Anemone rivularis inhibits pyruvate dehydrogenase kinase activity and tumor growth.
BACKGROUND
Anemone rivularis Buch.-Ham. ex DC. (Ranunculaceae) have been used as a traditional remedy for treatment of inflammation and cancer. However, there is no report demonstrating experimental evidence on anti-tumor action of A. rivularis.


OBJECTIVE
The Warburg's effect, preference of aerobic glycolysis rather than oxidative phosphorylation (OXPHOS) even in oxygen rich condition, is focused as one of major characteristics of malignant tumor. Thus, we investigated the effect of A. rivularis on the Pyruvate dehydrogenase (PDH) kinases (PDHKs), a major molecular targets for reducing aerobic glycolysis.


METHODS
The ethanol extract of whole plant of A. rivularis (ARE), fingerprinted by high performance liquid chromatography (HPLC), was applied to in vitro and cell-based PDHK activity assays. The effect of ARE on cell viabilities of several tumor cells was estimated by MTT assay. The expression of phosphor-PDH, PDH and PDHK1 were measured by Western blot analysis. The production of reactive oxygen species (ROS) and apoptosis was measured by fluorescence-activated cell sorting analysis, using 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and Annexin V/propidium iodide (PI) staining, respectively. Mitochondrial membrane potential was examined by tetramethylrhodamine methyl ester (TMRM) staining. In vivo anti-tumor efficacy of ARE was estimated by means of tumor volume and weight using allograft injection of murine Lewis lung carcinoma (LLC) cells to dorsa of C57BL/6 mice.


RESULTS
ARE inhibited the viabilities of several cancer cells, including MDA-MB321, K562, HT29, Hep3B, DLD-1, and LLC. ARE suppressed PDHK activity in in vitro kinase assay, and also inhibited aerobic glycolysis by reducing phosphorylation of PDHA in human DLD-1 colon cancer and murine LLC cells. The expression of PDHK1, a major isoform of PDHKs in cancer, was not affected by ARE treatment. Moreover, ARE increased the both ROS production and mitochondrial damage. In addition, ARE suppressed the in vitro tumor growth through mitochondria-mediated apoptosis. The growth rates of allograft LLC cells were also reduced by ARE treatment.


CONCLUSIONS
Here, we firstly report that ARE inhibits PDHK activity and growth of tumor in both in vitro and in vivo experiments. Therefore, we suggest ARE as a potential candidate for developing anti-cancer drugs.
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Tae-Wook
TW
Chung
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Jung Hee
JH
Lee
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Hee-Jung
HJ
Choi
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Mi-Ju
MJ
Park
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Eun-Yeong
EY
Kim
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea.
Jung Ho
JH
Han
Department of Molecular Biology, College of Natural Science, Pusan National University, Busan, Republic of Korea.
Se Bok
SB
Jang
Department of Food Science and Technology, Keimyung University, Daegu, Republic of Korea.
Syng-Ook
SO
Lee
Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk 34141, Republic of Korea.
Sang Woo
SW
Lee
Yunnan Academy of Agricultural Sciences, Yunnan 650223, China.
Jin
J
Hang
Yunnan Academy of Agricultural Sciences, Yunnan 650223, China.
Li Wan
LW
Yi
Department of Korean Medical Science, School of Korean Medicine and Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongnam, Republic of Korea. Electronic address: hagis@pusan.ac.kr.
Ki-Tae
KT
Ha
D031883:Anemone / Q000737:chemistry*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D018827:Carcinoma, Lewis Lung / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000473:pathology; D006019:Glycolysis / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D020014:K562 Cells; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D010936:Plant Extracts / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017382:Reactive Oxygen Species / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D010936:Plant Extracts; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017382:Reactive Oxygen Species; D017346:Protein Serine-Threonine Kinases
Anemone rivularis; Apoptosis; Mitochondria; Pyruvate dehydrogenase kinase; Warburg's effect