Compound Z526 alleviates chemotherapy-induced cachectic muscle loss by ameliorating oxidative stress-driven protein metabolic imbalance and apoptosis.

Chemotherapy is one of the primary and indispensable intervention against cancers though it is always accompanied by severe side effects especially cachexia. Cachexia is a fatal metabolic disorder syndrome, mainly characterized by muscle loss. Oxidative stress is the key factor that trigger cachectic muscle loss by inducing imbalance in protein metabolism and apoptosis. Here, we showed an oral compound (Z526) exhibited potent alleviating effects on C2C12 myotube atrophy induced by various chemotherapeutic agents in vitro as well as mice muscle loss and impaired grip force induced by oxaliplatin in vivo. Furthermore, Z526 also could ameliorate C2C12 myotube atrophy induced by the combination of chemotherapeutic agents with conditioned medium of various tumor cells in vitro as well as mice muscle atrophy of C26 tumor-bearing mice treated with oxaliplatin. The pharmacological effects of Z526 were based on its potency in reducing oxidative stress in cachectic myocytes and muscle tissues, which inhibited the activation of NF-κB and STAT3 to decrease Atrogin-1-mediated protein degradation, activated the AKT/mTOR signaling pathway to promote protein synthesis, regulated Bcl-2/BAX ratio to reduce Caspase-3-triggered apoptosis. Our work suggested Z526 to be an optional strategy for ameliorating cachexia muscle atrophy in the multimodality treatment of cancers.

Paeoniflorin alleviated muscle atrophy in cancer cachexia through inhibiting TLR4/NF-κB signaling and activating AKT/mTOR signaling.

Cancer cachexia is a progressive wasting syndrome, which is mainly characterized by systemic inflammatory response, weight loss, muscle atrophy, and fat loss. Paeoniflorin (Pae) is a natural compound extracted from the dried root of Paeonia lactiflora Pallas, which is featured in anti-inflammatory, antioxidant, and immunoregulatory pharmacological activities. While, the effects of Pae on cancer cachexia had not been reported before. In the present study, the effects of Pae on muscle atrophy in cancer cachexia were observed both in vitro and in vivo using C2C12 myotube atrophy cell model and C26 tumor-bearing cancer cachexia mice model. In the in vitro study, Pae could alleviate myotubes atrophy induced by conditioned medium of C26 colon cancer cells or LLC Lewis lung cancer cells by decreasing the expression of Atrogin-1 and inhibited the decrease of MHC and MyoD. In the in vivo study, Pae ameliorated weight loss and improved the decrease in cross-sectional area of muscle fibers and the impairment of muscle function in C26 tumor-bearing mice. The inhibition of TLR4/NF-κB pathway and the activation of AKT/mTOR pathway was observed both in C2C12 myotubes and C26 tumor-bearing mice treated by Pae, which might be the main basis of its ameliorating effects on muscle atrophy. In addition, Pae could inhibit the release of IL-6 from C26 tumor cells, which might also contribute to its ameliorating effects on muscle atrophy. Overall, Pae might be a promising candidate for the therapy of cancer cachexia.

Carnosol analogue WK-63 alleviated cancer cachexia by inhibiting NF-κB and activating AKT pathways in muscle while inhibiting NF-κB and AMPK pathways in adipocyte.

Cancer cachexia is a systemic metabolic disorder syndrome characterized by severe wasting of muscle and adipose tissues while is lack of effective therapeutic approaches. Carnosol (CS) was found in our previous study to exhibit ameliorating effects on cancer cachexia. In the present study, we designed and synthesized 49 CS analogues by structural modification of CS. Results of activity screening revealed that, among the analogues, WK-63 exhibited better effects than CS in ameliorating atrophy of C2C12 myotubes induced by conditioned medium of C26 tumor cells. WK-63 could also dose-dependently alleviate adipocyte lipolysis of mature 3 T3-L1 cells induced by C26 tumor cell conditioned medium. WK-63 alleviated myotube atrophy by inhibiting Nuclear Factor kappa-B (NF-κB) and activating the Protein Kinase B (AKT) signaling pathway, and also alleviated fat loss by inhibiting NF-κB and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways. Results of pharmacokinetic (PK) assay showed that, compared with other analogues, WK-63 exhibited longer half-life (T1/2) and mean residence time (MRTs), as well as a larger concentration curve area (AUC0-t). These findings suggested that WK-63 might exert optimal effects in vivo. In the C26 tumor-bearing mice model, administration of WK-63 ameliorated the body weight loss and also improved the weight loss of epididymal adipose tissue. WK-63 is expected to be a novel therapeutic option for the treatment of cancer cachexia.

Corylifol A ameliorates muscle atrophy by inhibiting TAOK1/p38-MAPK/FoxO3 pathway in cancer cachexia.

BACKGROUND: Corylifol A (CYA) is one of the main active components of Psoralea corylifolia L. CYA had been reported to have ameliorating effects on dexamethasone-induced atrophy of C2C12 mouse skeletal myotubes, but its effects on cancer cachexia were unclear. Here, we checked the influence of CYA on muscle atrophy in cancer cachexia mice and tried to clarify its mechanisms. METHODS: C26 tumour-bearing mice were applied as the animal model to examine the effects of CYA in attenuating cachexia symptoms. The in vitro cell models of TNF-α-induced C2C12 myotubes or ad-mRFP-GFP-LC3B-transfected C2C12 myotubes were used to check the influence of CYA on myotube atrophy based on both ubiquitin proteasome system (UPS) and autophagy-lysosome system. The possible direct targets of CYA were searched using the biotin-streptavidin pull-down assay and then confirmed using the Microscale thermophoresis binding assay. The levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting and immunocytochemical assay. RESULTS: The administration of CYA prevented body weight loss and muscle wasting in C26 tumour-bearing mice without affecting tumour growth. At the end of the experiment, the body weight of mice treated with 30 mg/kg of CYA (23.59 ± 0.94 g) was significantly higher than that of the C26 model group (21.66 ± 0.56 g) with P < 0.05. The values of gastrocnemius muscle weight/body weight of mice treated with 15 or 30 mg/kg CYA (0.53 ± 0.02% and 0.54 ± 0.01%, respectively) were both significantly higher than that of the C26 model group (0.45 ± 0.01%) with P < 0.01. CYA decreased both UPS-mediated protein degradation and autophagy in muscle tissues of C26 tumour-bearing mice as well as in C2C12 myotubes treated with TNF-α. The thousand-and-one amino acid kinase 1 (TAOK1) was found to be the direct binding target of CYA. CYA inhibited the activation of TAOK1 and its downstream p38-MAPK pathway thus decreased the level and nuclear location of FoxO3. siRNA knockdown of TAOK1 or regulation of the p38-MAPK pathway using activator or inhibitor could affect the ameliorating effects of CYA on myotube atrophy. CONCLUSIONS: CYA ameliorates cancer cachexia muscle atrophy by decreasing both UPS degradation and autophagy. The ameliorating effects of CYA on muscle atrophy might be based on its binding with TAOK1 and inhibiting the TAOK1/p38-MAPK/FoxO3 pathway.

Atractylenolide I ameliorates cancer cachexia through inhibiting biogenesis of IL-6 and tumour-derived extracellular vesicles.

BACKGROUND: Atractylenolide I (AI) is a natural sesquiterpene lactone isolated from Atractylodes macrocephala Koidz, known as Baizhu in traditional Chinese medicine. AI has been found to ameliorate cancer cachexia in clinic cancer patients and in tumour-bearing mice. Here, we checked the influence of AI on biogenesis of IL-6 and extracellular vesicles (EVs) in cancer cachexia mice and then focused on studying mechanisms of AI in inhibiting the production of tumour-derived EVs, which contribute to the ameliorating effects of AI on cancer cachexia. METHODS: C26 tumour-bearing BALB/c mice were applied as animal model to examine the effects of AI (25 mg/kg) in attenuating cachexia symptoms, serum IL-6 and EVs levels. IL-6 and EVs secretion of C26 tumour cells treated with AI (0.31-5 µM) was further observed in vitro. The in vitro cultured C2C12 myotubes and 3T3-L1 mature adipocytes were used to check the potency of conditioned medium of C26 cells treated with AI (0.625-5 µM) in inducing muscle atrophy and lipolysis. The glycolysis potency of C26 cells under AI (0.31-5 µM) treatment was evaluated by measuring the extracellular acidification rate using Seahorse XFe96 Analyser. Levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting to study the possible mechanisms. STAT3 overexpression or knockout C26 cells were also used to confirm the effects of AI (5 µM). RESULTS: AI ameliorated cancer cachexia symptoms (P < 0.05), improved grip strength (P < 0.05) and decreased serum EVs (P < 0.05) and IL-6 (P < 0.05) levels of C26 tumour-bearing mice. AI directly inhibited EVs biogenesis (P < 0.001) and IL-6 secretion (P < 0.01) of cultured C26 cells. The potency of C26 medium in inducing C2C12 myotube atrophy (+59.54%, P < 0.001) and 3T3-L1 adipocyte lipolysis (+20.73%, P < 0.05) was significantly attenuated when C26 cells were treated with AI. AI treatment inhibited aerobic glycolysis and the pathway of STAT3/PKM2/SNAP23 in C26 cells. Furthermore, overexpression of STAT3 partly antagonized the effects of AI in suppressing STAT3/PKM2/SNAP23 pathway, EVs secretion, glycolysis and the potency of C26 medium in inducing muscle atrophy and lipolysis, whereas knockout of STAT3 enhanced the inhibitory effect of AI on these values. The inhibition of AI on STAT3/PKM2/SNAP23 pathway was also observed in C26 tumour tissues. CONCLUSIONS: AI ameliorates cancer cachexia by decreasing the production of IL-6 and EVs of tumour cells. The decreasing effects of AI on EVs biogenesis are based on its inhibition on STAT3/PKM2/SNAP23 pathway.

GDF-15 in tumor-derived exosomes promotes muscle atrophy via Bcl-2/caspase-3 pathway.

Tumor-derived exosomes are emerging mediators of cancer cachexia, a kind of multifactorial syndrome characterized by serious loss of skeletal muscle mass and function. Our previous study had showed that microRNAs in exosomes of C26 colon tumor cells were involved in induction of muscle atrophy. Here, we focus on studying proteins in tumor-derived exosomes which might also contribute to the development of cancer cachexia. Results of comparing the protein profiles of cachexic C26 exosomes and non-cachexic MC38 exosomes suggested that growth differentiation factor 15 (GDF-15) was rich in C26 exosomes. Western blotting analysis confirmed the higher levels of GDF-15 in C26 cells and C26 exosomes, compared with that of MC38 cells. Results of animal study also showed that GDF-15 was rich in tumor tissues, serum exosomes, and gastrocnemius (GA) muscle tissues of C26 tumor-bearing mice. GDF-15 protein could directly induce muscle atrophy of cultured C2C12 myotubes via regulating Bcl-2/caspase-3 pathways. What's more, overexpression of GDF-15 in MC38 cells could increase the potency of MC38 conditioned medium or exosomes in inducing muscle atrophy. Knockdown of GDF-15 in C26 cells decreased the potency of C26 conditioned medium or exosomes in inducing muscle atrophy. These results suggested that GDF-15 in tumor-derived exosomes could contribute to induction of muscle atrophy and also supported the possibility of targeting GDF-15 in treatment of cancer cachexia.

The critical role of STAT3 in biogenesis of tumor-derived exosomes with potency of inducing cancer cachexia in vitro and in vivo.

Tumor-derived exosomes are emerging mediators of cancer cachexia. Clarifying the regulation of exosome biogenesis and finding possible targets for cancer cachexia therapy are important and necessary. In the present study, systemic analysis of the roles of STAT3 in controlling exosome biogenesis of murine C26 colon tumor cells and its contribution to the development of cancer cachexia is conducted. The genetic manipulation of STAT3 expression, STAT3 knockout (KO) or overexpression (OE), significantly affected the exosome biogenesis and also the potency of C26 conditioned medium (CM) in inducing muscle atrophy and lipolysis in vitro. The genetic manipulation of STAT3 expression caused change in phosphorylation of PKM2 and glycolysis. PKM2/SNAP23 pathway was involved in regulation of exosome biogenesis by STAT3 genetic manipulation as well as by STAT3 inhibitors in C26 cells. Mice inoculated with STAT3 knockout or overexpression C26 cells exhibited ameliorated or aggravated cancer cachexia symptoms, with a positive correlation with the serum exosome and IL-6 levels. The STAT3/PKM2/SNAP23 pathway was affected in C26 tumor tissues with genetic manipulation of STAT3 expression. The capacity of exosome biogenesis of different human cancer cells also exhibited a positive correlation with the activation of STAT3/PKM2/SNAP23 pathway. The research presented here confirms that STAT3 plays a critical role in regulating biogenesis of tumor-derived exosomes which could contribute to cancer cachexia development.

Bile acid metabolism dysregulation associates with cancer cachexia: roles of liver and gut microbiome.

BACKGROUND: Cancer cachexia is a multifactorial metabolic syndrome in which bile acid (BA) metabolism might be involved. The aim of the present study was to clarify the contribution of liver and gut microbiota to BA metabolism disturbance in cancer cachexia and to check the possibility of targeting BA metabolism using agents such as tauroursodeoxycholic acid (TUDCA) for cancer cachexia therapy. METHODS: The BA profiles in liver, intestine, and serum of mice with cancer cachexia induced by inoculation of colon C26 tumour cells were analysed using metabolomics methods and compared with that of control mice. Proteomic analysis of liver protein expression profile and 16S rRNA gene sequencing analysis of gut microbiota composition in cancer cachexia mice were conducted. Expression levels of genes related to farnesoid X receptor (FXR) signalling pathway in the intestine and liver tissues were analysed using RT-PCR analysis. The BA profiles in serum of clinical colon cancer patients with or without cachexia were also analysed and compared with that of healthy volunteers. The effects of TUDCA in treating cancer cachexia mice were observed. RESULTS: In the liver of cancer cachexia mice, expression of BA synthesis enzymes was inhibited while the amount of total BAs increased (P < 0.05). The ratios of conjugated BAs/un-conjugated BAs significantly increased in cancer cachexia mice liver (P < 0.01). Gut microbiota dysbiosis such as decrease in Lachnospiraceae and increase in Enterobacteriaceae was observed in the intestine of cancer cachexia mice, and microbial metabolism of BAs was reduced. Increase in expression of FGF15 in intestine (P < 0.01) suggested the activation of FXR signalling pathway which might contribute to the regulation of BA synthesis enzymes, transporters, and metabolic enzymes. Increase in the BA conjugation was observed in the serum of cancer cachexia mice. Results of clinical patients showed changes in BA metabolism, especially the increase in BA conjugation, and also suggested compensatory mechanism in BA metabolism regulation. Oral administration of 50 mg/kg TUDCA could significantly ameliorate the decrease in body weight (P < 0.001), muscle loss (P < 0.001), and atrophy of heart and liver (P < 0.05) in cancer cachexia mice without influence on tumour growth. CONCLUSIONS: Bile acid metabolism dysregulation such as decrease in BA synthesis, increase in BA conjugation, and decrease in BA microbial metabolism was involved in development of cancer cachexia in mice. Targeting BA metabolism using agents such as TUDCA might be helpful for cancer cachexia therapy.

miR-92b-3p Regulates Cell Cycle and Apoptosis by Targeting CDKN1C, Thereby Affecting the Sensitivity of Colorectal Cancer Cells to Chemotherapeutic Drugs.

Colorectal cancer (CRC) is the third most common malignant tumor in the world and the second leading cause of cancer death. Multidrug resistance (MDR) has become a major obstacle in the clinical treatment of CRC. The clear molecular mechanism of MDR is complex, and miRNAs play an important role in drug resistance. This study used small RNAomic screens to analyze the expression profiles of miRNAs in CRC HCT8 cell line and its chemoresistant counterpart HCT8/T cell line. It was found that miR-92b-3p was highly expressed in HCT8/T cells. Knockdown of miR-92b-3p reversed the resistance of MDR HCT8/T cells to chemotherapeutic drugs in vitro and in vivo. Paclitaxel (PTX, a chemotherapy medication) could stimulate CRC cells to up-regulate miR-92b-3p expression and conferred cellular resistance to chemotherapeutic drugs. In studies on downstream molecules, results suggested that miR-92b-3p directly targeted Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, which encodes a cell cycle inhibitor p57Kip2) to inhibit its expression and regulate the sensitivity of CRC cells to chemotherapeutic drugs. Mechanism study revealed that the miR-92b-3p/CDKN1C axis exerted a regulatory effect on the sensitivity of CRC cells via the regulation of cell cycle and apoptosis. In conclusion, these findings showed that miR-92b-3p/CDKN1C was an important regulator in the development of drug resistance in CRC cells, suggesting its potential application in drug resistance prediction and treatment.

Cancer-derived exosome miRNAs induce skeletal muscle wasting by Bcl-2-mediated apoptosis in colon cancer cachexia.

Cancer cachexia is a kind of whole-body metabolic disorder syndrome accompanied by severe wasting of muscle tissue in which cancer exosomes may be involved. Analysis of clinical samples showed that the serum exosome concentrations were correlated with the development of cancer cachexia. Exosomes secreted by C26 cells could decrease the diameter of C2C12 myotubes in vitro and decrease mouse muscle strength and tibialis anterior (TA) muscle weight in vivo. GW4869, an inhibitor of exosome excretion, ameliorated muscle wasting in C26 tumor-bearing mice. MicroRNA (miRNA) sequencing (miRNA-seq) analysis suggested that miR-195a-5p and miR-125b-1-3p were richer in C26 exosomes than in exosomes secreted from MC38 cells (non-cachexic). Both miR-195a-5p and miR-125b-1-3p mimics could induce atrophy of C2C12 myoblasts. Downregulation of Bcl-2 and activation of the apoptotic signaling pathway were observed in C2C12 myoblasts transfected with miR-195a-5p and miR-125b-1-3p mimics, in the gastrocnemius muscle of C26 tumor-bearing mice and in the TA muscle injected with C26 exosomes. Results of dual-luciferase assay confirmed the targeting of miR-195a-5p/miR-125b-1-3p to Bcl-2. Overexpression of Bcl-2 successfully reversed atrophy of C2C12 myoblasts induced by the two miRNA mimics. These results suggested that cancer exosome enriched miRNAs might induce muscle atrophy by targeting Bcl-2-mediated apoptosis.

Autophagy inhibition and microRNA­199a­5p upregulation in paclitaxel­resistant A549/T lung cancer cells.

Multidrug resistance (MDR) is one of the major reasons for the clinical failure of cancer chemotherapy. Autophagy activation serves a crucial role in MDR. However, the specific molecular mechanism linking autophagy with MDR remains unknown. The results of the present study demonstrated that autophagy was inhibited and microRNA (miR)­199a­5p levels were upregulated in MDR model lung cancer cells (A549/T and H1299/T) compared with those in the parental cell lines. Paclitaxel (PTX) treatment increased the expression levels of miR­199a­5p in parental lung cancer cells compared with those in PTX­untreated cells, and these expression levels were negatively correlated with PTX sensitivity of the cells. miR­199a­5p knockdown in A549/T cells induced autophagy and resensitized cells to multiple chemotherapeutic drugs including PTX, taxotere, topotecan, SN38, oxaliplatin and vinorelbine. By contrast, miR­199a­5p overexpression in A549 cells suppressed autophagy and desensitized cells to these chemotherapeutic drugs. Mechanistically, the results of the present study demonstrated that miR­199a­5p blocked autophagy by activating the PI3K/Akt/mTOR signaling pathway and inhibiting the protein expression of autophagy­related 5. Furthermore, p62 protein was identified as a direct target of miR­199a­5p; miR­199a­5p bound to p62 mRNA to decrease its mRNA and protein expression levels. In conclusion, the results of the present study suggested that miR­199a­5p may contribute to MDR development in lung cancer cells by inhibiting autophagy and targeting p62. The regulatory effect of miR­199a­5p on autophagy may provide novel insights for future multidrug­resistant lung cancer chemotherapy.

Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by cancer cachexia.

BACKGROUND: Cancer cachexia is a multifactorial debilitating syndrome that directly accounts for more than 20% of cancer deaths while there is no effective therapeutic approach for treatment of cancer cachexia. Carnosol (CS) is a bioactive diterpene compound present in Lamiaceae spp., which has been demonstrated to have antioxidant, anti-inflammatory, and anticancer properties. But its effects on cancer cachexia and the possible mechanism remain a mystery. METHODS: The in vitro cell models of C2C12 myotube atrophy and 3T3-L1 mature adipocyte lipolysis were used to check the activities of CS and its synthesized analogues. C26 tumour-bearing BALB/c mice were applied as the animal model to examine their therapeutic effects on cancer cachexia in vivo. Levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting to study the possible mechanisms. RESULTS: Carnosol and its analogues [dimethyl-carnosol (DCS) and dimethyl-carnosol-D6 (DCSD)] alleviated myotube atrophy of C2C12 myotubes and lipolysis of 3T3-L1 adipocytes in vitro. Interestingly, CS and its analogues exhibited stronger inhibitive effects on muscle atrophy induced by tumour necrosis factor-α (TNF-α) (CS, P < 0.001; DCS, P < 0.001; DCSD, P < 0.001) in C2C12 myoblasts than on muscle atrophy induced by IL-6 (CS, P < 0.05; DCS, P = 0.08; DCSD, P < 0.05). In a C26 tumour-bearing mice model, administration of CS or its analogue DCSD significantly prevented body weight loss without affecting tumour size. At the end of the experiment, the body weight of mice treated with CS and DCSD was significantly increased by 11.09% (P < 0.01) and 11.38% (P < 0.01) compared with that of the C26 model group. CS and DCSD also improved the weight loss of epididymal adipose tissue in C26 model mice by 176.6% (P < 0.01) and 48.2% (P < 0.05) increase, respectively. CS and DCSD treatment partly preserved gastrocnemius myofibres cross-sectional area. CS treatment decreased the serum level of TNF-α (-95.02%, P < 0.01) but not IL-6 in C26 tumour-bearing mice. Inhibition on NF-κB and activation of Akt signalling pathway were involved in the ameliorating effects of CS and its analogues on muscle wasting both in vitro and in vivo. CS and its analogues also alleviated adipose tissue loss by inhibiting NF-κB and AMPK signalling pathways both in vitro and in vivo. CONCLUSIONS: CS and its analogues exhibited anticachexia effects mainly by inhibiting TNF-α/NF-κB pathway and decreasing muscle and adipose tissue loss. CS and its analogues might be promising drug candidates for the treatment of cancer cachexia.

Binding of RNA m6A by IGF2BP3 triggers chemoresistance of HCT8 cells via upregulation of ABCB1.

The overexpression of ATP-binding cassette transporters subfamily B member 1 (ABCB1) is known to be the primary trigger of multidrug resistance (MDR) in colorectal cancer (CRC), leading to chemotherapy failure. However, factors that regulate chemoresistance in CRC cells are largely unknown. To identify proteins involved in MDR in CRC, we used proteomics and transcriptomics approaches to analyze HCT8/T cells and parental HCT8 cells. Results showed that the expression of insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was upregulated in HCT8/T cells, and siIGF2BP3 remarkably elevated the sensitivity of HCT8/T cells to DOX. Overexpression of IGF2BP3 promoted ABCB1 expression, and reduced the sensitivity to ABCB1 substrates. Conversely, knockdown of IGF2BP3 reduced ABCB1 expression, and increased the sensitivity to ABCB1 substrates in vitro and in vivo. This phenomeon was further confirmed by the strong association of IGF2BP3 and ABCB1 expression with DOX sensitivity. Mechanistically, IGF2BP3, as a N6-methyladenosine (m6A) reader, directly bound to the m6A-modified region of ABCB1 mRNA, thereby promoting the stability and expression of ABCB1 mRNA. Overall, the results showed that IGF2BP3 bound to the m6A modification region of ABCB1 mRNA, and conferred chemoresistance in CRC cells via upregulation of ABCB1. These findings suggest that IGF2BP3 might be a potential biomarker for predicting the development of MDR in CRC. Targeting IGF2BP3 might be an important chemotherapeutic strategy for preventing MDR development in CRC.

Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo.

OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.

Design, synthesis, and structure activity relationship (SAR) studies of novel imidazo[1,2-a] pyridine derivatives as Nek2 inhibitors.

Never in mitosis (NIMA) related kinase 2 (Nek2) is involved in multiple cellular processes such as cell cycle checkpoint regulation, cell division, DNA damage response and cell apoptosis. Nek2 has been reported to be overexpressed in various tumors and correlated with poor prognosis. Herein, a series of imidazo[1,2-a] pyridines Nek2 inhibitors were designed, synthesized, and their biological activities were investigated. Besides, structure activity relationship analysis of these compounds were performed in the MGC-803 cell. The screening results are promising, and compound 28e shows good proliferation inhibitory activity with an IC50 of 38 nM. The results would be helpful to design and develop more effective Nek2 inhibitors for the treatment of gastric cancer.

Optimized HSP90 mediated fluorescent probes for cancer-specific bioimaging.

Cancer-specific bioimaging has been correlated with fluorescence-guided tumor therapy, garnering extensive interest from researchers. Herein, a highly efficient tumor-targeting fluorescent probe (NP-001), which is integrated with 4-hydroxy-1,8-naphthalimide and NVP-AUY922, for tumor imaging has been established. 4-Hydroxy-1,8-naphthalimide is a fluorescent molecule with remarkable imaging compatibility. NVP-AUY922 is a heat shock protein 90 (HSP90) inhibitor with preferential tumor selectivity that is conjugated to 4-hydroxy-1,8-naphthalimide as a tumor-targeting ligand. NP-002, a resorcinol-blocked probe which prevented binding with an amino acid residue of the HSP90 ATP binding pocket, was also synthesized as a control. In vitro and ex vivo assays showed that NP-001 could arrest cell proliferation, induce apoptosis and accumulate to inhibit HSP90. Confocal laser scanning microscopy (CLSM) also confirmed that NP-001 could be selectively internalized by tumor cells for cancer-specific bioimaging. Moreover, pharmacokinetic studies and histological analysis also indicated that NP-001 had a relatively longer retention time and showed no major organ-related toxicities. Overall, these encouraging data suggest that NP-001 is a promising new candidate for the early diagnosis of metastatic disease as well as targeted tumor imaging.

Recombinant cucurmosin-based immunotoxin targeting HER-2 with potent in vitro anti-cancer cytotoxicity.

Trastuzumab is a humanized monoclonal antibody against HER2 approved by FDA for breast and gastric cancer therapy. However, only a quarter of patients have the potential to benefit from it, and most of them develop resistance within therapy. The main purpose of this study is to broaden trastuzumab's therapeutic window by conjugating trastuzumab with recombinant cucurmosin to form an immunotoxin called T-CUS245C. T-CUS245C was chemically conjugated and the purification of T-CUS245C was evaluated by SDS-PAGE. SRB tests showed a remarkable cytotoxicity of T-CUS245C with IC50 values in picomolar range on HER2 positive cancer cells without significantly proliferation inhibition on HER2 negative cells (P < 0.01). Confocal microscopy verified the time-dependent internalization effects of T-CUS245C and revealed that the lethal efficacy can be increased by provoking the internalization. These results indicate the therapeutic potential of T-CUS245C for the HER-2 targeted therapy.

Two new phenylspirodrimanes from the deep-sea derived fungus Stachybotrys sp. MCCC 3A00409.

Two new phenylspirodrimanes, stachybotrin H (1) and stachybotrysin H (9) together with eleven known analogues (2-8, 10-13) were isolated from deep-sea derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 9-12 showed weak cytotoxicity against three human cancer cell lines K562, Hela and HL60 with IC50 in the range of 18.5-52.8 µM.

Novel recombinant immunotoxin of EGFR specific nanobody fused with cucurmosin, construction and antitumor efficiency in vitro.

Epidermal growth factor receptor (EGFR) overexpression is related to the increased aggressiveness, metastases, and poor prognosis in various cancers. In this study, we successfully constructed a new EGFR nanobody-based immunotoxin rE/CUS containing cucurmosin (CUS), The immunotoxin was expressed by prokaryotic system and we obtained a yield of 5 mg protein per liter expression medium. The percentage of it's binding ability totumor cell lines A549, HepG2, SW116, which highly expressed EGFR was 55.6%, 79.6% and 97.1%, respectively, but SW620 was only 4.45%. rE/CUS has the ability to bind A549, HepG2, SW116 cells specifically, and the antigen binding capability was not affected because of extra part of CUS component. The rE/CUS significantly inhibited the cell viability against EGFR over expression tumor cell lines in a dose-and time-dependent manner. Moreover, rE/CUS also induced apoptosis of HepG2 and A549 mightily. Our results demonstrate that rE/CUS is a potential therapeutic strategy for treating EGFR-positive solid tumors.