Antibacterial efficacy of Citrus hystrix (makrut lime) essential oil against clinical multidrug-resistant methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates.

The increasing incidence of methicillin-resistant S. aureus is a major public health concern. Recently, the performance of Citrus hystrix essential oil (CHEO) has been shown to contain broad-spectrum antibacterial activity. Therefore, this study aims to determine the antibacterial activity of CHEO alone and in combination with gentamicin against panels of clinical isolates of methicillin-susceptible S. aureus (MSSA, n = 45) and methicillin-resistant S. aureus (MRSA, n = 40). Antibiotic susceptibility testing revealed multidrug-resistant (MDR) patterns among 3 MSSA isolates and 39 MRSA isolates, indicating that the clinical MRSA isolates were associated with MDR (p < 0.05). For the drug resistant isolates, resistance was observed toward most antibiotics, except for chloramphenicol, trimethoprim-sulfamethoxazole, linezolid, and vancomycin. Antibacterial screening by disk diffusion demonstrated that CHEO alone had certain antibacterial activity toward all MSSA isolates (IZD: 16.0 ± 4.7 mm) and MRSA isolates (IZD: 16.5 ± 4.2 mm) (p > 0.05). The MIC values of CHEO are 18.3 ± 6.1 mg/mL in MSSA isolates and 17.9 ± 6.9 mg/mL in MRSA isolates (p > 0.05). The antibacterial activity of CHEO demonstrated the bactericidal effect with MIC index 1.0-1.4. Time-killing kinetics revealed that CHEO at 1 × MIC completely killed MSSA and MRSA within 12 h. Moreover, the checkerboard titration demonstrated the synergistic and additive interactions of CHEO with gentamicin with FIC index 0.012-0.625. CHEO against human epidermal keratinocyte; HaCaT cell line demonstrated the IC50 value at 2.15 mg/mL. The use of CHEO as an alternative antibacterial agent would reduce the emergence of resistant bacteria, especially MDR MRSA.

In Silico Target Identification of Galangin, as an Herbal Flavonoid against Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies in the bile duct, which proliferates aggressively. CCA is highly prevalent in Northeastern Thailand wherein it is associated with liver fluke infection, or Opisthorchis viverrini (OV). Most patients are diagnosed in advanced stages, when the cancer has metastasized or severely progressed, thereby limiting treatment options. Several studies investigate the effect of traditional Thai medicinal plants that may be potential therapeutic options in combating CCA. Galangin is one such herbal flavonoid that has medicinal properties and exhibits anti-tumor properties in various cancers. In this study, we investigate the role of Galangin in inhibiting cell proliferation, invasion, and migration in OV-infected CCA cell lines. We discovered that Galangin reduced cell viability and colony formation by inducing apoptosis in CCA cell lines in a dose-dependent manner. Further, Galangin also effectively inhibited invasion and migration in OV-infected CCA cells by reduction of MMP2 and MMP9 enzymatic activity. Additionally, using proteomics, we identified proteins affected post-treatment with Galangin. Enrichment analysis revealed that several kinase pathways were affected by Galangin, and the signature corroborated with that of small molecule kinase inhibitors. Hence, we identified putative targets of Galangin using an in silico approach which highlighted c-Met as candidate target. Galangin effectively inhibited c-Met phosphorylation and subsequent signaling in in vitro CCA cells. In addition, Galangin was able to inhibit HGF, a mediator of c-Met signaling, by suppressing HGF-stimulated invasion, as well as migration and MMP9 activity. This shows that Galangin can be a useful anti-metastatic therapeutic strategy in a subtype of CCA patients.

Porcine placenta extract improves high-glucose-induced angiogenesis impairment.

BACKGROUND: High glucose (HG)-induced reactive oxygen species (ROS) overproduction impairs angiogenesis that is one pivotal factor of wound healing process. Angiogenesis impairment induces delayed wound healing, whereby it eventually leads to amputation in cases of poorly controlled diabetes with diabetic ulceration. Porcine placenta extract (PPE) is a natural waste product that comprises plenty of bioactive agents including growth factors and antioxidants. It was reported as an effective compound that prevents ROS generation. The goal of this study was to investigate the in vitro effect of PPE on HG-induced ROS-mediated angiogenesis impairment. METHODS: Primary endothelial cells (HUVECs) and endothelial cell line (EA.hy926) were treated with HG in the presence of PPE. The endothelial cells (ECs) viability, intracellular ROS generation, migration, and angiogenesis were determined by MTT assay, DCFDA reagent, wound healing assay, and tube formation assay, respectively. Additionally, the molecular mechanism of PPE on HG-induced angiogenesis impairment was investigated by Western blot. The angiogenic growth factor secretion was also investigated by the sandwich ELISA technique. RESULTS: HG in the presence of PPE significantly decreased intracellular ROS overproduction compared to HG alone. HG in the presence of PPE significantly increased ECs viability, migration, and angiogenesis compared to HG alone by showing recovery of PI3K/Akt/ERK1/2 activation. HG in the presence of PPE also decreased ECs apoptosis compared to HG alone by decreasing p53/Bax/cleaved caspase 9/cleaved caspase 3 levels and increasing Bcl 2 level. CONCLUSION: PPE attenuated HG-induced intracellular ROS overproduction that improved ECs viability, proliferation, migration, and angiogenesis by showing recovery of PI3K/Akt/ERK1/2 activation and inhibition of ECs apoptosis. This study suggests PPE ameliorated HG-induced ROS-mediated angiogenesis impairment, whereby it potentially provides an alternative treatment for diabetic wounds.

Gadd45ß silencing impaired viability and metastatic phenotypes in cholangiocarcinoma cells by modulating the EMT pathway.

Growth arrest and DNA damage-inducible-ß (Gadd45ß) is a stress-response protein involved in a number of processes, including cell cycle control, DNA repair, survival and death control, and stress signaling, depending on its interactions. Gadd45ß expression is dysregulated in numerous types of cancer, functioning as either a tumor promoter or a tumor suppressor. However, the functions of Gadd45ß in cholangiocarcinoma (CCA), particularly in metastasis, has not been studied. The immunohistochemical analysis of Gadd45ß expression revealed that 75% of histological specimens from patients with CCA expressed high levels of Gadd45ß, and that high Gadd45ß expression was associated with metastasis. The role of Gadd45ß in CCA was examined using siRNA-mediated gene knockdown in HuCCA-1, a human CCA cell line established from a Thai patient. The effects of Gadd45ß downregulation upon cell viability and death, invasion, migration, matrix metalloproteinase (MMP) activity and epithelial-mesenchymal transition (EMT) marker expression were investigated. Gadd45ß knockdown impaired cell viability, which was associated with the induction of apoptosis. In addition, there was a marked reduction in invasion and migration, although MMP activity was unaffected. Impairment of these metastatic properties was accompanied by the decreased expression of EMT markers, including Slug, vimentin, claudin-1 and zona occludens protein 1, whereas E-cadherin expression was increased. The present study suggests that Gadd45ß is involved in regulating the viability and the metastatic potential of CCA cells, which may be mediated by the modulation of the EMT pathway.

Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient.

AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient. METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham's F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods. CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.

Involvement of c-Met/hepatocyte growth factor pathway in cholangiocarcinoma cell invasion and its therapeutic inhibition with small interfering RNA specific for c-Met.

BACKGROUND: Hepatocyte growth factor receptor (c-Met) plays an important role in many functions of cancer cells. We examined the roles of c-Met and its downstream signaling molecules in cholangiocarcinoma cell lines RMCCA1 and HuCCA1. MATERIALS AND METHODS: The expression of c-Met and their signaling cascades were determined in RMCCA1 and HuCCA1 cholangiocarcinoma cell lines by Western blotting. Small interfering RNA (siRNA) specific for c-Met was used to suppress the expression of c-Met. The proliferation, migration and invasion assay were tested in these cholangiocarcinoma cells treated with hepatocyte growth factor (HGF). RESULTS: Activation of c-Met with HGF triggered the signaling via the ERK cascade mediated by sequential phosphorylation of MEK1/2 and MAPK and induction of cholangiocarcinoma cell invasion. The expression of c-Met in cholangiocarcinoma cells was suppressed by treatment with small interfering RNA (siRNA) specific for c-Met, and resulted in decrease in phosphorylation of MEK1/2. Furthermore, treatment with siRNA specific for c-Met or MEK inhibitor U0126 inhibited cholangiocarcinoma cell invasion induced by HGF. CONCLUSIONS: These results indicated that HGF and c-Met involved in the mechanism of cholangiocarcinoma cell invasion. It implies a potential role for the inhibition of c-Met in the treatment of cholangiocarcinoma.

Recrudescence in artesunate-treated patients with falciparum malaria is dependent on parasite burden not on parasite factors.

Artemisinin derivatives are first-line antimalarial drugs in Thailand. No firm evidence of clinically relevant artemisinin resistance exists. When used as monotherapy, artesunate has been associated with a high treatment failure (recrudescence) rate, which could be due to low-level artemisinin resistance. To understand the causes of recrudescence, we retrospectively studied a cohort of 104 malaria patients treated with artesunate monotherapy, 32 of whom recrudesced. There was no difference in in vitro artesunate sensitivities between 6 nonrecrudescent isolates and 16 paired admission and recrudescent isolates. Paired admission and recrudescent isolates from 10 patients were genotyped; only 3 had pfmdr1 mutations. Patients with admission parasitemias >10,000 per microl had a 9-fold higher likelihood of recrudescence (adjusted odds ratio) compared with patients with lower parasitemias. This study suggests (1) recrudescence after treatment with artesunate is not the result of inherent parasite resistance, and (2) admission parasitemia may be useful in choosing therapeutic options.