Chutima Kaewpiboon

Studies of the in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities of selected Thai medicinal plants

BackgroundTraditional folk medicinal plants have recently become popular and are widely used for primary health care. Since Thailand has a great diversity of indigenous (medicinal) plant species, this research investigated 52 traditionally used species of Thai medicinal plants for their in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities.MethodsThe 55 dried samples, derived from the medicinally used parts of the 52 plant species were sequentially extracted by hexane, dichloromethane, ethanol and water. These 220 extracts were then screened for in vitro (i) cytotoxicity against four cell lines, derived from human lung (A549), breast (MDA-MB-231), cervical (KB3-1) and colon (SW480) cancers, using the MTT cytotoxicity assay; (ii) antioxidant activity, analyzed by measuring the scavenging activity of DPPH radicals; (iii) lipase inhibitory activity, determined from the hydrolytic reaction of p-nitrophenyllaurate with pancreatic lipase; and (iv) antimicrobial activity against three Gram-positive and two Gram-negative bacteria species plus one strain of yeast using the disc-diffusion method and determination of the minimum inhibitory concentration by the broth micro-dilution assay.ResultsThe crude dichloromethane and/or ethanol extracts from four plant species showed an effective in vitro cytotoxic activity against the human cancer cell lines that was broadly similar to that of the specific chemotherapy drugs (etoposide, doxorubicin, vinblastine and oxaliplatin). In particular, this is the first report of the strong in vitro cytotoxic activity of Bauhinia strychnifolia vines. The tested tissue parts of only six plant species (Allium sativum, Cocoloba uvifera, Dolichandrone spathacea, Lumnitzera littorea, Sonneratia alba and Sonneratia caseolaris) showed promising potential antioxidant activity, whereas lipase inhibitory activity was only found in the ethanol extract from Coscinum fenestratum and this was weak at 17-fold lower than Orlistat, a known lipase inhibitor. The highest antimicrobial activity was observed in the extracts from S. alba and S. caseolaris against Pseudomonas aeruginosa and Candida albicans, respectively.ConclusionThe Thai medicinal plant B. strychnifolia is first reported to exert strong in vitro cytotoxic activities against human cancer cell lines and warrants further enrichment and characterization. The broad spectrum of the biological activities from the studied plant extracts can be applied as the guideline for the selection of Thai medicinal plant species for further pharmacological and phytochemical investigations.

Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress.

Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-induced ER stress, suggesting that protein synthesis is involved in ASK-induced ER stress. Moreover, we showed using salubrinal, an ER stress inhibitor that reactive oxygen species production, JNK activation, and Nur77 upregulation and its translocation to cytoplasm are necessary for ER-induced stress. Interestingly, we found that JNK inactivation suppresses ASK-induced ER stress, whereas Nur77 siRNA treatment does not, indicating that JNK is required for ASK-induced ER stress. Accordingly, we report that ASK induces ER stress, which is prerequisite for apoptosis of HBX-expressing hepatocellular carcinoma cells.

Upregulation of Stat1-HDAC4 confers resistance to etoposide through enhanced multidrug resistance 1 expression in human A549 lung cancer cells

Despite efforts to develop efficient chemotherapeutic drug strategies to treat cancer, acquired drug resistance is a commonly encountered problem. In the present study, to investigate this phenomenon, human A549 lung cancer cells resistant to the topoisomerase inhibitor etoposide (A549RT‑eto) were used and compared with A549 parental cells. A549RT‑eto cells demonstrated increased resistance to etoposide‑induced apoptosis when compared with A549 parental cells. Notably, A549RT‑eto cells were observed to exhibit greater levels of histone deacetylase 4 (HDAC4), phospho‑Stat1 and P‑glycoprotein [P‑gp; encoded by the multidrug resistance 1 (MDR1) gene], compared with A549 cells. To address whether HDAC4 protein is involved in etoposide resistance in A549 cells, A549RT‑eto cells were treated with trichostatin A (TSA; an HDAC inhibitor) during etoposide treatment. The combined treatment was demonstrated to enhance etoposide‑induced apoptosis and reduce expression levels of HDAC4, P‑gp and phospho‑Stat1. In addition, the suppression of Stat1 with siRNA enhanced etoposide‑induced apoptosis and reduced the expression levels of HDAC4 and P‑gp, suggesting that Stat1 is essential in the regulation of resistance to etoposide, and in the upregulation of P‑gp. Notably, TSA treatment reduced P‑gp transcript levels but Stat1 siRNA treatment did not, suggesting that P‑gp is regulated by HDAC at the transcriptional level and by Stat1 at the post‑transcriptional level. These results suggest that the upregulation of Stat1 and HDAC4 determines etoposide resistance through P‑gp expression in human A549 lung cancer cells.

Extract of Bryophyllum laetivirens reverses etoposide resistance in human lung A549 cancer cells by downregulation of NF-κB

Since multidrug resistance (MDR) is one of the main reasons for failure in cancer treatment, its suppression may increase the efficacy of cancer therapy. In the present study we attempted to identify a new and effective anticancer drug against MDR cancer cells. We first found that lung cancer A549 cells resistant to etoposide (A549RT-eto) exhibit upregulation of NF-κB and SIRT1 in comparison to A549 parental cells. During a search for anticancer drug candidates from medicinal plant sources, we found that an extract fraction (F14) of Bryophyllum laetivirens leaves downregulated expression of NF-κB and SIRT1, sensitizing the levels of A549RT-eto cells to apoptosis through downregulation of P-glycoprotein (P-gp), which is encoded by the MDR1 gene. To address whether NF-κB is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with Bay11-7802, an inhibitor of NF-κB. We then observed that Bay11-7802 treatment reduced P-gp expression levels, and furthermore combined treatment with the F14 extract and Bay11-7802 accelerated apoptosis through a decrease in P-gp levels, suggesting that NF-κB is involved in MDR. To address whether upregulation of SIRT1 is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with SIRT1 siRNA or nicotinamide (NAM), an inhibitor of SIRT1. we found that suppression of SIRT1 did not reduce P-gp levels. furthermore, the combined treatment with the F14 extract, and SIRT1 siRNA or NAM did not accelerate apoptosis, indicating that SIRT1 is not involved in the regulation of P-gp levels in A549RT-eto cells. Taken together, we suggest that upregulation of NF-κB determines etoposide resistance through P-gp expression in human A549 lung cancer cells. We herein demonstrated that B. laetivirens extract reverses etoposide resistance in human A549 lung cancer cells through downregulation of NF-κB.

Feroniellin A-induced autophagy causes apoptosis in multidrug-resistant human A549 lung cancer cells

During the screening of natural chemicals that can reverse multidrug resistance in human A549 lung cancer cells resistant to etoposide (A549RT-eto), we discovered that Feroniellin A (FERO), a novel furanocoumarin, shows toxicity toward A549RT-eto cells in a dose- and time-dependent manner. FERO reduced the expression of NF-κB, leading to downregulation of P-glycoprotein (P-gp), encoded by MDR1, which eventually sensitized A549RT-eto cells to apoptosis. FERO specifically diminished transcription and promoter activity of MDR1 but did not inhibit the expression of other multidrug resistance genes MRP2 and BCRP. Moreover, co-administration of FERO with Bay11-7802, an inhibitor of NF-κB, accelerated apoptosis of A549RT-eto cells through decreased expression of P-gp, indicating that NF-κB is involved in multidrug resistance. Conversely, addition of Z-VAD, a pan-caspase inhibitor, blocked FERO-induced apoptosis in A549RT-eto cells but did not block downregulation of P-gp, indicating that a decrease in P-gp expression is necessary but not sufficient for FERO-induced apoptosis. Interestingly, we found that FERO also induces autophagy, which is characterized by the conversion of LC3 I to LC3 II, induction of GFP-LC3 puncta, enhanced expression of Beclin-1 and ATG5, and inactivation of mTOR. Furthermore, suppression of Beclin-1 by siRNA reduced FERO-induced apoptosis in A549RT-eto cells and activation of autophagy by rapamycin accelerated FERO-induced apoptosis, suggesting that autophagy plays an active role in FERO-induced apoptosis. Herein, we report that FERO reverses multidrug resistance in A549RT-eto cells and exerts its cytotoxic effect by induction of both autophagy and apoptosis, which suggests that FERO can be a useful anticancer drug for multidrug-resistant lung cancer.

Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4.

Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.

Effect of three fatty acids from the leaf extract of Tiliacora triandra on P-glycoprotein function in multidrug-resistant A549RT-eto cell line

Background: Cancer cells have the ability to develop resistance to chemotherapy drugs, which then leads to a reduced effectiveness and success of the treatment. Multidrug resistance (MDR) involves the resistance in the same cell/tissue to a diverse range of drugs of different structures. One of the characteristics of MDR is an overexpression of P-glycoprotein (P-gp), which causes the efflux of the accumulated drug out of the cell. The MDR human non-small cell lung carcinoma cell line with a high P-gp expression level (A549RT-eto) was used to investigate the bioactive compounds capable of reversing the etoposide resistance in this cell line. Materials and Methods: The leaves of Tiliacora triandra were sequentially extracted with hexane, dichloromethane, methanol and water. Only the hexane extract reduced the etoposide resistance of the A549RT-eto cell line, and was further fractionated by column chromatography using the TLC-pattern and the restoration of etoposide sensitivity as the selection criteria. Results: The obtained active fraction (F22) was found by nuclear magnetic resonance and gas chromatography-mass spectroscopy analyses to be comprised of a 49.5:19.6:30.9 (w/w/w) mixture of hexadecanoic: octadecanoic acid: (Z)-6-octadecenoic acids. This stoichiometric mixture was recreated using pure fatty acids (MSFA) and gave a similar sensitization to etoposide and enhanced the relative rate of rhodamine-123 accumulation to a similar extent as F22, supporting the action via reducing P-gp activity. In contrast, the fatty acids alone did not show this effect. Conclusion: This is the first report of the biological activity from the leaves of T. triandra as a potential source of a novel chemosensitizer.

STAT1‑HDAC4 signaling induces epithelial‑mesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2

Our previous studies have shown that the novel oncogene, cancer upregulated gene 2 (CUG2), activates STAT1, which is linked to anticancer drug resistance, induces epithelial-mesenchymal transition (EMT) and cancer stem cell-like phenotypes as determined by MTT, migration and sphere formation assays. We thus aimed to ascertain whether the activation of STAT1 by CUG2 is involved in these malignant phenotypes besides drug resistance. Here, we showed that STAT1 suppression decreased the expression of N-cadherin and vimentin, biomarkers of EMT, which led to inhibition of the migration and invasion of human lung A549 cancer cells stably expressing CUG2, but did not recover E-cadherin expression. STAT1 siRNA also diminished CUG2-induced TGF-β signaling, which is critical in EMT, and TGF-β transcriptional activity. Conversely, inhibition of TGF-β signaling reduced phosphorylation of STAT1, indicating a crosstalk between STAT1 and TGF-β signaling. Furthermore, STAT1 silencing diminished sphere formation, which was supported by downregulation of stemness-related factors such as Sox2, Oct4, and Nanog. Constitutive suppression of STAT1 also inhibited cell migration, invasion and sphere formation. As STAT1 acetylation counteracts STAT1 phosphorylation, acetylation of STAT1 by treatment with trichostatin A, an inhibitor of histone deacetylases (HDACs), reduced cell migration, invasion, and sphere formation. As HDAC4 is known to target STAT1, its role was investigated under CUG2 overexpression. HDAC4 suppression resulted in inhibition of cell migration, invasion, and sphere formation as HDAC4 silencing hindered TGF-β signaling and decreased expression of Sox2 and Nanog. Taken together, we suggest that STAT1-HDAC4 signaling induces malignant tumor features such as EMT and sphere formation in CUG2-overexpressing cancer cells.

N-Benzyl-N-methyl-dodecan-1-amine, a novel compound from garlic, exerts anti-cancer effects on human A549 lung cancer cells overexpressing cancer upregulated gene (CUG)2

ABSTRACT Dietary garlic has been suggested to possess anticancer properties, and several attempts have been made to isolate the anticancer compounds. In this study, we efficiently synthesized N‐benzyl‐N‐methyl‐dodecan‐1‐amine (BMDA) by the reductive amination method. We evaluated the potential anticancer activities of BMDA against A549 lung cancer cells with cancer stem cell‐like phenotypes due to the overexpression of cancer upregulated gene (CUG)2. N‐Benzyl‐N‐methyl‐dodecan‐1‐amine treatment sensitized A549 cells overexpressing CUG2 (A549‐CUG2) to apoptosis and autophagy compared with those of the control cells. The treatment with BMDA also reduced tumor development in xenografted nude mice. Furthermore, BMDA inhibited cell migration, invasion, and sphere formation in A549‐CUG2 cells, in which TGF‐&bgr; signaling is involved. Further analysis showed that BMDA hindered TGF‐&bgr; promoter activity, protein synthesis, and phosphorylation of Smad2, thus decreasing the expression of TGF‐&bgr;‐targeted proteins, including Snail and Twist. N‐Benzyl‐N‐methyl‐dodecan‐1‐amine also decreased Twist expression in vivo. In addition, BMDA inhibited Akt‐ERK activities, &bgr;‐catenin expression, and its transcriptional activity. These results suggest that BMDA can be a promising anticancer agent against cancer cells overexpressing CUG2.

Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells

Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4.