Targeting TAOK1 with resveratrol inhibits esophageal squamous cell carcinoma growth in vitro and in vivo.

The worldwide incidence and mortality rates of esophageal squamous cell carcinoma (ESCC) have increased over the last decade. Moreover, molecular targets that may benefit the therapeutics of patients with ESCC have not been fully characterized. Our study discovered that thousand and one amino-acid protein kinase 1 (TAOK1) is highly expressed in ESCC tumor tissues and cell lines. Knock-down of TAOK1 suppresses ESCC cell proliferation in vitro and patient-derived xenograft or cell-derived xenograft tumors growth in vivo. Moreover, TAOK1 overexpression promotes ESCC growth in vitro and in vivo. Additionally, we identified that the natural small molecular compound resveratrol binds to TAOK1 directly and diminishes the kinase activity of TAOK1. Targeting TAOK1 directly with resveratrol significantly inhibits cell proliferation, induces cell cycle arrest and apoptosis, and suppresses tumor growth in ESCC. Furthermore, the silencing of TAOK1 or the application of resveratrol attenuated the activation of TAOK1 downstream signaling effectors. Interestingly, combining resveratrol with paclitaxel, cisplatin, or 5-fluorouracil synergistically enhanced their therapeutic effects against ESCC. In conclusion, this work illustrates the underlying oncogenic function of TAOK1 and provides a theoretical basis for the application of targeting TAOK1 therapy to the clinical treatment of ESCC.

Costunolide is a dual inhibitor of MEK1 and AKT1/2 that overcomes osimertinib resistance in lung cancer.

EGFR-TKI targeted therapy is one of the most effective treatments for lung cancer patients harboring EGFR activating mutations. However, inhibition response is easily attenuated by drug resistance, which is mainly due to bypass activation or downstream activation. Herein, we established osimertinib-resistant cells by stepwise dose-escalation in vitro and an osimertinib-resistant patient-derived xenograft model through persistent treatment in vivo. Phosphorylated proteomics identified that MEK1 and AKT1/2 were abnormally activated in resistant cells compared with parental cells. Likewise, EGFR inhibition by osimertinib induced activation of MEK1 and AKT1/2, which weakened osimertinib sensitivity in NSCLC cells. Consequently, this study aimed to identify a novel inhibitor which could suppress resistant cell growth by dual targeting of MEK1 and AKT1/2. Based on computational screening, we identified that costunolide could interact with MEK1 and AKT1/2. Further exploration using in vitro kinase assays validated that costunolide inhibited the kinase activity of MEK1 and AKT1/2, which restrained downstream ERK-RSK2 and GSK3ß signal transduction and significantly induced cell apoptosis. Remarkably, the combination of osimertinib and costunolide showed synergistic or additive inhibitory effects on tumor growth in osimertinib-resistant cell lines and PDX model. Hence, this study highlights a potential therapeutic strategy for osimertinib-resistant patients through targeting of MEK1 and AKT1/2 by costunolide.

COVID-19 and Strategies for Its Therapeutics.

Currently the epidemic of SARS-CoV-2-caused COVID-19 is a major threat to global public health. The latest clinical data, laboratory results, and autopsy information are summarized herein to provide a brief review of the significant issues surrounding SARS-CoV-2 and COVID-19. In this review, we also cover research on the ways in which the virus enters the human body, general clinical symptoms, immunopathological responses in severe cases of COVID-19, and the issues surrounding the potential therapeutic responses to the illness.

Expression of PTEN-long mediated by CRISPR/Cas9 can repress U87 cell proliferation.

PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN-long is an alternative translation variant, with an additional 173 amino acids added to the N-terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN-long is secreted into serum and can re-enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells. As an alternative approach, if a therapeutic protein can be directly delivered to target cell of interest, it should theoretically function well within the cells, particularly for genes that are deficiently expressed in vivo. Most therapeutic proteins are incapable of efficiently permeating the cell membrane. In this study, we have employed CRISPR/Cas9 gene editing tool combined with single-stranded template to edit CTG of PTEN-long to ATG in the genome. Two guide RNAs close to CTG site were found to have similar efficiency in driving PTEN-long expression. Furthermore, we detected PTEN-long expression in transfected whole-cell lysate and in concentrated culture media in Western blot. Interestingly, the culture media of PTEN-long expression can reduce Akt phosphorylation level and repress U87 cell proliferation compared to wild-type U87 or control media. Taken together, PTEN-long driven by CRISPR/Cas9 imports and exports cells and represses nearby cell proliferation, indicating the PTEN-long generated by CRISPR/Cas9 has potential to be an alternative strategy for PTEN gene therapy.

Intratumoural delivery of TRAIL mRNA induces colon cancer cell apoptosis.

Novel strategies in intratumoral injection and emerging immunotherapies have heralded a new era of precise cancer treatments. The affinity of SARS-CoV-2 to ACE2 receptors, a feature which facilitates virulent human infection, is leveraged in this research. Colon cancer cells, with their high ACE2 expression, provide a potentially strategic target for using this SARS-CoV-2 feature. While the highly expression of ACE2 is observed in several cancer types, the idea of using the viral spike protein for targeting colon cancer cells offers a novel approach in cancer treatment. Intratumoral delivery of nucleic acid-based drugs is a promising alternative to overcoming the limitations of existing therapies. The increasing importance of nucleic acids in this realm, and the use of Lipid Nanoparticles (LNPs) for local delivery of nucleic acid therapeutics, are important breakthroughs. LNPs protect nucleic acid drugs from degradation and enhance cellular uptake, making them a rapidly evolving nano delivery system with high precision and adaptability. Our study leveraged a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with a receptor-binding domain from the SARS-CoV-2 spike protein, encapsulated in LNPs, to target colon cancer cells. Our results indicated that the TRAIL fusion-mRNA induced apoptosis in vitro and in vivo. Collectively, our findings highlight LNP-encapsulated TRAIL fusion-mRNA as a potential colon cancer therapy.

Repurposing pentamidine for cancer immunotherapy by targeting the PD1/PD-L1 immune checkpoint.

Immunotherapy has emerged as an effective therapeutic approach to several cancer types. The reinvigoration of tumor-infiltrating lymphocyte-mediated immune responses via the blockade of immune checkpoint markers, such as program cell death-1 (PD-1) or its cognate ligand PD-L1, has been the basis for developing clinically effective anticancer therapies. We identified pentamidine, an FDA-approved antimicrobial agent, as a small-molecule antagonist of PD-L1. Pentamidine enhanced T-cell-mediated cytotoxicity against various cancer cells in vitro by increasing the secretion of IFN-γ, TNF-α, perforin, and granzyme B in the culture medium. Pentamidine promoted T-cell activation by blocking the PD-1/PD-L1 interaction. In vivo administration of pentamidine attenuated the tumor growth and prolonged the survival of tumor-bearing mice in PD-L1 humanized murine tumor cell allograft models. Histological analysis of tumor tissues showed an increased number of tumor-infiltrating lymphocytes in tissues derived from pentamidine-treated mice. In summary, our study suggests that pentamidine holds the potential to be repurposed as a novel PD-L1 antagonist that may overcome the limitations of monoclonal antibody therapy and can emerge as a small molecule cancer immunotherapy.

Challenge and countermeasures for EGFR targeted therapy in non-small cell lung cancer.

Lung cancer causes the highest mortality compared to other cancers in the world according to the latest WHO reports. Non-small cell lung cancer (NSCLC) contributes about 85% of total lung cancer cases. An extensive number of risk factors are attributed to the progression of lung cancer. Epidermal growth factor receptor (EGFR), one of the most frequently mutant driver genes, is closely involved in the development of lung cancer through regulation of the PI3K/AKT and MAPK pathways. As a representative of precision medicine, EGFR-tyrosine kinase inhibitors (TKIs) targeted therapy significantly relieves the development of activating mutant EGFR-driven NSCLC. However, treatment with TKIs facilitates the emergence of acquired resistance that continues to pose a significant hurdle with respect to EGFR targeted therapy. In this review, the development of current approved EGFR-TKIs as well as the related supporting clinical trials are summarized and discussed. Mechanisms of action and resistance were addressed respectively, which serve as important guides to understanding acquired resistance. We also explored the corresponding combination treatment options according to different resistance mechanisms. Future challenges include more comprehensive characterization of unclear resistance mechanisms in different populations and the development of more efficient and precision synthetic therapeutic strategies.

The phosphatase of regenerating liver-3 protein (PRL-3) promotes glioma cell invasiveness by interacting with ß3 -tubulin.

PRL-3 is a tyrosine phosphatase linked with tumor metastasis. It is detected high expression in different kinds of cancers, including colorectal, gastric, ovarian, and liver cancer. Its high expression is positively correlated with the progression of tumors and negatively with survivals of patients. However, the detailed mechanism underlying PRL-3 in tumor metastasis still remains unclear. In the present study, we found that PRL-3 is able to bind to ß3-tubulin in pull-down and co-immunoprecipitation assays. Furthermore, overexpression of PRL-3 dephosphorylated ß3-tubulin, a component of cytoskeleton, which plays critical role in cell shape formation and migration. Using cell wound healing and matrigel invasion assays, we found that PRL-3 could promote the migration and invasion of glioma cells. Taken together, our study revealed that PRL-3 may be involved in migration and invasion of glioma by dephosphorylating ß3-tubulin. It is tempting to speculate that dephosphorylation of ß3-tubulin by PRL-3 results in assembly of the cytoskeleton and facilitates cell migration and/or tumor metastasis.

A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy.

Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand-1 (PD-L1), have achieved enormous success in the treatment of several cancers. However, monoclonal antibodies are expensive to produce, have poor tumor penetration, and may induce autoimmune side effects, all of which limit their application. Here, we demonstrate that PDI-1 (also name PD1/PD-L1 inhibitor 1), a small molecule antagonist of PD-1/PD-L1 interactions, shows potent anti-tumor activity in vitro and in vivo and acts by relieving PD-1/PD-L1-induced T cell exhaustion. We show that PDI-1 binds with high affinity to purified human and mouse PD-1 and PD-L1 proteins and is a competitive inhibitor of human PD-1/PD-L1 binding in vitro. Incubation of ex vivo activated human T cells with PDI-1 enhanced their cytotoxicity towards human lung cancer and melanoma cells, and concomitantly increased the production of granzyme B, perforin, and inflammatory cytokines. Luciferase reporter assays showed that PDI-1 directly increases TCR-mediated activation of NFAT in a PD-1/PD-L1-dependent manner. In two syngeneic mouse tumor models, the intraperitoneal administration of PDI-1 reduced the growth of tumors derived from human PD-L1-transfected mouse lung cancer and melanoma cells; increased and decreased the abundance of tumor-infiltrating CD8+ and FoxP3+ CD4+ T cells, respectively; decreased the abundance of PD-L1-expressing tumor cells, and increased the production of inflammatory cytokines. The anti-tumor effect of PDI-1 in vivo was comparable to that of the anti-PD-L1 antibody atezolizumab. These results suggest that the small molecule inhibitors of PD-1/PD-L1 may be effective as an alternative or complementary immune checkpoint inhibitor to monoclonal antibodies.

Periplogenin suppresses the growth of esophageal squamous cell carcinoma in vitro and in vivo by targeting STAT3.

The mortality rate of esophageal squamous cell carcinoma (ESCC) is higher than that of other cancers worldwide owing to a lack of therapeutic targets and related drugs. This study aimed to find new drugs by targeting an efficacious therapeutic target in ESCC patients. Signal transducer and activator of transcription 3 (STAT3) is hyperactive in ESCC. Herein, we identified a novel STAT3 inhibitor, periplogenin, which strongly inhibited phosphorylation of STAT3 at Tyr705. Docking models and pull-down assays revealed that periplogenin bound directly and specifically to STAT3, leading to significant suppression of subsequent dimerization, nuclear import, and transcription activities. In addition, STAT3 knockdown cell lines were insensitive to periplogenin, whereas in contrast, STAT3-overexpressing cells were more sensitive to periplogenin, indicating that STAT3 was a target of periplogenin. Intraperitoneally administered periplogenin exhibited efficacious therapeutic effects in ESCC patient-derived xenograft models and dramatically impaired the phosphorylation of STAT3 and expression levels of STAT3-mediated downstream genes. Thus, our study demonstrated that periplogenin acted as a new STAT3 inhibitor, suppressing the growth of ESCC in vitro and in vivo, providing a basis for its potential application in ESCC treatment and prevention.

Characterization of lamin B receptor of Sf9 cells and its fate during Autographa californica nucleopolyhedrovirus infection.

Baculovirus nucleocapsids egress from the nuclear membrane during infection. However, details of alternation of nuclear membrane structure during baculovirus egress are unknown. In this study, we examined the changes of lamin B receptor (LBR), a main inner nuclear membrane component, during Autographa californica nucleopolyhedrovirus (AcMNPV) infection. Firstly, the open reading frame (Orf) of Sf9 lbr was cloned by reverse transcription PCR, and the distribution of LBR in Sf9 cells were observed by fusing LBR with the red fluorescence protein mcherry. Besides, the amount of endogenous LBR during AcMNPV infection was detected by western blotting. Moreover, the distribution of LBR after AcMNPV infection was observed under the confocal fluorescence microscopy. Furthermore, the effects of protein kinase C (PKC) inhibitor on stability of LBR and release of budded virus (BVs) were determined. The results showed that Sf9 lbr contains an Orf of 2040 nucleotides (NTs), which encodes a predicted protein of 679 amino acids (AAs). Fluorescence microscopy showed that LBR is localized to the nuclear membrane. Western blotting result showed that the amount of endogenous LBR is significantly reduced after AcMNPV infection. Transfection and infection assay demonstrated that the fluorescence of LBR nearly completely disappeared after viral infection. PKC inhibitor can suppress the degradation of LBR induced by AcMNPV, resulting in the reduction of viral titer of progeny viruses. The electron microscopy analysis demonstrated that PKC inhibitor did not influence virion entry, uncoating, and assembly, but may partially protect the nuclear membrane from disruption by AcMNPV. Taken together, AcMNPV infection can distort the expression of LBR, which may promote the egress of nucleocapsids.

Cytokine Signature Induced by SARS-CoV-2 Spike Protein in a Mouse Model.

Although COVID-19 has become a major challenge to global health, there are currently no efficacious agents for effective treatment. Cytokine storm syndrome (CSS) can lead to acute respiratory distress syndrome (ARDS), which contributes to most COVID-19 mortalities. Research points to interleukin 6 (IL-6) as a crucial signature of the cytokine storm, and the clinical use of the IL-6 inhibitor tocilizumab shows potential for treatment of COVID-19 patient. In this study, we challenged wild-type and adenovirus-5/human angiotensin-converting enzyme 2-expressing BALB/c mice with a combination of polyinosinic-polycytidylic acid and recombinant SARS-CoV-2 spike-extracellular domain protein. High levels of TNF-α and nearly 100 times increased IL-6 were detected at 6 h, but disappeared by 24 h in bronchoalveolar lavage fluid (BALF) following immunostimulant challenge. Lung injury observed by histopathologic changes and magnetic resonance imaging at 24 h indicated that increased TNF-α and IL-6 may initiate CSS in the lung, resulting in the continual production of inflammatory cytokines. We hypothesize that TNF-α and IL-6 may contribute to the occurrence of CSS in COVID-19. We also investigated multiple monoclonal antibodies (mAbs) and inhibitors for neutralizing the pro-inflammatory phenotype of COVID-19: mAbs against IL-1α, IL-6, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibitors of p38 and JAK partially relieved CSS; mAbs against IL-6, TNF-α, and GM-CSF, and inhibitors of p38, extracellular signal-regulated kinase, and myeloperoxidase somewhat reduced neutrophilic alveolitis in the lung. This novel murine model opens a biologically safe, time-saving avenue for clarifying the mechanism of CSS/ARDS in COVID-19 and developing new therapeutic drugs.

CRISPR/Cas9 - An evolving biological tool kit for cancer biology and oncology.

The development of genetic engineering in the 1970s marked a new frontier in genome-editing technology. Gene-editing technologies have provided a plethora of benefits to the life sciences. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/ Cas9) system is a versatile technology that provides the ability to add or remove DNA in the genome in a sequence-specific manner. Serious efforts are underway to improve the efficiency of CRISPR/Cas9 targeting and thus reduce off-target effects. Currently, various applications of CRISPR/Cas9 are used in cancer biology and oncology to perform robust site-specific gene editing, thereby becoming more useful for biological and clinical applications. Many variants and applications of CRISPR/Cas9 are being rapidly developed. Experimental approaches that are based on CRISPR technology have created a very promising tool that is inexpensive and simple for developing effective cancer therapeutics. This review discusses diverse applications of CRISPR-based gene-editing tools in oncology and potential future cancer therapies.

A quantitative method for measuring the transfection efficiency of CD19-directed chimeric antigen receptor in target cells.

BACKGROUND: Adoptive cell therapy (ACT) based on chimeric antigen receptors (CARs) expressed on the surface of T cells shows a remarkable clinical outcome, particularly for B-cell malignancies. However, toxicity and side effects of CD19-redirected CAR T cells have been observed concurrently in most cases due to cytokine release and tumor cell lysis. Therefore, strictly controlling the amount of valid T cells re-transfused to patients seems to be an important step in reducing toxicity and side effects of CAR T cells. Transfection efficiency via lentiviral particles varies widely in different cases. OBJECTIVES: The aim of this study was to accurately calculate and control the number of valid CAR T cells through ACT because the restriction antibiotics gene or the fluorescence gene are not suitable for tracking or screening for valid transfected T cells. MATERIAL AND METHODS: We expressed and purified a GFP-CD19 fusion protein as a probe to measure the expression efficiency of CD19-redirected CAR on the cell surface in adherent and suspension cell lines. RESULTS: We can precisely calculate the transfected efficiency of lentiviral particles by counting the number of GFP-labeled cells under a microscope, as well as calculate the percentage by comparing the number of GFP-labeled cells to total cells. CONCLUSIONS: We propose a method to control the number of valid cells in ACT and to reduce toxicity and side effects in clinical use - a convenient technique for monitoring the dosage of CAR T cells for patients.