Search

Background: Prostate cancer (PCa) is one of the causes of death in men worldwide. Although treatment strategies have been developed, the recurrence of the disease and consequential side effects remain an essential concern. Diospyros rhodocalyx Kurz, a traditional Thai medicine, exhibits diverse therapeutic properties, including anti-cancer activity. However, its anti-cancer activity against prostate cancer has not been thoroughly explored. This study aims to evaluate the anti-cancer activity and underlying mechanisms of the ethyl acetate extract of D. rhodocalyx Kurz (EADR) related to apoptosis induction in the LNCaP human prostate cancer cell line. Methods: Ethyl acetate was employed to extract the dried bark of D. rhodocalyx Kurz. The cytotoxicity of EADR on both LNCaP and WPMY-1 cells (normal human prostatic myofibroblast cell line) was evaluated using MTS assay. The effect of EADR on the cell cycle, apoptosis induction, and alteration in mitochondrial membrane potential (MMP) was assessed by the staining with propidium iodide (PI), Annexin V-FITC/PI, and JC-1 dye, respectively. Subsequent analysis was conducted using flow cytometry. The expression of cleaved caspase-3, BAX, and Bcl-2 was examined by Western blotting. The phytochemical profiling of the EADR was performed using gas chromatography-mass spectrometry (GC-MS). Results: EADR exhibited a dose-dependent manner cytotoxic effect on LNCaP cells, with IC50 values of 15.43 and 12.35 µg/mL after 24 and 48 h, respectively. Although it also exhibited a cytotoxic effect on WPMY-1 cells, the effect was comparatively lower, with the IC50 values of 34.61 and 19.93 µg/mL after 24 and 48 h of exposure, respectively. Cell cycle analysis demonstrated that EADR did not induce cell cycle arrest in either LNCaP or WPMY-1 cells. However, it significantly increased the sub-G1 population in LNCaP cells, indicating a potential induction of apoptosis. The Annexin V-FITC/PI staining indicated that EADR significantly induced apoptosis in LNCaP cells. Subsequent investigation into the underlying mechanism of EADR-induced apoptosis revealed a reduction in MMP as evidenced by JC-1 staining. Moreover, Western blotting demonstrated that EADR treatment resulted in the upregulation of BAX, downregulation of BCL-2, and elevation of caspase-3 cleavage in LNCaP cells. Notably, the epilupeol was a prominent compound in EADR as identified by GC-MS. Conclusion: The EADR exhibits anti-cancer activity against the LNCaP human prostate cancer cell line by inducing cytotoxicity and apoptosis. Our findings suggest that EADR promotes apoptosis by upregulating pro-apoptotic BAX, whereas downregulation of anti-apoptotic Bcl-2 results in the reduction of MMP and the activation of caspase-3. Of particular interest is the presence of epilupeol, a major compound identified in EADR, which may hold promise as a candidate for the development of therapeutic agents for prostate cancer. [ABSTRACT FROM AUTHOR]

Citrobacter spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported in Citrobacter spp., which leads to the postulation that Citrobacter spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant Citrobacter spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as Citrobacter sedlakii and Citrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that Citrobacter sedlakii and Citrobacter amalonaticus harbored mcr-3.5 gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (Tn3-IS1-DTnAs2-mcr-3.5-dgkA-IS91) and exhibited high transferring frequencies ranging from 1.03×10-4 - 4.6×10-4 CFU/recipient cell Escherichia coli J53. Colistin-MICs of transconjugants increased = 16-fold suggesting that mcr-3.5 on these plasmids can be expressed in other species. However, beside mcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that mcr gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that Citrobacter could be a reservoir for spreading of antimicrobial resistant genes. To the best of ourknowledge, this is the first report that discovered human-derived Citrobacter spp. that harbored mcr but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of mcr gene in C. sedlakii and mcr-3 in C. amalonaticus. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV), a re-emerging infectious arbovirus, causes Chikungunya fever that is characterized by fever, skin rash, joint pain, arthralgia and occasionally death. Despite it has been described for 66 years already, neither potential vaccine nor a specific drug is available yet. During CHIKV infection, interferon type I signaling pathway is stimulated and releases hundreds of interferon stimulated genes (ISGs). Our previous study reported that IFI16, a member of ISGs, is up-regulated during CHIKV virus infection and the suppression of the gene resulted in increased virus replication. Furthermore, our group also found that inflammasome activation can inhibit CHIKV infection in human foreskin cells (HFF1). Concomitantly, it has been reported that IFI16 activates the inflammasome to suppress virus infection. Therefore, we have hypothesized that IFI16 could be involved in CHIKV infection. In this study, we confirmed the expression level of IFI16 by Western blotting analysis and found that IFI16 was up-regulated following CHIKV infection in both HFF1 and human embryonic kidney cells. We next investigated its antiviral activity and found that forced expression of IFI16 completely restricted CHIKV infection while endogenous silencing of the gene markedly increased virus replication. Furthermore, we have discovered that IFI16 inhibited CHIKV replication, at least, in cell-to-cell transmission as well as the diffusion step. Interestingly, IFI16 also exerted its antiviral activity against Zika virus (ZIKV) infection, the global threat re-emerging virus can cause microcephaly in humans. Taken together, this study provides the first evidence of an antivirus activity of IFI16 during in vitro arbovirus infection, thus expanding its antiviral spectrum that paves the way to further development of antiviral drugs and vaccines.

Paraquat (PQ; a widely used herbicide) exerts its harmful effect to human, mammals and microorganisms upon intracellular conversion to superoxide radical. Cellular responses against toxic paraquat remain not fully understood, especially on the adaptive metabolic changes as a consequence of oxidative burden. In this study, alterations of metabolic processes of Escherichia coli ( E. coli ) by paraquat were systematically investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF). In host cells, the first line mechanism was scrutinized by a remarkable induction of endogenous superoxide dismutase ( E. coli SOD). The second line involved in the metabolic adaptation and compensation for energy production by up- or down-regulation of the enzymes implicated in glycolysis and tricarboxylic acid cycle. Notably, down-regulation of aconitase enzyme and changes of enzyme isoform from the acidic ( p I~5.29) to the higher basidic form ( p I~5.59) were detected. Meanwhile, up-regulation of fumarase approximately 4-5 folds were observed. Importantly, overexpression of human manganese superoxide dismutase (human Mn-SOD) in E. coli cells could in turn down-regulate the expression of fumarase enzyme. This observation was not found when the cells expressing human catalase were tested. Other mechanisms such as changes of purine nucleoside phosphorylase and protein transporters (D-ribose-binding protein and oligopeptide binding protein) were also accounted. However, among all the differentially expressed proteins, the fumarase enzyme is evidenced to be a major target responsible for superoxide-generating paraquat, which may further be applied as a potential biomarker for paraquat toxicity in the future.

Superoxide radical (O2*-) is a toxic byproduct of oxidative metabolism that extensively damages cellular macromolecules and organelles. Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide (H2O2) and molecular oxygen (O2) thus providing a biological defense against oxygen toxicity. The structural gene of human manganese superoxide dismutase (hMnSOD) was successfully cloned into the pET46Ek/LIC by using a Ligation Independent Cloning (LIC) technique. The recombinant human MnSOD was expressed in E. coli strain BL21(DE3)pLysS and purified to homogeneity by Ni2+ -NTA. Supplementation of Mn2+ in the bacterial growth media was proven to be crucial for production of enzymatically active hMnSOD. The recombinant enzyme revealed a specific activity up to 2,857 U mg(-1) as measured by inhibition of photoreduction of nitroblue tetrazolium (NBT). The molecular weight of each subunit was estimated to be 22 kDa by SDS-PAGE. More interestingly, E. coli expressing hMnSOD provides resistance against oxidative stress induced by the herbicide paraquat up to 1.2 mM. These findings gain insights into the biochemical characterization and significant roles of oxidative-protection of the hMnSOD in biological systems.