Ganoderic acid T, a Ganoderma triterpenoid, modulates the tumor microenvironment and enhances the chemotherapy and immunotherapy efficacy through downregulating galectin-1 levels.

Ganoderic acid T (GAT), a triterpenoid molecule of Ganoderma lucidum, exhibits anti-cancer activity; however, the underlying mechanisms remain unclear. Therefore, in this study, we aimed to investigate the anti-cancer molecular mechanisms of GAT and explore its therapeutic applications for cancer treatment. GAT exhibited potent anti-cancer activity in an ES-2 orthotopic ovarian cancer model in a humanized mouse model, leading to significant alterations in the tumor microenvironment (TME). Specifically, GAT reduced the proportion of α-SMA+ cells and enhanced the infiltration of tumor-infiltrating lymphocytes (TILs) in tumor tissues. After conducting proteomic analysis, it was revealed that GAT downregulates galectin-1 (Gal-1), a key molecule in the TME. This downregulation has been confirmed in multiple cancer cell lines and xenograft tumors. Molecular docking suggested a theoretical direct interaction between GAT and Gal-1. Further research revealed that GAT induces ubiquitination of Gal-1. Moreover, GAT significantly augmented the anti-cancer effects of paclitaxel, thereby increasing intratumoral drug concentrations and reducing tumor size. Combined with immunotherapy, GAT enhanced the tumor-suppressive effects of the anti-programmed death-ligand 1 antibody and increased the proportion of CD8+ cells in the EMT6 syngeneic mammary cancer model. In conclusion, GAT inhibited tumor growth, downregulated Gal-1, modulated the TME, and promoted chemotherapy and immunotherapy efficacy. Our findings highlight the potential of GAT as an effective therapeutic agent for cancer.

Preliminary study on the anti-CO2 stress and growth ability of hypsizygus marmoreus mutant strain HY68.

BACKGROUND: A high concentration of CO2 will stagnate the development of the newly formed primordia of Hypsizygus marmoreus, hinder the development of the mushroom cap, thereby inhibiting the normal differentiation of the fruiting body. Moreover, in the previous experiment, our research group obtained the mutant strain HY68 of H. marmoreus, which can maintain normal fruiting under the condition of high concentration of CO2. Our study aimed to evaluate the CO2 tolerance ability of the mutant strain HY68, in comparison with the starting strain HY61 and the control strain HY62. We analyzed the mycelial growth of these strains under various conditions, including different temperatures, pH levels, carbon sources, and nitrogen sources, and measured the activity of the cellulose enzyme. Additionally, we identified and predicted ß-glucosidase-related genes in HY68 and analyzed their gene and protein structures. RESULTS: Our results indicate that HY68 showed superior CO2 tolerance compared to the other strains tested, with an optimal growth temperature of 25 °C and pH of 7, and maltose and beef paste as the ideal carbon and nitrogen sources, respectively. Enzyme activity assays revealed a positive correlation between ß-glucosidase activity and CO2 tolerance, with Gene14147 identified as the most closely related gene to this activity. Inbred strains of HY68 showed trait segregation for CO2 tolerance. CONCLUSIONS: Both HY68 and its self-bred offspring could tolerate CO2 stress. The fruiting period of the strains resistant to CO2 stress was shorter than that of the strains not tolerant to CO2 stress. The activity of ß-GC and the ability to tolerate CO2 were more closely related to the growth efficiency of fruiting bodies. This study lays the foundation for understanding how CO2 regulates the growth of edible fungi, which is conducive to the innovation of edible fungus breeding methods. The application of the new strain HY68 is beneficial to the research of energy-saving production in factory cultivation.

ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors.

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.

A cysteine-reactive alkyl hydroquinone modifies topoisomerase IIα, enhances DNA breakage, and induces apoptosis in cancer cells.

We previously reported that the anticancer activity of a botanical compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was attributed to topoisomerase (Topo) IIα poisoning and the induction of oxidative damage. HQ17(3) irreversibly inhibits Topo IIα activity in vitro and is more cytotoxic in leukemia HL-60 cells than in Topo IIα-deficient variant HL-60/MX2 cells, which suggests that Topo IIα is a cellular target of HQ17(3). This study further characterizes the molecular mechanisms of the anticancer activity of HQ17(3). Proteomic analyses indicated that HQ17(3) reacted with Cys-427, Cys-733, and Cys-997 of recombinant Topo IIα in vitro, whereas it reacted with Cys-427 of cellular Topo IIα in Huh7 hepatoma cells. The modification of HQ17(3) inhibited Topo IIα catalytic activity, increased the Topo IIα-DNA cleavage complex, and caused the accumulation of DNA breakage. In Huh7 cells, HQ17(3) treatment caused prompt inhibition of DNA synthesis and consequently induced the expression of DNA damage-related genes DDIT3, GADD45A, and GADD45G. Topo IIα inhibition, apoptosis, and oxidative stress were found to account for cytotoxicity caused by HQ17(3). Pretreatment of Huh7 cells with N-acetylcysteine (NAC) partially attenuated mitochondrial membrane damage, DNA breakage, and caspase activation. However, NAC pretreatment did not diminish HQ17(3)-induced cell death. These results suggest that the anticancer activity of HQ17(3) is attributed significantly to Topo IIα poisoning. The structural feature of HQ17(3) can be used as a model for the design of Topo IIα inhibitors and anticancer drugs.

Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor.

RATIONALE: Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown. OBJECTIVES: To examine the role of EMT regulators in resistance to gefitinib. METHODS: The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells. CONCLUSIONS: Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Arachidin-1, a peanut stilbenoid, induces programmed cell death in human leukemia HL-60 cells.

The stilbenoids, arachidin-1 (Ara-1), arachidin-3, isopentadienylresveratrol, and resveratrol, have been isolated from germinating peanut kernels and characterized as antioxidant and anti-inflammatory agents. Resveratrol possesses anticancer activity, and studies have indicated that it induces programmed cell death (PCD) in human leukemia HL-60 cells. In this study, the anticancer activity of these stilbenoids was determined in HL-60 cells. Ara-1 had the highest efficacy in inducing PCD in HL-60 cells, with an approximately 4-fold lower EC50 than resveratrol. Ara-1 treatment caused mitochondrial membrane damage, activation of caspases, and nuclear translocation of apoptosis-inducing factor, resulting in chromosome degradation and cell death. Therefore, Ara-1 induces PCD in HL-60 cells through caspase-dependent and caspase-independent pathways. Ara-1 demonstrates its efficacy as an anticancer agent by inducing caspase-independent cell death, which is an alternative death pathway of cancer cells with mutations in key apoptotic genes. These findings indicate the merits of screening other peanut stilbenoids for anticancer activity.

Identification of an Alkylhydroquinone from Rhus succedanea as an Inhibitor of Tyrosinase and Melanogenesis.

The alkylhydroquinone 10'(Z)-heptadecenylhydroquinone [HQ17(1)], isolated from the sap of the lacquer tree Rhus succedanea, was found to inhibit the activity of tyrosinase and to suppress melanin production in animal cells. The IC50 of HQ17(1) as a tyrosinase inhibitor was 37 microM versus 70 microM for hydroquinone (HQ), a known inhibitor of tyrosinase and melanogenesis. For the inhibition of melanin production in mouse B16 melanoma cells, the EC50 of HQ17(1) was 40 microM versus 124 microM for HQ. HQ17(1) induced much less oxidative stress than did HQ. The effectiveness in inhibiting melanin production could be mimicked by intermittent exposure of cells to HQ17(1). The potent inhibitory effects of HQ17(1) on tyrosinase activity and melanin production are likely due to its heptadecenyl chain, which facilitates retention of the compound in cell membrane compartments and may impede oxidation of the hydroquinone ring. As tyrosinase activity accounts for postharvest browning of botanical products and animal skin melanogenesis, HQ17(1) could be useful for the preservation of these products or as a skin-whitening cosmetic.

Bioengineering a humanized acne microenvironment model: proteomics analysis of host responses to Propionibacterium acnes infection in vivo.

Acne is a human disease of the sebaceous hair follicle. Unlike humans, most animals produce little or no triglycerides in hair follicles to harbor Propionibacterium acnes a fact that has encumbered the development of novel treatments for acne lesions. Although genetic mutant mice with acne-like skins have been used for screening anti-acne drugs, the mice generally have deficits in immune system that turns out to be inappropriate to generate antibodies for developing acne vaccines. Here, we employed a bioengineering approach using a tissue chamber integrated with a dermis-based cell-trapped system (DBCTS) to mimic the in vivo microenvironment of acne lesions. Human sebocyte cell lines were grown in DBCTS as a scaffold and inserted into a perforated tissue chamber. After implantation of a tissue chamber bearing human sebocytes into ICR mice, P. acnes or PBS was injected into a tissue chamber to induce host immune response. Infiltrated cells such as neutrophils and macrophages were detectable in tissue chamber fluids. In addition, a proinflammatory cytokine macrophage-inflammatory protein-2 (MIP-2) was elevated after P. acnes injection. In tissue chamber fluids, 13 proteins including secreted proteins and cell matrix derived from mouse, human cells or P. acnes were identified by proteomics using isotope-coded protein label (ICPL) coupled to nano-LC-MS analysis. After P. acnes infection, four proteins including fibrinogen, alpha polypeptide, fibrinogen beta chain, S100A9, and serine protease inhibitor A3K showed altered concentrations in the mimicked acne microenvironment. The bioengineered acne model thus provides an in vivo microenvironment to study the interaction of host with P. acnes and offers a unique set-up for screening novel anti-acne drugs and vaccines.

Antibodies elicited by inactivated propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris.

Propionibacterium acnes is a key pathogen involved in the progression of inflammation in acne vulgaris. We examined whether vaccination against P. acnes suppressed P. acnes-induced skin inflammation. Inactivation of P. acnes with heat was employed to create a P. acnes-based vaccine. Intranasal immunization in mice with this inactivated vaccine provoked specific antibodies against P. acnes. Most notably, immunization with inactivated vaccines generated in vivo protective immunity against P. acnes challenge and facilitated the resolution of ear inflammation in mice. In addition, antibodies elicited by inactivated vaccines effectively neutralized the cytotoxicity of P. acnes and attenuated the production of proinflammatory cytokine IL-8 in human sebocyte SZ95 cells. Intranasal immunization using heat-inactivated P. acnes-based vaccines provided a simple modality to develop acne vaccines. These observations highlight the concept that development of vaccines targeting microbial products may represent an alternative strategy to conventional antibiotic therapy.

Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris.

BACKGROUND: Acne vulgaris afflicts more than fifty million people in the United State and the severity of this disorder is associated with the immune response to Propionibacterium acnes (P. acnes). Systemic therapies for acne target P. acnes using antibiotics, or target the follicle with retinoids such as isotretinoin. The latter systemic treatment is highly effective but also carries a risk of side effects including immune imbalance, hyperlipidemia, and teratogenicity. Despite substantial research into potential new therapies for this common disease, vaccines against acne vulgaris are not yet available. METHODS AND FINDINGS: Here we create an acne vaccine targeting a cell wall-anchored sialidase of P. acnes. The importance of sialidase to disease pathogenesis is shown by treatment of a human sebocyte cell line with recombinant sialidase that increased susceptibility to P. acnes cytotoxicity and adhesion. Mice immunized with sialidase elicit a detectable antibody; the anti-sialidase serum effectively neutralized the cytotoxicity of P. acnes in vitro and P. acnes-induced interleukin-8 (IL-8) production in human sebocytes. Furthermore, the sialidase-immunized mice provided protective immunity against P. acnes in vivo as this treatment blocked an increase in ear thickness and release of pro-inflammatory macrophage inflammatory protein (MIP-2) cytokine. CONCLUSIONS: Results indicated that acne vaccines open novel therapeutic avenues for acne vulgaris and other P. acnes-associated diseases.

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning.

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo IIalpha activity through the accumulation of Topo II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC50 of 0.9 microM, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC50 of 9.6 microM, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 microM. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC50 about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design.

A novel immunogenic spore coat-associated protein in Bacillus anthracis: characterization via proteomics approaches and a vector-based vaccine system.

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An Escherichia coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines.

Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes.

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time-consuming task to examine a protein's immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet-c) was used as a representative antigen to establish this platform. A cell wall-anchoring sialidase-like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector-based vaccine by overexpressing Tet-c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet-c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector-based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet-c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV-irradiated E. coli vector-based vaccines. The antibody production of Tet-c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes-associated diseases.

Quantitative proteomes and in vivo secretomes of progressive and regressive UV-induced fibrosarcoma tumor cells: mimicking tumor microenvironment using a dermis-based cell-trapped system linked to tissue chamber.

The alterations of tumor proteome and/or in vivo secretome created by host-tumor cell interaction may be crucial factors for tumors to undergo progression or regression in a host system. Two UV-induced fibrosarcoma tumor cell lines (UV-2237 progressive cells and UV-2240 regressive cells) were used as models to address this issue. Hundreds of proteins including in vivo secretome have been identified and quantified via an isotope-coded protein label (ICPL) in conjunction with high-throughput NanoLC-LTQ MS analysis. A newly designed technology using a dermis-based cell-trapped system was employed to encapsulate and grow 3-D tumor cells. A tissue chamber inserted with a tumor cell-trapped dermis was implanted into mice to mimic the tumor microenvironment. The in vivo secretome created by host-tumor interaction was characterized from samples collected from tissue chamber fluids via ICPL labeling mass spectrometric analysis. Twenty-five proteins including 14-3-3 proteins, heat shock proteins, profilin-1, and a fragment of complement C3 with differential expression in proteomes of UV-2237 and UV-2240 cells were revealed. Three secreted proteins including myeloperoxidase, alpha-2-macroglobulin, and a vitamin D-binding protein have different abundances in the in vivo secretome in response to UV-2237 and UV-2240 cells. Differential tumor proteomes and in vivo secretome were thus accentuated as potential therapeutic targets to control tumor growth.

Ganoderiol F, a ganoderma triterpene, induces senescence in hepatoma HepG2 cells.

Ganoderiol F (GolF), a tetracyclic triterpene, was isolated from Ganoderma amboinense and found to induce senescence of cancer cell lines. GolF induced growth arrest of cancer cell lines HepG2, Huh7 and K562, but exerted much less effect in hepatoma Hep3B cells and normal lung fibroblast MRC5 cells, and no effect on peripheral blood mononuclear cells. GolF treatment of the cancer cells, with the exception of Hep3B, resulted in prompt inhibition of DNA synthesis and arrest of cell progression cycle in G1 phase. Short-term exposure of HepG2 cells to GolF temporarily arrested progression of the cell cycle; cell growth was recovered if the drug was withdrawn from the medium after a 24-h exposure. After 18 days of continuous treatment of HepG2 cells with 30 muM GolF, over 50% of cells were found to be enlarged and flattened, and were beta-galactosidase positive phenotypes of senescent cells. GolF was found to inhibit activity of topoisomerases in vitro, which may contribute to the inhibition of cellular DNA synthesis. Activation of the mitogen-activated protein kinase EKR and up-regulation of cyclin-dependent kinase inhibitor p16 were found in early stages of GolF treatment and were presumed to cause cell-cycle arrest and trigger premature senescence of HepG2 cells. The growth-arrest and senescence induction capability on cancer cells suggest anticancer potential of GolF.

Antioxidative and cytotoxic compounds extracted from the sap of Rhus succedanea.

Two new antioxidative and cytotoxic compounds, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone (1) and 10'(Z),13'(E)-heptadecadienylhydroquinone (2), as well as the known 10'(Z)-heptadecenylhydroquinone (3), were isolated from an EtOH extract of the sap of Rhus succedanea. The structures were elucidated by spectral analyses. These compounds showed antioxidative and cytotoxic activities against five cancer cell lines.

A tumor cell growth inhibitor from Saposhnikovae divaricata.

In the present study, we tested ethanolic extracts from 10 Chinese herbs for their effects on K562, Raji, Wish, HeLa, Calu-1, and Vero tumor cells proliferation. On a percentage basis, panaxynol purified from Saposhnikovae divaricata had the highest inhibitory activity on various tumor cells proliferation. Cell-cycle analysis indicated that panaxynol arrested the cell cycle progression of tumor cells from the G1 transition to the S phase. In an attempt to further localize the point in the cell cycle where arrest occurred, gene expression of cyclin E, a key regulatory event leading to the G1/S boundary was examined. Results indicated that the levels of cyclin E mRNA in various tumor cells were decreased by panaxynol. Thus, the suppressant effects of panaxynol on proliferation of various tumor cells appeared to be mediated, at least in part, through impairments of cyclin E mRNA levels and arresting cell cycle progression in the cells.