baohuoside-i and Melanoma

Metastatic melanoma is the most aggressive skin cancer. Although BRAF inhibitor treatment has achieved great success in melanoma, resistance develops within 12 months. Icariside II (IS), a natural compound extracted from Herba Epimedii, exerts anticancer properties. In the present study, we determined by MTT, flow cytometry and western blotting, respectively that IS potentiated the PLX4032‑induced downregulation of cell viability and increase in apoptosis and autophagy in BRAF inhibitor‑resistant melanoma. In addition, we also revealed by flow cytometry and western blotting, respectively, that IS combined with PLX4032 increased mitochondrial and intracellular reactive oxygen species (ROS) generation and subsequently promoted depolarization of mitochondria and release of apoptotic proteins. N‑acetyl cysteine (NAC) and glutathione (GSH), ROS scavengers, reversed the IS‑induced enhancement of the response to PLX4032. Microphthalmia‑associated transcription factor (MITF) and tyrosine‑protein kinase Met (c‑Met) are well‑known factors that contribute to BRAF inhibitor resistance. Furthermore, c‑Met is a direct transcriptional target of MITF in melanocytes and melanoma cells. It was also revealed that IS markedly inhibited MITF and c‑Met expression partially by increasing ROS production in BRAF inhibitor‑resistant melanoma cells.

Topics: Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Membrane Potential, Mitochondrial; Microphthalmia-Associated Transcription Factor; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Reactive Oxygen Species; Vemurafenib

Baohuoside-I was reported to induce apoptosis in non-small-cell lung cancer and inhibit the growth of multiple myeloma cells. The antitumour potential of baohuoside-I has not been demonstrated in melanoma yet.. To investigate the potential antitumour activity of baohuoside-I against melanoma and elucidate its underlying molecular mechanism.. Cell viability was evaluated by MTT assay. The malignant invasion capacity was measured with trans-well assay. The relative expression change of microRNAs was profiled with microarray. TargetScan was utilized for prediction of target gene of miR-144. Regulatory effect of miR-144 on SMAD1 was determined by dual luciferase reporter assay. Endogenous SMAD1 protein in response to ectopic expression of miR-144 was determined by immunoblotting. Xenograft mice were employed to evaluate antitumour potential of baohuoside-I (25 mg/kg by tail intravenous injection every two days) in vivo.. Baohuoside-I significantly inhibited proliferation (45 ± 4% reduction in M14 and 35 ± 3% reduction in MV3 at 24 h) and migration (70 ± 4% reduction in M14 and 72 ± 3% reduction in MV3) in melanoma cells. Mechanistically, baohuoside-I up-regulated miR-144 expression levels (3 ± 0.2-fold). Silence of miR-144 reversed the inhibition of baohuoside-I in melanoma. We have identified that SMAD1 was the novel target of miR-144. Moreover, baohuoside-I suppressed melanoma in vivo (52 ± 8% reduction in xenograft tumour size at day 20).. Our data suggested significant antitumour potential of baohuoside-I against melanoma both in vitro and in vivo, which warrants further laboratory investigation and clinical trial.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drugs, Chinese Herbal; Female; Flavonoids; Humans; Melanoma; Mice; Mice, Nude; MicroRNAs; Up-Regulation; Xenograft Model Antitumor Assays

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, many melanoma cells show weak responses to TRAIL. Here, we investigated whether Icariside II (IS), an active component of Herba Epimedii, could potentiate antitumor effects of TRAIL in melanoma cells. Melanoma cells were treated with IS and/or TRAIL and cell death, apoptosis and signal transduction were analyzed. We showed that IS promoted TRAIL-induced cell death and apoptosis in A375 melanoma cells. Mechanistically, IS reduced the expression levels of cFLIP in a phospho-STAT3 (pSTAT3)-dependent manner. Ectopic expression of STAT3 abolished IS-induced cFLIP down-regulation and the associated potentiation of TRAIL-mediated cell death. Moreover, IS-induced reactive oxygen species (ROS) production preceded down-regulation of pSTAT3/cFLIP via activating AKT, and the consequent sensitization of cells to TRAIL. We also found that IS treatment down-regulated cFLIP via ROS-mediated NF-κB pathway. In addition, IS converted TRAIL-resistant melanoma MeWo and SK-MEL-28 cells into TRAIL-sensitive cells. Taken together, our results indicated that IS potentiated TRAIL-induced apoptosis through ROS-mediated down-regulation of STAT3/cFLIP signaling.

Topics: Antineoplastic Combined Chemotherapy Protocols; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Flavonoids; Humans; Melanoma; Reactive Oxygen Species; Signal Transduction; STAT3 Transcription Factor; TNF-Related Apoptosis-Inducing Ligand

Icariside II (IS) is a metabolite of icariin, which is derived from Herba Epimedii. In the present study, the antiproliferative effects of IS on A375 human melanoma cells were examined in vitro and a possible mechanism through the ROS-p38-p53 pathway is discussed. A cell WST-8 assay revealed that treatment with IS markedly reduced cell viability from 77 to 21% (25 and 100 µM, respectively), and cell counting demonstrated that IS treatment reduced cell proliferation. IS treatment also induced cell cycle arrest of A375 cells at the G0/G1 and G2/M transitions and inhibited the expression of cell-cycle related proteins, including cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin B1 and phosphorylated cyclin-dependent kinase 1 (P-CDK1). In this study, it was determined that IS inhibits cell proliferation and induces cell cycle arrest through the generation of reactive oxygen species and activation of p38 and p53. These findings were further supported by the evidence that pretreatment with N-acetyl-L-cysteine, SB203580 or pifithrin-α significantly blocked IS-induced reduction of cell viability, increase of cell death and cell cycle arrest. In conclusion, IS inhibits cell proliferation and induces cell cycle arrest. Crucially, it was confirmed that these effects were mediated at least in part by activating the ROS-p38-p53 pathway.

Topics: Antineoplastic Agents, Phytogenic; Cell Cycle Checkpoints; Cell Line, Tumor; Flavonoids; Humans; Melanoma; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53

This study evaluated the antitumor effects of icariside II (IS), isolated from Herba Epimedii, on in vitro and in vivo models of melanoma and determined its mechanism of apoptosis. Mouse (B16) and human (A375, SK-MEL-5) melanoma cell lines were treated with IS at different concentrations (0-100 μM). Cell viability and proliferation was detected by WST-1 assay and with the xCELLigence system, respectively. Apoptosis was measured by the annexin-V/PI flow cytometric assay. Western blot was used to measure cleaved caspase 3, survivin, P-STAT3, P-ERK and P-AKT. B16 and A375 cells were injected subcutaneously into C57BL/6J and BALB/c-nu mice, respectively. After 1 wk, IS solution at (50 mg/kg, 100 mg/kg) was administered by intraperitoneal injection 3 times for a week. Tumor size was measured with an electronic digital caliper. IS inhibited the proliferation of melanoma cells in a dose- and time-dependent manner. Treatment of A375 cells with IS resulted in an increased number of apoptotic cells ranging from 5.6% to 26.3% mirrored by increases in cleaved caspase-3 and a decrease in survivin expression. IS significantly inhibited the activation of the JAK-STAT3 and MAPK pathways but promoted an unsustained activation peak of the PI3K-AKT pathway. IS administration (50 mg/kg) resulted in a 47.5% decreased tumor volume in A375 bearing mice. Furthermore, IS administration (50 mg/kg, 100 mg/kg) resulted in 41% and 49% decreased tumor volume in B16 bearing mice, respectively. IS dramatically inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. These effects demonstrate the ability of IS to effectively overcome the survival signals of tumor cells, which support further preclinical evaluation of IS in cancer as a new potential chemotherapeutic agent.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Humans; Inhibitor of Apoptosis Proteins; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Survivin

Combination therapy of paclitaxel (taxol) with natural anti-tumor agents that are capable of inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Our previous study showed that icariside II (IS), derived from Herba Epimedii, inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. In this report, the combination effects of paclitaxel and IS were investigated in human melanoma A375 cells. As compared to the treatment with paclitaxel alone, the co-administration of IS and paclitaxel resulted in an enhancement of apoptosis as revealed by WST-8 and PI assays. Meanwhile, Western blot analysis showed that the co-administration of IS and paclitaxel resulted in increases of cleaved caspase-3, one of the terminal pro-apoptotic proteins. In melanoma, IL-8 and VEGF are positively correlated with disease stage and a high probability of progression. We demonstrated that treatment of A375 cells with IS in combination with paclitaxel resulted in a significant decrease in the production of IL-8 and VEGF, compared with paclitaxel alone. Recent studies suggest that TLR4-MyD88-ERK signaling may be a novel target for reversing chemoresistance to paclitaxel. Our flow cytometry and Western blot data showed that paclitaxel activated TLR4-MyD88-ERK signaling and that IS treatment could effectively inhibit this paclitaxel-induced activation of TLR4-MyD88-ERK signaling. In conclusion, this study demonstrated for the first time that IS could potentiate paclitaxel-induced apoptosis in melanoma cells. These effects were mediated, at least in part, by inhibiting the activation of the TLR4 signal transduction pathways. These findings support further preclinical evaluation of IS as a new potential anti-tumor agent.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Line, Tumor; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Flavonoids; Flow Cytometry; Humans; Interleukin-8; Melanoma; Paclitaxel; Signal Transduction; Toll-Like Receptor 4; Vascular Endothelial Growth Factor A