Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death

Background

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.

Principal Findings

This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.

Conclusions

Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.     

Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy

The alkylating agent temozolomide (TMZ) is the major chemotherapeutic drug used clinically in the treatment of malignant gliomas. This study investigated the mechanism behind TMZ-induced cell death and the possibility that resveratrol might increase TMZ efficacy. TMZ induced both apoptotic cell death and cytoprotective autophagy through a reactive oxygen species (ROS) burst and extracellular signal-regulated kinase (ERK) activation, which was suppressed by resveratrol, resulting in a decrease in autophagy and an increase in apoptosis, suggesting that the ROS/ERK pathway plays a crucial role in the fate of cells after TMZ treatment. Isobolographic analysis indicated that the combination of TMZ and resveratrol has a synergistic effect. Moreover, an in vivo mouse xenograft study also showed that coadministration of resveratrol and TMZ reduced tumor volumes by suppressing ROS/ERK-mediated autophagy and subsequently inducing apoptosis. Taken together, our data indicate that TMZ-induced ROS/ERK-mediated autophagy protected glioma cells from apoptosis, and the combination of resveratrol with TMZ could improve the efficacy of chemotherapy for brain tumors.     

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 microM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.     

β-elemene combined with temozolomide in treatment of brain glioma

Temozolomide (TMZ) is a widely used chemotherapeutic agent for malignant glioma. β-Elemene has been reported to have the ability of passing through the blood-brain barrier and reverse multidrug resistance. In the present study, transport of drugs through the in vitro blood-brain barrier (BBB) model also suggested that β-elemene can assist in TMZ transport to the brain. Plasma and brain pharmacokinetics demonstrated that when β-elemene is used in combination with TMZ, the metabolic rate of TMZ in plasma is slowed, and mean residence time (MRT) in brain is prolonged. The brain tissue distribution at 1 h indicated that the combination of TMZ and β-elemene promotes the distribution of β-elemene in the brain but slightly reduces the distribution of TMZ in the brain. Furthermore the antitumor effect and toxicity in vivo were also investigated. The combination of β-elemene and TMZ was well tolerated and significantly inhibited tumor growth in glioma xenografts. In summary, the present study indicates a synergistic antitumor effect of β-elemene and TMZ in glioma.     

维生素C联合替莫唑胺对胶质瘤细胞的毒性作用及其机制*

目的:探讨维生素C(VC)联合替莫唑胺(TMZ)对胶质瘤细胞活力的毒性作用及其机制。方法:在体外条件下培养人胶质瘤细胞BMG-1和SHG44细胞,设对照组(不施加VC与TMZ)、TMZ组(0.2 mmol/L)、VC(0.5 mmol/L)+TMZ(0.2 mmol/L)组,TMZ(0.2 mmol/L TMZ)+U0126(10 μmol/L)组,每组实验重复3次。采用MTT实验检测细胞生存率;流式细胞术和Annexin V-FITC/PI染色检测细胞凋亡情况; ROS检测试剂盒检测活性氧簇(ROS)水平, Western blot检测与凋亡、自噬及ERK通路相关蛋白的表达。结果:与对照组比较,TMZ组胶质瘤细胞的存活率显著下降(P＜0.05)。与TMZ组比较,VC+TMZ组胶质细胞瘤细胞的存活率显著下降(P＜0.01),VC+TMZ组中细胞凋亡率显著升高,且Bax、Cleaved caspase-3及Cleaved PARP蛋白表达显著增加,Bcl-2表达显著降低,而ROS水平及细胞自噬率显著降低,LC3-II/LC3-1表达显著降低,p62表达显著增加(P均＜0.05)。同时,联用可降低BMG-1和SHG44细胞中的p-ERK1/2相关蛋白的表达水平,且提高细胞凋亡率(P均＜0.05)。结论:VC联合TMZ能够增强对胶质瘤细胞的毒性,而这一作用是通过ERK信号通路来促进细胞凋亡并抑制替莫唑胺所介导的自噬作用。     

Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.     

Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.     

Guanosine promotes cytotoxicity via adenosine receptors and induces apoptosis in temozolomide-treated A172 glioma cells

Gliomas are a malignant tumor group whose patients have survival rates around 12 months. Among the treatments are the alkylating agents as temozolomide (TMZ), although gliomas have shown multiple resistance mechanisms for chemotherapy. Guanosine (GUO) is an endogenous nucleoside involved in extracellular signaling that presents neuroprotective effects and also shows the effect of inducing differentiation in cancer cells. The chemotherapy allied to adjuvant drugs are being suggested as a novel approach in gliomas treatment. In this way, this study evaluated whether GUO presented cytotoxic effects on human glioma cells as well as GUO effects in association with a classical chemotherapeutic compound, TMZ. Classical parameters of tumor aggressiveness, as alterations on cell viability, type of cell death, migration, and parameters of glutamatergic transmission, were evaluated. GUO (500 and 1000 μM) decreases the A172 glioma cell viability after 24, 48, or 72 h of treatment. TMZ alone or GUO plus TMZ also reduced glioma cell viability similarly. GUO combined with TMZ showed a potentiation effect of increasing apoptosis in A172 glioma cells, and a similar pattern was observed in reducing mitochondrial membrane potential. GUO per se did not elevate the acidic vesicular organelles occurrence, but TMZ or GUO plus TMZ increased this autophagy hallmark. GUO did not alter glutamate transport per se, but it prevented TMZ-induced glutamate release. GUO or TMZ did not alter glutamine synthetase activity. Pharmacological blockade of glutamate receptors did not change GUO effect on glioma viability. GUO cytotoxicity was partially prevented by adenosine receptor (A₁R and A_2AR) ligands. These results point to a cytotoxic effect of GUO on A172 glioma cells and suggest an anticancer effect of GUO as a putative adjuvant treatment, whose mechanism needs to be unraveled.     

Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells

Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca(2+) signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies.     

4th International Symposium on Autophagy: Exploiting the Frontiers of Autophagy Research

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.     

Cancer cell death by design: Apoptosis,autophagy and glioma virotherapy

Autophagy has been defined as a mechanism by which oncolytic adenoviruses mediate cell killing in some cancers, including malignant glioma. Until recently, however, adenovirus replication was regarded as a process that induced classical apoptosis in the infected cell. We have assessed the method of conditionally replicating adenovirus (CRAd) death in a model of malignant glioma, considering both autophagy and apoptosis as possible mechanisms of virally-induced cell death. Our initial investigations indicated that autophagy was the predominant system in CRAd-induced cell death in glioma. This appeared to be the case in vitro; however, further investigation in vivo shows that CRAds are capable of inducing both apoptotic and autophagic cell death. In this punctum, we summarize our latest research to uncover the method of oncolytic adenovirus-induced cell death in malignant glioma. Elucidating the relationship between autophagy and apoptosis in glioma virotherapy has significant implications for the design of optimal viral vectors.     

CD147 confers temozolomide resistance of glioma cells via the regulation of β-TrCP/Nrf2 pathway

Background: Drug resistance is one of the biggest challenges in cancer therapy. temozolomide (TMZ) represents the most important chemotherapeutic option for glioma treatment. However, the therapeutic efficacy of TMZ remains very limited due to its frequent resistance in glioma, and the underlying mechanisms were not fully addressed. Herein, we demonstrate that the elevated expression of CD147 contributes to TMZ resistance in glioma cells, potentially through the post-translational regulation of Nrf2 expression.Methods: Cell-based assays of CD147 triggered drug resistance were performed through Edu-incorporation assay, CCK8 assay, TUNEL staining assay and flow cytometric assay. Luciferase reporter assay, protein stability related assays, co-immunoprecipitation assay were used to determine CD147 induction of Nrf2 expression through β-TrCP dependent ubiquitin system. Finally, the effect of the CD147/Nrf2 signaling on glioma progression and TMZ resistance were evaluated by functional experiments and clinical samples.Results: Based on the analysis of clinical glioma tissues, CD147 is highly expressed in glioma tissues and positively associated with tumor malignancy. Suppression of CD147 expression increased the inhibitory effect of TMZ on cell survival in both U251 and T98G cells, whereas the gain of CD147 function blocked TMZ-induced ROS production and cell death. Mechanistic study indicates that CD147 inhibited GSK3β/β-TrCP-dependent Nrf2 degradation by promoting Akt activation, and subsequently increased Nrf2-mediated anti-oxidant gene expressions. Supporting the biological significance, the reciprocal relationship between CD147 and Nrf2 was observed in glioma tissues, and associated with patient outcome.Conclusions: Our data provide the first evidence that glioma resistance to TMZ is potentially due to the activation of CD147/Nrf2 axis. CD147 promotes Nrf2 stability through the suppression of GSK3β/β-TrCP dependent Nrf2 protein degradation, which results in the ablation of TMZ induced ROS production. As such, we point out that targeting CD147/Nrf2 axis may provide a new strategy for the treatment of TMZ resistant gliomas.     

Minocycline inhibits the growth of glioma by inducing autophagy

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.     

A novel approach to overcome temozolomide resistance in glioma and melanoma: Inactivation of MGMT by gene therapy

Malignant glioma is the most common primary brain tumor. Malignant melanoma is the most malignant of skin tumor. The two malignancies are poorly responsive to conventional treatment regimens such as chemotherapy. Temozolomide (TMZ) is a DNA-alkylating agent used for the treatment of glioma, astrocytoma, and melanoma. Resistance to alkylating agents such as TMZ correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Several studies in animal models have demonstrated that decreasing MGMT level with gene therapy could overcome TMZ resistance and enhance tumor cell death. In the present review, we provide an overview of recent advances in this field.     

Hsa_circ_0110757 upregulates ITGA1 to facilitate temozolomide resistance in glioma by suppressing hsa-miR-1298-5p

Temozolomide (TMZ) is the internationally recognized and preferred drug for glioma chemotherapy treatment. However, TMZ resistance in glioma appears after long-term use and is an urgent problem that needs to be solved. Circular RNAs (circRNAs) are noncoding RNAs and play an important role in the pathogenesis and progression of tumors. Hsa_circ_0110757 was identified in TMZ-resistant glioma cells by high-throughput sequencing analysis and was derived from reverse splicing of myeloid cell leukemia-1 (Mcl-1) exons. The role of hsa_circ_0110757 in TMZ-resistant glioma was evaluated both in vitro and in vivo. It was found that hsa_circ_0110757 and ITGA1 are more highly expressed in TMZ-resistant glioma than in TMZ-sensitive glioma. The overexpression of hsa_circ_0110757 in glioma patients treated with TMZ was obviously associated with tumor invasion. This study indicates that hsa_circ_0110757 inhibits glioma cell apoptosis by sponging hsa-miR-1298-5p to promote ITGA1 expression. Thus, hsa_circ_0110757/hsa-miR-1298-5p/ITGA could be a potential therapeutic target for reversing the resistance of glioma to TMZ.Subject terms: Chemotherapy, Tumour-suppressor proteins     

Integrated multi-omics analysis reveals unique signatures of paclitaxel-loaded poly(lactide-co-glycolide) nanoparticles treatment of head and neck cancer cells

The use of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as carriers for chemotherapeutic drugs is regarded as an actively targeted nano-therapy for the specific delivery of anti-cancer drugs to target cells. However, the exact mechanism by which PLGA NPs boost anticancer cytotoxicity at the molecular level remains largely unclear. This study employed different molecular approaches to define the response of carcinoma FaDu cells to different types of treatment, specifically: paclitaxel (PTX) alone, drug free PLGA NPs, and PTX-loaded PTX-PLGA NPs. Functional cell assays revealed that PTX-PLGA NPs treated cells had a higher level of apoptosis than PTX alone, whereas the complementary, UHPLC-MS/MS (TIMS-TOF) based multi-omics analyses revealed that PTX-PLGA NPs treatment resulted in increased abundance of proteins associated with tubulin, as well as metabolites such as 5-thymidylic acid, PC(18:1(9Z)/18:1(9Z0), vitamin D, and sphinganine among others. The multi-omics analyses revealed new insights about the molecular mechanisms underlying the action of novel anticancer NP therapies. In particular, PTX-loaded NPs appeared to exacerbate specific changes induced by both PLGA-NPs and PTX as a free drug. Hence, the PTX-PLGA NPs’ molecular mode of action, seen in greater detail, depends on this synergy that ultimately accelerates the apoptotic process, resulting in cancer cell death.     

Oroxylin A increases the sensitivity of temozolomide on glioma cells by hypoxia-inducible factor 1α/hedgehog pathway under hypoxia

Microenvironmental hypoxia-mediated drug resistance is responsible for the failure of cancer therapy. To date, the role of the hedgehog pathway in resistance to temozolomide (TMZ) under hypoxia has not been investigated. In this study, we discovered that the increasing hypoxia-inducible factor 1α (HIF-1α) activated the hedgehog pathway in hypoxic microenvironment by promoting autocrine secretion of sonic hedgehog protein (Shh), and then upregulating transfer of Gli1 to the nucleus, finally contributed to TMZ resistance in glioma cells. Oroxylin A (C16H12O5), a bioactive flavonoid, could induce HIF-1α degradation via prolyl-hydroxylases–VHL signaling pathway, resulting in the inactivation of the hedgehog. Besides, oroxylin A increased the expression of Sufu, which is a negative regulator of Gli1. By this mechanism, oroxylin A sensitized TMZ on glioma cells. U251 intracranial transplantation model and GL261 xenograft model were used to confirm the reversal effects of oroxylin A in vivo. In conclusion, our results demonstrated that HIF-1α/hedgehog pathway conferred TMZ resistance under hypoxia, and oroxylin A was capable of increasing the sensitivity of TMZ on glioma cells in vitro and in vivo by inhibiting HIF-1α/hedgehog pathway and depressing the activation of Gli1 directly.     

Autophagy regulation by inorganic,organic, and organic/inorganic hybrid nanoparticles: Organelle damage,regulation factors,and potential pathways

The rapid development of nanotechnology requires a more thorough understanding of the potential health effects caused by nanoparticles (NPs). As a programmed cell death, autophagy is one of the biological effects induced by NPs, which maintain intracellular homeostasis by degrading damaged organelles and removing aggregates of defective proteins through lysosomes. Currently, autophagy has been shown to be associated with the development of several diseases. A significant number of research have demonstrated that most NPs can regulate autophagy, and their regulation of autophagy is divided into induction and blockade. Studying the autophagy regulation by NPs will facilitate a more comprehensive understanding of the toxicity of NPs. In this review, we will illustrate the effects of different types of NPs on autophagy, including inorganic NPs, organic NPs, and organic/inorganic hybrid NPs. The potential mechanisms by which NPs regulate autophagy are highlighted, including organelle damage, oxidative stress, inducible factors, and multiple signaling pathways. In addition, we list the factors influencing NPs-regulated autophagy. This review may provide basic information for the safety assessment of NPs.