The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Ligustrazine inhibits platelet activation via suppression of the Akt pathway.

Aberrant activation of platelets has a critical role in thrombotic vascular events, including atherosclerosis, arterial thrombosis and myocardial infarction. The process of platelet activation is associated with multiple intracellular signaling pathways, including the phosphoinositide 3­kinase/AKT serine/threonine kinase (Akt) pathway. The well­known medicinal herb Rhizoma Ligusticum Wallichii (RLW) has long been used in China to clinically treat various cardiovascular disorders. As the most pharmacologically active component of RLW, ligustrazine has been demonstrated to possess a potent antiplatelet activity. However, the precise mechanisms mediating the bioactivities of ligustrazine have not been thoroughly elucidated. The present study evaluated the effects of ligustrazine hydrochloride (LH; the clinical­grade form of ligustrazine) on platelet activation and investigated the underlying molecular mechanisms. In vitro and ex vivo platelet activation models were used, established by stimulating rat platelet­rich plasma either with the platelet activator adenosine diphosphate (ADP) or with the specific Akt pathway activator insulin­like growth factor­1 (IGF­1). The results demonstrated that treatment with LH significantly and dose­dependently inhibited ADP­induced platelet aggregation, in addition to thromboxane A2 (TXA2) secretion and intracellular Ca2+ mobilization in platelets, in vitro and ex vivo. In addition, LH markedly suppressed ADP­induced Akt phosphorylation in vitro and ex vivo. Furthermore, LH markedly inhibited IGF­1­induced Akt phosphorylation, platelet aggregation, TXA2 formation and Ca2+ mobilization in vitro. Finally, LH was able to reverse adrenaline­induced shortening of bleeding time. Taken together, these results suggested that ligustrazine possesses a broad range of antiplatelet activities without apparent hemorrhagic side-effects, and suppression of Akt signaling may be one of the mechanisms by which ligustrazine exerts its antiplatelet activities.

Hedyotis diffusa Willd inhibits proliferation and induces apoptosis of 5­FU resistant colorectal cancer cells by regulating the PI3K/AKT signaling pathway.

Hedyotis diffusa Willd (HDW) is a major component frequently used in Traditional Chinese Medicine for the clinical treatment of colorectal cancer (CRC) and its associated drug resistance. However, the underlying mechanism of HDW circumventing drug resistance of cancer cells remains to be elucidated. Cancer cell resistance to apoptosis and activation of the phosphatidylinositol­3­kinase (PI3K)/protein kinase B (AKT) signaling pathway have been implicated as major factors in the acquired resistance to chemotherapeutic anti­cancer drugs. The present study investigated the effect and mechanisms of action of ethanol extract of Hedyotis diffusa Willd (EEHDW) on the proliferation and apoptosis of CRC 5­fluorouracil (5­FU) resistant (HCT­8/5­FU) cells. CRC HCT­8/5­FU cell viability following treatment with EEHDW was determined using MTT and colony formation assay. In addition, Annexin V/propidium iodide staining with flow cytometry analysis and 4',6­diamidino­2­phenylindole staining were used to determine the apoptosis of drug­resistant cancer cells following treatment with EEHDW. The mRNA and protein expression levels of B cell leukemia/lymphoma (Bcl­2), Bcl­2 associated X (Bax), cyclin dependent kinase 4 (CDK4), cyclin D1 and p21 were evaluated using reverse transcription­polymerase chain reaction and western blot analysis, respectively. Furthermore, activation of the PI3K/AKT signaling pathway and expression of phosphatase and tensin homolog (PTEN), PI3K, AKT and phosphorylated (p)­AKT were determined. EEHDW significantly reduced cell viability, inhibited cell colony formation and promoted apoptosis of HCT­8/5­FU cells. Furthermore, EEHDW significantly downregulated the expression of Bcl­2, cyclin D1 and CDK4 and upregulated the expression of Bax and p21. In addition, EEHDW inhibited the activation of the PI3K/AKT pathway by increasing expression of PTEN and suppressing the expression of PI3K and p­AKT. The present study provided the first direct evidence that EEHDW may overcome drug­resistance in human CRC cells by inhibiting PI3K/AKT signaling pathway and provides a basis for the improved therapeutic use of HDW in the clinical treatment of cancer.

Hedyotis diffusa Willd suppresses metastasis in 5­fluorouracil­resistant colorectal cancer cells by regulating the TGF­ß signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, and threatens the survival and health of patients with CRC. Chemotherapy remains one of the main therapeutic approaches for patients with CRC; however, drug resistance limits the long­term use. CRC cells with multi­drug resistance (MDR) exhibit increased survival times and metastatic potential, which may lead to the recurrence and metastasis of CRC. In addition, MDR is one of the major causes of chemotherapy failure in clinical treatment. Hedyotis diffusa Willd (HDW) has been used in the treatment of inflammation­associated diseases and malignant tumors, including CRC. The authors previously demonstrated that HDW could reverse MDR in CRC cells; however, its underlying mechanism, particularly in MDR­associated metastasis, remains to be elucidated. In the present study, the drug­resistant CRC cell line HCT­8/5­fluorouracil (5­FU) was used to investigate the effect of HDW on the growth and metastasis of cancer cells. Cell viability was assessed using the MTT assay. Cell adhesion potential was evaluated using adhesion experiments. Cell migration was assessed using wound healing and Transwell assays. The mRNA and protein expression levels of crucial factors in the transforming growth factor­ß (TGF­ß) signaling pathway, including TGF­ß, Mothers against decapentaplegic homolog 4 (SMAD4), neural (N)­cadherin, and epithelial (E)­cadherin, were analyzed using the reverse transcription­semi­quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that the HCT­8/5­FU cell line was more resistant to 5­FU and thus can be used as the resistant cell model. HDW was able to inhibit the viability, and adhesive, migratory and invasion potential of the HCT­8/5­FU cells. In addition, HDW was able to downregulate the expression of TGF­ß, SMAD4 and N­cadherin, and upregulate E­cadherin, at the gene and protein level. In conclusion, the results demonstrated that HDW may suppress the metastasis of 5­FU­resistant CRC cells via regulation of the TGF­ß signaling pathway, which was also considered to be one of the underlying mechanisms of its anti­CRC effect.

Scutellaria barbata D. Don inhibits 5-fluorouracil resistance in colorectal cancer by regulating PI3K/AKT pathway.

5-Fluorouracil (5-FU) resistance or multidrug resistance (MDR) has become a major obstacle in clinical treatment of cancers including colorectal cancer (CRC). Aberrant activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway may lead to unlimited growth and chemoresistance in CRC cells, which thus could be a promising therapeutic target. As a long-term used traditional Chinese folk-medicine, Scutellaria barbata D. Don (SB) processes specific anticancer activity, but its activity against cancer chemoresistance is less known. Therefore, using a 5-FU-resistant CRC cell line HCT-8/5-FU, in this study we evaluated the therapeutic efficacy of the ethanol extracts of SB (EESB) against 5-FU resistance and explored the possible molecular mechanisms. We found that EESB significantly suppressed proliferation and promoted apoptosis in HCT-8/5-FU cells. Additionally, EESB displayed remarkable effect enhancing the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine­123 (Rh­123) in HCT-8/5-FU cells. Furthermore, EESB obviously downregulated the expression of cyclin D1, Bcl-2 and ABCG2, while upregulated p21 and Bax expression. Moreover, EESB showed a prominent suppressive effect on the activation of PI3K/AKT pathway. The findings suggested that Scutellaria barbata D. Don was able to inhibit chemoresistance in colorectal cancer by suppression of the PI3K/AKT pathway.

Scutellaria barbata D. Don inhibits growth and induces apoptosis by suppressing IL-6-inducible STAT3 pathway activation in human colorectal cancer cells.

One of the most critical cellular signal transduction pathways known to malfunction in colorectal cancer is the interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) pathway. Scutellaria barbata D. Don (SB) is well-known traditional medicine in China that targets STAT3 signaling, and it has long been used to treat various types of cancer; however, the precise mechanism of its antitumor activity remains largely unclear. In order to further elucidate this underlying mechanism, an ethanol extract of SB (EESB) in cancer treatment. The aim of the present study was to evaluate the effects of EESB on the IL-6-inducible STAT3 pathway. We tested the dose-response association between EESB, IL-6-induced proliferaion and apoptosis using an MTT assay, colony formation and flow cytometry analysis in vitro. In addition, caspase-9 and caspase-3 activation was determined using a colorimetric assay, the activity of IL-6-induced STAT3 pathway was evaluated using western blot analysis, and the expression levels of cyclin D1, cyclin-dependent kinase 4, Bcl2 and Bcl2-associated X were determined using reverse transcription-polymerase chain reaction and western blot analysis. In the present study it was found that EESB could significantly inhibit the IL-6-mediated increase in STAT3 phosphorylation levels and transcriptional activity in HT-29 human colon carcinoma cells, resulting in the suppression of cell proliferation and the induction of apoptosis. In addition, treatment with EESB markedly inhibited the IL-6-induced upregulation of cyclin D1 and B-cell lymphoma-2, two key target genes of the STAT3 pathway. These results suggest that treatment with EESB could effectively inhibit the proliferation and promote the apoptosis of human colon carcinoma cells via modulation of the IL-6/STAT3 signaling pathway and its target genes.

Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2.

Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC.

Pien Tze Huang inhibits liver metastasis by targeting TGF-ß signaling in an orthotopic model of colorectal cancer.

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth in vitro and in vivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis model of colon cancer using microsurgical techniques. Mice were intragastrically administered 234 mg/kg/day dose of either PZH or saline for 14 days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-ß signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the in vivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis (5/6), whereas one case was found in the PZH group (1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-ß, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-ß signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.