SARS-CoV-2 NSP2 as a Potential Delivery Vehicle for Proteins.

The development of biomolecule delivery systems is essential for the treatment of various diseases such as cancer, immunological diseases, and metabolic disorders. For the first time, we found that SARS-CoV-2-encoded nonstructural protein 2 (NSP2) can be secreted from the cells, where it is synthesized. Brefeldin A and H89, inhibitors of ER/Golgi secretion pathways, did not inhibit NSP2 secretion. NSP2 is likely secreted via an unconventional secretory pathway. Moreover, both secreted and purified NSP2 proteins were able to traverse the plasma membrane barrier and enter both immortalized human umbilical vein endothelial cells and tumor cell lines. After entry, the NSP2 protein was localized in only the cytoplasm. Cytochalasin D, a potent inhibitor of actin polymerization, inhibited the entry of NSP2. NSP2 can carry other molecules into cells. Burkholderia lethal factor 1, a monomeric toxin from the intracellular pathogen Burkholderia pseudomallei, has demonstrated antitumor activity by targeting host eukaryotic initiation translation factor 4A. An NSP2-BLF1 fusion protein was translocated across the cellular membranes of Huh7 cells and mediated cell killing. By using different approaches, including protein purification, chemical inhibition, and cell imaging, we confirm that NSP2 is able to deliver heterologous proteins into cells. NSP2 can act as a potential delivery vehicle for proteins.

GJA1-20k attenuates Ang II-induced pathological cardiac hypertrophy by regulating gap junction formation and mitochondrial function.

Cardiac hypertrophy (CH) is characterized by an increase in cardiomyocyte size, and is the most common cause of cardiac-related sudden death. A decrease in gap junction (GJ) coupling and mitochondrial dysfunction are important features of CH, but the mechanisms of decreased coupling and energy impairment are poorly understood. It has been reported that GJA1-20k has a strong tropism for mitochondria and is required for the trafficking of connexin 43 (Cx43) to cell-cell borders. In this study, we investigated the effects of GJA1-20k on Cx43 GJ coupling and mitochondrial function in the pathogenesis of CH. We performed hematoxylin-eosin (HE) and Masson staining, and observed significant CH in 18-week-old male spontaneously hypertensive rats (SHRs) compared to age-matched normotensive Wistar-Kyoto (WKY) rats. In cardiomyocytes from SHRs, the levels of Cx43 at the intercalated disc (ID) and the expression of GJA1-20k were significantly reduced, whereas JAK-STAT signaling was activated. Furthermore, the SHR rats displayed suppressed mitochondrial GJA1-20k and mitochondrial biogenesis. Administration of valsartan (10 mg· [Formula: see text] d-1, i.g., for 8 weeks) prevented all of these changes. In neonatal rat cardiomyocytes (NRCMs), overexpression of GJA1-20k attenuated Ang II-induced cardiomyocyte hypertrophy and caused elevated levels of GJ coupling at the cell-cell borders. Pretreatment of NRCMs with the Jak2 inhibitor AG490 (10 µM) blocked Ang II-induced reduction in GJA1-20k expression and Cx43 gap junction formation; knockdown of Jak2 in NRCMs significantly lessened Ang II-induced cardiomyocyte hypertrophy and normalized GJA1-20k expression and Cx43 gap junction formation. Overexpression of GJA1-20k improved mitochondrial membrane potential and respiration and lowered ROS production in Ang II-induced cardiomyocyte hypertrophy. These results demonstrate the importance of GJA1-20k in regulating gap junction formation and mitochondrial function in Ang II-induced cardiomyocyte hypertrophy, thus providing a novel therapeutic strategy for patients with cardiomyocyte hypertrophy.

Nitric oxide affects cisplatin cytotoxicity oppositely in A2780 and A2780-CDDP cells via the connexin32/gap junction.

Chemoresistance is a main obstacle in ovarian cancer therapy and new treatment strategies and further information regarding the mechanism of the medication cisplatin are urgently needed. Nitric oxide has a critical role in modulating the activity of chemotherapeutic drugs. Our previous work showed that connexin32 contributed to cisplatin resistance. However, whether nitric oxide is involved in connexin32-mediated cisplatin resistance remains unknown. In this study, using A2780 and A2780 cisplatin-resistant cells, we found that S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, attenuated cisplatin toxicity by decreasing gap junctions in A2780 cells. Enhancement of gap junctions using retinoic acid reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin toxicity. In A2780 cisplatin-resistant cells, however, S-nitroso-N-acetyl-penicillamine enhanced cisplatin toxicity by decreasing connexin32 expression. Downregulation of connexin32 expression by small interfering RNA exacerbated the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity and upregulation of connexin32 expression by pcDNA transfection reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity. Our study suggests for the first time that combining cisplatin with nitric oxide in clinical therapies for ovarian cancer should be avoided before cisplatin resistance emerges. The present study provides a productive area of further study for increasing the efficacy of cisplatin by combining cisplatin with the specific inhibitors or enhancers of nitric oxide in clinical treatment.

Inhibition of ubiquitin­specific protease 14 promotes connexin 32 internalization and counteracts cisplatin cytotoxicity in human ovarian cancer cells.

Although cisplatin is one of the most accepted therapies for ovarian cancer, recurrence and drug resistance remain problematic. Both the ubiquitin­proteasome system (UPS) and connexin (Cx) are closely related to tumor progression. However, the role of ubiquitin­specific protease 14 (USP14) and Cx in mediating drug resistance remains unclear. In the present study, we aimed to determine whether USP14 is involved in cisplatin resistance and modulates the internalization of connexin 32 (Cx32) in ovarian cancer. The results of the deubiquitinase (DUB) trap assay and western blot analysis revealed that the expression and activity levels of USP14 were downregulated in A2780 cisplatin­resistant cells (A2780­CDDP) relative to these levels in A2780 cisplatin­sensitive cells (A2780). CCK­8 assay results showed that inhibition of USP14 by a specific inhibitor or siRNA decreased cisplatin cytotoxicity in A2780 cells. Additionally, USP14 inhibition increased the expression of Cx32 without changing its mRNA and ubiquitination levels, as showed by Real­time qPCR and immunoprecipitation assay respectively. Cisplatin resistance induced by USP14 inhibition was counteracted by Cx32 knockdown. Moreover, USP14 inhibition contributed to Cx32 internalization, as determined by western blot analysis and a reduction in gap junction intercellular communication (GJIC), as showed by parachute dye­coupling assay. Collectively, these data suggest that Cx32 internalization by USP14 inhibition modulates the cisplatin resistance in ovarian cancer cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, USP14 can also be used as a marker to monitor the development of cisplatin resistance in ovarian cancer treatment.

Cx32 mediates norepinephrine-promoted EGFR-TKI resistance in a gap junction-independent manner in non-small-cell lung cancer.

The second-generation EGFR-TKI Afatinib is an irreversible ErbB family blocker used to treat patients with non-small-cell lung cancer (NSCLC). Unfortunately, resistance to this drug develops over time, and patients are always under great psychological pressure. A previous study showed that chronic stress hormones participate in EGFR-TKI resistance via ß2 -AR signaling via an IL-6 dependent mechanism. Our study further explores a novel potential underlying mechanism. In the present study, we show that the stress hormone norepinephrine (NE) promotes Afatinib resistance by upregulating Cx32 expression. Furthermore, we, for the first time, find that Cx32 is a target gene for transcription factor CREB and NE enhances Cx32 mRNA expression by activation of CREB. We also demonstrate that Cx32 promotes Afatinib resistance by decreasing the degradation of EGFR-TKI resistance-associated proteins (MET, IGF-1R) and by increasing their transcription levels. Together, these results reveal that the stress hormone NE accelerates Afatinib resistance by increasing the expression of Cx32, which augments MET and IGF-1R levels in cancer cells and provides a promising therapeutic strategy against EGFR-TKI Afatinib resistance in NSCLC.

Pattern of cell-to-cell transfer of microRNA by gap junction and its effect on the proliferation of glioma cells.

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18-27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA-34a. Additionally, these effects of microRNA-34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.