Glutamatergic neurons in ventral pallidum modulate heroin addiction via epithalamic innervation in rats.

Glutamatergic neurons in ventral pallidum (VPGlu) were recently reported to mediate motivational and emotional behavior, but its role in opioid addiction still remains to be elucidated. In this study we investigated the function of VPGlu in the context-dependent heroin taking and seeking behavior in male rats under the ABA renewal paradigm. By use of cell-type-specific fiber photometry, we showed that the calcium activity of VPGlu were inhibited during heroin self-administration and context-induced relapse, but activated after extinction in a new context. The drug seeking behavior was accompanied by the decreased calcium signal of VPGlu. Chemogenetic manipulation of VPGlu bidirectionally regulated heroin taking and seeking behavior. Anterograde tracing showed that the lateral habenula, one of the epithalamic structures, was the major output region of VPGlu, and its neuronal activity was consistent with VPGlu in different phases of heroin addiction and contributed to the motivation for heroin. VPGlu axon terminals in LHb exhibited dynamic activity in different phases of heroin addiction. Activation of VPGlu-LHb circuit reduced heroin seeking behavior during context-induced relapse. Furthermore, the balance of excitation/inhibition from VP to LHb was shifted to enhanced glutamate transmission after extinction of heroin seeking motivation. Overall, the present study demonstrated that the activity of VPGlu was involved in the regulation of heroin addiction and identified the VPGlu-LHb pathway as a potential intervention to reduce heroin seeking motivation.

Reversal of subtype-selectivity and function by the introduction of a para-benzamidyl substituent to N-cyclopropylmethyl nornepenthone.

The discovery and development of novel µ-opioid receptor (MOR) antagonists is a significant area to combat Opioid Use Disorder (OUD). In this work, a series of para-substituted N-cyclopropylmethyl-nornepenthone derivatives were designed and synthesized and pharmacologically assayed. Compound 6a was identified as a selective MOR antagonist both in vitro and in vivo. Its molecular basis was elucidated using molecular docking and MD simulations. A subpocket on the extracellular side of the TM2 domain of MOR, in particular the residue Y2.64, was proposed to be responsible for the reversal of subtype selectivity and functional reversal of this compound.

Up-regulation of BDNF/TrkB signaling by δ opioid receptor agonist SNC80 modulates depressive-like behaviors in chronic restraint-stressed mice.

Depressive disorder is a psychiatric disease characterized by its main symptoms of low mood and anhedonia. Due to its complex etiology, current clinical treatments for depressive disorder are limited. In this study, we assessed the role of the δ opioid receptor (δOR) system in the development of chronic-restraint-stressed (CRS)-induced depressive behaviors. We employed a 21-day CRS model and detected the c-fos activation and protein levels' changes in enkephalin (ENK)/δOR. It was found that the hippocampus and amygdala were involved in CRS-induced depression. The expression of pro-enkephalin (PENK), the precursors of the endogenous ligand for δOR, was significantly decreased in the hippocampus and amygdala following CRS. We then treated the mice with SNC80, a specific δOR agonist, to examine its anti-depressant effects in the tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT). SNC80 administration significantly reversed depressive-like behaviors, and this antidepressant effect could be blocked by a TrkB inhibitor: ANA-12. Although ANA-12 treatment had no significant effect on the expression of ENK/δOR, it blocked the promoting effects of brain-derived neurotrophic factor (BDNF)/tyrosine kinase B(TrkB) signaling by SNC80 in the hippocampus and amygdala. Therefore, the present study demonstrates that SNC80 exerts anti-depressant effects by up-regulating the BDNF/TrkB signaling pathway in the hippocampus and amygdala in CRS-induced depression and provides evidence that δOR's agonists may be potential anti-depressant therapeutic agents.

Glycolysis is essential for chemoresistance induced by transient receptor potential channel C5 in colorectal cancer.

BACKGROUND: Elevated intracellular Ca2+ ([Ca2+] i ) level could lead to [Ca2+] i overload and promote apoptosis via different pathways. In our previously study, up-regulated expression of transient receptor potential canonical channel (TRPC5) was proven to increase [Ca2+] i level, and resulted in chemoresistance whereas not apoptosis in human colorectal cancer (CRC) cells. The ATP-dependent homeostatic maintenance of resting [Ca2+] i should be important in this process. Increased glycolysis was found to be an important adenosine triphosphate (ATP) source in cancer. This study aimed to explore the potential mechanism of aerobic glycolysis in transient receptor potential channel TRPC5 induced chemoresistance. METHODS: In this study, we examined glucose transporter 1 (GLUT1) expression, glucose consumption and celluar ATP production to determine glycolytic activity. Real-time PCR and western blot were analyzed to determine TRPC5 expression at the mRNA and protein levels in human CRC cells (HCT-8, LoVo), and fluorouracil (5-Fu) resistant CRC cells (HCT-8/5-Fu, LoVo/5-Fu). 3-bromopyruvate (3-BP) and 2-Deoxy-D-glucose (2DG) were used to inhibit glycolysis. Glycolytic activity, intracellular Ca2+ ([Ca2+] i ) and the half maximal inhibitory concentration of 5-Fu (5-Fu IC50) were measured. Western blot was analyzed to determine cleaved Caspase-3 protein level. Flow cytometry was performed to detect the apoptosis rates. Immunohistochemistry staining was performed to determine TRPC5 and GLUT1 expression level in human CRC tissues. RESULTS: Overproduced of TRPC5 and increased glycolysis were found in HCT-8/5-Fu and LoVo/5-Fu than in HCT-8 and LoVo cells. Compared to HCT-8 cells, the HCT-8/5-Fu cells showed higher [Ca2+] i levels which decreased after treated with TRPC5-specific shRNA. Furthemore, inhibition of glycolysis resulted in decreased ATP production, elevation of [Ca2+] i level and cleaved caspase-3, increased apoptotic cells rate, and a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu cells, while showed no effect in HCT-8 cells. BAPTA-AM, a [Ca2+] i chelator, could reduce the elevation of cleaved caspase-3 and increased apoptotic cells rate due to glycolysis inhibition. Advanced CRC patients with high expression of TRPC5/GLUT1 displayed poorer chemotherapy outcome, and notably, the significant association between high TRPC5 expression and chemoresistance is GLUT1 expression level dependent. CONCLUSIONS: We demonstrated the essential role of glycolysis in TRPC5 induced chemoresistance in human CRC cells via maintaining [Ca2+] i homeostasis.

S100A16 is a prognostic marker for colorectal cancer.

BACKGROUND: S100 is a superfamily of calcium-binding proteins that regulate multiple biological processes and are involved in many diseases. S100A16 has recently been identified to be involved in several cancers such as bladder cancer, lung cancer, and oral squamous cell carcinoma. However, the role of S100A16 expression in the colorectal cancer (CRC) has not been investigated. METHODS: S100A16 protein expression was detected by immunohistochemistry in 296 cases of CRC. Kaplan-Meier survival analysis and Cox regression analysis were performed to evaluate the prognostic significance of S100A16. RESULT: The results showed that the overall survival (OS) of patients with low membrane S100A16 expression was significantly shorter than patients with high expression (P < 0.05). Chi-square analysis showed that S100A16 expression had a positive correlation with tumor grade (P = 0.02). Multivariate analysis identified membrane S100A16 expression as an independent prognostic marker for OS in CRC patients. (P < 0.05). Univariate analysis showed no significant association between cytoplasmic/nuclear S100A16 expression and OS. CONCLUSION: Membrane S100A16 is associated with the prognosis of CRC patients, indicating that S100A16 may be a potential prognostic biomarker and therapeutic target for CRC.

Inhibition of B7-H3 reverses oxaliplatin resistance in human colorectal cancer cells.

B7-H3, an immunoregulatory protein, has been found highly expressed in several cancer types, and involved in cancer cell migration and invasion. Here, we investigated the role of B7-H3 in oxaliplatin resistance in colorectal cancer (CRC) cells. Transient silencing of B7-H3 enhanced oxaliplatin sensitivity by increasing oxaliplatin-induced DNA damage. The overexpression of B7-H3 increased oxaliplatin resistance reducing the formation of phosphorylated histone H2AX (γH2AX) loci. The silencing of X-ray repair cross complementing group 1 (XRCC1), upregulated in B7-H3 overexpressing cells, induced an increase in cell death following oxaliplatin treatment. Finally, the upregulation of XRCC1 expression induced by B7-H3 involved PI3K-AKT pathway. In conclusion, B7-H3 promotes the oxaliplatin resistance in CRC cells upregulating the expression of XRCC1 via PI3K-AKT pathway.

UCH-L1-containing exosomes mediate chemotherapeutic resistance transfer in breast cancer.

BACKGROUND: Chemotherapy resistance has become a serious challenge in the treatment of breast cancer. Previous studies showed cells can transfer proteins, including those responsible for drug resistance to adjacent cells via exosomes. METHODS: The switches of drug resistance via exosomes transfer were assessed by CellTiter-Blue Viability assay, flow cytometry, and immunostaining analysis. Relative protein levels of Ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), P-glycoprotein (P-gp), extracellular-signal regulated protein kinase1/2 (ERK1/2), and phospho-extracellular-signal regulated protein kinase1/2 (p-ERK1/2) were measured by Western blot. Immunohistochemistry was performed on 93 breast cancer samples to assess the associations of UCH-L1 levels with immunofluorescence value of UCH-L1 in circulating exosomes. RESULT: The Adriamycin-resistant human breast cancer cells (MCF7/ADM) secreted exosomes carrying UCH-L1 and P-gp proteins into the extracellular microenvironment then integrated into Adriamycin-sensitive human breast cancer cells (MCF7/WT) in a time-dependent manner, transferring the chemoresistance phenotype. Notably, in blood samples from patients with breast cancer, the level of exosomes carrying UCH-L1 before chemotherapy was significantly negatively correlated with prognosis. CONCLUSION: Our study demonstrated that UCH-L1-containing exosomes can transfer chemoresistance to recipient cells and these exosomes may be useful as non-invasive diagnostic biomarkers for detection of chemoresitance in breast cancer patients, achieving more effective and individualized chemotherapy.

Increasing circulating exosomes-carrying TRPC5 predicts chemoresistance in metastatic breast cancer patients.

Chemoresistance, the major obstacle in breast cancer chemotherapy, results in unnecessary chemotherapy and wasting of medical resources. No feasible method has been available to predict chemoresistance before chemotherapy. In our previous study, elevated expression of transient receptor potential channel TRPC5 was found to be an essential element for chemoresistance in breast cancer cells, and it was determined that it could be transferred to chemosensitive breast cancer cells through releasing extracellular vesicles (EV) containing TRPC5 from chemoresistant cells, resulting in acquired chemoresistance. Exosomes, a type of EV, are secreted membrane-enclosed vesicles of 50-150-nm diameter. In this study we found that circulating exosomes in peripheral blood from breast cancer patients carried TRPC5. In the present study, circulating exosome-carrying TRPC5 (cirExo-TRPC5) level was significantly correlated with TRPC5 expression level in breast cancer tissues and tumor response to chemotherapy. Furthermore, increased cirExo-TRPC5 level after chemotherapy preceded progressive disease (PD) based on imaging examination and strongly predicted acquired chemoresistance. Taken together, our study demonstrated that cirExo-TRPC5 might act as a noninvasive chemoresistance marker and might serve as an adjuvant to the current imaging examination-based chemoresistance.

Elevated expression of TrpC5 and GLUT1 is associated with chemoresistance in colorectal cancer.

Reprogramming of energy metabolism (aerobic glycolysis) is thought to play an essential role in cancer. Compared to oxidative phosphorylation, aerobic glycolysis consumes more glucose through the upregulation of glucose transporters, notably glucose transporter 1 (GLUT1). Elevated glycolysis occurs in chemoresistant cancer cells, but the detailed mechanism is not well understood. The upregulation of the Ca2+-permeable transient receptor potential channel 5 (TrpC5) activates the Wnt/ß-catenin signaling pathway in 5-fluorouracil (5-Fu)-resistant human colorectal cancer (CRC) HCT-8 (HCT-8/5-Fu) cells. In the present study, TrpC5 was overexpressed at the mRNA and protein levels along with GLUT1 in HCT-8/5-Fu cells. Suppression of TrpC5 expression with a TrpC5-specific shRNA reduced the induction of GLUT1 in the HCT-8 cells. The inhibition of the Wnt/ß-catenin signaling pathway with XAV939 resulted in a decreased GLUT1 and nuclear c-Myc expression. Further study using clinical specimens validated the positive correlation between TrpC5 and GLUT1 protein levels and showed that a high TrpC5/GLUT1 expression was significantly correlated with chemoresistance. Taken together, we demonstrated the essential role of TrpC5 in GLUT1 induction and revealed that a high TrpC5/GLUT1 expression is associated with chemoresistance in human CRC.

B7-H3 upregulates BRCC3 expression, antagonizing DNA damage caused by 5-Fu.

5-fluorouracil (5-Fu) is still recognized as the mainstay in colorectal cancer chemotherapy, but the response rate of 5-Fu in colorectal cancer is less than 50%. Our previous mRNA microarray data revealed that BRCC3, a component of the BRCA1-BRCA2-BRCC3 DNA repair complex, had a direct relationship with B7-H3, an immunoglobulin that is upregulated in tumor tissue and associated with metastasis and poor prognosis. Real-time PCR and western blot analysis confirmed that the expression of both BRCC3 mRNA and protein, respectively, were elevated following B7-H3 overexpression in SW480 cells; likewise, BRCC3 expression decreased after B7-H3 was knocked down in HCT-8 cells. DNA comet assay results indicate an inverse correlation between the extent of 5-Fu-induced DNA damage and the expression level of B7-H3 in both SW480- and HCT-8-based cell lines. In SW480 cells that overexpress B7-H3, knockdown of BRCC3 similarly permitted greater 5-Fu-induced DNA damage. Altogether, results suggest that BRCC3 may play a role in B7-H3-induced 5-Fu resistance, such that B7-H3 upregulates BRCC3 expression, enhancing DNA repair in colorectal cancer cells.

[Construction and expression of eukaryotic expression plasmid pcDNA3.1/hIL-18].

AIM: To construct an eukaryotic expression plasmid pcDNA3.1/hIL-18 and express it in mammalian cells. METHODS: cDNA encoding mature hIL-18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM-T/hIL-18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL-18. Then the constructed plasmid was transfected into COS-7 and Rlc310 cells by liposome-mediated gene transfer method. hIL-18 expressed in transfected COS-7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL-18 mRNA in transfected Rlc310 cells was assayed by RT-PCR. RESULTS: A recombinant eukaryotic expression plasmid pcDNA3.1/hIL-18 was successfully constructed and expressed transiently in COS-7 cells and stably in Rlc310 cells. CONCLUSION: The construction and expression of pcDNA3.1/hIL-18 have been achieved successfully, which lays a foundation for further research on anti-tumor effect of IL-18.

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.