Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells.

BACKGROUND: Indigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents. METHODS: Cytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach. RESULTS: We showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine. CONCLUSIONS: ISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Low Geriatric Nutritional Risk Index Is Associated with Poorer Prognosis in Elderly Diffuse Large B-Cell Lymphoma Patients Unfit for Intensive Anthracycline-Containing Therapy: A Real-World Study.

Nutritional assessments, including the Geriatric Nutritional Risk Index (GNRI), have emerged as prediction tools for long-term survival in various cancers. This study aimed to investigate the therapeutic strategy and explore the prognostic factors in the elderly patients (≥65 years) with diffuse large B cell lymphoma (DLBCL). The cutoff value of the GNRI score (92.5) was obtained using the receiver operating characteristic curve. Among these patients (n = 205), 129 (62.9%) did not receive standard R-CHOP chemotherapy. Old age (≥80 years), poor performance status, low serum albumin level, and comorbidities were the major factors associated with less intensive anti-lymphoma treatment. Further analysis demonstrated that a lower GNRI score (<92.5) was linked to more unfavorable clinical features. In the patients who received non-anthracycline-containing regimens (non-R-CHOP), multivariate analysis showed that a low GNRI can serve as an independent predictive factor for worse progression-free (HR, 2.85; 95% CI, 1.05-7.72; p = 0.039) and overall survival (HR, 2.98; 95% CI, 1.02-8.90; p = 0.045). In summary, nutritional evaluation plays a role in DLBCL treatment and the GNRI score can serve as a feasible predictive tool for clinical outcomes in frail elderly DLBCL patients treated with non-anthracycline-containing regimens.

A water-based topical Chinese traditional medicine (Zicao) for wound healing developed using 2-hydroxypropyl-ß-cyclodextrin.

Zicao is a traditional Chinese herbal medicine that has been used for the topical treatment of wounds in the form of oil-based ointment for several hundred years. To overcome the disadvantages of oil-based ointment such as irritation, discomfort, and difficulty in cleaning, this study developed a water-based topical formulation of Zicao. An ethanol extract of Zicao was included in 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to form a water-soluble Zicao-HP-ß-CD complex. The formation of the Zicao-HP-ß-CD complex was determined using LC-MS, 1H NMR, ROSEY, and solubility analysis. The bioactivity of Zicao-HP-ß-CD complex in aqueous solution was evaluated using cellular uptake in vitro and experimental excision wounds in vivo. The LC-MS, 1H NMR, ROESY, and solubility analyses results show that Zicao extract was successfully included by the HP-ß-CD. The results of the cellular uptake in vitro and wound healing in vivo suggest that the effect of Zicao was enhanced following the formation of the Zicao-HP-ß-CD complex. Therefore, we concluded that complexation with HP-ß-CD might provide a potential method for developing an effective water-based topical solution of Zicao.

Dithiothreitol enhanced arsenic-trioxide-induced cell apoptosis in cultured oral cancer cells via mitochondrial dysfunction and endoplasmic reticulum stress.

Arsenic is naturally occurring toxic metalloid and drinking As2 O3 containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As2 O3 has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As2 O3 action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As2 O3 alone or combined with dithiothreitol (DTT). The results showed that 0.5 µM As2 O3 plus 20 µM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As2 O3 plus DTT upregulated Bax and Bak, downregulated Bcl-2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As2 O3 also triggered endoplasmic reticulum stress and increased the levels of glucose-regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As2 O3 on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 17-27, 2017.

Inhibitory Effects of AVEMAR on Proliferation and Metastasis of Oral Cancer Cells.

Oral cancer is keeping its 4th rank on the death causing cancers among Taiwan males, and its metastatic and recurrent rates remain high and a life-threatening issue to the citizens. Fermented wheat germ extract (AVEMAR) is used in clinical cancer nutritional therapy in gastrointestinal cancers but not in oral cancer yet. In this study, the potential of AVEMAR to inhibit tumor proliferation and metastasis of oral cancer was first investigated. Antiproliferative activity of AVEMAR was determined in human oral squamous carcinoma SCC-4 cells by MTT methodology. Wound-healing migration, transwell invasion, and Western blotting assays were carried out to examine the in vitro antimetastatic effects and involved signaling molecules for AVEMAR in oral cancer cells. AVEMAR at 0.2-1.6 mg/ml significantly inhibited the cell viability with IC50 values of 1.19 and 0.98 mg/ml for 24-h and 48-h treatment. Furthermore, AVEMAR could induce cell apoptosis and inhibit the migration and invasion of metastatic SCC-4 cells at a similar dose range. Notably, AVEMAR suppressed the expression of matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator (u-PA), but not MMP-1 or MMP-9, in SCC-4 cells. These results strongly support the antiproliferation and in vitro antimetastatic capacity of AVEMAR which may extend its contributions from cancer nutrition supplements to preventive agent for oral cancer.

Inhibitory effect of Angelica sinensis extract in the presence of 2-hydroxypropyl-ß-cyclodextrin.

Angelica sinensis (AS) is a traditional Chinese medicinal herb. Its ethanolic extract contains active ingredients, such as ferulic acid, ligustilide, and butylidenephthalide, which are hydrophobic and have inhibitory effects on hepatoma cells. To increase the aqueous solubility/dispersibility of AS extract and study the consequent inhibitory effect on hepatoma cells, the ethanolic extract of AS was complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity. The AS-HP-ß-CD complex (weight ratio of AS extract: HP-ß-CD=1:5) was prepared and characterized. The effect of complexing the AS extract with HP-ß-CD on Hep3B cell growth was investigated by analyzing cytotoxicity. Our results showed that cytotoxicity inhibition of AS-HP-ß-CD complex was up to 94% and higher than that of AS extract (about 68%). These observations suggested that the use of HP-ß-CD to stabilize AS extract in aqueous solution was possible for herbal medicine application.

Enhancement of rhubarb extract solubility and bioactivity by 2-hydroxypropyl-ß-cyclodextrin.

Rhubarb is a traditional Chinese medicinal herb, and the ethanolic extract of rhubarb consists of active anthraquinones, which are hydrophobic and have antiproliferative effects on hepatoma cell lines. To increase the aqueous solubility of rhubarb and study the consequent bioavailability, the ethanolic extract of rhubarb was complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity, to form a rhubarb-HP-ß-CD complex. This complex was characterized by performing nuclear magnetic resonance spectroscopy, two-dimensional rotating frame spectroscopy and thin layer chromatography to confirm the inclusion of anthraquinones from rhubarb extract in HP-ß-CD (weight ratio of rhubarb extract:HP-ß-CD=1:9). We investigated the effects of complexing rhubarb extract with HP-ß-CD on the growth of Huh7 and HepG2 cells by performing cytotoxicity analysis, cellular uptake test, and colony formation assay. Our results showed that complexation of rhubarb extract with HP-ß-CD increased the aqueous solubility and bioavailability of rhubarb and thus enhanced its effect on hepatoma cells.

Bufalin induces G2/M phase arrest and triggers autophagy via the TNF, JNK, BECN-1 and ATG8 pathway in human hepatoma cells.

Liver cancer is the fifth most common cause of cancer death worldwide. The study of more effective anti-hepatoma drugs is urgently required. Bufalin has been isolated from a traditional Chinese medicine and possesses less toxicity to normal cells. However, it has been found to inhibit growth of cancer cells. In this study, we aimed to investigate the efficacy and mechanism of bufalin in Huh7, Hep3B and HA22T human hepatoma cells. The three cell lines were treated with bufalin, the proliferation was detected by WST-1 assay and cell cycle was detected by flow cytometry analysis. The results showed that bufalin inhibited the proliferation of hepatoma cells and regulated the hepatoma cell death program in a dose- and time-dependent manner without typical features of apoptosis. RT-PCR arrays were used to investigate the autophagy transcriptional response triggered by bufalin and 13 genes were altered and further confirmed by real-time PCR. The translation levels of selected genes were examined by western blot analysis to reveal the bufalin-induced autophagy cascade. Bufalin synergized with the JNK pathway to induce autophagy of hepatoma cells and is closely associated with the upregulation of TNF, BECN-1, MAPK and ATG8, together with the downregulation of Bcl-2 and Bid. Our study provided a multi-angle evaluation system for anti-hepatoma pharmacology for pre-clinical drug investigation. In this case, bufalin was capable of inducing hepatoma cell autophagy, suggesting a potential regimen for single or combined chemotherapy to overcome hepatoma in clinical practice.