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Background Gastric cancer is one of the most leading causes of cancer death worldwide. Therefore, treatment studies have been being conducted, one of which is screening of novel agents from medicinal herbs. Elephantopus mollis Kunth (EM) belonging to Asteraceae family is a perennial herb with several therapeutic properties including anticancer activity. However, the effect of this species on gastric cancer has not been reported yet. In this study, cytotoxicity of different EM crude extracts was investigated on AGS gastric cancer cell line. Besides, the effects of extract on nuclear morphology, caspase-3 activation, and gene expression were also explored. Results The results showed that ethyl acetate extract exhibited a remarkably inhibitory ability (IC50 = 27.5 μg/ml) on the growth of AGS cells, while causing less toxicity to normal human fibroblasts. The extract also induced apoptotic deaths in AGS cells as evidenced by cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, caspase-3 activation, and the upregulation of BAK and APAF-1 pro-apoptotic genes related to mitochondrial signaling pathway. Specifically, BAK and APAF-1 mRNA expression levels showed 2.57 and 2.71-fold increases respectively. Conclusions The current study not only proved the anti-gastric cancer activity of EM ethyl acetate extract but also proposed its molecular mechanism. The extract could be a potential candidate for further investigation.

Recombinant granulocyte colony–stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Background: The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (∼97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

MTT assay has been applied widely in stimulation and inhibition tests for adherent cells. However, reading step in regular MTT assays requires medium removal to dissolve formazan by absolute solvents results are usually affected by incomplete formazan dissolution and protein precipitation in diluted solvents. Therefore, most of current MTT assay procedures have difficulties in application on suspension cell lines. In this study, we demonstrated a modified MTT assay in which formazan dissolution method was improved by using the combination of DMSO and SDS-lysis solution. Importantly, the modified MTT assay did not require medium removal, thus it can be applicable for both suspension and adherent cell lines. We also verified that the modified MTT procedure could be effectively applied in bioactivity assays such as cancer cell inhibition and fibroblast stimulation assays. Besides the ease of use, our data regarding nonlinear regression model fitting, data variation, and separation clearly demonstrated that the sensitivity, stability and precision of modified assay were higher than those of common MTT procedures using isopropanol or DMSO as solvents. This study indicated that the modification described here can broaden the MTT assay application on suspension cell lines and also simplify the MTT protocol on adherent cell lines.

Ethnopharmacological relevance Elephantopus mollis Kunth (EM), which belongs to Asteraceae family, has been used as a folk medicine with diverse therapeutic properties. Previous studies reported that crude extracts of this plant could inhibit several cancer cell lines, including breast carcinoma MCF-7, liver carcinoma HepG2, colorectal carcinoma DLD-1, lung carcinoma NCI–H23, etc. Aim In this study, the anticancer activity and associated molecular mechanism of EM which is distributed in Vietnam were investigated. Materials and methods The cytotoxicity of various EM extracts was evaluated on different cell lines by MTT assay. In addition, the effects of EM extracts on cell growth, cell morphology, nuclear morphology, caspase-3 activation, and mRNA expression levels of apoptosis-related genes were also examined. Results Our results demonstrated that ethyl acetate extract (EM-EA) caused proliferative inhibition and apoptotic induction towards A549 lung cancer cells (IC50 = 18.66 μg/ml, SI = 5.8) and HL60 leukemia cells (IC50 = 7.45 μg/ml, SI = 14.5) while petroleum ether extract (EM-PE) showed high toxicity to HL60 cell line (IC50 = 11.14 μg/ml, SI = 6.7). Notably, Raji lymphoma cells were also affected by these extracts (IC50 4), which has not been reported yet. Furthermore, mechanisms of EM extracts were elucidated. The significant downregulation of PCNA mRNA level induced by EM-EA/PE extracts contributed to the cell-growth restraint. EM-EA extract might activate apoptosis in A549 cells through both extrinsic and intrinsic signaling pathways by causing a 1.55-fold increase in BID, 3.65-fold increase in BAK and 3.11-fold decrease in BCL-2 expression level. Meanwhile, with EM-EA-extract treatment, HL60 cells might encounter P53-dependent apoptotic deaths. Conclusions The combination of antiproliferation and apoptosis activation contributed to the high efficacy of EM extracts. These findings not only proved the anticancer potential of EM but also provided further insights into the mechanisms of EM extracts.

Vanillin is a phenolic aldehyde compound generated during the saccharification pretreatment of the lignocellulosic biomass in bioethanol production. It has been reported that vanillin inhibits yeast growth, represses translation and induces the formation of ribonucleoprotein granules such as P-bodies and stress granules in Saccharomyces cerevisisae. I herein demonstrated that vanillin causes oxidative stress and induces mitochondrial fragmentation in yeast cells, providing some new information regarding the effects of vanillin on yeast. Additionally, I found that the null mutant of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in the pentose phosphate pathway (PPP), was more susceptible to vanillin than the wild-type, suggesting the importance of G6PDH and PPP in vanillin tolerance. It has been known that vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7 in vitro. In this study, I verified the roles of these proteins in vanillin tolerance in vivo. I also showed that although both the ADH6 and ADH7 genes were transcriptionally upregulated, the protein levels of Adh7 but not Adh6 were significantly increased in the presence of high concentration of vanillin, indicating that the ADH7 mRNA was preferentially translated under severe vanillin stress. In addition to the ADH7 gene, we found that the expression of VIE1 gene, coding an unknown protein, was also significantly induced upon vanillin treatment. Both the ADH7 and VIE1 promoter can drive the expression of non-native genes under severe vanillin stress despite the overall translation repression, suggesting that these promoters are useful to overexpress genes in bioethanol production. Importantly, the VIE1 promoter also enabled the increased expression of genes in the presence of furfural and 2-hydroxymethylfurfural, other fermentation inhibitors derived from lignocellulosic biomass. Therefore, the VIE1 promoter would be more practical in modification of gene expression in order to improve the efficiency of ethanol production from lignocellulosic biomass.