Int J Mol Med. 2013 Sep;32(3):577-84. doi: 10.3892/ijmm.2013.1441. Epub 2013 Jul 12.

Extracts of Koelreuteria henryi Dummer induce apoptosis and autophagy by inhibiting dihydrodiol dehydrogenase, thus enhancing anticancer effects.

Chiang YY1, Wang SL1, Yang CL2, Yang HY2, Yang HC3, Sudhakar JN2, Lee CK4, Huang HW5, Chen CM5, Chiou SH3, Chiang SF3, Fang HY6, Chen CY6, Shieh SH7, Chow KC2

1Department of Dental Laboratory Technology, Central Taiwan University of Science and Technology; 2Graduate Institute of Biomedical Sciences; 3Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan; 4College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan; 5Endemic Species Research Institute, Council of Agriculture, Executive Yuan, Chi-Chi, Taiwan; 6Departments of Surgery, China Medical University Hospital; 7Departments of Health Services Administration, China Medical University, Taichung, Taiwan.

 

Abstract

Dihydrodiol dehydrogenase (DDH) is frequently detected in cancer cells, and its overexpression correlates with drug resistance, down-regulation of DNA repair mechanism, increased frequency of tumor recurrence, cancer cell metastasis and poor prognosis. Silencing of DDH expression using siRNA, on the other hand, reduced drug resistance and cancer cell mobility. These results suggested that DDH could be an oncogene-related protein. However, no specific DDH inhibitor has been identified. In this study, we used DDH as a target enzyme in a live-cell enzyme-linked immunosorbent assay to screen Chinese medicinal herbal extracts (CMHE) for finding a DDH inhibitor. Using this method, we found 49 among 796 CMHE that inhibited DDH expression. We then selected three potential extracts, which had the highest activity against DDH, for further fractionation using high-performance liquid chromatography. The active ingredient was identified by immunoblotting. The function of the active ingredient was characterized by cell function studies. Our results showed that the purified compounds from CMHE could target at DDH to induce autophagy, and to reduce DNA repair, which in turn enhanced cytoxic effect of anticancer drugs and irradiation.

Keywords: dihydrodiol dehydrogenase, oncogene, drug detoxification, lipid flow, autophagy

PMID: 23857115

 

Supplement:

As noted above, overexpression of dihydrodiol dehydrogenase (DDH) is frequently detected in cancer cells, including bladder, esophageal, gastric, NSCLC, ovarian, prostate, and uterine cervical cancers, and its overexpression correlates with drug resistance. The DDH detected in cancer cells were mostly aldo-keto reductase (AKR) 1C1, and to a lesser extent AKR1C2 (for the nomenclature of respective enzymes, please refer to http://www.med.upenn.edu/akr/). The nature of DDH to catabolize xenobiotic compounds, e.g., polycyclic aromatic hydrocarbons (PAH) and the derivatives, in association with cytochrome p450, family 1, member A1 (cyp1A1) and microsomal epoxide hydrolase, indicate that the enzyme is able to deactivate anticancer drugs with similar polycyclic structures (Figure 1), and renders cancer cells drug resistant. Therefore, inhibiting DDH expression could then increase drug sensitivity in cancer cells. However, protein structure analysis (by web software’s PSORT II, http://psort.hgc.jp/form2.html and SOSUI, http://bp.nuap.nagoya-u.ac.jp/sosui/) shows that the enzyme per se is a soluble protein, which is distributed in the cytoplasm. Chemical configuration of substrates, on the other hand, indicates the extreme hydrophobicity, suggesting that these compounds could be embedded in the lipid structure, e.g., the endoplasmic reticulum (ER), microsomes and the Golgi apparatus, or they could be located in the recessive pocket of carrier proteins. These facts, however, expose a dilemma between the enzyme and its substrates; in particular, the ubiquitous presence of waters in the cytoplasm will become impassible hindrances that block the direct enzyme-substrate interaction. Our current immunofluorescence microscopic findings suggested that DDH was located on the ER, microsomes, mitochondria-associated membrane (MAM) and the Golgi apparatus via a yet to be determined mechanism. These data resolve the direct enzyme-substrate interaction problem, and shed further light on the oncogenic essence of the enzyme in cancer progression. Interestingly, silencing of DDH expression simultaneously reduced protein levels of dynamin-related protein 1 (DRP1, a GTPase) and the ATPase family, AAA domain containing 3A (ATAD3A, an ATPase), the two essential proteins that were co-overexpressed in cancer cells and involved in inter-organelle material transport as well as flow of membrane lipids (Figure 2), including phospholipids and sphingolipids. Silencing of DRP1 or ATAD3A gene expression would not only accumulate the newly synthesized proteins and toxic lipids, e.g., ceramide, in the previous organelle or inter-organelle transport vesicles to interrupt cytoplasmic material flow, which would then induce aberrant conversion of LC3-I to LC3-II and increase autophagosomal formation to diminish the function of tumor progression.

KCChow-fig1

Figure 1 Roles of DDH in the metabolic conversion of polycyclic aromatic hydrocarbons (PAH) and the derivatives (modified from Burczynski ME & Penning TM. Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor. Cancer Res. 2000;60(4):908-15).

KCChow-fig2Figure 2. A simplified sketch of the importation route for apoptosis-inducing factor (AIF) to the mitochondria. The importation route for AIF is from the ER, the MAM, and the transport vesicles (TV) to the mitochondria (left-side of the Figure). In DRP1 knockdown (DRP1kd) cells, because the budding-off mechanism of TV is inhibited, the accumulated cargo proteins and phospholipids, which are detained in the ER and MAM, will cause the enlargement of the MAM (upper center of the Figure). In ATAD3Akd cells, TV are formed around the dilated ER/MAM, and the lack of ATAD3A (ATPase, for providing movement energy of the TV) will idle the movement of TV towards mitochondria. Insufficient supply of the newly synthesized proteins and phospholipids would induce mitochondrial fragmentation (middle center of the Figure). Similarly, in MFN2kd cells, the TV cannot fuse with the mitochondria, and the presence of TV around mitochondria is more evident (lower center of the Figure). Silencing of either DRP1 or ATAD3A could interrupt cytoplasmic material flow and increase autophagosomal formation (Modified from Chiang SF, et al. An alternative import pathway of AIF to the mitochondria. Int J Mol Med 2012;29:365-372)

Acknowledgements: This study was supported by grants from the Department of Health, Executive Yuan, Taipei, Taiwan to the China Medical University Hospital, Cancer Research of Excellence program (DOH102-TD-C-111-005), Taichung, Taiwan, and the National Science Council (NSC 101-2320-B-005-002), Taipei, Taiwan.

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