shikonin and Lung-Neoplasms

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Gefitinib; Glucose; Lung Neoplasms; Mice; Oxidoreductases; Pyruvate Kinase

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Topics: Activating Transcription Factor 3; Cell Line, Tumor; Ferroptosis; Histones; Humans; Lipids; Lung Neoplasms; Naphthoquinones; Reactive Oxygen Species; Small Cell Lung Carcinoma

Topics: Alkaloids; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Enzyme Inhibitors; G2 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Matrines; Molecular Docking Simulation; Naphthoquinones; Protein Binding; Quinolizines; Telomerase

Shikonin, a naturally occurring naphthoquinone with potent anti-tumor activity, has been reported to induce cancer cell death via targeting selenoenzyme thioredoxin reductase 1 (TrxR1; TXNRD1). However, the interaction between shikonin and TrxR1 remains unclear, and the roles of the cellular antioxidant system in shikonin induced cell death are obscure. Here, we found that shikonin modified the Sec

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; Naphthoquinones; Necroptosis; NF-E2-Related Factor 2; Oxidation-Reduction; Oxygen; Reactive Oxygen Species; Thioredoxin Reductase 1; Thioredoxin-Disulfide Reductase

Overexpressed survivin is associated with worse survival of several types of human tumors. In this study, the antitumor activity of shikonin in non-small-cell lung cancer (NSCLC) by regulating survivin pathway was investigated. Results showed that shikonin inhibited the NSCLC H1299 cell proliferation in a dose-dependent manner. Moreover, shikonin fits well with survivin by molecular docking. Shikonin also inhibited the mRNA expression and protein level of survivin in H1299 cells. Shikonin arrested H1299 cell cycle at the G0/G1 phase by regulating CDK/cyclin family members. In addition, shikonin regulated the expression of X-linked inhibitor of apoptosis- (XIAP-) mediated caspases 3 and 9, thus leading to the damage of mitochondrial membrane potential and induction of H1299 cell apoptosis. Overall, shikonin inhibited H1299 cell growth by inducing apoptosis and blocking the cell cycle. The underlying mechanism involves targeting survivin, which subsequently regulates the protein expression of XIAP/caspase 3/9, CDK2/4, and cyclin E/D1. Thus, shikonin, a survivin inhibitor, is a promising therapeutic strategy in NSCLC treatment.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Naphthoquinones; Signal Transduction; Survivin

Lung cancer is one of the most lethal diseases and therefore poses a significant threat to human health. The Warburg effect, which is the observation that cancer cells predominately produce energy through glycolysis, even under aerobic conditions, is a hallmark of cancer. 6‑phosphofructo‑2‑kinase/fructose‑2,6‑biphosphatase 2 (PFKFB) is an important regulator of glycolysis. Shikonin is a Traditional Chinese herbal medicine, which has been reported to exert antitumor effects. The present study aimed to investigate the anticancer activity of shikonin in lung cancer. Cell Counting Kit‑8 (CCK‑8) and colony formation assays were used to analyze proliferation in A549 and H446 cells. Wound healing and Transwell assays were used to measure migration and invasion in A549 and H446 cells. Cell apoptosis was analyzed using flow cytometry. Lactate levels, glucose uptake and cellular ATP levels were measured using their corresponding commercial kits. Western blotting was performed to analyze the protein expression levels of key enzymes involved in aerobic glucose metabolism. Reverse transcription‑quantitative PCR was used to analyze the mRNA expression levels of PFKFB2. The results of the present study revealed that PFKFB2 expression levels were significantly upregulated in NSCLC tissues. Shikonin treatment decreased the proliferation, migration, invasion, glucose uptake, lactate levels, ATP levels and PFKFB2 expression levels and increased apoptosis in lung cancer cells in a dose‑dependent manner. The overexpression of PFKFB2 increased the proliferation, migration, glucose uptake, lactate levels and ATP levels in lung cancer cells, while the knockdown of PFKFB2 expression exerted the opposite effects. Moreover, there were no significant differences in lung cancer cell migration, apoptosis, glucose uptake, lactate levels and ATP levels between cells with knocked down PFKFB2 expression or treated with shikonin and the knockdown of PFKFB2 in cells treated with shikonin. In conclusion, the results of the present study revealed that shikonin inhibited the Warburg effect and exerted antitumor activity in lung cancer cells, which was associated with the downregulation of PFKFB2 expression.

Topics: Aged; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Lung Neoplasms; Male; Middle Aged; Naphthoquinones; Phosphofructokinase-2; Up-Regulation; Warburg Effect, Oncologic

Topics: A549 Cells; Apoptosis; Cell Survival; Drug Synergism; Humans; Lung Neoplasms; Metal Nanoparticles; Naphthoquinones; Silver

NSCLC is the major type of lung cancer and the survival rates of NSCLC patients remain low. AZD9291 is a third-generation EGFR-TKI and approved to treat NSCLC patients harboring EGFR T790M mutation and common targetable activating EGFR mutations, but it has a limited effect for wtEGFR NSCLC.. The current study investigated whether shikonin could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells.. SRB and colony formation assay were used to detect the proliferation of NSCLC cells, propidium iodide staining was performed to detect the apoptosis, ROS was analyzed using DCFH-DA staining, and western blot was used to detect the expression of indicated proteins.. We demonstrated that shikonin, a natural ROS inducer, could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells. In addition, shikonin increased AZD9291-induced apoptosis accompanying with the generation of ROS and activation of ER stress. Furthermore, ROS inhibition by NAC or GSH reversed the apoptosis induced by shikonin plus AZD9291, and recovered the ER stress activated by combination treatment, indicating that ROS mediated ER stress played a vital role in this combination therapy. Moreover, shikonin increased the anticancer activity of AZD9291 in primary wtEGFR NSCLC cells through ROS-mediated ER stress.. Our study suggests that combining shikonin with AZD9291 is a promising therapeutic strategy for treating wtEGFR NSCLC patients.

Topics: A549 Cells; Acrylamides; Aniline Compounds; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Synergism; Endoplasmic Reticulum Stress; ErbB Receptors; Humans; Lung Neoplasms; Naphthoquinones; Protein Kinase Inhibitors; Reactive Oxygen Species

The development of paclitaxel-resistance led to the tumor relapse and treatment failure of non-small cell lung cancer. Shikonin has been demonstrated to show anti-cancer activity in many cancer types. The present study aimed to investigate the anti-cancer activity of shikonin in paclitaxel-resistant non-small cell lung cancer treatment.. MTT, clonogenic assay, apoptotic cell death analysis, western blot, qRT-PCR, gene knockdown and overexpression, xenograft experiment, immunohistochemistry were performed.. Shikonin decreased paclitaxel-resistant NSCLC cell viability and inhibited the growth of xenograft tumor. Shikonin induced apoptotic cell death of paclitaxel-resistant NSCLC cell lines and suppressed the level of NEAT1 and Akt signaling of paclitaxel-resistant NSCLC cell lines and xenograft tumors. Either low dose or high dose of shikonin considerably suppressed the cell growth and induced the cell apoptotic death in NEAT1 knockdown A549/PTX cells, and p-Akt expression was decreased.. Shikonin could be a promising candidate for paclitaxel-resistant NSCLC treatment.

Topics: A549 Cells; Animals; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Naphthoquinones; Paclitaxel; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Xenograft Model Antitumor Assays

Increasing evidence indicates that the inflammatory tumor microenvironment can lead to cancer cell metastasis. Shikonin, which is extracted from the Chinese herb Zicao (the dried root of Lithospermum erythrorhizon), possesses various pharmacological effects, but its effect on tumor metastasis in the inflammatory microenvironment remains unknown. In the present study, we aimed to investigate the potential effect of shikonin on tumor metastasis in an inflammatory microenvironment as well as the underlying molecular mechanisms. It was found that, in the inflammatory microenvironment simulated by THP‑1 cell conditioned medium (THP‑1‑CM) in vitro, shikonin significantly inhibited the epithelial‑mesenchymal transition (EMT), migration and invasion of human lung adenocarcinoma cell lines A549 and H1299. In addition, we found that interleukin‑6 (IL‑6), which is expressed in THP‑1‑CM, promoted the EMT of lung adenocarcinoma cells, and shikonin markedly inhibited IL‑6‑induced EMT and cell motility. Moreover, shikonin inhibited IL‑6‑induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), prevented phosphorylated STAT3 (p‑STAT3) translocation into the nucleus, and suppressed p‑STAT3 transactivation activity. Additionally, it was found that shikonin inhibited lung metastasis, EMT and expression of p‑STAT3 of A549 cells in vivo. Furthermore, IL‑6 levels in human lung adenocarcinoma tissues were significantly associated with tumor‑node‑metastasis stage and lymph node metastasis, and its expression was correlated with tumor‑associated macrophage (TAM) infiltration. Together, these results suggest that shikonin suppresses the migration and invasion of human lung adenocarcinoma cells in an inflammatory microenvironment involving the IL‑6/STAT3 signaling pathway.

Topics: A549 Cells; Adenocarcinoma of Lung; Cell Movement; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Female; Humans; Interleukin-6; Lung; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Signal Transduction; STAT3 Transcription Factor; THP-1 Cells; Tumor Microenvironment; Tumor-Associated Macrophages; Xenograft Model Antitumor Assays

Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo.. CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo.. The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1.. These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Naphthoquinones; Quinazolines; Signal Transduction; Sincalide; STAT3 Transcription Factor; Thyroid Hormone-Binding Proteins; Thyroid Hormones

As patients with non‑small cell lung cancer (NSCLC) and wild‑type epidermal growth factor receptor (EGFR) are resistant to treatment with erlotinib or gefitinib, potential chemosensitizers are required to potentiate wild‑type EGFR NSCLC cells to erlotinib/gefitinib treatment. The present study reported that shikonin could sensitize the anticancer activity of erlotinib/gefitinib in wild‑type EGFR NSCLC cells. Furthermore, shikonin could potentiate mitochondrial‑mediated apoptosis induced by erlotinib/gefitinib in wild‑type EGFR NSCLC cells. In addition, the present study demonstrated that shikonin could induce apoptosis by activating reactive oxygen species (ROS)‑mediated endoplasmic reticulum (ER) stress, and that erlotinib/gefitinib may also induce ER stress in wild‑type EGFR NSCLC cells; however, shikonin plus erlotinib/gefitinib was more effective in activating ER stress than either agent alone. This indicated that ROS‑mediated ER stress may be associated with enhanced mitochondrial apoptosis induced by shikonin plus erlotinib/gefitinib. In addition, shikonin may promote the transition of cytoprotective ER stress‑inducing EGFR‑tyrosine kinase inhibitor tolerance to apoptosis‑promoting ER stress. Furthermore, shikonin may enhance the anti‑NSCLC activity of erlotinib/gefitinib in vivo. The data of the present study indicated that shikonin may be a potential sensitizer to enhance the anti‑cancer efficacy of erlotinib/gefitinib in wild‑type EGFR NSCLC cells resistant to erlotinib/gefitinib treatment.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Lithospermum; Lung Neoplasms; Mice, Nude; Naphthoquinones; Protein Kinase Inhibitors; Reactive Oxygen Species

Topics: Afatinib; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Lithospermum; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Epithelial-to-mesenchymal transition (EMT) is a major process to regulate cell migration and invasion. Inhibition of epidermal growth factor receptor (EGFR)-mediated EMT by tyrosine kinase inhibitors (TKIs) is a strategy to prevent lung cancer invasion. However, drug resistance is emerged and accelerated invasion through other signaling bypassing EGFR after TKIs therapy. c-Met signaling pathway is highly activated in EGFR-mutated lung cancer cells. Targeting c-Met signaling pathway may be a strategy to suppress EGFR-independent migration and invasion for lung cancer therapy. Therefore, we examined the anti-migration and anti-invasion abilities of shikonin, an active compound from Lithospermum erythrorhizon, in highly and ligand-induced c-Met activation lung cancer cells. Our results revealed that cell viability and cell cycle progression did not change under 1 μM of shikoinin treatment in highly c-Met expressive HCC827 lung cancer cells. Endogenous c-Met activation was dose-dependently inhibited and the migration and invasion activity of HCC827 cells were suppressed by shikonin treatment. Induction of E-cadherin expression and inhibition of vimentin, slug, and snail expression by shikonin was through c-Met-mediated PI3K/Akt and ERK signaling suppression. Furthermore, hepatocyte growth factor (HGF)-induced migration, invasion and EMT marker change were reversed by shikonin in low c-Met expressive A549 lung cancer cells. Inhibition of HGF-induced c-Met, PI3K/Akt and MEK/ERK activation were observed in shikonin-treated cells. Co-treatment of PI3K/Akt inhibitor or ERK inhibitor with shikonin enhanced shikonin-reversed HGF-regulated EMT marker expression. Taken together, the results suggested that the anti-migration and anti-invasion activities of shikonin was through c-Met inhibition and following by EMT suppression in lung cancer. J. Cell. Biochem. 118: 4639-4651, 2017. © 2017 Wiley Periodicals, Inc.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; MAP Kinase Signaling System; Naphthoquinones; Neoplasm Invasiveness; Proto-Oncogene Proteins c-met

Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.. Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.. Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.. Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Topics: A549 Cells; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 8; Cell Line, Tumor; Gene Silencing; Humans; Imidazoles; Indoles; Lithospermum; Lung Neoplasms; Macrolides; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation; RNA, Small Interfering; X-Ray Microtomography

Non-small cell lung cancer (NSCLC) is the dominant type of lung cancer. Molecular targeting has highly improved the treatment efficacy of lung cancer, but new challenges have emerged, such as gefitinib-resistance and cancer recurrence. Therefore, new chemotherapeutic agents and treatment strategies are urgently needed. Shikonin is the main active component of a Chinese medicinal plant 'Zi Cao', which has been shown to exhibit powerful anti-cancer activity in certain types of cancer; however, its activity in gefitinib-resistant lung cancer has never been addressed. In this study, we used a high-throughput screening assay for epidermal growth factor receptor (EGFR) inhibitors and discovered that Shikonin is a potent inhibitor of EGFR. The cytotoxicity of Shikonin and its anti-cancer mechanism in NSCLC was deeply explored. Shikonin exhibited selective cytotoxicity among two NSCLC cell lines (H1975 and H1650) and one normal lung fibroblast cell line (CCD-19LU). Shikonin significantly increased the activity of caspases and poly (ADP-ribosyl) polymerase (PARP), which are indicators of apoptosis, and the intensity of ROS by greater than 10-fold. NAC, an inhibitor of ROS, completely blocked apoptosis, caspase and PARP activation induced by Shikonin. Shikonin remarkably suppressed the phosphorylation of EGFR and led to EGFR degradation. The enhancement of ROS generation in H1650 and H1975 gefitinib-resistant NSCLC cells leads to impairment of growth and induction of apoptosis, whereas modulation of EGFR degradation and its downstream signalling pathways by Shikonin contributes to its anti-tumour properties in H1975 gefitinib-resistant NSCLC cells (with T790M and L858R activating mutations). Shikonin-induced cell apoptosis is closely associated with ROS elevation in the cells. These findings indicate that Shikonin can be an effective small molecule treating gefitinib-resistant NSCLC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; High-Throughput Screening Assays; Humans; Lung Neoplasms; Mutation; Naphthoquinones; Neoplasm Recurrence, Local; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; Thioredoxin-Disulfide Reductase

Topics: A549 Cells; Acetylation; Animals; Antineoplastic Agents; Apoptosis; Bcl-2-Like Protein 11; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; E1A-Associated p300 Protein; Early Growth Response Protein 1; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Sirtuin 1; Time Factors; Transfection; Xenograft Model Antitumor Assays

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, can exert inhibitory effect on tumor cell growth. However, little has been known concerning the effect of shikonin on lung adenocarcinoma cell and underlying mechanisms. In the present study, we investigated the effect of shikonin on the proliferation, cell cycle arrest, and apoptosis in human lung adenocarcinoma cells. We found that shikonin significantly suppressed the proliferation of lung adenocarcinoma cells compared with control in dose- and time-dependent manner (P < 0.05). In the meantime, our results showed that shikonin markedly increased the proportion of A549 cells at stage G1 as well as induced apoptosis in A549 cells. Furthermore, suppressed CCND1 and elevated caspase3 and caspase7 expression levels at mRNA were found in this study, indicating that shikonin may inhibit the growth of lung adenocarcinoma cell by changing cell cycle and promoting cell apoptosis through the regulation of CCND1, caspase3, and caspase7. Although more studies are needed, this study suggests that shikonin has the potential to be used as an anti-cancer agent in the treatment of lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Naphthoquinones; Resting Phase, Cell Cycle

Integrin β1 facilitates cancer cell adhesion, migration and metastasis by activating intracellular signaling pathways including the ERK and PI3K signaling pathways. In previous studies, shikonin, an active naphthoquinone isolated from the Chinese medicine Zi Cao (gromwell), showed effective anticancer activity both in vivo and in vitro. However, the mechanisms underlying shikonin's anticancer activity are not fully elucidated. Increasing evidence indicates that shikonin inhibits tumor metastasis, but little is known about the effect of shikonin on lung cancer cells. To better understand the anti-metastatic role of shikonin in lung cancer, in this study we sought to investigate the effect of shikonin on lung cancer cell proliferation, adhesion to extracellular matrices (ECM), migration and invasion in non-small cell lung cancer A549 cells. We also sought to investigate the molecular mechanisms underlying shikonin's anticancer effects. Here we showed that when non-small cell lung cancer A549 cells were treated with shikonin for 24h, 8.0μM shikonin significantly inhibited cell proliferation, while cells treated with less than 2.0μM shikonin for 24h significantly suppressed cell adhesion to the ECM, invasion and migration in a dose-dependent manner. Moreover, real-time PCR and Western blot analysis showed that shikonin led to a reduction in the expression of integrin β1 at the mRNA and protein levels. Further elucidation of the mechanisms involved revealed that shikonin repressed the phosphorylation of extracellular signal-regulated kinase (ERK1/2). Taken together, our findings provide new evidence that shikonin suppresses lung cancer invasion and metastasis by inhibiting integrin β1 expression and the ERK1/2 signaling pathway.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Lung Neoplasms; MAP Kinase Signaling System; Naphthoquinones

Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.. MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.. The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).. Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Naphthoquinones; Necrosis; Neoplasm Transplantation; Nuclear Pore Complex Proteins; Osteosarcoma; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Shikonin has been reported to induce apoptosis and inhibit angiogenesis in vivo and in vitro. 6-(1-propoxyiminoalkyl)-5,8-dimethoxyoxy 1,4-naphtoquinone S-64 (DMNQ S-64) was synthesized as a shikonin derivative. In this article, the underlying apoptotic mechanism of DMNQ S-64 was examined. DMNQ S-64 exerted cytotoxicity against A549 lung carcinoma cells with IC(50) of 27.3 microM. Apoptotic bodies were observed in DMNQ S-64-treated A549 cells by 4'-6-diamidino-2-phenylindole (DAPI) staining assay. DMNQ S-64 also increased sub-G1 DNA portion in a concentration-dependent manner by flow cytometric analysis. Western blotting has revealed that DMNQ S-64 effectively activates the expression of caspase 8, 9, and 3, cleaves poly (ADP-ribose) polymerase, and increases the ratio of Bax/Bcl-2. Furthermore, cytochrome c was released in a concentration-dependent manner by DMNQ S-64. Similarly, DMNQ S-64 significantly increased caspase 3 activity by enzyme-linked immunosorbent assay (ELISA). It also significantly inhibited the level of prostaglandin E2 (PGE(2)) by ELISA and downregulated the expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner. Taken together, DMNQ S-64 may exhibit cytotoxicity against A549 cells via caspase activation and COX-2 inhibition.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Enzyme Activation; Humans; Hydroxylamines; Lung Neoplasms; Membrane Proteins; Naphthoquinones

The shikonin mixture was used for 19 cases of later-stage lung cancer who were not the candidates for operation, radiotherapy and chemotherapy. The clinical observation showed that shikonin mixture could inhibit the growth of lung cancer and improve the immune function of the body. The tumors were reduced over 25% in diameter. The effective rate was 63.3%, remission rate 36.9%, survival rate of one year 47.3%. The intermedium survival period was about 10 months, including adenocarcinoma 10 months, squamous carcinoma 12 months. After treatment the life quality of patients were greatly improved. The patients got better appetite and their body weights were increased. They could manage themselves in daily life. The Karnofsky scores were enhanced by 20. The authors also observed that shikonin mixture could relieve such symptoms as cough, bloody sputum and chest pain caused by lung cancer. The levels of cells and interleukin-2 were increased (P less than 0.001). It had no harmful effects on peripheral blood picture, heart, kidney and liver. Shikonin mixture is safe and effective for later-stage cancer.

Topics: Adenocarcinoma; Adult; Antineoplastic Agents, Phytogenic; Carcinoma, Bronchogenic; Carcinoma, Squamous Cell; Female; Ginsenosides; Humans; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Naphthoquinones; Saponins