Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synt... more Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. Maspin mRNA and micromolar amounts of the protein were found in all three laye...
This paper describes a new approach to computer based testing where lecturers submit questions vi... more This paper describes a new approach to computer based testing where lecturers submit questions via word document which is processed to produce an examination, with student results analyzed and reported in a spreadsheet. The overall process starts with lecturers sending question files in word document format via email to the service provider. The questions are passed through the approval process using the editing system and then transferred to the examination system. The examination system directly accesses information from the question files to create a test, which students complete by inserting their answers directly into the spreadsheet file. Finally, the data are analyzed using spreadsheet formulas and the report system sends the results to students' emails. The document based approach helps the system implementation to be simpler and well accepted by the users while consistent with organizational requirements of moving towards electronic data management.
This paper describes a new approach to computer based testing where lecturers submit questions vi... more This paper describes a new approach to computer based testing where lecturers submit questions via word document which is processed to produce an examination, with student results analyzed and reported in a spreadsheet. The overall process starts with lecturers sending question files in word document format via email to the service provider. The questions are passed through the approval process using the editing system and then transferred to the examination system. The examination system directly accesses information from the question files to create a test, which students complete by inserting their answers directly into the spreadsheet file. Finally, the data are analyzed using spreadsheet formulas and the report system sends the results to students' emails. The document based approach helps the system implementation to be simpler and well accepted by the users while consistent with organizational requirements of moving towards electronic data management.
Nowadays, Thai herbal plants are widely accepted in alternative medicine for treatment patients s... more Nowadays, Thai herbal plants are widely accepted in alternative medicine for treatment patients suffering deleterious diseases such as cancer. Having a variety of indications, several herbal formulas including Triphala have been routinely used as health tonic in Thai traditional and Ayurvedic medicines. The formulation of Triphala is a mixture of fruits of three plants: Phyllanthus emblica Linn., Terminalia chebula Retz. and Terminalia bellerica (Gaertn.) Roxb., all of which were reported to inhibit the growth and induce the death of cancer cells effectively. Therefore, anticancer activities inevitably turn out to be one of the essential properties of Triphala formula as well. It is likely that a number of active compounds in the formula, especially tannins, are the key agents that induce the apoptotic cell death via free radical production in cancer cells. On the other hand, all three fruits of these plants also contain high levels of antioxidants, capable of protecting normal cell...
Expression of recombinant proteins using a secretion system can minimize co-purification of conta... more Expression of recombinant proteins using a secretion system can minimize co-purification of contaminating host proteins. Production of His-tagged recombinant proteins in the yeast alpha-factor secretion system has previously required a fermenter system to control the growth conditions such as pH of the yeast culture. We describe an inexpensive non-fermenter system for the production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae that uses a buffered low peptone YP glycerol medium, which does not interfere with immobilized metal affinity chromatography. Maspin, a tumor suppressor serpin, was expressed as a secreted N-terminal His/FLAG-tagged protein. Purification of the soluble active recombinant protein only requires centrifugation, concentration by ultrafiltration, and Ni2+ affinity chromatography. Purified protein yields of this system are 3-5 mg/L culture medium.
To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia belle... more To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia bellerica (T. bellerica) extracts on human hepatocellular carcinoma (HepG2), and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin. HepG2 and A549 cells were treated with P. emblica and T. bellerica extracts either alone or in combination with doxorubicin or cisplatin and effects on cell growth were determined using the sulforhodamine B (SRB) assay. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between plant extracts and drugs. P. emblica and T. bellerica extracts demonstrated growth inhibitory activity, with a certain degree of selectivity against the two cancer cell lines tested. Synergistic effects (CI < 1) for P. emblica/doxorubicin or cisplatin at different dose levels were demonstrated in A549 and HepG2 cells. The T. bellerica/cisplatin or doxorubicin also showed synergistic effects in A549 an...
Dactyloctenium aegyptium (L.) P.B. (Yaa paak khwaai) and Eleusine indica (L.) Gaerth. (Yaa teen-k... more Dactyloctenium aegyptium (L.) P.B. (Yaa paak khwaai) and Eleusine indica (L.) Gaerth. (Yaa teen-ka) have long been used in traditional Thai medicine because of their diuretic, anti-inflamatory, and antipyretic effects. The present study examined the antiproliferative and cytotoxic effects of the hexane and butanolic extracts of these two grass species. All the grass extracts exhibited selective growth inhibition effect on human lung cancer (A549) and cervical cancer (HeLa) cells relative to normal human lung MRC-5 fibroblasts with IC50 values in a range of 202 to 845 mg/ml. Apparently, HeLa cellswere more sensitive to the extracts than A549 cells. Moreover, all the extracts induced lethality in both cancer cell lines atconcentrations close to 1,000 mg/ml, indicating their selective cytotoxicity effects. ELISA assay showed that only the hexaneextract of D. aegyptium (L.) P.B. and E. indica (L.) Gaerth. significantly increased the apoptotic level in extract-treatedA549 cells. However,...
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2012
Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vit... more Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin&amp;amp;amp;amp;#39;s action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.
Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is ... more Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is strongly induced by type I interferons (IFN-α/β), bacterial endotoxin, and cellular stress. Up-regulation of ISG15 is observed in several cancer cell types and is associated with cancer progression. As many cytokines can influence all stages of tumorigenesis, the elevated expression of ISG15 system may be regulated in cancer cells by inflammatory cytokines. In this study, we showed that TNF-α, but not TGF-β and IL-6, up-regulates levels of both ISG15 and its conjugates in human lung carcinoma A549 and human squamous carcinoma HSC4 cell lines. Induction of ISG15 and its conjugates by TNF-α was dose-dependent and required mediation of p38 MAP kinase and Jak1 through up-regulation of endogenous type I interferon expression. SB202190 (p38 MAPK inhibitor) and Jak1 inhibitor suppressed TNF-α-induced expression of ISG15 and its conjugates. However, only SB202190 inhibited the expression of type I interferons by TNF-α. Although B18R, a soluble type I interferon receptor, totally abolished the effect of exogenous IFN-β, it was unable to inhibit completely the TNF-α-induced ISG15 production. In addition, the initiation of ISG15 induction by TNF-α was detected earlier than that of IFN-β induction. Taken together, TNF-α elicits the induction of ISG15 and ISG15 conjugates not only via the autocrine stimulation of type I interferon expression, but also through a type I interferon-independent pathway. These data provide a possible link between inflammatory response and cancer progression.
Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix m... more Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;, amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.
In vitro study of antiproliferation and cytotoxicity of the hexane and butanolic extracts of the ... more In vitro study of antiproliferation and cytotoxicity of the hexane and butanolic extracts of the three grass species, namely Leersia hexandra (L.) Sw. ("Yaa sai"), Panicum repens Linn. ("Yaa channakaat"), and Brachiaria mutica (Forsk.) Stapf ("Yaa khon") demonstrated selective antiproliferative properties in both human lung cancer A549 and cervical cancer HeLa cell lines relative to normal human lung fibroblasts MRC-5,
Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synt... more Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. Maspin mRNA and micromolar amounts of the protein were found in all three laye...
This paper describes a new approach to computer based testing where lecturers submit questions vi... more This paper describes a new approach to computer based testing where lecturers submit questions via word document which is processed to produce an examination, with student results analyzed and reported in a spreadsheet. The overall process starts with lecturers sending question files in word document format via email to the service provider. The questions are passed through the approval process using the editing system and then transferred to the examination system. The examination system directly accesses information from the question files to create a test, which students complete by inserting their answers directly into the spreadsheet file. Finally, the data are analyzed using spreadsheet formulas and the report system sends the results to students' emails. The document based approach helps the system implementation to be simpler and well accepted by the users while consistent with organizational requirements of moving towards electronic data management.
This paper describes a new approach to computer based testing where lecturers submit questions vi... more This paper describes a new approach to computer based testing where lecturers submit questions via word document which is processed to produce an examination, with student results analyzed and reported in a spreadsheet. The overall process starts with lecturers sending question files in word document format via email to the service provider. The questions are passed through the approval process using the editing system and then transferred to the examination system. The examination system directly accesses information from the question files to create a test, which students complete by inserting their answers directly into the spreadsheet file. Finally, the data are analyzed using spreadsheet formulas and the report system sends the results to students' emails. The document based approach helps the system implementation to be simpler and well accepted by the users while consistent with organizational requirements of moving towards electronic data management.
Nowadays, Thai herbal plants are widely accepted in alternative medicine for treatment patients s... more Nowadays, Thai herbal plants are widely accepted in alternative medicine for treatment patients suffering deleterious diseases such as cancer. Having a variety of indications, several herbal formulas including Triphala have been routinely used as health tonic in Thai traditional and Ayurvedic medicines. The formulation of Triphala is a mixture of fruits of three plants: Phyllanthus emblica Linn., Terminalia chebula Retz. and Terminalia bellerica (Gaertn.) Roxb., all of which were reported to inhibit the growth and induce the death of cancer cells effectively. Therefore, anticancer activities inevitably turn out to be one of the essential properties of Triphala formula as well. It is likely that a number of active compounds in the formula, especially tannins, are the key agents that induce the apoptotic cell death via free radical production in cancer cells. On the other hand, all three fruits of these plants also contain high levels of antioxidants, capable of protecting normal cell...
Expression of recombinant proteins using a secretion system can minimize co-purification of conta... more Expression of recombinant proteins using a secretion system can minimize co-purification of contaminating host proteins. Production of His-tagged recombinant proteins in the yeast alpha-factor secretion system has previously required a fermenter system to control the growth conditions such as pH of the yeast culture. We describe an inexpensive non-fermenter system for the production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae that uses a buffered low peptone YP glycerol medium, which does not interfere with immobilized metal affinity chromatography. Maspin, a tumor suppressor serpin, was expressed as a secreted N-terminal His/FLAG-tagged protein. Purification of the soluble active recombinant protein only requires centrifugation, concentration by ultrafiltration, and Ni2+ affinity chromatography. Purified protein yields of this system are 3-5 mg/L culture medium.
To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia belle... more To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia bellerica (T. bellerica) extracts on human hepatocellular carcinoma (HepG2), and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin. HepG2 and A549 cells were treated with P. emblica and T. bellerica extracts either alone or in combination with doxorubicin or cisplatin and effects on cell growth were determined using the sulforhodamine B (SRB) assay. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between plant extracts and drugs. P. emblica and T. bellerica extracts demonstrated growth inhibitory activity, with a certain degree of selectivity against the two cancer cell lines tested. Synergistic effects (CI < 1) for P. emblica/doxorubicin or cisplatin at different dose levels were demonstrated in A549 and HepG2 cells. The T. bellerica/cisplatin or doxorubicin also showed synergistic effects in A549 an...
Dactyloctenium aegyptium (L.) P.B. (Yaa paak khwaai) and Eleusine indica (L.) Gaerth. (Yaa teen-k... more Dactyloctenium aegyptium (L.) P.B. (Yaa paak khwaai) and Eleusine indica (L.) Gaerth. (Yaa teen-ka) have long been used in traditional Thai medicine because of their diuretic, anti-inflamatory, and antipyretic effects. The present study examined the antiproliferative and cytotoxic effects of the hexane and butanolic extracts of these two grass species. All the grass extracts exhibited selective growth inhibition effect on human lung cancer (A549) and cervical cancer (HeLa) cells relative to normal human lung MRC-5 fibroblasts with IC50 values in a range of 202 to 845 mg/ml. Apparently, HeLa cellswere more sensitive to the extracts than A549 cells. Moreover, all the extracts induced lethality in both cancer cell lines atconcentrations close to 1,000 mg/ml, indicating their selective cytotoxicity effects. ELISA assay showed that only the hexaneextract of D. aegyptium (L.) P.B. and E. indica (L.) Gaerth. significantly increased the apoptotic level in extract-treatedA549 cells. However,...
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2012
Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vit... more Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin&amp;amp;amp;amp;#39;s action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.
Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is ... more Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is strongly induced by type I interferons (IFN-α/β), bacterial endotoxin, and cellular stress. Up-regulation of ISG15 is observed in several cancer cell types and is associated with cancer progression. As many cytokines can influence all stages of tumorigenesis, the elevated expression of ISG15 system may be regulated in cancer cells by inflammatory cytokines. In this study, we showed that TNF-α, but not TGF-β and IL-6, up-regulates levels of both ISG15 and its conjugates in human lung carcinoma A549 and human squamous carcinoma HSC4 cell lines. Induction of ISG15 and its conjugates by TNF-α was dose-dependent and required mediation of p38 MAP kinase and Jak1 through up-regulation of endogenous type I interferon expression. SB202190 (p38 MAPK inhibitor) and Jak1 inhibitor suppressed TNF-α-induced expression of ISG15 and its conjugates. However, only SB202190 inhibited the expression of type I interferons by TNF-α. Although B18R, a soluble type I interferon receptor, totally abolished the effect of exogenous IFN-β, it was unable to inhibit completely the TNF-α-induced ISG15 production. In addition, the initiation of ISG15 induction by TNF-α was detected earlier than that of IFN-β induction. Taken together, TNF-α elicits the induction of ISG15 and ISG15 conjugates not only via the autocrine stimulation of type I interferon expression, but also through a type I interferon-independent pathway. These data provide a possible link between inflammatory response and cancer progression.
Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix m... more Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;, amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.
In vitro study of antiproliferation and cytotoxicity of the hexane and butanolic extracts of the ... more In vitro study of antiproliferation and cytotoxicity of the hexane and butanolic extracts of the three grass species, namely Leersia hexandra (L.) Sw. ("Yaa sai"), Panicum repens Linn. ("Yaa channakaat"), and Brachiaria mutica (Forsk.) Stapf ("Yaa khon") demonstrated selective antiproliferative properties in both human lung cancer A549 and cervical cancer HeLa cell lines relative to normal human lung fibroblasts MRC-5,
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