DYRK1A inhibition suppresses STAT3/EGFR/Met signalling and sensitizes EGFR wild‐type NSCLC cells to AZD9291

DYRK1A is considered a potential cancer therapeutic target, but the role of DYRK1A in NSCLC oncogenesis and treatment requires further investigation. In our study, high DYRK1A expression was observed in tumour samples from patients with lung cancer compared with normal lung tissues, and the high levels of DYRK1A were related to a reduced survival time in patients with lung cancer. Meanwhile, the DYRK1A inhibitor harmine could suppress the proliferation of NSCLC cells compared to that of the control. As DYRK1A suppression might be effective in treating NSCLC, we next explored the possible specific molecular mechanisms that were involved. We showed that DYRK1A suppression by siRNA could suppress the levels of EGFR and Met in NSCLC cells. Furthermore, DYRK1A siRNA could inhibit the expression and nuclear translocation of STAT3. Meanwhile, harmine could also regulate the STAT3/EGFR/Met signalling pathway in human NSCLC cells. AZD9291 is effective to treat NSCLC patients with EGFR‐sensitivity mutation and T790 M resistance mutation, but the clinical efficacy in patients with wild‐type EGFR remains modest. We showed that DYRK1A repression could enhance the anti‐cancer effect of AZD9291 by inducing apoptosis and suppressing cell proliferation in EGFR wild‐type NSCLC cells. In addition, harmine could enhance the anti‐NSCLC activity of AZD9291 by modulating STAT3 pathway. Finally, harmine could enhance the anti‐cancer activity of AZD9291 in primary NSCLC cells. Collectively, targeting DYRK1A might be an attractive target for AZD9291 sensitization in EGFR wild‐type NSCLC patients.     

AZD9291 promotes autophagy and inhibits PI3K/Akt pathway in NSCLC cancer cells

AZD9291, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is highly selective against EGFR T790M-mutant non–small cell lung cancer (NSCLC). On investigating the growth inhibitory effects of AZD9291 on NSCLC and the underlying mechanism, we found that AZD9291 can trigger autophagy-mediated cell death in both A549 and H1975 cells by increasing the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3) and decreasing the expression of p62. In the presence of the autophagy inhibitor chloroquine, the AZD9291-induced increase in LC3 level was further augmented. AZD9291 decreased the levels of phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt. AZD9291-induced cell death was enhanced by Akt knockdown, and the levels of both EGFR and phosphorylated EGFR were decreased by AZD9291. AZD9291 was also found to significantly suppress the tumor growth in H1975 xenograft nude mice. Thus, AZD9291 was found to induce autophagy, decrease in EGFR levels, and show a strong inhibitory effect on NSCLC both in vitro and in vivo. Furthermore, the PI3K/Akt signaling pathway was found to play a critical role in AZD9291-induced cell death.     

Pristimerin induces apoptosis and tumor inhibition of oral squamous cell carcinoma through activating ROS-dependent ER stress/Noxa pathway

BackgroundPristimerin (Pri), a natural quinone methide triterpenoid isolated from Celastraceae and Hippocrateaceae, exhibits potent antitumor activity against various cancers. However, the mechanism of apoptosis induction by Pri in oral squamous cell carcinoma (OSCC) and its anti-OSCC effect in vivo has not been widely studied.PurposeThis study aimed to investigate the anti-OSCC activities of Pri in vitro and in vivo and addressed the potential mechanisms of Pri-induced apoptosis.MethodsThe effects of Pri on OSCC cells were analyzed by cell viability, colony formation and flow cytometry assays. Western blotting and qRT-PCR assays were chosen to detect the expression of proteins and genes. The anti-OSCC efficacy of Pri in vivo was evaluated by CAL-27 xenografts.ResultsWe showed that Pri inhibited the proliferation of human OSCC cell lines. Additionally, Pri induced apoptosis by upregulating Noxa expression. Furthermore, Pri treatment triggered excessive endoplasmic reticulum (ER) stress activation and subsequently induced c-Jun N-terminal kinase (JNK) signaling. ROS scavengers and ER stress inhibitors significantly attenuated Pri-induced OSCC cell apoptosis. Finally, Pri suppressed tumor growth in CAL-27 xenografts, accompanied ER stress activation and cell apoptosis.ConclusionThese results reveal that Pri suppressed tumor growth and triggered cell apoptosis through ER stress activation in OSCC cells and xenografts, suggesting that Pri may serve as a therapeutic agent for OSCC.     

Identification of osimertinib (AZD9291) as a lysine specific demethylase 1 inhibitor

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In this study, osimertinib was characterized as a LSD1 inhibitor for the first time with an IC₅₀ of 3.98 ± 0.3 μM and showed LSD1 inhibitory effect at cellular level. These findings provide new molecular skeleton for dual inhibitor for LSD1 and EGFR. Osimertinib could serve as a lead compound for further development for anti-NSCLC drug discovery with dual targeting.     

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.     

Effect of pristimerin on apoptosis through activation of ROS/ endoplasmic reticulum (ER) stress-mediated noxa in colorectal cancer

BackgroundPristimerin, a natural quinonemethid triterpenoid found in different spp. of Celastraceae and Hippocrateaceae families, has been reported to exhibit potent antitumor activities against colorectal cancer (CRC). However, the mechanisms underlying pristimerin-induced apoptosis in CRC is not clear.PurposeThis study aimed to investigate the mechanisms of pristimerin-induced apoptosis against CRC in vitro and in vivo.MethodsCell viability and cell apoptosis analyses were conducted to assess the effects of pristimerin on CRC. Western blotting was performed to detect the expression of proteins affected by pristimerin in vitro and in vivo. HCT116 colon cancer xenografts and APC^min/+ mouse models were used to evaluate the anti-CRC effect of pristimerin in vivo.ResultsOur data showed that pristimerin induced apoptosis by regulating proapoptotic proteins of which Noxa showed higher expression. Pristimerin triggered reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress signaling activation. Pristimerin significantly elevated the expression of ER stress-related proteins, resulting in activation of the IRE1α and c-Jun N-terminal kinase (JNK) signal pathway through the formation of the IRE1α-TRAF2-ASK1 complex. Pristimerin exhibited apoptosis-inducing activities in HCT116 colon cancer xenografts and APC^min/+ mice.ConclusionBoth in vitro and in vivo data demonstrated that pristimerin induced Noxa expression and apoptosis through activation of the ROS/ER stress/JNK axis in CRC. Thus, pristimerin may be a promising antitumor agent for CRC.     

Insight into binding mechanisms of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations

Abstract

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors.

Communicated by Ramaswamy H. Sarma     

The tyrphostin AG1478 augments oridonin-induced A431 cell apoptosis by blockage of JNK MAPK and enhancement of oxidative stress

Abstract

Oridonin, a diterpenoid compound, extracted and purified from Rabdosia rubescen has been reported to have cytotoxic effect on tumour cells through apoptosis, and tyrosine kinase pathways are involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478 was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant human epidermoid carcinoma A431 cells. Inhibition of EGFRaugmented oridonin-induced A431 cell apoptosis, while the changes of expression of downstream proteins, Bcl-2, Bcl-xL, Bax, cytochrome c, pro-caspase-3, Fas, FADD and pro-caspase-8 suggested that both the intrinsic and extrinsic apoptotic pathways are involved in these processes. Pretreatment with AG1478 aggravated oridonin-induced loss of mitochondrial membrane potential (MMP) and increased ROS generation in A431 cells, while a ROS scavenger, N-acetylcysteine (NAC) completely reversed oridonin- and AG1478-induced ROS generation and apoptosis. Therefore, AG1478 augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478 decreased the expression of downstream MAPK proteins ERK, JNK and P38 and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38 inhibitors, only JNK inhibitor SP600125 effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonised apoptosis and exerted a protective effect in A431 cells, and both AG1478 and SP600125 decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis and this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonising apoptosis and ROS generation.     

Discovery of selective EGFR modulator to inhibit L858R/T790M double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC₅₀ values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index.     

Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.     

Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets

AimsObesity is associated with hypertriglyceridemia and elevated circulating free fatty acids (FFA), resulting in endothelial dysfunction. Endoplasmic reticulum (ER) stress has been implicated in many of these processes. To determine if ER stress participates in palmitate-induced apoptosis, we investigated the effects of diet-induced obesity and palmitate on mouse aortic endothelial cells (MAEC) in vivo and in vitro.Main methodsMale C57BL/6 mice were fed standard chow diets (SCD) or high-calorie and high-cholesterol diets (HCD) for 3 months. Insulin resistance was detected, and the serum, including proinflammatory indices and markers of endothelial function, was also analyzed. The ultrastructure and apoptosis of the endothelial cells in the thoracic aorta were observed. The primary MAEC were separated and treated with palmitate at different concentrations or different times respectively to observe any changes in cellular proliferation, intracellular reactive oxygen species (ROS) levels and apoptosis. Finally, the ER stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were analyzed.Key findingsHCD-fed obese mice became inflammation-activated and insulin-resistant. Swollen mitochondria, expanded ER and apoptosis in the endothelial cells of the thoracic aorta were observed in HCD-fed mice. Palmitate inhibited cell proliferation, increased production of ROS and induced apoptosis in MAEC. CHOP was overexpressed and shifted into the nucleus (mainly), while the expression of GRP78 was upregulated in the palmitate-treated MAEC.SignificanceOur results indicate that diet-induced obesity results in endothelial dysfunction in vivo, and that oxidative and ER stress may be involved in apoptosis induced by the palmitate in vitro.     

Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

ObjectivesTargeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.Materials and MethodsThe effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca²⁺ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting.ResultsPR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca²⁺ and then stimulated autophagy through Ca²⁺‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619.ConclusionsOur findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.     

Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts

PurposeThe stem of Marsdenia tenacissima (Roxb.) Wight et Arn. has long been used as a medicine to treat cancer in China. Our previous in vitro results showed that Marsdenia tenacissima extract (MTE) overcomes gefitinib resistance in non-small cell lung cancer (NSCLC) cells. However, it is unknown whether MTE could enhance gefitinib efficacy in vivo. The present study was intended to investigate the in vivo anti-tumour activity of MTE combined with gefitinib.MethodsHuman NSCLC H460 (K-ras mutation) or H1975 cells (EGFR T790M mutation) were subcutaneously inoculated into nude mice. Tumour volume and body weight were measured regularly. Resected tumours were weighed after the animals were sacrificed. Immunoblotting or immunohistochemistry was used to assess the cellular proliferation and apoptosis in xenograft tumour tissue. Expression of the EGFR downstream pathways and c-Met were measured with western blot analysis to explore possible mechanisms.ResultsMTE (5, 10, 20 g/kg) dose-dependently reduced tumour growth and induced cell apoptosis. MTE suppressed EGFR related signals, and 20 g/kg was the most effective dose. Low-dose MTE (5 g/kg) significantly enhanced gefitinib efficacy in resistant H460 and H1975 xenografts. The combination inhibited tumour proliferation and induced cell apoptosis in both resistant NSCLC xenografts. Constitutive activation of the PI3K/Akt and MEK/ERK pathways is related to EGFR-TKI resistance. Accordingly, phosphorylation of PI3K/Akt/mTOR and ERK1/2 was suppressed after combination treatment. Simultaneously, cross-talked c-Met and EGFR were also prominently lowered in the presence of MTE combined with gefitinib.ConclusionThe present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.     

Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy

Background

Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.

Methods/Principal Findings

After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.

Conclusions/Significance

Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.     

Gambogenic acid induces Noxa-mediated apoptosis in colorectal cancer through ROS-dependent activation of IRE1α/JNK

BackgroundGambogenic acid (GNA), an active component of Garcinia hanburyi Hook.f. (Clusiaceae) (common name gamboge), exerts anti-inflammatory and antitumor properties. However, the underlying mechanism of GNA in colorectal cancer (CRC) is still not well understood.PurposeThis study aimed to investigate the antitumor effects and mechanisms of GNA on CRC in vitro and in vivo.MethodsCell viability, colony formation and cell apoptosis assays were performed to determine the antitumor effects of GNA. qRT-PCR and Western blotting were performed to evaluate the expression of genes or proteins affected by GNA in vitro and in vivo. HCT116 colon cancer xenografts and the APC^min/+ mice model were used to confirm the antitumor effects of GNA on CRC in vivo.ResultsGNA induced Noxa-mediated apoptosis by inducing reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation. Moreover, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme-1α (IRE1α) leading to JNK phosphorylation. ROS scavenger attenuated GNA-induced IRE1α activation and JNK phosphorylation. Knockdown of IRE1α also prevented GNA-induced JNK phosphorylation. In vivo, GNA suppressed tumor growth and progression in HCT116 colon cancer xenografts and the APC^min/+ mices model.ConclusionThese findings revealed that GNA induced Noxa-mediated apoptosis by activating the ROS/IRE1α/JNK signaling pathway in CRC both in vitro and in vivo. GNA is therefore a promising antitumor agent for CRC treatment.     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway

Background

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results

The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion

The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0127-1) contains supplementary material, which is available to authorized users.     

Shikonin Kills Glioma Cells through Necroptosis Mediated by RIP-1

Background and Purpose

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.

Methods

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.

Results

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.

Conclusions

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.     

β-lapachone significantly increases the effect of ionizing radiation to cause mitochondrial apoptosis via JNK activation in cancer cells

Background

β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.

Methodology/Principal Findings

β-lap enhanced the effect of IR to cause clonogenic cells in NQO1⁺-MDA-MB-231 cells but not in NQO1⁻-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1⁺ cells and not in NQO1⁻ cells and it markedly increased IR-induced apoptosis only in NQO1⁺ cells. Combined treatment of NQO1⁺ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.

Conclusions/Significance

β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1⁺ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.