Curcumin induced the cell death of immortalized human keratinocytes (HaCaT) through caspase-independent and caspase-dependent pathways.

Curcumin is a diketone compound found in turmeric. It is used as food additives and spices, and has anti-proliferation and anti-cancer properties. However, the effect of curcumin on human keratinocytes (KCs) is still unclear. In this study, curcumin dramatically inhibited the cell growth of immortalized human KCs (HaCaT) and arrested the cells at the G2/M phase, with an apoptosis rate of 33.95% after 24 µM curcumin treatment. HaCaT cells showed changes in typical apoptotic morphology and the configuration of nuclear matrix-intermediate filaments (NM-IFs) after treatment with curcumin. We identified 16 differentially expressed nuclear matrix (NM) proteins, including apoptosis inducing factor (AIF) and caspase 3, by 2-DE and MALDI-TOF/TOF mass spectrometry. The expression of AIF decreased in the mitochondria and increased in the nucleus. Immunofluorescence assays showed that AIF was released from the mitochondria to the nucleus. AIF silencing and caspase inhibitor (z-vad-fmk) both lead to HaCaT cells being insensitive to apoptosis induced by curcumin. Meanwhile, after curcumin treatment, mitochondrial membrane depolarization led to cytochrome c release from the mitochondria to the cytoplasm, and the ratio of Bax to Bcl-2 in HaCaT cells was also increased, which subsequently initiated the activation of caspase-3. These results suggest that curcumin-induced apoptosis of HaCaT cells occurs not only through the caspase-dependent pathway but also through the caspase-independent pathway. This discovery enhances the development and utilization of curcumin and provides possible evidence for the treatment of proliferative skin diseases, including skin cancer.

Proanthocyanidins isolated from the leaves of Photinia × fraseri block the cell cycle and induce apoptosis by inhibiting tyrosinase activity in melanoma cells.

Tyrosinase is considered a molecular marker of melanoma, and few natural antitumor drugs targeting tyrosinase have been identified. In this study, proanthocyanidins (PAs) were isolated from the leaves of Photinia × fraseri and their structures were characterized by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the effects of antityrosinase activity were investigated. The results showed that the basic structural units of PAs are composed of catechin and epicatechin and that oligomer is the main component. PAs exhibited better antityrosinase activity via chelation of copper ions and by disturbing o-quinone production. Furthermore, analyses of the cell cycle, apoptosis rate, and regulation of melanin protein expression revealed preliminarily that PAs could affect melanin production by downregulating microphthalmia transcription factor (MITF) expression and by inhibiting the activities of tyrosinase and tyrosinase related protein 1 (TRP-1), leading to cell cycle arrest and apoptosis of melanoma cells. Collectively, our study demonstrated that PAs are potential tyrosinase inhibitors and have good antimelanoma effects. These findings provide a theoretical support for the application of tyrosinase inhibitors and for further drug development.

Inhibitory effects and molecular mechanism on mushroom tyrosinase by condensed tannins isolation from the fruit of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow.

The structure of extracted condensed tannin (CT) from the fruit of Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow) and the molecular mechanisms by which CT inhibits the activity of mushroom tyrosinase were investigated. The structure of CT was characterized by high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The kinetic assays were used to detect inhibition effect, type and mechanism. UV scanning, fluorescence quenching, copper interacting, o-quinone interaction and molecular docking assays were also used to reveal the molecular mechanisms by which CT inhibit tyrosinase. The results showed the structural units of CT containing afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin. Kinetic analysis showed that CT inhibits both the monophenolase and diphenolase activities of tyrosinase and exhibits reversible, mixed type mechanism. The fruit CT interacts primarily with the copper ions and specific amino acid residue (Asn191, Thr203, Ala202, Ser206, Met201, His194, His54, Glu182 and Ile42) in the active site of tyrosinase to disturb oxidation of substrates by tyrosinase. These results suggested the sour jujube fruit is a potential natural source of tyrosinase inhibitors, and has a potential to be used in food preservation, whitening cosmetics.

Effects of isoimperatorin on proliferation and apoptosis of human gastric carcinoma cells.

Resistance to apoptosis is an characteristic of cancer cells that serves a critical function in tumor development and represents a target for antitumor therapy. Isoimperatorin (ISOIM), a coumarin compound, exhibits antitumor functions in multiple types of tumor cells. However, its antitumor effects and molecular mechanisms with respect to gastric cancer have not been elucidated. The present study assessed the anti-proliferative and apoptotic effects of ISOIM on human BGC-823 gastric cancer cells and elucidated its underlying molecular mechanisms. Cell proliferation was evaluated using MTT assays. Analysis of cell morphology was performed by hematoxylin and eosin, Hoechst 33258 and acridine orange/ethidium bromide staining. In addition, cell cycle and apoptosis was evaluated using flow cytometry analysis; expression of apoptosis-associated proteins was studied by western blotting. The results of the present study revealed that ISOIM significantly inhibited cell proliferation by arresting the cell cycle at the G2/M phase and induced apoptosis by increasing Bcl-2-associated X (Bax) expression with a concomitant decrease in Bcl-2 expression, resulting in a decreased Bcl-2/Bax ratio compared with the control. In addition, ISOIM treatment also resulted in cytochrome c translocating from the mitochondria to the cytosol. Furthermore, caspase-3 was significantly activated in response to treatment with ISOIM, suggesting that apoptosis in BGC-823 cells is induced in the mitochondrial pathway. Taken together, the results of the present study indicate that ISOIM may significantly induce apoptosis in BGC-823 cells and that the pro-apoptotic mechanisms of ISOIM could be associated with the mitochondrial pathway.