important way to reduce recurrence rate. Radiation therapy and chemotherapy can be used as adjuvant therapy in patients with recurrent or unresectable or inoperable disease.