Anti-cancer Effect of Xao Tam Phan Paramignya trimera Methanol Root Extract on Human Breast Cancer Cell Line MCF-7 in 3D Model.

BACKGROUND: Cancer is one of the leading causes of death in the world. A great deal of effort has been made to discover new agents for cancer treatment. Xao tam phan (Paramignya trimera) is a traditional medicine of Vietnam used in cancer treatment for a long time, yet there is not much scientific evidence proving its anticancer potency. The study aimed to evaluate the toxicity of Paramignya trimera extract (PTE) on multicellular tumor spheres (MCTS) of MCF-7 cells using hanging drop technique. METHODS: Firstly, MCF-7 cells were seeded on hanging drop plates, spheroid size was tracked, and growth curve was measured by MTT assay and AlamarBlue® assay. The necrotic core of MCTS was evaluated by propidium iodide (PI) staining. Toxicity of doxorubicin (DOX) and tirapazamine (TPZ) was then tested on 3D model compared to 2D culture condition. RESULTS: The results showed that the IC50 of DOX on 3D MCF-7 cells was nearly 50 times greater than monolayer MCF-7 cells. In contrast, TPZ (an agent which is specifically toxic under hypoxic conditions) had significantly lower IC50 in 3D condition than in 2D. The toxicity tests for PTE showed that PTE strongly inhibited MCF-7 cells in both 2D and 3D conditions. Interestingly, the IC50 of PTE in 3D model was remarkably lower than in 2D (IC50 value was 168.9 ± 11.65 µg/ml compared to 260.8 ± 16.54 µg/ml, respectively). The invasion assay showed that PTE completely inhibited invasion of MCF-7 cells at 250 µg/mL concentration. Also, flow cytometry results indicated that PTE effectively induced apoptosis in MCF-7 spheroids in 3D condition at 250 µg/mL concentration. CONCLUSION: The results from this study emphasize the promise of PTE in cancer therapy.

Paratrimerin I, cytotoxic acridone alkaloid from the roots of Paramignya trimera.

Bioactivity-guided fractionation of the CHCl3-soluble extract of the roots of Paramignya trimera was carried out to obtain a new acridone alkaloid, paratrimerin I. Its structure was elucidated based on NMR spectroscopic data interpretation. Paratrimerin I showed noteworthy cytotoxicity against the HepG2 human hepatocellular and MCF-7 human breast carcinoma cell lines, with the submicromolar IC50 values of 0.43 and 0.26 µM, respectively. The N-methyl, C-4 methoxy, and C-5 hydroxy groups in the acridone skeleton can be proposed as a structural feature for good cytotoxicity.

Adipose-Derived Stem Cells Can Replace Fibroblasts as Cell Control for Anti-Tumor Screening Assay.

Anti-tumor activity screening is a typical process used in anti-tumor drug discovery. The ideal anti-tumor drug candidates are extracts or compounds that can inhibit the proliferation of cancer cells via apoptosis, while exerting minimal effects on normal somatic cells. For a long time, fibroblasts were used as normal cells for all anti-tumor screening assays. However, the fibroblasts exhibited several limitations as cell controls for anti-tumor screening. This study aimed to compare the usage of dermal fibroblasts (DFs) and adipose-derived stem cells (ADSCs) as normal cell controls in anti-tumor screening protocols. The DFs and ADSCs were prepared per the published protocols. The IC50 values of doxorubicin on hepatocellular carcinoma cells HepG2, breast cancer cells MCF-7, DFs and ADSCs were determined via the Alamar blue assay. The side effect indexes (SEIs) were calculated as the ratio of IC50 values of drugs on cancer cells and IC50 values of drugs on DFs, and on ADSCs. The stability of the anti-tumor assay was investigated when carried out on DFs and ADSCs from different passages. The results showed that the IC50 values, as well as SEI values, were not significantly different between using DFs or ADSCs as normal cell controls when DFs and ADSCs were at passage 3. However, for DFs at passage 6 to 12, the IC50 values of doxorubicin were significantly different between DFs and ADSCs. The IC50 values of doxorubicin on DFs were strongly reduced due to the senescence of DFs, while the values were more constant in ADSCs. The SEI values of doxorubicin on DFs, compared to HepG2 and MCF-7 cells, were also changed during passage 3 to 12 of the DFs. However, these values were only slightly changed for ADSCs from the 3rd to 12th passages. ADSCs can replace DFs as a normal cell control for anti-tumor activity screening.

Hopea odorata extract inhibits hepatocellular carcinoma via induction of caspase-dependent apoptosis.

INTRODUCTION: Cancer is a disease with a global burden and is a major and increasing threat to public health. The demand for new modalities to treat and prevent cancer is high. Given the toxic side effects of standard treatments, such as chemotherapy, there is greater research interest in naturally derived compounds due to their selective toxicity to cancer cells. This study aimed to test the anticancer activity of a crude extract of Hopea odorata on hepatocellular carcinoma (HCC) HepG2 cell line. METHODS: Methanol extracts of H. odorata were prepared from the bark of H. odorata plants (H. odorata extract). The in vitro cytotoxicity of H. odorata extracts on human HCC cell line HepG2 compared to normal human fibroblasts (HFs) was assessed by Alamar Blue assay. Caspase-3/7 was detected using a reagent that consists of DEVD peptide conjugated to a nucleic acid-binding dye. Apoptosis induction by the H. odorata plant extract on HepG2 was evaluated by Annexin V/7-AAD using flow cytometry. Disintegrated nuclei of plant-treated cells were observed under a fluorescent microscope using Hoechst and propidium iodide (PI) staining. In addition, using the Hoechst/PI staining technique, the ratio of dead to total cells was determined by distinguishing Hoechst and PI fluorescent signals. RESULTS: We found that the IC50 value of H. odorata extract on HepG2 was 12.67±5 µg/mL and on HF was 44±3 µg/mL. The IC50 value of doxorubicin on HepG2 was 153.3±15 ng/mL and on HF was 6.3±0.6 ng/mL. The selectivity index (SI) of H. odorata extract for HepG2 cells was ~3.48, while the SI of doxorubicin for HepG2 cells was ~0.04. The ratio of dead to total cells increased in a dose-dependent manner for HepG2 cells when observed under a fluorescent microscope, while the ratio of dead to total cells barely changed for HF cells. The H. odorata extract inhibited HepG2 cells via the activation of caspase-3/7. At 250 µg/mL concentration of the H. odorata extract, 35% of HepG2 cells were induced into apoptosis, and the cells exhibited disintegrated nuclei under a fluorescent microscope. CONCLUSION: These findings demonstrate that the methanolic bark extracts of H. odorata plant induce apoptosis and selective cytotoxicity toward HepG2 but not HF. Therefore, purification of compounds from H. odorata bark extracts may be useful as anticancer agents, and thus, more studies are warranted to investigate the anticancer properties of H. odorata.

Targeting breast cancer stem cells by dendritic cell vaccination in humanized mice with breast tumor: preliminary results.

BACKGROUND: Breast cancer (BC) is one of the leading cancers in women. Recent progress has enabled BC to be cured with high efficiency. However, late detection or metastatic disease often renders the disease untreatable. Additionally, relapse is the main cause of death in BC patients. Breast cancer stem cells (BCSCs) are considered to cause the development of BC and are thought to be responsible for metastasis and relapse. This study aimed to target BCSCs using dendritic cells (DCs) to treat tumor-bearing humanized mice models. MATERIALS AND METHODS: NOD/SCID mice were used to produce the humanized mice by transplantation of human hematopoietic stem cells. Human BCSCs were injected into the mammary fat pad to produce BC humanized mice. Both hematopoietic stem cells and DCs were isolated from the human umbilical cord blood, and immature DCs were produced from cultured mononuclear cells. DCs were matured by BCSC-derived antigen incubation for 48 hours. Mature DCs were vaccinated to BC humanized mice with a dose of 10(6) cells/mice, and the survival percentage was monitored in both treated and untreated groups. RESULTS: The results showed that DC vaccination could target BCSCs and reduce the tumor size and prolong survival. CONCLUSION: These results suggested that targeting BCSCs with DCs is a promising therapy for BC.

Targeting specificity of dendritic cells on breast cancer stem cells: in vitro and in vivo evaluations.

Breast cancer is a leading cause of death in women, and almost all complications are due to chemotherapy resistance. Drug-resistant cells with stem cell phenotypes are thought to cause failure in breast cancer chemotherapy. Dendritic cell (DC) therapy is a potential approach to eradicate these cells. This study evaluates the specificity of DCs for breast cancer stem cells (BCSCs) in vitro and in vivo. BCSCs were enriched by a verapamil-resistant screening method, and reconfirmed by ALDH expression analysis and mammosphere assay. Mesenchymal stem cells (MSCs) were isolated from allogeneic murine bone marrow. DCs were induced from bone marrow-derived monocytes with 20 ng/mL GC-MSF and 20 ng/mL IL-4. Immature DCs were primed with BCSC- or MSC-derived antigens to make two kinds of mature DCs: BCSC-DCs and MSC-DCs, respectively. In vitro ability of BCSC-DCs and MSC-DCs with cytotoxic T lymphocytes (CTLs) to inhibit BCSCs was tested using the xCELLigence technique. In vivo, BCSC-DCs and MSC-DCs were transfused into the peripheral blood of BCSC tumor-bearing mice. The results show that in vitro BCSC-DCs significantly inhibited BCSC proliferation at a DC:CTL ratio of 1:40, while MSC-DCs nonsignificantly decreased BCSC proliferation. In vivo, tumor sizes decreased from 18.8% to 23% in groups treated with BCSC-DCs; in contrast, tumors increased 14% in the control group (RPMI 1640) and 47% in groups treated with MSC-DCs. The results showed that DC therapy could target and be specific to BCSCs. DCs primed with MSCs could trigger tumor growth. These results also indicate that DCs may be a promising therapy for treating drug-resistant cancer cells as well as cancer stem cells.

A simple in vitro method for evaluating dendritic cell-based vaccinations.

BACKGROUND: Dendritic cell (DC) therapy is a promising therapy for cancer-targeting treatments. Recently, DCs have been used for treatment of some cancers. We aimed to develop an in vitro assay to evaluate DC therapy in cancer treatment using a breast cancer model. METHODS: DCs were induced from murine bone marrow mononuclear cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Immature DCs were primed with breast cancer stem cell (BCSC)-derived antigens. BCSCs were sorted from 4T1 cell lines based on aldehyde dehydrogenase expression. A mixture of DCs and cytotoxic T lymphocytes (CTLs) were used to evaluate the inhibitory effect of antigen-primed DCs on BCSCs. BCSC proliferation and doubling time were recorded based on impedance-based cell analysis using the xCELLigence system. The specification of inhibitory effects of DCs and CTLs was also evaluated using the same system. RESULTS: The results showed that impedance-based analysis of BCSCs reflected cytotoxicity and inhibitory effects of DCs and CTLs at 72 hours. Differences in ratios of DC:CTL changed the cytotoxicity of DCs and CTLs. CONCLUSION: This study successfully used impedance-based cell analysis as a new in vitro assay to evaluate DC efficacy in cancer immunotherapy. We hope this technique will contribute to the development and improvement of immunotherapies in the near future.