Screening of Crude Drugs Used in Japanese Kampo Formulas for Autophagy-Mediated Cell Survival of the Human Hepatocellular Carcinoma Cell Line

Abstract

Background: Autophagy is a catabolic process through which dysfunctional proteins and organelles are degraded, and that is associated with the proliferation of cancer cells. The aim of this study was to screen approximately 130 kinds of crude drugs used in Japanese Kampo formulas to identify crude drugs that would regulate the proliferation through autophagy of human hepatocellular carcinoma HepG2 cells. Methods: Extracts of each crude drug were prepared using methanol. Protein levels were determined using Western blotting. Cell viability was measured by MTT assay. Results: Among the 130 crude extracts, 24 of them increased LC3-II expression. Among these, Goboshi (burdock fruit), Soboku (sappan wood), Mokko (saussurea root), Rengyo (forsythia fruit), and Hikai (dioscorea) notably suppressed the proliferation of HepG2 cells and increased p62 expression levels, which suggested that these five extracts downregulate the autophagic activity resulting in the accumulation of p62. On the other hand, Hishinomi (water chestnut), Biwayo (loquat leaf), and Binroji (areca) induced cell growth and decreased or were uninvolved with p62 expression levels, which implied that these three extracts might induce autophagy modulators for cell growth. Conclusions: The results suggest that the compounds contained in the crude drugs selected for this study could control cell viability by regulating autophagic activity in HepG2 cells. The isolation and identification of the active compounds in these drugs might lead to the development of agents for autophagy research and cancer chemoprevention.

Steroidal Saponins Isolated from the Rhizome of Dioscorea tokoro Inhibit Cell Growth and Autophagy in Hepatocellular Carcinoma Cells

“…Cell viability was measured using a previously described method [ 20 , 21 , 22 ]. Briefly, cells (0.9 × 10 4 cells/well) were seeded in 96-well plates.…”

“…We previously screened approximately 130 crude drugs used in Kampo formulas in a comprehensive search to identify bioactive compounds that regulate autophagy in hepatocellular carcinoma HepG2 cells [ 20 ]. We demonstrated that arctigenin, which is a lignan found in the fruits of Arctium lappa and the fruits of Forsythia suspensa , inhibited cell proliferation and attenuated autophagy in HepG2 cells [ 21 ].…”

“…Interestingly, among approximately 130 crude drugs that we screened, the MeOH extract of the rhizome of Dioscorea tokoro exhibited the strongest antiproliferative effect on HepG2 cells. Furthermore, the extract significantly increased LC3-II expression and p62 accumulation [ 20 ]. D. tokoro , a perennial plant belonging to the family Dioscoreaceae, is distributed in the sunny mountains and fields of Japan and in the central to southern part of China.…”

Sphk1 involves in the regulation of autophagy process in cardiac myocyte cells at high glucose condition

“…For examples, a mostly recent study performed by Yang and co-authors indicated that autophagy was capable of protecting nucleus pulposus cells from the apoptosis induced cyclic mechanical tension [25]. Autophagy is also reported to potently increase human hepatocellular carcinoma HepG2 cell proliferation, by using this notion, Okubo et al screened approximately 130 kinds of crude drugs to discovery effective crude drugs supporting cell growth via regulating autophagy process [26].…”

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan

“…In addition to the aforementioned expectation of future clinical application, knowledge of the detailed mechanisms by which Kampo interferes with autophagy will provide insight into the regulation of autophagy. To date, very few studies have focused on the relationship between Kampo and autophagy [11], [12]. We took advantages of our original tf-LC3 screening system, which could quantitatively estimate the effect of each drug on autophagy [13].…”