Induction of apoptosis by ginsenoside Rk1 in SK-MEL-2-human melanoma.

Ginsenosides are active compounds isolated from Panax ginseng Meyer. Among these ginsenosides, less polar ginsenosides such as ginsenoside Rg3 and ginsenoside Rh2 have been demonstrated to have tumor inhibitory effects because of their cytotoxicity. In this study, we evaluated the apoptotic effects of ginsenoside Rk1 in SK-MEL-2 human melanoma. Ginsenoside Rk1 isolated from red ginseng is one of the novel ginsenosides that shows strong cytotoxicity compared to ginsenoside Rg3 in dose- and time-dependent manners. The results of DNA fragmentation, 4',6-diamidino-2-phenylindole staining, and flow cytometric analysis are corroborated that ginsenoside Rk1 induced apoptosis in SK-MEL-2 cells. Western blot analysis revealed up-regulation of Fas, FasL, and Bax protein expression and down-regulation of procaspase-8, procaspase-3, mutant p53 and Bcl-2 protein expression. These findings suggest that ginsenoside Rk1 might be a promising compound to induce apoptosis through both extrinsic and intrinsic pathways in SK-MEL-2 cells.

Comparative study of diabetes mellitus resolution according to reconstruction type after gastrectomy in gastric cancer patients with diabetes mellitus.

BACKGROUND: This study was conducted to investigate diabetes mellitus (DM) resolution after gastrectomy according to reconstruction type in gastric cancer patients. METHODS: Two hundred twenty-nine gastric cancer patients with DM who underwent gastrectomy with curative intent from May 2003 to December 2009 were enrolled. Changes in fasting blood sugar concentration and the dosage of oral hyperglycemic agents or insulin were compared between reconstruction types. RESULTS: The numbers of patients who underwent distal gastrectomy with a Billroth I (BI), Billroth II (BII), Roux-en-Y gastrojejunostomy (RYGJ), or total gastrectomy with Roux-en-Y esophagojejunostomy (RYEJ) were 119 (51.7%), 54 (23.5%), 40 (17.4%), and 16 (6.9%), respectively. DM remitted in 45 (19.7%) patients: 18 BI patients (15.1%), 11 BII patients (20.3%), 8 RYGJ patients (20.0%), and 8 RYEJ patients (50.0%). DM improved in 85 (37.1%) patients: 41 BI patients (34.4%), 25 BII patients (46.2%), 15 RYGJ patients (37.5%), and 4 RYEJ patients (25.0%). The DM remission or improvement rate was higher in the duodenal bypass group (BII, RYGJ, RYEJ) than in the BI group (67.2% vs. 49.5%, P = 0.022), and the DM remission rate was higher in the RYEJ group than in the distal gastrectomy group (50.0% vs. 17.3%, P = 0.002). CONCLUSIONS: Many gastric cancer patients with DM who received a gastrectomy showed remission or improvement of DM. The duodenal bypass group had higher DM remission or improvement rate than the BI group, and the RYEJ group had the highest DM remission rate.

Involvement of T-cell immunoregulation by ochnaflavone in therapeutic effect on fungal arthritis due to Candida albicans.

Arthritis due to pathogenic fungi is a serious disease causing rapid destruction of the joint. In the pathogenesis of arthritis, T lymphocytes are considered to be one of the major immune cells. In present study, we examined the T cell immunoregulatory effect by ochnaflavone (Och), a biflavonoid, on arthritis caused by Candida albicans that is the most commonly associated with fungal arthritis. To examine the effects of ochnaflavonon Candida albicans-caused septic arthritis, an emulsified mixture of C. albicans cell wall and complete Freund's adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route on days -3, -2, and -1. On Day 0, Och at 1 or 2 mg/dose/time was intratraperitoneally given to mice with the swollen footpad every other day for 3 times. The footpad-edema was measured for 20 days. Results revealed that Och reduced the edema at all dose levels and furthermore, there was app. 45% reduction of the edema in animals given 2 mg-dose at the peak of septic arthritis (p < 0.05). This anti-arthritic effect was accompanied by the diminishing of the DTH (delayed type hypersensitivity) activity against the CACW and by the provoking of the dominant T helper 2 (Th2) type cytokines production (IL-4 and Il-10), which appeared to result in a suppression of T helper 1 cytokines (IFN-γ and IL-2). Besides the T cell immunoregulatory activity, Och inhibited T cells activation as evidenced by the IL-2 reduction from PMA/ionomycin-stimulated Jurkat cell line and in addition, the compound killed macrophages in a dose-dependent manner (p < 0.05). However, Och caused no hemolysis (p < 0.05). These data implicate that Och, which has anti-arthritic activity based on the Th2 dominance as well as macrophage removal, can be safely administered into the blood circulation for treatment of the arthritis caused by C. albicans. Thus, it can be concluded that Och would be an ideal immunologically evaluated agent for treating of Candida arthritis.

Antiarthritic effect of lonicerin on Candida albicans arthritis in mice.

Fungal arthritis is a potentially serious disease resulting in rapid destruction of the joint. Among the various Candida species, Candida albicans is the most commonly associated with fungal arthritis. In the present study, we examined the effect of lonicerin, a flavonoid isolated from Lonicerae Flos, on an arthritis caused by C. albicans cell wall (CACW) in mice. To examine the effect, an emulsified mixture of CACW and complete Freund's adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route on days -3, -2, and -1. On Day 0, mice with the swollen footpad received lonicerin at 1 or 2 mg/dose/time intraperitoneally 3 times every other day. The footpad-swelling was measured for 20 days. Results showed that the lonicerin treatment reduced the edema at all dose levels, and, furthermore, there was app. 54% edema reduction in animals given the 2 mg-dose at the peak (day 10) of septic arthritis (p < 0.05). Since the peak, the edema was reduced in similar rates. This antiarthritic activity appeared to be mediated by lonicerin's ability to suppress T cell proliferation, nitric oxide production from macrophages, and shift of cellular immunity from Th1- toward Th2-type responses, all of which are beneficial to treat arthritis. In addition, the flavonoid had anticandidal activity (p < 0.01). These data suggest that lonicerin alone, which has both anti-arthritic and antifungal activities, can result in a combination therapy for the treatment of fungal arthritis due to C. albicans infection.

Combination immunotherapy of MAb B6.1 with fluconazole augments therapeutic effect to disseminated candidiasis.

We recently reported that IgM MAb B6.1, specific for ß-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection - a condition of developing partial therapeutic activity - enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combination-received mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.

A case of anaphylactic shock attributed to latex allergy during gastric cancer surgery.

Latex allergy is a known cause of allergic contact dermatitis. It produces mild symptoms, including skin rash and itching, which usually subside in a few days. However, latex allergy can also induce anaphylaxis, a severe type I hypersensitivity reaction that can cause urticaria, angioedema, hypotension, tachycardia, and bronchospasm. We report a case of anaphylactic shock during gastric cancer surgery in a patient with no previous allergic history. Surgery was suspended when hypotension, tachycardia, and wheezing developed. A thorough workup revealed that the patient had a latex allergy. The patient subsequently underwent curative gastrectomy performed with latex-free procedures.

Liquiritigenin, a licorice flavonoid, helps mice resist disseminated candidiasis due to Candida albicans by Th1 immune response, whereas liquiritin, its glycoside form, does not.

Licorice (the root of Glycyrrhizae plant) has been used as an oriental herbal medicine for thousands of years. The licorice flavonoid components are reported to possess immunomodulatory activities. In this present study, we investigated the immunomodulatory effects of liquiritigenin (LG) and liquiritin (LQ), licorice flavonoid components, against disseminated candidiasis due to Candida albicans, a dimorphic fungus, that causes severe disease via hematogenous dissemination and local diseases such as vaginitis and thrush. Results showed that direct interaction of LG or LQ with C. albicans yeast cells resulted in no growth-inhibition, in vitro. When tested in a murine model of disseminated candidiasis, mice given LQ intraperitoneally before intravenous challenge with live C. albicans yeast cells had similar mean survival times (MST) as untreated mice groups. On the contrary, mice given LG in the same manner as LQ above had longer MST than the untreated mice groups (P < 0.05). In one experiment, 3 out of 5 LG-treated mice survived during the entire period of the 55-day observation. Furthermore, the 3 survivors were cured -- shown by a lack of CFU (colony forming unit) in the kidneys. This protection was nulled when mice were pretreated with anti-CD4+ antibody before LG-treatment and challenge with the yeast. However, the protection was transferable by the CD4+ T cells isolated from LG-treated mice not infected with the yeast. In addition, mice given CD4+ T cells that were pre-treated with LG, in vitro were also protected against disseminated candidiasis. ELISA analysis revealed that in LG-treated mice IFNgamma and IL-2 were dominantly produced compared to IL-4 and IL-10. When LG-given mice were treated with anti-mouse IFNgamma, the protection was again nulled. Combined together, these results indicate that LG protects mice against disseminated candidiasis by the CD4+ Th1 immune response.

Chlorogenic acid, a polyphenolic compound, treats mice with septic arthritis caused by Candida albicans.

There has been an increasing number of studies concerning the multiple biological activities of polyphenolic compounds. In this present study, we determined the effect of chlorogenic acid (CRA), a polyphenolic compound, on septic arthritis caused by Candida albicans, a major etiological agent that causes fungal arthritis. To induce septic arthritis, an emulsified mixture of C. albicans cell wall and Complete Freund's Adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route once a day for 3 days. Twenty-four hours after the final injection, in order to determine CRA effect, mice having the swollen footpad were given CRA (1 mg/dose/mouse) intraperitoneally every other day three times. The footpad edema was measured for 15 days. Results showed that the CRA treatment reduced approximately 40% of the edema at the peak of septic arthritis (P<0.05). This anti-arthritic activity appeared to be mediated by a complete inhibition of nitric oxide (NO) production from macrophages and suppression of T-cells proliferation. Furthermore, CRA also inhibited growth of C. albicans yeast cells (P<0.01) and caused no hemolysis. These data indicate that CRA, which has antifungal and anti-arthritic effects, can safely be administered into the blood circulation for treatment of septic arthritis due to C. albicans. In addition, in respect to antiseptic arthritis, it can be suggested that the anti-candidal effect of CRA may be helpful as an all-in-one treatment of the candidal arthritis.

Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage.

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.

Immunoregulatory activity by daucosterol, a beta-sitosterol glycoside, induces protective Th1 immune response against disseminated Candidiasis in mice.

In the present study, we investigated immunomodulatory effect of daucosterol, a beta-sitosterol glycoside, against disseminated candidiasis caused by Candida albicans. Results showed that direct interaction of daucosterol with C. albicans yeast cells resulted in no growth-inhibition by in vitro susceptibility analysis. In contrast, mice given daucosterol (DS) intraperitoneally before intravenous challenge with live C. albicans yeast cells survived longer than DS-untreated control mice against disseminated candidiasis (P<0.05). By assessment of the fungal CFU in kidneys, DS-treated mice before the challenge developed about 81% fewer kidney CFU than untreated controls. This protection was removable by pretreatment of mice with anti-CD4+ antibody before the DS-treatment and challenge with the yeast. However, the protection was transferable by the CD4+ T cells from DS-treated mice not infected with the yeast. ELISA analysis revealed there were predominant production of IFNgamma and IL-2 cytokines as compared to IL-4, and IL-10 productions in DS-treated mice. By treatment of DS-given mice with anti-mouse IFNgamma, the protection was also abolished. Our studies show that DS protects mice against disseminated candidiasis by the CD4+ Th1 immune response.

Ginsenoside Rg1 helps mice resist to disseminated candidiasis by Th1 type differentiation of CD4+ T cell.

Ginsenosides, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rg1 is known to have various immune-modulating activities such as increase of immune activity of T helper (Th) cells. In this present work, we investigated the effect of the Rg1 on Candida albicans growth. Results showed that direct interaction of the Rg1 to C. albicans yeast cells resulted in no growth inhibition as tested by agar diffusion susceptibility method. Reversely, mice given the Rg1 intraperitoneally (i.p.) before intravenous (i.v.) challenge with live C. albicans yeast cells protected the mice to experimental disseminated candidiasis. By kidney candidal CFU (colony forming unit) determination, the disease severity of the Rg1-treated mice was confirmed far less than Rg1-untreated control mice. The protection was transferable by CD4+ T cells (RGCD4T) isolated from Rg1-treated mice. ELISA analysis revealed that there were cytokine inductions of IFNgamma, IL-2, IL-4 and IL-10 from the RGCD4T, demonstrating the Th1-lineage development of predominant IFNgamma and IL-2 production. Anti-mouse IFNgamma antibody treatment of Rg1-treated mice abolished the protection to disseminated disease. Our studies show that ginsenoside Rg1 helps the host resists disseminated candidiasis by the CD4(+) T cell-mediated immune response led from a Th1-dominant cytokine response.

Candida albicans can utilize siderophore during candidastasis caused by apotransferrin.

Ability of iron acquisition of pathogenic microorganisms functions as a virulence factor. Candida albicans, a fungal pathogen that requires iron for growth, is susceptible to growth retardation by high-affinity iron binding proteins such as transferrin. Recently, we reported that C. albicans could utilize the heme as a part of heme-containing proteins dissociated by heme oxygenase, CaHMX1. In search of another pathway that C. albicans can use to bypass the growth regulation produced by iron limitation, this present study examined utilization of non-candidal siderophores such as Desferal and rhodotorulic acid (RA) for acquisition of inorganic iron by the fungus. C. albicans secreting no siderophores was cultured in iron-free (pretreated with apotransferrin for 24 h) (culture medium). Once growth of the yeast reached stasis from iron starvation, a siderophore was added to the culture media. Results showed that cultures containing apotransferrin within a dialysis membrane recovered growth to the level of untreated controls, whereas C. albicans yeast cells in direct contact with soluble iron-free (apo) transferrin recovered growth only partially. When static growth from iron limitation was reached, the addition of siderophore-apotransferrin complex to culture medium also permitted the yeast to recover growth from apotransferrin growth regulation. All the data show that C. albicans can utilize the non-candidal siderophores for iron acquisition under transferrin regulation as can pathogenic bacteria.

Berberine synergy with amphotericin B against disseminated candidiasis in mice.

In present study, we investigated the synergic effect of berberine against disseminated candidiasis caused by the pathogenic fungus, Candida albicans. Berberine inhibited the growth of C. albicans under in-vitro condition. The broth susceptibility revealed the synergic effect of berberine with amphotericin B (Amp B). To confirm these results under the in-vivo condition, the effect was examined in mice against disseminated candidiasis. Results showed mice that were given diluent (negative control), Amp B (0.5 mg/kg of body weight), or berberine (1 mg/kg of body weight) had mean survival times (MST) of approximately 12, 14, and 17 d, respectively. On the contrary, mice that were treated using a combination of the two agents at the same concentrations resulted in a MST value of 36 d, surviving at an average of 22 d longer than the mice group treated only with the Amp B. This MST value was almost same as MST value from the mice that were given four times the Amp B dose. These data indicate that the combination of Amp B and berberine could reduce approximately 75% of the Amp B dose, implying that berberine indeed has synergy with Amp B against the disseminated disease.