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Dihydrodiol dehydrogenase (DDH) is frequently detected in cancer cells, and its overexpression correlates with drug resistance, the downregulation of DNA repair mechanisms, increased frequency of tumor recurrence, cancer cell metastasis and poor prognosis. The silencing of DDH expression using siRNA, on the other hand, reduces drug resistance and cancer cell mobility. These data suggest that DDH may be an oncogene-related protein. However, no specific DDH inhibitor has been identified to date. Thus, in this study, we used DDH as a target enzyme in a live-cell enzyme-linked immunosorbent assay to screen Chinese medicinal herb extracts (CMHEs) with the aim of identifying a DDH inhibitor. Using this method, we found 49 among 796 CMHEs that inhibited DDH expression. We 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 immunoblot analysis. The function of the active ingredient was characterized by cell function analysis. Our results revealed that the CMHE-purified compounds targeted DDH, inducing autophagy and reducing DNA repair, which in turn enhanced the cytotoxic effects of the anticancer drugs and irradiation.

In eukaryotic cells, transport of the newly synthesized proteins and phospholipids to the appropriate subcellular target compartments is essential for maintaining organelle morphology and cell survival. In animal cells, mitochondria are major organelles containing DNA genome that encodes only for a small fraction of their proteins, which are required for the organelle function. Most mitochondrial proteins are encoded by the nuclear genes and imported to the mitochondria following protein synthesis. Apoptosis-inducing factor (AIF), an essential FAD-dependent NADH oxidase for the oxidative phosphorylation, is located in the intermembranous space and contains mitochondrial localization signals. However, the import mechanism of AIF to the mitochondria is not yet studied. Using sucrose gradient ultracentrifugation and immunoblotting, AIF was detected in fractions of the endoplasmic reticulum, mitochondria-associated membranes (MAM) and mitochondria, and AIF from these fractions was resistant to trypsin in the absence of digitonin, suggesting that AIF could be protected by phospholipids. Knockdown of dynamin-related protein 1 (DRP1kd) expression reduced AIF levels in the mitochondria, but increased AIF concentrations in the MAM. Knockdown of mitofusin-2 (Mfn-2kd) or ATPase family AAA domain containing 3A (ATAD3Akd) expression, however, reduced AIF levels in the mitochondria and increased the number of transport vesicles that contained AIF in the cytosol, indicating that ATAD3A and Mfn-2 were respectively essential for the import and fusion of transport vesicles into the mitochondria. Here we show that AIF is imported from the endoplasmic reticulum to the mitochondria via mitochondria-associated membranes and transport vesicles.

Expression of thymidylate synthase (TS) in tumor cells is frequently suggested as an important prognostic factor for patients scheduled for chemotherapy with 5-fluorouracil (5-FU). However, clinical evidence does not fully support such an anticipation. We studied the expression of rTSbeta, a reverse orientation gene of TS, as a 5-FU resistance marker in patients with primary breast cancer. Expression of rTSbeta was examined in 129 patients with newly diagnosed breast cancer and five breast cancer cell lines by immunohistochemistry, immunocytochemistry and immunoblotting. Clinically, expression of rTSbeta was found to correlate with survival of the patients (p=0.023) when patients received chemotherapeutic regimen containing 5-FU. In vitro, rTSbeta expression was found to correlate with 5-FU resistance in breast cancer cell lines. Notably, in the 5-FU-resistant cells, rTSbeta was identified in the nucleus, whereas in the 5-FU-sensitive cells, rTSbeta was found in the cytoplasm. Nuclear localization of rTSbeta was further found to be associated with protein farnesylation. Therefore, nuclear expression of rTSbeta could be a novel 5-FU resistance marker in patients with primary breast cancer.

By combining suppression subtractive hybridization and microarray to examine gene expressions between metastatic and non-metastatic non-small cell lung cancer (NSCLC), we have identified differential expression spectra of matrix metalloproteinases (MMP). Among MMPs, expressions of MMP-13, -14, -15 and -24 decreased, those of MMP-9, -11, -12, -16, -17, -19 and -23B did not change, and those of MMP-1, -2, -7, -8 and -10 increased dramatically. Overexpressions of MMP-1, -2, -7 and -10 were confirmed by reverse transcription-polymerase chain reaction. In this study we further assessed the clinical significance of MMP-1, -2, -7 and -10. Specimens from 472 patients with completely resected NSCLC were examined by immunohistochemistry. The median follow-up period was 38 months (range, 2-113 months). Overexpression of MMP-1 was observed in 72.9% (n=344) of 472 patients, that of MMP-2 was 77.9% (n=352), MMP-7 63.3% (n=299) and that of MMP-10 was 27.1% (n=128). For patients with lymph node metastasis, MMP-1 and -2 overexpressions were not only independent prognostic factors for unfavorable outcome, but also associated with decreased survival (p=0.0015, and p=0.011 respectively). The present study showed that MMP expression spectrum in NSCLC was heterogeneous: expression of some MMP increased, some unchanged, while some decreased. Therefore, it should be worth determining MMP expression pattern as a regimen reference for NSCLC patients who were scheduled to receive MMP inhibitor, which was class-specific, as adjuvant therapeutic agent.