Shikonin-induced Apoptosis Research Articles

Shikonin Induces Autophagy and Apoptosis in Esophageal Cancer EC9706 Cells by Regulating the AMPK/mTOR/ULK Axis.

Shikonin is a plant medicine extracted from Lithospermum, which dominate influential antioxidant and antitumor effect. Here, we report that shikonin was capable of inducing human esophageal cancer EC9706 cell apoptosis and autophagy, in a time- and dose-dependent manner. Shikonin exposure repressed cell viability and migration and invasion capabilities and caused EC9706 cell autophagy and apoptosis by activating the AMPK/mTOR/ULK axis. Autophagy inhibition secured EC9706 cells against shikonin-induced autophagy and apoptosis and reversed the upregulation of AMPK and ULK phosphorylation and downregulation of mTOR phosphorylation provoked by shikonin. In summary, shikonin instigates EC9706 cell apoptosis and autophagy using the target AMPK/mTOR/ULK signal pathway axis, which provides a potential new target to treat human esophageal cancer.

The Endoplasmic Reticulum Stress Response Mediates Shikonin-Induced Apoptosis of 5-Fluorouracil-Resistant Colorectal Cancer Cells.

Resistance to chemotherapeutic drugs is a significant problem in the treatment of colorectal cancer, resulting in low response rates and decreased survival. Recent studies have shown that shikonin, a naphthoquinone derivative, promotes apoptosis in colon cancer cells and cisplatin-resistant ovarian cells, raising the possibility that this compound may be effective in drug-resistant colorectal cancer. The aim of this study was to characterize the molecular mechanisms underpinning shikonin-induced apoptosis, with a focus on endoplasmic reticulum (ER) stress, in a 5-fluorouracil–resistant colorectal cancer cell line, SNU-C5/5-FUR. Our results showed that shikonin significantly increased the proportion of sub-G1 cells and DNA fragmentation and that shikonin-induced apoptosis is mediated by mitochondrial Ca2+ accumulation. Shikonin treatment also increased the expression of ER-related proteins, such as glucose regulatory protein 78 (GRP78), phospho-protein kinase RNA-like ER kinase (PERK), phospho-eukaryotic initiation factor 2 (eIF2α), phospho-phosphoinositol-requiring protein-1 (IRE1), spliced X-box–binding protein-1 (XBP-1), cleaved caspase-12, and C/EBP-homologous protein (CHOP). In addition, siRNA-mediated knockdown of CHOP attenuated shikonin-induced apoptosis, as did the ER stress inhibitor TUDCA. These data suggest that ER stress is a key factor mediating the cytotoxic effect of shikonin in SNU-C5/5-FUR cells. Our findings provide an evidence for a mechanism in which ER stress leads to apoptosis in shikonin-treated SNU-C5/5-FUR cells. Our study provides evidence to support further investigations on shikonin as a therapeutic option for 5-fluorouracil–resistant colorectal cancer.

Shikonin differentially regulates glucose metabolism via PKM2 and HIF1α to overcome apoptosis in a refractory HCC cell line

AimsIn tumor cells, shikonin treatment has been reported to inhibit glycolysis by suppressing the activity of pyruvate kinase M2 (PKM2) and to induce apoptosis by increasing reactive oxygen species (ROS) production. However, hepatocellular carcinoma (HCC) shows variable sensitivity to shikonin treatment, and the mechanism for these differences remains unclear. We evaluated the effects of shikonin on metabolic and oxidative pathways in sensitive and refractory HCC cell lines to identify mechanisms of differential sensitivity. Main methodsCell viability and apoptosis were evaluated by MTT assay, PI/Annexin V and JC-1 staining. Mitochondrial function was further evaluated by measurements of ROS and mitochondrial mass. Oxygen consumption rates, NAD+/NADH, ATP and lactate were measured as indicators of energy metabolism and glycolysis. Protein expression associated with glycolysis and apoptosis was evaluated by western blotting, RT-qPCR and immunofluorescence staining. Key findingsThe sensitivity to shikonin treatment was significantly higher for HepG2 cells than for HCCLM3 cells, with less dramatic effects in HCCLM3 cells on apoptosis, ROS, and oxidative phosphorylation. Shikonin up-regulated mitochondrial biogenesis to increase mitochondrial oxidative phosphorylation in HepG2 cells, but displayed the opposite trend in HCCLM3 cells. Mechanistically, shikonin promoted nuclear expression of PKM2 and HIF1α in HCCLM3 cells, with upregulation of glycolysis-related gene transcription and glycolysis. SignificanceThese results suggest that PKM2 rewires glucose metabolism, which explains the differential sensitivity to shikonin-induced apoptosis in HCC cells. Our findings elucidate mechanisms for differential responses to shikonin, provide potential biomarkers, and indicate a theoretical basis for targeting glycolytic enzymes in refractory HCC.

Shikonin suppresses trophoblast cell growth via regulation of GLI1, and p62 mediated caspase 8 activation

Unruptured ectopic pregnancy (UEP) is a common cause of morbidity and, occasionally, of mortality in women of reproductive age. Pharmacological intervention is a common therapeutic approach for early-stage UEP. Herein, we investigated the cytotoxic effect and novel mechanism of shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, in human trophoblast cells. These data demonstrated that shikonin suppressed proliferation and induced apoptosis in a time-dependent manner in HTR-8/SVneo cells. Shikonin blocked autophagic flux and promoted p62 interaction with caspase 8, resulting in caspase 8 activation. Moreover, shikonin suppressed GLI1 expression, and GLI1 overexpression attenuated shikonin-induced cell apoptosis. Although silencing GLI1 slightly promoted cell apoptosis, p62 overexpression enhanced GLI1 silencing-induced cell apoptosis by activating caspase 8. Furthermore, rapamycin increased shikonin-induced cell apoptosis in HTR-8/SVneo cells, whereas 3-MA attenuated the cytotoxic effect of shikonin. In conclusion, shikonin suppressed trophoblast cell growth by silencing GLI1 and increasing p62 co-mediated activation of caspase 8, which suggested a potential novel therapeutic target for UEP.

Shikonin-induced Apoptosis of Colon Cancer Cells Is Reduced by Peroxiredoxin V Expression.

Colon cancer is the second most common deadliest malignancy in the world and better understanding of its underlying mechanisms is needed to improve clinical management. Natural plant extracts are gaining attention in the development of new therapeutic strategies against various cancer types. Shikonin is a naturally extracted naphthoquinone pigment with effects against cancer, including colon cancer. In this study, we conducted a series of in vitro experiments to show the effects of Shikonin on colon cancer cell apoptosis. A colon cancer cell line with overexpression of peroxiredoxin V (PrxV) was constructed and the relationship of PrxV expression with Shikonin-induced cell apoptosis was investigated. Shikonin induced colon cancer cell apoptosis via regulation of mammalian target of rapamycin signaling. Shikonin-induced cell apoptosis was abrogated by overexpression of PrxV. According to the results obtained in this study, targeting PrxV may provide new insight for the successful management of colon cancer by inducing cell apoptosis.

Shikonin induces apoptosis and prosurvival autophagy in human melanoma A375 cells via ROS-mediated ER stress and p38 pathways

Shikonin, a botanical drug extracted from Lithospermum erythrorhizon, exhibits anti-cancer effects in various cancer cell lines. However, the mechanisms underlying these effects have not been completely elucidated yet. Here, we showed that Shikonin induces apoptosis and autophagy in A375 cells and inhibits their proliferation. Shikonin caused G2/M phase arrest through upregulation of p21 and downregulation of cyclin B1. Shikonin significantly triggered ER stress-mediated apoptosis by upregulating the expression of p-eIF2α, CHOP, and cleaved caspase-3. It also induced protective autophagy by activating the p38 pathway, followed by an increase in the levels of p-p38, LC3B-II, and Beclin 1. Upon suppression of autophagy by 3-methyladenine, Shikonin-induced apoptosis was enhanced in A375 cells. Moreover, after pretreatment with N-acetyl-cysteine, Shikonin increased the production of reactive oxygen species that are involved in regulating ER stress-mediated apoptosis and p38-activated autophagy, as evidenced by the reversion of cell viability and apoptosis and a decrease in p-eIF2α, CHOP, p-p38, LC3B-II, and Beclin 1 levels. Thus, we demonstrated that Shikonin induced apoptosis and autophagy in A375 cells via the activation of ROS-mediated ER stress and p38 pathways, indicating that Shikonin can serve as a potential agent for human melanoma therapy.

Shikonin Exerts Cytotoxic Effects in Human Colon Cancers by Inducing Apoptotic Cell Death via the Endoplasmic Reticulum and Mitochondria-Mediated Pathways.

The apoptotic effects of shikonin (5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methylpent-3-enyl]naphthalene-1,4-dione) on the human colon cancer cell line SNU-407 were investigated in this study. Shikonin showed dose-dependent cytotoxic activity against SNU-407 cells, with an estimated IC50 value of 3 µM after 48 h of treatment. Shikonin induced apoptosis, as evidenced by apoptotic body formation, sub-G1 phase cells, and DNA fragmentation. Shikonin induced apoptotic cell death by activating mitogen-activated protein kinase family members, and the apoptotic process was mediated by the activation of endoplasmic reticulum (ER) stress, leading to activation of the PERK/elF2α/CHOP apoptotic pathway, and mitochondrial Ca2+ accumulation. Shikonin increased mitochondrial membrane depolarization and altered the levels of apoptosis-related proteins, with a decrease in B cell lymphoma (Bcl)-2 and an increase in Bcl-2-associated X protein, and subsequently, increased expression of cleaved forms of caspase-9 and -3. Taken together, we suggest that these mechanisms, including MAPK signaling and the ER-and mitochondria-mediated pathways, may underlie shikonin-induced apoptosis related to its anticancer effect.

Shikonin suppresses proliferation and induces apoptosis in endometrioid endometrial cancer cells via modulating miR-106b/PTEN/AKT/mTOR signaling pathway.

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, which exerts anticancer effects in various cancers. However, the molecular mechanisms underlying the therapeutic effects of shikonin against endometrioid endometrial cancer (EEC) have not yet been fully elucidated. Herein, we investigated anticancer effects of shikonin on EEC cells and explored the underlying molecular mechanism. We observed that shikonin inhibits proliferation in human EEC cell lines in a dose-dependent manner. Moreover, shikonin-induced apoptosis was characterized by the up-regulation of the pro-apoptotic proteins cleaved-Caspase-3 and Bax, and the down-regulation of the anti-apoptotic protein Bcl-2. Microarray analyses demonstrated that shikonin induces many miRNAs’ dysregulation, and miR-106b was one of the miRNAs being most significantly down-regulated. miR-106b was identified to exert procancer effect in various cancers, but in EEC remains unclear. We first confirmed that miR-106b is up-regulated in EEC tissues and cells, and knockdown of miR-106b suppresses proliferation and promotes apoptosis. Meanwhile, our results validated that the restored expression of miR-106b abrogates the antiproliferative and pro-apoptotic effects of shikonin. We also identified that miR-106b targets phosphatase and tensin homolog (PTEN), a tumor suppressor gene, which in turn modulates AKT/mTOR signaling pathway. Our findings indicated that shikonin inhibits proliferation and promotes apoptosis in human EEC cells by modulating the miR-106b/PTEN/AKT/mTOR signaling pathway, suggesting shikonin could act a potential therapeutic agent in the EEC treatment.

Shikonin causes apoptosis by disrupting intracellular calcium homeostasis and mitochondrial function in human hepatoma cells.

Shikonin is known to suppress proliferation and induce apoptosis in a variety of cancer cell lines. In the present study, SMMC-7721 human hepatocellular carcinoma cells were treated with shikonin (1, 2 or 4 µM) for 12–48 h. Cell morphological alterations and DNA damage were determined. Furthermore, changes in cell cycle, mitochondrial transmembrane potential, calcium homeostasis and levels of reactive oxygen species were measured. Shikonin-treated SMMC-7721 cells exhibited morphological changes and DNA damage. Shikonin inhibited cell proliferation causing cell cycle arrest at the G0/G1 phase and induced apoptosis in a dose- and time-dependent manner. Shikonin-induced apoptosis was associated with activation of caspases-3, −8 and −9, elevated levels of intracellular Ca2+ and reactive oxygen species, reduced mitochondrial membrane potential and enhanced efflux of Ca2+ and K+. Gene expression B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), p53 and caspase-3 was up-regulated, whereas Bcl-2 expression was downregulated. Shikonin caused apoptosis by inhibiting cell cycle progression, disrupting Ca2+ homeostasis, inducing oxidative stress and triggering mitochondrial dysfunction. Activation of caspases-3, −8 and −9, K+ efflux, and regulation of Bax, Bcl-2, p53 and caspase-3 expression are involved in the process. These results provide in-depth insight into the mechanisms of action of shikonin in the treatment of cancer.

Shikonin induces ROS-based mitochondria-mediated apoptosis in colon cancer.

Colon cancer is the third most common malignancy worldwide, and chemotherapy is a widely used strategy in clinical therapy. Chemotherapy-resistant of colon cancer is the main cause of recurrence and progression. Novel drugs with efficacy and safety in treating colon cancer are urgently needed. Shikonin, a naphthoquinone derived from the roots of the herbal plant Lithospermum erythrorhizon, has been determined to be a potent anti-tumor agent. The aim of the present study was to detect the underlying anti-tumor mechanism of shikonin in colon cancer. We found that shikonin suppressed the growth of colon cancer cells in a dose-dependent manner in vitro and in vivo. Shikonin induced mitochondria-mediated apoptosis, which was regulated by Bcl-2 family proteins. Shikonin increased the generation of intracellular ROS, which played an upstream role in shikonin-induced apoptosis. Our data indicated that generation of ROS, down-regulated expression of Bcl-2 and Bcl-xL, depolarization of the mitochondrial membrane potential and activation of the caspase cascade were components of the programmed event of shikonin-induced apoptosis in colon cancer cells. In addition, shikonin presented minimal toxicity to non-neoplastic colon cells and no liver injury in xenograft models, showing safety in the control of colon cancer cell growth in vitro and in vivo. Taken together, our findings suggest that shikonin might serve as a potential novel therapeutic drug in the treatment of human colon cancer.

Shikonin inhibits gefitinib-resistant non-small cell lung cancer by inhibiting TrxR and activating the EGFR proteasomal degradation pathway

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.

Anti-Leukemic Activity of Shikonin: Role of ERP57 in Shikonin Induced Apoptosis in Acute Myeloid Leukemia

Background/Aims: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy. Methods: Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells. Results: Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis. Conclusions: Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.

Shikonin causes apoptosis by up-regulating p73 and down-regulating ICBP90 in human cancer cells

Shikonin, a natural naphthoquinone isolated from the Chinese traditional medicine Zi Cao (purple gromwell), is known to suppress the growth of several cancer cell types. In this study, we evaluated the pro-apoptotic effects of shikonin on MCF-7 and HeLa cells, and investigated the underlying mechanism. Shikonin-induced apoptosis was associated with activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, up-regulation of p73, and down-regulation of BCL-2. Shikonin also induced up-regulation of the tumor suppressor gene, p16INK4A. Increasing transcriptional activity of p16INK4A by shikonin treatment, we observed in luciferase promoter assay, reflects reduced promoter binding by down-regulation of ICBP90 (inverted CCAAT box binding protein, 90 kDa), which are involved in down-regulation of its partner, DNMT1 (DNA methyltransferase 1). On the basis of these results, we conclude that shikonin causes apoptosis via a p73-related, caspase-3–dependent pathway.

Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR-NF-κB signalling pathway in human epidermoid carcinoma A431 cells.

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR–NF-κB signaling pathways.

Mechanism Underlying Shikonin-induced Apoptosis and Cell Cycle Arrest on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

Mechanism Underlying Shikonin-induced Apoptosis and Cell Cycle Arrest on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

Cbl-b-regulated extracellular signal-regulated kinase signaling is involved in the shikonin-induced apoptosis of lung cancer cells in vitro.

Shikonin (SK), a naturally occurring naphthoquinone, exhibits antitumor activity. However, its precise mechanisms of action are unknown. In the present study, the effects of SK on NCI-H460 human lung cancer cells were investigated. It was found that SK reduced cell viability and induced apoptosis in the NCI-H460 cells. Additionally, SK inhibited extracellular signal-regulated kinase (ERK) activity, which indicates that inhibition of the ERK pathway is probably one of the mechanisms by which SK induced NCI-H460 cell apoptosis. The expression of Cbl-b was significantly increased by treatment with SK for 4 h, and gradually increased to a maximal level at 24 h; the time taken for the upregulation of Cbl-b protein was in accordance to that required for the downregulation of phospho (p)-ERK protein. The Cbl inhibitor Ps341 reversed the SK-induced downregulation of p-ERK and apoptosis of NCI-H460 cells. These results indicate that Cbl-b potentiates the apoptotic action of SK by inhibiting the ERK pathway in lung cancer cells.

Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression.

Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1–2.5 μg/mL) cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5–10 μg/mL) precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.

Nec-1 Enhances Shikonin-Induced Apoptosis in Leukemia Cells by Inhibition of RIP-1 and ERK1/2

Necrostatin-1 (Nec-1) inhibits necroptosis by allosterically inhibiting the kinase activity of receptor-interacting protein 1 (RIP1), which plays a critical role in necroptosis. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and the phosphorylation of mitogen activated protein kinases (MAPKs). NF-κB, ROS and MAPKs all play important roles in apoptotic signaling. Nec-1 was regarded as having no effect on apoptosis. Here, we report that Nec-1 increased the rate of nuclear condensation and caspases activation induced by a low concentration of shikonin (SHK) in HL60, K562 and primary leukemia cells. siRNA-mediated knockdown of RIP1 significantly enhanced shikonin-induced apoptosis in K562 and HL60 cells. Shikonin treatment alone could slightly inhibit the phosphorylation of ERK1/2 in leukemia cells, and the inhibitory effect on ERK1/2 was significantly augmented by Nec-1. We also found that Nec-1 could inhibit NF-κB p65 translocation to the nucleus at a later stage of SHK treatment. In conclusion, we found that Nec-1 can promote shikonin-induced apoptosis in leukemia cells. The mechanism by which Nec-1 sensitizes shikonin-induced apoptosis appears to be the inhibition of RIP1 kinase-dependent phosphorylation of ERK1/2. To our knowledge, this is the first study to document Nec-1 sensitizes cancer cells to apoptosis.

Novel Multiple Apoptotic Mechanism of Shikonin in Human Glioma Cells

Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells. Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis. Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment. These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.