Homodimerized cytoplasmic domain of PD-L1 regulates its complex glycosylation in living cells.

Whether membrane-anchored PD-L1 homodimerizes in living cells is controversial. The biological significance of the homodimer waits to be expeditiously explored. However, characterization of the membrane-anchored full-length PD-L1 homodimer is challenging, and unconventional approaches are needed. By using genetically incorporated crosslinkers, we showed that full length PD-L1 forms homodimers and tetramers in living cells. Importantly, the homodimerized intracellular domains of PD-L1 play critical roles in its complex glycosylation. Further analysis identified three key arginine residues in the intracellular domain of PD-L1 as the regulating unit. In the PD-L1/PD-L1-3RE homodimer, mutations result in a decrease in the membrane abundance and an increase in the Golgi of wild-type PD-L1. Notably, PD-1 binding to abnormally glycosylated PD-L1 on cancer cells was attenuated, and subsequent T-cell induced toxicity increased. Collectively, our study demonstrated that PD-L1 indeed forms homodimers in cells, and the homodimers play important roles in PD-L1 complex glycosylation and T-cell mediated toxicity.

2,6-DMBQ suppresses cell proliferation and migration via inhibiting mTOR/AKT and p38 MAPK signaling pathways in NSCLC cells.

2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ) is the major bioactive compound found in fermented wheat germ extract. Although fermented wheat germ extract has been reported to show anti-proliferative and anti-metabolic effects in various cancers, the anticancer potential and molecular mechanisms exerted by 2,6-DMBQ have not been investigated in non-small cell lung cancer (NSCLC) cells. Here, we report that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibiting activation of AKT and p38 MAPK. 2,6-DMBQ significantly suppressed anchorage-dependent and independent cell growth. Additionally, 2,6-DMBQ induced G2 phase cell cycle arrest through inhibiting the expression and phosphorylation of cyclin B1 and CDC2, respectively. Furthermore, 2,6-DMBQ strongly suppressed NSCLC cell migration through induction of E-cadherin expression. To determine the molecular mechanism(s) exerted by 2,6-DMBQ upon NSCLC cell lines, various signaling kinases were screened; the results indicate that 2,6-DMBQ strongly inhibits the phosphorylation of AKT and p38 MAPK. Additionally, the growth kinetics of cells treated with an AKT or p38 MAPK inhibitor in combination with 2,6-DMBQ indicate that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibition of AKT and p38 MAPK. Taken together, our results suggest that 2,6-DMBQ is a potential anticancer reagent against NSCLC cells and could be useful for treating lung cancer patients.

2,6-DMBQ is a novel mTOR inhibitor that reduces gastric cancer growth in vitro and in vivo.

BACKGROUND: Fermented wheat germ extract has been reported to exert various pharmacological activities, including anti-oxidant, anti-cell growth and cell apoptosis in various cancer cells. Although 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ) is a benzoquinone compound and found in fermented wheat germ extract, its anticancer effects and molecular mechanism(s) against gastric cancer have not been investigated. METHODS: Anticancer effects of 2,6-DMBQ were determined by MTT, soft agar, cell cycle and Annexin V analysis. Potential candidate proteins were screened via in vitro kinase assay and Western blotting. mTOR knockdown cell lines were established by lentiviral infection with shmTOR. The effect of 2,6-DMBQ on tumor growth was assessed using gastric cancer patient-derived xenograft models. RESULTS: 2,6-DMBQ significantly reduced cell growth and induced G1 phase cell cycle arrest and apoptosis in gastric cancer cells. 2,6-DMBQ reduced the activity of mTOR in vitro. The inhibition of cell growth by 2,6-DMBQ is dependent upon the expression of the mTOR protein. Remarkably, 2,6-DMBQ strongly reduced patient-derived xenograft gastric tumor growth in an in vivo mouse model. CONCLUSIONS: 2,6-DMBQ is an mTOR inhibitor that can be useful for treating gastric cancer. It has therapeutic implications for gastric cancer patients.

Knockdown of Pyruvate Kinase M Inhibits Cell Growth and Migration by Reducing NF-kB Activity in Triple-Negative Breast Cancer Cells.

Altered genetic features in cancer cells lead to a high rate of aerobic glycolysis and metabolic reprogramming that is essential for increased cancer cell viability and rapid proliferation. Pyruvate kinase muscle (PKM) is a rate-limiting enzyme in the final step of glycolysis. Herein, we report that PKM is a potential therapeutic target in triple-negative breast cancer (TNBC) cells. We found that PKM1 or PKM2 is highly expressed in TNBC tissues or cells. Knockdown of PKM significantly suppressed cell proliferation and migration, and strongly reduced S phase and induced G2 phase cell cycle arrest by reducing phosphorylation of the CDC2 protein in TNBC cells. Additionally, knockdown of PKM significantly suppressed NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity by reducing the phosphorylation of p65 at serine 536, and also decreased the expression of NF-kB target genes. Taken together, PKM is a potential target that may have therapeutic implications for TNBC cells.

Scutellarin Suppresses Patient-Derived Xenograft Tumor Growth by Directly Targeting AKT in Esophageal Squamous Cell Carcinoma.

Scutellarin is a flavonoid compound that is found in Scutellaria barbata It has been reported to exhibit anticancer and anti-inflammation activities. However, the anticancer properties of scutellarin and its molecular targets have not been investigated in esophageal squamous cell carcinoma (ESCC). In the current study, we report that scutellarin is a potential AKT inhibitor that suppresses patient-derived xenograft ESCC tumor growth. To identify possible molecular targets of scutellarin, potential candidate proteins were screened by an in vitro kinase assay and Western blotting. We found that scutellarin directly binds to the AKT1/2 proteins and inhibits activities of AKT1/2 in vitro The AKT protein is activated in ESCC tissues and knockdown of AKT significantly suppresses growth of ESCC cells. Scutellarin significantly inhibits anchorage-dependent and independent cell growth and induces G2 phase cell-cycle arrest in ESCC cells. The inhibition of cell growth by scutellarin is dependent on the expression of the AKT protein. Notably, scutellarin strongly suppresses patient-derived xenograft ESCC tumor growth in an in vivo mouse model. Taken together, our data suggest that scutellarin is a novel AKT inhibitor that may prevent progression of ESCC.

Lapachol is a novel ribosomal protein S6 kinase 2 inhibitor that suppresses growth and induces intrinsic apoptosis in esophageal squamous cell carcinoma cells.

Lapachol is a 1,4-naphthoquinone that is isolated from the Bignoniaceae family. It has been reported to exert anti-inflammatory, antibacterial, and anticancer activities. However, the anticancer activity of lapachol and its molecular mechanisms against esophageal squamous cell carcinoma (ESCC) cells have not been fully investigated. Herein, we report that lapachol is a novel ribosomal protein S6 kinase 2 (RSK2) inhibitor that suppresses growth and induces intrinsic apoptosis in ESCC cells. We found that lapachol strongly attenuates downstream signaling molecules of RSK2 in ESCC cells and also directly inhibits RSK2 activity in vitro. The RSK protein is highly activated in ESCC cells and knockdown of RSK2 significantly suppresses anchorage-dependent and anchorage-independent growth of ESCC cells. Additionally, lapachol inhibits anchorage-dependent and anchorage-independent growth of ESCC cells, and the inhibition of cell growth by lapachol is dependent on the expression of RSK2. We also found that lapachol induces mitochondria-mediated cellular apoptosis by activating caspases-3, -7, and PARP, inducing the expression of cytochrome c and BAX by inhibiting downstream molecules of RSK2. Overall, lapachol is a potent RSK2 inhibitor that might be used for chemotherapy against ESCC.

Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo.

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.

3,3'-Diindolylmethane inhibits patient-derived xenograft colon tumor growth by targeting COX1/2 and ERK1/2.

3,3'-Diindolymethane (DIM) is a dimeric condensation product of indole-3-carbinol (I3C) that is found in broccoli and cabbage. Although DIM has been reported to exhibit anticancer properties against multiple tumor types, the direct target proteins of DIM have not been fully investigated. In the present study, we report that DIM is a novel COX1/2 and ERK1/2 inhibitor that suppresses growth of colon cancer in vitro and in vivo. To identify possible molecular targets of DIM, 11 potential candidate proteins were validated by an in vitro kinase or enzyme assay. We found that DIM directly inhibits COX1/2 and ERK1/2 protein activities in vitro. Additionally, the PGE2 production (COX-mediated metabolite) and phosphorylated RSK expression (ERK1/2 direct downstream kinase) were strongly suppressed by DIM in colon cancer cells. The inhibition of cell growth by DIM is dependent on the expression of COX1/2 or ERK1/2 proteins. Notably, oral administration of DIM suppressed patient-derived xenograft colon tumor growth in an in vivo mouse model. Overall these results suggest that DIM is a potent and dual COX1/2 and ERK1/2 inhibitor that might be used for chemotherapy against colon cancer.

Ethyl gallate as a novel ERK1/2 inhibitor suppresses patient-derived esophageal tumor growth.

Ethyl gallate (EG) is a phenolic compound that is isolated from walnut kernels, euphorbia fischeriana, and galla rhois. It has been reported to exhibit antioxidant and anticancer activities. However, EG's effects on esophageal cancer have not yet been investigated. In the present study, we report that EG is a novel ERK1/2 inhibitor that suppresses esophageal cancer growth in vitro and in vivo. EG suppressed anchorage-dependent and -independent esophageal cancer cell growth. The results of in vitro kinase assays and cell-based assays indicated that EG directly binds to and inhibits ERK1 and ERK2 activities and their downstream signaling. Additionally, EG's inhibitory effect on cell growth is resistant to the re-activation of ERK1/2. EG increased G2/M phase cell cycle by reducing the expression of cyclin A2 and cyclin B1. The compound also stimulated cellular apoptosis through the activation of caspases 3 and 7 and inhibition of BCL2 expression. Notably, EG inhibited patient-derived esophageal tumor growth in an in vivo mouse model. These results indicate that EG is an ERK1/2 inhibitor that could be useful for treating esophageal cancer.

Gossypetin is a novel MKK3 and MKK6 inhibitor that suppresses esophageal cancer growth in vitro and in vivo.

Gossypetin as a hexahydroxylated flavonoid found in many flowers and Hibiscus. It exerts various pharmacological activities, including antioxidant, antibacterial and anticancer activities. However, the anticancer capacity of gossypetin has not been fully elucidated. In this study, gossypetin was found to inhibit anchorage-dependent and -independent growth of esophageal cancer cells. To identify the molecular target(s) of gossypetin, various signaling protein kinases were screened and results indicate that gossypetin strongly attenuates the MKK3/6-p38 signaling pathway by directly inhibiting MKK3 and MKK6 protein kinase activity in vitro. Mechanistic investigations showed that arginine-61 in MKK6 is critical for binding with gossypetin. Additionally, the inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Gossypetin caused G2 phase cell cycle arrest and induced intrinsic apoptosis by activating caspases 3 and 7 and increasing the expression of BAX and cytochrome c. Notably, gossypetin suppressed patient-derived esophageal xenograft tumor growth in an in vivo mouse model. Our findings suggest that gossypetin is an MKK3 and MKK6 inhibitor that could be useful for preventing or treating esophageal cancer.

Gossypin inhibits gastric cancer growth by direct targeting of AURKA and RSK2.

Gossypin is a flavone extracted from Hibiscus vitifolius, which has been reported to exhibit anti-inflammatory, antioxidant, and anticancer activities. However, the anticancer properties of gossypin and its molecular mechanism of action against gastric cancer have not been fully investigated. In the present study, we report that gossypin is an Aurora kinase A (AURKA) and RSK2 inhibitor that suppresses gastric cancer growth. Gossypin attenuated anchorage-dependent and anchorage-independent gastric cancer cell growth as well as cell migration. Based on the results of in vitro screening and cell-based assays, gossypin directly binds to and inhibits AURKA and RSK2 activities and their downstream signaling proteins. Gossypin decreased S phase and increased G2/M phase cell cycle arrest by reducing the expression of cyclin A2 and cyclin B1 and the phosphorylation of the CDC protein. Additionally, gossypin also induced intrinsic apoptosis by activating caspases and PARP and increasing the expression of cytochrome c. Our results demonstrate that gossypin is an AURKA and RSK2 inhibitor that could be useful for treating gastric cancer.