Polymorphism of Polymeric Amino Acid Regions in Fungal Proteins and Correlation with Altered Echinocandin and Azole Susceptibility.

Polymorphism of polymeric amino acid (polyX) regions within fungal proteins represents a potential mechanism for rapid genotypic adaptation to environmental pressures, including antifungal exposure. Polyglutamine (polyQ) was the most abundant repeat in the proteomes of 8 diverse fungal species and was preferentially found in regulatory proteins. In Candida glabrata, polyX polymorphisms were characterized in 36 proteins implicated in azole or echinocandin susceptibility. General transcriptional repressor Tup1A exhibited Q44/Q45 polymorphism, and Hog1 signaling component Ssk2 exhibited N44/N45 polymorphism in phylogenetically matched echinocandin- and azole-susceptible/resistant strains, respectively.

Cryptolepine inhibits melanoma cell growth through coordinated changes in mitochondrial biogenesis, dynamics and metabolic tumor suppressor AMPKα1/2-LKB1.

Dysregulated mitochondrial dynamics and biogenesis have been associated with various pathological conditions including cancers. Here, we assessed the therapeutic effect of cryptolepine, a pharmacologically active alkaloid derived from the roots of Cryptolepis sanguinolenta, on melanoma cell growth. Treatment of human melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with cryptolepine (1.0, 2.5, 5.0 and 7.5 µM) for 24 and 48 h significantly (P < 0.001) inhibited the growth of melanoma cells but not normal melanocytes. The inhibitory effect of cryptolepine was associated with loss of mitochondrial membrane potential and reduced protein expression of Mfn1, Mfn2, Opa1 and p-Drp1 leading to disruption of mitochondrial dynamics. A decrease in the levels of ATP and mitochondrial mass were associated with activation of the metabolic tumor suppressor AMPKα1/2-LKB1, and a reduction in mTOR signaling. Decreased expression of SDH-A and COX-I demonstrated that cryptolepine treatment reduced mitochondrial biogenesis. In vivo treatment of A375 xenograft-bearing nude mice with cryptolepine (10 mg/Kg body weight, i.p.) resulted in significant inhibition of tumor growth, which was associated with disruption of mitochondrial dynamics and a reduction in mitochondrial biogenesis. Our study suggests that low toxicity phytochemicals like cryptolepine may be tested for the treatment of melanoma.

Honokiol inhibits ultraviolet radiation-induced immunosuppression through inhibition of ultraviolet-induced inflammation and DNA hypermethylation in mouse skin.

Ultraviolet (UV) radiation exposure induces immunosuppression, which contributes to the development of cutaneous malignancies. We investigated the effects of honokiol, a phytochemical found in plants of the genus Magnolia, on UVB-induced immunosuppression using contact hypersensitivity (CHS) as a model in C3H/HeN mice. Topical application of honokiol (0.5 and 1.0 mg/cm2 skin area) had a significant preventive effect on UVB-induced suppression of the CHS response. The inflammatory mediators, COX-2 and PGE2, played a key role in this effect, as indicated by honokiol inhibition of cyclooxygenase-2 (COX-2) expression and PGE2 production in the UVB-exposed skin. Honokiol application also inhibited UVB-induced DNA hypermethylation and its elevation of the levels of TET enzyme, which is responsible for DNA demethylation in UVB-exposed skin. This was consistent with the restoration of the CHS response in mice treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine, after UVB exposure. There was no significant difference in the levels of inhibition of UVB-induced immunosuppression amongst mice that were treated topically with available anti-cancer drugs (imiquimod and 5-fluorouracil). This study is the first to show that honokiol has the ability to inhibit UVB-induced immunosuppression in preclinical model and, thus, has potential for use as a chemopreventive strategy for UVB radiation-induced malignancies.

Dietary grape seed proanthocyanidins inactivate regulatory T cells by promoting NER-dependent DNA repair in dendritic cells in UVB-exposed skin.

Ultraviolet B (UVB) radiation induces regulatory T cells (Treg cells) and depletion of these Treg cells alleviates immunosuppression and inhibits photocarcinogenesis in mice. Here, we determined the effects of dietary grape seed proanthocyanidins (GSPs) on the development and activity of UVB-induced Treg cells. C3H/HeN mice fed a GSPs (0.5%, w/w)-supplemented or control diet were exposed to UVB (150 mJ/cm2) radiation, sensitized to 2,4-dinitrofluorobenzene (DNFB) and sacrificed 5 days later. FACS analysis indicated that dietary GSPs decrease the numbers of UVB-induced Treg cells. ELISA analysis of cultured sorted Treg cells indicated that secretion of immunosuppressive cytokines (interleukin-10, TGF-ß) was significantly lower in Treg cells from GSPs-fed mice. Dietary GSPs also enhanced the ability of Treg cells from wild-type mice to stimulate production of IFNγ by T cells. These effects of dietary GSPs on Treg cell function were not found in XPA-deficient mice, which are incapable of repairing UVB-induced DNA damage. Adoptive transfer experiments revealed that naïve recipients that received Treg cells from GSPs-fed UVB-irradiated wild-type donors that had been sensitized to DNFB exhibited a significantly higher contact hypersensitivity (CHS) response to DNFB than mice that received Treg cells from UVB-exposed mice fed the control diet. There was no significant difference in the CHS response between mice that received Treg cells from UVB-irradiated XPA-deficient donors fed GSPs or the control diet. Furthermore, dietary GSPs significantly inhibited UVB-induced skin tumor development in wild-type mice but not in XPA-deficient mice. These results suggest that GSPs inactivate Treg cells by promoting DNA repair in dendritic cells in UVB-exposed skin.

Dietary proanthocyanidins prevent ultraviolet radiation-induced non-melanoma skin cancer through enhanced repair of damaged DNA-dependent activation of immune sensitivity.

Numerous plant products have been used to prevent and manage a wide variety of diseases for centuries. These products are now considered as promising options for the development of more effective and less toxic alternatives to the systems of medicine developed primarily in developed countries in the modern era. Grape seed proanthocyanidins (GSPs) are of great interest due to their anti-carcinogenic effects that have been demonstrated using various tumor models including ultraviolet (UV) radiation-induced non-melanoma skin cancer. In a pre-clinical mouse model supplementation of a control diet (AIN76A) with GSPs at concentrations of 0.2% and 0.5% (w/w) significantly inhibits the growth and multiplicity of UVB radiation-induced skin tumors. In this review, we summarize the evidence that this inhibition of UVB-induced skin tumor development by dietary GSPs is mediated by a multiplicity of coordinated effects including: (i) Promotion of the repair of damaged DNA by nuclear excision repair mechanisms, and (ii) DNA repair-dependent stimulation of the immune system following the functional activation of dendritic cells and effector T cells. Dietary GSPs hold promise for the development of an effective alternative strategy for the prevention of excessive solar UVB radiation exposure-induced skin diseases including the risk of non-melanoma skin cancer in humans.

Loss of INK4a/Arf gene enhances ultraviolet radiation-induced cutaneous tumor development.

The CDKN2A locus encodes for tumor suppressor genes p16INK4a and p14Arf which are frequently inactivated in human skin tumors. The purpose of this study was to determine the relationship between loss of INK4a/Arf activity and inflammation in the development of ultraviolet (UV) radiation-induced skin tumors. Panels of INK4a/Arf-/- mice and wild-type (WT) mice were treated with a single dose of UVB (200 mJ/cm2 ). For long-term studies, these mice were irradiated with UVB (200 mJ/cm2 ) three times weekly for 30 weeks. At the end of the experiment, tissues were harvested from mice and assayed for inflammatory biomarkers and cytokines. A single dose of UVB resulted in a significant increase in reactive oxygen species (ROS) and 8-dihydroxyguanosine (8-oxo-dG) lesions in INK4a/Arf-/- mice compared to WT mice. When subjected to chronic UVB, we found that 100% of INK4a/Arf-/- mice had tumors, whereas there were no tumors in WT controls after 24 weeks of UVB exposure. The increase in tumor development correlated with a significant increase in nuclear factor (NF)-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2 ) and its receptors both in UVB-exposed skin and in the tumors. A significant increase was seen in inflammatory cytokines in skin samples of INK4a/Arf-/- mice following treatment with chronic UVB radiation. Furthermore, significantly more CD11b+ Gr1+ myeloid cells were present in UVB-exposed INK4a/Arf-/- mice compared to WT mice. Our data indicate that by targeting UVB-induced inflammation, it may be possible to prevent UVB-induced skin tumors in individuals that carry CDKN2A mutation.

Crosstalk Among UV-Induced Inflammatory Mediators, DNA Damage and Epigenetic Regulators Facilitates Suppression of the Immune System.

The suppression of the immune system by overexposure to ultraviolet (UV) radiation has been implicated in the initiation and progression of photocarcinogenesis. Numerous changes occur in the skin on UVB exposure, including the generation of inflammatory mediators, DNA damage, epigenetic modifications, and migration and functional alterations in the antigen-presenting dendritic cells. Although each of these alterations can elicit a cascade of events that have the potential to modulate immune sensitivity alone, there is emerging evidence that there is considerable crosstalk between these cascades. The development of an understanding of UV-induced changes in the skin that culminate in UV-induced immunosuppression, which has been implicated in the risk of nonmelanoma skin cancer, as a network of events has implications for the development of more effective chemopreventive strategies. In the current review article, we discuss the evidence of interactions between the various molecular targets and signaling mechanisms associated with UV-induced immunosuppression.

Interstitial Lung Disease in India. Results of a Prospective Registry.

RATIONALE: Interstitial lung disease (ILD) is a heterogeneous group of acute and chronic inflammatory and fibrotic lung diseases. Existing ILD registries have had variable findings. Little is known about the clinical profile of ILDs in India. OBJECTIVES: To characterize new-onset ILDs in India by creating a prospective ILD using multidisciplinary discussion (MDD) to validate diagnoses. METHODS: Adult patients of Indian origin living in India with new-onset ILD (27 centers, 19 Indian cities, March 2012-June 2015) without malignancy or infection were included. All had connective tissue disease (CTD) serologies, spirometry, and high-resolution computed tomography chest. ILD pattern was defined by high-resolution computed tomography images. Three groups independently made diagnoses after review of clinical data including that from prompted case report forms: local site investigators, ILD experts at the National Data Coordinating Center (NDCC; Jaipur, India) with MDD, and experienced ILD experts at the Center for ILD (CILD; Seattle, WA) with MDD. Cohen's κ was used to assess reliability of interobserver agreement. MEASUREMENTS AND MAIN RESULTS: A total of 1,084 patients were recruited. Final diagnosis: hypersensitivity pneumonitis in 47.3% (n = 513; exposure, 48.1% air coolers), CTD-ILD in 13.9%, and idiopathic pulmonary fibrosis in 13.7%. Cohen's κ: 0.351 site investigator/CILD, 0.519 site investigator/NDCC, and 0.618 NDCC/CILD. CONCLUSIONS: Hypersensitivity pneumonitis was the most common new-onset ILD in India, followed by CTD-ILD and idiopathic pulmonary fibrosis; diagnoses varied between site investigators and CILD experts, emphasizing the value of MDD in ILD diagnosis. Prompted case report forms including environmental exposures in prospective registries will likely provide further insight into the etiology and management of ILD worldwide.

Cryptolepine, a Plant Alkaloid, Inhibits the Growth of Non-Melanoma Skin Cancer Cells through Inhibition of Topoisomerase and Induction of DNA Damage.

Topoisomerases have been shown to have roles in cancer progression. Here, we have examined the effect of cryptolepine, a plant alkaloid, on the growth of human non-melanoma skin cancer cells (NMSCC) and underlying mechanism of action. For this purpose SCC-13 and A431 cell lines were used as an in vitro model. Our study reveals that SCC-13 and A431 cells express higher levels as well as activity of topoisomerase (Topo I and Topo II) compared with normal human epidermal keratinocytes. Treatment of NMSCC with cryptolepine (2.5, 5.0 and 7.5 µM) for 24 h resulted in marked decrease in topoisomerase activity, which was associated with substantial DNA damage as detected by the comet assay. Cryptolepine induced DNA damage resulted in: (i) an increase in the phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX; (ii) activation of p53 signaling cascade, including enhanced protein expressions of p16 and p21; (iii) downregulation of cyclin-dependent kinases, cyclin D1, cyclin A, cyclin E and proteins involved in cell division (e.g., Cdc25a and Cdc25b) leading to cell cycle arrest at S-phase; and (iv) mitochondrial membrane potential was disrupted and cytochrome c released. These changes in NMSCC by cryptolepine resulted in significant reduction in cell viability, colony formation and increase in apoptotic cell death.

Emerging Phytochemicals for the Prevention and Treatment of Head and Neck Cancer.

Despite the development of more advanced medical therapies, cancer management remains a problem. Head and neck squamous cell carcinoma (HNSCC) is a particularly challenging malignancy and requires more effective treatment strategies and a reduction in the debilitating morbidities associated with the therapies. Phytochemicals have long been used in ancient systems of medicine, and non-toxic phytochemicals are being considered as new options for the effective management of cancer. Here, we discuss the growth inhibitory and anti-cell migratory actions of proanthocyanidins from grape seeds (GSPs), polyphenols in green tea and honokiol, derived from the Magnolia species. Studies of these phytochemicals using human HNSCC cell lines from different sub-sites have demonstrated significant protective effects against HNSCC in both in vitro and in vivo models. Treatment of human HNSCC cell lines with GSPs, (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic component of green tea or honokiol reduced cell viability and induced apoptosis. These effects have been associated with inhibitory effects of the phytochemicals on the epidermal growth factor receptor (EGFR), and cell cycle regulatory proteins, as well as other major tumor-associated pathways. Similarly, the cell migration capacity of HNSCC cell lines was inhibited. Thus, GSPs, honokiol and EGCG appear to be promising bioactive phytochemicals for the management of head and neck cancer.

Honokiol, an Active Compound of Magnolia Plant, Inhibits Growth, and Progression of Cancers of Different Organs.

Honokiol (C18H18O2) is a biphenolic natural product isolated from the bark and leaves of Magnolia plant spp. During the last decade or more, honokiol has been extensively studied for its beneficial effect against several diseases. Investigations have demonstrated that honokiol possesses anti-carcinogenic, anti-inflammatory, anti-oxidative, anti-angiogenic as well as inhibitory effect on malignant transformation of papillomas to carcinomas in vitro and in vivo animal models without any appreciable toxicity. Honokiol affects multiple signaling pathways, molecular and cellular targets including nuclear factor-κB (NF-κB), STAT3, epidermal growth factor receptor (EGFR), cell survival signaling, cell cycle, cyclooxygenase and other inflammatory mediators, etc. Its chemopreventive and/or therapeutic effects have been tested against chronic diseases, such as cancers of different organs. In this chapter, we describe and discuss briefly the effect of honokiol against cancers of different organs, such as melanoma, non-melanoma, lung, prostate, breast, head and neck squamous cell carcinoma, urinary bladder cancer, gastric cancer, and neuroblastoma, etc. and describe its mechanism of action including various molecular and cellular targets. Although more rigorous in vivo studies are still needed, however it is expected that therapeutic effects and activities of honokiol may help in the development and designing of clinical trials against chronic diseases in human subjects.

Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression.

Lung cancer and its metastasis is the leading cause of cancer-related mortality world-wide. Non-small cell lung cancer (NSCLC) accounts for about 90% of total lung cancer cases. Despite advancements in therapeutic approaches, only limited improvement has been achieved. Therefore, alternative strategies are required for the management of lung cancer. Here we report the chemotherapeutic effect of silymarin, a phytochemical from milk thistle plant (Silybum marianum L. Gaertn.), on NSCLC cell migration using metastatic human NSCLC cell lines (A549, H1299 and H460) together with the molecular targets underlying these effects. Using an in vitro cell migration assay, we found that treatment of human NSCLC cells (A549, H1299 and H460) with silymarin (0, 5, 10 and 20 µg/mL) for 24 h resulted in concentration-dependent inhibition of cell migration, which was associated with the inhibition of histone deacetylase (HDAC) activity and reduced levels of class 1 HDAC proteins (HDAC1, HDAC2, HDAC3 and HDAC8) and concomitant increases in the levels of histone acetyltransferase activity (HAT). Known HDAC inhibitors (sodium butyrate and trichostatin A) exhibited similar patterns of therapeutic effects on the lung cancer cells. Treatment of A549 and H460 cells with silymarin reduced the expression of the transcription factor ZEB1 and restored expression of E-cadherin. The siRNA knockdown of ZEB1 also reduced the expression of HDAC proteins and enhanced re-expression of the levels of E-cadherin in NSCLC cells. MicroRNA-203 (miR-203) acts as a tumor suppressor, regulates tumor cell invasion and is repressed by ZEB1 in cancer cells. Silymarin treatment restored the levels of miR-203 in NSCLC cells. These findings indicate that silymarin can effectively inhibit lung cancer cell migration and provide a coherent model of its mechanism of action suggesting that silymarin may be an important therapeutic option for the prevention or treatment of lung cancer metastasis when administered either alone or with standard cancer therapeutic drugs.