Glycoprotein isolated from Solanum nigrum L. kills HT-29 cells through apoptosis.
Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We previously isolated glycoprotein from SNL and observed that it decreased viable HT-29 cell numbers at a low concentration (60 microg/mL). This study investigated the apoptotic signal pathway triggered by glycoprotein isolated from SNL in HT-29 cells. Treatment of HT-29 cells with SNL glycoprotein (60 microg/mL) for 4 hours resulted in a cytotoxic effect of more than 60%, compared with the control. To explain the apoptotic effects of SNL glycoprotein, we investigated its effects on 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated protein kinase C (PKC) alpha activity and DNA-binding activity of nuclear factor (NF) kappaB in HT-29 cells, using western blot analysis and electrophoretic mobility shift assays. Results from these experiments showed that SNL glycoprotein has remarkable inhibitory effects on the activities of TPA (100 nM)-stimulated PKCalpha and NF-kappaB in HT-29 cells. They also substantiated that PKCalpha is a part of the TPA-activated upstream signal pathway of NF-kappaB, since NF-kappaB activity was inhibited by staurosporine (a PKC inhibitor) and pyrrolidine dithiocarbamate (an NF-kappaB inhibitor) in a western blot analysis. Furthermore, to verify the triggering of apoptosis by the SNL glycoprotein, we performed DNA fragmentation, nuclear staining, and protein expression assays of apoptotic-related proteins. The amount of DNA fragmentation and apoptotic cell numbers increased in a dose-dependent manner after treatment with SNL glycoprotein. Apoptosis-related protein assays demonstrated that SNL glycoprotein-induced apoptosis is associated with the regulation of bcl-2 and Bax expression. Taken together, the results of this study showed that the activation of PKCalpha, NF-kappaB, and Bax expression by SNL glycoprotein is possibly involved in the apoptotic process. Consequently, these results indicate that SNL glycoprotein causes HT-29 cell death through apoptosis by its ability to modulate anti-apoptotic signals. We suggest that SNL glycoprotein is a natural anti-cancer agent due to its potential to induce apoptosis in HT-29 cells.
Lim
Kye-Taek
KT
#521 Molecular Biochemistry Laboratory, Institute of Biotechnology, Chonnam National University, Yongbong Dong, Kwangju, Korea. ktlim@chonnanm.ac.kr
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D032324:Solanum nigrum / Q000737:chemistry*; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; D006023:Glycoproteins; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha