Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

Background

Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear.

Methodology/Principal Findings

In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis.

Conclusions

These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies.     

Inhibition of Nrf2 promotes the antitumor effect of Pinelliae rhizome in papillary thyroid cancer

We previously reported that Xiaotan Sanjie (XTSJ) decoction can prevent the progression of gastric cancer in vitro and in vivo. Pinelliae rhizome (PR), one component of XTSJ decoction, has an inhibitory effect on the growth and proliferation of tumor cells. The present study investigated the underlying mechanisms of action of PR. Using the human papillary thyroid cancer cell lines, TPC-1 and BCPAP, we found that XTSJ decoction and PR alone decreased cell viability to a similar extent in both cell lines, whereas treatment with XTJS decoction without PR [PR (−)] had a lesser effect. PR treatment inhibited the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in a dose-dependent manner. To investigate the role of Nrf2 in the PR-mediated effects of XTSJ, knockdown of Nrf2 in the tumor cell lines using Nrf2 siRNA (siNrf2) was performed and transfected cells were treated with PR. Silencing of Nrf2 amplified the effects on autophagy, cell viability, apoptosis, and colony formation. Similar results were obtained following treatment with the autophagy inhibitor 3-methyladenine (3-MA). Furthermore, treatment with PR, siNrf2, and/or 3-MA inhibited the MAPK pathway, and analysis of the MAPK pathway components confirmed the role of this pathway in the PR-mediated cellular effects. In mice implanted with siNrf2-transfected cells, the effects of PR were amplified. Taken together, these findings indicate that PR is critical for the inhibitory effects of XTSJ decoction on tumor cell viability and that downregulation of Nrf2 promotes the antitumor effects of PR on papillary thyroid cancer cells.     

Daphnetin triggers ROS-induced cell death and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway in ovarian cancer

BackgroundOvarian cancer is one of the most common gynecological malignancies in the world. Daphnetin (Daph) was previously reported to possess antitumor potential, but its potential and molecular mechanisms in ovarian cancer remain poorly understood.PurposeIn the current study, we aimed to explore the antitumor effect and detailed mechanisms of Daph in ovarian cancer cells.MethodsThe cytotoxic effect of Daph on ovarian cells was determined in vitro and in vivo. Cell growth, proliferation, apoptosis and ROS generation were measured by CCK8 assays, colony formation assays and flow cytometry. Western blotting was used to evaluate the related signal proteins. Immunofluorescence and transmission electron microscopy were used to evaluate markers of autophagy and autophagic flux. The antitumor effects were observed in the A2780 xenograft model. Moreover, Daph-induced autophagy was observed by enhanced LC3-II accumulation and endogenous LC3 puncta, and an autophagy inhibitor further enhanced the antitumor efficacy of Daph, which indicated that the cytoprotective role of autophagy in ovarian cancer.ResultsWe found that Daph exhibited antitumor effects by inducing ROS-dependent apoptosis in ovarian cancer, which could be reversed by N-acetyl cysteine (NAC). The AMPK/Akt/mTOR pathway was involved in Daph-mediated cytoprotective autophagy, and when Daph-mediated the expression level of AMPK and autophagy were blocked, there was robust inhibition of cell proliferation and induction of apoptosis. In addition, in the A2780 xenograft model, combined treatment with Daph and an autophagy inhibitor showed obvious synergetic effects on the inhibition of cell viability and promotion of apoptosis, without any side effects.ConclusionOur results suggest that Daph triggers ROS-induced cell apoptosis and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway. Moreover, the combination of Daph and autophagy inhibitor may be a potential therapeutic strategy for ovarian cancer.     

GMI,an immunomodulatory protein from Ganoderma microsporum,induces autophagy in non-small cell lung cancer cells

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.     

Autophagy inhibition enhances celecoxib-induced apoptosis in osteosarcoma

Osteosarcoma (OS) is the most prevalent bone malignancy in childhood and adolescence, with highly aggressive and early systemic metastases. Here, we reported that celecoxib, a selective COX-2 inhibitor in the NSAID class, exhibits strong antitumor activity in dose dependent manner in two OS cell lines-143B and U2OS. We showed that celecoxib inhibits OS cell growth, causes G0/G1-phase arrest, modulates apoptosis and autophagy and reduces migration in OS cells. In addition, the results of fluorescent mitochondrial probe JC-1 test indicated that the mitochondrial pathway mediates celecoxib-induced apoptosis. Significantly, the autophagy inhibitor CQ combined with celecoxib causes greater cell proliferation inhibition and apoptosis. Pharmacologic inhibition of autophagy with another potent autophagy inhibitor SAR405 also enhances celecoxib-mediated suppression of cell viability. These results were confirmed with shRNAs targeting the autophagy-related gene Atg5. In OS tumor xenografts in vivo, celecoxib also presents antitumor activity. Taken together, our results shed light on the function and mechanism of antitumor action of celecoxib for treatment of OS patients.     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.     

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Dual inhibition of autophagy and the AKT pathway in prostate cancer

Genetic inactivation of PTEN through either gene deletion or mutation is common in metastatic prostate cancer, leading to activation of the phosphoinositide 3-kinase (PI3K-AKT) pathway, which is associated with poor clinical outcomes. The PI3K-AKT pathway plays a central role in various cellular processes supporting cell growth and survival of tumor cells. To date, therapeutic approaches to develop inhibitors targeting the PI3K-AKT pathway have failed in both pre-clinical and clinical trials. We showed that a novel AKT inhibitor, AZD5363, inhibits the AKT downstream pathway by reducing p-MTOR and p-RPS6KB/p70S6K. We specifically reported that AZD5363 monotherapy induces G2 growth arrest and autophagy, but fails to induce significant apoptosis in PC-3 and DU145 prostate cancer cell lines. Blocking autophagy using pharmacological inhibitors (3-methyladenine, chloroquine and bafilomycin A₁) or genetic inhibitors (siRNA targeting ATG3 and ATG7) enhances cell death induced by AZD5363 in these prostate cancer cells. Importantly, the combination of AZD5363 with chloroquine significantly reduces tumor volume compared with the control group, and compared with either drug alone in prostate tumor xenograft models. Taken together, these data demonstrate that AKT inhibitor AZD5363, synergizes with the lysosomotropic inhibitor of autophagy, chloroquine, to induce apoptosis and delay tumor progression in prostate cancer models that are resistant to monotherapy, with AZD5363 providing a new therapeutic approach potentially translatable to patients.     

Protective effects of Cinnamomum verum,Cinnamomum cassia and cinnamaldehyde against 6-OHDA-induced apoptosis in PC12 cells

Cinnamon (Cinnamomum verum and C. cassia) is a medicinal plant, widely-used as a culinary spice. It possesses various therapeutic effects and can slow down the progression of neurological disorders impressively. In this article, the effects of hydro-alcohol extract and essential oil of C. verum and C. cassia and its main bioactive component cinnamaldehyde, has been examined on 6-OHDA-exposed PC12 cells as an in vitro model of Parkinson's disease. The cytotoxicity and cell apoptosis has been induced by 6-OHDA in PC12 cells. The protective effect was determined by measuring cell viability, the amount of reactive oxygen species (ROS), and apoptosis. Cell viability and apoptosis were assessed using resazurin assay, flow cytometry of propidium iodide (PI) stained cells, and western blot analysis. 6-OHDA resulted in the death and apoptosis of cells while, pretreatment with the extract and essential oil of C. verum and C. cassia at 20 µg/ml and cinnamaldehyde at 5 and 10 µM for 24 h could significantly increase the viability (p?<?0.001), and decrease ROS content (p?<?0.05). Pretreatment with the extracts increased survivin and decreased cyt-c whereas, pretreatment with the essential oil decreased cyt-c, increased survivin, and reduced P-p44/42/p44/42 levels to a level near that of the related control. The extract and essential oil of C. verum and C. cassia can be effective against 6-OHDA cytotoxicity. It is suggested that, the synergistic effects of cinnamaldehyde and other components of extract and essential oil promote cinnamon’s medicinal properties.

     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

We investigated the effects of KML001 (NaAsO₂, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-_L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-_L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

A concomitant ATP-depleting strategy markedly enhances anticancer agent activity

Most anticancer agents effect DNA damage which initiate the cell death pathways of necrosis and apoptosis, but cancer cells of lesser sensitivity are only sublethally injured, and recover. The two death pathways and their interelationships in the presence of endogenous inhibitors of apoptosis and genetic deletions that facilitates only sublethal damage, are reviewed.Both ATP and pyrimidine levels in the sublethally injured cancer cells are reduced but not to low levels insuffient to sustain cell viability. However, this sublethal damage by the anticancer agent creates a therapeutic opportunity for further reduction of these key metabolites to lower levels that will not support life. Data in tumor-bearing animals is reviewed demonstrating that a combination of ATP-depleting agents plus a de novo pyrimidine inhibitor (PALA) administered concomitantly with each of nine different anticancer agents markedly enhances tumor regression rates, and even produces some cures. It is necessary to deplete tumor ATP levels seveerely (>85%) by a combination of agents that block both synthesis(6-methylmercaptopurine riboside, a purine de novo synthesis inhibitor) and generation of ATP(6-aminonicotin-amide, an inhibitor of glycolysis.) Cell viability cannot be sustained if the intracellular ATP level is reduced to 15% of normal or below. In vivo data employing this novel therapeutic strategy with cisplatin is presented. The potential significance of these findings to the improvement of cancer treatment is discussed.     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.Key words: EGFR, cetuximab, autophagy, apoptosis, cancer therapy     

Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl₂), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) – surprisingly – autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl₂. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

Novel combination of Celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Celecoxib (Celebrex®) exhibits antitumor effects in human HCC cells, and its mechanism of action is mediated either by its ability to inhibit cyclooxygenase 2 (COX-2) or by a number of various other COX-2 independent effects. Proteasome inhibitors (PIs) can exert cell growth inhibitory and apoptotic effects in different tumor cell types, including HCC cells. The present study examined the interaction between celecoxib and the PI MG132 in two human liver tumor cell lines HepG2 and HA22T/VGH. Our data showed that each inhibitor reduced proliferation and induced apoptosis in a dose-dependent manner in both cell lines. Moreover, the combination of celecoxib with MG132 synergistically inhibited cell viability and increased apoptosis, as documented by caspase 3 and 7 activation, PARP cleavage, and down-regulation of Bcl-2. Celecoxib and MG132, both alone and synergistically in combination, induced expression of the endoplasmic reticulum (ER) stress genes ATF4, CHOP, TRB3 and promoted the splicing of XBP1 mRNA. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased combination-induced cell death in HA22T/VGH cells, whereas it increased combination-induced cell death in HepG2 cells, suggesting that activation of the ER stress response might have either a detrimental or a protective role in liver tumor cell survival. In conclusion, our data indicate that combination treatment with celecoxib and MG132 resulted in synergistic antiproliferative and proapoptotic effects against liver cancer cells, providing a rational basis for the clinical use of this combination in the treatment of liver cancer.     

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.