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Chronic stress has been reported to be associated with tumor initiation and progression. But the underlying mechanism and the specific role of tumor immunity in this process are still unknown. Herein, we applied the repeated restrain stress model in C57BL/6J mice and found that the tumor growth in stressed mice was accelerated compared with that in control mice. In addition, serotonin, also called 5-hydroxytryptamine (5-HT), in the serum of stressed mice was also elevated. Sertraline, a selective serotonin reuptake inhibitor used in the clinic, can restore the serum 5-HT level in stressed mice and restrain tumor growth. We further explored the distribution of major immune cells, including B lymphocytes cells, T lymphocytes, natural killer cells, dendritic cells, tumor-associated macrophages (TAM) and regulatory T cells (Treg). We found that the infiltration of CD8 + T cells in the tumor microenvironment (TME) decreased significantly in stressed mice. And the extra 5-HT treatment could further decrease the infiltration of CD8 + T cells in the TME. The expression of IFN-γ and Granular enzyme B (GzmB) in CD8 + T cells were also dropped in the stressed mice group, whereas the expression of programmed cell death protein 1 (PD-1) on CD8 + T cells was increased. The T cell deficiency induced by stress can be reversed by sertraline, indicating its promising role in strengthening the efficacy of anti-PDL1/PD-1 immunotherapy. The present study provides new mechanistic insights into the impact of chronic stress on antitumor immunity and implicates a novel combined immunotherapy strategy for cancer patients with chronic stress.

p38δ is a member of p38 mitogen-activated protein kinases (MAPKs) family that displays cell- and tissue-specific expression patterns. Recent studies demonstrate that p38δ is centrally involved in several pathologic events, such as diabetes, neurodegeneration diseases, inflammatory diseases, and cancer, and suggest that it may be a potential target for diagnosis and therapy of specific diseases. A nanobody is a new type of antibody that exhibits high antigen-binding activity, solubility, stability, and easy production. This study utilized phage display to isolate nanobodies specifically against p38δ from a fully synthetic nanobody library. Two of them, nanobodies Nb13-6 and Nb13-1, display high binding activity to p38δ, less cross-reactivity with other p38 MAPKs, and high thermal and pH stabilities. Modeling and docking analysis indicated that Nb13-6 is mostly linked to the activation loop of p38δ. Furthermore, detailed studies revealed that Nb13-6 inhibited the protein kinase activity of p38δ and the growth of cancer cells. Therefore, this study provides p38δ-specific nanobodies that are promisingly exploited for diagnosing and treating p38δ-associated diseases.

The synergistic effect of trametinib and dipyridamole in CRC cells.

Both the MAPK pathway and mevalonate (MVA) signaling pathway play an increasingly significant role in the carcinogenesis of colorectal carcinoma, whereas the cross-talk between these two pathways and its implication in targeted therapy remains unclear in colorectal carcinoma. Here, we identified that HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1), the rate-limiting enzyme of the MVA pathway, is overexpressed in colon cancer tissues and positively regulates the cell proliferation, migration, and invasion of colon cancer cells. In addition, HMGCS1 could enhance the activity of pERK independent of the MVA pathway, and the suppression of HMGCS1 could completely reduce the EGF-induced proliferation of colon cancer cells. Furthermore, we found that trametinib, a MEK inhibitor, could only partially abolish the upregulation of HMGCS1 induced by EGF treatment, while combination with HMGCS1 knockdown could completely reverse the upregulation of HMGCS1 induced by EGF treatment and increase the sensitivity of colon cancer cells to trametinib. Finally, we combined trametinib and dipyridamole, a common clinically used drug that could suppress the activity of SREBF2 (sterol regulatory element-binding transcription factor 2), a transcription factor regulating HMGCS1 expression, and identified its synergistic effect in inhibiting the proliferation and survival of colon cancer cells in vitro as well as the in vivo tumorigenic potential of colon cancer cells. Together, the current data indicated that HMGCS1 may be a novel biomarker, and the combination of targeting HMGCS1 and MEK might be a promising therapeutic strategy for patients with colon cancer.

Targeting ferroptosis, which provokes lipid peroxidation in cancer cells, presents potentially new avenues for anticancer therapy. Recent studies have begun to explore how immune cells in the tumor microenvironment (TME) respond and adapt to lethal lipid peroxides (LPOs). A better understanding of this process in the TME is likely to uncover another side of ferroptosis in cancer immunity and promote the development of ferroptosis-targeted therapy. This Opinion article overviews the main metabolic processes in ferroptosis, summarizes the emerging roles of ferroptosis not only in immune cells in the TME but also in the crosstalk between tumor cells and immune cells, and presents a perspective on the targeting of ferroptosis in cancer immunotherapy.

A novel protein-polysaccharide conjugate NFP-A4 was isolated and purified from water extracts of Nervilia fordii. The polysaccharide chain of NFP-A4 was found to contain D-galacturonic acid, D-gala...

Immunotherapy targeting the PD‐L1/PD‐1 pathway is a novel type of clinical cancer treatment, but only small subsets of patients can benefit from it because of multiple factors. PD‐L1/PD‐1 expression is a biomarker for predicting the efficacy of anti‐PD‐L1/PD‐1 therapy, which highlights the importance of understanding the regulatory mechanisms of PD‐L1 expression in cancer cells. Casp8 is an apical caspase protease involved in mediating cell apoptosis, but it also has multiple nonapoptotic functions. Casp8 mutations are associated with increased risks of cancer, and low expression of Casp8 is closely connected with poor prognosis in patients with cancer. In addition, mutations of Casp8 in lymphocytes also lead to human immunodeficiency, thereby causing dysfunction of the innate immune system, but the roles of Casp8 in antitumor immunity remain unclear. Here, we found that knocking down Casp8 in mouse melanoma cells promoted tumor progression in an immune system–dependent manner. Mechanistically, Casp8 induced PD‐L1 degradation by upregulating TNFAIP3 (A20) expression, a ubiquitin‐editing enzyme that results in PD‐L1 ubiquitination. In addition, compared with Casp8fl/fl mice, mice with conditional deletion of Casp8 in natural killer (NK) cells (Ncr1iCre/+Casp8fl/fl mice) showed a decreased frequency of IFN‐γ+ and CD107a+ NK cells but an increased frequency of PD‐1+ and CTLA‐4+ NK cells. Melanoma cells with Casp8 knocked down exhibited sensitivity to anti‐PD‐1 or anti‐CTLA‐4 antibody treatments, particularly in Ncr1iCre/+Casp8fl/fl mice. Together, the results indicate that Casp8 induces PD‐L1 degradation by upregulating A20 expression and that decreased Casp8 expression is a potential biomarker for predicting the sensitivity to anti‐PD‐L1/PD‐1 immunotherapy., Casp8 induces PD‐L1 degradation by upregulating A20 expression, and decreased Casp8 expression is a potential biomarker for predicting the sensitivity to anti‐PD‐L1/PD‐1 immunotherapy.

Sertraline and fluoxetine are the two most commonly used selective serotonin reuptake inhibitors (SSRIs) in the treatment of depression. Accumulating evidence has revealed that SSRIs can reduce the risk of hepatocellular carcinoma (HCC), but their therapeutic effects in HCC have not yet been elucidated. Previous studies have reported that sertraline and fluoxetine can suppress the growth of gastric carcinoma, melanoma and nonsmall cell lung cancers by inhibiting the mammalian target rapamycin (mTOR) activity. In this study, we found that sertraline and fluoxetine blocked the protein kinase B (AKT)/mTOR pathway and suppressed the growth of HCC cells in vitro, in xenografts and in diethylnitrosamine/carbon tetrachloride (DEN/CCL4)-induced primary liver mouse model. Sertraline and fluoxetine can synergize with sorafenib, the first approved standard therapy for advanced HCC, to inhibit the viability of HCC cells in vitro and in vivo. In addition, the combination of sorafenib and SSRIs synergistically inhibited the effects of the AKT/mTOR pathway. These results reveal novel therapeutic effects of a combination of SSRIs and sorafenib in HCC.

Mitomycin (MMC)/5-fluoroural (5-FU) with concurrent radiation is the standard treatment of anal squamous cell carcinoma (ASCC). The aim of this study is to evaluate the efficacy and safety of cisplatin/capecitabine (XP) as an alternative with intensity-modulated radiation therapy (IMRT) in ASCC setting.We retrospectively screened all patients with stage I-IV ASCC from January 2010 to June 2019. The records of patients who received definitive chemoradiation with cisplatin/capecitabine (XP) and IMRT were collected and analyzed.The first patient was treated with XP in 2017, so totally 11 patients were included in our study from January 2017 to June 2019. All patients have experienced clinical complete response (cCR). After a median follow-up of 30 months (range, 18-39 months), no patient had local recurrence or distant metastasis. Two-year colostomy-free survival (CFS) and two-year disease-free survival (DFS) were both 100%. The median overall survival (OS) has not reached. Grade 3 acute toxicities included leukopenia (1, 9.1%), neutropenia (2, 18.2%) and thrombocytopenia (2, 18.2%). No grade 4 acute adverse events occurred.In our study, cisplatin/capecitabine combined with IMRT was safe in ASCC patients, with favorable efficacy as an alternative, and is expected to be explored in study with larger sample.

Nearly one-fifth of patients diagnosed with gastrointestinal stromal tumors (GISTs) simultaneously experience a second primary tumor. In particular, coexistence of gastric GISTs and gastric cancer is relatively more common. However, the optimal treatment for advanced GIST with gastric cancer is largely unknown. We report a case of simultaneous occurrence of gastric GIST and gastric cancer that benefited from apatinib. After first-line imatinib and S-1 treatment for 6 months, the GIST and the gastric cancer both progressed. The patient was then treated with apatinib, exhibiting a partial response (PR) both in the GIST and the gastric cancer at 7 months, and continuous PR so far with well-controlled toxic effects of hypertension. Progression-free survival reached 10 months. In view of the relatively high incidence of advanced GIST with synchronous gastric cancer, therapy to simultaneously treat the two kinds of tumors is urgently needed. Apatinib provides promising and well-tolerated therapy for GISTs with synchronous gastric cancer refractory to chemotherapy combined with imatinib.

Protein aggregation and degradation via autophagy (aggrephagy) are major strategies adopted by cells to remove misfolded polypeptides when there is proteasome dysfunction. The functional protein complex consisting of heat shock protein 70 (Hsp70), cochaperone ubiquitin ligase carboxyl-terminal of Hsp70/Hsp90 interacting protein (CHIP), and co-chaperone Bcl-2-associated athanogene 3 (BAG3) has been associated with the activation of protein aggregation. However, data on the mechanisms of action of the complex in the protein degradation remains scant. Here, we report that upon proteasome stress, the M2 isoform of pyruvate kinase (PKM2) promotes the aggregation of ubiquitinated proteins and its knockout or knockdown aggravates the sensitivity of cells to proteasome inhibitors. Besides, following proteasome inhibition, PKM2 promotes the interaction of BAG3 with CHIP and HSP70. Interestingly, re-expression of loss-of-function mutants in PKM2-knockout cells showed that the regulatory function of PKM2 in this progress does not depend on the activity of glycolytic enzymes or protein kinases. Taken together, these findings demonstrate that PKM2 mediates the formation of the CHIP-HSP70-BAG3 protein complex and promotes the aggregation of ubiquitinated misfolded proteins, thus compensating for proteasome stress in cells.

Background: The Hippo/YAP pathway plays an important role in the development of cancers. Previous studies have reported that bile acids can activate YAP (Yes Associated Protein) to promote tumorigenesis and tumor progression. Ursodeoxycholic acid (UDCA) is a long-established old drug used for cholestasis treatment. So far, the effect of UDCA on YAP signaling in colorectal cancer (CRC) is not well defined. The study means to explore relationship of UDCA and YAP in CRC.Methods: The effect of UDCA on CRC cells proliferation was examined by MTT and clonogenic assay, western blotting (WB), real-time PCR, seperation of nucleoprotein and cytoplasmic protein and immunofluorescence (IF) assays were used to detect expression of YAP, WB, real-time PCR, GST-pull down assay and rhodamine-labeled phalloidin staining assays were used to detect activity of RhoA. After transfection of YAP5SA plasmids and RhoAV14 plasmids respectively, MTT, clonogenic assay, EdU incorporation assays and WB were performed. WB and ELISA were used to detect activity of cAMP/PKA axis, after blocking cAMP/PKA axis with siRNA and inhibitors, MTT, WB, IF and clonogenic assay were performed to test effect of UDCA on CRC cells. WB, IF and real-time PCR assays were used to detect expression of TGR5 in CRC cells after UDCA treatment, and then TGR5 agonist, TGR5 antagonist and siRNA were used to detect effect of UDCA on CRC cells. AOM/DSS-induced primary model was used to detect effect of UDCA, WB and Immunohistochemistry (IHC) were used to detect expression of related proteins.Results: UDCA suppressed YAP signaling by activating the membrane G‐protein‐coupled bile acid receptor (TGR5). TGR5 mainly regulated cAMP/PKA signaling pathway to inhibit RhoA activity, thereby suppressing YAP signaling. Moreover, the restoration of YAP expression alleviated the inhibitory effect of UDCA on CRC cell proliferation. In AOM/DSS-induced CRC model, UDCA inhibited tumor growth in a concentration-dependent manner and decreased expression of YAP and Ki67.Conclusion: UDCA plays a distinguished role in regulating YAP signaling and CRC growth from the primary bile acids and partial secondary bile acids, demonstrating the importance of maintaining normal intestinal bile acid metabolism in cancer patients. It also presents a potential therapeutic intervention for CRC.

Both the MAPK pathway and mevalonate (MVA) signaling pathway play an increasingly significant role in the carcinogenesis of colorectal carcinoma, whereas the cross-talk between these two pathways and its implication in targeted therapy remains unclear in colorectal carcinoma. Here, we identified that HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1), the rate-limiting enzyme of the MVA pathway, is overexpressed in colon cancer tissues and positively regulates the cell proliferation, migration, and invasion of colon cancer cells. In addition, HMGCS1 could enhance the activity of pERK independent of the MVA pathway, and the suppression of HMGCS1 could completely reduce the EGF-induced proliferation of colon cancer cells. Furthermore, we found that trametinib, a MEK inhibitor, could only partially abolish the upregulation of HMGCS1 induced by EGF treatment, while combination with HMGCS1 knockdown could completely reverse the upregulation of HMGCS1 induced by EGF treatment and increase the sensitivity of colon cancer cells to trametinib. Finally, we combined trametinib and dipyridamole, a common clinically used drug that could suppress the activity of SREBF2 (sterol regulatory element-binding transcription factor 2), a transcription factor regulating HMGCS1 expression, and identified its synergistic effect in inhibiting the proliferation and survival of colon cancer cells in vitro as well as the in vivo tumorigenic potential of colon cancer cells. Together, the current data indicated that HMGCS1 may be a novel biomarker, and the combination of targeting HMGCS1 and MEK might be a promising therapeutic strategy for patients with colon cancer.

Background Colon cancer is one of the most common cancers worldwide. IFN-stimulated gene 15 (ISG15), a ubiquitin-like molecule, is strongly up-regulated by type I interferon as a crucial response to a variety of microbial and cellular stress stimuli. However, the role of ISG15 in colon cancer remains unclear. Methods The expression of ISG15 in colon cancer tissues and cell lines was detected by using Western blotting and immunohistochemistry. ISG15 expression levels of colon cancer cells treated with trametinib was verified by using the data downloaded from the Gene Expression Omnibus (GEO) databases, quantitative real-time PCR analysis and Western blots assays. ISG15-siRNA was used to silence ISG15 in colon cancer cell line to determine the roles of ISG15 in colon cancer cell proliferation. Results ISG15 was highly expressed in colon cancer tissues and ISG15 upregulation was closely associated with poor prognoses in colon cancer patients. Enhanced ISG15 expression promoted the migration and proliferation of colon cancer cells in vitro, while ISG15 knockdown decreased cell proliferation and metastasis. In addition, we first found that the mRNA and protein expression of ISG15 were up-regulated following trametinib treatment. Further investigation showed that ISG15 knockdown could enhance the anti-cancer effect of trametinib in colon cancer cells. Conclusion We proposed an interesting possibility that ISG15 may be a prognostic bio-marker, and the combined targeting of ISG15 and MEK might be a promising therapeutic strategy for colon cancer.

Background Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide and has a poor prognosis. Sorafenib, the only targeted therapeutic agent for HCC, is a multiple kinase inhibitor with targets including RAF and VEGFR-2/3 that display a very limited ability to extend the survival of patients with advanced metastatic HCC for approximately three months. MEK inhibitors including trametinib and selumetinib have shown promising efficacy in combination with sorafenib in clinical trials. However, the mechanisms about the combined effect of these drugs remain unclear. Methods Two HCC cell lines (Bel7402 and SMMC7721) were used in the experiments. The protein expression of HCC cell lines was quantified via western blotting. Cell viability was examined by cell counting kit-8 and colony formation assays. Drug interactions between sorafenib and trametinib/selumetinib were determined by the combination index (CI) value. Results In this study, we found that short-term sorafenib treatment could inhibit the downstream RAF effectors phosphorylated (p)-MEK and p-ERK in Bel7402 and SMMC7721 cells, while long-term sorafenib treatment could induce a rebound of p-MEK and p-ERK expression in these two human HCC cell lines. We then tested the effect of sorafenib combined with two different FDA-approved MEK inhibitors, trametinib and selumetinib, in the two cell lines. Western blot analysis showed that trametinib/selumetinib could abolish the ERK activation caused by long-term sorafenib treatment. Cell counting kit-8 and colony formation assays indicated that the use of sorafenib or trametinib/selumetinib alone produced a minor effect on the proliferation of these HCC cell lines, while the combination therapies induced strong growth inhibition. CI assays using CompuSyn software indicated that the combined therapies could produce a synergistic effect in these two cell lines. In addition, mechanistic studies revealed that the combination therapies could synergistically reduce the expression of proliferation-related proteins, including cyclin D1 and c-Myc. Conclusions Taken together, our study showed that the rebound of p-ERK induced by long-term sorafenib treatment might limit the benefit of sorafenib monotherapy, and the MEK inhibitors trametinib and selumetinib could enhance the efficacy of sorafenib in HCC cells.

Tumours manage to survive the ablation of mutant KRAS, despite the development of KRAS-targeted drugs. Here we describe that inhibition of mutant KRAS promotes MEK nuclear localization as an alternative mechanism of KRAS-targeted drugs resistance. Tissue microarray analysis in colon tumours shows that aberrant MEK nuclear localization is closely related to YAP levels and tumour malignancy. MEK nuclear localization could sequester β-TrCP from cytoplasmic inactive YAP, then stabilizing YAP. Mutant KRAS restrains MEK within the cytoplasm via IQGAP1, inhibiting MEK nuclear translocation. Trametinib, an allosteric MEK inhibitor, could prevent MEK nuclear localization and subsequently promote YAP degradation. In vitro and in vivo results suggests that inhibition of MEK nuclear localization by trametinib synergizes with KRAS knockdown or deltarasin treatment in suppressing the viability of KRAS mutant colon cancer cells. Our study provides new insights into the mechanisms of resistance to KRAS ablation, and suggests novel strategies for the treatment of KRAS-mutant colon cancers.

Ferroptosis, a new form of programmed necrosis characterized by iron-dependent lethal accumulation of lipid hydroperoxides, is associated with many human diseases. Targeting amino acid (AA) availability can selectively suppress tumor growth and has been a promising therapeutic strategy for cancer therapy. Compelling studies have indicated that AA metabolism is also involved in ferroptosis, closely regulating its initiation and execution. This manuscript systematically summarizes the latest advances of AA metabolism in regulating ferroptosis and discusses the potential combination of therapeutic strategies that simultaneously target AA metabolism and ferroptosis in cancer to eliminate tumors or limit their invasiveness.

Serotonin (5-hydroxytryptamine, 5-HT) metabolism has long been linked to tumorigenesis and tumor progression. Numerous studies have shown the functions of 5-HT and its metabolites in the regulation of tumor biological processes like cell proliferation, invasion, metastasis, tumor angiogenesis and immunomodulatory through multi-step complex mechanisms. Reprogramming of 5-HT metabolism has been revealed in various tumors paving way for development of drugs that target enzymes, metabolites or receptors involved in 5-HT metabolic pathway. However, information on the role of 5-HT metabolism in cancer is scanty. This review briefly describes the main metabolic routes of 5-HT, the role of 5-HT metabolism in cancer and systematically summarizes the most recent advances in 5-HT metabolism-targeted cancer therapy.

Lipid homeostasis plays a fundamental role in the development of hepatocellular carcinoma (HCC). However, the mechanisms that regulate lipid homeostasis to avoid lipotoxicity in HCC remain elusive. Here, we found high-fat diet (HFD) improved the expression of sterol o-acyltransferase1 (SOAT1) and carnitine palmitoyltransferase 1A (CPT1A) in diethylnitrosamine-induced HCC. Bioinformatic analysis showed that SOAT1-mediated fatty acid storage and CPT1A-mediated fatty acids oxidation (FAO) formed a double-negative feedback loop in HCC. We verified that SOAT1 inhibition enhanced CPT1A protein, which shuttled the released fatty acids into the mitochondria for oxidation in vivo and in vitro. Besides, we further confirmed that CPT1A inhibition converted excess fatty acids into lipid drops by SOAT1 in vitro. Simultaneously targeting SOAT1 and CPT1A by the small-molecule inhibitors avasimibe and etomoxir had synergistic anticancer efficacy in HCC in vitro and in vivo. Our study provides new mechanistic insights into the regulation of lipid homeostasis and suggests the combination of avasimibe and etomoxir is a novel strategy for HCC treatment.

Background Serotonin signaling has been associated with tumorigenesis and tumor progression. Targeting the serotonin transporter to block serotonin cellular uptake confers antineoplastic effects in various tumors, including colon cancer. However, the antineoplastic mechanism of serotonin transporter inhibition and serotonin metabolism alterations in the absence of serotonin transporter have not been elucidated, especially in colon cancer, which might limit anti-tumor effects associating with targeting serotonin transporter. Methods The promotion in the uptake and catabolism of extracellular tryptophan and targeting serotonin transporter was detected by using quantitative reverse-transcription polymerase chain reaction, western blotting and liquid chromatography tandem mass spectrometry. Western blotting Immunoprecipitation and immunofluorescence was utilized to research the serotonylation of mTOR by serotonin and serotonin transporter inhibition. The primary mouse model, homograft model and tissue microarry was used to explore the tryptophan pathway in colon cancer. The cell viability assay, western blotting, xenograft and primary colon cancer mouse model were used to identify whether the combination of sertraline and tryptophan restriction had a synergistic effect. Results Targeting serotonin transporter through genetic ablation or pharmacological inhibition in vitro and in vivo induced a compensatory effect by promoting the uptake and catabolism of extracellular tryptophan in colon cancer. Mechanistically, targeting serotonin transporter suppressed mTOR serotonylation, leading to mTOR inactivation and increased tryptophan uptake. In turn, this process promoted serotonin biosynthesis and oncogenic metabolite kynurenine production through enhanced tryptophan catabolism. Tryptophan deprivation, or blocking its uptake by using trametinib, a MEK inhibitor, can sensitize colon cancer to selective serotonin reuptake inhibitors. Conclusions The present study elucidated a novel feedback mechanism involved in the regulation of serotonin homeostasis and suggested innovative strategies for selective serotonin reuptake inhibitors-based treatment of colon cancer.

Additional file 12: Table S1. Primer sequence.

Background: Pancreatic acinar cell carcinoma (PACC) is a rare tumor, accounting for about 1% of all pancreatic exocrine cancers. Consensus on the management of metastatic PACC remains unclear. Case presentation: Starting from April 2019, a patient first received chemotherapy with two cycles of gemcitabine and nab-paclitaxel and two cycles of SOX regimen. After progression of disease evaluated based on RECIST 1.1, toripalimab and SOX regimen was administered because of PD-L1–positive expression, high tumor mutation burden (TMB), and somatic FANCA deletion in the tumor. Both the primary and metastatic tumor mass shrank significantly after two courses. The patient exhibited sustained partial response for at least six courses with well-controlled toxic effects. Then the treatment had to be stopped for 2 months because of the coronavirus disease 2019 pandemic. Computed tomography scan in March 2020 showed disease progression. Time from initiating treatment to tumor progression on toripalimab and SOX regimen treatment took up to at least 8 months. Conclusions: We present the first case report where a PD-L1 positive, high TMB, and FANCA-deleted pancreatic acinar cell carcinoma was treated using chemotherapy combined with immunotherapy, in which the patient exhibited satisfactory response and tolerance.

Viburnum taitoense Hayata has been used as folk medicine by the minority people in Southwestern China for a long history, especially in Guangxi Zhuang Autonomous Region. The minority in Guangxi including Zhuang, Miao and Yao people use the ethanol extract of V. taitoense Hayata to treat the fracture, kill the pain of rheumatism because of its definite therapeutic effects.So far, the scientific investigation of V. taitoense Hayata is done very little. Here, we first prepared the ethyl acetate extract of V. taitoense (EEVt), secondly measured the contents of phenols, flavonoids, and terpenoids in EEVt, and thirdly, the anti-inflammatory and analgesic activities of EEVt were investigated by invitro model of RAW 264.7 cells and invivo models of inflammation and pain in rats and mice.The contents of phenols, flavonoids, and terpenoids in EEVt were determined by UV spectrophotometry, respectively. The anti-inflammatory effect of EEVt (5, 25, 50, 100, and 200 μg/mL) in vitro was tested by determining its inhibitory effect on the nitric oxide production of RAW264.7 cells activated by lipopolysaccharide (LPS). The anti-inflammatory and analgesic effects of EEVt in vivo were investigated in the following experimental rats and mice models: carrageenan-induced paw edema, corton-oil-induced ear edema, acetic acid writhing test, and formalin pain test.The contents of total phenolic, total flavonoids, and total triterpenoids in V. taitoense were measured to be 3.46 ± 0.04%, 2.38 ± 0.04%, and 14.96 ± 0.17%, respectively. In vitro test showed that EEVt at different tested dosages (5, 25, 50, 100, and 200 μg/mL) had no significant toxicity to RAW264.7 macrophages. At dosages of 37.5 and 75 μg/mL of EEVt significant inhibitory (p 0.001) on the productions of nitric oxide (NO). High dosage (200 μg/mL) of EEVt displayed highly significant inhibitory (p 0.001) on the productions of proinflammatory cytokines IL-6, IL-1β, and TNF-α from the LPS-induced RAW264.7 macrophages. EEVt showed obvious anti-inflammatory activity at different time points after carrageenan injection (p 0.05) in vivo test, and its anti-inflammatory activity reached the strongest 4 h. Similarly, through the ear swelling test, EEVt (200 mg/kg) showed significant (p 0.05) anti-inflammatory activity. Besides, formalin and acetic acid writhing experiments also showed that EEVt has significant (p 0.05) analgesic activity.EEVt was confirmed to be definite anti-inflammatory and analgesic effects, and the phytochemicals of EEVt was disclosed to be rich in triterpenoids, which was worthy to be further investigated.

Cholesterol and its metabolites (precursors and derivatives) play an important role in cancer. In recent years, numerous studies have reported the functions of cholesterol metabolism in the regulation of tumor biological processes, especially oncogenic signaling pathways, ferroptosis, and tumor microenvironment. Preclinical studies have over the years indicated the inhibitory effects of blocking cholesterol synthesis and uptake on tumor formation and growth. Besides, some new cholesterol metabolic molecules such as SOAT1, SQLE, and NPC1 have recently emerged as promising drug targets for cancer treatment. Here, we systematically review the roles of cholesterol and its metabolites, and the latest advances in cancer therapy targeting cholesterol metabolism.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Many studies have shown a significant increase in the marker signal of the receptor for advanced glycation end-products (RAGE) with the malignant progression of tumor growth, metastasis and recurrence of breast cancer, including TNBC of primary tumors and lymph node metastases. Azeliragon is a RAGE inhibitor and it has been shown to actively inhibit the TNBC cell line, SUM149 (IC50 = 5.292 ± 0.310 μM). In order to develop a new anti-TNBC agent, we designed, synthesized and screened 26 Azeliragon triazole analogues to determine their anti-TNBC activities in vitro. The most active compound was KC-10 with an IC50 value of 0.220 ± 0.034 μM.

Five previously undescribed lanostane-type triterpenoids, including two triterpenoids with a rearranged side chain (applanoic acids E and F), one C21 nortriterpenoid (16,17-dehydroapplanone E), as well as two highly oxygenated lanostane triterpenoids (methyl applaniate B and applanoic acid G), were isolated from the fruiting bodies of Ganoderma applanatum (Pers.) Pat. Their structures were elucidated on the basis of spectroscopic analysis, X-ray crystallography and ECD data. Applanoic acid E, 16,17-dehydroapplanone E, and methyl applaniate B showed inhibitory effects on the release of NO by LPS-induced BV-2 cells.

Background: 5-hydroxytryptamine (5-HT, serotonin), a well-known neurotransmitter, plays a vital role in the physiological and pathological regulation of the central nervous system and gastrointestinal tract. Recently, emerging evidences indicate that 5-HT also plays an important role in tumorigenesis, but little is known about its role in colorectal cancer. Methods: By using western blot, immunofluorescent staining, GST pulldown assay, gene interference and LC-MS/MS assays, we investigated the effect and mechanism of 5-HT on the malignancies of the two colon cell lines (SW480 and SW1116 cells) in vitro. We then evaluated the role of 5-HT in tumorigenesis in vivo and the clinical significance of two pivotal proteins of 5-HT signal in human colon cancer tissues. Findings: 5-HT could be transported into cells via SERT, enhancing YAP expression and promoting colon cancer cell growth in vitro and in vivo. Further study revealed that intracellular 5-HT transported by SERT serotonylated and activated RhoA under the catalysation of TG2, subsequently through activating the RhoA-ROCK1/2 signalling to upregulate YAP expression in SW480 and SW1116 cells. Blocking SERT with citalopram reversed the 5-HT-induced YAP expression and cell proliferation, and inhibited the effects of 5-HT on tumour formation in mice. Moreover, SERT expression was correlated with YAP in pathological human colorectal cancer samples. Interpretation: Our findings suggested that 5-HT could enter cells via SERT transportation to activate the RhoA/ROCK/YAP signalling to promote the carcinogenesis of colon cancer. Thus, targeting 5-HT-SERT-YAP axis may be a potential therapeutic strategy for colon cancer. Funding Statement: This work was supported by grants from the National Key R&D Program of China(No. 2016YFC1303200/2016YFC1303203), the National Natural Science Foundation of China(No. 81872020, 81621003). Declaration of Interests: The authors declare no conflict of interest associated with this manuscript. Ethics Approval Statement: The animal studies were carried out in accordance to guidelines established by the State Key Laboratory of Biotherapy Institutional Animal Experimental Ethics Committee (approval no. 20160017)

Background: Both the mitogen-activated protein kinase pathway and mevalonate signaling pathway play a increasingly significant role in the carcinogenesis of colorectal carcinoma (CRC). whereas, the crosstalk between these two pathways and its implication in targeted therapy remains unclear in CRC. Methods: The expression of HMGCS1 in colon cancer tissues and cell lines was detected by using western blotting and immunohistochemistry. A cell viability assay, clonogenic assay, western blot assay were employed to study the interaction between si-RNA of HMGCS1 and EGF treatment. Xenograft studies were used to identify whether the combination of trametinib and dipyridamole had a synergistic effect. Findings: We identified that HMGCS1 is overexpressed in colon cancer tissues and positively regulates the cell proliferation, migration and invasion of colon cancer cells. In addition, HMGCS1 could enhance the activity of pERK independent of the MVA pathway, and the suppression of HMGCS1 could completely reduce the EGF-induced proliferation of colon cancer cells. Furthermore, we found that trametinib could only partially abolish the upregulation of HMGCS1 induced by EGF treatment, while combination with HMGCS1 knockdown could completely reverse the upregulation of HMGCS1 induced by EGF treatment and increase the sensitivity of colon cancer cells to trametinib. Finally, we found that dipyridamole could enhance the cytotoxicities of the trametinib against colon cancer cells in vitro and in vivo. Interpretation: The present data indicated that HMGCS1 may be a novel biomarker, and the combination of targeting HMGCS1 and MEK might be a promising therapeutic strategy for colon cancer patients. Funding Statement: This study was supported by the National Natural Science Foundation of China (81572731). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The animals were handled according to the guidelines of the China Animal Welfare Legislation, as provided by the Committee on Ethics in the Care and Use of Laboratory Animals of Sichuan University West China Hospital. The human colon cancer tissues as provided by the Ethics Committee of Taizhou Hospital of Zhejiang Province.

The aim of this article is to study the regulatory feedback loop between β-catenin and IQ motif containing GTPase activating protein 1 (IQGAP1), as well as the effect of this regulation loop in colon cancer cell proliferation. Western blot was used to detect the expression of IQGAP1 and β-catenin after changing their expression respectively by transfection in SW1116 cells. CCK-8 cell proliferation assay was used to detect the effect of IQGAP1 involved in the proliferation of SW1116 cells promoted by β-catenin. The results of Western blot indicated that β-catenin could positively regulate IQGAP1, while IQGAP1 silencing could up-regulate β-catenin, forming a negative feedback loop. The results of CCK-8 showed that IQGAP1 silencing inhibited β-catenin-mediated proliferation in SW1116 cells. In conclusion, our research reveals a negative regulatory feedback loop between β-catenin and IQGAP1 which has a remarkable effect on the proliferation ability of colon cancer cells.本文主要研究 β 连环蛋白（β-catenin）和含 IQ 基序的 GTP 激酶活化蛋白 1（IQGAP1）的相互调控，及其在肠癌细胞增殖中的功能。利用细胞转染技术改变肠癌细胞 SW1116 中的 β-catenin 或者 IQGAP1 表达后，通过蛋白质印迹法分别检测 SW1116 细胞中 IQGAP1 和 β-catenin 的表达情况。利用 CCK-8 细胞增殖实验检测干扰下调 IQGAP1 对 β-catenin 促进 SW1116 细胞增殖的影响。结果发现在 SW1116 细胞中干扰 β-catenin 可以下调 IQGAP1 表达，过表达 β-catenin 可以上调 IQGAP1 并加强有丝分裂原激活蛋白激酶（MAPK）通路；干扰 IQGAP1 在 SW1116 细胞中反而引起 β-catenin 上升；同时 CCK-8 细胞增殖实验发现干扰 IQGAP1 减弱了 β-catenin 的促肠癌细胞增殖作用。本研究表明 β-catenin 与 IQGAP1 形成负向反馈调控环路，在调控肠癌细胞的增殖中发挥着重要功能。.

Figure S5. a HMGCR is higher in carcinomatous tissues. HMGCR were identified and confirmed by Western blot analysis in 12 pairs of colon cancer tissues. (F:distal normal tissues;C:colon cancer tissues). (PDF 946 kb)

Figure S4. Antitumour effect of the combination of trametinib and simvastatin. a The CI and Fa of the HCT116 and SW480 cells treated with DMSO or 10 μM simvastatin (or/and 50 nM trametinib) for 48 h by CompuSyn software. b The tumours were analysed for proliferation (c-Myc, cyclin D1) and proapoptotic (Bax) proteins by an immunoblot assay. GAPDH was used as the protein loading control. (PDF 947 kb)

Figure S6. Quantitative RT–PCR analysis for interactions between drugs. a Quantitative real-time PCR analysis of ERRα, IDH3A and CytC in the SW480 cells treated with shNC or shERRα#2 (or/and 20 ng/μl EGF) for 2 d. b QPCR for ERRα, IDH3A and CytC in the SW480 cells treated with DMSO or trametinib (10 nM) (or/and 20 ng/μl EGF) for 2 d. c SW480 cells cultured with DMSO or 50 nM trametinib (or/and shERRα#2) at day 2 by qPCR assay. d Quantitative real-time PCR analysis of ERRα, IDH3A and CytC in the SW480 cells treated with 10 μM simvastatin (or/and 50 nM trametinib) for 2d. (PDF 949 kb)

Figure S3. Simvastatin decreases the transcriptional activity of ERRα in colon cancer cells. a Cell proliferation assays at day 3 for the SW1116 cells cultured with simvastatin (5 μM and 10 μM) using the Cell Counting Kit-8. b Clonogenic assays and qualitative analysis of the SW1116 cells cultured with DMSO or 5 μM simvastatin (or/and 20 ng/μl EGF) at day 7. (PDF 947 kb)

Figure S2. Suppression of ERRα completely reduces the EGF treatment-induced cell proliferation and enhances the cytotoxicity of trametinib. a WB for ERRα, c-Myc, cyclin D1, pERK and ERK in the SW1116 cells treated with EGF (20/μl) at the indicated times (0.5, 2, 4, 6 and 8 h) in serum-free medium. b CCK-8 assay for the HCT116 and SW480 cells cultured with si-NC or si-ERRα (or/and 20 ng/μl EGF) for 3 d (* P

Acetylcholine (ACh), known as a neurotransmitter, regulates the functions of numerous fundamental central and peripheral nervous system. Recently, emerging evidences indicate that ACh also plays an important role in tumorigenesis. However, little is known about the role of ACh in gastric cancer. Here, we reported that ACh could be auto-synthesized and released from MKN45 and BGC823 gastric cancer cells. Exogenous ACh promoted cell proliferation in a does-dependent manner. The M3R antagonist 4-DAMP, but not M1R antagonist trihexyphenidyl and M2/4 R antagonist AFDX-116, could reverse the ACh-induced cell proliferation. Moreover, ACh, via M3R, activated the EGFR signaling to induce the phosphorylation of ERK1/2 and AKT, and blocking EGFR pathway by specific inhibitor AG1478 suppressed the ACh induced cell proliferation. Furthermore, the M3R antagonist 4-DAMP and darifenacin could markedly inhibit gastric tumor formation in vivo. 4-DAMP could also significantly enhance the cytotoxic activity of 5-Fu against the MKN45 and BGC823 cells, and induce the expression of apoptosis-related proteins such as Bax and Caspase-3. Together, these findings indicated that the autocrine ACh could act through M3R and the EGFR signaling to promote gastric cancer cells proliferation, targeting M3R or EGFR may provide us a potential therapeutic strategy for gastric cancer treatment.