Antifungal Activity of Gallic Acid In Vitro and In Vivo

Gallic acid (GA) is a polyphenol natural compound found in many medicinal plant species, including pomegranate rind (Punica granatum L.), and has been shown to have antiinflammatory and antibacterial properties. Pomegranate rind is used to treat bacterial and fungal pathogens in Uyghur and other systems of traditional medicine, but, surprisingly, the effects of GA on antifungal activity have not yet been reported. In this study, we aimed to investigate the inhibitory effects of GA on fungal strains both in vitro and in vivo. The minimal inhibitory concentration (MIC) was determined by the NCCLS (M38‐A and M27‐A2) standard method in vitro, and GA was found to have a broad spectrum of antifungal activity, with MICs for all the tested dermatophyte strains between 43.75 and 83.33 μg/mL. Gallic acid was also active against three Candida strains, with MICs between 12.5 and 100.0 μg/mL. The most sensitive Candida species was Candida albicans (MIC = 12.5 μg/mL), and the most sensitive filamentous species was Trichophyton rubrum (MIC = 43.75 μg/mL), which was comparable in potency to the control, fluconazole. The mechanism of action was investigated for inhibition of ergosterol biosynthesis using an HPLC‐based assay and an enzyme linked immunosorbent assay. Gallic acid reduced the activity of sterol 14α‐demethylase P450 (CYP51) and squalene epoxidase in the T. rubrum membrane, respectively. In vivo model demonstrated that intraperitoneal injection administration of GA (80 mg/kg d) significantly enhanced the cure rate in a mice infection model of systemic fungal infection. Overall, our results confirm the antifungal effects of GA and suggest a mechanism of action, suggesting that GA has the potential to be developed further as a natural antifungal agent for clinical use. Copyright © 2017 John Wiley & Sons, Ltd.     

Protective Effects of HemoHIM on Immune and Hematopoietic Systems Against γ‐Irradiation

We examined the effect of HemoHIM on the protective efficacy of hematopoietic stem cells and on the recovery of immune cells against sublethal doses of ionizing radiation. Two‐month‐old mice were exposed to γ‐rays at a dose of 8, 6.5, or 5 Gy for a30‐day survival study, endogenous spleen colony formation, or other experiments, respectively. HemoHIM was injected intraperitoneally before and after irradiation. Our results showed that HemoHIM significantly decreased the mortality of sublethally irradiated mice. The HemoHIM administration decreased the apoptosis of bone marrow cells in irradiated mice. On the other hand, HemoHIM increased the formation of endogenous spleen colony in irradiated mice. In irradiated mice, the recovery of total leukocytes in the peripheral blood and lymphocytes in the spleen were enhanced significantly by HemoHIM. Moreover, the function of B cells, T cells, and NK cells regenerated in irradiated mice were significantly improved by the administration of HemoHIM. HemoHIM showed an ideal radioprotector for protecting hematopoietic stem cells and for accelerating the recovery of immune cells. We propose HemoHIM as a beneficial supplement drug during radiotherapy to alleviate adverse radiation‐induced effects for cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC‐3 human prostate cancer. BSN exerted a significant dose‐dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen‐independent PC‐3 cells as compared to androgen‐dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down‐regulation of antiapoptotic and proliferative proteins, activation of caspase‐3, and cleavage of PARP. Additionally, BSN could block broad‐spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3‐myr‐HA‐Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl‐2 also attenuated BSN‐mediated apoptosis in PC‐3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

HPV circulating tumor DNA to monitor the efficacy of anti‐PD‐1 therapy in metastatic squamous cell carcinoma of the anal canal: A case report

Squamous cell carcinoma of the anal canal (SCCA) is a rare HPV‐associated cancer with limited sensitivity to standard chemotherapy. In a phase 2 study, nivolumab, an anti PD‐1 immune checkpoint inhibitor, demonstrated significant efficacy as single‐agent therapy in metastatic SCCA patients. Nevertheless, imaging assessment by standard RECIST criteria of the efficacy of immune therapy can be difficult in some patients due to tumor immune cell infiltration, and biomarkers of treatment efficacy are needed. We have previously developed a quantitative droplet digital PCR (ddPCR) technique to detect HPV circulating tumor DNA (HPV ctDNA), with excellent sensitivity and specificity. Here, we report, for the first time, the kinetics of HPV ctDNA during therapy in a patient with metastatic SCCA, who obtained sustained partial response to single‐agent nivolumab. We observed an early and very significant decrease of HPV ctDNA during therapy from the baseline level of 3713 copies/ml plasma to 564 copies/ml plasma at 4 weeks, and 156 copies/ml at 6 weeks, followed by a plateau. This observation provides proof‐of‐concept that HPV ctDNA can be used as a noninvasive early dynamic biomarker to monitor the efficacy of new immunotherapy agents.     

Characterization of the GNMT‐HectH9‐PREX2 tripartite relationship in the pathogenesis of hepatocellular carcinoma

The pathogenesis of hepatocellular carcinoma (HCC) involves many molecular pathways. Glycine N‐methyltransferase (GNMT) is downregulated in almost all HCC and its gene knockout mice developed HCC with high penetrance. We identified PREX2, a novel PTEN inhibitor, as a GNMT‐interacting protein. Such interaction enhanced degradation of PREX2 through an E3 ligase HectH9‐mediated proteasomal ubiquitination pathway. Depletion of GNMT or HectH9 resulted in AKT activation in a PREX2 dependent manner and enhanced cell proliferation. An elevated PREX2 protein expression accompanied by activation of AKT was observed in the liver of Gnmt knockout mice. PREX2 protein expression was upregulated in 54.9% of human HCC samples, while its mRNA level was comparable in tumor and tumor‐adjacent tissue, suggesting a post‐translational alteration of PREX2 expression. Higher level of PREX2 in the tumor tissues was associated with poorer survival. These results reveal a novel mechanism in which GNMT participates in AKT signaling and HCC tumorigenesis by promoting HectH9‐mediated PREX2 degradation.     

ADAMTS4 and its proteolytic fragments differentially affect melanoma growth and angiogenesis in mice

The metalloproteinase ADAMTS4 (ADAMTS, a disintegrin‐like and metalloproteinase with thrombospondin motif)/aggrecanase‐1 is highly expressed in cartilage and has been implicated in human arthritis. Although abundantly expressed in many types of cancer, its role in cancer remains unknown. In this work, we demonstrate for the first time that full‐length ADAMTS4 and its catalytically more active N‐terminal 53 kDa autocatalytic fragment both promote B16 melanoma growth and angiogenesis in mice. In contrast, overexpression of its catalytically inactive E362A mutant or truncated fragments containing only the C‐terminal ancillary domains suppresses melanoma growth and angiogenesis under similar conditions. Structure–function mapping revealed that the single thrombospondin‐type 1 repeat domain is essential and sufficient for the antitumorigenic activity displayed by the catalytically inactive ADAMTS4 isoforms. Suppression of tumor growth and angiogenesis in mice is accompanied by a significant increase in tumor cell apoptosis, whereas tumor cell proliferation is not affected. Importantly, we identified and demonstrated the presence of novel proteolytic fragments of ADAMTS4 containing essentially only the C‐terminal ancillary domains in cultured cells, and also in human cancer tissues, coexisting with full‐length and catalytically active N‐terminal fragments. The contrasting functions toward tumor growth in mice by the wild‐type proteinase and its catalytically inactive mutant correlate with their contrasting influences on angiogenesis signaling pathway molecules in B16 melanoma in mice. Our results suggest a complex role for ADAMTS4 in cancer with the functional balance of protumorigenic and antitumorigenic isoforms likely to act as an important parameter in determining the net influence of this metalloproteinase on tumor growth in vivo.