As the most intensively studied initiator caspase, caspase-9 is a key player in the intrinsic or mitochondrial pathway which is involved in various stimuli, including chemotherapies, stress agents and radiation. Caspase-9 is activated on the apoptosome complex to remain catalytic status and is thought of involving homo-dimerization monomeric zymogens. Failing to activate caspase-9 has profound physiological and pathophysiological outcomes, leading to degenerative and developmental disorders even cancer. To govern the apoptotic commitment process appropriately, plenty of proteins and small molecules involved in regulating caspase-9. Therefore, this review is to summarize recent pertinent literature on the comprehensive description of the molecular events implicated in caspase-9 activation and inhibition, as well as the clinical trials in progress to give deep insight into caspase-9 for suppressing cancer. We hope that our concerns will be helpful for further clinical studies addressing the roles of caspase-9 and its regulators demanded to identify more effective solutions to overcome intrinsic apoptosis-related diseases especially cancer.

Emerging evidence indicates that reprogramming of energy metabolism involving disturbances in energy production from a defect in cellular respiration with a shift to glycolysis is a core hallmark of cancer. Alterations in cancer cell energy metabolism are linked to abnormalities in mitochondrial function. Mitochondrial dysfunction of cancer cells includes increased glycolysis, decreased apoptosis, and resistance to radiotherapy. The study was designed for two main points: firstly, to investigate whether exogenous functional mitochondria can transfer into glioma cells and explore the underlying molecular mechanisms from the perspective of endocytosis; secondly, to further verify whether the mitochondrial transplantation is able to rescue aerobic respiration, attenuate the Warburg effect and enhance the radiosensitivity of gliomas. Methods: Mitochondria were isolated from normal human astrocytes (HA) and immediately co-incubated with starved human glioma cells (U87). Confocal microscopy and gene sequencing were performed to evaluate the ability of isolated mitochondria internalization into U87 cells. The interaction between endocytosis and isolated mitochondria transfer were captured by 3D tomographic microscopy and transmission electron microscopy. NAD + , CD38, cADPR and Ca 2+ release were determined by commercial kits, western blot, HLPC-MS and Fluo-3 AM respectively. PCR array expression profiling and Seahorse XF analysis were used to evaluate the effect of mitochondrial transplantation on energy phenotypes of U87 cells. U87 cells and U87 xenografts were both treated with mitochondrial transplantation, radiation, or a combination of mitochondrial transplantation and radiation. Apoptosis in vitro and in vivo were detected by cytochrome C, cleaved caspase 9 and TUNEL staining. Results: We found that mitochondria from HA could be transferred into starved U87 cells by simple co-incubation. Starvation treatment slowed the rate of glycolysis and decreased the transformation of NAD + to NADH in U87 cells. A large amount of accumulated NAD + was released into the extracellular space. CD38 is a member of the NAD + glycohydrolase family that catalyzes the cyclization of extracellular NAD + to intracellular cADPR. cADPR triggered release of Ca 2+ to promote cytoskeleton remodeling and plasma membrane invagination. Thus, endocytosis involving isolated mitochondria internalization was mediated by NAD + -CD38-cADPR-Ca 2+ signaling. Mitochondrial transfer enhanced gene and protein expression related to the tricarboxylic acid (TCA) cycle, increased aerobic respiration, attenuated glycolysis, reactivated the mitochondrial apoptotic pathway, inhibited malignant proliferation of U87 cells. Isolated mitochondria injected into U87...

Abstract:The aim of the present study was to investigate the radiosensitizing effect of genistein, and the corresponding mechanisms of action on breast cancer cells with different estrogen receptor (ER) status. Human breast cancer cell lines such as MCF-7 (ER-positive, harboring wild-type p53) and MDA-MB-231 (ER-negative, harboring mutant p53) were irradiated with X-rays in the presence or absence of genistein. Cell survival, DNA damage and repair, cell cycle distribution, cell apoptosis, expression of proteins related to G 2 /M cell cycle checkpoint and apoptosis were measured with colony formation assays, immunohistochemistry, flow cytometry and western blot analysis, respectively. Genistein showed relatively weak toxicity to both cell lines at concentrations in the range of 5-20 μM. Using the dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at a 10% cell survival (IC 10 ) were 1.43 for MCF-7 and 1.36 for MDA-MB-231 cells, respectively. Significantly increased DNA damages, arrested cells at G 2 /M phase, decreased homologous recombination repair protein Rad51 foci formation and enhanced apoptotic rates were observed in both cell lines treated by genistein combined with X-rays compared with the irradiation alone. The combined treatment obviously up-regulated the phosphorylation of ATM, Chk2, Cdc25c and Cdc2, leading to permanent G 2 /M phase OPEN ACCESSMolecules 2013, 18 13201 arrest, and up-regulated Bax and p73, down-regulated Bcl-2, finally induced mitochondriamediated apoptosis in both cell lines. These results suggest that genistein induces G 2 /M arrest by the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway and ultimately enhances the radiosensitivity of both ER+ and ER-breast cancer cells through a mitochondria-mediated apoptosis pathway.

Non-small cell lung cancer (NSCLC) cells often possess a hypermethylated Keap1 promoter, which decreases Keap1 mRNA and protein expression levels, thus impairing the Nrf2-Keap1 pathway and thereby leading to chemo- or radio-resistance. In this study, we showed that genistein selectively exhibited a radiosensitizing effect on NSCLC A549 cells but not on normal lung fibroblast MRC-5 cells. Genistein caused oxidative stress in A549 cells rather than MRC-5 cells, as determined by the oxidation of the ROS-sensitive probe DCFH-DA and oxidative damage marked by MDA, PCO or 8-OHdG content. In A549 instead of MRC-5 cells, genistein reduced the level of methylation in the Keap1 promoter region, leading to an increased mRNA expression, thus effectively inhibited the transcription of Nrf2 to the nucleus, which suppressed the Nrf2-dependent antioxidant and resulted in the upregulation of ROS. Importantly, when combined with radiation, genistein further increased the ROS levels in A549 cells whereas decreasing the radiation-induced oxidative stress in MRC-5 cells, possibly via increasing the expression levels of Nrf2, GSH and HO-1. Moreover, radiation combined with genistein significantly increased cell apoptosis in A549 but not MRC-5 cells. Together, the results herein show that the intrinsic difference in the redox status of A549 and MRC-5 cells could be the target for genistein to selectively sensitize A549 cells to radiation, thereby leading to an increase in radiosensitivity for A549 cells.

No abstract

During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways.

Unfolded protein response (UPR) is comprised of complex and conserved stress pathways that function as a short-term adaptive mechanism to reduce levels of unfolded or misfolded proteins and maintain homeostasis in the endoplasmic reticulum (ER). UPR can be triggered by prolonged or persistent ER stress under many physiological or pathological conditions, including radiation exposure. Radiation-induced ER stress elicits autophagy and apoptosis in cancer cells, where C/EBP homologous protein (CHOP) and c-Jun NH2-terminal kinase (JNK) may play crucial roles. However, the specific mechanisms that regulate autophagy and apoptosis through CHOP and JNK after radiation exposure and how the balance of these activities determines the cellular radiosensitivity remain largely unclear. In this study, we found that exposure to X-ray radiation induced ER stress, UPR and high expression of CHOP and JNK. Furthermore, autophagy and apoptosis occurred in sequential order when breast cancer MDA-MB-231 and MCF-7 cells were exposed to X-ray radiation. CHOP gene knockdown with RNA interference inhibited autophagy and enhanced radiosensitivity in MDA-MB-231 cells, while impacting apoptosis and subsequently increasing radioresistance in MCF-7 cells. However, treatment with JNK inhibitor decreased autophagy while promoting apoptosis, thereby leading to radiosensitivity in both cell lines. Our results indicate that CHOP mediates radiation-induced autophagy and apoptosis in a cellular environment. Importantly, the functional consistency of regulating apoptosis and autophagy in these two irradiated breast cancer cell lines suggests that JNK may be more useful as a potential target for maximizing the efficacy of radiation therapy for breast cancers.

Heavy-ion radiotherapy has a potential advantage over conventional radiotherapy due to improved dose distribution and a higher biological effectiveness in cancer therapy. However, there is a little information currently available on the cellular and molecular basis for heavy-ion irradiation-induced cell death. Autophagy, as a novel important target to improve anticancer therapy, has recently attracted considerable attention. In this study, the effect of autophagy induced by high linear energy transfer (LET) carbon ions was examined in various tumor cell lines. To our knowledge, our study is the first to reveal that high-LET carbon ions could induce autophagy in various tumor cells effectively, and the autophagic level in the irradiated cells increased in a dose- and LET-dependent manner. The ability of carbon ions to inhibit the activation of the PI3K/Akt pathway rose with increasing their LET. Moreover, modulation of autophagy in tumor cells could modify their sensitivity to high-LET radiation, and inhibiting autophagy accelerated apoptotic cell death, resulting in an increase in radiosensitivity. Our data imply that targeting autophagy might enhance the effectiveness of heavy-ion radiotherapy.