Down‐regulation of Notch‐1 is associated with Akt and FoxM1 in inducing cell growth inhibition and apoptosis in prostate cancer cells

Although many studies have been done to uncover the mechanisms by which down‐regulation of Notch‐1 exerts its anti‐tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch‐1 down‐regulation and also assessed the molecular consequence of Notch‐1‐mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down‐regulation of Notch‐1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down‐regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch‐1 signaling. Moreover, we found that a “natural agent” (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down‐regulation of Notch‐1, Akt, and FoxM1. These results suggest that down‐regulation of Notch‐1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa. J. Cell. Biochem. 112: 78–88, 2011. © 2010 Wiley‐Liss, Inc.     

Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells

Lysosomes are acidic organelles that have a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, was reported as an autophagy activator. Here, in contrast with previous studies, we show that Tet is a potent lysosomal deacidification agent and is able to block autophagic flux in the degradation stage. Single-agent Tet induces significant apoptosis both in vitro and in xenograft models. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and an increased level of microtubule-associated protein 1 light chain 3, type II (LC3-II). However, Tet increased the level of sequestosome 1 and decreased the turnover of LC3, indicating the blockade of autophagic flux in the degradation stage. As blockade of autophagic flux decreases the recycling of cellular fuels, Tet reduces the uptake of glucose in cancer cells. These effects lead to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. Blunting autophagosome formation using 3-methyladenine or genetic knockdown of Beclin-1 failed to rescue cells upon Tet treatment. By contrast, addition of methyl pyruvate to supplement TCA substrates protected Tet-treated tumor cells. These results demonstrate that energetic impairment is required in Tet-induced apoptosis. Tet, as a potent lysosomal inhibitor, is translatable to the treatment of malignant tumor patients.     

Anti‐cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition,Beclin‐1 for autophagy induction

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti‐apoptosis engineering. Recently, autophagy has received attention as a new anti‐cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti‐apoptosis and pro‐autophagy in CHO cells (DG44) was attempted by co‐overexpressing an anti‐apoptotic protein, Bcl‐2, and a key regulator of autophagy pathway, Beclin‐1, respectively. Co‐overexpression of Bcl‐2 and Beclin‐1 exhibited a longer culture period as well as higher viability during serum‐free suspension culture, compared with the control (without co‐overexpression of Bcl‐2 and Beclin‐1) and Bcl‐2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl‐2 overexpression, Beclin‐1 overexpression successfully induced the increase in the autophagic marker protein, LC3‐II, and autophagosome formation with the decrease in mTOR activity. Co‐immunoprecipitation and qRT‐PCR experiments revealed that the enforced expression of Beclin‐1 increased Ulk1 expression and level of free‐Beclin‐1 that did not bind to the Bcl‐2 despite the Bcl‐2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co‐overexpression of Bcl‐2 and Beclin‐1 also protected the cells from cell death more efficiently than Bcl‐2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro‐autophagy engineering together with anti‐apoptosis engineering yields a synergistic effect and successfully enhances the anti‐cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195–2207. © 2013 Wiley Periodicals, Inc.     

Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: The cellular mechanism

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti‐tumor properties. To better understand berberine‐induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97‐L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine‐induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3‐methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97‐L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97‐L cells through activation of Beclin‐1 and inhibition of the mTOR‐signaling pathway by suppressing the activity of Akt and up‐regulating P38 MAPK signaling. This is the first study to describe the role of Beclin‐1 activation and mTOR inhibition in berberine‐induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms. J. Cell. Biochem. 111: 1426–1436, 2010. © 2010 Wiley‐Liss, Inc.     

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G₁/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G₁/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G₁/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.     

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G₁/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G₁/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G₁/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.     

Inhibition of cell growth and induction of apoptosis in non-small cell lung cancer cells by delta-tocotrienol is associated with notch-1 down-regulation

Lung cancer is the leading cause of death among all cancers. Non-small cell lung cancer accounts for 80% of lung cancer with a 5-year survival rate of 16%. Notch pathway, especially Notch-1 is up-regulated in a subgroup of non-small cell lung cancer patients. Since Notch-1 signaling plays an important role in cell proliferation, differentiation, and apoptosis, down-regulation of Notch-1 may exert anti-tumor effects. The objective of this study was to investigate whether delta-tocotrienol, a naturally occurring isoform of Vitamin E, inhibits non-small cell lung cancer cell growth via Notch signaling. Treatment with delta-tocotrienol resulted in a dose and time dependent inhibition of cell growth, cell migration, tumor cell invasiveness, and induction of apoptosis. Real-time RT-PCR and western blot analysis showed that antitumor activity by delta-tocotrienol was associated with a decrease in Notch-1, Hes-1, Survivin, MMP-9, VEGF, and Bcl-XL expression. In addition, there was a decrease in NF-κB-DNA binding activity. These results suggest that down-regulation of Notch-1, via inhibition of NF-κB signaling pathways by delta-tocotrienol, could provide a potential novel approach for prevention of tumor progression in non-small cell lung cancer.     

Induction of apoptotic but not autophagic cell death by Cinnamomum cassia extracts on human oral cancer cells

Cinnamomum cassia has been widely studied in different fields to reveal its antidiabetic, antidepressive, antiviral, anti-inflammatory, antiosteoporotic, and anticancer effects. Its antimalignant activities have been explored in lung cancer, breast cancer, colorectal cancer, and even oral cancer, but the detailed signaling mechanism and effects of this plant on animal models need to be clarified. In the current study, C. cassia extract (CCE) was used to investigate the antitumorigenesis mechanism in vitro and in vivo. The major constituents of CCE used in this study were coumarin, cinnamic acid, and cinnamic aldehyde. CCE reduced the viability, number, and colony formation of human oral cancer cells, and induced their apoptosis. Caspase-3 activation, Bcl-2 reduction, and phosphatidylserine inversion were involved in CCE-stimulated apoptosis. CCE also enhanced the expression of autophagic markers, including acidic vesicular organelle, microtubule-associated protein 1 light chain 3-I, autophagy-related protein 14, rubicon, and p62. The combined treatment of CCE and caspase inhibitor significantly restored mitochondrial membrane potential (Δ ψ _m) and cell viability. However, the combined treatment of CCE and autophagy inhibitor further reduced the cell viability indicating that autophagy might be a survival pathway of CCE-treated SASVO3 cells. In contrast, CCE treatment for 12 days did not adversely affect SASVO3 tumor-bearing nude mice. CCE also elicited dose-dependent effects on the decrease in tumor volume, tumor weight, and Ki-67 expression. These results suggested that CCE showed the potential for the complementary treatment of oral caner.     

Cannabinoid receptor 1 modulates the autophagic flux independent of mTOR‐ and BECLIN1‐complex

Cannabinoid Receptor 1 (CB1) has been initially described as the receptor for Delta‐9‐Tetrahydrocannabinol in the central nervous system (CNS), mediating retrograde synaptic signaling of the endocannabinoid system. Beside its expression in various CNS regions, CB1 is ubiquituous in peripheral tissues, where it mediates, among other activities, the cell's energy homeostasis. We sought to examine the role of CB1 in the context of the evolutionarily conserved autophagic machinery, a main constituent of the regulation of the intracellular energy status. Manipulating CB1 by siRNA knockdown in mammalian cells caused an elevated autophagic flux, while the expression of autophagy‐related genes remained unaltered. Pharmacological inhibition of CB1 activity using Rimonabant likewise caused an elevated autophagic flux, which was independent of the mammalian target of rapamycin complex 1, a major switch in the control of canonical autophagy. In addition, knocking down coiled‐coil myosin‐like BCL2‐interacting protein 1, the key‐protein of the second canonical autophagy control complex, was insufficient to reduce the elevated autophagic flux induced by Rimonabant. Interestingly, lysosomal activity is not altered, suggesting a specific effect of CB1 on the regulation of autophagic flux. We conclude that CB1 activity affects the autophagic flux independently of the two major canonic regulation complexes controlling autophagic vesicle formation.