Astragaloside IV inhibits colorectal cancer metastasis by reducing extracellular vesicles release and suppressing M2-type TAMs activation.

Ethnopharmacological relevance: Tumour-derived extracellular vesicles (TEVs) have been confirmed to facilitate colorectal cancer (CRC) metastasis by remodelling the tumour microenvironment (TME). Drugs targeted TEVs is considered as a promising therapeutic strategy for cancer treatment. Traditional Chinese medicine (TCM) plays a vital role in improving the prognosis of CRC patients and eventually CRC patients with distant metastasis. Although the anti-tumour effects of active compounds from TCM prescriptions are observed widely, the molecular mechanisms remain unknown. Aim of the study: This study aims to investigate the effects of active compounds in our library of TCM on preventing CRC metastasis, and also explore the potential mechanisms from the perspective of TEVs. Materials and methods: The effects of active compounds on the proliferation of CRC cells were determined by CCK-8 assay. TEVs were extracted from MC38 cells by ultracentrifugation and characterized by electron microscopy, Nanosight NS300 and western blotting. The TEV particles were quantified by Nanosight NS300. The potential mechanism by which astragaloside IV (ASIV) reduced TEV secretion was determined by western blotting. RAW264.7 cells were cocultured with the conditioned medium (CM) of MC38 cells treated with or without ASIV, and the activation of tumour-associated macrophages (TAMs) was assessed by immunofluorescence and quantitative polymerase chain reaction (qPCR). The migration of CRC cells was measured by wound healing and Transwell assay. A spleen-to-liver metastasis model of colorectal cancer was used to confirm the efficiency of ASIV in vivo. Liver metastatic tumours of the mice were used for liver weight measures and H&E staining. Immunofluorescence was applied to observe the infiltration of TAMs, the expression of neutral sphingomyelinase 2 (nSMase2) and Rab27a. Results: By screening our TCM monomer library, we found that ASIV, which is mainly extracted from Radix Astragali, reduced the release of TEVs from CRC cells in a time- and concentration-dependent manner. Mechanistically, ASIV inhibited the production and secretion of TEVs by downregulating nSMase2 and Rab27a expression in CRC cells. CM from ASIV-treated CRC cells reshaped the polarization of TAMs by decreasing M2-type polarization, increasing M1-type polarization. Consequently, the repolarization of M2-type to M1-type macrophages led to reduced invasion and migration of CRC cells. Moreover, we confirmed that ASIV inhibited the liver metastasis of CRC, reduced M2-type macrophage infiltration and decreased the expression of nSMase2 and Rab27a in liver metastases. Conclusions: ASIV inhibited CRC metastasis by reducing EVs release and suppressing M2-type TAMs activation. All these findings reveal a new insight into the mechanisms of ASIV in preventing CRC progression and provide a promising approach for anti-tumour therapy.

RSPO3 induced by Helicobacter pylori extracts promotes gastric cancer stem cell properties through the GNG7/ß-catenin signaling pathway.

BACKGROUND: Helicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)-like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored. METHODS: Spheroid-formation experiments were performed to explore the self-renewal capacity of GC cells. The expression of R-spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter-4 (OCT-4), SRY-box transcription factor 2 (SOX-2), CD44, Akt, glycogen synthase kinase-3ß (GSK-3ß), p-Akt, p-GSK-3ß, ß-catenin, and G protein subunit gamma 7 (GNG7) were detected by RT-qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co-immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus-based RNA interference constructed short hairpin (sh)-RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The in vivo mechanism was verified using a tumor peritoneal seeding model in nude mice. RESULTS: H. pylori extracts promoted a CSC-like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC-like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/ß-catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3-induced activation of ß-catenin. CoIP and LC-MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK-3ß, and the expression of ß-catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC-like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding in vivo. IHC staining also showed that RSPO3, CD44, OCT-4, and SOX-2 were elevated in H. pylori GC tissues. CONCLUSION: RSPO3 enhanced the stemness of H. pylori extracts-infected GC cells through the GNG7/ß-catenin signaling pathway.

Kaempferol inhibits colorectal cancer metastasis through circ_0000345 mediated JMJD2C/ß-catenin signalling pathway.

BACKGROUND: Recurrence and metastasis are the main causes of disease deterioration in colorectal cancer (CRC) patients, yet efficient therapeutic strategies are lacking. Natural compounds for efficient antitumour therapeutics are becoming increasingly prominent. Kaempferol, one of the main components of flavonoids in plants, displays a variety of pharmacological activities. Our preliminary experiments suggested that kaempferol could inhibit CRC metastasis and is significantly associated with the ß-catenin signalling pathway. Moreover, we also defined the regulatory roles of JMJD2C in ß-catenin signalling in our previous work. PURPOSE: This study aims to reveal the mechanism by which kaempferol inhibits CRC progression and regulates the JMJD2C/ß-catenin signalling pathway. METHODS: The migratory capabilities of CRC cells after kaempferol intervention were measured by scratch wound healing and transwell assays. Circ_0000345 knockdown CRC stable cell lines were generated by lentivirus infection. The possible mechanism of kaempferol on circ_0000345 was verified by molecular-protein docking and verification program cellular thermal shift assay (CETSA). A dual luciferase reporter gene assay was carried out for the targeting relationship among circ_0000345, miR-205-5p and JMJD2C. Fluorescence in situ hybridization (FISH) was performed to determine the expression of circ_0000345 in tumour tissues. A pulmonary metastatic model of CRC in vitro was built to assess the antimetastatic effect and mechanism of kaempferol in vivo. RESULTS: In vitro, kaempferol inhibits the ability to migrate of CRC cells by reducing the activation of the JMJD2C/ß-catenin signalling pathway. MiR-205-5p is a key bridge for kaempferol to inhibit the expression of JMJD2C. The function of miR-205-5p is impeded by circ_0000345, which shows higher expression levels in human metastatic CRC tissues than nonmetastatic CRC tissues, and its formation is regulated by the RNA-binding proteins HNRNPK and HNRNPL. Mechanistically, kaempferol physically interacts with HNRNPK and HNRNPL to suppress JMJD2C by downregulating the expression of circ_0000345. In vivo, kaempferol suppresses CRC lung metastasis. Kaempferol inhibits the activation of JMJD2C/ß-catenin signalling through reducing the expression of circ_0000345 in the CRC lung metastasis model. CONCLUSION: Circ_0000345 enhances activation of the JMJD2C/ß-catenin signalling pathway through miR-205-5p to promote CRC metastasis. Kaempferol inhibits CRC metastasis through the circ_0000345-mediated JMJD2C/ß-catenin signalling pathway, and this effect is influenced as a direct consequence of the binding of kaempferol with HNRNPK and HNRNPL. This provides promising therapeutic and/or adjuvant agents for advanced CRC and sheds light on the multifaceted role of phytomedicine in cancer.

Circulating extracellular vesicle-derived MARCKSL1 is a potential diagnostic non-invasive biomarker in metastatic colorectal cancer patients.

Extracellular vesicle-derived proteins are closely related to colorectal cancer metastasis, and early detection and diagnosis of colorectal cancer metastasis is very important to improve the prognosis. In this study, we evaluated the clinical significance of plasma EV-derived MARCKSL1 in differentiating patients with metastatic and nonmetastatic CRC. This study included 78 patients, including 40 patients with nonmetastatic colorectal cancer, 38 patients with metastatic colorectal cancer, and 15 healthy volunteers. The extracellular vesicles extracted from the participants' plasma were characterized through transmission electron microscopy, nanoparticle tracking analysis and western blotting. MARCKSL1 protein expression in the EVs was detected by ELISA, and the diagnostic efficacy of MARCKSL1 alone or in combination with CA125 and lymphocyte levels was evaluated by receiver operating characteristic curve (ROC) analysis. Pearson's correlation test was performed to detect the correlation between MARCKSL1, CA125, lymphocyte level and clinicopathological characteristics of tumors. The present study demonstrated that the level of circulating EV-derived MARCKSL1 in patients with metastatic colorectal cancer was significantly higher than that in patients with nonmetastatic colorectal cancer and healthy people. Combined with CA125 and lymphocyte levels, the best diagnostic effect was achieved, and the area under the ROC curve was 0.7480. Together, our findings indicated that circulating EV-derived MARCKSL1 could be used as a new potential diagnostic biomarker for metastatic CRC.

Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway.

Background: Oxaliplatin (L-OHP) is a common chemotherapy drug used in the treatment of colorectal cancer (CRC). Our previous work showed that Zuo Jin Wan (ZJW), a traditional Chinese medicine prescription, could improve sensitivity to L-OHP in the treatment of CRC, but the detailed mechanism is not clear. In previous mechanistic studies, we found that the miR-200s expression in CRC is associated with L-OHP sensitivity through regulation of MDR1/p-gp and the downstream c-JunN-terminal kinase (JNK) signaling pathway. Moreover, lncRNA-MALAT1 offers great potential in the regulation of drug resistance by interacting with miR-200s. Therefore, in this work, we explored whether ZJW could reverse L-OHP resistance in CRC by regulating MALAT1, miR-200s, and the downstream signaling pathway. Methods: Cell Counting Kit-8 and flow cytometry were used to detect the effects of ZJW combined with L-OHP on chemotherapy tolerance and cell apoptosis of HCT116/L-OHP cells. Western blotting and quantitative real-time PCR (qRT-PCR) were used to detect the activation of the JNK signaling pathway and the protein and mRNA expression levels of the drug resistance-related MDR1/ABCB1 gene in HCT116/L-OHP cells treated with ZJW. The binding sites of MALAT1 and miR-200s were predicted by bioinformatics tools and confirmed by qRT-PCR. qRT-PCR was used to detect the expression of miR-200s and MALAT1 in HCT116/L-OHP cells treated with ZJW. A xenograft model of CRC in nude mice was established to observe the effect of ZJW combined with L-OHP on the growth of subcutaneously transplanted tumors. Apoptosis in tumor cells was detected by TUNEL staining. The activation of the JNK signaling pathway and the expression of drug resistance-related proteins were detected by immunohistochemistry and immunofluorescence. qRT-PCR was used to detect the expression of miR-200s and the MALAT1 gene in the tumors. Results: Our study showed that ZJW could significantly decrease the proliferation and promote apoptosis of HCT116/L-OHP cells treated with L-OHP. We further proved that ZJW could reverse the drug resistance of HCT116/L-OHP cells by reducing MALAT1, indirectly upregulating miR-200s, alleviating the activation of the JNK signaling axis, and downregulating the expression of resistance proteins such as MDR1/ABCB1 and ABCG2. ZJW combined with L-OHP inhibited the growth of subcutaneously transplanted tumors and induced apoptosis in nude mice. ZJW reduced the expression of MALAT1 and upregulated the expression of miR-200s in transplanted tumors. In addition, ZJW also alleviated the activation of the JNK signaling pathway while reducing the expression of MDR1/ABCB1 and ABCG2. Conclusions: Our study identified that MALAT1 promotes colorectal cancer resistance to oxaliplatin by reducing the miR-200s expression. ZJW may reverse chemoresistance by inhibiting the expression of MALAT1 and regulating the miR-200s/JNK pathway, providing an experimental basis for the clinical application of ZJW in relieving chemotherapy resistance.

Tumor-Associated Macrophages Regulate PD-1/PD-L1 Immunosuppression.

Anti-programmed cell death 1 (PD-1) or anti-PD-ligand (L) 1 drugs, as classic immune checkpoint inhibitors, are considered promising treatment strategies for tumors. In clinical practice, some cancer patients experience drug resistance and disease progression in the process of anti-PD-1/PD-L1 immunotherapy. Tumor-associated macrophages (TAMs) play key roles in regulating PD-1/PD-L1 immunosuppression by inhibiting the recruitment and function of T cells through cytokines, superficial immune checkpoint ligands, and exosomes. There are several therapies available to recover the anticancer efficacy of PD-1/PD-L1 inhibitors by targeting TAMs, including the inhibition of TAM differentiation and re-education of TAM activation. In this review, we will summarize the roles and mechanisms of TAMs in PD-1/PD-L1 blocker resistance. Furthermore, we will discuss the therapies that were designed to deplete TAMs, re-educate TAMs, and intervene with chemokines secreted by TAMs and exosomes from M1 macrophages, providing more potential options to improve the efficacy of PD-1/PD-L1 inhibitors.

Jianpi Jiedu Recipe inhibits colorectal cancer liver metastasis via regulating ITGBL1-rich extracellular vesicles mediated activation of cancer-associated fibroblasts.

BACKGROUND: Extracellular vesicles (EVs) contribute greatly to the formation of pre-metastatic niche and tumor metastasis. Our previous study has revealed that tumor-derived ITGBL1 (integrin beta- like 1)-rich EVs activate fibroblasts through the NF-κB signaling to promote colorectal cancer (CRC) metastasis. Targeting ITGBL1-loaded EVs may be a new and effective therapy for treating CRC metastasis. Simultaneously, our preliminary clinical trial has demonstrated that Jianpi Jiedu Recipe (JPJDR) was an ideal alternative traditional Chinese medicine for the prevention and treatment of CRC metastasis. However, the underlying mechanism of JPJDR in the prevention of CRC metastasis is not clear. In this study, we will investigate the regulatory effect of JPJDR on ITGBL1 levels in CRC-derived EVs, and to detect how JPJDR regulate ITGBL1-rich EVs mediated activation of fibroblasts to inhibit CRC metastasis. METHODS: EVs derived from CRC cells with/without JPJDR treatment were obtained by ultracentrifugation, following by characterization with electron microscopy, LM10 nanoparticle characterization system and western blot. The migration and growth of CRC cells were tested by transwell assay, wound healing assay and colony formation assay. The effect of JPJDR on the fibroblasts-activation associated inflammatory factors including IL-6, IL-8 and α-SMA was detected by real-time PCR. The levels of IL-6, IL-8 and α-SMA in the cell culture supernatant were detected by ELISA. The protein expressions of TNFAIP3, ITGBL1, p-NF-κB, IκBα and ß-actin were detected by western blot. Liver metastasis model in mice was established by injecting MC38 single cell suspension into the spleen of mice to observe the effect of JPJDR on CRC liver metastasis. Immunohistochemistry were applied to detect the expression of ITGBL1 and TNFAIP3 in the liver metastatic tissues. Tissue immunofluorescence detection was performed to observe the regulatory effect of JPJDR on the ITGBL1-NF-κB signaling pathway. Cancer-associated fibroblasts (CAFs) in the liver metastatic tissues were sorted and characterized by platelet-derived growth factor receptor ß (PDGFRß) with flow cytometry, following by the detection of inflammatory factors including IL-6, IL-8 and α-SMA using real-time PCR. RESULTS: JPJDR reduced the ITGBL1 levels in CRC cells-derived EVs. JPJDR inhibited the migration and growth of CRC cells via regulating ITGBL1-rich EVs mediated fibroblasts activity. Mechanically, JPJDR decreased fibroblasts activation by regulating ITGBL1-rich EVs mediated TNFAIP3-NF-κB signaling. Further in vivo experiments demonstrated that JPJDR reduced CRC liver metastasis by regulating ITGBL1-rich EVs secretion from CRC and blocked the fibroblasts activation by regulating ITGBL1-TNFAIP3- NF-κB signaling. CONCLUSION: Our research demonstrated that JPJDR preventd CRC liver metastasis via down-regulating CRC-derived ITGBL1-loaded EVs mediated activation of CAFs, providing the experimental evidence for the clinical application of JPJDR in the prevention and treatment of CRC metastasis. More importantly, our study confirmed the great benefits of therapeutic targeting the EVs-mediated metastasis and warranted future clinical validation.