Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death.

BACKGROUND: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained. METHODS: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers. RESULTS: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death. CONCLUSIONS: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.

Apoptosis of human cholangiocarcinoma cell lines induced by ß-escin through mitochondrial caspase-dependent pathway.

The study aimed to evaluate the effects of ß-escin on human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and to explore its mechanisms. Cell growth, cell cycle and apoptosis were investigated, respectively, by MTT assay, single PI and FITC/PI double-staining flow cytometry, and fluorescence microscopy. The protein expression was determined by western blotting. The study revealed that ß-escin inhibited cholangiocarcinoma cell growth in a dose- and time-dependent manner, and the cell cycle of QBC939 and Sk-ChA-1 cells was arrested in the G2/M phase, and MZ-ChA-1 cells in G1 phase. Apoptosis of the three cholangiocarcinoma cell lines induced by ß-escin was associated with the collapse of the mitochondrial membrane potential and the activation of caspase-3. The apoptotic effect of ß-escin was suppressed by pancaspase inhibitor z-VAD-fmk. Molecular dissection revealed that the antiapoptotic protein bcl-2 was down-regulated after cholangiocarcinoma cell lines were treated with ß-escin, while the protein levels of bax and p53 were unchanged. Apoptosis was accompanied by an increase in reactive oxygen species (ROS). These results suggest that ß-escin induces apoptosis of cholangiocarcinoma cells through an intrinsic mitochondrial caspase-dependent pathway, and the increase in the bax/bcl-2 ratio and ROS may play important roles in ß-escin-induced apoptosis of cholangiocarcinoma cells.

Tanshinone IIA inhibits Hep-J5 cells by increasing calreticulin, caspase 12 and GADD153 protein expression.

Tanshinone IIA (Tan-IIA) is extracted from Danshen, Salviae miltiorrhizae Radix, which has been widely adopted in traditional herbal medicine to treat cardiovascular and hepatic diseases. Tan-IIA induces apoptosis and inhibits proliferation in human hepatocellular carcinoma (HCC) cells. However, the molecular mechanisms of Tan-IIA on human HCC cells are not understood clearly. In the present study, the cytotoxicity of Tan-IIA as well as its molecular mechanisms in human HCC J5 cells was investigated. The cytotoxicity was assayed by MTT. The protein expression of p53, p21, Bax, Bcl-2, Cdc25c, Cdc2, calreticulin, caspase 12, GADD153, caspase 3 and beta-actin in J5 cells were determined by Western blotting. The cell cycles were analyzed by FACS. The protein expression of caspase 12, GADD1533 and caspase 3 were detected by immunocytochemical staining. The results showed that Tan-IIA inhibited J5 cells in a dose- and time-dependent manner. The protein expression of p53, p21, Bax, calreticulin, caspase 12, caspase 3 and GADD153 were increased, but Bcl-2, Cdc25c and Cdc2 were decreased in J5 cells. In addition, the results also showed that Tan-IIA arrested J5 cells in the G2/M phase. Immunocytochemistry staining showed that J5 cells treated with Tan-IIA up-regulated the protein expression of caspase 12, 3 and GADD153. Taken together, the findings suggest that Tan-IIA inhibits and induces apoptosis in J5 cells through novel molecular targets, calreticulin, caspase 12 and GADD153.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells.

BACKGROUND: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. METHODS: In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. RESULTS: Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3. CONCLUSION: These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells.

AIM: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. METHODS: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis. RESULTS: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3. CONCLUSION: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

Acetone extract of Angelica sinensis inhibits proliferation of human cancer cells via inducing cell cycle arrest and apoptosis.

Angelica sinensis (Oliv.) Diels, a traditional Chinese medicine, has been widely prescribed in treatment of gynecological diseases. Bio-based assays for extracts of Angelica sinensis showed that the acetone extract (AE-AS) had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells. The IC50 values of AE-AS on mentioned cancer cells ranged from 35 to 50 microg/ml after 24 h of treatment. After 72 h of exposure, AE-AS (40 microg/ml) significantly reduced A549 cell proliferation to 24 +/- 3.2% of control. In A549 cells, the cell cycle analysis showed that AE-AS induced a significant increase in the number of cells in G0/G1, with a concomitant decrease in the number of cells in S phase. AE-AS-induced chromatin changes and apoptosis of A549 cells were confirmed by Hoechst 33342 DNA staining and annexin V staining. A549 cells treated with AE-AS caused activation of caspase-9 and -3, and AE-AS-induced apoptosis could be inhibited by the broad-spectrum caspase inhibitor, z-VAD-fmk. The Western blot indicated the AE-AS-triggered apoptosis is mediated via suppression of Bcl-2 oncoprotein expression rather than p53 or Bax. Besides, AE-AS decreased the levels of cdk4 protein was observed. These results indicate that the AE-AS could induce G1/S arrest and activate the mechanism of apoptosis in human cancer cells. Extracts obtained from different methods of fractionation might possess distinct bioactivity. These results prompted us to further evaluate the in vivo anticancer effects and elucidate the chemical composition profile of AE-AS.