A synthetic retinoic acid receptor γ antagonist (7C)-loaded nanoparticle enhances bone morphogenetic protein-induced bone regeneration in a rat spinal fusion model.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) have potent osteoinductivity and have been applied clinically for challenging musculoskeletal conditions. However, the supraphysiological doses of BMPs used in clinical settings cause various side effects that prevent widespread use, and therefore the BMP dosage needs to be reduced. PURPOSE: To address this problem, we synthesized 7C, a retinoic acid receptor γ antagonist-loaded nanoparticle (NP), and investigated its potential application in BMP-based bone regeneration therapy using a rat spinal fusion model. STUDY DESIGN: An experimental animal study. METHODS: Fifty-three male 8-week-old Sprague-Dawley rats underwent posterolateral spinal fusion and were divided into the following five treatment groups: (1) no recombinant human (rh)BMP-2 and blank-NP (Control), (2) no rhBMP-2 and 1 µg 7C-NP (7C group), (3) low-dose rhBMP-2 (0.5 µg) and 1 µg blank-NP (L-BMP group), (4) low-dose rhBMP-2 (0.5 µg) and 1 µg 7C-NP (L-BMP + 7C group), and (5) high-dose rhBMP-2 (5.0 µg) and 1 µg blank-NP (H-BMP group). Micro-computed tomography and histologic analysis were performed 2 and 6 weeks after the surgery. RESULTS: The spinal fusion rates of the Control and 7C groups were both 0%, and those of the L-BMP, L-BMP + 7C, and H-BMP groups were 55.6%, 94.4%, and 100%, respectively. The L-BMP + 7C group markedly promoted cartilaginous tissue formation during BMP-induced endochondral bone formation that resulted in a significantly better spinal fusion rate and bone formation than in the L-BMP group. Although spinal fusion was slower in the L-BMP + 7C group, the L-BMP + 7C group formed a spinal fusion mass with better bone quality than the spinal fusion mass in the H-BMP group. CONCLUSIONS: The combined use of 7C-NP with rhBMP-2 in a rat posterolateral lumbar fusion model increased spinal fusion rate and new bone volume without deteriorating the quality of newly formed bone. CLINICAL SIGNIFICANCE: 7C-NP potentiates BMP-2-induced bone regeneration and has the potential for efficient bone regeneration with low-dose BMP-2, which can reduce the dose-dependent side effects of BMP-2 in clinical settings.

Zinc deficiency impairs axonal regeneration and functional recovery after spinal cord injury by modulating macrophage polarization via NF-κB pathway.

Background: Spinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target. Methods: We created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination, axonal regeneration, and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test. Results: In macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function. Conclusion: We demonstrated that zinc affected axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

Diminishing acetyl-CoA carboxylase 1 attenuates CCA migration via AMPK-NF-κB-snail axis.

Cholangiocarcinoma (CCA), a cancer of the biliary tract, is a significant health problem in Thailand. Reprogramming of cellular metabolism and upregulation of lipogenic enzymes have been revealed in CCA, but the mechanism is unclear. The current study highlighted the importance of acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme in de novo lipogenesis, on CCA migration. ACC1 expression in human CCA tissues was determined by immunohistochemistry. The results demonstrated that increased ACC1 was related to the shorter survival of CCA patients. Herein, ACC1-deficient cell lines (ACC1-KD) were generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (cas9) system and were used for the comparative study. The ACC1 levels in ACC1-KD were 80-90 % lower than in parental cells. Suppression of ACC1 significantly reduced intracellular malonyl-CoA and neutral lipid contents. Two-fold growth retardation and 60-80 % reduced CCA cell migration and invasion were observed in ACC1-KD cells. The reduced 20-40 % of intracellular ATP levels, AMPK activation, lowered NF-κB p65 nuclear translocation, and snail expression were emphasized. Migration of ACC1-KD cells was restored by supplementation with palmitic acid and malonyl-CoA. Altogether, the importance of rate-limiting enzyme in de novo fatty acid synthesis, ACC1, and AMPK-NF-κB-snail axis on CCA progression was suggested herein. These might be the novel targets for CCA drug design. (ACC1, AMPK, Cholangiocarcinoma, De novo lipogenesis, NF-κB, Palmitic acid).

Antitumor effect of polyphyllin D on liver metastases of neuroblastoma.

PURPOSE: We previously reported that polyphyllin D, a main component of the traditional Chinese medicinal herb Paris polyphylla, exhibited anticancer effects in vitro against human neuroblastoma cells. The aims of this investigation was to examine the presence or absence of in vivo anti-metastasis effects of polyphyllin D were to establish a liver metastasis model of neuroblastoma and to evaluate the anti-metastasis effects of polyphyllin D. METHODS: Subcutaneous and intraperitoneal tumors, and metastasis models were established in immune-deficient BALB/c nude and BALB/c Rag-2/Jak3 double-deficient (BRJ) mice using the human neuroblastoma cell lines IMR-32, LA-N-2, or NB-69. For evaluating polyphyllin D activity, we used a mouse model of liver metastasis with the IMR-32 cells line injected through the tail vein. We analyzed the livers number and area of liver tumors in of the phosphate buffer solution- and polyphyllin D-treated groups. RESULTS: Liver metastasis and intraperitoneal dissemination models were successfully established in immune-deficient BRJ mice using the three human neuroblastoma cell lines. In the liver metastasis, the model of IMR-32 cells, we found that polyphyllin D suppressed both the number and total area of metastatic foci the average number of metastatic foci, average focus areas, and number of cleaved caspase-3-positive cells were significantly lower in the polyphyllin D group (p = 0.016, 0.020, 0.043, respectively). CONCLUSIONS: We developed a mouse models of neuroblastoma metastasis and demonstrated for the first time that polyphyllin D has an antitumor effect on neuroblastoma liver metastases.

Effects of rhBMP-2-loaded hydroxyapatite granules/beta-tricalcium phosphate hydrogel (HA/ß-TCP/hydrogel) composite on a rat model of caudal intervertebral fusion.

The effects and inflammation-related side effects of bone morphogenetic protein (BMP)-2 on posterior lumbar interbody fusion are controversial. One of the potential causes for the inconsistent results is the uncontrolled release of BMP-2 from the collagen carrier. Therefore, BMP delivery systems that support effective bone regeneration while attenuating the side effects are strongly sought for. We developed NOVOSIS putty (NP), a novel composite material of hydroxyapatite (HA), beta-tricalcium phosphate (ß-TCP)/hydrogel, and BMP-2, which can sustainably release BMP-2 over 2 weeks. This study was aimed at comparing the effects and side effects of NP and collagen sponge (CS) containing BMP-2 using a rat caudal intervertebral fusion model. The fusion rates of NP with low and high doses of BMP-2 were significantly higher than those of an iliac bone (IB) graft, but those of CS with low and high doses of BMP-2 were not different from those of the IB graft. Furthermore, the incidences of ectopic bone formation and soft tissue swelling were significantly lower in the NP group than in the CS group. The HA/ß-TCP/hydrogel carrier enabled superior bone induction with low-dose BMP-2 and decreased the incidence of side effects caused by high-dose BMP-2 vis-à-vis the collagen carrier.

Antitumor effect of acanthoic acid against primary effusion lymphoma via inhibition of c-FLIP.

Acanthoic acid (AA) is an active substance that is extracted from Croton oblongifolius Roxb., a traditional plant in Thailand. The antiinflammatory effect of AA on NF-κB pathway has been exclusively reported, however, its anticancer effect is still lacking. PEL is a B cell lymphoma that is mostly found in HIV patients. The prognosis and progression of PEL patients are terribly poor with a median survival time less than 6 months, so the new effective treatment is urgently needed. In this study, we found that AA effectively inhibited PEL cell proliferation with IC50s at 120-130 µM in well-representative cells, while the IC50s of AA in PBMC were higher (>200 µM). AA increased percentages of Annexin V/PI positive cells, whereas adding of caspase inhibitor (Q-VD-OPh) prevented AA-induced cell death. The antiapoptotic protein, c-FLIP, was downregulated by AA which leading to the activation of caspase-8 and -3. Combination of AA and TRAIL dramatically enhanced apoptotic cell death. In PEL xenograft model, AA at the dose of 250 mg/kg effectively inhibited PEL tumor growth without detectable toxicities assessed by mice weight and appearance.

Cucurbitacin B induces apoptosis of primary effusion lymphoma via disruption of cytoskeletal organization.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin lymphoma that develops especially in AIDS patients and immunocompromised patients infected with human herpes virus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a poor prognosis in patients despite conventional chemotherapeutic treatment, and a safe and efficient therapy is required. PURPOSE: To examine the effects on PEL of cucurbitacin B (CuB), a triterpene found in plants of the Cucurbitaceae family that has several anti-cancer activities. STUDY DESIGN: We evaluated the anti-cancer activities of CuB in vitro and in vivo. METHODS: Cell proliferation of PEL cell lines was measured by MTT assay. Cleaved caspases and signaling transduction associated proteins were analyzed by western blotting. Wright and Giemsa staining and immunofluorescence staining were carried out to observe cell morphology. Cell cycles were analyzed by flow cytometry. RT-PCR was performed to detect viral gene expressions. A xenograft mouse model was employed to evaluate the anti-cancer activity of CuB in vivo. RESULTS: CuB inhibited cell proliferation of PEL cell lines (BCBL-1, BC-1, GTO and TY-1) in a dose-dependent manner (0-50 nM) and induced apoptosis of BCBL-1 cells via caspase activation in a dose- and time-dependent manner. In addition, CuB caused cell-shape disruption by inducing actin aggregation and suppressing the p-cofilin level, resulting in BCBL-1 cell arrest at the G2/M phase. In contrast, CuB showed almost no suppression of p-STAT3 and p-Akt activation, which were constitutively activated by KSHV-derived proteins. Furthermore, CuB (0.5 mg/kg) via intraperitoneal injection significantly (p < 0.05) suppressed solid tumor growth in the xenograft mouse model. CONCLUSION: This study suggests that CuB is a promising agent for PEL treatment.

Novel Analytical Platform For Robust Identification of Cell Migration Inhibitors.

Wound healing assay is a simple and cost-effective in vitro assay for assessing therapeutic impacts on cell migration. Its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. In this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. Finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. Our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.

Antimicrobial resistance of Enterococcus spp. isolated from Thai fermented pork in Chiang Rai Province, Thailand.

OBJECTIVES: The aim of this study was to evaluate the prevalence of antimicrobial-resistant phenotypes and genes of Enterococcus spp. in order to explore the range of resistance profiles from Thai traditional fermented pork. METHODS: A total of 120 Thai fermented pork specimens were collected in Chiang Rai, Thailand. Antimicrobial resistance among isolated enterococci to 11 antimicrobial agents was determined by the agar disk diffusion method. Antibiotic resistance genes from resistant phenotypes and virulence genes were observed. RESULTS: A total of 119 enterococci were found contaminating the collected samples. The most prevalent species was Enterococcus faecalis (68.9%), followed by Enterococcus hirae (16.0%), Enterococcus faecium (13.4%) and Enterococcus gallinarum (1.7%). The highest percentage of resistance was to ciprofloxacin (97.5%), followed by erythromycin (78.2%) and tetracycline (67.2%), whilst high-level gentamicin- and streptomycin-resistant isolates were of lower frequency (7.6% and 22.7%, respectively). All isolates were susceptible to the clinically important agents vancomycin and teicoplanin. Overall, a relatively high frequency of multidrug-resistant (MDR) enterococci was observed (76.2%). Antimicrobial-resistant phenotypes were found to carry aacA-aphD, addE, erm(B), mefA/E, cat, tet(L) and tet(M) resistance genes. Virulence genes were also evaluated and the gelE gene was found to be the most common (37.8%). CONCLUSIONS: These data highlight the importance of MDR enterococci in fermented pork in Thailand. This is the first report to detect the unusual species E. hirae carrying the mefA/E macrolide resistance gene. These clinically important and unusual enterococci isolates from Thai fermented pork could be a source of transferable resistance genes to other bacteria.

In vitro and in vivo antitumor activity of tiliacorinine in human cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a fatal cancer with poor prognosis and less than 10% of CCA patients can be offered surgical cure. Conventional chemotherapy results in unfavorable outcomes. At present, plant-derived compounds are gaining interest as potential cancer therapeutics, particularly for treatment-refractory cancers. In this study, antitumor activity of tiliacorinine, the major alkaloid isolated from a tropical plant, on CCA was first demonstrated. Antiproliferative effects of tiliacorinine on human CCA cell lines were investigated using SRB assays. Acridine orange/ethidium bromide staining, flow cytometric analysis and DNA laddering assays were used for apoptotic determination. Apoptosis-related proteins were verified by Western blotting and antitumor activity of tiliacorinine in vivo was demonstrated in CCA xenografted mice. Tiliacorinine significantly inhibited proliferation of human CCA cell lines with IC50 4.5-7 µM by inducing apoptosis through caspase activation, up- regulation of BAX, and down-regulation of BclxL and XIAP. Tiliacorinine considerably reduced tumor growth in CCA xenografted mice. These results demonstrated antitumor effects of tiliacorinine on human CCA in vitro and in vivo. Tiliacorinine may be an effective agent for CCA treatment.

The effects of Salacia reticulata on anti-cellular oxidants and melanogenesis inhibition in alpha-MSH-stimulated and UV irradiated B16 melanoma cells.

The purposes of this study were to investigate the inhibitory effects of Salacia reticulata Tul. root extract on cellular oxidants and melanogenesis in B16 melanoma cells. Cells treated with non-toxic doses of S. reticulata root extract were investigated for their effects on melanogenesis, cellular tyrosinase activity and cellular oxidant scavenging activity. The results indicated that S. reticulata extract inhibited melanin synthesis and tyrosinase activity in alpha-MSH-induced or UV-irradiated B16 melanoma cells in a dose dependent manner. Additionally, the extract also exhibited anti-cellular oxidants in UV-induced radical melanoma cells. Altogether, these results suggested that S. reticulata root extract has roles in suppression of melanogenesis and oxidant inhibition. S. reticulata root extract may be a potential source for the development of pharmaceutical products for treatment of skin hyperpigmentation disorders.

Potential use of the complex of doxorubicin with folate-conjugated methyl-ß-cyclodextrin for tumor-selective cancer chemotherapy.

In a recent study, we attempted to confer a tumor-selective cytotoxic activity to methyl-ß-cyclodextrin (M-ß-CyD), we synthesized folate-conjugated M-ß-CyD (FA-M-ß-CyD), and demonstrated the potential of FA-M-ß-CyD as a novel anticancer agent at a high dose. In the present study, to expand the application of FA-M-ß-CyD for cancer chemotherapy, we evaluated the potential of FA-M-ß-CyD as a tumor-targeting anticancer drug carrier at a low dose. FA-M-ß-CyD formed an inclusion complex with doxorubicin (DOX) with a high-stability constant (3.0 × 105 M-1). Antitumor activity of DOX was increased by the complexation with FA-M-ß-CyD, but not with folate-conjugated ß-CyD (FA-ß-CyD) or M-ß-CyD in KB cells, a folate receptor-α (FR-α)-expressing cell line. Also, FA-M-ß-CyD increased antitumor activity of paclitaxel, a class IV compound in the biopharmaceutical classification system (BCS), but not 5-fluorouracil, a class III compound in the BCS. Furthermore, FA-M-ß-CyD enhanced cellular uptake of DOX through a complexation in KB cells (FR-α (+)), compared to FA-ß-CyD and M-ß-CyD. The DOX/FA-M-ß-CyD complex showed markedly high antitumor activity, compared to DOX alone and DOX/M-ß-CyD complex, after an intravenous administration to FR-α-expressing tumor cell-bearing mice. These findings suggest that FA-M-ß-CyD could be useful as a tumor-selective carrier for anticancer drugs.

Establishment of an allo-transplantable hamster cholangiocarcinoma cell line and its application for in vivo screening of anti-cancer drugs.

Opisthorchis viverrini (O. viverrini) is a well-known causative agent of cholangiocarcinoma (CCA) in humans. CCA is very resistant to chemotherapy and is frequently fatal. To understand the pathogenesis of CCA in humans, a rodent model was developed. However, the development of CCA in rodents is time-consuming and the xenograft-transplantation model of human CCA in immunodeficient mice is costly. Therefore, the establishment of an in vivo screening model for O. viverrini-associated CCA treatment was of interest. We developed a hamster CCA cell line, Ham-1, derived from the CCA tissue of O. viverrini-infected and N-nitrosodimethylamine-treated Syrian golden hamsters. Ham-1 has been maintained in Dulbecco's Modified Essential Medium supplemented with 10% fetal bovine serum for more than 30 subcultures. These cells are mostly diploid (2n=44) with some being polyploid. Tumorigenic properties of Ham-1 were demonstrated by allograft transplantation in hamsters. The transplanted tissues were highly proliferative and exhibited a glandular-like structure retaining a bile duct marker, cytokeratin 19. The usefulness of this for in vivo model was demonstrated by berberine treatment, a traditional medicine that is active against various cancers. Growth inhibitory effects of berberine, mainly by an induction of G1 cell cycle arrest, were observed in vitro and in vivo. In summary, we developed the allo-transplantable hamster CCA cell line, which can be used for chemotherapeutic drug testing in vitro and in vivo.

Antitumor effect of berberine against primary effusion lymphoma via inhibition of NF-κB pathway.

Primary effusion lymphoma (PEL) is an infrequent and distinct entity among the aggressive non-Hodgkin B cell lymphomas that occurs predominantly in patients with advanced AIDS. It shows serous lymphomatous effusion in body cavities, and is resistant to conventional chemotherapy with a poor prognosis. Thus, the optimal treatment for PEL is not well defined and there is a need for novel agents. PEL has been recognized as the tumor caused by Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), and nuclear factor (NF)-κB activation plays a critical role in the survival and growth of PEL cells. In this study, we assessed the antitumor effect of berberine, a naturally occurring isoquinoline alkaloid, on this pathway. The methylthiotetrazole assay showed that cell proliferation in the PEL cell lines was inhibited by berberine. Berberine also induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IκB kinase (IKK) phosphorylation, IκB phosphorylation and IκB degradation, upstream targets of the NF-κB pathway, in PEL cells. In a xenograft mouse model that showed ascites and diffuse organ invasion of PEL cells, treatment with berberine inhibited the growth and invasion of PEL cells significantly compared with untreated mice. These results show that the suppression of NF-κB is a molecular target for treating PEL, and berberine is a potential antitumor agent for PEL.

The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement.

Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.

22-oxa-1,25-dihydroxyvitamin D3 efficiently inhibits tumor growth in inoculated mice and primary histoculture of cholangiocarcinoma.

BACKGROUND: It is well known that 1α,25-Dihydroxyvitamin D3 (1,25[OH]2 D3) restrains cell proliferation and induces differentiation and apoptosis in normal and tumor cells. The authors of this report recently demonstrated that 1,25(OH)2 D3 effectively inhibits the proliferation of cholangiocarcinoma (CCA) cell lines. The antitumor activity and the underlying mechanism of 22-oxa-D3, an analog of vitamin D, in mice and in tissue cultures from patients with CCA were further explored in the current study. METHODS: Cell growth and cell cycle distribution were examined in CCA cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Mice were injected subcutaneously with 4×10(6) CCA cells at both flank sides and intraperitoneal injections with phosphate-buffered saline or 22-oxa-D3 (15 µg/kg/day) for 17 days thereafter. Tumors were removed the next day. The expression levels of cyclin D1 and the cyclin-dependent kinase inhibitor p21 were determined by Western blot analysis and immunohistochemistry. Growth inhibition of 22-oxa-D3 in fresh tissue samples from patients with CCA was analyzed by using a histodrug response assay. RESULTS: 22-oxa-D3 effectively suppressed the growth of CCA cell lines in a time-dependent and dose-dependent manner. 22-oxa-D3 arrested CCA cells at G1 phase to S phase by the suppression of cyclin D1 expression and the up-regulation of p21. Supplementation of 22-oxa-D3 to CCA-inoculated mice significantly inhibited tumor growth without hypercalcemia or serious side effects. The treatment also induced cellular apoptosis in tissue samples from patients with CCA. CONCLUSIONS: 22-oxa-D3 effectively suppressed tumor growth in CCA-inoculated mice and induced cellular apoptosis in tissue samples from patients with CCA. The current data encourage further investigation of 1,25(OH)2 D3 or its analogues as therapeutic agents in the treatment of patients with CCA.

Cepharanthine exerts antitumor activity on cholangiocarcinoma by inhibiting NF-kappaB.

Cholangiocarcinoma (CCA) is a major cause of cancer deaths in northeast Thailand. It is aggressive, highly metastatic, and responds poorly to traditional chemotherapy. We demonstrated the potential for Cepharanthine (CEP), a biscoclaurine alkaloid extracted from Stephania cepharantha, to treat CCA. CEP significantly inhibited growth of human CCA cell lines in a dose- and time-dependent manner, regardless of the histologic type of tumor origin. Increasing cell apoptosis via caspase-3 and capase-9 activation was demonstrated in CEP-treated cells. We found that CEP controlled the growth of CCA cells through nuclear factor-kappa B (NF-kappaB) inactivation by inhibiting nuclear translocation. CEP treatment effectively reduced tumor size in CCA-inoculated mice without serious side effects. CEP also increased cell apoptosis in primary histocultures of CCA patients' tissues; this was demonstrated by immunohistochemistry using TUNEL staining. Our results suggest that CEP possesses therapeutic potential against human CCA.

Ephedrae herba, a component of Japanese herbal medicine Mao-to, efficiently activates the replication of latent human immunodeficiency virus type 1 (HIV-1) in a monocytic cell line.

The persistence of latent human immunodeficiency virus type 1 (HIV-1)-infected cellular reservoirs, despite prolonged treatment with highly active antiretroviral therapy (HAART), represents a major hurdle to virus eradication. In this study, we evaluated the effect of Japanese herbal medicine on the induction of HIV-1 replication in latently infected monocytic cell line, U1, in order to eradicate virus efficiently. We found that Mao-to was able to induce HIV-1 replication either alone or in combination with tumor necrosis factor-alpha (TNF-alpha). Among the four components of Mao-to, only Ephedrae herba had strong effects in inducing HIV-1 replication. Analysis by Western blotting revealed that Ephedrae herba induced the nuclear translocation of nuclear factor-kappa B (NF-kappaB). Reporter assay data also showed that Ephedrae herba and, slightly, Mao-to activated the NF-kappaB promoter, indicating that these herbal agents may induce HIV-1 replication through NF-kappaB activation. These findings suggest that Mao-to and its component, Ephedrea herba, may be good candidates to augment HAART by inducing the expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in individuals infected with HIV-1.

Heat shock suppresses human NK cell cytotoxicity via regulation of perforin.

Human natural killer (NK) cell, which is an important lymphocyte for immune surveillance, is highly sensitive to heat, but the nature of its response to and its mechanistic regulation by heat remain unclear. Here we determined the effect of in vitro heat shock and in vivo hyperthermia on human NK cell cytotoxicity. Human peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were subjected to heat shock in vitro (42 degrees C, 1 h). PBMC from cancer patients receiving intentional hyperthermia (42 degrees C, 1 h) for cancer therapy were also obtained. NK cytolytic activity was determined in these samples. NK cell cytotoxicity was down-regulated by heat shock in vitro at 5 h, but at 24 h after heat shock, the NK cytotoxicity was comparable to that with its respective control. Furthermore, we observed that the mRNA and protein expression levels of perforin, which is the cytolytic granule of NK cells, were regulated by heat shock in a similar manner as NK cytotoxicity at 5 h and at 24 h after heat shock. Heat regulation involved the perforin protein in CD56(dim) but not in CD56(bright) NK cell subset. Heat shock neither induced cell death nor altered the expression of some NK activating receptors and adhesion molecules. Moreover, whole-body hyperthermia at 42 degrees C for 1 h of cancer patients also suppressed the cytotoxicity of NK cells but recovered to basal level 1 week after hyperthermia. Heat shock in vitro and in vivo temporarily represses the cytotoxicity of human NK cells.