Sexual Crossing, Chromosome-Level Genome Sequences, and Comparative Genomic Analyses for the Medicinal Mushroom Taiwanofungus Camphoratus (Syn. Antrodia Cinnamomea, Antrodia Camphorata).

Taiwanofungus camphoratus mushrooms are a complementary and alternative medicine for hangovers, cancer, hypertension, obesity, diabetes, and inflammation. Though Taiwanofungus camphoratus has attracted considerable biotechnological and pharmacological attention, neither classical genetic nor genomic approaches have been properly established for it. We isolated four sexually competent monokaryons from two T. camphoratus dikaryons used for the commercial cultivation of orange-red (HC1) and milky-white (SN1) mushrooms, respectively. We also sequenced, annotated, and comparatively analyzed high-quality and chromosome-level genome sequences of these four monokaryons. These genomic resources represent a valuable basis for understanding the biology, evolution, and secondary metabolite biosynthesis of this economically important mushrooms. We demonstrate that T. camphoratus has a tetrapolar mating system and that HC1 and SN1 represent two intraspecies isolates displaying karyotypic variation. Compared with several edible mushroom model organisms, T. camphoratus underwent a significant contraction in the gene family and individual gene numbers, most notably for plant, fungal, and bacterial cell-wall-degrading enzymes, explaining why T. camphoratus mushrooms are rare in natural environments, are difficult and time-consuming to artificially cultivate, and are susceptible to fungal and bacterial infections. Our results lay the foundation for an in-depth T. camphoratus study, including precise genetic manipulation, improvements to mushroom fruiting, and synthetic biology applications for producing natural medicinal products. IMPORTANCETaiwanofungus camphoratus (Tc) is a basidiomycete fungus that causes brown heart rot of the aromatic tree Cinnamomum kanehirae. The Tc fruiting bodies have been used to treat hangovers, abdominal pain, diarrhea, hypertension, and other diseases first by aboriginal Taiwanese and later by people in many countries. To establish classical genetic and genomic approaches for this economically important medicinal mushroom, we first isolated and characterized four sexually competent monokaryons from two dikaryons wildly used for commercial production of Tc mushrooms. We applied PacBio single molecule, real-time sequencing technology to determine the near-completed genome sequences of four monokaryons. These telomere-to-telomere and gapless haploid genome sequences reveal all genomic variants needed to be studied and discovered, including centromeres, telomeres, retrotransposons, mating type loci, biosynthetic, and metabolic gene clusters. Substantial interspecies diversities are also discovered between Tc and several other mushroom model organisms, including Agrocybe aegerita, Coprinopsis cinerea, and Schizophyllum commune, and Ganoderma lucidum.

Fabrication and characterization of Rhizochitosan and its incorporation with platelet concentrates to promote wound healing.

Composite dressing composed of Rhizochitosan and Regenplex™ to promote wound healing were assessed. Rhizochitosan was fabricated by deacetylation of Rhizochitin, which obtained by simply depigmenting sporangium-free mycelial mattress produced from Rhizopus stolonifer F6. Physicochemical characterizations of Rhizochitosan demonstrated that it contained 13.5% chitosan with a water-absorption ability of 35-fold dry weight and exhibiting hydrogel nature after hydration. In a wound-healing study on SD rats with full-thickness injury, the composite dressing had a better healing effect than those for each individual components and control group and wound even healed as functional tissue instead of scar tissue. The underlying mechanism of the composite beneficial to wound remodeling is likely attributable to a more reduction level of matrix metalloproteinase (MMP)-9 expression in early stage and a higher MMP-2 expression level in a later stage of healing process. Conclusively, the composite dressing demonstrated to be highly beneficial to the healing of full-thickness injury wound.

Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Taiwanofungus camphoratus Combined With Amphotericin B for Metastatic Cancer Patients Unresponsive to or Unwilling to Undergo Chemotherapy: A Pilot Study.

CONTEXT: Taiwanofungus camphoratus is a parasitic mushroom found in the heartwood of Cinnamomum kanehirai and is used as a nutritional supplement. It has an anticancer action, both alone and synergistically with amphotericin B (AmB). OBJECTIVE: The study intended to assess the efficacy of a T camphoratus ethanol extract (TCEE) combined with AmB for patients with metastatic cancer whose cancer did not respond to multiline chemotherapy or who were unwilling to receive chemotherapy. DESIGN: The research team performed a retrospective analysis as a pilot study. SETTING: The study took place at a single hospital (Taipei Medical University Hospital, Taipei, Taiwan). PARTICIPANTS: Participants were 9 patients at the hospital who were terminally ill with metastatic cancer. INTERVENTIONS: The participants had received daily doses of 2-3 g of the TCEE in combination with a weekly dose of 20-25 mg of AmB in 500 cc of 5% glucose water, given intravenously in 4-6 h. OUTCOME MEASURES: Outcome measures included (1) a primary evaluation index measuring the efficacy of the treatment; (2) a measure of tumor burden that was estimated using the response evaluation criteria in solid tumors (RECIST 1.1), (3) a secondary evaluation index measuring survival duration, and (4) safety. RESULTS: The mean treatment time was 54.4 ± 18.3 wk. At the end of the study, 2 patients showed a continued complete response, 1 patient had a continued partial response, and 1 patient showed a stable disease. The other 5 participants had times to progression ranging from 24 to 48 wk, with a mean of 35.6 wk. The mean survival time was 57.8 ± 18.5 wk, and 5 patients were still alive at the end of the study. CONCLUSIONS: For patients whose metastatic cancer did not respond to multiline chemotherapy or who were unwilling to receive chemotherapy, the use of TCEE as an adjuvant therapy to AmB resulted in tumor suppression and a delay in time to disease progression. The preliminary results reported here can be used to guide a future, more extensive clinical study of the combination.

Combination treatment of ergosterol followed by amphotericin B induces necrotic cell death in human hepatocellular carcinoma cells.

The incidence of liver cancer, the second leading cause of cancer-related deaths has increased over the past few decades. Although recent treatments such as sorafenib are promising in patients with advanced hepatocellular carcinoma (HCC), the response rates remain poor thereby warranting the identification of novel therapeutic agents against liver cancer. Herein, we investigated the anti-cancer effect of ergosterol (a secondary metabolite in medicinal fungus) pretreatment followed by amphotericin B (AmB) treatment on liver cancer cell lines. We demonstrated that pretreatment with a nontoxic dose of ergosterol synergistically enhanced the cytotoxicity of AmB in both Hep3B and HepJ5 cells. The combination treatment-mediated suppression of cancer cell viability occurred through necrosis characterized by disrupted cell membrane and significant amounts of debris accumulation. In addition, we also observed a concomitant increase in reactive oxygen species (ROS) and LC3-II levels in HepJ5 cells treated with ergosterol and AmB. Our results suggest that ergosterol-AmB combination treatment effectively induced necrotic cell death in cancer cells, and deserves further evaluation for development as an anti-cancer agent.

Increased risk of chronic obstructive pulmonary disease among patients with Helicobacter pylori infection: a population-based cohort study.

BACKGROUND: Increasing evidence suggests that Helicobacter pylori infection (HPI) may have extragastric manifestations, including the respiratory system. This study investigated the role of HPI in increasing the subsequent risk of chronic obstructive pulmonary disease (COPD) in a nationwide population. METHODS: We conducted this retrospective cohort study using data from the Longitudinal Health Insurance Database, which is derived from the Taiwanese National Health Insurance Research Database. A total of 5941 adults who were newly diagnosed with HPI between 2005 and 2006 were selected. Healthy patients without HPI were selected from the general population and frequency matched as a ratio of 4:1, according to age, sex, and index years. Both cohorts were followed up from the index date to the end of 2011 to measure the incidence of COPD. Cox proportional hazard regression analysis was used to assess the hazard ratio (HR) of COPD between the HPI cohort and non-HPI cohorts. RESULTS: The overall HR of COPD was 1.84 (95% confidence intervals = 1.57-2.17) for the HPI cohort, compared with the non-HPI cohort, after adjusting for age, sex, and comorbidities. Although the incidence of COPD was substantially higher in the elderly participants (age, ≥ 65 years) than that in younger participants, the highest HR (4.05, 95% confidence intervals = 1.39-11.8) of COPD was observed in the youngest (age, 20-49 years) participants. CONCLUSION: In this study, the patients with HPI exhibited a significantly higher risk of COPD than those without HPI did.

Fluconazole induces rapid high-frequency MTL homozygosis with microbiological polymorphism in Candida albicans.

BACKGROUND: Candida albicans, a common fungal pathogen that can cause opportunistic infections, is regarded as an apparently asexual, diploid fungus. A parasexual cycle was previously found between homozygotes with opposite mating type-like loci (MTLa/α). Fluconazole-resistant strains had a higher proportion of MTL homozygotes, whereas MTL homozygous C. albicans was found in only about 3.2% of clinical strains. MTL heterozygotes had a low frequency (1.4 × 10-4) of white-opaque switching to MTL homozygotes in nature. METHODS: Here, a reference C. albicans strain (SC5314) was used in a fluconazole-induced assay to obtain standard opaque MTL homozygous strains and first-generation daughter strains from the fluconazole inhibition zone. Further separation methods were employed to produce second- and third-generation daughter strains. Polymerase chain reaction analysis based on MTL genes was used to define MTL genotypes, and microscopic observations, a flow-cytometric assay, and an antifungal E-test were used to compare microbiological characteristics. RESULTS: MTL homozygotes were found at a high frequency (17 of 35; 48.6%) in fluconazole-induced first-generation daughter strains, as were morphological polymorphisms, decreased DNA content, and modified antifungal drug susceptibility. High-frequency MTL homozygosity was identified inside the fluconazole inhibition zone within 24 hours. The DNA content of fluconazole-induced daughter strains was reduced compared with their progenitor SC5314 and standard MTL homozygous strains. CONCLUSION: Treatment with fluconazole, commonly used to treat invasive candidiasis, inhibited the growth of C. albicans and altered its microbiological characteristics. Our results suggest that fluconazole treatment induces the high frequency of loss of heterozygosity and microbiological polymorphism in C. albicans.

The Ethanolic Extract of Taiwanofungus camphoratus (Antrodia camphorata) Induces Cell Cycle Arrest and Enhances Cytotoxicity of Cisplatin and Doxorubicin on Human Hepatocellular Carcinoma Cells.

Taiwanofungus camphoratus (synonym Antrodia camphorata) is a widely used medicinal fungus in the folk medicine of Taiwan with several pharmacological features such as anti-inflammatory, liver protection, antihypertensive, and antioxidative activities. The ethanolic extract of T. camphoratus (TCEE) which contains abundant bioactive compounds including triterpenoids and polysaccharides also has antitumor effects in various human cancer cell lines. The aims of this study are to clarify the antitumor effects of TCEE on human hepatocellular carcinoma cells and also evaluate the combination drug effects with conventional chemotherapy agents, cisplatin and doxorubicin. In the present study, the TCEE treatment induced cell cycle arrest and suppressed cell growth on both Hep3B and HepJ5 cells. Expression of cell cycle inhibitors, P21 and P27, and activation of apoptosis executer enzyme, caspase-3, were also induced by TCEE. In combination with the chemotherapy agents, TCEE treatment further enhanced the tumor suppression efficiency of cisplatin and doxorubicin. These results together suggested that TCEE is a potential ingredient for developing an integrated chemotherapy for human liver cancer.

Association of inflammatory bowel disease with asthma risk: A nationwide cohort study.

OBJECTIVE: We evaluated the risk of asthma development in adult patients with inflammatory bowel disease (IBD) in a nationwide population. METHODS: A retrospective cohort study was conducted by using data retrieved from the Taiwan National Health Insurance Research Database. Patients, ages 20 year or older, with newly diagnosed IBD between 2000 and 2005 were identified and randomly frequency-matched (based on sex, age, and index year) with four times the number of enrollees without IBD from the general population. Both cohorts were followed up until the end of 2011 to examine the incidence of asthma. Cox proportional hazard regression analysis was used to measure the hazard ratios (HR) of asthma in the IBD cohort compared with that in the non-IBD cohort. RESULTS: The IBD and non-IBD cohorts comprised 5260 patients with IBD and 21,040 participants, respectively. After adjustment for covariates, the IBD cohort exhibited a 1.50-fold increased risk for asthma (HR 1.50, [95% confidence interval {CI}, 1.32-1.71]). Further analysis according to the two major forms of IBD revealed that the adjusted HR of asthma was 1.46 (95% CI, 1.03-2.07) and 1.50 (95% CI, 1.31-1.72) in patients with ulcerative colitis and Crohn's disease, respectively, compared with the non-IBD cohort. CONCLUSION: After adjustment for comorbidities, patients with IBD were associated with a higher subsequent risk of asthma.

Mycelial Mattress from a Sporangia Formation-Delayed Mutant of Rhizopus stolonifer as Wound Healing-Enhancing Biomaterial.

A mycelial mattress of Rhizopus stolonifer obtained from a liquid static culture was utilized for wound dressing and biomedical use. Following screening of mutants induced by UV radiation, F6, exhibiting delayed sporangium formation was selected because its sporangium maturation exhibited a 5-day delay without significant loss of mycelial weight compared to the wild type. The sporangium-free mycelial mattress from the sporangiospore culture of F6 was treated with 1N sodium hydroxide NaOH at 85°C for 2 h to produce a sponge-like membrane named Rhizochitin. The trifluoroacetic acid hydrolysate of Rhizochitin contained 36% N-acetylglucosamine and 53% hexose respectively detected by the Elson-Morgen and phenol-sulfuric acid methods. Results indicated the wound area in rats covered with Rhizochitin was 40% less than that of the uncovered group. Rhizochitin decreased the expression of PDGF in the proliferation stage, increased the expression of TGF-ß in the inflammation and proliferation stages, and increased the expression of VEGF in the inflammation and proliferation stages. Rhizochitin inhibited secretion of matrix metalloproteinase-9 on days 1, 7, 9, and 12 and matrix metalloproteinase-2 on days 3, 7, 9, and 12. It was concluded that Rhizochitin has beneficial properties of biocompatible, biodegradable, and wound healing.

Increased risk of dementia among patients with pulmonary tuberculosis: a retrospective population-based cohort study.

OBJECTIVE: We investigated and compared the risk of dementia development in a cohort of patients with tuberculosis (TB). METHODS: The study involved 6473 patient with newly diagnosed TB, and each patient was randomly frequency matched with 4 people without TB based on age, sex, and index year. The risk of dementia development was analyzed using Cox proportional hazards regression. RESULTS: Among the patients with TB, the overall risk of developing dementia was 1.21-fold significantly higher than the non-TB cohort. In the stratified analysis of dementia risks, only the patients with TB who were male or 50 to 64 years of age exhibited a significantly higher risk of dementia development compared with those without TB. An analysis of the follow-up duration revealed that patients with TB had a 1.78-fold increased risk within 1 year of follow-up. CONCLUSION: Patients with TB have a significantly higher risk of developing dementia than that of the general population.

Adult asthma increases dementia risk: a nationwide cohort study.

BACKGROUND: Studies on the association between adult asthma and dementia are few. We investigated the risk of dementia in patients diagnosed with adult asthma compared with that of people without asthma who were age and sex matched to the study patients. METHODS: We used data from the National Health Insurance Research Database. A total of 12 771 patients with newly diagnosed asthma between 2001 and 2003 were evaluated and 51 084 people without asthma were used as the comparison cohort. Cox proportional hazard regression analysis was used to measure the HR of dementia for the asthmatic cohort, compared with that of the non-asthmatic cohort. RESULTS: The HR of dementia was 1.27 (95% confidence interval (CI) 1.15 to 1.41) for the asthmatic cohort, compared with the non-asthmatic cohort after adjusting for age, sex, comorbidities, annual outpatient department visits and medicine used. The HR of dementia development increased substantially as frequency of asthma exacerbation and hospitalisation increased. CONCLUSIONS: This nationwide cohort study suggests that the risk of dementia development is significantly increased in patients with asthma compared with that of the general population. In addition, dementia risk increases substantially with asthma exacerbation and hospitalisation frequency increases.

Short-term exposure to fluconazole induces chromosome loss in Candida albicans: an approach to produce haploid cells.

Candida albicans is considered to be an obligate diploid fungus. Here, we describe an approach to isolate aneuploids or haploids induced by the short-term (12-16 h) exposure of diploid reference strains SC5314 and CAI4 to the most commonly used antifungal drug, fluconazole, followed by repeated single-cell separation among small morphologically distinct colonies in the inhibition zone. The isolated strains had altered cell morphology and LOH events in the MTL and other marker alleles of the analyzed loci at 8 chromosomes of C. albicans with decreased DNA content. The present study employed next-generation sequencing (NGS) combined flow cytometry analysis of the DNA content to analyze the haploid, autodiploid, and aneuploid strains that arose from the fluconazole treatment instead of using the conventional single nucleotide polymorphism/comparative genome hybridization (SNP/CGH) method. A multiple-alignment tool was also developed based on sequenced data from NGS to establish haplotype mapping for each chromosome of the selected strains. These findings revealed that C. albicans experiences 'concerted chromosome loss' to form strains with homozygous alleles and that it even has a haploid status after short-term exposure to fluconazole. Additionally, we developed a new platform to analyze chromosome copy number using NGS.

Dried fruit of the Luffa sponge as a source of chitin for applications as skin substitutes.

LUFFACHITIN obtained from the residue of the sponge-like dried fruit of Luffa aegyptiaca was developed as a weavable skin substitute in this study. A chemical analysis revealed that LUFFACHITIN was composed of a copolymer containing N-acetyl-glucosamine (~40%) as a major monomer with a filamentary structure as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue of the sponge-like dried fruit of Luffa aegyptiaca after treatment was then woven into a thin, porous membrane by filtration and lyophilization as a skin substitute for conducting wound-healing study on rats. The results indicated that the LUFFACHITIN membrane showed significant wound-healing enhancement (25 days to complete healing) compared to cotton gauze (>30 days), but not inferior to that of SACCHACHITIN. Furthermore, the LUFFACHITIN membrane had advantages of having a high yield, better physical properties for fabrication, and a more attractive appearance.

Quantifying membrane permeability of amphotericin B ion channels in single living cells.

Recently, the structure-function relationships between amphotericin B (AmB) and ergosterol have been solved using synthetic techniques that require a mycosamine-mediated direct binding interaction between AmB and ergosterol to form AmB ion channels. However, studies to directly probe the AmB-induced membrane permeability changes have not been conducted. In the present work, we investigate the following fundamental question: does AmB induce concentration- and time-dependent permeability changes across ergosterol-containing membranes? Herein, we employ fluorescent dyes of known average diameter to quantify the diameters of AmB ion channels. In addition, we take a single-particle tracking approach to define the intracellular microrheology in the absence and presence of AmB ion channels. Present results show that increasing AmB concentration tends to increase the preferential accumulation of AmB ion channels in the presence of the excess membrane-embedded ergosterol. We found that AmB induces time-dependent membrane permeability; increases approaching 50% in both the velocity fluctuations and diffusion coefficients of vesicles occur on the same time scale as the efflux of potassium ions (≅30min). Furthermore, we propose a two-dimensional, semi-regular tessellation model to geometrically assess the pore size of the AmB ion channels in response to the AmB dose. This approach offers one possibility for the design of AmB ion channels with tunable aqueous pore size, which could provide an opportunity to replace damaged membrane water channels of the aquaporin family in future applications.

Damage from periorbital ageing to the multilayered structures and resilience of the skin in Chinese population.

Ageing dynamically disrupts the multilayered supporting components of the skin that are held together by cell adhesion molecules (CAMs). Skin specimens from 33 female Chinese patients undergoing lower blepharoplasty were divided into three age groups and examined by haematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and Elastica-van Gieson (EVG) stains, western blotting, surface electron microscopy (SEM) and biomechanical tension analysis. The SEM density (skin surface topology) showed a negative linear relationship with age. The triangular pattern of the skin surface in the younger group gradually broke down into quadrangular and irregular patterns in the older group. Collagens and elastic fibres in the dermis showed anisotropy and decreased density in the older groups compared with the younger group, especially in the papillary dermis. Anisotropy means that physical properties differ according to the direction of measurement. E-cadherin and integrin αv (whose functions are to bind epidermal and dermal elements respectively) increased and decreased, respectively, in the oldest group. Skin resilience decreased significantly in this group under repetitive stress. In conclusion, a loss of skin surface textures, integrin αv expressions, epidermal-dermal connections and dermal compactness led to the multilayered structure of the skin becoming separated. This in turn decreased resilience during ageing. These findings may therefore explain why aged skins cannot tolerate repetitive facial expressions, and why this action produces further dynamic wrinkles.

Dynamic analysis of modified transcutaneous lower blepharoplasty based on biochemical and biophysical principles.

In this study, we analyzed the key parameters of modified transcutaneous lower blepharoplasty based on multidisciplinary principles (biochemical findings and biophysical wrinkling theory). A total of 408 female patients received our subciliary lower blepharoplasty between March 2002 and January 2010. The severity of the eyebags (dynamic wrinkle numbers and prolapse) was evaluated through preoperative and postoperative photography, whereas the excised lower eyelid skin specimens from 56 patients were investigated with hematoxylin and eosin staining. The modified techniques produced significant improvements in the severity of eyebags in all age groups (P < 0.001). Poor surgical outcome was found to correlate significantly with preoperative dynamic wrinkle numbers (P < 0.001). Age, dynamic wrinkle numbers, and prolapse correlated significantly with dermal fiber density (P = 0.004, 0.000, and 0.000, respectively) but not epidermal, rete ridge, and dermal thickness or the number of rete ridges. In conclusion, modified transcutaneous lower blepharoplasty provides significant improvement to dynamic wrinkles and prolapse in the eyebags. Periorbital aging progressively disturbs the dermal compactness (fiber density) until the structure can no longer hold its integrity at the critical age (around the age of 40).

The 'obligate diploid' Candida albicans forms mating-competent haploids.

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast-hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

Taiwanofungus camphoratus (Syn Antrodia camphorata) extract and amphotericin B exert adjuvant effects via mitochondrial apoptotic pathway.

The use of multiple drugs in cancer therapy increases the efficacy of the potential therapeutic effects. In this study, the authors investigated the adjuvant effects of an ethanol extract of solid-state cultivated Taiwanofungus camphoratus (TCEE) and amphotericin B (AmB) in the human cancer cell lines RPMI7951 and MG63. Taiwanofungus camphoratus is a well-known Chinese medicine in Taiwan, and AmB is a widely used antifungal agent. The authors demonstrated that TCEE pretreatment followed by AmB treatment effectively inhibited cell growth. The combination of sublethal doses of TCEE and AmB revealed a significant growth inhibitory effect in both cell lines. The combination of TCEE and AmB but not AmB alone induced phosphatidylserine externalization and loss of mitochondrial membrane potential. Cell cycle analyses revealed that combination of TCEE and AmB triggered G2/M arrest and significant apoptosis after 48 hours. These effects were greater than those achieved using TCEE or AmB alone. Furthermore, the authors demonstrated that the drugs increased the levels of p21(Cip1/Waf1) and pro-apoptotic protein Bax and reduced the level of anti-apoptotic protein Bcl-2. Taken together, the results showed that the combination treatment of TCEE and AmB displays strong adjuvant effects, which are indicated by the inhibition of cell proliferation in 2 human cancer cell lines, RPMI7951 and MG63. These findings suggest possible therapeutic applications and alternative medicines using this drug combination.