shikonin and Necrosis

Shikonin, the main active ingredient of Lithospermum, and its derivatives have been proved to have antitumor effects, and the anti-tumor mechanisms involve multiple targets. Based on recent literatures, this review focuses on the antitumor effects and its mechanisms. More emphases are given on the aspects of induction of apoptosis, induction of necrosis, acting on matrix metalloproteinase, acting on the protein tyrosine kinase and antiangiogenesis. The current status and problems of shikonin derivatives in antitumor effects are simply summarized and lookout for the development of antitumor drugs with shikonin as leading compounds.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drugs, Chinese Herbal; Humans; Lithospermum; Matrix Metalloproteinase 9; Naphthoquinones; Necrosis; Neoplasms; Neovascularization, Pathologic; Plants, Medicinal; Protein-Tyrosine Kinases; Reactive Oxygen Species

Our previous studies showed that Shikonin (SK) had a strong anti-Candida albican (C. albicans) activity, especially against some fluconazole-resistant strains, which is probably due to the oxidative damage of SK to C. albicans.. In this study, we expanded the antifungal spectrum and evaluate the toxicity of SK. The results indicated that SK also exhibited potent invitro antifungal activities against other pathogenic fungi such as other Candida, Aspergillus, Cryptococcus, and Dermatophytes, but did not display apparent toxicity to the mammalian cells, suggesting that SK is safe to be a potential antifungal drug. Furtherly, we analyze the exact mechanism of SK against C. albicans. We found that SK could induce a series of apoptosis characteristics, including phosphatidylserine externalization, chromatin condensation and fragmentation, decreased cytochrome c oxidase activity as well as caspase activation.. In summary, this study highlighted the antifungal activity and mechanism of SK against C. albicans, providing a potential therapeutic strategy for C. albicans infection.

Topics: Animals; Antifungal Agents; Apoptosis; Candida; Candida albicans; Humans; Mammals; Microbial Sensitivity Tests; Necrosis

Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after.. We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro.. Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin.. Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP.. Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.

Topics: Apoptosis; Cell Line, Tumor; Cysteine; Humans; Melanoma; Naphthoquinones; Necrosis; Reactive Oxygen Species

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

Topics: Alarmins; Animals; Apoptosis; Autophagy; Cell Communication; Cell Line, Tumor; Chloroquine; Dendritic Cells; Female; Immunization; Immunologic Surveillance; Mice, Inbred BALB C; Models, Biological; Naphthoquinones; Necrosis; Neoplasm Metastasis; Up-Regulation

Shikonin, a natural small agent, has shown inhibitory effect in many kinds of cells, which increases intracellular reactive oxygen species (ROS) level and causes mitochondrial injury. In this study, shikonin showed good inhibitory effect on nasopharyngeal carcinoma CNE-2Z cells in vivo and vitro. The results presented here revealed that ROS levels increased markly after shikonin treated. The electron microscopy displays the change in ultrastructure of CNE-2Z cells after treatment for shikonin, which indicated that shikonin induced necroptosis. Shikonin-induced cell death was inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the activity was unaffected by the caspase inhibitor z-VAD-fmk. Furthermore, we have demonstrated that the activation of receptor-interacting kinase (RIP) led to necroptosis. Meanwhile, shikonin also significantly inhibited tumor growth in a CNE-2Z xenograft mouse model. Taken together, shikonin induced CNE-2Z cells death by producing ROS as a necroptosis inducer. It could serve as a new therapeutic agent for treating CNE-2Z cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Cell Line, Tumor; Heterografts; Humans; Mice; Mice, Nude; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Necrosis; Reactive Oxygen Species

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

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 μmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioma; Humans; Mitochondria; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Oxidative Stress; Protein Serine-Threonine Kinases; Rats; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Signal Transduction; Time Factors

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Death; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Humans; Imidazoles; Indoles; Multiple Myeloma; Naphthoquinones; Necrosis; Proteasome Inhibitors; Purine Nucleosides; Pyrazines; Regulatory Factor X Transcription Factors; Transcription Factors; X-Box Binding Protein 1

Breast cancer, the most common cancer in the women, is the leading cause of death. Necrotic signaling pathways will enable targeted therapeutic agents to eliminate apoptosis-resistant cancer cells. In the present study, the effect of shikonin on the induction of cell necroptosis or apoptosis was evaluated using the T-47D breast cancer cell line. The cell death modes, caspase-3 and 8 activities and the levels of reactive oxygen species (ROS) were assessed. Cell death mainly occurred through necroptosis. In the presence of Nec-1, caspase-3 mediated apoptosis was apparent in the shikonin treated cells. Shikonin stimulates ROS generation in the mitochondria of T-47D cells, which causes necroptosis or apoptosis. Induction of necroptosis, as a backup-programmed cell death pathway via ROS stimulation, offers a new strategy for the treatment of breast cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Caspase 3; Cell Proliferation; Female; Humans; Mitochondria; Naphthoquinones; Necrosis; Reactive Oxygen Species; Tumor Cells, Cultured

The effects of shikonin on gastric cancer cells were investigated in this study. Exposure to shikonin reduced the viability of gastric cancer cells in a time- and dose-dependent manner. However, apoptosis was not observed in gastric cancer cell treatment with different concentrations of shikonin for 6h. By contrast, treatment with shikonin for 24h significantly induced apoptosis, as evidenced by the results of TUNEL assay and flow cytometry analysis in proportion to the concentration. Disruption of the mitochondrial membrane potential was observed in gastric cancer cells that were treated with shikonin for 6 and 24h. Pretreatment with necrostatin-1 recovered cell death and mitochondrial membrane potential in the 6h shikonin treatment, but not in the 24h shikonin treatment. Western blot results reveal enhanced p38 phosphorylation, downregulated AKT phosphorylation, and increased caspase3 and PARP cleavage in cells that were treated with shikonin for 24h, but not in cells treated for 6h. Shikonin also triggered reactive oxygen species (ROS) generation both in the 6 and 24h treatments. Pretreatment with N-acetylcysteine blocked shikonin-induced cell death. In summary, our findings suggest that shikonin, which may function as a promising agent in the treatment of gastric cancers, sequentially triggered necrosis or apoptosis through ROS generation in gastric cancer cells.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Flow Cytometry; Free Radical Scavengers; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Necrosis; Phosphorylation; Reactive Oxygen Species; Stomach Neoplasms; Time Factors

Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1μM after 6h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10μM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1μM SHK-induced apoptosis and converted 10μM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1μM and 85 genes by 10μM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1μM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10μM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Death; Drugs, Chinese Herbal; Gene Expression; Gene Expression Profiling; Glutathione; Humans; Naphthoquinones; Necrosis; Oxidative Stress; U937 Cells

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.. Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.. Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.. We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Glioma; Humans; Medicine, Chinese Traditional; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Rats; Reactive Oxygen Species; RNA-Binding Proteins

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

Degterev et al. previously demonstrated that death receptor mediated apoptosis could be diverted to necroptosis when apoptosis signaling was blocked, suggesting that necroptosis may function as a backup mechanism to insure the elimination of damaged cells under certain conditions when apoptosis was inhibited. Here, we show that shikonin-induced necroptosis can be reverted to apoptosis in the presence of necrostatin-1 (Nec-1), a specific necroptosis inhibitor and that the death mode switch is at least partially due to the conversion from mitochondrial inner membrane permeability to mitochondrial outer membrane permeability, which is associated with Bax translocation. The data combined with the previous reports support a notion that apoptosis and necroptosis may function as reciprocal backup mechanisms of cellular demise. To the best of our knowledge, this is the first study to document a conversion from necroptosis to apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Imidazoles; Indoles; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Naphthoquinones; Necrosis; Permeability

Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cell Death; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Mice; Mice, Nude; Naphthoquinones; Necrosis; Neoplasm Transplantation